US20040200909A1 - Apparatus and method for cell disruption - Google Patents
Apparatus and method for cell disruption Download PDFInfo
- Publication number
- US20040200909A1 US20040200909A1 US10/759,741 US75974104A US2004200909A1 US 20040200909 A1 US20040200909 A1 US 20040200909A1 US 75974104 A US75974104 A US 75974104A US 2004200909 A1 US2004200909 A1 US 2004200909A1
- Authority
- US
- United States
- Prior art keywords
- chamber
- cartridge
- wall
- transducer
- container
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
Images
Classifications
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L7/00—Heating or cooling apparatus; Heat insulating devices
- B01L7/52—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
- C12M47/06—Hydrolysis; Cell lysis; Extraction of intracellular or cell wall material
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
- B01L2200/10—Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/06—Auxiliary integrated devices, integrated components
- B01L2300/0627—Sensor or part of a sensor is integrated
- B01L2300/0654—Lenses; Optical fibres
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/06—Auxiliary integrated devices, integrated components
- B01L2300/0681—Filter
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/08—Geometry, shape and general structure
- B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
- B01L2300/087—Multiple sequential chambers
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/14—Means for pressure control
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2400/00—Moving or stopping fluids
- B01L2400/04—Moving fluids with specific forces or mechanical means
- B01L2400/0475—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure
- B01L2400/0487—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure fluid pressure, pneumatics
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2400/00—Moving or stopping fluids
- B01L2400/06—Valves, specific forms thereof
- B01L2400/0622—Valves, specific forms thereof distribution valves, valves having multiple inlets and/or outlets, e.g. metering valves, multi-way valves
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2400/00—Moving or stopping fluids
- B01L2400/06—Valves, specific forms thereof
- B01L2400/0633—Valves, specific forms thereof with moving parts
- B01L2400/0644—Valves, specific forms thereof with moving parts rotary valves
Definitions
- the present invention relates to an apparatus and method for rapidly disrupting cells or viruses.
- nucleic acid may be used for genetic analysis, e.g., sequencing, pathogen identification and quantification, nucleic acid mutation analysis, genome analysis, gene expression studies, pharmacological monitoring, storing of DNA libraries for drug discovery, etc.
- the genetic analysis typically involves nucleic acid amplification and detection using known techniques.
- known polynucleotide amplification reactions include polymerase chain reaction (PCR), ligase chain reaction (LCR), QB replicase amplification (QBR), self-sustained sequence replication (3SR), strand-displacement amplification (SDA), “branched chain” DNA amplification, ligation activated transcription (LAT), nucleic acid sequence-based amplification (NASBA), repair chain reaction (RCR), and cycling probe reaction (CPR).
- PCR polymerase chain reaction
- LCR ligase chain reaction
- QBR QB replicase amplification
- SDA self-sustained sequence replication
- LAT ligation activated transcription
- NASBA nucleic acid sequence-based amplification
- RCR repair chain reaction
- CPR cycling probe reaction
- the extraction of nucleic acids from cells or viruses is generally performed by physical or chemical methods.
- Chemical methods typically employ lysing agents (e.g., detergents, enzymes, or strong organics) to disrupt the cells and release the nucleic acid, followed by treatment of the extract with chaotropic salts to denature any contaminating or potentially interfering proteins.
- lysing agents e.g., detergents, enzymes, or strong organics
- Such chemical methods are described in U.S. Pat. No. 5,652,141 to Henco et al. and U.S. Pat. No. 5,856,174 to Lipshutz et al.
- One disadvantage to the use of harsh chemicals for disrupting cells is that the chemicals are inhibitory to subsequent amplification of the nucleic acid. In using chemical disruption methods, therefore, it is typically necessary to purify the nucleic acid released from the cells before proceeding with further analysis. Such purification steps are time consuming, expensive, and reduce the amount of nucleic acid recovered for analysis.
- Physical methods for disrupting cells often do not require harsh chemicals that are inhibitory to nucleic acid amplification (e.g., PCR). These physical methods, however, also have their disadvantages.
- one physical method for disrupting cells involves placing the cells in a solution and heating the solution to a boil to break open the cell walls. Unfortunately, the heat will often denature proteins and cause the proteins to stick to the released nucleic acid. The proteins then interfere with subsequent attempts to amplify the nucleic acid.
- Another physical method is freeze thawing in which the cells are repeatedly frozen and thawed until the cells walls are broken. Unfortunately, freeze thawing often fails to break open many structures, most notably certain spores and viruses that have extremely tough outer layers.
- Another physical method for disrupting cells is the use of a pressure instrument.
- a solution of mycobacterial microorganisms is passed through a very small diameter hole under high pressure.
- the mycobacteria are broken open by the mechanical forces and their internal contents are spilled into solution.
- Such a system is large, expensive and requires a cooling system to prevent excessive heat from building up and damaging the contents of the lysed cells.
- the instrument needs to be cleaned and decontaminated between runs and a large containment system is required when infectious material is handled.
- a further disadvantage to this system is that the solution must contain only particles having substantially the same size, so that it may not be used to process many untreated clinical or biological specimens.
- cells can be lysed by subjecting the cells to ultrasonic agitation. Typically, the cells are disrupted by placing an ultrasonic probe directly into a volume of liquid containing the cells. Since the probe is in direct contact with a sample liquid, cross contamination and cavitation-induced foaming present serious complications.
- the present invention overcomes the disadvantages of the prior art by providing an improved apparatus and method for disrupting cells or viruses.
- the present invention provides for the rapid and effective disruption of cells or viruses, including tough spores, without requiring the use of harsh chemicals.
- the disruption of the cells or viruses can often be completed in 5 to 10 seconds.
- the apparatus and method of the present invention provide for highly consistent and repeatable lysis of cells or viruses, so that consistent results are achieved from one use of the apparatus to the next.
- the apparatus comprises a container having a chamber for holding the cells or viruses.
- the container includes at least one flexible wall defining the chamber.
- the apparatus also includes a transducer, such as an ultrasonic horn, for impacting an external surface of the flexible wall to generate dynamic pressure pulses or pressure waves in the chamber.
- the apparatus also includes a pressure source for increasing the pressure in the chamber. The pressurization of the chamber ensures effective coupling between the transducer and the flexible wall.
- the apparatus may also include beads in the chamber for rupturing the cells or viruses.
- the cells or viruses to be disrupted are placed in the chamber of the container.
- a liquid is also placed in the chamber.
- the cells or viruses are placed in the chamber by capturing the cells or viruses on at least one filter positioned in the chamber.
- the liquid placed in the chamber is usually a lysis buffer added to the chamber after the cells or viruses have been captured.
- the liquid placed in the chamber contains the cells or viruses to be disrupted (e.g., the liquid is a sample containing the cells or viruses) so that the liquid and cells are placed in the chamber simultaneously.
- the transducer is placed against the external surface of the flexible wall, and the static pressure in the chamber is increased. Disruption of the cells is accomplished by impacting the flexible wall with the transducer to generate dynamic pressure pulses or pressure waves in the chamber. Beads may also be agitated in the chamber to rupture the cells or viruses.
- FIG. 1 is an isometric view of a cartridge for analyzing a fluid sample according to a first embodiment of the invention.
- FIG. 2 is a lower isometric view of the cartridge of FIG. 1.
- FIG. 3 is an exploded view of the cartridge of FIG. 1.
- FIG. 4 is another exploded view of the cartridge of FIG. 1.
- FIG. 5 is a partially cut away view of an ultrasonic horn coupled to a wall of a lysing chamber formed in the cartridge of FIG. 1.
- FIG. 6 is an exploded view of a filter stack positioned in the lysing chamber of the cartridge of FIG. 1.
- FIG. 7 is a top plan view of the cartridge of FIG. 1.
- FIG. 8 is a bottom plan view of the cartridge of FIG. 1.
- FIG. 9 is a schematic block diagram of the cartridge of FIG. 1.
- FIG. 10 is an isometric view of an instrument into which the cartridge of FIG. 1 is placed for processing.
- FIG. 11 is an isometric view of the cartridge of FIG. 1 in the instrument of FIG. 10.
- FIG. 12 is a partially cut-away view of the cartridge of FIG. 1 in the instrument of FIG. 10.
- FIG. 13 is a schematic, plan view of optical sensors positioned to detect liquid levels in the cartridge of FIG. 1.
- FIG. 14 is a partially cut away, schematic, side view of a slotted optical sensor positioned to detect the liquid level in a sensor chamber of the cartridge of FIG. 1.
- FIG. 15A is a cross-sectional view of a portion of the body of the cartridge of FIG. 1 illustrating two different types of valves in the cartridge.
- FIG. 15B is a cross-sectional view of the valves of FIG. 15A in a closed position.
- FIG. 16A is another cross-sectional view of one of the valves of FIG. 15A in an open position.
- FIG. 16B is a cross-sectional view of the valve of FIG. 16A in a closed position.
- FIGS. 17-19 illustrate a valve actuation system for opening and closing the valves of FIG. 15A.
- FIG. 20 is a cross sectional view of alternative valve actuators for opening and closing the valves in the cartridge of FIG. 1.
- FIG. 20 also shows a pressure delivery nozzle sealed to a pressure port formed in the cartridge of FIG. 1.
- FIG. 21 is a partially exploded, isometric view of a reaction vessel of the cartridge of FIG. 1.
- FIG. 22 is a front view of the vessel of FIG. 21.
- FIG. 23 is a side view of the vessel of FIG. 21 inserted between two heater plates.
- FIG. 24 is a front view of one of the heater plates of FIG. 23.
- FIG. 25 is a front view of an alternative reaction vessel according to the present invention.
- FIG. 26 is a front view of another reaction vessel according to the present invention.
- FIG. 27 is another front view of the vessel of FIG. 21.
- FIG. 28 is a front view of the vessel of FIG. 21 inserted into a heat-exchanging module of the instrument of FIG. 10.
- FIG. 29 is an exploded view of a support structure for holding the plates of FIG. 23.
- FIGS. 30-31 are assembled views of the support structure of FIG. 29.
- FIG. 32 is an isometric view showing the exterior of one the optics assemblies in the heat-exchanging module of FIG. 28.
- FIG. 33 is an isometric view of the plates of FIG. 23 in contact with the optics assembly of FIG. 32.
- FIG. 34 is a partially cut away, isometric view of the reaction vessel of FIG. 21 inserted between the plates of FIG. 23. Only the lower portion of the vessel is included in the figure.
- FIG. 35 is a schematic block diagram of the electronics of the heat-exchanging module of FIG. 28.
- FIG. 36 is an isometric view of an apparatus for disrupting cells or viruses according to another embodiment of the invention.
- FIG. 37 is a cross sectional view of the apparatus of FIG. 36.
- FIG. 38 is an exploded view of a container used in the apparatus of FIG. 36.
- FIG. 39 is a cross sectional view of the container of FIG. 38.
- FIG. 40 is a schematic block diagram of a fluidic system incorporating the apparatus of FIG. 36.
- FIG. 41 is a cross sectional view of another container for use in the apparatus of FIG. 36.
- An ultrasonic horn is in contact with a wall of the container that curves outwardly towards the horn.
- FIG. 42 is a cross-sectional view of the wall of FIG. 41.
- FIGS. 43A-43B are isometric views of opposite sides of another wall suitable for use in a container for holding cells or viruses to be disrupted.
- FIG. 44 is a partially cut-away, isometric view of a container incorporating the wall of FIGS. 43A-43B.
- FIG. 45 is a bottom plan view of the container of FIG. 44.
- FIG. 46 is a partially exploded, isometric view of a container for holding cells or viruses to be disrupted according to another embodiment of the invention.
- FIG. 47 is a front view of the container of FIG. 46.
- FIG. 48 is another schematic, front view of the container of FIG. 46.
- FIG. 49 is a side view of the container of FIG. 46.
- FIG. 50 is a view of a pipette inserted into the container of FIG. 46.
- the container is holding beads for rupturing cells or viruses.
- FIG. 51 is an isometric view of the container of FIG. 46 inserted into an apparatus for disrupting cells or viruses.
- FIG. 52 is a different isometric view of the container of FIG. 46 inserted into the apparatus of FIG. 51.
- FIG. 53 is a partially cut-away, isometric view of the apparatus of FIG. 51.
- FIG. 54 is an isometric view of a holder for holding the container of FIG. 46.
- FIG. 55 is another isometric view of the apparatus of FIG. 51 in which several parts of the apparatus have been removed to show an ultrasonic horn contacting the container of FIG. 46.
- FIG. 56 is a schematic side view of the container of FIG. 46 inserted into the apparatus of FIG. 51 for disruption of the cells or viruses contained in the container.
- the present invention provides an apparatus and method for analyzing a fluid sample.
- the invention provides a cartridge for separating a desired analyte from a fluid sample and for holding the analyte for a chemical reaction.
- The-fluid sample may be a solution or suspension.
- the sample may be a bodily fluid (e.g., blood, urine, saliva, sputum, seminal fluid, spinal fluid, mucus, or other bodily fluids).
- the sample may be a solid made soluble or suspended in a liquid or the sample may be an environmental sample such as ground or waste water, soil extracts, pesticide residues, or airborne spores placed in a fluid.
- the sample may be mixed with one or more chemicals, reagents, diluents, or buffers.
- the sample may be pretreated, for example, mixed with chemicals, centrifuged, pelleted, etc., or the sample may be in a raw form.
- the desired analyte is typically intracellular material (e.g., nucleic acid, proteins, carbohydrates, lipids, bacteria, or intracellular parasites).
- the analyte is nucleic acid which the cartridge separates from the fluid sample and holds for amplification (e.g., using PCR) and optical detection.
- nucleic acid refers to any synthetic or naturally occurring nucleic acid, such as DNA or RNA, in any possible configuration, i.e., in the form of double-stranded nucleic acid, single-stranded nucleic acid, or any combination thereof.
- FIG. 1 shows an isometric view of a cartridge 20 according to the preferred embodiment.
- the cartridge 20 is designed to separate nucleic acid from a fluid sample and to hold the nucleic acid for amplification and detection.
- the cartridge 20 has a body comprising a top piece 22 , a middle piece 24 , and a bottom piece 26 .
- An inlet port for introducing a fluid sample into the cartridge is formed in the top piece 22 and sealed by a cap 30 .
- Six pressure ports 32 are also formed in the top piece 22 .
- the pressure ports 32 are for receiving nozzles from pressure sources, e.g., pumps or vacuums.
- the cartridge also includes alignment legs 28 extending from the bottom piece 26 for positioning the cartridge 20 in an instrument (described below with reference to FIG. 10).
- Indentations or depressions 38 A, 38 B, and 38 C are formed in the top and middle pieces 22 , 24 .
- the indentations are for receiving optical sensors that detect fluid flow in the cartridge 20 .
- the cartridge 20 further includes vents 34 , 36 .
- Each pressure port and vent preferably includes a hydrophobic membrane that allows the passage of gas but not liquid into or out of the vents and pressure ports.
- Modified acrylic copolymer membranes are commercially available from, e.g., Gelman Sciences (Ann Arbor, Mich.) and particle-track etched polycarbonate membranes are available from Poretics, Inc. (Livermore, Calif.).
- FIG. 2 is an isometric view showing the underside of the cartridge 20 .
- Nine holes 60 are formed in the bottom piece 26 for receiving valve actuators that open and close valves in the cartridge 20 .
- a hole 62 is also formed in the bottom piece 26 for receiving a transducer (described in detail below with reference to FIG. 5).
- the cartridge 20 also includes a reaction vessel 40 extending outwardly from the body of the cartridge.
- the vessel 40 has a reaction chamber 42 for holding a reaction mixture (e.g., nucleic acid mixed with amplification reagents and fluorescent probes) for chemical reaction and optical detection.
- a reaction mixture e.g., nucleic acid mixed with amplification reagents and fluorescent probes
- One of the flow paths in the cartridge carries the reaction mixture to the chamber 42 for chemical reaction and optical detection.
- the vessel 40 extends outwardly from the body of the cartridge 20 so that the vessel 40 may be inserted between a pair of opposing thermal plates (for heating and cooling the chamber 42 ) without the need for decoupling the vessel 40 from the rest of the cartridge 20 . This greatly reduces the risk of contamination and/or spilling.
- the vessel 40 may be integrally formed with the body of the cartridge (e.g., integrally molded with middle piece 24 ). It is presently preferred, however, to produce the vessel 40 as a separate element that is coupled to the body during manufacture of the cartridge.
- FIGS. 3-4 show exploded views of the cartridge.
- the middle piece 24 has multiple chambers formed therein.
- the middle piece 24 includes a sample chamber 65 for holding a fluid sample introduced through the inlet port 64 , a wash chamber 66 for holding a wash solution, a reagent chamber 67 for holding a lysing reagent, a waste chamber 68 for receiving used sample and wash solution, a neutralizer chamber 70 for holding a neutralizer, and a master mix chamber 71 for holding a master mix (e.g., amplification reagents and fluorescent probes) and for mixing the reagents and probes with analyte separated from the fluid sample.
- a master mix e.g., amplification reagents and fluorescent probes
- the sample chamber 65 optionally includes a side compartment 155 having slightly lower walls than the sample chamber 65 .
- the side compartment 155 is for visually indicating to a user when sufficient sample has been added to the sample chamber 65 , i.e., when the liquid level in the chamber 65 is high enough to spill over into the compartment 155 .
- the top piece 22 includes the vents 34 , 36 and the six pressure ports 32 , as previously described.
- An elastomeric membrane or gasket 61 is positioned and squeezed between the pieces 22 , 24 to seal the various channels and chambers formed in the pieces.
- the middle piece 24 preferably includes multiple sealing lips to ensure that the gasket 61 forms an adequate seal.
- the middle piece 24 preferably includes sealing lips 73 surrounding each of the chambers 65 , 66 , 67 , 68 . 70 , and 71 .
- the middle piece 24 also includes support walls 75 around the perimeter, and intermediate sealing lips 76 . The sealing lips 73 , 76 and support walls 75 locally compress the gasket 61 and achieve a seal.
- the middle piece 24 has formed in its underside various channels, one of which leads to a lysing chamber 86 .
- the chamber 86 is aligned with the hole 62 in the bottom piece 26 so that a transducer (e.g., an ultrasonic horn) may be inserted through the hole 62 to generate dynamic pressure pulses or pressure waves in the lysing chamber 86 .
- the middle piece 24 also has nine valve seats 84 formed in its bottom surface. The valve seats 84 are aligned with the nine holes 60 in the bottom piece 26 so that valve actuators may be inserted through the holes 60 into the valve seats 84 .
- An elastomeric membrane or gasket 61 is positioned and squeezed between the pieces 24 , 26 to seal the various channels, valve seats, and chamber formed in the middle piece 24 .
- the middle piece 24 preferably includes multiple sealing lips to ensure that the gasket 63 forms an adequate seal.
- the middle piece 24 preferably includes sealing lips 73 surrounding the lysing chamber 86 , valve seats 84 , and various channels.
- the middle piece 24 also includes support walls 75 around its perimeter, and intermediate sealing lips 76 . The sealing lips 73 , 76 and support walls 75 locally compress the gasket 63 and achieve a seal.
- the gasket 63 also functions as a valve stem by compressing, when actuated through one of the holes 60 , into a corresponding valve seat 84 , thus shutting one of the flow channels in the middle piece 24 . This valve action is discussed in greater detail below with reference to FIGS. 15-16.
- the gasket 63 also forms the bottom wall of the lysing chamber 86 against which a transducer is placed to effect disruption of cells or viruses in the chamber 86 .
- Each of the gaskets 61 , 63 is preferably composed of an elastomer. Suitable gasket materials are silicone rubber, neoprene, EPDM, or any other compliant material.
- Each of the gaskets 61 , 63 preferably has a thickness in the range of 0.005 to 0.125 inches (0.125 to 3.175 mm), and more preferably in the range of 0.01 to 0.06 inches (0.25 to 1.5 mm), with a presently preferred thickness of 0.031 inches (0.79 mm). The thickness is selected to ensure that the gasket is sufficiently compliant to seal the channels and chambers, to compress into the valve seats 84 when forced, and to expand under pressure to contact the transducer.
- the middle piece 24 includes a slot 79 through which the reaction vessel 40 is inserted during assembly of the cartridge.
- the vessel 40 has two fluid ports 41 , 43 for adding and removing fluid from the vessel.
- the ports 41 , 43 are placed into fluidic communication with channels 80 , 81 , respectively, that are formed in the top piece 22 (see FIG. 4).
- the gasket 61 seals the respective fluidic interfaces between the ports 41 , 43 and the channels 80 , 81 .
- the top, middle, and bottom pieces 22 , 24 , 26 are preferably injection molded parts made of a polymeric material such as polypropylene, polycarbonate, or acrylic.
- top, middle, and bottom pieces 22 , 24 , 26 may be machined.
- the pieces 22 , 24 , 26 may be held together by screws or fasteners.
- ultrasonic bonding, solvent bonding, or snap fit designs could be used to assemble the cartridge.
- FIG. 4 also shows a filter ring 88 .
- the filter ring 88 compresses and holds a stack of filters in the lysing chamber 86 .
- FIG. 6 shows an exploded view of a filter stack 87 .
- the purpose of the filter stack 87 is to capture cells or viruses from a fluid sample as the sample flows through the lysing chamber 86 . The captured cells or viruses are then disrupted (lysed) in the chamber 86 .
- the cells may be animal or plant cells, spores, bacteria, or microorganisms.
- the viruses may be any type of infective agents having a protein coat surrounding an RNA or DNA core.
- the filter stack 87 comprises a gasket 93 , a first filter 94 , a gasket 95 , a second filter 97 having a smaller pore size than the first filter 94 , a gasket 98 , a third filter 100 having a smaller pore size than the second filter 97 , a gasket 101 , a woven mesh 102 , and a gasket 103 .
- the filter stack also preferably includes a first set of beads 96 disposed between the first and second filters 94 and 97 and a second set of beads 99 disposed between the second and third filters 97 and 100 .
- the filter ring 88 compresses the filter stack 87 into the lysing chamber 86 so that the gasket 93 is pressed against the filter 94 , the filter 94 is pressed against the gasket 95 , the gasket 95 is pressed against the filter 97 , the filter 97 is pressed against the gasket 98 , the gasket 98 is pressed against the filter 100 , the filter 100 is pressed against the gasket 101 , the gasket 101 is pressed against the mesh 102 , the mesh 102 is pressed against the gasket 103 , and the gasket 103 is pressed against the outer perimeter of the bottom wall of the lysing chamber 86 .
- the gasket 95 is thicker than the average diameter of the beads 96 so that the beads are free to move in the space between the filters 94 and 97 .
- the gasket 98 is thicker than the average diameter of the beads 99 so that the beads 99 are free to move in the space between the filters 97 and 100 .
- a fluid sample flowing through the channel 106 into the lysing chamber 86 first flows through filter 94 , then through filter 97 , next through filter 100 , and lastly through the mesh 102 . After flowing through the filter stack 87 , the sample flows along flow ribs. 91 formed in the top of the lysing chamber 86 and through an outlet channel (not shown in FIG. 6).
- the cells or viruses captured in the filter stack are lysed by coupling a transducer 92 (e.g., an ultrasonic horn) directly to the wall of the lysing chamber 86 .
- a transducer 92 e.g., an ultrasonic horn
- the wall of the lysing chamber 86 is formed by the flexible gasket 63 .
- the transducer 92 should directly contact an external surface of the wall.
- the term “external surface” is intended to mean a surface of the wall that is external to the lysing chamber 86 .
- the transducer 92 is a vibrating or oscillating device that is activated to generate dynamic pressure pulses or pressure waves in the chamber 86 .
- the transducer for contacting the wall of the lysing chamber 86 may be an ultrasonic, piezoelectric, magnetostrictive, or electrostatic transducer.
- the transducer may also be an electromagnetic device having a wound coil, such as a voice coil motor or a solenoid device.
- the actuator be an ultrasonic transducer, such as an ultrasonic horn. Suitable horns are commercially available from Sonics & Materials, Inc. having an office at 53 Church Hill, Newton., Conn. 06470-1614 USA.
- the ultrasonic transducer may comprise a piezoelectric disk or any other type of ultrasonic transducer that may be coupled to the container. It is presently preferred to use an ultrasonic horn because the horn structure is highly resonant and provides for repeatable and sharp frequency of excitation and large motion of the horn tip.
- the filter stack includes a gasket at both of its ends.
- the middle cartridge piece 24 has a sealing lip 90 against which the gasket at one end of the filter stack is compressed.
- the gasket at the other end of the filter stack is compressed by the filter ring 88 to form a seal.
- the gasket material may expand into the relief area outside of the sealing lip 90 .
- the width of the sealing lip 90 is small (typically 0.5 mm) so that an excessive amount of force is not required to achieve a sufficient seal.
- the filter ring 88 is held between the filter stack and the cartridge gasket 63 .
- the cartridge gasket 63 is held between the middle piece 24 and the bottom piece 26 by a sealing lip 406 . Force is therefore transferred from the bottom piece 26 through the gasket 63 to the filter ring 88 and finally to the filter stack.
- the filter ring 88 contains a contact lip 404 that contacts the gasket 63 .
- the contact lip 404 is not a primary sealing lip (though it will seal) but a force transfer mechanism.
- the width of the contact lip 404 is larger than the width of the sealing lip 90 to ensure that deformation and sealing action occurs in the filter stack and not taken up in squeezing the cartridge gasket 63 .
- the cartridge middle piece 24 also has a sealing lip 406 that surrounds the filter ring 88 .
- This is an active sealing area that should not be compromised by the presence of the filter ring 88 .
- the gap 407 is provided to allow the gasket 63 to extrude into the gap 407 as it is compressed by the sealing lip 406 and the contact lip 404 . If the contact lip 404 comes to a different elevation than the sealing lip 406 , the seal will not be compromised because of the gap 407 and the distance between the lips 404 and 406 .
- the filter stack 87 is effective for capturing cells or viruses as a fluid sample flows through the stack 87 without clogging of any of the filters 94 , 97 , 100 in the stack.
- the first filter 94 (having the largest pore size) filters out coarse material such as salt crystals, cellular debris, hair, tissue, etc.
- the second filter 97 (having the medium pore size) captures cells or viruses in the fluid sample.
- the third filter 100 (having the smallest pore size) captures smaller cells or viruses in the sample.
- the filter stack 87 thus enables the simultaneous capture of differently sized sample components without clogging of the filters.
- the average pore size of the first filter 94 is selected to be small enough to filter coarse material from the fluid sample (e.g., salt crystals, cellular debris, hair, tissue) yet large enough to allow the passage of the target cells or viruses containing the desired analyte (e.g., nucleic acid or proteins).
- the pore size of the first filter 94 should be in the range of about 2 to 25 ⁇ m, with a presently preferred pore size of about 5 ⁇ m.
- the average pore sizes of the second and third filters are selected in dependence upon the average size of the target cells or viruses that contain the desired analyte(s).
- the filter stack 87 is used to capture gonorrhea (GC) and chlamydia (Ct) organisms to determine the presence of the diseases in the fluid sample.
- GC and Ct organisms have different average diameters, about 1 to 2 ⁇ m for GC organisms and about 0.3 ⁇ m for Ct organisms.
- the second filter 97 has an average pore size of about 1.2 ⁇ m while the third filter 100 has an average pore size of about 0.22 ⁇ m so that most of the GC organisms are captured by the second filter 97 while most of the Ct organisms are captured by the third filter 100 .
- the filter stack thus enables the simultaneous capture of differently sized target organisms and does so without clogging of the filters.
- the pore sizes of the filters 97 , 100 may be selected to capture desired cells or viruses of any size, and the scope of the invention is not limited to the specific example given.
- the filter stack 87 is also useful for disrupting the captured cells or viruses to release the intracellular material (e.g., nucleic acid) therefrom.
- the first and second sets of beads 96 , 99 serve two useful purposes in this regard. First, the beads are agitated by dynamic pressure pulses or pressure waves generated by the transducer. The movement of the beads ruptures the captured cells or viruses. Second, the beads may shear the nucleic acid released from the lysed cells or viruses so that the strands of nucleic acid are sufficiently short to flow through the filters and out of the lysing chamber 86 .
- Suitable beads for rupturing cells or viruses include borosilicate glass, lime glass, silica, and polystyrene beads.
- the beads may be porous or non-porous and preferably have an average diameter in the range of 1 to 200 ⁇ m.
- the average diameter of the beads 96 , 99 is selected in dependence upon the intended target cells or viruses to be ruptured by the beads.
- the average diameter of the beads 96 in the first set may be equal to the average diameter of the beads 99 in the second set.
- the beads 96 are 20 ⁇ m diameter borosilicate glass beads for rupturing the GC organisms and the beads 99 are 106 ⁇ m diameter soda lime glass beads for rupturing the Ct organisms.
- Each of the silicone gaskets 95 , 98 should be sufficiently thick to allow room for the beads 96 , 99 to move and rupture the cells or viruses.
- the mesh 102 also serves two useful purposes. First the mesh provides support to the filter stack 87 . Second, the mesh breaks up air bubbles so that the bubbles can be channeled through the flow ribs 91 and out of the lysing chamber 86 . To effectively break up or reduce the size of the air bubbles, the mesh 102 preferably has a small pore size. Preferably, it is a woven polypropylene mesh having an average pore size of about 25 ⁇ m. To ensure that the air bubbles can escape from the lysing chamber 86 , it is desirable to use the cartridge in an orientation in which liquid flows up (relative to gravity) through the filter stack 87 and the lysing chamber 86 . The upward flow through the chamber 86 aids the flow of air bubbles out of the chamber 86 . Thus, the inlet port for entry of fluids into the chamber 86 should generally be at the lowest point in the chamber, while the exit port should be at the highest.
- the filter stack has only two filters and one set of beads disposed between the filters.
- the first filter has the largest pore size (e.g., 5 ⁇ m) and filters out coarse material such as salt crystals, cellular debris, hair, tissue, etc.
- the second filter has a pore size smaller than the first filter and slightly smaller than the target cells or viruses to be captured.
- the filter having the largest pore size is positioned in a filter chamber (not shown) that is positioned upstream of the lysing chamber 86 .
- a channel connects to the filter chamber to the lysing chamber 86 .
- a fluid sample flows first through the coarse filter in the filter chamber and then through a second filter in the lysing chamber to trap the target cells or viruses in the lysing chamber.
- the beads in the filter stack may have a binding affinity for target cells or viruses in the fluid sample to facilitate capture of the target cells or viruses.
- antibodies or certain receptors may be coated onto the surface of the beads to bind target cells in the sample.
- the lysing chamber 86 may contain two different types of beads for interacting with target cells or viruses.
- the lysing chamber may contain a first set of beads coated with antibodies or receptors for binding target cells or viruses and a second set of beads (intermixed with the first set) for rupturing the captured cells or viruses.
- the beads in the lysing chamber 86 may also have a binding affinity for the intracellular material (e.g., nucleic acid) released from the ruptured cells or viruses.
- the lysing chamber may contain silica beads to isolate DNA or cellulose beads with oligo dT to isolate messenger RNA for RT-PCR.
- the lysing chamber 86 may also contain beads for removing unwanted material (e.g., proteins, peptides) or chemicals (e.g., salts, metal ions, or-detergents) from the sample that might inhibit PCR.
- the chamber 86 may contain ion exchange beads for removing proteins.
- beads having metal ion chelators such as iminodiacetic acid will remove metal ions from biological samples.
- FIGS. 21-22 illustrate the reaction vessel 40 in greater detail.
- FIG. 21 shows a partially exploded view of the vessel 40
- FIG. 22 shows a front view of the vessel 40 .
- the vessel 40 includes the reaction chamber 42 (diamond-shaped in this embodiment) for holding a reaction mixture.
- the vessel 40 is designed for optimal heat transfer to and from the reaction mixture and for efficient optical viewing of the mixture.
- the thin shape of the vessel contributes to optimal thermal kinetics by providing large surfaces for thermal conduction and for contacting thermal plates.
- the walls of the vessel provide optical windows into the chamber 42 so that the entire reaction mixture can be optically interrogated.
- the reaction vessel 40 includes a rigid frame 46 that defines the side walls 57 A, 57 B, 59 A, 59 B of the reaction chamber 42 .
- the frame 46 also defines an inlet port 41 and a channel 50 connecting the port 41 to the chamber 42 .
- the frame 46 also defines an outlet port 43 and a channel 52 connecting the port 43 to the chamber 42 .
- the inlet port 41 and channel 50 are used to add fluid to the chamber 42
- the channel 52 and outlet port 43 are used for exit of fluid from the chamber 42 .
- Alignment prongs 44 A, 44 B are used to position the vessel 40 correctly during assembly of the cartridge.
- the vessel 40 also includes thin, flexible sheets attached to opposite sides of the rigid frame 46 to form opposing major walls 48 of the chamber.
- the major walls 48 are shown in FIG. 1 exploded from the rigid frame 46 for illustrative clarity).
- the reaction chamber 42 is thus defined by the rigid side walls 57 A, 57 B, 59 A, 59 B of the frame 46 and by the opposing major walls. 48 .
- the opposing major walls 48 are sealed to opposite sides of the frame 46 such that the side walls 57 A, 57 B, 59 A, 59 B connect the major walls 48 to each other.
- the walls 48 facilitate optimal thermal conductance to the reaction mixture contained in the chamber 42 .
- Each of the walls 48 is sufficiently flexible to contact and conform to a respective thermal surface, thus providing for optimal thermal contact and heat transfer between the thermal surface and the reaction mixture contained in the chamber 42 . Furthermore, the flexible walls 48 continue to conform to the thermal surfaces if the shape of the surfaces changes due to thermal expansion or contraction during the course of the heat-exchanging operation.
- the thermal surfaces for contacting the flexible walls 48 are preferably formed by a pair of opposing plates 190 A, 190 B positioned to receive the chamber 42 between them.
- the inner surfaces of the plates contact the walls 48 and the flexible walls conform to the surfaces of the plates.
- the plates are preferably spaced a distance from each other equal to the thickness T of the chamber 42 as defined by the thickness of the frame 46 . In this position, minimal or no gaps are found between the plate surfaces and the walls 48 .
- the plates may be heated and cooled by various thermal elements to induce temperature changes within the chamber 42 , as is described in greater detail below.
- the walls 48 are preferably flexible films of polymeric material such as polypropylene, polyethylene, polyester, or other polymers.
- the films may either be layered, e.g., laminates, or the films may be homogeneous. Layered films are preferred because they generally have better strength and structural integrity than homogeneous films.
- layered polypropylene films are presently preferred because polypropylene is not inhibitory to PCR.
- the walls 48 may comprise any other material that may be formed into a thin, flexible sheet and that permits rapid heat transfer.
- the thickness of each wall 48 is preferably between about 0.003 to 0.5 mm, more preferably between 0.01 to 0.15 mm, and most preferably between 0.025 to 0.08 mm.
- the vessel 40 also preferably includes optical windows for in situ optical interrogation of the reaction mixture in the chamber 42 .
- the optical windows are the side walls 57 A, 57 B of the rigid frame 46 .
- the side walls 57 A, 57 B are optically transmissive to permit excitation of the reaction mixture in the chamber 42 through the side wall 57 A and detection of light emitted from the chamber 42 through the side wall 57 B.
- Arrows A represent illumination beams entering the chamber 42 through the side wall 57 A and arrows B represent emitted light (e.g., fluorescent emission from labeled analytes in the reaction mixture) exiting the chamber 42 through the side wall 57 B.
- the side walls 57 A, 57 B are preferably angularly offset from each other. It is usually preferred that the walls 57 A, 57 B are offset from each other by an angle of about 90° .
- a 90° angle between excitation and detection paths assures that a minimum amount of excitation radiation entering through the wall 57 A will exit through wall 57 B.
- the 90° angle permits a maximum amount of emitted light (e.g. fluorescence) to be collected through wall 57 B.
- the walls 57 A, 57 B are preferably joined to each other to form a “V” shaped intersection at the bottom of the chamber 42 .
- the angled walls 57 A, 57 B need not be directly joined to each other, but may be separated by an intermediary portion, such as another wall or various mechanical or fluidic features which do not interfere with the thermal and optical performance of the vessel.
- the walls 57 A, 57 B may meet at a port which leads to another processing area in communication with the chamber 42 , such as an integrated capillary electrophoresis area.
- a locating tab 58 extends from the frame 46 below the intersection of walls 57 A, 57 B. The tab 58 is used to properly position the vessel 40 in a heat-exchanging module described below with reference to FIG. 28.
- Optimum optical sensitivity may be attained by maximizing the optical path length of the light beams exciting the labeled analyte in the reaction mixture and the emitted light that is detected, as represented by the equation:
- I o is the illumination output of the emitted light in volts, photons or the like
- C is the concentration of analyte to be detected
- I i is the input illumination
- L is the path length
- A is the intrinsic absorptivity of the dye used to label the analyte.
- the thin, flat reaction vessel 40 of the present invention optimizes detection sensitivity by providing maximum optical path length per unit analyte volume.
- the vessel 40 is preferably constructed such that each of the sides walls 57 A, 57 B, 59 A, 59 B of the chamber 42 has a length L in the range of 1 to 15 mm, the chamber has a width W in the range of 1.4 to 20 mm, the chamber has a thickness T in the range of 0.5 to 5 mm, and the ratio of the width W of the chamber to the thickness T of the chamber is at least 2:1.
- the average optical path length of the chamber 42 is the distance from the center of the side wall 57 A to the center of the chamber 42 plus the distance from the center of the chamber 42 to the center of the side wall 57 B.
- the vessel 40 is constructed such that each of the sides walls 57 A, 57 B, 59 A, 59 B of the chamber 42 has a length L in the range of 5 to 12 mm, the chamber has a width W in the range of 7 to 17 mm, the chamber has a thickness T in the range of 0.5 to 2 mm, and the ratio of the width W of the chamber to the thickness T of the chamber is at least 4:1.
- These ranges are more preferable because they provide a vessel having both a larger average optical path length (i.e., 5 to 12 mm) and a volume capacity in the range of 12 to 100 ⁇ l while still maintaining a chamber sufficiently thin to permit extremely rapid heating and cooling of a reaction mixture.
- the relatively large volume capacity provides for increased sensitivity in the detection of low concentration analytes, such as nucleic acids.
- the reaction vessel 40 has a diamond-shaped chamber 42 defined by the side walls 57 A, 57 B, 59 A, 59 B, each of the side walls has a length of about 10 mm, the chamber has a width of about 14 mm, the chamber has a thickness T of 1 mm as defined by the thickness of the frame 46 , and the chamber has a volume capacity of about 100 ⁇ l.
- This reaction vessel provides a relatively large average optical path length of 10 mm through the chamber 42 .
- the thin chamber allows for extremely rapid heating and/or cooling of the reaction mixture contained therein.
- the diamond-shape of the chamber 42 helps prevent air bubbles from forming in the chamber as it is filled with the reaction mixture and also aids in optical interrogation of the mixture.
- the frame 46 is preferably made of an optically transmissive material, e.g., a polycarbonate or clarified polypropylene, so that the side walls 57 A, 57 B are optically transmissive.
- optically transmissive means that one or more wavelengths of light may be transmitted through the walls.
- the optically transmissive walls 57 A, 57 B are substantially transparent.
- one or more optical elements may be present on the optically transmissive side walls 57 A, 57 B.
- the optical elements may be designed, for example, to maximize the total volume of solution which is illuminated by a light source, to focus excitation light on a specific region of the chamber 42 , or to collect as much fluorescence signal from as large a fraction of the chamber volume as possible.
- the optical elements may comprise gratings for selecting specific wavelengths, filters for allowing only certain wavelengths to pass, or colored lenses to provide filtering functions.
- the wall surfaces may be coated or comprise materials such as liquid crystal for augmenting the absorption of certain wavelengths.
- the optically transmissive walls 57 A, 57 B are substantially clear, flat windows having a thickness of about 1 mm.
- the side walls 59 A, 59 B preferably includes reflective faces 56 which internally reflect light trying to exit the chamber 42 through the side walls 59 A, 59 B.
- the reflective faces 56 are arranged such that adjacent faces are angularly offset from each other by about 90°.
- the frame 46 defines open spaces between the side walls 59 A, 59 B and the support ribs 53 .
- the open spaces are occupied by ambient air that has a different refractive index than the material composing the frame (e.g., plastic). Due to the difference in the refractive indexes, the reflective faces 56 are effective for internally reflecting light trying to exit the chamber through the walls 59 A, 59 B and provide for increased detection of optical signal through the walls 57 A, 57 B.
- the optically transmissive side walls 57 A, 57 B define the bottom portion of the diamond-shaped chamber 42
- the retro-reflective side walls 59 A, 59 B define the top portion of the chamber.
- the reaction vessel 40 may be fabricated by first molding the rigid frame 46 using known injection molding techniques.
- the frame 46 is preferably molded as a single piece of polymeric material, e.g., clarified polypropylene. After the frame 46 is produced, thin, flexible sheets are cut to size and sealed to opposite sides of the frame 46 to form the major walls 48 of the chamber 42 .
- the major walls 48 are preferably cast or extruded films of polymeric material, e.g., polypropylene films, that are cut to size and attached to the frame 46 using the following procedure. A first piece of film is placed over one side of the frame 46 .
- the frame 46 preferably includes a tack bar 47 for aligning the top edge of the film.
- the film is placed over the bottom portion of the frame 46 such that the top edge of the film is aligned with the tack bar 47 and such that the film completely covers the bottom portion of the frame 46 below the tack bar 47 .
- the film should be larger than the bottom portion of the frame 46 so that it may be easily held and stretched flat across the frame.
- the film is then cut to size to match the outline of the frame by clamping to the frame the portion of the film that covers the frame and cutting away the portions of the film that extend past the perimeter of the frame using, e.g., a laser or die.
- the film is then tack welded to the frame, preferably using a laser.
- the film is then sealed to the frame 46 , preferably by heat sealing.
- Heat sealing is presently preferred because it produces a strong seal without introducing potential contaminants to the vessel as the use of adhesive or solvent bonding techniques might do.
- Heat sealing is also simple and inexpensive.
- the heat sealing may be performed using, e.g., a heated platen.
- An identical procedure may be used to cut and seal a second sheet to the opposite side of the frame 46 to complete the chamber 42 .
- the film is stretched across the bottom portion of the frame 46 and then sealed to the frame prior to cutting the film to size. After sealing the film to the frame, the portions of the film that extend past the perimeter of the frame are cut away using, e.g., a laser or die.
- the frame 46 may be molded from polycarbonate, which has good optical transparency, while the rest of the frame is molded from polypropylene, which is inexpensive and compatible with PCR.
- the separate pieces can be attached together in a secondary step.
- the side walls 57 A, 57 B may be press-fitted and/or bonded to the remaining portion of the frame 46 .
- the flexible walls 48 may then be attached to opposite sides of the frame 46 as previously described.
- a gasket 61 to seal the ports 41 , 43 of the vessel 40 to corresponding channels 80 , 81 (FIG. 4) in the cartridge body.
- fluidic seals may be established using a luer fitting, compression fitting, or swaged fitting.
- the cartridge body and frame of the vessel 40 are molded as a single part, and the flexible major walls of the vessel are heat-sealed to opposite sides of the frame.
- the chamber 42 is filled by forcing liquid (e.g., a reaction mixture) to flow through the port 41 and the channel 50 into the chamber 42 .
- the liquid may be forced to flow into the chamber 42 using differential pressure (i.e., either pushing the liquid through the inlet port 41 or aspirating the liquid by applying a vacuum to the outlet port 43 ).
- differential pressure i.e., either pushing the liquid through the inlet port 41 or aspirating the liquid by applying a vacuum to the outlet port 43 .
- the liquid displaces air in the chamber.
- the displaced air exits the chamber 42 through the channel 52 and the port 43 .
- the chamber should not contain air bubbles.
- the connection between the chamber 42 and the outlet channel 52 should be at the highest point (with respect to gravity) in the chamber 42 . This allows air bubbles in the chamber 42 to escape without being trapped.
- the vessel 40 is designed to be used in the vertical orientation shown in FIG. 22.
- FIG. 25 shows another vessel 206 designed to be used in a horizontal orientation.
- the vessel 206 has an inlet port 41 and an inlet channel 50 connecting the inlet port 41 to the bottom of the chamber 42 .
- the vessel also has an outlet port 43 and an outlet channel 50 connecting the outlet port 43 to the top of the chamber 42 .
- any air bubbles in the chamber 42 may escape through the outlet channel 52 without becoming trapped.
- FIG. 26 shows another vessel 207 having two inlet ports 41 , 45 and one outlet port 43 .
- Inlet channels 50 , 54 connect the respective inlet ports 41 , 45 to the chamber 42
- outlet channel 52 connects the chamber 42 to outlet port 43 .
- Many other different embodiments of the vessel are also possible. In each embodiment, it is desirable to evacuate the chamber 42 from the highest point (with respect to gravity) in the chamber and to introduce liquid into the chamber from a lower point.
- FIGS. 15A-15B illustrate two types of valves used in the cartridge.
- the first valve uses a cone-shaped or conical valve seat 160 formed in the middle cartridge piece 24 .
- the valve seat 160 is a depression, recess, or cavity molded or machined in the middle piece 24 .
- the valve seat 160 is in fluid communication with a chamber 167 through a port or channel 157 that intersects the center of the conical valve seat 160 .
- FIG. 15A there are two types of fundamental concepts to the valve action, and hence two types of valves.
- the first valve uses a cone-shaped or conical valve seat 160 formed in the middle cartridge piece 24 .
- the valve seat 160 is a depression, recess, or cavity molded or machined in the middle piece 24 .
- the valve seat 160 is in fluid communication with a chamber 167 through a port or channel 157 that intersects the center of the conical valve seat 160 .
- a valve actuator 164 having a spherical surface is forced against the elastic membrane 63 and into the valve seat 160 , establishing a circular ring of contact between the membrane 63 and the valve seat 160 .
- the kinematic principle is that of a ball seated into a cone.
- the circular seal formed by the membrane 63 and valve seat 160 prevents flow between the channel 157 (and hence the chamber 167 ) and a side channel 158 extending from a side of the valve seat 160 .
- the side channel 158 is defined by the membrane 63 and the middle cartridge piece 24 .
- the other type of valve controls the cross flow between the channel 158 and another side channel 159 formed between the membrane 63 and the middle cartridge piece 24 .
- the second valve comprises a recess depression or cavity 161 formed in the middle cartridge piece 24 .
- the cavity 161 separates the channels 158 , 159 from each other.
- An end of the channel 158 is positioned on one side of the cavity 161
- an end of the channel 159 is positioned on the opposite side of the cavity 161 .
