MINIATURIZED DNA MICROARRAY FOR SMALL-VOLUME SAMPLE PROCESSING

Abstract

Miniaturized DNA microarrays are described to be used in conjunction with microfluidic channels or microcentrifuge tubes and microcentrifuge filters to reduce sample size, incubation time and to increase overall binding efficiency.

Claims

1. A miniaturized DNA microarray assembly, comprising: a) a high-density array of oligonucleotide probes printed on a solid substrate; b) an inert gasket surrounding the sides of the solid substrate, wherein the gasket has a fluid inlet port and a fluid outlet/exit port which allows fluid to flow over and contact the oligonucleotide probes of the array; and c) an optically transparent top surface sealed to the gasket.

2. The microarray assembly of claim 1, wherein the transparent top surface comprises a serpentine channel in contact with the inlet and outlet ports of the gasket thus allowing controlled flow of fluid over the oligonucleotide probes of the array.

3. The microarray assembly of claim 1, wherein the dimensions of the solid support of the array are about 11.0 mm by about 6.75 mm.

4. A miniaturized DNA microarray assembly, comprising: a) a high-density array of oligonucleotide probes printed on a solid substrate; b) an inert enclosure surrounding sides and bottom of the solid substrate, wherein the enclosure has a fluid inlet port and a fluid outlet/exit port which allows fluid to flow over and contact the oligonucleotide probes of the array; and c) an optically transparent top surface sealed to the enclosure.

5. The enclosure of claim 4, wherein one of the sides of the enclosure comprises a rupturable or removable barrier.

6. The microarray assembly of claim 4, wherein the dimensions of the solid support of the array are about 11.0 mm by about 6.75 mm.

7. A miniaturized DNA microarray assembly comprising: a) a high-density array of oligonucleotide probes printed on a solid substrate; and b) a microcentrifuge tube adapted to support the array during centrifugation.

8. The microcentifuge tube of claim 7, wherein the support adaptation comprises a bracket affixed to, or formed on, the inside walls of the tube.

9. A miniaturized DNA microarray assembly comprising: a) a high-density array of oligonucleotide probes printed on a solid substrate; b) a microcentrifuge tube; and c) a microcentrifuge tube filter apparatus adapted to support the array during centrifugation.

10. The microcentifuge tube filter apparatus of claim 9, wherein the support adaptation comprises a bracket affixed to, or formed on, the inside walls of the filter apparatus.

11. The microarray assembly of claim 9, wherein the dimensions of the solid support of the array are about 11.0 mm by about 6.75 mm.

12. A method of assaying a sample for one, or more, nucleotide sequences of interest, the method comprising: a) contacting the sample with a miniaturized DNA microarray of claim 1, under conditions sufficient for the specific hybridization of the nucleotide sequences of interest; and b) detecting the presence, or absence of hybridization on the array, wherein detection of hybridization indicates the presence of the nucleotides of interest in the sample.

13. A microfluidic device comprising: a) a miniaturized oligonucleotide microarray comprising a high-density array of oligonucleotide probes printed on a solid substrate; b) a fluid channel formed on top of the microarray with at least one inlet and at least one outlet port for sample and reagent flow; c) means for controlling the sample and reagent flow over the miniaturized microarray to allow a hybridization reaction to occur; d) means for controlling the hybridization reaction temperature in the device; and e) means for detecting the hybridization reaction.

14. The device of claim 13, wherein the microarray solid substrate is a glass slide.

15. A method of assaying a sample for one, or more, nucleotide sequences of interest, the method comprising: a) contacting the sample with a miniaturized DNA microarray of claim 4, under conditions sufficient for the specific hybridization of the nucleotide sequences of interest; and b) detecting the presence, or absence of hybridization on the array, wherein detection of hybridization indicates the presence of the nucleotides of interest in the sample.

