WO2007082480A1

WO2007082480A1 - Cartridge-based microfluidic analyzer

Info

Publication number: WO2007082480A1
Application number: PCT/CN2007/000198
Authority: WO
Inventors: Min Guo; Xiaosheng Guan; Cheng Zhou; Dongxu Weng; Yuming Hu; Wanjie Zhang; Yonggang Guo; Guanbin Zhang; Jing Cheng
Original assignee: Capitalbio Corporation; Tsinghua University
Priority date: 2006-01-19
Filing date: 2007-01-18
Publication date: 2007-07-26
Also published as: CN101004423A; CN101004423B

Abstract

An integrated polynucleotide amplification and nucleotide sequence identification system comprises an expendable microfluidic cartridge and a reusable external unit. Sample fluids and reagents are confined to the expendable microfluidic cartridge for processing. The reusable external unit comprises pumps, valves, sensors, and temperature controllers. Polynucleotide sample amplification through polymerase chain reaction (PCR), and nucleotide sequence identification using oligonucleotide arrays can be conducted in an automatic, integrated process.

Description

CARTRIDGE-BASED MICROFLUIDIC ANALYZER

Technical Field

[0001] Embodiments of the present invention relate to fluidic analyzers that use removable cartridges for fluid containment and analysis. In particular, some embodiments relate to cartridge- based, microfluidic nucleic acid analyzers.

Background Art

[0002] Microfluidic devices have emerged as a new approach for improving the performance and functionality of systems for chemical and biochemical synthesis, as well as chemical, biochemical, and medical analysis. Miniaturization and new effects in micro-scale provide completely new solutions in these fields. Dimension reduction results in faster processes with reduced reagent and sample consumption rates. The small size scale also encourages parallel processing, in which more compounds can be produced and/or analyzed simultaneously. Massively parallel processing can speed DNA, RNA, protein, immunologic, and other tests to reduce time intervals for drug discovery and medical diagnosis. Currently, microfluidic based microanalysis systems for such applications typically have fluid channel dimensions on the order of tenths of millimeters to several millimeters, although future trends are to further reduce channel dimensions. Various microfluidic components have also been demonstrated on the same size scale, for example: micro-valves, micro-pumps, micro-flow sensors, micro-filters, micro-mixers, micro-reactors, micro-separators, and micro-dispensers, to name just a few. The book, FUNDAMENTALS AND APPLICATIONS OF MICROFLUIDICS by Nam-Trung Nguyen and Steven T. Werely, published by Artech House of Boston, U.S.A., in 2002 provides an overview of some microfluidic technologies and applications.

[0003] System integration and automation is an effective means to eliminate contamination in and improve efficiency over manually performed nucleic acid assays. A desired integrated nucleic acid analysis system may comprise of two essential parts according to their respective functions: sample residing part and process control part. The sample residing part provides a physical body for nucleic acid analysis, and can further be composed of different functional units. Nucleic acid samples and reagents required for analyses are to be stored, flowed, and reacted within the sample residing part. The process control part manipulates fluid motions within or between different functional units of the sample residing part, regulates temperature conditions suitable for biochemical reactions, and detects results of assays.

[0004] One approach to integration is, by using various microfabrication methods, to form some of the process control part onto the sample residing part, such as micro-pumps, micro-valves, temperature sensors, electric circuits, and so on. Since their fabrication is still costly imposed by current technologies and they are not meant for reusage after being contacted by sample fluids, this approach increases ultimate end-use costs and creates difficulties for many prototype systems having developed in this manner to successfully hit on market. A different strategy is to separate the sample residing part as much as possible from the process control part. It is desirable for the sample residing part to comprise of only passive microfluidic structures, such as microchannels and microchambers, while leaving out any moving mechanical parts, so that it can be made cheap enough to achieve a great economy for disposable usage. Meanwhile, the process control part can be made separately and reusable for many times. When the process control part is linked with the sample residing part through appropriate interfaces, a complete system for integrated and automated nucleic acid analyses is obtained.

[0005] Even if physically the sample residing part and the process control part may be separated, they are not totally independent of each other in their respective designs. For instance, in what manner the fluid samples are to be driven, be it by a constant pressure source, or by a pump that can be turned on and off, or by an electroosmotic force, will determine the corresponding microfluidic structures; vice versus, purposely designed microfluidics, such as dimensional changes or surface modifications of microchannels affect selection of pump and valve types, as well as their placement and an overall control scheme. Moreover, locations for desired biochemical reactions to take place, be it on surfaces or in the interior regions of the microchannels, requirements of special temperature conditions, or specific usage of biomarkers, determine the temperature regulation and assay detection methods; meanwhile, the size and configurations of microfluidic structures must be compatible with the specific forms of temperature sensors and detection elements employed.

[0006] Microfludic devices with cartridge systems have been previously described in U.S. Pat. Nos. 6,615,856 and 6,197,595, and U.S. Pat. Appl. No. 20050153430.

[0007] All references described herein are incorporated by reference in their entirety.

Summary of Embodiments of the Invention

[0008] The present invention provides microfluidic devices and uses thereof. [0009] In one aspect, there are provided microfluidic devices comprising a cartridge and a separate control unit. In some embodiments, there is provided a microfluidic device comprising a cartridge comprising at least one microchannel and a separate control unit comprising at least a pump and a valve, wherein the at least one end of the microchannel is coupled to the pump and at least one end of the microchannel is coupled to the valve, wherein the pump controls movement of fluid along the microchannel, and wherein the valve controls direction of fluid flow along the microchannel. In some embodiments, the control unit further comprises at least one sensor, wherein the sensor senses the passage of fluid flow front and provides a feedback signal to control the action of the pump and the valve. In some embodiments, the microfluidic device does not comprise an air duct. In some embodiments, the microfluidic device is a closed-loop system.

[0010] In some embodiments, there is provided a control unit comprising at least a pump and at least a valve, wherein the pump is configured to be coupled to at least one end of a microchannel and controls fluid flow along the microchannel, wherein the valve is configured to be coupled to at least one end of a microchannel and controls direction of fluid flow in the microchannel. In some embodiments, the control unit further comprises a sensor, wherein the sensor is configured to sense the passage of fluid flow front along a microchannel and provide a feedback signal to control the action of the pump and th,e valve. In some embodiments, the control unit is reusable.

[0011] In some embodiments, there is provided a cartridge comprising at least one microchannel, wherein at least one end of the microchannel can be coupled to a pump that controls fluid flow along the microchannel, wherein at least one end of the microchannel can be coupled to a valve that controls the direction of fluid flow along the microchannel. In some embodiments, the passage of fluid flow front in the microchannel can be sensed by at least one sensor. In some embodiments, change of the flow of a fluid in the microchannel can be controlled by a feedback signal generated from the sensor. In some embodiments, fluid flow in the microchannel of the cartridge does not rely on an air duct. In some embodiments, the cartridge does not comprise an air duct. In some embodiments, the cartridge is disposable.

[0012] In some embodiments, the control unit (either alone or in the context of the microfluidic device) further comprises a controller for controlling the action of the pump and/or the valves. In some embodiments, the controller also receives and processes information from the sensor and provides a feedback control mechanism to change the actions of the pump and/or the valve. In some embodiments, the control unit further comprises a temperature control module. In some embodiments, the control unit further comprises one of the following: a magnetic element for generating a magnetic field, an electric element for generating an electric field.

[0013] In some embodiments, the control unit further comprises a magnetic element for generating a magnetic field, wherein the magnetic field is capable of moving magnetic particles in a fluid sample along the microchannel. hi some embodiments, the control unit further comprises an electric element for generating an electric field, wherein the electric field is capable of moving charged particles in a fluid sample along the microchannel.

[0014] The sensors described herein may comprise an electromagnetic signal transmitting element and an electromagnetic signal receiving element, and the intensity or frequency of said electromagnetic signal or the combination thereof changes as a flow front of fluid passes the location to be sensed by the sensor, hi some embodiments, the sensor comprises an electric element, wherein the impedance of the electric element is sensitive to the wetness of the environment, and wherein the electric element generates a response as a flow front of fluid passes the location to be sensed by the sensor.

[0015] In some embodiments, the cartridge (either alone or in the context of the microfluidic device) further comprises a wicking inhibitor. In some embodiments, the cartridge further comprises a sample injector port (such as a sealable sample injector port). In some embodiments, the sample injector port (such as the sealable sample injector port) is a bubble resistant injector port. In some embodiments, the cartridge does not comprise an air duct. In some embodiments, the cartridge further comprises a heating element for increasing the temperature of one or more locations on the cartridge. In some embodiments, the cartridge further comprises a temperature sensor for sensing the temperature of one or more locations on the cartridge.

[0016] In some embodiments, the cartridge comprises a nucleic acid extraction unit, a nucleic acid amplification unit, and a nucleic acid detection unit. In some embodiments, the cartridge comprises within the microchannel a structure or surface that is effective for a specific nucleic acid adsorption under one condition, and effective for nucleic acid desorption under another condition, hi some embodiments, the structure has a large surface to volume ratio, and wherein the structure comprises particles, microspheres, fibers, membranes, array of micro-pillars or frits. In some embodiments, the surface is organic, inorganic, coated, surface-modified, or a combination thereof.

