WO2007089464A2

WO2007089464A2 - Self assembled monolayer surface patterning using a molding technique

Info

Publication number: WO2007089464A2
Application number: PCT/US2007/001600
Authority: WO
Inventors: Ieong Wong
Original assignee: The Regents Of The University Of California
Priority date: 2006-01-20
Filing date: 2007-01-22
Publication date: 2007-08-09
Also published as: WO2007089464A3

Abstract

A high performance, high throughput technique for patterning protein- and cell-repellant molecule (e.g. poly(ethylene glycol) (PEG)) self-assembled monolayers (SAM) in a submicron/nano scale is provided for generating ultra-high density protein/cell arrays on silica substrates.

Description

SELF ASSEMBLED MONOLAYER SURFACE PATTERNING USING A -

MOLDING TECHNIQUE

CROSS-REFERENCE OF RELATED APPLICATION [0001] This application claims priority to U.S. Provisional Application No. 60/760,509, filed January 20, 2006, the entire contents of which are hereby incorporated b reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with Government support of Grant No. NCC2-1364, awarded by the NASA and Grant No. DE15018 awarded by the NIH. The Government has certain rights in this invention.

FIELD OF INVENTION

[0003] The present invention relates to methods for fabricating devices in the field of biomaterial patterning and bio-devices manufactured by the methods. Specifically, the present invention provides methods for producing self-assembled monolayers on substrates such that portions of the monolayer are comprised of materials that are non-adherent for proteins and cells and other portions which are adherent for proteins and cells.

BACKGROUND [0004] The field of biomaterial (e.g. protein, cell, and biomolecules) patterning has applications in miniature biosensors, cellular patterning for cell- cell and cell-substrate interaction studies in tissue engineering, and protein microarrays for proteomics and pharmacology screening. One way to significantly enhance these applications is to improve the patterning resolution. A simple and inexpensive patterning technique is needed to replace the labor-intensive and expensive micro/nano electronic fabrication processes while retaining comparable patterning quality.

[0005] Conventional patterning methods include photolithography, photochemistry, soft lithography, and direct spotting. Photolithography-based patterning techniques are a well-established technology. In photolithography, ultraviolet (UV) light is exposed through a mask to pattern the photoresist. Then adhesion-promoting silanes are used to silanize the exposed region of the substrate while the area covered by the photoresist is protected from being silanized. After stripping the photoresist, the non-sϋanized area is silanized with an adhesion-resistant silane, forming a mixed monolayer interface where biomolecules only bind to the adhesion-promoting regions. However, photolithography requires clean room facilities which are very expensive to build and maintain. Moreover, residual organic solvents and photoresist after the photolithography process tends to denature proteins and thus affect cell activity. [0006] Besides photolithography, the photochemical method is another widely exploited protein patterning method. In the photochemical method, photoactive chemicals are spun on the substrate followed by UV exposure through a mask to selectively activate certain regions of the surface with reactive functional groups which allows covalent binding of protein resistant molecules. The whole surface Is then exposed to UV light again to activate the remaining region for subsequent protein binding. Nonetheless, this technique also requires clean room facilities for the UV exposure process. Moreover, many intricacies in the fabrication process, such as smearing of the surface by the mask or scattering of irradiation from backmasking, are the main obstacles keeping this method from becoming popular. Additionally, a labor-intensive characterization process is also needed for robust manufacturing.

[0007] More recently, soft lithography has become one of the most common techniques to generate patterns of self-assembled monolayers (SAM) for cell and protein patterning. Soft lithography is done by using an elastomer stamp made of polydimethylsiloxane (PDMS) inked with alkanethiol molecules. The stamp is brought into contact with a gold surface where alkanethiol molecules can self-assemble forming a pattern of SAM. The remaining area is subsequently derivatized with poly(ethylene glycol) (PEG)- terminated alkanethiol blocking the protein from binding. This method is inexpensive because no clean room facilities are required, however, soft lithography SAM patterning is most often performed on gold surfaces using alkanethiol derivative molecules. Occasionally soft lithography is used to pattern SAM on glass surfaces with high pattern fidelity. In addition, only organic solvent-based chemicals can be used for printing due to the dewetting effect of aqueous solutions on the hydrophobic elastomeric mold. Therefore, aqueous solutions (such as, PEG-amine solutions) cannot be used for soft lithography contact printing.

