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WO2006062745A2 - Procede d'isolement de micro-reactions chimiques independantes et paralleles mettant en oeuvre un filtre poreux - Google Patents

Procede d'isolement de micro-reactions chimiques independantes et paralleles mettant en oeuvre un filtre poreux Download PDF

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Publication number
WO2006062745A2
WO2006062745A2 PCT/US2005/042522 US2005042522W WO2006062745A2 WO 2006062745 A2 WO2006062745 A2 WO 2006062745A2 US 2005042522 W US2005042522 W US 2005042522W WO 2006062745 A2 WO2006062745 A2 WO 2006062745A2
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WIPO (PCT)
Prior art keywords
membrane
membrane reactor
wells
planar array
array layer
Prior art date
Application number
PCT/US2005/042522
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English (en)
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WO2006062745A3 (fr
Inventor
Said Attiya
Vinod B. Makahijani
Ming Lei
Yi-Ju Chen
John Simpson
G. Thomas Roth
Chun Heen Ho
Yu Pengguang
Original Assignee
454 Life Sciences Corporation
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Publication date
Priority claimed from US11/016,942 external-priority patent/US20060019264A1/en
Priority claimed from US11/217,194 external-priority patent/US20060088857A1/en
Application filed by 454 Life Sciences Corporation filed Critical 454 Life Sciences Corporation
Publication of WO2006062745A2 publication Critical patent/WO2006062745A2/fr
Publication of WO2006062745A3 publication Critical patent/WO2006062745A3/fr

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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
    • B01L3/5025—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures for parallel transport of multiple samples
    • B01L3/50255—Multi-well filtration
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J19/0046—Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00279—Features relating to reactor vessels
    • B01J2219/00281—Individual reactor vessels
    • B01J2219/00286—Reactor vessels with top and bottom openings
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00279—Features relating to reactor vessels
    • B01J2219/00306—Reactor vessels in a multiple arrangement
    • B01J2219/00313—Reactor vessels in a multiple arrangement the reactor vessels being formed by arrays of wells in blocks
    • B01J2219/00315—Microtiter plates
    • B01J2219/00317—Microwell devices, i.e. having large numbers of wells
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00351—Means for dispensing and evacuation of reagents
    • B01J2219/00353—Pumps
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00351—Means for dispensing and evacuation of reagents
    • B01J2219/00414—Means for dispensing and evacuation of reagents using suction
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00351—Means for dispensing and evacuation of reagents
    • B01J2219/00423—Means for dispensing and evacuation of reagents using filtration, e.g. through porous frits
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00457—Dispensing or evacuation of the solid phase support
    • B01J2219/00459—Beads
    • B01J2219/00466—Beads in a slurry
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00457—Dispensing or evacuation of the solid phase support
    • B01J2219/00459—Beads
    • B01J2219/00468—Beads by manipulation of individual beads
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00497—Features relating to the solid phase supports
    • B01J2219/005—Beads
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00596—Solid-phase processes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/0068—Means for controlling the apparatus of the process
    • B01J2219/00702—Processes involving means for analysing and characterising the products
    • B01J2219/00704—Processes involving means for analysing and characterising the products integrated with the reactor apparatus
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00718—Type of compounds synthesised
    • B01J2219/0072—Organic compounds
    • B01J2219/00722—Nucleotides
    • 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
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
    • B01L2200/0668—Trapping microscopic beads
    • 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
    • B01L2300/0819—Microarrays; Biochips
    • 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
    • B01L2400/0487—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure fluid pressure, pneumatics
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing

Definitions

  • the invention describes methods and apparatuses for conducting densely packed, independent chemical reactions in parallel in a membrane reactor with mobile supports disposed thereon.
  • One approach for conducting chemical reactions in a high throughput manner includes performing larger numbers of independent steps in parallel, and specifically conducting simultaneous, independent reactions with a multi-reactor system.
  • a common format for conducting parallel reactions at high throughput levels comprises two-dimensional (2-D) arrays of individual reactor vessels, such as the 96-well or 384-well microtiter plates widely used in molecular biology, cell biology, and other areas. Individual reagents, solvents, catalysts, and the like are added sequentially and/or in parallel to the appropriate wells in these arrays, and multiple reactions subsequently proceed in parallel.
  • Individual wells may be further isolated from adjacent wells and/or from the environment by sealing means (e.g., a tight-fitting cover or adherent plastic sheet) or they may remain open.
  • sealing means e.g., a tight-fitting cover or adherent plastic sheet
  • the base of the wells in such microtiter plates may or may not be provided with filters of various pore sizes.
  • Yet another widely applied approach for conducting miniaturized and independent reactions in parallel involves spatially localizing or immobilizing at least some of the participants in a chemical reaction on a surface.
  • Reagents immobilized in such a manner include chemical reactants, catalysts, other reaction auxiliaries, and adsorbent molecules capable of selectively binding to complementary molecules.
  • Microarray techniques involving immobilization on planar surfaces have been commercialized for the hybridization of oligonucleotides (e.g. by Affymetrix, Inc.) and for target drugs (e.g. by Graffmity, AB).
  • a major obstacle to creating microscopic, discrete centers for localized reactions is that restricting unique reactants and products to a single, desired reaction center is frequently difficult.
  • the first is that "unique" reagents - i.e., reactants and other reaction auxiliaries that are meant to differ from one reaction center to the next — must be dispensed or otherwise deployed to particular reaction centers and not to their nearby neighbors.
  • Such "unique” reagents are to be distinguished from “common” reagents like solvents, which frequently are meant to be brought into substantial contact with all the reaction centers simultaneously and in parallel.
  • the second aspect of this problem has to do with restricting reaction products to the vicinity of the reaction center where they were created - i.e., preventing them from traveling to other reaction centers with attendant loss of reaction fidelity.
  • reaction centers can consists of discrete microwells with the micro vessel walls (and cover, if provided) designed to prevent fluid contact with adjacent microwells.
  • delivery of reagents to individual microwells can be difficult, particularly if the wells are especially small.
  • a reactor measuring 100 ⁇ m X 100 ⁇ m X 100 ⁇ m has a volume of only 1 nanoliter. This can be considered a relatively large reactor volume in many types of applications. Even so, reagent addition in this case requires that sub-nanoliter volumes be dispensed with a spatial resolution and precision of at least ⁇ 50 ⁇ m.
  • reaction centers can be brought into contact with a common fluid, e.g., such that microwells all open out onto a common volume of fluid at some point during the reaction or subsequent processing steps.
  • a common fluid e.g., such that microwells all open out onto a common volume of fluid at some point during the reaction or subsequent processing steps.
  • this can cause the reaction products (and excess and/or unconverted reactants) originating in one reaction microwell or vessel to travel and contaminate adjacent reaction microwells.
  • Such cross-contamination of reaction centers can occur (i) via bulk convection of solution comprising reactants and products from the vicinity of one well to another, (ii) by diffusion (especially over reasonably short distances) of reactant and/or product species, or (iii) by both processes occurring simultaneously.
  • the individual chemical compounds that are produced at the discrete reaction centers are themselves the desired objective of the process (e.g., as is the case in combinatorial chemistry).
  • any reactant and/or product cross- contamination that may occur will reduce the yield and ultimate chemical purity of this library of discrete products.
  • the reaction process is conducted with the objective of obtaining information of some type, e.g., information as to the sequence or composition of DNA, RNA, or protein molecules.
  • information e.g., information as to the sequence or composition of DNA, RNA, or protein molecules.
  • the integrity, fidelity, and signal-to-noise ratio of that information may be compromised by chemical "cross-talk" between adjacent or even distant microwells.
  • the invention encompasses novel membrane-based arrays that allow for effective trapping of mobile supports (e.g., beads or particles), fast reagent exchange, and controlled microfluidic flow.
  • the invention further encompasses novel methods for densely packing mobile supports. This technique provides not only dense packing of reaction sites, microvessels, and reaction wells, but also provides for efficient delivery of reagents and removal of products by convective flow rather than by diffusion alone. This latter feature permits much more rapid delivery of reagents and other reaction auxiliaries. In addition, it permits faster and more complete removal of reaction products and by-products than has heretofore been possible using methods and apparatus described in the prior art.
  • the invention pertains generally to microfluidic devices, membrane engineering, microfabrication, and convective flow methods. The present invention finds use in numerous applications including DNA sequencing, drug discovery, microimaging, microchemical reactions, substrate treatment, and high throughput screening.
  • One embodiment of the invention is directed to a membrane reactor comprising a porous membrane layer attached to a planar mesh array.
  • the planar mesh comprises a plurality of openings with reactant- or reagent-carrying mobile supports of an appropriate size disposed in the openings.
  • an appropriate size is one whereby the mobile supports are retained in the openings of the mesh.
  • the mesh array is permeable to an aqueous fluid, such as a fluid or reagent used in sequencing but the mesh array is not permeable to the reagent- or reactant-carrying mobile supports.
  • the planar mesh array is weaved from individual fibers with a spacing of less than about 100 ⁇ m center to center.
  • the weaving may be made from two sets of parallel fibers that intersect at right angles. In other words, the weaving may be similar to the strings on a tennis racket at a microscopic scale.
  • Another embodiment of the invention is directed to a membrane reactor comprising a porous membrane and a planar array which is fabricated above the top surface of the membrane.
  • the planar array comprises a plurality of wells for trapping mobile supports.
  • the pores in the membrane are sufficiently sized such that the membrane is permeable to fluids but impermeable to the mobile supports.
  • Each well in the array has an opening of less than about 40 ⁇ m. That is, for an array with a well size of 40 ⁇ m, each mobile support should be somewhat smaller than 40 ⁇ m in diameter, hi a preferred embodiment, the mobile supports are 2 - 3 ⁇ m smaller than the well width. This relationship between mobile support size and well size also ensures that only one or fewer mobile supports are immobilized to a well.
  • a plurality of wells in the planar fabricated array comprise one or fewer mobile supports.
  • the array is in direct or indirect contact with the top surface of the porous supporting membrane.
  • the array is contacted with a fluidic stream (e.g., vertical or near-vertical) to maintain the mobile supports in the wells by convective force.
  • the fiuidic stream also carries reagents for reacting with chemical groups on the mobile supports.
  • Micropores in the membrane allow flow-through and provide flow resistance for the membrane reactor.
  • the wells comprise sidewalls and bottoms to reduce physical and chemical cross-talk between the wells. Opaque sidewalls in the wells prevent optical crosstalk, while opaque bottoms prevent optical bleeding between the wells.
  • the sidewalls and bottoms for the wells also concentrate the optical signal generated by the mobile support. The signals generated by reactions in the wells are detected by optical or electronic means.