[0017] In some embodiments, the portion of the microchannel has its interior surface or interior region immobilized with chemical molecules, which are capable of binding with specific nucleic acids or derivatives thereof. In some embodiments, the chemical molecules are immobilized in an array and can each be identified by their respective location in the array.

[0018] In another aspect, there are provided methods of controlling fluid movement in a microfluidic device described herein. In some embodiments, the method comprises: a) introducing a fluidic sample to the cartridge, and b) using the pump and the valve to move the fluid in a desired direction along the microchannel. Li some embodiments, the method comprises: a) introducing a fluidic sample to the cartridge, b) using the pump and the valve to move the fluid in a desired direction along the microchannel, and c) sensing the passage of fluid front at least in one location on the microchannel downstream of the sample injector port via the sensor, wherein the sensor generates a feedback signal for controlling the action of the pump and the valve. In some embodiments, the cartridge comprises a sample injector port (such as a sealable sample injector port, for example a bubble resistant sample injector port), and the fluid sample is introduced through the sample injector port.

[0019] In some embodiments, there is provided a method of controlling fluid movement in a cartridge by use of a control unit separated from the cartridge comprising: a) providing a control unit separated from said cartridge, said control unit further comprising of at least a pump, a valve, and a sensor; b) providing a microfabricated cartridge, said cartridge further comprising of at least one microchannel, said at least one microchannel having at least one end coupled to said pump in said control unit, at least one end coupled to said valve in said control unit, and at least a sealable sample injection point between said ends; c) opening a valve downstream of said sample injection point, introducing a fluid sample to said cartridge via said sample injection point, then closing said sample injection point; d) turning on/off said pumps and opening/closing said valves selectively to move said fluid sample to a desired direction in said microchannel; e) sensing of passage of fluid flow front at least in one location on said microchannel downstream of said sample injection point, generating a feedback signal to selectively turn on/off said pumps and open/close said valves to change movement of said fluid sample.

[0020] Also provided are kits comprising the microfluidic device and/or control units and cartridges for use in the microfluidic device, as well as methods of using the microfluidic devices described herein.

[0021] Other features and aspects of the invention will become apparent from the following detailed description, taken in conjunction with the accompanying drawings, which illustrate, by way of example, the features in accordance with embodiments of the invention. The summary is not intended to limit the scope of the invention, which is defined solely by the claims attached hereto.

Brief Description of the Drawings

[0022] Figure 1 presents an exploded view of an operation of an embodiment of the invention.

[0023] Figure 2 is a block diagram of the invention according to an embodiment.

[0024] Figure 3 is a top view of a cartridge, according to an embodiment of the invention.

[0025] Figure 4 is a bottom view of a cartridge, according to the embodiment of Figure 3.

[0026] Figure 5 is a side view of a cartridge, according to the embodiment of Figure 3.

[0027] Figure 6 is a top view of a cartridge fixture, according to an embodiment of the invention.

[0028] Figure 7 is an internal, top view of a cartridge showing internal structure, according to the embodiment of Figure 3.

[0029] Figure 8 provides a top view of an exemplary device according to one embodiment of the present invention.

[0030] Figure 9 presents an analysis result obtained using an embodiment of the invention.

[0031] Some of the figures are labeled with coordinate axes that cross reference orientations and views among the figures. When the text herein refers to "top," it refers to a drawing aspect presenting itself as viewed from the positive y-axis direction. When the text refers to "bottom," it refers to a drawing aspect presenting itself as viewed from the negative y-axis direction. Although the axes shown are in particular orientations in the drawings, the actual physical structures illustrated may be rotated to any particular orientation without performance impact, as long as component alignments are maintained and unless otherwise stated.

[0032] The figures provided are merely representational and may not be drawn to scale. Certain proportions thereof may be exaggerated, while others may be minimized. The figures are intended to illustrate various implementations of the invention that can be understood and appropriately carried out by those of ordinary skill in the art. Commonly designated elements among the various figures refer to common or equivalent elements in the depicted embodiments. The figures are not intended to be exhaustive or to limit the invention to the precise form disclosed. It should be understood that the invention can be practiced with modification and alteration, and that the invention be limited only by the claims and the equivalents thereof. Detailed Description of Embodiments of the Invention

[0033] The present invention provides a simple and integrated microfluidic device comprising a cartridge and a separate control unit. The cartridge comprises microchannels for moving and processing fluidic samples and reagents. The control unit comprises a pump, a valve, and optionally a sensor for controlling movement of fluid in the cartridge. The device allows for automatic methods of manipulating fluidic samples in the microfluidic device, and is useful for various applications such as polynucleotide sample amplification through polymerase chain reaction (PCR), and nucleotide sequencing, and nucleic acid hybridization.

[0034] In one aspect, there are provided a cartridge comprising a microchannel; a control unit comprising a pump, a valve, and a sensor; and microfluidic devices comprising both.

[0035] In another aspect, there are provided methods (such as automated methods) for controlling fluidic movement in microfluidic devices described herein.

[0036] Also provided are kits and methods of using the microfluidic devices described herein.

[0037] As used herein, the word "microstructure" generally refers to structural features on a microfluidic substrate component with walls having at least one dimension in the range of about 0.1 micrometer to about 1000 micrometers. These features may be, but are not limited to, microchannels, micropumps, and micro valves.

[0038] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which this invention belongs. AU patents, applications, published applications and other publications referred to herein are incorporated by reference in their entirety. If a definition set forth in this section is contrary to or otherwise inconsistent with a definition set forth in applications, published applications and other publications that are herein incorporated by reference, the definition set forth in this section prevails over the definition that is incorporated herein by reference.

[0039] As used herein, "a" or "an" means "at least one" or "one or more."

Microfluidic devices, cartridges, and control units

[0040] Provided herein are microfluidic devices, as well as cartridges and control units for use in the microfluidic device.

[0041] In some embodiments, there is provided a microfluidic device comprising a cartridge comprising at least one microchannel and a separate control unit comprising at least a pump and a valve, wherein the at least one end of the microchannel is coupled to the pump and at least one end of the microchannel is coupled to the valve, wherein the pump controls movement of fluid along the microchannel, and wherein the valve controls direction of fluid flow along the microchannel. In some embodiments, the control unit further comprises at least one sensor, wherein the sensor senses the passage of fluid flow front and provides a feedback signal to control the action of the pump and the valve.

[0042] The pump and valve in the control units are directly coupled to the ends of microchannel(s). Rather than relying on pneumatic air barrier to control fluidic movement, movement of fluidic sample in the microchannel of the present invention is directly controlled by the pump and the valve at the ends of the microchannel. The present invention thud does not rely on the use of air ducts.

[0043] In some embodiments, there is provided a control unit comprising at least a pump and at least a valve, wherein the pump is configured to be coupled to at least one end of a microchannel and controls fluid flow along the microchannel, wherein the valve is configured to be coupled to at least one end of a microchannel and controls direction of fluid flow in the microchannel. In some embodiments, the control unit further comprises a sensor, wherein the sensor is configured to sense the passage of fluid flow front along one microchannel and provide a feedback signal to control the action of the pump and the valve.

[0044] In some embodiments, there is provided a cartridge comprising at least one microchannel, wherein at least one end of the microchannel can be coupled to a pump that controls fluid flow along the microchannel, wherein at least one end of the microchannel can be coupled to a valve that controls the direction of fluid flow along the microchannel. In some embodiments, the passage of fluid flow front in the microchannel can be sensed by at least one sensor. In some embodiments, change of the flow of a fluid in the microchannel can be controlled by a feedback signal generated from the sensor.

[0045] In some embodiments, the control unit (either alone or in the context of the microfluidic device) further comprises a controller for controlling the action of the pump and/or the valves. In some embodiments, the controller also receives and processes information from the sensor and provides a feedback control mechanism to change the actions of the pump and/or the valve. In some embodiments, the control unit further comprises a temperature control module. In some embodiments, the control unit further comprises one of the following: a magnetic element for generating a magnetic field, an electric element for generating an electric field.

[0046] In some embodiments, the cartridge (either alone or in the context of the microfluidic device) further comprises a wicking inhibitor. In some embodiments, the cartridge further comprises a sample injector port (such as a bubble resistant sample injector port). In some embodiments, the cartridge does not comprise an air duct. In some embodiments, the cartridge further comprises a heating element for increasing the temperature of one or more locations on the cartridge. In some embodiments, the cartridge further comprises a temperature sensor for sensing the temperature of one or more locations on the cartridge.

[0047] In some embodiments, the cartridge comprises a nucleic acid extraction unit, a nucleic acid amplification unit, and a nucleic acid detection unit. In some embodiments, the cartridge comprises within the microchannel a structure or surface that is effective for nucleic acid adsorption under one condition and effective for nucleic acid desorption under another condition. In some embodiments, the cartridge comprises within the microchannel surfaces or regions that are immobilized with chemicals (such as probes that are capable of binding with specific nucleic acids).