[0008] Direct spotting is commonly used to produce protein microarrays where protein microspots are placed on the surface with tiny needles. Direct spotting is the most widely used method to create protein microarrays wherein nanoliters of each different protein are directly spotted on the surface by an array of tiny needles. After spotting, the surface is then incubated with a protein blocking agent, such as non-fat milk or bovine serum albumin, to prevent target analytes binding to non-spotted area of the surface. Due to constraints from the size of the needle tips, the smallest protein spot sizes are in the range of a hundred microns, preventing this method from creating ultra- high density protein arrays (with subrnicron feature size) on a single chip.

[0009] Self-assembled monolayers are attractive for surface-related nanotechnology and biotechnology applications due to their spontaneous formation and thermodynamic stability. Protein- and cell-resistant SAMs are some of the most intensively studied candidates for biomedical applications such as drug discovery, biomaterials, and biosensors. Poly(ethylene glycol) (PEG) is a well known and powerful biomaterial providing protein- and cell- repellant properties, as well as having low toxicity and biological inertness. Therefore, the capability to construct a high quality PEG SAM is important for biomedical applications. [0010] PEG SAM patterning is often performed on gold surfaces using micro contact printing (μCP) of alkanethiol-terrninated PEG derivative molecules. However, the non-transparent optical property of the gold surface is incompatible with optical measurement. Very few studies have been reported of patterning SAMs on oxide surfaces due to difficulties with the intrinsic properties of the silane coupling chemistry. Therefore, the ability to form SAM patterns directly on transparent glass surfaces is highly desirable. PEG SAM can be patterned by linking biotin-PEG molecules through streptavidin-biotin interaction on alkanethiol or alkaπesilane precoated gold or oxide surfaces. An alternative is to graft PEG on oxide surfaces using poly-L- lysine-g-PEG copolymers via electrostatic interaction between the positively charged poly-L-lysine chain and the negatively charged oxide surface. Another method to pattern SAM is to use the well-characterized photolithography lift-off technique to selectively pattern PEG-derivative silane molecules onto oxide substrate.

SUMMARY

[0011] A high performance, high throughput, low cost, and simple technique for patterning protein- and cell-repellant molecules (e.g. poly(ethylene glycol) (PEG)) self-assembled monolayers (SAM) in a submicron/nano scale can be provided by embodiments of the current invention for generating ultra-high density protein/cell arrays on silica substrates.

[0012] A molding technique is employed to generate a poly(ethylene glycol) (PEG) self-assembled monolayer on a Si/SiO₂ substrate for accurate protein patterning. The monolayer shows significant reduction, >99%, of nonspecific protein adsorption compared to bare Si/SiO₂ substrate. Large areas, in the order of centimeters, of a high-density array (500 * 500) of 2μm features can be resolved with high fidelity. This method can alleviate the difficulties and complexities in SAM patterning using micro contact printing and photolithography.

[0013] In one embodiment of the present invention, a method of patterning the surface of a solid substrate is provided comprising forming one or more than one self-assembled monolayer (SAM) on the solid substrate by a molding technique to form a surface-modified substrate; and contacting the surface-modified substrate with at least one biomaterial.

[0014] In an embodiment of the present invention, the solid substrate is selected from the group consisting of silica, glass, polymers and metals.

[0015] In another embodiment of the present invention, at least one of the one or more than one SAM is comprised of a biomaterial-adherent material. In yet another embodiment, at least one of said one or more than one SAM is ^■comprised of a biomaterial-nonadherent material. [0016] In another embodiment of the present invention, the one or more than one SAM comprises a first SAM and a second SAM. In yet another embodiment, the patterned substrate comprises two SAMs wherein one of the first SAM or the second SAM is comprised of a biomaterial-adherent material and the other of the first SAM or the second SAM is comprised of a biomaterial-nonadherent material.

[0017] In still another embodiment of the present invention, the first SAM comprises a silane including, but not limited to, epoxysilane, dimethoxysilane, trichlorosilane and triethylsilane. In one embodiment the epoxysilane is glycidoxypropyl trimethoxysilane.

