JP2004511761A - Method and apparatus for producing microarray - Google Patents

Abstract

Methods and apparatus are provided for making multiple identical microarrays of biological samples simultaneously. The present invention utilizes a plurality of substrates each having a top surface, a bottom surface, and a fixed pattern of through holes. Each through-hole has a larger cross-sectional area at the top and a smaller cross-sectional area at the lower part, with a step or plateau at the transition between them. When multiple substrates are stacked, the through holes are registered and tunnels are formed through the stack of stacked substrates. The desired reagent flows through this tunnel and attaches to a step or plateau. A barrier layer may be provided to prevent leakage between adjacent through holes. Once the desired reagents have been deposited, the substrate is separated. Thus, a series of microarrays are formed simultaneously, each of which can contain biological samples such as hundreds or thousands of cDNA fragments.

Description

[0001]
BACKGROUND OF THE INVENTION
Field of the invention
The present invention relates to a method and apparatus that can simultaneously produce a series of identical microarrays, each microarray comprising hundreds to thousands of analyte assay areas on a solid support, Each region contains, for example, an analyte-specific reagent useful for detecting labeled cDNA in a hybridization assay.
[0002]
Description of related technology
Microarrays consisting of hundreds to thousands of biological analyte assay areas are widely used for biological analysis. Small droplets, each containing a different known reagent, usually a well-defined polynucleotide or polypeptide-type biopolymer, such as a known DNA fragment, are placed in a defined array on a solid substrate such as a microscope glass slide ( Column) and immobilized. The dried array of droplets is exposed to a solution containing an unknown, eg, complementary DNA (cDNA), pre-labeled with a fluorescent or radiochemical tag. If the complementary sequence polynucleotides of the array and the cDNA match, a binding reaction or hybridization occurs. After that, by optical or radiation sensitive scanning, which spots contain the tag, complementary compounds present in solution are identified.
[0003]
Although microarrays provide a useful tool for rapid biological analysis, methods for producing microarrays remain time consuming and expensive.
For example, from U.S. Pat. No. 5,807,522 (Shalon et al.), It is known to use various shapes of capillary pens to print or drop droplets on a substrate, but one substrate at a time. is there. A large number (typically 8 or 16) pens can be used simultaneously, often under the control of a robot, but each pen or group of pens is filled with only one reagent per pen. Can not. Thereafter, the pen is successively touched from one substrate to the next to deposit substantially identical droplets on each substrate. After depositing the first set of reagent droplets on each substrate of the set of substrates to be prepared (typically dozens to hundreds), wash and dry the set of pens and the next set of reagents. Is refilled and the next set of droplets is printed at adjacent locations on the same substrate. This operating procedure is time consuming and requires expensive and precise equipment to achieve accuracy and speed.
[0004]
Another known method uses long flexible capillaries to carry liquid from multiple sets of storage wells to the tip of the capillary, which in turn is applied to the substrate in a manner similar to Shalon et al. Go. This method has all of the same disadvantages as Shalon et al., And requires the storage of significant amounts of expensive reagents in capillaries.
[0005]
Yet another known method is disclosed in US Pat. No. 5,800,992 (Fodor et al.), Which relates to combinatorial chemistry for the synthesis of oligonucleotides on a substrate by a series of chemical reactions (Affymetrix). I do. This method is limited to oligonucleotides and is not suitable for long strand cDNAs. Also, the sequence of the oligonucleotide must be known in advance. This method also has the disadvantage that it requires complicated and expensive equipment and involves a time-consuming reaction step.
[0006]
To amplify a fluorescent or radioactive signal indicative of a binding reaction, US Pat. No. 5,843,767 (Beattie) discloses a multiplicity of discrete channels running through a substrate, arranged in groups. , With a binding reagent immobilized on the wall of the channel. The channels increase the amount of substrate surface area available for binding, and thus theoretically increase detection sensitivity and efficiency. However, in practice, it has been found that the improvement is not as great as expected, since the detection optics are still limited to direct reception only from the projected area.
