Microstamping on an Activated Polymer Surface: Patterning Biotin and

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Microstamping on an Activated Polymer Surface: Patterning Biotin and Streptavidin onto Common Polymeric Biomaterials Jinho Hyun,† Yingjie Zhu,‡ Andrea Liebmann-Vinson,§ Thomas P. Beebe, Jr.,‡ and Ashutosh Chilkoti*,† Department of Biomedical Engineering, Duke University, Durham, North Carolina 27708-0281, Department of Chemistry, University of Utah, Salt Lake City, Utah 84112, and Becton-Dickinson Technologies, 21 Davis Drive, P.O. Box 12016, Research Triangle Park, North Carolina 27709-2016 Received May 9, 2001. In Final Form: July 23, 2001 Microstamping on an activated polymer surface (MAPS) is a methodology that enables biomolecules to be patterned on polymers with micrometer spatial resolution. MAPS combines homogeneous surface derivatization of a polymer to introduce a reactive functional group followed by reactive microcontact printing (µCP) of a biological ligand of interest, linked to an appropriate reactive group. We demonstrate here that polyethylene, polystyrene, poly(methyl methacrylate), and poly(ethylene terephthalate) films can be successfully modified to introduce COOH groups on their surfaces, which can be subsequently patterned by reactive µCP of amine-terminated biotin after derivatization of the COOH groups with pentafluorophenol. X-ray photoelectron spectroscopy and time-of-flight secondary ion mass spectrometry (TOF-SIMS) confirmed the chemistry of MAPS at each stage of the derivatization of the polymer surfaces and reactive µCP of biotin. Micropatterned biotin surfaces fabricated by MAPS were patterned with streptavidin by exploiting molecular recognition between biotin and streptavidin. The formation of streptavidin patterns was examined by fluorescence microscopy of Alexa488-labeled streptavidin and by TOF-SIMS imaging of 15N-labeled recombinant streptavidin, bound to biotin patterns. The contrast in the streptavidin micropatterns was optimized by examining the effect of blocking agents and streptavidin incubation time. Maximum contrast was obtained for binding of 0.1 µM streptavidin from a buffer containing 0.02% (v/v) Tween 20 detergent for an incubation time of 1 min.

1. Introduction The ability to spatially pattern biomolecules on surfaces has become increasingly important for the development of cellular biosensors, biomaterials, and genomic arrays.1,2 An ensemble of techniques, collectively termed “soft lithography”,3 and complementary photolithographic4-8 techniques have been developed to pattern molecules on surfaces. In particular, microcontact printing (µCP), a soft lithographic technique, is an attractive method to pattern biological molecules because of its simplicity and flexibility, but its use has largely been restricted to self-assembled monolayers (SAMs) on gold or silicon.9 In µCP, an * To whom correspondence should be addressed. Tel: (919) 6605373. Fax: (919) 660-5362. E-mail: [email protected]. † Duke University. ‡ University of Utah. § Becton-Dickinson Technologies. (1) Blawas, A. S.; Reichert, W. M. Biomaterials 1998, 19, 595-609. (2) Mrksich, M.; Whitesides, G. M. Trends Biotechnol. 1995, 13, 228235. (3) Kumar, A.; Abbott, N.; Kim, E.; Biebuyck, H.; Whitesides, G. Acc. Chem. Res. 1995, 28, 219. Xia, Y.; Whitesides, G. M. Angew. Chem., Int. Ed. 1998, 37, 550-575. (4) Mooney, J. F.; Hunt, A. J.; McIntosh, J. R.; Librerko, C. A.; Walba, D. M.; Rogers, C. T. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 1228712291. (5) Wybourne, M. N.; Yan, M.; Keana, J. K. W.; Wu, J. C. Nanotechnology 1996, 7, 302-305. (6) Hengsakul, M.; Cass, A. E. G. Bioconjugate Chem. 1996, 7, 249254. (7) Schwarz, A.; Rossier, J. S.; Roulet, E.; Mermod, N.; Roberts, M. A.; Girault, H. H. Langmuir 1998, 14, 5526-5531. (8) Dewez, J. L.; Lhoest, J. B.; Detrait, E.; Berger, V.; Dupont-Gillain, C. C.; Vincent, L. M.; Schneider, Y. J.; Bertrand, P.; Rouxhet, P. G. Biomaterials 1998, 19, 1441-1445. (9) Lahiri, J.; Ostuni, E.; Whitesides, G. M. Langmuir 1999, 15, 20552060.