[0018] In another embodiment of the present invention, the second SAM comprises a poly(ethylene glycol) (PEG), such as a PEG derivative having a functional group selected from the group consisting of amines, thiols, carboxyls, trichlorosilanes, maleimides, aldehydes and NHS. In one embodiment the PEG derivative is a methoxy-terminated PEG-amine.

[0019] In an embodiment of the present invention, a method of patterning the surface of a solid substrate is provided comprising forming one or more than one. self-assembled monolayer (SAM) on the solid substrate by a molding technique to form a surface-modified substrate; and contacting the surface-modified substrate with at least one biomaterial wherein said forming step comprises cleaning the substrate; immersing the cleaned substrate in an silane and allowing silanization to occur on the substrate surface to form a first SAM; cleaning the silanized substrate; applying a PEG to the surface of the silanized substrate; bringing a mold into conformal contact with the silanized surface and allowing the second SAM to form; drying the substrate; removing the mold; and rinsing the substrate to form a surface-modified substrate.

[0020] In another embodiment of the present invention, the biomaterial is selected from the group consisting of proteins, peptides, carbohydrates, lipids, nucleic acids, chemicals and cells.

[0021] A bio-device according to embodiments of the current invention is produced by a method of the current invention. [0022] A bio-device according to an embodiment of the current invention has a substrate; a self-assembled monolayer formed on the substrate; a pattern of biomaterial formed on one of the self-assembled monolayer or exposed portions of the substrate to provide a bio-device having a biomaterial patterned substrate.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figures 1A-G depict the fabrication process flow of the poly(ethylene glycol) (PEG) self-assembled monolayer (SAM) patterning technique according to an embodiment of the present invention. [0024] Figure 2 depicts a scanning electron micrograph (SEM) image of the dry solid PEG structure after the polydimethylsiloxane (PDMS) mold is peeled off according to an embodiment of the present invention. The microwells have a diameter of 3μm. Wavy distortion of the rim on the PEG layer is produced during PDMS peeling (inset). [0025] Figure 3 depicts atomic force microscopy (AFM) images of the dry solid PEG microstructure after the PDMS is peeled off according to an embodiment of the present invention. The size of the wells (3μm) can be determined by the width of the bottom of the valley in the inset. The height of the structure is ~200nm. [0026] Figures 4A-B depict contact angle measurements of the two SAM layers according to an embodiment of the present invention. The 3- glycidoxypropyl trimethoxysilane (GPTS) SAM layer has a contact angle of 52° before PEG coating (A); after PEG coating, the contact angle changes to 32° (B). [0027] Figures 5A-C depict fluorescence microscopy images of high density array patterning of both bovine serum albumin (BSA) (A) and poly-L- lysine (PLL) (B) according to an embodiment of the present invention. Protein adsorption on the PEG surface is substantially reduced to 1 % for BSA and 3% for PLL (C). Each bright dot is 3μm in diameter. [0028] Figures 6(a) and 6(b) provide an example illustration some possible resolutions according to an embodiment of the current invention. Figure 6(a) is an example of nano protein patterning observed by fluorescence imaging of the patterned protein (fluorescent conjugated Bovine Serum Albumin). Figure 6(b) is bright field imaging of the mold corresponding to the example of Figure 6(a). The scale of the bar is 5μm. DETAILED DESCRIPTION

[0029] Self-assembled monolayer (SAM) patterning by molding is counterintuitive because molding generally refers to the patterning of microstructures of polymeric materials with certain surface topographies, instead of interfacial patterning at the molecular level. An innovation of the present invention combines the merits of soft-molding, i.e., photolithography-less imprint lithography-quality microfabrication processes, and the virtue of self- assembled covalent coupling chemistry. This advancement also proves the feasibility of using micro-topographical molding techniques to create surface patterns at the nanomolecular level. [0030] The present invention generates submicron to nanometer sized SAM patterns of protein/cell repellant molecules (e.g. PEG) in a large area, in the order of centimeters, using a molding technique. The methods of the present invention can lower the manufacturing cost by using inexpensive commercially available chemicals, non-destructively reusable molds, and simple batch molding techniques. Moreover, by bypassing the photolithography or nanolithography processes, the method of the present invention also can eliminate the concerns of protein denaturation by residual organic solvent and photoresist left on the surface of the substrate.

