WO2009149115A1

WO2009149115A1 - Cartridge for conducting biochemical assays

Info

Publication number: WO2009149115A1
Application number: PCT/US2009/046016
Authority: WO
Inventors: Mark Wobken; Laurence Sampson
Original assignee: Vectrant Technologies Inc.
Priority date: 2008-06-04
Filing date: 2009-06-02
Publication date: 2009-12-10

Abstract

Provided is a cartridge that is configured with multiple deformable chambers that carry out at least one biochemical assay within the cartridge. The cartridge is designed to accept a biological sample and to keep the sample, reagents, and assay products within the cartridge in a fully sealed manner. Biochemical assays that may be performed with the cartridge include molecular diagnostic assays, such as amplification assays. The deformable chambers of the cartridge can carry out a real time PCR assay in singleplex and multiplex format using a single deformable chamber of the thermocycling chambers to quantify the PCR product. The measurement chamber can be further used to conduct a melting curve analysis to identify target genes or molecules in a singleplex or multiplex diagnostic assay.

Description

CARTRIDGE FOR CONDUCTING BIOCHEMICAL ASSAYS

STATEMENT OF GOVERNMENT SUPPORT

[0001] This invention was made with Government support under Contract No. 2004*H838109*000 awarded by the Central Intelligence Agency. The Government has certain rights in the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 61/058,882, filed June 4, 2008, which is incorporated herein by reference in its entirety. Priority in the U.S. is claimed under 35 U.S.C. § 119(e).

TECHNICAL FIELD

[0003] The present invention relates generally to devices for use in diagnostic assays, and more particularly to a cartridge used for carrying out biochemical assays, such as immunoassays and amplification assays.

BACKGROUND OF THE INVENTION

[0004] The ability to quantitatively measure a wide variety of physiologically active compounds is important as an adjunct to diagnosis and therapy. The medical industry has become increasingly dependent on the ability to carry out diagnostic assays that measure various physiological parameters in a biological sample, such as for example, antigen levels, antibody levels, protein and/or peptide levels, viral load, bacterial infection, genomic sequences, and the presence of legal and/or illegal active agents in an individual's system. Examples of biological samples that are required in order to run the diagnostic assays include without limitation, blood, blood plasma, epidermal cells, mucosal cells, urine, and saliva. [0005] Diagnostic assays of biological samples have traditionally been performed in sophisticated laboratories that have a substantial investment in equipment and human resources. Recently, there has been an increasing focus on being able to carry out diagnostic assays in smaller and/or less sophisticated laboratories that do not require the degree of investment required of the larger laboratories. [0006] Co-owned Patent Publication Nos. US 2006/0165558 and WO 2006/069328 to Witty and Castanon disclose a cartridge for conducting diagnostic assays that contains biological fluids within the cartridge in a sealed manner. The cartridge is configured to carry out a biological assay on the fluid as it flows through the cartridge. Data on the fluids is transmitted to a host computer that is capable of transmitting the information to an off-site laboratory or hospital clinic. Co-owned Patent Application Nos. US 11/933,900 and PCT/US2007/083381 to Witty, Wobken, and Dreismann disclose an improved cartridge that is capable of performing at least two biochemical assays on a single biological sample, such as for example a molecular assay and an immunoassay. Both the single assay and the dual assay cartridges use pumps and valves to push the fluid through the cartridge and chambers to store reagents for the assay procedures.

[0007] In order for smaller and/or less sophisticated laboratories to perform the diagnostic assays required by the medical industry, there is a need in the art for cost effective equipment that may be used to carry out single or multiple diagnostic assays on biological samples quickly and efficiently and in such a way that the technician conducting the assays is not exposed to biohazards that accompany the handling of biological samples.

SUMMARY OF THE INVENTION

[0008] The present invention satisfies the need in the art by providing a molecular cartridge equipped with deformable chambers that serve as both reagent chambers and bidirectional pumps to drive the fluids through the cartridge.

[0009] In one aspect of the invention, there is provided a cartridge for conducting biochemical assays comprising multiple deformable chambers, wherein each of the multiple chambers are connected to each other via fluid channels.

[0010] In another aspect of the invention, there is provided a cartridge for conducting molecular assays comprising multiple deformable chambers, wherein fluid is capable of passing directly back and forth between at least two of the deformable chambers. [0011] In a further aspect of the invention, there is provided a cartridge for conducting molecular assay comprising multiple deformable chambers, wherein the chambers serve as reagent chambers and bidirectional pumps to drive fluids through the cartridge. [0012] In one embodiment of the invention, the multiple deformable chambers are used to conduct at least one biochemical assay within the cartridge. [0013] In another embodiment of the invention, the biochemical assays are selected from the group consisting of immunoassays, molecular diagnostic assays, molecular binding assays, protein binding assays, electrolyte assays, coagulation assays, routine chemistry assays, and hematology assays.

[0014] In a further embodiment of the invention, the molecular diagnostic assay is an amplification assay.

[0015] In another embodiment of the invention, the amplification assay is selected from real time PCR and real time RT-PCR.

[0016] In a further embodiment of the invention, the real time PCR and RT-PCR assays are multiplex assays.

[0017] In another embodiment of the invention, the real time PCR and real time RT-PCR assays are followed with a melting curve analysis.

[0018] In a further embodiment of the invention, the thermocycling for the real time PCR and real time RT-PCR assays is carried out in at least two deformable chambers. [0019] In another embodiment of the invention, at least one of the two deformable chambers is a measurement chamber for quantification of PCR product and temperature measurement for the melting curve analysis.

[0020] In still another embodiment of the invention, the cartridge further comprising a instrument that is configured to accept the cartridge, wherein the instrument is coupled or integrated to a host computer.

[0021] In a further embodiment of the invention, the instrument is configured with at least one actuator to assist in carrying out the biochemical assays.

[0022] In still another embodiment of the invention, the instrument is configured with at least one magnet to assist in carrying out the biochemical assays.

[0023] In a further embodiment of the invention, the deformable chambers are comprised of a thin expandable material selected from the group consisting of cellulose polymers, silicon polymers, polymeric films, phenolic resins, polypropylenes, liquid crystalline polymers, and cyclic olefin polymers.

[0024] The cellulose polymers may be selected from the group consisting of nitrocellulose, cellulose acetate, and hydroxypropylcellulose.

[0025] The silicon polymers may be selected from polydimethylsiloxane (PDMS) and iron- poly dimethylsiloxane (Fe-PDMS). [0026] The polymeric films may be selected from nylon, polyvinyl chloride (PVC), polystyrene, polycacrylonitrile, polyvinyl butyral, and polyetherimide.

[0027] The thin expandable materials will typically have a thickness in the range of 2-5 microns.

[0028] In another embodiment of the invention, the biological assays are conducted on biological samples that are maintained in the cartridge in a fully sealed manner.

[0029] In a further embodiment of the invention, the cartridge is disposable.

[0030] Additional aspects, advantages and features of the invention will be set forth, in part, in the description that follows, and, in part, will become apparent to those skilled in the art upon examination of the following, or may be learned by practice of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0031] FIG. 1 is a schematic representation of the front or the top of a two assay cartridge of the present invention designed to run an immunoassay and amplification assay.

[0032] FIG. 2 is a schematic representation of the back or the bottom of the two assay cartridge of FIG. 1.

[0033] FIG. 3 is a schematic representation of a single assay molecular cartridge of the present invention designed to run an amplification assay.

[0034] FIG. 4 is a schematic representation of an actuator of the instrument of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

[0035] Set forth below is a description of what are currently believed to be the preferred embodiments and best examples of the claimed invention. Any alternates or modifications in function, purpose, or structure are intended to be covered by the claims of this application. [0036] In describing and claiming the present invention, the following terminology the following definitions are used for the purpose of describing particular embodiments only, and is not intended to be limiting. As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0037] As used herein, the term "deformable chambers" is meant to refer to the collapsible chambers of the cartridge of the present invention. The deformable chambers are comprised in part by the non-deformable material of the cartridge and in part by flexible material (see, FIG. 4). The deformable chambers may have various purposes, for example, they may house reagents used for initiating biochemical reagents or they may house washing buffers or lysis buffers or they may act as mixing chambers, degassing chambers, waste chambers, incubation chambers, or reconstitution chambers. The deformable chambers may have any suitable shape, for example, they may be spherical or dome shaped. [0038] The thin membrane of the deformable chambers may be made of any suitable flexible material. For example, the membrane may be comprised of a thin expandable material selected from the group consisting of cellulose polymers, silicon polymers, polymeric films, phenolic resins, polypropylenes, liquid crystalline polymers, and cyclic olefin polymers. Examples of cellulose polymers are nitrocellulose, cellulose acetate, and hydroxypropylcellulose. Examples of silicon polymers are polydimethylsiloxane (PDMS) and iron-poly dimethylsiloxane (Fe-PDMS). Examples of polymeric films are nylon, polyvinyl chloride (PVC), polystyrene, polycacrylonitrile, polyvinyl butyral, and polyetherimide. One example of a polypropylene material that may be used to manufacture the deformable chambers include without limitation textured polypropylene. Examples of textured polypropylene materials include without limitation woven polypropylene, metallized polypropylene, or air jet textured polypropylene. The thin expandable materials will typically have a thickness in the range of 2-5 microns.

[0039] The body of the cartridge itself is comprised of a non-expandable polymer material, such as for example, polypropylene, polystyrene, and polymethylacyrlate (PMA). The body of the cartridge will typically have a thickness in the range of 1-4 mm. [0040] The flexible materials will be connected to the cartridge using methods known in the art, such as for example, laser welding, sonic welding, and thermal resistance heating. [0041] As used herein, the term "agitation" in the context of the description of the deformable chambers is used to refer to the procedure by which air from an inflated deformable chamber is forcefully expelled into a sample/reagent chamber to agitate the contents therein.

[0042] As used herein, the term "mixing" is the context of the description of the deformable chambers is used to refer to the two-way pump procedure, wherein the contents of a sample/reagent chamber is transferred to a deflated empty deformable chamber. [0043] As used herein, the term "motivation force" is meant to refer to any force that may inflate the deformable chambers of the present invention. Suitable motivation forces for use with the present invention include without limitation, air, liquids, and/or mechanical forces, such as for example, pumps. Examples of motivation force pumps include without limitations, mechanical pumps, pressure pumps, vacuum pumps, centrifugal pumps, air operated pumps, and fluid pumps.

[0044] As used herein, the term "instrument" is meant to refer to a device that is suitable for docking the cartridge and housing the actuators, magnets, sensing means, thermal control means, and other equipment required to interact with the cartridge and to run the biochemical assays of the invention. The instrument will be coupled and/or integrated with a host computer, which will have an interface for user operation.

[0045] The cartridge of the present invention is a self-contained disposable unit that is designed to facilitate the running of biochemical assays in a completely sealed manner.

Biochemical assays that may be performed with the cartridge of the present invention include without limitation, immunoassays, molecular diagnostic assays, molecular binding assays, protein binding assays, electrolyte assays, coagulation assays, routine chemistry assays, and hematology assays.

[0046] Upon completion of the assays, the cartridge is intended to be disposed of in an appropriate biohazard disposal waste container. At no time during the running of the assays with the cartridge of the present invention is the operator exposed to the sample or to the reagents used to run the sample or to the products of the assays or any materials that come in contact with any of the above, e.g., fluids or gases etc.

[0047] The cartridge of the present invention is equipped with fluid channels that direct the fluid through the deformable chambers where the individual steps of the assay process take place. Entry and exit of the fluid from the deformable chambers into the fluid channels is regulated via activatable valves that are adjacently positioned to each of the deformable chambers.

[0048] In order to run the assays, the deformable chambers will be pre -treated with reagents required to run appropriate assays. The reagents may be incorporated into the deformable chambers in liquid form or they may be dry (e.g., by vacuum lyophilization or heat drying).

[0049] In its initial state, the deformable chambers of the cartridge are collapsed (i.e., in inverted form) and all of the valves are closed. When the cartridge is ready for use, a sample is placed into the cartridge, the valves to appropriate chambers are opened, and the motivation force is applied to the cartridge. Where the motivation force is a displacement vacuum pump, the vacuum will simultaneously pull the sample into a fluid channel of the cartridge while inflating each of the deformable chambers having opened valves. As the assay progresses, the sample will make its way through the fluid channels to appropriate deformable chambers where the steps of the assays will take place. Those of skill in the art will appreciate that the amount of feree (i.e., pressure) required to push the fluids within the cartridge will be pre-determined prior to the running of the assay based upon the motivation force that will be used; thus, for example, the amount of feree used to move the fluid through the cartridge when a vacuum pump is the motivation force may be different from the amount of force used to move the fluid through the cartridge when a fluid pump is the motivation force.

[0050] It is to be appreciated that the where the reagents are liquid, the liquid reagent chambers will need to be inflated so that the contents can be used to run the assays. Where the reagents are dry, the reagent chambers will remain deflated while the reconstitution chambers will need to be inflated so that the buffers in the reconstitution chambers may reconstitute the reagents in the lyophilized reagent chambers.

[0051] To conduct the assays, the cartridge of the present invention has the capacity to use empty chambers to agitate and/or mix the contents of full chambers. The empty chambers may be used as waste chambers to collect used reagents and/or sample. [0052] In one embodiment of the invention, the deformable chambers may be used as agitation chambers by expelling air into reagent chambers to agitate the contents therein. In this embodiment, an empty chamber is filled with air either upon initiation of the motivation force to pull the sample into the cartridge or at a later stage during the assay procedure. Where the motivation force is applied after initiation of the sample into the cartridge, it is important that the valves to any chambers not requiring additional pressure are closed. To implement the agitation, valves to both the empty chamber (filled with air) and the sample/reagent chamber are opened causing the air to be forced into the chamber housing the sample and reagent thus agitating the contents of the chamber housing the sample and reagent mixture. In this embodiment, it is important that the chamber housing the sample/reagent is capable of withstanding the increased pressure being expelled from the inflated empty chamber. The air from the empty chamber may be forced out of the empty chamber by applying pressure from an actuator to the empty chamber to force the air out of it (see, FIG.

4).

[0053] In another embodiment of the invention, the deformable chambers operate as bidirectional or two-way pumps by passing fluid (such as for example a mixed sample and reagent) back and forth between two or more chambers. In this embodiment, an empty chamber adjacent to a reagent chamber will be used as a mixing chamber. To facilitate the mixing process, the empty chamber will remain deflated upon initiation of the motivation force to the cartridge to pull the sample. During the course of the assay, the fluids will be pushed into and out of the empty and full adjacent deformable chambers by repeatedly changing the pressure differential within the chambers and their fluid channels. For example, a positive pressure differential (increased pressure within the deformable chamber versus the fluid channel) pushes the air out of the deformable chambers (and thereby collapsing the chamber) while a negative pressure differential (decreased pressure within the deformable chamber versus the fluid channel) pushes air into the deformable chamber (thereby increasing the volume of the chamber). In operation, the valves to the sample/reagent and empty chambers are simultaneously opened causing the pressure and contents from the sample/reagent chamber to be transferred to the empty chamber. The contents may be moved back and forth between the two chambers as many times as required by the assay procedure. It will be appreciated by those of skill in the art that the reconstitution of the lyophilized reagents will use this mixing technique. The contents of each of the chambers may be forced out by applying pressure from an actuator to the filled chamber to force out the contents. [0054] It is to be understood that the cartridge of the present invention is designed to facilitate the configuration of fluid passageways and deformable chambers that will maximize the efficiency and accuracy of the biochemical assays that are to be run in any particular cartridge.

[0055] The cartridge of the present invention is intended to be placed into a instrument equipped with a source for the motivation force as well as detection means for processing data from the assays. Detection means that may be found within the instrument may include without limitation means for detecting any of the following in combination or alone: fluorescence, absorbance, luminescence, electrochemical changes, and magnetic pull. For example, the instrument may be equipped with one or more photo-optic arrays, which may be used to detect optical changes in the sample as it progresses through the assay process. In one embodiment of the invention, the instrument is further equipped with one or more actuators that facilitate the emptying of the inflated deformable chambers (see, FIG. 4). The instrument of the present invention is in turn coupled to or integrated with a host computer (not shown) equipped with a user interface. [0056] Examples of molecular assays that may be run with the cartridge of the present invention include without limitation amplification assays. Examples of amplification assays that can be run with the cartridge of the present invention include without limitation: thermocycling amplification reactions, such as polymerase chain reaction (PCR) and isothermal amplification reactions such as linear isothermal amplification, exponential isothermal amplification (EXPAR), ligase chain reaction (LCR), loop-mediated amplification (LAMP), strand displacement amplification (SDA), and helicase dependent amplification (HDA).

[0057] Depending on the type of thermal cycling reaction that is to be run, the cartridge may be configured with one or more deformable chambers. Three or four chamber PCR assays that can be performed with the cartridge of the present invention include traditional PCR (with DNA as the starting material), reverse transcriptase PCR (RT-PCR) (with RNA as the starting material), real time PCR (PCR amplification and simultaneous quantification in real time with DNA as the starting material), real time RT-PCR (with RNA as the starting material).

[0058] With respect to amplification assays, the cartridge and instrument of the present invention are further equipped to run a melting curve analysis. It is the inventors' belief that the incorporation of melting curve processing in a cartridge/instrument configuration has never been done prior to the present invention. As is known to those of skill in the art, melting curve analysis is a method for analyzing PCR products by slowing heating the PCR product. Each double stranded DNA has its own specific melting temperature (Tm), which is the temperature at which 50% of the DNA become single stranded. By measuring the Tm, the DNA can be identified. For a three chamber real time PCR assay with three temperatures, a denaturing temperature of 95°C, an annealing temperature of 56°C, and an extension temperature of 76°C, each amplification step is preferably measured in the extension temperature chamber. Co-owned U.S. Patent Nos. 7,189,573; 7, 192,777; and 7,217,393 to Witty and Case disclose ways to monitor changes within a measurement chamber using optical means. After the reaction is complete, the PCR product in the extension temperature well will be heated slowly to run the melting curve analysis. Examples 1-3 show the use of the cartridge of the present invention to run melting curve analysis after real time PCR assays.

[0059] To conduct a melting curve analysis, the amplification assay must include a saturating DNA binding dye (preferably a fluorescent dye) that does not interfere with amplification at high concentrations. Melting curve raw data is generally represented by plotting fluorescence over temperature. Melting curve analyses have significant utility in the identification of a particular gene (Example 1) or a single nucleotide polymorphisms (SNPs) (Example 2). Melting curve analysis exploits the fact that even a single mismatch between the labeled probe and the amplicon will significantly reduce the Tm; thus, probe/amplicon heteroduplexes containing destabilized mismatches, such as SNPs melt off at lower temperatures than probes bound to a perfectly matched wild type target DNA. Using melting curve analyses, data derived from homozygous wild types, heterozygous mutants, and heterozygous samples can be identified on a melting curve by comparing peak number, peak position, or a combination of both. The ability of the cartridge to conduct a melting curve analysis permits the cartridge to be an effective tool to conduct multiplex assays (see, Example 3).

[0060] FIGS. 1 and 2, which show a cartridge 10 that is configured to perform two assays: an immunoassay and a thermocycling amplification assay. FIG. 3 shows a cartridge that is configured to perform a single linear amplification assay. It is to be understood that the cartridges shown in FIGS. 1-3 are merely illustrative of an exemplary use of the cartridge of the present invention and is not meant to be limiting with respect to the number and/or the types of biochemical assays that the cartridge of the present invention may perform. One of skill in the art will appreciate that the cartridge of FIG. 3, may be refined to include a purification step prior to the linear amplification. Where the cartridge is being used to conduct two assays, upon entry of the sample into the cartridge, the sample will be split such that a portion of the sample will go into fluid channels that will conduct a first assay and a portion of the sample will go into fluid channels that will conduct a second assay. For example, in FIG. 1, a portion of the sample will be used to run the molecular assay and another portion of the sample will be used to run the molecular amplification assay. [0061] Operation of the cartridge of the present invention will be described with reference to FIGS. 1 and 2. With reference to FIG. 1, the cartridge 10 includes an input port 11 having an internal cavity that is sized to removably receive a sample tube (not shown), such as a venipuncture tube. It is to be understood that the cartridge of the present invention is not limited to use with a sample tube, but rather, it may be configured to accept sample directly from a primary collection tube using a device such as for example, a pipette. [0062] As noted above, in its initial state, the deformable chambers of the cartridge of the present invention are in collapsed form. In order to prepare the cartridge 10 for the immuno and molecular assays, the cartridge 10 with the sample tube in place in the input port 11 is placed into the instrument (not shown), which is equipped with a motivation force for simultaneously pulling the sample into the cartridge and for inflating the deformable chambers, such as for example, a positive displacement pump.

[0063] Upon entry of the cartridge into the instrument, the vacuum pump within the instrument makes contact with the port 12 on the cartridge, which is configured with an activatable valve 13 (all valves in FIGS. 1-3 are shown as small round dots), which may be mechanically or manually opened and closed via means for doing so on the instrument. Upon initiation of the motivation force, the port valve 13 is opened as are the valves for the deformable chambers that will be used conduct the steps of the immunoassay and molecular assays. As indicate above, the valves to any liquid-containing chambers should be opened at this time. The result of the simultaneous opening of the valves is that the vacuum pump will inflate the accessible deformable chambers while at the same time pulling sample from the sample tube into fluid channels (not shown) that lead toward the accessible (and inflated) deformable chambers.

[0064] In one embodiment of the invention, as noted above, the instrument may be equipped with one or multiple actuators that assist and/or facilitate the emptying of the contents of the deformable chambers by applying appropriate pressure to the membrane of the deformable chambers such that the contents of the deformable chambers are expelled at a desired rate (see, FIG. 4).

[0065] In another embodiment of the invention, the deformable chambers of the cartridge 10 of the present invention are equipped with a single valve such that the fluid enters and leaves the chambers from a single channel. In another embodiment of the invention, the deformable chambers of the cartridge 10 are equipped with two valves, i.e., a fluid entry valve and a fluid exit valve (see, valves 15/16 associated with deformable chamber 14). [0066] As discussed above, to conduct the assays, the deformable chambers of the cartridge 10 will contain appropriate reagents to run the respective assays. [0067] For the immunoassay, upon initiation of the motivation force, a portion of the sample enters into fluid channels towards the inflated immunoassay incubation chamber 14, which has its entry valve opened 15 (the exit valve 16 is closed). Once the fluid has fully entered into the immunoassay incubation chamber 14, the entry valve 15 closes so that the antigens in the fluid may react with the label housed within the immunoassay incubation chamber 14. The fluid exit valve 16 of the immunoassay incubation chamber 14 remains closed while the labeling proceeds such that the immunoassay incubation chamber 14 is completely sealed from the remainder of the channels and chambers within the cartridge 10. In another embodiment of the invention, which is not shown, the cartridge may include an additional empty agitation and/or mixing chamber to facilitate the agitation and/or mixing of the sample and label in the immunoassay incubation chamber. Upon completion of the labeling process, the exit valve 16 to the immunoassay incubation chamber 14 is opened thus releasing the contents of the immunoassay incubation chamber 14 (i.e., the labeled sample and the pressure) directly into the immunoassay capture zone 17, where the labeled antigen will react with bound antibody. Suitable materials for use in the immunoassay capture zone are microporous materials, one example being nitrocellulose.

[0068] In order to remove unbound labeled antigen from the capture zone 17, the contents of the immunoassay wash chamber 18 will be washed over the immunoassay capture zone. The capture zone 17 is equipped with inmobilized reactants for detection by the sensing means of the instrument (not shown) that will record the fluorescence of the antigen-antibody conjugates and transmit the data to the host computer, which is coupled to the instrument that houses the cartridge.

[0069] For the amplification assay, upon imitation of the motivation force, a portion of the sample enters into fluid channels towards the inflated lysis chamber 20 via the opened lysis chamber valve 21, which is closed upon entry of the sample into the deformable chamber. Once within the lysis chamber 20, lysis chamber valve 21 is closed and the sample is allowed to react with the lysis buffer to release DNA from the sample. Examples of lysis buffers are well known to those of skill in the art. Examples of lysis buffers include without limitation sodium dodecyl sulfate (SDS) and guanidinium hydrochloride (GndHCl). Mixing of the contents of the lysis buffer chamber may be carried out by agitation and/or mixing of the contents with empty chamber 22 (and associated valve 23) as previously described. If desired, the empty chamber 22 may be used first for agitation and subsequently for mixing. [0070] Upon completion of the lysis reaction, the lysed DNA sample is released into the upper capture chamber 24, which is a deformable chamber located within the cartridge and shown in FIG. 2. The upper capture chamber 24 houses functionalized or non-functionalized particles, which will bind to nucleic acids in the lysed sample. The contents of the upper capture chamber may be mixed by once again using the empty chamber 22 to transfer the contents between the two deformable chambers 22, 24. [0071] Once the nucleic acids have bound to the particles, wash buffer from the first wash chamber 25 is released into the upper capture chamber 22 via the first wash chamber valve 26. Used wash buffer may be released from the upper capture chamber 22 to a waste chamber (not shown) or alternatively, the used wash buffer may be released back into the first wash buffer chamber 25.

[0072] Where the particles are functionalized paramagnetic particles, in order to ensure that the particles stay within the chamber after the washing step and do not leave the chamber along with the used wash buffer, the instrument (not shown) may be equipped with a magnet, which is positioned above the appropriate wash chamber and which when activated holds the particles in place while the used wash buffer is moved from the upper capture chamber 22 to the first wash chamber 25. The instrument may be equipped with as many magnets as necessary in order to facilitate the multiple washings of the particles, which will be described in more detail below.

[0073] Where the particles are not paramagnetic, other means may be used to keep the particles in place, such as for example, a filter and/or openings between the chambers that are proportioned to prevent passage of the particles.

[0074] The bound sample in the upper capture chamber 22 may be washed multiple times by releasing wash buffer from additional wash chambers. As is known to those of skill in the art, thorough washing of a sample can frequently reduce background noise that may interfere with the analytical reading of a sample. In FIG. 1, the repeat washing is carried out with additional buffer from a second wash buffer chamber 27 via the associated second wash chamber valve 28. An advantage of the two chamber 24, 29 system described herein relates to the cleansing of the particles prior to the amplification step of the molecular assay. Mixing can be carried out between the two chambers 24, 27 as previously discussed. Additional wash chambers with associated valves may be added to the cartridge 10 as desired. By washing the particles multiple times in the upper capture chamber, the harsh chemicals of the lysis buffer may isolated so that they do not interfere with the milder chemicals that are required to amplify the isolated nucleic acids.

[0075] Returning to FIG. 1 , upon release of the last wash buffer into the upper capture chamber 24, the wash buffer is not sent back to the second wash chamber 27 nor sent to a waste chamber; rather, it remains mixed with the bound nucleic acid and is transferred from the upper capture chamber 24 into the lower capture chamber 29 (for elution of the nucleic acid) via pressure that is applied to the upper capture chamber 22 from the instrument. The particles and buffer travel from the upper capture chamber 22 to the lower capture chamber 29 via a fluid channel 30 configured with valves 31, 32 on either end of the fluid channel. [0076] Upon entry of the particles (and wash buffer) into the lower capture chamber 29, the valve 32 to the lower capture chamber 29 is closed thus sealing the particles and wash buffer within the lower capture chamber 29. In order to elute nucleic acid from the particles, a magnetic is placed over the lower capture chamber 29 and activated to hold the particles in place while the wash buffer is removed from the lower capture chamber 29 into a waste chamber (not shown) or into an empty deformable chamber 33 equipped with its own valve 34. Prior to elution, the particles are preferably subjected to an additional wash in order to ensure that the particles are free of any harsh chemicals from the lysis step. After washing, the particles may be dried by forcing air from an empty chamber 41 (with associated valve 42) into the lower capture chamber 29. Elution buffer is then released from the elution chamber 35, which is configured with an elution chamber valve 36 and the particles are released from the magnetic hold to mix with the elution buffer within the lower capture chamber 29. Elution buffers for extracting nucleic acids from functionalized or non- functionalized particles are well-known to those of skill in the art and are readily available through commercial sources. In order to mix the contents of the lower capture chamber 29, the sample/buffer mixture may be moved back and forth between the lower capture chamber 29 and the elution chamber 35.

[0077] When the particles have been sufficiently mixed with the elution buffer to cause elution of the nucleic acid from the particles, the magnet is activated to remove the particles from the mixture so that the eluted nucleic acid may be mixed with the amplification reagents that are released from the master mix chamber 39 via the master mix chamber valve 40. Mixing of the eluted nucleic acid with the amplification mix is carried out by moving the eluted sample/master mixture between the lower capture chamber 29 and the master mix chamber 39.

[0078] As is known to those of skill in the art, the amplification mix will preferable include a label so that the progress of the amplification reaction may be monitored. In order to ensure that the mixture in the lower capture chamber 29 is completely particle free, a third magnet may be activated to remove any stray particles from the nucleic acid-amplification mixture. [0079] The instrument of the present invention may be equipped with one magnet or with multiple magnets. Where one magnetic is used, the instrument will be equipped with drivers that will allow the magnetic to change position over the appropriate section of the cartridge as is appropriate. Where more than one magnet is used, the magnets may be stationary within the instrument. As described herein, the instrument of the present invention may be equipped with as many as three separate magnets. An embodiment having a movable magnet or alternatively up to three individual magnets as described above.

[0080] After the nucleic acid has been thoroughly mixed with the amplification buffer, the mixture is ready for amplification. The mixture is released from the lower capture chamber 29 into a fluid channel 43 (equipped with two valves on either side of the channel 44, 45), which empties into the first of the thermocycling chambers. Those of skill in the art are familiar with thermocycling amplification procedures. In FIG. 1, three amplification chambers 46, 47, 48 with three different temperatures are used: a first high denaturation temperature (typically 90-95⁰C) to denature dsDNA, a second annealing temperature (typically 45-55°C) to anneal the primers in the amplification mix to the ssDNA, and a third extension temperature (typically 75-80⁰C) to initiate DNA synthesis with a thermostable DNA polymerase (typically Taq polymerase). The rate of primer extension by Taq polymerase is about 50-100 nucleotides/second; thus, the time and number of cycles required for primer extension will depends on the length of the sequence to be amplified.. Typically, thermocycling PCR assays can be completed in 20-30 cycles. In FIG. 1, the three amplification chambers are connected with fluid channels; however, it is to be understood that the amplification chambers may be designed such that the fluid flows directly from one chamber to another without the need of passing into a fluid channel.

[0081] It is to be understood that the cartridge of the present invention may be configured to house as many temperature chambers as is necessary to carry out the thermal cycling and/or isothermal reactions. For example, for isothermal reactions, only one temperature chamber may be necessary to run the amplification reaction. For some PCR and RT-PCR amplifications, three or four temperature chambers may be required in order to efficiently run the hot start, denaturation, annealing, and extension reactions required of the amplification reactions. For other reactions, it may be necessary to design a cartridge with more than four temperature chambers.

[0082] In another embodiment of the invention, the amplification chambers are connected to a trap chamber 49, which is an empty chamber that is designed to capture gases that are released during the amplification procedure. For example, in FIG. 1, the fluid channels may be comprised of a membrane that allows any gases in the fluids to escape into the trap chamber 46 during flow of the fluid between the three amplification chambers 43, 44, 45. Suitable membrane materials will typically be microporous hydrophobic membranes (MHMs), which block liquids, while allowing air to flow through the membrane. Examples of suitable MHMS include without limitation, PTFE (polytetrafluoroethylene), polypropylene, PVDF (polyvinylidene difluoride), and acrylic copolymer. [0083] As discussed above, the cartridge and instrument of the present invention are configured such that the data from the amplification reaction is used to conduct a melt curve analysis during and/or after the amplification reaction.

[0084] Where the amplification reagents include a label, detection means in the instrument may record the progress of the reaction by measuring a signal from the reaction products. For example, where the label is a fluorescent dye, the instrument will record an increased fluorescent signal each time an amplification cycle is complete. In this way, the progress of the reaction can be tracked by measuring the intensity of the signal. The cartridge of the present invention may be designed so that the amplified sample remains sealed in the cartridge and is discarded along with the disposable container or alternatively, the cartridge may be designed so that the amplified sample may be removed for further testing. [0085] It is to be understood that while the invention has been described in conjunction with the preferred specific embodiments thereof, that the foregoing description as well as the examples that follow are intended to illustrate and not limit the scope of the invention. Other aspects, advantages and modifications within the scope of the invention will be apparent to those skilled in the art to which the invention pertains.

[0086] All patents and publications mentioned herein are incorporated by reference in their entireties.

EXPERIMENTAL

[0087] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the compositions of the invention. The examples are intended as non- limiting examples of the invention.

EXAMPLE 1 GENOTYPING HLA FOR LIVING-RELATED STEM CELL TRANSPLANTATION

[0088] A phlebotomist takes blood from two siblings: an ill sibling requiring a hematopoietic stem cell transplant and a healthy sibling that is a potential stem cell donor. The blood samples are obtained via venipuncture. Upon completion of the sample collection, the phlebotomist discards the venipuncture needle in a biohazard box and hands the venipuncture tube to a laboratory worker for molecular analysis using a cartridge of the present invention.

[0089] For hematopoietic stem cell transplantation, HLA genotype identity has been shown to be critical to survival of the graft; accordingly, the laboratory worker will run a molecular test of the two samples for HLA genotypes. Identical melting curves obtained from the two samples will confirm HLA genotype identity and the healthy sibling will be a suitable stem cell donor. If the two samples have different melting curves, then the healthy sibling will not be a good donor of hematopoietic stems cells for the ill sibling.

[0090] To perform the test, the cartridges are prepared with primers and reagents required to run a real time PCR assay for HLA gene at the A locus. To run the melting curve analysis, the reagents must include a fluorescent dye. Primers and PCR conditions and reagents that may be used to run the HLA-A PCR are disclosed in Zhou et al., High-resolution DNA melting curve analysis to establish HLA genotypic identity, TISSUE ANTIGENS 64(2): 156-164, 157 (2004).

[0091] Upon completion of the PCR amplification, the PCR product is heated from 50⁰C to 100⁰C at a rate of 0.2-0.5°C/s and fluorescence and temperature measurements are taken every 25 -50ms over 2 minutes. The raw data from each sample is plotted as fluorescence v. temperature and the melting curve is calculated from normalization of the data to percent of fluorescence between linear fits of the raw fluorescence before the melting transition (the 100% line) and after the transition (the 0% line).

[0092] To insure accuracy of the results, a third sample having a 1 :1 mixture of the two siblings may be run through the PCR and melting curve analysis. If the two samples are true homozygotes, then the three melting curves of the three samples will be the same. In this same vein, if the samples are heterozygotes, then the melting curves of the three samples will be different.

EXAMPLE 2

SNP GENOTYPING FOR APOE GENES

[0093] One of the genes associated with Alzheimer's disease is apolipoprotein E (ApoE). Each individual inherits one maternal copy of ApoE and one paternal copy of ApoE. ApoE contains two SNPs that result in three possible alleles for the gene: E2, E3, and E4. Each allele differs by one DNA base and the protein product of each gene differs by one amino acid. Research has shown that an individual who inherits at least one E4 allele will have a greater chance of getting Alzheimer's. By contrast, research shows that inheriting the E2 allele indicate that an individual is less likely to develop Alzheimer's. See, The Human Genome Project Information SNP Fact Sheet at www.ornl.gov/sci/techresources/Huma^Genome/faq/snps.shtml (May 20, 2008). [0094] A patient with a long familial history of what appears to be E4-related Alzheimer's disease wants to know if he has inherited one or two ApoE4 alleles. The patient's doctor approves the patient's request and places an order for the test. Prior to the test, the lab prepares a homozygous ApoE4 control, a heterozygous ApoE4/E2 control, and a homozygous ApoE2 control by obtaining samples of human blood from Alzheimer patients that have tested positive for the three genotypes. Using a cartridge of the present invention prepared with the appropriate primers and PCR reagents for a real time PCR assay, the laboratory worker runs the PCR amplification assays and subsequent melting curve analyses for the two samples. Primers and PCR conditions and reagents that may be used to run an ApoE PCR amplification assays are disclosed in PCT Publication No. WO 1995/016791 to McGiIl University.

[0095] A sample of the patient's blood is taken via venipuncture and the venipuncture tube is placed into the entry port of a cartridge of the present invention, which has been prepared with the same primers and reagents that were used on the control sample. The laboratory worker runs the PCR reaction and melting curve analysis using the identical conditions that were used for the control sample.

[0096] A melting curve that is identical to the melting curve of the homozygous ApoE4 control or the homozygous ApoE2 control will mean that the patient is carrying two ApoE4 alleles or two ApoE4 alleles, respectively. Similarly, a melting curve that is identical to the heterozygous ApoE4/E2 control will mean that the patient is carrying the ApoE4 gene on one allele and the ApoE2 gene on the other allele. If the melting curve of the patient is different from all three of the samples, then further SNP genotyping may be necessary to determine which of the other known ApoE SNP genotypes the patient is carrying. EXAMPLE 3 MULTIPLEX ASSAY FOR IDENTIFYING RSV, INFLUENZA A, AND INFLUENZA B

IN A SINGLE SAMPLE

[0097] A patient with a viral infection is drawn by a phlebotomist into a venipuncture tube, which is transferred to a laboratory where a real time PCR multiplex assay will be conducted on the blood for RSV (respiratory syncytial virus, Influenza A, and Influenza B using a cartridge of the present invention.

[0098] Controls for each of the three infections are prepared by obtaining control samples from a commercial source and amplifying the control samples by running them through a 30 cycle real time PCR using the cartridge of the present invention and running a melting curve analysis on the PCR product.

[0100] To run the multiplex assay, the cartridge is prepared with primers for all three of the viral species in the amplification reagent chamber, which further includes a fluorescent dye and additional reagents to run the real time PCR assay. Primers and reagents used to run the real time PCR assay are all obtained from commercial sources. The real time PCR assay will be a 30 cycle thermocycling reaction carried out in three deformable chambers having the following temperatures: 95°C for denaturation, 56°C for annealing, and 76°C for extension. The quantification of the viruses will be carried out via measurement from the 76°C deformable chamber. The melting curve analysis will be carried out by slowing heating the sample from the 76° C measurement chamber after completion of the amplification assay. The melting curve of the sample will be compared against the melting curves of the controls to determine which virus has infected the patient. Melting curve analysis to identify which of the viral species is in the sample.

Claims

WE CLAIM:

1. A cartridge for conducting biochemical assays comprising multiple deformable chambers, wherein each of the multiple chambers are connected to each other via fluid channels.

2. A cartridge for conducting molecular assays comprising multiple deformable chambers, wherein fluid is capable of passing directly back and forth between at least two of the deformable chambers.

3. A cartridge for conducting molecular assay comprising multiple deformable chambers, wherein the chambers serve as reagent chambers and bidirectional pumps to drive fluids through the cartridge.

4. The cartridge of claims 1, 2, or 3, wherein the multiple deformable chambers are used to conduct at least one biochemical assay within the cartridge.

5. The cartridge of claims 4, wherein the biochemical assays are selected from the group consisting of immunoassays, molecular diagnostic assays, molecular binding assays, protein binding assays, electrolyte assays, coagulation assays, routine chemistry assays, and hematology assays.

6. The cartridge of claim 4, wherein the molecular diagnostic assay is an amplification assay.

7. The cartridge of claim 6, wherein the amplification assay is selected from real time PCR and real time RT-PCR.

8. The cartridge of claim 7, wherein the real time PCR and RT-PCR assays are multiplex assays.

9. The cartridge of claim 8, wherein the real time PCR and real time RT-PCR assays are followed with a melting curve analysis.

10. The cartridge of claims 7 and 9, wherein thermocycling for the real time PCR and real time RT-PCR assays is carried out in at least two deformable chambers.

11. The cartridge of claim 10, wherein at least one of the two deformable chambers is a measurement chamber for quantification of PCR product and temperature measurement for the melting curve analysis.

12. The cartridge of claims 1, 2, or 3, further comprising a instrument that is configured to accept the cartridge, wherein the instrument is coupled or integrated to a host computer.

13. The cartridge of claim 12, wherein the instrument is configured with at least one actuator to assist in carrying out the biochemical assays.

14. The cartridge of claim 12, wherein the instrument is configured with at least one magnet to assist in carrying out the biochemical assays.

15. The cartridge of claims 1, 2, or 3, wherein the deformable chambers are comprised of a thin expandable material selected from the group consisting of cellulose polymers, silicon polymers, polymeric films, phenolic resins, polypropylenes, liquid crystalline polymers, and cyclic olefin polymers.

16. The cartridge of claim 15, wherein the cellulose polymers are selected from the group consisting of nitrocellulose, cellulose acetate, and hydroxypropylcellulose.

17. The cartridge of claim 15, wherein the silicon polymers are selected from polydimethylsiloxane (PDMS) and iron-polydimethylsiloxane (Fe-PDMS).

18. The cartridge of claim 15, wherein the polymeric films are selected from nylon, polyvinyl chloride (PVC), polystyrene, polycacrylonitrile, polyvinyl butyral, and polyetherimide.

19. The cartridge of claim 15, wherein the thin expandable materials have a thickness in the range of 2-5 microns.

20. The cartridge of claims 1, 2, or 3, wherein the biological assays are conducted on biological samples, wherein the biological samples, reagents, and assay products are maintained in the cartridge in a fully sealed manner.

21. The cartridge of claims 1, 2, or 3, wherein the cartridge is disposable.