REFERENCE TO RELATED APPLICATION

The present application claims the priority to U.S. Application Serial No. 12/649,993, filed December 30, 2009, which is herein incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates generally to microarray chips, and more specifically to microarray chips comprising concave and convex microstructures.

BACKGROUND OF THE INVENTION

A single cell microarray system has been used for analyzing cellular response of individual cells, in which a single chip made from polystyrene contains microchambers to accommodate cells. See Yomamura et al. (2005) Anal. Chem. 77: 8050-8056. Microwells of microarray systems have been fabricated from agarose, acrylamide, polydimethylsiloxane (PDMS), and etc., to confine and control cells and their growth on the surface of a substrate. Conventional methods for fabricating microarray systems have involved preparing a porous substrate to increase the surface area of a microarray, and consequently the throughput capacity and sensitivity, wherein the pores serve as sites for attachment of one or more biomolecules.

Chin et al. used the photoresists SU-8 5 and SU-8 100 to construct microwells on a glass slide. See Chin et al. (2004) Biotechnology and Bioengineering 88 (3): 399-415. However, it had been reported that photoresist SU-8 does not adhere well to silicon dioxide. D C S Bien et al. (2003)

"Characterization of masking materials for deep glass micromachining" J. Micromech. Microeng. 13 S34-S40. A microarray fabricated with a photoresist that does not adhere well to a glass substrate may be problematic because the photoresist film having defined microwells may peel off from the glass easily when it is immersed in a culture medium, which may give unreliable experimental results.

Therefore, a heretofore unaddressed need exists in the art to address the aforementioned deficiencies and inadequacies, especially in connection with establishment of 3-dimensional chips comprising microstructures that exhibits minimal or no peel-off from the chip.

SUMMARY OF THE INVENTION

In one aspect, the invention relates to a three-dimensional (3-D) chip, which comprises: a) a substrate; and b) a photoresist material adhered onto one surface of the substrate, the photoresist material comprising a plurality of microstructures; wherein the substrate is one chosen from a silicon substrate and a quartz substrate. In another aspect, the invention relates to a three-dimensional (3-D) chip, which comprises: a) substrate; and b) a polymeric material adhered onto one surface of the substrate, the polymeric material comprising a plurality of convex microstructures.

Further in another aspect, the invention relates to a set of paired 3-D chips, which comprises: a) a first 3-D chip; and b) a second 3-D chip. The first 3-D chip in the paired set as aforementioned comprises: (i) a first substrate; and ii) a first photoresist material adhered onto one surface of the first substrate, the first photoresist material comprising a plurality of microstructures; each of the microstructure having a shape of a well. The second 3-D chip in the paired set comprises: (i) a second substrate; and (ii) a second photoresist material adhered onto one surface of the second substrate, the second photoresist material comprising a plurality of microstructures, each of the microstructures having a shape of a column, the column having a size that fits into the well of the first 3-D chip.

In one embodiment of the invention, the aforementioned substrate is replaced with a glass substrate with the proviso that the photoresist is not one chosen from SU-8 5, SU-8 100, and SU-8 2000 series, and wherein the photoresist material of the 3-D chip remains adhered to the glass substrate when immersed in a liquid or a culture medium for at least 3 days.

In another embodiment of the invention, each of the microstructures has a diameter of > 10 micrometer but < 10,000 micrometer. By > 10 μπτ but < 10,000 μιη, it meant that all integer unit amounts within the range are specifically disclosed as part of the invention. Thus, 10, 11, 12 . . . 997, 998, 999 and 10,000 μιη unit amounts are included as embodiment of this invention.

In another embodiment of the invention, the microstructures are either concave or convex structures.

In another embodiment of the invention, the photoresist material comprises an arrayed series of microstructures.

In another embodiment of the invention, the photoresist material forms the microstructures, and each of the microstructures has a shape of a column.

In another embodiment of the invention, the aforementioned 3-D chip may further comprise two or more than two different compounds; each of the compounds being located and/or coated on the microstructures, with each of the microstructures having a shape of a column, in a spatially discrete region of the 3-D chip.

In another embodiment of the invention, the plurality of microstructures are formed through the photoresist material, and each of the microstructures has a shape of a well. The well is distributed in and through the photoresist material, and surrounded by the photoresist material adhered onto one surface of the substrate, in which the bottom of the well is on the surface of the substrate. In another embodiment of the invention, the 3-D chip as aforementioned further comprises two or more than two different nucleic acids, each of the nucleic acids being located and/or coated on the microstructures (or microwells) in a spatially discrete region of the 3-D chip.

