US20030036054A1 - Biosensor and related method - Google Patents
Biosensor and related method Download PDFInfo
- Publication number
- US20030036054A1 US20030036054A1 US10/172,263 US17226302A US2003036054A1 US 20030036054 A1 US20030036054 A1 US 20030036054A1 US 17226302 A US17226302 A US 17226302A US 2003036054 A1 US2003036054 A1 US 2003036054A1
- Authority
- US
- United States
- Prior art keywords
- detection chamber
- fluid sample
- biosensor
- substrate
- method defined
- 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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
- G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
- G01N33/5438—Electrodes
Definitions
- the present invention relates to an integrated-chip-type biosensor and a related method for detection of pathogenic substances.
- the biosensor and method are particularly, but not exclusively, useful in detecting foodborne pathogens such as Listeria monocytogenes.
- E. coli O157:H7 and Listeria monocytogenes are the pathogens of most concern. Ground meat containing E. coli O157:H7 is now considered to be an adulterated food while Listeria monocytogenes has emerged as one of the most important food pathogens with a “zero tolerance” criterion for it in ready-to-eat processed (lunch) meats and dairy foods.
- the genus Listeria is comprised of six species, L. monocytogenes, L. ivanovii, L. seeligeri, L. innocua, L. welshimeri, and L. grayi. Of these species, only L. monocytogenes is harmful to humans. Consumption of contaminated food may cause meningitis, encephalitis, liver abscess, headache, fever and gastroenteritis (diarrhea) in immunologically challenged individuals and abortion in pregnant women. L. monocytogenes is ubiquitous in nature and can be found in meat, poultry, seafood, and vegetables. Occurrence of this organism could be as high as 32%. In a food sample, L. monocytogenes is often present in close association with other nonpathogenic Listeria species, thereby complicating the specific detection procedures. A successful detection method ideally detects only L. monocytogenes in the presence of overwhelming populations of nonpathogenic Listeria and other background resident bacteria.
- Ligand/receptor binding pairs used commonly in diagnostics include antigen-antibody, hormone-receptor, drug-receptor, cell surface antigen-lectin, biotin-avidin, substrate/enzyme, and complementary nucleic acid strands.
- the analyte to be detected may be either member of the binding pair; alternatively, the analyte may be a ligand analog that competes with the ligand for binding to the complement receptor.
- a variety of devices for detecting ligand/receptor interactions are known. The most basic of these are purely chemical/enzymatic assays in which the presence or amount of analyte is detected by measuring or quantitating a detectable reaction product, such as a detectable marker or reporter molecule or ligand. Ligand/receptor interactions can also be detected and quantitated by radiolabel assays.
- Quantitative binding assays of this type involve two separate components: a reaction substrate, e.g., a solid-phase test strip and a separate reader or detector device, such as a scintillation counter or spectrophotometer.
- a reaction substrate e.g., a solid-phase test strip
- a separate reader or detector device such as a scintillation counter or spectrophotometer.
- the substrate is generally unsuited to multiple assays, or to miniaturization, for handling multiple analyte assays from a small amount of body-fluid sample.
- biochip has been used in various contexts but can be defined as a “microfabricated device that is used for delivery, processing, and analysis of biological species (molecules, cells, etc.).” Such devices have been used, among other things, for the direct interrogation of the electric properties and behavior of cells (Borkholder et al. “Planar Electrode Array Systems for Neural Recording and Impedance Measurements”, IEEE Journal of Microelectromechanical Systems, vol 8(1), pp.
- biochips One of the most interesting uses of biochips is for the detection of small quantities of pathogenic bacteria or toxigenic substances in food, bodily fluids, tissue samples, soil, etc. In applications such as the screening of food products for the presence of pathogenic bacteria, it would be beneficial to detect between 100 and 1000 microorganisms per milliliter of sample, with a sample volume of a couple of milliliters. Not counting the fact that bacteria are substantially larger than single biomolecules ( ⁇ 2 ⁇ m vs.
- 1000 cells are approximately equivalent to a 10 ⁇ 5 femto-moles of cells, which gives an idea of the difficulty in directly detecting such a small number suspended in a volume of 1 or 2 ml, along with large numbers of food debris, proteins, carbohydrates, oils, and other bacteria. Additionally, in many cases the screening technique must be able to discern between viable and dead cells. Many bacteria will not produce toxins when not viable and consequently will not be pathogenic in that state. DNA detection methods, which search for DNA sequences specific to the pathogen of interest, can be extremely sensitive because they rely on the very specific binding of complementary DNA strands, often coupled with Polymerase Chain Reaction (PCR) for amplification.
- PCR Polymerase Chain Reaction
- Impedimetric detection works by measuring impedance changes produced by the binding of target molecules to receptors (antibodies, for example) immobilized on the surface of microelectrodes. Amperometric devices measure the current generated by electrochemical reactions at the surfaces of microelectrodes, which are commonly coated with enzymes.
- bacterial sensors determine whether the bacterium of interest is indeed live or dead.
- a technique that has been widely reported to detect the viability of bacteria on a macro-scale relies on measuring the conductance/impedance changes of a medium in which the microbes are cultured. Such a method is recognized by the Association of Official Analytical Chemists International (AOAC) as a standard technique for the detection of Salmonella in food. This is possible because bacterial metabolism changes the electrolyte concentration in the suspension medium, significantly altering the electrical characteristics of the medium.
- AOAC Association of Official Analytical Chemists International
- a more specific object of the present invention is to provide a method and/or an associated device for a more rapid detection of foodborne pathogens, particularly including, but not necessarily limited to, Listeria monocytogenes.
- An even more specific object of the present invention is to provide such a method and/or device which detects pathogens in a few hours or less, possibly within minutes.
- a further specific object of the present invention is to provide such a method and/or device which is capable of detecting a relatively small number of instances of a pathogen such as a bacterium.
- Another specific object of the present invention is to provide such a method and/or device which is able to distinguish between a sample of live bacteria and a sample of dead bacteria of the same type.
- Another object of the present invention is to provide a method for manufacturing a biosensor, particularly a microscale biosensor.
- the present invention is directed in part to a microscale biosensor for use in the detection of target biological substances including molecules and cells.
- a preferred embodiment of a biosensor pursuant to the present invention is a microfluidic system with integrated electronics, inlet-outlet ports and interface schemes, high sensitivity detection of pathogen specificity, and processing of biological materials at semiconductor interfaces.
- the present invention is also directed in part to a fabrication process for a microfluidic biochip that is used for impedance spectroscopy of biological species.
- Key features of the device include an all top-side processing for the formation of fluidic channels, planar fluidic interface ports, integrated metal electrodes for impedance measurements, and a glass cover sealing the non-planar topography of the chip using spin-on-glass as an intermediate bonding layer.
- the total volume of the fluidic path in the device is on the order of 30 nl.
- a method in accordance with the present invention for detecting a microbiological substance utilizes a microfabricated biosensor chip including integrated detection elements.
- the method comprises delivering a fluid sample to the biosensor chip and thereafter separating at least some contaminants or debris from the fluid sample to at least partially isolate and retain instances of a predetermined target type of microbiological material, a material to be detected, on the biosensor chip.
- the separating of the contaminants takes place at least in part on the biosensor chip itself.
- the detection elements are operated to determine whether the separated fluid sample contains microbiological material of the predetermined target type.
- This method may further comprise carrying out a bioseparations process on the fluid sample prior to the delivering of the fluid sample to the biosensor chip.
- the bioseparations process includes adding to the fluid sample a plurality of microscopic carrier elements each provided with a multiplicity of binding agents for coupling the microbiological material to the carrier elements. These carrier elements preferably take the form of beads or microspheres.
- the separating of contaminants from the fluid sample on the biosensor chip preferably includes trapping the carrier elements with the coupled microbiological material in a detection chamber on the biosensor chip while flushing remaining portions of the fluid sample from the chamber.
- This trapping of the carrier elements with the coupled microbiological material in a detection chamber serves in part to concentrate the microbiological material of interest and thus enhance the sensitivity of the detection technique.
- the trapping of the carrier elements may be implemented in part by providing a filter barrier or retention structure at an outlet of the detection chamber. Such a barrier or retention structure preferably takes the form of a microfabricated filter grid or post array.
- the trapping of the carrier elements, where the carrier elements are made of a magnetic material in a magnetic field generated in the detection chamber.
- the bioseparations process includes subjecting a the fluid sample (prior to delivery to the biosensor chip, to a bioactive surface taken from the group consisting of a cation exchange resin and an anion exchange resin.
- the cation exchange resin may include Amberlyst 35 while the anion exchange resin includes IRA 400.
- the present invention is especially effective in detecting microbiological material in the form of a pathogenic strain of bacteria such as Listeria monocytogenes.
- the methodology includes extracting the fluid sample from a food product prior to delivering of the fluid sample to the biosensor chip.
- the detection of Listeria monocytogenes is implemented in part by attaching antibodies to a capture surface in the detection chamber of the biosensor. That capture surface may be on an electrode or oxide surface in the detection chamber. Alternatively, the capture surface may be on a bead or microsphere floating in the detection chamber. It will be apparent to one of ordinary skill in the art that virtually any microorganism may be detected by the method of the present invention simply by attaching an appropriate antibody to a capture surface as described herein.
- Antibodies and their associated antigens on the cell membranes of various microorganisms are well documented in the art. It will also be apparent to one skilled in the art that species other than bacteria may be detected by the methodology of the present invention. Various proteins, peptide groups, nucleic acid chains, and other molecules may be detected by the selection of suitable binding agents and the attachment of those binding agents to a capture surface in a detection chamber of a biosensor.
- a biosensor in accordance with the present invention comprises a substrate microfabricated to include, as integrated components, a detection chamber, a first channel segment extending to an inlet of the detection chamber, a second channel segment extending from an outlet of the chamber, and a retention structure for holding, in the detection chamber, a carrier element entraining a target microbiological species and for permitting the passage from the detection chamber of contaminants or debris in a fluid sample containing the carrier element and the target microbiological species.
- the retention structure may take the form of a filter grid or grating disposed on the substrate on an upstream side of the outlet.
- the retention structure may include a magnetic field generating element such as an electromagnet.
- the retention structure on the biosensor enables the concentration of a target microbiological species at the point of measurement. This facilitates and enhances the detection process.
- the small size of the detection chamber less than 100 microliters and preferably between about 1 picoliter and 1 microliter, also increases the sensitivity of the detection process.
- Yet another factor contributing to the efficacy of the present methodology is the use of a low conductivity buffer as the fluid matrix in which the microbiological species of interest is entrained in the detection chamber.
- the detection chamber is provided with at least one pair of electrodes, preferably with interdigitated finger parts, and has a volume of less than approximately one microliter.
- the volume of a fluid sample in the device may be substantially less than one microliter, even down to about 1 picoliter.
- the electrodes are spaced from each other by 1 to 100 microns and, more preferably, by 2 to 50 microns.
- a biosensor in accordance with another embodiment of the present invention comprises a substrate microfabricated to include, as integrated components, a detection chamber and a channel extending to an inlet of the detection chamber.
- the biosensor further comprises a wicking element connected at one end to the substrate so as to be in communication with the channel, for drawing a fluid sample by capillary action to the channel for delivery to the detection chamber.
- the wicking element may be attached at the one end by an adhesive to the substrate.
- the substrate is microfabricated to include an inlet groove or trench substantially coplanar with the channel and the detection chamber, the one end of the wicking element is disposed in the inlet groove or trench, so that the wicking element is coplanar at the one end with the channel and the detection chamber.
- An integrated microscale biosensor in accordance with a further embodiment of the present invention comprises a substrate microfabricated to include, as integrated components, a detection chamber, a channel extending to an inlet of the detection chamber, and an inlet groove or trench substantially coplanar with the channel and the detection chamber.
- the biosensor further comprises an elongate fluid delivery member having a downstream end disposed in the inlet groove or trench.
- the fluid delivery member is connected at the downstream end to inlet groove or trench so that at least the downstream end of the fluid delivery member is coplanar with the channel and the detection chamber.
- the elongate fluid delivery member may take the form of a microbore tube or a wicking element.
- a biosensor chip in accordance with the present invention is top-side processed only.
- This structure facilitates the manufacturing process, in part by obviating alignment requirements between the cover plate and the substrate.
- the cover attached to the substrate over the detection chamber, the channel, the inlet groove, and the downstream end of the fluid delivery member can be an integral or continuous member, devoid of holes or apertures. Such holes or apertures would be required, for instance, where a feed tube was to be inserted through the cover.
- a method for manufacturing a biosensor comprises, in accordance with the present invention, providing a substrate, processing the substrate to generate a detection chamber and a channel extending to the detection chamber, further processing the substrate to provide at least one pair of electrodes in the detection chamber, and exposing the processed substrate to BSA (bovine serum albumin) and avidin to adsorb the avidin to the electrodes in the presence of the BSA.
- BSA bovine serum albumin
- This manufacturing method may further comprise subjecting the exposed processed substrate to a fluid containing a biotinylated antibody specific to a preselected antigen, thereby attaching the antibody to the electrodes via a biotin-avidin link.
- the biotinylated antibody is specific to an antigen on a cell membrane of Listeria monocytogenes .
- Monoclonal antibody producing clones of C11E9 and EM-7G1 producing antibodies specific for Listeria monocytogenes
- Antibodies are harvested from culture supernatants by salt (ammonium sulfate) precipitation. After an initial concentration step, carried out by known techniques, high quality antibodies are obtained by further purification through size exclusion chromatography followed by protein-A affinity chromatography in an FPLC system.
- a method for manufacturing a biosensor comprises, pursuant to another embodiment of the present invention, processing a substrate to create a shallow detection chamber and a channel extending to the detection chamber, thereafter further processing the substrate to deposit at least one pair of electrodes in the detection chamber, and subsequently processing the substrate to create at least deep groove at a periphery of the substrate, for receiving an elongate fluid delivery element, the channel communicating with the deep groove. A downstream end of the fluid delivery element is inserted into and attached to the deep groove.
- This method may further comprise attaching a cover to the substrate over the detection chamber, the channel, the deep groove and the downstream end of the fluid delivery element.
- the attaching of the cover to the substrate includes placing a spin-on-glass composition on the glass, subsequently contacting the substrate with the spin-on-glass composition, and heating the substrate, the cover, and the spin-on-glass composition to enabling a flow of the spin-on-glass composition into interstitial spaces on the substrate and form a fluid-tight seal.
- a method for detecting a microorganism comprises, in accordance with the present invention, preparing a fluid sample containing at least one microorganism of a preselected type, the fluid sample having a buffer of a low conductivity liquid, the fluid sample also containing a nonionic nutrient.
- the fluid sample is disposed in or delivered to a detection chamber having a volume between about 1 picoliter and approximately 1 microliter.
- the fluid sample is maintained at a predetermined temperature in the detection chamber and an electrical parameter of an electrical circuit incorporating the detection chamber and the fluid sample therein is measured.
- the electrical parameter is an impedance measure taken from the group consisting of a magnitude and phase.
- the method is effective in the detection of living Listeria monocytogenes cells.
- the buffer may be a low conductivity Tris-Glycine buffer.
- the measuring of the electrical parameter includes measuring the impedance parameter at a plurality of frequencies within a range from 100 Hz to 1 MHz.
- a method for testing a food product for the presence of a predetermined type of pathogenic bacteria comprises, in accordance with the present invention, extracting a fluid sample from the food product, feeding the extracted fluid sample providing an integrated microscale biosensor, subjecting the fluid sample to a bioseparations process to remove extraneous particles including proteins and kinds of bacteria other than the predetermined type of pathogenic bacteria, binding bacteria of the predetermined type in the fluid sample to at least one substrate body, and, after the feeding of the extracted fluid sample to the chamber, the subjecting of the fluid sample to the bioseparations process, and the binding of the predetermined type of bacteria to the at least one substrate body, measuring an electrical parameter of an electrical circuit incorporating the detection chamber and the fluid sample therein to detect the presence in the fluid sample of living instances of the predetermined type of bacteria.
- the binding of the predetermined type of bacteria may be to beads or microspheres floating in the fluid sample.
- the binding of the predetermined type of bacteria may be to electrodes in the biosensor. Subjecting of the fluid sample to the bioseparations process may take place at least partially after feeding of the fluid sample to the biosensor.
- the sensitivity to biological pathogens of a biosensor chip in accordance with the present invention is based on the placement of protein receptors, derived through biotechnology processes, on a surface of the biosensor. A tiny amount of fluid taken from a specimen such as a processed meat or dairy product is then delivered to the biosensor. If a target bacterium such as Listeria monocytogenes is present, it will bind to the receptor and cause a measurable electronic signal to be generated in no more than several hours and possibly within minutes.
- the present invention provides a method and an associated device for the relatively rapid detection of biological pathogens such as bacteria.
- the method and device can detect small numbers of bacteria such as Listeria monocytogenes in time intervals short enough to enable removal of contaminated products from the stream of commerce before consumption of the products by individuals.
- Biosensors or biosensors as disclosed herein improve the quality of life by providing cost-effective means for probing biological materials for pathogenic organisms and molecules in manufacturing facilities, the environment, hospitals, doctors' offices, and ultimately in the home.
- the present invention provides a method and an associated device for a relatively rapid detection of foodborne pathogens.
- the present invention obviates the time-consuming steps of culturing and transferring cells, if present, to increase their numbers or genetic material to the detectible levels required by conventional detection techniques.
- the present technique solves some of these problems.
- the present methodology inherently detects only live microorganisms, which is very important for certain applications, especially in food safety (many microorganisms present in food are not pathogenic if they are dead).
- the method of the present invention also relies exclusively on electrical signals, making the related equipment less expensive and smaller than others. Additionally, the absence of a lengthy growth step makes detection possible in a couple of hours instead of days.
- Instruments for the analysis of the conductivity or impedance of an incubated bacterial suspension have been available for a number of years, but they suffer from two limitations. First, their selectivity is very poor because they rely on the composition of the growth medium for encouraging the proliferation of the microorganism of interest, while suppressing the proliferation of others. The second limitation is related to the scale in which the assay is performed. The available equipment uses volumes of bacterial suspension in the milliliter range and above, which requires large numbers of bacteria to provide a discernible signal.
- the method of the present invention eliminates the first limitation by selectively capturing the bacteria using antibodies prior to the measurement, and increases the sensitivity for very small numbers of microorganisms (1 to 1000) by confining them to an extremely small volume (1 picoliter to 1 microliter). Additionally, the method of the present invention uses a low conductivity buffer, which increases even further the sensitivity. Even very small amounts of ions released by the microorganisms can produce a large change in impedance (in relative terms), since the ionic concentration of the low conductivity buffer is very low.
- FIG. 1 is a schematic top plan view of a biosensor in accordance with the present invention.
- FIG. 2 is a photograph showing, in top plan view, an integrated microscale biosensor in accordance with the present invention.
- FIG. 3 is a photomicrograph, on a larger scale, of a portion of the biosensor of FIG. 2.
- FIG. 4 is a photomicrograph, on an even larger scale, of another portion of the biosensor of FIG. 2.
- FIGS. 5A through 5F are schematic cross-sectional views, on an enlarged scale, showing successive steps in a manufacturing process in accordance with the present invention.
- FIG. 6 is a circuit diagram modeling electrical activity in a biosensor as illustrated in FIG. 1 or FIGS. 2 - 4 .
- FIG. 7 is a pair of graphs showing measured complex impedance (magnitude and angle) of different microorganism-containing samples injected into a biochip in accordance with the present invention.
- the numbers of cells in the legend correspond to the numbers present in a detection chamber of the biochip where the measurement was performed.
- FIG. 9 is a pair of graphs of complex impedance (magnitude and angle vs. frequency), showing a fit between the circuit model of FIG. 6 and the measured complex impedance of the microorganism-containing samples at a concentration of ⁇ 10 5 cells/ml.
- FIG. 10 is a bar graph showing normalized differences of three measurement parameters for each microorganism-containing sample injected into the biochip of the present invention.
- FIG. 11 is a bar graph similar to FIG. 10, showing normalized differences of three measurement parameters for each of several live-microorganism-containing samples and each of several dead-microorganism-containing samples injected into the biochip of the present invention, demonstrating an ability to distinguish between live and dead microorganisms.
- FIG. 12 is a schematic cross-sectional view of a pipette tip with structure for preparing a biological sample for testing with a biosensor in accordance with the present invention.
- FIG. 13 is a schematic cross-sectional view of an ancillary pipette tip with respective structure for preparing a biological sample for testing with a biosensor in accordance with the present invention.
- FIGS. 14A through 14C are diagrams showing successive stages in manufacturing and testing processes for automated detection of microorganisms with a biosensor or biochip as illustrated in FIG. 1 or FIGS. 2 - 4 .
- FIG. 15 is a pair of histogram graphs plotting fluorescence emission from surfaces of a biosensor or biochip, in accordance with the present invention, incubated in 1 mg/mL avidin at room temperature for 18 hours and at 37° C. for 15 hours and then rinsed in DI water followed by drying with compressed air.
- FIG. 16 is a pair of histogram graphs plotting fluorescence emission from surfaces of biochips in accordance with the present invention, where a first biochip was unprocessed, a second biochip was processed for avidin adsorption at 37° C. for 15 hours without ammonium sulfate, and a third biochip was processed for avidin adsorption at 37° C. for 15 hours with ammonium sulfate. Histogram data labeled “control” correspond to untreated surfaces.
- FIG. 17 is a pair of histogram graphs plotting fluorescence emission from surfaces of biochips in accordance with the present invention, where a first biochip was unprocessed, a second biochip was treated with labeled avidin, a third biochip was treated with unlabeled BSA and labeled avidin, and a fourth biochip was treated with labeled BSA.
- FIG. 18 is a pair of histogram graphs plotting fluorescence emission from surfaces of a reference biochip that was not contacted with any protein solution, a second biochip contacted with BSA and labeled avidin, a third biochip contacted with BSA and labeled biotin, and a fourth biochip contacted with BSA followed by unlabeled avidin and labeled biotin, as indicated.
- FIG. 19 is a schematic cross-sectional view of an alternative biochip design in accordance with the present invention.
- FIG. 20 is a FIG. 21 is a partial schematic top plan view of a biosensor or biochip in accordance with the present invention, showing a detection chamber with interdigitated electrodes, heating element and temperature sensor.
- FIG. 22 is a diagram illustrating steps in another bioseparation procedure utilized in a biodetection process in accordance with the present invention.
- Avidin is a protein with four identical subunits and a total molecular weight of 67,000-68,000 daltons.
- Biotin is a vitamin (B-6) having a molecular weight of 244 daltons.
- the avidin-biotin system is well-established and extensively used as a biological probe.
- biotinylated is used herein to generically describe a preselected molecule, generally a protein, which has been derivatized with biotin. Where avidin, has been adsorbed to a capture surface such as a surface of an electrode in a detection chamber, the biotin functions to secure the preselected molecule to the capture surface via the avidin-biotin linkage.
- binding agent is used herein to denote a chemical structure such as an antibody or a molecular complex (two or more molecules coupled together) capable of latching onto or capturing a target microbiological species or material which is to be detected in a biochip sensor pursuant to the techniques described herein.
- a biotinylated antibody bound to avidin on a capture surface of an electrode serves as a binding agent for a target bacterium having a cell membrane carrying the antigen of the biotinylated antibody.
- biosensor and “biochip” as used herein refer to microelectronic-inspired construction of devices that are used for processing (delivery, analysis or detection) of biological molecules and cellular species.
- a biosensor or biochip as described herein is a microfluidic system with integrated electronics, inlet-outlet ports and interface schemes, high sensitivity detection of pathogen specificity, and processing of biological materials at semiconductor interfaces.
- bioseparation or “bioseparations” as used herein refers to a process for removing contaminants and detritus from a fluid sample possibly containing a target microbiological species.
- capture surface refers to a surface in a biochip sensor or in a preseparation process which is prepared with a binding agent for purposes of latching onto and holding, at least for the duration of a detection process, a target substance, whether that target consists of a molecule such as a protein, an antibodies, an antigens, or an enzyme; a molecular fragment such as a peptide or a DNA sequence; or a cell such as a muscle cell or a bacterium; a virus; etc.
- a target substance whether that target consists of a molecule such as a protein, an antibodies, an antigens, or an enzyme; a molecular fragment such as a peptide or a DNA sequence; or a cell such as a muscle cell or a bacterium; a virus; etc.
- carrier refers to movable structures to which binding agents are attached for securing, anchoring or attaching target microbiological materials.
- One kind of carrier is a microsphere or bead made of magnetic or nonmagnetic material.
- contaminants and “detritus” are used herein to describe various microscopic and submicroscopic cells, cellular fragments, molecules, molecular fragments, which are of no interest to a biosensor detection process in accordance with the present invention. Contaminants can be disruptive of the detection process, for example, by causing noise to electrical detection.
- the term “detection chamber” is used herein to generally designate a space provided with sensors for measuring a change in a predetermined parameter owing to the presence of a target microbiological species in the detection chamber.
- the term “detection chamber” is used to designate a small well or cavity produced by microfabrication techniques in a wafer and provided with sensing elements such as electrodes for sensing a change in an electrical characteristic or parameter (such as resistance or phase) in the chamber owing to the presence of the target microbiological species.
- This specific detection chamber has a small volume, no more than 100 microliters, and preferably no more than 1 microliter, and even more preferably, in a range about 1 to 10 nanoliters.
- low conductivity is used herein with reference to a buffer solution which has a sufficiently low concentration of charge carriers (e.g., ions) to enable detection of a difference in an impedance parameter, such as magnitude or phase, between a bacteria-containing sample and a reference sample free of bacteria.
- charge carriers e.g., ions
- microbiological species or “microbiological material” is used herein to denote any microscopic or submicroscopic entity of interest to researchers or commerce.
- the term encompasses molecules such as proteins, antibodies, antigens, and enzymes; molecular fragments such as peptides and DNA sequences; cells such as muscle cells or bacteria; viruses; fungi; etc.
- microfabricated or “microfabrication” as used herein refers to the utilization of photolithography, X-ray lithography, acid etching, and other silicon treatment processes developed in the semiconductor industry to manufacture integrated circuits and solid state components such as microprocessor chips.