  • Another embodiment of the invention is directed to a method of loading a membrane reactor with mobile supports, hi the method, a membrane that is substantially permeable to a fluid but substantially impermeable to a population of mobile supports is provided.
  • a planar array comprising wells is positioned above this membrane.
  • a fluid comprising a suspension of said population of mobile supports is introduced onto the surface of the array.
  • the mobile supports may be linked to a sample (e.g., nucleic acid or peptide) or they may be unlinked.
  • the mobile supports are settled onto the wells of the array, preferably using a pump or negative pressure or suction. Settling may be performed, for example by allowing the mobile supports to slowly settle out of solution under gravity. Another method of settling may involve centrifugation.
  • the fluid is drawn through the array and membrane. Since the mobile supports are larger than the pores of the membrane, they are trapped (loaded) in the wells of the array as the fluid is drawn through.
  • Another embodiment of the invention is directed to a method of identifying a base at a target position (e.g., sequencing) in one or more sample nucleic acid, preferably DNA.
  • the sequencing reaction is a pyrophosphate sequencing reaction, hi one aspect of the method, the sample DNA is immobilized on a mobile support on the membrane reactor.
  • An extension primer is used to hybridize to the sample DNA immediately adjacent to the target position.
  • the extension primer is subjected to a polymerase reaction in the presence of a deoxynucleotide or dideoxynucleotide so that the deoxynucleotide or dideoxynucleotide will only become incorporated and release pyrophosphate (PP 1 ) if it is complementary to the base in the target position.
  • PP 1 pyrophosphate
  • the light emissions are generated directly or through a chemical pathway involving additional chemical steps or amplification steps.
  • the sequencing reagents including the deoxynucleotides or dideoxynucleotides, are contacted to the nucleic acid by a flow of reagent that is normal (i.e., orthogonal, perpendicular) to the plane of the membrane reactor. Because the flow is normal to the plane of the mobile supports, each fluid stream will only contact one mobile support or one species of nucleic acid before it is disposed into a waste container. Such reagent flow is useful for reducing or eliminating cross contamination between wells in the array.
  • the deoxynucleotides or dideoxynucleotides are added successively to the sample- primer mixture and subjected to the polymerase reaction to indicate which deoxynucleotide or dideoxynucleotide is incorporated.
  • Another embodiment of the invention is directed to a microimaging system for imaging a light emission (e.g., from a pyrophosphate sequencing reaction) from a membrane reactor.
  • the system comprises one or more lens groups.
  • the first lens group is the front lens group which is positioned closer to the light source to be detected to collect the light that is emitted.
  • the second lens group is the rear lens group which is positioned closer to the light detector such as a CCD detection device to image the light onto the detector, hi a preferred embodiment, the lens groups comprise 50 mm lenses with an aperture larger than or equal to 2.8 (e.g., 2.0, 1.8, 1.4, 1.0, etc.).
  • the larger apertures are expressed by a smaller aperture value so that, for example, an aperture of 1 is larger than an aperture of 2.
  • the cartridge comprises a flow chamber for enclosing an above described membrane reactor.
  • a membrane supporting structure inside the flow chamber separates the flow chamber into two subchambers.
  • the first subchamber comprises the membrane reactor and also comprises an inlet and a first outlet for controlling a fluid flow tangential to the membrane reactor.
  • the first subchamber also comprises a window, covered with a transparent material such as glass or crystal, to allow the optical examination of the membrane reactor.
  • the second subchamber without the membrane reactor comprises a second outlet allowing fluid to flow normally (i.e., orthogonally) from the inlet, through the membrane reactor, and out through the second outlet, m this manner, both the tangential and normal flow of reagent through the membrane reactor may be regulated.
  • Another embodiment of the invention is directed to a method of amplifying a sample nucleic acid on a mobile support and then loading the mobile support on a membrane reactor.
  • one or more nucleic acid templates to be amplified are individually attached to separate mobile supports to form a population of nucleic acid template-carrying supports.
  • the template-carrying supports are suspended in an amplification reaction solution comprising reagents necessary to perform nucleic acid amplification.
  • An emulsion is formed to encapsulate the plurality of said template-carrying supports with PCR reaction solution to form a plurality of microreactors (see, e.g., U.S. Application Serial No. 60/476,504, filed June 6, 2003; U.S. Application Serial No.
  • nucleic acid templates in fluidic isolation from each other are then amplified to form multiple copies of nucleic acid templates.
  • the amplified nucleic acid templates, still in fluidic isolation, are attached to the mobile supports.
  • the mobile supports are loaded into the membrane reactor.
  • Another embodiment of the invention is directed to a method of producing a membrane reactor by providing one or more nucleic acid templates to be amplified, wherein a plurality of nucleic acid templates are individually attached to separate mobile supports to form a population of nucleic acid template-carrying supports.
  • the mobile supports are loaded onto the membrane reactor.
  • the template-carrying supports are contacted to an amplification reaction solution comprising reagents necessary to perform nucleic acid amplification.
  • the nucleic acid template is amplified in fluidic isolation from other templates to form amplified nucleic acid. Fluidic isolation may be achieved, for example, by removing most fluids from the membrane reactor and allowing amplification on the fluids that is still in contact with the mobile supports.
  • oil may be added to the membrane reactor to prevent evaporation during amplification, and then removed by organic solvents such as hexane.
  • Immobilization may be arranged to take place on any number of substrates, including planar surfaces and/or high surface area and sometimes porous support media such as beads or gels.
  • FIG. 1 represents an integral or physical composite of a microchannel array and a porous membrane barrier forming a membrane reactor.
  • the flow of fluid through the membrane reactor carries reaction participants along with the fluid.
  • FIG. 2 shows a schematic of one version of the experimental set-up for the convective flow sequencing apparatus described herein.
  • FIG. 3 shows a membrane reactor comprising a nylon mesh membrane useful for trapping reagent- or reactant-carrying mobile supports and one embodiment of the mobile supports. Shown here are Sepharose beads.
  • FIGS. 4A-4B show the size of the Sepharose beads relative to the membrane pores.
  • FIG. 4A the beads are shown to be swollen in liquid.
  • FIG. 4B shows how the beads are shrunken when dry.
  • FIG. 5A represents a membrane holder with a circular optical window; a flow chamber with an inlet port and a first outlet port; a nylon membrane with sepharose beads; a fine pore nylon membrane; a membrane support structure with 1.02 mm holes, 1.35 pitch; and a funnel-shaped collector with a second outlet port.
  • FIG. 5B represents a support structure.
  • FIG. 6 A shows a membrane support structure with a 50 mm capillary plate with 10 ⁇ m holes and a 12 ⁇ m pitch for supporting the membrane reactor array.
  • FIG. 6B shows 5X magnification of the membrane.
  • FIG. 6C shows 4OX magnification of the membrane.
  • FIG. 7 shows a schematic of a pyrophosphate-based sequencing method with photon detection.
  • FIG. 8 shows an automated convective sequencing apparatus.
  • FIG. 9 shows a sequencing pyrogram indicating the results of sequence analysis.
  • the pyrogram sequence top; SEQ ID NO:5) and signal intensity sequence (bottom; SEQ ID NO:10) are shown.
  • FIGS. 10A- 1OB show a sequencing graph.
  • FIG. 1OA shows the results for the negative control (no template added), where no sequence was detected.
  • FIG. 1OB shows the results for the template, where the correct sequence was detected (SEQ ID NO:11).
  • FIG. HA shows a schematic for the coupling of amine-primer and amine-biotin to NHS activated sepharose beads.
  • FIG. HB shows a schematic for the addition of biotinylated sulfurylase and luciferase.
  • FIG. 12 represents a side-view of a single well of the planar array layer of the membrane reactor.
  • the well encloses one bead and comprises opaque sidewalls and an opaque or reflective bottom. .
  • the bead is positioned over a porous membrane layer which is permeable to a microfluidic flow path.
  • FIG. 13 A represents a side-view of the porous membrane layer.
  • FIG. 13B depicts a top view of the porous membrane layer.
  • FIG. 13C depicts a porous membrane layer being positioned with forceps.
  • FIGS. 14A-14B show schematic steps for the construction of a membrane reactor. Features are not drawn to scale.
  • FIG. 14A depicts metal deposition on the porous membrane layer.
  • FIG. 14B depicts photoresist coating on the porous membrane layer.
  • FIG. 14C depicts the photolithography process.
  • FIG. 14D depicts development of the photoresist.
  • FIG. 14E depicts the electroplating process.
  • FIG. 14F depicts removal of the photoresist.
  • FIG. 14G depicts optional gold, cliromium, or titanium etching.
  • FIG. 14H depicts bead loading and trapping.
  • FIG. 141 depicts the fluid convention process.
  • FIG. 15 represents a top view of two wells of a planar array layer positioned over a porous membrane layer. Features are not shown to scale.
  • FIGS. 16A-16D represent various well configurations.
  • FIG. 17 shows the optical paths in a well in a planar array layer.
  • the sidewalls of the well concentrate light and reduce optical bleeding.
  • the bottom of the well reduces light scattering and increases reflection.
  • FIGS. 18A-18B shows an example of a mold plating system for producing electroplated microstructures (see, e.g., J.B. Lee, University of Texas).
  • FIG. 18A depicts the mold.
  • FIG. 18B depicts the electroplated microstructure.
  • FIG. 19A represents a membrane reactor with sidewalls and structural support beams.
  • a post structure is shown with a square cross-section, arranged in hexagonal shape.
  • FIGS 19B- 19C depict different membrane configurations.
  • the cross-sections of the posts are in diamond or circular shapes, while the array is square or hexagonal.
  • FIG. 19C a reversed structure is shown in which wells are formed on substrate surface or in a built-on top layer.
  • FIG. 20 represents a membrane reactor in contact with a CCD imaging screen.
  • FIG. 21 A represents a cross-section of a membrane reactor showing the planar array layer and porous membrane layer.
  • FIG. 21B shows schematic steps for the construction of a membrane reactor including metal deposition (e.g., chromium) on the substrate, photolithography, electroplating, and removal of the photoresist and chromium layers.
  • metal deposition e.g., chromium
  • FIG. 22 represents various gaskets, holders, rivets, and supports for the planar array and the porous membrane for the membrane reactor.
  • FIG. 23A depicts a membrane reactor comprising sloped wells and beads.
  • FIG. 23B represents a cross-section of the sloped wells and beads.
  • the membrane reactor is a general term that describes both the confined membrane reactor array (CMRA) and the unconfined membrane reactor array (UMRA) as described by U.S. Application Serial No. 10/191,438 filed July 8, 2002, the entire contents of which are incorporated herein by reference. Methods and apparatuses are described here for providing a. dense array of discrete reaction sites, microreactor vessels, and/or microwells (see Fig. 2) and for charging such microreactors with reaction participants by affecting a convective flow of fluid normal to the plane of and through the array of reaction sites or microvessels.
