Protein patterning

Biomaterials 19 (1998) 595 — 609

REVIEW

Protein patterning A.S. Blawas, W.M. Reichert* NSF/ERC Center For Emerging Cardiovascular Technologies, Department of Biomedical Engineering, Duke University, Durham, NC 27708, USA Received 24 April 1997; accepted 6 July 1997

Abstract The current technology available for patterning proteins is reviewed. Examples of two-dimensional protein patterning using conventional photolithographic methods, photochemistry, and self-assembled monolayers are given. Consideration is also given to some major issues affecting protein patterning, including non-specific binding, protein pattern uniformity, and measurement techniques. S-layer nanopatterning and three-dimensional biochip patterning are discussed in possible future directions. In addition, a discussion of the impact of protein patterning technology on the field of biomaterials is discussed. ( 1998 Published by Elsevier Science Ltd. All rights reserved Keywords: Protein patterning; Biochip; Photolithography; Self-assembling proteins; Photochemistry

1. Introduction

1.1. Background

Crucial to the development of biologically integrated devices is the ability to organize multiple biomolecules on surfaces with resolutions from the micron to the nanometer scale. Several techniques have been examined for creating micron-level two-dimensional arrays of proteins on surfaces, including the use of conventional photoresist lithography, photochemistry and selfassembled monolayers. While each of these methods may be useful for some applications, each has its inherent limitations, particularly in the areas of multiple protein binding, non-specific binding, and in the ability to immobilize proteins while retaining their maximum activity. This review describes some of the techniques currently employed for creating two-dimensional protein arrays, and research results regarding their effectiveness. The impact of protein patterning on future applications of biomaterials, nanopatterning and three-dimensional protein patterning are also presented.

The field of protein patterning was originally conceived as a critical technology for the integration of biological molecules into miniature biological-electronic devices [1—3]. In 1978 MacAlear and Wehrung used photoresist technology from the semiconductor industry to create patterns on an underlying compressed proteinaceous layer [4, 5]. These patterns were intended for creating a bio-electronic microcircuit. Since then, there has been a growth of research done in the area of protein patterning. A current patent search has shown 19 patents filed since 1978 [4—21] and over 100 papers which address protein patterning. Table 1, summarizes some of the new developments in protein patterning techniques and applications. The variety of protein molecules and their functionality appeals to the creation of complex and miniaturized devices. Miyahara and Kimura used photoresist technology to pattern micro wells on ISFET (ion sensitive field-effect transistor) surfaces [22, 23] for the physical containment of immobilizing enzyme solutions. This led to the development of directly suspended enzymes in photopolymers and using photolithography to pattern the polymers onto the gate surface of an FET [24—26]. Simultaneously, Lowe and Early used photoactive nitroarylazide chemistry to create a patterned

* Corresponding author. Tel.: 00 919 660 5151; fax: 00 919 684 4488; e-mail: [email protected].

0142-9612/98/$19.00 ( 1998 Published by Elsevier Science Ltd. All rights reserved. PII S 0 1 4 2 - 9 6 1 2 ( 9 7 ) 0 0 2 1 8 - 4

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Table 1 Overview of recent protein patterning technology References

Patterning technique

Resolution

Application

Substrate

49

Photoresist technology with alkyl- and amino-terminated silanes Photoresist technology with methyland amino-terminated silanes

(10 lm

Controlled cellular growth

Silicon, SiO 2

(10 lm

Controlled cellular growth

Glass, fused silica

1.5 lm

Glass, fused silica

1.5 lm

Protein immobilization and enzymatic assay Controlled cellular growth; protein immobilization Multiple analyte immunoassay

350 lm

Controlled cellular growth

Polyvinyl alcohol

0.5 lm

Enzymatic assay

Polystyrene

2.5 lm 100 lm

DNA and peptide libraries Immunoassay

Glass Glass

300 lm (1 lm 100 lm 1 lm 100 lm 25 lm

Polystyrene TiO /SiO 2 2 Glass, fused silica, SiO 2 Glass, fused silica Glass Gold Gold

10 lm 10m5 m

Controlled cellular growth Immunoassay Controlled cellular growth Immunoassay Controlled cellular growth Immunoassay Immunoassays Protein adsorption; controlled cellular growth Controlled cellular growth Immunoassay

1 lm 50 lm 5 lm

Immunoassay Immunoassay Controlled cellular growth

\200 lm

DNA arrays

50, 53

27, 51, 52

79 80 81

Photoresist technology with methyl-, aminoand alkyl-terminated silanes Nitroarylazide photochemistry with biotin/avidin Nitroarylazide photochemistry with RGD peptide Perfluorophenylazide photochemistry with N-hydroxysuccinimide esters Nitrobenzyl photochemistry Nitrobenzyl photochemistry with biotin/streptavidin Diazirine photochemistry with oligopeptide Diazirine photochemistry with BSA/F(ab@) 2 Deep UV of silane SAMs with EDA and 13F Deep UV of silanes with OTS and EDA Deep UV of silanes with OTS and EDA Deep UV of thiol terminated silanes UV of alkane thiol SAMs Micro-manipulation and UV lithography of alkane thiol SAMs Microcontact printing of alkane thiols Electrochemical patterning of alkane thiol SAMs UV of benzophenone alkane thiol SAMs Laser vapor deposition of proteins RFGD functionalization of fluoropolymers