- target is used herein to mean a microbiological entity or species of interest.
- a target microbiological species is that which is to be detected by a biosensor or biochip as herein described.
- wicking element denotes any elongate guide capable of moving a liquid sample by capillary action, where the liquid sample include molecular and cellular material.
- the present invention is directed in part to a microfabricated biochip 20 illustrated schematically in FIG. 1.
- a silicon wafer substrate or body 22 having a size on the order of a postage stamp is formed with a plurality of receptacles or grooves 24 and 26 which receive ends of respective microbore tubes 28 and 30 made, for instance, of polytetrafluorethylene.
- Receptacles 24 and 26 communicate with opposite ends of a meandering microscale channel or groove 32 formed at intervals with cavities or wells 34 .
- Cavities 34 are provided with platinum electrodes 36 which may be coated, as described hereinafter, with molecular probes for selectively capturing target molecules such as antigens on the surfaces of a target bacterium such as Listeria monocytogenes.
- Electrodes 36 are connected to respective bonding pads or electrical terminals 38 via conductors or traces 40 .
- a glass cover 42 is positioned over receptacles 24 and 26 , the ends of tubes 28 and 30 , channel 32 and cavities 34 and is sealed to substrate 22 .
- Biochip 20 is thus a self-contained biosensor unit with integrated fluidic paths represented by channel 32 and cavities 34 and electrodes 36 useful in performing micro-scale electronic measurements of biological solutions.
- the electrodes 36 are spaced from each other by 1 to 100 microns and, more preferably, by 2 to 50 microns.
- FIG. 2 is a photograph of a microfluidic biochip 220 as actually manufactured.
- Biochip 220 includes a first area 202 having electrode-containing cavities 204 of 80 by 80 microns and a second area of electrode-containing cavities 206 of 850 by 530 microns, with a common depth of 10 microns.
- Cavities 204 are connected to one another and to a pair of tube receptacles or grooves 208 and 210 by a channel or series of channel segments 212 , while cavities 206 communicate with each other and with a respective pair of microbore-tube receptacles or in/out ports 214 and 216 via a channel or series of channel segments 218 .
- Cavities 204 contain simple electrodes 36 as shown schematically in FIG. 1, whereas cavities 206 contain electrodes (not designated) having several interdigitated segments.
- FIGS. 3 and 4 are scanning electron micrographs, on different scales, of a portion of biochip 220 .
- An inlet port or expanded inlet section 222 of channel 212 is disposed between a respective receptacle or groove 208 or 210 .
- cavities 204 and 204 and channels 212 and 218 were formed by anisotropic KOH-based etching. The process etches the (100) crystal planes about 400 times faster than the (111) planes, creating cavities with walls at an angle of 54.74 degrees, as discussed in greater detail hereinafter with reference to FIG. 5B. RF sputtering of chrome and platinum deposited the electrodes.
- Biochip 220 (or generically biochip 20 ) as disclosed herein has been used to detect and measure a change in conductance in nanoliter volumes of bacterial suspensions and to indicate the viability of the bacteria. Fluid flow through the chip was demonstrated using 2 ⁇ m fluorescently labeled beads imaged through a fluorescence microscope. Electrical impedance measurements demonstrate that the device can be used to distinguish between different concentrations of the bacterium Listeria innocua, a non-pathogenic strain of Listeria, by the change in conductance of the suspension produced by bacterial metabolism. These concentrations correspond to very few bacterial cells in the very small volumes (nanoliters) of the measurement chambers of the biochip.
- the photoresist layer (not shown) needs to be thick enough to keep it from cracking at the upper edges of the channel walls 50 due to the tension that builds up during baking.
- a metallization 52 is formed by RF-sputtering of an 800 ⁇ -thick layer of platinum over a 600 ⁇ -thick film of chromium, the latter serving as an adhesion layer.
- the sheet resistance of the metallization 52 is approximately 30 ⁇ cm (2.1 ⁇ /square for the given thickness).
- a 0.6 ⁇ m thick SiO 2 film 54 is deposited by Plasma-Enhanced Chemical Vapor Deposition (PECVD) to insulate the electrodes 36 and traces 40 .
- PECVD Plasma-Enhanced Chemical Vapor Deposition
- This film 54 is subsequently wet-etched to open windows 56 (FIG. 5C) and thereby define electrodes 36 and bonding pads 38 (FIG. 1) along a periphery (not designated) of wafer substrate 22 .
- FIGS. 3 and 4 show electron micrographs of a section of biochip 220 , where electrodes 36 are defined at the bottom of cavities 204 , and metal lines 54 cross the channels 212 .
- bonding cover 42 FIGS. 1 and 5E
- a rectangular piece of glass, 0.17 to 0.25 mm thick No.2 Dow Coming microscopy glass cover
- a satisfactory bond is achieved by using a low-melting-temperature Spin-On-Glass (SOG) as adhesive (FIG. 5E).
- SOG Spin-On-Glass
- This SOG is methylsilsesquioxane polymer (Methylsilsesquioxane 400F from Filmtronics Inc., Pennsylvania, U.S.A.) that flows at temperatures between 150° C. and 210° C.
- the flowing SOG fills the grooves in between the platinum traces 40 and any other surface irregularities, providing a perfectly hermetic seal, while the low flow temperature minimizes thermally induced stresses and damage to temperature-sensitive materials on the die or wafer substrate 22 .
- the glass cover 42 is first cut to the desired size in a diamond saw, thoroughly rinsed in DI water, dried, and cleaned in Ar/O 2 plasma for 20 minutes. After cleaning, the SOG is spun on the glass at 5000 rpm for 40 s and dried in a convection oven at 90° C. for 2 minutes. This process results in a SOG film approximately 3000 ⁇ thick according to the data-sheet provided by the manufacturer (Spin-on-Glass, 1998). The glass is then manually aligned onto the substrate 22 (SOG side down) and clamped in place. Subsequently, the clamped assembly is heated on a hot plate to 100° C. for 5 minutes, followed by 180° C. for 5 minutes, and 200° C. for at least 1 hour to cure the SOG film.
- the manufacturer indicates that the SOG must be cured at 400° C. for 30 minutes, extensive cracking of the SOG film was observed if the bonded assembly was exposed to temperatures above 300° C. Most likely, the cause of this cracking is the large mismatch between the thermal expansion coefficients of silicon substrate 22 , glass cover 42 , and the SOG. For this reason, to minimize the stress in the SOG film the curing temperature is kept at 200° C., which seems to be sufficient for a reliable bond. The curing time could be substantially increased to compensate for the lower temperature, but even a one hour cure produces a bond capable of withstanding the maximum pressures that have been applied to drive fluids through micro-channel 32 .
- connections for injecting samples into the device are created by etching receptacles or grooves 24 and 26 deeply running up to the edge of substrate 22 , so that microbore tubes 28 and 30 can be inserted horizontally or laterally as depicted in FIG. 1.
- This configuration has several advantages over the standard top connection through the sealing cover.
- the length of tube receptacles 24 and 26 can be adjusted to provide a large bonding surface which improves the robustness and reliability of the connection to the microbore tubes 28 and 30 ; in this case receptacles 24 and 26 were made 2 mm long and 700 ⁇ m wide.
- the receptacles 24 and 26 are created by a Deep Reactive Ion Etch (DRIE) system (Plasma Therm SLR770 system using the Bosch Etch process), to a depth of approximately 390 ⁇ m, with a 10 ⁇ m photoresist layer as mask (FIG. 5D).
- the etch-rate is about 1.6 ⁇ m/min with a selectivity to photoresist of approximately 75:1.
- a protrusion 58 at the edge between inlet section 222 of channel 212 and receptacle or groove 208 (FIG. 4) appears because the photoresist hardmask flows during the bake step prior to the DRIE.
- Tubes 28 and 30 are bonded into the trenches after the glass cover is attached to the device. Before bonding, the tips of the tubes 28 and 30 are treated with FluoroEtch (Acton Technologies Inc., Pennsylvania, U.S.A.) to improve their bondability (by forming a carbonaceous layer on the surface). Tubes 28 and 30 are cut at an angle at 60 to keep the respective bores from being blocked by the inner walls (not separately labeled) of receptacles 24 and 26 .
- FluoroEtch Acton Technologies Inc., Pennsylvania, U.S.A.
- Tubes 28 and 30 are inserted into receptacles 24 and 26 and the remaining voids in the receptacles are filled with biomedical grade epoxy adhesive 58 (Durabond M-121HP from Loctite Corp., Connecticut, U.S.A.), which penetrates into the receptacles 24 and 26 by capillarity (FIG. 5F). Even very rough handling of the tubes 28 and 30 does not compromise the integrity of the bond.
- biomedical grade epoxy adhesive 58 Durabond M-121HP from Loctite Corp., Connecticut, U.S.A.
- FIG. 6 A fairly simple circuit model of a pair of electrodes immersed in an electrolytic solution is shown in FIG. 6 (see Jacobs et al., “Impedimetric Detection of Nucleic Acid Hybrids,” Proceedings of the Second International Conference on Microreaction Technology, New La., pp. 223-229, 1998), where C di is the dielectric capacitance (incorporating contributions from all the materials surrounding the electrodes 36, including the solution), R S is the bulk solution resistance (charge transport across the bulk), and Z W is the interfacial impedance (the so-called Warburg impedance), which accounts for the changes in the electrolyte concentration gradient at the interface.
- n and B are parameters that depend on the properties of the electrolytes and of the electrodes. This equation assumes that the phase between voltage and current is constant at n ⁇ /2 rad. It is to be noted that the impedance Z is a measured parameter, whereas n and B are extracted parameters.
- the exposed area of each finger was 450 ⁇ m by 50 ⁇ m, and the distance between finger centers was 80 ⁇ m.
- a HP4284A LCR meter Hewlett Packard Corp., now Agilent Technologies, Palo Alto, Calif. measured the impedance of the interdigitated electrodes at 52 frequencies, logarithmically spaced between 100 Hz and 1 MHz, with a 50 mV (amplitude) voltage excitation. The impedance of the wiring and probes was automatically substracted from the measurements, so that only the impedance of the elements in the biochip 220 was recorded.
- Tris-Gly buffer contained 3.6 mM Tris, ⁇ 4.7 mM Glycine, plus 0.05%(vol/vol) Tween-20 (a detergent). This detergent is necessary to prevent sticking of the cells to each other and to biochip 220 , which would clog the microscopic channels 212 , 218 in the biochip.
- the nominal pH and conductivity of the buffer were 7.4 and 33.5 ⁇ S/cm, respectively.
- the Glycine concentration was modified around the nominal value to adjust the pH close to 7.4.
- Previous (unpublished) macro-scale experiments using live and heat-killed L. innocua had clearly indicated that the bacteria remain alive in the Tris-Gly buffer and that their metabolism does indeed change the buffer conductance. After washing, the bacteria were re-suspended at concentrations of ⁇ 10 5 , ⁇ 10 7 , ⁇ 10 8 , and ⁇ 10 9 , cells/ml in the Tris-Gly buffer. These concentrations resulted in approximately 0.53, 53, 530, and 5300 bacterial cells (in average) in the 5.27 nl chamber or cavity 206 (FIGS.
- FIG. 7 shows the complex impedance (magnitude and angle) as a function of frequency for six different samples injected into biochip 220 : De-ionized (DI) water with a conductivity of 0.06 ⁇ S/cm, Tris-Gly buffer with 2.2 mg/ml dextrose, and the four L. innocua suspensions mentioned above.
- FIG. 11 is a bar graph similar to FIG. 10 and shows normalized differences of the above three measurement parameters for four concentrations of live Listeria innocua and four like concentrations of dead Listeria innouca cells. These results indicate that the viability of a captured sample of microorganisms could be assessed by measuring the change in impedance of the electrodes 36 in the detection chamber or cavity 34 , 204 , 206 .
- ⁇ Z is sufficient in many cases to enable detection of a target microbiological species or substance. In other cases, ⁇ R s is a more sensitive value.
- impedance is significantly different between buffer alone, and buffer containing microorganisms. Detection of 10 to 100 microorganisms is feasible. Where the electrode spacing is small (e.g., about one to three microns), detection results not only directly from the presence of the microorganisms in effective contact with the electrodes, but also indirectly from a change in electrolyte concentration in the liquid matrix close about the microorganisms, owing to the metabolic activity of the organisms. Where the electrode spacing is 5 microns or larger, bulk impedance is measured to study the effect of bacterial metabolism in the environment of the chambers, as discussed in detail below. In both approaches, the measurement of impedance reflects the generation of metabolites of cells and therefore gives a measure of whether the cells are living or dead.
- a method utilizing biochip 20 or 220 to detect the presence of a target microbiological entity such as a pathogenic strain of bacteria preferably includes a purification or bioseparations process performed immediately prior to, and optionally upon, injection into biochip 20 or 220 .
- This bioseparations process serves to remove, from the fluid sample tested in the detection chamber or cavity 34 , 204 , 206 of the biochip 20 , 220 , molecular and cellular detritus which would impede the accurate detection of the target species.
- the removal of such molecular and cellular detritus increases the signal-to-noise ratio and thus improves the accuracy and reliability of the measurement process.
- a bioseparations process as contemplated herein may utilize bioactive fibers and/or surfaces 62 exemplarily of cotton (cellulose) and packaged into a micro-pipette tip 64 , as depicted schematically in FIG. 12.
- Pipette tip 64 is inserted into a specimen of bodily fluids, foodstuffs, soil, etc., and operated (e.g., via a suction source such as a syringe-type plunger) to aspirate a raw fluid from the specimen.
- Bioactive fibers and/or surfaces 62 function to remove colloidal particles and extraneous proteins and to that end are derivatized with cation and/or anion ion exchange groups (represented in FIG.
- Fibers and surfaces 62 are packed loosely enough to allow a raw fluid sample to be aspirated from a specimen.
- the ion exchanger and appropriate conditions are selected so that the targets cells do not bind (see Ladisch, 1997) and can be injected, as indicated by an arrow 65 , into a second pipette tip 66 (FIG. 13) containing polyclonal antibodies 68 for the concentration of both pathogenic and nonpathogenic bacteria 70 , for instance, different species of Listeria.
- Polyclonal antibodies 68 are either immobilized to a fiber 62 or fixed to the inner surfaces of pipette tip 66 .
- the sample is then rinsed with a buffer solution, e.g., a phosphate buffer solution, to remove extraneous fluid.
- a buffer solution e.g., a phosphate buffer solution
- This sample is then pH modified and injected into microbore tube 28 (FIG. 1) for measurement.
- the affinity binding of a target bacterium such as Listeria monocytogenes to an antibody may be effectuated inside the biochip 20 , 220 and more particularly inside the detection chambers or cavities 34 , 204 , 206 .
- the antibodies are attached to the electrodes 36 (FIG. 1) via avidin-biotin coupling.
- the antibodies are biotinylated, i.e., chemically bonded to the biotin and the biotin is in turn adhered to avidin adsorbed onto the surfaces of electrodes 36 .
- FIG. 14A depicts avidin molecules 74 adsorbed onto an electrode 36 , as well as onto surrounding silicon oxide 54 (see FIGS. 5 A- 5 F).
- FIG. 14B shows biotinylated antibodies 76 attached to the adsorbed avidin molecules 74 .
- target bacteria cells 78 are depicted coupled to the biotinylated antibodies 76 and hence to electrode 36 and oxide 54 .
- a difference in electrical measurements between electrode 36 and a reference electrode 80 indicates the presence of target cells 78 in the detection chamber or cavity.
- the adsorption of avidin molecules 74 onto electrodes 36 of biochip 20 or 220 may be implemented as follows.
- Avidin is dissolved in 10 mM HEPES buffer containing 0.1 M NaCl, to obtain a concentration of the avidin of 5 mg/mL.
- the buffer has a pH of 8.5 and contains 0.08% sodium azide to prevent microbial growth.
- the avidin solution is then diluted with PBS (phosphate buffer saline) at a ratio of 0.2 mL to 0.8 mL to obtain a final concentration of 1 mg/mL.
- PBS phosphate buffer saline
- the chip 20 , 220 is then immersed in this solution, e.g., 1 mL thereof, overnight at room temperature.
- the antibodies are derivatized with biotin using conventional techniques, while the biotinylated antibodies are applied to the biosensor chip using the same methodology as used in adsorbing the avidin. Finally, all chips are rinsed in PBS for 5 minutes at room temperature.
- total protein peak area For the screening study, three replicate chromatography runs of the hot-dog juice extract were used to calculate the “total protein peak area” or the 100% point on the plot. A total of 15 peaks were used as a benchmark for this comparison. The areas of these 15 peaks were totaled to give “total peak area.” Values for the three replicate runs were then averaged together for the initial total protein peak area. Only a single injection of each time course study sample was made. Areas for each of the 15 components corresponding to the original time “0” sample were added together and compared against time “0.” Plots show % total “Protein Peak Area” remaining at each time course point in the study.
- IRA 400 results from the four most promising sorbents are shown: IRA 400, A-35, IRA 120+ and IRN-150.
- the top cation exchanger A-35 and the top anion exchanger IRA 400 were chosen for the final study.
- the screening study designed to measure the time course of adsorption monitored the “total protein” peak area remaining after exposure to the series of sorbents.
- each of the 15 benchmark peaks in the hotdog juice extract were followed after a 30 minute exposure to the sorbents.
- a figure is provided to give a visual representation to the numeric data for the IRA 400 experiment. As shown, the IRA 400 drops the peak maximums from 0.25-0.5 absorbance units to less than 0.10 AU.
- the avidin solution contacted with the chip was prepared from 0.2 mL of 5 mg/ml protein stock solution in 10 mM HEPES, 0.15M NaCl, and 0.08% sodium azide buffer (as supplied by Pierce) diluted with 0.8 mL PBS, pH 7.4, to obtain a protein concentration of 1 mg/mL.
- the chip was then placed in 0.2 mL of this solution for 15 hours at either room temperature or 37° C.
- the chips were immersed (rinsed) 3 times in PBS for 5 min each time to remove excess protein.
- the protein remaining on the chip was then measured using fluorescence microscopy at room temperature.
- Histograms of fluorescence intensity from the surface of the chip generated using MatLab and image analysis software show that more protein binds on the platinum than the silicon oxide and that more adsorption occurs at 37° C. than at room temperature (FIG. 15).
- peaks on the right side coincided with highest emission.
- the protein adsorbed at room temperature is removed when the chip is rinsed (dipped) in deionized water for 30 sec and then dried by a blast of dry compressed (120 psig) air for 15 sec.
- the result confirms that avidin is readily be absorbed onto both the silicon dioxide (e.g., oxide) and platinum surfaces of a chip in a wet state and removed in a dry state.
- FIG. 16 shows the histograms of the emission intensity.
- the precipitation process induced by ammonium sulfate enhanced the deposition of avidin on platinum.
- the ammonium sulfate promotes a greater degree of adsorption of avidin on platinum (lower graph, triangles). The opposite effect is noted for the oxide (upper graph, triangles) (FIG. 16).
- a possible explanation for the enhanced protein adsorption relates to the position of the ammonium sulfate, (NH 4 ) 2 SO 4 , on the lytotropic series. It is a kosmotrope and hence promotes an ordered arrangement of water molecules around itself and attracts water molecules away from the hydration layer that surrounds a protein in aqueous solution. The water layer around a protein helps to keep it in solution. The decrease in the water layer, such as happens in the presence of ammonium sulfate, promotes hydrophobic interactions between protein molecules and leads to their reversible aggregation and precipitation.
- the avidin itself maintains an active conformation on the surface of the chip.
- the avidin binds its target molecule (i.e., biotin) as indicated by the fluorescent signal obtained when labeled biotin is added to a chip that has previously been treated with unlabeled avidin, or unlabeled avidin and BSA.
- a chip treated with unlabeled BSA, then unlabeled avidin and finally fluorescein labeled biotin (FIG. 18) gave an emission brighter than that for an experiment where only unlabeled avidin (no BSA) was adsorbed onto the chip followed by addition of labeled biotin (data not shown).
- the confirmation of avidin adsorption, and its ability to bind biotin provides a method for fixing a primary antibody, specific for Listeria monocytogenes, onto biochip 20 , 220 by forming a biotinylated antibody.
- the biotin associated with the primary antibody protein binds with the avidin, already fixed onto the chip's surface (on electrodes 36 and oxide surfaces 54 ), and thereby anchors the antibody to which it is attached to the chip's surfaces.
- This type of approach is used in the preparation of particulate supports for affinity chromatography, and provides a viable option for biochips as well.
- Binding can be determined by visual interpretation of fluorescence micrographs of emission patterns of labeled antibodies or cells.
- coli, and BSA deposited on the surface of the chip further enhances the selectivity of the chip for one type of bacteria over another, even in the absence of the primary antibody. While an antibody is still needed as the bio-recognition element, selective materials design of the chip enhances the signal to noise ratio, if the chip's surface has lower affinity for non-pathogenic bacteria compared to pathogenic ones. In some applications, this design may or may not require deposition of a protein such as BSA.
- the monoclonal antibody MAb C11E9 binds with L. monocytogenes but also show some cross-reaction with some strains of Listeria innocua. Antigens that bind with this MAb are the 52, 55, 66, and 76 kDa surface proteins of the IgG2b subclass.
- a second antibody, Mab EM-7G1 binds with L. monocytogenes, and specifically with a 66 kDa surface protein (IG1 subclass). Despite the lower specificity of C11 E9, its binding activity is attractive, since the antibody differentiates between living and dead cells.
- transporting of a fluid sample from a food product specimen to the biochip 20 , 220 may be implemented via a microfiber wick 82 taking the place of microbore tube 28 (FIG. 1).
- a microfiber wick 82 is approximately 20 to 100 microns in diameter.
- Preliminary experiments consisted of threading the wicks through pH paper and then holding the wicks vertically while they were placed in an acid solution. Transport of the fluid to the pH paper was indicated by a change in color of the paper.
- Tested materials were: (1) Zwicky-Trys 1189 100/3 Spun-Polyester (pink); (2) Wooly Nylon 1000m, YLI Corp 161W, Nylon 100% #283 (red); (3) 001 Richardson Silk A (light brown); (4) 100% polyester (light brown); (5) 100% Spun Polyester 0001 (white);and (6) Super Sheen, mercerized, 40 (white).
- the times required for the transport of the fluids through the wicks of the indicated lengths were: (1) 3 minutes/2 cm; (2) 5 hours/0.5 cm; (3) 45 seconds/2 cm; (4) 1 minute/2 cm; (5) minute/2 cm; and (6) 45 seconds/2 cm.
- Wick 82 may be placed inside of a support tube 84 as shown in FIG. 19.
- the utilization of microwick 82 instead of microbore tube 28 for transporting the fluid sample is attractive since there are no moving parts or mechanical energy needed to deliver the sample.
- This methodology would not be feasible for laboratory scale assays, but is attractive for a biochip, since the samples that must be delivered to the sensor are preferably less than about 100 microliters and more preferably less than 1 microliter.
- the channels 32 , 212 , 218 in the biochips 20 , 220 are small (on the order of 100 microns), and hence diffusive transport at the chip surfaces will be a controlling parameter.
- a fluid sample 86 taken from bodily fluids, foodstuffs, soil, etc. contains live microorganisms 88 such as bacteria or single-cell fungi.
- the sample also contains contaminant biological matter or detritus 90 , that is, biological material which is not targeted by the detection process.
- biological material includes protein molecules and non-pathogenic cells, as well as molecular and cellular fragments.
- the present electronic method using biochip 20 or 220 is based on the confinement of a small number (1 to 1000) of the microorganism or microorganisms 88 of interest into a very small volume, on the order of 1 picoliter to 1 microliter, and measuring the changes in the electrical characteristics of the fluid in which the microorganisms are suspended. These changes are produced by the release of byproducts of the microorganism's metabolism into the fluid (mainly by the ionic species released).
- the microorganisms 88 may be selectively collected from the raw sample 86 by means of beads or microspheres or beads 92 functionalized with antibodies 94 specific to the microorganism of interest, affinity chromatography (also using antibodies), filtration using synthetic or natural membranes, or any other technique than can selectively and controllably separate and concentrate some of the microorganisms from the original sample.
- the microorganisms are suspended in a liquid medium having a low conductivity (lower than 100 gS/cm), such as Tris-Glycine buffer (3.6 mM Tris, 4.7 mM Glycine).
- a single or multiple non ionic nutrients such as a sugars, and enough dissolved oxygen (in the case of aerobic microorganisms) are added to stimulate bacterial metabolism.
- These nutrients can be selected such that they can be more easily metabolized by the microorganism of interest, than by other microorganisms that might be present due to inefficiencies in the selective collection method used. In this way, the selectivity can be increased beyond what the collection step provides.
- a container 102 FIG. 21
- a sample of low conductivity medium with nutrients but no microorganisms is injected into another container 104 (FIG. 21), identical to the first one.
- Some means of heating the containers 102 and 104 and controlling their temperature should be provided, such that the temperatures of the two containers do not differ by more than ⁇ 0.1° C.
- the preferred but not exclusive way of accomplishing this is by having the containers in very close physical contact.
- a pair of metallic electrodes 110 , 112 are either suspended in each container 102 , 104 , or attached to the walls thereof, with the electrodes 110 in one container 102 being identical in structure and composition to those 112 in the other container 104 .
- the preferred form of these electrodes is an interdigitated structure.
- the temperature of the containers is raised to a level that will stimulate the metabolism of the microorganisms and maintained at that level for several hours. While the samples are at this temperature, the AC electrical impedance of the electrodes in each container is repeatedly measured at several frequencies, between 100 Hz and I MHz, at time intervals on the order of minutes.
- a circuit model of the electrode-liquid medium-electrode system is fitted to the resulting frequency vs. impedance curves to extract the parameters of the model.
- the parameters of the model fitted to the curves measured at the container with bacteria change over time.
- the parameters extracted from measuring the impedance of the electrodes in the container with no bacteria remain constant within the limits imposed by the noise inherent in the measurement, since no metabolic activity is taking place in this container. If a statistical analysis and comparison of the parameters extracted from measuring both containers indicates that their difference is statistically significant, it can be concluded that the bacteria present in the first container have been detected.