[0007]
SUMMARY OF THE INVENTION
In view of the aforementioned difficulties inherent in known methods and types of microarray fabrication, it is an object of the present invention to provide an improved method and apparatus for simultaneously producing multiple identical microarrays of a biological sample. .
[0008]
The present invention utilizes a plurality of substrates each having a top surface, a bottom surface, and a fixed pattern of through holes. Each through-hole has a larger cross-sectional area at the top and a smaller cross-sectional area at the lower part, with a transition or plateau preferably formed at the transition therebetween, parallel to the top surface of the substrate. . When multiple substrates are stacked, the corresponding through-holes register and form a tunnel through the stack of stacked substrates. The desired reagent flows through the tunnel and attaches to the step or plateau. As a result, all the substrates in the stack are spotted at the correct position and with the correct amount of reagent at the same time (liquid adhesion processing). In order to prevent leakage between adjacent through holes, a barrier layer may be provided between the substrates.
[0009]
Once the desired reagents have been deposited, the substrate is separated. Thus, a series of microarrays are formed simultaneously, each of which can contain biological samples such as hundreds or thousands of cDNA fragments.
[0010]
In order that the following detailed description of the invention may be better understood and the contribution of the invention to the art may be more fully understood, the following is a fairly broad overview of the more relevant and important aspects of the invention. Other aspects of the invention which form the subject of the claims of the invention will be described hereinafter. The technical ideas and specific embodiments disclosed herein can be easily used as a basis for improvement or, alternatively, for designing the microarray of the present invention and designing other methods and apparatuses for performing the same purpose. It will be understood by those skilled in the art. It will be readily apparent to those skilled in the art that such equivalent constructions do not depart from the spirit and scope of the invention as set forth in the appended claims.
[0011]
For a more complete understanding of the nature and objects of the present invention, reference should be had to the following detailed description taken in conjunction with the accompanying drawings.
[0012]
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to a method of forming a microarray of analyte assay regions on a solid support, wherein each region of the array has a known amount of a selected analyte-specific reagent. More broadly, there is provided a substrate for use in detecting binding of a labeled polynucleotide to an immobilized polynucleotide of one or more different sequences.
[0013]
Microarrays and the reagents used to make them are well known to those skilled in the art and need not be described in further detail here. The reagent is preferably a separate polynucleotide or polypeptide biopolymer immobilized on a substrate.
[0014]
The fluorescent reporter-labeled cDNA is contacted with a microarray of polynucleotides representing a plurality of known DNA fragments under conditions that cause hybridization of the labeled cDNA to complementary sequence polynucleotides in the array, and then fluorescently excited under fluorescent excitation conditions. Methods of using microarrays, such as performing tests by, are well known to those skilled in the art and need not be described in further detail here. See U.S. Patent Nos. 5,800,992 (Fodor et al.) And 5,807,522 (Brown et al.) For a description of this technique. The disclosures of these U.S. patents are incorporated herein by reference.
[0015]
The present invention specifically relates to methods and devices that can easily and quickly generate multiple identical microarrays of a biological sample.
An important and salient feature of the present invention is that it utilizes a plurality of substrates each having a top surface, a bottom surface, and a pattern of through holes. Each through-hole has a larger cross-sectional area at the top and a smaller cross-sectional area at the bottom, with transitions between them preferably forming steps or plateaus parallel to the top surface of the substrate. When multiple substrates are stacked, the corresponding through-holes register and form a tunnel through the stack of stacked substrates. The desired reagent flows through the tunnel and attaches to the step or plateau. As a result, all the substrates in the stack are simultaneously spotted (liquid adhesion processing) with a correct position and a correct amount of the reagent. In order to prevent leakage between adjacent through holes, a barrier layer may be provided between the substrates.
[0016]
Once the desired reagents have been deposited, the substrate is separated. Thus, a series of microarrays are formed simultaneously, each of which can contain biological samples such as hundreds or thousands of cDNA fragments.