  • the convective flow or delivery of reactants includes both the delivery of sequencing reactants (e.g., dNTPs) towards the reaction site, and convective removal of excess reactants away from the site.
  • Fluid flow sweeps the sequencing reaction products (e.g., pyrophosphate (PPj), ATP) through the reaction region in a normal direction thus countering back-diffusion and resultant contamination.
  • sequencing reactants e.g., dNTPs
  • PPj pyrophosphate
  • ATP pyrophosphate
  • Reaction participants that may be charged, concentrated, and contained within said reaction sites or microreactor vessels by methods of the present invention include high- molecular-weight reactants, catalysts, and other reagents and reaction auxiliaries.
  • high-molecular-weight reactants include, for example, oligonucleotides, longer DNA/RNA fragments, and constructs thereof.
  • These reactants may be free and unattached (if their molecular weight is sufficient to permit them to be contained by the method of the present invention), or they may be covalently bound to or otherwise associated with, e.g., high-molecular- weight polymers, high-surface-area mobile supports, or gels, or other supports known in the art.
  • reaction catalysts examples include enzymes, which may or may not be associated with or bound to solid phase supports such as porous or non-porous mobile supports (e.g., beads or particles).
  • enzymes such as polymerase may be attached to supports as a reagent, hi addition, additional polymerase may be delivered during a reaction to replenish, or supplement the bound polymerase.
  • a reagent e.g., polymerase
  • Any reagent or reactant of this invention may be both free and bound as described herein.
  • the present invention also includes a means for efficiently supplying relatively lower- molecular-weight reagents and reactants to said discrete reaction sites or microreactor vessels. Also included are means for efficiently removing unconverted reactants and reaction products from said reaction sites or microvessels. More particularly, efficient reagent delivery and product removal are accomplished in the present invention by arranging for at least some convective flow of solution to take place in a direction normal to the plane of the substantially two-dimensional array of reaction sites or microreactor vessels. This flow can lead past or through the discrete sites or microvessels, respectively, where chemical reaction takes place.
  • reactants and products will not necessarily be retained or concentrated at the reaction sites or within the reaction microvessels or microwells; indeed, it may be desired that certain reaction products be rapidly swept away from and/or out of said reaction sites or microvessels.
  • This invention also minimizes the amount of contamination among neighboring reaction sites or "blow-by" which typically occurs in diffusive sequencing.
  • diffusive sequencing reaction products from an upstream site have multiple chances to contaminate downstream sites. This contamination is a cumulative effect that may worsen if there are a large number of DNA fragments and multiplets in the upstream reaction sites, hi the present invention, the possibility of blow-by has been minimized such that any possible contamination is not cumulative.
  • each of the mobile supports is washed independently by downward flow of wash solution so that the washing of each reaction site (and any mobile support disposed therein) is independent of washing of neighboring reaction sites during the washing step.
  • the present invention also includes permselective, porous filter means capable of discriminating between large (i.e., high-molecular-weight) and small (i.e., low-molecular- weight) reaction participants.
  • This filter means is capable of selectively capturing or retaining certain reaction participants while permitting others to be flushed through and/or out the bottom of the microreactor array.
  • the apparatus of the present invention consists of an array of microreactor elements comprised of at least two functional elements that may take various physical or structural forms. These include: (i) a planar array layer comprised of an array of microchannels or microwells and on average no more than one mobile support (e.g., reagent- or reactant-carrying mobile support) disposed therein, and (ii) a porous membrane layer comprising, e.g., a porous film or membrane in the form of a sheet or thin layer. These two elements are arranged next to and in close proximity or contact with one another, with the plane of the microchannel/microvessel element parallel to the plane of the porous membrane element. In other words, the planar array layer and the porous membrane layer may be in contact with each other to form one sheet with two layers.
  • a planar array layer comprised of an array of microchannels or microwells and on average no more than one mobile support (e.g., reagent- or reactant-carrying mobile support) disposed there
  • the side of this composite structure containing the microchannel or microvessel array will be referred to hereinafter as the "top", while the side defined by the porous membrane will be referred to as the "bottom" of the structure.
  • contact between the planar array and the porous membrane may be tight, in which case a fluid cannot travel from the bottom side of one microchannel into another microchannel without entering and exiting the porous membrane element.
  • the contact may also be loose, in which case some fluid may travel from one microchannel to another on the bottom of the planar array layer without passage through the porous membrane element.
  • the flow of fluids in a direction normal to the plane of the membrane reactor would prevent significant cross contamination between microchannels even if the contacts were loose.
  • the membrane reactor of the invention allows increased trapping efficiency for mobile supports, high density deposition of mobile supports, and loading of one or fewer mobile supports per well.
  • the membrane reactor is amenable for use with automatic or semi-automatic deposition processes for mobile supports.
  • the membrane reactor allows variations in pitch and density of the planar array and adjustment of flow resistance, which can be used to improve microfluidic flow distribution around the mobile supports.
  • the membrane reactor optimizes stability and flatness to enhance imaging quality and improve high throughput screening.
  • the membrane reactor is easily assembled by batch fabrication processes, which are cost effective for small or large scale productions.
  • the membrane reactor is designed to reduce or eliminate optical or chemical cross-talk, and blow-by from reagents in adjacent wells.
  • the membrane reactor also allows tracking of locations for individual mobile supports location.
  • the planar array layer comprises individual wells (also called microchannels, microvessels, reaction chambers). Each well consists of a single microchannel.
  • the planar array layer comprises a plurality of wells, with the longitudinal axes of said wells being arranged in a substantially parallel manner, and with the downstream ends of said channels being in functional contact with a porous membrane (described below).
  • the aspect ratio of the microchannels i.e., their height- or length-to-diameter ratio
  • the effective well size of the array layer is comparable to or slightly larger than the diameter of the mobile supports that one desires to retain.
  • the array layer typically comprises at least 10,000 wells, at least 50,000 wells, at least 100,000 wells, or at least 250,00 wells, and in one preferred embodiment, between about 100,000 and 1,000,000 wells, and in another preferred embodiment, between about 250,000 and 750,000 wells.
  • the array layer comprises at least about 100, 100-1000, 1000-10,000, 10,000-20,000, 20,000-30,000, or 32,000 wells per mm 2 .
  • the array layer is typically constructed to have wells with a center-to- center (c-t-c) spacing less than 100 ⁇ m, preferably about 5 to 200 ⁇ m, preferably about 10 to 150 ⁇ m, even more preferably about 25 to 100 ⁇ m, and most preferably about 50 to 78 ⁇ m.
  • the center to center (c-t-c) spacing is less than or equal to about 58 ⁇ m, 64 ⁇ m, 68 ⁇ m, 70 ⁇ m, or 100 ⁇ m, respectively.
  • the c-t-c spacing is less than or equal to about 100 ⁇ m, 32 ⁇ m, 10 ⁇ m, 7 ⁇ m, 5.7 ⁇ m, or 5.6 ⁇ m.
  • each reaction chamber in the array layer has a well width in at least one dimension of between about 5 ⁇ m and 200 ⁇ m, preferably between about 10 ⁇ m and 150 ⁇ m, more preferably between about 15 ⁇ m and 100 ⁇ m, most preferably between about 20 ⁇ m and 35 ⁇ m.
  • the reaction chamber can be square and can have the above cited dimensions (or can be rectangular with those dimensions along one linear dimension of the rectangle).
  • the average size can include, e.g., about 15 to 100 ⁇ m in width, or preferably, about 20 to 35 ⁇ m in width.
  • the reaction chamber is square with well widths of about 25 ⁇ m, 28 ⁇ m, 30 ⁇ m, or 31 ⁇ m, respectively.
  • the array layer is selected from a nylon membrane with: (1) a c-t-c spacing of about 64 ⁇ m and a 31 ⁇ m well width; (2) a c-t-c spacing of about 58 ⁇ m and a 25 ⁇ m well width; (3) a c-t-c spacing of about 70 ⁇ m and a 30 ⁇ m well width; and (4) a c-t-c spacing of about 68 ⁇ m and a 28 ⁇ m well width.
  • a preferred well width is determined by bead size. For example, if a bead is about 25 ⁇ m in a diameter, then a preferred well diameter can be about 30 ⁇ m. As other examples, the bead diameter can be about 80%, 83%, 85%, 87%, 90%, 93%, or 95% of the well diameter.
  • the mobile supports of the invention can comprise one or more suitable materials, including glass, silica, dextrans, ceramics, metals, or plastics.
  • suitable materials include Sephadex, Sepharose, agarose, polysulfone, polypropylene, polyethylene, polycarbonate, polyethyleneterephthalate, polyethersulfone, polystyrene, polytetrafluoroethylene, carboxymethyl cellulose, cellulose acetate, cellulose butyrate, polyvinylidene fluoride, acrylonitrile PVC copolymer, polyaminemethylvinylether maleic acid copolymer, polystyrene/acrylonitrile copolymer, and any combination thereof.
  • Preferred beads include sizes of about 25 to 28 ⁇ m in diameter.
  • bead size can be, e.g., about 15 ⁇ m in a diameter.
  • a particular reactant molecule e.g., an oligonucleotide or construct thereof
  • This may be accomplished by immobilizing said reactants on particulate or colloidal supports (e.g., beads, particles), suspending the supports in a fluid, and then depositing or settling these onto the membrane reactor surface by drawing the fluid through the membrane reactor.
  • One method for depositing a mobile support is to place a fluid suspension of mobile supports on a membrane reactor and allow gravity to deposit the mobile supports into the individual wells. This process that can be accelerated by vibration or centrifugation. It is recognized that there may be infrequent times where more than one bead is disposed in a well but this is not preferred.
  • the mobile supports in the wells reduce the size of the wells but do not eliminate the opening in the wells.
  • the mobile supports are spherical while the wells are square (see Examples). The deposition of a round mobile support in a square well would still allow a flow of fluid through the well.
  • the mobile supports and the wells have irregular shapes that deviate, slightly or grossly, from a perfect sphere and a perfect circle.
  • the deposition of an imperfect spherical mobile support onto an imperfect circular well would not completely block the well.
  • the planar array layer is a fabricated or micromachined to comprise round or square wells.
  • the planar array can be constructed on the surface of a substrate using photolithography and electroplating techniques (e.g., Figs. 14A-14I).
  • the planar array can be produced by micromolding (e.g., Figs. 18A-18B).
  • the planar array can also be built on the surface of a substrate, such as a fiber bundle plate, wafer, film, or sheet.
  • a substrate such as a fiber bundle plate, wafer, film, or sheet.