86

Ink-jet based robotic printing

56, 83 13, 57—59 60, 61 12, 16, 17, 62, 64 63, 65 47 66 68—70 71 72 15, 18, 85 73 74, 75 76, 77 78

10 lm

1 lm

Glass, quartz Gold, SiO 2

Gold Laser ablated gold on glass Gold Glass Poly(tetrafluoroethylene-cohexa fluoropropylene Glass

Abbreviations: RGD: argenine-glycine-aspartic acid; BSA: bovine serum albumin; UV: ultraviolet; EDA: (N-(2-aminoethyl) (3-aminopropyl) trimethoxysilane; 13F: tridecafluoro-1,1,2,2-tetrahydrooctyl-l-dimethylchlorosilane; OTS: N-octadecyltrichlorosilane; RFGD: radio frequency glow discharge.

BIOCHEMFET [8]. Protein patterning is currently being used for the development of biosensors with multiple sensing regions. These sensors exploit the selectivity and sensitivity of antibodies, a particular subset of proteins which bind selected analytes. Several studies regarding cell patterning have also been included in this review because in most cases the patterned assembly of serum proteins is necessary to promote patterned cell growth [27]. Patterned cellular growth has many applications including the potential for aiding in the creation of engineered tissue and organ grafts. Other possible applications for protein patterning are DNA or peptide libraries [17], optical data storage, and image detection and processing [6]. 1.2. Scope Protein patterning is really just a physically defined form of protein immobilization. The simplest method

for immobilizing protein on surfaces is physical adsorption where mutual attraction between the solid surface and the protein results in coverage of the surface [28—31]. Protein adsorption results from attractive forces such as ionic, hydrophobic, or van der Waals [28], and is also entropically driven [32]. A more stable means of protein immobilization is to covalently link a protein to the surface via a chemical bond between the molecules of the solid support and the protein [33—37]. An example of this type of immobilization is the use of bifunctional crosslinkers such as silanes, silica-based linkers which bind at one end to a glass or metal surface via a silanol bond and at the other end have a variable pendant group for binding protein. Finally, researchers have used high-affinity ligand pairs to immobilize proteins to surfaces, such as lectins, avidinbiotin, protein G and protein A [38—41]. These ligands provide stable immobilization similar to covalent coupling.

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Beyond protein surface coverage, the field of protein immobilization takes two directions: protein orientation and protein patterning. The area of protein orientation is critically important in the development of immunodiagnostics, as the optimum orientation of an antibody can greatly increase the surface binding ability and therefore the sensitivity for biosensing applications [37]. Developments in this area include selectively orienting whole antibodies [42—44], and immobilizing antibody fragments. A relatively new direction in protein immobilization, is the creation of engineered proteins that occupy less space on a surface and may also contain critically placed amino acid sequences suitable for optimally orienting the protein with respect to the surface [45]. The emphasis of this review is protein patterning, which may be defined as protein immobilization within specific locations in a two- or three-dimensional space. Some helpful reviews in the field are [3, 27, 46—48]. To date, research in the field has concentrated on micron level patterning within two dimensions, primarily with a single protein per surface. Further study for the incorporation of multiple proteins onto a single surface is preliminary. Two emerging branches of the protein patterning field are nanopatterning and three-dimensional patterning. Nanopatterning is the concept of placing a single protein in a specific location by creating patterns on the order of nanometers, the same size as a protein. Work in the area of three-dimensional protein patterning has focused on developing components for a biological three-dimensional working circuit, generally referred to as a biochip. The actual means for construction of such a circuit are theoretical, if not speculative. Relevant references on these topics accompany the subsequent text.

2. Examples of two-dimensional protein patterning techniques and applications 2.1. Conventional microfabrication techniques Conventional photoresist technology used for metal patterning for microcircuits is readily adapted to protein patterning. For protein patterning, instead of depositing metals on selected regions of the exposed substrate, patterning is done using chemical linkers with different pendant groups to create a heterogeneous monolayer. Silane coupling agents [33] have been the reagent of choice for attaching proteins to silica or metal surfaces. They can withstand the harsh solvent systems required to remove photoresist; they do not interfere with the optical properties of quartz substrates; they can be used with metal substrates for electrical measurements; and they form a robust covalent bond with the substrate and protein. Fig. 1 shows how silanes are used in a positive photoresist scenario. The substrate is spun cast with photo-

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Fig. 1. Conventional photoresist technology applied to the assembly of silane SAMs.

resist, covered with a mask, and exposed to ultraviolet (UV) irradiation. UV light decomposes the photoresist, allowing it to be dissolved away, exposing defined regions of the substrate. An adhesion promoting silane, usually amino terminated, is bound within these exposed regions, the slide is then sonicated with acetone to remove the remaining photoresist thus exposing the rest of the substrate. The slide is incubated with a hydrophobic or adhesion resistant silane, typically a methyl or alkyl terminated silane, resulting in a mixed monolayer interface. The sequence with which silanes are immobilized also can be reversed. Several groups have worked with photoresist technology of silanes to control cellular growth. Some of the initial work was done by Kleinfeld et al. [49], who was interested in reestablishing an ordered cytoarchitecture to nervous tissue cells by creating a patterned in vitro substrate. They used two silanes, alkyl-trichlorosilane, which inhibited cell growth via a long-chain alkyl group, and amino-trihydroxysilane which promoted growth via a hydrophilic amine. Subsequently, the surfaces were

