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WO2008144054A1 - Dispositifs et procédés microfluidiques - Google Patents

Dispositifs et procédés microfluidiques Download PDF

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Publication number
WO2008144054A1
WO2008144054A1 PCT/US2008/006431 US2008006431W WO2008144054A1 WO 2008144054 A1 WO2008144054 A1 WO 2008144054A1 US 2008006431 W US2008006431 W US 2008006431W WO 2008144054 A1 WO2008144054 A1 WO 2008144054A1
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WIPO (PCT)
Prior art keywords
optical contrast
contrast layer
antigen
microfluidic device
layer
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PCT/US2008/006431
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English (en)
Inventor
Abraham Lee
Philip L. Felgner
Armando Tovar
Uland Liao
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The Regents Of The University Of California
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Publication of WO2008144054A1 publication Critical patent/WO2008144054A1/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
    • 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
    • B01L2300/00—Additional constructional details
    • B01L2300/04—Closures and closing means
    • B01L2300/046—Function or devices integrated in the closure
    • 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
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0627—Sensor or part of a sensor is integrated
    • B01L2300/0636—Integrated biosensor, microarrays
    • 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
    • B01L2300/0887—Laminated structure
    • 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
    • B01L2300/16—Surface properties and coatings
    • B01L2300/168—Specific optical properties, e.g. reflective coatings

Definitions

  • the field of the invention is diagnostic devices and methods, especially as it relates to microfluidic protein microarray devices.
  • microarray tests and test formats have been developed to allow analysis of several ten to several ten thousand analytes in a biological sample.
  • optical detection has become more and more complex.
  • most typically, currently known microarray tests require conventional lab-bench methods that often require whole-day processes and large amounts of user-handling confined to laboratory settings.
  • dedicated detection devices e.g., to detect fluorescence, luminescence, etc.
  • operate in conjunction with confocal or other magnifying optics rendering such devices less suitable for field use.
  • U.S. patent application 2005/031488 teaches an array of isolated proteins immobilized onto a membrane in a dot blot format where the membrane is suspended in a frame for simplified handling. While such array is relatively simple to handle, quantitative analysis is typically not possible and where large numbers of peptides are required, the size of such arrays is prohibitive to high-throughput screening. Still further, such arrays require isolated and purified protein, which adds substantial effort to the construction of the array. To increase density of the array, multiple fibers coated with selected and purified antigens can be bundled and sliced to produce an array as taught in U.S. Patent 6,887,701. Detection in these arrays can use various formats, and is typically done via fluorescence detection. Such array significantly increases the analyte density, however, is typically limited to qualitative detection of reagents. Moreover, the preparation of such arrays is labor intensive and typically requires isolated compounds.
  • individual clones of a nucleic acid library can be expressed in vitro as taught in U.S. application 2004/161748 or in situ as taught in U.S. application 2005/048580.
  • epitope arrays can be expressed in vitro as taught in U.S. application 2006/224329.
  • the recombinant protein is then immobilized to the array carrier using a tag or affinity peptide that is fused to the recombinant protein. While such methods significantly simplify array production and increase probe density, various difficulties nevertheless remain. For example, the recombinant protein preparations must be purified and so add substantial effort. Moreover, quantitative detection is typically limited to fluorescence and/or luminescence methods, which require dedicated and expensive devices.
  • the present invention provides significantly improved apparatus, systems and methods in which antibody-based protein array analysis allows for quantitative detection of antigens and/or antibodies from crude protein preparations using a precipitating or agglomerating dye that is visually detectable.
  • Particularly preferred systems are configured as micro fluidic systems and provide a dynamic range of at least three orders of magnitude, while analysis can be completed in less than one hour.
  • a method of performing an analytic test will include a step of providing a carrier having a surface that comprises an optical contrast layer, wherein a plurality of detergent-containing non-purified distinct antigen preparations are non-covalently and non-specifically coupled to the optical contrast layer at respective predetermined locations to form an antigen array having a density of at least 10 distinct antigen preparations per cm 2 .
  • the optical contrast layer has a thickness and composition sufficient to prevent confluence of the antigen preparations when the distinct antigen preparations are deposited onto the optical contrast layer.
  • the antigen array is contacted with a solution comprising an antibody (typically human blood or serum) under conditions to allow binding of the antibody to an antigen of at least one of the antigen preparations, hi a still further step, binding of the antibody is detected using a visually detectable, and precipitating or agglomerating dye.