[0017]
That is, as compared to prior art planar substrate arrays, which process one substrate at a time, with an apparatus designed to deposit reagents only on the surface of the substrate one after another, the present invention provides each of the same. Consisting of a stack of preferably identical substrates having through-holes in the pattern, one hole in each substrate corresponds to each spot of analyte-specific reagent intended in the final array. For example, if one array per substrate (one row) and one would like to produce 100 identical arrays, each array having 10,000 different spots, each array was fabricated with 10,000 through holes. One would use 100 identical substrates. Each through-hole is registered when the substrates are stacked.
[0018]
"Register" as used herein simply means that the through-holes of adjacent substrates in the stack are in communication. In a preferred embodiment of the invention, the stack is formed such that the horizontal steps or plateaus of each column of the through-hole appear to overlap one another. These horizontal steps or plateaus are referred to as separate assay areas or "spot zones" for simplicity. For interchangeable use of substrates with microarrays, the spot zone of each substrate is identical so that spotting of the assay can be performed by robotic means programmed to spot at specific xy coordinates. Is preferred. The smaller cross-sectional area is preferably provided on one side of the spot zone, i.e. close to one edge of the spot zone. More preferably, the small cross-sectional area of the through holes of even numbered slides in the stack is provided on one side (eg, right side) of the spot zone, and the small cross sectional area of the through holes of odd numbered slides in the same stack is spotted. Installed on the opposite side (eg, left side) of the zone, the reagent flowing in the tunnel flows back and forth in a slalom (zigzag), washing each spot zone with the reagent and ensuring that air bubbles are not trapped in the tunnel. To
[0019]
In one preferred embodiment of the present invention, the "spot zones" are patterned on the substrate in a pattern having 180 ° symmetry. That is, when the first substrate is rotated by 180 ° and then superimposed on the second substrate that has not been rotated, a tunnel is formed with the through-hole kept in register. This allows all substrates to be made using one and the same manufacturing technique, and every other numbered slide is simply rotated 180 ° when stacked, with odd numbered slides having a small cross-sectional area on one side. On the other hand, even-numbered slides can be provided with a small cross-sectional area on the opposite side.
[0020]
Although there is no theoretical limitation on the size of the substrate, the width may be 0.5 to 5 cm, the thickness may be 0.05 to 3 mm, and the size of an ordinary microscope slide may be used. Different sizes may be appropriate for different applications. The number, size and spacing of the spots and the number of substrates will depend on the number and amount of reagents to be used in the microarray.
[0021]
Lateral leakage from one tunnel to any other tunnel when the same substrates are arranged in a stack so that the through holes overlap one another and form a tunnel through the stack of substrates It is preferable to provide a barrier seal between the slides on the substrate so that the occurrence of the heat generation cannot occur. In addition, each through-hole in each substrate is accompanied by a cylindrical hole divergence (counterbore), a conical hole divergence (counter sink) or another form of (possibly eccentric) pocket provided in the substrate. You may. These pockets create a small volume beside the line of the through hole through the substrate when viewed in cross section. Also, these pockets provide a small area of substrate surface area that is substantially parallel to the overall surface of each substrate. Preferably, each through-hole has a wider upper cross-sectional area and a narrower lower cross-sectional area, with a step or plateau formed at its transition, preferably parallel to the top surface of the substrate.
[0022]
The sealing means is a hydrophobic, viscous material, such as grease, wax, a weak adhesive, or any other (inert) bonding or sealing material that is compatible with the specific chemical used in the array. Is fine. For other applications, a very thin elastomer layer (gasket) may be sufficient. It has also been found that the extremely smooth surface inherent in the micromachining (cutting) method proposed for fabricating the substrate makes the sealing relatively simple. In fact, for some applications, the adjacent slides may be in continuous contact with each other, except for the through-holes, so that the seal may rely entirely on the ultra-smooth surface of a substrate such as a glass slide. Can be
[0023]
The individual reagents used to create the array are injected at one end of each tunnel, typically at the top of the tunnel. Each tunnel receives (generally) a different reagent. The injections can be performed one at a time, or in groups, or preferably simultaneously for all tunnels. Infusion can be by syringe, tube, or other means; manually or automatically; using various pumps, capillary action, or reduced pressure.
