Light-Activated Affinity Micropatterning of Proteins on Self-Assembled

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Light-Activated Affinity Micropatterning of Proteins on Self-Assembled Monolayers on Gold Zhongping Yang,† Wolfgang Frey,† Tom Oliver,‡ and Ashutosh Chilkoti*,† Department of Biomedical Engineering, Box 90281, Duke University, Durham, North Carolina 27708, and Food and Drug Administration, Rockville, Maryland 20857 Received June 23, 1999 We describe a method to pattern proteins onto a photolabile “caged” biotin-derivatized self-assembled monolayer (SAM) on gold, which we term light-activated affinity micropatterning of proteins (LAMP). LAMP is a multistep patterning process with considerable flexibility in its implementation. First, a reactive SAM on gold is formed from a mixture of 11-mercaptoundecanol and 16-mercaptohexadecanoic acid. Next, the carboxylic acid end groups in the SAM are coupled to methyl R-nitropiperonyloxycarbonyl biotin succinimidyl ester (caged biotin ester) through a diamine linker. The caged biotin is then deprotected in regions irradiated by masked UV light, and subsequent incubation with streptavidin results in selective binding of streptavidin to the irradiated regions. Micropatterning of various proteins has been demonstrated with a spatial resolution of ∼6 µm by confocal microscopic imaging of fluorophore-labeled proteins, and a contrast ratio of ∼4:1 was determined by direct ellipsometric imaging of streptavidin. Immobilization of biotinylated antibodies on the streptavidin pattern indicates that LAMP can enable spatially resolved micropatterning of different biomolecules by repeated cycles of spatially defined photodeprotection of biotin, streptavidin incubation, followed by immobilization of the biotinylated moiety of interest.

1. Introduction Protein micropatterningsthe spatially resolved immobilization of proteinsshas diverse applications, which range from modulation of cell-substrate interactions in biomaterials and tissue engineering to the fabrication of multi-analyte biosensors, clinical assays, and genomic arrays.1-4 Patterned self-assembled monolayers (SAMs), which can be fabricated by photolithography,5,6 micromachining,7 microwriting,4 electrochemical stripping,8 and microcontact printing,9 are convenient molecular templates for protein patterning on gold and silicon oxide substrates. For example, patterned SAMs of a hydrophobic thiol (e.g., 16-hexadecanethiol on gold) and a SAM formed from a second alkanethiol (typically containing a terminal oligomeric ethylene glycol or hydroxyl moiety), which prevents nonspecific protein adsorption, allow proteins to be patterned by physical adsorption onto hydrophobic regions. This approach has been extended by Whitesides and co-workers to spatially localize biomolecular ligands onto a reactive SAM on gold by microcontact printing.10 * To whom correspondence should be addressed. Tel: (919) 6605373. FAX: (919) 660-5362. E-mail: [email protected]. † Duke University. ‡ Food and Drug Administration. (1) Mrksich, M.; Whitesides, G. M. Tibtech 1995, 13, 228-235. (2) Blawas, A. S.; Reichert, W. M. Biomaterials 1998, 19, 595-609. (3) Chen, C. S.; Mrksich, M.; Huang, S.; Whitesides, G. M.; Ingber, D. E. Biotechnol. Prog. 1998, 14, 356-363. (4) Vaidya, R.; Tender, L. M.; Bradley, G.; O’Brien, M. J., II, Cone, M.; Lopez, G. P. Biotechnol. Prog. 1998, 14, 371-377. (5) Mooney, J. F.; Hunt, A. J.; McIntosh, J. R.; Librerko, C. A.; Walba, D. M.; Rogers, C. T. Proc. Natl. Acad. Sci. 1996, 93, 12287-12291. (6) Knoll, W.; Liley, M.; Pisceivic, D.; Spinke, J.; Tarlov, M. J. Adv. Biophy. 1997, 34, 231-251. (7) Abbott, N. L.; Folkers, J. P.; Whitesides, G. M. Science 1992, 257, 1380-1382. (8) Tender, L. M.; Worley, R. L.; Fan, H. Y.; Lopez, G. P. Langmuir 1996, 12, 5515-5518. (9) Xia, Y.; Whitesides, G. M. Angew. Chem., Int. Ed. Engl. 1998, 37, 550-575. (10) Lahiri, J.; Ostuni, E.; Whitesides, G. M. Langmuir 1999, 15, 2055-2060.