  • an antibody typically human blood or serum
  • the contrast layer and the antigen preparations have a composition such that the step of detecting binding of the antibody allows detection over a dynamic range of at least three orders of magnitude, and/or the array comprises at least two distinct antigens from the same pathogen, most preferably with known quantified and known relative reactivities with respect to sera of a population infected with the pathogen.
  • contemplated dyes insoluble in the developing medium to precipitate in situ.
  • contemplated dyes therefore include 3-amino-9-ethylcarbazole, 5-bromo-4-chloro-3- indolylphosphate, 3-3'-diaminobenzidine tetrachloride, 3,3',5,5'-tetramethylbenzidine.
  • colloidal gold or other colloidal metals may be employed as a dye. Consequently, it should be appreciated that detection can be performed with the unaided eye using no further detection devices such as luminometers or fluorimeters.
  • the step of detecting is performed using a scanner or CCD detector.
  • a scanner or CCD detector Remarkably, using an ordinary commercially available flat bed scanner with a resolution of 1200 dpi, quantitative analysis at a dynamic range of at least three orders of magnitude can be achieved.
  • the step of detecting is performed from opposite sides of the optical contrast layer.
  • contemplated microfluidic devices will comprise an enclosed reaction volume formed by a carrier material such that an optical contrast layer is disposed within the reaction volume and opposite to a cavity layer within the reaction volume, wherein a plurality of distinct antigens are non- covalently and non-specifically coupled to the optical contrast layer in predetermined positions. Additionally, it is preferred that the distinct antigens are non-purified and further comprise a detergent.
  • the cavity layer has plurality of cavities (preferably opposite the contrast layer) that are sized and dimensioned to allow trapping of air in the plurality of cavities, wherein the number and/or size of the cavities is selected such that hybridization of an antibody to the antigens is substantially complete within less than 60 minutes upon mixing.
  • the carrier material is configured to allow quantitative detection of a visually detectable, and precipitating or agglomerating dye.
  • the optical contrast layer comprises nitrocellulose, and/or the carrier material comprises a glass and/or transparent synthetic polymer.
  • the cavities in the cavity layer in contemplated microfluidic devices are circular cavities and perpendicularly arranged at regular intervals along an x- and y-coordinate.
  • the ratio between the number of antigens and the number of cavities is at least 3:1, and/or the ratio between the area of an antigen spot and the cavity diameter is at least 1 :3.
  • the reaction volume is between 1 ⁇ l and 500 ⁇ l, and most typically between 1 ⁇ l and 50 ⁇ l.
  • the carrier material and the optical contrast layer are configured to allow quantitative detection over a dynamic range of at least three orders of magnitude, and quantitative detection is based on a dye that is insoluble in the detection solution (e.g., 3-amino-9-ethylcarbazole, 5-bromo-4-chloro-3- indolylphosphate, 3-3'-diaminobenzidine tetrachloride, 3,3',5,5'-tetramethylbenzidine, and a colloidal metal) to so form a visually detectable spot.
  • the carrier material and/or the optical contrast layer are configured to allow optical detection from opposite sides of the optical contrast layer.
  • Figure 1 schematically depicts an exemplary microfluidic device.
  • Figure 2 is an exemplary detail view of glass carrier onto which multiple and distinct nitrocellulose layers are coupled and where multiple and distinct protein antigens are arrayed on the nitrocellulose layers.
  • Figure 3 is a graph depicting resonance intensity for varying bubble cavity distances for an 800 micron bubble over a time scale of 5 seconds for a 400 micrometer distance.
  • Figure 4 is a graph depicting maximum resonance intensities for varying ratios of bubble cavity distances to cavity size.
  • Figure 5 is a graph depicting performance comparison of volumetric reaction chambers against conventional fluid handling methods.
  • Figure 6 is an exemplary scan of a protein array (Vaccinia, unpurif ⁇ ed) probed with anti-His and Anti-HA antibodies for which corresponding peptides are denoted in the adjacent table.
  • Figure 7 is another exemplary scan of a protein array (Vaccinia, unpurified) in which binding of human immunoglobulin was quantified using ProScanArray, and in which the highly reproducible corresponding signal intensities are depicted in the graph below.
  • Figure 8 is a graph depicting a comparison of binding sensitivity between contemplated microfluidic device hybridization and conventional hybridization using VIG antibody.
  • Figure 9 depicts exemplary scans and corresponding graph illustrating high correlation between test results obtained by fluorescence detection (top) and colorimetric dye detection (bottom) using two distinct secondary antibodies on a total of over 4600 antigens.