16. A method of assaying a sample for one, or more, nucleotide sequences of interest, the method comprising: a) contacting the sample with a miniaturized DNA microarray of claim 9, under conditions sufficient for the specific hybridization of the nucleotide sequences of interest; and b) detecting the presence, or absence of hybridization on the array, wherein detection of hybridization indicates the presence of the nucleotides of interest in the sample.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In the accompanying drawings, reference characters refer to the same parts throughout the different views. Some but not all drawings are to scale; emphasis has instead been placed upon illustrating the principles of the invention. The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings(s) will be provided by the Office upon request and payment of the necessary fee. Of the drawings:

[0020] FIGS. 1A and 1B show two isometric views (perspective views, looking down) of a DNA microarray to scale according to the present invention.

[0021] FIGS. 2A and 2B are isometric views of the chamber for enclosing the top of the microarray. The embodiments show inflow and outflow openings for sample. FIG. 2B is a transparent (optically clear) version of the chamber shown in FIG. 2A.

[0022] FIGS. 3A, 3B, 3C and 3D show isometric and cross-sectional views of the chamber fitted on top of the microarray. FIG. 3A shows the chamber being lowered for position on the microarray. FIG. 3B shows the microarray with the chamber positioned snuggly on top. FIG. 3C is a transparent chamber version of the FIG. 3B. FIG. 3D shows a cross-section of the assembly of FIG. 3B, cut parallel to the width of the assembly.

[0023] FIGS. 4A and 4B are isometric views showing (to scale) microfluidic channels constructed in the interior (bottom of cover) of the microfluidic chamber. Both figures show top and bottom views of the chamber top. FIG. 4B is a transparent version of FIG. 4A. The embodiments have inlets for inflow of sample and outlets for outflow of excess sample and other liquid. Some versions encompass outlets at the end of each serpentine channel.

[0024] FIGS. 5A, 5B, 5C and 5D show isometric and cross-sectional views of the serpentine chamber fitted on top of the microarray. FIG. 5A shows the chamber being lowered for position on the microarray. FIG. 5B shows the microarray with the chamber positioned snuggly on top. FIG. 5C is a transparent chamber version of the FIG. 5B. FIG. 5D shows a cross-section of the assembly of FIG. 5B, cut parallel to the width of the assembly.

[0025] FIGS. 6 A-E is an embodiment of the mini microarray inserted in a microcentrifuge tube filter apparatus using the microarray of FIGS. 1A and B. Incubation is in a microcentrifuge spin column. The microarray can be completely enclosed (serpentine or large chamber) and a gasket can be added to the column to hold the microarray firmly in place.

[0026] FIG. 7 is an embodiment of the DNA microarray positioned vertically inside a microcentrifuge filter apparatus for the incubation process.

[0027] FIGS. 8A and 8B are perspective views of a custom microcentrifuge filter insert inside a microcentrifuge filter apparatus for use in the embodiment of FIG. 4. A gasket (or space-filler) can be added to the microcentrifuge spin column to hold the microarray firmly in place and to reduce the internal volume of the column, allowing the entire incubation process to take place inside a microcentrifuge tube.

[0028] FIGS. 9 A-B show side views of an alternative gasket design that can be added to the 1.5 ml microcentrifuge spin column to hold the array firmly in place and to reduce the internal volume of the column. In this embodiment the microarray is inserted into the bracket and sample solution held in the upper half of the tube.

[0029] FIGS. 10A, 10B and 10C show exemplary steps of a mini-microarray assay in a microcentrifuge tube. The mini microarray is enclosed in a gasket and can be inserted in a vertical or horizontal position. The entire apparatus (DNA microarray with custom hybridization chamber) is prepared, filled with sample, left in a microcentrifuge tube for the duration of the hybridization process and then placed in a microcentrifuge tube to elute and collect the unused sample.

[0030] FIGS. 11A and 11B show a DNA microarray (multiple views) with epoxysilane on a glass substrate 11.0 mm×6.75 mm×0.7 mm.

[0031] FIG. 12 shows a flow chart for a sample protocol for spotting oligonucleotides onto a substrate.