[0024]
As the reagent flows through the tunnel through the substrate stack, having side pockets formed by the large diameter portion of the through-hole, the reagent reacts with and binds to the exposed surface of the side pocketed tunnel. This reaction or coupling depends on the reagents used and the surface chemistry and is similar to that which occurs in the prior art, where the droplets are physically attached to a flat surface. Drying can be performed after deposition, again in a manner similar to that which occurs in the prior art. Thus, all of the same chemical reactions and combinations currently used in the state of the art can be advantageously used for application with the method and apparatus of the present invention.
[0025]
Whatever reaction is desired for the entire stack, once the reaction is complete and the stack is dry, the stack may be separated into its individual substrates. This means that it is only necessary to peel off the sealing means, if any. At this point, a plurality of identical individual substrates can be used for hybridization and the like in a conventional manner. Moreover, instead of spotting each of tens or hundreds of slides, the spotting operation was performed only once.
[0026]
The same pattern of holes (and associated side pockets or large diameter portions) is preferably made using silicon or glass microlithography and micromachining techniques. This method is ideally suited for inexpensive production of many identical patterns of desired small size. However, other methods include, but are not limited to, laser machining, plasma etching, and conventional machining and abrading methods, and one is acid-etchable (channel glass) to accommodate the through holes to be formed A method can also be used in which a dissimilar glass material is placed, in the form of a fibrous material, the other of which is inert, followed by a chemical etch to remove the etchable glass.
[0027]
Using one side of each substrate, for example, a projection (convex portion) provided on the upper surface of the substrate and a corresponding concave portion provided on the opposite surface, for example, the lower surface, create a stack in which all holes are registered. The substrates may be aligned in a manner as described. Alternatively, the same purpose can be achieved by passing pins through alignment holes provided in all the substrates. As a further alternative, if the machining is sufficiently precise, the guide can be used to align all substrates (layers) with the guide so that the substrate is aligned with its edges. Other alignment methods will be apparent to those skilled in the art.
[0028]
It is important to note that during this initial fabrication process, the location of the spot is determined without relying on sample attachment by a robot. In this way, significant accuracy can be achieved at relatively low cost in the large-scale, central substrate fabrication stage. Subsequent injection steps do not require expensive equipment, and this step can be performed in many different laboratories using different reagents. The core fabrication process for substrate fabrication does not involve or determine the location or placement of various reagents or spots. They can be selected by the individual user's laboratory.
[0029]
Various schemes can be used to connect the reagent injection means to the stack of substrates. Simple passive microfunnels or arrays of channels can mediate the transition from relatively large injection means to relatively small array spacing. Alternatively, or in addition, a simple but accurate xy positioning device can be used to move the stack of arrays relative to the injection means. Since hundreds or more substrates are simultaneously implanted, it is possible to obtain a reasonable production rate without the expense of fast robots as used in the prior art.
[0030]
The present invention will now be described in more detail with reference to the embodiments shown in the accompanying drawings. In FIG. 1, a plurality of substrates 1 are stacked by an intermediate adhesive layer 4. The adhesive layer has a through hole corresponding to the hole provided in the base so that the reagent can flow through the tunnel, and a horizontal barrier for preventing leakage of the reagent between the tunnels. Act as Each substrate layer has an array of through-holes 3 that register with the through-holes of the adjacent substrate. The adhesive is applied to one or both of the upper and lower flat surfaces of the substrate so that the adhesive does not break at the holes and block the holes. Alternatively, the adhesive layer may be formed on the substrate prior to the step of forming holes in the substrate. In that case, the adhesive is removed from the same location as the hole at the same time as forming the hole.
[0031]
As shown in FIG. 1, each through-hole has a wide upper cross-sectional area and a smaller lower cross-sectional area, and preferably has a step or plateau 2 parallel to the upper surface of the base at the transition portion. This step or plateau 2 ultimately forms the indicator part of the analyte assay area. Locating pins 14 and / or bosses 15 may be used for initial alignment or alignment of the stack.