In another embodiment of the invention, the photoresist material is at least one chosen from SU-8 2-15, SU-8 50-100, SU-8 2000 series, SU-8 3000 series and KMPR ^® 1000 series.

In another embodiment of the invention, the photoresist material remains adhered onto the surface of the substrate when immersed in a culture medium for at least three days.

In another embodiment of the invention, the photoresist material remains adhered onto the surface of the substrate during air storage for more than one week.

In another embodiment of the invention, each of the convex microstructures in the 3-D chip as aforementioned has a shape of a column.

In another embodiment of the invention, the polymeric material comprises an arrayed series of micrcolumns.

In another embodiment of the invention, the polymeric material is at least one chosen from polydimethylsiloxane (PDMS), polyethylene glycol (PEG) and polyethylene glycol diacrylate (PEGDA).

In another embodiment of the invention, the 3-D chips further comprises two or more than two different compounds, each of the test compounds being located and/or coated on the

microstructures in a spatially discrete region of the 3-D chip.

Further in one embodiment of the invention, the second photoresist material in the paired set as aforementioned is replaced with a polymeric material.

Yet in another embodiment of the invention, the second 3-D chip in the paired set further comprises two or more than two different compounds, each of the compounds being located and/coated on the microstructures in a spatially discrete region of the second 3-D chip.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 A is a scanning electron microscopic image showing 3-dimensional morphology of a microwell (concave microstructure) chip.

FIG. IB is a scanning electron microscopic image showing 3-dimensional morphology of a microcolumn (convex microstructure) chip.

FIG. 2 shows phase contrast microscopic images of HeLa cells cultured in a 2592 -well cell chip at different magnifications (40x and lOOx).

FIG. 3 shows fluorescent microscopic images of HeLa cells cultured in a 2592-well cell chip at different magnifications (40x and lOOx).

FIG. 4 is a microscopic image of HeLa cells cultured in a 40098-well cell chip. FIG. 5 A is a phase contrast microscopic image of 293T cells in a 40098-well cell chip. FIG. 5B is a fluorescent microscopic image of 293T cells in a 40098-well cell chip.

FIG. 6A is a phase contrast microscopic image of HeLa cells transfected with siGLO green transfection indicator in a 2592-well chip.

FIG. 6B is a fluorescent microscopic image of HeLa cells transfected with siGLO green transfection indicator in a 2592-well cell chip.

FIG. 7A is a fluorescent microscopic image showing TNF-oc-induced subcellular localization of NF-κΒ in HeLa cells pretreated with PDTC.

FIG. 7B is a fluorescent microscopic image showing the cell nuclei of the cells in FIG. 7A. FIG. 7C is a fluorescent microscopic image showing T F-a-induced subcellular localization of NF-KB in non-pretreated HeLa cells;

FIG. 7D is a fluorescent microscopic image showing the cell nuclei of the cells in FIG. 7C; FIG. 7E is a fluorescent microscopic image showing TNF-a-induced subcellular localization of NF-KB in HeLa cells pretreated with Y294002.

FIG. 7F is a fluorescent microscopic image showing cell nuclei of the cells in FIG. 7E.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is more particularly described in the following examples that are intended as illustrative only since numerous modifications and variations therein will be apparent to those skilled in the art. Various embodiments of the invention are now described in detail. As used in the description herein and throughout the claims that follow, the meaning of "a", "an", and "the" includes plural reference unless the context clearly dictates otherwise. Also, as used in the description herein and throughout the claims that follow, the meaning of "in" includes "in" and "on" unless the context clearly dictates otherwise. Moreover, titles or subtitles may be used in the specification for the convenience of a reader, which shall have no influence on the scope of the present invention. Additionally, some terms used in this specification are more specifically defined below.

DEFINITIONS

The terms used in this specification generally have their ordinary meanings in the art, within the context of the invention, and in the specific context where each term is used. Certain terms that are used to describe the invention are discussed below, or elsewhere in the specification, to provide additional guidance to the practitioner regarding the description of the invention. For convenience, certain terms may be highlighted, for example using italics and/or quotation marks. The use of highlighting has no influence on the scope and meaning of a term; the scope and meaning of a term is the same, in the same context, whether or not it is highlighted. It will be appreciated that same thing can be said in more than one way. Consequently, alternative language and synonyms may be used for any one or more of the terms discussed herein, nor is any special significance to be placed upon whether or not a term is elaborated or discussed herein. Synonyms for certain terms are provided. A recital of one or more synonyms does not exclude the use of other synonyms. The use of examples anywhere in this specification including examples of any terms discussed herein is illustrative only, and in no way limits the scope and meaning of the invention or of any exemplified term. Likewise, the invention is not limited to various embodiments given in this specification.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In the case of conflict, the present document, including definitions will control.