Molecular recognition between the patterned ligand and its protein partner then permits protein patterning, mediated by molecular recognition between the protein and its ligand. A primary limitation, however, of most previous protein patterning studies is that they have been restricted to the patterning of a single protein on a substrate. Extension of current techniques to permit patterning of multiple proteins on the same substrate is a challenging task. A recent report has demonstrated the patterning of three different antibodies on the same substrate by direct microstamping.11 Alternatively, spatially resolved photochemical techniques have also been developed to pattern multiple proteins on the same substrate. This approach has been implemented by direct, covalent coupling of proteins to SAMs derivatized with a photoreactive moiety,12 or mediated by protein-ligand interactions.13,14 The biotin-avidin system is the prototypical protein-ligand system commonly used for protein micropatterning mediated by biomolecular recognition because: (1) both avidin and its bacterial homologue streptavidin bind biotin with high affinity (Keq ) 1013 and 1015 M-1 for streptavidin and avidin, respectively);15 (2) both proteins can be used as homobifunctional protein adapters because the 222 point symmetry of the streptavidin (and avidin) homotetramer positions two pairs of biotin binding sites on opposite faces of the protein,16 so that each protein molecule can simultaneously bind to a biotin-functionalized substrate and to biotinylated molecules on the opposite face exposed (11) Martin, B. D.; Gaber, B. P.; Patterson, C. H.; Turner, D. C. Langmuir 1998, 14, 3971-3975. (12) Delamarche, E.; Sundarababu, G.; Biebuyck, H.; Michel, B.; Gerber, C.; Sigrist, H.; Wolf, H.; Ringsdorf, H.; Xanthopoulos, N.; Mathieu, H. J. Langmuir 1996, 12, 1997-2006. (13) Pritchard, D. J.; Morgan, H.; Cooper, J. M. Angew. Chem., Int. Ed. Engl. 1995, 34, 91-92. (14) Hengsakul, M.; Cass, A. E. G. Bioconj. Chem. 1996, 7, 249-254. (15) Wilchek, M.; Bayer, E. Avidin-Biotin Technology; Methods in Enzymology, Academic Press: San Diego, CA, 1990; Vol. 184. (16) Weber, P. C.; Ohlendorf, D. H.; Wendoloski, J. J.; Salemme, F. R. Science 1989, 243, 85-88.

10.1021/la9908079 CCC: $19.00 © 2000 American Chemical Society Published on Web 12/17/1999

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Figure 1. Schematic of the preparation of a MeNPOC-biotin-SAM. (A) Mixed SAM of MUOH/MHA on gold; (B) PFP-activated SAM prepared by coupling PFP to the carboxylic acid group of the mixed SAM in the presence of EDAC. (C) DADOO-derivatized SAM prepared by coupling DADOO to the PFP-activated SAM; (D) MeNPOC-biotin-SAM prepared by coupling MeNPOC-biotin succinimidyl ester with terminal amino group in the DADOO-derivatized SAM.

to solution; and (3) both proteins are extremely stable to thermal denaturation and chaotropes.17 Pirrung and co-workers18,19 recently introduced a lightactivated patterning technique, in which a photolabile, caged biotin was immobilized onto silanized glass, deprotected by masked UV irradiation, and streptavidin was then immobilized to reconstituted biotin in irradiated regions. Subsequently, Reichert and co-workers20 extended this approach to a step-and-repeat patterning of multiple antibodies onto an adsorbed monolayer of BSA conjugated to caged biotin. Here, we report a complementary method to micropattern proteins onto SAMs on gold with a photolabile caged biotin derivative, which we term lightactivated affinity micropatterning of proteins (LAMP). There were several reasons that motivated us to implement LAMP on SAMs of alkanethiols on gold: (1) SAMs of alkanethiols on gold are easy to prepare and are reproducible;21 (2) alkanethiols with a diversity of terminal functional groups have been synthesized, which allow SAMs to be easily modified after their self-assembly from solution;21 (3) SAMs are densely packed and relatively (17) Bayer, E. A.; Ehrlich-Rogozinski, S.; Wilchek, M. Electrophoresis 1996, 17, 1319-1324. (18) Sundberg, S. A.; Barrett, R. W.; Pirrung, M.; Lu, A. L.; Kiangsoontra, B.; Holmes, C. P. J. Am. Chem. Soc. 1995, 117, 1205012057. (19) Pirrung, M. C.; Huang, C.-Y. Bioconj. Chem. 1996, 7, 317-321. (20) Blawas, A. S.; Oliver, T. F.; Pirrung, M. C.; Reichert, W. M. Langmuir 1998, 14, 4245-4250. (21) Dubois, L. H.; Nuzzo, R. G. Annu. Rev. Phys. Chem. 1992, 43, 437-463.