- the cavity 161 is defined by a first curved surface 162 A positioned adjacent the end of the channel 158 , a second curved surface 162 B positioned adjacent the end of the channel 159 , and a third surface 163 between the first and second curved surfaces 162 A, 162 B.
- the curved surfaces provide two valve seats that are the primary contact area for the membrane 63 to seal off the flow between the channels 158 and 159 .
- the kinematic principle is that of a ball (or spherical end on a valve actuator) held by three contact points, the upward force on the actuator and the two valve seats 162 A, 162 B.
- the first and second curved surfaces 162 A, 162 B are preferably concentric spherical surfaces.
- the valve actuator 164 has also has a spherical surface for pressing the membrane 63 tightly against the surfaces 162 A, 162 B.
- each of the surfaces 162 A, 162 B preferably has a spherical radius of curvature R 1 equal to the combined radius of curvature R 2 of the valve actuator 164 plus the thickness T of the membrane 63 .
- the radius of curvature R 2 of the surface of the valve actuator 164 is 0.094 inches and the membrane 63 has a thickness T of 0.031 inches, then the radius of curvature R 1 of each of the surfaces 162 A, 162 B is 0.125 inches.
- the size and radius of curvature of the valve seats is dependent upon the size of the channels in the cartridge. The valves are preferably made just large enough to effectively seal the channels but no larger so that dead volume in the cartridge is minimized.
- the third surface 163 is recessed from the first and second surfaces 162 A, 162 B to provide a gap 166 between the membrane 63 and the third surface 163 when the membrane 63 is pressed against the first and second surfaces 162 A, 162 B. Stated another way, the surfaces 162 A, 162 B are raised or elevated from the surface 163 .
- the gap 166 ensures that the membrane 63 contacts primarily the valve seats. 162 A, 162 B rather than the entire surface of the cavity 161 so that maximum pressure is applied to the valve seats 162 A and 162 B by the membrane 63 . This provides a very strong seal with minimal actuator force required.
- the respective kinematic principle defines the location of the mating parts.
- the ball or spherical shaped valve actuator is permitted to seek its own location as it is forced against the valve seat(s).
- There is a deliberate clearance e.g., 0.01 to 0.03 inches between the valve actuator and the hole in the bottom cartridge piece 26 in which the actuator 164 travels so that only the valve seat action defines the location of the mating pieces.
- valve actuators can be controlled by a variety of mechanisms.
- FIGS. 17-19 illustrate one such mechanism.
- a valve actuator 172 has a spherical surface for pressing the gasket 63 into a valve seat.
- the actuator 172 also has a flange 177 on its bottom portion.
- the cartridge includes an elastic body, such as a spring 174 , that pushes against a ledge in the lower cartridge piece 26 to bias the valve actuator against the gasket 63 .
- the spring 174 is sufficiently strong to close the valve unless a deliberate force is applied to pull down the actuator 172 .
- the valves in the cartridge may be kept closed in this manner for shipping and storage before the cartridge is used.
- the cartridge may be preloaded during manufacture with the necessary reagents and wash solutions to analyze a fluid sample without the fluids leaking out of the cartridge during shipping and storage.
- the actuator pull-down mechanism is usually located in an instrument into which the cartridge is placed for sample. analysis (one such instrument is described in detail below with reference to FIG. 10).
- the mechanism comprises a sliding guide 175 that rotates a hinged pull-down member 180 having a jaw 181 for receiving the flange 177 of the actuator 172 .
- the sliding guide 175 rotates the hinged pull-down member 180 until the flange 177 is positioned within the jaw 181 .
- a solenoid 146 pulls down the member 180 and thus the valve actuator 172 so that the gasket 63 is released from the valve seat, thus opening the valve and permitting fluid flow between the channels 170 and 171 .
- FIG. 20 illustrates the manner in which fluid flow into and out of the sample chamber, wash chamber, neutralizer chamber, and reagent chambers is controlled in the cartridge.
- Each of these chambers as illustrated by a chamber 414 in FIG. 20, is covered by a hydrophobic membrane 410 that allows the passage of gas but not liquid therethrough.
- the hydrophobic membrane 410 is positioned between the chamber 414 and a pressure port 32 .
- the pressure port 32 is formed in the upper cartridge piece 22 and positioned over the chamber 414 .
- the membrane 410 holds liquids in the chamber 414 during shipping and storage of the cartridge, even if the cartridge is turned upside down.
- the pressure port 32 is sized to receive a pressure nozzle 182 that is connected to a pressure source (e.g., a vacuum or pneumatic pump) usually located in the external instrument.
- the nozzle 182 includes an o-ring 184 and a flange 415 .
- a spring 185 pushes against the flange 415 to force the nozzle 182 into the pressure port 32 so that the o-ring 184 establishes a seal around the port 32 .
- positive air pressure or a vacuum is applied to the chamber 414 through the pressure port 32 to force liquids out of or into, respectively, the chamber 414 .
- a conical valve seat 160 (previously described with reference to FIGS. 15A-15B) is formed in the middle cartridge piece 24 below the chamber 414 to control the flow of liquid between the chamber 414 and a connecting channel 411 .
- the valve is opened and closed by a valve actuator 188 having a flange 187 and a spring 188 pressing against the flange to hold the valve closed until a downward force is applied to the actuator 186 .
- the downward force is preferably supplied by a solenoid that pulls down the actuator 186 to open the valve.
- the valve actuator 186 and solenoid are preferably located in the instrument.
- FIGS. 7-8 show top and bottom plan views, respectively, of the cartridge.
- FIG. 9 is a schematic block diagram of the cartridge.
- the cartridge includes a sample chamber 65 having a port for adding a fluid sample to the cartridge and a sample flow path extending from the sample chamber 65 .
- the sample flow path extends from the sample chamber 65 through a valve 107 and into a channel 106 .
- the channel 106 includes a sensor region 136 in which the channel 106 has a flat bottom enabling easy optical detection of the presence of liquid in the channel.
- the sample flow path continues from the channel 106 into the lysing chamber 86 and through the filter stack 87 .
- the sample flow path also includes a channel 109 for exit of fluid from the lysing chamber 86 , a channel 110 having a flat-bottomed detection region 137 , a valve 111 , and a channel 112 leading to the vented waste chamber 68 through a valve 114 .
- the cartridge also includes the wash chamber 66 for holding wash solution and the reagent chamber 67 for holding lysing reagent.
- the wash chamber 66 is connected to the lysing chamber 86 through a valve 115 , channel 117 , and channel 106 .
- the reagent chamber 67 is connected to the lysing chamber. 86 through a valve 119 , channel 117 , and channel 106 .
- Sample components e.g., cells or viruses in the sample
- target analyte e.g., nucleic acid
- the cartridge also includes an analyte flow path extending from the lysing chamber 86 for carrying the analyte separated from the fluid sample to the reaction vessel 40 for chemical reaction and optical detection.
- the analyte flow path extends from the chamber 86 through the channel 109 , channel 110 , and valve 111 . After passing through the valve 111 , the analyte flow path diverges from the sample flow path. While the sample flow path extends though channel 112 into the waste chamber 68 , the analyte flow path diverges into the U-shaped channel 122 . The analyte flow path then extends into and out of the neutralizer chamber 70 through a valve 124 .
- the analyte flow path also passes into and out of the master mix chamber 71 through a valve 126 . From the master mix chamber 71 , the analyte flow path extends along the channel 122 , through a valve 127 , through channel 80 , and into the reaction vessel 40 through the port 41 .
- the reaction vessel 40 includes the port 41 for adding a reaction mixture to the vessel, and the port 43 for exit of fluids (e.g., air or excess reaction mixture) from the vessel.
- the cartridge also includes channel 81 in fluid communication with the port 43 .
- the channel 81 includes a flat-bottomed detection region 130 for detecting the presence of liquid in the channel.
- the channel 81 connects to a channel 131 (channel 131 extends straight down perpendicular to the page in the top plan view of FIG. 7).
- Channel 131 connects to a channel 132 which in turn connects to a channel 134 through a valve 133 (channel 134 extends straight up perpendicular to the page in the top plan view of FIG. 7).
- the channel 134 leads to the vent 36 which has a hydrophobic membrane to permit the escape of gas but not liquid from the cartridge.
- the channels, vent and valve positioned downstream from the reaction vessel 40 are used to pressurize the chamber 42 of the vessel, as is described in the operation section below.
- the cartridge also includes a first pressure port 105 positioned above the sample chamber 65 , a second pressure port 116 positioned above the wash chamber 66 , a third pressure port 118 positioned above the reagent chamber 67 , a fourth pressure port 123 positioned above the neutralizer chamber 70 , a fifth pressure port 125 positioned above the master mix chamber 71 , and a sixth pressure port 128 positioned at the end of the U-shaped channel 122 .
- the cartridge further includes sensor chambers 120 and 121 in fluid communication with the waste chamber 68 . The sensor chambers 120 and 121 indicate when predetermined volumes of liquid have been received in the waste chamber 68 , as is described in detail below.
- the cartridge is preferably used in combination with an instrument 140 designed to accept one or more of the cartridges.
- an instrument 140 designed to accept one or more of the cartridges.
- the instrument 140 shown in FIG. 10 accepts just one cartridge. It is to be understood, however, that the instrument may be designed to process multiple cartridges simultaneously.
- the instrument 140 includes a cartridge nest 141 into which the cartridge is placed for processing.
- the instrument 140 also includes the transducer 92 (e.g., an ultrasonic horn) for generating dynamic pressure pulses or pressure waves in the lysing chamber of the cartridge, nine valve actuators 142 for actuating the nine valves in the cartridge, nine corresponding solenoids 146 for pulling down the valve actuators, and six pressure nozzles 145 for interfacing with six corresponding pressure ports formed in the cartridge.
- the instrument includes or is connected to one or more regulated pressure sources for supplying pressure to the cartridge through the pressure nozzles 145 . Suitable pressure sources include syringe pumps, compressed air sources, pneumatic pumps, or connections to external sources of pressure.
- the instrument further includes three slotted optical sensors 143 and three reflective optical sensors 144 .
- FIG. 13 illustrates the slotted optical sensors 143 positioned to detect liquid in the sensor chambers 120 , 121 and in the reagent chamber 67 .
- Each sensor 143 includes a built in LED and photodiode positioned on opposite sides of the sensor. The LED emits a beam that is detected by the photodiode if the beam is not substantially refracted.
- Such slotted optical sensors are commercially available from a number of suppliers. The cartridge is shaped so that the slotted optical sensors fit around the chambers 67 , 120 , and 121 . The operation of each sensor is as follows.
- the sensor surrounds, the beam from the LED is substantially refracted by air in the chamber and the curved inner walls of the chamber and only a weak signal, if any, is detected by the photodiode since air has an index of refraction that does not closely match that of the plastic cartridge. If there is liquid present in the chamber, however, the beam from the LED does not refract or is only slightly refracted and produces a much stronger signal detected by the photodiode since the liquid has an index of refraction closely matching that of the plastic cartridge.
- the optical sensors 143 are therefore useful for determining the presence or absence of liquid in the chambers 67 , 120 , and 121 .
- FIG. 14 shows a cut-away, schematic side view of the sensor chamber 120 in fluid communication with the waste chamber 68 and surrounded by the slotted optical sensor 143 .
- the sensor chamber 120 and sensor 143 are used to indicate when a predetermined volume of liquid is present in the waste chamber 68 .
- the sensor chamber 120 is partially separated from the waste chamber 68 by a wall 151 having a spillover rim 152 .
- the height of the wall is selected so that when the predetermined volume of liquid is received in the waste chamber 68 , the liquid spills over the spillover rim 152 and into the sensor chamber 120 .
- the liquid in the sensor chamber 120 is then detected by the sensor 143 .
- the cartridge may also include a second sensor chamber 121 in fluid communication with the waste chamber 68 .
- the second sensor chamber 121 is also separated from the waste chamber 68 by a wall 153 having a spillover rim.
- the wall 153 is taller than the wall 152 so that liquid does not spill over the wall 153 until a second predetermined volume of fluid in addition to the first predetermined volume of fluid has been received in the waste chamber 68 .
- the sensor chambers 120 , 121 and the optical sensors 143 are useful for controlling the operation of the cartridge.
- the height of the wall 152 is preferably selected such that when a fixed volume of fluid sample from the sample chamber 65 has flowed through the sample flow path to the waste chamber 68 , the sample liquid spills over into the sensor chamber 120 and is detected.
- the detection in chamber 120 triggers the release of wash solution from the wash chamber 66 which flows through the sample flow path to the waste chamber 68 .
- the detection of liquid in the chamber 121 then triggers the release of lysing reagent from the chamber 67 .
- the sensor 143 surrounding the chamber 67 may then be used to indicate when the chamber 67 is empty, triggering the start of ultrasonic lysis.
- the cartridge may have two waste chambers, one for sample and one for wash, with each waste chamber having a respective sensor chamber connected thereto.
- In-line reflective optical sensors 144 are used to determine the presence or absence of liquid in the flat-bottomed detection regions 130 , 136 , 137 , of channels 81 , 106 , and 110 , respectively (FIG. 7).
- Each sensor 144 has a built in emitter and detector that is positioned over a flat-bottomed detection region. The emitter emits a beam that is reflected from the cartridge and detected by the detector. The sensor detects a change in signal when as an air/liquid interface passes through the detection region.
- dual emitter reflective optical sensors may be used for a more reliable detection operation. Both types of reflective optical sensors are well known in the art and commercially available.
- the instrument 140 also includes a heat-exchanging module 147 having a slot 148 for receiving the reaction vessel of the cartridge.
- the module 147 is described in detail below with reference to FIG. 28.
- the instrument 140 further includes a latch mechanism 149 for latching a lid 150 over a cartridge.
- the cartridge nest 141 includes alignment holes 401 for receiving the legs of the cartridge. The alignment holes 401 ensure proper positioning of the cartridge in the nest 141 so that the pressure nozzles 145 , transducer 92 , and valve actuators 142 fit into the corresponding ports in the cartridge and so that the reaction vessel fits into the slot 148 .
- the transducer 92 should be positioned in the instrument 140 such that when the cartridge is placed in the nest 141 , the transducer contacts the bottom wall of the lysing chamber 86 , as shown in the cut-away view of FIG. 5.
- the instrument may include a spring or similar mechanism to bias the transducer 92 against the wall of the lysing chamber 86 .
- the instrument 140 also includes various conventional equipment not shown in FIG. 10 including a main logic board having a microcontroller for controlling the operation of the solenoids 146 , transducer 92 , heat-exchanging module 147 , and optical sensors 143 , 144 .
- the instrument also includes or is connected to a power supply for powering the instrument and a pneumatic pump for supplying air pressure through the nozzles 145 .
- the instrument 140 is preferably computer-controlled using, e.g., the microcontroller which is programmed to perform the functions described in the operation section below. Alternatively, the instrument may controlled by a separate computer, or controlled by a combination of a separate computer and an on-board microcontroller.
- FIG. 11 shows an isometric view of the cartridge 20 placed in the instrument 140 for processing.
- FIG. 11 shows a partial cut-away view of the instrument 140 with the lid 150 closed.
- a memory or microprocessor chip may optionally be incorporated as part of the cartridge 20 .
- This chip preferably contains information such as the type of cartridge, program information such as specific protocols for the processing of the cartridge, tolerances for accept and reject, serial numbers and lot codes for quality tracking, and provision for storing the results of the processing.
- Integrated electronic memory on the cartridge 20 allows for rapid, easy, and error-free set-up of the instrument 140 for different fluidic processing protocols.
- the instrument may electronically address the memory on the cartridge, and thus automatically receive the appropriate set of instructions for controlling the time-sequence of fluidic operations to be carried out with the inserted cartridge.
- the instrument 140 may simply sequentially retrieve and execute each step in the cartridge's memory, or download its contents so that the user may edit the sequence using, e.g., the controller computer.
- suitable memory such as writable memory (e.g., erasable programmable read-only memory (EPROM), electrically erasable programmable read-only memory (EEPROM), etc.
- EPROM erasable programmable read-only memory
- EEPROM electrically erasable programmable read-only memory
- intermediate and final results based on the sample introduced into the cartridge, could be written by the instrument into the cartridge's memory for co-located storage with the physical sample after processing. This is particularly advantageous in applications where archiving of samples and results is necessary, such as forensics.
- other information can be stored in the memory on the cartridge, in unalterable (or alterable) forms. For example, cartridge serial number, lot manufacture information, and related information could be pre-programmed and unalterable.
- FIG. 28 shows the heat-exchanging module 147 of the instrument into which the reaction vessel 40 is inserted for thermal processing and optical detection of target analyte(s) in the reaction mixture.
- the module 147 preferably includes a housing 208 for holding the various components of the module.
- the module 147 also includes the thermal plates 190 described above.
- the housing 208 includes a slot (not shown in FIG. 28) above the plates 190 so that the reaction chamber of the vessel 40 may be inserted through the slot and between the plates.
- the heat-exchanging module 147 also preferably includes a cooling system, such as a fan 212 .
- the fan 212 is positioned to blow cooling air past the surfaces of the plates 190 to cool the plates and hence cool the reaction mixture in the vessel 40 .
- the housing 208 preferably defines channels for directing the cooling air past the plates 190 and out of the module 147 .
- the heat-exchanging module 147 further includes an optical excitation assembly 216 and an optical detection assembly 218 for optically interrogating the reaction mixture contained in the vessel 40 .
- the excitation assembly 216 includes a first circuit board 220 for holding its electronic components, and the detection assembly 216 includes a second circuit board 222 for holding its electronic components.
- the excitation assembly 216 includes one or more light sources (e.g., an LED. laser, or light bulb) for exciting fluorescently-labeled analytes in the vessel 40 .
- the excitation assembly 216 also includes one or more lenses for collimating the light from the light sources, as well as filters for selecting the excitation wavelength ranges of interest.
- the detection assembly 218 includes one or more detectors (e.g., a photodiode, photomultiplier tube, or CCD) for detecting the light emitted from the vessel 40 .
- the detection assembly 218 also includes one or more lenses for focusing and collimating the emitted light, as well as filters for selecting the emission wavelength ranges of interest.
- Suitable optical excitation and detection assemblies for use in the heat-exchanging module 147 are described in International Publication Number WO 99/60380 (International Application Number PCT/US99/11182) published Nov. 25, 1999, the disclosure of which is incorporated by reference herein.
- the optics assemblies 216 , 218 are positioned in the housing 208 such that when the chamber of the vessel 40 is inserted between the plates 190 , the excitation assembly 216 is in optical communication with the chamber 42 through the optically transmissive side wall 57 A (see FIG. 22) and the detection assembly 218 is in optical communication with the chamber through the optically transmissive side wall 57 B (FIG. 22).
- FIG. 34 shows a partially cut-away, isometric view of the chamber of the vessel inserted between the plates 190 A, 190 B (the top portion of the vessel is cut away).
- the vessel preferably has an angled bottom portion (e.g., triangular) formed by the optically transmissive side walls 57 A, 57 B.
- Each of the plates 190 A, 190 B has a correspondingly shaped bottom portion.
- the bottom portion of the first plate 190 A has a first bottom edge. 250 A and a second bottom edge 2190 B.
- the bottom portion of the second plate 190 B has a first bottom edge 252 A and a second bottom edge 252 B.
- each plate is preferably angularly offset from each other by the same angle that the side walls 57 A, 57 B are offset from each other (e.g., 90°).
- the plates 190 A, 190 B are preferably positioned to receive the chamber of the vessel between them such that the first side wall 57 A is positioned substantially adjacent and parallel to each of the first bottom edges 250 A, 252 A and such that the second side wall 57 B is positioned substantially adjacent and parallel to each of the second bottom edges 2190 B, 252 B.
- This arrangement provides for easy optical access to the optically transmissive side walls 57 A, 57 B and hence to the chamber of the vessel.
- a gel or fluid may optionally be used to establish or improve optical communication between each optics assembly and the side walls 57 A, 57 B.
- the gel or fluid should have a refractive index close to the refractive indexes of the elements that it is coupling.
- the optics assemblies 216 , 218 are preferably arranged to provide a 90° angle between excitation and detection paths.
- the 90° angle between excitation and detection paths assures that a minimum amount of excitation radiation entering through the first side wall of the chamber exits through the second side wall.
- the 90° angle permits a maximum amount of emitted radiation to be collected through the second side wall.
- the vessel 40 includes a locating tab 58 (see FIG. 22) that fits into a slot formed between the optics assemblies 216 , 218 to ensure proper positioning of the vessel 40 for optical detection.
- the module 147 also preferably includes a light-tight lid (not shown) that is placed over the top of the vessel 40 and made light-tight to the housing 208 after the vessel is inserted between the plates 190 .
- optical communication may be established between the optics assemblies 216 , 218 and the walls of the vessel 40 via optical fibers, light pipes, wave guides, or similar devices.
- One advantage of these devices is that they eliminate the need to locate the optics assemblies 216 , 218 physically adjacent to the plates 190 . This leaves more room around the plates in which to circulate cooling air or refrigerant, so that cooling may be improved.
- the heat-exchanging module 147 also includes a PC board 226 for holding the electronic components of the module and an edge connector 224 for connecting the module 147 to the instrument 140 (FIG. 10).
- the heating elements and temperature sensors on the plates 190 , as well as the optical boards 220 , 222 , are connected to the PC board 226 by flex cables (not shown in FIG. 28 for clarity of illustration).
- the module 147 may also include a grounding trace 228 for shielding the optical detection circuit.
- the module 147 may optionally include an indicator, such as an LED 214 , for indicating to a user the current status of the module such as “heating,” “cooling,” “finished,” or “fault”.
- the housing 208 may be molded from a rigid, high-performance plastic, or other conventional material.
- the primary functions of the housing 208 are to provide a frame for holding the plates 190 , optics assemblies 216 , 218 , fan 212 , and PC board 226 .
- the housing 208 also preferably provides flow channels and ports for directing cooling air from the fan 212 across the surfaces of the plates 190 and out of the housing.
- the housing 208 comprises complementary pieces (only one piece shown in the schematic side view of FIG. 28) that fit together to enclose the components of the module 147 between them.
- the plates 190 A, 190 B may be made of various thermally conductive materials including ceramics or metals. Suitable ceramic materials include aluminum nitride, aluminum oxide, beryllium oxide, and silicon nitride. Other materials from which the plates may be made include, e.g., gallium arsenide, silicon, silicon nitride, silicon dioxide., quartz, glass, diamond, polyacrylics, polyamides, polycarbonates, polyesters, polyimides, vinyl polymers, and halogenated vinyl polymers, such as polytetrafluoroethylenes.
- Suitable ceramic materials include aluminum nitride, aluminum oxide, beryllium oxide, and silicon nitride.
- Other materials from which the plates may be made include, e.g., gallium arsenide, silicon, silicon nitride, silicon dioxide., quartz, glass, diamond, polyacrylics, polyamides, polycarbonates, polyesters, polyimides, vinyl polymers, and halogenated vinyl polymers, such as
- plate materials include chrome/aluminum, superalloys, zircaloy, aluminum, steel, gold, silver, copper, tungsten, molybdenum, tantalum, brass, sapphire, or any of the other numerous ceramic, metal, or polymeric materials available in the art.
- Suitable thermal elements may be employed to heat and/or cool the plates 190 A, 190 B and thus control the temperature of the reaction mixture in the chamber 42 .
- suitable heating elements for heating the plate include conductive heaters, convection heaters, or radiation heaters.
- conductive heaters include resistive or inductive heating elements coupled to the plates, e.g., resistors or thermoelectric devices.
- Suitable convection heaters include forced air heaters or fluid heat-exchangers for flowing fluids past the plates.
- Suitable radiation heaters include infrared or microwave heaters.
- various cooling elements may be used to cool the plates.
- various convection cooling elements may be employed such as a fan, peltier device, refrigeration device, or jet nozzle for flowing cooling fluids past the surfaces of the plates.
- various conductive cooling elements may be used, such as a heat sink, e.g. a cooled metal block, in direct contact with the plates.
- each plate 190 preferably has a resistive heating element 206 disposed on its outer surface.
- the resistive heating element 206 is preferably a thick or thin film and may be directly screen printed onto each plate 190 , particularly plates comprising a ceramic material, such as aluminum nitride or aluminum oxide. Screen-printing provides high reliability and low cross-section for efficient transfer of heat into the reaction chamber. Thick or thin film resistors of varying geometric patterns may be deposited on the outer surfaces of the plates to provide more uniform heating, for example by having denser resistors at the extremities and thinner resistors in the middle.
- a heating element may alternatively be baked inside of each plate, particularly if the plates are ceramic.
- the heating element 206 may comprise metals, tungsten, polysilicon, or other materials that heat when a voltage difference is applied across the material.
- the heating element 206 has two ends which are connected to respective contacts 204 which are in turn connected to a voltage source (not shown in FIG. 24) to cause a current to flow through the heating element.
- Each plate 190 also preferably includes a temperature sensor 192 , such as a thermocouple, thermistor, or RTD, which is connected by two traces 202 to respective ones of the contacts 204 .
- the temperature sensor 192 is be used to monitor the temperature of the plate 190 in a controlled feedback loop.
- each of the plates has a low thermal mass to enable rapid heating and cooling of the plates.
- each of the plates has a thermal mass less than about 5 J/° C., more preferably less than 3 J/° C., and most preferably less than 1 J/° C.
- the term thermal mass of a plate is defined as the specific heat of the plate multiplied by the mass of the plate.
- each plate should be large enough to cover a respective major wall of the reaction chamber.
- each of the plates has a width X in the range of 2 to 22 mm, a length Y in the range of 2 to 22 mm, and a thickness in the range of 0.5 to 5 mm.
- each plate is selected to be slightly larger than the width and length of the reaction chamber.
- each plate preferably has an angled bottom portion matching the geometry of the bottom portion of the reaction chamber, as previously described with reference to FIG. 34.
- each of the plates is made of aluminum nitride having a specific heat of about 0.75 J/g° C.
- the mass of each plate is preferably in the range of 0.005 to 5.0 g so that each plate has a thermal mass in the range of 0.00375 to 3.75 J/° C.
- the opposing plates 190 are positioned to receive the chamber of the vessel 40 between them such that the flexible major walls of the chamber contact and conform to the inner surfaces of the plates. It is presently preferred that the plates 190 be held in an opposing relationship to each other using, e.g., brackets, supports, or retainers. Alternatively, the plates 190 may be spring-biased towards each other as described in International Publication Number WO 98/38487, the disclosure of which is incorporated by reference herein. In another embodiment of the invention, one of the plates is held in a fixed position, and the second plate is spring-biased towards the first plate. If one or more springs are used to bias the plates towards each other, the springs should be sufficiently stiff to ensure that the plates are pressed against the flexible walls of the vessel with sufficient force to cause the walls to conform to the inner surfaces of the plates.
- FIGS. 29-30 illustrate a preferred support structure 209 for holding the plates 190 A, 190 B in an opposing relationship to each other.
- FIG. 29 shows an exploded view of the structure
- FIG. 30 shows an assembled view of the structure.
- the support structure 209 and plates 190 A, 190 B are shown upside down relative to their normal orientation in the heat-exchanging module of FIG. 28.
- the support structure 209 includes a mounting plate 210 having the slot 148 formed therein.
- the slot 148 is sufficiently large to enable the chamber of the vessel to be inserted through it.
- Spacing posts 230 A, 230 B extend from the mounting plate 210 on opposite sides of the slot 148 .
- Spacing post 230 A has indentations 232 formed on opposite sides thereof (only one side visible in the isometric view of FIG. 29), and spacing post 230 B has indentations 234 formed on opposite sides thereof (only one side visible in the isometric view of FIG. 29).
- the indentations 232 , 234 in the spacing posts are for receiving the edges of the plates 190 A, 190 B.
- the plates 190 A, 190 B are placed against opposite sides of the spacing posts 230 A, 230 B such that the edges of the plates are positioned in the indentations 232 , 234 .
- the edges of the plates are then held in the indentations using a suitable retention means.
- the plates are retained by retention clips 236 A, 236 B.
- the plates 190 A, 190 B may be retained by adhesive bonds, screws, bolts, clamps, or any other suitable means.
- the mounting plate 210 and spacing posts 230 A, 230 B are preferably integrally formed as a single molded piece of plastic.
- the plastic should be a high temperature plastic, such as polyetherimide, which will not deform of melt when the plates 190 A, 190 B are heated.
- the retention clips 230 A, 230 B are preferably stainless steel.
- the mounting plate 210 may optionally include indentations 240 A, 240 B for receiving flex cables 238 A, 238 B, respectively, that connect the heating elements and temperature sensors disposed on the plates 190 A, 190 B to the PC board 226 of the heat-exchanging module 147 (FIG. 28).
- the portion of the flex cables 238 A adjacent the plate 190 A is held in the indentation 240 A by a piece of tape 242 A, and the portion of the flex cables 238 B adjacent the plate 190 B is held in the indentation 240 B by a piece of tape 242 B.
- FIG. 31 is an isometric view of the assembled support structure 209 .
- the mounting plate 210 preferably includes tabs 246 extending from opposite sides thereof for securing the structure 209 to the housing of the heat-exchanging module.
- the housing 208 preferably includes slots for receiving the tabs to hold the mounting plate 210 securely in place.
- the mounting plate 210 may be attached to the housing 208 using, e.g., adhesive bonding, screws, bolts, clamps, or any other conventional means of attachment.
- the support structure 209 preferably holds the plates 190 A, 190 B so that their inner surfaces are angled very slightly towards each other.
- each of the spacing posts 230 A, 230 B has a wall 244 that is slightly tapered so that when the plates 190 A, 190 B are pressed against opposite sides of the wall, the inner surfaces of the plates are angled slightly towards each other.
- the inner surfaces of the plates 190 A, 190 B angle towards each other to form a slightly V-shaped slot into which the chamber 42 is inserted.
- the amount by which the inner surfaces are angled towards each other is very slight, preferably about 1° from parallel.
- the surfaces are angled towards each other so that, prior to the insertion of the chamber 42 between the plates 190 A, 190 B, the bottoms of the plates are slightly closer to each other than the tops. This slight angling of the inner surfaces enables the chamber 42 of the vessel to be inserted between the plates and withdrawn from the plates more easily.
- the inner surfaces of the plates 190 A, 190 B could be held parallel to each other, but insertion and removal of the vessel 40 would be more difficult.
- the inner surfaces of the plates 190 A, 190 B are preferably spaced from each other a distance equal to the thickness of the frame 46 .
- the centers of the inner surfaces are preferably spaced a distance equal to the thickness of the frame 46 and the bottoms of the plates are initially spaced a distance that is slightly less than the thickness of the frame 46 .
- the retention clips 236 A, 236 B should be sufficiently flexible to accommodate this slight outward movement of the plates 190 A, 190 B, yet sufficiently stiff to hold the plates within the recesses in the spacing posts 230 A, 230 B during insertion and removal of the vessel.
- the wedging of the vessel between the plates 190 A, 190 B provides an initial preload against the chamber and ensures that the flexible major walls of the chamber, when pressurized, establish good thermal contact with the inner surfaces of the plates.
- stops may be molded into the housings of optics assemblies 216 , 218 .
- the housing 249 of the optics assembly 218 includes claw-like stops 247 A, 247 B that extend outwardly from the housing.
- the housing 249 is positioned such that the bottom edges of the plates 190 A, 190 B are inserted between the stops 247 A, 247 B.
- the stops 247 A, 247 B thus prevent the plates 190 A, 190 B from spreading farther than a predetermined maximum distance from each other.
- the optics assembly 216 (see FIG. 28) has a housing with corresponding stops for preventing the other halves of the plates from spreading farther than the predetermined maximum distance from each other.
- the maximum distance that stops permit the inner surfaces of the plates 190 A, 190 B to be spaced from each other should closely match the thickness of the frame 46 .
- the maximum spacing of the inner surfaces of the plates 190 A, 190 B is slightly larger than the thickness of the frame 46 to accommodate tolerance variations in the vessel 40 and plates 190 A, 190 B.
- the maximum spacing is preferably about 0.1 to 0.3 mm greater than the thickness of the frame 46 .
- FIG. 35 is a schematic, block diagram of the electronic components of the heat-exchanging module 147 .
- the module includes a connector 224 or flex cable for connection to the main logic board of the instrument.
- the module also includes heater plates 190 A, 190 B each having a resistive heating element as described above.
- the plates 190 A, 190 B are wired in parallel to receive power input 253 from the instrument.
- the plates 190 A, 190 B also include temperature sensors 192 A, 192 B that output analog temperature signals to an analog-to-digital converter 264 .
- the converter 264 converts the analog signals to digital signals and routes them to the microcontroller in the instrument through the connector 224 .
- the heat-exchanging module also includes a cooling system, such as a fan 212 , for cooling the plates 190 A, 190 B and the reaction mixture contained in the vessel inserted between the plates.
- the fan 212 is activated by switching a power switch 272 , which is in turn controlled by a control logic block 270 that receives control signals from the microcontroller.
- the module further includes four light sources, such as LEDs 200 , for excitation of labeled analytes in the reaction mixture and four detectors 198 , preferably photodiodes, for detecting fluorescent emissions from the reaction mixture.
- the module also includes an adjustable current source 255 for supplying a variable amount of current (e.g., in the range of 0 to 30 mA) to each LED to vary the brightness of the LED.
- a digital-to-analog converter 260 is connected between the adjustable current source 255 and the microcontroller to permit the microcontroller to adjust the current source digitally.
- the adjustable current source 255 is preferably used to ensure that each LED has about the same brightness when activated. Due to manufacturing variances, many LEDs have different brightnesses when provided with the same amount of current. Therefore, it is presently preferred to test the brightness of each LED during manufacture of the heat-exchanging module and to store calibration data in a memory 268 of the module. The calibration data indicates the correct amount of current to provide to each LED. The microcontroller reads the calibration data from the memory 268 and controls the current source 255 accordingly.
- the module additionally includes a signal conditioning/gain select/offset adjust block 262 comprised of amplifiers, switches, electronic filters, and a digital-to-analog converter.
- the block 262 adjusts the signals from the detectors 198 to increase gain, offset, and reduce noise.
- the microcontroller controls block 262 through a digital output register 266 .
- the output register 266 receives data from the microcontroller and outputs control voltages to the block 262 .
- the block 262 outputs the adjusted detector signals to the microcontroller through the analog-to-digital converter 264 and the connector 224 .
- the module also includes the memory 268 , preferably a serial EEPROM, for storing data specific to the module, such as calibration data for the LEDs 200 , thermal plates 190 A, 190 B, and temperature sensors 192 A, 192 B.
- a fluid sample to be analyzed is added to the sample chamber 65 through the sample port 64 and the cap 30 screwed into the port 64 to seal the port shut.
- the cartridge 20 is then placed into the cartridge nest 141 of the instrument 140 for processing. All valves in the cartridge are initially closed when the cartridge is placed into the instrument 140 .
- the transducer 92 contacts an external surface of the flexible gasket 63 forming the bottom wall of the lysing chamber 86 , as shown in FIG. 5.
- the instrument 140 is preferably computer-controlled to perform the functions described in the following section, e.g., opening and closing valves in the cartridge using valve actuators 142 , providing pressure to the cartridge through nozzles 145 , activating the transducer 92 , sensing liquid presence or liquid levels using optical sensors 143 and 144 , and controlling the heat-exchanging and optical detection module 147 .
- a programmer having ordinary skill in the art will be able to program a microcontroller and/or computer to perform these functions based upon the following description.
- liquids are preferably forced to flow through the cartridge using differential pressure.
- positive pressure is described herein, negative pressure (vacuum) may also be used to control fluid flow in the cartridge.
- the maximum amount of positive pressure that can be applied is usually limited by the hydrophobic membranes which may reach liquid break-through pressure above 30 pounds per square inch (psi).
- the lower limit of pressure is limited by the need to move sample and other fluids through the cartridge sufficiently quickly to meet assay goals. Below 1 psi, for example, sample may not flow efficiently through the filter stack 87 . Pressure in the range of 6 to 20 psi is generally adequate.
- the sample flow rate through the cartridge is preferably in the range of 10 to 30 ml/minute.
- the wash flow rate may be slower, e.g. 6 to 18 ml/minute so that the wash effectively washes the lysing chamber 86 .
- the cartridge is preferably primed with wash solution from the wash chamber 66 before the fluid sample is forced to flow from the sample chamber 65 .
- valves 111 and 115 are opened and a pressure of 10 psi is applied to the chamber 66 through the pressure port 116 for about two seconds.
- a small portion of the wash solution flows through the channels 117 and 106 , through the lysing chamber 86 , through the channels 109 and 110 , into the U-shaped channel 122 , and all the way to the hydrophobic membrane below the pressure port 128 .
- valve 115 and pressure port 116 are closed and valves 107 and 114 are opened.
- a pressure of 20 psi is applied to the sample chamber 65 through the pressure port 105 for about 15 seconds to force the sample to flow through the channel 106 , through the filter stack 87 in the chamber 87 , through the channels 110 , 111 , 112 and into the vented waste chamber 68 .
- the reflective optical sensor 144 (FIG. 13) may be used to determine when the sample chamber 65 has been emptied. As the sample liquid flows through the filter stack 87 , target cells or viruses in the sample are captured.
- the steps in a predetermined protocol may simply be timed, e.g., applying predetermined pressures for predetermined durations of time to move known volumes of fluid at known flow rates.
- the flow-through design of the lysing chamber 86 permits target cells or viruses from a relatively large sample volume to be concentrated into a much smaller volume for amplification and detection. This is important for the detection of low concentration analyte in the sample, such as nucleic acid.
- the ratio of the volume of the sample forced to flow through the lysing chamber 86 to the volume capacity of the chamber 86 is preferably at least 2:1, and more preferably at least 5:1.
- the volume of sample forced to flow through the chamber 86 is preferably at least 100 ⁇ l, and more preferably at least 1 ml.
- a sample volume of 5 ml is forced to flow through the lysing chamber 86 , and the chamber 86 has a volume capacity of about 0.5 ml, so that the ratio is 10:1.
- the lysing chamber 86 may be sonicated (e.g., using an ultrasonic horn coupled to a wall of the chamber) as the sample is forced to flow through the chamber. Sonicating the chamber 86 helps to prevent clogging of the filter stack 87 , providing for more uniform flow through the chamber 86 .
- the sound waves help keep particulate matter or the beads in the filter stack from clogging one or more filters.
- valves 111 , 114 , 115 are opened and a pressure of 20 psi is applied to the wash chamber 66 for about seven seconds to force the wash solution to flow through the channels 117 and 106 into the lysing chamber 86 .
- the washing solution washes away PCR inhibitors and contaminants from the lysing chamber 86 and carries then through the channels 109 , 110 , and 112 into the waste chamber 68 .
- a variety of suitable wash solutions of varying pH, solvent composition, and ionic strength may be used for this purpose and are well known in the art.
- a suitable washing reagent is a solution of 80 mM potassium acetate, 8.3 mM Tris-HCl, pH 7.5, 40 uM EDTA, and 55% ethanol.
- the lysing chamber 86 may be sonicated (e.g., using an ultrasonic horn coupled to a wall of the chamber) while the wash solution is forced to flow through the chamber. Sonicating the chamber 86 helps to prevent clogging of the filter stack 87 , providing for more uniform flow through the chamber 86 as previously described. In addition, the sound waves may help loosen the material to be washed away. When the incremental volume of wash solution reaches the waste chamber 68 , some of the liquid spills over into the sensor chamber 121 , triggering the next step in the protocol.
- valve 115 is closed and valve 119 is opened while a pressure of 15 psi is applied to the reagent chamber 67 through the pressure port 118 for about three seconds.
- the pressure forces lysing reagent to flow from the chamber 67 through the channels 117 , 106 into the lysing chamber 86 , and into the channel 110 .
- the chamber 86 is thus filled with liquid.
- Suitable lysing reagents include, e.g., solutions containing a chaotropic salt, such as guanidine HCl, guanidine thiocyanate, guanidine isothiocyanate, sodium iodide, urea, sodium perchlorate, and potassium bromide.
- a lysing reagent that is not inhibitory to PCR is used.
- the lysing reagent comprises 10 mM tris, 5% tween-20, 1 mM tris (2-carboxyethyl phosphine hydrochloride), 0.1 mM Ethylene Glycol-bis (b-amino-ethyl ether)—N,N,N′,N′—tetracetic acid.
- the pressurization of the lysing chamber 86 is important because it ensures effective coupling between the transducer 92 and the flexible wall 63 of the lysing chamber 86 .
- the transducer 92 is activated (i.e., set into vibratory motion).
- the flexible wall 63 of the lysing chamber 86 transfers the vibratory motion of the transducer 92 to the liquid in the chamber 86 by allowing slight deflections without creating high stresses in the wall.
- the wall 63 may be formed by the elastomeric membrane as previously described.
- the wall may be a film or sheet of polymeric material (e.g., a polypropylene film) preferably having a thickness in the range of 0.025 to 0.1 mm.
- the transducer 92 is preferably an ultrasonic horn for sonicating the chamber 86 .
- the chamber 86 is preferably sonicated for 10 to 40 seconds at a frequency in the range of 20 to 60 kHz. In the exemplary protocol, the chamber is sonicated for 15 seconds at a frequency of 47 kHz.
- the amplitude of the horn tip is preferably in the range of 20 to 25 ⁇ m (measured peak to peak).
- the tip of the transducer 92 vibrates, it repeatedly impacts the flexible wall 63 .
- the tip of the transducer 92 pushes the wall 63 and creates a pressure pulse or pressure wave in the chamber 86 .
- the tip of the transducer 92 usually separates from the flexible wall 63 because the flexible wall 63 cannot move at the same frequency as the transducer.
- the tip of the transducer 92 On its next forward stroke, the tip of the transducer 92 once again impacts the wall 63 in a head-on collision as the tip and wall speed towards each other.
- the effective forward stroke of the transducer is less than its peak-to-peak amplitude.
- the effective forward stroke determines the level of sonication in the chamber 86 . It is therefore important to increase the static pressure in the lysing chamber 86 so that when the tip of the transducer 92 retreats, the flexible wall 63 is forced outwardly to meet the tip on its return stroke.
- the static pressure in the chamber 86 should be sufficient to ensure that the effective forward stroke of the transducer 92 generates pressure pulses or pressure waves in the chamber 86 . It is presently preferred to increase the static pressure in the chamber 86 to at least 5 psi above the ambient pressure external to the cartridge, and more preferably to a pressure in the range of 15 to 25 psi above the ambient pressure.
- the transducer 92 On each forward stroke, the transducer 92 imparts a velocity to the liquid in the chamber 86 , thus creating a pressure pulse that quickly sweeps across the chamber 86 .
- the beads in the filter stack 87 (FIG. 6) are agitated by the pressure pulses in the chamber 86 .
- the pressure pulses propel the beads into violent motion in the chamber 86 , and the beads mechanically rupture the cells or viruses to release the material (e.g., nucleic acid) therefrom.
- some types of cells such as blood cells, are relatively weak and may be disrupted using only pressure pulses without the use of beads. Other types of cells (particularly spores) have highly resistant cell walls and beads are generally required for effective lysis.
- valves 111 , 124 are opened and a pressure of 12 psi is delivered for about 4 seconds to the reagent chamber 67 through the pressure port 118 .
- the pressure forces the lysis reagent to elute the nucleic acid from the filter stack 87 and to flow with the nucleic acid into the neutralization chamber 70 .
- the lysing chamber 86 may be sonicated (e.g., using an ultrasonic horn coupled to a wall of the chamber) while the eluting the nucleic acid. Sonicating the chamber 86 may help prevent clogging of the filter stack 87 , as previously described.
- the chamber 420 is partially filled (e.g., half-filled) with neutralizer, such as detergent, for neutralizing the lysing reagent. If a lysing reagent non-inhibitory to PCR is used, the neutralizer is optional.
- valve 124 is closed to hold the lysing reagent, analyte, and neutralizer in the chamber 70 .
- the valve 114 is opened and a pressure of 15 psi is applied for about three seconds through the pressure port 128 to force any liquid in the U-shaped channel 122 to flow into the waste chamber 68 .
- valves 124 and 126 are opened and a pressure of 15 psi is applied for about five seconds through the pressure port 123 on top of the neutralizer chamber 70 .
- the pressure forces the neutralized lysing reagent and nucleic acid in the chamber 70 to flow into the channel 122 and into the master mix chamber 71 .
- the valve 126 to the master mix chamber 71 is then closed.
- the master mix chamber contains PCR reagents and fluorescent probes that mix with the neutralized lysing reagent and nucleic acid to form a reaction mixture.
- the channel 122 is cleared by opening valve 114 to waste chamber 68 and applying a pressure of 15 psi for about one second to pressure port 128 .
- the reaction mixture formed in the master mix chamber 71 is moved into the reaction vessel 40 as follows. Valves 126 , 127 , and 133 are opened and a pressure of 15 psi is applied for about six seconds to the pressure port 125 on top of the master mix chamber 71 to force the reaction mixture to flow through the channel 122 , valve 127 , and channel 80 into the reaction vessel 40 through the port 41 .
- the reaction mixture fills the chamber 42 of the vessel, displacing air in the chamber which exits through the outlet channel 52 .
- the air escaping through the outlet channel 52 travels in channel 81 past sensor region 130 and into channel 131 . From channel 131 , the air flows into channel 132 , through valve 133 , channel 134 , and exits the cartridge through the vent 36 . When a volume of reaction mixture sufficient to fill the chamber 42 has flowed into the vessel, excess reaction mixture exits the vessel through the outlet channel 52 . The excess reaction mixture flows into channel 81 and is optically detected in the sensor region 130 . When the reaction mixture is detected, valve 133 is closed while pressure from the pressure port 125 is applied to pressurize the reaction chamber 42 .
- the pressurization of the chamber 42 expands the flexible major walls 48 of the vessel.
- the pressure forces the major walls 48 to contact and conform to the inner surfaces of the plates 190 A, 190 B. This ensures optimal thermal conductance between the plates 190 A, 190 B and the reaction mixture in the chamber 42 .
- the chamber 42 is pressurized to a pressure in the range of 8 to 15 psi above ambient pressure. This range is more preferred because it is safely within the practical limits described above.
- the reaction mixture in the vessel 40 is thermally processed and optically interrogated to determine the presence or absence of a target analyte in the mixture.
- the reaction mixture is thermally processed between the plates 190 A, 190 B using standard proportional-integral-derivative (PID) control using target temperatures and feedback signals from the temperature sensors 192 A, 192 B.
- Proportioning may be accomplished either by varying the ratio of “on” time to “off” time, or, preferably with proportional analog outputs which decrease the average power being supplied either to the heating elements on the plates 190 A, 190 B or to the fan 212 as the actual temperature of the plates 190 A, 190 B approaches the desired set point temperature.