- the present technique solves some of these problems.
- the technique described above inherently detects only live microorganisms, which is very important for certain applications, especially in food safety (many microorganisms present in food are not pathogenic if they are dead). It also relies exclusively on electrical signals, making the related equipment less expensive and smaller than others. Additionally, the absence of a growth step makes detection possible in a couple of hours instead of days.
- the present technique utilizing biochip 20 , 220 bypasses the first limitation by requiring a selective separation prior to the assay, and increases the sensitivity for very small numbers of microorganisms (1 to 1000) by confining them to an extremely small volume (1 picoliter to 1 microliter). Additionally, the present use of a low conductivity buffer increases even further the sensitivity. Since the ionic concentration of the low conductivity buffer is very low, even very small amounts of ions released by the microorganisms can produce a large change in impedance. In addition, measuring the impedance over a large range of frequencies (100 Hz to 1 MHz) and fitting a model circuit to the measurements also improves the sensitivity of the technique.
- a detection device such as biochip 20 or 220 has two identical detection chambers or cavities 102 , 104 with volumes between 1 picoliter and 1 microliter. Some means of heating the chambers 102 , 104 and controlling their temperature may be provided, such that the temperatures of the two chambers do not differ by more than ⁇ 0.1° C. (the preferred but not exclusive way of accomplishing this is by having the chambers in very close physical contact).
- the temperature can be controlled by one or more resistive heaters 106 and temperature sensors 108 , 109 microfabricated within or adjacent to the detection chambers.
- a pair of metallic preferably interdigitated electrodes 110 , 112 are either suspended in each chamber or cavity 102 , 104 or attached to its walls, with the electrodes 110 in one chamber 102 being identical in structure and composition to those 112 in the other chamber 104 .
- the chambers or cavities 102 , 104 (or 34 , 204 , 206 ) are designed so that the antibody-functionalized microspheres or beads 92 (FIG. 20) can be trapped inside them, while allowing fluids to pass through.
- the beads 92 can be trapped by a microfabricated filter-like structure 114 such as a grid or series of gating posts, with orifices or passages 116 large enough for non-target bacteria and other biological material 90 present in the injected sample to go through, but small enough to prevent the beads 92 , with the attached target microorganisms 88 from flowing out.
- a magnetic field 118 FIG. 20
- the magnetic field 118 can be established by permanent magnets or electromagnets 120 microfabricated within or adjacent to the detection chamber.
- Microorganism collection can be performed in two slightly different ways, after the sample has been concentrated and cleaned to remove excess salt, food debris, and other unwanted material.
- the beads 92 are mixed with the sample 86 containing the microorganisms 88 , outside of the measuring volume, and the antibodies 94 are allowed to capture the bacteria 88 for a specific period of time. This time should be long enough to allow the antibodies 94 on the beads 92 to capture all of the microorganisms 88 of interest that might exist in the sample 86 .
- the beads 92 can be separated from the sample by filtration or magnetically (in the case of magnetic beads), and resuspended in a “washing” fluid to completely eliminate any unwanted material (unwanted microorganisms, food debris, excess salt, etc.) that might have been left after the initial cleaning step; this fluid can also help remove any species non-selectively bound to the antibodies.
- the beads 92 are injected into a detection chamber 102 and trapped there (along with the microorganisms 88 they carry) by magnetic field 118 , in the case of magnetic beads 92 , or by filter structure 114 previously described.
- the sample 86 plus beads 92 can be injected directly into the chamber 102 and the washing step could be performed after the beads have been trapped inside the chamber.
- the beads 92 are first injected into a detection or measuring chamber 122 or 124 and trapped there by magnetic field 118 , in the case of magnetic beads 92 , or by filter structure 114 described above.
- the sample 86 containing the microorganisms 88 (which could have been previously purified and concentrated) is then flowed through the chamber 122 or 124 containing the beads 92 at a rate that would allow for any and all of the microorganisms 88 of interest to be captured by the antibodies 94 on the beads.
- a “washing” fluid is passed through the chamber 102 to wash away, from the chamber and the beads 92 , any unwanted material (unwanted microorganisms, food debris, excess salt, etc.) that might have been left after the initial cleaning step; this fluid can also help remove any species non-selectively bound to the antibodies.
- This second technique is similar to the principle of affinity chromatography, with the measuring chambers acting as chromatographic columns.
- the collection step is performed for two samples 86 with two separate sets of beads 92 , one set for each sample.
- One sample is the test sample being analyzed for the presence of microorganisms, the other is a “dummy” or reference sample, artificially prepared to ensure that it does not contain any microorganisms 88 .
- Each set of beads 92 is injected into one of the chambers 102 , 104 (or 122 ) in the detection device, and trapped there by the means described earlier. This results in one chamber 104 containing beads 92 which are guaranteed not to have any microorganisms 88 attached to them. This latter chamber may be called “the reference chamber,” while the other chamber 102 , which could have the microorganisms 88 of interest if they were present in the original sample, will be called “the detection chamber.”
- the chambers are filled with a liquid medium having a low conductivity (lower than 100 gS/cm), such as Tris-Glycine buffer (0.2 mM Tris, 4.7 mM Glycine).
- This medium also contains a single or multiple nonionic nutrients, such as sugars, and enough dissolved oxygen (in the case of aerobic microorganisms) to stimulate the microorganism's metabolism. These nutrients can be selected such that they can be more easily metabolized by the microorganism of interest than by other microorganisms that might be present due to inefficiencies in the antibody-mediated capture.
- the selectivity can be increased beyond what the antibody-based collection step provides.
- the temperature of both chambers is raised to a level that will stimulate the metabolism of the microorganisms 88 and maintained at that level for several hours. While the samples are at this temperature, the AC electrical impedance of the electrodes 110 , 112 in each chamber 102 , 104 (or 122 ) is repeatedly measured at several frequencies, between 100 Hz and I MHz, at time intervals on the order of minutes.
- a computer, or microprocessor, or microcontroller, or digital signal processor acquires the measured impedance vs. frequency data and analyzes it to extract certain parameters that will be the basis for detection.
- the parameters extracted from the curves measured at the detection chamber change over time because the microorganisms metabolize the provided nutrients and release ionic species into the medium. These ionic species, in turn, change the electric properties of the liquid medium and hence change the impedance of the electrodes in contact with the liquid.
- the parameters extracted from the impedance of the electrodes 112 in the reference chamber 104 remain constant within the limits imposed by the noise inherent in the measurement, since no metabolic activity is taking place in this chamber (it was guaranteed from the beginning that no bacteria would be present in the reference chamber).
- the sensing of a target microbiological species such as a pathogenic bacterium in a detection chamber or cavity 34 , 204 , 206 may be implemented via circuit designs other than electrodes 36 ( 110 , 112 ).
- a binding agent such as an avidin-biotinylated antibody may be attached to a gate of a silicon MOSFET.
- the MOSFET is a charge sensor where charge changes induced on the gate by the coupling of a target microbiological species become mirrored in a channel region under the gate insulator.
- the device must be biased in the sub-threshold regime where the dI/dV G slope is the maximum, i.e., the drain to the source current (I DS ) is maximum as a function of voltage on the gate (V G ).
- the device can be biased in the appropriate regime using the back bias or a dual gated MOSFET where the threshold of the top gate is controlled by the bottom gate.
- the double layer interfacial capacitance changes with the binding of the antigen and the related conformation changes.
- the simple MOSFET of this detection structure is fabricated in silicon.
- the device has gate oxides of less than 150 ⁇ . Platinum is used as the gate material and the exposed gate area may vary from 100 ⁇ m ⁇ 100 ⁇ m to 2 ⁇ m ⁇ 2 ⁇ m.
- the fabrication of the MOSFET is standard and double gated MOSFETs may also be used.
- Each device has a source, drain and body terminal in addition to the open (exposed) gate terminal.
- the devices are packaged and biochemically treated with binding agents as described hereinabove.
- the main difference is that the measurement consists of source to drain current measured by a high-precision pico-ammeter, a semiconducter parameter analyzer, or a digital oscilloscope.
- the device is biased using a DC and AC signal and the measurements will be taken before, during and after the binding of the avidin to the biotinylated gate electrode. Only the binding event taking place on the gate electrode affects the source/drain current measurement.
- the substrate of a biochip may be of a material other than silicon, including but not limited to glass such as Coming 7740 , and polymers such as polyethylene based plastics and polytetrafluoroethylene.
- the bulk solution resistance R S may be determined directly using a four point probe sheet resistivity measurement.
- four electrodes are positioned in a detection chamber at comers of a quadrilateral such as a square. Current is conducted between two diagonally opposed electrodes, while voltage is measured across the other two diagonally disposed electrodes. The interfacial impedance Z W is automatically eliminated.
Abstract
A microscale biosensor for use in the detection of target biological substances including molecules and cells is a microfluidic system with integrated electronics, inlet-outlet ports and interface schemes, high sensitivity detection of pathogen specificity, and processing of biological materials at semiconductor interfaces. A fabrication process includes an all top-side processing for the formation of fluidic channels, planar fluidic interface ports, integrated metal electrodes for impedance measurements, and a glass cover sealing the non-planar topography of the chip using spin-on-glass as an intermediate bonding layer. Detection sensitivity is enhanced by small fluid volumes, use of a low-conductivity buffer, and electrical magnitude or phase measurements over a range of frequencies.
Description
- This application relies for priority purposes on U.S. provisional application No. 60/197,560 filed Apr. 17, 2000.
- [0002] This invention was made with Government support under a USDA cooperative agreement: CRIS number 1935-42000-035-00D, Agreement #58-1935-9-010. This invention was also partially funded through a NSF IGERT graduate student fellowship. The Government has certain rights in the invention.
- The present invention relates to an integrated-chip-type biosensor and a related method for detection of pathogenic substances. The biosensor and method are particularly, but not exclusively, useful in detecting foodborne pathogens such asListeria monocytogenes.
- Pathogenic bacteria in foods are the cause of 90% of the cases of reported foodborne illnesses. The Centers for Disease Control and Prevention estimate that there 76 million cases of foodborne illnesses each year in the United States, resulting in hospitalization of 325,000 people, 5,500 deaths, and an annular cost of $7 to $23 billion.E. coli O157:H7 and Listeria monocytogenes are the pathogens of most concern. Ground meat containing E. coli O157:H7 is now considered to be an adulterated food while Listeria monocytogenes has emerged as one of the most important food pathogens with a “zero tolerance” criterion for it in ready-to-eat processed (lunch) meats and dairy foods.
- The genus Listeria is comprised of six species,L. monocytogenes, L. ivanovii, L. seeligeri, L. innocua, L. welshimeri, and L. grayi. Of these species, only L. monocytogenes is harmful to humans. Consumption of contaminated food may cause meningitis, encephalitis, liver abscess, headache, fever and gastroenteritis (diarrhea) in immunologically challenged individuals and abortion in pregnant women. L. monocytogenes is ubiquitous in nature and can be found in meat, poultry, seafood, and vegetables. Occurrence of this organism could be as high as 32%. In a food sample, L. monocytogenes is often present in close association with other nonpathogenic Listeria species, thereby complicating the specific detection procedures. A successful detection method ideally detects only L. monocytogenes in the presence of overwhelming populations of nonpathogenic Listeria and other background resident bacteria.
- The food processing industry annually carries out more than 144 million microbial tests costing $5 to $10 each. About 24 million of these tests are for detection of food pathogens based on biochemical profile analysis, immunogenic tests (such as enzyme linked immuno-sorbent assays or ELISA), and DNA/RNA probes. These tests are reliable but most require two to seven days to complete because of the steps that are needed to resuscitate cells, increase cell numbers or amplify genetic material needed for detection. This time period is too long for real-time detection of contamination in a food plant and is sufficiently long for contaminated food to be formulated, processed, packaged, shipped, and purchased and eaten by the consumer. Current tests require at least several days to confirm presence ofListeria monocytogenes. The number of annual tests is only expected to increase due to heightened consumer concerns about food safety and the requirement of compulsory testing.
- In general, diagnostic tools used for detecting or quantitating biological analytes rely on ligand-specific binding between a ligand and a receptor. Ligand/receptor binding pairs used commonly in diagnostics include antigen-antibody, hormone-receptor, drug-receptor, cell surface antigen-lectin, biotin-avidin, substrate/enzyme, and complementary nucleic acid strands. The analyte to be detected may be either member of the binding pair; alternatively, the analyte may be a ligand analog that competes with the ligand for binding to the complement receptor.
- A variety of devices for detecting ligand/receptor interactions are known. The most basic of these are purely chemical/enzymatic assays in which the presence or amount of analyte is detected by measuring or quantitating a detectable reaction product, such as a detectable marker or reporter molecule or ligand. Ligand/receptor interactions can also be detected and quantitated by radiolabel assays.
- Quantitative binding assays of this type involve two separate components: a reaction substrate, e.g., a solid-phase test strip and a separate reader or detector device, such as a scintillation counter or spectrophotometer. The substrate is generally unsuited to multiple assays, or to miniaturization, for handling multiple analyte assays from a small amount of body-fluid sample.
- In recent years, there has been a merger of microelectronics and biological sciences to develop what are called “biochips.” The term “biochip” has been used in various contexts but can be defined as a “microfabricated device that is used for delivery, processing, and analysis of biological species (molecules, cells, etc.).” Such devices have been used, among other things, for the direct interrogation of the electric properties and behavior of cells (Borkholder et al. “Planar Electrode Array Systems for Neural Recording and Impedance Measurements”,IEEE Journal of Microelectromechanical Systems, vol 8(1), pp. 50-57, 1999); impedance-based detection of protein binding to surfaces, antigen-antibody binding, and DNA hybridization (DeSilva et al., “Impedance Based Sensing of the Specific Binding Reaction Staphylococcus Enterotoxin B and its Antibody on an Ultra-thin Platinum Film,” Biosensors & Bioelectronics, vol. B 44, pp 578-584, 1995); micro-scale capillary electrophoresis (Wooley et al., :Ultra High Speed DNA Sequencing Using Capillary Electrophoresis Chips,” Analytical Chemistry, vol. 67(20), pp. 3676-3680, 1995); and optical detection of DNA hybridization using fluorescence signals in the commercially available “DNA-chips” (Fodor et al., “Light-directed Spatially Addressable Parallel Chemical Synthesis,” Science, vol. 251, pp. 767-773).
- One of the most interesting uses of biochips is for the detection of small quantities of pathogenic bacteria or toxigenic substances in food, bodily fluids, tissue samples, soil, etc. In applications such as the screening of food products for the presence of pathogenic bacteria, it would be beneficial to detect between 100 and 1000 microorganisms per milliliter of sample, with a sample volume of a couple of milliliters. Not counting the fact that bacteria are substantially larger than single biomolecules (˜2 μm vs. ˜10-100 Å), 1000 cells are approximately equivalent to a 10−5 femto-moles of cells, which gives an idea of the difficulty in directly detecting such a small number suspended in a volume of 1 or 2 ml, along with large numbers of food debris, proteins, carbohydrates, oils, and other bacteria. Additionally, in many cases the screening technique must be able to discern between viable and dead cells. Many bacteria will not produce toxins when not viable and consequently will not be pathogenic in that state. DNA detection methods, which search for DNA sequences specific to the pathogen of interest, can be extremely sensitive because they rely on the very specific binding of complementary DNA strands, often coupled with Polymerase Chain Reaction (PCR) for amplification. But the detected DNA fragments cannot reveal whether the pathogen was viable or not. These are the main reasons why current methods of detection almost always involve a growth step, in which the bacteria are cultured to increase their numbers by several orders of magnitude. Once the bacteria are amplified to a large number, visual detection of colonies or Enzyme-Linked Immunosorbent Assays (ELISA) confirm their presence in the original sample. Even though bacteria can multiply very rapidly, this amplification by means of extended growth makes conventional detection methods extremely lengthy, taking anywhere from 2 to 7 days. Thus, one of the main goals of micro-scale detection is a reduced time of analysis, on the order of 2 to 4 hours, to be better than the more conventional methods like plate counts and ELISA.
- Numerous reports can be found in the literature on biosensors based on the impedimetric detection of biological binding events, or the amperometric detection of enzymatic reactions. (See DeSilva et al., “Impedance Based Sensing of the Specific Binding Reaction Staphylococcus Enterotoxin B and its Antibody on an Ultra-thin Platinum Film,”Biosensors & Bioelectronics, vol. B 44, pp 578-584, 1995; Mirsky et al., “Capacitive Monitoring of Protein Immobilization and Antigen-antibody Reactions on Monomolecular Alkylthiol films on Gold Electrodes,” Biosensors & Bioelectronics, vol. 112(9-10), pp. 977-989, 1997; Berggren et al., “An Immunilogical Interleukine-6 Capacitive Biosensor Using Perturbation with a Potentiostatic Step,” Biosensors & Bioelectronics, vol. 13, pp. 1061-1068, 1998; Van Gerwen et al., “Nanoscaled Impedimetric Sensors for Multiparameter Testing of Biochemical Samples,” Sensors and Actuators, vol.
B 49, pp. 73-80, 1998; Hoshi et al., “Electrochemical Deposition of Avidin on the Surface of a Platinum Electrode for Enzyme Sensor Applications,” Analytical Chimica Acta, vol. 289, pp. 321-327, 1994; Jobst et al., “Mass producible Miniaturized Flow Through a Device with a Biosensor Array,” Sensors and Actuators, vol. B 43, pp. 121-125, 1997; Towe et al., “A Microflow Amperometric Glucose Biosensor,” Biosensors & Bioelectronics, vol. 97(9), pp. 893-899, 1997.) Impedimetric detection works by measuring impedance changes produced by the binding of target molecules to receptors (antibodies, for example) immobilized on the surface of microelectrodes. Amperometric devices measure the current generated by electrochemical reactions at the surfaces of microelectrodes, which are commonly coated with enzymes. Both of these methods can be very sensitive, but preparation of the surfaces of the electrodes (immobilization of antibodies or enzymes) is a complex and sometimes unreliable process, they can be prone to drift, and tend to be very sensitive to noise produced by the multitude of species present in real samples (bodily fluids, food, soil, etc.). - Most, if not all, of the above-mentioned devices are not fully integrated biochips, and sometimes lack integrated electrodes and a sealed fluidic path for the injection and extraction of samples. The most common design of these sensors uses thin metal rods or wires as electrodes, immersed in a flow-through cell. And even those devices based on microfabricated biochips either have a fluidic system separately fabricated over the chip, or the samples are dropped over an open reservoir on the chip, or the whole chip is immersed in a vessel containing the fluids. Having a fully closed system permits the incorporation of sample pre-processing steps, like filtering and chromatography, onto the same chip as the detector.
- As mentioned earlier, one of the main goals of bacterial sensors is to determine whether the bacterium of interest is indeed live or dead. A technique that has been widely reported to detect the viability of bacteria on a macro-scale relies on measuring the conductance/impedance changes of a medium in which the microbes are cultured. Such a method is recognized by the Association of Official Analytical Chemists International (AOAC) as a standard technique for the detection of Salmonella in food. This is possible because bacterial metabolism changes the electrolyte concentration in the suspension medium, significantly altering the electrical characteristics of the medium.
- It is a general object of the present invention to provide a method and/or an associated apparatus for detecting whether a microbiological substance is present in a fluid sample.
- A more specific object of the present invention is to provide a method and/or an associated device for a more rapid detection of foodborne pathogens, particularly including, but not necessarily limited to,Listeria monocytogenes.
- An even more specific object of the present invention is to provide such a method and/or device which detects pathogens in a few hours or less, possibly within minutes.
- A further specific object of the present invention is to provide such a method and/or device which is capable of detecting a relatively small number of instances of a pathogen such as a bacterium.
- Another specific object of the present invention is to provide such a method and/or device which is able to distinguish between a sample of live bacteria and a sample of dead bacteria of the same type.
- Another object of the present invention is to provide a method for manufacturing a biosensor, particularly a microscale biosensor.
- These and other objects of the present invention will be apparent from the drawings and descriptions herein. Every object of the invention is considered to be attained by at least one embodiment of the invention. However, no embodiment necessarily meets every object set forth herein.
- The present invention is directed in part to a microscale biosensor for use in the detection of target biological substances including molecules and cells. A preferred embodiment of a biosensor pursuant to the present invention is a microfluidic system with integrated electronics, inlet-outlet ports and interface schemes, high sensitivity detection of pathogen specificity, and processing of biological materials at semiconductor interfaces.
- The present invention is also directed in part to a fabrication process for a microfluidic biochip that is used for impedance spectroscopy of biological species. Key features of the device include an all top-side processing for the formation of fluidic channels, planar fluidic interface ports, integrated metal electrodes for impedance measurements, and a glass cover sealing the non-planar topography of the chip using spin-on-glass as an intermediate bonding layer. In one embodiment of the biosensor chip, the total volume of the fluidic path in the device is on the order of 30 nl.
- A method in accordance with the present invention for detecting a microbiological substance utilizes a microfabricated biosensor chip including integrated detection elements. The method comprises delivering a fluid sample to the biosensor chip and thereafter separating at least some contaminants or debris from the fluid sample to at least partially isolate and retain instances of a predetermined target type of microbiological material, a material to be detected, on the biosensor chip. The separating of the contaminants takes place at least in part on the biosensor chip itself. After the separating of contaminants from the fluid sample, the detection elements are operated to determine whether the separated fluid sample contains microbiological material of the predetermined target type.
- This method may further comprise carrying out a bioseparations process on the fluid sample prior to the delivering of the fluid sample to the biosensor chip. In accordance with one embodiment of the present invention, the bioseparations process includes adding to the fluid sample a plurality of microscopic carrier elements each provided with a multiplicity of binding agents for coupling the microbiological material to the carrier elements. These carrier elements preferably take the form of beads or microspheres. The separating of contaminants from the fluid sample on the biosensor chip preferably includes trapping the carrier elements with the coupled microbiological material in a detection chamber on the biosensor chip while flushing remaining portions of the fluid sample from the chamber. This trapping of the carrier elements with the coupled microbiological material in a detection chamber serves in part to concentrate the microbiological material of interest and thus enhance the sensitivity of the detection technique. The trapping of the carrier elements may be implemented in part by providing a filter barrier or retention structure at an outlet of the detection chamber. Such a barrier or retention structure preferably takes the form of a microfabricated filter grid or post array. Alternatively, the trapping of the carrier elements, where the carrier elements are made of a magnetic material, in a magnetic field generated in the detection chamber.
- In accordance with another, more particular, feature of the present invention, the bioseparations process includes subjecting a the fluid sample (prior to delivery to the biosensor chip, to a bioactive surface taken from the group consisting of a cation exchange resin and an anion exchange resin. The cation exchange resin may include
Amberlyst 35 while the anion exchange resin includes IRA 400. - The present invention is especially effective in detecting microbiological material in the form of a pathogenic strain of bacteria such asListeria monocytogenes. In that case, the methodology includes extracting the fluid sample from a food product prior to delivering of the fluid sample to the biosensor chip. As discussed below, the detection of Listeria monocytogenes is implemented in part by attaching antibodies to a capture surface in the detection chamber of the biosensor. That capture surface may be on an electrode or oxide surface in the detection chamber. Alternatively, the capture surface may be on a bead or microsphere floating in the detection chamber. It will be apparent to one of ordinary skill in the art that virtually any microorganism may be detected by the method of the present invention simply by attaching an appropriate antibody to a capture surface as described herein. Antibodies and their associated antigens on the cell membranes of various microorganisms are well documented in the art. It will also be apparent to one skilled in the art that species other than bacteria may be detected by the methodology of the present invention. Various proteins, peptide groups, nucleic acid chains, and other molecules may be detected by the selection of suitable binding agents and the attachment of those binding agents to a capture surface in a detection chamber of a biosensor.
- A biosensor in accordance with the present invention comprises a substrate microfabricated to include, as integrated components, a detection chamber, a first channel segment extending to an inlet of the detection chamber, a second channel segment extending from an outlet of the chamber, and a retention structure for holding, in the detection chamber, a carrier element entraining a target microbiological species and for permitting the passage from the detection chamber of contaminants or debris in a fluid sample containing the carrier element and the target microbiological species. The retention structure may take the form of a filter grid or grating disposed on the substrate on an upstream side of the outlet. Alternatively or additionally, where the carrier element is made of magnetic material, the retention structure may include a magnetic field generating element such as an electromagnet.
- The retention structure on the biosensor enables the concentration of a target microbiological species at the point of measurement. This facilitates and enhances the detection process. The small size of the detection chamber, less than 100 microliters and preferably between about 1 picoliter and 1 microliter, also increases the sensitivity of the detection process. Yet another factor contributing to the efficacy of the present methodology is the use of a low conductivity buffer as the fluid matrix in which the microbiological species of interest is entrained in the detection chamber.
- The detection chamber is provided with at least one pair of electrodes, preferably with interdigitated finger parts, and has a volume of less than approximately one microliter. The volume of a fluid sample in the device may be substantially less than one microliter, even down to about 1 picoliter. The electrodes are spaced from each other by 1 to 100 microns and, more preferably, by 2 to 50 microns.
- A biosensor in accordance with another embodiment of the present invention comprises a substrate microfabricated to include, as integrated components, a detection chamber and a channel extending to an inlet of the detection chamber. The biosensor further comprises a wicking element connected at one end to the substrate so as to be in communication with the channel, for drawing a fluid sample by capillary action to the channel for delivery to the detection chamber. The wicking element may be attached at the one end by an adhesive to the substrate. Where the substrate is microfabricated to include an inlet groove or trench substantially coplanar with the channel and the detection chamber, the one end of the wicking element is disposed in the inlet groove or trench, so that the wicking element is coplanar at the one end with the channel and the detection chamber.
- An integrated microscale biosensor in accordance with a further embodiment of the present invention comprises a substrate microfabricated to include, as integrated components, a detection chamber, a channel extending to an inlet of the detection chamber, and an inlet groove or trench substantially coplanar with the channel and the detection chamber. The biosensor further comprises an elongate fluid delivery member having a downstream end disposed in the inlet groove or trench. The fluid delivery member is connected at the downstream end to inlet groove or trench so that at least the downstream end of the fluid delivery member is coplanar with the channel and the detection chamber. The elongate fluid delivery member may take the form of a microbore tube or a wicking element.