  • microarrays can be miniaturized to high densities, even when unpurified or partially purified proteins are used, and that such microarrays can be processed in various microfluidic devices at a heretofore not achieved speed while at the same time allowing for simple colorimetric signal generation and detection at a remarkably high dynamic range.
  • proteins corresponding to selected antigens of known pathogens are separately arrayed and non-covalently immobilized onto a nitrocellulose substrate that also acts as an optical contrast layer wherein the nitrocellulose is most preferably coupled to a glass carrier or other carrier that is optically transparent.
  • the so immobilized antigens are then used to specifically capture antibodies present within a serum sample.
  • the antigens for such arrays need not be purified at all (or may only be partially purified), and suitable antigen sources therefore include crude in vitro translation reactions (which may also be used in situ where desired).
  • unpurified antigen refers to an antigen that is present together the contents of a bacterial (E.coli), yeast (e.g., Pichia pasteuris), or eukaryotic cell ⁇ e.g., Sf9 or CHO cells), or components hereof suitable for in vitro translation.
  • unpurified antigens will typically be applied to the optical contrast layer as an aliquot of an in vitro or in situ translation/expression reaction.
  • partially purified antigen refers to an antigen preparation (typically the unpurified antigen as defined above) after the preparation was subjected to a step of filtration, fractionated precipitation, solvent extraction, and/or centrifugation.
  • electrophoretically separated antigens and affinity purified antigens are expressly excluded from the definition of the terms "unpurified antigen” and "partially purified antigen”. There are numerous methods of preparing such contemplated antigens known in the art, and all of the known methods are deemed suitable for use herein.
  • WO 02/097051 and WO 2006/088492 both of which are incorporated by reference herein may be used to produce relatively large libraries of antigens.
  • the antigens may be applied to the optical contrast layer in substantially purified form (i.e., at least 60%, and more typically at least 75% electrophoretic purity as evidenced by using bromophenol blue staining). Purification may be performed in numerous manners, and especially preferred manners include affinity and ion exchange chromatography.
  • suitable antigens may be recombinant and expressed as a fusion protein with affinity tag, or native and directly isolated from the pathogen.
  • the antigen may also comprise an isolated antigenic epitope of a known larger antigen, and even fusion constructs of multiple antigenic epitopes.
  • the antigen is combined with one or more detergents before the antigen is deposited onto the optical contrast layer.
  • the detergent renders the antigen more available to both non-specific and non-covalent binding to the optical contrast layer, and also more available within the other components of the unpurified antigen for antibody binding.
  • presence of the detergent is further thought to also render membrane bound and otherwise hydrophobic epitopes available for antibody binding.
  • the detergent is selected from the group of a polysorbate surfactant (e.g., Tween 80), an alkylglycoside (e.g., octylglycoside), an alkylsulfate (e.g., SDS), and various quaternary ammonium detergents (e.g., cetyl trimethylammonium bromide).
  • a polysorbate surfactant e.g., Tween 80
  • an alkylglycoside e.g., octylglycoside
  • an alkylsulfate e.g., SDS
  • various quaternary ammonium detergents e.g., cetyl trimethylammonium bromide.
  • the detergent will be present at a concentration of between about 0.01 wt% to about 1 wt%, and even more typically between about 0.05 wt% to about 0.1 wt%.
  • the antigen preparation is then applied onto the optical contrast layer using conventional methods such as pin spotting, or nano/picoliter syringe deposition.
  • Typical amounts of antigen deposited per spot will be between about 1 microliter and 1 picoliter, more typically between 100 nanoliter and 10 picoliter, and most typically between 50 nanoliter and 100 picoliter.
  • the layer provides an at least opaque surface (and even more preferably a non-transparent, white, or off-white surface) and that the contemplated that optical contrast layer also allows for non- specific and typically non-covalent coupling of the antigen to the optical contrast layer.
  • especially contemplated optical contrast layers will comprise nitrocellulose, polyvinylidenedifluoride (PVDF), or polyethersulfone (PES), preferably at a thickness between 10 and 100 microns, and most preferably at a thickness of between 10 and 20 microns (which allows deposition of antigen spots without confluence).
  • Preferred pore size of such membranes is typically between 0.2 micron and 0.5 micron.
  • the carrier may be rendered non-transparent using etching, staining, or sanding (where the carrier is glass or a transparent polymer).