defect free;21 (4) SAMs are robust enough for most technological applications of patterned proteins;22 and (5) gold is the preferred substrate for many contemporary analytical techniques used in biosensors and immunoassays, such as BIACORE surface plasmon resonance (SPR) instruments (BIACORE AB, Sweden), and other evanescent SPR platforms.23 Our implementation of LAMP on gold is a multistep patterning process, with considerable flexibility in its implementation: first, a gold substrate is functionalized with a binary mixture of a hydroxyl-terminated and a carboxylic acid-terminated thiol to provide a reactive SAM on gold (Figure 1a). The carboxylic acid end groups enable subsequent derivatization of the SAM, while the hydroxyl groups resist nonspecific protein adsorption.6 The carboxylic acid end groups in the mixed SAM are converted to a reactive pentafluorophenyl ester (Figure 1b), and coupled to a bifunctional amine-terminated oligo(ethylene glycol) linker (Figure 1c). The terminal amine groups in the oligo(ethylene glycol)-derivatized SAM are conjugated with a caged biotin NHS-ester (Figure 1d). Finally, caged biotin is deprotected by UV light through a photomask and subsequent incubation with streptavidin results in selective binding of streptavidin to the irradiated regions. LAMP can be extended to enable spatially resolved micropatterning of different biomolecules, by repeated (22) Bishop, A. R.; Nuzzo, A. R. Current Opin. Colloid Interface Sci. 1996, 1, 127-136. (23) Homola, J.; Yee, S. S.; Gauglitz, G. Sensors Actuators B 1999, 54, 3-15.

Light-Activated Pattern of Proteins on SAM/Au

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cycles of spatially defined photodeprotection of biotin, streptavidin incubation, followed by binding of the biotinylated biomolecule of interest. 2. Experimental Section 2.1. Materials. All materials and chemicals were used as received unless stated otherwise. 11-mercapto-1-undecanol (MUOH, 97%), 16-mercapto-1-hexadecanoic acid (MHA, 90%), 2,2′-(ethylenediooxyl)bis-(ethylamine) (DADOO) and anhydrous dimethylformamide (DMF) were purchased from Aldrich. MHA was recrystallized in hexane before use. 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDAC), and pentafluorophenol (PFP) were obtained from Sigma. Streptavidin was obtained from Boehringer Mannheim. D-biotin succinimidyl ester, 6-((6-((biotinoyl)amino)hexanoyl)amino)hexanoic acid, sulfosuccinimidyl ester, sodium salt (biotin-XX-ester), Alexa 488 protein labeling kit and Alexa 488 labeled anti-biotin mouse monoclonal antibody (anti-biotin), were purchased from Molecular Probes. Methyl R-nitropiperonyl-oxycarbonyl biotin (MeNPOC-biotin) succinimidyl ester was prepared as described previously.19,20 Absolute ethanol was purchased from AAPER Alcohol. HEPES buffered saline (HBS/BSA/P20) containing 10 mM 4-(2-hydroxyethyl)-1piperazineethane-sulfonic acid, 0.1 M NaCl, 0.02% (w/v) nonionic surfactant P20 and 0.1% (w/v) bovine serum albumin (BSA) was adjusted to pH 7.4, and used for protein binding experiments. Goat anti-human IgG was purchased from Sigma and labeled with both biotin-XX and Alexa 488. Fifty microliters of a solution of biotin-XX-ester (10 mg/mL) and Alexa 488 ester (10 mg/mL) in DMF were added to a solution of goat anti-human IgG (1 mg) in 0.5 mL of 0.1 M sodium bicarbonate (pH 8.3). After incubation for 1 h with stirring at room temperature, unreacted biotinXX-ester and Alexa 488 ester were inactivated by adding hydroxylamine, and separated from labeled IgG by gel filtration. The final labeling mole ratio of Alexa 488 to IgG was 4, determined by the absorption at 494 nm for Alexa 488 ( ) 71 000 M-1‚cm-1) and at 280 nm for IgG ( ) 203 000 M-1‚cm-1). 2.2. Preparation of Gold Substrates. Gold substrate (12 × 12 mm) for SPR, prepared by sputter deposition of an adhesion layer of chromium (1 nm) and gold (45 nm) onto glass coverslips, were obtained from Texas Instruments Inc. Gold substrates for ellipsometry were prepared in-house by thermal evaporation of an adhesion layer of chromium (3 nm) followed by gold (200 nm) at a pressure of 5 × 10-7 Torr. 2.3. Preparation of MeNPOC-Biotin Derivatized SAMs. The gold substrate was cleaned in Piranha solutionsa mixture of 30% H2O2 and 70% H2SO4 (v/v) at 80 °C for 10 min, and subsequently in a 5:1:1 (v/v) mixture of H2O, H2O2, and NH3 at 80 °C for 10 min. The mixed SAMs of MUOH and MHA were prepared by immersing a clean gold substrate into an ethanol solution of 0.1 mM MHA and 0.9 mM MUOH (unless stated otherwise) overnight (>16 h) at room temperature. After formation of mixed SAMs, the samples were rinsed in ethanol, followed by ultrasonication in ethanol for 10 min, and dried under a stream of nitrogen. The mixed MUOH/MHA SAMs were immersed in a DMF solution of 0.1 M EDAC and 0.2 M PFP for 20 min, rinsed with ethanol, dried under a stream of nitrogen, and immersed in an ethanol solution containing 10 mM DADOO for 20 min. The substrate was rinsed with ethanol and dried under a stream of nitrogen again. Subsequently, the substrate was covered with a solution of MeNPOC-biotin succinimidyl ester or D-biotin succinimidyl ester (5 mg/mL in DMF) for 2 h at room temperature. The substrate was rinsed with ethanol, dried under a stream of nitrogen, and stored in the dark prior to further use. 2.4. Ellipsometry. A manual null ellipsometer in PCSA configuration, built in-house, was used for ellipsometric measurement. A He-Ne laser (632.8 nm, Melles Griot) incident at an angle of 68.25° was used as the light source for intensity as well as for imaging ellipsometry. Polarizer angles were determined with a precision of 0.01° and the intensity was measured with a lock-in amplifier (Princeton Applied Research, Princeton, NJ). Ellipsometric constants of the substrate were measured before and after modification. The thicknesses of the SAMs were