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incubated with cerebellar and spinal cord cells in serum. It was demonstrated that both of these cell types could be grown within the amino-silanized regions on the scale of 50 lm. Additional work in this area [27, 50—52] uses the same photoresist technology with EDS (N-(2-aminoethyl)-3-aminopropyl-trimethoxysilane) and DMS (dimethyldichlorosilane). DMS was chosen over a longer alkyl chain terminated silanes, because dimethyl and amino groups are of similar size, therefore avoiding unintended topographic features which could influence cell behavior. DMS/EDS patterned surfaces have been used

extensively for studying cell growth phenomena, and have also been used to pattern proteins [52, 53]. 2.2. Photochemical techniques Photochemical protein patterning methods use chemically labile species, which can be activated upon UV irradiation, to bind target molecules, [47, 54, 55]. Conversely, deep UV irradiation can be used to ‘deactivate’ chemical species, such as the conversion of thiol groups to sulfonates [36]. In order to pattern protein molecules,

Fig. 2. Scenarios for photochemical protein patterning. (a) A substrate is derivitized with a photochemical species, and incubated with a protein solution. The solution-covered substrate is irradiated, activating localized regions on the substrate. The activated regions bind protein in solution. (b) A substrate is incubated with a photochemical species, and irradiated; activating species within localized regions. The activated species binds to the substrate within these regions, leaving a pendant group which can bind protein. The substrate is incubated with protein which binds in the localized regions. (c) A substrate is derivitized with a ‘caged’ species. The substrate is irradiated, which removes the caging group, and leaves localized active regions on the substrate. Protein binds within the localized active regions. (d) A substrate is incubated with a cross-linking polymer containing several photochemical species and a protein solution. The solution covered substrate is irradiated, activating polymer within localized regions on the substrate. The activated polymer binds to the substrate and the protein within localized regions. Note: Deep UV of silane SAMs is the reverse of scenario (a), in that the deep UV deactivates derivitized surfaces in localized irradiated regions, prohibiting protein binding.

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localized areas of reactivity can be created by selectively irradiating a photochemically derivitized surface using one of the four scenarios outlined in Fig. 2. The most commonly used photochemical methods are aryl azide chemistry, nitrobenzyl caging chemistry, and diazirine chemistry (Fig. 3). 2.2.1. Arylazide photochemistry Arylazide photochemistry is used to selectively immobilize proteins via an azide substituted aromatic group. Upon photolysis at the appropriate UV wavelength, an arylazide results in a reactive nitrene which can insert into C—H bonds (Fig. 3a) [55]. One application of arylazide chemistry was used by Pritchard et al. in the form of photoactive biotin: a biotin molecule with a nitroarylazide group attached to biotin tail [56]. The photobiotin is immobilized onto the surface via an adsorbed avidin molecule, and the derivitized substrate was

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then incubated with a protein solution. Upon masked irradiation of the solution-covered substrate, the free azide group was transformed to the active nitrene and inserted into a protein in the surrounding solution (Fig. 2a). This process was repeated with four additional protein solutions, until five antibodies were assembled into five localized regions. Matsuda et al. [57—59] describe in a recent paper the use of an arylazide to immobilize an octapeptide onto localized regions of a polyvinyl alcohol surface, in order to control cellular growth (Fig. 2b). The octapeptide contained an RGD (argenine—glycine—aspartic acid) sequence which has been found to be a critical amino acid sequence for adhesion within extracellular and adhesive proteins. Endothelial cells were cultured onto the patterned octapeptide surface, and ultimately migrated and proliferated within the localized RGD regions.

Fig. 3. Photochemistry reactions. (a) Arylazide chemistry. UV irradiation of an arylazide results in an active nitrene which can insert into C—H, C—C, C"C, N—H, O—H or S—H bonds. (b) Nitrobenzyl caging chemistry. UV irradiation of a ‘caged’ moiety results in a ketone, carbon dioxide and the active moiety. (c) Aryldiazirine chemistry. UV irradiation of an aryldiazirine results in an active carbene which can insert into C—H, C—C, C"C, N—H, O—H or S—H bonds (d) Benzophenone chemistry. UV irradiation of a benzophenone forms a biradical, then results in a C—C.

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Perfluorophenylazide (PFPA) photochemistry is analogous to arylazide chemistry, except the substitution of four fluorine atoms on the aryl ring, improves the nitrene insertion efficiency into C—H bonds. PFPA chemistry has been used to covalently attach N-hydroxysuccinimide (NHS) active ester groups to a polystyrene surface (Fig. 2b) [60]. To create patterned surfaces, a polystyrene film is created by spin casting a polystyrene solution onto a microcover glass, and a solution of the NHS-PFPA is spun cast onto the polystyrene. The film is photolyzed through a mask causing the PFPA to insert into the polystyrene in selected regions. The NHS ester reacted readily with amine groups on proteins, immobilizing them via a strong amide bond within the patterned areas [61].