  • the carrier is glass or a transparent polymer.
  • non- transparent layers are also deemed suitable for use herein.
  • the carrier and the optical contrast layers may form an integrated structure.
  • the carrier is a glass or polymer (optionally stained) slide and that the optical contrast layer is coupled to the slide using conventional methods.
  • Suitable array sizes will typically vary from test to test, however, it is generally preferred that the array is on a nitrocellulose (or other protein-binding) membrane having an area of less than 1 cm 2 , more typically less than 100 mm 2 , even more typically less than 50 mm 2 , and most typically less than 40 mm 2 .
  • Protein spot size will typically vary also, however, it is generally preferred that the spot size is between 10 ⁇ m and 1000 ⁇ m, more preferably between 50 ⁇ m and 500 ⁇ m, and most preferably between 100 ⁇ m and 300 ⁇ m (diameter or largest dimension). Therefore, contemplated arrays will have a density of protein spots between 1-1000 spots/cm 2 , more typically between 10-500 spots/cm 2 , and most typically between 100-300 spots/cm 2 .
  • a nitrocellulose membrane (12 micron thickness, 0.45 micron pore size) is configured as a square pad of approximately 6 mm by 6 mm in area, and is coupled to a glass slide.
  • Antigens typically unpurified antigens
  • the nitrocellulose layer is then coupled to a glass slide to form the bottom of a chamber, which is then covered by side walls and a top cavity layer ⁇ infra).
  • the chamber in such configurations will typically have a volume of between 3-4 microliter and is typically formed from the side walls and top cavity layer using standard soft lithography methods.
  • the antigen array is disposed within a closed chamber that is accessible only via fluid or reagent inflow port(s) and fluid or reagent outflow port(s).
  • fluid or reagent inflow port(s) and fluid or reagent outflow port(s) are accessible only via fluid or reagent inflow port(s) and fluid or reagent outflow port(s).
  • conventional Western blot lab-bench methods require pipettes to introduce reagent volumes atop an open-air chamber placed over the array. Reagent volumes in known methods are then removed via aspiration, thus allowing for ambient air contamination and reagent evaporation, which is typically resolved by incubating within a 4 °C environment.
  • the microfluidic approach contemplated herein uses a micro-scale platform enclosed within a low volume reaction chamber (e.g., made from poly-dimethylsiloxane (PDMS) and a glass slide) that reduces exposure and contamination to the outside environment as well as reagent evaporation. Reagent introduction is controllable and can be manipulated using a micro-syringe pump or other known micro fluidics methods.
  • a low volume reaction chamber e.g., made from poly-dimethylsiloxane (PDMS) and a glass slide
  • Reagent introduction is controllable and can be manipulated using a micro-syringe pump or other known micro fluidics methods.
  • antigen-antibody hybridization is additionally favored in preferred configurations by not only selecting a small chamber volume, but also by employing an active mixing configuration.
  • certain parameters e.g., ratio between mixing plate cavities, cavity volume and size, and array area.
  • active acoustic resonance 'on-chip' micromixing can be used enhance antibody-antigen binding collisions to overcome limitations associated with slow diffusion-based interactions.
  • the volume of contemplated chambers in which the nitrocellulose membrane is disposed it is generally preferred that the volume is between 0.1 ⁇ l and 100 ⁇ l, more preferably between 1 ⁇ l and 50 ⁇ l, and most preferably between 2 ⁇ l and 20 ⁇ l.
  • Suitable chambers will have at least one, and more typically two fluid ports for entry and exit of reagents and samples, and fluid entry may be parallel or perpendicular to the nitrocellulose membrane.
  • the chamber has a roof portion (typically opposite to the array) that is configured as a cavity layer to include a plurality of (most preferably circular) cavities with 100 micrometers in diameter and 25 micrometers in height, wherein the cavities are arrayed along the top surface of the inner chamber.
  • the cavities form air pockets during reagent introduction (to so permit bubble formation) that can act as a micromixer when acoustically resonated.
  • the chamber is closed by a cover that is preferably bonded to the structure over the pad using plasma oxidation surface treatment.
  • microfluidic device 100 has a transparent glass carrier 110 to which opaque optical contrast layer 120 is coupled.
  • Intermediate layer 130 (preferably transparent) is shaped to form the sidewalls of chamber 140 that defines the reaction volume.
  • Chamber 140 receives fluids from inlet port 152 (or internal reservoir, not shown) and delivers fluid from the chamber to an internal reservoir 132 that may be formed within the intermediate layer 130.