[0032]
As can be seen in FIG. 2, the portions of the holes 3 having a small cross-sectional area are in register with each other, forming a tunnel that runs completely through the stack of substrates from top to bottom, while also passing through the adhesive layer 4. It is connected as follows. Side pockets are formed in the step or plateau 2 portion. The adhesive may or may not be on the plateau portion substrate.
[0033]
FIG. 3 shows an apparatus which can be used to carry out the implantation in combination with a stack of substrates 1 according to the invention. The stack of substrates is clamped between the adapter plate 6 and the vacuum manifold 9. Clamping pressure is provided by clamp 10 and frame 8. The adapter plate has a large-diameter hole 7 at an upper portion to facilitate access of an injection means such as a pipette tip, and a tapered hole 7 at a lower portion to have a small diameter in register with a through-hole of the base. An implantation mask is formed. During the introduction of reagents, an amount of reagent is introduced into the adapter plate and allowed to flow down by gravity or capillary action, or to facilitate flow by differential pressure, as created by applying a slight vacuum at the bottom of each tunnel. Let it. Due to the cost of the reagents, care should be taken that enough reagents enter the tunnel to completely fill the tunnel, but that no or little excess reagent exits the bottom of the tunnel. This is usually adjusted so that the vacuum pressure is large enough to draw the reagent into the tunnel, but not so large as to overcome the capillary forces that tend to hold the reagent inside the tunnel. Can be achieved.
[0034]
FIG. 4 illustrates another embodiment of the invention, showing a stack of substrates 1 clamped between an upper vacuum manifold 9 and a lower tube adapter plate 13. The tubes 11 extend to match the spacing of the wells of the microtiter tray 12 and are in communication with the reagents contained in the wells of the microtiter tray 12.
[0035]
FIG. 5 shows another arrangement of the substrate stack similar to that shown in FIG. 2, except that the pattern of the through-holes 3 is zigzag or alternate, forming a zigzag reagent flow path. It is sectional drawing. As long as there is one continuous (connected) channel through each column of through-holes and pockets, the exact location of each need not be repeated exactly in adjacent or alternate layers. This alternating (zigzag) flow path may offer the advantage of better coverage of the pocket bottom, depending on the mixing conditions of the flow to be filled. It is simplest to place every other pattern between two different patterns, but it is also possible to repeat the pattern for every three slides or to make a random pattern. Also, to simplify subsequent observations and automatic data acquisition, it is simple to align all pockets, but that is not absolutely necessary for proper operation of the method of the present invention. Many possible modifications will be apparent to those skilled in the art.
[0036]
The invention can also be implemented using the embodiment shown in FIGS. 6a (exploded view) and 6b (assembly view). These figures show perhaps two dozens of alternately stacked substrates and spacers, dozens of. In this embodiment, the bases 17a and 17b are provided with only small-diameter through holes 18a and 18b penetrated by machining. This simplifies the fabrication process as compared to the stepped or countersunk substrate shown in FIGS. Each planar attachment area on the substrate is actually defined by holes 19a, 19b provided in the spacers 16a, 16b.
[0037]
The spacer may be separated from the substrate, or a spacer layer may be formed on the substrate by coating and then a through hole may be formed by etching, or the spacer layer may be formed on the substrate by silk screen printing, offset printing, or the like. The film may be formed by printing, or a film of a solid elastomer or the like in which through holes are formed in advance may be laminated on the base layer. To minimize reagent waste, it is preferable that the constituent material of the spacer does not absorb the reagent, and that the reagent such as plastic (preferably an elastomeric polymer), rubber, wax, glass, and metal does not adhere easily. More preferably, Any of the materials described above with respect to the first aspect of the invention can be used in the second aspect of the invention.