- Preferably a biosensor chip in accordance with the present invention is top-side processed only. In addition, there is no processing (e.g., cutting) of a cover plate. This structure facilitates the manufacturing process, in part by obviating alignment requirements between the cover plate and the substrate. Thus, the cover attached to the substrate over the detection chamber, the channel, the inlet groove, and the downstream end of the fluid delivery member can be an integral or continuous member, devoid of holes or apertures. Such holes or apertures would be required, for instance, where a feed tube was to be inserted through the cover.
- A method for manufacturing a biosensor comprises, in accordance with the present invention, providing a substrate, processing the substrate to generate a detection chamber and a channel extending to the detection chamber, further processing the substrate to provide at least one pair of electrodes in the detection chamber, and exposing the processed substrate to BSA (bovine serum albumin) and avidin to adsorb the avidin to the electrodes in the presence of the BSA.
- This manufacturing method may further comprise subjecting the exposed processed substrate to a fluid containing a biotinylated antibody specific to a preselected antigen, thereby attaching the antibody to the electrodes via a biotin-avidin link. In a particular embodiment of the invention, the biotinylated antibody is specific to an antigen on a cell membrane ofListeria monocytogenes. Monoclonal antibody producing clones of C11E9 and EM-7G1 (producing antibodies specific for Listeria monocytogenes) are cultured in growth media in a growth chamber. Antibodies are harvested from culture supernatants by salt (ammonium sulfate) precipitation. After an initial concentration step, carried out by known techniques, high quality antibodies are obtained by further purification through size exclusion chromatography followed by protein-A affinity chromatography in an FPLC system.
- A method for manufacturing a biosensor comprises, pursuant to another embodiment of the present invention, processing a substrate to create a shallow detection chamber and a channel extending to the detection chamber, thereafter further processing the substrate to deposit at least one pair of electrodes in the detection chamber, and subsequently processing the substrate to create at least deep groove at a periphery of the substrate, for receiving an elongate fluid delivery element, the channel communicating with the deep groove. A downstream end of the fluid delivery element is inserted into and attached to the deep groove.
- This method may further comprise attaching a cover to the substrate over the detection chamber, the channel, the deep groove and the downstream end of the fluid delivery element. Where the cover is made of glass, the attaching of the cover to the substrate includes placing a spin-on-glass composition on the glass, subsequently contacting the substrate with the spin-on-glass composition, and heating the substrate, the cover, and the spin-on-glass composition to enabling a flow of the spin-on-glass composition into interstitial spaces on the substrate and form a fluid-tight seal.
- A method for detecting a microorganism comprises, in accordance with the present invention, preparing a fluid sample containing at least one microorganism of a preselected type, the fluid sample having a buffer of a low conductivity liquid, the fluid sample also containing a nonionic nutrient. The fluid sample is disposed in or delivered to a detection chamber having a volume between about 1 picoliter and approximately 1 microliter. The fluid sample is maintained at a predetermined temperature in the detection chamber and an electrical parameter of an electrical circuit incorporating the detection chamber and the fluid sample therein is measured. The electrical parameter is an impedance measure taken from the group consisting of a magnitude and phase. The method is effective in the detection of livingListeria monocytogenes cells. The buffer may be a low conductivity Tris-Glycine buffer.
- In accordance with another feature of the present invention, the measuring of the electrical parameter includes measuring the impedance parameter at a plurality of frequencies within a range from 100 Hz to 1 MHz.
- A method for testing a food product for the presence of a predetermined type of pathogenic bacteria comprises, in accordance with the present invention, extracting a fluid sample from the food product, feeding the extracted fluid sample providing an integrated microscale biosensor, subjecting the fluid sample to a bioseparations process to remove extraneous particles including proteins and kinds of bacteria other than the predetermined type of pathogenic bacteria, binding bacteria of the predetermined type in the fluid sample to at least one substrate body, and, after the feeding of the extracted fluid sample to the chamber, the subjecting of the fluid sample to the bioseparations process, and the binding of the predetermined type of bacteria to the at least one substrate body, measuring an electrical parameter of an electrical circuit incorporating the detection chamber and the fluid sample therein to detect the presence in the fluid sample of living instances of the predetermined type of bacteria. The binding of the predetermined type of bacteria may be to beads or microspheres floating in the fluid sample. Alternatively or additionally, the binding of the predetermined type of bacteria may be to electrodes in the biosensor. Subjecting of the fluid sample to the bioseparations process may take place at least partially after feeding of the fluid sample to the biosensor.
- The sensitivity to biological pathogens of a biosensor chip in accordance with the present invention is based on the placement of protein receptors, derived through biotechnology processes, on a surface of the biosensor. A tiny amount of fluid taken from a specimen such as a processed meat or dairy product is then delivered to the biosensor. If a target bacterium such asListeria monocytogenes is present, it will bind to the receptor and cause a measurable electronic signal to be generated in no more than several hours and possibly within minutes.
- The present invention provides a method and an associated device for the relatively rapid detection of biological pathogens such as bacteria. The method and device can detect small numbers of bacteria such asListeria monocytogenes in time intervals short enough to enable removal of contaminated products from the stream of commerce before consumption of the products by individuals.
- Biosensors or biosensors as disclosed herein improve the quality of life by providing cost-effective means for probing biological materials for pathogenic organisms and molecules in manufacturing facilities, the environment, hospitals, doctors' offices, and ultimately in the home.
- The present invention provides a method and an associated device for a relatively rapid detection of foodborne pathogens. The present invention obviates the time-consuming steps of culturing and transferring cells, if present, to increase their numbers or genetic material to the detectible levels required by conventional detection techniques.
- The vast majority of the bacterial detection methods currently in use are based on fluorescent tagging of the bacteria, or on the detection of DNA fragments from the bacterial genome. Both techniques are unable to determine if the microorganism was dead or alive in the original sample, and both require extensive manipulations of the sample. Moreover, any fluorescence technique requires bulky and expensive optical apparatuses for excitation and detection of the fluorescence. Additionally, when the microorganism is present in very small concentrations (10 to 1000 cells per milliliter) a growth step is necessary to increase the concentration, but this can drive the total assay time to anywhere from 2 to 7 days.
- The present technique solves some of these problems. By its very nature, the present methodology inherently detects only live microorganisms, which is very important for certain applications, especially in food safety (many microorganisms present in food are not pathogenic if they are dead). The method of the present invention also relies exclusively on electrical signals, making the related equipment less expensive and smaller than others. Additionally, the absence of a lengthy growth step makes detection possible in a couple of hours instead of days.
- Instruments for the analysis of the conductivity or impedance of an incubated bacterial suspension have been available for a number of years, but they suffer from two limitations. First, their selectivity is very poor because they rely on the composition of the growth medium for encouraging the proliferation of the microorganism of interest, while suppressing the proliferation of others. The second limitation is related to the scale in which the assay is performed. The available equipment uses volumes of bacterial suspension in the milliliter range and above, which requires large numbers of bacteria to provide a discernible signal. The method of the present invention eliminates the first limitation by selectively capturing the bacteria using antibodies prior to the measurement, and increases the sensitivity for very small numbers of microorganisms (1 to 1000) by confining them to an extremely small volume (1 picoliter to 1 microliter). Additionally, the method of the present invention uses a low conductivity buffer, which increases even further the sensitivity. Even very small amounts of ions released by the microorganisms can produce a large change in impedance (in relative terms), since the ionic concentration of the low conductivity buffer is very low.
- FIG. 1 is a schematic top plan view of a biosensor in accordance with the present invention.
- FIG. 2 is a photograph showing, in top plan view, an integrated microscale biosensor in accordance with the present invention.
- FIG. 3 is a photomicrograph, on a larger scale, of a portion of the biosensor of FIG. 2.
- FIG. 4 is a photomicrograph, on an even larger scale, of another portion of the biosensor of FIG. 2.
- FIGS. 5A through 5F are schematic cross-sectional views, on an enlarged scale, showing successive steps in a manufacturing process in accordance with the present invention.
- FIG. 6 is a circuit diagram modeling electrical activity in a biosensor as illustrated in FIG. 1 or FIGS.2-4.
- FIG. 7 is a pair of graphs showing measured complex impedance (magnitude and angle) of different microorganism-containing samples injected into a biochip in accordance with the present invention. The numbers of cells in the legend correspond to the numbers present in a detection chamber of the biochip where the measurement was performed.
- FIG. 8 is a pair of graphs showing differences between the complex impedance of a Tris-Gly buffer and each one of the different samples injected into the biochip. Again, the numbers of cells in the legend correspond to the numbers present in a detection chamber of the biochip where the measurement was performed (vol. =5.27 nl).
- FIG. 9 is a pair of graphs of complex impedance (magnitude and angle vs. frequency), showing a fit between the circuit model of FIG. 6 and the measured complex impedance of the microorganism-containing samples at a concentration of ˜105 cells/ml.
- FIG. 10 is a bar graph showing normalized differences of three measurement parameters for each microorganism-containing sample injected into the biochip of the present invention.
- FIG. 11 is a bar graph similar to FIG. 10, showing normalized differences of three measurement parameters for each of several live-microorganism-containing samples and each of several dead-microorganism-containing samples injected into the biochip of the present invention, demonstrating an ability to distinguish between live and dead microorganisms.
- FIG. 12 is a schematic cross-sectional view of a pipette tip with structure for preparing a biological sample for testing with a biosensor in accordance with the present invention.
- FIG. 13 is a schematic cross-sectional view of an ancillary pipette tip with respective structure for preparing a biological sample for testing with a biosensor in accordance with the present invention.
- FIGS. 14A through 14C are diagrams showing successive stages in manufacturing and testing processes for automated detection of microorganisms with a biosensor or biochip as illustrated in FIG. 1 or FIGS.2-4.
- FIG. 15 is a pair of histogram graphs plotting fluorescence emission from surfaces of a biosensor or biochip, in accordance with the present invention, incubated in 1 mg/mL avidin at room temperature for 18 hours and at 37° C. for 15 hours and then rinsed in DI water followed by drying with compressed air.
- FIG. 16 is a pair of histogram graphs plotting fluorescence emission from surfaces of biochips in accordance with the present invention, where a first biochip was unprocessed, a second biochip was processed for avidin adsorption at 37° C. for 15 hours without ammonium sulfate, and a third biochip was processed for avidin adsorption at 37° C. for 15 hours with ammonium sulfate. Histogram data labeled “control” correspond to untreated surfaces.
- FIG. 17 is a pair of histogram graphs plotting fluorescence emission from surfaces of biochips in accordance with the present invention, where a first biochip was unprocessed, a second biochip was treated with labeled avidin, a third biochip was treated with unlabeled BSA and labeled avidin, and a fourth biochip was treated with labeled BSA.
- FIG. 18 is a pair of histogram graphs plotting fluorescence emission from surfaces of a reference biochip that was not contacted with any protein solution, a second biochip contacted with BSA and labeled avidin, a third biochip contacted with BSA and labeled biotin, and a fourth biochip contacted with BSA followed by unlabeled avidin and labeled biotin, as indicated.
- FIG. 19 is a schematic cross-sectional view of an alternative biochip design in accordance with the present invention.
- FIG. 20 is a FIG. 21 is a partial schematic top plan view of a biosensor or biochip in accordance with the present invention, showing a detection chamber with interdigitated electrodes, heating element and temperature sensor.
- FIG. 22 is a diagram illustrating steps in another bioseparation procedure utilized in a biodetection process in accordance with the present invention.
- Avidin is a protein with four identical subunits and a total molecular weight of 67,000-68,000 daltons. Biotin is a vitamin (B-6) having a molecular weight of 244 daltons. Each subunit of an avidin molecule binds one molecule of biotin. The binding action is pronounced: affinity of biotin to avidin is very strong (Ka=1015 M−1). The avidin-biotin system is well-established and extensively used as a biological probe.
- The word “biotinylated” is used herein to generically describe a preselected molecule, generally a protein, which has been derivatized with biotin. Where avidin, has been adsorbed to a capture surface such as a surface of an electrode in a detection chamber, the biotin functions to secure the preselected molecule to the capture surface via the avidin-biotin linkage.
- The term “binding agent” is used herein to denote a chemical structure such as an antibody or a molecular complex (two or more molecules coupled together) capable of latching onto or capturing a target microbiological species or material which is to be detected in a biochip sensor pursuant to the techniques described herein. A biotinylated antibody bound to avidin on a capture surface of an electrode serves as a binding agent for a target bacterium having a cell membrane carrying the antigen of the biotinylated antibody.
- The terms “biosensor” and “biochip” as used herein refer to microelectronic-inspired construction of devices that are used for processing (delivery, analysis or detection) of biological molecules and cellular species. Thus, a biosensor or biochip as described herein is a microfluidic system with integrated electronics, inlet-outlet ports and interface schemes, high sensitivity detection of pathogen specificity, and processing of biological materials at semiconductor interfaces.
- The word “bioseparation” or “bioseparations” as used herein refers to a process for removing contaminants and detritus from a fluid sample possibly containing a target microbiological species.
- The term “capture surface” as used herein refers to a surface in a biochip sensor or in a preseparation process which is prepared with a binding agent for purposes of latching onto and holding, at least for the duration of a detection process, a target substance, whether that target consists of a molecule such as a protein, an antibodies, an antigens, or an enzyme; a molecular fragment such as a peptide or a DNA sequence; or a cell such as a muscle cell or a bacterium; a virus; etc.
- The word “carrier” as used herein refers to movable structures to which binding agents are attached for securing, anchoring or attaching target microbiological materials. One kind of carrier is a microsphere or bead made of magnetic or nonmagnetic material.
- The words “contaminants” and “detritus” are used herein to describe various microscopic and submicroscopic cells, cellular fragments, molecules, molecular fragments, which are of no interest to a biosensor detection process in accordance with the present invention. Contaminants can be disruptive of the detection process, for example, by causing noise to electrical detection.
- The term “detection chamber” is used herein to generally designate a space provided with sensors for measuring a change in a predetermined parameter owing to the presence of a target microbiological species in the detection chamber. In a more specific embodiment of the invention, the term “detection chamber” is used to designate a small well or cavity produced by microfabrication techniques in a wafer and provided with sensing elements such as electrodes for sensing a change in an electrical characteristic or parameter (such as resistance or phase) in the chamber owing to the presence of the target microbiological species. This specific detection chamber has a small volume, no more than 100 microliters, and preferably no more than 1 microliter, and even more preferably, in a range about 1 to 10 nanoliters.
- The term “low conductivity” is used herein with reference to a buffer solution which has a sufficiently low concentration of charge carriers (e.g., ions) to enable detection of a difference in an impedance parameter, such as magnitude or phase, between a bacteria-containing sample and a reference sample free of bacteria.
- The term “microbiological species” or “microbiological material” is used herein to denote any microscopic or submicroscopic entity of interest to researchers or commerce. The term encompasses molecules such as proteins, antibodies, antigens, and enzymes; molecular fragments such as peptides and DNA sequences; cells such as muscle cells or bacteria; viruses; fungi; etc.
- The word “microfabricated” or “microfabrication” as used herein refers to the utilization of photolithography, X-ray lithography, acid etching, and other silicon treatment processes developed in the semiconductor industry to manufacture integrated circuits and solid state components such as microprocessor chips.
- The term “target” is used herein to mean a microbiological entity or species of interest. A target microbiological species is that which is to be detected by a biosensor or biochip as herein described.
- The term “wicking element” as used herein denotes any elongate guide capable of moving a liquid sample by capillary action, where the liquid sample include molecular and cellular material.
- The present invention is directed in part to a
microfabricated biochip 20 illustrated schematically in FIG. 1. A silicon wafer substrate orbody 22 having a size on the order of a postage stamp is formed with a plurality of receptacles orgrooves 24 and 26 which receive ends ofrespective microbore tubes Receptacles 24 and 26 communicate with opposite ends of a meandering microscale channel or groove 32 formed at intervals with cavities orwells 34.Cavities 34 are provided withplatinum electrodes 36 which may be coated, as described hereinafter, with molecular probes for selectively capturing target molecules such as antigens on the surfaces of a target bacterium such as Listeria monocytogenes.Electrodes 36 are connected to respective bonding pads orelectrical terminals 38 via conductors or traces 40. Aglass cover 42 is positioned overreceptacles 24 and 26, the ends oftubes channel 32 andcavities 34 and is sealed tosubstrate 22.Biochip 20 is thus a self-contained biosensor unit with integrated fluidic paths represented bychannel 32 andcavities 34 andelectrodes 36 useful in performing micro-scale electronic measurements of biological solutions. Theelectrodes 36 are spaced from each other by 1 to 100 microns and, more preferably, by 2 to 50 microns. - FIG. 2 is a photograph of a
microfluidic biochip 220 as actually manufactured.Biochip 220 includes afirst area 202 having electrode-containingcavities 204 of 80 by 80 microns and a second area of electrode-containingcavities 206 of 850 by 530 microns, with a common depth of 10 microns.Cavities 204 are connected to one another and to a pair of tube receptacles orgrooves channel segments 212, whilecavities 206 communicate with each other and with a respective pair of microbore-tube receptacles or in/outports channel segments 218.Cavities 204 containsimple electrodes 36 as shown schematically in FIG. 1, whereascavities 206 contain electrodes (not designated) having several interdigitated segments. - FIGS. 3 and 4 are scanning electron micrographs, on different scales, of a portion of
biochip 220. An inlet port or expandedinlet section 222 ofchannel 212 is disposed between a respective receptacle or groove 208 or 210. - In general,
cavities channels - Biochip220 (or generically biochip 20) as disclosed herein has been used to detect and measure a change in conductance in nanoliter volumes of bacterial suspensions and to indicate the viability of the bacteria. Fluid flow through the chip was demonstrated using 2 μm fluorescently labeled beads imaged through a fluorescence microscope. Electrical impedance measurements demonstrate that the device can be used to distinguish between different concentrations of the bacterium Listeria innocua, a non-pathogenic strain of Listeria, by the change in conductance of the suspension produced by bacterial metabolism. These concentrations correspond to very few bacterial cells in the very small volumes (nanoliters) of the measurement chambers of the biochip.
- The manufacture of biochips as disclosed herein will now be described with reference to
generic biochip 20. The manufacturing process consists solely of top-side processing to formtube receptacles 24 and 26,channel 32, and cavities orwells 34, onsilicon wafer substrate 22. A silicon wafer is used to facilitate a future integration of other electronic detectors or active electronic circuitry in later versions of the chip. The entire fabrication process is depicted in FIGS. 5A through 5F. Silicon wafer blanks 46 (FIG. 5A) with a thickness of 450 μm and (100) orientation are oxidized so as to be provided with 0.45 μm thick SiO2 layers 48, and a series ofrectangular cavities 34, connected bychannel 32, are etched into the oxide. Potassium Hydroxide (KOH) is used to etch the silicon surface to a depth of about 12 μm using the thermally grown SiO2 as a hard mask. This depth is still within the depth of focus of the mask aligner used, thus guaranteeing a good definition of patterns at the bottom of the etched areas. Since the surface of the wafer is a (100) plane, the etchedchannels 32 andcavities 34 have tapered walls 50 (FIG. 5B) forming an angle of 54,74° with respect to the oxide surface of thewafer blank 46. Such an angle permits the deposition of metal over thewalls 50, allowing for metal traces 40 (FIG. 1) to run into and across thechannels 32 without breaks. After the KOH etching, the SiO2 hard mask is completely removed and the wafer is oxidized at 1050° C. for 60 minutes in wet-O2 to form a 0.45 μmthick layer 51 of SiO2 (FIG. 2B).Electrodes 36 at the bottoms of thecavities 34, as well as metal conductors or traces 40 connecting them to thebonding pads 38 on the periphery of thewafer substrate 22, are defined over theoxide layer 48 by lift-off, using a 5 μm thick photoresist layer (AZ4620 from Clariant Corp., New Jersey, U.S.A.). The photoresist layer (not shown) needs to be thick enough to keep it from cracking at the upper edges of thechannel walls 50 due to the tension that builds up during baking. Ametallization 52 is formed by RF-sputtering of an 800 Å-thick layer of platinum over a 600 Å-thick film of chromium, the latter serving as an adhesion layer. The sheet resistance of themetallization 52 is approximately 30 Ωcm (2.1Ω/square for the given thickness). After themetal 52 is deposited and patterned, a 0.6 μm thick SiO2 film 54 is deposited by Plasma-Enhanced Chemical Vapor Deposition (PECVD) to insulate theelectrodes 36 and traces 40. Thisfilm 54 is subsequently wet-etched to open windows 56 (FIG. 5C) and thereby defineelectrodes 36 and bonding pads 38 (FIG. 1) along a periphery (not designated) ofwafer substrate 22. Thesewindows 56 leave only the upper, platinum surface exposed, which is fairly resistant to chemical attack, while keeping the chromium covered so that it does not interact with, or is not affected by any of the solutions that may flow through thechannel 32 andcavities 34. (FIGS. 3 and 4 show electron micrographs of a section ofbiochip 220, whereelectrodes 36 are defined at the bottom ofcavities 204, andmetal lines 54 cross thechannels 212.) Only after the formation ofchannel 32 andcavities 34 is thewafer substrate 22 etched to create tube receptacles orgrooves 24 and 26 (FIG. 5D). Thereafter,channel 32 andcavities 34 are hermetically sealed to the surface ofwafer substrate 22 by bonding cover 42 (FIGS. 1 and 5E), a rectangular piece of glass, 0.17 to 0.25 mm thick (No.2 Dow Coming microscopy glass cover). Anodic bonding ofglass cover 42 may also be possible. - A satisfactory bond is achieved by using a low-melting-temperature Spin-On-Glass (SOG) as adhesive (FIG. 5E). This SOG is methylsilsesquioxane polymer (Methylsilsesquioxane 400F from Filmtronics Inc., Pennsylvania, U.S.A.) that flows at temperatures between 150° C. and 210° C. The flowing SOG fills the grooves in between the platinum traces40 and any other surface irregularities, providing a perfectly hermetic seal, while the low flow temperature minimizes thermally induced stresses and damage to temperature-sensitive materials on the die or
wafer substrate 22. Theglass cover 42 is first cut to the desired size in a diamond saw, thoroughly rinsed in DI water, dried, and cleaned in Ar/O2 plasma for 20 minutes. After cleaning, the SOG is spun on the glass at 5000 rpm for 40 s and dried in a convection oven at 90° C. for 2 minutes. This process results in a SOG film approximately 3000 Å thick according to the data-sheet provided by the manufacturer (Spin-on-Glass, 1998). The glass is then manually aligned onto the substrate 22 (SOG side down) and clamped in place. Subsequently, the clamped assembly is heated on a hot plate to 100° C. for 5 minutes, followed by 180° C. for 5 minutes, and 200° C. for at least 1 hour to cure the SOG film. Although the manufacturer indicates that the SOG must be cured at 400° C. for 30 minutes, extensive cracking of the SOG film was observed if the bonded assembly was exposed to temperatures above 300° C. Most likely, the cause of this cracking is the large mismatch between the thermal expansion coefficients ofsilicon substrate 22,glass cover 42, and the SOG. For this reason, to minimize the stress in the SOG film the curing temperature is kept at 200° C., which seems to be sufficient for a reliable bond. The curing time could be substantially increased to compensate for the lower temperature, but even a one hour cure produces a bond capable of withstanding the maximum pressures that have been applied to drive fluids throughmicro-channel 32. A single pressurization test of one fullyfunctional biochip 20, with no flow, indicated a failure pressure of approximately 700 kPa. At this pressure the glass started to unbind from the chip and leaks appeared in the region around the input/output receptacles orgrooves 24 and 26. - One of the challenges that exists in the development of microfluidic biochips is creating reliable fluidic interfaces to the macro-world. For
biochip 20, connections for injecting samples into the device are created by etching receptacles orgrooves 24 and 26 deeply running up to the edge ofsubstrate 22, so thatmicrobore tubes tube receptacles 24 and 26 can be adjusted to provide a large bonding surface which improves the robustness and reliability of the connection to themicrobore tubes tubes silicon substrate 22 and sealingcover 42, which would be necessary if the input/output ports were oncover 42. Thereceptacles 24 and 26 are created by a Deep Reactive Ion Etch (DRIE) system (Plasma Therm SLR770 system using the Bosch Etch process), to a depth of approximately 390 μm, with a 10 μm photoresist layer as mask (FIG. 5D). The etch-rate is about 1.6 μm/min with a selectivity to photoresist of approximately 75:1. (Aprotrusion 58 at the edge betweeninlet section 222 ofchannel 212 and receptacle or groove 208 (FIG. 4) appears because the photoresist hardmask flows during the bake step prior to the DRIE.) -
Tubes tubes Tubes receptacles 24 and 26.Tubes receptacles 24 and 26 and the remaining voids in the receptacles are filled with biomedical grade epoxy adhesive 58 (Durabond M-121HP from Loctite Corp., Connecticut, U.S.A.), which penetrates into thereceptacles 24 and 26 by capillarity (FIG. 5F). Even very rough handling of thetubes - There have been publications describing the detection of pathogenic bacteria in food by monitoring the conductance or the impedance of a specially formulated culture medium innoculated with extracts from food samples. This is possible because bacterial metabolism changes the electrolyte concentration in the suspension medium, significantly altering the electrical characteristics of the medium. Most of these conductivity measurements are performed with DC signals, yielding no information about interfacial phenomena at the solution-electrode interfaces. But Felice et al. (“Impedance Microbiology: Quantification of Bactrial Content in Milk by Means of Capacitance Growth Curves,”Journal of Microbiological Methods, vol. 35, pp. 37-42, 1999) claim that measuring some of the interfacial parameters using an AC excitation (at a single frequency, or preferably at multiple frequencies) makes the technique more sensitive.