  • the flat and solid areas of the substrate are used as a foundation for building.
  • the substrate is coated by metal deposition (e.g., gold and titanium or chromium) with photoresist and the pattern of the structure is generated by a photomask.
  • the metal layer can be, e.g., about 0.05 ⁇ m, 0.07 ⁇ m, 0.1 ⁇ m, or 0.15 ⁇ m in thickness.
  • the photoresist layer can be, e.g., about 25 ⁇ m, 35 ⁇ m, 50 ⁇ m, or 57 ⁇ m in thickness.
  • the pattern is transferred from the photomask onto the photoresist coating using UV exposure.
  • the photoresist coating can comprise SU-8 film or other photosensitive materials.
  • the substrate is submerged in an electrolyte solution for electroplating. Metal deposition occurs only in exposed grooves. The thickness of plated structure is controlled by time and current density or voltage.
  • the photoresist layer is removed to produce sidewalls. The sidewalls form the wells of the planar array layer.
  • the pitch of the wells i.e., the distance between the centre of one well and the next
  • array patterns can be designed according to need.
  • additional metal thin film can be deposited onto the surface to produce a near black top layer.
  • This thin layer of metal can be blasted onto the surface by electroplating, thermal evaporation, or sputtering processes.
  • Metal thin film can remain at the bottom of the wells or be removed by additional etching.
  • the sidewalls form wells that are slightly wider than the mobile supports. This allows for trapping single mobile supports in the wells.
  • the wells can include opaque sidewalls and bottoms to prevent optical crosstalk, e.g., between mobile supports and between wells (see, e.g., Fig. 17).
  • the heights of the sidewalls are controlled during the electroplating process.
  • the sidewalls are higher than the mobile supports (e.g., the walls are higher than the diameter of the beads).
  • the sidewalls are only slightly higher than the mobile support. Sidewalls that are substantially higher than the mobile supports will allow multiple supports to load in each well.
  • the wells can be formed in patterns, such as regular arrays, or in an irregular 5 042522
  • the wells can comprise one or more shapes, e.g., substantially round, square, oval, rectangular, hexagonal, crescent, and/or star-shaped wells (e.g., Figs. 15, 16A-16D, 19, 20, and 21A).
  • the pitch of the well can be varied, for example, at least 15 ⁇ m, at least 35 ⁇ m, or at least 50 ⁇ m pitch.
  • Reference marks e.g., anchors
  • the sidewalls can be oriented at any angle on the surface of substrate. Angles can be varied on different membranes or on different areas of one membrane. Beams can be placed among plated structure to reinforce the membrane mechanical strength (see below).
  • the sidewalls can be composed of single metals, alloys, metal-plated materials and/or laminated layers, and can include porous, black, matte, shiny, reflective, or mirrored surfaces. Layered metals can be introduced with different colors, different surface morphologies, and different composites.
  • the sidewall can also be coated with one or more additional layers of materials, such as thin film metal, insulation coating, for example, Teflon and metal oxide for improving optical properties. Contemplated for use with the invention are commercially available materials.
  • Materials for the planar array layer of invention include nylon or nitrocellulose membranes and precision woven open mesh fabrics, especially monofilament open mesh fabrics, such as those available from Sefar, Inc. (Ruschlikon, Switzerland).
  • Non-limiting examples of such fabrics include Sefar Nitex (PA 6.6), e.g., Cat. # 03-25/14, 03-28/17, 03-30/18, 03-30/20, and 03-35/16.
  • Other materials for the planar array layer include woven nylon net filters such as those available from Millipore (Bedford, MA), including, but not limited to, Cat. # NY41 025 00, NY41 047 00, NY41 090 00, and NY41 000 10.
  • useful metals include, but are not limited to, copper (Cu), gold (Au), iron (Fe), nickel (Ni), silver (Ag), zinc (Zn), cadmium (Cd), tin (Ti), lead (Pb), antimony (Sb), cobalt (Co), and any alloys thereof, e.g., Ti/Pb.
  • Preferred metals for this aspect include nickel, chromium, and silver, as well as combinations comprising silver and chromium or silver and nickel. Gold as the top layer of coating is also preferred.
  • Membrane reactors without a porous high flow resistance membrane element may suffer from non-uniform flow of reagents.
  • a well with a mobile support would have reduced flow compared to a well without a mobile support.
  • a mixture of open wells and loaded wells in a membrane reactor would have uneven flow.
  • an uneven flow may cause some pores to receive reagents in a non-uniform fashion.
  • Non-uniform delivery of reagents may lead, at least, to a delay in performing reactions because a longer flow is necessary to deliver reagents to all the wells. More significantly, non-uniform delivery may cause errors in interpreting results.
  • some wells may receive more reagents than others and the excess or lack of reagents may change the results of a biochemical reaction.
  • Spatially uneven flow through the array layer may also result in the lateral diffusion of reaction products from a reactive well (i.e., one comprising a mobile support) to a neighboring empty well, which can lead to cross-contamination, or bleeding.
  • the problem with uneven (non-uniform) flow can be significantly reduced by the use of a porous high flow resistance membrane element.
  • the porous membrane layer can be positioned below the planar array layer to provide significant flow restriction in the membrane reactor. This flow restriction is useful in achieving uniform or near-uniform flow of reagents through the membrane reactor.
  • the membrane is substantially permeable to aqueous solutions but is substantially impermeable to the mobile supports. This is possible, for example, if the pores are smaller than the mobile supports so that mobile supports cannot flow through.
  • the porous membrane may be configured in any number of ways to provide satisfactory flow resistance in conjunction with the planar array layer.
  • the porous membrane may comprise pores that are less than one tenth (1/10) or less than one hundredth (1/100) the size of the wells in the planar array layer.
  • the porous high flow resistance membrane because of its small pores, will have a flow restriction that is about 10-fold or more, preferable about 100-fold or more, than that of the planar array layer. Because the porous high flow resistance membrane provides most of the flow restriction in a membrane reactor, the wells of the planar array layer, regardless of whether it comprises a mobile support, would provide only a small portion of the flow restriction.
  • the porous high flow resistance membrane has an average pore size of between about 0.01 ⁇ m and 10 ⁇ m, preferably between about 0.01 ⁇ m and 5 ⁇ m, more preferably between about 0.01 ⁇ m and 0.5 ⁇ m and even more preferably between about 0.1 ⁇ m and 1 ⁇ m, or less than 0.1 ⁇ m. This is particularly the case when a symmetric membrane is used.
  • the pore size is about 0.2 ⁇ m and in another embodiment, the pore size is about 0.02 ⁇ m.
  • the high flow resistance membrane has a pore diameter that is less than 10%, or less than 1%, of the well diameter of the planar array.
  • the porous membrane of the invention can comprise one or more suitable materials, including glass, quartz, ceramics, metal, and silicon as well as polymeric substrates, such as polyolefin, polyamide, polyimide, polyurea, polyether, polyether imides, polyether sulfone, polyurethane, polyethylene, polyester, polycarbonate, polyethyleneamine, polyethylene terephthalate, polyethylene naphthalate, polyglycol acrylate (PGA), polymethylmethacrylate, polyacrylonitrile, polyvinyl acetate, polyvinylchloride (PVC), polyvinylidene fluoride, vinyl polymer, polyvinylacetal resin, polydimethylsiloxane (PDMS), polysulfone, polypropylene, polybutadiene, phenol-formalin resin, cellulose acetate, regenerated cellulose, nitrocellulose, mel
  • membranes with altered surface chemistries may be used.
  • the porous membrane may restrict flow of aqueous materials by being composed of a hydrophobic material.
  • a hydrophobic porous high flow resistance membrane we contemplate using a 10 to 40 ⁇ m PTFE membrane.
  • the membrane has a pore size of less than or about 20 ⁇ m.
  • membranes with altered surface chemistries may be used (e.g., hydrophobic membranes).
  • the planar array is fabricated on a porous membrane.
  • the surface of the membrane is preferably flat, with pores distributed in the membrane (e.g., Figs. 13 A- 13C).
  • Commercially available membranes can be used. Pore size and density can be chosen from different commercial products and used to adjust flow resistance. In preferred aspects, the pores are about 0.2 to 12 ⁇ m in a diameter.
  • the pores can be oriented in a perpendicular direction to the membrane surface. Pores can be arranged uniformly, or in colonies or clusters at different areas on the membrane. Pores can also be distributed in a random manner.
  • One or more additional porous membranes can be placed underneath the original porous membrane to increase flow resistance. Other structures can be placed underneath the porous membrane for additional support (see below).
  • Each layer of the membrane reactor can be offset or aligned to produce different three- dimensional structures.
  • the structures for the planar array can be built into wedges or other tapered shape on the top surface of the membrane.
  • Metal deposition can be used to coat the surface of the membrane.
  • Structures for the planar array can be constructed on top and bottom sides of the porous membrane. The structures can be aligned in a single membrane or from membrane to membrane. In a later case, multiple membranes can be stacked together to form a more complex structure. Pores in the membrane can be partially blocked during metal deposition and electroplating. Pores can also be completely blocked in certainly areas with particular shapes.
  • the metal film can be applied to the membrane so as to avoid blocking the pores. For example, pores can be blocked by a masking process prior to metal deposition.
  • the conductive, thin metal film on the membrane can provide a platform for the planar array layer as described in detail above.
  • Preferred materials for the high-flow resistance membrane of the invention include nylon membrane filters such as those available from Millipore, including, but not limited to, Cat. # GNWP 025 00 and GNWP 047 00.
  • Other preferred materials for the high-flow resistance include membrane ceramic filters such as those available from Refractron Technologies Corp. (Newark, NY), including, but not limited to, alumina or silicon carbide filter plates with 15- 30 ⁇ m pores and 40-50% porosity (volume %).
  • Most preferred are track-etched membranes, such as Whatman CycloporeTM polyester or polycarbonate membranes. For polyester membranes, pore sizes are about 0.1 to 5 ⁇ m and thickness is about 10 to 23 ⁇ m.
  • pore sizes are about 0.1 to 12 ⁇ m and thickness is about 10 to 20 ⁇ m. Most preferably, pore sizes are about 0.5 to 12 ⁇ m and thickness is about 9 to 23 ⁇ m.
  • Metals for deposition include, but are not limited to, gold (Au), titanium (Ti), chromium (Cr), nickel (Ni), tin (Sn), copper (Cu), tantalum (TaN), aluminum (Al), palladium (Pd), platinum (Pt), zinc (Zn), silicon (Si), silver (Ag), and any alloy thereof, such as, Ag/Pd, Ag/Pt, Au/Sn, Ti/Pt/Au, TaN/Cu, and Al/Ti.