elastomer, typically poly(dimethylsiloxane) (PDMS) is cast against a microfabricated silicon or photoresist master, which has micrometer size features etched on the surface in contact with the PDMS.10 Curing the prepolymer and peeling it away from the master provides an elastomeric stamp, which contains a negative image of the features etched on the master. The stamp is then inked with the solution to be printed and pressed into contact with the substrate. The ink is transferred only from the raised areas on the stamp that come into contact with the substrate, thereby creating a spatially resolved pattern on the substrate. SAMs formed on gold or silicon are relatively easily patterned with biological ligands using µCP and other soft lithography techniques.3 These model systems are useful to interrogate biomolecular and cellular interactions on surfaces11 and also have technological utility in optical biosensing.12 There are many other applications, however, for which SAMs are less suitable substrates, such as for the fabrication of biomaterials. We are interested in patterning biological molecules on commonly used poly(10) Clarson, S. J.; Semlyen, J. A. Siloxane Polymers; Prentice Hall: Englewood Cliffs, NJ, 1993. (11) Singhvi, R.; Kumar, A.; Lopez, G. P.; Stephanopoulos, G. P.; Wang, D. I. C.; Whitesides, G. M.; Ingber, D. E. Science 1994, 264, 696-698. Mrksich, M.; Chen, C. S.; Xia, Y.; Dike, L. E.; Ingber, D. E.; Whitesides, G. M. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 10775-10778. Chen, C. S.; Mrksich, M.; Huang, S.; Whitesides, G. M.; Ingber, D. E. Science 1997, 276, 1425-1428. Patel, N.; Bhandari, R.; Shakesheff, K. M.; Cannizzaro, S. M.; Davies, M. C.; Langer, R.; Roberts, C. J.; Tendler, S. J. B.; Williams, P. M. J. Biomater. Sci., Polym. Ed. 2000, 11, 319331. (12) Mrksich, M.; Sigal, G. B.; Whitesides, G. M. Langmuir 1995, 11, 4383-4385. Kajikawa, K.; Hara, M.; Sasabe, H.; Knoll, W. Jpn. J. Appl. Phys., Part 2 1997, 36, L1116-L1119.

10.1021/la010695x CCC: $20.00 © 2001 American Chemical Society Published on Web 09/08/2001

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Figure 1. (A) Schematic of MAPS. (B) Chemical surface modification of PE, PS, PMMA, and PET to introduce COOH groups.

mers because of their wide use in cell culture and as biomaterials. To this end, we recently developed a new method, microstamping onto an activated polymer surface (MAPS), which enables polymer surfaces to be patterned with biological ligands and proteins with micrometer lateral resolution.13,14 A schematic of the steps in MAPS is shown in Figure 1A. In previous studies, we have shown that MAPS can be used to pattern a reactive biotin derivative onto carboxylic acid derivatized poly(ethylene terephthalate) (PET) with micrometer lateral resolution13,14 and subsequently with streptavidin, mediated by the high-affinity streptavidinbiotin interaction.15 This paper follows from our previous studies and has the following objectives. First, we wished to demonstrate that MAPS is not restricted to PET but can also be used to pattern biological ligands on a number of other polymeric biomaterials. In this study, we therefore examined the feasibility of creating biotin and streptavidin micropatterns on polyethylene (PE), polystyrene (PS), poly(methyl methacrylate) (PMMA), and PET, polymers that are commonly used as biomaterials and in tissue culture. The second and related objective of this paper was to optimize key steps in MAPS to achieve maximum contrast in the streptavidin patterns, with minimal binding to the background. The extension of these studies to the patterning of a cell-adhesive peptide and the (13) Yang, Z. P.; Belu, A. M.; Liebmann-Vinson, A.; Sugg, H.; Chilkoti, A. Langmuir 2000, 16, 7482-7492. (14) Yang, Z. P.; Chilkoti, A. Adv. Mater. 2000, 12, 413-417. (15) Wilchek, M.; Bayer, E. A. Avidin-Biotin Technology; Methods in Enzymology Vol. 184; Academic Press: San Diego, CA, 1990.