- PID control combines the proportional mode with an automatic reset function (integrating the deviation signal with respect to time) and rate action (summing the integral and deviation signal to shift the proportional band).
- Standard PID control is well known in the art and need not be described further herein.
- the reaction mixture may be thermally processed using a modified version of PID control described in International Publication Number WO 99/48608 (Application Number PCT/US99/06628) the disclosure of which is incorporated by reference herein.
- the reaction mixture is thermally cycled between the heater plates 190 A, 190 B to amplify one or more target nucleic acid sequences in the mixture
- the mixture is optically interrogated, preferably at the lowest temperature point in each cycle.
- Optical interrogation is accomplished by sequentially activating each of the LEDs 200 to excite different fluorescently-labeled analytes in the mixture and by detecting light emitted (fluorescent output) from the chamber 42 using detectors the 198 .
- excitation beams are preferably transmitted to the chamber 42 through the optically transmissive side wall 57 A, while fluorescent emission is detected through the side wall 57 B.
- the cartridge of the present invention allows the intracellular material from a relatively large volume of fluid sample, e.g. several milliliters or more, to be separated from the sample and concentrated into a much smaller volume of reaction fluid, e.g., 100 ⁇ L or less.
- the cartridge permits extraordinary concentration factors by efficiently extracting material from milliliter quantities of fluid sample.
- the sample chamber 65 preferably has a volume capacity in the range of 100 ⁇ l to 12 ml. More preferably, the sample chamber 65 has a volume capacity of at least 1 ml. The lower limit of 1 ml is preferred because at least 1 ml of sample should be analyzed to detect low concentration analytes such as nucleic acid.
- the upper limit of 12 ml is preferred because a sample volume greater than 12 ml would require a much larger cartridge and likely clog the filter stack.
- the sample chamber has a volume capacity of 5.5 ml for holding 5 ml of sample.
- the wash chamber 66 has a volume capacity proportional to the volume of the lysing chamber 86 .
- the wash chamber 66 preferably holds a volume of wash that is at least one to two times the volume of the lysing chamber 86 to ensure that there is enough wash solution to wash out PCR inhibitors and debris from the chamber 86 .
- the volume of the lysing chamber 86 is about 0.5 ml and the volume of the wash chamber 66 is 2.5 ml for holding 2 ml of wash solution.
- the lysing chamber volume of 0.5 ml is a compromise between a size large enough to avoid clogging of the filter stack 87 and a size small enough to concentrate analyte into a small volume for improved amplification and detection.
- the reagent chamber 67 preferably holds a volume of lysing reagent that is at least one to two times the volume of the lysing chamber 86 so that there is sufficient lysing reagent to pressurize the chamber and to elute nucleic acid from the chamber.
- the chamber 67 has a volume capacity of 1.5 ml for holding about 1 to 1.5 ml of lysing reagent.
- the waste chamber 68 has a volume capacity sufficient to hold the sample, wash solution, and unused lysing reagent.
- the waste chamber 68 is sized at 9.5 ml volume capacity in the preferred embodiment.
- the size of the neutralization chamber 70 is dependent upon the volume of the lysing chamber 86 since the neutralizer in the chamber 70 neutralizes the volume of lysing reagent that fills the lysing chamber 86 . It is currently preferred that the lysing chamber have a volume if 0.5 ml, so the chamber 70 has a volume capacity of 10.0 ml for holding about 0.5 ml of neutralizer that is mixed with 0.5 ml of the lysing reagent and eluted analyte.
- the volume capacity of the master mix chamber 71 should be sufficient to produce a reaction mixture to fill the vessel 40 and the channels 122 , 127 leading to the vessel.
- the master mix chamber has a volume capacity of 200 ⁇ l for holding an initial load of 100 ⁇ l of master mix to which is added 100 ⁇ l of neutralized lysing reagent and eluted analyte to form the reaction mixture.
- the flow channels in the cartridge are generally D-shaped in cross section (with the gasket 63 forming the flat side of the channel) and preferably have a width or diameter in the range of ⁇ fraction (1/64) ⁇ to 1 ⁇ 8 of an inch (0.4 to 3.2 mm), and more preferably a width of ⁇ fraction (1/32) ⁇ to ⁇ fraction (1/16) ⁇ of an inch (0.8 to 1.6 mm). These ranges are presently preferred to avoid having channels to narrow (which creates flow restriction) and to avoid having channels too wide (which yields unused volumes of liquid sitting in the flow path).
- the cartridge and instrument may be used to amplify nucleic acid sequences using any amplification method, including both thermal cycling amplification methods and isothermal amplification methods.
- Suitable thermal cycling methods include, but are not limited to, the Polymerase Chain Reaction (PCR; U.S Pat. Nos. 4,683,202, 4,683,195 and 4,965,188); Reverse Transcriptase PCR (RT-PCR); DNA Ligase Chain Reaction (LCR; International Patent Application No. WO 89/09835); and transcription-based amplification (D. Y. Kwoh et al. 1989, Proc.
- Suitable isothermal amplification methods useful in the practice of the present invention include, but are not limited to, Rolling Circle Amplification; Strand Displacement Amplification (SDA; Walker et al. 1992, Proc. Nati. Acad. Sci. USA 89, 392-396); Q-.beta. replicase (Lizardi et al. 1988, Bio/Technology 6, 1197-1202); Nucleic Acid-Based Sequence Amplification (NASBA; R. Sooknanan and L. Malek 1995, Bio/Technology 13, 563-65); and Self-Sustained Sequence Replication (3SR; Guatelli et al. 1990, Proc. Nati. Acad. Sci. USA 87, 1874-1878).
- the cartridge and instrument may be used to conduct chemical reactions other than nucleic acid amplification.
- fluorescence excitation and emission detection is preferred, optical detection methods such as those used in direct absorption and/or transmission with on-axis geometries may also be used to detect analyte in the cartridge.
- Another possible detection method is time decay fluorescence.
- the cartridge is not limited to detection based upon fluorescent labels. For example, detection may be based upon phosphorescent labels, chemiluminescent labels, or electrochemiluminescent labels.
- a fluid sample may be introduced into the cartridge by a variety of means, manual or automated.
- a measured volume of material may be placed into a receiving area of the cartridge through an input port and a cap is then placed over the port.
- a greater amount of sample material than required for the analysis can be added to the cartridge and mechanisms within the cartridge can effect the precise measuring and aliquoting of the sample needed for the specified protocol.
- tissue biopsy material such as tissue biopsy material, soil, feces, exudates, and other complex material
- a piece of tissue may be placed into the lumen of a secondary device that serves as the cap to the input port of the cartridge. When the cap is pressed into the port, the tissue is forced through a mesh that slices or otherwise divides the tissue.
- a syringe may be integrated into a device to provide a means for moving external fluidic samples directly into the cartridge.
- a venous puncture needle and an evacuated blood tube can be attached to the cartridge forming an assembly that can be used to acquire a sample of blood. After collection, the tube and needle are removed and discarded, and the cartridge is then placed in an instrument to effect processing. The advantage of such an approach is that the operator or the environment is not exposed to pathogens.
- the input port can be designed with a consideration of appropriate human factors as a function of the nature of the intended specimen.
- respiratory specimens may be acquired from the lower respiratory tract as expectorants from coughing, or as swab or brush samples from the back of the throat or the nares.
- the input port can be designed to allow the patient to cough directly into the cartridge or to otherwise facilitate spitting of the expectorated sample into the cartridge.
- brush or swab specimens the specimen is placed into the input port where features of the port and closure facilitate the breaking off and retaining of the end of the swab or brush in the cartridge receiving area.
- the cartridge includes input and output tubes that may be positioned in a sample pool of very large volume, such as a flowing stream of water, so that the sample material flows through the cartridge.
- a hydrophilic wicking material can serve as an interactive region so that the entire cartridge can be immersed directly into the specimen, and a sufficient amount of specimen is absorbed into the wicking material. The cartridge is then removed, and can be transported to the laboratory or analyzed directly using a portable instrument.
- tubing can be utilized so that one end of the tube is in direct communication with the cartridge to provide a fluidic interface with at least one interactive region and the other end is accessible to the external environment to serve as a receiver for sample.
- the tube can then be placed into a specimen and serve as a sipper.
- the cartridge itself may also serve as the actual specimen collection device, thereby reducing handling and inconvenience.
- the direct accessing of the test material into the fluidic cartridge is advantageous because the chain of custody is conveniently and reliably preserved.
- reagents may be exogenously introduced into the cartridge before use, e.g., through sealable openings in the reagent chamber 67 , neutralizer chamber 70 , and master mix chamber 71 .
- the reagents may be placed in the cartridge during manufacture, e.g., as aqueous solutions or dried reagents requiring reconstitution.
- the particular format is selected based on a variety of parameters, including whether the interaction is solution-phase or solid-phase, the inherent thermal stability of the reagent, speed of reconstitution, and reaction kinetics.
- Reagents containing compounds that are thermally unstable when in solution can be stabilized by drying using techniques such as lyophilization.
- Additives, such as simple alcohol sugars, methylcelluloses, and bulking proteins may be added to the reagent before drying to increase stability or reconstitutability.
- the reaction vessel 40 does not require two flexible sheets forming opposing major walls 48 of the reaction chamber 42 .
- the vessel 40 has only one flexible sheet forming a major wall of the chamber.
- the rigid frame 46 defines the other major wall of the chamber, as well as the side walls of the chamber.
- the major wall formed by the frame 46 should have a minimum thickness of about 0.05 inches (1.25 mm) which is typically the practical minimum thickness for injection molding, while the flexible sheet may be as thin as 0.0005 inches (0.0125 mm).
- the advantage to this embodiment is that the manufacturing of the reaction vessel 40 is simplified, and hence less expensive, since only one flexible sheet need be attached to the frame 46 .
- the disadvantage is that the heating and cooling rates of the reaction mixture are likely to be slower since the major wall formed by the frame 46 will probably not permit as high a rate of heat transfer as the thin, flexible sheet.
- the heat-exchanging module 147 only requires one thermal surface for contacting a flexible wall of the reaction vessel 40 and one thermal element for heating and/or cooling the thermal surface.
- the advantage to using one thermal surface and one thermal element is that the apparatus may be manufactured less expensively. The disadvantage is that the heating and cooling rates are likely to be about twice as slow.
- the thermal surfaces be formed by the thermally conductive plates 190 , each thermal surface may be provided by any rigid structure having a contact area for contacting a wall of the vessel 40 .
- the thermal surface preferably comprises a material having a high thermal conductivity, such as ceramic or metal.
- the thermal surface may comprise the surface of the thermal element itself.
- the thermal surface may be the surface of a thermoelectric device that contacts the wall to heat and/or cool the chamber.
- the transducer may be built into the cartridge.
- a piezoelectric disk may be built into the cartridge for sonicating the lysing chamber.
- a speaker or electromagnetic coil device may be built into the cartridge.
- the cartridge includes suitable electrical connectors for connecting the transducer to a power supply.
- the transducer should be prevented from contacting the fluid sample directly, e.g., the transducer should be laminated or separated from the sample by a chamber wall. Further, lysis of the cells or viruses may be performed using a heater in place of or in combination with a transducer.
- the heater may be a resistive heating element that is part of cartridge, or the heater could be built into the instrument that receives the cartridge.
- the cells or viruses are disrupted by heating the lysis chamber to a high temperature (e.g., 95° C.) to disrupt the cell walls.
- FIGS. 36-46 show another apparatus 350 for disrupting cells or viruses according to the present invention.
- FIG. 36 shows an isometric view of the apparatus 350
- FIG. 37 shows a cross sectional view of the apparatus 350 .
- the apparatus 350 includes a cartridge or container 358 having a chamber 367 for holding the cells or viruses.
- the container includes a flexible wall 440 defining the chamber 367 .
- the flexible wall 440 is the bottom wall of the chamber 367 .
- the flexible wall 440 is preferably a sheet or film of polymeric material (e.g., a polypropylene film) and the wall 440 preferably has a thickness in the range of 0.025 to 0.1 mm.
- the apparatus 350 also includes a transducer 314 , such as an ultrasonic horn, for contacting an external surface of the flexible wall 440 (i.e., a surface of the wall 440 that is external to the chamber 367 ).
- the transducer 314 should be capable of vibratory motion sufficient to create pressure pulses in the chamber 367 .
- Suitable transducers include ultrasonic, piezoelectric, magnetostrictive, or electrostatic transducers.
- the transducer may also be an electromagnetic device having a wound coil, such as a voice coil motor or a solenoid device.
- the apparatus 350 further includes a support structure 352 for holding the container 358 and the transducer 314 against each other such that the transducer 314 contacts the wall 440 of the chamber 367 and for applying a substantially constant force to the container 358 or to the transducer 314 to press together the transducer 314 and the wall 440 of the chamber.
- the support structure 352 includes a base structure 354 having a stand 356 .
- the transducer 314 is slidably mounted to the base structure 354 by a guide 364 .
- the guide 364 is either integrally formed with the base structure 354 or fixedly attached to the base structure.
- the support structure 352 also includes a holder 360 attached to the base structure 354 for holding the container 358 .
- the holder 360 has a U-shaped bottom portion providing access to the flexible wall 440 of the chamber 367 .
- the guide 364 and the holder 360 are arranged to hold the transducer 314 and the container 358 , respectively, such that the external surface of the wall 440 contacts the transducer 314 .
- the support structure 352 also includes a top retainer 362 for the container 358 .
- the retainer 362 is U-shaped to allow access to an exit port 444 formed in the container 358 .
- the support structure 352 further includes an elastic body, such as a spring 366 , for applying a force to the transducer 314 to press the transducer 314 against the wall 440 .
- an elastic body such as a spring 366
- the spring 366 is positioned between a spring guide. 372 and the base of a coupler 368 that supports the bottom of the transducer 314 .
- the coupler 370 preferably has a window 370 through which the power cord (not shown) of the transducer 314 may be placed.
- Bolts or screws 376 hold the spring guide 372 in adjustment grooves 374 formed in the base structure 354 .
- the magnitude of the force provided by the spring 366 may be adjusted by changing the preload on the spring. To adjust the preload on the spring 366 , the bolts 376 holding the spring guide 372 are loosened, the guide 372 is moved to a new position, and the bolts 376 are retightened to hold the guide 372 in the new position. Once the preload on the spring 366 is adjusted to provide a suitable coupling force between the transducer 314 and the wall 440 , it is desirable to keep the preload constant from one use of the apparatus 350 to the next so that valid comparisons can be made between different samples disrupted by the apparatus.
- the magnitude of the force provided by the spring 366 to press together the transducer 314 and the wall 440 is important for achieving a consistent transfer of energy between the transducer 314 and the chamber 367 . If the force is too light, the transducer 314 will only be held lightly against the wall 440 , leading to poor translation of vibratory movement from the transducer 314 to the wall 440 . If the force is too strong, the container 358 or wall 440 may be damaged during sonication. An intermediate force results in the most consistent and repeatable transfer of vibratory motion from the transducer 314 to the wall 440 . It is presently preferred that the spring 366 provide a force in the range of 2 to 5 lbs.
- FIG. 38 shows an exploded view of the container 358
- FIG. 39 shows an assembled view of the container 358
- the container 358 has a body comprising a to p piece 4 48 , a middle piece 450 , and a bottom piece 452 .
- the middle piece 450 defines an inlet port 442 to the chamber 367
- the top piece 448 defines an outlet port 444 to the chamber.
- the ports 442 , 444 are positioned to permit the continuous flow of a fluid sample through the chamber 367 .
- the flexible wall 440 is held between the middle and bottom pieces 450 , 452 using gaskets 453 , 454 .
- the flexible wall 440 may simply be heat sealed to the middle piece 450 so that the bottom piece 452 and gaskets 453 , 454 may be eliminated.
- the container 358 also includes a filter stack 446 in the chamber 367 for capturing sample components (e.g., target cells or viruses) as the sample flows through the chamber 367 .
- the filter stack comprises (from bottom to top in FIGS. 38-39) a gasket 456 , a first filter 458 , a gasket 460 , a second filter 464 having a smaller average pore size than the first filter 458 , and a gasket 466 .
- the filter stack is held between the top and middle pieces 448 , 450 of the container 358 .
- the filter stack also includes beads 462 disposed between the first and second filters 458 and 464 .
- the gasket 460 spaces the first filter 458 from the second filter 464 .
- the gasket 460 should be thick enough to permit the beads to move freely in the space between the filters 458 , 464 .
- a fluid sample flowing through the chamber 367 first flows through the filter 458 and then through the filter 466 . After flowing through the filter stack, the sample flows along flow ribs 468 (FIG. 38) formed in the portion of the top piece 448 that defines the top of the chamber and through the outlet port 444 (FIG. 39).
- the filter stack is effective for capturing cells or viruses as a fluid sample flows through the chamber 367 without clogging of the.
- the first filter 458 (having the largest pore size) filters out coarse material such as salt crystals, cellular debris, hair, tissue, etc.
- the second filter 464 (having a smaller pore size) captures target cells or viruses in the fluid sample.
- the average pore size of the first filter 458 is selected to be small enough to filter coarse material from the fluid sample (e.g., salt crystals, cellular debris, hair, tissue) yet large enough to allow the passage of the target cells or viruses.
- the average pore size of the first filter 458 should be in the range of about 2 to 25 ⁇ m, with a presently preferred pore size of about 5 ⁇ m.
- the average pore size of the second filter 464 is selected to be slightly smaller than the average size of the target cells or viruses to be captured (typically in the range of 0.2 to 5 ⁇ m).
- the beads 462 are useful for disrupting the captured cells or viruses to release the intracellular material (e g., nucleic acid) therefrom. Movement of the beads 462 ruptures the cells or viruses captured on the filter 464 .
- Suitable beads for rupturing cells or viruses include borosilicate glass, lime glass, silica, and polystyrene beads.
- the beads may be porous or non-porous and preferably have an average diameter in the range of 1 to 200 ⁇ m. In the presently preferred embodiment, the beads. 462 are polystyrene beads having an average diameter of about 100 ⁇ m.
- the beads 462 may have a binding affinity for target cells or viruses in the fluid sample to facilitate capture of the target cells or viruses.
- antibodies or certain receptors may be coated onto the surface of the beads 462 to bind target cells in the sample.
- the chamber 367 may contain two different types of beads for interacting with target cells or viruses.
- the chamber may contain a first set of beads coated with antibodies or receptors for binding target cells or viruses and a second set of beads (intermixed with the first set) for rupturing the captured cells or viruses.
- the beads in the chamber may also have a binding affinity for the intracellular material (e.g., nucleic acid) released from the ruptured cells or viruses.
- the chamber 367 may contain silica beads to isolate DNA or cellulose beads with oligo dT to isolate messenger RNA for RT-PCR.
- the chamber 367 may also contain beads for removing unwanted material (e.g., proteins, peptides) or chemicals (e.g., salts, metal ions, or detergents) from the sample that might inhibit PCR.
- the container 358 In an orientation in which liquid flows up (relative to gravity) through the filters 458 , 464 and the chamber 367 .
- the upward flow through the chamber 367 aids the flow of air bubbles out of the chamber.
- the inlet port 442 for entry of fluids into the chamber 367 should generally be at a lower elevation than the outlet port 444 .
- the volume capacity of the chamber 367 is usually in the range of 50 to 500 ⁇ l.
- the volume capacity of the chamber 367 is selected to provide for concentration of analyte separated from a fluid sample without the chamber being so small that the filters 458 , 464 become clogged.
- the pieces 448 , 450 , 452 forming the body of the container 358 are preferably molded polymeric parts (e.g., polypropylene, polycarbonate, acrylic, etc.). Although molding is preferred for mass production, it also possible to machine the top, middle, and bottom pieces 448 , 450 , 452 .
- the pieces 448 , 450 , 452 may be held together by screws or fasteners. Alternatively, ultrasonic bonding, solvent bonding, or snap fit designs could be used to assemble the container 358 .
- Another method for fabricating the container 358 is to mold the body as a single piece and heat seal the flexible wall 440 and the filters 458 , 464 to the body.
- FIG. 40 shows a fluidic system for use with the apparatus.
- the system includes a bottle 470 for holding lysis buffer, a bottle 472 containing wash solution, and a sample container 474 for holding a fluid sample.
- the bottles 470 , 472 and sample container 474 are connected via tubing to the valve ports of a syringe pump 476 .
- the inlet port of the container 358 is also connected to the syringe pump 476 .
- the outlet port of the container 358 is connected to the common port of a distribution valve 478 .
- the system also includes a collection tube 480 for receiving intracellular material removed from the sample, a waste container 482 for receiving waste, and a pressure source, such as a pump 484 .
- the collection tube 480 , waste container 482 , and pump 484 are connected to respective peripheral ports of the distribution valve 478 .
- a pressure regulator 486 regulates the pressure supplied by the pump 484 .
- the syringe pump 476 pumps a fluid sample from the sample container 474 through the container 358 and into the waste container 482 . As the fluid sample is forced to flow through the filters in the chamber 367 , coarse material is filtered by the filter 458 and target cells or viruses in the sample are captured by the filter 464 . The chamber 367 may be sonicated as the sample is forced to flow through the chamber to help prevent clogging of the filters.
- the syringe pump 476 pumps wash solution from the bottle 472 through the container 358 and into the waste container 482 . The washing solution washes away PCR inhibitors and contaminants from the chamber 367 .
- the syringe pump 476 pumps lysis buffer from the bottle 470 into the container 358 so that the chamber 367 is filled with liquid.
- the lysis buffer should be a medium through which dynamic pressure pulses or pressure waves can be transmitted.
- the lysis buffer may comprise deionized water for holding the cells or viruses in suspension or solution.
- the lysis buffer may include one or more lysing agents to aid in the disruption of the cells or viruses.
- One of the advantages of the present invention, however, is that harsh lysing agents are not required for successful disruption of the cells or viruses.
- the distribution valve of the syringe pump 476 is closed upstream of the container 358 , and the distribution valve 478 is opened.
- the pump 484 then pressurized the chamber 367 through the outlet port 444 , preferably to about 20 psi above the ambient pressure.
- the distribution valve 478 downstream of the container 358 is then closed.
- the static pressure in the chamber 367 is therefore increased to about 20 psi in preparation for the disruption of the cells or viruses trapped on the filter 464 .
- the pressurization of the chamber 367 is important because it ensures effective coupling between the transducer 314 and the flexible wall 440 .
- the transducer 314 is activated (i.e., set into vibratory motion).
- the flexible wall 440 transfers the vibrational motion of the transducer 314 to the liquid in the chamber 367 by allowing slight deflections without creating high stresses in the wall.
- the transducer 314 is preferably an ultrasonic horn for sonicating the chamber 367 .
- the chamber 367 is preferably sonicated for 10 to 40 seconds at a frequency in the range of 20 to 60 kHz. In the exemplary protocol, the chamber is sonicated for 15 seconds at a frequency of 40 kHz.
- the amplitude of the horn tip is preferably in the range of 20 to 25 ⁇ m (measured peak to peak).
- the effective forward stroke of the transducer is less than its peak-to-peak amplitude.
- the effective forward stroke determines the level of sonication in the chamber 367 . It is therefore important to increase the static pressure in the chamber 367 so that when the tip of the transducer 314 retreats, the flexible wall 440 is forced outwardly to meet the tip on its return stroke.
- the static pressure in the chamber 367 should be sufficient to ensure that the effective forward stroke of the transducer 314 generates the necessary pressure pulses or pressure waves in the chamber to effect cell disruption. It is presently preferred to increase the static pressure in the chamber 367 to at least 5 psi above the ambient pressure, and more preferably to a pressure in the range of 15 to 25 psi above the ambient pressure.
- the solid wall 488 should have a natural frequency that is higher than the vibrating frequency at which the transducer 314 is operated.
- the ratio of the natural frequency of the wall 488 to the vibrating frequency is at least 2:1, and more preferably the ratio is at least 4:1.
- the wall 488 should not be so rigid that it cannot transfer the vibratory motion of the transducer to the liquid in the chamber 367 . It is preferred that the wall 488 be capable of deflecting a distance in the range of 5 to 40 ⁇ m, and more preferably about 20 ⁇ m peak to peak when the transducer 314 applies a force in the range of 1 to 10 lbs. to the external surface of the wall 488 .
- the wall 488 be capable of deflecting a distance in the range of 5 to 40 ⁇ m, and more preferably about 20 ⁇ m peak to peak when the transducer 314 applies a force in the range of 2 to 5 lbs.
- the wall 488 is dome-shaped and convex with respect to the transducer 314 (i.e., the wall 488 curves outwardly towards the transducer).
- the advantage to the dome-shaped design of the wall 488 is that the dome shape increases the natural frequency of the wall (compared to a flat wall) without causing the wall to be so stiff that it cannot transfer the vibratory movements of the transducer 314 to the chamber 367 .
- FIG. 42 shows a cross sectional view of the wall 488 .
- the dome-shaped portion 495 of the wall preferably has a radius of curvature R in the range of 6.3 to 12.7 mm when the diameter D of the dome-shaped portion is about 11.1 mm. More preferably, the dome-shaped portion 495 of the wall preferably has a radius of curvature R of about 9.5 mm when the diameter D of the dome-shaped portion is about 11.1 mm.
- the wall 488 also includes a flat outer rim 497 for clamping the wall 488 in the container 358 .
- the wall 488 may be integrally molded with either of pieces 450 , 452 (FIG. 41).
- the thickness T of the wall is preferably in the range of 0.25 to 1 mm. If it is less than 0.25 mm thick, the wall 488 may be too weak. If the wall has a thickness greater than 1 mm, the wall may be too stiff to deflect properly in response to the vibratory movements of the transducer. In the presently preferred embodiment, the wall 488 has a thickness T of about 0.5 mm.
- the wall 488 is preferably a molded plastic part. Suitable materials for the wall 488 include Delrin® (acetal resins or polymethylene oxide), polypropylene, or polycarbonate.
- the interaction of the transducer 314 with the solid wall 488 will now be described with reference to FIG. 41.
- target cells or viruses are captured on the filter 490 by forcing a fluid sample to flow though the chamber 367 (e.g., using the fluidic system previously described with reference to FIG. 40).
- the chamber 367 is filled with a liquid (e.g., lysis buffer) as previously described.
- the chamber 367 does not require pressurization. Instead, it is preferred that ambient pressure is maintained in the chamber.
- the transducer 314 is placed in contact with the external surface of the wall 488 , preferably using a support structure as previously described with reference to FIG. 37.
- a spring preferably pushes the transducer against the wall 488 with a force in the range of 1 to 10 lbs., and more preferably in the range of 2 to 5 lbs.
- the transducer 314 is activated (i.e., induced into vibratory motion). As the tip of the transducer 314 vibrates, it deflects the wall 488 . On its forward stroke (in the upward direction in FIG. 41), the tip of the transducer 314 pushes the wall 488 and creates a pressure pulse or pressure wave in the chamber 367 . On its retreating stroke (downward in FIG. 41), the wall 488 remains in contact with the tip of the transducer 314 because the wall 488 has a natural frequency higher than the vibrating frequency of the transducer.
- the chamber 367 is preferably sonicated for 10 to 40 seconds at a frequency in the range of 20 to 40 kHz. In the exemplary protocol, the chamber is sonicated for 15 seconds at a frequency of 40 kHz.
- the amplitude of the horn tip is preferably in the range of 20 to 25 ⁇ m (measured peak to peak), and the natural frequency of the wall 488 should be greater than 40 kHz, preferably at least 80 kHz, and more preferably at least 160 kHz.
- One advantage to using the solid interface wall 488 is that strong pressure drops can be achieved in the chamber 367 as long as the static pressure in the chamber is low. For example, at atmospheric pressure, cavitation (the making and breaking of microscopic bubbles) can occur in the chamber 367 . As these bubbles or cavities grow to resonant size, they collapse violently, producing very high local pressure changes. The pressure changes provide a mechanical shock to the cells or viruses, resulting in their disruption. The disruption of the cells or viruses may also be caused by sharp pressure rises resulting from the vibratory movement of the transducer 314 . In addition, the disruption of the cells or viruses may be caused by the violent motion of the beads 462 in the chamber 367 .
- the beads are agitated by the dynamic pressure pulses in the chamber and rupture the cells or viruses.
- the applicants have found that it is usually necessary to use beads to disrupt certain types of cells (particularly spores) having highly resistant cell walls. Other types of cells, such as blood cells, are easier to disrupt and may often be disrupted without the use of the beads 462 .
- the transducer should be capable of creating pressure pulses or pressure waves in the chamber 367 .
- the transducer should be capable of providing high velocity impacts to the liquid in the chamber.
- Suitable transducers include ultrasonic, piezoelectric, magnetostrictive, or electrostatic transducer.
- the transducer may also be an electromagnetic device having a wound coil, such as a voice coil motor or a solenoid device.
- the vibrating frequency of the transducer may be ultrasonic (i.e., above 20 kHz) or below ultrasonic (e.g., in the range of 60 to 20,000 Hz).
- the advantage to using higher frequencies is that cell disruption is very rapid and can often be completed in 10 to 20 seconds.
- the disadvantage is that ultrasonic transducers are often more expensive than a simple mechanical vibrator, e.g., a speaker or electromagnetic coil device.
- the solid wall 488 is used in combination with a speaker or electromagnetic coil device that vibrates at an operating frequency in the range of 5 to 10 kHz.
- FIGS. 43A-43B illustrate another solid wall 500 for contacting a transducer according to the present invention.
- one side of the wall 500 has a central portion 502 and a plurality of stiffening ribs 504 extending radially from the central portion 502 .
- the wall also has recesses 506 formed between the ribs 504 .
- the other side of the wall 500 has a flat surface 508 .
- FIG. 44 shows a partially-cut away isometric view of the container 358 with the wall 500 .
- the wall 500 is preferably positioned so that the side of the wall having the flat surface is internal to the chamber 367 and such that the side of the wall having the ribs 504 is external to the chamber.
- the ribs 504 are advantageous because they increase the natural frequency of the wall without causing the wall to be so stiff that it cannot transfer the vibratory movements of the transducer to the chamber 367 .
- FIG. 45 shows a bottom plan view of the container 358 having the wall 500 .
- the central portion 502 provides the external surface of the wall 500 for contacting a transducer.
- the interaction of the wall 500 with the transducer is analogous to the interaction of the wall 488 with the transducer previously described with reference to FIG. 41.
- the wall 500 remains in contact with the tip of the transducer because the wall 500 has a natural frequency higher than the vibrating frequency of the transducer. Consequently, pressurization is not required, and cavitation may be achieved.
- the solid walls 488 , 500 described with reference to FIGS. 41-45 may be used in the container 358 or the walls 488 , 500 may be used in a fully integrated cartridge, such as the cartridge shown in FIG. 1.
- FIG. 46 shows a partially exploded view of a container 274 for holding cells or viruses to be disrupted according to another embodiment of the invention.
- FIG. 47 shows a front view of the container 274 .
- the container 274 has a chamber 277 for holding a liquid containing cells or viruses to be disrupted.
- the container 274 has a rigid frame 278 that defines the side walls 282 A, 282 B, 282 C, 282 D of the chamber 277 .
- the rigid frame 278 also defines a port 276 and a channel 288 that connects the port 276 to the chamber 277 .
- the container 274 also includes a plunger 284 that is inserted into the channel 288 after adding the cells or viruses to the chamber 277 .
- the plunger 284 compresses gas in the container 274 thereby increasing pressure in the chamber 277 .
- the gas compressed by the plunger 284 is typically air filling the channel 288 .
- the pressurization of the chamber 277 forces the flexible wall 280 A to conform to the surface of the transducer (not shown in FIGS. 46-47), as is discussed in greater detail below.
- the plunger 284 also closes the port 276 and seals the chamber 277 from the environment external to the container.
- the plunger 284 also preferably includes an alignment ring 294 encircling the stem for maintaining the plunger 284 in coaxial alignment with the channel 288 as the plunger is inserted into the channel.
- the alignment ring 294 is preferably integrally formed (e.g., molded) with the stem 290 .
- the stem 290 may optionally includes support ribs 293 for stiffening and strengthening the stem.
- the plunger 284 also includes a plunger cap 296 attached to the stem 290 . As shown in FIG. 47, the cap 296 includes a snap ring 297 and the container includes an annular recess 279 encircling the port 276 for receiving the snap ring 297 .
- the cap 296 may optionally include a lever portion 298 which is lifted to remove the plunger 284 from the channel 288 .
- the container 274 may also include finger grips 287 for manual handling of the container.
- FIG. 51 shows an isometric view of an apparatus 304 for disrupting cells or viruses.
- the apparatus 304 includes a transducer 314 , preferably an ultrasonic horn, for generating pressure pulses the chamber of the container 274 .
- the apparatus 304 also includes a support structure 306 for holding the transducer 314 and the container 274 against each other.
- the support structure 306 includes a base 308 and a first holder 310 attached to the base for holding the outer housing of the transducer 314 .
- the holder 310 includes a bore for receiving the transducer 314 and screws or bolts 312 that are tightened to clamp the outer housing of the horn firmly in the holder.
- the base 308 may optionally include bolt holes 320 for bolting the support structure 306 to a surface, e.g., a counter or bench top.
- the support structure 306 also includes a holder 316 for holding the container 274 .
- the holder 316 is slidably mounted to the base 308 by means of a guide 318 .
- the guide 318 may be fixedly attached to the base 308 or integrally formed with the base.
- the guide 318 has two guide pins 322
- the holder 316 has two guide slots 324 for receiving the guide pins 322 .
- the holder 316 may thus slide on the guide pins 322 .
- the holder 316 is designed to hold the container 274 such that the external surface of the flexible wall 280 A is exposed and accessible to the tip 326 of the transducer 314 .
- the guide 318 is appropriately aligned with the transducer 314 to slide the holder 316 into a position in which the external surface of the flexible wall 280 A contacts the tip 326 .
- FIG. 54 shows an isometric view of the holder 316 .
- the holder 316 has a body 317 in which are formed the guide slots 324 for receiving the guide pins.
- the body also has a recess 334 for receiving the container 274 .
- the shape of the recess 334 matches the shape of the lower portion of the frame 278 so that the frame fits securely in the recess 334 .
- the holder 316 also includes a retaining member 328 attached to the body 317 by screws or bolts 330 .
- the retaining member 328 and body 317 define a slot 332 through which the frame 278 is inserted when the frame is placed in the recess 334 .
- the retaining member 328 holds the frame 278 in the recess.
- the body 317 also has an opening 336 adjacent the recess 334 .
- the shape of the opening 336 corresponds to the shape of the chamber 277 .
- the opening 336 is positioned next to the flexible wall 280 B.
- the opening 336 is thus positioned to permit the flexible wall 280 B to expand outwardly into the opening.
- the holder 316 holds only the frame of the container 274 so that the flexible walls 280 A, 280 B are unrestrained by the holder.
- the flexible wall 280 A is therefore free to move inwardly and outwardly with the horn tip 326 as vibrational motion is transmitted from the tip 326 to the wall 280 A.
- the flexible wall 280 B is also free to move inwardly or outwardly as pressure pulses sweep through the chamber 277 .
- Venting of the opening 336 is provided by first and second bores 338 , 344 formed in the body of the holder 316 .
- One end of the narrower bore 338 is connected to the opening 336 and the other end is connected to the larger bore 344 .
- the bore 344 extends through the body of the holder 316 to permit the escape of gas (e.g., air) from the opening 336 .
- the venting prevents pressure from building in the opening 336 when the flexible wall 280 B expands into the opening. Such pressure would restrict the motion of the wall 280 B.
- the container 274 has a bulb-shaped tab 275 extending from the bottom of the frame 278 .
- the holder 316 has holes 340 formed in the body 317 adjacent the recess 334 .
- the tab 275 is positioned between the holes 340 .
- the holes 340 are for receiving retaining pins.
- the retaining pins 342 extend from the guide 318 (from which the guide pins have been removed for clarity in FIG. 55) and are positioned on opposite sides of the bulb-shaped tab 275 when the container 274 is moved into contact with the horn tip 326 .
- the spacing of the pins 342 is less than the width of the bulb so that the pins 342 hold down the tab 275 , and thus the container 274 , as ultrasonic energy is transmitted into the container from the transducer 314 . This ensures that the container 274 does not rise out of position due to the motion of the horn tip 326 .
- a collar or other suitable retention mechanism may be used to hold the container 274 in position.
- the support structure 306 also includes an elastic body, such as a spring 366 , for applying a force to the holder 316 to press the wall 280 A of the chamber 277 against the horn tip 326 .
- an elastic body such as a spring 366
- the spring 366 is positioned in the bore 344 .
- the holder 316 has an inner surface surrounding the junction of the larger bore 344 and the narrower bore 338 .
- One end of the spring 366 contacts the inner surface, and the other end of the spring contacts a rod 348 that extends from the guide 318 .
- the spring 366 is thus compressed between the surface of the holder 316 and the rod 348 so that it pushes the holder 316 , and thus the flexible wall 280 A of the container 274 , against the tip 326 .
- the magnitude of the force provided by the spring 366 may be adjusted by changing the preload on the spring.
- the support structure 306 includes a rod 348 that contacts one end of the spring.
- the guide 318 includes a first bore for receiving the rod 348 and a second bore for receiving a set screw 349 that holds the rod 348 in a fixed position.
- the screw 349 is loosened, the rod 348 is moved to a new position, and the screw 349 is retightened to hold the rod 348 in the new position.
- the rod 348 and set screw 349 thus provide a simple mechanism for adjusting the preload on the spring 366 .
- the preload on the spring 366 is adjusted to provide a suitable coupling force between the wall 280 A and the horn tip 326 , it is desirable to keep the preload constant from one use of the apparatus to the next so that valid comparisons can be made between different samples disrupted by the apparatus.
- the flexible wall 280 A facilitates the transfer of vibrating motion from the transducer 314 to the chamber 277 .
- the wall 280 A is sufficiently flexible to conform to the surface of the tip 326 of the transducer, ensuring good coupling between the tip 326 and the wall 280 A.
- the surface of the tip 326 that contacts the wall 280 A is preferably planar (e.g., flat) to ensure power coupling over the entire area of the surface.
- the tip 326 may have a slightly curved (e.g., spherical) surface for contacting the wall 280 A
- the opposite wall 280 B is preferably sufficiently flexible to move inwardly and outwardly as dynamic pressure pulses are generated in the chamber 277 . This permits the liquid within the chamber 277 greater freedom of movement as it receives the pressure pulses and thus enhances the action in the chamber 277 .
- the walls 280 A, 280 B are preferably flexible sheets or films of polymeric material such as polypropylene, polyethylene, polyester, or other polymers.
- the films may either be layered, e.g., laminates, or the films may be homogeneous. Layered films are preferred because they generally have better strength and structural integrity than homogeneous films.
- the walls 280 A, 280 B may comprise any other material that may be formed into a thin, flexible sheet.
- the thickness of each wall is preferably in the range of 0.01 to 0.2 mm, and more preferably in the range of 0.025 to 0.1 mm.
- the plunger 284 is inserted into the channel 288 after, adding the cells or viruses to the chamber 277 .
- the plunger 284 compresses air in the channel 288 , thereby increasing pressure in the chamber 277 .
- the pressurization of the chamber 277 ensures effective coupling between the wall 280 A and the tip of the transducer 314 .
- the chamber 277 It is presently preferred to pressurize the chamber 277 to a pressure in the range of 2 to 50 psi above ambient pressure. This range is presently preferred because 2 psi is generally enough pressure to ensure effective coupling between the flexible wall 280 A and the transducer 314 , while pressures above 50 psi may cause bursting of the walls 280 A, 280 B or deformation of the frame of the container 274 . More preferably, the chamber 277 is pressurized to a pressure in the range of 8 to 15 psi above ambient pressure. This range is more preferred because it is safely within the practical limits described above.
- beads 301 are placed in the chamber 277 of the container to enhance the disruption of the cells or viruses.
- the beads 301 may be composed of glass, plastic, polystyrene, latex, crystals, metals, metal oxides, or non-glass silicates.
- the beads 301 may be porous or non-porous and preferably have a diameter in the range of 1 to 200 ⁇ m. More preferably, the beads 301 are either polystyrene beads, borosilicate glass beads, or soda lime glass beads having an average diameter of about 100 ⁇ m.
- the beads 301 may be placed in the chamber 277 using a funnel.
- the funnel should be sufficiently long to extend from the port 276 through the channel 288 and into the chamber 277 . After inserting the funnel into the container 274 , the beads 301 are placed in the funnel and the container 274 is tapped lightly (e.g., against a bench top) until the beads 301 settle into the bottom of the chamber 277 . It is preferred that the funnel extend through the channel 288 and into the chamber 277 as the beads 301 are added to the chamber to prevent the beads from contaminating the channel. The presence of beads in the channel 288 would interfere with the subsequent stroke of the plunger into the channel.
- the quantity of beads 301 added to the chamber 277 is preferably sufficient to fill about 10% to 40% of the volume capacity of the chamber.
- the chamber 277 has a volume capacity of about 100 ⁇ l, and 30 to 40 mg of beads are placed into the chamber.
- the chamber is filled with a liquid containing the cells or viruses to be disrupted.
- the chamber 277 may be filled using a pipette having a pipette tip 300 (e.g., a standard 200 ⁇ l loading tip).
- the chamber 277 may be filled using a syringe or any other suitable injection system.
- the liquid should be a medium through which pressure waves or pressure pulses can be transmitted.
- the liquid may comprise deionized water or ultrasonic gel for holding the cells or viruses in suspension or solution.
- the liquid may comprise a biological sample containing the cells or viruses.
- Suitable samples include bodily fluids (e.g., blood, urine, saliva, sputum, seminal fluid, spinal fluid, mucus, etc) or environmental samples such as ground or waste water.
- the sample may be in raw form or mixed with diluents or buffers.
- the liquid or gel may also include one or more lysing agents to aid in the disruption of the cells or viruses.
- One of the advantages of the present invention is that harsh lysing agents are not required for successful disruption of the cells or viruses.
- the plunger 284 is inserted into the channel 288 to seal and pressurize the container 274 .
- the piston 292 compresses gas in the channel 288 to increase pressure in the chamber 277 , preferably to about 8 to 15 psi above ambient pressure, as previously described.
- the spring 366 applies to the holder 316 , and thus to the container 274 , a substantially constant force to press the wall 280 A against the horn tip 326 .
- the force provided by the spring 366 ensures effective coupling between the wall 280 A and horn tip 326 as energy is transmitted to the chamber 277 .
- the tab 275 slides between the retaining pins 342 .
- the pins 342 prevent the container from sliding upward in response to the vibratory motion of the tip 326 .
- an intermediate force results in the most consistent and repeatable transfer of energy from the transducer 314 to the chamber 277 . It is presently preferred that the spring 366 provide a force in the range of 0.25 to 4 lbs., with a force of about 1 lb. being the most preferred.
- the tip 326 vibrates to transmit ultrasonic energy into the chamber 277 .
- the coupling force between the wall 280 A and the tip 326 and the desired amplitude of the vibratory movements of the tip 326 .
- a balance can be sought between the coupling force and the amplitude.
- a light coupling force requires a greater amplitude to effect disruption of the cells or viruses, while a stronger coupling force requires less amplitude to effect disruption.
- the peak-to-peak amplitude of the vibratory movements should be in the range of 4 to 40 ⁇ m, with a preferred peak-to-peak amplitude of amount 15 ⁇ m.
- Ultrasonic waves are preferably transmitted to the chamber 277 at a frequency in the range of 20 to 50 kHz, with a frequency of about 40 kHz being preferred.
- the duration of time for which the chamber 277 is sonicated is preferably in the range of 5 to 30 seconds. This range is preferred because it usually takes at least 5 seconds to disrupt the cells or viruses in the chamber, while sonicating the chamber for longer than 30 seconds will most likely denature or shear the nucleic acid released from the disrupted cells or viruses. Extensive shearing of the nucleic acid could interfere with subsequent amplification or detection. More preferably, the chamber is sonicated for about 10-20 seconds to fall safely within the practical limits stated above. The optimal time that a particular type of cell sample should be subjected to ultrasonic energy may be determined empirically.
- the container 274 is removed from the holder 316 by pulling the holder 316 away from the tip 326 and withdrawing the container from the holder.
- the liquid or gel containing the disrupted cells and released nucleic acid is then removed from the container 274 . This may be accomplished by centrifuging the container 274 and removing the supernatant using, e.g., a pipette or syringe.
- the liquid may be removed from the container 274 by setting the container on edge and at an incline until the beads precipitate. The beads usually settle in about 15 to 20 seconds. When the beads have settled, the plunger is withdrawn from the container 274 and the liquid is removed using a syringe or pipette.
- the released nucleic acid contained in the liquid may then be amplified and detected using techniques well known in the art.
- One advantage of the apparatus and method of the present invention is that it provides for the rapid and effective disruption of cells or viruses, including tough spores, without requiring the use of harsh chemicals.
- the apparatus and method provide for highly consistent and repeatable lysis of cells or viruses, so that consistent results are achieved from one use of the apparatus to the next.
- the amount of energy that is absorbed by the liquid and beads held in the chamber 277 depends on the amplitude of the oscillations of the tip 326 , the mass of the contents of the chamber 277 , the pressure in the chamber 277 , and the coupling force between the tip 326 and the wall 280 A. All four of these parameters should be held substantially constant from one use of the apparatus to the next in order to achieve the same amount of disruption repeatably.
- the holder 316 may be slidably mounted to the base 308 by a variety of means, including rails, wheels, sliding in a groove, sliding in a cylinder, etc.
- the holder 316 may be fixedly attached to the base 308 and the transducer 314 slidably mounted to the base.
- an elastic body is positioned to apply a force to the transducer 314 (either directly or to a holder holding the horn) to press together the horn tip 326 and the wall 280 A.
- the elastic body may be positioned to either push or pull the transducer 314 or the container 274 towards each other.
- the spring 366 may be positioned to push or pull the holder 316 towards the horn tip 326 or to push or pull the transducer 314 towards the holder 316 .
- multiple elastic bodies may be employed to apply forces to both the container 274 and the transducer 314 to push or pull them towards each other. All of these embodiments are intended to fall within the scope of the present invention.
- a coil spring 366 is shown in FIGS. 37 and 56, it is to be understood that any type of elastic body may be used. Suitable elastic bodies include, but are not limited to, coil springs, wave springs, torsion springs, spiral springs, leaf spring, elliptic springs, half-elliptic springs, rubber springs, and atmospheric springs.
- the elastic body may also be compressed air or rubber.
- the elastic body is a coil spring. Coil springs are preferred because they are simple and inexpensive to place in the apparatus and because the have a low spring rate. A compressed air system is also effective, but considerably more expensive. In embodiments in which the elastic body is a spring, the spring should have a low spring rate, preferably less than 4 lb./in.