The diazirine/BSA and a F(ab@) were spun cast together 2 onto TiO /SiO surfaces, and then the surfaces were 2 2 selectively irradiated at 350 nm. This created a diazirine/BSA link between the TiO /SiO surface and the 2 2 antibody fragment (Fig. 2d). The most unique aspect of this work was the ability of the surfaces to be regenerated after conducting an immunoassay with the F(ab@) . 2 A treatment with glycine buffer at a pH of 2.3, released bound analyte, and the F(ab@) could be restored for 2 a subsequent assay. Diazirine chemistry has also been used to immobilize a synthetic oligiopeptide to mediate cell adhesion [47], similar to the experiments performed by Matsuda et al. [13]. 2.3. Self-assembled monolayers (SAMs)

2.2.2. Nitrobenzyl photochemistry Nitrobenzyl chemistry is often called caging chemistry because it involves attaching a labile chemical group to a molecule which prevents its normal activity. Two of these caging groups are: NVOC (nitroveratryloxycarbonyl) and MeNPOC (a-methyl-6-nitropiperonyloxycarbonyl) [12, 16, 17, 62—64]. Upon irradiation of the appropriate UV wavelength, the caging group is broken down to a ketone and carbondioxide, and the ‘freed’ molecule retains its normal activity (Fig. 3b) [54]. By attaching the caged moiety to molecules which can bind proteins or amino acids, discrete protein patterning can be produced by selectively removing the ‘cage’ via photolithography (Fig. 2c). In Fodor et al. [64] 1024 specific sequence pentapeptides were constructed in a 50 lm checkerboard pattern within 1.6 cm2 using this nitrobenzyl caging chemistry. The array was used to identify the key sequences of amino acids responsible for antibody binding that could potentially be useful for genetically engineering antibodies. Additional work in this area has resulted in the assembly of a DNA library [62]. Our work [63, 65] has examined patterning antibodies onto waveguide surfaces for a multiple analyte sensor, using caged biotin and streptavidin. Published experiments focus on immobilizing the caged biotin by conjugating the MeNPOC-biotin to BSA, and then adsorbing it to glass surfaces. Subsequent exposures and incubations with streptavidin, and then biotinylated antibody resulted in a sensing element. Fluorescence microscopy is used to image bound, fluorescently labeled antigen. 2.2.3. Diazirine photochemistry Diazirine chemistry has been used in a variety of applications for the immobilization of proteins [47]. Diazirines absorb light at 350 nm and form highly reactive carbenes, the carbenes in turn insert into covalent bonds (Fig. 3c). For example, a 3-[trifluoromethyl]-3-[m-isothiocyanophenyl] diazirine coupled to BSA has been used to immobilize F(ab@) fragments on surfaces [66]. 2

A third type of method for patterning proteins is selfassembled monolayer (SAM) technology. When alkylsilanes or alkane thiol molecules are exposed to a silica or metal surface, they assemble into organized layers [67]. One endgroup of the molecular chain binds to the surface and one endgroup remains free to interact at the interface. Variation of the reactive endgroup can change the binding or surface energy of the monolayer. By creating mixed monolayers on a surface, proteins can be patterned within the regions of hydrophilic or adhesion promoting SAMs. 2.3.1. Ultraviolet irradiation of SAMs Dulcey et al. [68] and Stenger et al. [69] both described using SAMs created with EDA ((N-(2aminoethyl) (3-aminopropyl) trimethoxysilane) to pattern proteins and cells onto silica and platinum surfaces. Hydrophilic EDA is deposited on a surface, and then selected areas are exposed to deep UV light at 193 nm. The UV removed the alkylamine portion of the silane and left a hydroxyl reactive group on the end. The surface is then exposed to 13F (tridecafluoro-1,1,2,2-tetrahydrooctyl-1-dimethylchlorosilane) which binds to the hydroxyl group and results in a hydrophobic region on the surface. The remaining EDA regions promote cell growth. This has been demonstrated using porcine endothelial cells and with neurons. Similar experiments have been performed with human endothelial cells [70]. An extension of this work employed deep UV irradiation of N-octadecyltrichlorosilane (OTS) which is chemically bonded to glass and fused silica substrates [71, 72]. The pendant octadecyl group is adhesion resistant to proteins and cells. After deep UV irradiation the octadecyl group is cleaved, leaving a silanol group. The surface is reacted with EDA, which binds to the silanol groups forming adhesion promoting regions. This technology has been successful for patterning neuroblastoma cells and functional antibodies. A similar technology is employed by knoll et al. [73] using alkanethiol SAMs on gold surfaces.