[0038]
The spacer is sandwiched between the base layers, and the holes provided in the separable spacer are larger in diameter than the through holes 18a and 18b provided in the bases 17a and 17b. The spacer layer is typically of a fixed thickness that is less than the substrate, thus forming a pocket between adjacent substrates, which allows the reagent to contact the substrate and adhere to the substrate during the flow of the reagent. It becomes possible. The through-hole option of the zig-zag substrate described above with respect to FIG. 5 can also be employed as desired in the embodiment of FIGS. 6a and 6b.
[0039]
FIG. 6b is a cross-sectional view of two layers of spacers and two layers of substrate, showing the assembled state of the two sets of layers according to FIG. 6a. Obviously, the reagent can flow continuously through the spacer holes 19a, 19b of the spacers 16a, 16b and the through holes 18a, 18b of the substrates 17a, 17b.
[0040]
FIG. 6c is a drawing of a portion of a microarray made using the assembly of FIGS. 6a and 6b, showing hundreds to thousands of analyte assay areas remaining on a solid support after removal of spacers 16a, 16b. 6 of them are shown. When the spacers are formed by coating or laminating on the substrate, one spacer layer adhered to each substrate remains on either the upper surface or the lower surface of the substrate. In practice, since the reagents coat both sides of the substrate, either side or surface of the substrate may be considered the top or usable side.
[0041]
It is preferred that the spacer be as thin as possible to minimize the amount of reagent required and minimize the amount of reagent attached to the spacer. The choice and thickness of the spacer and substrate materials are a matter of preference and can be readily determined by those working in this field.
[0042]
With respect to the above description, it will now be appreciated by those skilled in the art that the optimal dimensional relationships for some of the present invention, including changes in size, material, shape, form, function and mode of operation, assembly and use. It is to be understood that all that is readily apparent and obvious and that is equivalent to what is shown in the accompanying drawings and described herein is encompassed by the present invention.
[0043]
Therefore, the above description is considered only as illustrative of the principles of the present invention. In addition, many modifications and changes will readily occur to those skilled in the art, and it is not intended that the present invention be limited to the exact construction and operation shown and described, but all suitable modifications and equivalents will be made. Belongs to the present invention and is included within its scope.
[0044]
The present invention has been described above.
[Brief description of the drawings]
FIG.
FIG. 4 is an isometric view of a stack of substrates showing the matching of holes and pockets.
FIG. 2
FIG. 4 is a cross-sectional view through a portion of a row of holes in a stack of substrates.
FIG. 3
FIG. 4 is a cross-sectional view of a substrate stack in combination with a means for filling a substrate with a pipette. This figure also shows the clamping means.
FIG. 4
FIG. 3 is a cross-sectional view of a substrate stack combined with vacuum suction and means for filling the substrate using tubes (capillaries) into a microtiter tray.
FIG. 5
FIG. 3 is a sectional view similar to FIG. 2 but through a stack of substrates in another arrangement.
FIG. 6
FIG. 6a is an exploded view of a stacked arrangement of alternating layers of large pore gaskets and small pore substrates used in another aspect of the invention, but the layers are not drawn to scale.
FIG. 6b is a cross-sectional view through two gasket layers and two base layers according to FIG. 6a.
FIG. 6c shows a portion of a microarray prepared using the assemblies of FIGS. 6a and 6b, showing six out of hundreds to thousands of analyte assay areas formed on a solid support. is there.