As used herein, the term "substrate" shall generally mean a material having a rigid or semirigid surface or surfaces. A substrate generally has top and bottom sides, and each side has a surface. The surface on one of the sides of the substrate is used for coating. A substrate material includes, but not limited to, plastics plastics (e.g., polypropylene or polystyrene), ceramic, silicon, silica, glass, or quartz. A substrate that is transparent to light is useful for fabricating microarray chips for assays that involve optical detections. A non-transparent substrate may be used for analyses that involve non- optical detections, such as laser microarray scanner detections.

The terms "microcolumn chip" and "drug chip" are interchangeable.

The term "microwell chip" and "cell chip" are interchangeable.

The term "spatially discrete region", shall generally mean an area on a substrate that is distinct or separate from another area on the substrate. For example, in the case of a plurality of microwells that are arranged in spatially discrete regions, each microwell occupies a specific area on the substrate. The specific areas may be distributed on the substrate in, for example, a random or uniform distribution.

The term "concave" shall generally mean curving inwards.

The term "convex" shall generally mean curving outwards.

The term "well" shall generally mean a hole, a cavity or a space formed to contain a sample and/or a liquid.

The term "column" shall generally mean a rigid, relatively slender, upright support; any column like object or formation.

The shape of the microstructure includes but is not limited to a well (e.g. microwell), a depression, a recess, a hole, a groove, a cavity, a pit, a pore, a trench, a channel, a concaved region, a channel-connected well, and other similar shapes known to those skilled in the art. To design a drug array or chip, the shape of the microstructure includes but is not limited to a column (e.g. microcolumn), a protrusion, a post, a hump, a hill, a ridge, a bump, a bulge, a prominence, a projection, a convex region, and other similar shapes known to those skilled in the art.

The microstructures may be a plurality of microwells defined on a surface of a substrate to contain or hold cells or a compound. The microstructures may be a plurality of microcolumns defined on a surface of a substrate to provide areas for containing or holding probes, drugs or test compounds. The microstructures may be prepared by "patterning". The method of patterning includes a photolithographic exposure and development. During a photolithographic exposure, a light source of an appropriate wavelength is used to transfer a geometric pattern containing an image of desired microstructures from a photomask to a coating material adhered onto a surface of a substrate. Alternatively, a method of patterning may comprise embossing a coating material.

There may be both positive and negative tone photoresists used in the photolithographic process. For a positive resist, wherever the resist material is to be removed is exposed to UV light. The UV exposure makes the positive resist becomes more soluble in a developer. The exposed resist is washed away by a developer solution, leaving windows of bare underlying material. Therefore, the mask contains an exact copy of the pattern which is to be remained on the surface of a wafer. By contrast, exposure to UV light causes a negative resist to cross-link and become more difficult to dissolve. Therefore, the negative resist remains on the surface wherever it is exposed and a developer solution removes only the unexposed portions. A Mask used for a negative photoresist therefore contains an inverse (or photographic "negative") of the pattern to be transferred.

Two different patterns of photomasks were used in fabricating 3-D chips according the invention. To make a microwell chip, a photomask having a pattern of a white area containing black spots was used. Under UV light, the negative photoresist under the white area of the photomask was exposed to the light, which caused a cross-link reaction. The resist after cross-linking reaction was not dissolved by a subsequent developer. The resist under the black spots was unexposed to the UV, not cross-linked, and thus is dissolved by the developer, leaving holes formed in and through the resist film of the chip, i.e., resulting in microwell or cell chips. To make a microcolumn chip, a photomask having a pattern of a black area containing white spots was used. Under UV light, the negative photoresist under the black area of the photomask was unexposed to the light, no cross-link reaction resulted and thus was dissolved by a subsequent developer. The resist under the white spots was exposed to the UV, cross-linked and was thus not dissolved by the developer, resulting in convex structures formed from the resist, i.e., resulting in the formation of microcolumn or drug chips.