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Bhatia et al. [36] examined the use of thiol terminated SAM silane films to create patterns of proteins on surfaces. Upon deep UV irradiation, the pendant thiol groups are converted to sulfonate groups which inhibit protein adsorption. By masking certain surface regions, designated areas retain the thiol group which can either promote protein adsorption, or be used for covalent binding to a protein. For example, silica surfaces are derivitized with MTS (3-mercaptopropyltrimethyoxy silane), masked and irradiated with 193 nm UV light. The patterned surfaces are incubated with IgG, BSA, and glucose oxidase resulting in patterned proteins within the thiol regions. Out of the remaining thiol regions the sulfonate group works well to inhibit non-specific binding. These experiments demonstrated reductions of non-specific binding for the previously mentioned proteins by 87, 74 and 90%, respectively. Covalent attachment of proteins is achieved by exposing the irradiated surfaces to GMBS (N-(c-maleimidobutyryloxy) succinimide) a homobifunctional crosslinking agent which reacts with remaining thiol groups, and can subsequently bind proteins. 2.3.2. Alkanethiol SAMs on gold Lopez et al. [74] reported several methods for creating mixed SAMs of alkane thiols on gold surfaces. These included microwriting, micromachining, stamping and UV microlithography. Using scanning electron microscopy, they have shown that variation of the pendant group can alter the adsorption of protein within different regions on the order of 10 lm. Combinations of two or more u-substituted alkane thiols have also been used for observing the response of cellular adhesion and growth to various functionalized surfaces at different serum conditions [75]. Kumar et al. [76] described a microcontact printing technique for creating alkane thiol patterns, which was done with a stamp made by casting silicone rubber, polydimethylsiloxane into the desired pattern, and ‘inking’ the stamp with a thiol terminated alkane. The stamp is contacted with a gold surface, which caused the alkanethiol to self-assemble within the stamp pattern onto the gold. Subsequently, the surface was incubated with a second alkanethiol which self-assembled onto the rest of the clean gold surfaces left after stamping. This resulted in a pattern of the first and second alkane thiol (Fig. 4). Stamping of alkane thiol SAMs has been used to isolate single cells in culture [77]. Using a prepared silicone rubber stamp, a gold surface was stamped with 10 lm grid of hexadecanethiol. The remaining bare gold was derivitized with a polyethylene glycol (PEG) terminated alkanethiol which was adhesion resistant. The PEG inhibited the adsorption of laminin, a protein used to attach hepatocyte cells to gold surfaces. Rat hepatocytes were plated onto the SAM coated gold surface

Fig. 4. Micro-contact printing with a polydimethylsiloxane stamp and two alkane thiols, adapted from Kumar et al. [73].

and single-cells cultured within the hexadecanethiol islands. Two additional methods have been reported which have been successful in patterning alkane thiols on gold. Tender et al. [78] described a laser ablation technique which created gold patterns on glass substrates. Once the gold pattern has been created, alkane thiols are immobilized on the surface for protein patterning. For example, in one method (1-mercaptoundec-11-yl) hexa(ethylene glycol) is adsorbed to the entire surface, and then desorbed from the gold areas via voltage cycling. The surface is then incubated with hexadecanethiol which adsorbs to the clean gold pattern. Protein preferentially adsorbed on the hexanedecanethiol creating a protein pattern. Finally, Delamarche et al. [79] combined photochemistry with alkanethiol SAMs to create protein patterns on gold surfaces. The terminal active ester group of alkane thiols immobilized on gold surfaces are converted to the photoactivatable group benzophenone (Fig. 3d). The surfaces are subsequently incubated with the protein solution and irradiated with UV light. Irradiation causes biradical formation at the ketyl center of the photolabile group, which is followed by a C—C bond with the protein on radical recombination (Fig. 2a).

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2.4. Miscellaneous There are two techniques which do not necessarily fit within the above categories of patterning methods. One study, prepared by Morales et al. [80] has taken the novel approach of patterning proteins using vapor deposition. Vapor deposition is conventionally used to deposit thin molecular layer films onto substrate surfaces. In this case, vaporized and ionized biomolecules are directed to desired locations onto glass surfaces through masks. One of the most significant obstacles with this technique is the potential for destruction of the protein by the vaporization and ionization processes. Vargo et al. [81] micropattern silanes onto a fluoropolymer film. Using a radio frequency glow discharge plasma treatment, the fluoropolymer is refunctionalized with localized areas of amino terminated silane, essentially the same scenario as illustrated in Fig. 2b. These surfaces have been able to control growth of mouse neuroblastoma cells within the amino functionalized regions.

3. Discussion 3.1. Advantages and disadvantages of different two-dimensional patterning techniques The following section summarizes the benefits and some of the potential obstacles for each of the patterning techniques described. Unfortunately, the literature rarely provides accurate accounting of the caveats associated with a given technique (i.e. scientific boosterism). Therefore, a discussion of relative pros and cons is inherently subjective. The biggest advantage of photoresist-based protein patterning is that it builds upon a well-established technology. However, another advantage is the option to choose which silane will be the ‘background’ silane, meaning the surface regions where protein is not bound. This allows for flexibility in varying the pendant group for minimum background protein adsorption. The most apparent disadvantage is the effect of the patterning technique on the overall protein activity. Residual solvents and photoresist can denature proteins, decreasing their surface activity. In addition, silanization may result in incomplete surface coverage or multilayer coverage. Incomplete coverage of the substrate could result in sites for increased non-specific binding, or ‘holes’ in the pattern. The effect of multilayer coverage is less predictable. Finally, the use of this technology allows for a single incubation with the patterned silane surface. This prohibits it from being used with more than one protein, unless physical boundaries can be made on the patterned surface to contain different protein solutions.

