EP1325108A1 - Procede et dispositif de creation de micro-reseaux - Google Patents

Procede et dispositif de creation de micro-reseaux

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Publication number
EP1325108A1
EP1325108A1 EP00970705A EP00970705A EP1325108A1 EP 1325108 A1 EP1325108 A1 EP 1325108A1 EP 00970705 A EP00970705 A EP 00970705A EP 00970705 A EP00970705 A EP 00970705A EP 1325108 A1 EP1325108 A1 EP 1325108A1
Authority
EP
European Patent Office
Prior art keywords
substrates
substrate
stack
holes
reagent
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP00970705A
Other languages
German (de)
English (en)
Other versions
EP1325108A4 (fr
Inventor
Stephen B. Leighton
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
LEIGHTON, STEPHEN B.
Original Assignee
Beecher Instruments
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Beecher Instruments filed Critical Beecher Instruments
Priority claimed from PCT/US2000/027925 external-priority patent/WO2002031106A1/fr
Publication of EP1325108A1 publication Critical patent/EP1325108A1/fr
Publication of EP1325108A4 publication Critical patent/EP1325108A4/fr
Withdrawn legal-status Critical Current

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    • 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
    • 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
    • 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
    • B01J2219/00279—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
    • B01J2219/00319—Reactor vessels in a multiple arrangement the reactor vessels being formed by arrays of wells in blocks the blocks being mounted in stacked arrangements
    • 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
    • B01J2219/00416—Vacuum
    • 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
    • B01J2219/00527—Sheets
    • B01J2219/00529—DNA chips
    • 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
    • B01J2219/00585—Parallel 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
    • B01J2219/00583—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
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • 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
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • B01J2219/00608—DNA chips
    • 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
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00659—Two-dimensional 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
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00664—Three-dimensional arrays
    • B01J2219/00668—Two-dimensional arrays within three-dimensional 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
    • B01J2219/0068—Means for controlling the apparatus of the process
    • B01J2219/00686—Automatic
    • B01J2219/00691—Automatic using robots
    • 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
    • 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
    • B01J2219/00725—Peptides
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/10—Libraries containing peptides or polypeptides, or derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B60/00—Apparatus specially adapted for use in combinatorial chemistry or with libraries
    • C40B60/14—Apparatus specially adapted for use in combinatorial chemistry or with libraries for creating libraries