The patterning method is not limited to the photolithographic process as aforementioned, other patterning techniques known to those skilled in art to achieve the similar patterning results may be used. Drug chips containing microcolumns are not limited to those made from photoresist materials. They may be fabricated from other polymers which are not photoresists by micromachined or MEMS fabrication processes, which are known in the art. For example, three dimensional microstructures, such as microcolumns may be fabricated in optical gain medium using two photon induced photopolymerization technique described by Yokoyama et al. (2003) "Thin Solid Films " (438-439): 452-456 and Mendonca et al. (2008) Applied Physics A (90): 633-636. In the

photopolymerization process, femtosecond pulse laser may be used to machine any kind of material such as a metal, dielectric, semiconductor, or polymer. The processing is driven by a multiphoton absorption of energy from the pulse laser, resulting in the breaking of all bonds and the atomizing of materials. This laser processing is also capable of very high spatial resolution with the highest precision in the range of hundreds of nanometers. In the polymerization process, a photochemical reaction is initiated through a radical mechanism following two photon excitation of a photoinitiator. The photo-reactive resins that are most commonly used are acylate monomers or acrylic pre- polymers, which can be made to cross-link with the use of a radical photoinitiator molecule. Another example is that a polymeric material such as PDMS or PEG may be applied into the microwells of a cell chip and cover the microwell chip by forming a thin layer of polymer coat or film. A substrate is then applied onto the polymer coat or film and adhere to the polymer. The whole assembly containing the polymer-coated microwell chip and a substrate attached onto the polymer coat film was then treated heat for PDMS or UV for PEG. The substrate and the polymer coat film adhered to the substrate can then be separated from the assembly and results in a chip containing microcolumns with a pattern that mirrors the microwell chip used at the beginning of fabrication.

An automated spotting device such as Perkin Elmer Biochip Arrayer™ may be used for applying a sample to a microarray chip. Many contact and non-contact microarray printers are available and may be used to print a binding molecule, such as a probe, a ligand or an agonist, on a substrate. For example, non-contact printers are available from Perkin Elmer (BioChip Arrauer™), Labcyte and IMTE (TopSpot™). These devices utilize various approaches for non-contact spotting, including piezo electric dispension; touchless acoustic transfer; en bloc printing from multiple microchannels; and the like. Other approaches include ink jet-based printing and microfluidic platforms. Contact printers are commercially available from TeleChem International (Array It™). Non-contact printing may be adopted for the production of high-specificity cellular microarrays. With a non-contact printer, no solid printer part contacts the array surface. By utilizing a printer that does not physically contact the surface of the substrate, no aberrations or deformities are introduced onto the substrate surface, thereby preventing uneven or aberrant cellular capture at the site of spotted probe. Such printing methods find particular use with high specificity hydrogel substrates.

Since the separation between tips in the microarrayers can be custom-made to make compatible with multi-well chips, one can simultaneously print each load into several microwells. Printing into microwells can be done using both contact and non-contact technology, where the latter is also compatible with non-flat multi-well plates.

The cell chips according to the invention may be used for high throughput screening for ligands that are capable of causing changes in cells. For example, ligand binding may produce a phenotypic change, such as a change in cell morphology, cell survival, apoptosis, cell migration, specific organelle, protein subcellular localization, protein level, enzyme production, enzyme activity, nucleotide level, or nucleotide subcellular localization. The microarray chips according to the invention are also useful for high throughput detection of an analyte of interest in a sample using various candidate probes. The candidate probes may be labeled with a detectable substance such as a fluorescent molecule, a chemiluminescent fragment, or a radioactive molecule. The sample is delivered to microwells on a microarray chip according to the invention, in which probes are immobilized onto the surface of the substrate inside the microwells. Washing the microwells removes unbound sample, and the bound analyte is retained in the microwells, which can be detected, either directly or indirectly.

Fluorescent signals within the microwells of a microarray chip can be quantified by scanning the array with a confocal camera or with a CCD camera. Detection may also be label-free. For example, the surface Plasmon resonance or microring methods have been shown for detecting the binding of analytes to probes or the changes of cell morphology or cell volume. See Jordan et al. (1997) Anal. Chem. 69: 4939-4947, Ferreira et al. (2009) J. AM. CHEM. SOC. 131:436-437 and Peterson et al. (2009) BMC Cell Biology 10: 16.

Molecules or compounds may be immobilized either covalently (e.g., utilizing single reactive thiol groups of cysteine residues,) or non-covalently but specifically (e.g., via immobilized

antibodies, the biotin/streptavidin system, and the like) on the substrate, by any method known in the art. When covalent immobilization is contemplated, the substrate should be polyfunctional or be capable of being polyfunctionalized or activated with reactive groups capable of forming a covalent bond with the target to be immobilized (e.g. carboxylic acids, aldehydes, amino groups, cyano groups, ethylenic groups, hydroxyl groups, mercapto groups and the like).