[0048] The structures and functions of the control unit and the cartridge are further described below in the context of the integrated microfiuidic device. Control unit

[0049] Movement of a fluidic sample in the microchannel depends on the on or off state of the pump. When the pump is coupled to the upstream end of the microchannel (that is, the end the fluid is flowing away from), the pump provides a pushing force for moving the fluidic sample away from the pump. When the pump is coupled to the downstream end of the microchannel (that is, the end of the fluid is flowing towards), the pump provides a drawing force for drawing the fluidic sample towards the pump. The fluidic sample moves when the pump is turned on, and stops when the pump is turned off.

[0050] Pumps described herein include, but are not limited to, syringe pumps, diaphragm pumps, peristaltic pumps, or other types of pressure pumps that can be turned on and off. In some embodiments, the pumps are micropumps that are compatible with microchannels of the present invention.

[0051] The valves described herein control the direction of the fluid flow in the microchannels. For example, when the pump is at the upstream end and in an "on" state, opening of the valve coupled to a downstream end of the microchannel allows the fluidic sample to move towards the down stream end coupled to the valve. When the pump is turned off, the fluid flow stops, even though the valve at the downstream end is open. When the microchannel has two downstream ends, one closed and the other opened, the fluid will flow towards the end when the valve is opened. Opening and closing valves on the control unit thus allows fluid to flow in a desired direction. In some embodiments, the valves are microvalves that are compatible with microchannels of the present invention.

[0052] In some embodiments, the control unit further comprises a sensor (such as microsensors) for sensing the passing of the front of the fluidic sample along the microchannel. Various mechanisms for optical detection of fluid may be used. For example, the sensor may be configured so that sensed light level will drop when the fluid is present. This would be the case when an index of refraction mismatch in the absence of fluid causes most light to be reflected off the bottom surface of the top plate, and back into the sensor; conversely, when in the presence of fluid, more light would be transmitted through the bottom surface of the top plate and into the fluid, where it is scattered, thus causing a reduction in the reflected signal. In some embodiments, when the fluid to be sensed is fluorescent, light source would produce an excitation signal at a first wavelength, and the fluid, when present, would produce a fluorescence signal at a different wavelength, which would be sensed by detector. Moreover, in place of separate light source and detector, a single fiber containing both forward and reflected signals could be used, with the two signals being split off and sensed at a convenience place within the device.

[0053] Other types of sensors include magnetic, electric, capacitive, NMR, chemical, and acoustic sensors. The sensing electronics would be turned to detect a threshold signal, indicating the presence or absence of fluid at that particular point in the fluid circuit. In some embodiments, the sensor comprises an electromagnetic signal transmitting element and an electromagnetic signal receiving element, and the intensity or frequency of the electromagnetic signal or the combination thereof changes as a flow front of fluid passes the location to be sensed by the sensor. In some embodiments, the sensor comprises an electric element. The impedance of the electric element is sensitive to the wetness of the environment, and the electric element generates a response as a flow front of fluid passes the location to be sensed by the sensor. As further described blow, the sensor can provide feedback signal that controls the action of the pump and/or the valve. Upon sensing the passage of fluid at a specific location on the microchannel, the sensor can send the signal to a controller, which processes the information and changes the action of the pump and/or the valve based on the information.

[0054] In some embodiments, the microfluidic device further comprises a controller (either on the control unit or as a separate module that is separated from the control unit). The controller processes signals sent by the sensor and/or controls the action mode of the pump and/or valves. By turning on and off the pumps and the opening and closing the valves, fluid in the microchannel can be directed to flow and stop in a desired manner. In some embodiments, the controller comprises a CPU.

[0055] In some embodiments, the microfluidic device or the control unit of the microfludic device further comprises a magnetic element for generating a magnetic field, wherein the magnetic field is capable of moving magnetic particles in a fluid sample along the microchannel. Similarly, the microfluidic device or the control unit may further comprise an electric element for generating an electric field, and the electric field is capable of moving charged particles in a fluid sample along the microchannel.

[0056] In some embodiments, the microfluidic device further comprises a temperature control module for controlling temperature at one or more locations on the cartridge (either on the control unit or as a separate module that is separated from the control unit). For example, the microfluidic device further comprises a heating circuit for increasing the temperature at certain locations on the cartridge. In some embodiments, the microfluidic device further comprises a temperature sensing circuit for sensing the temperature on the cartridge. In some embodiments, both the heating circuit and the temperature sensing circuit are connected with a resistive heating element on the cartridge, as further described below.

Cartridge

[0057] The cartridge described herein comprises at least one microchannel, and at least one end of the microchannel can be coupled to the pumps and/or valves on the control unit. The microchannel allows fluids to flow within the cartridge, and can be of any desired shape. The microchannel comprises two or more ends. In some embodiments, the channels have a cross section of 0.5mm x 0.5mm, and shorter and even smaller channels located in several places are having a cross section of 0.25mm x 0.25mm.

[0058] In some embodiments, the cartridge further comprises a sample injector port (such as a sealable sample injector port) between two ends of the microchannel. In some embodiments, the sealable sample injector port is a bubble resistant sample injector port. In microfluidic systems (such as the microfluidic device of described herein), it is important to be able to inject sample solutions and reagents without introducing bubbles. The present invention follows the philosophy of segregating fluid processing functions in an expendable cartridge, whereas fluid flow actuation, temperature control, and sensing functions are performed by a reusable external unit that is coupled to the cartridge. A complete nucleic acid analysis can controlled by the external unit, which at least comprises of a pump that can be turned on and off, a valve that can be opened and closed, and a sensor that is used to detect fluid position. These elements can form a closed-loop system for moving fluid samples from one place to another in the cartridge. Note that choosing a pump which can be turned on and off, instead of a constant pressure source, as the fluid driving force makes it much easier to enable and disable differential pressures for moving fluids within the cartridge, and does away otherwise complex fluidic designs.

[0059] Special pumping actions, such as time-interleaving or time-pulsing, or micro perturbing structures on channel walls, are effective for micro-mixing without bubbling created by some other means, such as sonication. Other measures for suppressing air bubbles may include: replacing chambers of large volumes by serpentine microchannels whenever possible for the purpose of obtaining a stable liquid meniscus; allowing ends of liquid streams to extend outside of any thermal control regions in order to avoid bubble formation and liquid segmentation caused by evaporation and condensation.

[0060] Co pending PCT application BUBBLE-RESISTANT INJECTOR PORT FOR FLUIDIC DEVICES based on Chinese Patent Application No. CN 200510130706.0 (Attorney Number 51457-20042.40) incorporated herein by reference in its entirety, describes an approach that additionally uses passive valve structures for bubble free mixing. Specifically, the application describes a bubble-resistant injector port for fluid and microfluidic devices which include an air-exhaustion feature to reduce the inclusion of bubbles or voids in injection samples, such as samples injected by a micropipette. The air exhaustion feature comprises an air- exhaustion cavity in gas communication with the injector port through a narrowed channel that permits a flow of air into the cavity, while impeding a flow of injected liquid into the cavity.

[0061] The sample injector port may comprise: an injector-port cavity, defined by at least one injector-port cavity wall, wherein the injector-port cavity is configured to accept the insertion of a micropipette tip; a downstream channel, defined by at least one downstream-channel wall, and having a downstream-channel first end, wherein the downstream channel is configured to be in fluid communication with the injector-port cavity at the first end of the downstream channel; an air-exhaustion cavity, defined by at least one air-exhaustion cavity wall, wherein the air- exhaustion cavity is configured to be in fluid communication with an ambient atmosphere; an air- exhaustion channel, defined by at least one air-exhaustion channel wall, and having first and second air-exhaustion channel ends, wherein the first air-exhaustion channel end is configured to be in fluid communication with the injector-port cavity, wherein the second air-exhaustion channel end is configured to be in fluid communication with the air-exhaustion cavity, and wherein the air-exhaustion channel is configured to impede the transport of liquid more than it impedes the transport of gasses therethrough; and an upstream channel, defined by at least one upstream-channel wall, and having a upstream-channel first end, wherein the upstream channel is configured to be in fluid communication with the air-exhaustion cavity at the first-end of the upstream channel.

[0062] In some embodiments, at least a portion of the at least one air-exhaustion channel wall comprises a hydrophobic material. In some embodiments, an interface between the air-exhaustion channel and the air-exhaustion cavity comprises a passive valve, hi some embodiments, a connecting length of the air-exhaustion channel between the injector-port cavity and the air- exhaustion cavity is configured to be short enough to allow a liquid meniscus trapped within the air-exhaustion channel to be entrained and swept away by a liquid flow from the injector-port cavity to the downstream cavity.