[0032] FIGS. 13A and 13B show a gasket (multiple views) that is plastic with double-sided adhesive on both sides; 11.0×6.75 mm×0.12 mm with a 0.75 mm opening for elution of fluid.

[0033] FIGS. 14A and 14B show a lid (multiple views) of polycarbonate 11.0 mm×6.75 mm×0.25 mm with an about 0.9 mm hole/opening for delivery of fluid.

[0034] FIGS. 15A and 15B show an assembled hybridization well on a DNA array (exploded views).

[0035] FIGS. 16A and 16B show an assembled hybridization well on a DNA array (multiple views).

[0036] FIGS. 17 A-D show cross-section views of a hybridization well on a DNA array.

[0037] FIGS. 18 A-C show the steps of filling and emptying the hybridization well using a fluorescent sample. Sample recovery was about 98.7% (N=3) after spinning at 1000×g for 1 minute in a fixed rotor centrifuge.

[0038] FIG. 19 shows a flow chart for post-hybridization washing steps.

[0039] FIGS. 20 A-D show the steps of using a DNA microarray of the present invention. The device/apparatus (DNA microarray with custom hybridization chamber) is prepared, filled with 5.3 μl of sample, left in a humidity chamber for the duration of the hybridization process as determined for the specific hybridization assay, and then placed in a microcentrifuge tube to elute and collect the unused sample. Sample specifically hybridized to the probes of the DNA array can then be detected using standard means.

[0040] FIGS. 21 A-B show the results of a hybridization experiment using the claimed device. Fluorescently-labeled DNA is specifically bound to/hybridized with the oligonucleotide probes immobilized on the surface. Two fluorescent spots are visible on the surface of the glass substrate (FIG. 20 A and FIG. 20 B are the same image with coloring inverted.

[0041] FIGS. 22 A and B depict a chiplet fixture for imaging of the assembled microarrays having the same footprint as a 96-well microtiter plate.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0042] The invention now will be described more fully hereinafter with reference to the accompanying drawings, in which illustrative embodiments of the invention are shown. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

[0043] As used herein, the term “and/or” includes any and all combinations of one or more of the associated listed items. Further, the singular forms and the articles “a”, “an” and “the” are intended to include the plural forms as well, unless expressly stated otherwise. It will be further understood that the terms: includes, comprises, including and/or comprising, when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof. Further, it will be understood that when an element, including component or subsystem, is referred to and/or shown as being connected or coupled to another element, it can be directly connected or coupled to the other element or intervening elements may be present.

[0044] Embodiments of the present invention utilize an ultra-high-density DNA microarray of probe spots which is meant to interface with a small sample volume and to allow optical access to the array during the incubation period. As used herein, the term “sample” can encompass “sample of target species”, as well as “oligonucleotide of interest” or simply “target”. Note that from hereinafter, the term “microarray” and “array” will denote “mini-microarray”. A sample can be any fluid comprising the target of interest, such as a bodily fluid such as blood, plasma, serum, cerebral spinal fluid, urine obtained from a subject.

[0045] The array may be printed on one of a variety of materials, substrates providing solid support, which may be optically transparent or opaque, including glass, PMMA (poly-methylmethacrylate), COP (cyclo-olefin polymer) or COC (cyclic-olefin copolymer), among others. The materials may have a surface coating such as epoxysilane, to assist in stable probe spot formation and in probe binding. Other suitable surface coatings compatible with the substrate and with properties suitable for stabilizing spot formation are known to those of skill in the art and can include, for example streptavidin, polylysine and other suitable reagents. Optionally, a reflective layer such as a gold or sulfur reflective layer, can be included under the surface coating. The array comprises discretely spaced spots of specific oligonucleotide probes (either all of the same oligonucleotide or of different oligonucleotides). Individual spots on the array may be as small as 5 micrometers (μm) or as large as 1 millimeter (mm) in diameter with a center-to-center pitch as small as 10 μm or as large as 2 mm. The array may completely cover the surface of the substrate or may only cover a partial section of the available area. FIGS. 1A and 1B show two isometric views (perspective views, looking down) of a DNA mini-microarray 10 to scale according to the present invention. FIG. 1A is a schematic of the array, substrate 11 size of about 11.0 mm× about 6.75 mm. However, the dimensions can be larger or smaller. FIG. 1B shows an alternate orientation of the microarray 10.