[0031] One issue of biomaterial/biomolecule patterning is the non-specific binding of proteins or cells onto non-patterned areas. Non-specific binding refers to the indiscriminate binding of biomolecules onto both patterned and non-patterned locations due to the intrinsically adhesive properties of proteins to most surfaces. The present invention can produce SAM surfaces wherein

>99% of nonspecific-binding of biomolecules is reduced, forming very high fidelity biomaterial features.

[0032] Embodiments of the present invention can overcome most of the disadvantages in the prior art methods of conventional patterning methods including photolithography, photochemistry, soft lithography and direct spotting. Compared to photolithography and photochemical methods, the patterning methods of the present invention do not require clean room facilities, and therefore can provide reduced costs. Furthermore, since this method is similar to imprint lithography, much smaller features, down to nanometer size, can be achieved. Compared to the soft lithography process, the patterning. methods of the present invention can be used to generate SAM using both aqueous and organic solvent soluble molecules. By bypassing the non-robust silane patterning by directly grafting silane molecules on the surface, this method first forms a homogeneous silane monolayer on the surface followed by patterning a second layer of PEG molecules, specifically selected to be non-adherent for cells and proteins, covalently grafted on to the silane layer.

[0033] Apart from fabrication, the surface coverage of the molecules on the surface is important to ensure the quality of the protein repellant (nonadherent) surface. The patterning methods of the present invention are able to reduce nonspecific binding of proteins onto non-patterned area by more than 99%.

[0034] The methods of the present invention can be used to generate high-density protein micro/nano arrays for a variety of uses, including but not limited to, rapid proteomic and pharmacology screening wherein biomaterials including, but not limited to, proteins, peptides, carbohydrates, lipids, nucleic acids, chemicals and cells are required to be immobilized on a substrate. Additionally, the methods of the present invention can also generate a high- density cellular array, where single or small population of cells can be site- specifically localized onto the patterned area for single cell or cell-cell interaction studies. This cellular array can also be further integrated with automated microfluidic system for single cell or controlled-population dynamic and real-time cell monitoring. [0035] The present invention provides for methods of patterning solid substrates which allows for site-specific localization of biomaterials onto the substrate. The patterning methods of the present invention provide for one or more than one self-assembled monolayers (SAM) to be patterned onto a substrate. The one or more than one SAM can be comprised of the same materials or of different materials. In one embodiment, one SAM may have biomaterial-adherent properties and a second SAM may have biomaterial- repellant properties. In another embodiment, the one SAM may cover the entire substrate while a second SAM may cover only portions of the substrate, allowing for both SAMs to be exposed. Solid substrates useful in the methods of the present invention include, but are not limited to, silica, glass," polymers and metals.

[0036] A schematic of the capillary force lithography technique of the present invention is shown in Figure 1. The process starts with highly polished Si <100> wafers cut into pieces of 2 cm * 2 cm before any surface modification. The substrates were first plasma cleaned for 10 min to generate hydroxyl functional groups for the silane covalent procedure (Figure 1A). After the plasma cleaning, the substrates were immersed into a silane, including but not limited to exoxysilanes, dimethoxysilanes, trichlorosilanes, and triethylsilanes. In one embodiment, the epoxysilane is 3-glycidoxypropyl trimethoxysilane (GPTS) (Sigma-Aldrich) which is prepared in a solution in anhydrous toluene. The substrates were then left undisturbed for one to 24 hours, in one embodiment at least 16 hours, to allow silanization to take place on the oxide surface (Figure 1 B)- After the silanization step, the modified substrate was ultrasonically cleaned with ethanol and de-ionized (Dl) water and dried with nitrogen gas. As will be understood by persons skilled in the art, other solvents can be used for removing residual unreacted SAM-forming chemicals from the substrates, including but not limited to ethanol, acetone, methanol, toluene and isopropanol. Additionally, any gas which is inert and will not react with the SAM functional groups can be used to dry the modified substrate. At this point, the substrate was terminated by a highly ordered SAM of epoxysilane molecules (Figure 1 C). A small amount of a PEG or PEG derivative is then dispensed onto the dry silanized substrate (Figure 1 D) and a polydimethylsiloxane (PDMS) mold was immediately brought into conformal contact with the surface. PEG derivatives suitable for forming the SAMs of the present invention include, but are not limited to, PEG derivatives with functional groups which function to graft PEG molecules onto the substrate, including but not limited to, amine, thiol, carboxy, trichlorosilane, maleimide, aldehyde and NHS. In one embodiment of the present invention, the PEG derivative is methoxy-terminated PEG-amine (Nektar Therapeutics). The mold can additionally be fabricated from any soft or deformable material such as elastomers, silicone or rubber. The mold was left undisturbed at room temperature for a period of time sufficient to allow solvents to evaporate (Figure 1 E). During this period, aqueous solvents evaporate through the edges of the PDMS mold and leave behind a visible layer of dry solid PEG microstructures after the PDMS is peeled off (Figure 1 F). The surface was then rinsed with Dl water to dissolve all the PEG microstructures until no visible features can be seen on the substrate. At this point, the substrate was patterned with a densely covered PEG SAM (Figure 1 G).