  • the chamber 140 is also fluidly coupled to an outlet or venting port 154.
  • a cavity layer 160 is disposed on top of the intermediate layer 130 and has a plurality of cavities 162, wherein the cavity layer also forms the roof of the reaction volume 140.
  • the optical contrast layer 120 Opposite the cavity layer is the optical contrast layer 120 to which a plurality of distinct and typically unpurified antigens 170 are non-specifically and non-covalently coupled. Bound to at least one of the antigens is serum antibody 172 and secondary detection antibody 174 that comprises an enzyme or other entity suitable for enzymatic conversion of a chromogenic dye to a dye (not shown) that is insoluble in the detection medium to so form a visually detectable colored signal in situ.
  • the topmost layer 150 is preferably a transparent layer that concludes the architecture of the micro fluidics device. Of course, it should be appreciated that two or more of the distinct layers shown in the figure may be combined into a single layer.
  • Figure 2 depicts another exemplary device in which multiple nitrocellulose membranes are disposed on a glass slide and in which multiple protein antigens are arrayed in a horizontal line, and in which each line represents a different pathogen.
  • contemplated microfluidic devices will typically comprise an enclosed reaction volume formed by a carrier material such that an optical contrast layer is disposed within the reaction volume and opposite to a cavity layer within the reaction volume, wherein a plurality of distinct (typically non-purified) antigens are non-covalently and non-specifically coupled to the optical contrast layer in predetermined positions.
  • the cavity layer has plurality of cavities (preferably opposite the contrast layer) that are sized and dimensioned to allow trapping of air in the plurality of cavities, wherein the number and/or size of the cavities is selected such that hybridization of an antibody to the antigens is substantially complete within less than 120 minutes, more typically within less than 60 minutes, and most typically within less than 45 minutes upon mixing.
  • the cavities in the cavity layer in contemplated microfluidic devices are circular, and perpendicularly arranged at regular intervals along an x- and y-coordinate.
  • the ratio between the number of antigens and the number of cavities is at least 2:1, more preferably at least 3:1, and most preferably at least 4:1, and/or the ratio between the area of an antigen spot and the cavity diameter is at least 1 :2, more preferably at least 1 :3., and most preferably at least 1 :4.
  • the carrier material and the optical contrast layer are configured to allow quantitative detection over a dynamic range of at least three orders of magnitude, and quantitative detection is based on a dye that is insoluble in the detection solution to so form a visually detectable spot.
  • the device portion, and particularly the portion that is above and/or below the optical contrast layer will preferably be transparent.
  • detection of a hybridization event of an antibody to an antigen on the nitrocellulose membrane (or other surface of the optical contrast layer) is mediated by formation of a visually detectable dye, which most preferably precipitates or otherwise localizes to the site of dye formation. It is generally preferred that such dye formation is mediated by an enzyme that reacts with a soluble, chromogen to form a colored (or gray) and preferably precipitating dye.
  • detection may also involve local concentration of colloidal gold (or other metal) which also leads to formation of a visually detectable dye (can be detected with the unaided eye).
  • contemplated colorimetric detection systems provide several advantages over heretofore alternative, but very limited detection methods (e.g., fluorescence or lumincescence).
  • fluorometric reaction products require excitation at particular wavelengths and thus necessitate expensive and large-scale equipment that is both fragile and expensive.
  • luminometric detection requires highly sensitive detectors and often chemically labile reagents.
  • colorimetric detection produces a reaction product that is detectable at visible wavelengths and thus can be analyzed using ordinary optical methods yielding the potential for portability.
  • detection is performed using a CCD detector, which may be in a camera or scanner format.
  • the detection may include magnifying optics, which may be associated with the CCD detector and/or with the housing.
  • the housing may include a lens that provides magnification for the detector, or the detector is a scanner and the magnification is electronic (e.g., based on 1200 dpi or higher resolution).
  • especially preferred methods include those in which a carrier having a surface that comprises an optical contrast layer is provided, wherein a plurality of detergent- containing non-purified distinct antigen preparations are non-covalently and non-specifically coupled to the optical contrast layer at respective predetermined locations to form an antigen array having a density of at least 10 distinct antigen preparations per cm 2 .
  • the optical contrast layer has a thickness and composition sufficient to prevent confluence of the antigen preparations when the distinct antigen preparations are deposited onto the optical contrast layer.