Figure 2. Schematic representation of the protein patterning procedure onto MeNPOC-biotin-SAM on gold. (A) MeNPOCbiotin-SAM is irradiated with 350-360 nm UV light through a chrome-on-glass negative test target; (B) streptavidin is selectively bound to the irradiated regions. calculated using a parallel slab model24 with assumed refractive indices of 1.00 for air and 1.5 for the SAMs. For imaging ellipsometry, two lenses were inserted into the output beam and the detector was replaced with a CCD camera with fixed gain and black level (Dage-MTI). Images were recorded with a VCR for subsequent image processing. Because the sample under observation was tilted at an angle of 68.25° with respect to normal, only vertical stripes were in focus and the resolution was significantly different in the vertical and horizontal directions. The true magnification was about 9× in the vertical direction. 2.5. Contact Angle Goniometry. Liquid drop contact angles were determined with a goniometer (Model 100, Rame´-Hart Inc., Mountain Lakes, NJ) at room temperature and ambient relative humidity using water as the probe liquid. Static contact angles (θ) were measured by pipetting a 5 µL drop onto the surface. For each surface, both sides of two drops were measured (four data points total) and averaged to obtain θ. This procedure gave reproducible contact angles to within (1°. 2.6. Light-Activated Micropatterning of Proteins (LAMP). The MeNPOC-biotin derivatized SAMs were placed in a Petri dish containing 25 mM phosphate buffer solution (pH 7.4), 15 cm below the lamp housing (Figure 2). A 350 W Hg vapor ultraviolet lamp (ORIEL Model 66033) with a 3 in. diameter collimated beam passing through a 350-360 nm band-pass filter was used as the irradiation source. The photomask, a chrome-on-glass USAF negative test target (Melles Griot), consisting of lines ranging in width from 1 mm to 2.2 µm, was placed on the gold substrate, and exposed at an average power density of 10 ( 0.4 mW/cm2 in 0.1 M phosphate buffered solution (PBS, pH 7.4) for 20 min, unless stated otherwise. After UV irradiation, the substrates were rinsed with water, and dried under a stream of nitrogen. Substrates presenting a photodeprotected MeNPOCbiotin (biotin) pattern were incubated either in the HBS/BSA/ P20 containing streptavidin (1 µM), or Alexa 488 conjugated anti-biotin (0.7 µM) for 1 h, or sequentially in HBS/BSA/Tween 20 containing streptavidin (1 µM), and biotin-IgG-Alexa 488 conjugate (0.1 µM) for 1 h, respectively. Subsequently, the substrates were rinsed thoroughly with HBS buffer. The substrates presenting streptavidin and Alexa 488 labeled protein micropatterns were imaged on an ellipsometer and a confocal microscope, respectively. 2.7. Confocal Microscopy. The substrate presenting Alexa 488 labeled protein micropatterns were placed on a cover slip (24) Azzam, R. M. A.; Bashara, N. M. Ellipsometry and Polarized Light; Elsevier Science Publishers: North-Holland, The Netherlands, 1977.

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Figure 3. Characterization of mixed MUOH/MHA SAMs as a function of fMHA (mole fraction of MHA in solution): (A) thickness; (B) static contact angle of water. and incubated in HBS/BSA/Tween 20 buffer. Selected regions of the substrate were imaged using a Zeiss LSM 510 confocal microscope (Carl Zeiss Inc., Englewood, CO) with 10× and 63× objectives. 2.8. Surface Plasmon Resonance Measurements. The BIACORE X instrument (Biacore AB, Sweden) was used for SPR studies. Typically, the substrate, a biotin (or photodeprotected MeNPOC-biotin) derivatized SAM on a gold-coated glass cover slip was mounted in an empty BIACORE sensor cartridge using water-insoluble, double-sided sticky tape (3M). The sensor cartridge was docked into the BIACORE X instrument, and checked for the absence of leaks and baseline drift. SPR measurement of streptavidin binding to the biotin (or photodeprotected MeNPOC-biotin) derivatized SAMs was carried out by injecting 50 nM streptavidin, or biotin-saturated streptavidin, in HBS buffer at a constant flow rate of 10 µL/min for 8 min.