[0037] Surface Characterization of PEG SAM

[0038] To investigate the protein adsorption of the PEG SAM, fluorescence-labeled proteins were used in optical microscopy characterization. A few drops of a 50 μg/ml solution of Texas-Red labeled bovine serum albumin (BSA) (Invitrogen) and 1 mg/ml solution of fluorescein isothiocyanate-labeled poly-L-lysine (PLL) (Sigma) were distributed onto the patterned SAM surface and incubated at room temperature for 10 min. The surface was then washed with PBS and analyzed by fluorescence microscopy

(DMIRB, Leica).

[0039] The static contact angle of the SAM surface was measured by an optical contact angle measurement system (FTA 4000, First Ten Angstroms). A droplet of 3 μl of Dl water was placed on the surface and measurement was made within 30 sec.

[0040] Characterization of Dry PEG Structure

[0041] Atomic force microscopy (AFM) was used to characterize the topography of the dried PEG microstructure after the PDMS was¹ peeled off. Measurements were taken in tapping mode on a Nanoscope III Dimension instrument in air, using RTESP tips at a scan rate of 0.5Hz.

[0042] After the water solvent of the PEG solution dried out through evaporation from the edges of the PDMS mold, the PDMS mold WAS peeled off and a visible layer of dried PEG microstructures remained on the surface. A scanning electron microscope (SEM) image (Figure 2) demonstrates that the patterns of these PEG microstructures (array of 3μm recessed wells) form a negative replica of the PDMS mold (array of 3μm protruding cylinders), with high pattern fidelity. The image also shows a highly smooth exposed substrate surface at the bottom of the recessed wells where no PEG resides.

[0043] Scanning the PEG microstructure with AFM reveals that the microstructures exhibit a periodical wavy topography of PEG mass pileup (Figure 3), instead of a flat uniform topography as the PDMS mold. During the molding process, a small volume^" of solution was dispensed on the substrate and was squeezed into the void region by capillary force exerted by the PDMS mold. Due -to the dewetting of the water on the wall of the PDMS mold, the water meniscus detaches from the PDMS wall and tends to adhere to the relatively more hydrophilic GPTS surface. The cross section image (Figure 3 inset) of the AFM measurement clearly reveals the water dewetting phenomenon in the PDMS mold. According to the AFM measurement, the bottom exposed substrate of the recessed well has a diameter of 3 μm and a height (h in inset) around 200 nm.

[0044] Contact Angle Measurement [0045] To understand the SAM grafting mechanism, contact angle measurements were carried out to characterize the two layers of SAMs (GPTS and PEG) on the oxide substrate. As disclosed above, the Si oxide surface was first silanized with GPTS to terminate the surface with a layer of epoxy functional groups for subsequent grafting of the PEG SAM. Figures 4A and 4B demonstrates that the contact angles of the SAM surfaces of GPTS and PEG are 52° ± 1° and 31° ± 1°, respectively. The contact angle of GPTS confirms a highly ordered and densely packed GPTS SAM was produced. This assures the PEG SAM is built upon a high quality functionalized GPTS SAM platform. After the GPTS SAM is formed, the PEG SAM is subsequently grafted on top, and the contact angle is decreased from 52° to 32°. This contact angle change ensures a covalent epoxy/amine reaction takes place on the surface. A contact angle of 32° corresponds to a highly packed and well oriented PEG SAM.