Definitions

  • the invention relates to a method and device capable of simultaneously creating a series of identical micro-arrays, each micro-array comprising hundreds or thousands of analyte- assay regions on a solid support, each analyte-specific reagent useful, for example, in detecting labeled cDNA in hybridization assays .
  • Micro-arrays of hundreds or thousands of biological analyte-assay regions are widely used for biological analysis.
  • Tiny droplets each containing a different known reagent, usually distinct polynucleotide or polypeptide biopolymers such as known DNA fragments, are deposited and immobilized in a regular array on a solid substrate such as a glass microscope slide.
  • the array of dried droplets is exposed to a solution containing an unknown, for example complementary DNA (cDNA) fragments pre-labeled with fluorescent or radioactive chemical tags. Binding reactions or hybridizations occur wherever there is a match between the complementary sequence polynucleotides in the array and the cDNA.
  • Subsequent optical or radiosensitive scanning determines which spots contain tags, thereby identifying the complementary compounds present in the solution.
  • micro-arrays provide a useful tool for rapid biological analysis, the processes by which the micro-arrays are produced remain time consuming and expensive.
  • the set of pens is washed and dried, reloaded with the next set of reagents and the next set of droplets are printed onto the same substrates at adjacent locations. This procedure is time consuming and requires expensive and elaborate equipment to achieve precision and speed.
  • Another known method involves long flexible capillary tubes to carry fluid from sets of storage wells to the tips of the tubes, which tips are applied to one substrate after another in a manner similar to Shalon et al .
  • This method suffers all of the same disadvantages as Shalon et al . and also requires a significant volume of expensive reagent to be stored in the capillary tubes.
  • Still another known method is disclosed in US Patent 5,800,992 (Fodor et al . ) and involves combinatorial chemistry to synthesize oligonucleotides on the substrate with a series of chemical reactions (Affymetrix) .
  • This method is limited to oligonucleotides and is not suitable for long stranded cDNA's.
  • the sequence of the oligonucleotides must be known in advance. This method also suffers the disadvantages of requiring cumbersome expensive equipment and involving time consuming reaction steps .
  • US Patent 5,843,767 (Beattie) teaches the provision of a multiplicity of discrete channels running through the substrate and arranged in groups, and with binding reagent immobilized on the walls of the channels .
  • the channels increase the amount of surface area in the substrate available for the binding, thus theoretically improving detection sensitivity and efficiency.
  • improvements were not as great as expected, since the detection optics will still be limited to direct reception only from the projected area.
  • the invention utilizes a plurality of substrates, each of which having a top side, a bottom side, and a pattern of through-holes.
  • Each through-hole has a wider upper cross- section, a narrower lower cross-section, and preferably a step or plateau parallel to the top side of the substrate formed in the transition area.
  • the corresponding through-holes are in registry and form tunnels extending through the stack of substrates .
  • Reagents of interest are caused to flow through the tunnels and deposit on the step or plateau area. Thereby all substrates in the stack are " ⁇ spotted"' simultaneously, at precise locations and with a precise amount of reagent .
  • a barrier layer may be provided between substrates to prevent leak-through between neighboring holes .
  • the substrates are separated. In this manner a series of micro- arrays , each capable of containing hundreds or thousands of biological samples such as cDNA fragments, is formed simultaneously.
  • Fig. 1 is an isometric drawing of stack of substrates, showing the matching holes and pockets.
  • Fig. 2 is a cross-sectional view through part of one row of holes in a stack of substrates .
  • Fig. 3 is a cross-sectional view of a stack of substrates combined with a means for filling them using a pipette . This figure also shows a clamping means .
  • Fig. 4 is a cross-sectional view of a stack of substrates combined with a means for filling them using vacuum suction and tubes to a micro-titre tray.
  • Fig. 5 is a cross section through a stack of substrates as in Fig. 2 but in an alternative arrangement.
  • Fig. 6a is an exploded view of a stacking arrangement of alternating layers of wide aperture gasket and narrow aperture substrate, the layers not-to-scale, as used in an alternative embodiment of the invention.
  • Fig. 6b is a cross-sectional view through two gasket and two substrate layers according to Fig. 6a.
  • Fig. 6c is a view of a section of a micro-array prepared using the assembly of Figs. 6a and 6b, showing six of the hundreds or thousands of analyte-assay regions formed on a solid support.