[0063] The sample injector port may comprise a plurality of structural layers that are bonded together, either directly or adhesively. Layers can comprise polymer materials such as polydimethylsiloxane (PDMS), polymethylmethacrylate (PMMA); polycarbonate (PC); polyoxymethylene (POM); and polyamide (PA), and/or inorganic materials such as silicon and glass.

[0064] In some embodiment, the injector port comprises: first and second layers; wherein side and bottom walls for the upstream channel, air-exhaustion cavity, air-exhaustion channel, injector- port cavity, and downstream channel are formed in a first surface of the first layer; wherein a surface of the second layer that faces the first surface of the first layer, forms top walls for the upstream channel, air-exhaustion channel, and downstream channel; and wherein the second layer comprises first and second through holes configured to substantially align with the side walls of the injector-port cavity and the air-exhaustion cavity, respectively.

[0065] In some embodiment, the injector port comprises: first and second layers; wherein side and bottom walls for the upstream channel, air-exhaustion cavity, air-exhaustion channel, injector- port cavity, and downstream channel are formed in a first surface of the first layer; wherein a surface of the second layer that faces the first surface of the first layer, forms top walls for the upstream channel, air-exhaustion channel, and downstream channel; and wherein the second layer comprises a first through-hole configured to substantially align with the side walls of the air- exhaustion cavity, and a second through-hole configured with a perimeter to extend beyond the perimeter formed by the side walls of the injector port.

[0066] The cartridge of the present invention (or the microchannel in the cartridge) in some embodiments further comprises a wicking inhibitor. A requirement for the closed-loop control to work is that fluids must not generate air bubbles and thereby be undesirably segmented into multiple sections within a microfluidic channel. However, samples and reagents used in nucleic acid analyses commonly contain surfactants, which decrease the fluids contact angles on cartridge walls. As a result, it is difficult for fluids to maintain a stable meniscus at flow fronts. Air bubbles are, therefore, readily formed, which trigger erroneous signals to sensors monitoring fluid positions. This perhaps is a major reason for that prior systems of integrated nucleic acid analyses do not use closed-loop control. Still having to get rid of generated bubbles, these systems take the path of partial integration of an external unit to a sample residing part, often in form of a cartridge as well. Common practices include the use of hydrophobic membranes for passing air only, use of swell able hydro-gel particles to serve as one-time valves, introduction of one or more layers of soft elastomers as deflectable channel walls that can function as check valves or peristaltic pumps, and even use of much rarely used hydrophobic materials, such as COC, to fabricate cartridge bodies. All of these measures increase the cartridge complexity and its manufacturing cost in one way or another.

[0067] In fact, if assisted by a number of particularly designed microfluidic structures and schemes, a bubble-free liquid flow, hence a closed-loop control of liquid motion by monitoring its positions, is perfectly attainable. For example, there are anti-wicking microstructures for stabilizing liquid meniscus in microchannels. These microstructures may be in form of local recesses or local enlargements, which interrupt extension of channel edges that cause wicking. If appropriately designed, abrupt changes in channel dimension or bypassing fluidic paths can induce passive valving forces that, if used in conjunction with a closed-loop control scheme, are working beautifully for many microfluidic operations, such as merging of separate liquid streams without trapping air bubbles.

[0068] Also, in fluidic systems, and microfluidic systems in particular, it is often desirable to have flowing liquid segments with sharply defined frontal and trailing boundaries along channels. Such sharply defined boundaries minimize spatial dispersion during fluid flow and allow for more precisely defined timing for synthesis and/or analysis operations in such systems. "Wicking" as a result of capillary action between a working fluid and containment walls of a fluid transport channel, and in particular at the edges where containment walls meet, can spread out both frontal and trailing boundaries of the working fluid. Wicking tends to be exacerbated when a fluid and a channel wall have a higher degree of affinity for one another, for example in the case of an aqueous solution and a hydrophilic surface. [0069] Various approaches have been used to implement wicking inhibitor structures or "traps" to reduce wicking in channels and/or other structures. U.S. patent no. 6,919,958, issued to Per Anderson, et al. on July 19, 2005, discusses wicking traps that involve fluidic channel surface modification, and/or sidewall structural modification. U.S patent no. 6,776,965, STRUCTURES FOR PRECISELY CONTROLLED TRANSPORT OF FLUID issued to Wyzgol et al. on August 17, 2004 describes a wicking inhibitor in which a bottom wall of a microfluidic channel has a series of steps formed therein to reduce wicking by interrupting capillary action at their edges. Co pending PCT application WICKING INHIBITOR FOR FLUIDIC DEVICES based on CN 200510130707.5 (Attorney Docket Number 51457-20039.40), incorporated herein in its entirety by reference, describes an improved, structurally based wicking inhibitor that overcomes at least some of the drawbacks described in relation with the other approaches. For example, there is provided a wicking inhibitor configured for fluid communication with a fluidic channel, both being configured to accept the flow of a working fluid therethrough, comprising an interface between the wicking inhibitor and the fluidic channel wherein all edges of the interface are configured to have corner angles greater than ninety degrees. In some embodiments, at least one of the corner angles is configured to have a corner angle that is substantially two hundred seventy degrees. In some embodiments, the wicking inhibitor comprises: a) a first layer, the first layer further comprising a first recess formed therein defining first, second, and third walls of the fluidic channel, wherein the first and second walls are substantially parallel to one another and both are substantially perpendicular to the third wall, and the first layer further comprising a second recess formed therein defining a first cavity region for the wicking inhibitor, the first cavity region having a fourth wall and a fifth wall, the fifth wall having first and second segments that are substantially partially circumferential to the second recess and substantially perpendicular to the fourth wall, wherein the fourth wall of the second recess extends beyond the third wall of the first recess, wherein the first and second segments of the fifth wall adjoin the first and second walls, respectively ; and b) a second layer, further comprising a sixth wall to partially define the fluidic channel that is generally parallel to third wall, and further comprising a recess formed therein defining a second cavity region for the wicking inhibitor, the second cavity region comprising a sixth wall and a seventh wall, the seventh wall being substantially circumferential to the recess and substantially perpendicular to the sixth wall, wherein the seventh wall is configured to substantially align with the first and second segments of the fifth wall. In some embodiments, at least one of the first and second layers is formed of a hydrophilic material. In some embodiments, the first and second layers are bonded together. [0070] The cartridge contains no moving mechanical parts, and the movement of fluids in cartridge is accomplished by a separate control unit. The pumps and valves in the control unit control movement of fluid in the cartridge by the application of differential pressures. Chinese Patent Applications CN200610065951.2 and CN200610065952.7, incorporated herein in its entirety by reference, additionally teaches enhancing such differential pressure fluid control through the use of passive valve structures.

[0071] In some embodiments, the cartridge described herein further comprises a heating element for increasing the temperature of one or more locations on the cartridge. In some embodiments, the cartridge comprises a temperature sensor for sensing the temperature of one or more locations on the cartridge. In some embodiments, the heating element is a resistive heating element and is connected to a heating circuit and a temperature sensing circuit on the control unit or located outside of the control unit. Use of resistive heating element to increase temperature of microchannels and capillary tubes without the use of a separate temperature sensing element has been disclosed in co-pending PCT application METHODS AND DEVICES FOR CONTROLLING TEMPERATURE WITHOUT TEMPERATURE SENSOR based on CN200510135478.6 (Attorney Docket No. 51457-20049.40), incorporated herein by reference in its entirety.

[0072] In some embodiments, the cartridge comprises a nucleic acid extraction unit, a nucleic acid amplification unit, and a nucleic acid detection unit. These different units can be used to carry out different reactions on the cartridge.

[0073] In some embodiments, the cartridge comprises within the microchannel a structure or surface that is effective for a specific nucleic acid adsorption under one condition, and effective for nucleic acid desorption under another condition. The structure may have a large surface to volume ratio. For example, the structure may comprise particles, microspheres, fibers, membranes, array of micro-pillars or frits. Such structure can be useful, for example the nucleic acid extraction unit of the cartridge. In some embodiments, the microchannel comprises a surface that is organic, inorganic, coated, surface-modified, or a combination thereof.

[0074] In some embodiments, a portion of the microchannel has its interior surface or interior region immobilized with chemical molecules, which are capable of binding with specific nucleic acids or derivatives thereof. The chemical molecules may be immobilized in an array and can each be identified by their respective location in the array.

[0075] In some embodiments, the cartridge may further comprise chambers for reagents, such as reagents for amplification reactions, reagents for nucleotide sequencing, and other reagents. [0076] Also provided are methods of making the control units, the cartridge, and/or the microfluidic devices described herein. The control units, cartridges, and/or microfluidic devices described herein can be made using techniques that are commonly used to make microfluidic devices and systems. Such techniques span a variety of diverse materials, fabrication, and assembly methods. Materials can be organic or inorganic, and be hydrophobic or hydrophilic to differing extents. A combination of different materials can be used in the same microfluidic device or system. Fabrication methods can be specific to specific types of materials, and can include photolithography; physical, wet, and dry-chemical etching; subtractive and additive material transfer; non-optical transfer printing; contact molding; injection molding; casting; micro-stereo lithography; and micro-machining. Assembly methods can include: anodic, direct, adhesive, and eutectic bonding; and press-fit. The selection of fabrication and assembly methods can affect the choice of microfluidic device and system design variations, or vice versa. This will be discussed below in connection with various embodiments of the wicking inhibitor.