[0046] Since the PDMS mold was applied after the PEG solution was dispensed on the surface, a certain amount of PEG molecules immediately react with the surface and affect the accuracy of the PDMS patterning technique. However, the present inventor surprisingly determined that no change in contact angle on the GPTS surface is observed after a 20-min immersion in PEG-derivative solution. This slow reaction comes from the intrinsic chemical reaction kinetics between the epoxy and amine functional groups.

[0047] Fluorescence Microscopy

[0048] To demonstrate the protein repellant ability of the PEG SAM, the surfaces were incubated with two fluorescence-labeled proteins: BSA and PLL. Fluorescence microscopy images (Figures 5A and B) and quantitative analysis (Figure 5C) demonstrate that proteins are only selectively adsorped to the exposed glass substrate (bright circles) while only extremely low levels of protein adhesion (<3%) was present on the PEG SAM (dark area). As shown in Figures 5A and C, the adsorption of BSA was significantly reduced

^• by ^"PEG SAM (>99%) compared to the exposed glass control surface. The images also demonstrate that patterning with the method of the present invention can create micro protein features, as small as 3 μm, in a large area, in the order of centimeter, with high pattern fidelity.

[0049] Interestingly, increasing the protein concentration or the incubation time by 2-3 fold does not increase the protein nonspecific adsorption on the PEG SAM, demonstrating that the PEG surface is a highly ordered and densely packed PEG SAM surface. In addition, only a brief washing step (less than 10 sec) is required to rinse off the unbound proteins, compared to most conventional methods which require prolonged and harsher rinsing steps. Similar results were obtained from the tests on PLL (Figure 5B), wherein more than 97% of the PLL is repelled by the PEG SAM surface.

[0050] The present inventor has successfully demonstrated a high performance, high throughput, low cost, and simple technique for large area patterning of self-assembled monolayers (SAM) of PEG molecules on SiC»2 substrates for selective protein and cell adhesion using a molding technique. The patterned SAM surface can significantly reduce more than 99% of nonspecific protein adsorption compared to bare SiC>2 substrate. The SAM patterning method of the present invention can also alleviate many of the intricacies in conventional SAM patterning techniques, including soft lithography, imprint lithography, and photolithography. This high feature fidelity and high protein selective patterning technique of the present invention can substantially improve the conventional platforms for low density or single mammalian cell and bacteria studies.

[0051] Figures 6(a) and 6(b) provide another example to help illustrate some bio patterning resolutions that are possible according to embodiments of the current invention. One can produce imprint lithography-quality SAM patterns, not just in micro scale, but also in sub-50nm scale according to embodiments of the current invention. Nano protein patterning has attracted substantial research efforts in recent years due to its importance in many related applications in a currently fast-growing technology, bio-nano technology. The most common techniques include contact printing (μCP) and nanoimprint lithography. However, a disadvantage of contact printing is that it can only be employed on gold surfaces which significantly limits its applicability on various other materials, such as glass. Nanoimprint lithography offers advantages of high throughput and can provide a capability of creating nano-features as small as 10nm. However, this method requires sophisticated high pressure embossing machines followed by classical cleanroom processing.

[0052] Methods according to embodiments of the current invention can be used to generate sub-50nm protein patterns on silica surfaces without requiring any sophisticated instruments or cleanroom facilities. Stringent nano protein patterns can be generated in a high-throughput, in a simple, and very-low cost manner according to embodiments of the current invention.

[0053] Fluorescence microscopy image (Figure 6(a)) demonstrates that fluorescence conjugated proteins are accurately adsorbed to the exposed glass substrate (bright lines) with decent patterning ability while only a negligibly low level of protein (-1 %) resides on the PEG SAM surface (dark area). Lines counting from left to right are 400nm, 300nm, 200nm, 100nm, 75nm, and 50nm, each spaced 1μm apart. Figure 6(b) shows the bright field images of the master used in the molding process corresponding to the example of Figure 6(a).