  • the present invention is concerned with a method of forming a micro-array of analyte-assay regions on a solid support, where each region in the array has a known amount of a selected, analyte-specific reagent. More generally, there is provided a substrate for use in detecting binding of labeled polynucleotides to one or more of a plurality different- sequence, immobilized polynucleotides.
  • Micro-arrays, and reagents used in the formation thereof, are well known in the art and thus need not be described herein in any great detail.
  • the reagents are preferably distinct polynucleotide or polypeptide biopolymers fixed to the substrate .
  • Methods of using the micro-arrays such as by contacting fluorescent reporter-labeled cDNAs with a micro-array of polynucleotides representing a plurality of known DNA fragments under conditions that result in hybridization of the labeled cDNAs to complementary-sequence polynucleotides in the array followed by examination by fluorescence under fluorescence excitation conditions, are also well known in the art and thus need not be described herein in greater detail .
  • US Patent 5,800,992 Fluorescence et al .
  • US Patent 5,807,522 Borrown et al .
  • the present invention is specifically concerned with the method and device with which a plurality of identical micro- arrays of biological samples can be easily and quickly produced.
  • a significant and distinguishing feature of the present invention resides in the utilization of a plurality of substrates, each of which having a top side, a bottom side, and a pattern of through-holes .
  • Each through-hole has a wider upper cross-section, a narrower lower cross-section, and preferably a step or plateau parallel to the top side of the substrate formed in the transition area.
  • the corresponding through-holes are in registry and form tunnels extending through the stack of substrates .
  • Reagents of interest are caused to flow through the tunnels and deposit on the step or plateau area. Thereby all substrates in the stack are ⁇ spotted" simultaneously, at the precise location and with a precise amount of reagent .
  • a barrier layer may be provided between substrates to prevent leak-through between neighboring holes. After the desired reagents have been deposited, the substrates are separated. In this manner a series of micro- arrays , each capable of containing hundreds or thousands of biological samples such as cDNA fragments, is formed simultaneously.
  • the present invention comprises a stack of preferably identical substrates each having the same pattern of through-holes, with one hole in each substrate corresponding to each spot of analyte-specific reagent intended in the final array. For example, if it were desired to create 100 identical arrays with one array per substrate and with each array having 10,000 different spots, then 100 identical substrates will be used, each manufactured with 10,000 through- holes, the through-holes are in registry when the substrates are stacked.
  • registration 1 ' simply means that through-holes of adjacent substrates in the stack are in communication.
  • the stack is formed such that horizontal step or plateau areas of each column of through-holes appear to be superimposed. These horizontal step or plateau area can be referred to for brevity as a discrete assay region or a "spot zone ' ' .
  • spot zones on each substrate are identical, such that assay spotting can be carried out by robotic means programmed to spot at specific x,y coordinates.
  • the areas of narrower cross-section are preferably provided on one side of the spot zone, i.e., at one of the edges of the spot zone. More preferably, the area of narrower cross section of the through-hole of even numbered slides in a stack are provided on one side of the spot zone (e.g., right side), and the area of narrower cross section of the through-hole of odd numbered slides in the same stack are provided on the opposite side of the spot zone (e.g., left side), such that reagent flowing through the tunnel is caused to " " " " "slalom 1 ' back and forth, washing over each of the spot zones with reagent, and ensuring that no bubbles are trapped in the tunnel .
  • the ""spot zones" are patterned on a substrate in a pattern which has 180° symmetry, i.e., when a first substrate is rotated about 180° and stacked on top of a second, non-rotated substrate, through- holes remain in registry and tunnels are formed.
  • This makes it possible to form all substrates using a single manufacturing technique, and to provide the areas of narrow diameter on one side of odd numbered slides, and to provide the areas of narrow diameter on the opposite side of even numbered slides, by simply rotating alternate numbered slides about 180° while stacking.
  • the size of the substrates may typically be 0.5 to 5 cm in lateral dimensions, and 0.05 to 3 mm thick, and may be the size of a conventional microscope slide. Different sizes would be appropriate for different applications.
  • the number, size and spacing of spots, and the number of substrates will depend on the number and the amounts of reagent to be used in the array.
  • each hole in each substrate may be associated with a counterbore, countersink, or other (possibly eccentric) pocket in the substrate .
  • These pockets create tiny volumes to the side of the line of holes through the substrates, when seen in cross-section.