  • Preferred metals for this aspect include nickel, chromium, and silver, as well as combinations comprising silver and chromium or silver and nickel. Gold as the top layer of coating is also preferred. 22
  • the membrane reactor can optionally employ a structural support layer that is more permeable than the other two layers and that is, in various embodiments, placed against and/or attached to the porous membrane or placed atop and/or attached to the planar array.
  • This support layer can be used to provide mechanical support to membrane reactor. See, e.g., Figs. 5 and 19 where examples of this are shown.
  • the support layer may be made from any material such as glass, metals, polymers, silicon, and/or ceramics with holes formed during manufacture (e.g., sintering, drilled by laser, cracking, etching, bombardment, and the like). It is noted that while a nonreactive material is generally preferred for the support layer, a nonreactive material is not necessary as long as the flow of reagents from the planar array layer to the porous support layer is sufficiently fast to prevent back diffusion of any molecules from the support layer to the planar array layer.
  • the support layer comprises a metal mesh.
  • the support layer comprises plated metal beams to reinforce the top surface of the planar array layer. Multiple support layers can also be used, e.g., gaskets, rivets, and washers, to form a larger support structure (e.g., Fig. 22).
  • Preferred materials include stainless steel microfiltration meshes, such as Spectra/Mesh® from Spectrum Laboratories (Rancho Dominguez, CA), including, but not limited to, Cat. # 145827, 145936, 145826, and 145935.
  • the membrane reactor can be constructed in a number of different configurations.
  • the porous high flow resistance membrane may be positioned on the top or the bottom of the planar array layer.
  • two porous high flow resistance membranes may be utilized under the planar array layer or with the planar array layer between them, hi addition, the permeable structural support layer may be positioned in several different configurations, e.g., on the top, bottom or in the middle of the membrane reactor.
  • the structural support layer is optional and may not be required when the membrane reactor is configured with sufficient inherent support (e.g., when the membrane reactor is provided with additional support by being affixed to a circumferential support, much like a drum head).
  • the entire array assembly i.e., the combination of porous membrane element plus planar array element
  • the entire array assembly i.e., the combination of porous membrane element plus planar array element
  • the membrane reactors of the present invention will be seen to possess some of the general structural features and functional attributes of commercially available microtiter filter plates of the sort commonly used in biology laboratories, wherein porous filter disks are molded or otherwise incorporated into the bottoms of plastic wells in 96-well plates.
  • the membrane reactor is differentiated from these by the unparalleled high density of discrete reaction sites that it provides, by its unique construction, and by the novel and uniquely powerful way in which it can be operated to perform high throughput chemistries — for example, DNA amplification and/or DNA analysis.
  • the composite microreactor/filter structure (i.e., the membrane reactor of the present invention) can take several physical forms; as alluded to above. Two such forms are represented by physical composites and integral composites, respectively.
  • the two functional elements of the structure include the planar array layer and the porous high flow resistance membrane. These elements may be provided as separate parts or components that are merely laid side-by-side, pressed together, or otherwise attached in the manner of a sandwich or laminate. This structural embodiment will be referred to hereinafter as a "physical composite”. Additional permeable supports (e.g., fine wire mesh or very coarse filters or metal beams) and/or spacing layers may also be provided where warranted to provide mechanical support.
  • Plastic mesh, wire screening, molded or machined spacers, or similar structures may be provided atop the membrane reactor to help provide spatial separation between tangential flow of fluid across the top of the membrane reactor and the upper surface of the membrane reactor. Similar structures may be provided beneath the membrane reactor to provide a pathway for egress of fluid that has permeated across the membrane reactor.
  • the operation of the membrane reactors of the present invention employs a convective flow through the membrane reactor.
  • a pressure difference is applied from the top to the bottom surface of the membrane reactor sufficient to establish a controlled convective flux of fluid through the structure in a direction normal to the substantially planar surface of the structure. Fluid is thus made to flow first through the planar array element and then subsequently across the porous membrane element.
  • This convective flow enables the rapid delivery to the site of reaction of reagents and reactants and the efficient and complete removal of excess or unreacted components from the site of the reaction.
  • the convective flow serves to impede or substantially prevent the back-diffusion of reaction products out of the upstream ends of the microchannels, where otherwise they would be capable of contaminating adjacent or even distant microreactor vessels.
  • reaction systems of interest e.g., DNA analysis by pyrophosphate sequencing, as discussed in more detail below
  • DNA polymerase used in pyrophosphate sequencing is a case in point. It is believed that DNA polymerase should retain at least a certain degree of mobility if it is to function optimally.
  • a polymerase may be in a native form or "tagged" with a moiety that adheres to the mobile supports, such as biotin.
  • a polymerase we contemplate: a) placing polymerase in contact with template loaded mobile supports; b) flowing polymerase over the array; and c) both (i.e., employing (a) and (b) together).
  • the present invention thereby provides means for localizing this macromolecular reagent within the microchannels or microvessels of a membrane reactor without having to covalently immobilize it.
  • the membrane reactor is highly rigid and flat with precise control of well size, pitch, and reference anchors.
  • the flat surface provides a good platform for optical focusing during the imagining process.
  • the variation in well profile e.g., size, shape, pitch
  • the flow resistance of the membrane reactor is less than 10 psi.
  • Adjustable pitch and/or tapered wells also allow different loading densities for mobile supports (e.g., Figs. 23A-23B).
  • Precisely controlled sidewalls e.g., height and profile
  • Smooth sidewalls and bottoms concentrate and reflect light from the wells.
  • Opaque sidewalls and bottoms eliminate or reduce optical crosstalk.
  • the membrane reactor encompasses automatic or semi-automatic deposition of mobile supports. Plated metal beams are included to reinforce membrane strength and stability.
  • the membrane reactor is preferably made from commercially available materials using conventional micromachining methods.
  • the membrane reactor is produced by batch processing with small bench top instruments, and the reactor is reused or disposed after each use.
  • microfabrication facilities with clean rooms are used for at least part of the construction of the membrane reactor.
  • the photomasks can be designed and drawn using available software. Metal deposition, photolithography, and electroplating can be performed by commercial vendors.
  • each cavity or well of the array comprises reagents for analyzing a nucleic acid or protein. Not all wells are required to include a nucleic acid or protein target. Typically those wells that comprise a nucleic acid comprise only a single species of nucleic acid (i.e., a single sequence that is of interest). There may be a single copy of this species of nucleic acid in any particular well, or they may be multiple copies.
  • a well comprise at least 1,000,000 copies of the species of nucleic acid sequence of interest, preferably between about 2,000,000 and 20,000,000 copies, and most preferably between about 5,000,000 and 15,000,000 copies of the species of nucleic acid sequence of interest.
  • the nucleic acid species is amplified to provide the desired number of copies using polymerase chain reaction ("PCR") (preferred), rolling circle amplification (“RCA”), ligase chain reaction, other isothermal amplification, or other conventional means of nucleic acid amplification.
  • PCR polymerase chain reaction
  • RCA rolling circle amplification
  • ligase chain reaction other isothermal amplification
  • the nucleic acid is single stranded, hi other embodiments, the single stranded DNA is a concatamer with each copy covalently linked end to end.
  • the nucleic acid may be immobilized in the well, either by attachment to the well itself or preferably by attachment to a mobile support (e.g., a bead) that is delivered to the well.
  • a bioactive agent e.g., a sequencing enzyme
  • the array can also include a population of mobile supports disposed in the wells, each mobile support having one or more bioactive agents (e.g., nucleic acids or sequencing enzymes) attached thereto. The diameter of each mobile support can vary.
  • the diameter of the mobile support is such that only one mobile support is trapped within a single well in the planar array. Not every well in the planar array need comprise a mobile support.
  • a mobile support there are numerous contemplated embodiments; in one embodiment, at least 5% to 20% of the wells can have a mobile support; a second embodiment has about 20% to 60% of the reaction chambers can have a mobile support; and a third embodiment has about 50% to 100% of the reaction chambers with a mobile support.
  • the percentage of wells loaded with mobile supports is about 5%, 10%, or 25%.
  • mobile supports carried in the stream can be pushed into wells in the planar array. Excessive mobile supports on top of the array can be flushed away with a parallel flow of fluid along the surface of membrane.
  • the loading process for mobile supports can use perpendicular and parallel flows: the first flow pushes mobile supports into wells, while the latter flow pushes excessive mobile supports into empty well or off the array.
  • mobile supports can be loaded using fluidic streams, vibration, shaking, rocking, spinning, centrifugation, or any combination thereof.
  • a mobile support typically has at least one reagent or reactant immobilized thereon.
  • the reagent may be a polypeptide with sulfurylase or luciferase activity, or both.
  • enzymes such as hypoxanthine phosphoribosyltransferase, xanthine oxidase, uricase or peroxidase could be utilized (e.g., Jansson and Jansson (2002), incorporated herein by reference).
  • the mobile supports can be used in methods for dispersing over the array a plurality of mobile supports having one or more nucleic sequences or proteins or enzymes immobilized thereon.
  • the invention in another aspect, involves an apparatus for simultaneously monitoring the array of wells for light generation, indicating that a reaction is taking place at one or more particular sites.
  • the wells are sensors, adapted to comprise analytes and an enzymatic or fluorescent means for generating light in the wells. Such sensors are suitable for use in a biochemical or cell-based assays.
  • the apparatus also includes an optically sensitive device to detect light from a well at a particular region of the optically sensitive device.
  • the apparatus also includes means for determining the light levels detected at these
  • the instrument includes a light detection means having a light capture means and a fiber optic bundle for transmitting light to the light detecting means.
  • a light detection means having a light capture means and a fiber optic bundle for transmitting light to the light detecting means.
  • the fiber optic bundle is typically in optical contact with the array, such that light generated in an individual well is captured by a separate fiber or groups of separate fibers of the second fiber optic bundle for transmission to the light capture means.
  • the membrane reactor can be utilized to achieve highly parallel sequencing without electrophorectic separation of DNA fragments and associated sample preparation.
  • the membrane reactor can also be used for other uses, e.g., combinatorial chemistry.
  • an array of photodetectors is utilized for monitoring light producing reactions within the membrane reactor.
  • the array of photodetectors is a CCD camera.
  • Another method of detection of discrete reactions within the membrane reactor is to monitor changes in light absorption as an indicator of a chemical reaction in a membrane reactor using an array of photodetectors.
  • the methods and apparatuses described are generally useful for any application in which the identification of any particular nucleic acid sequence is desired.
  • the methods allow for identification of polymorphisms, including single nucleotide polymorphisms (SNPs) and haplotypes, and for transcript profiling.
  • Other uses include sequencing artificial DNA constructs to confirm or elicit their primary sequence, and identifying specific mutant clones from random mutagenesis screens.