[0054] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0055] The terms "a" and "an" and "the" and similar referents used in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g. "such as") provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0056] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0057] ^• Various - embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations on those embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventor expects skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above- described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context. [0058] Furthermore, numerous references have been made to patents and printed publications throughout this specification. Each of the above cited references and printed publications are herein individually incorporated by reference in their entirety. [0059] In closing, it is to be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the present invention. Other modifications that may be employed are within the scope of the invention. Thus, by way of example, but not of limitation, alternative configurations of the present invention may be utilized in accordance with the teachings herein. Accordingly, the present invention is not limited to that precisely as shown and described.

Claims

1 claim:

1. A method of patterning the surface of a solid substrate comprising: forming one or more than one self-assembled monolayer (SAM) on said solid substrate by capillary force lithography to form a surface- modified substrate; and contacting said surface-modified substrate with at least one biomaterial.

2. The method of claim 1 wherein said solid substrate is selected from the group consisting of silica, glass, polymers and metals.

3. The method of claim 2 wherein said solid substrate is silica.

4. The method of claim 1 wherein at least one of said one or more than one SAM is comprised of a biomaterial-adherent material.

5. The method of claim 1 wherein at least one of said one or more than one SAM is comprised of a biomaterial-nonadherent material.

6. The method of claim 1 wherein said one or more than one SAM comprises a first SAM and a second SAM.

7. The method of claim 6 wherein said patterned substrate comprises two SAMs wherein one of said first SAM or said second SAM is comprised of a biomaterial-adherent material and the other of said first SAM or said second SAM is comprised of a biomaterial-nonadherent material.

8. The method of claim 6 wherein said first SAM comprises an silane

9. The method of claim 8 wherein said silane is selected from the group consisting of epoxysilane, dimethoxysilane, trichlorosilane or triethylsilane.

10. The method of claim 9 wherein said epoxysilane is glycidoxypropyl trimethoxysilane.

11. The method of claim 6 wherein said second SAM comprises poly(ethylene glycol) (PEG).

12. The method of claim 11 wherein said PEG is a PEG derivative.

13. The method of claim 12 wherein said PEG derivative is a PEG derivative having a functional group selected from the group consisting of amines, thiols, carboxyls, trichlorosilanes, maleimides, aldehydes and NHS.

14. The method of claim 13 wherein said PEG derivative is a methoxy-terminated PEG-amine.

15. The method of claim 1 wherein said forming step comprises: cleaning said substrate; immersing said cleaned substrate in an epoxysilane and allowing silanization to occur on the substrate surface to form a first SAM; cleaning said silanized substrate; applying a PEG to the surface of said silanized substrate; bringing ^''a mold into^' conformal contact with said silanized surface and allowing a second SAM to form; drying said substrate; removing said mold; and rinsing said substrate to form a surface-modified substrate.

16. The method of claim 1 wherein said biomaterial is selected from the group consisting of proteins, peptides, carbohydrates, lipids, nucleic acids, chemicals and cells.

17. A bio-device having a bio-patterned substrate produced according to the method of any one of claims 1-16.

18. A bio-device, comprising: a substrate; a self-assembled monolayer formed on said substrate; a pattern of biomaterial formed on one of said self-assembled monolayer or exposed portions of said substrate to provide a bio-device having a biomaterial patterned substrate.

19. A bio-device according to claim 18, wherein said biomaterial patterned substrate comprises biomaterial patterned to a submicron resolution.

20. A bio-device according to claim 18, wherein said biomaterial patterned substrate comprises biomaterial patterned to a resolution of at least 100 nm.

21. A bio-device according to claim 18, wherein said biomaterial patterned substrate comprises biomaterial patterned to a resolution of at least 50 nm.

22. A bio-device according to claim 18, wherein said substrate consists essentially of a material selected from the group of materials consisting Of SiIiCa, glass, polymers, metals, and any combination thereof.

23. A bio-device according to claim 18, wherein said bio-material is selected from the group consisting of proteins, peptides, carbohydrates, lipids, nucleic acids, cells, and any combination thereof.