  • the antigen array is then contacted with a solution comprising an antibody (typically human blood or serum) under conditions that allow binding of the antibody to an antigen of at least one of the antigen preparations.
  • Binding of the antibody is detected using a visually detectable, and precipitating or agglomerating dye using secondary antibodies (or fragments thereof) that most typically comprise an enzyme or ligand suitable for a subsequent reaction that forms from a chromogen a precipitating dye.
  • Such methods advantageously allow concurrent, fast, and simple multiple pathogen detection using nothing more than a minute blood sample and reagents (which may be stored within one or more compartments of the device. Analysis can then be visually performed, and where quantitative analysis is desired, analysis may also include electronic image analysis with or without optical magnification.
  • preferred antigens are typically unpurified or only partially purified.
  • diagnostic applications it is particularly preferred that the array comprises at least two distinct antigens from the same pathogen, most preferably with known quantified and known relative reactivities with respect to sera of a population infected with the pathogen.
  • diagnostic devices will allow a person not only to ascertain presence or absence of a pathogen, but also to obtain information on the type (chronic, acute, etc.) and/or stage (early, late, treated, etc.) of a disease, and even prognosis of responsiveness to treatment.
  • PCR amplification of linear acceptor vector Plasmid pXT7 (10 ⁇ g; 3.2 kb, KanR) was linearized with BamHI (0.1 ⁇ g/ ⁇ l DNA/0.1 mg/ml BSA/0.2 units/ ⁇ l BamHI; 37 0 C for 4 hr; additional BamHI was added to 0.4 units/ ⁇ l at 37°C overnight). The digest was purified using a PCR purification kit (Qiagen, Valencia, CA), quantified by fluorometry using
  • Picogreen (Molecular Probes, Carlsbad, CA) according to the manufacturer's instructions, and verified by agarose gel electrophoresis (1 ⁇ g).
  • One ng of this material was used to generate the linear acceptor vector in a 50- ⁇ l PCR using 0.5 ⁇ M each of suitable primers, and 0.02 units/ ⁇ l Taq DNA polymerase (Fisher Scientific, buffer A)/0.1 mg/ml gelatin (Porcine, Bloom 300; Sigma, G-1890)/0.2 mM each dNTP with the following conditions: initial denaturation of 95°C for 5 min; 30 cycles of 95°C for 0.5 min, 50°C for 0.5 min, and 72°C for 3.5 min; and a final extension of 72°C for 10 min.
  • PCR amplification of ORF insert A total of 1-10 ng of genomic DNA (e.g., Plasmodium falciparum 3D7 strain) was used as template in a 50- ⁇ l PCR using suitable primers (0.5 ⁇ M each). PCR was carried out using regular Taq DNA polymerase: 0.02 units/ ⁇ l TaqDNA polymerase (buffer A, Fisher Scientific)/0.1 mg/ml gelatin (Bloom 300, Porcine; G-1890, Sigma)/0.2 mM each dNTP.
  • Conditions were as follows: initial denaturation of 95 0 C for 5 min; 30 cycles of 20 sec at 95°C, 30 sec at 50 0 C, and 60 sec/kb at 72°C (1-3 min on average, based on ORF size); and a final extension of 72°C for 10 min.
  • PCR products that were more difficult to produce were reamplified by using a 30 sec annealing time at 45°C or 40 0 C, instead of 30 sec at 50 0 C.
  • the extension temperature was decreased from 65-72°C to 5O 0 C.
  • PCR products were obtained using a Taq polymerase with improved proof-reading characteristics (Triplemaster from Eppendorf), increasing the efficiency of the PCR: 0.04 units/ ⁇ l Triple Master PCR system (high-fidelity buffer, Eppendorf)/0.4 mM each dNTP (Eppendorf).
  • Conditions were as follows: initial denaturation of 95 0 C for 3 min; 35 cycles of 15 sec at 95 0 C, 30 sec at 40 0 C, and 60 sec/kb at 50 0 C (1-3 min on average, based on ORF size); and a final extension of 50 0 C for 10 min.
  • PCR products that were difficult were reamplif ⁇ ed using 50 ng genomic DNA.
  • the PCR product was visualized by agarose gel electrophoresis (3 ⁇ l).
  • the product was purified (PCR purification kit, Qiagen) and quantified by fluorometry.