A drug or a ligand may be delivered to a cell sample using microcolumns or similar structures on a array chip. Microcolumns of an array chip (or a drug chip) are first applied with a drug or ligand. The drug-containing microcolumns are then inserted into microwells holding or containing the cell sample to allow the release of the test drug or ligand into the microwells. After an incubation period, the microcolumn chip may be removed and microwells are washed to remove unbound drug or ligand. The cellular target molecules in response to the drug or the ligand may be detected by using immunolabeling reagents and a fluorescent detector/quantifier with optical access to the microwells, either through a transparent or translucent substrate.

EXAMPLES

Without intent to limit the scope of the invention, exemplary instruments, apparatus, methods and their related results according to the embodiments of the present invention are given below. Note that titles or subtitles may be used in the examples for convenience of a reader, which in no way should limit the scope of the invention. Moreover, certain theories are proposed and disclosed herein; however, in no way they, whether they are right or wrong, should limit the scope of the invention so long as the invention is practiced according to the invention without regard for any particular theory or scheme of action.

EXAMPLE 1:

Array Chip Fabrication Using Quartz Wafers

Quartz wafers (Tekstarter, Taiwan) were cleaned according to a Piranha Clean procedure (See http://engineering.tufts.edu/microfab/index_files/SOP/Pirari haClean_SOP.pdf) dried, and coated with a photoresist material. Briefly, quartz wafers were placed in a TEFLON™ carrier, submerged in a bath of 96%H ₂ SO ₄ :30%H ₂ O ₂ solution (1: 1) for 10-20 minutes to remove all organic deposits, and rinsed in deionized (DI) water for 15 minute. The cleaned quartz wafers were blown dry with nitrogen or dried in an oven at 120°C or on a hotplate at 150°C and placed in a carrier box until ready for coating.

Commercially available photoresist material such as SU-8 100 (MicroChem, Newton MA) was spin-coated on quartz wafers as follow. Wafers were subjected to a spread cycle, during which the wafers were ramped to 500 rpm at an acceleration of 100 rpm/second, and held at 500 rpm for 10 seconds to allow the resist to cover the entire surface on one side of the wafer. The wafers then were subjected to a spin cycle, during which the wafers were ramped to 3000 rpm at an acceleration of 300 rpm/second and held at 3000 rpm for 30 seconds to obtain a photoresist coat or film having a thickness of about 100 μm. Changing the conditions of the spread and/or spin cycles would have impacts on the thickness of the photoresist coat/film on the wafers. For example, an increasing in the speed at a spin cycle would reduce the thickness of the resist coat/film on the wafers. The

photoresist-coated wafers were soft baked on a hot plate at 65°C for 10 minutes and then at 95 °C for 30 minutes. To form micro wells through the photoresist coat/film on the wafers, a standard

photolithography process was performed by using an UV light source. The EVG 620 Top Side Mask Aligner was used for aligning patterns on wafers to expose the resist at 650 mJ/cm ² and UV below 350nm was eliminated. After the UV exposure, the wafers coated with the photoresist SU-8 100 were baked on a hot plate at 65 °C for 3 minutes and 95 °C for 10 minutes. The patterns were developed using SU-8 developer (MicroChem, Newton MA) for 10 minutes. The SU-8 100 coat/film was rinsed with isopropyl alcohol after development, air-dried with nitrogen, and subjected to hard baking on a conventional oven at 150 °C for 15 min. FIG. 1 shows an electron microscopic image of a microarray chip having a plurality of wells (with each having a diameter of -500 μιη) defined on a quartz wafer.

EXAMPLE 2

Array Chip Fabrication Using Glass Wafers

Glass wafers were cleaned according to a Piranha clean procedure as described above, dried and coated with a photoresist. Briefly, quartz wafers were placed in a TEFLON™ carrier, submerged in a bath of 96%H ₂ SO ₄ :30%H ₂ O ₂ solution ( 1 : 1 ) for 10-20 minutes to remove all organic deposits, and rinsed in deionized (DI) water for 15 minute. ) After the Piranha Clean the glass wafer was blown dry with nitrogen and stored in a carrier box until ready for coating.

Commercially available photoresist such as SU-8 3050 (MicroChem, Newton MA) was spin- coated on glass wafers as described below. Briefly, during a spread cycle the wafers were ramped to 500 rpm at an acceleration of 100 rpm/second and held at 500 rpm for 10 seconds to allow the resist to uniformly coat on the surface on one side of each wafer. At a spin cycle, the wafers were ramped to 1000 rpm at an acceleration of 300 rpm/second and held at 1000 rpm for 30 seconds to obtain a photoresist coat or film having a thickness of about 100 μπι. The wafers were soft baked on a hot plate at 95 °C for 45 minutes.