[0046] In some embodiments both sides of the array substrate may be used to increase probe density, however, the substrate must then be opaque for detecting the hybridization reactions on both sides of the arrayed substrate.

[0047] FIGS. 2A and 2B show various embodiments (to scale) of a rectangular chamber 20 to fit snuggly on top of the microarray 10. In all embodiments the inflow opening/hole (IN) is for inserting sample for hybridization and is on top. The outflow opening/hole (OUT) for expelling unused sample and other liquids can be either on top and bottom. Both IN and OUT are along the smaller dimension, width, of the enclosing chamber. FIG. 2B shows that the chamber may be transparent (glass or plastic) for monitoring progress of hybridization. In FIG. 2B one can see the interior through the chamber 20 as the chamber top 25 may be optically transparent. The other sides (not just the top 25) of the chamber can also be transparent.

[0048] FIGS. 3A through 3D show the chamber 20 (with top 25) laid on top of the DNA microarray 10. The views are isometric and to scale. All embodiments show the outflow (OUT) in the bottom of the chamber, but the outflow can alternatively be on top.

[0049] FIG. 3A shows the chamber 20 being prepared to go on top of the DNA microarray 10. Although not shown a rectangular gasket may be inserted between the chamber 20 and the microarray 10 for leak proof fitting of the array and the chamber.

[0050] FIG. 3B shows the chamber 20 fitted over the microarray 10. FIG. 3C is a transparent version of the chamber 20 laid on top of the microarray. The advantage of a transparent chamber is that it allows for optical monitoring of the progress of hybridization. FIG. 3C shows the visible spots on the microarray through the transparent chamber top 25.

[0051] The sample will be loaded into the well (using fluid inlet IN) and will hybridize with probes on the DNA microarray surface. The small volume of the enclosure (about 1-to about 100 μl; preferably 1-10 μl, and for example, more specifically can be about 5.3 μl) will reduce the amount of target sample necessary, relative to state-of-the-art techniques, to achieve probe-target binding. The sample may sit unperturbed during the incubation period or closed-loop pressure-driven flow will enable the sample to be passed over the array in a cyclic fashion to maximize interaction between targets and nucleic acid probes. This will reduce incubation time and will increase overall binding efficiency. The high specificity of conventional DNA microarray assays will be maintained by the increased target-probe interaction. The closed nature of the system will prevent evaporative sample loss and will enable the recovery of unhybridized sample following the incubation period. In addition, the top/ceiling surface of the chamber, or, alternatively, the entire device surface, will be optically transparent to enable detection (read out) using standard optical techniques (i.e., fluorescence detection).

[0052] FIG. 3D shows an end view of the mini-microarray and the chamber.

[0053] Alternatively, as shown in FIGS. 4A and 4B, serpentine microfluidic channels are constructed to sit directly atop the DNA microarray to allow for even further reduction in the fluid volume. FIG. 4A shows the top of the serpentine chamber 35 with IN showing inflow for sample. Each serpentine tube has an outflow OUT exit. The second figure in FIG. 4A shows the bottom of the serpentine chamber which shows the serpentine channels more clearly. FIG. 4B is a transparent version of the two views shown in FIG. 4A.

[0054] FIG. 5A shows the chamber 35 being prepared to go on top of the DNA microarray 10. Although not shown a rectangular gasket may be inserted between the chamber 35 and the microarray 10 for leak proof fitting of the array and the chamber.

[0055] FIG. 5B shows the chamber 35 fitted over the microarray 10. FIG. 5C is a transparent version of the chamber 35 laid on top of the microarray. The advantage of a transparent chamber is that it allows for optical monitoring of the progress of hybridization. FIG. 5C shows the visible spots on the microarray through the transparent chamber top 35. FIG. 5D shows an end view of the mini-microarray and the chamber.