  • these pockets provide small areas of substrate surface area roughly parallel to the overall surface of each substrate.
  • each through-hole has a wider upper cross-section, a narrower lower cross-section, and preferably a step or plateau formed in the transition area parallel to the top side of the substrate.
  • the sealing means may be a hydrophobic viscous substance such as grease, wax, a weak adhesive, or any other bonding or sealing agent compatible with (inert to) the particular chemistry being used for the arrays.
  • a very thin elastomer layer gasket
  • the extremely smooth surfaces characteristic of the micro-machining processes proposed for manufacture of the substrates makes the sealing relatively simple. Indeed, for some applications it may be possible to rely entirely on the super-smooth surfaces of the substrates, such as glass slides, wherein adjacent slides are in continuous contact with each other with the exception of the through-holes .
  • the respective reagents used to create the array are injected at one end of each tunnel, generally the upper end of the tunnel, with each tunnel receiving (in general) a different reagent.
  • the injections may be done one at a time or in groups or, preferably, simultaneously to all tunnels.
  • the injection may be done with syringes, tubes, or other means; manually or automatically; with the aid of pumps of various sorts, with capillary action or with vacuum.
  • the reagents flow through the tunnels extending through the stack substrates, including the side pockets formed by the areas of the through-holes with greater diameter, they will react with, and bond to, the exposed surfaces of said tunnels with side pockets, dependent on the chemistry of the particular reagents and surface in use, in a manner analogous to that which occurs in the prior art when droplets are physically deposited on flat surfaces. Drying can occur after deposition, also in a manner analogous to that which occurs in the conventional techniques. Thus, all of the same chemistries and combinations now in use in the state of the art may be used to advantage for particular applications with the method and device of the present invention.
  • the stack may be separated into its individual substrates by simply releasing the sealing means, if any. At this point multiple identical individual substrates are available for hybridization, etc. as with the conventional techniques. However, instead of having spotted each of dozens or hundreds of slides, the spotting process was only carried out once .
  • the identical patterns of holes are preferably manufactured using silicon or glass micro-lithography and micro-machining techniques .
  • This technology is ideally suited for inexpensive production of multiple identical patterns in the small sizes desired.
  • other techniques including but not limited to laser machining, plasma etching, and conventional machining or abrading may be used, as well as a technique involving the arrangement of dissimilar glass materials, one of which is acid etchable (channel glass) and in the form of fibers corresponding to the through-holes to be formed, the other of which is inert, followed by chemical etching to remove the etchable glass.
  • Protrusions on one side of each substrate, for example on the top side of the substrate, and corresponding depressions on the opposite side, for example the bottom side, may be used to align the substrates to create a stack with all holes in registry.
  • pins can be placed through alignment holes provided in all of the substrates to achieve the same end.
  • the substrates can be aligned with reference to their edges by providing guides against which to rest all of the layers. Other methods for aligning will be obvious to anyone skilled in the art .
  • the central manuf cturing step for manufacturing the substrates does not involve or determine the various reagents or site-selection or arrangement of spots, which can be chosen by the individual user laboratories .
  • a variety of schemes can be used to connect a reagent injection means to the stack of substrates. Simple arrays of passive micro-funnels or channels can mediate the transition from a relatively coarse injection means to a relatively fine array spacing. Alternatively or additionally, simple but precise x-y positioning devices can be used to move the stack of arrays with respect to the injection means. Since hundreds or more substrates are being injected simultaneously, reasonable production rates are possible without the expense of fast robots as used in the prior art .
  • substrates 1 are stacked with intermediate adhesive layers 4.
  • the adhesive layers have through-holes corresponding to the holes in the substrates to permit reagent to flow through the tunnel, and serve as horizontal barriers to prevent leakage of reagent between tunnels .
  • Each substrate layer has an array of through-holes 3 in registry with the through holes of adjacent substrates.
  • the adhesive is coated onto one or both of the top and bottom planar surfaces of the substrate in a manner such that the adhesive is interrupted at the location of the holes .
  • the adhesive may be provided on the substrate prior to the step of forming the holes, in which case adhesive is removed at the same time and in the same areas in which the holes are formed.