The benefit of photochemical methods over conventional lithographic methods is that solvents, buffers, and reactants, can be tailored to minimize the negative effect on protein activity. Most importantly, these chemistries can be used repeatedly on the same surface with different proteins to create a multiple protein pattern. A concern with photochemical techniques is the limited ability to control the surface energy of the background region. The specific surface derivitization that is necessary to conduct the photochemical reaction dictates the background surface. The introduction of blocking proteins such as BSA [82] may alleviate this problem. The most difficult aspect of using photochemical methods is that the surfaces are sensitive to UV light. In order to retain maximum efficiency, the surfaces must be kept in subdued lighting until the ultimate protein binding step. Also, photolithographic masking must be done either via backmasking through a transparent substrate or through some air gap to avoid marring or smearing of the derivitized surface. This may cause scatter of the irradiation source as it must pass through the substrate of the derivitized surface. The primary disadvantage with arylazide, benzophenone, and diazirine chemistries as they have been applied to protein patterning is that the protein must be in intimate contact with the derivitized surface during the irradiation process, and therefore there is potential for reduction in protein activity by UV light. A NO or 2 perfluoro-substitution on the arylazide ring enables the compound to be activated at higher wavelength, eliminating the need for light in the UV range of 265—275 nm which is most likely to damage proteins [83]. In addition, activated species conjugation to proteins may be low due to the short half-life of the nitrene [84]. By incorporating an NHS ester as an intermediate protein binding group, the perfluorophenylazide technique described here [61] does not require protein to be present during the irradiation step. PFPA chemistry is also more efficient than arylazide chemistry due to the four fluorine substitutions on the phenyl ring. The primary disadvantage of this technique is that the insertion of the immobilized NHS ester is random. The reaction of the surface with a lysine group too close to the protein’s reactive site may interfere with its activity. Also, the presence of polystyrene may interfere with certain sensing modalities for biosensor application. Nitrobenzyl caging chemistry is different from the other photochemical techniques in that UV irradiation reactivates an existing moiety, by removal of the caging group. This is an advantage, as the caging group can be bound to a variety of molecules which allows for flexibility and specificity in surface immobilization techniques. For example, using this technique in conjunction with the well-exploited avidin—biotin pair [41] allows us to create a generic procedure for immobilizing several proteins on a single surface. During the irradiation step the caging group breaks down into harmless by-products

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which do not interfere with the protein binding, and protein is bound to the surface after irradiation, alleviating the danger of ultraviolet damage. Self-assembled monolayer techniques are useful for easily creating a monolayer with localized regions for protein immobilization [68, 76, 85]. Microwriting, micromachining, and stamping of alkane thiols have an advantage over microlithography, as the surface is never exposed to UV light. One disadvantage is that the surface is prepared ahead of time, and then the protein solution is incubated in a single step, this discourages the possibility of multiple protein patterning. The only means for binding multiple proteins would have to involve alkanes with selectively pendant groups that would only bind a single protein in a specific manner. Surfaces with areas of more than two substituted alkane thiols and areas of more than two mixed monolayer composition have been created using pinning and writing techniques. Multi-region alkane patterned surfaces have proved useful for comparing cellular and protein adsorption under different conditions. Deep UV treatment of silane SAMs has the potential for patterning multiple proteins using the technique described by Bhatia et al. [85], however, it does suffer from the same coverage issues as conventional photolithography of silanes. Overall, it appears that conventional photolithography and SAM surfaces are taking the lead as the preferred methods for controlled cellular growth studies. On the other hand, protein patterning for immuno- or enzymatic assay has been accomplished primarily via photochemical methods. This is probably due to the potential for applying these techniques to pattern multiple proteins on a single surface. For large-scale arrays of proteins, peptides, or oligionucleotides many of the techniques discussed suffer from the need for repeated processing steps. Recently, Schena et al. [86] have described a technique based on ink-jet printing which would allow for parallel assembly of these types of arrays. Such parallel techniques also may avoid cross contamination which is a problem for the serial techniques presented above. 3.2. Non-specific binding The most difficult issue to address for protein patterning is non-specific binding (NSB) of protein to the surface. NSB refers to the indiscriminate adhesion of proteins to a surface due to weak attractive interactions or entropic forces. The implication is that the protein will not only adhere within the desired location, but will attach to all regions of the patterned surface. For patterning of a single protein, non-specific binding in the background region can reduce the sensitivity of an assay or allow for random cellular adhesion. The problem is only further compounded when patterning multiple proteins, because more than one protein may be present within a given region. For example, if protein X is patterned in