[0056] The transparent DNA microarray forms the bottom surface of the channels. The sample will be loaded into the channels and exits out of the channels using a set of fluid ports placed in the roof of the enclosure. The channels will be arranged in a serpentine pattern and will be spaced such that the fluid path matches the center-to-center pitch of the microarray spots.

[0057] FIG. 6 A-E is an embodiment of incubation in microcentrifuge tube filter. The microarray may be completely enclosed (serpentine or large chamber) and a gasket may be added to the filter to hold the array firmly in place. (Microcentrifuge tube filter is shown only for illustrative purposes as the dimensions do not exactly match commercial filter as drawn.)

[0058] FIG. 7 is an embodiment of the DNA microarray positioned inside a microcentrifuge filter apparatus for the incubation process. In the figure the DNA microarray is constructed (similar to FIG. 1A) on a substrate that is small enough to fit inside of a standard microcentrifuge tube (e.g., a 1.5 ml Eppendorf tube). The DNA microarray is placed inside a microcentrifuge filter (FIGS. 8A, 8B and 9), which is placed inside the microcentrifuge tube (FIG. 9). Such microcentrifuge filters are known to those of skill in the art and are also commercially available. The filters can comprise any suitable material such as fiberglass, and further comprise a specific pore size suitable for separating unhybridized sample from the hybridized target. A hydrophobic membrane with a large pore size would keep the fluid stable above the membrane but still allow for elution of the entire sample after the incubation period and spin-down. Adhesion/adsorption tests will determine the best membrane material. The microcentrifuge filter can comprise an insert that holds the mini-microarray and reduces the interior space of the filter. Such an insert can be made out of any suitable inert/non-reactive material such as a plastic.

[0059] The target sample is then added to the well of the microcentrifuge filter to allow for incubation with the DNA microarray. Following the incubation period, the entire apparatus is loaded into a centrifuge and centrifugal force is used to draw the unhybridized sample away from the microarray, through the microcentrifuge filter, and into the base/bottom of the microcentrifuge tube where it can be collected for downstream processing. Unhybridized sample may also be collected on the surface of the microcentrifuge filter if the filter pore size is of suitable size. The DNA microarray is finally removed from the microcentrifuge tube for analysis.

[0060] The DNA mini-microarray with a sealed microfluidic well (the microcentrifuge filter apparatus) or the mini-microarray itself can be fit into a custom-made adapter/bracket (molded or 3D-printed) which allows the filter holding the mini-microarray, or the mini-microarray itself, to be snugly/securely fit into a cylindrical microcentrifuge filter (FIG. 9). Following the hybridization incubation period, the entire microcentrifuge tube is loaded into a centrifuge and centrifugal force is used to draw the unhybridized sample out of the sealed DNA microarray, through the microcentrifuge tube and, if present, through the filter, and into the base of the microcentrifuge tube where it can be collected for downstream processing. The DNA microarray is removed from the microcentrifuge tube for analysis.

[0061] In all of the embodiments, the mini-microarray assembly/apparatus is compact, requires a small sample volume, and enables collection of unused sample following the incubation period. The embodiments of FIGS. 3A-3D and 5A-5D will enable real-time optical readout to identify DNA hybridization on the array. The only required equipment for this readout is a microscope, camera, and computer. One embodiment of the present invention (FIGS. 6, 7 and 9) requires that the microarray to be removed from the microcentrifuge tube following the incubation period to optically assess DNA hybridization.

[0062] FIGS. 10A, 10B and 10C illustrate a hybridization assay using the mini-microarray assembly of the present invention. The mini-microarray glass slide with a gasket 10G (FIG. 10A) is lowered into the tube with the outlet port (in this case a funnel shape) toward the bottom of the tube. The centrifuge tube 30_1 is modified with an adapter/bracket to securely hold the mini-microarray assembly in place.