- A fairly simple circuit model of a pair of electrodes immersed in an electrolytic solution is shown in FIG. 6 (see Jacobs et al., “Impedimetric Detection of Nucleic Acid Hybrids,”Proceedings of the Second International Conference on Microreaction Technology, New Orleans, La., pp. 223-229, 1998), where Cdi is the dielectric capacitance (incorporating contributions from all the materials surrounding the
electrodes 36, including the solution), RS is the bulk solution resistance (charge transport across the bulk), and ZW is the interfacial impedance (the so-called Warburg impedance), which accounts for the changes in the electrolyte concentration gradient at the interface. The simplest model of the interfacial response to AC signals, yields the following expression for: -
- where n and B are parameters that depend on the properties of the electrolytes and of the electrodes. This equation assumes that the phase between voltage and current is constant at nπ/2 rad. It is to be noted that the impedance Z is a measured parameter, whereas n and B are extracted parameters.
- Preliminary experiments to study the effects of bacterial metabolism on the electrical properties of the suspension medium were carried out using biochip220 (FIGS. 2-4). The main purpose of these experiments was to determine whether impedance measurements in the microscale could provide information about the metabolic activity of a small number of bacteria. Metabolic activity could then be used as an indicator of bacterial viability. Impedance measurements were done in a chamber or
cavity 206 that was 530 μm wide by 850 μm long by 12 μm deep, for a total volume of 5.27 nl (taking into account that the walls of the well have a 54.74° angle). Thischamber 206 had two interdigitated platinum electrodes (not shown) with five fingers each. The exposed area of each finger was 450 μm by 50 μm, and the distance between finger centers was 80 μm. A HP4284A LCR meter (Hewlett Packard Corp., now Agilent Technologies, Palo Alto, Calif.) measured the impedance of the interdigitated electrodes at 52 frequencies, logarithmically spaced between 100 Hz and 1 MHz, with a 50 mV (amplitude) voltage excitation. The impedance of the wiring and probes was automatically substracted from the measurements, so that only the impedance of the elements in thebiochip 220 was recorded. -
biochip 220, which would clog themicroscopic channels biochip 220 using pressurized nitrogen, and were allowed to flow through the chip for 15 minutes, before measuring, to properly flush the whole fluidic path. -
- described above, and an additional series resistor Rt that accounts for the resistance of the metal traces on the biochip220 (FIGS. 2-4), connecting the bonding pads 38 (FIG. 1) to the
electrodes 36 in the chamber 206 (FIG. 2). Values of Rtr=2889 Ω and Cdi=17.98 pF were extracted from fitting to the Tris-Gly buffer data and held fixed when fitting the model to all other samples. FIG. 9 shows an example of how well the chosen model fits the measured impedances. The values of Rs, n, and B obtained from the fits to all the samples are contained in Table 1.TABLE 1 Parameters resulting from fitting the circuit model of FIG. 6 to the impedance data shown in FIG. 7. Number Bulk Solution of Cells Resistance Sample In 5.27 nl Rs [kΩ] n B [× 10−12] DI water 0 242.0 0.149 5.83 × 104 Tris- Glys Buffer 0 56.58 0.968 800.6 ˜105 cells/ml 0.527 55.26 0.961 857.6 ˜107 cells/ml 52.7 51.98 0.960 869.7 ˜108 cells/ml 52.7 35.54 0.952 915.5 ˜109 cells/ml 5270 15.01 0.945 1003.1 -
- At the lowest concentration, the difference in B is larger than that in Z and Rs, while above 107, cells/ml Rs shows the largest differences with respect to buffer. Detection can rely on both {overscore (ΔRs)} and {overscore (ΔB)} to increase the sensitivity at the lowest concentrations. These results indicate that if the small number of Listeria cells present in a food or soil sample can be captured and retained in a chamber in the biochip, their viability could be assessed by measuring the change in impedance of the
electrodes 36 in the detection chamber orcavity - FIG. 11 is a bar graph similar to FIG. 10 and shows normalized differences of the above three measurement parameters for four concentrations of liveListeria innocua and four like concentrations of dead Listeria innouca cells. These results indicate that the viability of a captured sample of microorganisms could be assessed by measuring the change in impedance of the
electrodes 36 in the detection chamber orcavity - The quantity ΔZ is sufficient in many cases to enable detection of a target microbiological species or substance. In other cases, ΔRs is a more sensitive value.
- It is to be noted that impedance is significantly different between buffer alone, and buffer containing microorganisms. Detection of 10 to 100 microorganisms is feasible. Where the electrode spacing is small (e.g., about one to three microns), detection results not only directly from the presence of the microorganisms in effective contact with the electrodes, but also indirectly from a change in electrolyte concentration in the liquid matrix close about the microorganisms, owing to the metabolic activity of the organisms. Where the electrode spacing is 5 microns or larger, bulk impedance is measured to study the effect of bacterial metabolism in the environment of the chambers, as discussed in detail below. In both approaches, the measurement of impedance reflects the generation of metabolites of cells and therefore gives a measure of whether the cells are living or dead.
- A
method utilizing biochip biochip cavity biochip - In general, a bioseparations process as contemplated herein may utilize bioactive fibers and/or
surfaces 62 exemplarily of cotton (cellulose) and packaged into amicro-pipette tip 64, as depicted schematically in FIG. 12.Pipette tip 64 is inserted into a specimen of bodily fluids, foodstuffs, soil, etc., and operated (e.g., via a suction source such as a syringe-type plunger) to aspirate a raw fluid from the specimen. Bioactive fibers and/or surfaces 62 function to remove colloidal particles and extraneous proteins and to that end are derivatized with cation and/or anion ion exchange groups (represented in FIG. 12 by “+” and “−” signs) using established technology (Ladisch et al., 1997, 1998). Fibers and surfaces 62 are packed loosely enough to allow a raw fluid sample to be aspirated from a specimen. The ion exchanger and appropriate conditions are selected so that the targets cells do not bind (see Ladisch, 1997) and can be injected, as indicated by anarrow 65, into a second pipette tip 66 (FIG. 13) containingpolyclonal antibodies 68 for the concentration of both pathogenic andnonpathogenic bacteria 70, for instance, different species of Listeria.Polyclonal antibodies 68 are either immobilized to afiber 62 or fixed to the inner surfaces ofpipette tip 66. The sample is then rinsed with a buffer solution, e.g., a phosphate buffer solution, to remove extraneous fluid. This sample is then pH modified and injected into microbore tube 28 (FIG. 1) for measurement. These preparation steps can be performed rapidly, within several minutes. - Alternatively or additionally, the affinity binding of a target bacterium such asListeria monocytogenes to an antibody may be effectuated inside the
biochip cavities electrodes 36. FIG. 14A depictsavidin molecules 74 adsorbed onto anelectrode 36, as well as onto surrounding silicon oxide 54 (see FIGS. 5A-5F). FIG. 14B showsbiotinylated antibodies 76 attached to the adsorbedavidin molecules 74. In FIG. 14C,target bacteria cells 78 are depicted coupled to thebiotinylated antibodies 76 and hence toelectrode 36 andoxide 54. A difference in electrical measurements betweenelectrode 36 and areference electrode 80 indicates the presence oftarget cells 78 in the detection chamber or cavity. - The adsorption of
avidin molecules 74 ontoelectrodes 36 ofbiochip chip - In the case ofListeria monocytogenes, highly selective antibodies that will bind the 66-kDA protein found on the surface of pathogenic Listeria monocytogenes cells are used as a binding agent or part of a binding agent. Monoclonal antibody producing clones of C11E9 and EM-7G1 (producing antibodies specific for Listeria monocytogenes) are cultured in growth media in a growth chamber. Antibodies are harvested from culture supernatants by salt (ammonium sulfate) precipitation. After an initial concentration step, carried out by known techniques, high quality antibodies are obtained by further purification through size exclusion chromatography followed by protein-A affinity chromatography in an FPLC system.
- Concentration of target pathogenic cells and removal of extraneous microbiological detritus from fluid samples are important for maximizing the signal to noise ratio on
biochip TABLE 2 Chromatographic Supports Tested for Protein Removal from Hotdog Juice Experiments Resin ID Functionality 1 DEAE 650M Weak anion exchanger 2 Super-Q-650M Strong anion exchanger 3 QAE-550C Strong anion exchanger 4 IRA 400 Strong anion exchanger 5 DEAE Cellulose Weak anion exchanger 6 IR-120+ Strong cation exchanger 7 Amberlite XAD-2 Polyaromatic adsorbent 8 Butyl-650s Hydrophobic interation support 9 Amberlite IRN-150 Mixed bed ion exchanger 10 Amberlyst 35Strong cation exchanger 11 Hydroxylapatite Inorganic adsorbent 12 Silica SiO2 Inorganic adsorbent 13 SP 550 Strong cation exchanger - HPLC analysis of the supernatant samples removed during the time course studies were used to generate the results shown in this report, Bradford protein assays were also run on the same samples in parallel. Assay results were inconclusive due to interference from non-protein compounds in the samples. UV absorbance was greater at 260 nm than 280 nm for many of the peaks suggesting the presence of DNA fragments and other UV absorbing non-protein compounds in the hot-dog juice extract. Chromatographic sorbents designated as optimal (A-35 and IRA 400) were shown to remove these other non-protein compounds in addition to the protein present in the hotdog juice extract.
- For the screening study, three replicate chromatography runs of the hot-dog juice extract were used to calculate the “total protein peak area” or the 100% point on the plot. A total of 15 peaks were used as a benchmark for this comparison. The areas of these 15 peaks were totaled to give “total peak area.” Values for the three replicate runs were then averaged together for the initial total protein peak area. Only a single injection of each time course study sample was made. Areas for each of the 15 components corresponding to the original time “0” sample were added together and compared against time “0.” Plots show % total “Protein Peak Area” remaining at each time course point in the study. Results from the four most promising sorbents are shown: IRA 400, A-35,
IRA 120+ and IRN-150. Of these four finalists, the top cation exchanger A-35 and the top anion exchanger IRA 400 were chosen for the final study. The screening study designed to measure the time course of adsorption monitored the “total protein” peak area remaining after exposure to the series of sorbents. In the final set of runs each of the 15 benchmark peaks in the hotdog juice extract were followed after a 30 minute exposure to the sorbents. A figure is provided to give a visual representation to the numeric data for the IRA 400 experiment. As shown, the IRA 400 drops the peak maximums from 0.25-0.5 absorbance units to less than 0.10 AU. Results from these three final experiments suggest that the IRA 400 alone is the best choice for cleaning up the hot-dog juice extract. Doubling the amount of IRA 400 from 5 to 10 grams approximately doubles the extent of protein removal.TABLE 3 Protein Removed by Cation and Anion Exchangers (10 g total wt. in 50 mL Hot-dog Juice. Analysis: Dynamax C18 Column 0-100% CAN in 35 min gradient Blank Hotdog Juice A35 IRA400 A35 + ini. (2 x) % (2 x) % IRA400 % Peak # RT (min) Peak Area Peak Area Reduc-tion Peak Area Reduc-tion Peak Area Reduc- tion 1 4.16 207575 133322 35.77 409904 80.25 104719 49.55 2 4.61 814650 673801 17.3 368994 54.71 585553 28.12 3 4.87 646233 376351 41.76 163097 74.76 360256 44.25 4 5.45 359038 153059 54.76 422584 88.26 142949 60.27 5 6.29 243742 205337 15.76 781038 68.0 937024 61.56 6 6.73 411003 212429 48.31 0 100.0 222436 45.88 7 7.41 456497 182578 60.00 0 100.0 76955 83.14 8 9.13 403089 240411 40.36 81659 79.74 37731 90.64 9 9.5 417685 268091 35.74 118727 71.54 312408 25.11 10 10.40 481247 392557 18.43 550753 0 326840 32.08 11 11.40 319132 0 100.0 88895 72.14 27663 91.33 12 11.80 222569 108530 51.24 28084 87.38 87965 60.48 13 12.5 326507 188755 42.19 43263 86.75 135401 58.53 14 13.5 105266 72316 31.3 17966 82.93 64559 38.67 15 14.0 117063 17293 85.23 17828 84.77 13650 88.34 Total 16,586,152 10,063,117 39.33 4,161,338 74.91 8,476,811 48.89 - In tests of avidin adsorbed onto electrodes and oxide surfaces of a biochip as described hereinabove, fluorescence microscopy confirmed binding of fluorescein labeled ImmunoPure Avidin (Pierce, Rockford, Ill., Cat.#21221, Lot#AI612511). Avidin is a glycoprotein (MW=68kDa) in egg white with an isoelectric point of about 10. Since adsorption is carried out below the isoelectric point, the avidin carries a positive charge at the conditions of these experiments. The avidin solution contacted with the chip was prepared from 0.2 mL of 5 mg/ml protein stock solution in 10 mM HEPES, 0.15M NaCl, and 0.08% sodium azide buffer (as supplied by Pierce) diluted with 0.8 mL PBS, pH 7.4, to obtain a protein concentration of 1 mg/mL. The chip was then placed in 0.2 mL of this solution for 15 hours at either room temperature or 37° C. The chips were immersed (rinsed) 3 times in PBS for 5 min each time to remove excess protein. The protein remaining on the chip was then measured using fluorescence microscopy at room temperature. Histograms of fluorescence intensity from the surface of the chip generated using MatLab and image analysis software, show that more protein binds on the platinum than the silicon oxide and that more adsorption occurs at 37° C. than at room temperature (FIG. 15). In the histogram graphs of FIG. 15, peaks on the right side coincided with highest emission. The protein adsorbed at room temperature is removed when the chip is rinsed (dipped) in deionized water for 30 sec and then dried by a blast of dry compressed (120 psig) air for 15 sec. The result confirms that avidin is readily be absorbed onto both the silicon dioxide (e.g., oxide) and platinum surfaces of a chip in a wet state and removed in a dry state.
- Another experiment showed that protein deposition on the
biochip - A possible explanation for the enhanced protein adsorption relates to the position of the ammonium sulfate, (NH4)2SO4, on the lytotropic series. It is a kosmotrope and hence promotes an ordered arrangement of water molecules around itself and attracts water molecules away from the hydration layer that surrounds a protein in aqueous solution. The water layer around a protein helps to keep it in solution. The decrease in the water layer, such as happens in the presence of ammonium sulfate, promotes hydrophobic interactions between protein molecules and leads to their reversible aggregation and precipitation. This rationale led to the experiment where ammonium sulfate was added to the PBS in order to promote precipitation of avidin on surface of
biochip - The hypothesis that BSA (bovine serum albumin) might preferentially bind onto the oxide surfaces54 (FIGS. 5A-5F) of
biochip microscale biochips electrodes 36 by blocking other sites on the chips using a second protein, such as BSA. Crystallized unlabeled BSA (Pierce, Cat. #77110, Lot #AD40111) and Fluorescein-labeled BSA (Pierce, Cat. #A-9771, Lot #89H7613) were purchased and dissolved in PBS to a concentration of 10 mg/ml. The chips were incubated in the BSA solutions at 40° C. for 2 hours. After the incubations, the chips were rinsed 3 times for 5 minutes each in PBS to remove excess unbound proteins. The chip was then stored wet at 4° C. for 15 hours before they were examined using fluorescence microscopy. The histograms generated from the resulting micrographs showed that more BSA binds onto the platinum than the oxide (compare peaks, squares, on lower and upper scales, respectively in FIG. 17). Furthermore, BSA binds to a greater extent than the avidin on both the oxide and platinum surfaces (FIG. 18). Avidin alone and BSA with avidin show similar emission and intensity peaks relative to each other (circles and triangles in lower scale of FIG. 18). Avidin binds to the surfaces on chips that have previously been treated with BSA. The BSA did not exhibit the postulated blocking effect. To the contrary, the data indicate that BSA unexpectedly promotes greater adsorption of the avidin onto the platinum surface and/or interacts with avidin in a manner that increases the intensity of fluorescence emission when avidin binds biotin (compare FIGS. 17 and 18). - The avidin itself maintains an active conformation on the surface of the chip. The avidin binds its target molecule (i.e., biotin) as indicated by the fluorescent signal obtained when labeled biotin is added to a chip that has previously been treated with unlabeled avidin, or unlabeled avidin and BSA. A chip treated with unlabeled BSA, then unlabeled avidin and finally fluorescein labeled biotin (FIG. 18) gave an emission brighter than that for an experiment where only unlabeled avidin (no BSA) was adsorbed onto the chip followed by addition of labeled biotin (data not shown).
- The confirmation of avidin adsorption, and its ability to bind biotin, provides a method for fixing a primary antibody, specific forListeria monocytogenes, onto
biochip electrodes 36 and oxide surfaces 54), and thereby anchors the antibody to which it is attached to the chip's surfaces. This type of approach is used in the preparation of particulate supports for affinity chromatography, and provides a viable option for biochips as well. - It is possible in some applications to have a selected antibody directly adsorbed on the biochip rather than anchoring it indirectly through avidin. For instance, the antibody Mab C11E9, Lot #00614. Secondary antibodies (KPL, Cat. #02-18-06, fluorescein-labeled antibody to mouse IgG (H+L) produced in goat, and negative antibody, FITC-conjugated goat F(ab)2 anti-human immunoglobulin-polyvalent). Binding can be determined by visual interpretation of fluorescence micrographs of emission patterns of labeled antibodies or cells.
- In the above-described experiments, in the case of primary antibody, a volume of 10 μL of the primary antibody was pipetted onto the chip and then incubated for 30 min at 37° C. The antibody solution was recovered from the chip's surface so that it could be reused. (Only a fraction of the protein was expected to adsorb). Subsequent contact of bacteria or antibody with the chip was carried out after the chip had been washed twice with 100 μL of 0.05% Tween in PBS. The cells were FTIC labeled and diluted to about 106 cells per mL before 100 μL of the cell suspension was pipetted onto the chip. Secondary antibodies were used to determine whether the primary antibody adsorbed on chips that had previously been treated with BSA. The experiments were designed to answer the following questions:
- 1. Does BSA blocking prevent the binding of bacteria to the chip's surface?
- 2. Does the primary antibody stick to the chip?
- 3. Does primary antibody on the chip bind heat-killedListeria monocytogenes?
- Incubation of the chips with BSA, primary antibody, and/or living and heat-killed bacteria, gave the answers to these questions. In the case of the bacterial binding, the numbers of bacteria that bound from one region to the next varied, although the patterns observed were sufficiently pronounced to interpret the micrographs with respect to any differences that may have resulted from different adsorption conditions. The data indicate that biochips treated only with buffer adsorb more heat-killed (at 80° C. for 20 min)Listeria monocytogenes on platinum than on oxide surfaces. E. coli cells do not show significant adsorption.
- It was demonstrated by these experiments that BSA adsorbed on the chips reduces the already low level ofE. coli binding but actually increases Listeria monocytogenes binding for Listeria monocytogenes. If BSA binds Listeria monocytogenes, then a larger population of Listeria monocytogenes should adsorb onto the platinum squares since BSA exhibits greater adsorption on platinum relative to oxide surfaces (FIG. 17). However, the image of distribution of heat-killed Listeria monocytogenes does not show such a pattern. These results are significant since they show that platinum has an affinity for Listeria monocytogenes, over E. coli, and BSA deposited on the surface of the chip further enhances the selectivity of the chip for one type of bacteria over another, even in the absence of the primary antibody. While an antibody is still needed as the bio-recognition element, selective materials design of the chip enhances the signal to noise ratio, if the chip's surface has lower affinity for non-pathogenic bacteria compared to pathogenic ones. In some applications, this design may or may not require deposition of a protein such as BSA.
- It was additionally demonstrated by these experiments that the primary antibody binds to the chip, with preference indicated for platinum surfaces. The fluorescent pattern results when unlabeled primary antibody is adsorbed, followed by a labeled secondary antibody that binds to the primary antibody. In comparison, there is little fluorescence detected for a chip that has been treated with buffer (rather than primary antibody) followed by BSA and the secondary antibody. An analogous result is obtained for primary antibody adsorption followed by buffer wash. Buffer followed by a secondary antibody gives no emission. BSA has no discernible effect on blocking binding.
- The monoclonal antibody MAb C11E9 binds withL. monocytogenes but also show some cross-reaction with some strains of Listeria innocua. Antigens that bind with this MAb are the 52, 55, 66, and 76 kDa surface proteins of the IgG2b subclass. A second antibody, Mab EM-7G1 binds with L. monocytogenes, and specifically with a 66 kDa surface protein (IG1 subclass). Despite the lower specificity of C11 E9, its binding activity is attractive, since the antibody differentiates between living and dead cells.
- As depicted in FIG. 19, transporting of a fluid sample from a food product specimen to the
biochip microfiber wick 82 taking the place of microbore tube 28 (FIG. 1). Experiments have shown that fluids can be transported for 2-cm distances in less than 3 minutes through such amicrofiber wick 82.Wick 82 is approximately 20 to 100 microns in diameter. Preliminary experiments consisted of threading the wicks through pH paper and then holding the wicks vertically while they were placed in an acid solution. Transport of the fluid to the pH paper was indicated by a change in color of the paper. Tested materials were: (1) Zwicky-Trys 1189 100/3 Spun-Polyester (pink); (2) Wooly Nylon 1000m, YLI Corp 161W,Nylon 100% #283 (red); (3) 001 Richardson Silk A (light brown); (4) 100% polyester (light brown); (5) 100% Spun Polyester 0001 (white);and (6) Super Sheen, mercerized, 40 (white). The times required for the transport of the fluids through the wicks of the indicated lengths were: (1) 3 minutes/2 cm; (2) 5 hours/0.5 cm; (3) 45 seconds/2 cm; (4) 1 minute/2 cm; (5) minute/2 cm; and (6) 45 seconds/2 cm. -
Wick 82 may be placed inside of a support tube 84 as shown in FIG. 19. The utilization ofmicrowick 82 instead ofmicrobore tube 28 for transporting the fluid sample is attractive since there are no moving parts or mechanical energy needed to deliver the sample. This methodology would not be feasible for laboratory scale assays, but is attractive for a biochip, since the samples that must be delivered to the sensor are preferably less than about 100 microliters and more preferably less than 1 microliter. Thechannels biochips - An additional or alternative bioseparations method will now be described with reference to FIG. 20. A
fluid sample 86 taken from bodily fluids, foodstuffs, soil, etc., containslive microorganisms 88 such as bacteria or single-cell fungi. The sample also contains contaminant biological matter ordetritus 90, that is, biological material which is not targeted by the detection process. Such biological material includes protein molecules and non-pathogenic cells, as well as molecular and cellular fragments. - The present electronic
method using biochip microorganisms 88 of interest into a very small volume, on the order of 1 picoliter to 1 microliter, and measuring the changes in the electrical characteristics of the fluid in which the microorganisms are suspended. These changes are produced by the release of byproducts of the microorganism's metabolism into the fluid (mainly by the ionic species released). Themicroorganisms 88 may be selectively collected from theraw sample 86 by means of beads or microspheres orbeads 92 functionalized withantibodies 94 specific to the microorganism of interest, affinity chromatography (also using antibodies), filtration using synthetic or natural membranes, or any other technique than can selectively and controllably separate and concentrate some of the microorganisms from the original sample. After collection, the microorganisms are suspended in a liquid medium having a low conductivity (lower than 100 gS/cm), such as Tris-Glycine buffer (3.6 mM Tris, 4.7 mM Glycine). To this medium, a single or multiple non ionic nutrients, such as a sugars, and enough dissolved oxygen (in the case of aerobic microorganisms) are added to stimulate bacterial metabolism. These nutrients can be selected such that they can be more easily metabolized by the microorganism of interest, than by other microorganisms that might be present due to inefficiencies in the selective collection method used. In this way, the selectivity can be increased beyond what the collection step provides. After the microorganisms are suspended in the low conductivity medium, they are injected into a container 102 (FIG. 21) which may take the form of detection chamber or cavity 34 (FIG. 1) or 204, 206 (FIG. 2) with a volume between 1 picoliter and 1 microliter. At the same time, a sample of low conductivity medium with nutrients but no microorganisms, is injected into another container 104 (FIG. 21), identical to the first one. Some means of heating thecontainers metallic electrodes container electrodes 110 in onecontainer 102 being identical in structure and composition to those 112 in theother container 104. The preferred form of these electrodes is an interdigitated structure. - After injection of the samples into the test containers, the temperature of the containers is raised to a level that will stimulate the metabolism of the microorganisms and maintained at that level for several hours. While the samples are at this temperature, the AC electrical impedance of the electrodes in each container is repeatedly measured at several frequencies, between 100 Hz and I MHz, at time intervals on the order of minutes. A circuit model of the electrode-liquid medium-electrode system is fitted to the resulting frequency vs. impedance curves to extract the parameters of the model. As the microorganisms metabolize the provided nutrients and release ionic species into the medium, the parameters of the model fitted to the curves measured at the container with bacteria change over time. At the same time, the parameters extracted from measuring the impedance of the electrodes in the container with no bacteria remain constant within the limits imposed by the noise inherent in the measurement, since no metabolic activity is taking place in this container. If a statistical analysis and comparison of the parameters extracted from measuring both containers indicates that their difference is statistically significant, it can be concluded that the bacteria present in the first container have been detected.
- The vast majority of the bacterial detection methods currently in use are based on fluorescent tagging of the bacteria, or on the detection of DNA fragments from the bacterial genome. Both techniques are unable to determine if the microorganism was dead or alive in the original sample, and both require extensive manipulations of the sample. Moreover, any fluorescence technique requires bulky and expensive optical apparatuses for excitation and detection of the fluorescence. Additionally, when the microorganism is present in very small concentrations (10 to 1000 cells per milliliter), a growth step is necessary to increase the concentration, but this can drive the total assay time to anywhere from 2 to 7 days.
- The present technique solves some of these problems. By its very nature, the technique described above inherently detects only live microorganisms, which is very important for certain applications, especially in food safety (many microorganisms present in food are not pathogenic if they are dead). It also relies exclusively on electrical signals, making the related equipment less expensive and smaller than others. Additionally, the absence of a growth step makes detection possible in a couple of hours instead of days.