  • the methods can also be used to determine cDNA sequences from single cells, whole tissues, or organisms from any developmental stage or environmental circumstance in order to determine a gene expression profile from that specimen.
  • the methods allow for the sequencing of
  • the DNA may be genomic DNA, cDNA, or recombinant DNA and may be derived from viral, bacterial, fungal, mammalian, or preferably human sources.
  • Pyrophosphate sequencing is a technique in which a complementary oligonucleotide is hybridized and extended using an unknown sequence (the sequence to be determined) as the template. This technique is also known as "sequencing by synthesis”. Each time a new nucleotide is polymerized onto the growing complementary strand, a pyrophosphate (PPO molecule is released. The release of pyrophosphate is then detected.
  • the method involves iterative addition of the four nucleotides (dATP, dCTP, dGTP, dTTP) or of analogs thereof ⁇ e.g., ⁇ -thio-dATP).
  • pyrophosphate can be detected via a coupled reaction in which pyrophosphate is used to generate ATP from adenosine 5'-phosphosulfate (APS) through the action of the enzyme ATP sulfurylase. The ATP is then detected photometrically via light released by the enzyme luciferase, for which ATP is a substrate. It may be noted that luciferase is capable of using dATP as a substrate.
  • a dATP analog such as ⁇ -thio-dATP can be substituted for dATP as a sequencing nucleotide.
  • the ⁇ - thio-dATP molecule can be incorporated into the growing DNA strand, but not used a substrate for luciferase.
  • Pyrophosphate sequencing can be performed in a membrane reactor in several different ways.
  • One such protocol follows: (1) A sample DNA is captured (preferably, many copies of a single sequence) onto beads and the beads are loaded onto the membrane reactor;
  • step (2) As a negative control, a separate set of beads is prepared as in step (1) with the exception that no DNA is captured.
  • the negative control is useful, at least, for determining background signal levels. All subsequent steps can be performed in parallel using DNA loaded beads and negative control beads;
  • dCTP dGTP, dTTP, ⁇ -thio-dATP, or any suitable dATP analog
  • dXTP dATP, dTTP, dGTP or dCTP
  • PPi PPi is produced in the region of the DNA being sequenced (with APS and luciferin flowing through);
  • ATP is produced from APS and PP; when PPi is brought into contact with the sulfurylase enzyme (with luciferin flowing through); and (c) Light is produced from ATP and luciferin in the vicinity of the luciferase enzyme.
  • a CCD camera can be optically coupled by a lens or other means to the membrane reactor. This can be used to monitor light production simultaneously from many wells or discrete reaction sites.
  • CCD cameras are available with millions of pixels, or photodetectors, arranged in a 2-D array.
  • Light originating from one well or discrete reaction site in or on a membrane reactor can be made to transmit a signal to one or a few pixels on the CCD.
  • each well or reaction site is arranged to comprise an independent sequencing reaction, each reaction can be monitored by one or at most a few CCD elements or photodetectors.
  • a CCD camera or other imaging means comprising millions of pixels, the progress of millions of independent sequencing reactions can be monitored simultaneously.
  • a plurality of wells or reaction sites can comprise amplification products from a single sample of DNA. If different wells (or mobile support disposed therein) hold the amplification products of DNA samples, then the simultaneous sequencing of millions of different samples of DNA is possible.
  • the distribution of DNA to be sequenced can be accomplished in many ways, two of which follow. In one approach, the amplification products of a single DNA strand are attached to a bead, and beads from many 005/042522
  • reagents or reactants are delivered to the membrane reactor immobilized or attached to a population of mobile supports, e.g., beads, particles, or microspheres.
  • the mobile support need not be spherical; in some aspects, hexagonal or irregular shaped beads may be used.
  • the beads are typically constructed from numerous substances, e.g., plastic, glass, or ceramic, and cross-linked agarose gel.
  • the mobile support of the invention may comprise various chemistries, such as, for example, methylstyrene, polystyrene, acrylic polymer, latex, paramagnetic, thoria sol, carbon graphite and titanium dioxide.
  • the construction or chemistry of the mobile support can be chosen to facilitate the attachment of the desired reagent or reactant.
  • the mobile supports are magnetic or paramagnetic.
  • Mobile support sizes depend on the well size and width of the well.
  • the diameter of each mobile support is chosen so that the mobile support cannot pass through the pores in the membrane layer.
  • the mobile supports may be smaller than the wells in the planar array.
  • the porous high flow resistance membrane layer can stop the mobile support from flowing through the membrane reactor.
  • the mobile supports are sized so that only one mobile support can fit within a single well and where the spatial separation between two adjacent reaction chambers has a linear dimension of between about 5 ⁇ m and 200 ⁇ m, preferably between about 10 ⁇ m and 150 ⁇ m, more preferably between about 25 ⁇ m and 100 ⁇ m, more preferably between about 50 ⁇ m and 75 ⁇ m, and most preferably between about 20 ⁇ m and 35 ⁇ m.
  • the mobile support diameter may be 31 ⁇ m and the well diameter may be 33 ⁇ m. Even though the 31 ⁇ m mobile support may flow through the 33 ⁇ m well, the porous high flow resistance membrane layer prevents the mobile support from flowing through.
  • a reagent immobilized to the mobile support can be a polypeptide with sulfurylase activity, a polypeptide with luciferase activity, or both on the same or different mobile supports, or a chimeric polypeptide having both sulfurylase and luciferase activity, hi one embodiment, it can be an ATP sulfurylase and luciferase fusion protein (see, e.g., U.S. Patent Application Serial No. 10/122,706, filed April 11, 2002, and U.S. Patent Application Serial No 10/154,515, filed May 23, 2002; which are incorporated herein by reference in their entirety).
  • Other sulfurylase and/or luciferase that may be used include those described in U.S.
  • Ultra-Glow luciferase (available from Promega) is also suitable for use with this invention.
  • both luciferase and sulfurylase are immobilized on the same mobile support. Since the product of the sulfurylase reaction is consumed by luciferase, proximity between these two enzymes may be achieved by covalently linking the two enzymes in the form of a fusion protein.
  • a fusion protein combining functional polymerase, sulfurylase and luciferase activity may be used.
  • a reactant immobilized to the mobile support can be a nucleic acid whose sequence is to be determined or analyzed. A DNA or RNA polymerase can be incubated with mobile supports that have nucleic acids attached thereto.
  • a membrane reactor device having normal cross-flow exhibits high levels of wash efficiency, hi some cases, the chamber cannot be washed efficiently within a reasonably short period of time. This can have a significant impact on the accuracy of pyrophosphate sequencing.
  • apyrase may be applied to degrade the leftover nucleotides after each nucleotide delivery.
  • the use of apyrase is typically at concentrations of 1 U/l to 100 U/l, preferably 4 U/l to 40 U/l, more preferably 8 U/l to 20 U/l, most preferably 8.5 U/l.
  • high fidelity but low processivity polymerase e.g., Klenow
  • polymerase may be present in the flow.
  • the flow rate of the membrane reactor device is about 0.15 ml/minute/cm 2 to 4 ml/minute/cm 2 , or about 0.1 ml/minute/cm 2 to 5 ml/minute/cm 2 .
  • the bioactive agents e.g., nucleic acids
  • the mobile supports are synthesized, and then covalently attached to the mobile supports.
  • the functionalization of solid support surfaces, e.g., polymers, with chemically reactive groups such as thiols, amines, carboxyls, etc., is generally known in the art. Accordingly, "blank" mobile supports may be used that have surface chemistries that facilitate the attachment of the desired functionality.
  • blank mobile supports include, but are not limited to, amino groups including aliphatic and aromatic amines, carboxylic acids, aldehydes, amides, chloromethyl groups, hydrazide, hydroxyl groups, sulfonates, and sulfates.
  • candidate agents comprising carbohydrates may be attached to an amino-functionalized support.
  • the aldehyde of the carbohydrate can be made using standard techniques.
  • the aldehyde can then be reacted with an amino group on the surface of the mobile support.
  • a sulfhydryl linker may be used.
  • sulfhydryl reactive linkers known in the art such as SPDP, maleimides, ⁇ -haloacetyls, and pyridyl disulfides (see for example the 1994 Pierce Chemical Company catalog, technical section on cross-linkers, pages 155-200, incorporated here by reference).
  • polystyrene resin can be used to attach proteinaceous agents comprising cysteine to the support.
  • an amino group on the candidate agent may be used for attachment to a suitable electrophilic moiety on the surface.
  • Such moieties include, but are not limited to, NHS esters.
  • a large number of stable bifunctional groups are well known in the art, including homobifunctional and heterobifunctional linkers (see Pierce Catalog and Handbook, pages 155-200).
  • carboxyl groups (either from the surface or from the candidate agent) may be derivatized using well known linkers (see Pierce catalog).
  • carbodiimides may be used to activate carboxyl groups for attack by nucleophiles such as amines (see Torchilin et al, Critical Rev.
  • Proteinaceous candidate agents may also be attached using other techniques known in the art, for example for the attachment of antibodies to polymers; see Slinkin et al, Bioconj. Chem. 2:342-348 (1991); Torchilin et al, supra; Trubetskoy et al, Bioconj. Chem. 3:323-327 (1992); King et al, Cancer Res. 54:6176-6185 (1994); and Wilbur 005/042522
  • the candidate agents may be attached in a variety of ways, including those listed above.
  • the manner of attachment does not significantly alter the functionality of the candidate agent. That is, the candidate agent should be attached in such a flexible manner as to allow its interaction with a target.
  • NH 2 surface chemistry beads can be used for immobilizing enzymes on beads.
  • Surface activation is achieved with a 2.5% glutaraldehyde in phosphate buffered saline (10 mM) providing a pH of 6.9 (138 mM NaCl, 2.7 niM KCl). This mixture is stirred on a stir bed for approximately 2 hours at room temperature.
  • the beads are then rinsed with ultrapure water plus 0.01% Tween 20 (surfactant), 0.02%, and rinsed again with a pH 7.7 PBS plus 0.01% Tween 20.
  • the enzyme is added to the solution, preferably after being prefiltered using a 0.45 ⁇ m AmiconTM micropure filter.
  • the mobile supports and bioactive agents are linked using a biotin/streptavidin linkages, which are well known to those skilled in the art.
  • the invention provides an apparatus for simultaneously monitoring an array of wells for light signals which indicate that one or more reactions are taking place at a particular well.
  • the reaction event e.g., photons generated by luciferase
  • the reaction event may be detected and quantified using a variety of detection apparatuses, e.g., a photomultiplier tube, a CCD, CMOS, absorbance photometer, luminometer, charge injection device (CID), or other solid state detector, as well as the apparatuses described herein.