  • Competent cells were prepared by growing DH5 ⁇ cells at 18°C in 500 ml of SOB (super optimal broth) medium (2% tryptone/0.5% yeast extract/10 mM NaCl/2.5 mM KCl/20 mM MgSO 4 ) to an OD of 0.5-0.7. The cells were washed and suspended in 10 ml of pre-chilled PCKMS buffer (10 mM Pipes/15 mM CaCl 2 /250 mM KCl/55 mM MnCl 2 /5% sucrose, pH 6.7) on ice, and 735 ⁇ l of DMSO was added dropwise with constant swirling.
  • SOB super optimal broth
  • PCKMS buffer 10 mM Pipes/15 mM CaCl 2 /250 mM KCl/55 mM MnCl 2 /5% sucrose, pH 6.7
  • the competent cells were frozen on dry ice-ethanol in 100- ⁇ l aliquots and stored at -80 0 C. Each transformation consisted of the following: 10 ⁇ l of competent DH5 ⁇ and 10 ⁇ l of DNA mixture (40 ng of PCR-generated linear vector/ 10 ng of PCR-generated ORF fragment; molar ratio, 1:1; vector, 1-kb ORF fragment). For transformation, the purification of PCR product was unnecessary.
  • the mixture was incubated on ice for 45 min, heat shocked at 42 0 C for 1 min, and chilled on ice for 1 min; mixed with 250 ⁇ l of SOC (super optimal catabolizer) medium (2% tryptone/0.55% yeast extract/10 mM NaCl/10 mM KCl/10 mM MgCl 2 AO mM MgS(V20 mM glucose); incubated at 37°C for 1 hr; diluted into 3 ml of LB medium supplemented with 50 ⁇ g of kanamycin per ml (LB Kan 50); and incubated with shaking overnight.
  • SOC super optimal catabolizer
  • Plasmid templates used for in vitro transcription/translation were prepared by using QIAprep Spin Miniprep kits (Qiagen), including the "optional” step, which contains protein denaturants to deplete RNase activity.
  • In vitro transcription/translation reactions RTS 100 Escherichia coliHY kits; Roche) were set up in 25 ⁇ l PCR 12-well strip tubes and incubated for 5 h at 30 0 C, according to the manufacturer's instructions.
  • Immuno-dot blots To assess relative efficiency of protein expression, 0.3 ⁇ l of whole rapid-translation system (RTS) reactions were spotted manually onto nitrocellulose and allowed to air dry before blocking in 5% nonfat milk powder in TBS containing 0.05% Tween 20. Blots were probed with hyperimmune sera diluted to 1:1,000 in blocking buffer with or without 10% E. coli lysate.
  • RTS rapid-translation system
  • dot blots were stained with both mouse anti- poly-HIS mAb (clone, HIS-I; H- 1029, Sigma) and rat anti-hemagglutinin (HA) mAb (clone, 3F10; 1 867 423, Roche), followed by alkaline phosphatase-conjugated goat anti-mouse IgG (H+L) (BioRad) or goat anti-rat IgG (H+L) (Jackson ImmunoResearch) secondary Abs, respectively. Bound human Abs were visualized with nitroblue tetrazolium (nitro-BT) developer to confirm the presence of protein.
  • nitro-BT nitroblue tetrazolium
  • Microarray chip printing For microarrays, 10 ⁇ l of 0.125% Tween 20 was mixed with 15 ⁇ l of RTS reaction (to a final concentration of 0.05 wt% Tween 20), and 15- ⁇ l volumes were transferred to 384-well plates. The plates were centrifuged at 1,600 x g to pellet any precipitate, and supernatant was printed without further purification onto nitrocellulose- coated FAST glass slides (Schleicher & Schuell; in general, microporous nitrocellulose coated onto glass at a thickness of about 12-14 micron, sufficiently flat to allow laminar flow, and enclosed in a reaction volume of less than 20 microliter) by using an OmniGrid 100 microarray printer (Genomic Solutions, Ann Arbor, MI). All ORFs were spotted in duplicate to enable statistical analysis of the data. Data values reported herein represent the average of pairs, hi addition, each chip contained an area printed with controls consisting of RTS reaction using no DNA.
  • a typical device can be constructed according to the following 4-step protocol: (1) A pre-spotted protein array nitrocellulose pad is coupled to a glass slide and the pad is masked using a substrate that is minimally affected by plasma oxidation (e.g., glass, PDMS, polycarbonate). (2) The pre-spotted protein array nitrocellulose pad is then placed in a plasma cleaner to activate the glass surface of the slide and the PDMS of a prefabricated microfluidics system (that provides the fluid channels, reservoirs, cavity layer, and remaining portion of the reaction volume) for irreversible binding. The two portions are placed into the plasma chamber for a set amount of time (typically 1.5 min) before binding takes place.