To define microwells through the photoresist coat film on the wafers, a standard

photolithography process was performed by using an UV light source. The EVG 620 Top Side Mask Aligner was used to align patterns on wafers to expose the resist at 375 mJ/cm ² and UV below 350nm was eliminated. After the UV exposure, the SU-8 3050 film was baked on a hot plate at 65 °C for 1 minute, 95 °C for 5 minutes. The patterns were developed with SU-8 developer (MicroChem, Newton MA) for 15 minutes. After the development, the SU-8 3050 film was rinsed with isopropyl alcohol, air-dried with nitrogen, and subjected to hard baking on a conventional oven at 150 °C for 15 min. A microarray chip having a plurality of wells (each having a diameter of about 500 μπι) defined on glass wafer was formed.

EXAMPLE 3 Drug Array Chip Fabrication Using Silicon Wafers

Silicon wafers were cleaned according to a Piranha clean procedure as described above, dried, and coated with a photoresist. Briefly, silicon wafers placed in a TEFLON™ carrier were submerged in H ₂ 0:H ₂ 0 ₂ :NH ₄ OH solution (5:1 : 1) for 10 minutes, rinsed in deionized (DI) water for 1 minute, submerged in H ₂ 0:HF solution (50: 1) for 15 seconds, rinsed in DI water for 1 minute, submerged in H ₂ 0:H ₂ 0 ₂ :HCl solution (6: 1 : 1) for 10 minutes and rinsed in DI water for 1 minute. The silicon wafers were blown dry with nitrogen and placed in a carrier box until ready for coating.

Commercially available negative photoresist such as SU-8 50 film (MicroChem, Newton MA) was spin-coated on silicon wafers as follow. During a spread cycle wafers were ramped to 500 rpm at an acceleration of 100 rpm/second and held at 500 rpm for 10 seconds to allow the resist to coat the surface on one side of each wafer. At a spin cycle, the wafer was ramped to 2000 rpm at an acceleration of 300 rpm/second and held at 2000 rpm for 30 seconds to obtain a photoresist coat or film having a thickness of having a thickness of about 50 μηι. The wafers were soft baked on a hot plate at 65°C for 6 minutes and 95 °C for 20 minutes.

To define microcolumns through the photoresist coat/film on the wafers, a standard photolithography process using an UV light source was performed. The EVG 620 Top Side Mask Aligner was used for aligning patterns on wafers to expose the resist at 375 mJ/cm ² and UV below 350nm was eliminated. After the UV exposure, the SU-8 50 film was baked on a hot plate at 65 °C for 1 minute and 95 °C for 5 minutes. The patterns were developed with SU-8 50 developer

(MicroChem, Newton MA) for 6 minutes. After the development, the SU-8 50 film was rinsed with isopropyl alcohol, air-dried with nitrogen and subjected to hard baking on a conventional oven at 150 °C for 15 min. As a result, a drug array chip having a plurality of columns (each having a diameter of about 350 μπι) defined on the silicon wafer was formed.

EXAMPLE 4

Adhesion Test

It has been reported that the photoresist SU-8 100 was used to fabricate microwells on the glass substrate of the microarray chip. See Chin et al. (2004) Biotechnology and Bioengineering 88 (3): 399-415. However, the inventor discovered that NANO™ SU-8 2-25, NANO™ SU-8 50-100 photoresist and SU-8 2000 series, such as NANO™ SU-8 2000.5-2015, NANO™ SU-8 2025-2075 or NANO™ SU-8 2100-2150 film, provided very weak adhesion to glass substrate. As a result, photoresist coat/film might peel off from the surface of the glass substrate when microarray chips were stocked in the air after one week at room temperature or immersed for in the culture medium during cell culture. During air storage, the resist SU-8 100 peeled off the grass substrate after a week (i.e., the stability lasted for only a week), and the resist SU-8 2000 peeled off the glass substrate after 3 days. When sitting in a cell culture medium, the resist SU-8 100 peeled off the glass slide within 2-3 days, and SU 8 2000 peeled off the glass slide within one day.

Various photoresist materials, such as SU-8 100, SU-8 2050 and SU-8 3050, MPR 1050 films, were tested for their adhesion against a set of substrates, such as silicon, quartz and glass substrates, using an adhesion tester (ROMULUS III universal tester) at National Nano Device

Laboratory (NDL). Aluminum nails were affixed onto a photoresist test film that was adhered onto a substrate. The tested film was baked on a hot plate at 150°C for an hour to allow the aluminum nails to adhere to the tested film, cooled down and mounted on a clamping device. A breaking point platform having a force system and force transducer was included in the adhesion tester to provide a 0 kg to 100 kg downward pulling force. The adhesion tester was semi-automated by a computer workstation to measure the maximal adhesion of the film. Any cracking on the tested film was checked to determine if the testing results were positive. Positive test results were recorded (Table 1). Otherwise, the test would be repeated on another test film.