3. Results and Discussion 3.1. Optimization of MeNPOC-biotin Derivatized SAMs. The formation of a mixed SAM of MUOH/MHA on gold was the first step in creating MeNPOC-biotin derivatized SAMs. MHA and MUOH were selected to create a mixed, reactive SAM on gold, because the terminal carboxylic acid group in MHA can be activated to directly conjugate proteins by their primary amines or through a bifunctional carboxy-reactive linker, and because the terminal hydroxyl moiety in SAMs of MUOH is known to reduce nonspecific adsorption of proteins.6 Although MUOH and MHA have been used previously to prepare pure SAMs,25 and mixed SAMs of MUOH or MHA with other alkanethiols26 have also been previously reported, to our knowledge, a mixed SAM of MUOH and MHA has not been reported in the literature. Figure 3A shows the thickness of mixed SAMs of MUOH and MHA measured by ellipsometry as a function of mole fraction of MHA in solution (fMHA). The thicknesses of pure (25) Bain, C. D.; Troughton, E. B.; Tao, Y. T.; Evall, J.; Whitesides, G. M.; Nuzzo, R. G. J. Am. Chem. Soc. 1989, 111, 321-335. (26) Bain, C. D.; Evall, J.; Whitesides, G. M. J. Am. Chem. Soc. 1989, 111, 7155-7164.

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Figure 4. (A) SPR sensorgrams of the binding of streptavidin (50 nM in HBS buffer, pH 7.4, flow rate: 10 µL/min) to biotinSAMs with fMHA: (1) 0.02; (2) 0.05; (3) 0.10; (4) 0.25, (5) 0.50, curve (6) is a control, DADOO-derivatized SAM with fMHA ) 0.10; (B) the maximum binding of streptavidin as a function of fMHA to (1) control, biotin derivatized SAMs, and (2) photodeprotected MeNPOC-biotin-SAMs (irradiation time ) 20 min).

SAMs of MUOH and MHA are 1.3 and 2.3 nm, respectively, consistent with previously reported values.25 The linear change in thickness of the mixed SAM, as a function of increasing fMHA, indicates an increase in mole fraction of MHA in the mixed SAM. Figure 3B shows the static contact angle of water on SAMs prepared from mixtures of MUOH and MHA with different fMHA. The pure SAMs of MUOH and MHA show wetting behavior with water (static contact angle: θ(H2O) ) 7° and 15°, respectively). The static contact angles of water for the mixed SAMs are higher than for the pure SAMs and reach a maximum at fMHA ) 0.4. The higher contact angle on the mixed SAM suggests disorder and possible exposure of nonpolar methylene groups in the terminal region of MHA.27 After formation of the mixed SAM, the terminal carboxylic acid groups were activated to pentafluorophenyl ester groups (Figure 1b) using PFP. PFP was chosen to convert the terminal carboxylic acid groups in the SAM to reactive esters in preference to the more commonly used N-hydroxysuccinimidyl ester (NHS ester) because pentafluorophenyl esters are more reactive than NHS ester.28,29 Next, the reactive ester on the mixed SAM was conjugated with a diamine linker, DADOO. DADOO was selected as a linker to couple the SAM with MeNPOCbiotin for two reasons. First, the ∼12 Å chain-length of DADOO presents the biotin ligand at an additional distance from the surface, making it more accessible to the biotin-binding site in streptavidin. This is because the biotin-binding site of streptavidin is buried ∼14 Å (27) Bain, C. D.; Whitesides, G. M. J. Am. Chem. Soc. 1989, 111, 7164-7175. (28) Adamczyk, M.; Fishpaugh, J. R.; Mattingly, P. G. Tetrahedron Lett. 1995, 36, 8345-8346. (29) Kovacs, J.; Mayers, G. L.; Johnson, R. H.; Cover, R. E.; Ghatak, U. R. J. Org. Chem. 1970, 35, 1810-1815.

Light-Activated Pattern of Proteins on SAM/Au

Figure 5. (A) SPR sensorgrams of the binding of streptavidin (50 nM in HBS buffer, pH 7.4, flow rate: 10 µL/min) to photodeprotected MeNPOC-biotin-SAMs, which were irradiated for different times: (1) 0 min; (2) 1 min; (3) 2 min; (4) 4 min; (5) 8 min; (6) 16 min; and (7) 32 min. Sensorgram (8) is an SPR sensorgram of the binding of biotin-saturated streptavidin to MeNPOC-biotin-SAM. (B) Streptavidin binding at steady state to reconstituted biotin on the photodeprotected MeNPOC-biotinSAMs as a function of irradiation time.