- Equipment for the analysis of the conductivity or impedance of an incubated bacterial suspension have been available for a number of years, but they suffer from two limitations. First, m their selectivity is poor because they rely on the composition of the growth medium for encouraging the proliferation of the microorganism of interest, while suppressing the proliferation of others. The second limitation is related to the scale in which the assay is performed. The available equipment uses volumes of bacterial suspension in the milliliter range and above, which requires large numbers of bacteria to provide a discernible signal. The present
technique utilizing biochip - As discussed above, a detection device such as
biochip cavities chambers resistive heaters 106 andtemperature sensors 108, 109 microfabricated within or adjacent to the detection chambers. A pair of metallic preferably interdigitatedelectrodes cavity electrodes 110 in onechamber 102 being identical in structure and composition to those 112 in theother chamber 104. As further illustrated in FIG. 21, the chambers orcavities 102, 104 (or 34, 204, 206) are designed so that the antibody-functionalized microspheres or beads 92 (FIG. 20) can be trapped inside them, while allowing fluids to pass through. Thebeads 92 can be trapped by a microfabricated filter-like structure 114 such as a grid or series of gating posts, with orifices orpassages 116 large enough for non-target bacteria and otherbiological material 90 present in the injected sample to go through, but small enough to prevent thebeads 92, with the attachedtarget microorganisms 88 from flowing out. If thebeads 92 are magnetic, a magnetic field 118 (FIG. 20) could be used to trap them inside the chamber, eliminating the need for the mentioned filter-like retention structure 114. Themagnetic field 118 can be established by permanent magnets orelectromagnets 120 microfabricated within or adjacent to the detection chamber. - Microorganism collection can be performed in two slightly different ways, after the sample has been concentrated and cleaned to remove excess salt, food debris, and other unwanted material. Pursuant to the first technique, depicted in FIG. 20, the
beads 92 are mixed with thesample 86 containing themicroorganisms 88, outside of the measuring volume, and theantibodies 94 are allowed to capture thebacteria 88 for a specific period of time. This time should be long enough to allow theantibodies 94 on thebeads 92 to capture all of themicroorganisms 88 of interest that might exist in thesample 86. After capture, thebeads 92 can be separated from the sample by filtration or magnetically (in the case of magnetic beads), and resuspended in a “washing” fluid to completely eliminate any unwanted material (unwanted microorganisms, food debris, excess salt, etc.) that might have been left after the initial cleaning step; this fluid can also help remove any species non-selectively bound to the antibodies. Subsequently, thebeads 92 are injected into adetection chamber 102 and trapped there (along with themicroorganisms 88 they carry) bymagnetic field 118, in the case ofmagnetic beads 92, or byfilter structure 114 previously described. Alternatively, thesample 86 plusbeads 92 can be injected directly into thechamber 102 and the washing step could be performed after the beads have been trapped inside the chamber. - In another technique of microorganism collection, depicted in FIG. 22, the
beads 92 are first injected into a detection or measuringchamber magnetic field 118, in the case ofmagnetic beads 92, or byfilter structure 114 described above. Thesample 86 containing the microorganisms 88 (which could have been previously purified and concentrated) is then flowed through thechamber beads 92 at a rate that would allow for any and all of themicroorganisms 88 of interest to be captured by theantibodies 94 on the beads. After capture, a “washing” fluid is passed through thechamber 102 to wash away, from the chamber and thebeads 92, any unwanted material (unwanted microorganisms, food debris, excess salt, etc.) that might have been left after the initial cleaning step; this fluid can also help remove any species non-selectively bound to the antibodies. This second technique is similar to the principle of affinity chromatography, with the measuring chambers acting as chromatographic columns. - The collection step is performed for two
samples 86 with two separate sets ofbeads 92, one set for each sample. One sample is the test sample being analyzed for the presence of microorganisms, the other is a “dummy” or reference sample, artificially prepared to ensure that it does not contain anymicroorganisms 88. Each set ofbeads 92 is injected into one of thechambers 102, 104 (or 122) in the detection device, and trapped there by the means described earlier. This results in onechamber 104 containingbeads 92 which are guaranteed not to have anymicroorganisms 88 attached to them. This latter chamber may be called “the reference chamber,” while theother chamber 102, which could have themicroorganisms 88 of interest if they were present in the original sample, will be called “the detection chamber.” - Once the
beads 92 are trapped inside thechambers 102, 104 (or 122), the chambers are filled with a liquid medium having a low conductivity (lower than 100 gS/cm), such as Tris-Glycine buffer (0.2 mM Tris, 4.7 mM Glycine). This medium also contains a single or multiple nonionic nutrients, such as sugars, and enough dissolved oxygen (in the case of aerobic microorganisms) to stimulate the microorganism's metabolism. These nutrients can be selected such that they can be more easily metabolized by the microorganism of interest than by other microorganisms that might be present due to inefficiencies in the antibody-mediated capture. In this way, the selectivity can be increased beyond what the antibody-based collection step provides. After injection of the samples, the temperature of both chambers is raised to a level that will stimulate the metabolism of themicroorganisms 88 and maintained at that level for several hours. While the samples are at this temperature, the AC electrical impedance of theelectrodes chamber 102, 104 (or 122) is repeatedly measured at several frequencies, between 100 Hz and I MHz, at time intervals on the order of minutes. A computer, or microprocessor, or microcontroller, or digital signal processor acquires the measured impedance vs. frequency data and analyzes it to extract certain parameters that will be the basis for detection. Ifmicroorganisms 88 were captured by thebeads 92 in the detection chamber 102 (122), the parameters extracted from the curves measured at the detection chamber change over time because the microorganisms metabolize the provided nutrients and release ionic species into the medium. These ionic species, in turn, change the electric properties of the liquid medium and hence change the impedance of the electrodes in contact with the liquid. At the same time, the parameters extracted from the impedance of theelectrodes 112 in thereference chamber 104 remain constant within the limits imposed by the noise inherent in the measurement, since no metabolic activity is taking place in this chamber (it was guaranteed from the beginning that no bacteria would be present in the reference chamber). If a statistical analysis and comparison of the parameters extracted from measuring both chambers indicates that their difference is statistically significant after a suitable incubation time, it can be concluded that themicroorganisms 88 present in thedetection chamber 102 have been detected. If no organisms are present in the detection chamber, no statistically significant difference in the extracted parameters will be observed. Also, if the microorganism of interest is present but dead, no change will be detected. The electronic detection method has been tested experimentally, demonstrating that 50 bacteria cells (Listeria innocua) confined into a volume of 5.3 nanoliters produce a detectable change in the impedance of a low conductivity medium. According to the experimental data, the limit in sensitivity seems to be somewhere between 1 and 50 cells in a 5.3 nl volume. - There are a multitude of parameters that could be extracted from the measured impedance vs. frequency data. One possibility is to fit a circuit model of the electrode-liquid electrode system to the data (using a least squares method, for example) to obtain values for the components of the circuit model. All or some of these values can be used as the detection parameters. Another method involves using only the phase of the impedance phasor as the detection parameter. Experiments indicate that changes in the phase of the impedance, at selected frequencies, are good indicators of bacterial metabolism. Additionally, the phase of the impedance can be measured with very high precision much more easily than the magnitude. It could also be possible to achieve detection by a DC measurement of the resistivity of the liquid inside the chambers, instead of using an AC measurement. The resistivity can be measured by a four-point-probe method, using four electrodes laid out in a Van der Pauw geometry in each chamber, in place of one pair of
interdigitated electrodes - The sensing of a target microbiological species such as a pathogenic bacterium in a detection chamber or
cavity - The simple MOSFET of this detection structure is fabricated in silicon. The device has gate oxides of less than 150 Å. Platinum is used as the gate material and the exposed gate area may vary from 100 μm×100 μm to 2 μm×2 μm. The fabrication of the MOSFET is standard and double gated MOSFETs may also be used. Each device has a source, drain and body terminal in addition to the open (exposed) gate terminal. The devices are packaged and biochemically treated with binding agents as described hereinabove. The main difference is that the measurement consists of source to drain current measured by a high-precision pico-ammeter, a semiconducter parameter analyzer, or a digital oscilloscope. The device is biased using a DC and AC signal and the measurements will be taken before, during and after the binding of the avidin to the biotinylated gate electrode. Only the binding event taking place on the gate electrode affects the source/drain current measurement.
- Although the invention has been described in terms of particular embodiments and applications, one of ordinary skill in the art, in light of this teaching, can generate additional embodiments and modifications without departing from the spirit of or exceeding the scope of the claimed invention. For example, the substrate of a biochip may be of a material other than silicon, including but not limited to glass such as Coming7740, and polymers such as polyethylene based plastics and polytetrafluoroethylene.
- It is to be noted that other methods of measuring electrical conductivity equivalent to the methods detailed herein may be used to detect the presence of a target microbiological species inside a microscale biochip. For instance, the bulk solution resistance RS may be determined directly using a four point probe sheet resistivity measurement. In this technique, four electrodes are positioned in a detection chamber at comers of a quadrilateral such as a square. Current is conducted between two diagonally opposed electrodes, while voltage is measured across the other two diagonally disposed electrodes. The interfacial impedance ZW is automatically eliminated.
- Accordingly, it is to be understood that the drawings and descriptions herein are proffered by way of example to facilitate comprehension of the invention and should not be construed to limit the scope thereof.
Claims (47)
1. A method for detecting a microbiological substance, comprising:
providing a microfabricated biosensor chip including integrated detection elements;
delivering a fluid sample to said biosensor chip;
after the delivering of said fluid sample to said biosensor chip, separating at least some contaminants from said fluid sample to at least partially isolate and retain instances of a predetermined type of microbiological material on said biosensor chip, the separating of said contaminants taking place on said biosensor chip; and
after the separating of contaminants from said fluid sample, operating said detection elements to determine whether the separated fluid sample contains microbiological material of said predetermined type.
2. The method defined in claim 1 , further comprising carrying out a bioseparations process on said fluid sample prior to the delivering of said fluid sample to said biosensor chip.
3. The method defined in claim 2 wherein said bioseparations process includes adding to said fluid sample a plurality of microscopic carrier elements each provided with a multiplicity of binding agents for coupling said microbiological material to said carrier elements.
4. The method defined in claim 3 wherein said carrier elements are beads or microspheres.
5. The method defined in claim 4 wherein the separating of contaminants from said fluid sample on said biosensor chip includes trapping said carrier elements with the coupled microbiological material in a detection chamber on said biosensor chip while flushing remaining portions of said fluid sample from said chamber.
6. The method defined in claim 5 wherein the trapping of said carrier elements includes providing a filter barrier at an outlet of said detection chamber.
7. The method defined in claim 2 wherein said bioseparations process includes subjecting a precursor of said fluid sample to a bioactive surface taken from the group consisting of a cation exchange resin and an anion exchange resin.
8. The method defined in claim 7 wherein said cation exchange resin includes Amberlyst 35 and said anion exchange resin includes IRA 400.
9. The method defined in claim 1 wherein said predetermined type of microbiological material is a pathogenic strain of bacteria.
10. The method defined in claim 9 wherein said strain of bacteria includes Listeria monocytogenes.
11. The method defined in claim 9 , further comprising extracting said fluid sample from a food product prior to delivering of said fluid sample to said biosensor chip.
12. A biosensor comprising a substrate microfabricated to include as integrated components:
a detection chamber;
a first channel segment extending to an inlet of said detection chamber;
a second channel segment extending from an outlet of said chamber; and
a retention structure for holding, in said chamber, carrier elements entraining a target microbiological species while permitting passage through said detection chamber of contaminant materials in a fluid stream.
13. The biosensor defined in claim 12 wherein said retention structure includes filter grid or grating on an upstream side of said outlet.
14. The biosensor defined in claim 12 wherein said retention structure includes a magnetic field generating element.
15. The biosensor defined in claim 12 wherein said detection chamber is provided with electrodes including interdigitated finger parts.
16. The biosensor defined in claim 12 wherein said detection chamber has a volume of less than approximately one microliter.
17. A biosensor comprising a substrate microfabricated to include as integrated components:
a detection chamber; and
a channel extending to an inlet of said detection chamber, further comprising a wicking element connected at one end to said substrate so as to be in communication with said channel, for drawing a fluid sample by capillary action to said channel for delivery to said detection chamber.
18. The biosensor defined in claim 17 wherein said wicking element is attached at said one end by an adhesive to said substrate.
19. The biosensor defined in claim 17 wherein said substrate is micro fabricated to include an inlet groove or trench substantially coplanar with said channel and said detection chamber, said one end of said wicking element being disposed in said inlet groove or trench, said wicking element being coplanar at said one end with said channel and said detection chamber.
20. An integrated microscale biosensor comprising a substrate microfabricated to include as integrated components:
a detection chamber;
a channel extending to an inlet of said detection chamber; and
an inlet groove or trench substantially coplanar with said channel and said detection chamber, further comprising an elongate fluid delivery member having a downstream end disposed in said inlet groove or trench, said fluid delivery member being connected at said downstream end to inlet groove or trench so that at least said downstream end of said fluid delivery member is coplanar with said channel and said detection chamber.
21. The biosensor defined in claim 20 wherein said elongate fluid delivery member is a microbore tube.
22. The biosensor defined in claim 20 wherein said elongate fluid delivery member is a wicking element.
23. The biosensor defined in claim 20 wherein said substrate is top-side processed only.
24. The biosensor defined in claim 20 , further comprising a cover attached to said substrate over said detection chamber, said channel, said inlet groove, and said downstream end of said fluid delivery member, said cover having an absence of holes or apertures.
25. A method for manufacturing a biosensor comprising:
providing a substrate;
processing said substrate to generate a detection chamber and a channel extending to said detection chamber;
further processing said substrate to provide at least one pair of electrodes in said detection chamber; and
exposing the processed substrate to BSA and avidin to adsorb said avidin to said electrodes in the presence of said BSA.
26. The method defined in claim 25 , further comprising subjecting the exposed processed substrate to a fluid containing a biotinylated antibody specific to a preselected antigen, thereby attaching the antibody to said electrodes via a biotin-avidin link.
27. The method defined in claim 26 wherein said biotinylated antibody is specific to an antigen on a cell membrane of Listeria monocytogenes.
28. The method defined in claim 27 wherein said antigen is a 66-kDA protein, further comprising:
culturing monoclonal antibody producing clones of C11E9 and EM-7G1 in growth media in a growth chamber;
harvesting antibodies from culture supernatants by salt precipitation; and
obtaining antibodies from the harvest by purification through size exclusion chromatography followed by protein-A affinity chromatography in an FPLC system.
29. The method defined in claim 25 , further comprising washing the exposed processed substrate after a predetermined time period.
30. A method for manufacturing a biosensor, comprising:
providing a substrate;
processing said substrate to create a shallow detection chamber and a channel extending to said detection chamber;
after the creation of said detection chamber and said channel, further processing said substrate to deposit at least one pair of electrodes in said detection chamber;
after the deposition of said electrodes, further processing said substrate to create at least one deep groove at a periphery of said substrate, for receiving an elongate fluid delivery element, said channel communicating with said deep groove;
inserting a downstream end of said fluid delivery element into said deep groove; and
attaching said downstream end of said fluid delivery element to said deep groove.
31. The method defined in claim 30 , further comprising attaching a cover to said substrate over said detection chamber, said channel, said deep groove and said downstream end of said fluid delivery element.
32. A method for detecting a microorganism, comprising:
preparing a fluid sample containing at least one microorganism of a preselected type, said fluid sample having a buffer of a low conductivity liquid, said fluid sample also containing a nonionic nutrient;
disposing said fluid sample in a detection chamber having a volume less than approximately 1 microliter;
maintaining said fluid sample at a predetermined temperature in said detection chamber; and
measuring an electrical parameter of an electrical circuit incorporating said detection chamber and the fluid sample therein
33. The method defined in claim 32 wherein said electrical parameter is an impedance measure taken from the group consisting of magnitude and phase.
34. The method defined in claim 32 wherein the measuring of said electrical parameter includes utilizing a four point probe to make a sheet resistivity measurement.
35. The method defined in claim 32 wherein said microorganism is Listeria Monocytogenes.
36. The method defined in claim 32 wherein said buffer is a Tris-Glycine buffer.
37. The method defined in claim 32 wherein said detection chamber has a volume of between about 1 picoliter and about 1 microliter.
38. The method defined in claim 32 wherein said electrical parameter is an impedance parameter, the measuring of said electrical parameter including measuring the impedance parameter at a plurality of frequencies within a range from 100 Hz to 1 MHz.
39. A method for testing a food product for the presence of a predetermined type of pathogenic bacteria, comprising:
extracting a fluid sample from the food product;
feeding the extracted fluid sample to an integrated microscale biosensor;
subjecting the fluid sample to a bioseparations process to remove extraneous particles including proteins and kinds of bacteria other than the predetermined type of pathogenic bacteria;
binding bacteria of said predetermined type in said fluid sample to at least one substrate body; and
after the feeding of the extracted fluid sample to said chamber, the subjecting of the fluid sample to the bioseparations process, and the binding of the predetermined type of bacteria to the at least one substrate body, measuring an electrical parameter of an electrical circuit incorporating said detection chamber and the fluid sample therein to detect the presence in the fluid sample of living instances of said predetermined type of bacteria.
40. The method defined in claim 39 wherein the binding of said predetermined type of bacteria is to beads or microspheres floating in said fluid sample.
41. The method defined in claim 39 wherein the binding of said predetermined type of bacteria is to electrodes in said biosensor.
42. The method defined in claim 39 wherein subjecting of said fluid sample to said bioseparations process takes place at least partially after feeding of the fluid sample to said biosensor.
43. The method defined in claim 39 wherein the binding of said predetermined type of bacteria is implemented via antibodies specific to said predetermined type of bacteria.
44. The method defined in claim 39 , further comprising concentrating said predetermined type of bacteria in said fluid sample prior to the measuring of said electrical parameter.
45. The method defined in claim 39 wherein said electrical parameter is an impedance parameter.
46. The method defined in claim 39 wherein said electrical parameter is phase.
47. An integrated microscale biosensor comprising a substrate microfabricated to include as integrated components:
a detection chamber;
a channel extending to an inlet of said detection chamber;
means for feeding a fluid sample to said detection chamber; and
at least one sensor attached to said substrate in operative communication with said detection chamber,
said detection chamber having a volume of between about 1 picoliter and 1 microliter.
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US10/172,263 US20030036054A1 (en) | 2000-04-17 | 2002-06-14 | Biosensor and related method |
US10/825,413 US7435579B2 (en) | 2000-04-17 | 2004-04-15 | Biosensor and related method |
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US19756000P | 2000-04-17 | 2000-04-17 | |
US09/817,541 US6716620B2 (en) | 2000-04-17 | 2001-03-26 | Biosensor and related method |
US10/172,263 US20030036054A1 (en) | 2000-04-17 | 2002-06-14 | Biosensor and related method |
Related Parent Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US09/817,541 Division US6716620B2 (en) | 2000-04-17 | 2001-03-26 | Biosensor and related method |
US10/825,413 Division US7435579B2 (en) | 2000-04-17 | 2004-04-15 | Biosensor and related method |
Related Child Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US10/825,413 Continuation-In-Part US7435579B2 (en) | 2000-04-17 | 2004-04-15 | Biosensor and related method |
Publications (1)
Publication Number | Publication Date |
---|---|
US20030036054A1 true US20030036054A1 (en) | 2003-02-20 |
Family
ID=22729906
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US09/817,541 Expired - Lifetime US6716620B2 (en) | 2000-04-17 | 2001-03-26 | Biosensor and related method |
US10/172,263 Abandoned US20030036054A1 (en) | 2000-04-17 | 2002-06-14 | Biosensor and related method |
Family Applications Before (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US09/817,541 Expired - Lifetime US6716620B2 (en) | 2000-04-17 | 2001-03-26 | Biosensor and related method |
Country Status (5)
Country | Link |
---|---|
US (2) | US6716620B2 (en) |
EP (1) | EP1283900A4 (en) |
AU (1) | AU2001252973A1 (en) |
CA (1) | CA2406133A1 (en) |
WO (1) | WO2001079529A1 (en) |
Cited By (39)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20030153023A1 (en) * | 1999-05-13 | 2003-08-14 | Starzl Timothy W. | Enumeration method of analyte detection |
US20040166555A1 (en) * | 1999-11-10 | 2004-08-26 | Rebecca Braff | Cell sorting apparatus and methods for manipulating cells using the same |
US20040171091A1 (en) * | 2003-02-27 | 2004-09-02 | Cell Work, Inc. | Standardized evaluation of therapeutic efficacy based on cellular biomarkers |
US20050042596A1 (en) * | 2000-06-09 | 2005-02-24 | Micronas Gmbh | Process for examining membrane enclosed biocompartments |