  • a substrate that is minimally affected by plasma oxidation e.g., glass, PDMS, polycarbonate.
  • the substrate used to mask the proteins is removed and the PDMS microsystem is aligned and placed in contact with the nitrocellulose coated glass substrate.
  • the contact between the glass and PDMS forms an irreversible seal after a plasma treatment has taken place. However, if an extended period of time occurs after plasma treatment and PDMS-glass contact is made then irreversible binding will not take place.
  • both parts can be placed in an inert gas, for example, for an extended binding window. Binding the two substrates will allow for an irreversible bound between the PDMS and glass even after long periods of time after plasma treatment. Through irreversible binding the PDMS microsystem to the glass substrate, it allows for an environmentally protected protein array within the system.
  • Bubble mixing parameters Characterization of bubble cavity distances and its effect on resonance was done on a polycarbonate substrate. Cavities approximately 800 microns in diameter were machine drilled at 400 microns deep in a 4 by 4 array, a total of 16 cavities. Results suggest that shorter cavity to cavity distances for the bubbles produce higher resonance intensities. This can be seen in Figure 3, showing a plot of resonance intensity for varying bubble cavity distances for an 800 micron bubble over a time scale of 5 seconds for the 400 micrometer distance. Similarly, Figure 4 depicts a plot of maximum resonance intensities for varying ratios of bubble cavity distances to cavity size of an 800 micron diameter bubble in which the ratio of 0.5, or 400 micrometer distance divided by 800 micron diameter, has the highest resonance.
  • Secondary reagent comprised of 0.5% anti-human IgA IgG IgM (H+L) alkaline phosphatase-co ⁇ jugated Affini Pure Goat diluted in blocking buffer.
  • Substrate solution comprised of 5-bromo-4-chloro-3 indolyl phosphate (BCIP) p-Toluidine salt, nitroblue tetrazolium (NBT), and AP developing buffer.
  • precipitating dyes include 3-amino-9-ethylcarbazole, 5-bromo-4-chloro-3-indolylphosphate, 3-3'- diaminobenzidine tetrachloride, 3,3',5,5'-tetramethylbenzidine, and even colloidal metals. Further suitable compounds are described in US 6,251,618, which is incorporated by reference herein.
  • a comparison test was done between conventional testing methods versus the microfluidic approach using analogous procedures, reagents, and fabrication setups as depicted in the tables below.
  • the microfluidic device's 'on-chip' acoustic micromixer was set at a frequency of 3.6 kHz and amplitude of 30Vpp (Volt peak-to-peak) and 20Vpp (Volt peak-to-peak) for step 2 and step 4, respectively.
  • Step 2 was performed in a flow and stop method in which flow was administered for 15 seconds then stopped for 30 seconds and restarted in a repetitive cycle totaling 10 minutes and 30 seconds.
  • Results for the following microfluidic design suggests a potential detection system for reducing reagent volumes and testing times while still maintaining high levels of sensitivity unachievable through conventional lab-bench processes.
  • Figure 9 exemplarily depicts the high correlation between test results obtained by fluorescence detection and colorimetric dye detection using over 4600 antigens.

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Abstract

La présente invention concerne des dispositifs et des procédés microfluidiques appliqués à des puces à protéines dans lesquels des préparations antigéniques distinctes et contenant un détergent sont déposées sur une couche de contraste optique de manière non spécifique et non covalente. La détection de liaison s'effectue en utilisant un colorant qui précipite ou s'agglomère de façon à former un signal visuellement détectable à gamme dynamique d'au moins trois ordres de grandeur.
PCT/US2008/006431 2007-05-18 2008-05-19 Dispositifs et procédés microfluidiques WO2008144054A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030108949A1 (en) * 2001-07-03 2003-06-12 Gang Bao Filtration-based microarray chip
US20070020678A1 (en) * 2002-10-30 2007-01-25 Dana Ault-Riche Methods for producing polypeptide-tagged collections and capture systems containing the tagged polypeptides

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030108949A1 (en) * 2001-07-03 2003-06-12 Gang Bao Filtration-based microarray chip
US20070020678A1 (en) * 2002-10-30 2007-01-25 Dana Ault-Riche Methods for producing polypeptide-tagged collections and capture systems containing the tagged polypeptides

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