from the solvent accessible surface of the protein,30 and previous studies have shown that optimizing the chain length of a linker attached to the valeric acid tail of biotin promotes the binding of streptavidin to immobilized biotin.31 Second, DADOO contains an (EG)2 moiety which is known to suppress nonspecific protein adsorption.32 In the final step, MeNPOC-biotin succinimidyl ester was coupled to the terminal amine group in the DADOOderivatized SAMs, resulting in MeNPOC-biotin derivatized SAMs (MeNPOC-biotin-SAMs). To examine the coupling of MeNPOC-biotin succinimidyl ester to the mixed MUOH/MHA SAMs, a control monolayer was similarly prepared by reacting biotin succinimidyl ester with the DADOO-derivatized SAMs, instead of MeNPOC-biotin succinimidyl ester (Figure 1). The chemical structure of our biotin-derivatized SAM prepared by multistep derivatization is identical to the mixed biotin/OH-SAMs formed directly by self-assembly from a solution of biotin- and OH-terminated alkanethiols, previously reported by Knoll and co-workers.6 Therefore, comparing streptavidin binding to our biotin-derivatized SAMs with those of Knoll et al. allows direct comparison of the density of the terminal biotin moiety in each SAM and thereby allows the overall efficiency of our derivatization scheme to be inferred. Streptavidin binding to the biotin-derivatized SAMs was examined by surface plasmon resonance spectroscopy (SPR). Figure 4A shows (30) Savage, M. D.; Mattson, G.; Desai, S.; Nielander, G.; Morgensen, S.; Conklin, E. Avidin-Biotin: A Handbook; Pierce Chemical Company: Rockford, IL, 1992. (31) Haugland, R. P.; You, W. W. Methods Mol. Biol. 1995, 45, 12333. (32) Prime, K. L.; Whitesides, G. M. Science 1991, 252, 1164-1167.

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SPR response curves (“sensorgrams”) of streptavidin binding to the biotin-derivatized SAMs as a function of fMHA. The amount of streptavidin bound at steady state displays a maximum for the monolayer with fMHA ) 0.1 and decreases for fMHA > 0.25 (Figure 4B, curve 1). These results are consistent with the previously reported maximum binding of streptavidin to the mixed biotin/ OH-SAM formed directly from solution with fbiotin ) 0.1 (mole fraction of the biotin-terminated alkanethiol).6 These results suggest that the optimal surface density of biotin ligand is obtained for fMHA ) 0.1 for the biotin-derivatized SAMs, and that a higher density of biotin in the biotinderivatized SAM sterically hinders the binding of streptavidin.6 Furthermore, the thickness of the bound streptavidin monolayer on our biotin-derivatized SAM with fMHA ) 0.1 was 2.9 nm, while a thickness of 3.8 nm was reported for the bound streptavidin monolayer on the mixed biotin/ OH-SAM (fbiotin ) 0.1) formed by self-assembly from solution.6 The thickness results indicate that the surface concentration of biotin in the biotin-derivatized SAM reported here is ∼80% of the biotin concentration on the mixed biotin/OH-SAM created by direct self-assembly from solution. We also examined the specificity of streptavidin binding to the mixed SAMs. Figure 4A (SPR sensorgram 6) shows no significant streptavidin binding to a control DADOO-derivatized SAM (fMHA ) 0.1). This result also confirmed that the OH-terminated alkanethiol, combined with the use of a DADOO linker, which contains an (EG)2 moiety, suppresses nonspecific binding of streptavidin. 3.2. Streptavidin Binding to MeNPOC-biotin Derivatized SAMs. Our objective in protein patterning was to maximize the binding of streptavidin to irradiated regions, which present a biotin ligand to achieve high signal, and to simultaneously minimize nonspecific binding of streptavidin to regions that were not irradiated, which present a MeNPOC-protected biotin ligand to achieve a high signal-to-noise ratio. Two variables were optimized to achieve this objective: (1) fMHA: the mole fraction of MHA to MUOH in solution, which determines the optimal concentration of MeNPOC-protected biotin; and (2) irradiation time, which controls the conversion of MeNPOC-protected biotin to active ligand. We examined the effect of fMHA upon streptavidin binding to photodeprotected MeNPOC-biotin-SAMs. MeNPOCbiotin succinimidyl ester was conjugated to DADOOmodified SAMs prepared with fMHA varying from 0 to 0.5, and the MeNPOC-biotin-SAMs were then irradiated with UV light for 20 min, thereby removing the MeNPOC moiety from the MeNPOC-biotin-SAMs. The amount of streptavidin bound at steady state determined by SPR (Figure 4B, curve 2), as a function of fMHA, is similar to that observed for the control biotin-derivatized SAMs. In particular, maximum streptavidin binding is observed for fMHA ) 0.1, but for all values of fMHA, the amount of streptavidin bound at steady state to the photodeprotected MeNPOC-biotin-SAMs is approximately half of that bound to the biotin-derivatized SAMs (Figure 4B, curve 1). There are at least two possible reasons for the decreased streptavidin binding to the photodeprotected MeNPOCbiotin SAMs relative to the control, biotin-derivatized SAMs: (1) UV deprotection of MeNPOC-biotin is incomplete even though it reaches a steady state for irradiation times exceeding 16 min (see below); (2) UV deprotection results in decreased affinity of photodeprotected biotin, possibly due to side reactions that modify the biotin headgroup. The cause of decreased binding of streptavidin to the photodeprotected MeNPOC-biotin-SAM (compared to the control, biotin-derivatized SAMs) is currently under