US20050048599A1 (en) * | 2003-07-12 | 2005-03-03 | Goldberg David A. | Sensitive and rapid determination of antimicrobial susceptibility |
US20050084865A1 (en) * | 2003-10-16 | 2005-04-21 | Hong Kong Dna Chips Limited | Apparatus and methods for detecting nucleic acid in biological samples |
US20060034493A1 (en) * | 2003-08-15 | 2006-02-16 | Toshishige Shimamura | Organism recognition system |
US20060057599A1 (en) * | 2002-08-26 | 2006-03-16 | The Regents Of The University Of California | System for autonomous monitoring of bioagents |
US20060147912A1 (en) * | 2002-05-30 | 2006-07-06 | Corbett John M | Dna amplification apparatus and method |
US20070026381A1 (en) * | 2005-04-05 | 2007-02-01 | Huang Lotien R | Devices and methods for enrichment and alteration of cells and other particles |
US20070026469A1 (en) * | 2005-07-29 | 2007-02-01 | Martin Fuchs | Devices and methods for enrichment and alteration of circulating tumor cells and other particles |
US20070160503A1 (en) * | 2003-06-13 | 2007-07-12 | Palaniappan Sethu | Microfluidic systems for size based removal of red blood cells and platelets from blood |
US20070218610A1 (en) * | 2001-04-23 | 2007-09-20 | Samsung Electronics Co., Ltd. | Methods of making a molecular detection chip having a metal oxide silicon field effect transistor on sidewalls of a micro-fluid channel |
US20070259424A1 (en) * | 2002-09-27 | 2007-11-08 | The General Hospital Corporation | Microfluidic device for cell separation and uses thereof |
US7341841B2 (en) | 2003-07-12 | 2008-03-11 | Accelr8 Technology Corporation | Rapid microbial detection and antimicrobial susceptibility testing |
US20090068638A1 (en) * | 2005-03-17 | 2009-03-12 | Biophage Inc. | Phage-based method for the detection of bacteria |
US20090152127A1 (en) * | 2005-09-14 | 2009-06-18 | Sumitomo Electric Industries, Ltd. | Biosensor Measurement Machine , Biosensor Measurement System and Biosensor Measurement Method |
US20090216082A1 (en) * | 2005-04-01 | 2009-08-27 | Elisha Rabinovitz | Device, System and Method for In Vivo Magnetic Immunoassay Analysis |
US20110097837A1 (en) * | 2006-11-08 | 2011-04-28 | Garcia Michael A | GaN-BASED NITRIC OXIDE SENSORS AND METHODS OF MAKING AND USING THE SAME |
US20110122551A1 (en) * | 2006-10-23 | 2011-05-26 | Acco Brands Usa Llc | Security Apparatus |
US20110151610A1 (en) * | 2009-12-23 | 2011-06-23 | Varian Semiconductor Equipment Associates, Inc. | Workpiece patterning with plasma sheath modulation |
US20110199102A1 (en) * | 2008-06-16 | 2011-08-18 | Garcia Michael A | Chemical sensors and methods for making and using the same |
US20110236924A1 (en) * | 2008-12-19 | 2011-09-29 | Halverson Kurt J | System and method for processing samples |
US20120122736A1 (en) * | 2009-02-11 | 2012-05-17 | Duke University | Sensors incorporating antibodies and methods of making and using the same |
US20130040848A1 (en) * | 2006-09-16 | 2013-02-14 | Medical Research Council | Methods and Devices for Detecting Structural Changes in a Molecule Measuring Electrochemical Impedance |
US8535945B2 (en) | 2008-12-19 | 2013-09-17 | 3M Innovative Properties Company | System and method for concentrating samples |
KR101465961B1 (en) * | 2007-10-09 | 2014-12-01 | 삼성전자주식회사 | A method and a device for detecting DNAs, etc. |
US9434937B2 (en) | 2011-03-07 | 2016-09-06 | Accelerate Diagnostics, Inc. | Rapid cell purification systems |
US9470612B2 (en) | 2011-06-30 | 2016-10-18 | 3M Innovative Properties Company | Systems and methods for detecting an analyte of interest in a sample using filters and microstructured surfaces |
US9488563B2 (en) | 2011-06-30 | 2016-11-08 | 3M Innovative Properties Company | Systems and methods for detecting an analyte of interest in a sample using microstructured surfaces |
WO2017015574A1 (en) * | 2015-07-23 | 2017-01-26 | Purdue Research Foundation | Rapid concentration, recovery and detection of pathogens in food samples |
US9657327B2 (en) | 2003-07-12 | 2017-05-23 | Accelerate Diagnostics, Inc. | Rapid microbial detection and antimicrobial susceptibility testing |
US9677109B2 (en) | 2013-03-15 | 2017-06-13 | Accelerate Diagnostics, Inc. | Rapid determination of microbial growth and antimicrobial susceptibility |
US10023895B2 (en) | 2015-03-30 | 2018-07-17 | Accelerate Diagnostics, Inc. | Instrument and system for rapid microogranism identification and antimicrobial agent susceptibility testing |
US10253355B2 (en) | 2015-03-30 | 2019-04-09 | Accelerate Diagnostics, Inc. | Instrument and system for rapid microorganism identification and antimicrobial agent susceptibility testing |
US10254204B2 (en) | 2011-03-07 | 2019-04-09 | Accelerate Diagnostics, Inc. | Membrane-assisted purification |
US20190256886A1 (en) * | 2009-10-02 | 2019-08-22 | The Curators Of The University Of Missouri | Rapid detection of viable bacteria system and method |
US10407716B2 (en) | 2014-03-13 | 2019-09-10 | Duke University | Electronic platform for sensing and control of electrochemical reactions |
US11346798B2 (en) | 2010-07-12 | 2022-05-31 | Arizona Board Of Regents On Behalf Of Arizona State University | Methods and device for tuning multiplexed markers for disease assay |
Families Citing this family (176)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
BR9814386B1 (en) * | 1997-12-22 | 2009-08-11 | apparatus and methods for determining the concentration of a medically significant component of a biological fluid. | |
US8071384B2 (en) | 1997-12-22 | 2011-12-06 | Roche Diagnostics Operations, Inc. | Control and calibration solutions and methods for their use |
US7407811B2 (en) * | 1997-12-22 | 2008-08-05 | Roche Diagnostics Operations, Inc. | System and method for analyte measurement using AC excitation |
US20050103624A1 (en) * | 1999-10-04 | 2005-05-19 | Bhullar Raghbir S. | Biosensor and method of making |
US6537433B1 (en) * | 2000-03-10 | 2003-03-25 | Applera Corporation | Methods and apparatus for the location and concentration of polar analytes using an alternating electric field |
US7485454B1 (en) * | 2000-03-10 | 2009-02-03 | Bioprocessors Corp. | Microreactor |
US7306924B2 (en) * | 2000-04-17 | 2007-12-11 | Purdue Research Foundation | Biosensor and related method |
US7435579B2 (en) * | 2000-04-17 | 2008-10-14 | Purdue Research Foundation | Biosensor and related method |
JP3636061B2 (en) * | 2000-11-08 | 2005-04-06 | 日本電気株式会社 | Data insertion apparatus and method |
US6764583B2 (en) * | 2000-12-13 | 2004-07-20 | The Regents Of The University Of California | Using impedance measurements for detecting pathogens trapped in an electric field |
US20020123134A1 (en) * | 2000-12-26 | 2002-09-05 | Mingxian Huang | Active and biocompatible platforms prepared by polymerization of surface coating films |
JP3974527B2 (en) * | 2001-01-16 | 2007-09-12 | ギブン・イメージング・リミテツド | System and method for determining body cavity status in vivo |
US20040058437A1 (en) * | 2001-04-10 | 2004-03-25 | Rodgers Seth T. | Materials and reactor systems having humidity and gas control |
US20040058407A1 (en) * | 2001-04-10 | 2004-03-25 | Miller Scott E. | Reactor systems having a light-interacting component |
DE10139742A1 (en) * | 2001-08-13 | 2003-03-06 | Univ Freiburg | Process for producing a "lab on chip" from photoresist material for medical diagnostic applications |
EP1448489B1 (en) * | 2001-11-16 | 2010-08-25 | Stefan Ufer | Flexible sensor and method of fabrication |
US20050026134A1 (en) * | 2002-04-10 | 2005-02-03 | Bioprocessors Corp. | Systems and methods for control of pH and other reactor environment conditions |
US20030232370A1 (en) * | 2002-04-22 | 2003-12-18 | Trifiro Mark A. | Glucose sensor and uses thereof |
ES2271565T3 (en) * | 2002-05-03 | 2007-04-16 | Dsm Ip Assets B.V. | PROCESS FOR THE MONITORING OF THE ELIMINATION OF POLLUTANTS DURING THE PROCESS OF PURIFICATION OF A PHARMACEUTICAL PRODUCT PRODUCED BY A HOSPEDERA CELL. |
US20050106714A1 (en) * | 2002-06-05 | 2005-05-19 | Zarur Andrey J. | Rotatable reactor systems and methods |
US7470533B2 (en) | 2002-12-20 | 2008-12-30 | Acea Biosciences | Impedance based devices and methods for use in assays |
US7560269B2 (en) | 2002-12-20 | 2009-07-14 | Acea Biosciences, Inc. | Real time electronic cell sensing system and applications for cytotoxicity profiling and compound assays |
DE60330022D1 (en) * | 2002-07-20 | 2009-12-24 | Acea Biosciences Inc | DEVICES FOR IMPEDANZBASIS AND METHOD FOR USE IN ASSAYS |
US8206903B2 (en) | 2002-12-20 | 2012-06-26 | Acea Biosciences | Device and method for electroporation-based delivery of molecules into cells and dynamic monitoring of cell responses |
US7468255B2 (en) * | 2002-12-20 | 2008-12-23 | Acea Biosciences | Method for assaying for natural killer, cytotoxic T-lymphocyte and neutrophil-mediated killing of target cells using real-time microelectronic cell sensing technology |
US7732127B2 (en) * | 2002-12-20 | 2010-06-08 | Acea Biosciences, Inc. | Dynamic monitoring of cell adhesion and spreading using the RT-CES system |
US8263375B2 (en) | 2002-12-20 | 2012-09-11 | Acea Biosciences | Dynamic monitoring of activation of G-protein coupled receptor (GPCR) and receptor tyrosine kinase (RTK) in living cells using real-time microelectronic cell sensing technology |
US7238496B2 (en) * | 2002-08-06 | 2007-07-03 | The Board Of Trustees Of The University Of Arkansas | Rapid and automated electrochemical method for detection of viable microbial pathogens |
WO2004014227A1 (en) | 2002-08-13 | 2004-02-19 | Given Imaging Ltd. | System for in vivo sampling and analysis |
US6877209B1 (en) * | 2002-08-28 | 2005-04-12 | Silicon Light Machines, Inc. | Method for sealing an active area of a surface acoustic wave device on a wafer |
US7303875B1 (en) * | 2002-10-10 | 2007-12-04 | Nanosys, Inc. | Nano-chem-FET based biosensors |
WO2004055199A2 (en) * | 2002-12-12 | 2004-07-01 | Chiron Corporation | A biological sample storage device and method for biological sample contamination testing |
US10551371B2 (en) | 2003-11-10 | 2020-02-04 | Acea Biosciences, Inc. | System and method for monitoring cardiomyocyte beating, viability and morphology and for screening for pharmacological agents which may induce cardiotoxicity or modulate cardiomyocyte function |
US10539523B2 (en) | 2002-12-20 | 2020-01-21 | Acea Biosciences, Inc. | System and method for monitoring cardiomyocyte beating, viability, morphology, and electrophysiological properties |
US11346797B2 (en) | 2002-12-20 | 2022-05-31 | Agilent Technologies, Inc. | System and method for monitoring cardiomyocyte beating, viability, morphology and electrophysiological properties |
US10215748B2 (en) | 2002-12-20 | 2019-02-26 | Acea Biosciences, Inc. | Using impedance-based cell response profiling to identify putative inhibitors for oncogene addicted targets or pathways |
US9612234B2 (en) | 2008-05-05 | 2017-04-04 | Acea Biosciences, Inc. | Data analysis of impedance-based cardiomyocyte-beating signals as detected on real-time cell analysis (RTCA) cardio instruments |
US7214528B1 (en) * | 2002-12-31 | 2007-05-08 | Oregon Health & Sciences University | Device for direct electrical detection of molecules and molecule-molecule interactions |
CN100389321C (en) * | 2003-02-19 | 2008-05-21 | 浙江大学 | Liquid level stabilizing device in micro-analysis chip liquid pool and method of use thereof |
EP1618206A4 (en) * | 2003-04-30 | 2006-11-29 | Biovitesse | Apparatus and method for detecting live cells with an integrated filter and growth detection device |
EP1473359A1 (en) * | 2003-05-02 | 2004-11-03 | LMB Technologie GmbH | Non-invasive fast assessment of bacterial load in blood and blood products |
KR100563834B1 (en) * | 2003-05-23 | 2006-03-28 | 주식회사 올메디쿠스 | Micor/nano fluidic 3 dimensional electrode system |
EP1628748A2 (en) * | 2003-06-05 | 2006-03-01 | Bioprocessors Corporation | Reactor with memory component |
US7604721B2 (en) * | 2003-06-20 | 2009-10-20 | Roche Diagnostics Operations, Inc. | System and method for coding information on a biosensor test strip |
US8071030B2 (en) | 2003-06-20 | 2011-12-06 | Roche Diagnostics Operations, Inc. | Test strip with flared sample receiving chamber |
US7452457B2 (en) | 2003-06-20 | 2008-11-18 | Roche Diagnostics Operations, Inc. | System and method for analyte measurement using dose sufficiency electrodes |
US7488601B2 (en) | 2003-06-20 | 2009-02-10 | Roche Diagnostic Operations, Inc. | System and method for determining an abused sensor during analyte measurement |
CN1846131B (en) | 2003-06-20 | 2012-01-18 | 霍夫曼-拉罗奇有限公司 | Method and reagent for producing narrow, homogenous reagent strips |
US8679853B2 (en) | 2003-06-20 | 2014-03-25 | Roche Diagnostics Operations, Inc. | Biosensor with laser-sealed capillary space and method of making |
US8206565B2 (en) * | 2003-06-20 | 2012-06-26 | Roche Diagnostics Operation, Inc. | System and method for coding information on a biosensor test strip |
US7645373B2 (en) | 2003-06-20 | 2010-01-12 | Roche Diagnostic Operations, Inc. | System and method for coding information on a biosensor test strip |
US7718439B2 (en) | 2003-06-20 | 2010-05-18 | Roche Diagnostics Operations, Inc. | System and method for coding information on a biosensor test strip |
US7645421B2 (en) | 2003-06-20 | 2010-01-12 | Roche Diagnostics Operations, Inc. | System and method for coding information on a biosensor test strip |
US8148164B2 (en) | 2003-06-20 | 2012-04-03 | Roche Diagnostics Operations, Inc. | System and method for determining the concentration of an analyte in a sample fluid |
US8058077B2 (en) | 2003-06-20 | 2011-11-15 | Roche Diagnostics Operations, Inc. | Method for coding information on a biosensor test strip |
EP1641390A4 (en) * | 2003-06-26 | 2008-06-04 | Given Imaging Ltd | Methods, device and system for in vivo detection |
US20050059105A1 (en) * | 2003-07-25 | 2005-03-17 | Board Of Trustees Of Michigan State University | Impedimetric biosensor and its use for rapid detection of bacterial pathogens in solution |
US7460896B2 (en) * | 2003-07-29 | 2008-12-02 | Given Imaging Ltd. | In vivo device and method for collecting oximetry data |
TW594007B (en) * | 2003-08-15 | 2004-06-21 | Prec Instr Dev Ct Nat | Biochemical detection device and method of magnetic fluid bead control combining digital fluid and electromagnetic field |
CA2550274A1 (en) | 2003-11-12 | 2005-05-26 | Acea Biosciences, Inc. | Real time electronic cell sensing systems and applications for cell-based assays |
US7341834B2 (en) * | 2003-12-15 | 2008-03-11 | Geneohn Sciences, Inc. | Multiplexed electrochemical detection system and method |
US20050142537A1 (en) * | 2003-12-24 | 2005-06-30 | Ronald Rieder | System and method for rapid detection of metabolic deviations of a biological sample |
KR100535817B1 (en) * | 2003-12-26 | 2005-12-12 | 한국전자통신연구원 | Plastic microfabricated structure for biochip, microfabricated thermal device, microfabricated reactor, microfabricated reactor array, and micro array using the same |
CA2834041C (en) * | 2003-12-31 | 2017-05-16 | President And Fellows Of Harvard College | Assay device and method |
US7407799B2 (en) * | 2004-01-16 | 2008-08-05 | California Institute Of Technology | Microfluidic chemostat |
US8030057B2 (en) * | 2004-01-26 | 2011-10-04 | President And Fellows Of Harvard College | Fluid delivery system and method |
ES2439225T3 (en) | 2004-01-26 | 2014-01-22 | President And Fellows Of Harvard College | System and method for fluid supply |
US7666285B1 (en) | 2004-02-06 | 2010-02-23 | University Of Central Florida Research Foundation, Inc. | Portable water quality monitoring system |
EP1725651A2 (en) * | 2004-03-15 | 2006-11-29 | Purdue Research Foundation | Cell concentration and pathogen recovery |
US8354066B2 (en) * | 2004-03-24 | 2013-01-15 | Technion Research & Development Foundation Ltd. | Artificial receptors |
CA2560760A1 (en) * | 2004-03-24 | 2005-09-29 | Technion Research And Development Foundation Ltd. | Electrode |
JP2008504845A (en) * | 2004-06-07 | 2008-02-21 | バイオプロセッサーズ コーポレイション | Reactor environmental condition control |
US7569126B2 (en) | 2004-06-18 | 2009-08-04 | Roche Diagnostics Operations, Inc. | System and method for quality assurance of a biosensor test strip |
US7504069B2 (en) * | 2004-06-21 | 2009-03-17 | Wisconsin Alumni Research Foundation | Micro device for high resolution delivery and monitoring of stimuli to a biological object, in vitro |
EP1789193A1 (en) * | 2004-09-03 | 2007-05-30 | Koninklijke Philips Electronics N.V. | Micro-fluidic system |
CN101019028B (en) * | 2004-09-10 | 2013-05-01 | 皇家飞利浦电子股份有限公司 | Compounds and methods for combined optical-ultrasound imaging |
US20090042280A1 (en) * | 2004-12-13 | 2009-02-12 | Geneohm Sciences, Inc., | Fluidic cartridges for electrochemical detection of dna |
CA2589976A1 (en) | 2004-12-16 | 2006-06-22 | Accelr8 Technology Corporation | Rapid microbial detection and antimicrobial susceptibility testing |
WO2006113727A2 (en) | 2005-04-19 | 2006-10-26 | President And Fellows Of Harvard College | Fluidic structures including meandering and wide channels |
US7749445B2 (en) * | 2005-05-02 | 2010-07-06 | Bioscale, Inc. | Method and apparatus for analyzing bioprocess fluids |
US7648844B2 (en) * | 2005-05-02 | 2010-01-19 | Bioscale, Inc. | Method and apparatus for detection of analyte using an acoustic device |
US7300631B2 (en) * | 2005-05-02 | 2007-11-27 | Bioscale, Inc. | Method and apparatus for detection of analyte using a flexural plate wave device and magnetic particles |
US7611908B2 (en) * | 2005-05-02 | 2009-11-03 | Bioscale, Inc. | Method and apparatus for therapeutic drug monitoring using an acoustic device |
NZ564141A (en) | 2005-05-09 | 2011-02-25 | Theranos Inc | Two way communication system for monitoring an analyte |
US20070072287A1 (en) * | 2005-05-23 | 2007-03-29 | Biovitesse, Inc. | Biomems cartridges |
US7265027B2 (en) * | 2005-06-14 | 2007-09-04 | Miradia Inc. | Bond method and structure using selective application of spin on glass |
US20070112796A1 (en) | 2005-11-17 | 2007-05-17 | Jung Edward K | Research in providing assistance related to health |
US8532938B2 (en) | 2005-11-17 | 2013-09-10 | The Invention Science Fund I, Llc | Testing-dependent administration of a nutraceutical |
US20070112592A1 (en) | 2005-11-17 | 2007-05-17 | Searete Llc, A Limited Liability Corporation Of The State Of Delaware | Payments in providing assistance related to health |
US10042980B2 (en) | 2005-11-17 | 2018-08-07 | Gearbox Llc | Providing assistance related to health |
US20080103746A1 (en) * | 2005-11-30 | 2008-05-01 | Searete Llc, A Limited Liability Corporation | Systems and methods for pathogen detection and response |
US7827042B2 (en) | 2005-11-30 | 2010-11-02 | The Invention Science Fund I, Inc | Methods and systems related to transmission of nutraceutical associated information |
US7974856B2 (en) | 2005-11-30 | 2011-07-05 | The Invention Science Fund I, Llc | Computational systems and methods related to nutraceuticals |
US8000981B2 (en) | 2005-11-30 | 2011-08-16 | The Invention Science Fund I, Llc | Methods and systems related to receiving nutraceutical associated information |
US8297028B2 (en) | 2006-06-14 | 2012-10-30 | The Invention Science Fund I, Llc | Individualized pharmaceutical selection and packaging |
US7927787B2 (en) | 2006-06-28 | 2011-04-19 | The Invention Science Fund I, Llc | Methods and systems for analysis of nutraceutical associated components |
US10296720B2 (en) | 2005-11-30 | 2019-05-21 | Gearbox Llc | Computational systems and methods related to nutraceuticals |
US8340944B2 (en) | 2005-11-30 | 2012-12-25 | The Invention Science Fund I, Llc | Computational and/or control systems and methods related to nutraceutical agent selection and dosing |
US7519409B2 (en) * | 2005-12-29 | 2009-04-14 | Medtronic, Inc. | Implantable cell/tissue-based biosensing device |
US8741230B2 (en) | 2006-03-24 | 2014-06-03 | Theranos, Inc. | Systems and methods of sample processing and fluid control in a fluidic system |
US11287421B2 (en) | 2006-03-24 | 2022-03-29 | Labrador Diagnostics Llc | Systems and methods of sample processing and fluid control in a fluidic system |
PL2244086T3 (en) * | 2006-04-08 | 2021-11-22 | F. Hoffmann-La Roche Ag | Analysis of optical data using histograms |
US8007999B2 (en) | 2006-05-10 | 2011-08-30 | Theranos, Inc. | Real-time detection of influenza virus |
EP2054525A1 (en) * | 2006-07-27 | 2009-05-06 | Koninklijke Philips Electronics N.V. | Device for molecular diagnosis |
US8236595B2 (en) * | 2006-08-11 | 2012-08-07 | Agency For Science, Technology And Research | Nanowire sensor, nanowire sensor array and method of fabricating the same |
US8050516B2 (en) * | 2006-09-13 | 2011-11-01 | Fluidigm Corporation | Methods and systems for determining a baseline during image processing |
US8055034B2 (en) * | 2006-09-13 | 2011-11-08 | Fluidigm Corporation | Methods and systems for image processing of microfluidic devices |
US8041515B2 (en) * | 2006-09-20 | 2011-10-18 | Acea Biosciences, Inc. | Use of impedance-based cytological profiling to classify cellular response profiles upon exposure to biologically active agents |
US20080113391A1 (en) | 2006-11-14 | 2008-05-15 | Ian Gibbons | Detection and quantification of analytes in bodily fluids |
WO2008127480A2 (en) * | 2006-12-21 | 2008-10-23 | Cornell Research Foundation, Inc. | Packed bed microreactors |
US10001496B2 (en) | 2007-01-29 | 2018-06-19 | Gearbox, Llc | Systems for allergen detection |
US8617903B2 (en) * | 2007-01-29 | 2013-12-31 | The Invention Science Fund I, Llc | Methods for allergen detection |
AT505106A1 (en) * | 2007-03-27 | 2008-10-15 | Arc Austrian Res Centers Gmbh | DEVICE, ESPECIALLY BIO-CHIP, FOR THE IDENTIFICATION OF MICRO-ORGANISMS |
DK2152417T3 (en) | 2007-05-04 | 2018-08-06 | Opko Diagnostics Llc | APPARATUS AND PROCEDURE FOR ANALYSIS IN MICROFLUID SYSTEMS |
EP2017618A1 (en) * | 2007-07-20 | 2009-01-21 | Koninklijke Philips Electronics N.V. | Methods and systems for detecting |
US8158430B1 (en) | 2007-08-06 | 2012-04-17 | Theranos, Inc. | Systems and methods of fluidic sample processing |
WO2009023857A1 (en) * | 2007-08-15 | 2009-02-19 | State Of Oregon By & Through The State Board Of Higher Education On Behalf Of Portland State Unv. | Impedance spectroscopy of biomolecules using functionalized nanoparticles |
WO2009033056A1 (en) * | 2007-09-06 | 2009-03-12 | Bioscale, Inc. | Reusable detection surfaces and methods of using same |
EP2197583A2 (en) * | 2007-09-19 | 2010-06-23 | Claros Diagnostics, Inc. | Liquid containment for integrated assays |
WO2009085356A2 (en) * | 2007-10-01 | 2009-07-09 | University Of Southern California Usc Stevens | Methods of using and constructing nanosensor platforms |
US20090137925A1 (en) * | 2007-11-23 | 2009-05-28 | Divya Cantor | Impedance Spectroscopy Cervix Scanning Apparatus and Method |
EP2285491A1 (en) | 2008-04-25 | 2011-02-23 | Claros Diagnostics, Inc. | Flow control in microfluidic systems |
CA2723223C (en) | 2008-05-05 | 2017-06-06 | Acea Biosciences, Inc. | Label-free monitoring of excitation-contraction coupling and excitable cells using impedance based systems with millisecond time resolution |
ES2401235T3 (en) * | 2008-05-07 | 2013-04-18 | University Of Strathclyde | System and method for cell characterization |
US8515507B2 (en) | 2008-06-16 | 2013-08-20 | Given Imaging Ltd. | Device and method for detecting in-vivo pathology |
AU2009298497B2 (en) | 2008-10-02 | 2013-12-19 | 12-15 Molecular Diagnostics, Inc. | Bionanosensor detection device |
US20100204062A1 (en) * | 2008-11-07 | 2010-08-12 | University Of Southern California | Calibration methods for multiplexed sensor arrays |
WO2010059687A2 (en) * | 2008-11-18 | 2010-05-27 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | A semiconductor for measuring biological interactions |
EP2376226B1 (en) | 2008-12-18 | 2018-09-12 | Opko Diagnostics, LLC | Improved reagent storage in microfluidic systems and related articles and methods |
EP2219033B1 (en) * | 2008-12-30 | 2016-05-25 | MicroCoat Biotechnologie GmbH | Device, instrument and process for detecting magnetically labeled analytes |
EP3278877B1 (en) | 2009-02-02 | 2020-06-03 | Opko Diagnostics, LLC | Structures for controlling light interaction with microfluidic devices |
US8449842B2 (en) * | 2009-03-19 | 2013-05-28 | Thermo Scientific Portable Analytical Instruments Inc. | Molecular reader |
WO2010115143A1 (en) * | 2009-04-03 | 2010-10-07 | University Of Southern California | Surface modification of nanosensor platforms to increase sensitivity and reproducibility |
EP2456713B1 (en) * | 2009-07-20 | 2024-03-27 | Monash University | Three-dimensional microfluidic systems |
MX2012004620A (en) | 2009-10-19 | 2012-06-25 | Theranos Inc | Integrated health data capture and analysis system. |
PT2504105T (en) | 2009-11-24 | 2021-03-31 | Opko Diagnostics Llc | Fluid mixing and delivery in microfluidic systems |
US8580569B2 (en) | 2010-04-16 | 2013-11-12 | Opko Diagnostics, Llc | Feedback control in microfluidic systems |
USD645971S1 (en) | 2010-05-11 | 2011-09-27 | Claros Diagnostics, Inc. | Sample cassette |
US8940148B2 (en) | 2010-06-22 | 2015-01-27 | International Business Machines Corporation | Nano-fluidic field effective device to control DNA transport through the same |
US8598018B2 (en) | 2010-06-22 | 2013-12-03 | International Business Machines Corporation | Forming an electrode having reduced corrosion and water decomposition on surface using a custom oxide layer |
US8354336B2 (en) | 2010-06-22 | 2013-01-15 | International Business Machines Corporation | Forming an electrode having reduced corrosion and water decomposition on surface using an organic protective layer |
CN102487118B (en) * | 2010-12-05 | 2014-04-30 | 北京德锐磁星科技有限公司 | Magnetic biosensor and preparation method thereof |
WO2012141605A1 (en) * | 2011-02-16 | 2012-10-18 | Centrul International De Biodinamica | Systems and method for detection and quantitation of analytes using impedance analysis |
EP2554213A1 (en) * | 2011-08-01 | 2013-02-06 | Centro de Estudios e Investigaciones Tecnicas (CEIT) | Intelligent subcutaneous venous access port and method for detecting biolayer |