[0077] As discussed above, various materials, fabrication methods, and assembly techniques can be used in the fabrication of microfluidic devices. The present discussion will focus on a subset of these in relation to embodiments of the current invention for the sake of focus and brevity, although further equivalent embodiments using other materials, fabrication methods, and assembly techniques would be apparent to one of ordinary skill in the art after reading the disclosure.

[0078] Inorganic materials include silicon, glasses, metals, and metal alloys. Glass is principally amorphous silicon dioxide (SiO₂) with varying amounts of additional elements in different types of glass. Among the desirable properties of glass for microfluidic device substrates are mechanical strength, dimensional stability, and low cost. A substrate of glass can form an active layer by having channels and other microfluidic structure formed in its surface, or it may merely serve as a mechanical support for active layers of other materials. Surface structures may be formed in glass by wet or dry chemical etching, mechanical ablation or milling, molding, and micromachining. Glass surfaces tend to be hydrophilic.

[0079] Typical polymer materials for other microfluidic device layers include thermosetting polymers such as polydimethylsiloxane (PDMS), as well as thermoplastic polymers such as: (i) polymethylmethacrylate (PMMA); (ii) polycarbonate (PC); (iii) polyoxymethylene (POM); and polyamide (PA).

[0080] PDMS has an inorganic siloxane backbone with organic methyl groups attached to the silicon. Both prepolymers and curing agents are commercially available. PMDS has a low interfacial free energy, which provides a relatively chemically unreactive, hydrophobic surface, although this can be modified with plasma treatment. PDMS is stable against temperature and humidity. PDMS is transparent, allowing for the visual examination of microfluidic structures and their operations. PDMS is flexible, so it can conform to nonplanar structures. PDMS is optically curable, so micro-stereo lithography can be used to form PDMS microfluidic structures, although PDMS structures can also be cast molded by applying a prepolymer solution to a mold, curing at an elevated temperature, and subsequently peeling the PMDS structure from the mold. The cast molding technique is capable of fabricating relief features down to the order of tens of microns across and deep, and is particularly low cost and does not require large capital investments in manufacturing equipment.

[0081] Structures can be formed in the thermoplastic polymers by using compression molding, injection molding, or micro-stereo lithography. Compression molding involves heating the polymer above its glass transition temperature and pressing it against a mold to form relief features, similar to the cast molding technique described in the previous paragraph. Injection molding involves heating the polymer above its glass transition temperature and pressure injecting it into a mold. After cooling, the mold is dismantled, and the molded part is removed.

[0082] All of the above fabrication techniques tend to create microfluidic layers with surface features formed in relief. Thus blind holes can be formed, but through holes can require further processing. Through holes (and other through structures) can be drilled by a variety of techniques, such as: (i) laser micro-machining using excimer, Nd: YAG, or CO₂ lasers; (ii) focused ion beam; (iii) micro-electric discharge; (iv) powder blasting; (v) ultrasonic micro-machining; or (vi) reduced-scale mechanical machining, all of which are well known to one of ordinary skill in the art.

[0083] Layers and substrate layers as discussed above can be assembled into microfluidic devices and systems using direct or adhesive bonding.

[0084] For direct bonding, the surfaces of layers to be bonded are cleaned and the layers are aligned relative to one another and pressed together to form a sandwiched structure. Thermoplastic polymers can be bonded together by heating to temperatures above their glass transition temperature. In cases of thermosetting polymers with low surface energy such as PMDS, layers can be bonded together under pressure at room temperature. PMDS layers can also bond to glass under similar conditions. Another method to bond layers together is wet bonding. In wet bonding, the surfaces to be bonded are wetted with a solvent, and then pressed together. Bonding is accomplished after evaporating the solvent. [0085] Adhesive bonding uses an intermediate layer to glue layers together. Depending on substrate and layer materials, the intermediate adhesive layer can comprise epoxies, photoresists, or other polymers. The intermediate adhesive layer can be applied to a surface to be bonded, through a removable mask, in order to exclude adhesive from microfluidic structures, as necessary. Techniques for such selective application are well known to one of ordinary skill in the art. Some adhesive layers can be cured by ultraviolet light, while other adhesive layers can be chemically cured, or cured at elevated temperatures.

Methods of controlling fluid flow in the microfluidic devices

[0086] Also provided are methods of controlling fluid flow in the microfluidic devices described herein. In some embodiments, there is provided a method of controlling fluid movement in a microfluidic device described herein, comprising: a) introducing a fluidic sample to the cartridge, b) using the pump and the valve to move the fluid in a desired direction along the microchannel, and c) sensing the passage of fluid front at least in one location on the microchannel downstream of the sample injector port via the sensor, wherein the sensor generates a feedback signal for controlling the action of the pump and the valve. In some embodiments, the cartridge comprises a sample injector port (such as a sealable sample injector port), and the fluid sample is introduced through the sample injector port. In some embodiments, one or more steps of the method are carried out automatically.

[0087] In some embodiments, there is provided a method of controlling fluid movement in a cartridge by use of a control unit separated from the cartridge comprising: a) providing a control unit separated from said cartridge, said control unit further comprising of at least a pump, a valve, and a sensor; b) providing a microfabricated cartridge, said cartridge further comprising of at least one microchannel, said at least one microchannel having at least one end coupled to said pump in said control unit, at least one end coupled to said valve in said control unit, and at least a sealable sample injection point between said ends; c) opening a valve downstream of said sample injection point, introducing a fluid sample to said cartridge via said sample injection point, then closing said sample injection point; d) turning on/off said pumps and opening/closing said valves selectively to move said fluid sample to a desired direction in said microchannel; e) sensing of passage of fluid flow front at least in one location on said microchannel downstream of said sample injection point, generating a feedback signal to selectively turn on/off said pumps and open/close said valves to change movement of said fluid sample. Methods of using the microfluidic devices

[0088] The microfluidic devices described herein are useful for a number of applications. For example, the microfluidic device may provide an integrated DNA amplification and analysis system, as well as systems for handling other types of macromolecules such as RNA, polypeptides, and polysaccharides.

[0089] A DNA analysis system can be used for diagnostic applications and de novo sequencing applications. For diagnostic applications, DNA analysis data may be used in a variety of ways, including nucleic acid sequencing which is directed toward a particular disease causing agent, such as viral or bacterial infections, e.g., AIDS₅ malaria, etc., or genetic disorders, e.g., sickle cell anemia, cystic fibrosis, Fragile X syndrome, Duchenne muscular dystrophy, and the like. Alternatively, the device can be employed in de novo sequencing applications to identify the nucleic acid sequence of a previously unknown sequence.

[0090] A step that precedes DNA analysis is often DNA amplification to increase the amount of DNA available for analysis. A variety of amplification methods are suitable for use in the methods and device of the present invention, including for example, the polymerase chain reaction method or (PCR), the ligase chain reaction (LCR), self sustained sequence replication (3SR), and nucleic acid based sequence amplification (NASBA). PCR techniques are well known in the art. See PCR Protocols: A Guide to Methods and Applications (Innis, M., Gelfand, D., Sninsky, J. and White, T., eds.) Academic Press (1990). PCR amplification generally involves the use of one strand of the target nucleic acid sequence as a template for producing a large number of complements to that sequence. Generally, two primer sequences complementary to different ends of a segment of the complementary strands of the target sequence hybridize with their respective strands of the target sequence, and in the presence of polymerase enzymes and nucleoside triphosphates, the primers are extended along the target sequence. The extensions are melted from the target sequence and the process is repeated, this time with the additional copies of the target sequence synthesized in the preceding steps. PCR amplification typically involves repeated cycles of denaturation, hybridization and extension reactions to produce sufficient amounts of the target nucleic acid. The first step of each cycle of the PCR involves the separation of the nucleic acid duplex formed by the primer extension. Once the strands are separated, the next step in PCR involves hybridizing the separated strands with primers that flank the target sequence. The primers are then extended to form complementary copies of the target strands. For successful PCR amplification, the primers are designed so that the position at which each primer hybridizes along a duplex sequence is such that an extension product synthesized from one primer, when separated from the template (complement), serves as a template for the extension of the other primer. The cycle of denaturation, hybridization, and extension is repeated as many times as necessary to obtain the desired amount of amplified nucleic acid.

[0091] In PCR methods, strand separation is normally achieved by heating the reaction to a sufficiently high temperature for a sufficient time to cause the denaturation of the duplex but not to cause an irreversible denaturation of the polymerase enzyme (see U.S. Pat. No. 5,965,188, incorporated herein by reference). Typical heat denaturation involves temperatures ranging from about 8O.degree. C. to 105. degree. C. for times ranging from seconds to minutes. Strand separation, however, can be accomplished by any suitable denaturing method including physical, chemical, or enzymatic means. Strand separation may be induced by a helicase, for example, or an enzyme capable of exhibiting helicase activity. For example, the enzyme RecA has helicase activity in the presence of ATP. The reaction conditions suitable for strand separation by helicases are known in the art (see Kulin Hoffman-Berling, 1978, CSH-Quantitative Biology, 43:63-67; and Radding, 1982, Ann. Rev. Genetics 16:405-436, each of which is incorporated herein by reference). Other embodiments may achieve strand separation by application of electric field across the sample. For example, published PCT application Ser. Nos. WO 92/04470 and WO 92/25177, incorporated herein by reference, describe electrochemical methods of denaturing double stranded DNA by application of an electric field to a sample containing the DNA. Structures for carrying out this electrochemical denaturation include a working electrode, counter electrode and reference electrode arranged in a potentiostat arrangement across a reaction chamber (See, Published PCT application Ser. Nos. WO 92/04470 and WO 95/25177, each of which is incorporated herein by reference). Such devices may be readily miniaturized for incorporation into the devices of the present invention utilizing the microfabrication techniques described herein. Template-dependent extension of primers in PCR is catalyzed by a polymerizing agent in the presence of adequate amounts of at least 4 deoxyribonucleotide triphosphates (typically selected from dATP, dGTP, dCTP, dUTP and dTTP) in a reaction medium which comprises the appropriate salts, metal cations, and pH buffering system. Reaction components and conditions are well known in the art (See PRC Protocols: A Guide to Methods and Applications (Innis, M., Gelfand, D., Sninsky, J. and White, T., eds.) Academic Press (1990), previously incorporated by reference). Suitable polymerizing agents are enzymes known in catalyze template-dependent DNA synthesis.

[0092] Published PCT application Ser. No. WO 94/05414, to Northrup and White, discusses the use of a microPCR chamber which incorporates microheaters and micropumps in the thermal cycling and mixing during the PCR reactions. The amplification reaction chamber of the device may comprise a sealable opening for the addition of the various amplification reagents. However, in preferred aspects, the amplification chamber will have an effective amount of the various amplification reagents described above, predisposed within the amplification chamber, or within an associated reagent chamber whereby the reagents can be readily transported to the amplification chamber upon initiation of the amplification operation. By "effective amount" is meant a quantity and/or concentration of reagents required to carry out amplification of a targeted nucleic acid sequence. These amounts are readily determined from known PCR protocols. See e.g., Sambrook, et al. Molecular Cloning: A Laboratory Manual, (2nd ed.) VoIs. 1-3, Cold Spring Harbor Laboratory, (1989) and PCR Protocols: A Guide to Methods and Applications (Innis, M., Gelfand, D., Sninsky, J. and White, T., eds.) Academic Press (1990), both of which are incorporated herein by reference for all purposes in their entirety. For those embodiments where the various reagents are predisposed within the amplification or adjacent chamber, it will often be desirable for these reagents to be lyophilized forms, to provide maximum shelf life of the overall device. Introduction of the liquid sample to the chamber then reconstitutes the reagents in active form, and the particular reactions may be carried out.

In some aspects, the polymerase enzyme may be present within the amplification chamber, couples to a suitable solid support, or to the walls and surfaces of the amplification chamber. Suitable sold supports include those that are well known in the art, e.g., agarose, cellulose, silica, divinylbenzene, polystyrene, etc. Coupling of enzymes to solid supports has been reported to impart stability to the enzyme in question, which allows for storage of days, weeks or even months without a substantial loss in enzyme activity, and without the necessity of lyophilizing the enzyme. The 94 kd, single subunit DNA polymerase from Thermus aquaticus (or taq polymerase) is particularly suited for the PCR based amplification methods used in the present invention, and is generally commercially available from, e.g., Promega, Inc., Madison, Wis. In particular, monoclonal antibodies are available which bind the enzyme without affecting its polymerase activity. Consequently, covalent attachment of the active polymerase enzyme to a solid support, or the walls of the amplification chamber can be carried out by using the antibody as a linker between the enzyme and the support.

[0093] Following amplification, the DNA sample can be subjected to one or more analysis operations. Particularly preferred analysis operations include, e.g., sequence based analyses using an oligonucleotide array. In one aspect, following sample preparation, the nucleic acid sample is probed using an array of oligonucleotide probes. Oligonucleotide arrays generally include a substrate having a large number of positionally distinct oligonucleotide probes attached to the substrate. These oligonucleotide arrays, also described as "Genechip™ arrays," have been generally described in the art, for example, U.S. Pat. No. 5,143,854 and PCT patent publication Ser. Nos. WO 90/15070 and 92/10092. These pioneering arrays may be produced using mechanical or light directed synthesis methods which incorporate a combination of photolithographic methods and solid phase oligonucleotide synthesis methods. See Fodor et al., Science, 251 :767-777 (1991), Pirrung et al., U.S. Pat. No. 5,143,854 (see also PCT application Ser. No. WO 90/15070) and Fodor et al., PCT Publication Ser. No. WO92/10092, all incorporated herein by reference. These references disclose methods of forming vast arrays of peptides, oligonucleotides and other polymer sequences using, for example light-directed synthesis techniques. Techniques for the synthesis of these arrays using mechanical synthesis strategies are described in, e.g., PCT Publication Ser. No. 93/09668 and U.S. Pat. No. 5,384,261, each of which is incorporated herein by reference in its entirety for all purposes. Incorporation of these arrays in injection molded polymeric casings has been described in Published PCT application Ser. No. 95/33846.

[0094] The basic strategy for light directed synthesis of oligonucleotide arrays is as follows. The surface of a solid support, modified with photosensitive protecting groups is illuminated through a photolithographic mask, yielding reactive hydroxyl groups in the illuminated regions. A selected nucleotide, typically in the form of a 3'-O-phosphoramidite-activated deoxynucleoside (protected at the 5' hydroxyl with a photosensitive protecting group), is then presented to the surface and coupling occurs at the sites that were exposed to light. Following capping and oxidation, the substrate is rinsed and the surface is illuminated through a second mask, to expose additional hydroxyl groups for coupling. A second selected nucleotide (e.g., 5'-protected, 3'-O- phosphoramidite-activated deoxynucleoside) is presented to the surface. The selective deprotection and coupling cycles are repeated until the desired set of products is obtained. Since photolithography is used, the process can be readily miniaturized to generate high density arrays of oligonucleotide probes. Furthermore, the sequence of the oligonucleotides at each site is known. See, Pease, et al. Mechanical synthesis methods are similar to the light directed methods except involving mechanical direction of fluids for deprotection and addition in the synthesis steps.

[0095] Typically, the arrays used in the present invention will have a site density of greater than 100 different probes per cm.sup.2. Preferably, the arrays will have a site density of greater than 500/cm.sup.2, more preferably greater than about lOOO/cm.sup.2, and most preferably, greater than about 10,000/cm.sup.2. Preferably, the arrays will have more than 100 different probes on a single substrate, more preferably greater than about 1000 different probes still more preferably, greater than about 10,000 different probes and most preferably, greater than 100,000 different probes on a single substrate.

[0096] For some embodiments, oligonucleotide arrays may be prepared having all possible probes of a given length. Such arrays may be used in such areas as sequencing or sequence checking applications, which offer substantial benefits over traditional methods. The use of oligonucleotide arrays in such applications is described in, e.g., U.S. patent application Ser. No. 08/505,919, filed JuI. 24, 1995, now abandoned, and U.S. patent application Ser. No. 08/284,064, filed Aug. 2, 1994, now abandoned, each of which is incorporated herein by reference in its entirety for all purposes. These methods typically use a set of short oligonucleotide probes of defined sequence to search for complementary sequences on a longer target strand of DNA. The hybridization pattern of the target sequence on the array is used to reconstruct the target DNA sequence. Hybridization analysis of large numbers of probes can be used to sequence long stretches of DNA.

[0097] One strategy of de novo sequencing can be illustrated by the following example. A 12- mer target DNA sequence is probed on an array having a complete set of octanucleotide probes. Five of the 65,536 octamer probes will perfectly hybridize to the target sequence. The identity of the probes at each site is known. Thus, by determining the locations at which the target hybridizes on the array, or the hybridization pattern, one can determine the sequence of the target sequence. While these strategies have been proposed and utilized in some applications, there has been difficulty in demonstrating sequencing of larger nucleic acids using these same strategies. Accordingly, in preferred aspects, SBH methods utilizing the devices described herein use data from mismatched probes, as well as perfectly matching probes, to supply useful sequence data, as described in U.S. patent application Ser. No. 08/505,919, now abandoned, incorporated herein by reference.