As shown in Table 1, a glass substrate did not have good adhesion with most of the

photoresist films tested except KMPR 1050. The SU-8 2050 film was found to peel off from the glass substrate as soon as it was fabricated thereon even before the adhesion test was conducted. By contrast, both silicon and quartz substrates have good adhesion with most of the photoresist films tested.

Adhesion tests were also conducted on fabricated chips in a cell culture environment. Briefly, the photoresist film coated on the surface of the glass or quartz wafer was defined and patterned by a photolithpgraphic process to form a plurality of microwells on the wafer. The wafer was diced using a dicing saw (a precision dicing system) into chips of a standard microscope slide size (75mm x 25mm). After storage for about 2 weeks, the fabricated chips were sterilized and placed in culture dishes (10 cm diameter each). The Minimum Essential Medium (MEM) was added into the dishes and incubated at 37°C. The fabricated chips in the culture dishes were examined with naked eyes 2 days after the incubation, and results are shown in Table 2.

Table 2

As shown in Tables 1 and 2, silicon and quartz substrates had good adhesion to all the photoresist tested and thus can provide a stable environment for cell microarray chips. By contrast, the glass substrate had poor adhesion to both photoresit SU-8 100 and SU-8 2050. The photoresist SU-8 100 film and photoresist SU-8 3050 film showed similar adhesion to glass in the physical adhesion test (Table 1), however, the microwells constructed through the SU-8 100 film were found to peel off from the glass surface during the cell culture. Only the microwells constructed through the photoresist SU-8 3050 were intact, adhering to the surface of the glass for long term maintenance of cell culture in the microarray chip (Table 2).

EXAMPLE 5

Microarray analysis of cells transfected with siRNA

Human cervical cancer cell line (HeLa cells) and Human embryonic kidney cell line (293T cells) commonly used for transfection assay were selected for transfection experiments. The HeLa cells were grown in MEM and 293T cells grown in Dulbecco's Modified Eagle's Medium at 37°C in a 5% C0 ₂ incubator. Both MEM and DMEM were supplemented with 10% FCS and 100 units/ml penicillin/streptomycin.

The cultured cells were collected as cell suspensions. The HeLa cells were transferred to each microwell of a 2,592-well or 40,098-well cell chips fabricated according to Examples 1 or 2. The 293T cells were transferred to each microwell of a 40,098-well microarray cell chip. The living cells were observed under a phase contrast microscope and the images shown are shown in FIG. 2 and 4, 5A. Cells were also labeled with fluorescent cell markers and detected using a fluorescent confocal microscope and the images are shown in FIGs. 3 (HeLa cells) and 5B (293T cells).

The HeLa cells were transfected with siGLO green transfection indicator according to the manufacturer's instruction (Dharmacon Inc.). Briefly, fluorescent RNA duplexes (siRNA) were spotted into a 2,592-well microarray cell chip at 0.001 pmole/well. Next, 1,575 μL of rehydration solution (25 uL of DharmaFECT and 1,550 μL of RNase-free water) was dispensed onto the chip. The DharmaFECT transfection reagent was allowed to complex with the RNA duplexes (siRNA) at room temperature for 20 minutes. The cell chip having microwells containing siGLO green transfection indicator was transferred into a culture dish, to which 12mL of cell suspension (6.25 x 10 ⁵ cells per mL) were added. The cells were incubated at 37°C, 5% CO ₂ to allow transfection to occur and observed at 48 h posttransfection using a phase contrast microscope (FIG. 6A) and a fluorescent microscope (FIG. 6B).

EXAMPLE 6

Effects of Compounds on Subcellular Localization of NF-κΒ using Microarray analysis

In this experiment, an assembly comprising a cell chip (FIG. IB) and a drug chip (FIG. 1C) was used. The cell chip comprises an arrayed series of thousands of microwells and the drug chip comprises an arrayed series of thousands of microcolumns. The microcolumns of the drug chip were complementary to the microwells of the cell chip in terms of the arrayed pattern and size. The size of each mcirocolumn is no larger than each microwell in the diameter and the height. Below illustrates one of the utilities of the drug chip according to the invention.