- a low spring rate minimizes the effect that any variations in the thickness of the chamber 277 (due to small variations in manufacturing, filling, or pressurizing the container) will have on the magnitude of the force provided by the spring to press together the wall 280 A and the horn tip 326 .
- the chamber 277 holds the cells or viruses in a thin volume of liquid that can be uniformly sonicated easily.
- each of the sides walls 282 A, 282 B, 282 C, 282 D of the chamber has a length L in the range of 5 to 20 mm
- the chamber has a width W in the range of 7 to 30 mm
- the chamber has a thickness T in the range of 0.5 to 5 mm.
- the chamber 277 preferably has a width W greater than its thickness T.
- the ratio of the width W of the chamber to the thickness T of the chamber is preferably at least 2:1.
- the ratio of the width W of the chamber to the thickness T of the chamber is at least 4:1. These ratios are preferred to enable the entire volume of the chamber 277 to be rapidly and uniformly sonicated.
- the volume capacity of the chamber 277 is preferably in the range of 0.02 to 1 ml.
- the transducer 314 is preferably an ultrasonic horn.
- the thickness of the chamber 277 (and thus the spacing between the walls 280 A and 280 B) is preferably less than half of the diameter of the tip 326 of the horn. This relationship between the thickness of the chamber 277 and the diameter of the tip 326 ensures that the ultrasonic energy received from the transducer 314 is substantially uniform throughout the volume of the chamber 277 .
- the tip 326 has a diameter of 6.35 mm and the chamber 277 has a thickness of about 1.0 mm.
- the major wall 280 A should be slightly larger than the surface of the horn tip 326 that presses against the wall 280 A. This allows the flexible wall 280 A to flex in response to the vibratory motion of the horn tip 326 .
- the container 274 may be fabricated by first molding the rigid frame 278 using known injection molding techniques.
- the frame 278 is preferably molded as a single piece of polymeric material, e.g., polypropylene or polycarbonate. After the frame 278 is produced, thin, flexible sheets are cut to size and sealed to opposite sides of the frame 278 to form the major walls 282 A, 282 B of the chamber 277 .
- the major walls 282 A, 282 B are preferably cast or extruded films of polymeric material, e.g., polypropylene films, that are cut to size and attached to the frame 278 using the following procedure.
- a first piece of film is placed over one side of the bottom portion of the frame 278 .
- the frame 278 preferably includes a tack bar 299 for aligning the top edge of the film.
- the film is placed over the bottom portion of the frame 278 such that the top edge of the film is aligned with the tack bar 299 and such that the film completely covers the bottom portion of the frame 278 below the tack bar 299 .
- the film should be larger than the bottom portion of the frame 278 so that it may be easily held and stretched flat across the frame.
- the film is then cut to size to match the outline of the frame by clamping to the frame the portion of the film that covers the frame and cutting away the portions of the film that extend past the perimeter of the frame using, e.g., a laser or die.
- the film is then tack welded to the frame, preferably using a laser.
- the film is then sealed to the frame 278 , preferably by heat sealing.
- Heat sealing is presently preferred because it produces a strong seal without introducing potential contaminants to the container as the use of adhesive or solvent bonding techniques might do.
- Heat sealing is also simple and inexpensive.
- the film should be completely sealed to the surfaces of the side walls 282 A, 282 B, 282 C, 282 D. More preferably, the film is additionally sealed to the surfaces of the support ribs 295 and tack bar 299 .
- the heat sealing may be performed using, e.g., a heated platen.
- An identical procedure may be used to cut and seal a second sheet to the opposite side of the frame 278 to complete the chamber 277 .
- the plunger 284 is also preferably molded from polymeric material (e.g., polypropylene or polycarbonate) using known injection molding techniques. As shown in FIG. 46, the frame 278 , plunger 284 , and leash 286 connecting the plunger to the frame may all be formed in the same mold to form a one-piece part.
- This embodiment of the container is especially suitable for manual use in which a human operator fills the container and inserts the plunger 284 into the channel 288 . The leash 286 ensures that the plunger 284 is not lost or dropped on the floor.
- the plunger 284 is presently preferred as a simple, effective, and inexpensive mechanism for increasing pressure in the chamber 277 and for sealing the chamber 277 from the external environment. It is to be understood, however, that the scope of the invention is not limited to this embodiment. There are many other suitable techniques for sealing and pressurizing the container. In addition, any suitable pressure source may be used to pressurize the chamber. Suitable pressure sources include syringe pumps, compressed air sources, pneumatic pumps, or connections to external sources of pressure.
- the container for holding the cells or viruses need not be one of the specialized containers described in the various embodiments above. Any type of container having a chamber for holding the cells or viruses may be used to practice the invention. Suitable containers include, but are not limited to, reaction vessels, cuvettes, cassettes, and cartridges. The container may have multiple chambers and/or channels for performing multiple sample preparation functions, or the container may have only a single chamber for holding cells or viruses for disruption.
- the support structure for pressing the transducer and the wall of the container against each other may have many alternative forms.
- the support structure includes a vise or clamp for pressing the transducer and container against each other.
- the apparatus includes a pressure system for applying air pressure to press together the transducer and the container.
- magnetic or gravitational force may be used to press together the transducer and the container.
- force may be applied to the transducer, to the container, or to both the transducer and the container.
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Wood Science & Technology (AREA)
- Clinical Laboratory Science (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Cell Biology (AREA)
- Microbiology (AREA)
- Sustainable Development (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- General Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Analytical Chemistry (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
Abstract
An apparatus for disrupting cells or viruses comprises a container having a chamber for holding the cells or viruses. The container includes at least one flexible wall defining the chamber. The apparatus also includes a transducer for impacting an external surface of the flexible wall to generate pressure waves in the chamber. The apparatus also includes a pressure source for increasing the pressure in the chamber. The pressurization of the chamber ensures effective coupling between the transducer and the flexible wall. The apparatus may also include beads in the chamber for rupturing the cells or viruses.
Description
- This application claims priority from provisional application Ser. No. 60/136,703 filed May 28, 1999. This application is also a continuation in part of U.S. application Ser. No. 09/469,724 filed Dec. 21, 1999. These applications are incorporated by reference herein for all purposes.
- The present invention relates to an apparatus and method for rapidly disrupting cells or viruses.
- The extraction of nucleic acid from cells or viruses is a necessary task for many applications in the fields of molecular biology and biomedical diagnostics. Once released from the cells, the nucleic acid may be used for genetic analysis, e.g., sequencing, pathogen identification and quantification, nucleic acid mutation analysis, genome analysis, gene expression studies, pharmacological monitoring, storing of DNA libraries for drug discovery, etc. The genetic analysis typically involves nucleic acid amplification and detection using known techniques. For example, known polynucleotide amplification reactions include polymerase chain reaction (PCR), ligase chain reaction (LCR), QB replicase amplification (QBR), self-sustained sequence replication (3SR), strand-displacement amplification (SDA), “branched chain” DNA amplification, ligation activated transcription (LAT), nucleic acid sequence-based amplification (NASBA), repair chain reaction (RCR), and cycling probe reaction (CPR).
- The extraction of nucleic acids from cells or viruses is generally performed by physical or chemical methods. Chemical methods typically employ lysing agents (e.g., detergents, enzymes, or strong organics) to disrupt the cells and release the nucleic acid, followed by treatment of the extract with chaotropic salts to denature any contaminating or potentially interfering proteins. Such chemical methods are described in U.S. Pat. No. 5,652,141 to Henco et al. and U.S. Pat. No. 5,856,174 to Lipshutz et al. One disadvantage to the use of harsh chemicals for disrupting cells is that the chemicals are inhibitory to subsequent amplification of the nucleic acid. In using chemical disruption methods, therefore, it is typically necessary to purify the nucleic acid released from the cells before proceeding with further analysis. Such purification steps are time consuming, expensive, and reduce the amount of nucleic acid recovered for analysis.
- Physical methods for disrupting cells often do not require harsh chemicals that are inhibitory to nucleic acid amplification (e.g., PCR). These physical methods, however, also have their disadvantages. For example, one physical method for disrupting cells involves placing the cells in a solution and heating the solution to a boil to break open the cell walls. Unfortunately, the heat will often denature proteins and cause the proteins to stick to the released nucleic acid. The proteins then interfere with subsequent attempts to amplify the nucleic acid. Another physical method is freeze thawing in which the cells are repeatedly frozen and thawed until the cells walls are broken. Unfortunately, freeze thawing often fails to break open many structures, most notably certain spores and viruses that have extremely tough outer layers.
- Another physical method for disrupting cells is the use of a pressure instrument. With this method, a solution of mycobacterial microorganisms is passed through a very small diameter hole under high pressure. During passage through the hole, the mycobacteria are broken open by the mechanical forces and their internal contents are spilled into solution. Such a system, however, is large, expensive and requires a cooling system to prevent excessive heat from building up and damaging the contents of the lysed cells. Moreover, the instrument needs to be cleaned and decontaminated between runs and a large containment system is required when infectious material is handled. A further disadvantage to this system is that the solution must contain only particles having substantially the same size, so that it may not be used to process many untreated clinical or biological specimens.
- It is also known that cells can be lysed by subjecting the cells to ultrasonic agitation. Typically, the cells are disrupted by placing an ultrasonic probe directly into a volume of liquid containing the cells. Since the probe is in direct contact with a sample liquid, cross contamination and cavitation-induced foaming present serious complications.
- Another method for cell disruption is disclosed by Murphy et al. in U.S. Pat. No. 5,374,522. According to the method, solutions or suspensions of cells are placed in a container with small beads. The container is then placed in an ultrasound bath until the cells disrupt, releasing their cellular components. This method has several disadvantages. First, the distribution of ultrasonic energy in the bath is not uniform, so that a technician must locate a high energy area within the bath and place the container into that area. The non-uniform distribution of ultrasonic energy also produces inconsistent results. Second, the ultrasound bath does not focus energy into the container so that the disruption of the cells often takes several minutes to complete, a relatively long period of time when compared to the method of the present invention. Third, it is not practical to carry an ultrasound bath into the field for use in biowarfare detection, forensic analysis, or on-site testing of environmental samples.
- The present invention overcomes the disadvantages of the prior art by providing an improved apparatus and method for disrupting cells or viruses. In contrast to the prior art methods described above, the present invention provides for the rapid and effective disruption of cells or viruses, including tough spores, without requiring the use of harsh chemicals. The disruption of the cells or viruses can often be completed in 5 to 10 seconds. In addition, the apparatus and method of the present invention provide for highly consistent and repeatable lysis of cells or viruses, so that consistent results are achieved from one use of the apparatus to the next.
- According to a first embodiment, the apparatus comprises a container having a chamber for holding the cells or viruses. The container includes at least one flexible wall defining the chamber. The apparatus also includes a transducer, such as an ultrasonic horn, for impacting an external surface of the flexible wall to generate dynamic pressure pulses or pressure waves in the chamber. The apparatus also includes a pressure source for increasing the pressure in the chamber. The pressurization of the chamber ensures effective coupling between the transducer and the flexible wall. The apparatus may also include beads in the chamber for rupturing the cells or viruses.
- In operation, the cells or viruses to be disrupted are placed in the chamber of the container. A liquid is also placed in the chamber. In one embodiment, the cells or viruses are placed in the chamber by capturing the cells or viruses on at least one filter positioned in the chamber. In this embodiment, the liquid placed in the chamber is usually a lysis buffer added to the chamber after the cells or viruses have been captured. In an alternative embodiment, the liquid placed in the chamber contains the cells or viruses to be disrupted (e.g., the liquid is a sample containing the cells or viruses) so that the liquid and cells are placed in the chamber simultaneously. In either embodiment, the transducer is placed against the external surface of the flexible wall, and the static pressure in the chamber is increased. Disruption of the cells is accomplished by impacting the flexible wall with the transducer to generate dynamic pressure pulses or pressure waves in the chamber. Beads may also be agitated in the chamber to rupture the cells or viruses.
- A greater understanding of the invention may be gained by considering the following detailed description and the accompanying drawings.
- FIG. 1 is an isometric view of a cartridge for analyzing a fluid sample according to a first embodiment of the invention.
- FIG. 2 is a lower isometric view of the cartridge of FIG. 1.
- FIG. 3 is an exploded view of the cartridge of FIG. 1.
- FIG. 4 is another exploded view of the cartridge of FIG. 1.
- FIG. 5 is a partially cut away view of an ultrasonic horn coupled to a wall of a lysing chamber formed in the cartridge of FIG. 1.
- FIG. 6 is an exploded view of a filter stack positioned in the lysing chamber of the cartridge of FIG. 1.
- FIG. 7 is a top plan view of the cartridge of FIG. 1.
- FIG. 8 is a bottom plan view of the cartridge of FIG. 1.
- FIG. 9 is a schematic block diagram of the cartridge of FIG. 1.
- FIG. 10 is an isometric view of an instrument into which the cartridge of FIG. 1 is placed for processing.
- FIG. 11 is an isometric view of the cartridge of FIG. 1 in the instrument of FIG. 10.
- FIG. 12 is a partially cut-away view of the cartridge of FIG. 1 in the instrument of FIG. 10.
- FIG. 13 is a schematic, plan view of optical sensors positioned to detect liquid levels in the cartridge of FIG. 1.
- FIG. 14 is a partially cut away, schematic, side view of a slotted optical sensor positioned to detect the liquid level in a sensor chamber of the cartridge of FIG. 1.
- FIG. 15A is a cross-sectional view of a portion of the body of the cartridge of FIG. 1 illustrating two different types of valves in the cartridge.
- FIG. 15B is a cross-sectional view of the valves of FIG. 15A in a closed position.
- FIG. 16A is another cross-sectional view of one of the valves of FIG. 15A in an open position.
- FIG. 16B is a cross-sectional view of the valve of FIG. 16A in a closed position.
- FIGS. 17-19 illustrate a valve actuation system for opening and closing the valves of FIG. 15A.
- FIG. 20 is a cross sectional view of alternative valve actuators for opening and closing the valves in the cartridge of FIG. 1. FIG. 20 also shows a pressure delivery nozzle sealed to a pressure port formed in the cartridge of FIG. 1.
- FIG. 21 is a partially exploded, isometric view of a reaction vessel of the cartridge of FIG. 1.
- FIG. 22 is a front view of the vessel of FIG. 21.
- FIG. 23 is a side view of the vessel of FIG. 21 inserted between two heater plates.
- FIG. 24 is a front view of one of the heater plates of FIG. 23.
- FIG. 25 is a front view of an alternative reaction vessel according to the present invention.
- FIG. 26 is a front view of another reaction vessel according to the present invention.
- FIG. 27 is another front view of the vessel of FIG. 21.
- FIG. 28 is a front view of the vessel of FIG. 21 inserted into a heat-exchanging module of the instrument of FIG. 10.
- FIG. 29 is an exploded view of a support structure for holding the plates of FIG. 23.
- FIGS. 30-31 are assembled views of the support structure of FIG. 29.
- FIG. 32 is an isometric view showing the exterior of one the optics assemblies in the heat-exchanging module of FIG. 28.
- FIG. 33 is an isometric view of the plates of FIG. 23 in contact with the optics assembly of FIG. 32.
- FIG. 34 is a partially cut away, isometric view of the reaction vessel of FIG. 21 inserted between the plates of FIG. 23. Only the lower portion of the vessel is included in the figure.
- FIG. 35 is a schematic block diagram of the electronics of the heat-exchanging module of FIG. 28.
- FIG. 36 is an isometric view of an apparatus for disrupting cells or viruses according to another embodiment of the invention.
- FIG. 37 is a cross sectional view of the apparatus of FIG. 36.
- FIG. 38 is an exploded view of a container used in the apparatus of FIG. 36.
- FIG. 39 is a cross sectional view of the container of FIG. 38.
- FIG. 40 is a schematic block diagram of a fluidic system incorporating the apparatus of FIG. 36.
- FIG. 41 is a cross sectional view of another container for use in the apparatus of FIG. 36. An ultrasonic horn is in contact with a wall of the container that curves outwardly towards the horn.
- FIG. 42 is a cross-sectional view of the wall of FIG. 41.
- FIGS. 43A-43B are isometric views of opposite sides of another wall suitable for use in a container for holding cells or viruses to be disrupted.
- FIG. 44 is a partially cut-away, isometric view of a container incorporating the wall of FIGS. 43A-43B.
- FIG. 45 is a bottom plan view of the container of FIG. 44.
- FIG. 46 is a partially exploded, isometric view of a container for holding cells or viruses to be disrupted according to another embodiment of the invention.
- FIG. 47 is a front view of the container of FIG. 46.
- FIG. 48 is another schematic, front view of the container of FIG. 46.
- FIG. 49 is a side view of the container of FIG. 46.
- FIG. 50 is a view of a pipette inserted into the container of FIG. 46. The container is holding beads for rupturing cells or viruses.
- FIG. 51 is an isometric view of the container of FIG. 46 inserted into an apparatus for disrupting cells or viruses.
- FIG. 52 is a different isometric view of the container of FIG. 46 inserted into the apparatus of FIG. 51.
- FIG. 53 is a partially cut-away, isometric view of the apparatus of FIG. 51.
- FIG. 54 is an isometric view of a holder for holding the container of FIG. 46.
- FIG. 55 is another isometric view of the apparatus of FIG. 51 in which several parts of the apparatus have been removed to show an ultrasonic horn contacting the container of FIG. 46.
- FIG. 56 is a schematic side view of the container of FIG. 46 inserted into the apparatus of FIG. 51 for disruption of the cells or viruses contained in the container.
- The present invention provides an apparatus and method for analyzing a fluid sample. In a first embodiment, the invention provides a cartridge for separating a desired analyte from a fluid sample and for holding the analyte for a chemical reaction. The-fluid sample may be a solution or suspension. In a particular use, the sample may be a bodily fluid (e.g., blood, urine, saliva, sputum, seminal fluid, spinal fluid, mucus, or other bodily fluids). Alternatively, the sample may be a solid made soluble or suspended in a liquid or the sample may be an environmental sample such as ground or waste water, soil extracts, pesticide residues, or airborne spores placed in a fluid. Further, the sample may be mixed with one or more chemicals, reagents, diluents, or buffers. The sample may be pretreated, for example, mixed with chemicals, centrifuged, pelleted, etc., or the sample may be in a raw form.
- The desired analyte is typically intracellular material (e.g., nucleic acid, proteins, carbohydrates, lipids, bacteria, or intracellular parasites). In a preferred use, the analyte is nucleic acid which the cartridge separates from the fluid sample and holds for amplification (e.g., using PCR) and optical detection. As used herein, the term “nucleic acid” refers to any synthetic or naturally occurring nucleic acid, such as DNA or RNA, in any possible configuration, i.e., in the form of double-stranded nucleic acid, single-stranded nucleic acid, or any combination thereof.
- FIG. 1 shows an isometric view of a
cartridge 20 according to the preferred embodiment. Thecartridge 20 is designed to separate nucleic acid from a fluid sample and to hold the nucleic acid for amplification and detection. Thecartridge 20 has a body comprising atop piece 22, amiddle piece 24, and abottom piece 26. An inlet port for introducing a fluid sample into the cartridge is formed in thetop piece 22 and sealed by acap 30. Sixpressure ports 32 are also formed in thetop piece 22. Thepressure ports 32 are for receiving nozzles from pressure sources, e.g., pumps or vacuums. The cartridge also includesalignment legs 28 extending from thebottom piece 26 for positioning thecartridge 20 in an instrument (described below with reference to FIG. 10). Indentations ordepressions middle pieces cartridge 20. Thecartridge 20 further includesvents - FIG. 2 is an isometric view showing the underside of the
cartridge 20. Nineholes 60 are formed in thebottom piece 26 for receiving valve actuators that open and close valves in thecartridge 20. Ahole 62 is also formed in thebottom piece 26 for receiving a transducer (described in detail below with reference to FIG. 5). Thecartridge 20 also includes areaction vessel 40 extending outwardly from the body of the cartridge. Thevessel 40 has areaction chamber 42 for holding a reaction mixture (e.g., nucleic acid mixed with amplification reagents and fluorescent probes) for chemical reaction and optical detection. One of the flow paths in the cartridge carries the reaction mixture to thechamber 42 for chemical reaction and optical detection. Thevessel 40 extends outwardly from the body of thecartridge 20 so that thevessel 40 may be inserted between a pair of opposing thermal plates (for heating and cooling the chamber 42) without the need for decoupling thevessel 40 from the rest of thecartridge 20. This greatly reduces the risk of contamination and/or spilling. Thevessel 40 may be integrally formed with the body of the cartridge (e.g., integrally molded with middle piece 24). It is presently preferred, however, to produce thevessel 40 as a separate element that is coupled to the body during manufacture of the cartridge. - FIGS. 3-4 show exploded views of the cartridge. As shown in FIG. 3, the
middle piece 24 has multiple chambers formed therein. In particular, themiddle piece 24 includes asample chamber 65 for holding a fluid sample introduced through theinlet port 64, awash chamber 66 for holding a wash solution, areagent chamber 67 for holding a lysing reagent, awaste chamber 68 for receiving used sample and wash solution, aneutralizer chamber 70 for holding a neutralizer, and amaster mix chamber 71 for holding a master mix (e.g., amplification reagents and fluorescent probes) and for mixing the reagents and probes with analyte separated from the fluid sample. Thesample chamber 65 optionally includes aside compartment 155 having slightly lower walls than thesample chamber 65. Theside compartment 155 is for visually indicating to a user when sufficient sample has been added to thesample chamber 65, i.e., when the liquid level in thechamber 65 is high enough to spill over into thecompartment 155. - The
top piece 22 includes thevents pressure ports 32, as previously described. An elastomeric membrane orgasket 61 is positioned and squeezed between thepieces middle piece 24 preferably includes multiple sealing lips to ensure that thegasket 61 forms an adequate seal. In particular, themiddle piece 24 preferably includes sealinglips 73 surrounding each of thechambers middle piece 24 also includessupport walls 75 around the perimeter, andintermediate sealing lips 76. The sealinglips support walls 75 locally compress thegasket 61 and achieve a seal. - As shown in FIG. 4, the
middle piece 24 has formed in its underside various channels, one of which leads to a lysingchamber 86. Thechamber 86 is aligned with thehole 62 in thebottom piece 26 so that a transducer (e.g., an ultrasonic horn) may be inserted through thehole 62 to generate dynamic pressure pulses or pressure waves in the lysingchamber 86. Themiddle piece 24 also has ninevalve seats 84 formed in its bottom surface. The valve seats 84 are aligned with the nineholes 60 in thebottom piece 26 so that valve actuators may be inserted through theholes 60 into the valve seats 84. - An elastomeric membrane or
gasket 61 is positioned and squeezed between thepieces middle piece 24. Themiddle piece 24 preferably includes multiple sealing lips to ensure that thegasket 63 forms an adequate seal. In particular, themiddle piece 24 preferably includes sealinglips 73 surrounding the lysingchamber 86, valve seats 84, and various channels. Themiddle piece 24 also includessupport walls 75 around its perimeter, andintermediate sealing lips 76. The sealinglips support walls 75 locally compress thegasket 63 and achieve a seal. In addition to sealing various channels and chambers, thegasket 63 also functions as a valve stem by compressing, when actuated through one of theholes 60, into acorresponding valve seat 84, thus shutting one of the flow channels in themiddle piece 24. This valve action is discussed in greater detail below with reference to FIGS. 15-16. - The
gasket 63 also forms the bottom wall of the lysingchamber 86 against which a transducer is placed to effect disruption of cells or viruses in thechamber 86. Each of thegaskets gaskets - As shown in FIG. 3, the
middle piece 24 includes aslot 79 through which thereaction vessel 40 is inserted during assembly of the cartridge. Thevessel 40 has twofluid ports top piece 22 is sealed to themiddle piece 24 via thegasket 61, theports channels gasket 61 seals the respective fluidic interfaces between theports channels bottom pieces bottom pieces pieces - FIG. 4 also shows a
filter ring 88. Thefilter ring 88 compresses and holds a stack of filters in the lysingchamber 86. FIG. 6 shows an exploded view of afilter stack 87. The purpose of thefilter stack 87 is to capture cells or viruses from a fluid sample as the sample flows through the lysingchamber 86. The captured cells or viruses are then disrupted (lysed) in thechamber 86. The cells may be animal or plant cells, spores, bacteria, or microorganisms. The viruses may be any type of infective agents having a protein coat surrounding an RNA or DNA core. - The
filter stack 87 comprises agasket 93, afirst filter 94, agasket 95, asecond filter 97 having a smaller pore size than thefirst filter 94, agasket 98, athird filter 100 having a smaller pore size than thesecond filter 97, agasket 101, awoven mesh 102, and agasket 103. The filter stack also preferably includes a first set ofbeads 96 disposed between the first andsecond filters beads 99 disposed between the second andthird filters filter ring 88 compresses thefilter stack 87 into the lysingchamber 86 so that thegasket 93 is pressed against thefilter 94, thefilter 94 is pressed against thegasket 95, thegasket 95 is pressed against thefilter 97, thefilter 97 is pressed against thegasket 98, thegasket 98 is pressed against thefilter 100, thefilter 100 is pressed against thegasket 101, thegasket 101 is pressed against themesh 102, themesh 102 is pressed against thegasket 103, and thegasket 103 is pressed against the outer perimeter of the bottom wall of the lysingchamber 86. Thegasket 95 is thicker than the average diameter of thebeads 96 so that the beads are free to move in the space between thefilters gasket 98 is thicker than the average diameter of thebeads 99 so that thebeads 99 are free to move in the space between thefilters channel 106 into the lysingchamber 86 first flows throughfilter 94, then throughfilter 97, next throughfilter 100, and lastly through themesh 102. After flowing through thefilter stack 87, the sample flows along flow ribs. 91 formed in the top of the lysingchamber 86 and through an outlet channel (not shown in FIG. 6). - Referring to FIG. 5, the cells or viruses captured in the filter stack (not shown in FIG. 5 for illustrative clarity) are lysed by coupling a transducer92 (e.g., an ultrasonic horn) directly to the wall of the lysing
chamber 86. In this embodiment, the wall of the lysingchamber 86 is formed by theflexible gasket 63. Thetransducer 92 should directly contact an external surface of the wall. The term “external surface” is intended to mean a surface of the wall that is external to the lysingchamber 86. Thetransducer 92 is a vibrating or oscillating device that is activated to generate dynamic pressure pulses or pressure waves in thechamber 86. The pressure waves agitate thebeads 96, 99 (FIG. 6), and the movement of the beads ruptures the captured cells or viruses. In general, the transducer for contacting the wall of the lysingchamber 86 may be an ultrasonic, piezoelectric, magnetostrictive, or electrostatic transducer. The transducer may also be an electromagnetic device having a wound coil, such as a voice coil motor or a solenoid device. It is presently preferred that the actuator be an ultrasonic transducer, such as an ultrasonic horn. Suitable horns are commercially available from Sonics & Materials, Inc. having an office at 53 Church Hill, Newton., Conn. 06470-1614 USA. Alternatively, the ultrasonic transducer may comprise a piezoelectric disk or any other type of ultrasonic transducer that may be coupled to the container. It is presently preferred to use an ultrasonic horn because the horn structure is highly resonant and provides for repeatable and sharp frequency of excitation and large motion of the horn tip. - As previously described in FIG. 6, the filter stack includes a gasket at both of its ends. As shown in FIG. 5, the
middle cartridge piece 24 has a sealinglip 90 against which the gasket at one end of the filter stack is compressed. The gasket at the other end of the filter stack is compressed by thefilter ring 88 to form a seal. The gasket material may expand into the relief area outside of the sealinglip 90. The width of the sealinglip 90 is small (typically 0.5 mm) so that an excessive amount of force is not required to achieve a sufficient seal. - The
filter ring 88 is held between the filter stack and thecartridge gasket 63. Thecartridge gasket 63 is held between themiddle piece 24 and thebottom piece 26 by a sealinglip 406. Force is therefore transferred from thebottom piece 26 through thegasket 63 to thefilter ring 88 and finally to the filter stack. Thefilter ring 88 contains acontact lip 404 that contacts thegasket 63. Thecontact lip 404 is not a primary sealing lip (though it will seal) but a force transfer mechanism. The width of thecontact lip 404 is larger than the width of the sealinglip 90 to ensure that deformation and sealing action occurs in the filter stack and not taken up in squeezing thecartridge gasket 63. Thecartridge middle piece 24 also has a sealinglip 406 that surrounds thefilter ring 88. This is an active sealing area that should not be compromised by the presence of thefilter ring 88. For this reason, there is agap 407 between the sealinglip 406 and thecontact lip 404 on thefilter ring 88. Thegap 407 is provided to allow thegasket 63 to extrude into thegap 407 as it is compressed by the sealinglip 406 and thecontact lip 404. If thecontact lip 404 comes to a different elevation than the sealinglip 406, the seal will not be compromised because of thegap 407 and the distance between thelips - Referring again to FIG. 6, the
filter stack 87 is effective for capturing cells or viruses as a fluid sample flows through thestack 87 without clogging of any of thefilters filter stack 87 thus enables the simultaneous capture of differently sized sample components without clogging of the filters. The average pore size of thefirst filter 94 is selected to be small enough to filter coarse material from the fluid sample (e.g., salt crystals, cellular debris, hair, tissue) yet large enough to allow the passage of the target cells or viruses containing the desired analyte (e.g., nucleic acid or proteins). In general, the pore size of thefirst filter 94 should be in the range of about 2 to 25 μm, with a presently preferred pore size of about 5 μm. - The average pore sizes of the second and third filters are selected in dependence upon the average size of the target cells or viruses that contain the desired analyte(s). For example, in one embodiment, the
filter stack 87 is used to capture gonorrhea (GC) and chlamydia (Ct) organisms to determine the presence of the diseases in the fluid sample. The GC and Ct organisms have different average diameters, about 1 to 2 μm for GC organisms and about 0.3 μm for Ct organisms. In this embodiment, thesecond filter 97 has an average pore size of about 1.2 μm while thethird filter 100 has an average pore size of about 0.22 μm so that most of the GC organisms are captured by thesecond filter 97 while most of the Ct organisms are captured by thethird filter 100. The filter stack thus enables the simultaneous capture of differently sized target organisms and does so without clogging of the filters. The pore sizes of thefilters - The
filter stack 87 is also useful for disrupting the captured cells or viruses to release the intracellular material (e.g., nucleic acid) therefrom. The first and second sets ofbeads chamber 86. Suitable beads for rupturing cells or viruses include borosilicate glass, lime glass, silica, and polystyrene beads. - The beads may be porous or non-porous and preferably have an average diameter in the range of 1 to 200 μm. The average diameter of the
beads beads 96 in the first set may be equal to the average diameter of thebeads 99 in the second set. Alternatively, when the first set ofbeads 96 is used to rupture a type of target cell or virus that differs from the type of cell or virus to be ruptured by the second set ofbeads 99, it is advantageous to select the average diameter of the beads such that the average diameter of thebeads 96 in the first set differs from the average diameter of thebeads 99 in the second set. For example, when the filter stack is used to capture GC and Ct cells as described above, thebeads 96 are 20 μm diameter borosilicate glass beads for rupturing the GC organisms and thebeads 99 are 106 μm diameter soda lime glass beads for rupturing the Ct organisms. Each of thesilicone gaskets beads - The
mesh 102 also serves two useful purposes. First the mesh provides support to thefilter stack 87. Second, the mesh breaks up air bubbles so that the bubbles can be channeled through theflow ribs 91 and out of the lysingchamber 86. To effectively break up or reduce the size of the air bubbles, themesh 102 preferably has a small pore size. Preferably, it is a woven polypropylene mesh having an average pore size of about 25 μm. To ensure that the air bubbles can escape from the lysingchamber 86, it is desirable to use the cartridge in an orientation in which liquid flows up (relative to gravity) through thefilter stack 87 and the lysingchamber 86. The upward flow through thechamber 86 aids the flow of air bubbles out of thechamber 86. Thus, the inlet port for entry of fluids into thechamber 86 should generally be at the lowest point in the chamber, while the exit port should be at the highest. - Many different embodiments of the filter stack are possible. For example, in one alternative embodiment, the filter stack has only two filters and one set of beads disposed between the filters. The first filter has the largest pore size (e.g., 5 μm) and filters out coarse material such as salt crystals, cellular debris, hair, tissue, etc. The second filter has a pore size smaller than the first filter and slightly smaller than the target cells or viruses to be captured. Such a filter stack is described below with reference to FIG. 38. In another embodiment of the cartridge, the filter having the largest pore size (for filtering the coarse material) is positioned in a filter chamber (not shown) that is positioned upstream of the lysing
chamber 86. A channel connects to the filter chamber to the lysingchamber 86. In this embodiment, a fluid sample flows first through the coarse filter in the filter chamber and then through a second filter in the lysing chamber to trap the target cells or viruses in the lysing chamber. - Further, the beads in the filter stack may have a binding affinity for target cells or viruses in the fluid sample to facilitate capture of the target cells or viruses. For example, antibodies or certain receptors may be coated onto the surface of the beads to bind target cells in the sample. Moreover, the lysing
chamber 86 may contain two different types of beads for interacting with target cells or viruses. For example, the lysing chamber may contain a first set of beads coated with antibodies or receptors for binding target cells or viruses and a second set of beads (intermixed with the first set) for rupturing the captured cells or viruses. The beads in the lysingchamber 86 may also have a binding affinity for the intracellular material (e.g., nucleic acid) released from the ruptured cells or viruses. Such beads are useful for isolating target nucleic acid for subsequent elution and analysis. For example, the lysing chamber may contain silica beads to isolate DNA or cellulose beads with oligo dT to isolate messenger RNA for RT-PCR. The lysingchamber 86 may also contain beads for removing unwanted material (e.g., proteins, peptides) or chemicals (e.g., salts, metal ions, or-detergents) from the sample that might inhibit PCR. For example, thechamber 86 may contain ion exchange beads for removing proteins. Alternatively beads having metal ion chelators such as iminodiacetic acid will remove metal ions from biological samples. - FIGS. 21-22 illustrate the
reaction vessel 40 in greater detail. FIG. 21 shows a partially exploded view of thevessel 40, and FIG. 22 shows a front view of thevessel 40. Thevessel 40 includes the reaction chamber 42 (diamond-shaped in this embodiment) for holding a reaction mixture. Thevessel 40 is designed for optimal heat transfer to and from the reaction mixture and for efficient optical viewing of the mixture. The thin shape of the vessel contributes to optimal thermal kinetics by providing large surfaces for thermal conduction and for contacting thermal plates. In addition, the walls of the vessel provide optical windows into thechamber 42 so that the entire reaction mixture can be optically interrogated. In more detail to FIGS. 21-22, thereaction vessel 40 includes arigid frame 46 that defines theside walls reaction chamber 42. Theframe 46 also defines aninlet port 41 and achannel 50 connecting theport 41 to thechamber 42. Theframe 46 also defines anoutlet port 43 and achannel 52 connecting theport 43 to thechamber 42. Theinlet port 41 andchannel 50 are used to add fluid to thechamber 42, and thechannel 52 andoutlet port 43 are used for exit of fluid from thechamber 42. Alignment prongs 44A, 44B are used to position thevessel 40 correctly during assembly of the cartridge. - As shown in FIG. 21, the
vessel 40 also includes thin, flexible sheets attached to opposite sides of therigid frame 46 to form opposingmajor walls 48 of the chamber. (Themajor walls 48 are shown in FIG. 1 exploded from therigid frame 46 for illustrative clarity). Thereaction chamber 42 is thus defined by therigid side walls frame 46 and by the opposing major walls. 48. The opposingmajor walls 48 are sealed to opposite sides of theframe 46 such that theside walls major walls 48 to each other. Thewalls 48 facilitate optimal thermal conductance to the reaction mixture contained in thechamber 42. Each of thewalls 48 is sufficiently flexible to contact and conform to a respective thermal surface, thus providing for optimal thermal contact and heat transfer between the thermal surface and the reaction mixture contained in thechamber 42. Furthermore, theflexible walls 48 continue to conform to the thermal surfaces if the shape of the surfaces changes due to thermal expansion or contraction during the course of the heat-exchanging operation. - As shown in FIG. 23, the thermal surfaces for contacting the
flexible walls 48 are preferably formed by a pair of opposingplates chamber 42 between them. When thechamber 42 of thevessel 40 is inserted between theplates walls 48 and the flexible walls conform to the surfaces of the plates. The plates are preferably spaced a distance from each other equal to the thickness T of thechamber 42 as defined by the thickness of theframe 46. In this position, minimal or no gaps are found between the plate surfaces and thewalls 48. The plates may be heated and cooled by various thermal elements to induce temperature changes within thechamber 42, as is described in greater detail below. - The
walls 48 are preferably flexible films of polymeric material such as polypropylene, polyethylene, polyester, or other polymers. The films may either be layered, e.g., laminates, or the films may be homogeneous. Layered films are preferred because they generally have better strength and structural integrity than homogeneous films. In particular, layered polypropylene films are presently preferred because polypropylene is not inhibitory to PCR. Alternatively, thewalls 48 may comprise any other material that may be formed into a thin, flexible sheet and that permits rapid heat transfer. For good thermal conductance, the thickness of eachwall 48 is preferably between about 0.003 to 0.5 mm, more preferably between 0.01 to 0.15 mm, and most preferably between 0.025 to 0.08 mm. - Referring again to FIG. 22, the
vessel 40 also preferably includes optical windows for in situ optical interrogation of the reaction mixture in thechamber 42. In the preferred embodiment, the optical windows are theside walls rigid frame 46. Theside walls chamber 42 through theside wall 57A and detection of light emitted from thechamber 42 through theside wall 57B. Arrows A represent illumination beams entering thechamber 42 through theside wall 57A and arrows B represent emitted light (e.g., fluorescent emission from labeled analytes in the reaction mixture) exiting thechamber 42 through theside wall 57B. - The
side walls walls wall 57A will exit throughwall 57B. In addition, the 90° angle permits a maximum amount of emitted light (e.g. fluorescence) to be collected throughwall 57B. Thewalls chamber 42. Alternatively, theangled walls walls chamber 42, such as an integrated capillary electrophoresis area. In the presently preferred embodiment, a locatingtab 58 extends from theframe 46 below the intersection ofwalls tab 58 is used to properly position thevessel 40 in a heat-exchanging module described below with reference to FIG. 28. - Optimum optical sensitivity may be attained by maximizing the optical path length of the light beams exciting the labeled analyte in the reaction mixture and the emitted light that is detected, as represented by the equation:
- I o /I i =C*L*A,
- where Io is the illumination output of the emitted light in volts, photons or the like, C is the concentration of analyte to be detected, Ii is the input illumination, L is the path length, and A is the intrinsic absorptivity of the dye used to label the analyte.