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region 1, and subsequently protein Y is incubated with the surface for patterning in region 2, there is the potential that protein X will non-specifically bind within region 2 and protein Y will non-specifically bind in region 1. The effect is that analyte binding within regions 1 and 2 cannot be attributed only to proteins X and Y, respectively. For a multiple sensing element, this error would be multiplied by the number of sensing regions. There are several steps which can be taken to reduce the NSB effect. Patterning with silane or alkane monolayers gives the flexibility of varying the pendant group of an SAM to reduce NSB within the background regions [85]. One can also use a blocking protein such as BSA or casein [82]. These ‘sticky’ proteins will adsorb to bare regions on the substrate and block other proteins from adhering. A more elaborate solution would be to include surface repulsion chemistry, such as using polyethylene glycol (PEG) groups [77, 87] in conjunction with the patterning chemistry. An example, would be to attach a photochemical species to a long-chain PEG group on the surface. The PEG occupies large volumes of space above the substrate preventing proteins from reaching the surface. The photochemical reaction would then take place at the end of these groups. The ability to functionalize typically adhesion resistant surfaces such as fluoropolymers, as demonstrated by Vargo et al. [81] is an innovative way to reduce NSB. 3.3. Measurement techniques In order to develop and apply patterned protein surfaces, there must be an effective means of visualizing these patterns. The imaging technique should ideally be nondestructive for quality assurance purposes as well as quantitative. Most of the protein patterns presented in recent studies have been developed with a fluorescently tagged protein with fluorescence microscopy. This has shown to be an effective means of non-destructively visualizing the protein pattern, however, without the proper calibration steps, this may not be a quantitative means of measuring bound protein. In addition, this technique is limited by fluorescent tag specificity, efficiency and potential chemical perturbation [81]. Conventional scanning electron microscopy is also a useful technique for imaging protein patterns. Although it is destructive, it requires no labels and can be used to look at protein coverage and pattern defects [74]. A relatively new means of visualizing protein patterns is by atomic force microscopy (AFM) [88, 89]. The difficulty is that this technique can be destructive as the sensing tip is rastered across the sample. Mazzola et al. [90] have successfully used AFM to image bimolecular arrays. By reducing the tip force and the tip profile, successful imaging of streptavidin on photoactivated biotin patterns have been produced. Additional examples of AFM images of protein patterns and SAMs are found

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in Gao et al. [91], Morgan et al. [92], and Kumar et al. [76]. Time of flight-secondary ion mass spectrometry (ToF-SIMS) also has been used to image silane patterns, and may hold potential for protein imaging. Healy et al. [51] presented a characterization of patterned silane surfaces by ToF-SIMS, as did Vargo et al. [81]. Accurate measurement is also important because it is critical to know how much protein is immobilized within a patterned region. In some examples of patterned proteins, the surfaces appear mottled and coverage is nonuniform, this can result in inaccuracies when performing a comparative assay. For example, consider the patterning of two antibodies X and Y on to separate regions, where the density of antibody X in region 1 is twice the density of antibody Y in region 2. Unless this difference in surface coverage is accounted for, then region 2 can give a falsely lower reading on analyte concentration. This variability can be accounted for through on-sensor calibration using reference patterns to map the patterned surface with fluorescence microscopy. As necessity drives patterning resolution to smaller features, non-uniformity will become a major obstacle.

4. Future technology

by Sleytr et al. [98], S-layers have the potential for technological applications as they can be purified, suspended in aqueous solution and adsorbed to solid surfaces while maintaining their intact lattice. S-layers may be used to immobilize organized monolayers of proteins because each subunit within the periodic array contains the same chemical composition. Therefore, a single active moiety within a subunit can be targeted to covalently immobilize a protein molecule. Douglas et al. have created metal masks with nanometers features using the S-layer as a template [92]. These masks can then be used for photolithography in conjunction with photochemistry, SAMs or conventional photoresist technology [99]. Chilkoti and coworkers are currently examining the method to pattern nano-structured protein arrays [100]. Another approach is being pursued by Van Duyne and co-workers [101] who used single- and dual-layer assemblies of polystyrene nanospheres to pattern nanometer sized various metal islands on metal and glass substrates. After metal deposition, the beads are removed chemically by dissolution or mechanically via tape lift off. The remaining metal features are on the order of 40 nm wide and 22 nm in depth. Theoretically, these arrays could be used to pattern biological materials directly into gold islands using thiol chemistry.

4.1. Nanopatterning 4.2. Three-dimensional protein patterning techniques One direction where protein patterning will play a significant role is in the field of nanotechnology: the science of creating structures on the submicron level. Currently, protein patterning has been limited to micron or tenths of micron size features, however, the ability to pattern on the order of nanometers, at the scale of biological molecules, would allow for increased miniaturization and complexity for many applications [48, 93, 94]. An interesting review of nanoscience is presented by Rohrer [95] where he describes how solid-state technology has focused on miniaturization, while chemistry has developed towards increasingly larger macromolecular structures. The meeting of these two disciplines leads to nanopatterning. Some of the techniques already described in the previous sections have achieved patterning on the order of hundreds of nanometers [60, 76]. Electron beam lithography has resulted in the production of features down to several nanometers in photoresist [96], however, it has not been applied to protein patterning. An alternative method for creating nanometer scale features is to exploit self-assembling biologically derived systems that contain nanometer patterns. S-layer proteins are two-dimensional lattice proteins which are found in the cell wall of many bacteria. These protein layers exhibit several types of periodic lattice patterns on the nanometer scale. For example, the hexagonal pattern of the S-layer of ¹hermoproteus tenax exhibits channels of 6 nm in diameter [97]. As discussed