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Figure 6. Streptavidin bound to a photodeprotected MeNPOC-biotin-SAM pattern. The photomask features are: (5) 40 µm; (6) 35 µm. (A) Ellipsometric image of photodeprotected and protected regions. Contrast in this image is indicative of the different coverage of bound streptavidin in irradiated and nonirradiated regions of the SAM. Note that due to the shallow angle of incidence, the vertical direction in the image is compressed. (B) Confocal image of biotin-IgG-Alexa 488 conjugate bound to patterned streptavidin.

investigation by surface spectroscopic characterization of SAM formation, post-assembly derivatization, and UV deprotection. We next examined the effect of irradiation time on the amount of streptavidin bound, at a constant mole fraction of fMHA ) 0.1. MeNPOC-biotin-SAMs were irradiated as a function of time to monitor the activationsremoval of the MeNPOC moiety from the MeNPOC-biotin-SAMss by the ability of the SAM to bind streptavidin. SPR sensorgrams for streptavidin binding (Figure 5A, sensorgrams 1-7) showed that the amount of streptavidin bound to the MeNPOC-biotin-SAM increased with increasing irradiation time, and reached steady state at an irradiation time of 16 min. This result suggests that the terminal MeNPOC moiety on the mixed SAMs is gradually removed by photolysis,19 liberating biotin at the surface as a function of irradiation time. Because the amount of streptavidin bound at steady state converges for irradiation times >16 min (Figure 5B), we concluded that an irradiation time g20 min is sufficient to ensure maximal activation of the MeNPOC-biotin-SAM with fMHA ) 0.1. Streptavidin was not expected to bind to MeNPOCbiotin-SAMs because the MeNPOC moiety was designed

to block streptavidin binding.19 Significant streptavidin binding to the mixed MeNPOC-biotin-SAM is, however, observed by SPR with a fast dissociation rate (Figure 5A, sensorgram 1). To investigate the origin of this interaction, biotin-saturated streptavidin (holostreptavidin) was injected over the MeNPOC-biotin-SAM (Figure 5A, sensorgram 8). No binding between holostreptavidin and the MeNPOC-biotin-SAM was observed by SPR. The binding of apostreptavidin to MeNPOC-biotin-SAMs, but not of holostreptavidin, indicated that there is a weak, though specific interaction between the MeNPOC-moiety and the biotin-binding site in streptavidin. Tarlov et al.33 have reported that a large dose of UV irradiation (3 W/cm2, 1 h) in air destroyed alkanethiol SAMs on gold because alkanethiolates are photooxidized to the corresponding alkanesulfonates. To test the stability of our SAMs to UV irradiation at ∼355 nm typically used in this study, the MeNPOC-biotin-SAMs were examined by X-ray photoelectron spectroscopy (XPS) before and after UV irradiation under optimal deprotection conditions (10 (33) Tarlov, M. J.; Burgess, D. R. F., Jr.; Gillen, G. J. Am. Chem. Soc. 1993, 115, 5305-5306.

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Figure 7. (A) Confocal image of Alexa 488 labeled anti-biotin IgG bound on the photodeprotected MeNPOC-biotin-SAM pattern. (B) Ratio of feature spacing (s) to feature width (w) of protein patterns as a function of the photomask dimension (x).

mW/cm2 for 20 min in PBS, pH 7.4). No change was observed in the XPS S2p spectrum after UV irradiation, which indicated that the low UV exposure of the SAM did not result in oxidation of the thiolate (results not shown). Detailed XPS and time-of-flight secondary ion mass spectrometry (ToF-SIMS) characterization of SAM formation, derivatization, and UV-deprotection will be published elsewhere. 3.3. LAMP on MeNPOC-biotin Derivatized SAMs. A MeNPOC-biotin-SAM on gold (fMHA ) 0.1) was deprotected by UV irradiation for 20 min through a photomask

with feature sizes varying from 2 µm to 1 mm. After irradiation, the substrate, presenting a spatially resolved, reconstituted biotin pattern, was incubated with streptavidin (1 µM) for 1 h. Figure 6A shows an ellipsometric image of patterned streptavidin with a photomask feature of 40 and 35 µm (features 5 and 6 in Figure 6, respectively). Ellipsometric imaging is an attractive complement to fluorescence microscopy because it does not require fluorescently labeled protein to image the pattern. It also provides information on the coverage density of the protein micropattern, based on an effective refractive index or an