- The thin,
flat reaction vessel 40 of the present invention optimizes detection sensitivity by providing maximum optical path length per unit analyte volume. Referring to FIGS. 23 and 27, thevessel 40 is preferably constructed such that each of thesides walls chamber 42 has a length L in the range of 1 to 15 mm, the chamber has a width W in the range of 1.4 to 20 mm, the chamber has a thickness T in the range of 0.5 to 5 mm, and the ratio of the width W of the chamber to the thickness T of the chamber is at least 2:1. These parameters are presently preferred to provide a vessel having a relatively large average optical path length through the chamber, i.e. 1 to 15 mm on average, while still keeping the chamber sufficiently thin to allow for extremely rapid heating and cooling of the reaction mixture contained therein. The average optical path length of thechamber 42 is the distance from the center of theside wall 57A to the center of thechamber 42 plus the distance from the center of thechamber 42 to the center of theside wall 57B. - More preferably, the
vessel 40 is constructed such that each of thesides walls chamber 42 has a length L in the range of 5 to 12 mm, the chamber has a width W in the range of 7 to 17 mm, the chamber has a thickness T in the range of 0.5 to 2 mm, and the ratio of the width W of the chamber to the thickness T of the chamber is at least 4:1. These ranges are more preferable because they provide a vessel having both a larger average optical path length (i.e., 5 to 12 mm) and a volume capacity in the range of 12 to 100 μl while still maintaining a chamber sufficiently thin to permit extremely rapid heating and cooling of a reaction mixture. The relatively large volume capacity provides for increased sensitivity in the detection of low concentration analytes, such as nucleic acids. - In the preferred embodiment, the
reaction vessel 40 has a diamond-shapedchamber 42 defined by theside walls frame 46, and the chamber has a volume capacity of about 100 μl. This reaction vessel provides a relatively large average optical path length of 10 mm through thechamber 42. Additionally, the thin chamber allows for extremely rapid heating and/or cooling of the reaction mixture contained therein. The diamond-shape of thechamber 42 helps prevent air bubbles from forming in the chamber as it is filled with the reaction mixture and also aids in optical interrogation of the mixture. - Referring again to FIG. 22, the
frame 46 is preferably made of an optically transmissive material, e.g., a polycarbonate or clarified polypropylene, so that theside walls transmissive walls transmissive side walls chamber 42, or to collect as much fluorescence signal from as large a fraction of the chamber volume as possible. - In alternative embodiments, the optical elements may comprise gratings for selecting specific wavelengths, filters for allowing only certain wavelengths to pass, or colored lenses to provide filtering functions. The wall surfaces may be coated or comprise materials such as liquid crystal for augmenting the absorption of certain wavelengths. In the presently preferred embodiment, the optically
transmissive walls - The
side walls chamber 42 through theside walls frame 46 defines open spaces between theside walls support ribs 53. The open spaces are occupied by ambient air that has a different refractive index than the material composing the frame (e.g., plastic). Due to the difference in the refractive indexes, the reflective faces 56 are effective for internally reflecting light trying to exit the chamber through thewalls walls transmissive side walls chamber 42, and the retro-reflective side walls - A preferred method for fabricating the
reaction vessel 40 will now be described with reference to FIGS. 21-22. Thereaction vessel 40 may be fabricated by first molding therigid frame 46 using known injection molding techniques. Theframe 46 is preferably molded as a single piece of polymeric material, e.g., clarified polypropylene. After theframe 46 is produced, thin, flexible sheets are cut to size and sealed to opposite sides of theframe 46 to form themajor walls 48 of thechamber 42. Themajor walls 48 are preferably cast or extruded films of polymeric material, e.g., polypropylene films, that are cut to size and attached to theframe 46 using the following procedure. A first piece of film is placed over one side of theframe 46. Theframe 46 preferably includes atack bar 47 for aligning the top edge of the film. The film is placed over the bottom portion of theframe 46 such that the top edge of the film is aligned with thetack bar 47 and such that the film completely covers the bottom portion of theframe 46 below thetack bar 47. The film should be larger than the bottom portion of theframe 46 so that it may be easily held and stretched flat across the frame. The film is then cut to size to match the outline of the frame by clamping to the frame the portion of the film that covers the frame and cutting away the portions of the film that extend past the perimeter of the frame using, e.g., a laser or die. The film is then tack welded to the frame, preferably using a laser. - The film is then sealed to the
frame 46, preferably by heat sealing. Heat sealing is presently preferred because it produces a strong seal without introducing potential contaminants to the vessel as the use of adhesive or solvent bonding techniques might do. Heat sealing is also simple and inexpensive. The heat sealing may be performed using, e.g., a heated platen. An identical procedure may be used to cut and seal a second sheet to the opposite side of theframe 46 to complete thechamber 42. Many variations to this fabrication procedure are possible. For example, in an alternative embodiment, the film is stretched across the bottom portion of theframe 46 and then sealed to the frame prior to cutting the film to size. After sealing the film to the frame, the portions of the film that extend past the perimeter of the frame are cut away using, e.g., a laser or die. - Although it is presently preferred to mold the
frame 46 as a single piece, it is also possible to fabricate the frame from multiple pieces. For example, theside walls side walls frame 46. Theflexible walls 48 may then be attached to opposite sides of theframe 46 as previously described. - Referring again to FIG. 3, it is presently preferred to use a
gasket 61 to seal theports vessel 40 to correspondingchannels 80, 81 (FIG. 4) in the cartridge body. Alternatively, fluidic seals may be established using a luer fitting, compression fitting, or swaged fitting. In another embodiment, the cartridge body and frame of thevessel 40 are molded as a single part, and the flexible major walls of the vessel are heat-sealed to opposite sides of the frame. - Referring again to FIG. 22, the
chamber 42 is filled by forcing liquid (e.g., a reaction mixture) to flow through theport 41 and thechannel 50 into thechamber 42. The liquid may be forced to flow into thechamber 42 using differential pressure (i.e., either pushing the liquid through theinlet port 41 or aspirating the liquid by applying a vacuum to the outlet port 43). As the liquid fills thechamber 42, it displaces air in the chamber. The displaced air exits thechamber 42 through thechannel 52 and theport 43. For optimal detection of analyte in thechamber 42, the chamber should not contain air bubbles. To help prevent the trapping of air bubbles in thechamber 42, the connection between thechamber 42 and theoutlet channel 52 should be at the highest point (with respect to gravity) in thechamber 42. This allows air bubbles in thechamber 42 to escape without being trapped. Thus, thevessel 40 is designed to be used in the vertical orientation shown in FIG. 22. - FIG. 25 shows another
vessel 206 designed to be used in a horizontal orientation. Thevessel 206 has aninlet port 41 and aninlet channel 50 connecting theinlet port 41 to the bottom of thechamber 42. The vessel also has anoutlet port 43 and anoutlet channel 50 connecting theoutlet port 43 to the top of thechamber 42. Thus, any air bubbles in thechamber 42 may escape through theoutlet channel 52 without becoming trapped. FIG. 26 shows anothervessel 207 having twoinlet ports outlet port 43.Inlet channels respective inlet ports chamber 42, andoutlet channel 52 connects thechamber 42 tooutlet port 43. Many other different embodiments of the vessel are also possible. In each embodiment, it is desirable to evacuate thechamber 42 from the highest point (with respect to gravity) in the chamber and to introduce liquid into the chamber from a lower point. - FIGS. 15A-15B illustrate two types of valves used in the cartridge. As shown in FIG. 15A, there are two types of fundamental concepts to the valve action, and hence two types of valves. The first valve uses a cone-shaped or
conical valve seat 160 formed in themiddle cartridge piece 24. Thevalve seat 160 is a depression, recess, or cavity molded or machined in themiddle piece 24. Thevalve seat 160 is in fluid communication with achamber 167 through a port orchannel 157 that intersects the center of theconical valve seat 160. As shown in FIG. 15B, avalve actuator 164 having a spherical surface is forced against theelastic membrane 63 and into thevalve seat 160, establishing a circular ring of contact between themembrane 63 and thevalve seat 160. The kinematic principle is that of a ball seated into a cone. The circular seal formed by themembrane 63 andvalve seat 160 prevents flow between the channel 157 (and hence the chamber 167) and aside channel 158 extending from a side of thevalve seat 160. Theside channel 158 is defined by themembrane 63 and themiddle cartridge piece 24. - As shown in FIG. 15A, the other type of valve controls the cross flow between the
channel 158 and anotherside channel 159 formed between themembrane 63 and themiddle cartridge piece 24. In this case, a circular ring of contact would be ineffective. Instead, the second valve comprises a recess depression orcavity 161 formed in themiddle cartridge piece 24. Thecavity 161 separates thechannels channel 158 is positioned on one side of thecavity 161, and an end of thechannel 159 is positioned on the opposite side of thecavity 161. Thecavity 161 is defined by a firstcurved surface 162A positioned adjacent the end of thechannel 158, a secondcurved surface 162B positioned adjacent the end of thechannel 159, and athird surface 163 between the first and secondcurved surfaces membrane 63 to seal off the flow between thechannels valve seats - As shown in FIG. 16A, the first and second
curved surfaces valve actuator 164 has also has a spherical surface for pressing themembrane 63 tightly against thesurfaces surfaces valve actuator 164 plus the thickness T of themembrane 63. For example, if the radius of curvature R2 of the surface of thevalve actuator 164 is 0.094 inches and themembrane 63 has a thickness T of 0.031 inches, then the radius of curvature R1 of each of thesurfaces - As shown in FIG. 16B, the
third surface 163 is recessed from the first andsecond surfaces gap 166 between themembrane 63 and thethird surface 163 when themembrane 63 is pressed against the first andsecond surfaces surfaces surface 163. Thegap 166 ensures that themembrane 63 contacts primarily the valve seats.162A, 162B rather than the entire surface of thecavity 161 so that maximum pressure is applied to thevalve seats membrane 63. This provides a very strong seal with minimal actuator force required. - Referring again to FIG. 15B, in both types of valves the respective kinematic principle defines the location of the mating parts. In both the ball-in-cone concept and the ball-against-two-spherical-surfaces concept, the ball or spherical shaped valve actuator is permitted to seek its own location as it is forced against the valve seat(s). There is a deliberate clearance (e.g., 0.01 to 0.03 inches) between the valve actuator and the hole in the
bottom cartridge piece 26 in which theactuator 164 travels so that only the valve seat action defines the location of the mating pieces. - The valve actuators can be controlled by a variety of mechanisms. FIGS. 17-19 illustrate one such mechanism. As shown in FIG. 17, a
valve actuator 172 has a spherical surface for pressing thegasket 63 into a valve seat. Theactuator 172 also has aflange 177 on its bottom portion. The cartridge includes an elastic body, such as aspring 174, that pushes against a ledge in thelower cartridge piece 26 to bias the valve actuator against thegasket 63. Thespring 174 is sufficiently strong to close the valve unless a deliberate force is applied to pull down theactuator 172. The valves in the cartridge may be kept closed in this manner for shipping and storage before the cartridge is used. Thus, the cartridge may be preloaded during manufacture with the necessary reagents and wash solutions to analyze a fluid sample without the fluids leaking out of the cartridge during shipping and storage. - The actuator pull-down mechanism is usually located in an instrument into which the cartridge is placed for sample. analysis (one such instrument is described in detail below with reference to FIG. 10). The mechanism comprises a sliding
guide 175 that rotates a hinged pull-down member 180 having ajaw 181 for receiving theflange 177 of theactuator 172. As shown in FIG. 18, the slidingguide 175 rotates the hinged pull-down member 180 until theflange 177 is positioned within thejaw 181. As shown in FIG. 19, asolenoid 146 pulls down themember 180 and thus thevalve actuator 172 so that thegasket 63 is released from the valve seat, thus opening the valve and permitting fluid flow between thechannels - FIG. 20 illustrates the manner in which fluid flow into and out of the sample chamber, wash chamber, neutralizer chamber, and reagent chambers is controlled in the cartridge. Each of these chambers, as illustrated by a
chamber 414 in FIG. 20, is covered by ahydrophobic membrane 410 that allows the passage of gas but not liquid therethrough. Thehydrophobic membrane 410 is positioned between thechamber 414 and apressure port 32. Thepressure port 32 is formed in theupper cartridge piece 22 and positioned over thechamber 414. Themembrane 410 holds liquids in thechamber 414 during shipping and storage of the cartridge, even if the cartridge is turned upside down. Thepressure port 32 is sized to receive apressure nozzle 182 that is connected to a pressure source (e.g., a vacuum or pneumatic pump) usually located in the external instrument. Thenozzle 182 includes an o-ring 184 and aflange 415. Aspring 185 pushes against theflange 415 to force thenozzle 182 into thepressure port 32 so that the o-ring 184 establishes a seal around theport 32. In operation, positive air pressure or a vacuum is applied to thechamber 414 through thepressure port 32 to force liquids out of or into, respectively, thechamber 414. - A conical valve seat160 (previously described with reference to FIGS. 15A-15B) is formed in the
middle cartridge piece 24 below thechamber 414 to control the flow of liquid between thechamber 414 and a connectingchannel 411. The valve is opened and closed by avalve actuator 188 having aflange 187 and aspring 188 pressing against the flange to hold the valve closed until a downward force is applied to theactuator 186. The downward force is preferably supplied by a solenoid that pulls down theactuator 186 to open the valve. Thevalve actuator 186 and solenoid are preferably located in the instrument. - FIGS. 7-8 show top and bottom plan views, respectively, of the cartridge. FIG. 9 is a schematic block diagram of the cartridge. As shown in any of FIGS. 7-9, the cartridge includes a
sample chamber 65 having a port for adding a fluid sample to the cartridge and a sample flow path extending from thesample chamber 65. The sample flow path extends from thesample chamber 65 through avalve 107 and into achannel 106. Thechannel 106 includes asensor region 136 in which thechannel 106 has a flat bottom enabling easy optical detection of the presence of liquid in the channel. The sample flow path continues from thechannel 106 into the lysingchamber 86 and through thefilter stack 87. The sample flow path also includes achannel 109 for exit of fluid from the lysingchamber 86, achannel 110 having a flat-bottomeddetection region 137, avalve 111, and achannel 112 leading to the ventedwaste chamber 68 through avalve 114. - The cartridge also includes the
wash chamber 66 for holding wash solution and thereagent chamber 67 for holding lysing reagent. Thewash chamber 66 is connected to the lysingchamber 86 through avalve 115,channel 117, andchannel 106. Thereagent chamber 67 is connected to the lysing chamber. 86 through avalve 119,channel 117, andchannel 106. Sample components (e.g., cells or viruses in the sample) are captured in thefilter stack 87 and lysed in thechamber 86 to release target analyte (e.g., nucleic acid) from the sample components. The cartridge also includes an analyte flow path extending from the lysingchamber 86 for carrying the analyte separated from the fluid sample to thereaction vessel 40 for chemical reaction and optical detection. The analyte flow path extends from thechamber 86 through thechannel 109,channel 110, andvalve 111. After passing through thevalve 111, the analyte flow path diverges from the sample flow path. While the sample flow path extends thoughchannel 112 into thewaste chamber 68, the analyte flow path diverges into theU-shaped channel 122. The analyte flow path then extends into and out of theneutralizer chamber 70 through avalve 124. The analyte flow path also passes into and out of themaster mix chamber 71 through avalve 126. From themaster mix chamber 71, the analyte flow path extends along thechannel 122, through avalve 127, throughchannel 80, and into thereaction vessel 40 through theport 41. - The
reaction vessel 40 includes theport 41 for adding a reaction mixture to the vessel, and theport 43 for exit of fluids (e.g., air or excess reaction mixture) from the vessel. The cartridge also includeschannel 81 in fluid communication with theport 43. Thechannel 81 includes a flat-bottomeddetection region 130 for detecting the presence of liquid in the channel. Thechannel 81 connects to a channel 131 (channel 131 extends straight down perpendicular to the page in the top plan view of FIG. 7).Channel 131 connects to achannel 132 which in turn connects to achannel 134 through a valve 133 (channel 134 extends straight up perpendicular to the page in the top plan view of FIG. 7). Thechannel 134 leads to thevent 36 which has a hydrophobic membrane to permit the escape of gas but not liquid from the cartridge. The channels, vent and valve positioned downstream from thereaction vessel 40 are used to pressurize thechamber 42 of the vessel, as is described in the operation section below. - The cartridge also includes a
first pressure port 105 positioned above thesample chamber 65, asecond pressure port 116 positioned above thewash chamber 66, athird pressure port 118 positioned above thereagent chamber 67, afourth pressure port 123 positioned above theneutralizer chamber 70, afifth pressure port 125 positioned above themaster mix chamber 71, and asixth pressure port 128 positioned at the end of theU-shaped channel 122. The cartridge further includessensor chambers waste chamber 68. Thesensor chambers waste chamber 68, as is described in detail below. - Referring to FIG. 10, the cartridge is preferably used in combination with an
instrument 140 designed to accept one or more of the cartridges. For clarity of illustration, theinstrument 140 shown in FIG. 10 accepts just one cartridge. It is to be understood, however, that the instrument may be designed to process multiple cartridges simultaneously. Theinstrument 140 includes acartridge nest 141 into which the cartridge is placed for processing. Theinstrument 140 also includes the transducer 92 (e.g., an ultrasonic horn) for generating dynamic pressure pulses or pressure waves in the lysing chamber of the cartridge, ninevalve actuators 142 for actuating the nine valves in the cartridge, nine correspondingsolenoids 146 for pulling down the valve actuators, and sixpressure nozzles 145 for interfacing with six corresponding pressure ports formed in the cartridge. In addition, the instrument includes or is connected to one or more regulated pressure sources for supplying pressure to the cartridge through thepressure nozzles 145. Suitable pressure sources include syringe pumps, compressed air sources, pneumatic pumps, or connections to external sources of pressure. The instrument further includes three slottedoptical sensors 143 and three reflectiveoptical sensors 144. - FIG. 13 illustrates the slotted
optical sensors 143 positioned to detect liquid in thesensor chambers reagent chamber 67. Eachsensor 143 includes a built in LED and photodiode positioned on opposite sides of the sensor. The LED emits a beam that is detected by the photodiode if the beam is not substantially refracted. Such slotted optical sensors are commercially available from a number of suppliers. The cartridge is shaped so that the slotted optical sensors fit around thechambers optical sensors 143 are therefore useful for determining the presence or absence of liquid in thechambers - FIG. 14 shows a cut-away, schematic side view of the
sensor chamber 120 in fluid communication with thewaste chamber 68 and surrounded by the slottedoptical sensor 143. Thesensor chamber 120 andsensor 143 are used to indicate when a predetermined volume of liquid is present in thewaste chamber 68. Thesensor chamber 120 is partially separated from thewaste chamber 68 by awall 151 having aspillover rim 152. The height of the wall is selected so that when the predetermined volume of liquid is received in thewaste chamber 68, the liquid spills over thespillover rim 152 and into thesensor chamber 120. The liquid in thesensor chamber 120 is then detected by thesensor 143. - Referring again to FIG. 13, the cartridge may also include a
second sensor chamber 121 in fluid communication with thewaste chamber 68. Thesecond sensor chamber 121 is also separated from thewaste chamber 68 by awall 153 having a spillover rim. Thewall 153 is taller than thewall 152 so that liquid does not spill over thewall 153 until a second predetermined volume of fluid in addition to the first predetermined volume of fluid has been received in thewaste chamber 68. Thesensor chambers optical sensors 143 are useful for controlling the operation of the cartridge. The height of thewall 152 is preferably selected such that when a fixed volume of fluid sample from thesample chamber 65 has flowed through the sample flow path to thewaste chamber 68, the sample liquid spills over into thesensor chamber 120 and is detected. The detection inchamber 120 triggers the release of wash solution from thewash chamber 66 which flows through the sample flow path to thewaste chamber 68. When an incremental volume of the wash solution is received in thechamber 68, liquid spills over thewall 153 into thesensor chamber 121 and is detected. The detection of liquid in thechamber 121 then triggers the release of lysing reagent from thechamber 67. Thesensor 143 surrounding thechamber 67 may then be used to indicate when thechamber 67 is empty, triggering the start of ultrasonic lysis. In an alternative embodiment, the cartridge may have two waste chambers, one for sample and one for wash, with each waste chamber having a respective sensor chamber connected thereto. - In-line reflective
optical sensors 144 are used to determine the presence or absence of liquid in the flat-bottomeddetection regions channels sensor 144 has a built in emitter and detector that is positioned over a flat-bottomed detection region. The emitter emits a beam that is reflected from the cartridge and detected by the detector. The sensor detects a change in signal when as an air/liquid interface passes through the detection region. Optionally, dual emitter reflective optical sensors may be used for a more reliable detection operation. Both types of reflective optical sensors are well known in the art and commercially available. - Referring again to FIG. 10, the
instrument 140 also includes a heat-exchangingmodule 147 having aslot 148 for receiving the reaction vessel of the cartridge. Themodule 147 is described in detail below with reference to FIG. 28. Theinstrument 140 further includes alatch mechanism 149 for latching alid 150 over a cartridge. Thecartridge nest 141 includes alignment holes 401 for receiving the legs of the cartridge. The alignment holes 401 ensure proper positioning of the cartridge in thenest 141 so that thepressure nozzles 145,transducer 92, andvalve actuators 142 fit into the corresponding ports in the cartridge and so that the reaction vessel fits into theslot 148. Thetransducer 92 should be positioned in theinstrument 140 such that when the cartridge is placed in thenest 141, the transducer contacts the bottom wall of the lysingchamber 86, as shown in the cut-away view of FIG. 5. In addition, the instrument may include a spring or similar mechanism to bias thetransducer 92 against the wall of the lysingchamber 86. - The
instrument 140 also includes various conventional equipment not shown in FIG. 10 including a main logic board having a microcontroller for controlling the operation of thesolenoids 146,transducer 92, heat-exchangingmodule 147, andoptical sensors nozzles 145. Theinstrument 140 is preferably computer-controlled using, e.g., the microcontroller which is programmed to perform the functions described in the operation section below. Alternatively, the instrument may controlled by a separate computer, or controlled by a combination of a separate computer and an on-board microcontroller. - FIG. 11 shows an isometric view of the
cartridge 20 placed in theinstrument 140 for processing. FIG. 11 shows a partial cut-away view of theinstrument 140 with thelid 150 closed. Referring again to FIG. 11, a memory or microprocessor chip may optionally be incorporated as part of thecartridge 20. This chip preferably contains information such as the type of cartridge, program information such as specific protocols for the processing of the cartridge, tolerances for accept and reject, serial numbers and lot codes for quality tracking, and provision for storing the results of the processing. Integrated electronic memory on thecartridge 20 allows for rapid, easy, and error-free set-up of theinstrument 140 for different fluidic processing protocols. When thecartridge 20 is inserted into theinstrument 140, the instrument may electronically address the memory on the cartridge, and thus automatically receive the appropriate set of instructions for controlling the time-sequence of fluidic operations to be carried out with the inserted cartridge. Theinstrument 140 may simply sequentially retrieve and execute each step in the cartridge's memory, or download its contents so that the user may edit the sequence using, e.g., the controller computer. - If suitable memory is included on the cartridge, such as writable memory (e.g., erasable programmable read-only memory (EPROM), electrically erasable programmable read-only memory (EEPROM), etc., intermediate and final results, based on the sample introduced into the cartridge, could be written by the instrument into the cartridge's memory for co-located storage with the physical sample after processing. This is particularly advantageous in applications where archiving of samples and results is necessary, such as forensics. In addition, other information can be stored in the memory on the cartridge, in unalterable (or alterable) forms. For example, cartridge serial number, lot manufacture information, and related information could be pre-programmed and unalterable. User data, technician identification number, date of test, location of test and instrument serial number could be unalterably written into the cartridge. This allows for easy identification of the “chain of custody” in the handling of a specimen. Engineers skilled in the art of data storage will recognize that other memory means than electronic can be used, such as optically-addressed printed regions (e.g., ink-jet or thermal), magnetic strips, etc.
- FIG. 28 shows the heat-exchanging
module 147 of the instrument into which thereaction vessel 40 is inserted for thermal processing and optical detection of target analyte(s) in the reaction mixture. Themodule 147 preferably includes ahousing 208 for holding the various components of the module. Themodule 147 also includes thethermal plates 190 described above. Thehousing 208 includes a slot (not shown in FIG. 28) above theplates 190 so that the reaction chamber of thevessel 40 may be inserted through the slot and between the plates. The heat-exchangingmodule 147 also preferably includes a cooling system, such as afan 212. Thefan 212 is positioned to blow cooling air past the surfaces of theplates 190 to cool the plates and hence cool the reaction mixture in thevessel 40. Thehousing 208 preferably defines channels for directing the cooling air past theplates 190 and out of themodule 147. - The heat-exchanging
module 147 further includes anoptical excitation assembly 216 and anoptical detection assembly 218 for optically interrogating the reaction mixture contained in thevessel 40. Theexcitation assembly 216 includes afirst circuit board 220 for holding its electronic components, and thedetection assembly 216 includes asecond circuit board 222 for holding its electronic components. Theexcitation assembly 216 includes one or more light sources (e.g., an LED. laser, or light bulb) for exciting fluorescently-labeled analytes in thevessel 40. Theexcitation assembly 216 also includes one or more lenses for collimating the light from the light sources, as well as filters for selecting the excitation wavelength ranges of interest. Thedetection assembly 218 includes one or more detectors (e.g., a photodiode, photomultiplier tube, or CCD) for detecting the light emitted from thevessel 40. Thedetection assembly 218 also includes one or more lenses for focusing and collimating the emitted light, as well as filters for selecting the emission wavelength ranges of interest. Suitable optical excitation and detection assemblies for use in the heat-exchangingmodule 147 are described in International Publication Number WO 99/60380 (International Application Number PCT/US99/11182) published Nov. 25, 1999, the disclosure of which is incorporated by reference herein. - The
optics assemblies housing 208 such that when the chamber of thevessel 40 is inserted between theplates 190, theexcitation assembly 216 is in optical communication with thechamber 42 through the opticallytransmissive side wall 57A (see FIG. 22) and thedetection assembly 218 is in optical communication with the chamber through the opticallytransmissive side wall 57B (FIG. 22). In the preferred embodiment, theoptics assemblies optics assemblies plates 190 so that when the chamber of the vessel is placed between the plates, theoptics assemblies - FIG. 34 shows a partially cut-away, isometric view of the chamber of the vessel inserted between the
plates transmissive side walls plates first plate 190A has a first bottom edge. 250A and a second bottom edge 2190B. Similarly, the bottom portion of thesecond plate 190B has a firstbottom edge 252A and a secondbottom edge 252B. The first and second bottom edges of each plate are preferably angularly offset from each other by the same angle that theside walls plates first side wall 57A is positioned substantially adjacent and parallel to each of the firstbottom edges second side wall 57B is positioned substantially adjacent and parallel to each of thesecond bottom edges 2190B, 252B. This arrangement provides for easy optical access to the opticallytransmissive side walls side walls - Referring again to FIG. 28, the
optics assemblies vessel 40 includes a locating tab 58 (see FIG. 22) that fits into a slot formed between theoptics assemblies vessel 40 for optical detection. For improved detection, themodule 147 also preferably includes a light-tight lid (not shown) that is placed over the top of thevessel 40 and made light-tight to thehousing 208 after the vessel is inserted between theplates 190. - Although it is presently preferred to locate the
optics assemblies plates 190, many other arrangements are possible. For example, optical communication may be established between theoptics assemblies vessel 40 via optical fibers, light pipes, wave guides, or similar devices. One advantage of these devices is that they eliminate the need to locate theoptics assemblies plates 190. This leaves more room around the plates in which to circulate cooling air or refrigerant, so that cooling may be improved. - The heat-exchanging
module 147 also includes aPC board 226 for holding the electronic components of the module and anedge connector 224 for connecting themodule 147 to the instrument 140 (FIG. 10). The heating elements and temperature sensors on theplates 190, as well as theoptical boards PC board 226 by flex cables (not shown in FIG. 28 for clarity of illustration). Themodule 147 may also include agrounding trace 228 for shielding the optical detection circuit. Themodule 147 may optionally include an indicator, such as anLED 214, for indicating to a user the current status of the module such as “heating,” “cooling,” “finished,” or “fault”. - The
housing 208 may be molded from a rigid, high-performance plastic, or other conventional material. The primary functions of thehousing 208 are to provide a frame for holding theplates 190,optics assemblies fan 212, andPC board 226. Thehousing 208 also preferably provides flow channels and ports for directing cooling air from thefan 212 across the surfaces of theplates 190 and out of the housing. In the preferred embodiment, thehousing 208 comprises complementary pieces (only one piece shown in the schematic side view of FIG. 28) that fit together to enclose the components of themodule 147 between them. - Referring again to FIG. 23, the
plates - Ceramic plates are presently preferred because their inside surfaces may be conveniently machined to very high smoothness for high wear resistance, high chemical resistance, and good thermal contact to the flexible walls of the reaction vessel. Ceramic plates can also be made very thin, preferably between about 0.6 and 1.3 mm, for low thermal mass to provide for extremely rapid temperature changes. A plate made from ceramic is also both a good thermal conductor and an electrical insulator, so that the temperature of the plate may be well controlled using a resistive heating element coupled to the plate.
- Various thermal elements may be employed to heat and/or cool the
plates chamber 42. In general, suitable heating elements for heating the plate include conductive heaters, convection heaters, or radiation heaters. Examples of conductive heaters include resistive or inductive heating elements coupled to the plates, e.g., resistors or thermoelectric devices. Suitable convection heaters include forced air heaters or fluid heat-exchangers for flowing fluids past the plates. Suitable radiation heaters include infrared or microwave heaters. Similarly, various cooling elements may be used to cool the plates. For example, various convection cooling elements may be employed such as a fan, peltier device, refrigeration device, or jet nozzle for flowing cooling fluids past the surfaces of the plates. Alternatively, various conductive cooling elements may be used, such as a heat sink, e.g. a cooled metal block, in direct contact with the plates. - Referring to FIG. 24, each
plate 190 preferably has aresistive heating element 206 disposed on its outer surface. Theresistive heating element 206 is preferably a thick or thin film and may be directly screen printed onto eachplate 190, particularly plates comprising a ceramic material, such as aluminum nitride or aluminum oxide. Screen-printing provides high reliability and low cross-section for efficient transfer of heat into the reaction chamber. Thick or thin film resistors of varying geometric patterns may be deposited on the outer surfaces of the plates to provide more uniform heating, for example by having denser resistors at the extremities and thinner resistors in the middle. Although it is presently preferred to deposit a heating element on the outer surface of each plate, a heating element may alternatively be baked inside of each plate, particularly if the plates are ceramic. Theheating element 206 may comprise metals, tungsten, polysilicon, or other materials that heat when a voltage difference is applied across the material. Theheating element 206 has two ends which are connected torespective contacts 204 which are in turn connected to a voltage source (not shown in FIG. 24) to cause a current to flow through the heating element. Eachplate 190 also preferably includes atemperature sensor 192, such as a thermocouple, thermistor, or RTD, which is connected by twotraces 202 to respective ones of thecontacts 204. Thetemperature sensor 192 is be used to monitor the temperature of theplate 190 in a controlled feedback loop. - The plates have a low thermal mass to enable rapid heating and cooling of the plates. In particular, it is presently preferred that each of the plates has a thermal mass less than about 5 J/° C., more preferably less than 3 J/° C., and most preferably less than 1 J/° C. As used herein, the term thermal mass of a plate is defined as the specific heat of the plate multiplied by the mass of the plate. In addition, each plate should be large enough to cover a respective major wall of the reaction chamber. In the presently preferred embodiment, for example, each of the plates has a width X in the range of 2 to 22 mm, a length Y in the range of 2 to 22 mm, and a thickness in the range of 0.5 to 5 mm. The width X and length Y of each plate is selected to be slightly larger than the width and length of the reaction chamber. Moreover, each plate preferably has an angled bottom portion matching the geometry of the bottom portion of the reaction chamber, as previously described with reference to FIG. 34. Also in the preferred embodiment, each of the plates is made of aluminum nitride having a specific heat of about 0.75 J/g° C. The mass of each plate is preferably in the range of 0.005 to 5.0 g so that each plate has a thermal mass in the range of 0.00375 to 3.75 J/° C.
- The opposing
plates 190 are positioned to receive the chamber of thevessel 40 between them such that the flexible major walls of the chamber contact and conform to the inner surfaces of the plates. It is presently preferred that theplates 190 be held in an opposing relationship to each other using, e.g., brackets, supports, or retainers. Alternatively, theplates 190 may be spring-biased towards each other as described in International Publication Number WO 98/38487, the disclosure of which is incorporated by reference herein. In another embodiment of the invention, one of the plates is held in a fixed position, and the second plate is spring-biased towards the first plate. If one or more springs are used to bias the plates towards each other, the springs should be sufficiently stiff to ensure that the plates are pressed against the flexible walls of the vessel with sufficient force to cause the walls to conform to the inner surfaces of the plates. - FIGS. 29-30 illustrate a
preferred support structure 209 for holding theplates support structure 209 andplates support structure 209 includes a mountingplate 210 having theslot 148 formed therein. Theslot 148 is sufficiently large to enable the chamber of the vessel to be inserted through it. Spacing posts 230A, 230B extend from the mountingplate 210 on opposite sides of theslot 148. Spacing post 230A hasindentations 232 formed on opposite sides thereof (only one side visible in the isometric view of FIG. 29), and spacingpost 230B hasindentations 234 formed on opposite sides thereof (only one side visible in the isometric view of FIG. 29). Theindentations plates plates indentations retention clips plates - The mounting
plate 210 andspacing posts plates plate 210 may optionally includeindentations flex cables plates PC board 226 of the heat-exchanging module 147 (FIG. 28). The portion of theflex cables 238A adjacent theplate 190A is held in theindentation 240A by a piece oftape 242A, and the portion of theflex cables 238B adjacent theplate 190B is held in theindentation 240B by a piece oftape 242B. - FIG. 31 is an isometric view of the assembled
support structure 209. The mountingplate 210 preferably includestabs 246 extending from opposite sides thereof for securing thestructure 209 to the housing of the heat-exchanging module. Referring again to FIG. 28, thehousing 208 preferably includes slots for receiving the tabs to hold the mountingplate 210 securely in place. Alternatively, the mountingplate 210 may be attached to thehousing 208 using, e.g., adhesive bonding, screws, bolts, clamps, or any other conventional means of attachment. - Referring again to FIG. 29, the
support structure 209 preferably holds theplates wall 244 that is slightly tapered so that when theplates plates chamber 42 is inserted. The amount by which the inner surfaces are angled towards each other is very slight, preferably about 1° from parallel. The surfaces are angled towards each other so that, prior to the insertion of thechamber 42 between theplates chamber 42 of the vessel to be inserted between the plates and withdrawn from the plates more easily. Alternatively, the inner surfaces of theplates vessel 40 would be more difficult. - In addition, the inner surfaces of the
plates frame 46. In embodiments in which the inner surfaces are angled towards each other, the centers of the inner surfaces are preferably spaced a distance equal to the thickness of theframe 46 and the bottoms of the plates are initially spaced a distance that is slightly less than the thickness of theframe 46. When thechamber 42 is inserted between theplates rigid frame 46 forces the bottom portions of the plates apart so that thechamber 42 is firmly sandwiched between the plates. The distance that theplates frame 46 is usually very small, e.g., about 0.035 mm if the thickness of the frame is 1 mm and the inner surfaces are angled towards each other by 1°. - Referring again to FIG. 30, the retention clips236A, 236B should be sufficiently flexible to accommodate this slight outward movement of the
plates plates - Referring again to FIG. 28, to limit the amount that the
plates 190 can spread apart due to the pressurization of thevessel 40, stops may be molded into the housings ofoptics assemblies housing 249 of theoptics assembly 218 includes claw-like stops housing 249 is positioned such that the bottom edges of theplates stops stops plates plates frame 46. Preferably, the maximum spacing of the inner surfaces of theplates frame 46 to accommodate tolerance variations in thevessel 40 andplates frame 46. - FIG. 35 is a schematic, block diagram of the electronic components of the heat-exchanging
module 147. The module includes aconnector 224 or flex cable for connection to the main logic board of the instrument. The module also includesheater plates plates power input 253 from the instrument. Theplates temperature sensors 192A, 192B that output analog temperature signals to an analog-to-digital converter 264. Theconverter 264 converts the analog signals to digital signals and routes them to the microcontroller in the instrument through theconnector 224. - The heat-exchanging module also includes a cooling system, such as a
fan 212, for cooling theplates fan 212 is activated by switching apower switch 272, which is in turn controlled by acontrol logic block 270 that receives control signals from the microcontroller. The module further includes four light sources, such asLEDs 200, for excitation of labeled analytes in the reaction mixture and fourdetectors 198, preferably photodiodes, for detecting fluorescent emissions from the reaction mixture. The module also includes an adjustablecurrent source 255 for supplying a variable amount of current (e.g., in the range of 0 to 30 mA) to each LED to vary the brightness of the LED. A digital-to-analog converter 260 is connected between the adjustablecurrent source 255 and the microcontroller to permit the microcontroller to adjust the current source digitally. - The adjustable
current source 255 is preferably used to ensure that each LED has about the same brightness when activated. Due to manufacturing variances, many LEDs have different brightnesses when provided with the same amount of current. Therefore, it is presently preferred to test the brightness of each LED during manufacture of the heat-exchanging module and to store calibration data in amemory 268 of the module. The calibration data indicates the correct amount of current to provide to each LED. The microcontroller reads the calibration data from thememory 268 and controls thecurrent source 255 accordingly. - The module additionally includes a signal conditioning/gain select/offset adjust
block 262 comprised of amplifiers, switches, electronic filters, and a digital-to-analog converter. Theblock 262 adjusts the signals from thedetectors 198 to increase gain, offset, and reduce noise. The microcontroller controls block 262 through adigital output register 266. Theoutput register 266 receives data from the microcontroller and outputs control voltages to theblock 262. Theblock 262 outputs the adjusted detector signals to the microcontroller through the analog-to-digital converter 264 and theconnector 224. The module also includes thememory 268, preferably a serial EEPROM, for storing data specific to the module, such as calibration data for theLEDs 200,thermal plates temperature sensors 192A, 192B. - The operation of the cartridge and instrument will now be described. As shown in FIG. 3, a fluid sample to be analyzed is added to the
sample chamber 65 through thesample port 64 and thecap 30 screwed into theport 64 to seal the port shut. Referring to FIG. 10, thecartridge 20 is then placed into thecartridge nest 141 of theinstrument 140 for processing. All valves in the cartridge are initially closed when the cartridge is placed into theinstrument 140. When the cartridge is placed in the instrument, thetransducer 92 contacts an external surface of theflexible gasket 63 forming the bottom wall of the lysingchamber 86 , as shown in FIG. 5. - Referring again to FIG. 10, the
instrument 140 is preferably computer-controlled to perform the functions described in the following section, e.g., opening and closing valves in the cartridge usingvalve actuators 142, providing pressure to the cartridge throughnozzles 145, activating thetransducer 92, sensing liquid presence or liquid levels usingoptical sensors optical detection module 147. A programmer having ordinary skill in the art will be able to program a microcontroller and/or computer to perform these functions based upon the following description. - Referring to FIG. 9, liquids are preferably forced to flow through the cartridge using differential pressure. Although positive pressure is described herein, negative pressure (vacuum) may also be used to control fluid flow in the cartridge. The maximum amount of positive pressure that can be applied is usually limited by the hydrophobic membranes which may reach liquid break-through pressure above30 pounds per square inch (psi). The lower limit of pressure is limited by the need to move sample and other fluids through the cartridge sufficiently quickly to meet assay goals. Below 1 psi, for example, sample may not flow efficiently through the
filter stack 87. Pressure in the range of 6 to 20 psi is generally adequate. The sample flow rate through the cartridge is preferably in the range of 10 to 30 ml/minute. The wash flow rate may be slower, e.g. 6 to 18 ml/minute so that the wash effectively washes the lysingchamber 86. - A specific protocol will now be described with reference to FIG. 9 to illustrate the operation of the cartridge. It is to be understood that this is merely an example of one possible protocol and is not intended to limit the scope of the invention. To begin, the cartridge is preferably primed with wash solution from the
wash chamber 66 before the fluid sample is forced to flow from thesample chamber 65. To prime the cartridge,valves chamber 66 through thepressure port 116 for about two seconds. A small portion of the wash solution flows through thechannels chamber 86, through thechannels U-shaped channel 122, and all the way to the hydrophobic membrane below thepressure port 128. - Following priming,
valve 115 andpressure port 116 are closed andvalves sample chamber 65 through thepressure port 105 for about 15 seconds to force the sample to flow through thechannel 106, through thefilter stack 87 in thechamber 87, through thechannels waste chamber 68. As the sample passes thedetection region 136 in thechannel 106, the reflective optical sensor 144 (FIG. 13) may be used to determine when thesample chamber 65 has been emptied. As the sample liquid flows through thefilter stack 87, target cells or viruses in the sample are captured. When a predetermined volume of sample reaches thewaste chamber 68, some of the liquid spills over into thesensor chamber 120, triggering the next step in the protocol. Alternatively, instead of using feedback from optical sensors to trigger events, the steps in a predetermined protocol may simply be timed, e.g., applying predetermined pressures for predetermined durations of time to move known volumes of fluid at known flow rates. - The flow-through design of the lysing
chamber 86 permits target cells or viruses from a relatively large sample volume to be concentrated into a much smaller volume for amplification and detection. This is important for the detection of low concentration analyte in the sample, such as nucleic acid. In particular, the ratio of the volume of the sample forced to flow through the lysingchamber 86 to the volume capacity of thechamber 86 is preferably at least 2:1, and more preferably at least 5:1. The volume of sample forced to flow through thechamber 86 is preferably at least 100 μl, and more preferably at least 1 ml. In the presently preferred embodiment, a sample volume of 5 ml is forced to flow through the lysingchamber 86, and thechamber 86 has a volume capacity of about 0.5 ml, so that the ratio is 10:1. In addition, the lysingchamber 86 may be sonicated (e.g., using an ultrasonic horn coupled to a wall of the chamber) as the sample is forced to flow through the chamber. Sonicating thechamber 86 helps to prevent clogging of thefilter stack 87, providing for more uniform flow through thechamber 86. In particular, the sound waves help keep particulate matter or the beads in the filter stack from clogging one or more filters. - In the next step,
valves wash chamber 66 for about seven seconds to force the wash solution to flow through thechannels chamber 86. The washing solution washes away PCR inhibitors and contaminants from the lysingchamber 86 and carries then through thechannels waste chamber 68. A variety of suitable wash solutions of varying pH, solvent composition, and ionic strength may be used for this purpose and are well known in the art. For example, a suitable washing reagent is a solution of 80 mM potassium acetate, 8.3 mM Tris-HCl, pH 7.5, 40 uM EDTA, and 55% ethanol. The lysingchamber 86 may be sonicated (e.g., using an ultrasonic horn coupled to a wall of the chamber) while the wash solution is forced to flow through the chamber. Sonicating thechamber 86 helps to prevent clogging of thefilter stack 87, providing for more uniform flow through thechamber 86 as previously described. In addition, the sound waves may help loosen the material to be washed away. When the incremental volume of wash solution reaches thewaste chamber 68, some of the liquid spills over into thesensor chamber 121, triggering the next step in the protocol. - In the next step,
valve 115 is closed andvalve 119 is opened while a pressure of 15 psi is applied to thereagent chamber 67 through thepressure port 118 for about three seconds. The pressure forces lysing reagent to flow from thechamber 67 through thechannels chamber 86, and into thechannel 110. Thechamber 86 is thus filled with liquid. Suitable lysing reagents include, e.g., solutions containing a chaotropic salt, such as guanidine HCl, guanidine thiocyanate, guanidine isothiocyanate, sodium iodide, urea, sodium perchlorate, and potassium bromide. In the presently preferred embodiment, a lysing reagent that is not inhibitory to PCR is used. The lysing reagent comprises 10 mM tris, 5% tween-20, 1 mM tris (2-carboxyethyl phosphine hydrochloride), 0.1 mM Ethylene Glycol-bis (b-amino-ethyl ether)—N,N,N′,N′—tetracetic acid. After the lysingchamber 86 is filled with lysing reagent, thevalves Valve 119 remains open and a pressure of 20 psi is applied topressure port 118. The static pressure in thelysis chamber 86 is therefore increased to 20 psi in preparation for the lysis of the cells or viruses trapped in thefilter stack 87. - Referring again to FIG. 5, the pressurization of the lysing
chamber 86 is important because it ensures effective coupling between thetransducer 92 and theflexible wall 63 of the lysingchamber 86. To disrupt the cells or viruses in thechamber 86, thetransducer 92 is activated (i.e., set into vibratory motion). Theflexible wall 63 of the lysingchamber 86 transfers the vibratory motion of thetransducer 92 to the liquid in thechamber 86 by allowing slight deflections without creating high stresses in the wall. Thewall 63 may be formed by the elastomeric membrane as previously described. Alternatively, the wall may be a film or sheet of polymeric material (e.g., a polypropylene film) preferably having a thickness in the range of 0.025 to 0.1 mm. Thetransducer 92 is preferably an ultrasonic horn for sonicating thechamber 86. Thechamber 86 is preferably sonicated for 10 to 40 seconds at a frequency in the range of 20 to 60 kHz. In the exemplary protocol, the chamber is sonicated for 15 seconds at a frequency of 47 kHz. The amplitude of the horn tip is preferably in the range of 20 to 25 μm (measured peak to peak). - As the tip of the
transducer 92 vibrates, it repeatedly impacts theflexible wall 63. On its forward stroke (in the upward direction in FIG. 6), the tip of thetransducer 92 pushes thewall 63 and creates a pressure pulse or pressure wave in thechamber 86. On its retreating stroke (downward in FIG. 5), the tip of thetransducer 92 usually separates from theflexible wall 63 because theflexible wall 63 cannot move at the same frequency as the transducer. On its next forward stroke, the tip of thetransducer 92 once again impacts thewall 63 in a head-on collision as the tip and wall speed towards each other. Because thetransducer 92 and thewall 63 separate as thetransducer 92 vibrates, the effective forward stroke of the transducer is less than its peak-to-peak amplitude. The effective forward stroke determines the level of sonication in thechamber 86. It is therefore important to increase the static pressure in the lysingchamber 86 so that when the tip of thetransducer 92 retreats, theflexible wall 63 is forced outwardly to meet the tip on its return stroke. The static pressure in thechamber 86 should be sufficient to ensure that the effective forward stroke of thetransducer 92 generates pressure pulses or pressure waves in thechamber 86. It is presently preferred to increase the static pressure in thechamber 86 to at least 5 psi above the ambient pressure external to the cartridge, and more preferably to a pressure in the range of 15 to 25 psi above the ambient pressure. - On each forward stroke, the
transducer 92 imparts a velocity to the liquid in thechamber 86, thus creating a pressure pulse that quickly sweeps across thechamber 86. The beads in the filter stack 87 (FIG. 6) are agitated by the pressure pulses in thechamber 86. The pressure pulses propel the beads into violent motion in thechamber 86, and the beads mechanically rupture the cells or viruses to release the material (e.g., nucleic acid) therefrom. It should be noted that some types of cells, such as blood cells, are relatively weak and may be disrupted using only pressure pulses without the use of beads. Other types of cells (particularly spores) have highly resistant cell walls and beads are generally required for effective lysis. - Referring again to FIG. 9, following disruption of the cells or viruses,
valves reagent chamber 67 through thepressure port 118. The pressure forces the lysis reagent to elute the nucleic acid from thefilter stack 87 and to flow with the nucleic acid into theneutralization chamber 70. The lysingchamber 86 may be sonicated (e.g., using an ultrasonic horn coupled to a wall of the chamber) while the eluting the nucleic acid. Sonicating thechamber 86 may help prevent clogging of thefilter stack 87, as previously described. The chamber 420 is partially filled (e.g., half-filled) with neutralizer, such as detergent, for neutralizing the lysing reagent. If a lysing reagent non-inhibitory to PCR is used, the neutralizer is optional. - In the next step, the
valve 124 is closed to hold the lysing reagent, analyte, and neutralizer in thechamber 70. Thevalve 114 is opened and a pressure of 15 psi is applied for about three seconds through thepressure port 128 to force any liquid in theU-shaped channel 122 to flow into thewaste chamber 68. Next,valves pressure port 123 on top of theneutralizer chamber 70. The pressure forces the neutralized lysing reagent and nucleic acid in thechamber 70 to flow into thechannel 122 and into themaster mix chamber 71. Thevalve 126 to themaster mix chamber 71 is then closed. The master mix chamber contains PCR reagents and fluorescent probes that mix with the neutralized lysing reagent and nucleic acid to form a reaction mixture. - In the next step, the
channel 122 is cleared by openingvalve 114 to wastechamber 68 and applying a pressure of 15 psi for about one second to pressureport 128. In the next step, the reaction mixture formed in themaster mix chamber 71 is moved into thereaction vessel 40 as follows.Valves pressure port 125 on top of themaster mix chamber 71 to force the reaction mixture to flow through thechannel 122,valve 127, andchannel 80 into thereaction vessel 40 through theport 41. The reaction mixture fills thechamber 42 of the vessel, displacing air in the chamber which exits through theoutlet channel 52. The air escaping through theoutlet channel 52 travels inchannel 81past sensor region 130 and intochannel 131. Fromchannel 131, the air flows intochannel 132, throughvalve 133,channel 134, and exits the cartridge through thevent 36. When a volume of reaction mixture sufficient to fill thechamber 42 has flowed into the vessel, excess reaction mixture exits the vessel through theoutlet channel 52. The excess reaction mixture flows intochannel 81 and is optically detected in thesensor region 130. When the reaction mixture is detected,valve 133 is closed while pressure from thepressure port 125 is applied to pressurize thereaction chamber 42. - Referring again to FIG. 23, the pressurization of the
chamber 42 expands the flexiblemajor walls 48 of the vessel. In particular the pressure forces themajor walls 48 to contact and conform to the inner surfaces of theplates plates chamber 42. It is presently preferred to pressurize thechamber 42 to a pressure in the range of 2 to 30 psi above ambient pressure. This range is presently preferred because 2 psi is generally enough pressure to ensure conformity between thewalls 48 and the surfaces of theplates walls 48, deformation of theframe 46 orplates chamber 42 is pressurized to a pressure in the range of 8 to 15 psi above ambient pressure. This range is more preferred because it is safely within the practical limits described above. When thechamber 42 is pressurized, the reaction mixture in thevessel 40 is thermally processed and optically interrogated to determine the presence or absence of a target analyte in the mixture. - Referring again to FIG. 35, the reaction mixture is thermally processed between the
plates temperature sensors 192A, 192B. Proportioning may be accomplished either by varying the ratio of “on” time to “off” time, or, preferably with proportional analog outputs which decrease the average power being supplied either to the heating elements on theplates fan 212 as the actual temperature of theplates - As the reaction mixture is thermally cycled between the
heater plates LEDs 200 to excite different fluorescently-labeled analytes in the mixture and by detecting light emitted (fluorescent output) from thechamber 42 using detectors the 198. Referring again to FIG. 22, excitation beams are preferably transmitted to thechamber 42 through the opticallytransmissive side wall 57A, while fluorescent emission is detected through theside wall 57B. - One advantage of the cartridge of the present invention is that it allows the intracellular material from a relatively large volume of fluid sample, e.g. several milliliters or more, to be separated from the sample and concentrated into a much smaller volume of reaction fluid, e.g., 100 μL or less. The cartridge permits extraordinary concentration factors by efficiently extracting material from milliliter quantities of fluid sample. In particular, the
sample chamber 65 preferably has a volume capacity in the range of 100 μl to 12 ml. More preferably, thesample chamber 65 has a volume capacity of at least 1 ml. The lower limit of 1 ml is preferred because at least 1 ml of sample should be analyzed to detect low concentration analytes such as nucleic acid. The upper limit of 12 ml is preferred because a sample volume greater than 12 ml would require a much larger cartridge and likely clog the filter stack. In the presently preferred embodiment, the sample chamber has a volume capacity of 5.5 ml for holding 5 ml of sample. - The
wash chamber 66 has a volume capacity proportional to the volume of the lysingchamber 86. In particular, thewash chamber 66 preferably holds a volume of wash that is at least one to two times the volume of the lysingchamber 86 to ensure that there is enough wash solution to wash out PCR inhibitors and debris from thechamber 86. In the presently preferred embodiment, the volume of the lysingchamber 86 is about 0.5 ml and the volume of thewash chamber 66 is 2.5 ml for holding 2 ml of wash solution. The lysing chamber volume of 0.5 ml is a compromise between a size large enough to avoid clogging of thefilter stack 87 and a size small enough to concentrate analyte into a small volume for improved amplification and detection. - The
reagent chamber 67 preferably holds a volume of lysing reagent that is at least one to two times the volume of the lysingchamber 86 so that there is sufficient lysing reagent to pressurize the chamber and to elute nucleic acid from the chamber. In the presently preferred embodiment, thechamber 67 has a volume capacity of 1.5 ml for holding about 1 to 1.5 ml of lysing reagent. Thewaste chamber 68 has a volume capacity sufficient to hold the sample, wash solution, and unused lysing reagent. Thewaste chamber 68 is sized at 9.5 ml volume capacity in the preferred embodiment. - The size of the
neutralization chamber 70 is dependent upon the volume of the lysingchamber 86 since the neutralizer in thechamber 70 neutralizes the volume of lysing reagent that fills the lysingchamber 86. It is currently preferred that the lysing chamber have a volume if 0.5 ml, so thechamber 70 has a volume capacity of 10.0 ml for holding about 0.5 ml of neutralizer that is mixed with 0.5 ml of the lysing reagent and eluted analyte. The volume capacity of themaster mix chamber 71 should be sufficient to produce a reaction mixture to fill thevessel 40 and thechannels - The flow channels in the cartridge are generally D-shaped in cross section (with the
gasket 63 forming the flat side of the channel) and preferably have a width or diameter in the range of {fraction (1/64)} to ⅛ of an inch (0.4 to 3.2 mm), and more preferably a width of {fraction (1/32)} to {fraction (1/16)} of an inch (0.8 to 1.6 mm). These ranges are presently preferred to avoid having channels to narrow (which creates flow restriction) and to avoid having channels too wide (which yields unused volumes of liquid sitting in the flow path). - Many modifications to the structure and operation of the cartridge and instrument are possible in alternative embodiments. For example, although amplification by PCR is presently preferred, the cartridge and instrument may be used to amplify nucleic acid sequences using any amplification method, including both thermal cycling amplification methods and isothermal amplification methods. Suitable thermal cycling methods include, but are not limited to, the Polymerase Chain Reaction (PCR; U.S Pat. Nos. 4,683,202, 4,683,195 and 4,965,188); Reverse Transcriptase PCR (RT-PCR); DNA Ligase Chain Reaction (LCR; International Patent Application No. WO 89/09835); and transcription-based amplification (D. Y. Kwoh et al. 1989, Proc. Natl.