[0063] Sample solution, i.e., target oligonucleotides, in solution, is added to the tube. The lid L is closed and the centrifuge tube with lid closed (30_2) is incubated with agitation, typically in the horizontal position as in FIG. 10B (although incubation in the vertical position is also an option). Another possibility for mixing/incubation could be ultrasonic excitation of the tube. In some implementations, electrophoretic forces can be used to bring the target sample closer to the surface of the microarray. This would require the integration of electrodes into the microfluidic device.

[0064] Following the incubation period, the entire apparatus is loaded into a centrifuge and centrifugal force is used to draw the unhybridized sample away from the microarray, through the exit port, and into the base of the centrifuge tube where it can be collected for downstream processing (FIG. 10C).

[0065] As shown in FIG. 10A in the vertical positioning, the tube is filled to completely submerge the probe area of the mini-microarray assembly with target sample solution. In the horizontal positioning, shown in FIG. 10B, only the top of the array assembly needs to be covered with the sample solution. Sections of the tube may be filled to reduce unnecessary volume, e.g., use of a filter insert or other insert that would convert the cylindrical section of the interior to a rectangular shape.

[0066] Vertically orienting the tube and allowing for partial coverage of the chip is best if one has a small number of probes. Preferably, the backside of the chip can also be used. For a full array of probes, one needs to cover the chip entirely and a thin film of sample with the tube in the horizontal position would be optimal.

[0067] There are advantages and disadvantages to both orientations of the tube. In the vertical orientation, more sample will be required, but the mixing of probe and target molecules will be more thorough during incubation reaction period. In the horizontal position, less sample is necessary with the accompanying risk of less than thorough mixing between the sample and the probe molecules.

Example 1: Fabrication of a Miniaturized DNA Microarray

[0068] The microarray can be fabricated as described herein, although some variation can be incorporated into the fabrication procedure as known to those of skill in the art. In particular, the microarray is fabricated on 0.7 mm-thick glass substrates which are cut to dimensions of 11.0 mm in length and 6.75 mm in width. The substrates are treated with epoxysilane to create a surface to which amine-modified oligonucleotide probes can bind. (FIGS. 1A-B) The epoxysilane coating process proceeds as follows. First, the glass substrates are plasma treated with oxygen in a plasma asher (see for example, the Nordsom March plasma asher at nordson.com/en-us/divisions/march) for two minutes at 80 W power. The plasma-cleaned substrates are then immediately immersed in a freshly made solution of 2% (3-glycidyloxypropyl)trimethoxysilane in 95% ethanol and incubated for 16-18 hours at 37° C. with mixing. Following the incubation period, the substrates are removed from the solution and rinsed three times in pure ethanol. Finally, the substrates are dried in a stream of nitrogen and stored in a desiccator until ready for use. Once the substrates have been treated with epoxysilane, amine-modified oligonucleotide probes diluted in 3×SSC to a concentration of 40 uM are spotted on the surface. The probes are incubated at room temperature for 30 minutes followed by a snap-drying step at 50° C. for 2-5 minutes which allows for probe-substrate binding. The substrates can then be washed to remove any non-specifically bound probes by submerging them in a series of wash buffers which could include 0.1% Triton X-100, 1 mM HCl, 100 mM KCl, and diH.sub.2O. The substrates are then blocked by submerging them in an ethanolamine solution which is made up of 0.1% SDS added freshly to 50 mM ethanolamine in 0.1 M Tris (pH 9.0). The substrates are incubated in the blocking buffer for 15 minutes at room temperature. Following the incubation period, the substrates are rinsed in deionized water and dried in a stream of nitrogen. The blocking step helps to reduce non-specific binding of analytes to the epoxysilane surface. All of the spotting, washing and blocking buffers and protocols are standard and known to those of skill in the art. For example, after the substrates are treated with epoxysilane, as described herein, the assay steps follow a protocol provided by one company that makes commercial epoxysilane slides (Schott Technical Glass Solutions, and the product is called Nexterion Epoxysilane Coating, or “Nexterion Slide E”). The protocol describes the steps starting from probe immobilization onto the epoxysilane surface (See FIG. 12) and continue through post-hybridization washes. (See FIG. 19). It is understood that variations of the protocol resulting in optimization of specific microarray assays can be determined by those skilled in the art.