A persistent paradigm for 3-D protein patterning is the biochip. The term biochip is a confusing one, because it has been applied to a variety of inventions from a simple electronic device containing biological entities, such as a enzyme impregnated ISFET, to futuristic chips which can enter the human body to repair or supplement cell function [1, 3]. Excellent reviews on this subject are readily available [1—3, 102, 103]. Several potential elements of the biochip have been developed [2, 104—106], what remains is the synthesis of these components into a working architecture [107—109]. Although biochips have been far more talk than action, they represent a conceptual discussion for pushing the upper limits of protein patterning. The problem of how to assemble biochip components in three-dimensional space still remains the major obstacle due to the fragility and flexibility of biological molecules [48]. One of the most promising techniques proposed for creating a three-dimensional patterned structure is to encode the architecture into DNA, and have the components self-assemble as they are generated by a bacterial or human cell [110], as tissues and organs do in a growing fetus. Protein organization can be encouraged by creating energetically favorable positioning or creating binding pairs on corresponding components. Some initial work in the area of protein architecture is presented by Ghandiri et al. [111]. An insightful review

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Fig. 5. Single cells grown in designated shapes on a patterned polyHEMA (polyhydroxyethylmethacrylate) substrates reproduced with permission from [113].

by Whitesides [46] discusses the benefits of self-assembling materials as potential building blocks for microcircuits and biomimetic membranes. He discusses phospholipids, SAMs, and ‘buckytubes’ as potential three-dimensional structural elements. In addition, it has been observed that S-layers can self-assemble to form cylindrical structures and sheets which may prove to be useful for nanopatterning in three dimensions [112]. 4.3. Impact on biomaterials It would be satisfying to write a critical review of how protein patterning will impact the field of biomaterials, as is done primarily for sensing in Section 3.1. However, the impact of this technology on the development of biomaterials remains speculative. In order to examine the potential effect of patterning on biomaterials let us use cellular adhesion to a substrate as a model. It has been demonstrated that patterning techniques can be used to determine the shape of a cell (Fig. 5) [77, 113], as well as control the area of cell adhesion and growth (Fig. 6) [27, 49]. It is also known that cells will rearrange proteins on surfaces into ‘patterns’ such as focal contacts [114]. An outstanding example of how the micron-level patterning of cell adhesion proteins affects cell function comes from Chen et al. [116]. In this work, an array of 5 lm islands of fibronectin separated by 10 lm spaces were patterned on a SAM using microcontact printing. Endothelial cells plated onto the patterned fibronectin array extended our membrane processes, literally reaching from island to island, and forming local contacts as indicated by vinculin staining. The problem is that these

patterns are static, and regularly spaced in a registered manner. The practical question is: can protein patterning technology be used to improve cellular adhesion to a substrate, and if so, will it promote normal cell function and improve biocompatibility? One hypothesis is that the most effective way to adhere cells to a substrate is to artificially create the cell’s own adhesive mechanism: the focal contact. This would require patterning ligands with submicron resolution as well as having a priori knowledge of the most judicious pattern juxtaposition and composition. Submicron patterning may be obtainable by either employing photochemistry with nanometer feature masks or by substituting specific ligands on an SAM components to create synthetic focal contact areas. Assuming that this can be facilitated, the next requirement would be that the pattern is fluid. Cells in vivo are exposed to a variety of environmental factors which dictate their subsequent growth and behavior. If the pattern ‘sentences’ the cell to a set position, then the material is likely doomed for failure. A potential solution would be to pattern a limited number of high affinity adhesion sites for strong initial attachment that degrades with time, along with lower affinity patterned ‘footprints’ to induce the cell to move in a directed course. One approach that we are investigating [115] is creating heterogeneous surfaces that consist of coimmobilized streptavidin and fibronectin. When biotinylated cells are introduced they adhere very rapidly and spread. However, Biotin (vitamin H) and streptavidin are rapidly metabolized and removed, leaving behind only the

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biodegradable matrix, patterned regions gradually receded, then the cells may be allowed to assume their natural function unencumbered by the preset patterned substrate. It appears that for the present, patterning technology for biomaterials remains an instrument of the laboratory, exploited for comparing a variety of surface derivitizations and ligands in a single pass. At this time, our knowledge of what a cell needs to function in vivo for long periods of time is too complex to recreate with the existing patterning technology. Acknowledgements We gratefully acknowledge support from NIH grant HL 32132, and the NSF sponsored Duke/North Carolina Center for Emerging Cardiovascular Technologies. In addition, we would like to thank Dr. Ashutosh Chilkoti from the Duke Center for Cellular and Biosurface Engineering for his contributions. We also extend appreciation to Dr. Frances Ligler of NRL and to anonymous reviewers for many insightful comments. References

Fig. 6. Multi-cellular patterning on: (a) EDS/HDS (hexadecylsilane); (b) EDS/DMS; and (c) EDS/BSA patterned substrates reproduced with permission from [27].

natural adhesion mechanism. To date we have not examined whether patterning the streptavidin makes any difference. Where a cell establishes its focal contacts remain indeterminate, therefore our best option may be to direct growth on a multicellular level, i.e. in a given direction. For example pre-aligning endothelial cells in the direction of flow of a vascular graft, via aligned patterns of cell adhesion ligand may give the cells a ‘head start’ on the desired in vivo morphology. If by some mechanism, i.e.

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