Acad. Sci. USA 86, 1173-1177). Suitable isothermal amplification methods useful in the practice of the present invention include, but are not limited to, Rolling Circle Amplification; Strand Displacement Amplification (SDA; Walker et al. 1992, Proc. Nati. Acad. Sci. USA 89, 392-396); Q-.beta. replicase (Lizardi et al. 1988, Bio/Technology 6, 1197-1202); Nucleic Acid-Based Sequence Amplification (NASBA; R. Sooknanan and L. Malek 1995, Bio/Technology 13, 563-65); and Self-Sustained Sequence Replication (3SR; Guatelli et al. 1990, Proc. Nati.Acad. Sci. USA 87, 1874-1878). - Moreover, the cartridge and instrument may be used to conduct chemical reactions other than nucleic acid amplification. Further, although fluorescence excitation and emission detection is preferred, optical detection methods such as those used in direct absorption and/or transmission with on-axis geometries may also be used to detect analyte in the cartridge. Another possible detection method is time decay fluorescence. Additionally, the cartridge is not limited to detection based upon fluorescent labels. For example, detection may be based upon phosphorescent labels, chemiluminescent labels, or electrochemiluminescent labels.
- A fluid sample may be introduced into the cartridge by a variety of means, manual or automated. For manual addition, a measured volume of material may be placed into a receiving area of the cartridge through an input port and a cap is then placed over the port. Alternatively, a greater amount of sample material than required for the analysis can be added to the cartridge and mechanisms within the cartridge can effect the precise measuring and aliquoting of the sample needed for the specified protocol. It may be desirable to place certain samples, such as tissue biopsy material, soil, feces, exudates, and other complex material into another device or accessory and then place the secondary device or accessory into the cartridge. For example, a piece of tissue may be placed into the lumen of a secondary device that serves as the cap to the input port of the cartridge. When the cap is pressed into the port, the tissue is forced through a mesh that slices or otherwise divides the tissue.
- For automated sample introduction, additional design features of the cartridge are employed and, in many cases, impart specimen accession functionality directly into the cartridge. With certain samples, such as those presenting a risk of hazard to the operator or the environment, such as human retrovirus pathogens, the transfer of the sample to the cartridge may pose a risk. Thus, in one embodiment, a syringe may be integrated into a device to provide a means for moving external fluidic samples directly into the cartridge. Alternatively, a venous puncture needle and an evacuated blood tube can be attached to the cartridge forming an assembly that can be used to acquire a sample of blood. After collection, the tube and needle are removed and discarded, and the cartridge is then placed in an instrument to effect processing. The advantage of such an approach is that the operator or the environment is not exposed to pathogens.
- The input port can be designed with a consideration of appropriate human factors as a function of the nature of the intended specimen. For example, respiratory specimens may be acquired from the lower respiratory tract as expectorants from coughing, or as swab or brush samples from the back of the throat or the nares. In the former case, the input port can be designed to allow the patient to cough directly into the cartridge or to otherwise facilitate spitting of the expectorated sample into the cartridge. For brush or swab specimens, the specimen is placed into the input port where features of the port and closure facilitate the breaking off and retaining of the end of the swab or brush in the cartridge receiving area.
- In another embodiment, the cartridge includes input and output tubes that may be positioned in a sample pool of very large volume, such as a flowing stream of water, so that the sample material flows through the cartridge. Alternatively, a hydrophilic wicking material can serve as an interactive region so that the entire cartridge can be immersed directly into the specimen, and a sufficient amount of specimen is absorbed into the wicking material. The cartridge is then removed, and can be transported to the laboratory or analyzed directly using a portable instrument. In another embodiment, tubing can be utilized so that one end of the tube is in direct communication with the cartridge to provide a fluidic interface with at least one interactive region and the other end is accessible to the external environment to serve as a receiver for sample. The tube can then be placed into a specimen and serve as a sipper. The cartridge itself may also serve as the actual specimen collection device, thereby reducing handling and inconvenience. In the case of specimens involved in legal disputes or criminal investigations, the direct accessing of the test material into the fluidic cartridge is advantageous because the chain of custody is conveniently and reliably preserved.
- Referring again to FIG. 9, reagents may be exogenously introduced into the cartridge before use, e.g., through sealable openings in the
reagent chamber 67,neutralizer chamber 70, andmaster mix chamber 71. Alternatively, the reagents may be placed in the cartridge during manufacture, e.g., as aqueous solutions or dried reagents requiring reconstitution. The particular format is selected based on a variety of parameters, including whether the interaction is solution-phase or solid-phase, the inherent thermal stability of the reagent, speed of reconstitution, and reaction kinetics. Reagents containing compounds that are thermally unstable when in solution can be stabilized by drying using techniques such as lyophilization. Additives, such as simple alcohol sugars, methylcelluloses, and bulking proteins may be added to the reagent before drying to increase stability or reconstitutability. - Referring again to FIG. 21, the
reaction vessel 40 does not require two flexible sheets forming opposingmajor walls 48 of thereaction chamber 42. For example, in one alternative embodiment, thevessel 40 has only one flexible sheet forming a major wall of the chamber. Therigid frame 46 defines the other major wall of the chamber, as well as the side walls of the chamber. In this embodiment, the major wall formed by theframe 46 should have a minimum thickness of about 0.05 inches (1.25 mm) which is typically the practical minimum thickness for injection molding, while the flexible sheet may be as thin as 0.0005 inches (0.0125 mm). The advantage to this embodiment is that the manufacturing of thereaction vessel 40 is simplified, and hence less expensive, since only one flexible sheet need be attached to theframe 46. The disadvantage is that the heating and cooling rates of the reaction mixture are likely to be slower since the major wall formed by theframe 46 will probably not permit as high a rate of heat transfer as the thin, flexible sheet. - Referring to FIG. 28, the heat-exchanging
module 147 only requires one thermal surface for contacting a flexible wall of thereaction vessel 40 and one thermal element for heating and/or cooling the thermal surface. The advantage to using one thermal surface and one thermal element is that the apparatus may be manufactured less expensively. The disadvantage is that the heating and cooling rates are likely to be about twice as slow. Further, although it is presently preferred that the thermal surfaces be formed by the thermallyconductive plates 190, each thermal surface may be provided by any rigid structure having a contact area for contacting a wall of thevessel 40. The thermal surface preferably comprises a material having a high thermal conductivity, such as ceramic or metal. Moreover, the thermal surface may comprise the surface of the thermal element itself. For example, the thermal surface may be the surface of a thermoelectric device that contacts the wall to heat and/or cool the chamber. - It is presently preferred to build the transducer into the
instrument 140. In another embodiment, however, the transducer may be built into the cartridge. For example, a piezoelectric disk may be built into the cartridge for sonicating the lysing chamber. Alternatively, a speaker or electromagnetic coil device may be built into the cartridge. In these embodiments, the cartridge includes suitable electrical connectors for connecting the transducer to a power supply. In embodiments in which the transducer is built into the cartridge, the transducer should be prevented from contacting the fluid sample directly, e.g., the transducer should be laminated or separated from the sample by a chamber wall. Further, lysis of the cells or viruses may be performed using a heater in place of or in combination with a transducer. The heater may be a resistive heating element that is part of cartridge, or the heater could be built into the instrument that receives the cartridge. In this embodiment, the cells or viruses are disrupted by heating the lysis chamber to a high temperature (e.g., 95° C.) to disrupt the cell walls. - FIGS. 36-46 show another
apparatus 350 for disrupting cells or viruses according to the present invention. FIG. 36 shows an isometric view of theapparatus 350, and FIG. 37 shows a cross sectional view of theapparatus 350. As shown in FIGS. 36-37, theapparatus 350 includes a cartridge orcontainer 358 having achamber 367 for holding the cells or viruses. The container includes aflexible wall 440 defining thechamber 367. In this embodiment, theflexible wall 440 is the bottom wall of thechamber 367. Theflexible wall 440 is preferably a sheet or film of polymeric material (e.g., a polypropylene film) and thewall 440 preferably has a thickness in the range of 0.025 to 0.1 mm. Theapparatus 350 also includes atransducer 314, such as an ultrasonic horn, for contacting an external surface of the flexible wall 440 (i.e., a surface of thewall 440 that is external to the chamber 367). Thetransducer 314 should be capable of vibratory motion sufficient to create pressure pulses in thechamber 367. Suitable transducers include ultrasonic, piezoelectric, magnetostrictive, or electrostatic transducers. The transducer may also be an electromagnetic device having a wound coil, such as a voice coil motor or a solenoid device. - The
apparatus 350 further includes asupport structure 352 for holding thecontainer 358 and thetransducer 314 against each other such that thetransducer 314 contacts thewall 440 of thechamber 367 and for applying a substantially constant force to thecontainer 358 or to thetransducer 314 to press together thetransducer 314 and thewall 440 of the chamber. Thesupport structure 352 includes abase structure 354 having astand 356. Thetransducer 314 is slidably mounted to thebase structure 354 by aguide 364. Theguide 364 is either integrally formed with thebase structure 354 or fixedly attached to the base structure. Thesupport structure 352 also includes aholder 360 attached to thebase structure 354 for holding thecontainer 358. Theholder 360 has a U-shaped bottom portion providing access to theflexible wall 440 of thechamber 367. Theguide 364 and theholder 360 are arranged to hold thetransducer 314 and thecontainer 358, respectively, such that the external surface of thewall 440 contacts thetransducer 314. Thesupport structure 352 also includes atop retainer 362 for thecontainer 358. Theretainer 362 is U-shaped to allow access to anexit port 444 formed in thecontainer 358. - The
support structure 352 further includes an elastic body, such as aspring 366, for applying a force to thetransducer 314 to press thetransducer 314 against thewall 440. When thetransducer 314 is in contact with thewall 440, the force provided by the spring. 366 is constant, providing for consistent coupling between thetransducer 314 and thewall 440. Thespring 366 is positioned between a spring guide.372 and the base of acoupler 368 that supports the bottom of thetransducer 314. As shown in FIG. 36, thecoupler 370 preferably has awindow 370 through which the power cord (not shown) of thetransducer 314 may be placed. Bolts or screws 376 hold thespring guide 372 inadjustment grooves 374 formed in thebase structure 354. The magnitude of the force provided by thespring 366 may be adjusted by changing the preload on the spring. To adjust the preload on thespring 366, thebolts 376 holding thespring guide 372 are loosened, theguide 372 is moved to a new position, and thebolts 376 are retightened to hold theguide 372 in the new position. Once the preload on thespring 366 is adjusted to provide a suitable coupling force between thetransducer 314 and thewall 440, it is desirable to keep the preload constant from one use of theapparatus 350 to the next so that valid comparisons can be made between different samples disrupted by the apparatus. - The magnitude of the force provided by the
spring 366 to press together thetransducer 314 and thewall 440 is important for achieving a consistent transfer of energy between thetransducer 314 and thechamber 367. If the force is too light, thetransducer 314 will only be held lightly against thewall 440, leading to poor translation of vibratory movement from thetransducer 314 to thewall 440. If the force is too strong, thecontainer 358 orwall 440 may be damaged during sonication. An intermediate force results in the most consistent and repeatable transfer of vibratory motion from thetransducer 314 to thewall 440. It is presently preferred that thespring 366 provide a force in the range of 2 to 5 lbs. - FIG. 38 shows an exploded view of the
container 358, and FIG. 39 shows an assembled view of thecontainer 358. As shown in FIGS. 38-39, thecontainer 358 has a body comprising a top piece 4 48, amiddle piece 450 , and abottom piece 452. Themiddle piece 450 defines aninlet port 442 to thechamber 367, and thetop piece 448 defines anoutlet port 444 to the chamber. Theports chamber 367. Theflexible wall 440 is held between the middle andbottom pieces gaskets flexible wall 440 may simply be heat sealed to themiddle piece 450 so that thebottom piece 452 andgaskets - The
container 358 also includes afilter stack 446 in thechamber 367 for capturing sample components (e.g., target cells or viruses) as the sample flows through thechamber 367. The filter stack comprises (from bottom to top in FIGS. 38-39) agasket 456, afirst filter 458, agasket 460, asecond filter 464 having a smaller average pore size than thefirst filter 458, and agasket 466. The filter stack is held between the top andmiddle pieces container 358. The filter stack also includesbeads 462 disposed between the first andsecond filters gasket 460 spaces thefirst filter 458 from thesecond filter 464. Thegasket 460 should be thick enough to permit the beads to move freely in the space between thefilters chamber 367 first flows through thefilter 458 and then through thefilter 466. After flowing through the filter stack, the sample flows along flow ribs 468 (FIG. 38) formed in the portion of thetop piece 448 that defines the top of the chamber and through the outlet port 444 (FIG. 39). - The filter stack is effective for capturing cells or viruses as a fluid sample flows through the
chamber 367 without clogging of the. The first filter 458 (having the largest pore size) filters out coarse material such as salt crystals, cellular debris, hair, tissue, etc. The second filter 464 (having a smaller pore size) captures target cells or viruses in the fluid sample. The average pore size of thefirst filter 458 is selected to be small enough to filter coarse material from the fluid sample (e.g., salt crystals, cellular debris, hair, tissue) yet large enough to allow the passage of the target cells or viruses. In general, the average pore size of thefirst filter 458 should be in the range of about 2 to 25 μm, with a presently preferred pore size of about 5 μm. The average pore size of thesecond filter 464 is selected to be slightly smaller than the average size of the target cells or viruses to be captured (typically in the range of 0.2 to 5 μm). - The
beads 462 are useful for disrupting the captured cells or viruses to release the intracellular material (e g., nucleic acid) therefrom. Movement of thebeads 462 ruptures the cells or viruses captured on thefilter 464. Suitable beads for rupturing cells or viruses include borosilicate glass, lime glass, silica, and polystyrene beads. The beads may be porous or non-porous and preferably have an average diameter in the range of 1 to 200 μm. In the presently preferred embodiment, the beads. 462 are polystyrene beads having an average diameter of about 100 μm. - The
beads 462 may have a binding affinity for target cells or viruses in the fluid sample to facilitate capture of the target cells or viruses. For example, antibodies or certain receptors may be coated onto the surface of thebeads 462 to bind target cells in the sample. Moreover, thechamber 367 may contain two different types of beads for interacting with target cells or viruses. For example, the chamber may contain a first set of beads coated with antibodies or receptors for binding target cells or viruses and a second set of beads (intermixed with the first set) for rupturing the captured cells or viruses. The beads in the chamber may also have a binding affinity for the intracellular material (e.g., nucleic acid) released from the ruptured cells or viruses. Such beads may be useful for isolating target nucleic acid for subsequent elution and analysis. For example, thechamber 367 may contain silica beads to isolate DNA or cellulose beads with oligo dT to isolate messenger RNA for RT-PCR. Thechamber 367 may also contain beads for removing unwanted material (e.g., proteins, peptides) or chemicals (e.g., salts, metal ions, or detergents) from the sample that might inhibit PCR. - To ensure that the air bubbles can escape from the
chamber 367, it is desirable to use thecontainer 358 in an orientation in which liquid flows up (relative to gravity) through thefilters chamber 367. The upward flow through thechamber 367 aids the flow of air bubbles out of the chamber. Thus, theinlet port 442 for entry of fluids into thechamber 367 should generally be at a lower elevation than theoutlet port 444. The volume capacity of thechamber 367 is usually in the range of 50 to 500 μl. The volume capacity of thechamber 367 is selected to provide for concentration of analyte separated from a fluid sample without the chamber being so small that thefilters - The
pieces container 358 are preferably molded polymeric parts (e.g., polypropylene, polycarbonate, acrylic, etc.). Although molding is preferred for mass production, it also possible to machine the top, middle, andbottom pieces pieces container 358. Another method for fabricating thecontainer 358 is to mold the body as a single piece and heat seal theflexible wall 440 and thefilters - FIG. 40 shows a fluidic system for use with the apparatus. The system includes a
bottle 470 for holding lysis buffer, abottle 472 containing wash solution, and asample container 474 for holding a fluid sample. Thebottles sample container 474 are connected via tubing to the valve ports of asyringe pump 476. The inlet port of thecontainer 358 is also connected to thesyringe pump 476. The outlet port of thecontainer 358 is connected to the common port of adistribution valve 478. The system also includes acollection tube 480 for receiving intracellular material removed from the sample, awaste container 482 for receiving waste, and a pressure source, such as apump 484. Thecollection tube 480,waste container 482, and pump 484 are connected to respective peripheral ports of thedistribution valve 478. Apressure regulator 486 regulates the pressure supplied by thepump 484. - A specific protocol will now be described with reference to FIGS. 39-40 to illustrate the operation of the
container 358. It is to be understood that this is merely an example of one possible protocol and is not intended to limit the scope of the invention. Thesyringe pump 476 pumps a fluid sample from thesample container 474 through thecontainer 358 and into thewaste container 482. As the fluid sample is forced to flow through the filters in thechamber 367, coarse material is filtered by thefilter 458 and target cells or viruses in the sample are captured by thefilter 464. Thechamber 367 may be sonicated as the sample is forced to flow through the chamber to help prevent clogging of the filters. Next, thesyringe pump 476 pumps wash solution from thebottle 472 through thecontainer 358 and into thewaste container 482. The washing solution washes away PCR inhibitors and contaminants from thechamber 367. - In the next step, the
syringe pump 476 pumps lysis buffer from thebottle 470 into thecontainer 358 so that thechamber 367 is filled with liquid. The lysis buffer should be a medium through which dynamic pressure pulses or pressure waves can be transmitted. For example, the lysis buffer may comprise deionized water for holding the cells or viruses in suspension or solution. Alternatively, the lysis buffer may include one or more lysing agents to aid in the disruption of the cells or viruses. One of the advantages of the present invention, however, is that harsh lysing agents are not required for successful disruption of the cells or viruses. Next, the distribution valve of thesyringe pump 476 is closed upstream of thecontainer 358, and thedistribution valve 478 is opened. Thepump 484 then pressurized thechamber 367 through theoutlet port 444, preferably to about 20 psi above the ambient pressure. Thedistribution valve 478 downstream of thecontainer 358 is then closed. The static pressure in thechamber 367 is therefore increased to about 20 psi in preparation for the disruption of the cells or viruses trapped on thefilter 464. - Referring again to FIG. 37, the pressurization of the
chamber 367 is important because it ensures effective coupling between thetransducer 314 and theflexible wall 440. To disrupt the cells or viruses in thechamber 367, thetransducer 314 is activated (i.e., set into vibratory motion). Theflexible wall 440 transfers the vibrational motion of thetransducer 314 to the liquid in thechamber 367 by allowing slight deflections without creating high stresses in the wall. Thetransducer 314 is preferably an ultrasonic horn for sonicating thechamber 367. Thechamber 367 is preferably sonicated for 10 to 40 seconds at a frequency in the range of 20 to 60 kHz. In the exemplary protocol, the chamber is sonicated for 15 seconds at a frequency of 40 kHz. The amplitude of the horn tip is preferably in the range of 20 to 25 μm (measured peak to peak). - As the tip of the
transducer 314 vibrates, it repeatedly impacts theflexible wall 440. On its forward stroke (in the upward direction in FIG. 37), the tip of thetransducer 314 pushes thewall 440 and creates a pressure pulse or pressure wave in thechamber 367. On its retreating stroke (downward in FIG. 37), the tip of thetransducer 314 usually separates from theflexible wall 440 because theflexible wall 440 cannot move at the same frequency as the transducer. On its next forward stroke, the tip of thetransducer 314 once again impacts thewall 440 in a head-on collision as the tip and wall speed towards each other. Because thetransducer 314 and thewall 440 separate as thetransducer 314 vibrates, the effective forward stroke of the transducer is less than its peak-to-peak amplitude. The effective forward stroke determines the level of sonication in thechamber 367. It is therefore important to increase the static pressure in thechamber 367 so that when the tip of thetransducer 314 retreats, theflexible wall 440 is forced outwardly to meet the tip on its return stroke. The static pressure in thechamber 367 should be sufficient to ensure that the effective forward stroke of thetransducer 314 generates the necessary pressure pulses or pressure waves in the chamber to effect cell disruption. It is presently preferred to increase the static pressure in thechamber 367 to at least 5 psi above the ambient pressure, and more preferably to a pressure in the range of 15 to 25 psi above the ambient pressure. - On each forward stroke, the
transducer 314 imparts a velocity to the liquid in thechamber 367, thus creating a pressure pulse or pressure wave that quickly sweeps across the chamber. Thebeads 462 in the filter stack 446 (FIG. 38) are agitated by the pressure pulses in thechamber 367. The pressure pulses propel the beads into violent motion, and the beads mechanically rupture the cells or viruses to release the analyte (e.g., nucleic acid) therefrom. Referring again to FIG. 40, following disruption of the cells or viruses, thesyringe pump 476 pumps the released intracellular material from thecontainer 358 into thecollection tube 480. - FIG. 41 shows another embodiment of the invention in which the
container 358 has asolid wall 488 for contacting thetransducer 314. Thesolid wall 488 differs from theflexible wall 440 previously described with reference to FIG. 37. Whereas the flexible wall is typically a thin film that bends under its own weight and does not hold its shape unless held on its edges, thesolid wall 488 holds it shape when unsupported. The advantage of using a solid wall to contact thetransducer 314 is that there is no need to pressurize thechamber 367 to ensure effective coupling between thewall 488 and thetransducer 314. The elastic restoring force of thesolid wall 488 provides the necessary coupling between the wall and thetransducer 314. However, the proper design of thesolid wall 488 is necessary so that the wall is not damaged (e.g., melted) by the vibratory movements of thetransducer 314. - In particular, the
solid wall 488 should have a natural frequency that is higher than the vibrating frequency at which thetransducer 314 is operated. Preferably, the ratio of the natural frequency of thewall 488 to the vibrating frequency is at least 2:1, and more preferably the ratio is at least 4:1. In addition, thewall 488 should not be so rigid that it cannot transfer the vibratory motion of the transducer to the liquid in thechamber 367. It is preferred that thewall 488 be capable of deflecting a distance in the range of 5 to 40 μm, and more preferably about 20 μm peak to peak when thetransducer 314 applies a force in the range of 1 to 10 lbs. to the external surface of thewall 488. It is more preferable that thewall 488 be capable of deflecting a distance in the range of 5 to 40 μm, and more preferably about 20 μm peak to peak when thetransducer 314 applies a force in the range of 2 to 5 lbs. To achieve these criteria, thewall 488 is dome-shaped and convex with respect to the transducer 314(i.e., thewall 488 curves outwardly towards the transducer). The advantage to the dome-shaped design of thewall 488 is that the dome shape increases the natural frequency of the wall (compared to a flat wall) without causing the wall to be so stiff that it cannot transfer the vibratory movements of thetransducer 314 to thechamber 367. - FIG. 42 shows a cross sectional view of the
wall 488. The dome-shapedportion 495 of the wall preferably has a radius of curvature R in the range of 6.3 to 12.7 mm when the diameter D of the dome-shaped portion is about 11.1 mm. More preferably, the dome-shapedportion 495 of the wall preferably has a radius of curvature R of about 9.5 mm when the diameter D of the dome-shaped portion is about 11.1 mm. Thewall 488 also includes a flatouter rim 497 for clamping thewall 488 in thecontainer 358. Alternatively, thewall 488 may be integrally molded with either ofpieces 450, 452 (FIG. 41). The thickness T of the wall is preferably in the range of 0.25 to 1 mm. If it is less than 0.25 mm thick, thewall 488 may be too weak. If the wall has a thickness greater than 1 mm, the wall may be too stiff to deflect properly in response to the vibratory movements of the transducer. In the presently preferred embodiment, thewall 488 has a thickness T of about 0.5 mm. Thewall 488 is preferably a molded plastic part. Suitable materials for thewall 488 include Delrin® (acetal resins or polymethylene oxide), polypropylene, or polycarbonate. - The interaction of the
transducer 314 with thesolid wall 488 will now be described with reference to FIG. 41. Prior to activating the transducer, target cells or viruses are captured on thefilter 490 by forcing a fluid sample to flow though the chamber 367 (e.g., using the fluidic system previously described with reference to FIG. 40). In addition, thechamber 367 is filled with a liquid (e.g., lysis buffer) as previously described. Unlike the previously described embodiments, however, thechamber 367 does not require pressurization. Instead, it is preferred that ambient pressure is maintained in the chamber. Thetransducer 314 is placed in contact with the external surface of thewall 488, preferably using a support structure as previously described with reference to FIG. 37. In particular, a spring preferably pushes the transducer against thewall 488 with a force in the range of 1 to 10 lbs., and more preferably in the range of 2 to 5 lbs. - To disrupt the cells or viruses in the
chamber 367, thetransducer 314 is activated (i.e., induced into vibratory motion). As the tip of thetransducer 314 vibrates, it deflects thewall 488. On its forward stroke (in the upward direction in FIG. 41), the tip of thetransducer 314 pushes thewall 488 and creates a pressure pulse or pressure wave in thechamber 367. On its retreating stroke (downward in FIG. 41), thewall 488 remains in contact with the tip of thetransducer 314 because thewall 488 has a natural frequency higher than the vibrating frequency of the transducer. In embodiments in which the transducer is an ultrasonic horn for sonicating thechamber 367, thechamber 367 is preferably sonicated for 10 to 40 seconds at a frequency in the range of 20 to 40 kHz. In the exemplary protocol, the chamber is sonicated for 15 seconds at a frequency of 40 kHz. The amplitude of the horn tip is preferably in the range of 20 to 25 μm (measured peak to peak), and the natural frequency of thewall 488 should be greater than 40 kHz, preferably at least 80 kHz, and more preferably at least 160 kHz. - One advantage to using the
solid interface wall 488 is that strong pressure drops can be achieved in thechamber 367 as long as the static pressure in the chamber is low. For example, at atmospheric pressure, cavitation (the making and breaking of microscopic bubbles) can occur in thechamber 367. As these bubbles or cavities grow to resonant size, they collapse violently, producing very high local pressure changes. The pressure changes provide a mechanical shock to the cells or viruses, resulting in their disruption. The disruption of the cells or viruses may also be caused by sharp pressure rises resulting from the vibratory movement of thetransducer 314. In addition, the disruption of the cells or viruses may be caused by the violent motion of thebeads 462 in thechamber 367. The beads are agitated by the dynamic pressure pulses in the chamber and rupture the cells or viruses. In experimental testing, the applicants have found that it is usually necessary to use beads to disrupt certain types of cells (particularly spores) having highly resistant cell walls. Other types of cells, such as blood cells, are easier to disrupt and may often be disrupted without the use of thebeads 462. - Although the use of an ultrasonic transducer has been described as a preferred embodiment, it is to be understood that different types of transducers may be employed in the practice of the present invention. The transducer should be capable of creating pressure pulses or pressure waves in the
chamber 367. In addition, the transducer should be capable of providing high velocity impacts to the liquid in the chamber. Suitable transducers include ultrasonic, piezoelectric, magnetostrictive, or electrostatic transducer. The transducer may also be an electromagnetic device having a wound coil, such as a voice coil motor or a solenoid device. The vibrating frequency of the transducer may be ultrasonic (i.e., above 20 kHz) or below ultrasonic (e.g., in the range of 60 to 20,000 Hz). The advantage to using higher frequencies is that cell disruption is very rapid and can often be completed in 10 to 20 seconds. The disadvantage is that ultrasonic transducers are often more expensive than a simple mechanical vibrator, e.g., a speaker or electromagnetic coil device. In one alternative embodiment, for example, thesolid wall 488 is used in combination with a speaker or electromagnetic coil device that vibrates at an operating frequency in the range of 5 to 10 kHz. - FIGS. 43A-43B illustrate another
solid wall 500 for contacting a transducer according to the present invention. As shown in FIG. 43A, one side of thewall 500 has acentral portion 502 and a plurality of stiffeningribs 504 extending radially from thecentral portion 502. The wall also hasrecesses 506 formed between theribs 504. As shown in FIG. 43B, the other side of thewall 500 has aflat surface 508. FIG. 44 shows a partially-cut away isometric view of thecontainer 358 with thewall 500. Thewall 500 is preferably positioned so that the side of the wall having the flat surface is internal to thechamber 367 and such that the side of the wall having theribs 504 is external to the chamber. Theribs 504 are advantageous because they increase the natural frequency of the wall without causing the wall to be so stiff that it cannot transfer the vibratory movements of the transducer to thechamber 367. - FIG. 45 shows a bottom plan view of the
container 358 having thewall 500. Thecentral portion 502 provides the external surface of thewall 500 for contacting a transducer. The interaction of thewall 500 with the transducer is analogous to the interaction of thewall 488 with the transducer previously described with reference to FIG. 41. In particular, thewall 500 remains in contact with the tip of the transducer because thewall 500 has a natural frequency higher than the vibrating frequency of the transducer. Consequently, pressurization is not required, and cavitation may be achieved. Thesolid walls container 358 or thewalls - FIG. 46 shows a partially exploded view of a
container 274 for holding cells or viruses to be disrupted according to another embodiment of the invention. FIG. 47 shows a front view of thecontainer 274. As shown in FIGS. 46-47, thecontainer 274 has achamber 277 for holding a liquid containing cells or viruses to be disrupted. Thecontainer 274 has arigid frame 278 that defines theside walls chamber 277. Therigid frame 278 also defines aport 276 and achannel 288 that connects theport 276 to thechamber 277. The container also includes thin, flexible sheets attached to opposite sides of therigid frame 278 to form two spaced-apart, opposingmajor walls major walls rigid frame 278 for illustrative clarity. When thecontainer 274 is assembled, themajor walls frame 278, as is described in detail below. Thechamber 277 is thus defined by the spaced apart, opposingmajor walls 280A, 202B and by the rigid sidewalls side walls - The
container 274 also includes aplunger 284 that is inserted into thechannel 288 after adding the cells or viruses to thechamber 277. Theplunger 284 compresses gas in thecontainer 274 thereby increasing pressure in thechamber 277. The gas compressed by theplunger 284 is typically air filling thechannel 288. The pressurization of thechamber 277 forces theflexible wall 280A to conform to the surface of the transducer (not shown in FIGS. 46-47), as is discussed in greater detail below. Theplunger 284 also closes theport 276 and seals thechamber 277 from the environment external to the container. - In general, the plunger may comprise any device capable of establishing a seal with the walls of the
channel 288 and of compressing gas in the container. Such devices include, but are not limited to, pistons, plugs, or stoppers. Theplunger 284 of the preferred embodiment includes astem 290 and apiston 292 on the stem. When theplunger 284 is inserted into thechannel 288, thepiston 292 establishes a seal with the inner walls of the channel and compresses air in the channel. Thepiston 292 is preferably a cup integrally formed (e.g., molded) with thestem 290. Alternatively, thepiston 292 may be a separate elastomeric piece attached to the stem. - The
plunger 284 also preferably includes analignment ring 294 encircling the stem for maintaining theplunger 284 in coaxial alignment with thechannel 288 as the plunger is inserted into the channel. Thealignment ring 294 is preferably integrally formed (e.g., molded) with thestem 290. Thestem 290 may optionally includessupport ribs 293 for stiffening and strengthening the stem. Theplunger 284 also includes aplunger cap 296 attached to thestem 290. As shown in FIG. 47, thecap 296 includes asnap ring 297 and the container includes anannular recess 279 encircling theport 276 for receiving thesnap ring 297. Thecap 296 may optionally include alever portion 298 which is lifted to remove theplunger 284 from thechannel 288. Thecontainer 274 may also include finger grips 287 for manual handling of the container. - FIG. 51 shows an isometric view of an
apparatus 304 for disrupting cells or viruses. Theapparatus 304 includes atransducer 314, preferably an ultrasonic horn, for generating pressure pulses the chamber of thecontainer 274. Theapparatus 304 also includes asupport structure 306 for holding thetransducer 314 and thecontainer 274 against each other. Thesupport structure 306 includes abase 308 and afirst holder 310 attached to the base for holding the outer housing of thetransducer 314. Theholder 310 includes a bore for receiving thetransducer 314 and screws orbolts 312 that are tightened to clamp the outer housing of the horn firmly in the holder. The base 308 may optionally include bolt holes 320 for bolting thesupport structure 306 to a surface, e.g., a counter or bench top. - As shown in FIG.52, the
support structure 306 also includes aholder 316 for holding thecontainer 274. Theholder 316 is slidably mounted to thebase 308 by means of aguide 318. Theguide 318 may be fixedly attached to the base 308 or integrally formed with the base. Theguide 318 has twoguide pins 322, and theholder 316 has twoguide slots 324 for receiving the guide pins 322. Theholder 316 may thus slide on the guide pins 322. As shown in the partially cut-away view of FIG. 53, theholder 316 is designed to hold thecontainer 274 such that the external surface of theflexible wall 280A is exposed and accessible to thetip 326 of thetransducer 314. Theguide 318 is appropriately aligned with thetransducer 314 to slide theholder 316 into a position in which the external surface of theflexible wall 280A contacts thetip 326. - FIG. 54 shows an isometric view of the
holder 316. Theholder 316 has abody 317 in which are formed theguide slots 324 for receiving the guide pins. The body also has arecess 334 for receiving thecontainer 274. The shape of therecess 334 matches the shape of the lower portion of theframe 278 so that the frame fits securely in therecess 334. Theholder 316 also includes a retainingmember 328 attached to thebody 317 by screws orbolts 330. The retainingmember 328 andbody 317 define aslot 332 through which theframe 278 is inserted when the frame is placed in therecess 334. The retainingmember 328 holds theframe 278 in the recess. Thebody 317 also has anopening 336 adjacent therecess 334. The shape of theopening 336 corresponds to the shape of thechamber 277. - As shown in the cross sectional view of FIG. 56, when the
container 274 is inserted into theholder 316, theopening 336 is positioned next to theflexible wall 280B. Theopening 336 is thus positioned to permit theflexible wall 280B to expand outwardly into the opening. Theholder 316 holds only the frame of thecontainer 274 so that theflexible walls flexible wall 280A is therefore free to move inwardly and outwardly with thehorn tip 326 as vibrational motion is transmitted from thetip 326 to thewall 280A. Theflexible wall 280B is also free to move inwardly or outwardly as pressure pulses sweep through thechamber 277. This permits the liquid within thechamber 277 to move more freely as it receives the dynamic pressure pulses and thus enhances the cell disruption in thechamber 277. Venting of theopening 336 is provided by first andsecond bores holder 316. One end of thenarrower bore 338 is connected to theopening 336 and the other end is connected to thelarger bore 344. Thebore 344 extends through the body of theholder 316 to permit the escape of gas (e.g., air) from theopening 336. The venting prevents pressure from building in theopening 336 when theflexible wall 280B expands into the opening. Such pressure would restrict the motion of thewall 280B. - Referring again to FIG. 54, the
container 274 has a bulb-shapedtab 275 extending from the bottom of theframe 278. Theholder 316 hasholes 340 formed in thebody 317 adjacent therecess 334. When theframe 278 is inserted into therecess 334, thetab 275 is positioned between theholes 340. Theholes 340 are for receiving retaining pins. As shown in FIG. 55, the retaining pins 342 extend from the guide 318 (from which the guide pins have been removed for clarity in FIG. 55) and are positioned on opposite sides of the bulb-shapedtab 275 when thecontainer 274 is moved into contact with thehorn tip 326. The spacing of thepins 342 is less than the width of the bulb so that thepins 342 hold down thetab 275, and thus thecontainer 274, as ultrasonic energy is transmitted into the container from thetransducer 314. This ensures that thecontainer 274 does not rise out of position due to the motion of thehorn tip 326. Alternatively, a collar or other suitable retention mechanism may be used to hold thecontainer 274 in position. - Referring to FIG. 56, the
support structure 306 also includes an elastic body, such as aspring 366, for applying a force to theholder 316 to press thewall 280A of thechamber 277 against thehorn tip 326. When thewall 280A is in contact with thehorn tip 326, the force provided by the spring is constant, providing for consistent coupling and transfer of power between thetransducer 314 and thecontainer 274. Thespring 366 is positioned in thebore 344. Theholder 316 has an inner surface surrounding the junction of thelarger bore 344 and thenarrower bore 338. One end of thespring 366 contacts the inner surface, and the other end of the spring contacts arod 348 that extends from theguide 318. Thespring 366 is thus compressed between the surface of theholder 316 and therod 348 so that it pushes theholder 316, and thus theflexible wall 280A of thecontainer 274, against thetip 326. - The magnitude of the force provided by the
spring 366 may be adjusted by changing the preload on the spring. Thesupport structure 306 includes arod 348 that contacts one end of the spring. Theguide 318 includes a first bore for receiving therod 348 and a second bore for receiving aset screw 349 that holds therod 348 in a fixed position. To adjust the preload on thespring 366, thescrew 349 is loosened, therod 348 is moved to a new position, and thescrew 349 is retightened to hold therod 348 in the new position. Therod 348 and setscrew 349 thus provide a simple mechanism for adjusting the preload on thespring 366. Once the preload on thespring 366 is adjusted to provide a suitable coupling force between thewall 280A and thehorn tip 326, it is desirable to keep the preload constant from one use of the apparatus to the next so that valid comparisons can be made between different samples disrupted by the apparatus. - The
flexible wall 280A facilitates the transfer of vibrating motion from thetransducer 314 to thechamber 277. Thewall 280A is sufficiently flexible to conform to the surface of thetip 326 of the transducer, ensuring good coupling between thetip 326 and thewall 280A. The surface of thetip 326 that contacts thewall 280A is preferably planar (e.g., flat) to ensure power coupling over the entire area of the surface. Alternatively, thetip 326 may have a slightly curved (e.g., spherical) surface for contacting thewall 280A Theopposite wall 280B is preferably sufficiently flexible to move inwardly and outwardly as dynamic pressure pulses are generated in thechamber 277. This permits the liquid within thechamber 277 greater freedom of movement as it receives the pressure pulses and thus enhances the action in thechamber 277. - Referring again to FIG. 46, the
walls walls plunger 284 is inserted into thechannel 288 after, adding the cells or viruses to thechamber 277. Theplunger 284 compresses air in thechannel 288, thereby increasing pressure in thechamber 277. The pressurization of thechamber 277 ensures effective coupling between thewall 280A and the tip of thetransducer 314. - It is presently preferred to pressurize the
chamber 277 to a pressure in the range of 2 to 50 psi above ambient pressure. This range is presently preferred because 2 psi is generally enough pressure to ensure effective coupling between theflexible wall 280A and thetransducer 314, while pressures above 50 psi may cause bursting of thewalls container 274. More preferably, thechamber 277 is pressurized to a pressure in the range of 8 to 15 psi above ambient pressure. This range is more preferred because it is safely within the practical limits described above. - A preferred method for disrupting cells or viruses using the
apparatus 304 will now be described with reference to FIGS. 46-56. Referring to FIG. 50,beads 301 are placed in thechamber 277 of the container to enhance the disruption of the cells or viruses. In general, thebeads 301 may be composed of glass, plastic, polystyrene, latex, crystals, metals, metal oxides, or non-glass silicates. Thebeads 301 may be porous or non-porous and preferably have a diameter in the range of 1 to 200 μm. More preferably, thebeads 301 are either polystyrene beads, borosilicate glass beads, or soda lime glass beads having an average diameter of about 100 μm. Thebeads 301 may be placed in thechamber 277 using a funnel. The funnel should be sufficiently long to extend from theport 276 through thechannel 288 and into thechamber 277. After inserting the funnel into thecontainer 274, thebeads 301 are placed in the funnel and thecontainer 274 is tapped lightly (e.g., against a bench top) until thebeads 301 settle into the bottom of thechamber 277. It is preferred that the funnel extend through thechannel 288 and into thechamber 277 as thebeads 301 are added to the chamber to prevent the beads from contaminating the channel. The presence of beads in thechannel 288 would interfere with the subsequent stroke of the plunger into the channel. The quantity ofbeads 301 added to thechamber 277 is preferably sufficient to fill about 10% to 40% of the volume capacity of the chamber. For example, in the presently preferred embodiment, thechamber 277 has a volume capacity of about 100 μl, and 30 to 40 mg of beads are placed into the chamber. - After the
beads 301 are placed in thechamber 277, the chamber is filled with a liquid containing the cells or viruses to be disrupted. Thechamber 277 may be filled using a pipette having a pipette tip 300 (e.g., a standard 200 μl loading tip). Alternatively, thechamber 277 may be filled using a syringe or any other suitable injection system. The liquid should be a medium through which pressure waves or pressure pulses can be transmitted. For example, the liquid may comprise deionized water or ultrasonic gel for holding the cells or viruses in suspension or solution. Alternatively, the liquid may comprise a biological sample containing the cells or viruses. Suitable samples include bodily fluids (e.g., blood, urine, saliva, sputum, seminal fluid, spinal fluid, mucus, etc) or environmental samples such as ground or waste water. The sample may be in raw form or mixed with diluents or buffers. The liquid or gel may also include one or more lysing agents to aid in the disruption of the cells or viruses. One of the advantages of the present invention, however, is that harsh lysing agents are not required for successful disruption of the cells or viruses. - After the
container 274 is filled with the liquid, theplunger 284 is inserted into thechannel 288 to seal and pressurize thecontainer 274. As theplunger 284 is inserted, thepiston 292 compresses gas in thechannel 288 to increase pressure in thechamber 277, preferably to about 8 to 15 psi above ambient pressure, as previously described. - Referring to FIG. 56, the
holder 316 is then pushed or pulled away from thetip 326 of the transducer 314 (in the direction of the rod 348) so that thecontainer 274 can be inserted into the holder. Thecontainer 274 is then placed in theholder 316. During the insertion of thecontainer 274, theholder 316 should be held a sufficient distance from the retainingpins 342 to provide clearance between thepins 342 and thetab 275. After thecontainer 274 is inserted into theholder 316, the holder is gently released and thespring 366 pushes theholder 316 along theguide 318 until thewall 280A contacts and conforms to the surface of thehorn tip 326. When thewall 280A is coupled to thehorn tip 326, thespring 366 applies to theholder 316, and thus to thecontainer 274, a substantially constant force to press thewall 280A against thehorn tip 326. The force provided by thespring 366 ensures effective coupling between thewall 280A andhorn tip 326 as energy is transmitted to thechamber 277. As shown in FIG. 55, when thecontainer 274 is moved into contact with thetip 326, thetab 275 slides between the retaining pins 342. Thepins 342 prevent the container from sliding upward in response to the vibratory motion of thetip 326. - Referring again to FIG. 56, the cells or viruses in the
chamber 277 are then disrupted by the pressure pulses and resulting bead movement in thechamber 277 generated by the vibration of thetip 326 against theflexible wall 280A. The magnitude of the force provided by thespring 366 to press together thewall 280A and thetip 326 is important for achieving a consistent transfer of energy between thetransducer 314 and thechamber 277. If the force is too light, thewall 280A will only be held lightly against thetip 326, leading to poor transmission of the vibratory movement of thetransducer 314. If the force is too strong, thecontainer 274 orwall 280A may be damaged during sonication. An intermediate force results in the most consistent and repeatable transfer of energy from thetransducer 314 to thechamber 277. It is presently preferred that thespring 366 provide a force in the range of 0.25 to 4 lbs., with a force of about 1 lb. being the most preferred. - When the
transducer 314 is activated, thetip 326 vibrates to transmit ultrasonic energy into thechamber 277. There is a relationship between the coupling force between thewall 280A and thetip 326 and the desired amplitude of the vibratory movements of thetip 326. A balance can be sought between the coupling force and the amplitude. Generally, a light coupling force requires a greater amplitude to effect disruption of the cells or viruses, while a stronger coupling force requires less amplitude to effect disruption. For the range of coupling forces presently preferred (0.25 to 4 lbs.), the peak-to-peak amplitude of the vibratory movements should be in the range of 4 to 40 μm, with a preferred peak-to-peak amplitude of amount 15 μm. - Ultrasonic waves are preferably transmitted to the
chamber 277 at a frequency in the range of 20 to 50 kHz, with a frequency of about 40 kHz being preferred. The duration of time for which thechamber 277 is sonicated is preferably in the range of 5 to 30 seconds. This range is preferred because it usually takes at least 5 seconds to disrupt the cells or viruses in the chamber, while sonicating the chamber for longer than 30 seconds will most likely denature or shear the nucleic acid released from the disrupted cells or viruses. Extensive shearing of the nucleic acid could interfere with subsequent amplification or detection. More preferably, the chamber is sonicated for about 10-20 seconds to fall safely within the practical limits stated above. The optimal time that a particular type of cell sample should be subjected to ultrasonic energy may be determined empirically. - Following disruption of the cells or viruses, the
container 274 is removed from theholder 316 by pulling theholder 316 away from thetip 326 and withdrawing the container from the holder. The liquid or gel containing the disrupted cells and released nucleic acid is then removed from thecontainer 274. This may be accomplished by centrifuging thecontainer 274 and removing the supernatant using, e.g., a pipette or syringe. Alternatively, the liquid may be removed from thecontainer 274 by setting the container on edge and at an incline until the beads precipitate. The beads usually settle in about 15 to 20 seconds. When the beads have settled, the plunger is withdrawn from thecontainer 274 and the liquid is removed using a syringe or pipette. The released nucleic acid contained in the liquid may then be amplified and detected using techniques well known in the art. - One advantage of the apparatus and method of the present invention is that it provides for the rapid and effective disruption of cells or viruses, including tough spores, without requiring the use of harsh chemicals. In addition, the apparatus and method provide for highly consistent and repeatable lysis of cells or viruses, so that consistent results are achieved from one use of the apparatus to the next. The amount of energy that is absorbed by the liquid and beads held in the
chamber 277 depends on the amplitude of the oscillations of thetip 326, the mass of the contents of thechamber 277, the pressure in thechamber 277, and the coupling force between thetip 326 and thewall 280A. All four of these parameters should be held substantially constant from one use of the apparatus to the next in order to achieve the same amount of disruption repeatably. - Many different modifications to the apparatus shown in FIG. 56 are possible. For example, the
holder 316 may be slidably mounted to thebase 308 by a variety of means, including rails, wheels, sliding in a groove, sliding in a cylinder, etc. Alternatively, theholder 316 may be fixedly attached to thebase 308 and thetransducer 314 slidably mounted to the base. In this embodiment, an elastic body is positioned to apply a force to the transducer 314 (either directly or to a holder holding the horn) to press together thehorn tip 326 and thewall 280A. In addition, in each of these embodiments, the elastic body may be positioned to either push or pull thetransducer 314 or thecontainer 274 towards each other. For example, thespring 366 may be positioned to push or pull theholder 316 towards thehorn tip 326 or to push or pull thetransducer 314 towards theholder 316. Further, multiple elastic bodies may be employed to apply forces to both thecontainer 274 and thetransducer 314 to push or pull them towards each other. All of these embodiments are intended to fall within the scope of the present invention. - Although a
coil spring 366 is shown in FIGS. 37 and 56, it is to be understood that any type of elastic body may be used. Suitable elastic bodies include, but are not limited to, coil springs, wave springs, torsion springs, spiral springs, leaf spring, elliptic springs, half-elliptic springs, rubber springs, and atmospheric springs. The elastic body may also be compressed air or rubber. Preferably, the elastic body is a coil spring. Coil springs are preferred because they are simple and inexpensive to place in the apparatus and because the have a low spring rate. A compressed air system is also effective, but considerably more expensive. In embodiments in which the elastic body is a spring, the spring should have a low spring rate, preferably less than 4 lb./in. A low spring rate minimizes the effect that any variations in the thickness of the chamber 277 (due to small variations in manufacturing, filling, or pressurizing the container) will have on the magnitude of the force provided by the spring to press together thewall 280A and thehorn tip 326. - Another advantage of the
container 274 is that thechamber 277 holds the cells or viruses in a thin volume of liquid that can be uniformly sonicated easily. Referring to FIGS. 48-49, it is presently preferred to construct thecontainer 274 such that each of thesides walls chamber 277 preferably has a width W greater than its thickness T. In particular, the ratio of the width W of the chamber to the thickness T of the chamber is preferably at least 2:1. More preferably, the ratio of the width W of the chamber to the thickness T of the chamber is at least 4:1. These ratios are preferred to enable the entire volume of thechamber 277 to be rapidly and uniformly sonicated. In general, the volume capacity of thechamber 277 is preferably in the range of 0.02 to 1 ml. - Referring again to FIG. 56, the
transducer 314 is preferably an ultrasonic horn. The thickness of the chamber 277 (and thus the spacing between thewalls tip 326 of the horn. This relationship between the thickness of thechamber 277 and the diameter of thetip 326 ensures that the ultrasonic energy received from thetransducer 314 is substantially uniform throughout the volume of thechamber 277. As a specific example, in the presently preferred embodiment, thetip 326 has a diameter of 6.35 mm and thechamber 277 has a thickness of about 1.0 mm. In addition, themajor wall 280A should be slightly larger than the surface of thehorn tip 326 that presses against thewall 280A. This allows theflexible wall 280A to flex in response to the vibratory motion of thehorn tip 326. - A preferred method for fabricating the
container 274 will now be described with reference to FIGS. 46-47. Thecontainer 274 may be fabricated by first molding therigid frame 278 using known injection molding techniques. Theframe 278 is preferably molded as a single piece of polymeric material, e.g., polypropylene or polycarbonate. After theframe 278 is produced, thin, flexible sheets are cut to size and sealed to opposite sides of theframe 278 to form themajor walls chamber 277. - The
major walls frame 278 using the following procedure. A first piece of film is placed over one side of the bottom portion of theframe 278. Theframe 278 preferably includes atack bar 299 for aligning the top edge of the film. The film is placed over the bottom portion of theframe 278 such that the top edge of the film is aligned with thetack bar 299 and such that the film completely covers the bottom portion of theframe 278 below thetack bar 299. The film should be larger than the bottom portion of theframe 278 so that it may be easily held and stretched flat across the frame. The film is then cut to size to match the outline of the frame by clamping to the frame the portion of the film that covers the frame and cutting away the portions of the film that extend past the perimeter of the frame using, e.g., a laser or die. The film is then tack welded to the frame, preferably using a laser. - The film is then sealed to the
frame 278, preferably by heat sealing. Heat sealing is presently preferred because it produces a strong seal without introducing potential contaminants to the container as the use of adhesive or solvent bonding techniques might do. Heat sealing is also simple and inexpensive. At a minimum, the film should be completely sealed to the surfaces of theside walls support ribs 295 andtack bar 299. The heat sealing may be performed using, e.g., a heated platen. An identical procedure may be used to cut and seal a second sheet to the opposite side of theframe 278 to complete thechamber 277. - The
plunger 284 is also preferably molded from polymeric material (e.g., polypropylene or polycarbonate) using known injection molding techniques. As shown in FIG. 46, theframe 278,plunger 284, andleash 286 connecting the plunger to the frame may all be formed in the same mold to form a one-piece part. This embodiment of the container is especially suitable for manual use in which a human operator fills the container and inserts theplunger 284 into thechannel 288. Theleash 286 ensures that theplunger 284 is not lost or dropped on the floor. - The
plunger 284 is presently preferred as a simple, effective, and inexpensive mechanism for increasing pressure in thechamber 277 and for sealing thechamber 277 from the external environment. It is to be understood, however, that the scope of the invention is not limited to this embodiment. There are many other suitable techniques for sealing and pressurizing the container. In addition, any suitable pressure source may be used to pressurize the chamber. Suitable pressure sources include syringe pumps, compressed air sources, pneumatic pumps, or connections to external sources of pressure. - Although the above description contains many specificities, these should not be construed as limitations on the scope of the invention, but merely as examples of some of the presently preferred embodiments. Many modifications or substitutions may be made to the apparatus and methods described without departing from the scope of the invention. For example, the container for holding the cells or viruses need not be one of the specialized containers described in the various embodiments above. Any type of container having a chamber for holding the cells or viruses may be used to practice the invention. Suitable containers include, but are not limited to, reaction vessels, cuvettes, cassettes, and cartridges. The container may have multiple chambers and/or channels for performing multiple sample preparation functions, or the container may have only a single chamber for holding cells or viruses for disruption.