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effective thickness for the protein layer, by independently minimizing the intensity for patterned and background regions in the image. We determined an effective streptavidin thickness of 2.1 and 0.5 nm for the photodeprotected and protected regions, assuming a refractive index of 1.45 for a densely packed monolayer such as a two-dimensional protein crystal.34 The effective thickness of streptavidin bound to the photodeprotected MeNPOC-biotin-SAM is approximately half the thickness of streptavidin bound to the same biotin-derivatized SAM by direct self-assembly from solution.6 The effective thickness obtained by imaging ellipsometry are also consistent with the SPR results of streptavidin binding to each of these SAMs. We also independently examined the patterning of biotinylated proteins onto streptavidin micropatterns created by LAMP using fluorescence microscopy. A streptavidin pattern was incubated for 1 h with a 1 µM solution of biotin-IgG-Alexa 488 conjugate, washed with buffer, and imaged on a confocal microscope. The fluorescence image (Figure 6B) confirms that the streptavidin pattern created by LAMP can be subsequently used to immobilize antibodies on gold. This result also suggests that LAMP can be further extended to enable spatially resolved micropatterning of multiple biomolecules by repeated cycles of spatially defined activation, streptavidin incubation, followed by binding of the biotinylated biomolecule of interest. 3.4. Resolution of LAMP on MeNPOC-biotin Derivatized SAMs. The spatial resolution of LAMP was investigated by binding Alexa 488 labeled anti-biotin to a biotin pattern created by photodeprotection of a MeNPOC-biotin-SAM, using the USAF negative test target for front masking. Figure 7A shows a 10× magnification confocal image, which demonstrates selective binding of anti-biotin to regions of MeNPOC-biotin that were deprotected by UV irradiation. The contrast ratio (ratio of signal in the bright bars to background) in this image is 5, which is consistent with the contrast ratio independently determined by imaging ellipsometry. We also observed that the UV irradiated, patterned features were broader than the features shadowed by the photomask. We believe that feature broadening is caused by the combined effect of divergence of the collimated incident light passing through the 2 mm thick photomask, diffraction at the feature borders, and by reflection between mask and gold substrate. Figure 7B shows the ratio of observed antibody feature spacing (s) to the observed antibody feature width (w), as obtained from 10× and 63× (not shown here) magnification fluorescence images, as a function of the (34) Darst, S. A.; Ahlers, M.; Meller, P. H.; Kubalek, E. W.; Blankenburg, R.; Ribi, H. O.; Ringsdorf, H.; Kornberg, R. D. Biophys. J. 1991, 59, 387-396.

Yang et al.

feature size of the photomask (x). The results in Figure 7B indicate that the anti-biotin pattern exhibits significant broadening when the photomask dimensions are e6 µm, resulting in a minimum feature resolution of ∼6 µm (s/w ) 0.5). The resolution of the protein pattern is significantly improved by the use of front masking in our implementation of LAMP as compared to the resolution obtained for backside masking in previous studies,18,20 presumably because light scattering through the transparent substrate in backside masking, which degrades resolution, is circumvented by the use of front masking. 4. Conclusions We have described a convenient methodology to generate protein micropatterns onto SAMs on gold, a method we term light-activated affinity micropatterning (LAMP). This methodology, which involves stepwise surface derivatization to couple a photolabile, biomolecular ligand to a reactive SAM on gold, has several attractive features. First, with the exception of the caged biotin derivative, it uses commercially available organic thiols, bifunctional linkers, and reactive biotin derivatives, and therefore minimizes organic synthesis. Second, it involves stepwise surface modification, which provides considerable flexibility in its implementation. For example, the types of terminal functional groups in the SAM can be specified by the choice of the two organic thiols that are used in the initial self-assembly on gold, and their concentration can also be carefully controlled from solution. Also, the use of different bifunctional linkers with different structures and chain lengths (we used only one linker, DADOO in this study, though others may be substituted) provides additional flexibility in optimizing the chemistry of the derivatized SAM. Third, UV deprotection of the photolabile caged biotin ligand through a photomask allows spatially resolved deprotection of biotin, which permits different biotinylated proteins or other molecules to be patterned on the same substrate, mediated by a homotetramer streptavidin adapter. Acknowledgment. This research was supported by North Carolina Biotechnology Center, grant 9705-ARG0026. We thank F. Guilak (Department of Orthopedics, Duke University Medical Center) for the use of the laser scanning confocal microscope and B. Neilsen for providing technical assistance in its use. We also thank G.P. Lopez (University New Mexico, Albuquerque) for sharing his results on the coupling of DADOO to silane-modified glass prior to publication, and A. Liebmann-Vinson and H. W. Sugg (Becton Dickinson Research Center, Research Triangle Park, NC) for the use of their XPS facility. LA9908079