  • each through-hole has a wider upper cross-section, a narrower lower cross-section, and preferably a step or plateau 2 parallel to the top side of the substrate formed in the transition area. This step or plateau 2 ultimately forms the presentation area of the analyte-assay regions .
  • Locating pins 14 and/or bosses 15 may be used for initial alignment or to maintain alignment of the stack.
  • the areas of the holes having the narrower cross-section 3 are in registry and connect to form a tunnel which extends completely through the stack from top to bottom, including extending through the adhesive layers 4.
  • Side pockets are formed in the area of the step or plateau 2.
  • Adhesive may or may not be on the substrate in the area of the plateau.
  • Fig. 3 shows a device which can be used for injecting in conjunction with a stack of the substrates 1 of the present invention.
  • the stack of substrates is clamped between an adapter plate 6 and a vacuum manifold 9. Clamping pressure is provided by clamp 10 and frame 8.
  • the adapter plate forms an injection mask with tapered holes 7 having a larger upper diameter for easy access for the injecting means such as a pipette tip and narrow lower diameter in registry with the through-holes .
  • an aliquot of reagent is introduced into the adapter plate and permitted to flow downwards by gravity or capillary action, or the flow is assisted by a pressure differential such as created by application of a slight vacuum to the lower end of each tunnel.
  • Fig. 4 represents an alternative embodiment of the invention, and shows a stack of substrates 1 clamped between an upper vacuum manifold 9 and a lower tubing adapter plate 13.
  • Tubes 11 spread out to adapt to the spacing of wells in a micro-titre tray 12, and are in communication with the reagent provided in the wells of the micro-titre tray 12.
  • Fig. 5 is a cross section through a stack of substrates as in Fig. 2 but in an alternative arrangement wherein the pattern of through-holes 3 is staggered or alternating, defining a slalom path for the reagent.
  • This alternating path may have the advantage in some situations of mixing the filling flow for better coverage of the bottoms of the pockets . It may be simplest to alternate between two different patterns, but it is also possible to have repeating patterns every three slides or random patterns . It is also simple to have the pockets all in line for the greatest simplicity in later observation and automated data acquisition, but it is not absolutely necessary for the proper operation of the method. Many possible variations will be obvious to one skilled in the art.
  • the invention may also be carried out using the embodiment shown in Figs. 6a (exploded) and 6b (assembled), with only two of potentially dozens of stacked alternating substrates and spacers being shown.
  • the spacers may be separable from the substrates , or alternatively a spacer layer may coated onto a substrate followed by etching of through-holes, or a spacer layer may be silk screen printed, offset printed, or otherwise printed onto the substrate, or a solid elastomeric or other film with preformed through-holes may be laminated onto a substrate layer.
  • the spacer is comprised of a material which does not absorb reagent, and more preferably resists deposition of reagent, such as plastic (preferably an elastomeric polymer) , rubber, wax, glass, and metal. Any of the materials discussed above with respect to the first embodiment of the invention can be used in the second embodiment of the invention.
  • the spacers are interposed between the substrate layers, with the holes in the separable spacers being larger than the through holes 18a, 18b in the substrate 17a, 17b.
  • the spacer layer is of finite thickness, usually thinner than the substrate, thus creating a pocket between adjacent substrates, the pocket allowing reagent to contact and be deposited on the substrate as it flows through.
  • the staggered substrate through-hole option as discussed above with respect to Fig. 5 may optionally used in the embodiment of Figs. 6a and 6b.
  • Fig. 6b is a cross-sectional view through two spacer and two substrate layers, showing the two sets of layers according to Fig. 6a in the assembled condition. It is apparent that reagent can flow continuously through spacer apertures 19a, 19b in the spacer 16a, 16b and the through holes 18a, 18b in the substrate 17a, 17b.
  • Fig. 6c is a view of a section of a micro-array prepared using the assembly of Figs. 6a and 6b, showing six of the hundreds or thousands of analyte-assay regions remaining on the solid support after removal of the spacer 16a, 16b.
  • the spacer is coated or laminated onto the substrate, one spacer layer would remain adhered to each substrate, either on the top or on the bottom of the substrate.
  • the reagent will coat both sides of the substrate, either side of the substrate can be considered the top or useable side.
  • the spacer be kept as thin as possible in order to minimize the amount of reagent required and to minimize reagent deposition on the spacer.
  • the selection and thickness of the spacer and substrate materials is a matter of preference and can be readily determined by those working in this art .