CN104364788B (en) | 2012-03-05 | 2018-02-06 | 阿克蒂克合伙公司 | Predict prostate cancer risk and the device of prostate gland volume |
GB2525800A (en) | 2013-02-14 | 2015-11-04 | Paul Weber | Systems, apparatus and methods for tissue dissection |
CA2900708C (en) | 2013-03-13 | 2021-06-15 | Opko Diagnostics, Llc | Mixing of fluids in fluidic systems |
ITLE20130011A1 (en) * | 2013-08-01 | 2015-02-02 | Ekuberg Pharma Srl | '' IMPEDENZIOMETRIC BIOCHIP FOR THE CONTEMPORARY DIAGNOSIS OF CANDIDA ALBICANS, CHLAMYDIA TRACHOMATIS AND STREPTOCOCCUS AGALACTIAE '' |
KR101474972B1 (en) * | 2013-10-31 | 2014-12-22 | 한국기계연구원 | A coupling device for a biochip |
EA038479B1 (en) | 2014-12-12 | 2021-09-03 | Опкоу Дайагностикс, Ллк | Device for performing analysis of an assay and method of operating said device |
US10274492B2 (en) * | 2015-04-10 | 2019-04-30 | The Curators Of The University Of Missouri | High sensitivity impedance sensor |
CN108027335B (en) | 2015-06-25 | 2021-05-04 | 罗斯韦尔生物技术股份有限公司 | Biomolecule sensor and method |
USD804682S1 (en) | 2015-08-10 | 2017-12-05 | Opko Diagnostics, Llc | Multi-layered sample cassette |
WO2017100457A1 (en) | 2015-12-11 | 2017-06-15 | Opko Diagnostics, Llc | Fluidic systems involving incubation samples and/or reagents |
JP7080489B2 (en) | 2016-01-28 | 2022-06-06 | ロズウェル バイオテクノロジーズ,インコーポレイテッド | Ultra-parallel DNA sequencer |
EP3408220A4 (en) | 2016-01-28 | 2019-09-04 | Roswell Biotechnologies, Inc | Methods and apparatus for measuring analytes using large scale molecular electronics sensor arrays |
WO2017139493A2 (en) | 2016-02-09 | 2017-08-17 | Roswell Biotechnologies, Inc. | Electronic label-free dna and genome sequencing |
US10597767B2 (en) | 2016-02-22 | 2020-03-24 | Roswell Biotechnologies, Inc. | Nanoparticle fabrication |
US9829456B1 (en) | 2016-07-26 | 2017-11-28 | Roswell Biotechnologies, Inc. | Method of making a multi-electrode structure usable in molecular sensing devices |
US11389096B2 (en) * | 2016-09-10 | 2022-07-19 | Ecole Polytechnique Federale De Lausanne (Epfl) | Bio-fluid collection and sensing device, system and method |
EP3529348A1 (en) * | 2016-11-24 | 2019-08-28 | Sensirion AG | Microorganism test system |
CA3052062A1 (en) | 2017-01-10 | 2018-07-19 | Roswell Biotechnologies, Inc. | Methods and systems for dna data storage |
KR20230158636A (en) | 2017-01-19 | 2023-11-20 | 로스웰 바이오테크놀로지스 인코포레이티드 | Solid state sequencing devices comprising two dimensional layer materials |
WO2018161063A1 (en) | 2017-03-03 | 2018-09-07 | Acea Biosciences, Inc. | METHODS AND SYSTEMS FOR FUNCTIONAL MATURATION OF iPSC AND ESC DERIVED CARDIOMYOCYTES |
US11525152B2 (en) | 2017-04-07 | 2022-12-13 | Acenxion Biosystems, Inc. | System and method for rapid detection of viable microorganisms in liquid media |
CN110546276A (en) | 2017-04-25 | 2019-12-06 | 罗斯威尔生命技术公司 | Enzyme circuit for molecular sensors |
US10508296B2 (en) | 2017-04-25 | 2019-12-17 | Roswell Biotechnologies, Inc. | Enzymatic circuits for molecular sensors |
EP3622086A4 (en) | 2017-05-09 | 2021-04-21 | Roswell Biotechnologies, Inc | Binding probe circuits for molecular sensors |
US10787695B2 (en) | 2017-06-01 | 2020-09-29 | General Electric Company | Systems and methods for rapidly sensing microbial metabolism |
KR20200039795A (en) | 2017-08-30 | 2020-04-16 | 로스웰 바이오테크놀로지스 인코포레이티드 | Progressive enzyme molecular electronic sensors for DNA data storage |
WO2019075100A1 (en) | 2017-10-10 | 2019-04-18 | Roswell Biotechnologies, Inc. | Methods, apparatus and systems for amplification-free dna data storage |
TWI645186B (en) * | 2017-12-29 | 2018-12-21 | 南臺學校財團法人南臺科技大學 | Impedance chip's detection system for biological testing |
WO2019173264A1 (en) * | 2018-03-05 | 2019-09-12 | Board Of Trustees Of Michigan State University | Wireless detection of electrically or magnetically labeled analytes |
EP3862415A1 (en) * | 2020-02-04 | 2021-08-11 | Sensirion AG | Method, device, sensor cartridge and kit of parts for culturing and detecting microorganisms |
USD941488S1 (en) | 2020-02-07 | 2022-01-18 | Agilent Technologies, Inc. | Instrument for analyzing biological cells |
CN112505029B (en) * | 2020-11-26 | 2022-12-09 | 北京市农林科学院 | Colorimetric sensing detection method for pathogenic bacteria and application thereof |
Citations (10)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US3984766A (en) * | 1974-10-15 | 1976-10-05 | Bactomatic Inc. | Digital apparatus for rapidly detecting the growth of and identifying micro-biological organisms |
US4009078A (en) * | 1975-01-24 | 1977-02-22 | The United States Of America As Represented By The Administrator Of The National Aeronautics And Space Administration | Detecting the presence of microorganisms |
US4072578A (en) * | 1975-11-19 | 1978-02-07 | Bactomatic Corporation | Multi-chambered module for use in monitoring growth of microorganisms |
US4156180A (en) * | 1977-06-24 | 1979-05-22 | Bactomatic, Inc. | Apparatus and method for detecting metabolic activity |
US5187096A (en) * | 1991-08-08 | 1993-02-16 | Rensselaer Polytechnic Institute | Cell substrate electrical impedance sensor with multiple electrode array |
US5432086A (en) * | 1992-11-18 | 1995-07-11 | Sy-Lab Vertriebsgellschaft M.B.H. | Apparatus for the automatic monitoring of microorganism culture |
US5726026A (en) * | 1992-05-01 | 1998-03-10 | Trustees Of The University Of Pennsylvania | Mesoscale sample preparation device and systems for determination and processing of analytes |
US5789191A (en) * | 1993-08-26 | 1998-08-04 | Beiersdorf Ag | Method of detecting and counting microorganisms |
US5824494A (en) * | 1994-05-01 | 1998-10-20 | Sirotech Ltd. | Method for enumerating bacterial populations |
US5981268A (en) * | 1997-05-30 | 1999-11-09 | Board Of Trustees, Leland Stanford, Jr. University | Hybrid biosensors |
Family Cites Families (19)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US3676775A (en) * | 1971-05-07 | 1972-07-11 | Ibm | Method for measuring resistivity |
GB1585067A (en) | 1976-10-19 | 1981-02-25 | Nat Res Dev | Detection of bacterial activity |
JPS612060A (en) * | 1984-06-15 | 1986-01-08 | Matsushita Electric Works Ltd | Biosensor |
US5192507A (en) * | 1987-06-05 | 1993-03-09 | Arthur D. Little, Inc. | Receptor-based biosensors |
US5770029A (en) * | 1996-07-30 | 1998-06-23 | Soane Biosciences | Integrated electrophoretic microdevices |
US5643742A (en) | 1990-04-03 | 1997-07-01 | Cellstat Technologies, Inc. | System for electronically monitoring and recording cell cultures |
DE4318519C2 (en) * | 1993-06-03 | 1996-11-28 | Fraunhofer Ges Forschung | Electrochemical sensor |
US5571410A (en) * | 1994-10-19 | 1996-11-05 | Hewlett Packard Company | Fully integrated miniaturized planar liquid sample handling and analysis 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 |
US6143247A (en) * | 1996-12-20 | 2000-11-07 | Gamera Bioscience Inc. | Affinity binding-based system for detecting particulates in a fluid |
US20010055812A1 (en) * | 1995-12-05 | 2001-12-27 | Alec Mian | Devices and method for using centripetal acceleration to drive fluid movement in a microfluidics system with on-board informatics |
US5837196A (en) * | 1996-01-26 | 1998-11-17 | The Regents Of The University Of California | High density array fabrication and readout method for a fiber optic biosensor |
US6165335A (en) | 1996-04-25 | 2000-12-26 | Pence And Mcgill University | Biosensor device and method |
US6130037A (en) | 1996-04-25 | 2000-10-10 | Pence And Mcgill University | Biosensor device and method |
US5954931A (en) * | 1997-01-24 | 1999-09-21 | Motorola, Inc. | Electrophoresis apparatus and method involving parallel channels |
US6169394B1 (en) | 1998-09-18 | 2001-01-02 | University Of The Utah Research Foundation | Electrical detector for micro-analysis systems |
US6149787A (en) * | 1998-10-14 | 2000-11-21 | Caliper Technologies Corp. | External material accession systems and methods |
US6416642B1 (en) * | 1999-01-21 | 2002-07-09 | Caliper Technologies Corp. | Method and apparatus for continuous liquid flow in microscale channels using pressure injection, wicking, and electrokinetic injection |
US6300141B1 (en) * | 1999-03-02 | 2001-10-09 | Helix Biopharma Corporation | Card-based biosensor device |
-
2001
- 2001-03-26 WO PCT/US2001/009745 patent/WO2001079529A1/en active Application Filing
- 2001-03-26 EP EP01926440A patent/EP1283900A4/en not_active Withdrawn
- 2001-03-26 US US09/817,541 patent/US6716620B2/en not_active Expired - Lifetime
- 2001-03-26 AU AU2001252973A patent/AU2001252973A1/en not_active Abandoned
- 2001-03-26 CA CA002406133A patent/CA2406133A1/en not_active Abandoned
-
2002
- 2002-06-14 US US10/172,263 patent/US20030036054A1/en not_active Abandoned
Patent Citations (10)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US3984766A (en) * | 1974-10-15 | 1976-10-05 | Bactomatic Inc. | Digital apparatus for rapidly detecting the growth of and identifying micro-biological organisms |
US4009078A (en) * | 1975-01-24 | 1977-02-22 | The United States Of America As Represented By The Administrator Of The National Aeronautics And Space Administration | Detecting the presence of microorganisms |
US4072578A (en) * | 1975-11-19 | 1978-02-07 | Bactomatic Corporation | Multi-chambered module for use in monitoring growth of microorganisms |
US4156180A (en) * | 1977-06-24 | 1979-05-22 | Bactomatic, Inc. | Apparatus and method for detecting metabolic activity |
US5187096A (en) * | 1991-08-08 | 1993-02-16 | Rensselaer Polytechnic Institute | Cell substrate electrical impedance sensor with multiple electrode array |
US5726026A (en) * | 1992-05-01 | 1998-03-10 | Trustees Of The University Of Pennsylvania | Mesoscale sample preparation device and systems for determination and processing of analytes |
US5432086A (en) * | 1992-11-18 | 1995-07-11 | Sy-Lab Vertriebsgellschaft M.B.H. | Apparatus for the automatic monitoring of microorganism culture |
US5789191A (en) * | 1993-08-26 | 1998-08-04 | Beiersdorf Ag | Method of detecting and counting microorganisms |
US5824494A (en) * | 1994-05-01 | 1998-10-20 | Sirotech Ltd. | Method for enumerating bacterial populations |
US5981268A (en) * | 1997-05-30 | 1999-11-09 | Board Of Trustees, Leland Stanford, Jr. University | Hybrid biosensors |
Cited By (92)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20030153023A1 (en) * | 1999-05-13 | 2003-08-14 | Starzl Timothy W. | Enumeration method of analyte detection |
US20040166555A1 (en) * | 1999-11-10 | 2004-08-26 | Rebecca Braff | Cell sorting apparatus and methods for manipulating cells using the same |
US7090996B2 (en) * | 2000-06-09 | 2006-08-15 | Micronas Gmbh | Process for examining membrane enclosed biocompartments |
US20050042596A1 (en) * | 2000-06-09 | 2005-02-24 | Micronas Gmbh | Process for examining membrane enclosed biocompartments |
US7863140B2 (en) * | 2001-04-23 | 2011-01-04 | Samsung Electronics Co., Ltd. | Methods of making a molecular detection chip having a metal oxide silicon field effect transistor on sidewalls of a micro-fluid channel |
US20070218610A1 (en) * | 2001-04-23 | 2007-09-20 | Samsung Electronics Co., Ltd. | Methods of making a molecular detection chip having a metal oxide silicon field effect transistor on sidewalls of a micro-fluid channel |
US9089851B2 (en) | 2002-05-30 | 2015-07-28 | Qiagen Instruments Ag | DNA amplification apparatus and method |
US20060147912A1 (en) * | 2002-05-30 | 2006-07-06 | Corbett John M | Dna amplification apparatus and method |
US20060057599A1 (en) * | 2002-08-26 | 2006-03-16 | The Regents Of The University Of California | System for autonomous monitoring of bioagents |
US11052392B2 (en) | 2002-09-27 | 2021-07-06 | The General Hospital Corporation | Microfluidic device for cell separation and uses thereof |
US8304230B2 (en) | 2002-09-27 | 2012-11-06 | The General Hospital Corporation | Microfluidic device for cell separation and uses thereof |
US8986966B2 (en) | 2002-09-27 | 2015-03-24 | The General Hospital Corporation | Microfluidic device for cell separation and uses thereof |
US10081014B2 (en) | 2002-09-27 | 2018-09-25 | The General Hospital Corporation | Microfluidic device for cell separation and uses thereof |
US8372579B2 (en) | 2002-09-27 | 2013-02-12 | The General Hospital Corporation | Microfluidic device for cell separation and uses thereof |
US20070259424A1 (en) * | 2002-09-27 | 2007-11-08 | The General Hospital Corporation | Microfluidic device for cell separation and uses thereof |
US8895298B2 (en) | 2002-09-27 | 2014-11-25 | The General Hospital Corporation | Microfluidic device for cell separation and uses thereof |
US20040171091A1 (en) * | 2003-02-27 | 2004-09-02 | Cell Work, Inc. | Standardized evaluation of therapeutic efficacy based on cellular biomarkers |
US20070160503A1 (en) * | 2003-06-13 | 2007-07-12 | Palaniappan Sethu | Microfluidic systems for size based removal of red blood cells and platelets from blood |
US9657327B2 (en) | 2003-07-12 | 2017-05-23 | Accelerate Diagnostics, Inc. | Rapid microbial detection and antimicrobial susceptibility testing |
US7341841B2 (en) | 2003-07-12 | 2008-03-11 | Accelr8 Technology Corporation | Rapid microbial detection and antimicrobial susceptibility testing |
US8460887B2 (en) | 2003-07-12 | 2013-06-11 | Accelerate Diagnostics, Inc. | Sensitive and rapid determination of antimicrobial susceptibility |
US8895255B1 (en) | 2003-07-12 | 2014-11-25 | Accelerate Diagnostics, Inc. | Sensitive and rapid determination of antimicrobial susceptibility |
US20080241858A1 (en) * | 2003-07-12 | 2008-10-02 | Metzger Steven W | Rapid microbial detection and antimicrobial susceptibiility testing |
US7687239B2 (en) | 2003-07-12 | 2010-03-30 | Accelrs Technology Corporation | Sensitive and rapid determination of antimicrobial susceptibility |
US20100136570A1 (en) * | 2003-07-12 | 2010-06-03 | Goldberg David A | Sensitive and rapid determination of antimicrobial susceptibility |
US9841422B2 (en) | 2003-07-12 | 2017-12-12 | Accelerate Diagnostics, Inc. | Sensitive and rapid determination of antimicrobial susceptibility |
US8071319B2 (en) | 2003-07-12 | 2011-12-06 | Accelr8 Technology Corporation | Rapid microbial detection and antimicrobial susceptibiility testing |
US20050048599A1 (en) * | 2003-07-12 | 2005-03-03 | Goldberg David A. | Sensitive and rapid determination of antimicrobial susceptibility |
US11054420B2 (en) | 2003-07-12 | 2021-07-06 | Accelerate Diagnostics, Inc. | Sensitive and rapid determination of antimicrobial susceptibility |
US7548636B2 (en) * | 2003-08-15 | 2009-06-16 | Nippon Telegraph And Telephone Corporation | Organism recognition system |
US20060034493A1 (en) * | 2003-08-15 | 2006-02-16 | Toshishige Shimamura | Organism recognition system |
US7390622B2 (en) | 2003-10-16 | 2008-06-24 | Hai Kang Life Corporation Limited | Apparatus and methods for detecting nucleic acid in biological samples |
US20080242562A1 (en) * | 2003-10-16 | 2008-10-02 | Hai Kang Life Corporation Limited | Apparatus and methods for detecting nucleic acid in biological samples |
US7888109B2 (en) | 2003-10-16 | 2011-02-15 | Hai Kang Life Corporation Limited | Apparatus and methods for detecting nucleic acid in biological samples |
US20050084865A1 (en) * | 2003-10-16 | 2005-04-21 | Hong Kong Dna Chips Limited | Apparatus and methods for detecting nucleic acid in biological samples |
US20090068638A1 (en) * | 2005-03-17 | 2009-03-12 | Biophage Inc. | Phage-based method for the detection of bacteria |
US20090216082A1 (en) * | 2005-04-01 | 2009-08-27 | Elisha Rabinovitz | Device, System and Method for In Vivo Magnetic Immunoassay Analysis |
US8585971B2 (en) | 2005-04-05 | 2013-11-19 | The General Hospital Corporation | Devices and method for enrichment and alteration of cells and other particles |
US10786817B2 (en) | 2005-04-05 | 2020-09-29 | The General Hospital Corporation | Devices and method for enrichment and alteration of cells and other particles |
US20070026381A1 (en) * | 2005-04-05 | 2007-02-01 | Huang Lotien R | Devices and methods for enrichment and alteration of cells and other particles |
US8021614B2 (en) | 2005-04-05 | 2011-09-20 | The General Hospital Corporation | Devices and methods for enrichment and alteration of cells and other particles |
US9956562B2 (en) | 2005-04-05 | 2018-05-01 | The General Hospital Corporation | Devices and method for enrichment and alteration of cells and other particles |
US9174222B2 (en) | 2005-04-05 | 2015-11-03 | The General Hospital Corporation | Devices and method for enrichment and alteration of cells and other particles |
US8921102B2 (en) | 2005-07-29 | 2014-12-30 | Gpb Scientific, Llc | Devices and methods for enrichment and alteration of circulating tumor cells and other particles |
US20070026469A1 (en) * | 2005-07-29 | 2007-02-01 | Martin Fuchs | Devices and methods for enrichment and alteration of circulating tumor cells and other particles |
AU2006290025B2 (en) * | 2005-09-14 | 2011-10-20 | National Institute Of Advanced Industrial Science And Technology | Bio-sensor measurement machine, bio-sensor measurement system, and bio-sensor measurement method |
US20090152127A1 (en) * | 2005-09-14 | 2009-06-18 | Sumitomo Electric Industries, Ltd. | Biosensor Measurement Machine , Biosensor Measurement System and Biosensor Measurement Method |
KR101299275B1 (en) * | 2005-09-14 | 2013-08-23 | 내셔날 인스티튜트 오브 어드밴스드 인더스트리얼 사이언스 앤드 테크놀로지 | Bio-sensor measuring device, bio-sensor measuring system, and bio-sensor measuring method |
US8282813B2 (en) * | 2005-09-14 | 2012-10-09 | Sumitomo Electric Industries, Ltd. | Biosensor measurement machine, biosensor measurement system and biosensor measurement method |
TWI396842B (en) * | 2005-09-14 | 2013-05-21 | Sumitomo Electric Industries | Biosensor measuring machine, biosensor measuring system and biosensor measuring method |
US9494583B2 (en) * | 2006-09-16 | 2016-11-15 | University Of Leeds | Methods and devices for detecting structural changes in a molecule measuring electrochemical impedance |
US20130040848A1 (en) * | 2006-09-16 | 2013-02-14 | Medical Research Council | Methods and Devices for Detecting Structural Changes in a Molecule Measuring Electrochemical Impedance |
US8842422B2 (en) | 2006-10-23 | 2014-09-23 | ACCO Brands Corporation | Security apparatus |
US9791894B2 (en) | 2006-10-23 | 2017-10-17 | ACCO Brands Corporation | Security apparatus |
US10146264B2 (en) | 2006-10-23 | 2018-12-04 | ACCO Brands Corporation | Security apparatus |
US10031558B2 (en) | 2006-10-23 | 2018-07-24 | ACCO Brands Corporation | Security apparatus |
US9423823B2 (en) | 2006-10-23 | 2016-08-23 | ACCO Brands Corporation | Security apparatus for securing a portable electronic device |
US10928861B2 (en) | 2006-10-23 | 2021-02-23 | ACCO Brands Corporation | Security apparatus |
US10656682B2 (en) | 2006-10-23 | 2020-05-19 | ACCO Brands Corporation | Security apparatus |
US20110122551A1 (en) * | 2006-10-23 | 2011-05-26 | Acco Brands Usa Llc | Security Apparatus |
US11392177B2 (en) | 2006-10-23 | 2022-07-19 | ACCO Brands Corporation | Security apparatus |
US10520985B2 (en) | 2006-10-23 | 2019-12-31 | ACCO Brands Corporation | Security apparatus |
US8471294B2 (en) | 2006-11-08 | 2013-06-25 | Duke University | GaN-based nitric oxide sensors and methods of making and using the same |
US20110097837A1 (en) * | 2006-11-08 | 2011-04-28 | Garcia Michael A | GaN-BASED NITRIC OXIDE SENSORS AND METHODS OF MAKING AND USING THE SAME |
KR101465961B1 (en) * | 2007-10-09 | 2014-12-01 | 삼성전자주식회사 | A method and a device for detecting DNAs, etc. |
US9970897B2 (en) | 2008-06-16 | 2018-05-15 | Duke University | Chemical sensors and methods for making and using the same |
US20110199102A1 (en) * | 2008-06-16 | 2011-08-18 | Garcia Michael A | Chemical sensors and methods for making and using the same |
US20110236924A1 (en) * | 2008-12-19 | 2011-09-29 | Halverson Kurt J | System and method for processing samples |
US8535945B2 (en) | 2008-12-19 | 2013-09-17 | 3M Innovative Properties Company | System and method for concentrating samples |
US9388448B2 (en) | 2008-12-19 | 2016-07-12 | 3M Innovative Properties Company | System and method for processing samples |
US8647508B2 (en) | 2008-12-19 | 2014-02-11 | 3M Innovative Properties Company | System and method for processing samples |
US20120122736A1 (en) * | 2009-02-11 | 2012-05-17 | Duke University | Sensors incorporating antibodies and methods of making and using the same |
US9958442B2 (en) * | 2009-02-11 | 2018-05-01 | Duke University | Sensors incorporating antibodies and methods of making and using the same |
US20190256886A1 (en) * | 2009-10-02 | 2019-08-22 | The Curators Of The University Of Missouri | Rapid detection of viable bacteria system and method |
US20110151610A1 (en) * | 2009-12-23 | 2011-06-23 | Varian Semiconductor Equipment Associates, Inc. | Workpiece patterning with plasma sheath modulation |
US11346798B2 (en) | 2010-07-12 | 2022-05-31 | Arizona Board Of Regents On Behalf Of Arizona State University | Methods and device for tuning multiplexed markers for disease assay |
US10254204B2 (en) | 2011-03-07 | 2019-04-09 | Accelerate Diagnostics, Inc. | Membrane-assisted purification |
US10202597B2 (en) | 2011-03-07 | 2019-02-12 | Accelerate Diagnostics, Inc. | Rapid cell purification systems |
US9714420B2 (en) | 2011-03-07 | 2017-07-25 | Accelerate Diagnostics, Inc. | Rapid cell purification systems |
US9434937B2 (en) | 2011-03-07 | 2016-09-06 | Accelerate Diagnostics, Inc. | Rapid cell purification systems |
US9909969B2 (en) | 2011-06-30 | 2018-03-06 | 3M Innovative Properties Company | Systems and methods for detecting an analyte of interest in a sample using microstructured surfaces |
US9488563B2 (en) | 2011-06-30 | 2016-11-08 | 3M Innovative Properties Company | Systems and methods for detecting an analyte of interest in a sample using microstructured surfaces |
US9470612B2 (en) | 2011-06-30 | 2016-10-18 | 3M Innovative Properties Company | Systems and methods for detecting an analyte of interest in a sample using filters and microstructured surfaces |
US9677109B2 (en) | 2013-03-15 | 2017-06-13 | Accelerate Diagnostics, Inc. | Rapid determination of microbial growth and antimicrobial susceptibility |
US11603550B2 (en) | 2013-03-15 | 2023-03-14 | Accelerate Diagnostics, Inc. | Rapid determination of microbial growth and antimicrobial susceptibility |
US10407716B2 (en) | 2014-03-13 | 2019-09-10 | Duke University | Electronic platform for sensing and control of electrochemical reactions |
US10669566B2 (en) | 2015-03-30 | 2020-06-02 | Accelerate Giagnostics, Inc. | Instrument and system for rapid microorganism identification and antimicrobial agent susceptibility testing |
US10619180B2 (en) | 2015-03-30 | 2020-04-14 | Accelerate Diagnostics, Inc. | Instrument and system for rapid microorganism identification and antimicrobial agent susceptibility testing |
US10273521B2 (en) | 2015-03-30 | 2019-04-30 | Accelerate Diagnostics, Inc. | Instrument and system for rapid microorganism identification and antimicrobial agent susceptibility testing |
US10253355B2 (en) | 2015-03-30 | 2019-04-09 | Accelerate Diagnostics, Inc. | Instrument and system for rapid microorganism identification and antimicrobial agent susceptibility testing |
US10023895B2 (en) | 2015-03-30 | 2018-07-17 | Accelerate Diagnostics, Inc. | Instrument and system for rapid microogranism identification and antimicrobial agent susceptibility testing |
WO2017015574A1 (en) * | 2015-07-23 | 2017-01-26 | Purdue Research Foundation | Rapid concentration, recovery and detection of pathogens in food samples |
Also Published As
Publication number | Publication date |
---|---|
CA2406133A1 (en) | 2001-10-25 |
AU2001252973A1 (en) | 2001-10-30 |
US6716620B2 (en) | 2004-04-06 |
EP1283900A4 (en) | 2006-02-08 |
US20010053535A1 (en) | 2001-12-20 |
EP1283900A1 (en) | 2003-02-19 |
WO2001079529A1 (en) | 2001-10-25 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US6716620B2 (en) | Biosensor and related method | |
US7306924B2 (en) | Biosensor and related method | |
US7435579B2 (en) | Biosensor and related method | |
Díaz‐González et al. | Recent advances in electrochemical enzyme immunoassays | |
US6887362B2 (en) | Dielectrophoretic separation and immunoassay methods on active electronic matrix devices | |
RU2530718C2 (en) | Device and methods of detecting analytes in saliva | |
Moina et al. | Fundamentals and applications of immunosensors | |
US20100075340A1 (en) | Electrical Detection Of Biomarkers Using Bioactivated Microfluidic Channels | |
CN102112877B (en) | Sensor | |
CN104969069B (en) | For the apparatus and method for the dynamic range for identifying hook effect and expansion point of care immunoassays | |
US20050059105A1 (en) | Impedimetric biosensor and its use for rapid detection of bacterial pathogens in solution | |
US20060286549A1 (en) | Microfluidic system for identifying or sizing individual particles passing through a channel | |
US20120021934A1 (en) | Methods and Devices for Active Bioassay | |
CN104998700A (en) | Multi-compartment device with magnetic particles | |
KR20050044559A (en) | Method for detecting analyte(s) using magnetic colloidal particles | |
CN104777298A (en) | Detection apparatus | |
AU6127899A (en) | Immuno-diagnostic test method for veterinary disease | |
WO2000060354A1 (en) | Lipopolysaccharide immunoassay and test device | |
US7456028B2 (en) | Electrochemical method for detecting water born pathogens | |
González-Cortés | Electrochemical impedance spectroscopy | |
NL2007328C2 (en) | A nanopore sensor and method for selective detection of analytes in a sample. | |
KR102433429B1 (en) | Nanomagnetic antibody labelled with carboxylated iron oxide nanoparticle and the preparation method thereof | |
JP4856854B2 (en) | Mass transfer device and mass transfer method | |
Johnson et al. | Biosensors | |
Salam | Development of immunosensors for Salmonella typhimurium |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
STCB | Information on status: application discontinuation |
Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION |