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Universal sample preparation system and use in an integrated analysis system — Integenx Inc. (US9663819B2)

Integenx Inc. · Google Patents

Google Patents · Patents · License: Open Access

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integenxinc.

patent, google patents, intellectual property, US9663819B2, Integenx Inc., Stevan B. Jovanovich, en, 2017

ABSTRACT

Abstract

The invention provides a system that can process a raw biological sample, perform a biochemical reaction and provide an analysis readout. For example, the system can extract DNA from a swab, amplify STR loci from the DNA, and analyze the amplified loci and STR markers in the sample. The system integrates these functions by using microfluidic components to connect what can be macrofluidic functions. In one embodiment the system includes a sample purification module, a reaction module, a post-reaction clean-up module, a capillary electrophoresis module and a computer. In certain embodiments, the system includes a disposable cartridge for performing analyte capture. The cartridge can comprise a fluidic manifold having macrofluidic chambers mated with microfluidic chips that route the liquids between chambers. The system fits within an enclosure of no more than 10 ft 3 . and can be a closed, portable, and/or a battery operated system. The system can be used to go from raw sample to analysis in less than 4 hours.

Description

CROSS-REFERENCE

This application is a continuation application of U.S. patent application Ser. No. 13/967,957, filed on Aug. 15, 2013, which is a continuation application of U.S. patent application Ser. No. 13/717,585, filed on Dec. 17, 2012 and issued as U.S. Pat. No. 8,562,918 on Oct. 22, 2013, which is a divisional application of U.S. patent application Ser. No. 12/795,515, filed on Jun. 7, 2010 and issued as U.S. Pat. No. 8,394,642 on Mar. 12, 2013, which is a continuation application of International Patent Application No. PCT/US2010/037545, filed on Jun. 4, 2010, and claims priority to and the benefit of U.S. Provisional Patent Application No. 61/184,759, filed on Jun. 5, 2009, U.S. Provisional Patent Application No. 61/235,664, filed on Aug. 20, 2009, and U.S. Provisional Patent Application No. 61/349,680, filed on May 28, 2010. All of the aforementioned patent applications are incorporated herein by reference in their entirety for all purposes.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with Government support under Contract No. 2004*H838109*000 awarded by the Central Intelligence Agency. The Government may have certain rights in this invention.

BACKGROUND OF THE INVENTION

Sample preparation is a ubiquitous problem in biological analytical systems. The issue of providing sufficiently purified targets from diverse raw sample types to reliably perform downstream analytical assays is pervasive and covers cell biology, genomics, proteomics, metabolomics, food biology, molecular diagnostics, and many other biological and medical assays. While many advances in sample preparation have been made the chief solution has been to develop reagents that are used manually or in robotic systems that use rectilinear stages or multi-axis arms to manipulate samples.

Microfluidics and nanofluidics allow miniaturized sample volumes to be prepared for analysis. Advantages include the nanoscale consumption of reagents to reduce operating costs and full automation to eliminate operator variances. Microfluidic sample preparation can either interface with existing or future detection methods or be part of a completely integrated system. In the present application, methods and apparatuses are disclosed that integrate full volume sample preparation with volumes over 10 mL with microliter and smaller volumes for sample preparation and analysis.

Starting from the sample, the present invention can be applied to concentrate, and pre-separate components for further processing to detect and classify organisms in matrices comprising aerosol samples, water, liquids, blood, stools, nasal, buccal and other swabs, bodily fluids, environmental samples with analysis by ELISA, PCR or other nucleic acid amplification techniques, single molecule detection, protein arrays, mass spectroscopy, and other analytical methods well known to one skilled in the art.

Microfluidic nucleic acid purification can be performed to prepare the sample for nucleic acid assays. For DNA analysis, PCR amplification is one current method. Microarray DNA, RNA and protein analysis also requires extensive sample preparation before the sample can be applied to the microarray for reaction and readout.

Samples can be obtained by a wide variety of substrates and matrices. The matrix may contain complex mixtures including inhibitory compounds such as hemes, indigo, humic acids, divalent cations, and proteins etc that interfere with DNA-based amplification. Aerosols can contain large amounts of molds, metals, and soils humic and other acids that all interfere with PCR amplificationâthe gold standard.

Early work showed that as few as three seeded organisms could be detected from diluted samples of soil extracts followed by PCR amplification of two 16S ribosomal gene fragments. Low-melting-temperature agarose has been used to extract DNA from soil samples for 165 and 18S rDNA PCR amplification using universal primers. Spun separation gels in column format can be used, such as Sephadex columns. Multistep purifications such as organic extractions combined with Sephadex columns were developed. Bead beating was found to be an effective way to prepare samples for high numbers of organisms and grinding in liquid nitrogen to detect low numbers of organisms. While these methods are effective they were best suited for research laboratory environments.

Solid phase extractions to columns, beads, and surfaces can be used to purify DNA before DNA analysis. Proteinase K followed by a Qiagen QIA Amp silica-gel membrane columns and IsoCode Stix, an impregnated membrane-based technology, followed by heating, washing and a brief centrifugation were compared for B. anthracis Sterne vegetative cells in buffer, serum, and whole blood and spores in buffer and found to work well.

A variety of separations can be performed using the devices and methods of the invention. For example, the devices and methods of the invention can be used to perform chromatography, phase-based or magnetic-based separation, electrophoresis, distillation, extraction, and filtration. For example, a microfluidic channel or a capillary can be used for chromatography or electrophoresis. As well, beads, such as magnetic beads can be used for phase-based separations and magnetic-based separations. The beads, or any other surfaces described herein, can be functionalized with binding moieties that exhibit specific or non-specific binding to a target. The binding can be based on electrostatics, van der Walls interactions, hydrophobicity, hydrophilicity, hydrogen bonding, ionic interactions, as well as partially covalent interactions like those exhibited between gold and sulfur. In preferred embodiments, the devices and methods of the invention utilize immunomagnetic separations.

Immunomagnetic separation (IMS) is a powerful technology that allows targets to be captured and concentrated in a single step using a mechanistically simplified format that employs paramagnetic beads and a magnetic field (see Grodzinski P, Liu R, Yang J, Ward M D. Microfluidic system integration in sample preparation microchip-setsâa summary. Conf Proc IEEE Eng Med Biol Soc. 2004; 4:2615-8, Peoples M C, Karnes H T. Microfluidic immunoaffinity separations for bioanalysis. J Chromatogr B Analyt Technol Biomed Life Sci. 2007 Aug. 30, and Stevens K A, Jaykus L A. Bacterial separation and concentration from complex sample matrices: a review. Crit. Rev Microbiol. 2004; 30(1):7-24.). IMS can be used to capture, concentrate, and then purify specific target antigens, proteins, toxins, nucleic acids, cells, and spores. While IMS as originally used referred to using an antibody, we generalize its usage to include other specific affinity interactions including lectins, DNA-DNA, DNA-RNA, biotin-streptavidin, and other affinity interactions that are coupled to a solid phase. IMS works by binding a specific affinity reagent, typically an antibody or DNA, to paramagnetic beads which are only magnetic in the presence of an external magnetic field. The beads can be added to complex samples such as aerosols, liquids, bodily fluids, or food. After binding of the target to the affinity reagent (which itself is bound to the paramagnetic bead) the bead is captured by application of a magnetic field. Unbound or loosely bound material is removed by washing with compatible buffers, which purifies the target from other, unwanted materials in the original sample. Because beads are small (about 1 nm to about 1 um) and bind high levels of target, when the beads are concentrated by magnetic force they typically form bead beds of between 1 mL and 1 uL, thus concentrating the target at the same time it is purified. The purified and concentrated targets can be conveniently transported, denatured, lysed or analyzed while on-bead, or eluted off bead for further sample preparation, or analysis.

Immunomagnetic separations are widely used for many applications including the detection of microorganisms in food, bodily fluids, and other matrices. Paramagnetic beads can be mixed and manipulated easily, and are adaptable to microscale and microfluidic applications. This technology provides an excellent solution to the macroscale-to-microscale interface: beads are an almost ideal vehicle to purify samples at the macroscale and then concentrate to the nanoscale (100's of nL) for introduction into microfluidic or nanofluidic platforms. Immunomagnetic separations are commonly used as an upstream purification step before real-time PCR, electrochemiluminescence, and magnetic force discrimination.

The ability to move fluids on microchips is a quite important. This invention describes technologies in sample capture and purification, micro-separations, micro-valves, -pumps, and -routers, nanofluidic control, and nano-scale biochemistry. A key component of the technology is Micro-robotic On-chip Valves (MOVe) technology (an example of which is shown in FIGS. 1A, 1B and 1C ) and its application to miniaturize and automate complex workflows. Collectively the MOVe valves, pumps, and routers and the instrumentation to operate them can be referred to as a microchip fluid processing platform.

The heart of the microchip fluid processing platform technology are MOVe pumps, valves, and routers that transport, process, and enable analysis of samples. These novel externally actuated, pneumatically-driven, on-chip valves, pumps, and routers, originally developed in the Mathies laboratory at the University of California at Berkeley (U. C. Berkeley) (Grover, W. H. A. M. Skelley, C. N. Liu, E. T. Lagally, and R. M. Mathies. 2003 . Sensors and Actuators B89:315-323; Richard A. Mathies et al., United States Patent Application, 20040209354 A1 Oct. 21, 2004; all of which are herein incorporated by reference in their entirety) can control fluidic flow at manipulate volumes from 20 nL to 10 Î¼L.

The MOVe valves and pumps ( FIGS. 1A, 1B and 1C ) can combine two glass and/or plastic microfluidic layers with a polydimethyl siloxane (PDMS) deformable membrane layer that opens and closes the valve, and a pneumatic layer to deform the membrane and actuate the valve. The microfluidic channel etched in the top glass fluidic wafer is discontinuous and leads to a valve seat which is normally closed ( FIG. 1A ). When a vacuum is applied to the pneumatic displacement chamber by conventional-scale vacuum and pressure sources, the normally closed PDMS membrane lifts from the valve seat to open the valve ( FIG. 1B ). FIG. 1C shows a top view of the valve a similar scale as the other panels.

Three microvalves can be used to make a micropump on a microchip to move fluids from the Input area to the Output area on Microchip A. The fluids are moved by three or more valves. The valves can be created actuation of a deformable structure. In some implementations a valve seat is created and in other embodiments no valve seat may be needed. FIG. 2 shows MOVe devices from top to bottom: valve, router, mixer, bead capture. Self-priming MOVe pumps ( FIG. 2 , top) are made by coordinating the operation of three valves and can create flow in either direction. Routers are made from three or more MOVe valves ( FIG. 2 , top middle panel). Mixing has been a holy grail for microfluidics: MOVe mixers ( FIG. 2 , bottom middle panel) rapidly mix samples and reagents. MOVe devices work exquisitely with magnetic beads to pump or trap sets of beads ( FIG. 2 , bottom panel).

The normally closed MOVe valves, pumps, and routers are durable, easily fabricated at low cost, can operate in dense arrays, and have low dead volumes. Arrays of MOVe valves, pumps, and routers are readily fabricated on microchips. Significantly, all the MOVe valves, pumps, and routers on a microchip are created at the same time in a simple manufacturing process using a single sheet of PDMS membraneâit costs the same to make 5 MOVe micropumps on a microchip as to create 500. This innovative technology offers for the first time the ability to create complex micro- and nanofluidic circuits on microchips.

Patents and applications which discuss the use and design of microchips include U.S. Pat. No. 7,312,611, issued on Dec. 25, 2007; U.S. Pat. No. 6,190,616, issued on Feb. 20, 2001; U.S. Pat. No. 6,423,536, issued on Jul. 23, 2002; U.S. patent Ser. No. 10/633,171 Mar. 22, 2005; U.S. Pat. No. 6,870,185, issued on Mar. 22, 2005 US Application No. US 2001-0007641, filed on Jan. 25, 2001; US Application US20020110900, filed on Apr. 18, 2002; US patent application 20070248958, filed Sep. 15, 2005; US patent application US 20040209354, filed on Dec. 29, 2003; US patent application US2006/0073484, filed on Dec. 29, 2003; US20050287572, filed on May 25, 2005; US patent application US20070237686, filed on Mar. 21, 2007; US 20050224352 filed on Nov. 24, 2004; US 20070248958, filed on, Sep. 15, 2005; US 20080014576, filed on Feb. 2, 2007; and, US application US20070175756, filed on Jul. 26, 2006; all of which are herein incorporated by reference in their entirety.

SUMMARY OF THE INVENTION

The invention provides a system that can process a raw biological sample, perform a biochemical reaction and provide an analysis readout in multiplex. For example, the system can extract DNA from a swab, amplify STR loci from the DNA, and analyze the amplified loci and STR markers in the sample. The system integrates these functions by using microfluidic components to connect what can be macrofluidic functions. In one embodiment the system includes a sample purification module, a reaction module, a post-reaction clean-up module, a capillary electrophoresis module and a computer. In certain embodiments, the system includes a disposable cartridge for performing analyte capture. The cartridge can comprise a fluidic manifold having macrofluidic chambers mated with microfluidic chips that route the liquids between chambers. The system fits within an enclosure of no more than 10 ft 3 and can be a closed, portable, and/or battery operated system. The system can be used to go from sample to analysis in less than 4 hours.

In one aspect, this invention provides a system that fits within an enclosure of no more than 10 ft 3 , the system comprising: (a) a sample preparation module adapted to capture an analyte from a non-microfluidic volume on a capture particle and route the captured analyte through a microfluidic channel; (b) a reaction module comprising a reaction chamber in fluidic communication with the microfluidic channel adapted to immobilized the captured analyte and perform a biochemical reaction on the analyte in a non-microfluidic volume to produce a reaction product; (c) and an analysis module in fluidic communication with the reaction chamber adapted to perform an analysis on the reaction product. In one embodiment, the system is configured to capture the analyte, perform a biochemical reaction on the analyte, and perform an analysis on the product in less than 4 hours, in less than 3 hours, or even in less than 2 hours. In one embodiment, the system further comprises a data analysis module configured to receive data about the analysis from the analysis module and comprising executable code that transforms the data and outputs a result of the analysis. In another embodiment, the system further comprises a processing module in fluidic communication with the reaction chamber and the analysis module and adapted to (1) route the reaction product through a second microfluidic channel into a non-microfluidic processing chamber; (2) process the reaction product and (3) route the processed reaction product into the analysis module. In one particular embodiment, the system fits within an enclosure of no more than 8 ft 3 , no more than 5 ft 3 or no more than 2Â½ ft 3 . In another embodiment of the system, the sample preparation module is adapted to release the analyte from a cell. In another embodiment of the system, the capture particle is a magnetically responsive capture particle and a reaction module comprises a source of magnetic force configured to immobilize the captured analyte. In another embodiment of the system, the reaction module is adapted to perform thermal cycling. In another embodiment of the system, the system is a closed system and/or battery operated.

CROSS-REFERENCE

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with Government support under Contract No. 2004*H838109*000 awarded by the Central Intelligence Agency. The Government may have certain rights in this invention.

BACKGROUND OF THE INVENTION

SUMMARY OF THE INVENTION

In another aspect, this invention provides a system comprising a cartridge cover, a cartridge and a pneumatic manifold wherein the cartridge can be releasably engaged with the cartridge cover and the pneumatic manifold, wherein the cartridge comprises one or more pneumatically actuated valves and one or more microfluidic channels, wherein the pneumatic manifold and the cartridge cover are each fluidically connected to at least one pressure source, and wherein the pneumatic manifold and the cartridge cover are each adapted to control fluid flow within the cartridge. In one embodiment, the pneumatic manifold is adapted to actuate the pneumatically actuated valves and the cartridge cover is adapted to apply pressure to one or more chambers in the cartridge.

In another aspect, this invention provides a system comprising: (a) a disposable cartridge comprising at least one set of fluidic chambers including a sample chamber, a mixing chamber and a thermal cycling chamber in fluid communication with each other, and a reagent card comprising reagents for performing a chemical reaction involving thermal cycling, wherein the reagent card is configured to be carried on the cartridge in a closed configuration and to be moved into fluid communication with the at least one set of fluidic chambers; (b) an actuator assembly configured to move fluids between chambers when the cartridge is engaged with the actuator assembly; (c) a thermal cycler configured to cycle temperature in the thermal cycling chamber when the cartridge is engaged with the actuator assembly; (d) a capillary electrophoresis assembly configured to accept a sample from cartridge when the cartridge is engaged with the actuator assembly and to perform capillary electrophoresis on the sample; and (e) a computerized control system configured to control the actuator assembly, the thermal cycler and the capillary electrophoresis assembly.

In another aspect, this invention provides a cartridge comprising: (a) a fluidic manifold comprising a fluidic side and a reagent card side wherein the fluidic manifold comprises: (i) at least one set of fluidic chambers, each chamber comprising a port on the fluidic side; (ii) at least one of thermal cycling chamber comprising at least one port; (iii) at least one of exit port; (iv) a slot on the reagent card side adapted to engage a reagent card, wherein the slot comprises a plurality of slot channels comprising cannulae on the reagent card side and communicating between the two sides; (b) at least one microfluidic chip comprising: at least one fluidic circuit; (ii) a plurality of ports in fluid communication with the fluidic circuit; (iii) at least one pneumatically activated diaphragm valve configured to regulate fluid flow within the fluidic circuit; wherein the at least one chip is engaged with the fluidic manifold so that the ports in the at least one chip are in fluid communication with the ports of the chambers and the slot channels wherein each fluidic chamber is in fluid communication with at least one other fluidic chamber and each cannula is in communication with a fluidic chamber; and (c) a reagent card engaged with the slot, wherein the card comprises a plurality of reagent chambers comprising reagents, each aligned with at least one of the cannulae and adapted to take a first engagement position wherein the reagent chambers are not punctured by the cannulae and a second engagement position wherein the reagent chambers are punctured by the cannulae, thereby putting the reagent chambers in fluid communication with the fluidic circuit. In one embodiment the reagents comprise reagents for performing PCR. In another embodiment, the reagents comprise primers for amplifying a plurality of short tandem repeats. In another embodiment, the at least one set of fluidic chambers is a plurality of sets of fluidic chambers. In yet another embodiment the cartridge is such that the fluidics manifold further comprises at least one auxiliary fluidic channel on the fluidic side of the manifold, the at least one chip is a plurality of chips and fluidic circuits in each of the plurality of chips are in fluidic communication with fluidic circuits of at least one other chip through the auxiliary fluidic channel. In this embodiment, the cartridge can further comprise a gasket between the chips and the manifold, wherein the gasket seals the channels on the manifold. In another embodiment of the cartridge, the fluidic chambers comprise a distribution chamber, a capture chamber, a sample chamber, and a clean-up chamber. In another embodiment of the cartridge, at least one fluidic chamber comprises magnetically responsive particles. In another embodiment of the cartridge, the at least one set of fluidic chambers is at least 4 sets or at least 8 sets. In another embodiment of the cartridge, the chips comprise at least one diaphragm valve.

In another aspect, the invention provides a system comprising: (a) a pneumatic assembly comprising: (i) a pneumatic manifold adapted to removably engage the cartridge on the fluidic side, wherein the pneumatic manifold comprises a plurality of pneumatic ports configured to engage pneumatic channels in the at least one microfluidic chip and activate the diaphragm valves; and (ii) a pressure source configured to supply positive or negative pressure to the pneumatic channels; (b) a cartridge activation assembly adapted to engage the cartridge on the reagent card side; wherein the cartridge activation assembly comprises: (i) a reagent pneumatic manifold comprising a pneumatic side and reagent card side, wherein the reagent pneumatic manifold comprises reagent pneumatic manifold channels communicating between the two sides and comprising a cannula on the reagent card side; (ii) a pressure source configured to supply positive or negative pressure to the reagent pneumatic manifold channels; and (iii) a clamp configured to move the reagent card from the first engagement position to the second engagement position, wherein clamping results in the cannulae of the reagent pneumatic manifold puncturing the reagent chambers and putting the reagent chambers in communication with the pressure source; (c) a thermal cycler configured to cycle temperature in the at least one thermal cycling chamber when the cartridge is clamped; (d) a capillary electrophoresis assembly comprising: (i) at least one separation channel fluidically engaged with the exit port when the cartridge is clamped; and (ii) an optical sub-assembly configured to detect signal from the at least one separation channel; and (e) a computerized control system configured to control pneumatic assembly, the cartridge activation assembly, the thermal cycler and the capillary electrophoresis assembly. In one embodiment of this system, clamping seals the chamber ports and the slot channels with the at least one microfluidic chip. In another embodiment of this system, the cartridge activation assembly further comprises at least one heater configured to heat at least one of the fluidic chambers when the reagent pneumatic manifold is engaged with the cartridge. In another embodiment of this system, the cartridge activation assembly further comprises movable magnets configured to move into and out of a position wherein the magnets exert a magnetic force on at least one fluidic chamber. In another embodiment of this system, the cartridge activation assembly further comprises sensors configured to detect the presence of a sample in a sample chamber of the fluidic manifold. In another embodiment of this system, the thermal cycler comprises a Peltier device. In one embodiment the system is comprised in a portable case. In one specific embodiment, the case has an internal volume of no more than 10 ft 3 or no more than 2Â½ ft 3 . In a related embodiment of system, the invention provides for an article in computer readable form comprising code for the operating system.

In another aspect the invention provides a method comprising: producing, from a sample comprising at least one cell comprising DNA, a computer file identifying a plurality of STR markers in the DNA, wherein the method is performed in less than 4 hours. In one embodiment the method is performed in less than 3 hours. In another embodiment the method is performed in less than 2 hours. In a related embodiment, the producing comprises extracting the DNA from the at least one cell, amplifying the STR markers from the DNA, performing capillary electrophoresis on the amplified markers, detecting the amplified markers, and performing computer analysis on the detected amplified markers to identify the markers. In one embodiment, the plurality of STR markers is at least 5 STR markers. In another embodiment the plurality of markers are CODIS STR markers. In a related embodiment, the plurality of STR markers is at least 5, 10, or 13 CODIS STR markers. In these embodiments, the at least one cell can be a plurality of cells. In some embodiments, the sample is a forensic sample. In specific embodiments, the method is performed at the site of a sample collection. The sample can comprise blood, or can comprise a cheek swab. The method can be carried out by any system described herein.

In a related aspect, the invention provides a system configured to perform a method, wherein the method comprises: producing, from a sample comprising at least one cell comprising DNA, a computer file identifying a plurality of STR markers in the DNA, wherein the method is performed in less than 4 hours.

In another aspect, this invention provides a method comprising: (a) providing a system comprising: (i) a disposable cartridge comprising at least one set of fluidic chambers including a sample chamber, a mixing chamber and a thermal cycling chamber in fluid communication with each other, and a reagent card comprising reagents for performing a chemical reaction involving thermal cycling, wherein the reagent card is configured to be carried on the cartridge in a closed configuration and to be moved into fluid communication with the at least one set of fluidic chambers; (ii) an actuator assembly configured to move fluids between chambers when the cartridge is engaged with the actuator assembly; (iii) a thermal cycler configured to cycle temperature in the thermal cycling chamber when the cartridge is engaged with the actuator assembly; (iv) a capillary electrophoresis assembly configured to accept a sample from cartridge when the cartridge is engaged with the actuator assembly and to perform capillary electrophoresis on the sample; and (v) a computerized control system configured to control the actuator assembly, the thermal cycler and the capillary electrophoresis assembly; (b) moving of the reagent card into fluid communication with at least one set of fluidic chambers; (c) providing a sample comprising a nucleic acid molecule to a sample chamber; and (d) operating the system to amplify and detect at least one nucleic acid sequence in the sample. In one embodiment the time it takes to go from step (b) to step (d) is less than 4 hours. In one embodiment, the method comprises providing each of a plurality of samples to a different sample chamber. In another embodiment, the method comprises amplifying and detecting a plurality of nucleic acid sequences in the sample. In a related embodiment, the plurality of nucleic acid sequences comprise short tandem repeats (STRs). In a specific embodiment, the short tandem repeats comprise a plurality of Combined DNA Index System (CODIS) markers. In another embodiment the CODIS markers comprise a plurality of markers selected from AMEL, D3S1358, THO1, D21s11, D18s51, D5s818, D13s317, D7s820, D16s539, CSF1PO, vWA, D8S1179, TPDX and FGA. In one embodiment, the system comprises: (a) a pneumatic assembly comprising: (i) a pneumatic manifold adapted to removably engage the cartridge on the fluidic side, wherein the pneumatic manifold comprises a plurality of pneumatic ports configured to engage pneumatic channels in the at least one microfluidic chip and activate the diaphragm valves; and (ii) a pressure source configured to supply positive or negative pressure to the pneumatic channels; (b) a cartridge activation assembly adapted to engage the cartridge on the reagent card side; wherein the cartridge activation assembly comprises: (i) a reagent pneumatic manifold comprising a pneumatic side and reagent card side, wherein the reagent pneumatic manifold comprises reagent pneumatic manifold channels communicating between the two sides and comprising a cannula on the reagent card side; (ii) a pressure source configured to supply positive or negative pressure to the reagent pneumatic manifold channels; and (iii) a clamp configured to move the reagent card from the first engagement position to the second engagement position, wherein clamping results in the cannulae of the reagent pneumatic manifold puncturing the reagent chambers and putting the reagent chambers in communication with the pressure source; (c) a thermal cycler configured to cycle temperature in the at least one thermal cycling chamber when the cartridge is clamped; (d) a capillary electrophoresis assembly comprising: (i) at least one separation channel fluidically engaged with the exit port when the cartridge is clamped; and (ii) an optical sub-assembly configured to detect signal from the at least one separation channel; and (e) a computerized control system configured to control pneumatic assembly, the cartridge activation assembly, the thermal cycler and the capillary electrophoresis assembly. In another embodiment, the system is any system named herein. In one embodiment, the sample is a forensic sample. In a related embodiment, the sample is selected from a buccal swab, blood, hair or semen. In another embodiment, the sample is a raw sample. In some embodiments, the method further comprises transporting the system to a forensic site.

In another aspect, the invention provides an optical system comprising: (a) a plurality of optically transparent channels; (b) a light source configured to direct to the plurality of optically transparent channels; (c) a dispersive element that disperses light passing through the optically transparent channels in a wavelength dependent manner; and (d) a detector configured to receive the dispersed light. In one embodiment the plurality of optically transparent channels comprises at least eight capillaries. In another embodiment the optically transparent channels are aligned in a first plane and the dispersive element disperses light along a second plane, wherein the first plane and the second plane are different. In another embodiment, the first plane is orthogonal to the second plane.

In another aspect, the invention provides an optical system comprising: (a) an excitation source configured to direct excitation light to an object; (b) a carrier for an object, wherein the object emits light other than excitation light when excited by the excitation energy; (c) a rejection filter configured to filter out excitation energy and to allow transmission of the emitted light; (d) an imaging lens configured to focus the emitted light; (e) a dichroic mirror substantially transparent to the excitation energy and configured to reflect emitted light to a detector; (f) a focusing system comprising at least one lens configured to focus light reflected from the dichroic mirror; and (g) a photodetector (CCD camera) configured to receive the reflected light. In one embodiment of the optical system, the excitation light comprises light of a wavelength between 03 microns and 1 micron. In another embodiment, the carrier comprises an array of capillary tubes and the object comprises a fluorescent species. In another embodiment, the mirror reflects emitted light and an angle between about 5 degrees and about 10 degrees off an incident angle. In another embodiment, the dichroic mirror further comprises a portion that transmits substantially all light. In another embodiment, the focusing system comprises at least one folding mirror. In another embodiment, the photodetector comprises a CCD camera. In a related embodiment, the optical system can further comprise a prism located between the object and the imaging lens.

In another aspect, the invention provides an optical system comprising: (a) an array of capillary tubes aligned substantially parallel and substantially in a plane; (b) an excitation assembly comprising an excitation source and configured to deliver excitation light from the excitation source to the array, wherein the light delivery assembly is configured (i) to deliver a thin band of light that covers the array and (ii) to deliver the light to the array at an angle other than 90 degrees to the plane; (c) a collection lens configured to collect light emitted from the array by objects in the array excited by the excitation light; wherein the excitation assembly and the collection lens are configured with respect to the array so that excitation light passing through the array substantially avoids collection by the collection lens. In one embodiment, the angle is between about 10 degrees and about 85 degrees.

In another aspect, the invention provides an instrument comprising: (a) a microfluidic component comprising a plurality of intersecting microfluidic channels and at least one controllable valve configured to regulate flow of fluid between the intersecting channels; and (b) a non-microfluidic component comprising a plurality of non-microfluidic chambers, wherein each non-microfluidic chamber is fluidically connected to at least one of the microfluidic channels; wherein the instrument is configured to flow fluid from at least one non-microfluidic chamber into another non-microfluidic chamber through a microfluidic channel and flow is regulated by at least one valve. In one embodiment of the instrument, the plurality of non-microfluidic chambers comprises at least three chambers and the at least one valve selectively directs fluid from one chamber to either of the at least two other chambers. In another embodiment, the instrument further comprises a pump to pump fluid from a non-microfluidic chamber into a microfluidic channel. In another embodiment of the instrument the at least one valve is a diaphragm valve. In a related embodiment, the pump is a diaphragm pump comprising a series of three diaphragm valves. In another embodiment, the microfluidic component comprises a monolithic device. IN another embodiment, the combination of the microfluidic component and the non-microfluidic component define a fluidic circuit and the instrument comprises a plurality of fluidic circuits. In another embodiment, the non-microfluidic component further comprises a particulate capture agent. In another embodiment, the particles are responsive to a magnetic field and the instrument further comprises a magnet configured to immobilize the particles. In another embodiment, the invention provides a device comprising a plurality of non-microfluidic chambers fluidically connected to a common microfluidic channel.

In another aspect, the invention provides a method comprising: (a) providing a device comprising: (i) a microfluidic component comprising a plurality of intersecting microfluidic channels and at least one controllable valve configured to regulate flow of fluid between the intersecting channels; and (ii) a non-microfluidic component comprising a plurality of non-microfluidic chambers, wherein each non-microfluidic chamber is fluidically connected to at least one of the microfluidic channels; wherein the instrument is configured to flow fluid from at least one non-microfluidic chamber into another non-microfluidic chamber through a microfluidic channel and flow is regulated by at least one valve; and wherein a first non-microfluidic chamber comprises a first volume of sample comprising an analyte; (b) providing an amount of particulate capture agent in the first non-microfluidic chamber to bind a selected amount of analyte from the sample; (c) moving the particulate capture agent bound to the analyte through a microfluidic channel in the microfluidic device to a second non-microfluidic chamber; (d) contacting the particulate capture agent bound to the analyte with a reagent in a second non-microfluidic chamber; and (e) performing a chemical reaction on the analyte using the reagent. In one embodiment of the method the contacting comprises flowing the reagent from a third non-microfluidic chamber through a microfluidic channel in the microfluidic device into the second non-microfluidic chamber. In another embodiment of the method, the particles are response to magnetic force and the method further comprises immobilizing the particulate capture agent bound to the analyte in the instrument with a magnetic force. In another embodiment, the method comprises suspending the particulate capture agent bound to the analyte in the instrument in a volume at least an order of magnitude smaller than the sample volume.

In another aspect, the invention provides a method comprising: (a) performing a first chemical reaction on an analyte in a first chamber which is a non-microfluidic chamber to produce a first reaction product; and (b) moving the first reaction product through a microfluidic channel into a second chamber which is a microfluidic chamber and performing a second chemical reaction on the first product to create a second reaction product. In a related aspect of the invention, provided herein is a method comprising: (a) performing a first chemical reaction on an analyte in a first chamber which is a microfluidic chamber to produce a first reaction product; and (b) moving the first reaction product through a microfluidic channel into a second chamber which is a non-microfluidic chamber and performing a second chemical reaction on the first product to create a second reaction product. In one embodiment, the methods of these related aspects comprise cleaning the first reaction product before moving it to the second chamber. In another embodiment, the methods of these related aspects comprise at least once, moving a reaction product through a microfluidic channel into a subsequent non-microfluidic chamber and performing a subsequent chemical reaction on the reaction product to create a subsequent reaction product. In another embodiment, the methods of these related aspects comprise at least once and before moving a reaction product into a non-microfluidic chamber, moving a reaction product through a microfluidic channel into a microfluidic chamber and performing a subsequent chemical reaction on the reaction product to create a subsequent reaction product.

In another aspect of the invention, provided herein is a device comprising: (a) a sample channel having a channel inlet and a channel outlet; (b) an electrophoresis capillary having a capillary inlet and a capillary outlet, wherein the capillary comprises an electrically conductive medium and is in communication with the sample channel at a point of connection; (c) an anode and a cathode configured to apply a voltage across the capillary inlet and capillary outlet, wherein one of the anode or cathode comprises a forked electrode wherein the forks are in electrical communication with the sample channel on different sides of the point of connection; and (d) a second electrode in electrical communication with the sample channel substantially opposite the point of connection. In one embodiment of the device, the second electrode is comprised as a third fork in the forked electrode.

INCORPORATION BY REFERENCE

All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

FIGS. 1A, 1B and 1C depict an example of a microscale on-chip valve (MOVe).

FIG. 2 shows a MOVe microvalve, a microrouter, a MOVe mixer, and bead capture on microchips.

FIG. 3 shows a fluidic cartridge with MOVe microvalves.

FIG. 4 shows a fluidic cartridge with ports to a microfluidic microchip with microvalves.

FIG. 5 shows a microfluidic microchip with MOVe valves that controls flows in a cartridge.

FIG. 6 shows a cartridge connected to reaction chamber and detector with downstream MOVe pumps and reagents.

FIG. 7 shows a temperature control device that can thermal cycle and incorporates magnetic capture, pinch clamps and the capability of cycling seven reactions simultaneously.

FIG. 8 shows a temperature control device that can thermal cycle and incorporates magnetic capture, pinch clamps and the capability of cycling seven reactions simultaneously.

FIG. 9 shows PowerPlex 16 STR (Short tandem repeat) amplification reaction performed in a passive, Teflon (PTFE) based Tube reaction chamber.

FIG. 10 shows purification of DNA from 25 uL of blood at 69â², 23.5â², 10.5â², and 4.5â²; yield in ng is shown on the bars.

FIG. 11 shows a schematic of using microvalves to capture beads on a microchip.

FIG. 12 shows bead capture from a cartridge on a microchip using a MOVe microvalve.

FIG. 13 shows bead capture from a cartridge on a microchip using a MOVe microvalve.

FIG. 14 shows a capture and reaction microchip using MOVe microvalves.

FIG. 15 shows a capture and reaction microchip using MOVe microvalves.

FIG. 16 shows a four module assembly.

FIG. 17 shows an example of STR reactions on microchips.

FIG. 18 shows a universal sample preparation workflow to prepare nucleic acids and toxins.

FIG. 19 shows purification of samples in a cartridge using paramagnetic beads.

FIG. 20 shows an integrated pneumatic manifold to operate the MOVe microvalves in cartridge.

FIG. 21 shows a cartridge mounted on a computer controlled apparatus.

FIG. 22 shows a cartridge mounted on a computer controlled apparatus.

FIG. 23 shows a reagent distribution manifold based on MOVe technology that can distribute five reagents to five extraction/isolation or other devices.

FIG. 24 shows a reagent distribution manifold based on MOVe technology that can distribute five reagents to five extraction/isolation or other devices.

FIG. 25 shows a distribution manifold with sample loops and MOVe microvalves.

FIG. 26 shows a pneumatic manifold, top panel shows the top side and the lower panel the bottom side.

FIG. 27 shows detection of E. coli by immunomagnetic separation, followed by alkaline lysis and PEG-facilitated capture on magnetic beads, and analyzed by real-time PCR.

FIG. 28 shows application of a cartridge with three chambers that can be used to construct genomic libraries and other applications.

FIG. 29 shows the workflow to prepare genomic libraries using the cartridge.

FIG. 30 shows a forked injector for microchip based electrophoresis.

FIG. 31 shows sample stacking with a forked injector.

FIG. 32 shows a forked injector coupled to MOVe microvalves.

FIG. 33 shows a forked cathode injector coupled with a MOVe microchip.

FIG. 34 shows a photograph of a microchip with the forked injector.

FIG. 35 shows a photograph of a microchip with the forked injector.

FIG. 36 shows an electropherogram of a single color from a DNA sequencing trace from a forked cathode injector.

FIG. 37 shows STR separations on a forked cathode injection system.

FIG. 38 shows a forked cathode with MOVe microfluidics for shuttle loading.

FIG. 39 shows an integrated system for nucleic acid isolation, amplification(s), separation and detection.

FIG. 40 depicts a device with a cartridge, microfluidic microchip, and a magnet.

FIG. 41 depicts a microfluidic microchip with a fluidics layer, an elastomeric layer, and a pneumatics layer.

FIG. 42 depicts a fluidics layer made of two layers of material.

FIG. 43 depicts a fluidics layer made of a single layer of material.

FIG. 44 depicts a reaction scheme for amplifying mRNA.

FIG. 45 shows a microfluidic microchip with MOVe valves that controls flows in a cartridge.

FIG. 46 shows a forked electrode.

FIG. 47 shows a forked electrode, a forked electrode with a wire run electrode, and a forked electrode with a cannular electrode.

FIG. 48 shows sample injection into a separation channel.

FIG. 49 shows a device for mating a separation capillary with injection tubing.

FIG. 50 shows a device for mating separation capillaries with four injection tubings.

FIG. 51 shows a thermocycler with an Ultem pinch clamp.

FIG. 52 shows a diagram indicating movement of reagents between components of a four channel parallel processing device.

FIG. 53 shows a four-channel parallel reagent delivery device: the Chip C microchip design is shown on the top left, a fluidic manifold is shown on the bottom left, and the fabricated and assembled device is shown on the right.

FIG. 54 shows a four-channel sample preparation device on the left and a four-channel sample preparation device mounted on a monolithic pneumatic manifold on the right.

FIG. 55 shows MOVe microchip designs of the four-channel sample preparation device.

FIG. 56 shows IdentiFiler STR profiles of DNA samples prepared on the four-channel sample preparation device, where STR amplifications were performed using fast protocols (1.5 hrs) on a STR Reaction subsystem thermocycler.

FIG. 57 shows a four-channel post amplification device combined with an Chip A microchip with a fluidics manifold: the Chip A microchip design is shown on the left, the fabricated microchip is shown in the center, and the assembled fluidic manifold and microchip is shown on the right.

FIG. 58 shows a post-amplification STR clean-up subsystem with the post-amplification device.

FIG. 59 shows the Chip E microchip design, which can be used in the post-amplification device.

FIG. 60 shows a diagram of a mixer.

FIG. 61 shows a diagram of a mixer.

FIG. 62 shows results of using a mixer to lyse cells.

FIG. 63 depicts components of an integrated analysis system.

FIG. 64 depicts hardware components of an integrated analysis system.

FIG. 65 depicts software components of an integrated analysis system.

FIG. 66 depicts consumable components of an integrated analysis system.

FIG. 67 depicts documentation components of an integrated analysis system.

FIG. 68 shows a picture of an encased integrated analysis system.

FIG. 69 shows a schematic of an encased integrated analysis system.

FIG. 70 shows a schematic of an encased and portable integrated analysis system.

FIG. 71 shows a schematic of an encased and portable integrated analysis system and a polymer injection system.

FIG. 72 shows a schematic of an optical detection system and pneumatic system.

FIG. 73 shows a diagram of an integrated analysis system.

FIG. 74 shows a schematic of a cartridge cover and pneumatic components.

FIG. 75 shows a schematic of a cartridge cover, cartridge, pneumatic manifold, and thermocycler.

FIG. 76 depicts a cartridge with reagent cassette.

FIG. 77 depicts a cartridge with reagent cassette.

FIG. 78 depicts a top view of a cartridge with reagent cassette.

FIG. 79 depicts a top view of a cartridge without reagent cassette.

<div id

CLAIMS

Claims ( 20 )

What is claimed is:

1. A method of positioning a sample comprising an analyte for injection into a separation channel, comprising:

a) providing an electrophoresis assembly comprising:

(i) a fluidic conduit having a fluidic conduit inlet and a fluidic conduit outlet;

(ii) a separation channel having a separation channel inlet and a separation channel outlet, wherein said separation channel comprises an electrically conductive separation medium, and wherein said separation channel inlet is in fluidic communication and electrical communication with said fluidic conduit at a point of connection; and

(iii) an anode and a cathode defining an electrical path and configured to apply a voltage across said separation channel inlet and said separation channel outlet, wherein said anode or said cathode is in or adjacent to said fluidic conduit at or adjacent to said point of connection;

b) positioning a bolus of a material downstream of said sample in said fluidic conduit, wherein said material has an electrical conductivity that differs from an electrical conductivity of electrophoresis buffer or said sample;

c) moving said bolus of said material and said sample in said fluidic conduit in a direction of said separation channel;

d) detecting a change in current or voltage that corresponds to a movement of said bolus of said material into said electrical path; and

e) based on said change in current or voltage detected in d), moving said sample into said electrical path.

2. The method of claim 1 , wherein said fluidic conduit comprises a tube.

3. The method of claim 1 , wherein said bolus of said material comprises air.

4. The method of claim 1 , wherein said cathode is in said fluidic conduit at or adjacent to said point of connection with said separation channel.

5. The method of claim 1 , wherein said cathode or said anode comprises a forked electrode, and wherein a plurality of forks of said cathode is positioned in said fluidic conduit adjacent to said point of connection.

6. The method of claim 5 , wherein said plurality of forks is in electrical communication with said fluidic conduit and positioned on different sides of said point of connection.

7. The method of claim 5 , wherein said forked electrode comprises metallic conductors.

8. The method of claim 5 , further comprising concentrating said analyte in said sample using said forked electrode before moving said sample into said electrical path.

9. The method of claim 1 , wherein d) further comprises detecting a decrease in said current or voltage that corresponds to said movement of said bolus of said material into said electrical path.

10. The method of claim 1 , wherein said positioning comprises moving said sample through said fluidic conduit with a pump.

11. The method of claim 10 , wherein said pump comprises a micro-robotic on-chip valve (MOV) pump.

12. The method of claim 1 , further comprising f) providing a voltage with said cathode to inject said sample into said separation channel.

13. The method of claim 1 , wherein said separation channel comprises an electrophoresis capillary.

14. The method of claim 1 , wherein said separation channel comprises a separation polymer.

15. The method of claim 1 , wherein said analyte comprises a nucleic acid.

16. The method of claim 15 , wherein said analyte comprises deoxyribonucleic acid (DNA).

17. The method of claim 16 , wherein said analyte comprises short tandem repeat (STR) amplicons.

18. The method of claim 1 , wherein said electrophoresis assembly further comprises an additional electrode for reducing injection of gas or bubbles into said separation channel.

19. The method of claim 18 , wherein said additional electrode comprises a single wire run electrode or a cannular run electrode.

20. The method of claim 19 , wherein said cannular run electrode comprises a cannula having an inner diameter greater than or equal to about 1/64 inches.

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