[0069] Following the fabrication of the DNA microarray, a custom hybridization chamber is assembled and affixed to the array's surface. (FIGS. 13-17). The hybridization chamber is comprised of a piece of 120 micron-thick double-side-adhesive-backed plastic (SecureSeal Adhesive, Grace Bio-Labs, Bend, Oreg.) which is laser-cut to form a gasket, and a piece of about 250 micron-thick polycarbonate which is laser-cut to form a lid. The gasket has outer dimensions which match those of the microarray substrate and an inner rectangular cutout of dimension of about 9.0 mm in length and about 4.75 mm in width. There is also a cutout along one side of the gasket of dimension of about 0.75 mm, to allow for sample elution from the hybridization chamber. The polycarbonate lid has outer dimensions which match those of the microarray substrate and a single circular cutout of diameter of about 0.9 mm to allow for sample delivery to the hybridization chamber. The assembled hybridization chamber has an interior volume of about 5.3 microliters.

[0070] Once the custom hybridization chamber has been affixed to the DNA microarray, a sample may be added to the chamber. The sample is delivered by using a micropipette to dispense through the circular cutout in the lid of the chamber. The entire apparatus is then left to incubate in a humidity chamber for about 60 minutes at room temperature. Following the incubation period the apparatus is then placed into a standard microcentrifuge tube and spun in a centrifuge at 500×g for five minutes. The centrifugation process causes the sample to be eluted out of the cutout in the base of the gasket and it is collected at the bottom of the microcentrifuge tube. Initial tests using a sample of fluorescein diluted in water show a sample collection efficiency of 98.7% (n=3) (See FIGS. 18A-C).

[0071] Post-hybridization washing steps can then be performed in a similar fashion by pipetting the desired wash buffer (5.3 uL) into the inlet port, then placing the microarray into a microcentrifuge tube and spinning to remove the wash buffer. For example, a low stringency wash buffer such as 2×SSC+0.2% SDS can be used first, followed by increasing stringency wash buffers such as 2×SSC and 0.2×SSC (from Schott Slide E Protocol, see FIG. 19). After each wash buffer is added and incubated for 10 minutes at room temperature, the microarray is put into a microcentrifuge tube and spun at 500×g for 5 minutes in a tabletop centrifuge. This process may be repeated as necessary to remove unbound DNA. The microarray is then removed from the centrifuge tube and may be used for further downstream processing (e.g., imaging or sequencing). The recovered sample can be reused for additional analyses. Demonstration of the use of a DNA microarray as described herein is shown in FIGS. 20 A-D.

Example 2: DNA Hybridization Using the Mini Micro Array

[0072] In one example, the DNA which is hybridized to the probes on the array is fluorescently labeled with a 5′ Alexa Fluor 488 fluorophore. The DNA may be visualized on the array using a fluorescence microscope. One such example is show in FIGS. 21 A-B. As shown in the figures, there are two identical probes on the array surface and fluorescently-labeled complementary DNA is hybridized to the probes. The locations of successful hybridization are imaged using a fluorescence imaging system. Such imagining systems are well known to those of skill in the art.

[0073] To enable ease of handling and imaging of the assembled microarrays, a custom fixture (chiplet) as shown in FIGS. 22 A-B was designed and constructed. The fixture has the same footprint as that of a 96-well plate, enabling compatibility with many standard laboratory instruments (e.g., microscopy stages). The fixture is comprised of two anodized aluminum plates and a rubber gasket. The plates have 36 rectangular cutouts in which assembled microarrays may be secured in place. Similar fixtures can be designed and constructed for various embodiments of the assembled microarrays as described herein.

[0074] While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.