[0098] While oligonucleotide probes may be prepared having every possible sequence of length n, it will often be desirable in practicing the present invention to provide an oligonucleotide array which is specific and complementary to a particular nucleic acid sequence. For example, in particularly preferred aspects, the oligonucleotide array will contain oligonucleotide probes which are complementary to specific target sequences, and individual or multiple mutations of these. Such arrays are particularly useful in the diagnosis of specific disorders which are characterized by the presence of a particular nucleic acid sequence. For example, the target sequence may be that of a particular exogenous disease causing agent, e.g., human immunodeficiency virus (see, U.S. application Ser. No. 08/284,064, now abandoned, previously incorporated herein by reference), or alternatively, the target sequence may be that portion of the human genome which is known to be mutated in instances of a particular disorder, i.e., sickle cell anemia (see, e.g., U.S. application Ser. No. 08/082,937, now abandoned, previously incorporated herein by reference) or cystic fibrosis.

[0099] In such an application, the array generally comprises at least four sets of oligonucleotide probes, usually from about 9 to about 21 nucleotides in length. A first probe set has a probe corresponding to each nucleotide in the target sequence. A probe is related to its corresponding nucleotide by being exactly complementary to a subsequence of the target sequence that includes the corresponding nucleotide. Thus, each probe has a position, designated an interrogation position, that is occupied by a complementary nucleotide to the corresponding nucleotide in the target sequence. The three additional probe sets each have a corresponding probe for each probe in the first probe set, but substituting the interrogation position with the three other nucleotides. Thus, for each nucleotide in the target sequence, there are four corresponding probes, one from each of the probe sets. The three corresponding probes in the three additional probe sets are identical to the corresponding probe from the first probe or a subsequence thereof that includes the interrogation position, except that the interrogation position is occupied by a different nucleotide in each of the four corresponding probes.

Some arrays have fifth, sixth, seventh and eighth probe sets. The probes in each set are selected by analogous principles to those for the probes in the first four probe sets, except that the probes in the fifth, sixth, seventh and eighth sets exhibit complementarity to a second reference sequence. In some arrays, the first set of probes is complementary to the coding strand of the target sequence while the second set is complementary to the noncoding strand. Alternatively, the second reference sequence can be a subsequence of the first reference sequence having a substitution of at least one nucleotide.

[00100] In some applications, the target sequence has a substituted nucleotide relative to the probe sequence in at least one undetermined position, and the relative specific binding of the probes indicates the location of the position and the nucleotide occupying the position in the target sequence.

[00101] Following amplification, the nucleic acid sample is incubated with the oligonucleotide array in the hybridization chamber. Hybridization between the sample nucleic acid and the oligonucleotide probes upon the array is then detected, using, e.g., epifluorescence confocal microscopy. Typically, sample is mixed during hybridization to enhance hybridization of nucleic acids in the sample to polynucleotide probes on the array. Again, mixing may be carried out by the methods described herein, e.g., through the use of piezoelectric elements, electrophoretic methods, or physical mixing by pumping fluids into and out of the hybridization chamber, i.e., into an adjoining chamber.

[00102] Gathering data from the oligonucleotide arrays, will typically be carried out using methods known in the art. For example, the arrays may be scanned using lasers to excite fluorescently labeled targets that have hybridized to regions of probe arrays, which can then be imaged using charged coupled devices ("CCDs") for a wide field scanning of the array. Alternatively, another particularly useful method for gathering data from the arrays is through the use of laser confocal microscopy which combines the ease and speed of a readily automated process with high resolution detection.

[00103] Following the data gathering operation, the data will typically be reported to a data analysis operation. To facilitate the sample analysis operation, the data obtained by the reader from the device will typically be analyzed using a digital computer. Typically, the computer will be appropriately programmed for receipt and storage of the data from the device, as well as for analysis and reporting of the data gathered, i.e., interpreting fluorescence data to determine the sequence of hybridizing probes, normalization of background and single base mismatch hybridizations, ordering of sequence data in SBH applications, and the like, as described in, e.g., U.S. patent application Ser. No. 08/327/525, filed Oct. 21, 1994, now U.S. Pat. No. 5,295,716, and incorporated herein by reference.

Exemplary embodiments of the invention

[00104] The microfluidic device of the present invention is further illustrated in conjunction with figures provided herein. Some of the figures are labeled with coordinate axes that cross reference orientations and views among the figures. When the text herein refers to "top," it refers to a drawing aspect presenting itself as viewed from the positive y-axis direction. When the text refers to "bottom," it refers to a drawing aspect presenting itself as viewed from the negative y- axis direction. Although the axes shown are in particular orientations in the drawings, the actual physical structures illustrated may be rotated to any particular orientation without performance impact, as long as component alignments are maintained and unless otherwise stated.

[00105] Figure 1 presents an exploded view of an operation of an embodiment of the invention. 102 refers to a base unit containing fluid drivers, flow sensors, temperature sensors, temperature controllers as is shown in more detail in Figures 2 and 6. Cartridge unit 101 is provisioned to interface mechanically, electrically, and electromagnetically. Optical objective 104 can be positioned above a oligonucleotide, or other array within cartridge unit 101 to read test results. Micropipette 103 can introduce samples into various apertures within cartridge unit 101.

[00106] Figure 2 is a block diagram of the invention illustrating an embodiment of the interface between base unit 102 and cartridge 101. Control circuitry 202 drives pumps 205, 206, 207, 209, and 211 — as well as pressure release valves 208, 210, and 212 that interface with corresponding fluid ports 701 through 708 on cartridge 101. Interface circuit 203 couples (761, 762) with resistive heater 760 of PCR reactor section 751 of cartridge 101. Interface circuit 204 couples with heater/temperature sensor pair 642 which during operation is in thermal contact with oligonucleotide section 742. Interface circuit 215 couples with flow detectors 621 through 627, which during operation are proximate to respective flow detection areas within cartridge 1. Control circuits 202, 203, 204, and 215 are provisioned for coupling with a control logic 201, control logic 101 being capable of directing operations to perform an analysis.

[00107] Figure 3 is a top view of a cartridge, according to an embodiment of the invention. 711-713 are sample/reagent injection ports for use with an Eppendorf-type micropipette. 714 is an exhaust port. Optical window 742 allows for scanning of an array chip within the cartridge. PCR reactor section 751 of the cartridge comprises reactor tube 750 (glass), reactor tube heating winding 751, and reactor tube temperature sensor 770.

[00108] Figure 4 is a bottom view of a cartridge, according to the embodiment of Figure 3. fluid ports 701-708 are configured for coupling with respective ports on the base unit. 721-727 are fluid flow sensing areas configured to be proximate to corresponding fluid flow sensors in the base unit. 761 and 762 are electrical connections to PCR reactor tube heater 760. Electrical connectors 771 and 772 are coupled to the PCR reactor tube temperature sensor. Note that in this embodiment, PCR reactor tube heater 760 is thermally isolated from the bulk of cartridge 101 by an air gap to reduce thermal mass and promote faster thermal cycling. Thermally conductive area 742 is configured to be in thermal contact with the corresponding heater/temperature sensor in the base unit, permitting temperature control of the bioarray within the cartridge.

[00109] Figure 5 is a side, partial X-ray view of a cartridge, according to the embodiments of Figure 3 and 4.

[00110] Figure 6 is a top view of a cartridge interface section. The labeled features are described above [00111] Figure 7 is an internal, top view of a cartridge showing internal structure. Labelled features are as described, above, with the addition of the fluid channels and the fluid junctions. The structure and operation is as described in the invention disclosure.

[00112] A number of microstructures, such as channels, chambers, injection holes, etc., can be CNC-machined onto the surface of a PMMA substrate, where primary channels are having a cross section of 0.5mm x 0.5mm, and shorter and even smaller channels located in several places are having a cross section of 0.25mm x 0.25mm. Other structures are drawn in-scale with the primary channels, so that their in-plane dimensions will not be described further in here. Their depths, which are not shown in Figure 7, can be different from each other. For example, in the case shown in Figure 7, the hybridization chamber has a depth of 0.1mm, while the waste reservoir has a depth of 2.5mm. The microfabricated PMMA substrate is to be cleaned, and then thermal bonded with a second piece of PMMA so as to form a cartridge.

[00113] It is seen in Figure 7 that some ends of channels are to be coupled to pumps, and some ends of channels are to be coupled to valves. The pumps and valves are all located in the external unit.

[00114] Figure 8 provides a similar diagram as the one in Figure 7. There are four sample injection holes in the cartridge, which are marked by arrows in Figure 8. They are used to introduce PCR reagent, template solution, hybridization buffer, and microsphere suspension for labeling purpose, respectively, by conventional pipetting. The sample injection holes are subsequently sealed by a scotch tape after sample introduction. There are several points on channels that need to be monitored for detection of fluid passage, in order to realize a feedback control of fluid movement, and they are marked as black spots in Figure 8. One of detection methods is positioning a pair of infra-red sensors (IR-O ~ IR-7), one being transmitter, the other being receiver, at each of those points, and wiring them to the control unit.