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  • Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
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Abstract

L'invention concerne un procédé et un dispositif permettant de créer simultanément plusieurs micro-réseaux identiques d'échantillons biologiques. Elle concerne plusieurs substrats, chacun présentant un côté supérieur et un côté inférieur et un motif de trous de passage. Chaque trou de passage présente une section transversale supérieure large et une section transversale inférieure étroite, ainsi qu'un niveau ou un plateau formé dans la zone de transition. Lorsque les substrats sont empilés, les trous de passage sont en communication et forment des tunnels traversant la pile de substrats. Les réactifs d'intérêt traversent les tunnels et se déposent dans la zone du niveau ou du plateau. On peut installer une couche barrière pour empêcher des fuites entre des trous voisins. Une fois les réactifs voulus déposés, on sépare les substrats. Ainsi, on peut former simultanément une série de micor-réseaux capables de contenir chacun des centaines, voire des milliers, d'échantillons biologiques, tels que des fragments d'ADNc.
EP00970705A 2000-10-10 2000-10-10 Procede et dispositif de creation de micro-reseaux Withdrawn EP1325108A4 (fr)

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PCT/US2000/027925 WO2002031106A1 (fr) 1999-10-07 2000-10-10 Procede et dispositif de creation de micro-reseaux

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EP1325108A4 EP1325108A4 (fr) 2005-08-31

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JP (1) JP2004511761A (fr)
KR (1) KR100891217B1 (fr)
CN (1) CN1242047C (fr)
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KR100839390B1 (ko) * 2004-08-12 2008-06-19 도꾸리쯔 교세이호징 노우교 · 쇼쿠힝 산교 기쥬쯔 소고 겡뀨 기꼬우 마이크로 채널 어레이
US8569046B2 (en) * 2009-02-20 2013-10-29 Massachusetts Institute Of Technology Microarray with microchannels
TWI756384B (zh) * 2017-03-16 2022-03-01 美商康寧公司 用於大量轉移微型led的方法及製程
US10512911B1 (en) * 2018-12-07 2019-12-24 Ultima Genomics, Inc. Implementing barriers for controlled environments during sample processing and detection
CN111751539A (zh) * 2020-06-18 2020-10-09 东南大学深圳研究院 一种高通量垂直流免疫试纸分析微阵列
CN114684782A (zh) * 2022-03-30 2022-07-01 湖南超亟检测技术有限责任公司 一种基于单分子微阵列芯片基片的盲孔状微孔制作方法
KR102715737B1 (ko) * 2022-05-19 2024-10-11 한국기계연구원 마이크로 채널 반응기의 제조를 위한 전열판 접합 방법 및 전열판 정렬 구조

Citations (5)

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Publication number Priority date Publication date Assignee Title
WO1996003212A1 (fr) * 1994-07-26 1996-02-08 Sydney Brenner Dispositif multidimensionnel a canaux destine a la synthese d'une banque combinatoire
WO1997045455A1 (fr) * 1996-05-30 1997-12-04 Smithkline Beecham Corporation Synthetiseur a reacteurs multiples et procede de chimie combinatoire
WO1999032219A1 (fr) * 1997-12-19 1999-07-01 Glaxo Group Limited Systemes et procedes de synthese parallele en phase solide
DE19809477A1 (de) * 1998-03-06 1999-09-16 Schueth Ferdi Anordnung zum Testen der katalytischen Aktivität von einem Reaktionsgas ausgesetzten Feststoffen
EP0983795A2 (fr) * 1998-09-04 2000-03-08 Hitachi Software Engineering Co., Ltd. Element porteur de sondes et méthode pour sa production

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US5108704A (en) * 1988-09-16 1992-04-28 W. R. Grace & Co.-Conn. Microfiltration apparatus with radially spaced nozzles

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996003212A1 (fr) * 1994-07-26 1996-02-08 Sydney Brenner Dispositif multidimensionnel a canaux destine a la synthese d'une banque combinatoire
WO1997045455A1 (fr) * 1996-05-30 1997-12-04 Smithkline Beecham Corporation Synthetiseur a reacteurs multiples et procede de chimie combinatoire
WO1999032219A1 (fr) * 1997-12-19 1999-07-01 Glaxo Group Limited Systemes et procedes de synthese parallele en phase solide
DE19809477A1 (de) * 1998-03-06 1999-09-16 Schueth Ferdi Anordnung zum Testen der katalytischen Aktivität von einem Reaktionsgas ausgesetzten Feststoffen
EP0983795A2 (fr) * 1998-09-04 2000-03-08 Hitachi Software Engineering Co., Ltd. Element porteur de sondes et méthode pour sa production

Non-Patent Citations (1)

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Title
See also references of WO0231106A1 *

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AU2000280042B2 (en) 2007-03-01
KR100891217B1 (ko) 2009-04-01
KR20040007402A (ko) 2004-01-24
EP1325108A4 (fr) 2005-08-31
AU8004200A (en) 2002-04-22
CN1454253A (zh) 2003-11-05
CA2425634A1 (fr) 2002-04-18
CN1242047C (zh) 2006-02-15
JP2004511761A (ja) 2004-04-15
CA2425634C (fr) 2008-03-18

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