  • the quantitation of the emitted photons is accomplished by the use of a CCD camera fitted with a fused fiber optic bundle
  • the quantitation of the emitted photons is accomplished by the use of a CCD camera fitted with a microchannel plate intensifier.
  • a back-thinned CCD can be used to increase sensitivity.
  • CCD detectors are described in, e.g., Bronks, et ah, 1995. Anal. Chem. 65: 2750-2757.
  • the CCD sensitivity may be enhanced by the known method of chilling the CCD during exposure.
  • An exemplary CCD system is a Spectral Instruments, Inc. (Tucson, AZ) Series 600 4-port camera with a Lockheed-Martin LM485 CCD chip and a 1-1 fiber optic connector (bundle) with 6-8 ⁇ m individual fiber diameters.
  • This system has 4096 x 4096, or greater than 16 million pixels, and has a quantum efficiency ranging from 10% to > 40%. Thus, depending 5 042522
  • the invention also provides a microimaging system for imaging one or more light emissions (e.g., from a pyrophosphate sequencing reaction) from a membrane reactor.
  • the system comprises two lens groups.
  • the first lens group is the front lens group which is positioned closer to the light source to be detected to collect the light emitted.
  • the second lens group is the rear lens group that is positioned closer to the light detector such as a CCD detection device to image the light onto the detector.
  • the front lens group and rear lens group are identical.
  • the lens group comprises 50 mm lens with an aperture larger than 2.8 (e.g., 2.0, 1.8, 1.4, 1.0, etc.).
  • the lens group has a focal length of at least 30 mm, at least 50 mm, or at least 70 mm.
  • the lens group has an aperture brighter than or equal to 4.0, or brighter than or equal to 2.8.
  • the lens group has a numerical aperture larger than 0.1, 0.2, or 0.3. It should be noted that the larger apertures are expressed by a smaller aperture value so that, for example, an aperture of 1 is larger than an aperture of 2.
  • An exemplary imaging system is shown in Fig. 2.
  • the data from the optical detection device can be analyzed instantaneously or stored electronically (e.g., by computers, hard drives, optical drives, solid state memories) for subsequent analysis by methods known to those of skill in the art.
  • a fluorescent moiety can be used as a label and the detection of a reaction event can be carried out using a confocal scanning microscope.
  • the microscope can be used to scan the surface of an array with a laser or other techniques such as scanning near-field optical microscopy (SNOM) which are capable of smaller optical resolution, thereby allowing the use of "more dense" arrays.
  • SNOM scanning near-field optical microscopy
  • individual polynucleotides may be distinguished when separated by a distance of less than 100 nm, e.g., 10 nm x 10 nm.
  • scanning tunneling microscopy (Binning et ah, Helvetica Physica Acta, 55:726-735, 1982) and atomic force microscopy (Hanswa et al, Annu Rev Biophys Biomol Struct, 23:115-139, 1994) can be used.
  • N- hydroxysuccinimide N- hydroxysuccinimide
  • Beads to be used for this purpose are supplied (Amersham) in 100% isopropanol to preserve the activity prior to coupling. Twenty-five microliters of 1 mM amine-labeled BGEG primer are dissolved in coupling buffer (200 mM NaHCO 3 , 0.5 M NaCl, pH 8.3). Beads were activated by adding 1 ml of ice cold 1 mM HCl. Beads were washed two times with ice cold coupling buffer.
  • Amine labeled primers and amine labeled biotin are added to the beads and incubated for 15 to 30 minutes at room temperature with rotation (to allow coupling to happen). Amine-labeled biotin is added. After coupling the emulsion PCR, the streptavidin is added to be coupled to the biotin. Then the biotinylated sulfurylase and luciferase (454 Life Sciences) are coupled to the streptavidin. Then the beads were washed one time with coupling buffer. The beads were washed two times with Acetate buffer (0.1 M sodium acetate, 0.5 M NaCl, pH 4).
  • the beads were washed three times with coupling buffer (0.5 M ethanolamine, 0.5 M NaCl, pH 8.3).
  • the beads were incubated with 500 ⁇ l of blocking buffer for one hour with rotation at room temperature to allow for deactivation or blocking of any leftover active groups.
  • the beads were washed with (a) coupling buffer and then with (b) acetate buffer. This wash ((a) then (b)) was repeated three times.
  • the beads were washed two times in IX annealing buffer. 2
  • the annealing buffer also serves as the storage buffer. This procedure is illustrated in Fig. 11.
  • Streptavidin-sepharose beads were size-selected by filtering to obtain diameter between 30-36 ⁇ m.
  • the primers and target DNA included: MMP7A sequencing primer (5'-ccatctgttc cctcctgtc-3'; SEQ ID NO:6); target DNA, termed UATF9 (3'-atgccgcaaa aacgcaaac gcaaacgcaa cgcatacctc tccgcgtagg cgctcgttgg tccagcagag gcggccgccc ttgcgagc agaatggcgg tagggggtct agctgcgtct cgggg-5'; SEQ ID NO:7); biotinylated primer and PCR reverse primer, termed Bio-Heg-MMPIB (5'-5Bio//iSpl8//iSpl8//iSpl8//iS
  • Immobilized PCR product was incubated in 0.10 M NaOH for 10 min, and the supernatant was removed to obtain single-stranded DNA.
  • the beads containing single-stranded DNA were washed 3 times with 100 ⁇ l of IX Annealing Buffer, pH 7.5 (30 mM Tris-HCl, 3 mM magnesium acetate, from Fisher). The beads were pelleted by centrifugation for 1 min at a maximum speed of 13,000 rpm. Supernatant was removed and the beads were suspended in 25 ⁇ l of IX Annealing Buffer. Five microliter of 100 pmol sequencing primer was added to mixture. The beads were incubated at 65 0 C for 5 min and cooled to room temperature. The beads were washed 3 times with 100 ⁇ l of IX Annealing Buffer and resuspended in final volume of 100 ⁇ l. Loading the Beads into the Membrane and Assembly into the Loading Jig
  • the beads were resuspended at a concentration of about 3,500 beads per microliter in IX Annealing Buffer. Following this, 25 ⁇ l of the suspension was added to 200 ⁇ l of IX Assay Buffer, pH 7.8 (25 mM tricine (Fisher), 5 mM magnesium acetate (Fisher), 1 mM dithiothreitol, 0.4 mg/ml polyvinylpyrrolidone, 0.01% Tween 20, 1 mg/ml BSA, all from Sigma (St. Louis, MO)). In the loading jig, a 0.2 ⁇ m nylon membrane was placed below the 30 ⁇ m pore nylon membrane (Sefar).
  • the loading jig was then attached to a peristaltic pump having a flow rate of 1 ml per min.
  • 200 ⁇ l of IX Assay Buffer was used to wash the membrane.
  • 200 ⁇ l of the bead suspension was added to the membrane. Negative pressure was applied to the membrane along with 500 ⁇ l of IX Assay Buffer to force the beads onto the pores of the membrane.
  • the membrane was washed with 500 ⁇ l of IX Assay Buffer.
  • the membrane was disassembled and placed in IX Assay Buffer (e.g., 20 ml) in a test tube (e.g., Falcon) for storage.
  • the following solutions were mixed in a container: 500 ⁇ l of biotinylated ATP Sulfurylase enzyme at 1 mg/ml (454 Life Sciences); 500 ⁇ l of biotinylated Luciferase at 3 mg/ml (454 Life Sciences); and 500 ⁇ l of IX Assay Buffer.
  • the bead-loaded nylon membrane was placed in the enzyme mixture. The mixture was rotated with the nylon membrane for 20 min at room temperature at a speed of one rotation per two seconds.
  • the membrane was then placed in 20 ml of IX Assay Buffer and swirled for 2 minutes. This wash step was repeated once.
  • a solution of 970 ⁇ l IX Assay Buffer with 30 ⁇ l of Bst Polymerase enzyme (New England Bio Labs) was prepared.
  • the membrane was immersed in this solution, and the solution/membrane was rotated for 25 min at room temperature at a speed of one rotation per two seconds. After the rotations, the membrane was washed two times in 20 ml of IX Assay buffer.
  • a 0.2 ⁇ m nylon membrane was immersed in 20 ml of IX Assay Buffer and placed above the wire mesh on the sequencing Jig holder.
  • a 30 ⁇ m membrane containing the DNA beads was placed on top of the 0.2 ⁇ m nylon membrane.
  • a sequence Jig cover with an optical glass window (13 mm) was placed on top of the membranes. The Jig cover was tightly attached to the lower part of the membrane holder. The cover and holder were threaded and tightening was performed by screwing the two parts together.
  • the sequencing Jig was placed on the z- translation stage below the CCD camera. The acquisition time on the camera was set to 7 sec and the read out time was set to 0.25 sec.
  • the inlet of the membrane holder was connected to the outlet of the pump, which was connected to the Valco valve (Valco Instruments, Houston, TX).
  • the lower part of the membrane holder was connected to the second peristaltic pump.
  • the outlet of the sequencing chamber was connected to the waste.
  • the substrate (25 mM tricine, 5 mM magnesium acetate, both from Fisher; 1 mM dithiothreitol, 0.4 mg/ml polyvinylpyrrolidone, 0.01% Tween 20, all from Sigma; 300 ⁇ M D- Luciferin, from Regis; 2.5 ⁇ M adenosine-5'-O-phosphosulfate, from Axxora, Inc.) was flowed for 2 min to prime the flow chamber and expel any air bubbles. This allowed the DNA on the nylon membrane to be equilibrated with substrate.
  • the reagents were flowed through the chamber in the following order: 1) dCTP; 2) substrate; 3) nucleotide Sp-dATP- ⁇ -thio; 4) substrate; 5) dGTP; 6) substrate; 7) dTTP; and 8) substrate. This cycle was repeated 20 times.
  • the flow rates for the lateral and vertical flow were controlled as follows.
  • the nucleotide flow (for steps (1), (3), (5), and (7), above) was at 2 ml/min lateral for 21 sec and then 0.5 ml/min for 7 sec.
  • the vertical flow was 0.5 ml/min for the same time. Total time was 28 sec.
  • the substrate flow (for steps (2), (4), (6), and (8), above) was at 2 ml/min lateral flow and 1 ml/min vertical flow 1 ml/min for 77 sec.
  • the results of the sequencing reaction are shown in Fig. 9.
  • Example 3 PCR on Nylon Membrane Containing Beads and Sequencing Using a Pyrophosphate Sequencer
  • the sequencing step was used to confirm the fidelity of the amplified template.
  • the primers and probe included:
  • the sepharose beads were treated as in Example 2, with a concentration of 3,500 beads per microliter.