Claims (22)

A substrate for producing a microarray, the substrate having a top surface, a bottom surface, and a plurality of through holes communicating between the top surface and the bottom surface of the substrate, each through hole having a large cross-sectional area portion, A substrate having a small cross-sectional area and a plateau formed at the transition. 2. The substrate according to claim 1, wherein the substrate comprises a water-impermeable sealing means provided on at least one surface of the substrate and surrounding the through hole. 3. The substrate of claim 2, wherein said sealing means is selected from the group consisting of a hydrophobic viscous material, a weak adhesive, and a polymeric elastomer. The base is 1 cm³ 2. The substrate according to claim 1, wherein the substrate has at least 100 through holes per hole. At least a first analyte-specific reagent is provided on at least a first plateau, and a second analyte-specific reagent different from the first analyte-specific reagent is provided on at least a second plateau. The substrate according to claim 1. 6. The substrate of claim 5, wherein said analyte-specific reagents are capable of detecting labeled cDNA in a hybridization assay. The substrate according to claim 1, wherein the substrate is glass or silicon. A substrate stack for stacking substrates according to claim 1 for making microarrays, the stack comprising at least two substrates, each substrate communicating with a top surface, a bottom surface, and a top surface and a bottom surface of the substrate. Having a plurality of through-holes, each through-hole having a large cross-sectional area, a small cross-sectional area, and a plateau formed at a transition portion thereof,
In this substrate stack, the through holes at corresponding locations are registered such that they form a continuous tunnel through the substrate stack. 9. The substrate stack of claim 8, further comprising a barrier layer between the substrates that allows fluid flow through the tunnels but blocks fluid flow between adjacent tunnels. 9. The stack of claim 8, wherein said stack comprises at least 10 substrates. Apparatus for simultaneously producing a set of identical microarrays, comprising:
Releasable holding means,
A substrate stack, comprising a stack of substrates, held by the releasable holding means, wherein the stack includes at least two substrates, each substrate having a top surface, a bottom surface, and a base material having a top surface and a bottom surface. A plurality of through-holes communicating therewith, each through-hole having a large cross-sectional area, a small cross-sectional area, and a plateau formed at a transition portion thereof; Means for introducing reagents into the individual through-holes from the top or bottom surface of the substrate stack, together and forming a continuous tunnel through the substrate stack. The apparatus according to claim 11, further comprising a decompression means communicating with the side of the substrate stack opposite to the side where the reagent is introduced. 13. The apparatus of claim 12, wherein said means for introducing a reagent into an individual through-hole comprises two or more tubular channels, each channel communicating with a supply well containing a reagent. A method for simultaneously producing a series of identical microarrays comprising the following steps:
A substrate stack comprising a stack of substrates, the stack including at least two substrates, each substrate having a top surface, a bottom surface, and a plurality of through holes communicating between the top and bottom surfaces of the substrates. The through-hole has a large cross-sectional area, a small cross-sectional area, and a plateau formed at its transition, wherein the through holes are in register and continuous through the substrate stack. Forming a substrate stack forming a tunnel,
At least a first reagent is introduced into at least a first tunnel and at least a second reagent is introduced into a second tunnel, and the substrate is separated. 15. The method of claim 14, further comprising applying a reduced pressure to a side of the substrate stack opposite the side where the reagents are introduced. A method for simultaneously producing a series of identical microarrays comprising the following steps:
(A) a stack of alternating spacers and substrates, the stack including at least two spacers and at least two substrates, each substrate having a top surface, a bottom surface, and a space between the top and bottom surfaces of the substrate. Each spacer has a top surface, a bottom surface, and a plurality of through holes communicating between the top surface and the bottom surface of the spacer, and the through hole of the spacer is a through hole of the base. Larger in diameter, the through-holes in the spacer are in register with the through-holes in the substrate, and the aligned spacers and through-holes in the substrate form a continuous flow through the stack of alternating stacked spacers and substrates. Forming a column with channels, wherein the spacers are adapted to form a barrier separating the through-holes of adjacent columns,
(B) flowing a reagent through the continuous flow path to deposit the reagent on the substrate, and (c) separating the substrate so that a microarray of reagents appears on each of the at least two substrates. 17. The method of claim 16, further comprising separating the spacer from the substrate. 17. The method of claim 16, wherein at least two different reagents are flowed through at least two different columns. 17. The method of claim 16, wherein said spacer is formed from a material selected from the group consisting of plastic, rubber, wax, glass and metal. 17. The method of claim 16, wherein the diameter of the through hole in the spacer is at least three times the diameter of the through hole in the substrate. 17. The method of claim 16, wherein the stack comprises at least ten alternating removable spacers and a substrate. 17. The method of claim 16, wherein at least 100 columns extend through said stack.

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