- Further, the support structure for pressing the transducer and the wall of the container against each other may have many alternative forms. For example, in one alternative embodiment, the support structure includes a vise or clamp for pressing the transducer and container against each other. In another embodiment, the apparatus includes a pressure system for applying air pressure to press together the transducer and the container. Alternatively, magnetic or gravitational force may be used to press together the transducer and the container. In each embodiment of the invention, force may be applied to the transducer, to the container, or to both the transducer and the container.
- Therefore, the scope of the invention should be determined by the following claims and their legal equivalents.
Claims (4)
1-82. (canceled)
83. A micro-sonicator for spore or cell lysis comprising:
a container having a cavity therein adapted to contain spore or cell samples,
a quantity of ultrasonic transmission media in said cavity,
at least a membrane positioned to cover said cavity,
piezoelectric material positioned on said membrane, and
means for causing flexing of said piezoelectric material and vibration of said membrane thereby causing ultrasonic excitation of the transmission media adapted to cause lysis of spore or cell samples in said cavity.
84. In a micro-fluidic system, the improvement comprising:
a micro-sonicator for spore and cell lysis using ultrasonic excitation of the spore or cell, said micro-sonicator including a container having a cavity therein and containing ultrasonic transmission media, a membrane positioned over said cavity, and means for causing vibration of said membrane producing ultrasonic excitation of spore or cell located in said cavity.
85. A device for spore or cell lysis comprising:
a container having a chamber for holding spore or cell samples,
a quantity of ultrasonic transmission media in said chamber,
a membrane forming at least one wall of the chamber, and
an ultrasonic transducer in contact with said membrane for transmitting ultrasonic waves to the chamber.
Priority Applications (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US10/759,741 US20040200909A1 (en) | 1999-05-28 | 2004-01-15 | Apparatus and method for cell disruption |
US11/234,656 US8268603B2 (en) | 1999-05-28 | 2005-09-22 | Apparatus and method for cell disruption |
US11/234,634 US8815521B2 (en) | 2000-05-30 | 2005-09-22 | Apparatus and method for cell disruption |
US11/233,629 US9073053B2 (en) | 1999-05-28 | 2005-09-22 | Apparatus and method for cell disruption |
US14/737,853 US9943848B2 (en) | 1999-05-28 | 2015-06-12 | Apparatus and method for cell disruption |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US13670399P | 1999-05-28 | 1999-05-28 | |
US09/469,724 US6431476B1 (en) | 1999-12-21 | 1999-12-21 | Apparatus and method for rapid ultrasonic disruption of cells or viruses |
US58432700A | 2000-05-30 | 2000-05-30 | |
US10/759,741 US20040200909A1 (en) | 1999-05-28 | 2004-01-15 | Apparatus and method for cell disruption |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US58432700A Continuation | 1999-05-28 | 2000-05-30 |
Related Child Applications (3)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US11/233,629 Continuation US9073053B2 (en) | 1999-05-28 | 2005-09-22 | Apparatus and method for cell disruption |
US11/234,634 Continuation US8815521B2 (en) | 1999-05-28 | 2005-09-22 | Apparatus and method for cell disruption |
US11/234,656 Continuation US8268603B2 (en) | 1999-05-28 | 2005-09-22 | Apparatus and method for cell disruption |
Publications (1)
Publication Number | Publication Date |
---|---|
US20040200909A1 true US20040200909A1 (en) | 2004-10-14 |
Family
ID=33135862
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US10/759,741 Abandoned US20040200909A1 (en) | 1999-05-28 | 2004-01-15 | Apparatus and method for cell disruption |
US11/234,656 Expired - Fee Related US8268603B2 (en) | 1999-05-28 | 2005-09-22 | Apparatus and method for cell disruption |
Family Applications After (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US11/234,656 Expired - Fee Related US8268603B2 (en) | 1999-05-28 | 2005-09-22 | Apparatus and method for cell disruption |
Country Status (1)
Country | Link |
---|---|
US (2) | US20040200909A1 (en) |
Cited By (98)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20050106713A1 (en) * | 2003-09-03 | 2005-05-19 | Phan Brigitte C. | Personal diagnostic devices and related methods |
US20060019379A1 (en) * | 2000-05-30 | 2006-01-26 | Cepheid | Apparatus and method for cell disruption |
US20060027686A1 (en) * | 1999-05-28 | 2006-02-09 | Cepheid | Apparatus and method for cell disruption |
US20060108012A1 (en) * | 2002-11-14 | 2006-05-25 | Barrow David A | Microfluidic device and methods for construction and application |
US20060166347A1 (en) * | 2005-01-27 | 2006-07-27 | Applera Corporation | Sample preparation devices and methods |
KR100695160B1 (en) | 2004-10-19 | 2007-03-14 | 삼성전자주식회사 | Method and apparatus for the rapid disruption of cells or viruses using LASER and micro magnetic beads |
US20070166192A1 (en) * | 2006-01-17 | 2007-07-19 | Thomas Ehben | Module for processing a biological sample, biochip kit, and use of the module |
US20080014627A1 (en) * | 2005-12-02 | 2008-01-17 | Cabochon Aesthetics, Inc. | Devices and methods for selectively lysing cells |
US20080070245A1 (en) * | 2006-09-15 | 2008-03-20 | Gilles Reza George Monif | Method for herd management of domestic ruminants to control infection due to mycobacterium avium subspecies paratuberculosis |
US20080159915A1 (en) * | 2006-12-27 | 2008-07-03 | Steve Jia Chang Yu | Instrument for cassette for sample preparation |
WO2008082432A1 (en) * | 2007-01-04 | 2008-07-10 | The Regents Of The University Of California | Sorting, amplification, detection, and identification of nucleic acid subsequences in a complex mixture |
US20080193961A1 (en) * | 2004-09-29 | 2008-08-14 | Easley Christopher J | Localized Control of Thermal Properties on Microdevices and Applications Thereof |
US20080195036A1 (en) * | 2005-12-02 | 2008-08-14 | Cabochon Aesthetics, Inc. | Devices and methods for selectively lysing cells |
US20080197517A1 (en) * | 2005-12-02 | 2008-08-21 | Cabochon Aesthetics, Inc. | Devices and methods for selectively lysing cells |
US20080200863A1 (en) * | 2005-12-02 | 2008-08-21 | Cabochon Aesthetics, Inc. | Devices and methods for selectively lysing cells |
US20080200864A1 (en) * | 2005-12-02 | 2008-08-21 | Cabochon Aesthetics, Inc. | Devices and methods for selectively lysing cells |
US20080248554A1 (en) * | 2005-12-02 | 2008-10-09 | Cabochon Aesthetics, Inc. | Devices and methods for selectively lysing cells |
US20080277387A1 (en) * | 2004-12-22 | 2008-11-13 | Landers James P | Use of Microwaves For Thermal and Non-Thermal Applications in Micro and Nanoscale Devices |
US20090170092A1 (en) * | 2005-10-12 | 2009-07-02 | Landers James P | Integrated microfluidic analysis systems |
US20090217993A1 (en) * | 2005-08-23 | 2009-09-03 | Easley Christopher J | Passive components for micro-fluidic flow profile shaping and related method thereof |
US20090269248A1 (en) * | 2008-04-23 | 2009-10-29 | Bioscale, Inc. | Method and apparatus for analyte processing |
US20100197008A1 (en) * | 2003-07-31 | 2010-08-05 | Handylab, Inc. | Processing particle-containing samples |
US20100233792A1 (en) * | 2005-08-01 | 2010-09-16 | Matthew R Begley | Microdevices for Chemical Sensing and Chemical Actuation |
US20100239471A1 (en) * | 2005-10-19 | 2010-09-23 | Jesus Ching | Cassette for sample preparation |
US20110033922A1 (en) * | 2005-10-04 | 2011-02-10 | Landers James P | Microchip-based acoustic trapping or capture of cells for forensic analysis and related method thereof |
US20110101136A1 (en) * | 2008-04-25 | 2011-05-05 | Qiagen Gmbh | Method and device for breaking down biological material |
US7967763B2 (en) | 2005-09-07 | 2011-06-28 | Cabochon Aesthetics, Inc. | Method for treating subcutaneous tissues |
US20110160744A1 (en) * | 2005-05-31 | 2011-06-30 | Xiaojing Zhang | Optically-implemented microsurgery system and approach |
US8056881B2 (en) | 2004-10-13 | 2011-11-15 | University Of Virginia Patent Foundation | Electrostatic actuation for management of flow in micro-total analysis systems (μ-TAS) and related method thereof |
US8372340B2 (en) | 2005-10-19 | 2013-02-12 | Luminex Corporation | Apparatus and methods for integrated sample preparation, reaction and detection |
US8441629B2 (en) | 2009-04-15 | 2013-05-14 | Biocartis Sa | Optical detection system for monitoring rtPCR reaction |
US8439940B2 (en) | 2010-12-22 | 2013-05-14 | Cabochon Aesthetics, Inc. | Dissection handpiece with aspiration means for reducing the appearance of cellulite |
US8512637B2 (en) | 2009-05-06 | 2013-08-20 | Biocartis Sa | Device for cutting a sample carrier |
US8518069B2 (en) | 2005-09-07 | 2013-08-27 | Cabochon Aesthetics, Inc. | Dissection handpiece and method for reducing the appearance of cellulite |
WO2013153176A1 (en) * | 2012-04-11 | 2013-10-17 | Stat-Diagnostica & Innovation, S.L. | Fluidically integrated rotary bead beater |
USD692162S1 (en) | 2011-09-30 | 2013-10-22 | Becton, Dickinson And Company | Single piece reagent holder |
US8617905B2 (en) | 1995-09-15 | 2013-12-31 | The Regents Of The University Of Michigan | Thermal microvalves |
US8641971B2 (en) | 2009-04-14 | 2014-02-04 | Biocartis Sa | HIFU induced cavitation with reduced power threshold |
US8685341B2 (en) | 2001-09-12 | 2014-04-01 | Handylab, Inc. | Microfluidic devices having a reduced number of input and output connections |
US8703069B2 (en) | 2001-03-28 | 2014-04-22 | Handylab, Inc. | Moving microdroplets in a microfluidic device |
US8710211B2 (en) | 2007-07-13 | 2014-04-29 | Handylab, Inc. | Polynucleotide capture materials, and methods of using same |
US8709787B2 (en) | 2006-11-14 | 2014-04-29 | Handylab, Inc. | Microfluidic cartridge and method of using same |
US8734733B2 (en) | 2001-02-14 | 2014-05-27 | Handylab, Inc. | Heat-reduction methods and systems related to microfluidic devices |
US8768517B2 (en) | 2001-03-28 | 2014-07-01 | Handylab, Inc. | Methods and systems for control of microfluidic devices |
US8852862B2 (en) | 2004-05-03 | 2014-10-07 | Handylab, Inc. | Method for processing polynucleotide-containing samples |
US8883490B2 (en) | 2006-03-24 | 2014-11-11 | Handylab, Inc. | Fluorescence detector for microfluidic diagnostic system |
US8894947B2 (en) | 2001-03-28 | 2014-11-25 | Handylab, Inc. | Systems and methods for thermal actuation of microfluidic devices |
US8895311B1 (en) | 2001-03-28 | 2014-11-25 | Handylab, Inc. | Methods and systems for control of general purpose microfluidic devices |
US20140377146A1 (en) * | 2009-11-23 | 2014-12-25 | Cyvek, Inc. | Microfluidic Assay Operating System and Methods of Use |
US9011473B2 (en) | 2005-09-07 | 2015-04-21 | Ulthera, Inc. | Dissection handpiece and method for reducing the appearance of cellulite |
US9040288B2 (en) | 2006-03-24 | 2015-05-26 | Handylab, Inc. | Integrated system for processing microfluidic samples, and method of using the same |
US9080207B2 (en) | 2006-03-24 | 2015-07-14 | Handylab, Inc. | Microfluidic system for amplifying and detecting polynucleotides in parallel |
US9079182B2 (en) | 2009-04-15 | 2015-07-14 | Biocartis Nv | Protection of bioanalytical sample chambers |
US9186677B2 (en) | 2007-07-13 | 2015-11-17 | Handylab, Inc. | Integrated apparatus for performing nucleic acid extraction and diagnostic testing on multiple biological samples |
AU2014201790B2 (en) * | 2005-10-19 | 2015-12-17 | Luminex Corporation | Cassette for Sample Preparation |
US9216412B2 (en) | 2009-11-23 | 2015-12-22 | Cyvek, Inc. | Microfluidic devices and methods of manufacture and use |
US9222954B2 (en) | 2011-09-30 | 2015-12-29 | Becton, Dickinson And Company | Unitized reagent strip |
US9229001B2 (en) | 2009-11-23 | 2016-01-05 | Cyvek, Inc. | Method and apparatus for performing assays |
US9238223B2 (en) | 2007-07-13 | 2016-01-19 | Handylab, Inc. | Microfluidic cartridge |
US9248422B2 (en) | 2010-02-23 | 2016-02-02 | Luminex Corporation | Apparatus and methods for integrated sample preparation, reaction and detection |
US9259734B2 (en) | 2007-07-13 | 2016-02-16 | Handylab, Inc. | Integrated apparatus for performing nucleic acid extraction and diagnostic testing on multiple biological samples |
US9272124B2 (en) | 2005-12-02 | 2016-03-01 | Ulthera, Inc. | Systems and devices for selective cell lysis and methods of using same |
US9347586B2 (en) | 2007-07-13 | 2016-05-24 | Handylab, Inc. | Automated pipetting apparatus having a combined liquid pump and pipette head system |
US9358033B2 (en) | 2005-09-07 | 2016-06-07 | Ulthera, Inc. | Fluid-jet dissection system and method for reducing the appearance of cellulite |
US9358064B2 (en) | 2009-08-07 | 2016-06-07 | Ulthera, Inc. | Handpiece and methods for performing subcutaneous surgery |
US9486274B2 (en) | 2005-09-07 | 2016-11-08 | Ulthera, Inc. | Dissection handpiece and method for reducing the appearance of cellulite |
US9500645B2 (en) | 2009-11-23 | 2016-11-22 | Cyvek, Inc. | Micro-tube particles for microfluidic assays and methods of manufacture |
AU2015221472B2 (en) * | 2010-02-23 | 2017-01-19 | Luminex Corporation | Apparatus and methods for integrated sample preparation, reaction and detection |
US9618139B2 (en) | 2007-07-13 | 2017-04-11 | Handylab, Inc. | Integrated heater and magnetic separator |
USD787087S1 (en) | 2008-07-14 | 2017-05-16 | Handylab, Inc. | Housing |
US9651568B2 (en) | 2009-11-23 | 2017-05-16 | Cyvek, Inc. | Methods and systems for epi-fluorescent monitoring and scanning for microfluidic assays |
US9700889B2 (en) | 2009-11-23 | 2017-07-11 | Cyvek, Inc. | Methods and systems for manufacture of microarray assay systems, conducting microfluidic assays, and monitoring and scanning to obtain microfluidic assay results |
AU2016201550B2 (en) * | 2005-10-19 | 2017-07-27 | Luminex Corporation | Cassette for sample preparation |
WO2017125521A1 (en) * | 2016-01-21 | 2017-07-27 | Robert Bosch Gmbh | Apparatus and method for detecting cellular parts of a sample potentially containing cells |
US9759718B2 (en) | 2009-11-23 | 2017-09-12 | Cyvek, Inc. | PDMS membrane-confined nucleic acid and antibody/antigen-functionalized microlength tube capture elements, and systems employing them, and methods of their use |
US9765389B2 (en) | 2011-04-15 | 2017-09-19 | Becton, Dickinson And Company | Scanning real-time microfluidic thermocycler and methods for synchronized thermocycling and scanning optical detection |
US9789481B2 (en) | 1999-05-28 | 2017-10-17 | Cepheid | Device for extracting nucleic acid from a sample |
US9855735B2 (en) | 2009-11-23 | 2018-01-02 | Cyvek, Inc. | Portable microfluidic assay devices and methods of manufacture and use |
US9987576B2 (en) | 2012-12-10 | 2018-06-05 | University Of Virginia Patent Foundation | Frequency-based filtering of mechanical actuation using fluidic device |
US10065403B2 (en) | 2009-11-23 | 2018-09-04 | Cyvek, Inc. | Microfluidic assay assemblies and methods of manufacture |
US10179910B2 (en) | 2007-07-13 | 2019-01-15 | Handylab, Inc. | Rack for sample tubes and reagent holders |
US10228367B2 (en) | 2015-12-01 | 2019-03-12 | ProteinSimple | Segmented multi-use automated assay cartridge |
US10548659B2 (en) | 2006-01-17 | 2020-02-04 | Ulthera, Inc. | High pressure pre-burst for improved fluid delivery |
WO2020140003A1 (en) * | 2018-12-27 | 2020-07-02 | Hall Labs, Llc | Liquid sample capture apparatus |
CN111366434A (en) * | 2013-03-15 | 2020-07-03 | 赛拉诺斯知识产权有限责任公司 | Automated assay device for disrupting cells in a sample |
US20200240902A1 (en) * | 2019-01-24 | 2020-07-30 | Nirrin Technologies, Inc. | Flow cell device and bioreactor product monitoring system and method |
US10822644B2 (en) | 2012-02-03 | 2020-11-03 | Becton, Dickinson And Company | External files for distribution of molecular diagnostic tests and determination of compatibility between tests |
US10900066B2 (en) | 2006-03-24 | 2021-01-26 | Handylab, Inc. | Microfluidic system for amplifying and detecting polynucleotides in parallel |
CN113174329A (en) * | 2021-01-15 | 2021-07-27 | 北京中科生仪科技有限公司 | Continuous reaction device based on nucleic acid extraction, purification and amplification |
US11096708B2 (en) | 2009-08-07 | 2021-08-24 | Ulthera, Inc. | Devices and methods for performing subcutaneous surgery |
CN113528317A (en) * | 2020-04-15 | 2021-10-22 | 拓原合壹(宁波)生物技术有限公司 | Nucleic acid extraction method |
US20220229050A1 (en) * | 2019-05-30 | 2022-07-21 | Beckman Coulter, Inc. | Methods and systems for immobilizing a biological specimen for microscopic imaging |
US11453906B2 (en) | 2011-11-04 | 2022-09-27 | Handylab, Inc. | Multiplexed diagnostic detection apparatus and methods |
CN115181669A (en) * | 2022-09-09 | 2022-10-14 | 鲲鹏基因(北京)科技有限责任公司 | Breaking device for cell or virus lysis |
EP4159053A1 (en) * | 2020-04-06 | 2023-04-05 | Shaheen Innovations Holding Limited | Cell lysis sytems and methods |
US11667979B2 (en) | 2020-06-01 | 2023-06-06 | Shaheen Innovations Holding Limited | Infectious disease screening device |
US11806718B2 (en) | 2006-03-24 | 2023-11-07 | Handylab, Inc. | Fluorescence detector for microfluidic diagnostic system |
US11946844B2 (en) | 2020-06-01 | 2024-04-02 | Shaheen Innovations Holding Limited | Infectious disease screening system |
Families Citing this family (21)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP3450019A1 (en) | 2006-07-28 | 2019-03-06 | Diagnostics for the Real World, Ltd | Device and system for processing a sample |
US8153064B2 (en) * | 2007-03-22 | 2012-04-10 | Doebler Ii Robert W | Systems and devices for isothermal biochemical reactions and/or analysis |
EP2191897B1 (en) | 2007-06-21 | 2014-02-26 | Gen-Probe Incorporated | Instrument and receptacles for performing processes |
WO2009024773A1 (en) | 2007-08-17 | 2009-02-26 | Diagnostics For The Real World, Ltd | Device, system and method for processing a sample |
EP2232259B1 (en) * | 2008-01-09 | 2016-10-12 | Keck Graduate Institute | System, apparatus and method for material preparation and/or handling |
US20110130560A1 (en) * | 2009-05-29 | 2011-06-02 | Bio-Rad Laboratories, Inc. | Sonication cartridge for nucleic acid extraction |
EP2446047B1 (en) * | 2009-06-26 | 2017-10-18 | Claremont Biosolutions LLC | Capture and elution of bio-analytes via beads that are used to disrupt specimens |
US8798950B2 (en) | 2010-08-20 | 2014-08-05 | Bio-Rad Laboratories, Inc. | System and method for ultrasonic transducer control |
US8663974B2 (en) | 2010-12-23 | 2014-03-04 | Claremont Biosolutions Llc | Compositions and methods for capture and elution of biological materials via particulates |
AU2012222178B2 (en) | 2011-02-24 | 2014-12-18 | Gen-Probe Incorporated | Systems and methods for distinguishing optical signals of different modulation frequencies in an optical signal detector |
BR112013022889B8 (en) | 2011-03-08 | 2022-12-20 | Univ Laval | CENTRIPETAL FLUIDIC DEVICE FOR TESTING COMPONENTS OF A BIOLOGICAL MATERIAL IN A FLUID, TEST APPARATUS AND TESTING METHOD USING SUCH CENTRIPETAL FLUIDIC DEVICE |
US9604213B2 (en) | 2012-02-13 | 2017-03-28 | Neumodx Molecular, Inc. | System and method for processing and detecting nucleic acids |
US9637775B2 (en) | 2012-02-13 | 2017-05-02 | Neumodx Molecular, Inc. | System and method for processing biological samples |
CN104254595A (en) | 2012-02-13 | 2014-12-31 | 纽莫德克斯莫勒库拉尔公司 | Microfluidic cartridge for processing and detecting nucleic acids |
US11648561B2 (en) | 2012-02-13 | 2023-05-16 | Neumodx Molecular, Inc. | System and method for processing and detecting nucleic acids |
US11485968B2 (en) | 2012-02-13 | 2022-11-01 | Neumodx Molecular, Inc. | Microfluidic cartridge for processing and detecting nucleic acids |
EP2825309B1 (en) * | 2012-03-16 | 2018-05-16 | Stat-Diagnostica & Innovation, S.L. | A test cartridge with integrated transfer module |
US9063037B2 (en) | 2012-05-02 | 2015-06-23 | Stat-Diagnostica & Innovation, S.L. | Swab elution chamber in a test cartridge |
WO2014066376A1 (en) | 2012-10-25 | 2014-05-01 | Neumodx Molecular, Inc. | Method and materials for isolation of nucleic acid materials |
USD799715S1 (en) | 2015-10-23 | 2017-10-10 | Gene POC, Inc. | Fluidic centripetal device |
CN109468224B (en) * | 2018-12-18 | 2022-02-11 | 亳州学院 | Cell disruption instrument |
Citations (74)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US3799742A (en) * | 1971-12-20 | 1974-03-26 | C Coleman | Miniaturized integrated analytical test container |
US4426451A (en) * | 1981-01-28 | 1984-01-17 | Eastman Kodak Company | Multi-zoned reaction vessel having pressure-actuatable control means between zones |
US4642220A (en) * | 1981-04-10 | 1987-02-10 | Pharmacia Ab | Apparatus for carrying out analysis |
US4789628A (en) * | 1986-06-16 | 1988-12-06 | Vxr, Inc. | Devices for carrying out ligand/anti-ligand assays, methods of using such devices and diagnostic reagents and kits incorporating such devices |
US4855240A (en) * | 1987-05-13 | 1989-08-08 | Becton Dickinson And Company | Solid phase assay employing capillary flow |
US4874137A (en) * | 1988-08-01 | 1989-10-17 | Shigeru Chiba | Ultrasonic cell-destroyer |
US4891120A (en) * | 1986-06-06 | 1990-01-02 | Sethi Rajinder S | Chromatographic separation device |
US4895500A (en) * | 1988-04-08 | 1990-01-23 | Hoek Bertil | Micromechanical non-reverse valve |
US4908112A (en) * | 1988-06-16 | 1990-03-13 | E. I. Du Pont De Nemours & Co. | Silicon semiconductor wafer for analyzing micronic biological samples |
US4908318A (en) * | 1987-09-04 | 1990-03-13 | Integrated Genetics, Inc. | Nucleic acid extraction methods |
US4915812A (en) * | 1986-06-20 | 1990-04-10 | Molecular Devices Corporation | Zero volume cell |
US4918025A (en) * | 1987-03-03 | 1990-04-17 | Pb Diagnostic Systems, Inc. | Self contained immunoassay element |
US4921952A (en) * | 1988-01-21 | 1990-05-01 | The United States Of America As Represented By The United States Department Of Energy | Nucleic acid isolation process |
US4923978A (en) * | 1987-12-28 | 1990-05-08 | E. I. Du Pont De Nemours & Company | Process for purifying nucleic acids |
US4963498A (en) * | 1985-08-05 | 1990-10-16 | Biotrack | Capillary flow device |
US4983523A (en) * | 1988-04-08 | 1991-01-08 | Gene-Trak Systems | Methods for preparing sample nucleic acids for hybridization |
US5061446A (en) * | 1988-07-28 | 1991-10-29 | Jean Guigan | Device for performing biological analyses by immunoenzymatic detection of antibodies or antigens in a serum |
US5074474A (en) * | 1987-07-10 | 1991-12-24 | Veb Berlin-Kosmetik | Method and equipment for producing bioactive suspensions |
US5114858A (en) * | 1990-06-26 | 1992-05-19 | E. I. Du Pont De Nemours And Company | Cellular component extraction process in a disposable filtration vessel |
US5124444A (en) * | 1989-07-24 | 1992-06-23 | Microprobe Corporation | Lactam-containing compositions and methods useful for the extraction of nucleic acids |
US5155018A (en) * | 1991-07-10 | 1992-10-13 | Hahnemann University | Process and kit for isolating and purifying RNA from biological sources |
US5207988A (en) * | 1989-07-06 | 1993-05-04 | The Cleveland Clinic Foundation | Apparatus for detecting and quantifying clot lysis and establishing optimum medication and dosages |
US5223219A (en) * | 1992-04-10 | 1993-06-29 | Biotrack, Inc. | Analytical cartridge and system for detecting analytes in liquid samples |
US5229297A (en) * | 1989-02-03 | 1993-07-20 | Eastman Kodak Company | Containment cuvette for PCR and method of use |
US5234809A (en) * | 1989-03-23 | 1993-08-10 | Akzo N.V. | Process for isolating nucleic acid |
US5296375A (en) * | 1992-05-01 | 1994-03-22 | Trustees Of The University Of Pennsylvania | Mesoscale sperm handling devices |
US5304487A (en) * | 1992-05-01 | 1994-04-19 | Trustees Of The University Of Pennsylvania | Fluid handling in mesoscale analytical devices |
US5342931A (en) * | 1992-02-13 | 1994-08-30 | Becton, Dickinson And Company | Process for purifying DNA on hydrated silica |
US5351777A (en) * | 1991-05-17 | 1994-10-04 | Mazda Motor Corporation | Rear wheel turning system for vehicle and method of neutral adjustment of the same |
US5374522A (en) * | 1986-03-20 | 1994-12-20 | Gen-Probe Incorporated | Method for releasing RNA and DNA from cells |
US5374533A (en) * | 1988-05-10 | 1994-12-20 | Tetjin Limited | Method for determining chondrocalcin |
US5422241A (en) * | 1991-07-03 | 1995-06-06 | Ambion, Inc. | Methods for the recovery of nucleic acids from reaction mixtures |
US5427663A (en) * | 1993-06-08 | 1995-06-27 | British Technology Group Usa Inc. | Microlithographic array for macromolecule and cell fractionation |
US5438129A (en) * | 1993-09-27 | 1995-08-01 | Becton Dickinson And Company | DNA purification by solid phase extraction using partially fluorinated aluminum hydroxide adsorbant |
US5443590A (en) * | 1993-06-18 | 1995-08-22 | General Electric Company | Rotatable turbine frame |
US5458761A (en) * | 1992-08-26 | 1995-10-17 | Hitachi, Ltd. | Method for analyzing nucleic acid or protein and apparatus therefor |
US5500071A (en) * | 1994-10-19 | 1996-03-19 | Hewlett-Packard Company | Miniaturized planar columns in novel support media for liquid phase analysis |
US5534054A (en) * | 1991-10-31 | 1996-07-09 | Becton, Dickinson And Company | Silicon tetrahydrazide for purification of DNA |
US5543305A (en) * | 1989-10-18 | 1996-08-06 | Johnson & Johnson Clinical Diagnostics, Inc. | Methods of extracting deoxyribonucleic acids without using a proteolytic enzyme |
US5580435A (en) * | 1994-06-10 | 1996-12-03 | The Board Of Trustees Of The Leland Stanford Junior University | System for detecting components of a sample in electrophoretic separation |
US5580794A (en) * | 1993-08-24 | 1996-12-03 | Metrika Laboratories, Inc. | Disposable electronic assay device |
US5585069A (en) * | 1994-11-10 | 1996-12-17 | David Sarnoff Research Center, Inc. | Partitioned microelectronic and fluidic device array for clinical diagnostics and chemical synthesis |
US5587128A (en) * | 1992-05-01 | 1996-12-24 | The Trustees Of The University Of Pennsylvania | Mesoscale polynucleotide amplification devices |
US5605662A (en) * | 1993-11-01 | 1997-02-25 | Nanogen, Inc. | Active programmable electronic devices for molecular biological analysis and diagnostics |
US5616701A (en) * | 1993-09-27 | 1997-04-01 | Becton Dickinson And Company | DNA purification by solid phase extraction using a hydroxide-washed glass fiber membrane |
US5639428A (en) * | 1994-07-19 | 1997-06-17 | Becton Dickinson And Company | Method and apparatus for fully automated nucleic acid amplification, nucleic acid assay and immunoassay |
US5639423A (en) * | 1992-08-31 | 1997-06-17 | The Regents Of The University Of Calfornia | Microfabricated reactor |
US5641400A (en) * | 1994-10-19 | 1997-06-24 | Hewlett-Packard Company | Use of temperature control devices in miniaturized planar column devices and miniaturized total analysis systems |
US5652141A (en) * | 1990-10-26 | 1997-07-29 | Oiagen Gmbh | Device and process for isolating nucleic acids from cell suspension |
US5705018A (en) * | 1995-12-13 | 1998-01-06 | Hartley; Frank T. | Micromachined peristaltic pump |
US5707799A (en) * | 1994-09-30 | 1998-01-13 | Abbott Laboratories | Devices and methods utilizing arrays of structures for analyte capture |
US5716852A (en) * | 1996-03-29 | 1998-02-10 | University Of Washington | Microfabricated diffusion-based chemical sensor |
US5741639A (en) * | 1994-03-08 | 1998-04-21 | Ciba-Geigy Corp. | Device and method for combined bioaffinity assay and electrophoretic separation of multiple analytes |
US5842787A (en) * | 1997-10-09 | 1998-12-01 | Caliper Technologies Corporation | Microfluidic systems incorporating varied channel dimensions |
US5843767A (en) * | 1993-10-28 | 1998-12-01 | Houston Advanced Research Center | Microfabricated, flowthrough porous apparatus for discrete detection of binding reactions |
US5846727A (en) * | 1996-06-06 | 1998-12-08 | Board Of Supervisors Of Louisiana State University And Agricultural & Mechanical College | Microsystem for rapid DNA sequencing |
US5856174A (en) * | 1995-06-29 | 1999-01-05 | Affymetrix, Inc. | Integrated nucleic acid diagnostic device |
US5858188A (en) * | 1990-02-28 | 1999-01-12 | Aclara Biosciences, Inc. | Acrylic microchannels and their use in electrophoretic applications |
US5863801A (en) * | 1996-06-14 | 1999-01-26 | Sarnoff Corporation | Automated nucleic acid isolation |
US5863502A (en) * | 1996-01-24 | 1999-01-26 | Sarnoff Corporation | Parallel reaction cassette and associated devices |
US5869004A (en) * | 1997-06-09 | 1999-02-09 | Caliper Technologies Corp. | Methods and apparatus for in situ concentration and/or dilution of materials in microfluidic systems |
US5874046A (en) * | 1996-10-30 | 1999-02-23 | Raytheon Company | Biological warfare agent sensor system employing ruthenium-terminated oligonucleotides complementary to target live agent DNA sequences |
US5880071A (en) * | 1996-06-28 | 1999-03-09 | Caliper Technologies Corporation | Electropipettor and compensation means for electrophoretic bias |
US5921678A (en) * | 1997-02-05 | 1999-07-13 | California Institute Of Technology | Microfluidic sub-millisecond mixers |
US6042709A (en) * | 1996-06-28 | 2000-03-28 | Caliper Technologies Corp. | Microfluidic sampling system and methods |
US6068752A (en) * | 1997-04-25 | 2000-05-30 | Caliper Technologies Corp. | Microfluidic devices incorporating improved channel geometries |
US6090545A (en) * | 1995-03-10 | 2000-07-18 | Meso Scale Technologies, Llc. | Multi-array, multi-specific electrochemiluminescence testing |
US6100084A (en) * | 1998-11-05 | 2000-08-08 | The Regents Of The University Of California | Micro-sonicator for spore lysis |
US6156273A (en) * | 1997-05-27 | 2000-12-05 | Purdue Research Corporation | Separation columns and methods for manufacturing the improved separation columns |
US6168948B1 (en) * | 1995-06-29 | 2001-01-02 | Affymetrix, Inc. | Miniaturized genetic analysis systems and methods |
US20020019060A1 (en) * | 1999-05-28 | 2002-02-14 | Cepheid | Device for analyzing a fluid sample |
US20020039783A1 (en) * | 1998-12-24 | 2002-04-04 | Cepheid | Device and method for lysing cells, spores, or microorganisms |
US6431476B1 (en) * | 1999-12-21 | 2002-08-13 | Cepheid | Apparatus and method for rapid ultrasonic disruption of cells or viruses |
US6440725B1 (en) * | 1997-12-24 | 2002-08-27 | Cepheid | Integrated fluid manipulation cartridge |
Family Cites Families (21)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB938163A (en) | 1960-09-20 | 1963-10-02 | Boots Pure Drug Co Ltd | Improvements in or relating to particle size reduction or cellular disruption |
US3448890A (en) * | 1966-05-02 | 1969-06-10 | Lusteroid Container Co Inc | Plastic tube construction |
US4180383A (en) * | 1975-04-07 | 1979-12-25 | Becton, Dickinson And Company | Chamber holder for immobilized immunoadsorbent |
AU570439B2 (en) | 1983-03-28 | 1988-03-17 | Compression Labs, Inc. | A combined intraframe and interframe transform coding system |
US4615984A (en) * | 1984-02-23 | 1986-10-07 | Becton Dickinson & Company | Dissociation of ligand-binder complex using ultrasound |
US4981523A (en) * | 1986-05-09 | 1991-01-01 | Sherman Industries, Inc. | Vehicle cleaning system and method |
CH667599A5 (en) | 1986-12-11 | 1988-10-31 | Battelle Memorial Institute | ENCLOSURE FOR CONTAINING A LIQUID MEDIUM. |
AU642444B2 (en) | 1989-11-30 | 1993-10-21 | Mochida Pharmaceutical Co., Ltd. | Reaction vessel |
US5582829A (en) * | 1990-04-05 | 1996-12-10 | Rx Technologies, Inc. | Sonicated borrelia burgdorferi vaccine |
DE4126436A1 (en) | 1990-09-17 | 1992-03-19 | Abion Ohg | DISPOSABLE REACTION TUBE FOR SOLID-PHASE IMMUNAL ANALYTICS AND METHOD FOR MEASURING COMPONENTS DETERMINABLE BY IMMUNE REACTIONS |
US5352777A (en) | 1990-12-26 | 1994-10-04 | Pioneer Hi-Bred International, Inc. | DNA isolation from animal cells |
SE9100392D0 (en) | 1991-02-08 | 1991-02-08 | Pharmacia Biosensor Ab | A METHOD OF PRODUCING A SEALING MEANS IN A MICROFLUIDIC STRUCTURE AND A MICROFLUIDIC STRUCTURE COMPRISING SUCH SEALING MEANS |
US5264184A (en) | 1991-03-19 | 1993-11-23 | Minnesota Mining And Manufacturing Company | Device and a method for separating liquid samples |
US6953676B1 (en) * | 1992-05-01 | 2005-10-11 | Trustees Of The University Of Pennsylvania | Mesoscale polynucleotide amplification device and method |
IT1272932B (en) * | 1995-01-24 | 1997-07-01 | Pier Giorgio Righetti | IMMOBILIZED ENZYME REACTOR |
DE19601759A1 (en) * | 1995-01-26 | 1996-08-01 | Koenig Ag | Exhaust air cleaner for e.g. a tenter frame |
DE19519015C1 (en) | 1995-05-24 | 1996-09-05 | Inst Physikalische Hochtech Ev | Miniaturised multi-chamber thermo-cycler for polymerase chain reaction |
US6315957B1 (en) * | 1999-01-15 | 2001-11-13 | Pharmacopeia, Inc. | Article comprising a filter pocket-plate |
FR2791697B1 (en) * | 1999-04-01 | 2003-02-28 | Biomerieux Sa | APPARATUS AND METHOD FOR ULTRASOUND LYSIS OF BIOLOGICAL CELLS |
US6942169B2 (en) * | 2001-06-06 | 2005-09-13 | Integrated Sensing Systems | Micromachined lysing device and method for performing cell lysis |
US6739531B2 (en) * | 2001-10-04 | 2004-05-25 | Cepheid | Apparatus and method for rapid disruption of cells or viruses |
-
2004
- 2004-01-15 US US10/759,741 patent/US20040200909A1/en not_active Abandoned
-
2005
- 2005-09-22 US US11/234,656 patent/US8268603B2/en not_active Expired - Fee Related
Patent Citations (81)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US3799742A (en) * | 1971-12-20 | 1974-03-26 | C Coleman | Miniaturized integrated analytical test container |
US4426451A (en) * | 1981-01-28 | 1984-01-17 | Eastman Kodak Company | Multi-zoned reaction vessel having pressure-actuatable control means between zones |
US4642220A (en) * | 1981-04-10 | 1987-02-10 | Pharmacia Ab | Apparatus for carrying out analysis |
US4963498A (en) * | 1985-08-05 | 1990-10-16 | Biotrack | Capillary flow device |
US5374522A (en) * | 1986-03-20 | 1994-12-20 | Gen-Probe Incorporated | Method for releasing RNA and DNA from cells |
US4891120A (en) * | 1986-06-06 | 1990-01-02 | Sethi Rajinder S | Chromatographic separation device |
US4789628A (en) * | 1986-06-16 | 1988-12-06 | Vxr, Inc. | Devices for carrying out ligand/anti-ligand assays, methods of using such devices and diagnostic reagents and kits incorporating such devices |
US4915812A (en) * | 1986-06-20 | 1990-04-10 | Molecular Devices Corporation | Zero volume cell |
US4918025A (en) * | 1987-03-03 | 1990-04-17 | Pb Diagnostic Systems, Inc. | Self contained immunoassay element |
US4855240A (en) * | 1987-05-13 | 1989-08-08 | Becton Dickinson And Company | Solid phase assay employing capillary flow |
US5074474A (en) * | 1987-07-10 | 1991-12-24 | Veb Berlin-Kosmetik | Method and equipment for producing bioactive suspensions |
US4908318A (en) * | 1987-09-04 | 1990-03-13 | Integrated Genetics, Inc. | Nucleic acid extraction methods |
US4923978A (en) * | 1987-12-28 | 1990-05-08 | E. I. Du Pont De Nemours & Company | Process for purifying nucleic acids |
US4921952A (en) * | 1988-01-21 | 1990-05-01 | The United States Of America As Represented By The United States Department Of Energy | Nucleic acid isolation process |
US4895500A (en) * | 1988-04-08 | 1990-01-23 | Hoek Bertil | Micromechanical non-reverse valve |
US4983523A (en) * | 1988-04-08 | 1991-01-08 | Gene-Trak Systems | Methods for preparing sample nucleic acids for hybridization |
US5374533A (en) * | 1988-05-10 | 1994-12-20 | Tetjin Limited | Method for determining chondrocalcin |
US4908112A (en) * | 1988-06-16 | 1990-03-13 | E. I. Du Pont De Nemours & Co. | Silicon semiconductor wafer for analyzing micronic biological samples |
US5061446A (en) * | 1988-07-28 | 1991-10-29 | Jean Guigan | Device for performing biological analyses by immunoenzymatic detection of antibodies or antigens in a serum |
US4874137A (en) * | 1988-08-01 | 1989-10-17 | Shigeru Chiba | Ultrasonic cell-destroyer |
US5229297A (en) * | 1989-02-03 | 1993-07-20 | Eastman Kodak Company | Containment cuvette for PCR and method of use |
US5234809A (en) * | 1989-03-23 | 1993-08-10 | Akzo N.V. | Process for isolating nucleic acid |
US5207988A (en) * | 1989-07-06 | 1993-05-04 | The Cleveland Clinic Foundation | Apparatus for detecting and quantifying clot lysis and establishing optimum medication and dosages |
US5124444A (en) * | 1989-07-24 | 1992-06-23 | Microprobe Corporation | Lactam-containing compositions and methods useful for the extraction of nucleic acids |
US5543305A (en) * | 1989-10-18 | 1996-08-06 | Johnson & Johnson Clinical Diagnostics, Inc. | Methods of extracting deoxyribonucleic acids without using a proteolytic enzyme |
US5858188A (en) * | 1990-02-28 | 1999-01-12 | Aclara Biosciences, Inc. | Acrylic microchannels and their use in electrophoretic applications |
US5114858A (en) * | 1990-06-26 | 1992-05-19 | E. I. Du Pont De Nemours And Company | Cellular component extraction process in a disposable filtration vessel |
US5330916A (en) * | 1990-06-26 | 1994-07-19 | E. I. Du Pont De Nemours And Company | Cellular component extraction apparatus and disposable vessel useful therein |
US5652141A (en) * | 1990-10-26 | 1997-07-29 | Oiagen Gmbh | Device and process for isolating nucleic acids from cell suspension |
US5351777A (en) * | 1991-05-17 | 1994-10-04 | Mazda Motor Corporation | Rear wheel turning system for vehicle and method of neutral adjustment of the same |
US5422241A (en) * | 1991-07-03 | 1995-06-06 | Ambion, Inc. | Methods for the recovery of nucleic acids from reaction mixtures |
US5155018A (en) * | 1991-07-10 | 1992-10-13 | Hahnemann University | Process and kit for isolating and purifying RNA from biological sources |
US5534054A (en) * | 1991-10-31 | 1996-07-09 | Becton, Dickinson And Company | Silicon tetrahydrazide for purification of DNA |
US5342931A (en) * | 1992-02-13 | 1994-08-30 | Becton, Dickinson And Company | Process for purifying DNA on hydrated silica |
US5223219A (en) * | 1992-04-10 | 1993-06-29 | Biotrack, Inc. | Analytical cartridge and system for detecting analytes in liquid samples |
US5296375A (en) * | 1992-05-01 | 1994-03-22 | Trustees Of The University Of Pennsylvania | Mesoscale sperm handling devices |
US5304487A (en) * | 1992-05-01 | 1994-04-19 | Trustees Of The University Of Pennsylvania | Fluid handling in mesoscale analytical devices |
US5587128A (en) * | 1992-05-01 | 1996-12-24 | The Trustees Of The University Of Pennsylvania | Mesoscale polynucleotide amplification devices |
US5458761A (en) * | 1992-08-26 | 1995-10-17 | Hitachi, Ltd. | Method for analyzing nucleic acid or protein and apparatus therefor |
US5639423A (en) * | 1992-08-31 | 1997-06-17 | The Regents Of The University Of Calfornia | Microfabricated reactor |
US5427663A (en) * | 1993-06-08 | 1995-06-27 | British Technology Group Usa Inc. | Microlithographic array for macromolecule and cell fractionation |