  • 90 ⁇ l of sepharose beads were washed by resuspension in 200 ⁇ l of IX PCR buffer and this was followed by centrifugation for a total of three washes.
  • 200 ⁇ l of IX PCR buffer was placed on top of the beads pelleted by centrifugation.
  • 6 ⁇ l of 100 pmol/ ⁇ l biotinylated Pl probe was added to the top of the beads/PCR buffer.
  • the beads were resuspended and the tube containing the beads was placed on a rotator for 45 min at room temperature.
  • aqueous layer above the beads in Sample A (negative control) was removed and replaced with 50 ⁇ l of PCR mix (37.7 ⁇ l H 2 O, 5 ⁇ l 1OX PCR buffer, 1 ⁇ l dNTPs (10 mM each), 0.4 ⁇ l of 100 pmol/ ⁇ l Pl forward primer, 0.4 ⁇ l of 100 pmol/ ⁇ l P2 reverse primer, 5 ⁇ l Betaine (5 M), and 0.5 ⁇ l of Taq polymerase (5 U/ ⁇ l).
  • Nylon membranes were cut into 2 mm circles using a die cutter. The circles were pre-wetted by immersion in IX PCR buffer.
  • the aqueous layer above the beads in Sample B was replaced with 50 ⁇ l PCR mix (35.45 ⁇ l H 2 O, 5 ⁇ l 1OX PCR buffer, 1 ⁇ l dNTPs (10 mM each), 0.4 ⁇ l of 100 pmol/ ⁇ l Pl forward primer, 0.4 ⁇ l of 100 pmol/ ⁇ l P2 reverse primer, 5 ⁇ l Betaine (5 M), 0.5 ⁇ l Taq polymerase (5 U/ ⁇ l) and 2.25 ⁇ l of 1.67 attomol/ ⁇ l tf2 adeno fragment.
  • One attomole is defined as 1 x 10 "18 moles.
  • the estimated concentration of DNA per bead prior to amplification was 10 copies per bead.
  • the tube containing the nylon membrane with Sample A and the tube containing the nylon membrane with Sample B were placed in a thermocycler with the following reaction conditions.
  • Step 1 incubation at 96°C for 2 min;
  • Step 2 incubation at 96°C for 1 min;
  • Step 3 incubation at 58 0 C for 1 min;
  • Step 4 incubation at 72°C for 1 min, go to Step 2, 29 times;
  • Step 5 incubation at 72 0 C for 10 min;
  • Step 6 incubation at 14°C overnight or until the reaction was terminated.
  • the PCR tubes with the nylon membranes were removed from the thermocycler. The membranes were removed and placed into separate tubes containing 1 ml chloroform and 200 ⁇ l of IX Annealing Buffer.
  • the tubes were shaken several times.
  • the membranes were transferred to individual tubes containing 1 ml of chloroform and the tubes were rotated several times.
  • the membranes were then transferred to individual tubes with 200 ⁇ l of IX Annealing Buffer.
  • the tubes were then rotated several times. This procedure was repeated an additional two times with 200 ⁇ l of Annealing Buffer.
  • the membranes were transferred to individual tubes with 50 ⁇ l of IX Annealing Buffer.
  • the tubes were heated to 90 0 C for 2 min in a PCR thermocycler.
  • the membranes were transferred to individual tubes containing 50 ⁇ l of IX Annealing Buffer on ice.
  • the membranes were washed two times with 100 ⁇ l of IX Annealing Buffer.
  • the membranes were then incubated with a solution of 5 ⁇ l of 100 pmol of P2 primer mixed with 20 ⁇ l of IX Annealing Buffer.
  • the tubes were placed in a thermocycler and heated to 65°C.
  • the tubes were slowly cooled to room temperature to allow the P2 sequencing primer to anneal to the DNA template.
  • the membranes were washed two times with 100 ⁇ l of IX Annealing Buffer.
  • the reaction product was sequenced on the beads using a pyrophosphate sequencer (PSQ).
  • PSQ pyrophosphate sequencer
  • Methods of pyrophosphate sequencing are generally described, e.g., in U.S. Patents 6,274,320, 6258,568 and 6,210,891, incorporated herein by reference in toto. Briefly, the membranes were soaked for 30 sec in 50 ⁇ l of a mixture of ATP sulfurylase and luciferase enzymes (454 Life Sciences). Then, the membrane was placed into a well of the PSQ. The nucleotides were flowed into the PSQ plates in the order of G, A, C, and then T. This was repeated five times.
  • Example 4 Methods for Pyrophosphate Sequencing Any DNA may be sequenced using the procedure described herein. Briefly, beads are filtered to obtain a diameter of 25-30 ⁇ m and resuspended at a concentration of 3,500 beads/ ⁇ l, as described above. Next, 14 ⁇ l of the bead solution is placed into a tube for each sample to be sequenced. The beads are pelleted at 13,000 rpm.
  • the supernatant is replaced with 500 ⁇ l of a mixture of the three enzymes (6 ⁇ l of sulfurylase at 1 mg/ml, 6 ⁇ l of luciferase at 3 mg/ml, and 60 ⁇ l of Bst polymerase at 50 U/ ⁇ l) and 428 ⁇ l of IX Assay Buffer containing 1 mg/ml BSA.
  • the tube is placed in a rotator for 1 hr at room temperature, at about one turn every 2 sec. Then, the beads are pelleted by centrifugation at 2,000 rpm for 2.5 min. The beads are washed once with 200 ⁇ l of IX Assay Buffer without BSA. Then the beads are loaded onto a membrane with 30 ⁇ m pore for pyrophosphate sequencing.
  • Example 5 Bead Loading Methods
  • a membrane e.g., nitrocellulose membrane circle, as described herein
  • bead solution such as IX Assay Buffer.
  • the membrane is agitated to trap beads in the membrane pores.
  • the membrane/bead mixture is submerged in bead solution. This is stirred or vortexed to trap the beads in the membrane pores.
  • the membrane is used as a bead filter in a sieve, and the bead solution is drained through the membrane using gravity or centrifugal force.
  • the bead solution is introduced from the top and drained through the bottom by a pump.
  • the loading Jig can include multiple cavities for bead deposits onto different areas of the membrane for different samples or tests. This method can be combined with the loading Jig method as described herein.
  • the beads are loaded using a wicking effect.
  • the membrane is placed on top of other highly hydrophilic membrane or tissue, or other wicking material, and the bead solution is applied to the membrane.
  • the bead solution is flowed across the membrane in an enclosed chamber.
  • the chamber can be placed in any orientation, and the beads can be introduced by various means such as a syringe, pipette, duct, tubing, and the like.
  • the arrayed sample delivery devices can be used to deposit beads onto discrete regions or patches on the membrane.
  • wash buffers, sample DNA, bead solution, enzyme solutions, and sequencing reagents are prepared according to the layout of the automated sequencing system ( Figure 8). The sequencing and resistance membranes are incubated in
  • IX Assay Buffer AB containing 1% bovine serum albumin (BSA) for at least 15 min, preferably 30 min, to prevent PPj drop during a long sequencing run.
  • the sequence chamber system is assembled with the membranes and connected to pumps and reagents.
  • the beads are loaded with Pump 2 at a flow rate of about 1-2 ml/min depending on the chamber size.
  • Non-binding beads are 5 042522
  • a sulfurylase and luciferase mixture is loaded with both Pump 1 and Pump 2 running. This is incubated for 15 min with mixing by the reciprocal movement by Pump 2.
  • the chamber is washed with a wash buffer for polymerase with both pumps running for 5 min.
  • a Bst polymerase (New England Biolabs, Beverly, MA) solution is loaded and incubated for 30 min with mixing.
  • the chamber is washed with substrate for 5 min with both pumps running.
  • the Bst polymerase can be mixed with sulfurylase and luciferase for combined infusion with all three enzymes.
  • a 0.1 ⁇ M PPj solution is run for signal calibration with a flow rate of 1.5 ml/min for both pumps for 21 sec.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Health & Medical Sciences (AREA)
  • Analytical Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Hematology (AREA)
  • Clinical Laboratory Science (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Saccharide Compounds (AREA)

Abstract

La présente invention a trait à des procédés et des appareils pour la réalisation de réactions chimiques indépendantes à haute densité en parallèle dans des jeux ordonnés d'échantillons perméables aux fluides. Par conséquent, la présente invention a également trait à l'utilisation de tels jeux ordonnés d'échantillons pour des applications telles que le séquençage de l'ADN, de préférence le séquençage au pyrophosphate, et l'amplification d'ADN.
PCT/US2005/042522 2004-11-23 2005-11-22 Procede d'isolement de micro-reactions chimiques independantes et paralleles mettant en oeuvre un filtre poreux WO2006062745A2 (fr)

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
US11/016,942 US20060019264A1 (en) 2003-12-01 2004-11-23 Method for isolation of independent, parallel chemical micro-reactions using a porous filter
PCT/US2004/039208 WO2005054431A2 (fr) 2003-12-01 2004-11-23 Procede permettant d'isoler des micro-reactions chimiques paralleles independantes au moyen d'un filtre poreux
USPCT/US2004/39208 2004-11-23
US11/016,942 2004-11-23
US11/217,194 2005-09-01
US11/217,194 US20060088857A1 (en) 2003-12-01 2005-09-01 Method for isolation of independent, parallel chemical micro-reactions using a porous filter

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WO2006062745A3 WO2006062745A3 (fr) 2007-07-26

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US8266791B2 (en) 2007-09-19 2012-09-18 The Charles Stark Draper Laboratory, Inc. Method of fabricating microfluidic structures for biomedical applications
WO2014001459A1 (fr) * 2012-06-29 2014-01-03 Danmarks Tekniske Universitet Procédé de charge d'un porteur d'essai, et porteur d'essai
EP2694967A4 (fr) * 2011-04-06 2014-10-29 Ortho Clinical Diagnostics Inc Dispositif d'analyse ayant des saillies en forme de losange

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8266791B2 (en) 2007-09-19 2012-09-18 The Charles Stark Draper Laboratory, Inc. Method of fabricating microfluidic structures for biomedical applications
US9181082B2 (en) 2007-09-19 2015-11-10 The Charles Stark Draper Laboratory, Inc. microfluidic structures for biomedical applications
US10265698B2 (en) 2007-09-19 2019-04-23 The Charles Stark Draper Laboratory, Inc. Microfluidic structures for biomedical applications
EP2694967A4 (fr) * 2011-04-06 2014-10-29 Ortho Clinical Diagnostics Inc Dispositif d'analyse ayant des saillies en forme de losange
WO2014001459A1 (fr) * 2012-06-29 2014-01-03 Danmarks Tekniske Universitet Procédé de charge d'un porteur d'essai, et porteur d'essai

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