[00115] An integrated nucleic acid amplification and analysis can be performed as follows using a microfludic device of Figure 8: Phase 1

[00116] Start pump-1 to push template solution towards where IR-I is pointed at. IR-I is to trigger a signal to stop pump-1. The template solution will be sucked by capillary force into the microchannel 100, and stopped flowing at its end by a passive valving effect. Start pump-0 to push PCR reagent towards where IR-O is pointed at. IR-O is to trigger a signal to continue pump- O's action for a short time period so that the template is joined with the PCR reagent. After that, the control unit drives pump-0 and pump-1 in a time-interleaving fashion namely, one is running for 2 seconds then is stopped to start running the other for 2 seconds, and then switch back. This type of pumping action is known to be effective to mix two separate fluid streams. As IR-I detects the tail of the template solution, pump-1 is stopped, and pump-0 is continued to push the PCR reaction mix into the PCR capillary until IR-7 detects the flow front. Then the control unit enters thermal cycling for nucleic acid amplification. In this example, the PCR capillary is a glass tube covered by a layer of resistive heating element and is encapsulated in the cartridge by glues. Phase 2:

[00117] After amplification, start pump-2 to push hybridization buffer towards where IR-5 is pointed at. IR-5 is to trigger a signal to continue pump-2 's action for a short time period so that the hybridization buffer passes and partially enters the microchannel 101 ; meanwhile, the flow front of the hybridization buffer must stop somewhere before reaching the microchannel 102. Start pump-0 to push PCR product out of the PCR capillary towards where IR-6 is pointed at. IR- 6 is to trigger a signal to continue pump-0's action for a short time period so that the PCR product joins the hybridization buffer. After that, the two fluid streams are mixed by the same time- interleaving pumping action of pump-0 and pump-2. When IR-5 and IR-6 each detects the tail of corresponding fluid, pump-2 and pump-0 are correspondingly stopped. At this time, the hybridization chamber ought to be filled completely with fluid, and the control unit enters hybridization process. Specific molecular probes have been immobilized on either top or bottom wall of the hybridization chamber, so that the specific target molecules in fluid can bind with the probes. Depending on applications, the hybridization reaction may require a suitable temperature, and it can be accomplished by applying an external temperature control element to the local area of the hybridization chamber. The cartridge shown in Figure 1 further provides a mode of flowing hybridization. Specifically, before either pump-0 or pump-2 is stopped, the front of the hybridization reaction mix should exit the hybridization chamber from the other end, and passes the point where IR-4 is pointed at while partially entering the microchannel 103. After that, by a coordination of (pump-2, valve-0) and (pump-4, valve-2), the hybridization reaction mix can be flowed back and forth, which is switched by the signals of IR-4 and IR-5 when they detects the fore-front and back-front of the reaction mix, respectively. Phase 3

[00118] The cartridge shown in Figure 8 is also integrated with a detection means enabled by microsphere labeling. Its principle is based on specific bindings of streptavidin and biotin, which are respectively attached onto the microspheres' surface and one end of the target molecules. The hybridized target molecules will capture microspheres onto the spots of hybridization chamber wall where the specific probe molecules are initially immobilized, so that the microspheres serve as labels to facilitate direct observation or simple optical microscopy of either positive or negative hybridization result. For this purpose, first open valve-0 and start pump-4 to push hybridization reaction mix out of the hybridization chamber and towards the waste reservoir. The IR-5 is used to determine if the hybridization chamber has been emptied by detection of the reaction mix tail. Then, open valve-2 and start pump-3, push the microsphere suspension towards the microchannel 103 and join the residue, tiny amount of hybridization reaction mix left there. During this process, the air is expelled via the channel that is coupled to the valve-2. The microsphere suspension exited from the microchannel 103 partially enters the hybridization chamber, and partially enters the channel which is pointed at by IR-4. IR-4 is to trigger a signal to close valve-2 and stop pump- 3. The microsphere suspension is then allowed to stay in the hybridization chamber for a while for labeling purpose. Phase 4

[00119] Finally, start either pump-3 or pump-4 to push the microsphere suspension out of the hybridization chamber towards the waste reservoir, and the entire nucleic acid amplification and analysis is completed.

[00120] The labeling result is shown in Figure 9, where there is a hybridization microarray consisting of 6 rows and 6 columns. The first and sixth columns are streptavidin-biotin binding quality control, the second column is negative control, the third column is blank, the fifth column is positive control, and the fourth column is the result corresponding to the template sample.

[00121] It should be understood that the invention can be practiced with modification and alteration within the spirit and scope of the appended claims. The description is not intended to be exhaustive or to limit the invention to the precise form disclosed, nor to limit the invention to the exemplary uses described. It should therefore be understood that the invention can be practiced with modification and alteration and that the invention be limited only by the claims and the equivalents thereof.

Claims

1. A microfluidic device comprising a cartridge comprising at least one microchannel and a separate control unit comprising at least a pump that can be turned on and off, a valve, and a sensor, wherein at least one end of the microchannel is coupled to the pump and at least one end of the microchannel is coupled to the valve, wherein the pump controls movement of fluid along the microchannel, wherein the valve controls direction of fluid flow along the microchannel, wherein the sensor senses the passage of the fluid flow front and provides a feedback signal to control the action of the pump and the valve.

2. The microfluidic device of claim 1 , wherein the device does not comprise an air duct.

3. The microfluidic device of claim 1, further comprising a sealable sample injector port between two ends of the microchannel.

4. The microfluidic device of claim 1, wherein the sensor comprises an electromagnetic signal transmitting element and an electromagnetic signal receiving element, and wherein the intensity or frequency of said electromagnetic signal or the combination thereof changes as a flow front of fluid passes the location to be sensed by the sensor.

5. The microfluidic device of claim 1, wherein the sensor comprises an electric element, wherein the impedance of the electric element is sensitive to the wetness of the environment, and wherein the electric element generates a response as a flow front of fluid passes the location to be sensed by the sensor.

6. The microfluidic device of claim 1 , wherein the control unit further comprises a temperature control module for controlling temperature at one or more locations on the cartridge.

7. The microfluidic device of claim 1 , wherein the control unit further comprises a magnetic element for generating a magnetic field, wherein the magnetic field is capable of moving magnetic particles in a fluid sample along the microchannel.

8. The microfluidic device of claim 1, wherein the control unit further comprises an electric element for generating an electric field, wherein the electric field is capable of moving charged particles in a fluid sample along the microchannel.

9. The mcirofluidic device of claim 1, wherein the cartridge further comprises a heating element for increasing the temperature of one or more locations on the cartridge.

10. The microfluidic device of claim 1 , wherein the cartridge further comprises a temperature sensor for sensing the temperature of one or more locations on the cartridge.

11. The microfluidic device of claim 1 , wherein the cartridge comprises a nucleic acid extraction unit, a nucleic acid amplification unit, and a nucleic acid detection unit.

12. The microfluidic device of claim 1 , wherein the cartridge comprises within the microchannel a structure or surface that is effective for a specific nucleic acid adsorption under one condition, and effective for nucleic acid desorption under another condition.

13. The microfluidic device of claim 12, wherein the structure has a large surface to volume ratio, and wherein the structure comprises particles, microspheres, fibers, membranes, array of micro-pillars or frits.

14. The microfluidic device of claim 12, wherein the surface is organic, inorganic, coated, surface-modified, or a combination thereof.

15. The microfluidic device of claim 1 , wherein a portion of the microchannel has its interior surface or interior region immobilized with chemical molecules, which are capable of binding with specific nucleic acids or derivatives thereof.

16. The microfluidic device of claim 15, wherein the chemical molecules are immobilized in an array and can each be identified by their respective location in the array.

17. The microfluidic device of claim 3, wherein the sealable sample injector port is a bubble resistant injector port.

18. The microfluidic device of claim 1 , wherein the microchannel comprises a wicking inhibitor.

19. A method of controlling fluid movement in a microfluidic device of any of claims 2- 17, comprising: a) introducing a fluidic sample to the cartridge, b) using the pump and the valve to move the fluid in a desired direction along the microchannel, and c) sensing the passage of fluid front at least in one location on the microchannel downstream of the sample injector port via the sensor, wherein the sensor generates a feedback signal for controlling the action of the pump and the valve.

20. The method of claim 18, wherein the cartridge comprises a sealable sample injector port, and wherein the fluid sample is introduced through the sealable sample injector port.

21. A method of controlling fluid movement in a cartridge by use of a control unit separated from the cartridge comprising: a. providing a control unit separated from said cartridge, said control unit further comprising of at least a pump, a valve, and a sensor; b. providing a microfabricated cartridge, said cartridge further comprising of at least one microchannel, said at least one microchannel having at least one end coupled to said pump in said control unit, at least one end coupled to said valve in said control unit, and at least a sealable sample injection point between said ends; c. opening a valve downstream of said sample injection point, introducing a fluid sample to said cartridge via said sample injection point, then closing said sample injection point; d. Turning on/off said pumps and opening/closing said valves selectively to move said fluid sample to a desired direction in said microchannel; e. Sensing of passage of fluid flow front at least in one location on said microchannel downstream of said sample injection point, generating a feedback signal to selectively turn on/off said pumps and open/close said valves to change movement of said fluid sample.