HeLa cells were seeded in the microwells (60 x 10 ⁴ cells/well) of a 2,666-well cell chip (each well diameter and depth:— 500μm by—l00μm) and cultured overnight at 37 °C.

The drug chip was pre-treated with 0.01% poly-L-lysine (PLL) (Sigma) to coat the surfaces of the microcolumns formed from the photoresist material. Different concentrations of test drugs were individually spotted onto the microcolumns of a drug chip. For example, pyrrolidine dithiocarbamate (PDTC) at different concentrations was mixed with a diluted alginate solution. PDTC has been shown as an inhibitor of NF-κΒ. Alginate was used to absorb/retain the drug PDTC. In some experiments, gelatin instead of alginate was used. The drug/alginate mixtures were individually spotted onto the top of the microcolumns of a drug chip (each microcolumn diameter and height: ~350μm by ~50μm) using a pipetman. For a negative control, phosphoinositide 3- kinase inhibitor (LY294002) was spotted onto different rows of microcolumns of the drug chip. The final concentrations of all of the drugs applied onto the microcolumns were 2mM. The drug chip was then placed at 4 °C overnight to dry the drug mixtures.

On the second day, the drug chip was inserted into the microwells of the cell chip with the drug spots on the microcolumns facing down and entering the openings of the cell-containing microwells of the cell chip. The entire assembly of the drug chip and cell chip was placed in an incubator with humidity > 95% at 37°C for 4 hours, during which the PDTC or LY294002 was released from the microcolumns and treated the cells in the microwells. The drug chip was then removed afterwards. The cell chip was rinsed 3 times to remove the residual PDTC or LY294002 in the culture medium, followed by incubation of cells with TNF-a for 30 minutes. TNF-a has been known to induce NF- κΒ to translocate from the cytoplasm to the nucleus.

Immunoassay was performed to determine subcellular localization of NF-κΒ as follows. The cells on the cell chip were washed with PBS (10ml) in a dish (10 cm), fixed with paraformaldehyde (3.7%, 2ml) at room temperature for 20 minutes, washed twice with PBS (10ml). The cells were blocked with BSA (1%, 10 ml) at room temperature for 30 minutes, incubated with the primary antibody NF-κΒ antibody at room temperature for 1 hour, washed three times with PBS (10ml, 5min each) and then incubated with a secondary antibody conjugated with tetramethyl rhodamine iso- thiocyanate (Rhodamine (TRITC)-conjugated anti-rabbit secondary antibody), and 4'-6-diamidino-2- phenylindole (DAPI) at room temperature for 1 hour in the dark. The cells were washed three times with PBS (10ml) for 10 minutes each.

The subcellular localization of NF-κΒ within the cells was detected by observing the TRITC staining and DAPI staining using fluorescent microscopy. FIG. 7 top panel shows a cell chip containing an arrayed series of thousands of microwells, in which the regions labeled (i)-(iii) contained microwells having cell cultures that were pretreated with PDTC prior to TNF-a treatment. The regions labeled (iv)-(vi) contained microwells having cell cultures that were pretreated with LY294002 prior to TNF-a treatment. The area between the region (iii) and the region (iv) contained microwells having cell cultures that were not pretreated any drug prior to the exposure to TNF-a.

FIGs. 7A, 7C, 7E show TRITC staining of cells for NF- Β, and FIGs. 7B, 7D, 7F show corresponding DAPI staining for the cell nucleus. PDTC-pretreated cells (7A, 7B) in microwells from the region (i), showed a significantly less induction of nuclear localization of NF-κΒ by TNF-a, as compared to the non-drug-pretreated cells in microwells from the region between (iii) and (iv) (7C, 7D). Both the non-drug-pretreated cells in microwells from the region between (iii) and (iv) (7C, 7D) and the LY294002-pretreated cells in microwells from the region (vi) showed NF-κΒ staining localized in the nuclei (FIGs. 7C, 7E), the location of which were confirmed by DAPI staining (FIGs. 7D, 7F).

The foregoing description of the exemplary embodiments of the invention has been presented only for the purposes of illustration and description and is not intended to be exhaustive or to limit the invention to the precise forms disclosed. Many modifications and variations are possible in light of the above teaching.

The embodiments and examples were chosen and described in order to explain the principles of the invention and their practical application so as to enable others skilled in the art to utilize the invention and various embodiments and with various modifications as are suited to the particular use contemplated. Alternative embodiments will become apparent to those skilled in the art to which the present invention pertains without departing from its spirit and scope. Accordingly, the scope of the present invention is defined by the appended claims rather than the foregoing description and the exemplary embodiments described therein.