US5443590A (en) * | 1993-06-18 | 1995-08-22 | General Electric Company | Rotatable turbine frame |
US5580794A (en) * | 1993-08-24 | 1996-12-03 | Metrika Laboratories, Inc. | Disposable electronic assay device |
US5616701A (en) * | 1993-09-27 | 1997-04-01 | Becton Dickinson And Company | DNA purification by solid phase extraction using a hydroxide-washed glass fiber membrane |
US5438129A (en) * | 1993-09-27 | 1995-08-01 | Becton Dickinson And Company | DNA purification by solid phase extraction using partially fluorinated aluminum hydroxide adsorbant |
US5843767A (en) * | 1993-10-28 | 1998-12-01 | Houston Advanced Research Center | Microfabricated, flowthrough porous apparatus for discrete detection of binding reactions |
US5605662A (en) * | 1993-11-01 | 1997-02-25 | Nanogen, Inc. | Active programmable electronic devices for molecular biological analysis and diagnostics |
US5741639A (en) * | 1994-03-08 | 1998-04-21 | Ciba-Geigy Corp. | Device and method for combined bioaffinity assay and electrophoretic separation of multiple analytes |
US5580435A (en) * | 1994-06-10 | 1996-12-03 | The Board Of Trustees Of The Leland Stanford Junior University | System for detecting components of a sample in electrophoretic separation |
US5639428A (en) * | 1994-07-19 | 1997-06-17 | Becton Dickinson And Company | Method and apparatus for fully automated nucleic acid amplification, nucleic acid assay and immunoassay |
US5707799A (en) * | 1994-09-30 | 1998-01-13 | Abbott Laboratories | Devices and methods utilizing arrays of structures for analyte capture |
US5500071A (en) * | 1994-10-19 | 1996-03-19 | Hewlett-Packard Company | Miniaturized planar columns in novel support media for liquid phase analysis |
US5641400A (en) * | 1994-10-19 | 1997-06-24 | Hewlett-Packard Company | Use of temperature control devices in miniaturized planar column devices and miniaturized total analysis systems |
US5585069A (en) * | 1994-11-10 | 1996-12-17 | David Sarnoff Research Center, Inc. | Partitioned microelectronic and fluidic device array for clinical diagnostics and chemical synthesis |
US6090545A (en) * | 1995-03-10 | 2000-07-18 | Meso Scale Technologies, Llc. | Multi-array, multi-specific electrochemiluminescence testing |
US6168948B1 (en) * | 1995-06-29 | 2001-01-02 | Affymetrix, Inc. | Miniaturized genetic analysis systems and methods |
US5856174A (en) * | 1995-06-29 | 1999-01-05 | Affymetrix, Inc. | Integrated nucleic acid diagnostic device |
US5705018A (en) * | 1995-12-13 | 1998-01-06 | Hartley; Frank T. | Micromachined peristaltic pump |
US5863502A (en) * | 1996-01-24 | 1999-01-26 | Sarnoff Corporation | Parallel reaction cassette and associated devices |
US5716852A (en) * | 1996-03-29 | 1998-02-10 | University Of Washington | Microfabricated diffusion-based chemical sensor |
US5972710A (en) * | 1996-03-29 | 1999-10-26 | University Of Washington | Microfabricated diffusion-based chemical sensor |
US5846727A (en) * | 1996-06-06 | 1998-12-08 | Board Of Supervisors Of Louisiana State University And Agricultural & Mechanical College | Microsystem for rapid DNA sequencing |
US5863801A (en) * | 1996-06-14 | 1999-01-26 | Sarnoff Corporation | Automated nucleic acid isolation |
US6080295A (en) * | 1996-06-28 | 2000-06-27 | Caliper Technologies Corporation | Electropipettor and compensation means for electrophoretic bias |
US5880071A (en) * | 1996-06-28 | 1999-03-09 | Caliper Technologies Corporation | Electropipettor and compensation means for electrophoretic bias |
US6042709A (en) * | 1996-06-28 | 2000-03-28 | Caliper Technologies Corp. | Microfluidic sampling system and methods |
US5874046A (en) * | 1996-10-30 | 1999-02-23 | Raytheon Company | Biological warfare agent sensor system employing ruthenium-terminated oligonucleotides complementary to target live agent DNA sequences |
US5921678A (en) * | 1997-02-05 | 1999-07-13 | California Institute Of Technology | Microfluidic sub-millisecond mixers |
US6068752A (en) * | 1997-04-25 | 2000-05-30 | Caliper Technologies Corp. | Microfluidic devices incorporating improved channel geometries |
US6153073A (en) * | 1997-04-25 | 2000-11-28 | Caliper Technologies Corp. | Microfluidic devices incorporating improved channel geometries |
US6156273A (en) * | 1997-05-27 | 2000-12-05 | Purdue Research Corporation | Separation columns and methods for manufacturing the improved separation columns |
US5869004A (en) * | 1997-06-09 | 1999-02-09 | Caliper Technologies Corp. | Methods and apparatus for in situ concentration and/or dilution of materials in microfluidic systems |
US5842787A (en) * | 1997-10-09 | 1998-12-01 | Caliper Technologies Corporation | Microfluidic systems incorporating varied channel dimensions |
US6440725B1 (en) * | 1997-12-24 | 2002-08-27 | Cepheid | Integrated fluid manipulation cartridge |
US6100084A (en) * | 1998-11-05 | 2000-08-08 | The Regents Of The University Of California | Micro-sonicator for spore lysis |
US20020039783A1 (en) * | 1998-12-24 | 2002-04-04 | Cepheid | Device and method for lysing cells, spores, or microorganisms |
US20020019060A1 (en) * | 1999-05-28 | 2002-02-14 | Cepheid | Device for analyzing a fluid sample |
US6391541B1 (en) * | 1999-05-28 | 2002-05-21 | Kurt E. Petersen | Apparatus for analyzing a fluid sample |
US20020045246A1 (en) * | 1999-06-25 | 2002-04-18 | Cepheid | Device for lysing cells, spores, or microorganisms |
US20020055167A1 (en) * | 1999-06-25 | 2002-05-09 | Cepheid | Device incorporating a microfluidic chip for separating analyte from a sample |
US6431476B1 (en) * | 1999-12-21 | 2002-08-13 | Cepheid | Apparatus and method for rapid ultrasonic disruption of cells or viruses |
Cited By (247)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US8617905B2 (en) | 1995-09-15 | 2013-12-31 | The Regents Of The University Of Michigan | Thermal microvalves |
US20060027686A1 (en) * | 1999-05-28 | 2006-02-09 | Cepheid | Apparatus and method for cell disruption |
US9943848B2 (en) | 1999-05-28 | 2018-04-17 | Cepheid | Apparatus and method for cell disruption |
US9789481B2 (en) | 1999-05-28 | 2017-10-17 | Cepheid | Device for extracting nucleic acid from a sample |
US9073053B2 (en) | 1999-05-28 | 2015-07-07 | Cepheid | Apparatus and method for cell disruption |
US20060019379A1 (en) * | 2000-05-30 | 2006-01-26 | Cepheid | Apparatus and method for cell disruption |
US8815521B2 (en) | 2000-05-30 | 2014-08-26 | Cepheid | Apparatus and method for cell disruption |
US9528142B2 (en) | 2001-02-14 | 2016-12-27 | Handylab, Inc. | Heat-reduction methods and systems related to microfluidic devices |
US8734733B2 (en) | 2001-02-14 | 2014-05-27 | Handylab, Inc. | Heat-reduction methods and systems related to microfluidic devices |
US9051604B2 (en) | 2001-02-14 | 2015-06-09 | Handylab, Inc. | Heat-reduction methods and systems related to microfluidic devices |
US8894947B2 (en) | 2001-03-28 | 2014-11-25 | Handylab, Inc. | Systems and methods for thermal actuation of microfluidic devices |
US10619191B2 (en) | 2001-03-28 | 2020-04-14 | Handylab, Inc. | Systems and methods for thermal actuation of microfluidic devices |
US10351901B2 (en) | 2001-03-28 | 2019-07-16 | Handylab, Inc. | Systems and methods for thermal actuation of microfluidic devices |
US8895311B1 (en) | 2001-03-28 | 2014-11-25 | Handylab, Inc. | Methods and systems for control of general purpose microfluidic devices |
US8768517B2 (en) | 2001-03-28 | 2014-07-01 | Handylab, Inc. | Methods and systems for control of microfluidic devices |
US9259735B2 (en) | 2001-03-28 | 2016-02-16 | Handylab, Inc. | Methods and systems for control of microfluidic devices |
US10571935B2 (en) | 2001-03-28 | 2020-02-25 | Handylab, Inc. | Methods and systems for control of general purpose microfluidic devices |
US9677121B2 (en) | 2001-03-28 | 2017-06-13 | Handylab, Inc. | Systems and methods for thermal actuation of microfluidic devices |
US8703069B2 (en) | 2001-03-28 | 2014-04-22 | Handylab, Inc. | Moving microdroplets in a microfluidic device |
US9028773B2 (en) | 2001-09-12 | 2015-05-12 | Handylab, Inc. | Microfluidic devices having a reduced number of input and output connections |
US8685341B2 (en) | 2001-09-12 | 2014-04-01 | Handylab, Inc. | Microfluidic devices having a reduced number of input and output connections |
US7802591B2 (en) | 2002-11-14 | 2010-09-28 | Q Chip Limited | Microfluidic device and methods for construction and application |
US20060108012A1 (en) * | 2002-11-14 | 2006-05-25 | Barrow David A | Microfluidic device and methods for construction and application |
US10731201B2 (en) | 2003-07-31 | 2020-08-04 | Handylab, Inc. | Processing particle-containing samples |
US20100197008A1 (en) * | 2003-07-31 | 2010-08-05 | Handylab, Inc. | Processing particle-containing samples |
US9670528B2 (en) | 2003-07-31 | 2017-06-06 | Handylab, Inc. | Processing particle-containing samples |
US8679831B2 (en) | 2003-07-31 | 2014-03-25 | Handylab, Inc. | Processing particle-containing samples |
US10865437B2 (en) | 2003-07-31 | 2020-12-15 | Handylab, Inc. | Processing particle-containing samples |
US11078523B2 (en) | 2003-07-31 | 2021-08-03 | Handylab, Inc. | Processing particle-containing samples |
US9133024B2 (en) * | 2003-09-03 | 2015-09-15 | Brigitte Chau Phan | Personal diagnostic devices including related methods and systems |
US20050106713A1 (en) * | 2003-09-03 | 2005-05-19 | Phan Brigitte C. | Personal diagnostic devices and related methods |
US11737694B2 (en) | 2003-09-03 | 2023-08-29 | Life Patch International, Inc. | Personal diagnostic device having a plurality of tubules |
US9993189B2 (en) | 2003-09-03 | 2018-06-12 | Life Patch International | Personal diagnostic device having a fluidic circuit with a plurality of analysis chambers |
US10494663B1 (en) | 2004-05-03 | 2019-12-03 | Handylab, Inc. | Method for processing polynucleotide-containing samples |
US10364456B2 (en) | 2004-05-03 | 2019-07-30 | Handylab, Inc. | Method for processing polynucleotide-containing samples |
US11441171B2 (en) | 2004-05-03 | 2022-09-13 | Handylab, Inc. | Method for processing polynucleotide-containing samples |
US10604788B2 (en) | 2004-05-03 | 2020-03-31 | Handylab, Inc. | System for processing polynucleotide-containing samples |
US10443088B1 (en) | 2004-05-03 | 2019-10-15 | Handylab, Inc. | Method for processing polynucleotide-containing samples |
US8852862B2 (en) | 2004-05-03 | 2014-10-07 | Handylab, Inc. | Method for processing polynucleotide-containing samples |
US20080193961A1 (en) * | 2004-09-29 | 2008-08-14 | Easley Christopher J | Localized Control of Thermal Properties on Microdevices and Applications Thereof |
US8056881B2 (en) | 2004-10-13 | 2011-11-15 | University Of Virginia Patent Foundation | Electrostatic actuation for management of flow in micro-total analysis systems (μ-TAS) and related method thereof |
US8403294B2 (en) | 2004-10-13 | 2013-03-26 | University Of Virginia Patent Foundation | Electrostatic actuation for management of flow in micro-total analysis systems (μ-TAS) and related method thereof |
KR100695160B1 (en) | 2004-10-19 | 2007-03-14 | 삼성전자주식회사 | Method and apparatus for the rapid disruption of cells or viruses using LASER and micro magnetic beads |
US20080277387A1 (en) * | 2004-12-22 | 2008-11-13 | Landers James P | Use of Microwaves For Thermal and Non-Thermal Applications in Micro and Nanoscale Devices |
US20060166347A1 (en) * | 2005-01-27 | 2006-07-27 | Applera Corporation | Sample preparation devices and methods |
US20110160744A1 (en) * | 2005-05-31 | 2011-06-30 | Xiaojing Zhang | Optically-implemented microsurgery system and approach |
US8505544B2 (en) | 2005-05-31 | 2013-08-13 | The Board Of Trustees Of The Leland Stanford Junior University | Optically-implemented microsurgery system and approach |
US8343755B2 (en) | 2005-08-01 | 2013-01-01 | University Of Virginia Patent Foundation | Microdevices for chemical sensing and chemical actuation |
US20100233792A1 (en) * | 2005-08-01 | 2010-09-16 | Matthew R Begley | Microdevices for Chemical Sensing and Chemical Actuation |
US8220493B2 (en) | 2005-08-23 | 2012-07-17 | University Of Virginia Patent Foundation | Passive components for micro-fluidic flow profile shaping and related method thereof |
US20090217993A1 (en) * | 2005-08-23 | 2009-09-03 | Easley Christopher J | Passive components for micro-fluidic flow profile shaping and related method thereof |
US9050596B2 (en) | 2005-08-23 | 2015-06-09 | University Of Virginia Patent Foundation | Passive components for micro-fluidic flow profile shaping and related method thereof |
US9364246B2 (en) | 2005-09-07 | 2016-06-14 | Ulthera, Inc. | Dissection handpiece and method for reducing the appearance of cellulite |
US8366643B2 (en) | 2005-09-07 | 2013-02-05 | Cabochon Aesthetics, Inc. | System and method for treating subcutaneous tissues |
US8518069B2 (en) | 2005-09-07 | 2013-08-27 | Cabochon Aesthetics, Inc. | Dissection handpiece and method for reducing the appearance of cellulite |
US9011473B2 (en) | 2005-09-07 | 2015-04-21 | Ulthera, Inc. | Dissection handpiece and method for reducing the appearance of cellulite |
US9358033B2 (en) | 2005-09-07 | 2016-06-07 | Ulthera, Inc. | Fluid-jet dissection system and method for reducing the appearance of cellulite |
US9005229B2 (en) | 2005-09-07 | 2015-04-14 | Ulthera, Inc. | Dissection handpiece and method for reducing the appearance of cellulite |
US9486274B2 (en) | 2005-09-07 | 2016-11-08 | Ulthera, Inc. | Dissection handpiece and method for reducing the appearance of cellulite |
US9179928B2 (en) | 2005-09-07 | 2015-11-10 | Ulthera, Inc. | Dissection handpiece and method for reducing the appearance of cellulite |
US7967763B2 (en) | 2005-09-07 | 2011-06-28 | Cabochon Aesthetics, Inc. | Method for treating subcutaneous tissues |
US20110033922A1 (en) * | 2005-10-04 | 2011-02-10 | Landers James P | Microchip-based acoustic trapping or capture of cells for forensic analysis and related method thereof |
US8916375B2 (en) | 2005-10-12 | 2014-12-23 | University Of Virginia Patent Foundation | Integrated microfluidic analysis systems |
US20090170092A1 (en) * | 2005-10-12 | 2009-07-02 | Landers James P | Integrated microfluidic analysis systems |
US20100239471A1 (en) * | 2005-10-19 | 2010-09-23 | Jesus Ching | Cassette for sample preparation |
US8124024B2 (en) * | 2005-10-19 | 2012-02-28 | Genturadx, Inc. | Cassette for sample preparation |
US9074250B2 (en) | 2005-10-19 | 2015-07-07 | Luminex Corporation | Apparatus and methods for integrated sample preparation, reaction and detection |
US10646875B2 (en) * | 2005-10-19 | 2020-05-12 | Luminex Corporation | Apparatus and methods for integrated sample preparation, reaction and detection |
US9017617B2 (en) | 2005-10-19 | 2015-04-28 | Luminex Corporation | Cassette for sample preparation |
US9828598B2 (en) | 2005-10-19 | 2017-11-28 | Luminex Corporation | Cassette for sample preparation |
US9624531B2 (en) | 2005-10-19 | 2017-04-18 | Luminex Corporation | Cassette for sample preparation |
AU2014201790B2 (en) * | 2005-10-19 | 2015-12-17 | Luminex Corporation | Cassette for Sample Preparation |
US9539577B2 (en) | 2005-10-19 | 2017-01-10 | Luminex Corporation | Apparatus and methods for integrated sample preparation, reaction and detection |
US10472622B2 (en) * | 2005-10-19 | 2019-11-12 | Luminex Corporation | Cassette for sample preparation |
US8372340B2 (en) | 2005-10-19 | 2013-02-12 | Luminex Corporation | Apparatus and methods for integrated sample preparation, reaction and detection |
US10040071B2 (en) | 2005-10-19 | 2018-08-07 | Luminex Corporation | Apparatus and methods for integrated sample preparation, reaction and detection |
US20180318838A1 (en) * | 2005-10-19 | 2018-11-08 | Luminex Corporation | Apparatus and methods for integrated sample preparation, reaction and detection |
AU2016201550B2 (en) * | 2005-10-19 | 2017-07-27 | Luminex Corporation | Cassette for sample preparation |
US20080197517A1 (en) * | 2005-12-02 | 2008-08-21 | Cabochon Aesthetics, Inc. | Devices and methods for selectively lysing cells |
US20080195036A1 (en) * | 2005-12-02 | 2008-08-14 | Cabochon Aesthetics, Inc. | Devices and methods for selectively lysing cells |
US20080014627A1 (en) * | 2005-12-02 | 2008-01-17 | Cabochon Aesthetics, Inc. | Devices and methods for selectively lysing cells |
US9272124B2 (en) | 2005-12-02 | 2016-03-01 | Ulthera, Inc. | Systems and devices for selective cell lysis and methods of using same |
US20080200863A1 (en) * | 2005-12-02 | 2008-08-21 | Cabochon Aesthetics, Inc. | Devices and methods for selectively lysing cells |
US20080200864A1 (en) * | 2005-12-02 | 2008-08-21 | Cabochon Aesthetics, Inc. | Devices and methods for selectively lysing cells |
US20080248554A1 (en) * | 2005-12-02 | 2008-10-09 | Cabochon Aesthetics, Inc. | Devices and methods for selectively lysing cells |
US9248317B2 (en) | 2005-12-02 | 2016-02-02 | Ulthera, Inc. | Devices and methods for selectively lysing cells |
US10548659B2 (en) | 2006-01-17 | 2020-02-04 | Ulthera, Inc. | High pressure pre-burst for improved fluid delivery |
GB2434205B (en) * | 2006-01-17 | 2011-04-20 | Siemens Ag | Module for preparing a biological sample, biochip-assembly and use of the module |
US20070166192A1 (en) * | 2006-01-17 | 2007-07-19 | Thomas Ehben | Module for processing a biological sample, biochip kit, and use of the module |
US8771609B2 (en) * | 2006-01-17 | 2014-07-08 | Siemens Aktiengesellschaft | Module for processing a biological sample, biochip kit, and use of the module |
US10857535B2 (en) | 2006-03-24 | 2020-12-08 | Handylab, Inc. | Integrated system for processing microfluidic samples, and method of using same |
US10900066B2 (en) | 2006-03-24 | 2021-01-26 | Handylab, Inc. | Microfluidic system for amplifying and detecting polynucleotides in parallel |
US11666903B2 (en) | 2006-03-24 | 2023-06-06 | Handylab, Inc. | Integrated system for processing microfluidic samples, and method of using same |
US9802199B2 (en) | 2006-03-24 | 2017-10-31 | Handylab, Inc. | Fluorescence detector for microfluidic diagnostic system |
US9040288B2 (en) | 2006-03-24 | 2015-05-26 | Handylab, Inc. | Integrated system for processing microfluidic samples, and method of using the same |
US11141734B2 (en) | 2006-03-24 | 2021-10-12 | Handylab, Inc. | Fluorescence detector for microfluidic diagnostic system |
US9080207B2 (en) | 2006-03-24 | 2015-07-14 | Handylab, Inc. | Microfluidic system for amplifying and detecting polynucleotides in parallel |
US11085069B2 (en) | 2006-03-24 | 2021-08-10 | Handylab, Inc. | Microfluidic system for amplifying and detecting polynucleotides in parallel |
US10913061B2 (en) | 2006-03-24 | 2021-02-09 | Handylab, Inc. | Integrated system for processing microfluidic samples, and method of using the same |
US11142785B2 (en) | 2006-03-24 | 2021-10-12 | Handylab, Inc. | Microfluidic system for amplifying and detecting polynucleotides in parallel |
US10843188B2 (en) | 2006-03-24 | 2020-11-24 | Handylab, Inc. | Integrated system for processing microfluidic samples, and method of using the same |
US10821436B2 (en) | 2006-03-24 | 2020-11-03 | Handylab, Inc. | Integrated system for processing microfluidic samples, and method of using the same |
US11806718B2 (en) | 2006-03-24 | 2023-11-07 | Handylab, Inc. | Fluorescence detector for microfluidic diagnostic system |
US11959126B2 (en) | 2006-03-24 | 2024-04-16 | Handylab, Inc. | Microfluidic system for amplifying and detecting polynucleotides in parallel |
US10821446B1 (en) | 2006-03-24 | 2020-11-03 | Handylab, Inc. | Fluorescence detector for microfluidic diagnostic system |
US10695764B2 (en) | 2006-03-24 | 2020-06-30 | Handylab, Inc. | Fluorescence detector for microfluidic diagnostic system |
US10799862B2 (en) | 2006-03-24 | 2020-10-13 | Handylab, Inc. | Integrated system for processing microfluidic samples, and method of using same |
US8883490B2 (en) | 2006-03-24 | 2014-11-11 | Handylab, Inc. | Fluorescence detector for microfluidic diagnostic system |
US20080070245A1 (en) * | 2006-09-15 | 2008-03-20 | Gilles Reza George Monif | Method for herd management of domestic ruminants to control infection due to mycobacterium avium subspecies paratuberculosis |
US8709787B2 (en) | 2006-11-14 | 2014-04-29 | Handylab, Inc. | Microfluidic cartridge and method of using same |
US8765076B2 (en) | 2006-11-14 | 2014-07-01 | Handylab, Inc. | Microfluidic valve and method of making same |
US9815057B2 (en) | 2006-11-14 | 2017-11-14 | Handylab, Inc. | Microfluidic cartridge and method of making same |
US10710069B2 (en) | 2006-11-14 | 2020-07-14 | Handylab, Inc. | Microfluidic valve and method of making same |
US9745615B2 (en) * | 2006-12-27 | 2017-08-29 | Luminex Corporation | Instrument for cassette for sample preparation |
US7754148B2 (en) * | 2006-12-27 | 2010-07-13 | Progentech Limited | Instrument for cassette for sample preparation |
US9273344B2 (en) | 2006-12-27 | 2016-03-01 | Luminex Corporation | Instrument for cassette for sample preparation |
US8029746B2 (en) * | 2006-12-27 | 2011-10-04 | Genturadx, Inc. | Instrument for cassette for sample preparation |
US10047391B2 (en) | 2006-12-27 | 2018-08-14 | Luminex Corporation | Instrument for cassette for sample preparation |
US8900877B2 (en) * | 2006-12-27 | 2014-12-02 | Luminex Corporation | Instrument for cassette for sample preparation |
US20120003631A1 (en) * | 2006-12-27 | 2012-01-05 | Genturadx, Inc. | Instrument for cassette for sample preparation |
US9856517B2 (en) * | 2006-12-27 | 2018-01-02 | Luminex Corporation | Instrument for cassette for sample preparation |
US9434939B2 (en) * | 2006-12-27 | 2016-09-06 | Luminex Corporation | Instrument for cassette for sample preparation |
US20080159915A1 (en) * | 2006-12-27 | 2008-07-03 | Steve Jia Chang Yu | Instrument for cassette for sample preparation |
US20170321255A1 (en) * | 2006-12-27 | 2017-11-09 | Luminex Corporation | Instrument for cassette for sample preparation |
US20130115712A1 (en) * | 2006-12-27 | 2013-05-09 | Genturadx, Inc. | Instrument for cassette for sample preparation |
US20160348154A1 (en) * | 2006-12-27 | 2016-12-01 | Luminex Corporation | Instrument for cassette for sample preparation |
US10214767B2 (en) * | 2006-12-27 | 2019-02-26 | Luminex Corporation | Instrument for cassette for sample preparation |
US8168443B2 (en) * | 2006-12-27 | 2012-05-01 | Genturadx, Inc. | Instrument for cassette for sample preparation |
US20110158849A1 (en) * | 2006-12-27 | 2011-06-30 | Genturadx, Inc. | Instrument for cassette for sample preparation |
US7910062B2 (en) * | 2006-12-27 | 2011-03-22 | Genturadx, Inc. | Instrument for cassette for sample preparation |
US20100262303A1 (en) * | 2006-12-27 | 2010-10-14 | Steve Jia Chang Yu | Instrument for cassette for sample preparation |
WO2008082432A1 (en) * | 2007-01-04 | 2008-07-10 | The Regents Of The University Of California | Sorting, amplification, detection, and identification of nucleic acid subsequences in a complex mixture |
US8338166B2 (en) | 2007-01-04 | 2012-12-25 | Lawrence Livermore National Security, Llc | Sorting, amplification, detection, and identification of nucleic acid subsequences in a complex mixture |
US20080166793A1 (en) * | 2007-01-04 | 2008-07-10 | The Regents Of The University Of California | Sorting, amplification, detection, and identification of nucleic acid subsequences in a complex mixture |
WO2009006009A1 (en) * | 2007-06-29 | 2009-01-08 | Cabochon Aesthetics, Inc. | Devices and methods for selectively lysing cells |
US11266987B2 (en) | 2007-07-13 | 2022-03-08 | Handylab, Inc. | Microfluidic cartridge |
US10590410B2 (en) | 2007-07-13 | 2020-03-17 | Handylab, Inc. | Polynucleotide capture materials, and methods of using same |
US11060082B2 (en) | 2007-07-13 | 2021-07-13 | Handy Lab, Inc. | Polynucleotide capture materials, and systems using same |
US10179910B2 (en) | 2007-07-13 | 2019-01-15 | Handylab, Inc. | Rack for sample tubes and reagent holders |
US9701957B2 (en) | 2007-07-13 | 2017-07-11 | Handylab, Inc. | Reagent holder, and kits containing same |
US10875022B2 (en) | 2007-07-13 | 2020-12-29 | Handylab, Inc. | Integrated apparatus for performing nucleic acid extraction and diagnostic testing on multiple biological samples |
US10139012B2 (en) | 2007-07-13 | 2018-11-27 | Handylab, Inc. | Integrated heater and magnetic separator |
US9618139B2 (en) | 2007-07-13 | 2017-04-11 | Handylab, Inc. | Integrated heater and magnetic separator |
US10234474B2 (en) | 2007-07-13 | 2019-03-19 | Handylab, Inc. | Automated pipetting apparatus having a combined liquid pump and pipette head system |
US10844368B2 (en) | 2007-07-13 | 2020-11-24 | Handylab, Inc. | Diagnostic apparatus to extract nucleic acids including a magnetic assembly and a heater assembly |
US9238223B2 (en) | 2007-07-13 | 2016-01-19 | Handylab, Inc. | Microfluidic cartridge |
US11254927B2 (en) | 2007-07-13 | 2022-02-22 | Handylab, Inc. | Polynucleotide capture materials, and systems using same |
US10100302B2 (en) | 2007-07-13 | 2018-10-16 | Handylab, Inc. | Polynucleotide capture materials, and methods of using same |
US9259734B2 (en) | 2007-07-13 | 2016-02-16 | Handylab, Inc. | Integrated apparatus for performing nucleic acid extraction and diagnostic testing on multiple biological samples |
US9186677B2 (en) | 2007-07-13 | 2015-11-17 | Handylab, Inc. | Integrated apparatus for performing nucleic acid extraction and diagnostic testing on multiple biological samples |
US11466263B2 (en) | 2007-07-13 | 2022-10-11 | Handylab, Inc. | Diagnostic apparatus to extract nucleic acids including a magnetic assembly and a heater assembly |
US10071376B2 (en) | 2007-07-13 | 2018-09-11 | Handylab, Inc. | Integrated apparatus for performing nucleic acid extraction and diagnostic testing on multiple biological samples |
US10065185B2 (en) | 2007-07-13 | 2018-09-04 | Handylab, Inc. | Microfluidic cartridge |
US11549959B2 (en) | 2007-07-13 | 2023-01-10 | Handylab, Inc. | Automated pipetting apparatus having a combined liquid pump and pipette head system |
US11845081B2 (en) | 2007-07-13 | 2023-12-19 | Handylab, Inc. | Integrated apparatus for performing nucleic acid extraction and diagnostic testing on multiple biological samples |
US10717085B2 (en) | 2007-07-13 | 2020-07-21 | Handylab, Inc. | Integrated apparatus for performing nucleic acid extraction and diagnostic testing on multiple biological samples |
US9217143B2 (en) | 2007-07-13 | 2015-12-22 | Handylab, Inc. | Polynucleotide capture materials, and methods of using same |
US10632466B1 (en) | 2007-07-13 | 2020-04-28 | Handylab, Inc. | Integrated apparatus for performing nucleic acid extraction and diagnostic testing on multiple biological samples |
US10625261B2 (en) | 2007-07-13 | 2020-04-21 | Handylab, Inc. | Integrated apparatus for performing nucleic acid extraction and diagnostic testing on multiple biological samples |
US10625262B2 (en) | 2007-07-13 | 2020-04-21 | Handylab, Inc. | Integrated apparatus for performing nucleic acid extraction and diagnostic testing on multiple biological samples |
US9347586B2 (en) | 2007-07-13 | 2016-05-24 | Handylab, Inc. | Automated pipetting apparatus having a combined liquid pump and pipette head system |
US8710211B2 (en) | 2007-07-13 | 2014-04-29 | Handylab, Inc. | Polynucleotide capture materials, and methods of using same |
US9039722B2 (en) | 2007-10-09 | 2015-05-26 | Ulthera, Inc. | Dissection handpiece with aspiration means for reducing the appearance of cellulite |
US10220122B2 (en) | 2007-10-09 | 2019-03-05 | Ulthera, Inc. | System for tissue dissection and aspiration |
US8961902B2 (en) | 2008-04-23 | 2015-02-24 | Bioscale, Inc. | Method and apparatus for analyte processing |
US20090269248A1 (en) * | 2008-04-23 | 2009-10-29 | Bioscale, Inc. | Method and apparatus for analyte processing |
US8573518B2 (en) * | 2008-04-25 | 2013-11-05 | Qiagen, Gmbh | Method and device for breaking down biological material |
US20110101136A1 (en) * | 2008-04-25 | 2011-05-05 | Qiagen Gmbh | Method and device for breaking down biological material |
USD787087S1 (en) | 2008-07-14 | 2017-05-16 | Handylab, Inc. | Housing |
US8986612B2 (en) | 2009-04-14 | 2015-03-24 | Biocartis Nv | HIFU induced cavitation with reduced power threshold |
US8641971B2 (en) | 2009-04-14 | 2014-02-04 | Biocartis Sa | HIFU induced cavitation with reduced power threshold |
US9097626B2 (en) | 2009-04-14 | 2015-08-04 | Biocartis Nv | HIFU induced cavitation with reduced power threshold |
US8441629B2 (en) | 2009-04-15 | 2013-05-14 | Biocartis Sa | Optical detection system for monitoring rtPCR reaction |
US9079182B2 (en) | 2009-04-15 | 2015-07-14 | Biocartis Nv | Protection of bioanalytical sample chambers |
US8512637B2 (en) | 2009-05-06 | 2013-08-20 | Biocartis Sa | Device for cutting a sample carrier |
US9757145B2 (en) | 2009-08-07 | 2017-09-12 | Ulthera, Inc. | Dissection handpiece and method for reducing the appearance of cellulite |
US9044259B2 (en) | 2009-08-07 | 2015-06-02 | Ulthera, Inc. | Methods for dissection of subcutaneous tissue |
US9078688B2 (en) | 2009-08-07 | 2015-07-14 | Ulthera, Inc. | Handpiece for use in tissue dissection |
US11096708B2 (en) | 2009-08-07 | 2021-08-24 | Ulthera, Inc. | Devices and methods for performing subcutaneous surgery |
US10271866B2 (en) | 2009-08-07 | 2019-04-30 | Ulthera, Inc. | Modular systems for treating tissue |
US8906054B2 (en) | 2009-08-07 | 2014-12-09 | Ulthera, Inc. | Apparatus for reducing the appearance of cellulite |
US9510849B2 (en) | 2009-08-07 | 2016-12-06 | Ulthera, Inc. | Devices and methods for performing subcutaneous surgery |
US8900261B2 (en) | 2009-08-07 | 2014-12-02 | Ulthera, Inc. | Tissue treatment system for reducing the appearance of cellulite |
US8900262B2 (en) | 2009-08-07 | 2014-12-02 | Ulthera, Inc. | Device for dissection of subcutaneous tissue |
US9358064B2 (en) | 2009-08-07 | 2016-06-07 | Ulthera, Inc. | Handpiece and methods for performing subcutaneous surgery |
US10485573B2 (en) | 2009-08-07 | 2019-11-26 | Ulthera, Inc. | Handpieces for tissue treatment |
US8894678B2 (en) | 2009-08-07 | 2014-11-25 | Ulthera, Inc. | Cellulite treatment methods |
US10531888B2 (en) | 2009-08-07 | 2020-01-14 | Ulthera, Inc. | Methods for efficiently reducing the appearance of cellulite |
US11337725B2 (en) | 2009-08-07 | 2022-05-24 | Ulthera, Inc. | Handpieces for tissue treatment |
US8920452B2 (en) | 2009-08-07 | 2014-12-30 | Ulthera, Inc. | Methods of tissue release to reduce the appearance of cellulite |
US8979881B2 (en) | 2009-08-07 | 2015-03-17 | Ulthera, Inc. | Methods and handpiece for use in tissue dissection |
US9216412B2 (en) | 2009-11-23 | 2015-12-22 | Cyvek, Inc. | Microfluidic devices and methods of manufacture and use |
US20140377146A1 (en) * | 2009-11-23 | 2014-12-25 | Cyvek, Inc. | Microfluidic Assay Operating System and Methods of Use |
US10022696B2 (en) | 2009-11-23 | 2018-07-17 | Cyvek, Inc. | Microfluidic assay systems employing micro-particles and methods of manufacture |
US9700889B2 (en) | 2009-11-23 | 2017-07-11 | Cyvek, Inc. | Methods and systems for manufacture of microarray assay systems, conducting microfluidic assays, and monitoring and scanning to obtain microfluidic assay results |
US9759718B2 (en) | 2009-11-23 | 2017-09-12 | Cyvek, Inc. | PDMS membrane-confined nucleic acid and antibody/antigen-functionalized microlength tube capture elements, and systems employing them, and methods of their use |
US9651568B2 (en) | 2009-11-23 | 2017-05-16 | Cyvek, Inc. | Methods and systems for epi-fluorescent monitoring and scanning for microfluidic assays |
US9500645B2 (en) | 2009-11-23 | 2016-11-22 | Cyvek, Inc. | Micro-tube particles for microfluidic assays and methods of manufacture |
US9229001B2 (en) | 2009-11-23 | 2016-01-05 | Cyvek, Inc. | Method and apparatus for performing assays |
US10065403B2 (en) | 2009-11-23 | 2018-09-04 | Cyvek, Inc. | Microfluidic assay assemblies and methods of manufacture |
US9855735B2 (en) | 2009-11-23 | 2018-01-02 | Cyvek, Inc. | Portable microfluidic assay devices and methods of manufacture and use |
US9546932B2 (en) * | 2009-11-23 | 2017-01-17 | Cyvek, Inc. | Microfluidic assay operating system and methods of use |
KR101882940B1 (en) * | 2010-02-23 | 2018-07-30 | 루미넥스 코포레이션 | Apparatus and methods for integrated sample preparation, reaction and detection |
US9931636B2 (en) | 2010-02-23 | 2018-04-03 | Luminex Corporation | Apparatus and method for integrated sample preparation, reaction and detection |
KR102004319B1 (en) * | 2010-02-23 | 2019-07-26 | 루미넥스 코포레이션 | Apparatus and methods for integrated sample preparation, reaction and detection |
AU2015221472B2 (en) * | 2010-02-23 | 2017-01-19 | Luminex Corporation | Apparatus and methods for integrated sample preparation, reaction and detection |
EP4043546A1 (en) | 2010-02-23 | 2022-08-17 | Luminex Corporation | Apparatus and methods for integrated sample preparation, reaction and detection |
EP3150690A1 (en) * | 2010-02-23 | 2017-04-05 | Luminex Corporation | Apparatus and methods for integrated sample preparation, reaction and detection |
US9248422B2 (en) | 2010-02-23 | 2016-02-02 | Luminex Corporation | Apparatus and methods for integrated sample preparation, reaction and detection |
KR20180088489A (en) * | 2010-02-23 | 2018-08-03 | 루미넥스 코포레이션 | Apparatus and methods for integrated sample preparation, reaction and detection |
KR20170078857A (en) * | 2010-02-23 | 2017-07-07 | 루미넥스 코포레이션 | Apparatus and methods for integrated sample preparation, reaction and detection |
AU2017201579B2 (en) * | 2010-02-23 | 2018-05-17 | Luminex Corporation | Apparatus and methods for integrated sample preparation, reaction and detection |
US10603066B2 (en) | 2010-05-25 | 2020-03-31 | Ulthera, Inc. | Fluid-jet dissection system and method for reducing the appearance of cellulite |
US11213618B2 (en) | 2010-12-22 | 2022-01-04 | Ulthera, Inc. | System for tissue dissection and aspiration |
US8439940B2 (en) | 2010-12-22 | 2013-05-14 | Cabochon Aesthetics, Inc. | Dissection handpiece with aspiration means for reducing the appearance of cellulite |
US11788127B2 (en) | 2011-04-15 | 2023-10-17 | Becton, Dickinson And Company | Scanning real-time microfluidic thermocycler and methods for synchronized thermocycling and scanning optical detection |
US9765389B2 (en) | 2011-04-15 | 2017-09-19 | Becton, Dickinson And Company | Scanning real-time microfluidic thermocycler and methods for synchronized thermocycling and scanning optical detection |
US10781482B2 (en) | 2011-04-15 | 2020-09-22 | Becton, Dickinson And Company | Scanning real-time microfluidic thermocycler and methods for synchronized thermocycling and scanning optical detection |
US10076754B2 (en) | 2011-09-30 | 2018-09-18 | Becton, Dickinson And Company | Unitized reagent strip |
USD742027S1 (en) | 2011-09-30 | 2015-10-27 | Becton, Dickinson And Company | Single piece reagent holder |
USD831843S1 (en) | 2011-09-30 | 2018-10-23 | Becton, Dickinson And Company | Single piece reagent holder |
US9222954B2 (en) | 2011-09-30 | 2015-12-29 | Becton, Dickinson And Company | Unitized reagent strip |
USD905269S1 (en) | 2011-09-30 | 2020-12-15 | Becton, Dickinson And Company | Single piece reagent holder |
US9480983B2 (en) | 2011-09-30 | 2016-11-01 | Becton, Dickinson And Company | Unitized reagent strip |
USD692162S1 (en) | 2011-09-30 | 2013-10-22 | Becton, Dickinson And Company | Single piece reagent holder |
US11453906B2 (en) | 2011-11-04 | 2022-09-27 | Handylab, Inc. | Multiplexed diagnostic detection apparatus and methods |
US10822644B2 (en) | 2012-02-03 | 2020-11-03 | Becton, Dickinson And Company | External files for distribution of molecular diagnostic tests and determination of compatibility between tests |
AU2013246867B2 (en) * | 2012-04-11 | 2018-01-18 | Stat-Diagnostica & Innovation, S.L. | Fluidically integrated rotary bead beater |
CN104703683A (en) * | 2012-04-11 | 2015-06-10 | 统计诊断与创新有限公司 | Fluidically integrated rotary bead beater |
RU2638854C2 (en) * | 2012-04-11 | 2017-12-18 | Стат-Диагностика Энд Инновэйшн, С.Л. | Rotary mini-bead beater, built-in with formation of message through fluid |
JP2015515629A (en) * | 2012-04-11 | 2015-05-28 | スタット−ダイアグノスティカ アンド イノベーション, エス. エル. | Fluidically integrated rotating bead beater |
WO2013153176A1 (en) * | 2012-04-11 | 2013-10-17 | Stat-Diagnostica & Innovation, S.L. | Fluidically integrated rotary bead beater |
US9304066B2 (en) | 2012-04-11 | 2016-04-05 | Stat-Diagnostica & Innovation S.L. | Fluidically integrated rotary bead beader |
US9987576B2 (en) | 2012-12-10 | 2018-06-05 | University Of Virginia Patent Foundation | Frequency-based filtering of mechanical actuation using fluidic device |
CN111366434A (en) * | 2013-03-15 | 2020-07-03 | 赛拉诺斯知识产权有限责任公司 | Automated assay device for disrupting cells in a sample |
US10228367B2 (en) | 2015-12-01 | 2019-03-12 | ProteinSimple | Segmented multi-use automated assay cartridge |
WO2017125521A1 (en) * | 2016-01-21 | 2017-07-27 | Robert Bosch Gmbh | Apparatus and method for detecting cellular parts of a sample potentially containing cells |
WO2020140003A1 (en) * | 2018-12-27 | 2020-07-02 | Hall Labs, Llc | Liquid sample capture apparatus |
US20200240902A1 (en) * | 2019-01-24 | 2020-07-30 | Nirrin Technologies, Inc. | Flow cell device and bioreactor product monitoring system and method |
US20220229050A1 (en) * | 2019-05-30 | 2022-07-21 | Beckman Coulter, Inc. | Methods and systems for immobilizing a biological specimen for microscopic imaging |
EP4159053A1 (en) * | 2020-04-06 | 2023-04-05 | Shaheen Innovations Holding Limited | Cell lysis sytems and methods |
CN113528317A (en) * | 2020-04-15 | 2021-10-22 | 拓原合壹(宁波)生物技术有限公司 | Nucleic acid extraction method |
US11667979B2 (en) | 2020-06-01 | 2023-06-06 | Shaheen Innovations Holding Limited | Infectious disease screening device |
US11946844B2 (en) | 2020-06-01 | 2024-04-02 | Shaheen Innovations Holding Limited | Infectious disease screening system |
US11959146B2 (en) | 2020-06-01 | 2024-04-16 | Shaheen Innovations Holding Limited | Infectious disease screening device |
CN113174329A (en) * | 2021-01-15 | 2021-07-27 | 北京中科生仪科技有限公司 | Continuous reaction device based on nucleic acid extraction, purification and amplification |
CN115181669A (en) * | 2022-09-09 | 2022-10-14 | 鲲鹏基因(北京)科技有限责任公司 | Breaking device for cell or virus lysis |
Also Published As
Publication number | Publication date |
---|---|
US20060030038A1 (en) | 2006-02-09 |
US8268603B2 (en) | 2012-09-18 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US9943848B2 (en) | Apparatus and method for cell disruption | |
US8268603B2 (en) | Apparatus and method for cell disruption | |
US9073053B2 (en) | Apparatus and method for cell disruption | |
EP1180135B1 (en) | Apparatus and method for cell disruption | |
US9322052B2 (en) | Cartridge for conducting a chemical reaction | |
AU781122B2 (en) | Cartridge for conducting a chemical reaction |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
STCB | Information on status: application discontinuation |
Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION |