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WO2008000060A1 - Procédés microfluidiques pour le monitorage de l'acide nucléique - Google Patents

Procédés microfluidiques pour le monitorage de l'acide nucléique Download PDF

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Publication number
WO2008000060A1
WO2008000060A1 PCT/CA2007/000959 CA2007000959W WO2008000060A1 WO 2008000060 A1 WO2008000060 A1 WO 2008000060A1 CA 2007000959 W CA2007000959 W CA 2007000959W WO 2008000060 A1 WO2008000060 A1 WO 2008000060A1
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Prior art keywords
nucleic acid
clinical sample
pcr
chip
microfluidic
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Ceased
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PCT/CA2007/000959
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English (en)
Inventor
Linda Pilarski
Christopher Backhouse
Govind Kaigala
Ryan Husinks
Craig K. Sherburne
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University of Alberta
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University of Alberta
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Priority to CA002652626A priority Critical patent/CA2652626A1/fr
Publication of WO2008000060A1 publication Critical patent/WO2008000060A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/14—Process control and prevention of errors
    • B01L2200/143—Quality control, feedback systems
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/14—Process control and prevention of errors
    • B01L2200/143—Quality control, feedback systems
    • B01L2200/147—Employing temperature sensors
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0816—Cards, e.g. flat sample carriers usually with flow in two horizontal directions
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/18—Means for temperature control
    • B01L2300/1805—Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks
    • B01L2300/1827—Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks using resistive heater
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0475—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure
    • B01L2400/0481—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure squeezing of channels or chambers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/06—Valves, specific forms thereof
    • B01L2400/0633—Valves, specific forms thereof with moving parts
    • B01L2400/0655—Valves, specific forms thereof with moving parts pinch valves
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502738—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by integrated valves
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502753—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by bulk separation arrangements on lab-on-a-chip devices, e.g. for filtration or centrifugation
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L7/00—Heating or cooling apparatus; Heat insulating devices
    • B01L7/52—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples

Definitions

  • the present invention pertains to the field of viral detection and microfluidics, in particular relating to monitoring of viral load during immunosuppressive or immunomodulatory therapy.
  • BKVN BKV-associated interstitial nephritis or nephropathy
  • BK viremia is the best positive laboratory predictor of BKVN, viruria always precedes viremia, resulting in the recommendation that the detection of BK virura be used as an initial screening test.
  • urine cytology and quantitative PCR for detection of BK DNA have been used for screening.
  • Quantitative PCR to determine BKV load in urine and/or plasma has been shown to be sensitive and useful, but the need to purify DNA from urine or plasma introduces significant variability, and the cost of this test is relatively high.
  • the present art has suffered from the. inability to provide widespread monitoring of viral load in blood or urine, particularly for point-of-care use. This need is particularly relevant for patients undergoing immunomodulatory, immunosuppressive, or immunoablative therapy.
  • the present invention provides for a microfluidic device capable of providing fast, accurate and reproducible point-of-care analysis of viral presence in a clinical sample.
  • the clinical sample is selected from the group comprising urine, blood or tissue.
  • the present invention provides for a method of monitoring specific nucleic acids presence in a clinical sample. Ih another aspect, the present invention provides for method for monitoring viral nucleic acid presence in the blood or urine of a patient undergoing immunomodulatory, immunosuppressive, or immunoablative therapy.
  • the viral DNA monitored is JC virus. In a further embodiment, the viral DNA monitored is BK virus.
  • the present invention provides for an apparatus and method for detection and monitoring of JC virus in the blood or urine of a Multiple Sclerosis patient undergoing immunomodulatory, immunosuppressive, or immunoablative therapy.
  • the immunomodulatory, immunosuppressive, or immunoablative therapy is selected from the group consisting of natalizumab and rifuximab.
  • FIGURE ⁇ shows an exploded view of this tri-layer chip to illustrate the relative placement of the valves and the resistive element
  • FIGURE 2 shows a cartoon of the tri-layer microchip
  • FIGURE 3 shows representations of the side view of the tri-layer microchip comprising a top etched glass layer 301 (flow-layer), a bottom etched glass layer 303 (control layer), and a PDMS membrane 302 between these two glass layers, which is actuated (microcontroller controlled) by pressurized air or vacuum;
  • FIGURE 4 shows an electropherogram of an on-microchip PCR analyzed using the dual-layer microchip using two different sieving matrices (a) non-denaturing medium of GeneScan® polymer (5GS 10G) and (b) a denaturing POP6 sieving matrix;
  • FIGURE 5 shows quantitation of BKV on-microchip on the basis of serial dilution (a) concentration in sample: 1.78xlO 7 copies/ml (b) concentration in sample: 1.83x10 copies/ml;
  • FIGURE 6 shows quantitation of BKV on-microchip based on PCR cycle number
  • FIGURE 7 shows detection of BKV on an integrated microchip
  • FIGURE 8 shows on-microchip detection outcomes of varying concentration of viral loads
  • FIGURE 10 shows the section of the Immunoglobulin-H gene, along with the appropriate location of the primers
  • FIGURE 11 shows the single-step microchip RT-PCR integration demonstrating the capability of the present invention to detect transcripts using the ⁇ 2 microglobulin ( ⁇ 2M) gene with primers designed to amplify a 243 bp fragment from total RNA from MM+ KMS-34 cell line: (a) On- chip run (without size standard), (b) On-chip CE run (with size standard), (c) Conventional thermal cycler positive control;
  • ⁇ 2M microglobulin
  • FIGURE 12 shows on-microchip single step RT-PCR performed to detect a region specific the CDR2/CDR3 of the Immunoglobulin VDJ for the MM positive patient Pt 1 , and resulting in a 177 bp PCR product: (a) On-chip run (without size standard), (b) On-chip CE run (with size standard), (c) Conventional thermal cycler positive control;
  • FIGURE 13 shows on-microchip single step RT-PCR performed to detect a region specific the CDR2/CDR3 of the Immunoglobulin VDJ for the MM positive patient Pt 2, and resulting in a
  • FIGURE 14 shows the microchip single-step RT-PCR detection to identify Norovirus starting with RNA isolated from the Norovirus positive patient, Pt-3: (a) On-chip run (without size standard), (b) On-chip CE run (with size standard), (c) Conventional thermal cycler positive control;
  • FIGURE 15 shows the microchip single-step RT-PCR detection to identify Norovirus starting with RNA isolated from the Norovirus positive patient, Pt-4: (a) On-chip run (without size standard), (b) On-chip CE run (with size standard), (c) Conventional thermal cycler positive control;
  • FIGURE 16 shows the results of the traditionally used three-stage PCR (denaturation, annealing and extension) reduced to a two-stage PCR (with annealing and extension combined), using isolated RNA from the KMS-34 cell lines: (a) On-chip run (without size standard), (b) On-chip CE run (with size standard), (c) Conventional thermal cycler positive control; and
  • immunomodulatory therapy means the therapeutic administration of a compound or compounds which result in the response of the immune system to an immune challenge being different following therapy as compared to prior to therapy.
  • immunomodulatory therapy includes, but is not limited to administration of a compound or compounds which: effect a switch from Tl to T2 immune response, effect a switch from T2 to Tl immune response, alter immune cell trafficking within the body, sequester immune cells to a
  • immunosuppressive therapy means the therapeutic administration of a compound or compounds which result in the decreased ability of the immune system respond to an immune challenge. It is contemplated that an immunosuppressive therapy will also be an immunomodulatory therapy.
  • immunoablative therapy means the therapeutic administration of a compound or compounds which result in the removal, destruction, death or permanent inactivation of immune cells, a subset of immune cells, immune effector cells and/or their precursor cells.
  • immunoablative therapy includes, but is not limited to, chemotherapy or radiation therapy which results in the death of immune cells and/or their precursors and administration of antibodies, humanized or otherwise, which results in the removal, destruction, death or permanent inactivation of immune cells and/or their precursor cells. It is contemplated that an immunoablative therapy will also be an immunomodulatory therapy,
  • viral load means the presence of viral nucleic acid in a sample, as determined on a quantitative, semi-quantitative or qualitative basis.
  • the term "clinical sample” means a fluid or tissue originating from a human.
  • the sample may either be unmodified, or alternatively the sample may be processed before introduction into the devices of the present invention. Processing is contemplate to include, but not be limited to, pH alteration, ion removal, ion addition, cell separation, cell purification, cell removal, protein removal or cell lysis; all of which give rise to a sample for analysis which would enrich the sample for the nucleic acid of interest, if present.
  • immunomodulatory, immunosuppressive or immunoablative therapies are established for the treatment of autoimmune diseases.
  • a number of therapies result in a deleterious alteration of the immune system, either intentionally or as an ancillary effect of the therapy.
  • One skilled in the art would be capable of identifying those therapies which result in a modification of the immune system of a patient in which the immune system is compromised or otherwise
  • immunomodulatory, immunosuppressive or immunoablative therapies are relevant include but are not limited to aplastic anemia, ulcerative colitis, cancer, systemic lupus erythematosus, multiple sclerosis (MS), rheumatoid arthritis, myasthenia gravis and Crohn's disease.
  • Immunosuppressive therapy usually accompanies organ transplants, including bone marrow and blood, and is frequently accompanied by increased viral loads for multiple viruses during, for example, acute or chronic rejection phases.
  • immunomodulatory, immunosuppressive or immunoablative therapies include, but are not limited to, cyclophosphamide, methotrexate, azathioprine, mercaptopurine, dactinomycin, mitomycin C, bleomycin, mitramycin, antilymphocyte antibodies, antithymocyte antibodies, nataluzimab, rituximab, anti-CD25(IL-2) antibodies, anti-CD3 antibodies, basiliximab, daclizumab, tacrolimus, cyclosporine, sirolimus, Interferons such as interferon-beta, Tumor Necrosis Factor binding agents such as infliximab or etanercept or adalimumab, mycophenolate, FTY720 and glatiramer acetate.
  • MS Multiple Sclerosis
  • an immunomodulatory therapy is the use of the MS therapeutic nataluzimab, which is a monoclonal antibody thought to block the migration of leukocytes to the site of inflammation and demyelination which gives rise to the observed symptoms of MS.
  • JC virus is acquired in childhood and normally remains dormant in the bone marrow, kidney epithelia and spleen. The virus is spread to the central nervous system during a bout of viremia which occurs in the immunocompromised patient (Langer-Gould, A. et al N EnglJMed 353:375 (2005)).
  • a JC virus viremia during an immunomodulatory, immunosuppressive or immunoablative therapy in this case treatment with natalizumab alone or in association with an additional immunomodulatory therapeutic such as interferon Beta- l ⁇ , will allow a treating physician to identify a patient at increased risk of PML, The treating physician may then choose to discontinue the immunomodulatory therapy; increase vigilance for such diseases including, but not limited to, PML; or administer prophylactic antiviral therapy such as Highly Active Antiviral Therapy (HAART) which has shown efficacy in treating of PML in the past (Garcia, D et al JCUn Microbiol 31:124 (1999)).
  • HAART Highly Active Antiviral Therapy
  • nucleic acid into the blood or urine for example, through lytic events in JC virus infected cells
  • lytic events in JC virus infected cells which can be used to quickly identify, predict or corroborate potential PML events.
  • the hypothesized independence of JC virus viremia or viruria from disease presentation in patients undergoing immunomodulatory, immunosuppressive or immunoablative therapy does not diminish the utility associated with the monitoring of viral load in patients.
  • the monitoring of viral load on a frequent and ongoing basis during the administration of the immunomodulatory, immunoablative or immunosuppressive therapy and following the end of the therapy is further needed while the immune system is modified or otherwise compromised; this provides the medical practitioner with valuable insight into the state of the patient and the potential disease risks.
  • the presence and/or quantity of viral nucleic acid, and therefore the viral load is determined through analysis of urine, tissue or blood and use of the methods and apparatus disclosed herein.
  • the source of the immunocompromise may be an infection, such as Human Immunodeficiency Virus (HIV, a congenital disorder or an inherited disease, resulting in reduced T-cell or B-cell activity; it may also be a concurrent infection, for example measles, or a therapeutic treatment resulting in immunomodulatory or immunosuppressive effects, such as those discussed above.
  • HIV Human Immunodeficiency Virus
  • viral infections associated with reduced or otherwise altered immune activity include but are not limited to JC vims, BK virus, Herpes Simplex Viruses and Cytomegalovirus.
  • JC vims JC vims
  • BK virus Herpes Simplex Viruses
  • Cytomegalovirus a virus that has been modified by the production of JC virus.
  • One skilled in the art would be capable of identifying the appropriate primers to be used in place of the BK virus or JC virus specific primers, but otherwise using the apparatus and methods of the present invention.
  • Microfluidic microchip-based monitoring of viral presence in blood, urine or tissue holds promise for fast, low cost screening of patients.
  • Miniaturization of the device lends itself to portability leading to pomt-of-care devices that are highly sensitive.
  • significant further miniaturization of on-microchip PCR volumes is readily feasible, the current level of
  • miniaturization is optimal for detection of clinically relevant viral titers. Although single-use chips are inexpensive, if microchip reusability is clinically feasible, the assay cost may be further diminished. Additionally, with ongoing improvements in custom-made instrumentation, miniaturized PCR and nucleic acid detection will be performed in under three hours, and in the preferred embodiment of the present invention, under one hour. As such, patients could be monitored in a primary setting such as the clinic or even at home by untrained personnel. Inexpensive and frequent testing offers the possibility for rapid detection, faster clinical response to elevated viruria, and a more informed evaluation of the significance of viruria or viremia through more comprehensive surveillance.
  • the present invention is the first demonstration of a miniaturized diagnostic procedure for BKV, JC virus, or viral load screening and the automated microfluidic approach described here offers significantly improved capability for close surveillance of renal transplant recipients, or patients otherwise under immunomodulatory, immunosuppressive or immunoablative therapy.
  • the system and method of the present invention is based on dual-layer and tri-layer chip architecture.
  • the chamber for thermal cycling is etched in the fhiidic layer and is ⁇ 600 nL in volume. Although lower volume PCR is feasible, a 600nl volume is more suitable for detection at the commonly observed low levels of template, in clinical samples; particularly at early stages of disease.
  • Thermal cycling is performed using a thin-film platinum resistive element, with resistive element and fluidic chamber geometry optimized for uniform temperature distribution using finite element modeling.
  • the present invention demonstrates RT-PCR-CE integration using a tri-layer microchip and PCR-CE integration using a dual-layer microchip; with no manual inclusion of reagents between RT and PCR steps and PCR and CE steps, thus minimizing potential contamination and simultaneously reducing the complexity of the instrumentation for RT and PCR integration.
  • Inclusion of CE as the detection strategy supports the use of many molecular diagnostics-based microchip tests, including mutation detection or •
  • the present invention demonstrates an entirely microchip-based system in which an application is developed, validated against conventional methods and then demonstrated using clinical samples.
  • the present invention performs a nucleic acid analysis, including a viral load analysis, directly from samples of human bodily fluids, i.e. without off-chip sample preparation.
  • the system is optimized to be insensitive to any PCR inhibitors resident in the sample. Its limit of detection allows performance of the testing over the clinically relevant range of viral titers.
  • the present invention demonstrates integration of PCR and CE, and PC, CE and RT; for the amplification of viral or human derived DNA and separation of amplified PCR products in the same chip within a microfluidic platform.
  • the art is in need of a single-use or reusable assay with an optimal sample analysis volume for either individual or limited patient screening usage while using inexpensive, portable, peripheral instrumentation.
  • the present invention provides a system capable of consistently detecting as few as 5 copies of the viral genome in a sample volume, and can detect as few as 1-2 copies, with sensitivity comparable to real-time PCR-based analysis, the current "gold standard”.
  • On- chip PCR can offer either a yes/no, quantitative or semiquantitative result that correlates well with parallel real-time PCR quantitation for the same samples. Further, preliminary costing suggests that on-chi ⁇ testing for BKV will have a cost one-tenth of real time PCR, even in the current single-analysis paradigm.
  • the present invention further contemplates simultaneous high-
  • the present invention provides a point-of-care device for molecular analysis that can be used in the clinic.
  • Microchip RT-PCR All RT-PCR reactions were prepared in a total volume of 50 ⁇ l.
  • the mixture included 25 ⁇ l of 2X reaction mixture (a buffer containing 0.4 mM of each dNTP, 2.4 mM MgSO4), 1 ⁇ l of the enzyme mixture comprising of Superscript® III RT and high-fidelity Platinum® Taq polymerase (Invitrogen Life Technology, Burlington, ON, Canada), 15-20 ⁇ l 5mM MgSO4, l ⁇ l of each forward and reverse primer (10 uM), 1 ⁇ g of RNA template, and including double distilled water to reach a 50 ⁇ l volume.
  • the primers were labelled with VIC dye and synthesized by ABI (Applied Biosystems, Foster City, CA).
  • Thermal cycling conditions using the microchip were 45°C for 30 min, 94°C for 2 min, 35 cycles of 94 0 C for 15 s, 60°C for 30 s, 68 0 C for 30 s, and a final extension time of 7 min at 68°C, subsequently the on- chip product was transferred to the CE microchannel209, 1707 for analysis.
  • the PCR reaction mixture contained a final concentration of IX PCR buffer, 2.8 ⁇ M MgCl 2 , 200 nM dATP, dGTP, dCTP and dTTP, 1.2M betaine. Both the forward and reverse primers were found to be optimal at 30OnM. Amplification was carried out by IU Platinum® Taq DNA polymerase (Invitrogen Life Technology, Carlsbad, USA).
  • Thermocycling of the microfluidic chip was performed using custom-built instrumentation with: 300 s of denaturation at 94 "C, followed by 35 cycles of denaturation at 94 0 C for 40 s, annealing at 60 0 C for 50 ⁇ , and extension at 72 0 C for 40 s, and ending with an extension step of 72 0 C for 180 s. After the chip reached room temperature, the mixture was unloaded by the actuation of the servo-motors and the amplified product allowed to mix with the electrophoresis buffer.
  • the on-chip PCR could be performed on DNA resulting from purification, preparation, or a reverse transcription procedures. It is therefore contemplated that the present invention may be used to detect both DNA or RNA viruses.
  • the methods or preparation, purification or reverse transcription are known to those skilled in the art, and in particular are described in Sambrook et al. Molecular Cloning a Laboratory Manual Cold Spring Harbor Press 3 ed, (2001).
  • Sequencing of PCR product amplified on chip Sequencing was performed to verify the PCR product amplified on-chip amplified product using capillary sequencing on the ABI 3100. The resulting sequence was identical to that predicted and also to that obtained from sequencing PCR product amplified from the same urine samples using the same primers in a conventional PCR reaction.
  • the product sequence was SEQ ID NO. 3.
  • Fluid handling Servo-motor driven probes allow for pumping and valving of an internal PCR reservoir by the application of pressure to compress and close the PDMS microchannels.
  • the elimination of physical and electrokinetic means of fluid handling prevents any inter-run contamination and is also suitable for use with physiological fluids due to it being independent of
  • FIG 1 shows the general tri-layer microchip structure (absent the CE component, for illustration purposes only), the top glass layer (fluidic layer) 110 was etched to form fluidic chambers 101, 206 and discontinuous channels 107, 207 for fluid handling.
  • the bottom glass layer (control layer) 130 was etched to form valves at the discontinuities of the fluid layer.
  • External vacuum and pressure lines were coupled to the microchip 102 and were controlled by a microcontroller-based circuitry to actuate the PDMS membrane 120 that forms valves 105, 205 (more folly described below), with three such valves in series acting together as a pump 202.
  • the Pt resistive element 106, 204 acting as the heater and the temperature sensor, was patterned on the upper surface of the control layer 130.
  • FIG 2 shows the integrated tri-layer microchip.
  • the sample mix is prepared external to the chip and loaded into input well 201 of the chip. Subsequently, by appropriate sequence of actuations for the valves 202 abetting the PCR chamber 203, this RT-PCR mix is pumped into the reaction chamber for thermal cycling by regulation of the temperature of the platinum resistive element 204. Bubble-free loading of the fluid within the chip is consistently achieved, and this is planned for by design. After the genetic amplification is completed, the product is pumped out and into the input well 208 of the CE section of the microchip 209.
  • FIG 3 shows representations of the side view of the tri-layer microchip valves comprising of a top etched glass layer 301 (fluidic layer), a bottom etched glass layer 303 (control layer), and a PDMS membrane 302 between these two glass layers, which is being actuated (microcontroller controlled) by pressurized air or vacuum.
  • the valves are normally closed when no external
  • FIG 3b When external vacuum is coupled to a valve, (FIG 3a), the valve opens, providing continuity in the channel within the flow layer. When pressurized air is coupled to the valve (FIG 3b) the valve is sealed.
  • FIG 17 shows the dual-layer microchip architecture
  • the glass layer 1710 is irreversibly bonded to a layer of PDMS 1720 which contains the features of fluidic chambers 1701, 1702, 1703, 170S, 1706 and 1707; and discontinuous channels 1707 for fluid handling.
  • the dual-layer microchip contains a sample loading well 1706, valving points 1704, an enclosed PCR chamber 1704, sample loading well 1702, buffer well 1701, sample waste well 1702, buffer waste well 1708 and CE channel (separation channel) 1707.
  • servo driven probes at valving points 1704 allow for pumping and valving of fluids within the internal PCR reservoir 1705 by the application of pressure to compress and close the PDMS 1710 microchannels against the glass layer 1720.
  • the elimination of physical and electrokinetic means of fluid handling prevents any inter-run contamination, and is also suitable for use with clinical samples.
  • Platinum thin film resistive element design and chip optimization of the tri-layer microchip Platinum has a good thermal response and a resistivity that exhibits a highly linear dependence on temperature.
  • Pt when patterned, in addition to functioning as a heater, Pt can be used as a temperature sensor.
  • thin-film resistive elements were designed to maintain uniform temperature within the PCR chamber, while simultaneously acting as a temperature sensor that provides necessary feedback for temperature regulation of the heater.
  • Such a heater/sensor design eliminates the need for two resistive elements, greatly simplifying the microchip electrical interfacing and reducing space usage, an important benefit for a portable system. To achieve this, and to have the total resistance of the resistive element accurately reflect its temperature, the temperature distribution in the thin film was designed to be uniform by making use of finite element analysis (FEA).
  • FFA finite element analysis
  • a ring geometry was established for the heater/sensor coupled with the optimal connection width to electrode pads yielded temperature uniformity (less then I 0 C temperature gradients) in the heater/sensor.
  • IR imaging verified temperature distribution, and absolute temperature was calibrated using custom-built temperature responsive crystals for different set temperatures (Hallcrest, Glenview, IL, USA).
  • FEA placement of fluidic components in the vicinity of the heaters was also optimized to ensure the operation of other microchip components do not alter the temperature uniformity.
  • Microcontroller- based circuitry was used to operate the valves and pumps, as well as for controlling the thin film element for heating and for temperature sensing.
  • the heaters were annealed prior to the calibration process and to all other operations, in order to minimize the stresses within the films that typically build up during fabrication. This annealing stage ensures highly repeatable performance at elevated temperatures.
  • a DC current was applied to the resistive element, and the temperature of the element was subsequently computed from its resistance with apre-determined resistance vs. temperature function. With this temperature as the feedback, the applied current was controlled and adjusted to yield the desired temperature for thermal cycling.
  • a PID controller was in operation to precisely regulate the current flow for thermal cycling within the PCR chamber.
  • Proportional- derivative (PD) control was used to realize rapid transients between the PCR temperature stages, while proportional-integrative (PI) control was used to maintain a constant temperature during the stages themselves.
  • the resistive element heats up almost instantaneously when the desired current is applied via the control circuitry, and therefore, the temperature cycling rate of the PCR chamber 203 was limited primarily by the thermal conductivity between the resistive thin film and the chamber and by cooling.
  • Cooling was achieved via passive convection and hence is relatively slow, though other means of cooling known to the art; which includes but is not limited to Peletier cooling, thermally conductive heat sinks in direct or indirect thermal communication with the PCR chamber 203, is contemplated as part of the present invention.
  • the PDMS/Glass chips are comprised of a 1.2mm thick layer of molded PDMS (Sylgard 184, Dow Coming, NC, USA) and a 1.1 mm thick borofloat glass (Paragon Optical Company, PA, USA) substrate.
  • the soft-lithography replica molding approach of fabrication for PDMS is followed (Duffy, D.C., et al Anal Chem 70:4974 (1998).; Unger, M.A., et al. Science 288:113 (2000)). Briefly, first, a master with features in
  • AZ4620 photoresist (Shipley Microelectronics, MA, USA) is patterned.
  • the chip designs were drawn in L-Edit v3.0 (MEMS Pro 8, MEMS CAP, CA, USA) and transferred to a chromium mask wafer using a pattern generator (DWL 200, Heidelberg Instruments, CA, USA).
  • the 4 X 4" borofloat glass substrate was cleaned in a fresh Piranha solution and sputter coated with a layer of chromium to a thickness of ⁇ 200nm, spin-coated with AZ4620 photoresist at a spin speed of 500 rpm for 10 s and a spread speed of 2000 rpm for 25 s.
  • a hotplate set at 100 °C was used to bake the wafer and then was placed in a box with a damp cleanroom wipe, light and moisture sealed, and left for ⁇ 2 h.
  • UV exposure (30 s, 356 nm, and intensity of (19.2 mW/cm ) of the spin-coated substrate was performed through the chrome mask using a mask aligner (ABM Inc., CA, USA).
  • the substrate was then chemically developed with AZ400K. that was diluted AZ400K (1:4) (AZ400K:H 2 O) for about 80s resulting in the patterned mould. This was typically reused for ⁇ 20 times after which the glass substrate was reused after acetone cleaning.
  • this photoresist results in a feature height of ⁇ 14 ⁇ m.
  • Posts made of aluminum or brass with radii of ⁇ 0.75 mm and heights of 1 mm were cleaned by a brief immersion in a cold Piranha, rinsed, dried and placed on the master at the locations for molding the PCR chambers. After mixing the pre-polymer and the curing agent in proportions of 1 : 10 by weight this was degassed (in vacuum oven at 20 in.Hg for 20 min) and thermally cured for 90 minutes at 90 0 C.
  • the active surfaces (features) of PDMS and the glass substrate were then exposed to oxygen plasma face-up in a reactive ion etch (RIE) (Plasmalab, Plasma Technology, Bristol, UK) with 80% O 3 gas flow at a chamber pressure of 0.15 Torr, a power of 35 W and treated for 60 s.
  • RIE reactive ion etch
  • the surfaces of glass and PDMS were brought together and irreversibly bonded to form the microchip.
  • punched holes were made in the PDMS layer, these were 1.5 mm in diameter and able to contain a maximum volume of ⁇ 3.5 ⁇ l.
  • Tri-layer microchip fabrication Tri-layer microchip fabrication.
  • the microchip designs were drawn in L-Edit v3.0 (MEMS Pro 8, MEMS CAP, CA, USA) and transferred to a mask wafer using a pattern generator (DWL 200, Heidelberg Instruments, CA, USA).
  • the 4 inch by 4 inch borofloat glass substrate (Paragon Optical Company, PA, USA) was cleaned in a hot Piranha (3:1 of H2S04:H2O2) and sputter coated with 20 ⁇ m of Cr and 200 ⁇ m of Au.
  • HPR 504 photoresist was spin coated with a spin speed of 500 rpm for 10 s and a spread speed of 4000 rpm for 40 s.
  • the photoresist coated substrate was then baked in an oven set at 115"C for 30 minutes.
  • UV exposure (4 s, 356 nm wavelength, and intensity of (19.2 mW/cm2) of the spin-coated substrate was performed through the chrome mask using a mask aligner (ABM Inc., CA, USA).
  • the substrate was then chemically developed with Microposit 354 developer (Shipley Company Inc., Marlborough, MA, USA) for -25 s.
  • Glass etch was performed using hydrofluoric acid (HF) at an etch rate of ⁇ 1.1 ⁇ m/min.
  • the control layer was etched to 70 ⁇ m depth, and the flow layer 90 ⁇ m depth. Photoresist was removed by rinsing the substrate using acetone and isopropyl alcohol.
  • Au and Cr (Arch Chemicals Inc., Norwalk, Connecticut, USA) etchants were used to strip the metal, the etch time being ⁇ 45 s for Au and ⁇ 30 s for Cr. Holes in the flow layer were drilled using a Waterjet for accessing both the flow and the control layers of the chip.
  • the control layer requires by design the patterning of Pt films and this was performed via a lift-off technique.
  • the metal-stripped etched glass was cleaned in fresh Piranha, and 20 ⁇ m of Cr was then sputter deposited.
  • AZ 4620 photoresist was spin-coated for 10 s at a spread speed of 500 rpm and spin speed of 2000 rpm for 25 S, the substrate was soft-baked on a hot-plate for 90 s, and then hydrated for 2 h.
  • the photoresist was them UV exposed for 30 s and developed using AZ 400K developer for ⁇ 180 s, after which the Cr was completely stripped.
  • 20 am of Ti and 220 nm of Pt were sputter deposited, and using lift-off, the Pt/Ti electrodes were defined on the control layer.
  • the PDMS membrane was irreversibly bonded with the etched face of both the flow and the control layer.
  • Dual-layer microchip capillary electrophoresis is performed within the crosstchanneJ CE section of the microchip using a modified .procedure. employed for the. glass-based chips (Vahedi, G. et al. Electrophoresis 25:2346 (2004)). Fragment analysis (CE) of the amplified PCR mix is performed within the microfluidic tool kit ( ⁇ TK, Micralyne, Edmonton, Canada) (Vahedi, G. et al. Electrophoresis 25:2346 (2004)). The ⁇ TK provides similar the optical detection and high voltages needed to perform CE with confocal laser-induced fluorescence (LIF) detection.
  • LIF laser-induced fluorescence
  • the LIF system uses excitation at 532 nm and detection at 578 nm. Further details on the system and its use can be found elsewhere (Ma, R. et al Electrophoresis 26:2692 (2005)). Briefly, conditioning of the chip involves flushing the chip with doubly
  • a 0.3 ⁇ l aliquot PCR product was pumped from the enclosed PCR chamber into the open injection CE well where it was diluted in 0.1 X TBE to constitute a total volume of 3 ⁇ l.
  • LIF detection was performed at 76 mm from the channel intersection.
  • Sizing was performed by simultaneously loading 0.3 ⁇ l of a DNA ladder GeneScan® 500 TAMRA (Applied Biosystems, Foster City, CA). Chip quality and consistency were monitored on a regular basis via a calibration procedure (Pilarski, L.M. et al. Journal of Immunological Methods 305:94 (2005)).
  • Tri-layer microchip capillary electrophoresis (CE) equipment Tri-layer microchip capillary electrophoresis (CE) equipment. Fragment analysis of on-chip PCR product is performed within the cross-channel CE section of the integrated multi-layer microchip (FIG. 2) using a modification in the procedure for glass-only microchip (Vahcdi, G. et al Electrophoresis 25:2346 (2004)). Fragment analysis of the amplified PCR mix was performed within the microfluidic tool kit, ⁇ TK (Micralyne, Edmonton, Canada) (Ma, R. et al Electrophoresis 26:2692 (2005)), however this can also be readily performed within a portable CE system. The microchip platform and procedures described here can be readily integrated into a portable CE platform.
  • the ⁇ TK provides the optical detection and high voltages needed to perform CE with confocal laser-induced fluorescence (LlF) detection with excitation at 532 nm, and detection at 578 ran.
  • LIF laser-induced fluorescence
  • a pinch-off injection approach was used, with 0.4 kV applied for 60 s to inject the DNA sample and a separation voltage of 6 kV was used for separation through the CE channel 209.
  • LIF-based detection was performed at 24 mm from the channel intersection.
  • the on-chip PCR product was flushed out of the chip with 2 ⁇ l of 0.1X GABE (genetic analysis buffer with EDTA), 1.2 ⁇ l of HiDi formamide (ABI) and 0.5 ⁇ l of size standard (GS500) were
  • GeneScan® polymer Applied Biosystems
  • POP6 Applied Biosystems
  • Sizing was performed by simultaneously loading 0.3 ⁇ L of a DNA ladder GeneScan® 500 TAMRA (Applied Biosystems).
  • GeneScan® polymer the conditioning of the chip requires flushing the chip with doubly deionized water for at least 2 minutes, followed by rinsing with the running buffer IXTBE.
  • TBE was made using Tris (crystalline free base) and boric acid (Fisher Scientific, Fair Law, NJ, USA) and EDTA (Merck, Darmstadt, Germany).
  • GeneScan® polymer was mixed with glycerol (Sigma, St.
  • a 0.3 ⁇ L aliquot PCR product was pumped from the PCR chamber 203, 1705 into the open injection CE well 208, 1702 where it was diluted in 0. IxTBEl G to constitute a total volume of 3 ⁇ L.
  • the polymer is heated to 67 C C for 10 minutes, before being loaded into the CE channel 209, 1707. Heating was done to reduce the viscosity of the POP6 polymer, as well as ensuring the complete dissolution of any unprecipitated urea, and facilitates the loading of the polymer within the CE channel 209, 1707.
  • the PCR product was denatured for 4 minutes at 96°C and rapidly cooled to
  • BM samples were obtained at diagnosis or relapse of 2 patients with multiple myeloma (MM). BM was processed as previously described (Szczepek, K. et al Blood 92:2844 (1998)). With institutional review board approval, Norovirus was obtained from anonymous patients who provided stool samples to the Provincial Laboratories of Public Health, Edmonton. All samples were confirmed to be positive by conventional testing prior to testing on the microchip.
  • the microchip cleaning procedure for reusability can be used indefinitely with the reusablity procedure presented here.
  • the PDMS that is irreversibly bonded on both surfaces to glass was dissociated using Dynasolve 210 (Dynalloy, Indianapolis, IN, USA) when the microchips were left to soak for ⁇ 2 hours . This was followed by a hot Piranha cleaning (3 : 1 of H2SO4 to H 2 O 2 ) procedure to ensure that the disintegrated PDMS was cleaned from the surface.
  • PDMS layers were then rebonded to the cleaned glass slides to re-use the chip. We have found that this re-usability procedure has no detectable ill-effect on the outcome of the PCR or on CE.
  • the microchips can be used indefinitely with the reusablity protocol described here.
  • Example 1 Detection of BKV on dual-layer chip.
  • BKV is a small-enveloped virus within the polyomavirus family with a genome of approximately 5 Kbps of double stranded DNA37.
  • the primers were designed to amplify a 293 bp region of the BKV structural protein VPl.
  • the forward primer SEQ ID NO. 1 and the reverse primer sequence SEQ ID NO. 2 were used.
  • the primers are located at nucleotide 433 on strain MM genome and position 2274 on the Dunlop strain genome. Analysis using the NIH BLAST database showed no cross reactivity with any other commonly present viral gene sequences. Specificity of these
  • initial testing utilized a two chip system, having the PCR functionally on one microchip and the CE DNA fragment analysis of PCR product on a second chip with separation carried out using a Microfluidic Toolkit ( ⁇ TK) as described herein and elsewhere (Vahedi, G. et al. Electrophoresis 25:2346 (2004)).
  • the microfluidic chips are made of patterned poly(dimethyl)siloxane (PDMS) bonded to a glass substrate. Externally-actuated reusable diaphragm pumps and a pinch-off valve are integrated in the system (Pilarski, P.M. et al.
  • FIG. 17 shows a PDMS/glass hybrid microchip able to seamlessly perform integrated PCR and CE, with a PCR volume of ⁇ 2 ⁇ l on a single assay, an optimal volume for BKV load assessment.
  • Raw urine samples having known titers of BKV were diluted 100-fold, manually added (0.24 ⁇ l of diluted urine and 2 ⁇ l PCR master mixture) into the PCR sample loading well 1706 and driven into the PCR chamber 1705 by using the valve/pump 1704.
  • pinch-off valves 1704 immobilized the fluid until the amplified product was pumped to an open chamber and manually removed.
  • a DNA ladder size standard was added to the product, followed by manual introduction to a glass CE chip for separation using the ⁇ TK.
  • the amplified product was confirmed to be the expected 293 bp size as shown in the electropherogram of FIG. 4.
  • product detected on chip was also detected using the ABI 3100 DNA fragment analysis instrument for a "gold standard” comparison.
  • sequencing of on-chip PCR product was carried out externally using the ABI 3100 and was shown to be correct, confirming the specificity of the on-chip reaction.
  • a comparison was also made between the amplification of BKV from purified DNA and unprocessed urine, with equivalent results (data not shown), indicating that no loss of sensitivity occurs with the use of raw urine.
  • Chip-based CE was performed using two different sieving matrices in the injection and
  • GeneScan® polymer is known to induce sequence dependent migration (FIG. 4a) in addition to size-based separations during migration which means that the placement of the product peak (293 bp) is not as expected with reference to the DNA ladder peaks. To address this, the same PCR products were verified by sizing on the ABI 3100 and by sequencing to confirm product identity. In the GeneScan® polymer, the DNA fragments migrate as double strands, the repeatability in positioning of the PCR product peak in the electropherogram relative to the DNA ladder peaks is hence used to indicate the occurrence of BKV.
  • POP6 denaturing sieving polymer
  • Example 2 Semi-quantitative detection of BK viral load on dual-layer chip.
  • Quantitative or semi-quantitative results are essential for determining whether follow-up is required as well as to monitor the progress of the disease during and following intervention.
  • Two semi-quantitative techniques were employed to determine virus titers on chip: (a) patient urine samples were diluted from 1/10 to 1/10 7 in steps of 10-fold dilutions, and (b) three aliquots of the same reaction mixture were cycled for 35, 25 or 15 cycles of PCR.
  • FIG. 5 shows representative serial dilution analysis of two urine samples with clinically defined BKV titers, one with a titer of 1.78xlO 7 copies/ml and one with a titer of 1.83 xlO 10 copies/ml.
  • the x-axis represents the serial dilution factor and the y-axis represents the relative fluorescence units (rfu) after on-chip PCR runs were performed and analyzed using the ABI 3100.
  • rfu relative fluorescence units
  • detectable PCR product is amplified at the 1/100 (-360 copies/reaction) and 1/1000 dilution (-36 copies/reaction) but not at 1/10,000 (-3.6
  • FIG. 6 shows two representative samples. Progress in DNA amplification as captured by change in relative fluorescence (in ABI 3100) with cycle progression during PCR for different patient samples having different concentrations. Overall, five samples were amplified on-chip in duplicate, (a) Concentration of the sample: 1.78xlO 7 copies/ml, (b) Concentration of the sample; 1.83xlO io copies/mL.
  • the x-axis represents the number of PCR thermal cycles performed prior to unloading the product from the chip and the y-axis represents the relative fluorescence units (rfu) after on-chip PCR runs and analysis on the ABI 3100 as indicated for FIG. 5.
  • rfu relative fluorescence units
  • FIGS. 5 and 6 indicate that the present microchip-based approach can be adapted for semiquantitative PCR strategies that could be incorporated on-chip in an automated manner.
  • a predefined volume of the PCR mixture for analysis after a user- defined number of thermal cycles, repeated analysis at increasing cycle numbers could be performed.
  • This type of quantitation can be evaluated for utility in the clinic and the extent of precision required for informed decision making, possibly providing a low-cost alternative to real time PCR.
  • Example 3 Integrated PCR-CE detection of BKV on dual-layer microchip.
  • FIG. 7 shows amplification of a 293 bp BKV PCR product using the integrated PCR-CE chip. Electropherogram with x-axis depicting the time in seconds when DNA fragments are detected as seen by the relative fluorescence intensity (rfu) peaks (y-axis).
  • the 293 bp PCR product indicates of the presence of BK virus that was detected on the microchip using the PCR-CE integrated chip wherein both the functionalities of PCR and CE were performed seamlessly on a single assay.
  • Example 4 Sensitivity of BKV detection using dual-layer on-chip PCR.
  • Chip-based testing was performed on a panel of 55 randomly selected urine samples from renal transplant recipients, previously screened for BKV titre using routine clinical testing by real-time PCR. The 55 samples were tested in a total of 228 on-chip PCR reactions.
  • FIG. 7 shows that product amplification is a function of the level of viruria, and that the detection level on-chip is consistent with the clinically established viral titre (y-axis of FIG. 8).
  • Viruria was detected in urine samples from renal transplant recipients randomly selected for on- chip PCR analysis. The number of recipients is indicated by the x-axis. Values on the y-axis were the BKV titers reported by the clinical testing laboratory (Provincial Laboratories, Edmonton, Canada) by quantitative PCR using LightCycler®. A total of 55 samples were tested at a dilution of 1/100 in a 2 ⁇ l PCR reaction volume, with a total of 228 replicate analyses performed on microfluidic chips. For low titer samples, multiple replicate tests were performed as indicated in results. Each sample was analyzed 2-10 times with reproducible results. As predicted from the expected random distribution of copies, consistently positive results were obtained for samples containing more than 10 copies of the BKV in the on-chip PCR reaction mixture (5x10 5 copies/ml).
  • a set of 13 urine samples from transplant patients with clinically undetectable BKV were analyzed on chip in a total of 38 different reactions, with three positive results among replicate tests for two urines. This may reflect true positives in a Poisson distribution of templates that were below the levels required for detection with clinical real time PCR (as might be expected with microchip detection at the near-single copy level); 11/13 of these urine samples were consistently negative. These results demonstrate that the false positive rate for on-chip PCR is likely to be low or zero.
  • BK-negative urine samples from the transplant recipients were spiked with ⁇ 10 4 copies each of JC virus, cytomegalovirus (CMV) and Epstein Barr Virus (EBV) DNA, since these can be present in urine from transplant recipients.
  • CMV cytomegalovirus
  • EBV Epstein Barr Virus
  • Chip-based testing was performed on cloned JC virus samples using primers specific for the JC virus, SEQ ID NO. 4 and SEQ ID NO. 5.
  • the resulting product of 253 base pairs was identified with serial dilutions of cloned JC virus when using the chip-based procedure described herein. No product was observed when using conventional non-chip based PCR procedures.
  • FIG 9 shows a SYBR green stained acrylamide gel showing the product obtained form a chip-based amplification using the JC specific primers disclosed herein with the chip-based amplification process and apparatus of the present invention.
  • RNA was isolated from KMS-34, a myeloma cell line.
  • the primers SEQ ID NO. 11 and SEQ ID NO. 12 were designed to amplify a 243 bp fragment from RNA or from genomic DNA .
  • the on-chip RT-PCR amplified a fragment of the
  • Example 7 Microchip identification of clonotypic signatures using tri-layer microchip
  • MM is a cancer of the immune system characterized by a unique immunoglobulin gene rearrangement, the clonotypic IgH VDJ, and by IgH translocations that enable identification of the MM clone and are thus clinically valuable biomarkers.
  • Point-of-care testing for such biomarkers has value for quantitative and real time monitoring of tumor burden in individual patients, as well as for monitoring response to treatment and detecting emerging relapse. Such detection techniques permit identification of malignant cells that would otherwise be clinically cryptic.
  • the clonotypic IgH VDJ rearrangement was detected with primers specific to the complementarity determining regions (CDR2 and CDR3) of the clonotypic IgH VDJ for each individual patient (Szczkpek, AJ. et al. Blood 92:2844 (1998)).
  • FIG 10 shows the variable region (VH) contains framework regions (FRs) that maintain antibody structure and stability, and complementarity determining regions (CDRs) that vary in nucleotide sequence and confer specificity for distinct antigens, hence, patient specific primers are required to be designed.
  • CDR3 is composed of diversity (DH) and joining (JH) regions and is the most variable of the CDRs.
  • DH diversity
  • JH joining
  • a portion of the constant (CH) region is shown. The remainder of the downstream CH region and immunoglobulin light chain V gene, have not been presented.
  • Patient specific CDR primers are indicated as black arrows. Scale indicated at the top is in base pairs.
  • Example 8 ⁇ Yi-layer microchip Norovirus detection
  • Norovirus is a single-stranded RNA virus with a genome of approximately 7.5 kbp, that causes acute gastroenteritis in humans. NV is transmitted by fecally contaminated food, vomit, and person-to-person contact. Tests involving electron microscopy (EM) are laborious and relatively insensitive, lmmuno EM or ELISA tests are limited to the viral subtypes detected by the antibodies used. Conventionally, and as recommended by the Center for Disease Control (CDC) both nucleic acid hybridization and RT-PCR assays are used to detect the NV genome in clinical and environmental specimens as they are highly specific. These genetic amplification techniques detect as few as 102-104 viral particles/ml in stool. The present invention demonstrates the potential for screening of patients one at a time, thereby holding promise for real time detection in the community rather than in a central diagnostic facility using high- throughput approaches.
  • EM electron microscopy
  • ELISA tests are limited to the viral subtypes detected by the antibodies used.
  • CDC
  • NV was successfully detected by the on-chip RT-PCR system (patient 3, FIG. 14 and patient 4, FIG 15). All on-chip tests were performed in triplicate to ensure highly repeatable outcomes.
  • the microchip-amplified product was detected by DNA fragment analysis on a glass CE chip using a DNA ladder (GS500), as well as conventionally by sizing using gel electrophoresis and DNA fragment analysis using the ABI 3100 genetic analyzer. The product identity was confirmed by sequencing.
  • GS500 DNA ladder
  • Example 9 Two-stage fast PCR using tri-Iayer microchip
  • the annealing and the extension steps of Hie regular 3-step PCR thermal cycling were combined to realize two-step PCR cycling comprising a denaturation step and a combined annealing/extension step.
  • the reaction mix was as for the regular PCR (described above), however, the thermal program was modified as: 46°C initially for 10 minutes for the RT step, followed by 88°C for 1 min to deactivate the Superscript® III. This was followed by a 10 s denaturation at 88 0 C and a 20s annealing/extension for 20 s, this was repeated for 40 cycles. A final extension of 1 min at 68°C was then realized.
  • time and temperature optimization is a viable method for optimizing PCR to reduce the overall time from application of sample to test result.
  • the product was confirmed by DNA fragment analysis on chip CE as well as conventionally using gel electrophoresis and DNA fragment analysis, and confirmed by sequencing.

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Abstract

L'invention concerne un dispositif microfluidique destiné à MONITORER des acides nucléiques spécifiques contenus dans un échantillon clinique. Ledit dispositif comprend au moins une cuvette d'entrée capable de recevoir au moins un échantillon clinique, au moins une chambre capable de recevoir une partie du au moins un échantillon clinique, et au moins un canal microfluidique capable de séparer les acides nucléiques en fonction de leur taille, et capable de recevoir une partie du au moins un échantillon clinique. Lesdits au moins une cuvette d'entrée, au moins une chambre et au moins un canal microfluidique sont tous mis en communication fluidique par au moins deux deuxièmes canaux microfluidiques. En outre, ledit dispositif microfluidique comprend au moins deux valves capable d'empêcher le fluide de s'écouler à travers au moins un desdits deuxièmes canaux microfluidiques. Dans un mode de réalisation, l'acide nucléique est viral, et l'échantillon clinique provient de sang, d'urine ou de tissu humains. L'invention concerne également un procédé de surveillance d'un patient soumis à un traitement immunomodulateur, immunosuppresseur ou immunoablateur, qui utilise le dispositif.
PCT/CA2007/000959 2006-05-19 2007-05-18 Procédés microfluidiques pour le monitorage de l'acide nucléique Ceased WO2008000060A1 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008119190A1 (fr) * 2007-03-29 2008-10-09 The Governors Of The University Of Alberta Plate-formes microfluidiques pour génotypage
KR20160026292A (ko) 2014-08-29 2016-03-09 (주) 타우피엔유메디칼 승모판막 서클라지 관상정맥동 성형술에서 루프를 형성하기 위한 루프용 로프 및 이의 제조장치
EP3234560A4 (fr) * 2015-01-30 2018-10-17 Hewlett-Packard Development Company, L.P. Régulation d'une température microfluidique
US10639630B2 (en) 2015-01-30 2020-05-05 Hewlett-Packard Development Company, L.P. Microfluidic temperature control
WO2020242927A1 (fr) * 2019-05-29 2020-12-03 Lexagene, Inc. Systèmes à faible volume pour identification d'échantillon

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