interaction of the peptide patterns with mammalian cells will be reported elsewhere. 2. Experimental Section 2.1. Surface Chemical Modification of Polymers. The surface derivatization reactions for each polymer are shown in Figure 1B. Experimental details for each reaction can be found in the Supporting Information of this paper. 2.2. µCP of Biotin-Amine. The fabrication of elastomeric stamps with micrometer-size relief features has been described previously,14,16-18 as has the use of MAPS to µCP EZ-Link biotinPEO-LC-amine ((+)-biotinyl-3,6,9-trioxaundecanediamine) (biotin-amine) onto polymers.13,14 2.3. Patterning of Streptavidin. After printing biotinamine onto the activated polymers with MAPS, the surfaces of the polymers were incubated with 0.1 µM Alexa488-labeled streptavidin or with 15N-labeled recombinant streptavidin in HEPES buffered saline (HBS, pH 7.4). Details on the synthesis and purification of 15N-labeled streptavidin have been reported previously.19 The Alexa488-to-streptavidin molar ratio was ∼3.8, as determined by UV-visible spectrophotometry (Shimadzu, UV1601, Columbia, MD). The solution concentration of streptavidin was 0.1 µM, which was chosen based on previous studies.13,14 The incubation time was systematically varied for the optimization experiments. Unless otherwise noted, the buffer for streptavidin contained 0.02% (v/v) Tween 20 detergent. When bovine (16) Wilbur, J. L.; Kumar, A.; Kim, E.; Whitesides, G. M. Adv. Mater. 1994, 6, 600-604. (17) Kumar, A.; Whitesides, G. M. Appl. Phys. Lett. 1993, 63, 20022004. (18) Xia, Y.; Kim, E.; Zhao, X. M.; Rogers, J. A.; Prentiss, M.; Whitesides, G. M. Science 1996, 273, 347-349. (19) Belu, A. M.; Yang, Z. P.; Aslami, R.; Chilkoti, A. Anal. Chem. 2001, 73, 143-150.

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serum albumin (BSA) was added to the streptavidin solution, its concentration was 0.1% (w/v). 2.4. Fluorescence Microscopy and Surface Characterization. Fluorescence images of Alexa488-labeled streptavidin patterns were acquired on a confocal microscope (Carl Zeiss LSM 510, Thornwood, NY). X-ray photoelectron spectroscopy (XPS) was performed on a SSX-100 spectrometer (Surface Science Inc., Mountain View, CA). Survey scan spectra were acquired from 0 to 1000 eV to determine the elemental composition by XPS. Time-of-flight secondary ion mass spectrometry (TOF-SIMS) spectra and ion images were obtained on a TOF-SIMS IV instrument (ION-TOF, Mu¨nster, Germany). Details of the fluorescence microscopy, XPS, and TOF-SIMS are in the Supporting Information. 2.5. Image Analysis. For images acquired by fluorescence microscopy and imaging TOF-SIMS, the contrast is defined as the intensity ratio of pixels within the patterned region to that in the background area, Ipattern/Ibackground. For TOF-SIMS images, the mass spectra from these areas were reconstructed from the raw data, followed by normalization of intensity to the primary ion dose density and the area from which the data were acquired. To quantify the intensities in the pattern and background to compute the contrast for the fluorescence and TOF-SIMS images, histograms of pixel intensities were generated for each image. A bimodal distribution was obtained in all cases, where the mean intensity for the distribution at lower intensity corresponded to the background and the mean of the distribution at higher intensity corresponded to the patterned regions. This procedure used the data from every pixel in each image, as compared to the more typical use of cursor profiles of intensity where only the pixel intensities on that line are utilized to compute contrast. The error bars for both fluorescence images and TOF-SIMS images were calculated by Gaussian fitting of pixel intensity histograms of corresponding images as σ/(Npixels)1/2, where Npixels is the number of pixels in the fitted Gaussian mode corresponding to the peak or background and σ is the fitted standard deviation of the peak or background mode. The value of N in each image is typically on the order of 5 × 103 to 1 × 104. Errors in the contrast were not calculated in this manner for some images for which the intensities were low and histograms did not exhibit a clear Gaussian form; in these cases, the contrast is reported with no error bar and is estimated to be (10% of the reported value. The results were quantitatively compared using an unpaired t-test.

3. Results and Discussion 3.1. Overview of MAPS. In our implementation of protein patterning by MAPS, initial patterning of the surface is achieved by reactive µCP of biotin onto a derivatized polymer surface (Figure 1A), followed by the spatially selective formation of streptavidin micropatterns, driven by the high-affinity streptavidin-biotin interaction, as follows: first, each polymer was derivatized to introduce COOH groups in the surface region of the polymer (Figure 1B). We chose PE, PS, PMMA, and PET as candidate polymers for MAPS because these polymers and their derivatives are commonly used as biomaterials and in tissue culture.20 We chose to introduce carboxylic acid groups at the surface of these polymers because they are a convenient functional group for conjugation to a wide variety of biomolecules. After derivatization of each polymer to introduce COOH groups (the derivatized polymers are termed polymer-COOH for brevity, e.g., PE-COOH), the COOH groups were converted to reactive pentafluorophenyl esters by reaction with pentafluorophenol (PFP) and 1-ethyl-3-(dimethylamino)propylcarbodiimide (EDAC) from solution.21,22 These “activated” (20) Ratner, B. D. Biomaterials science: an introduction to materials in medicine; Academic Press: San Diego, CA, 1996. (21) Adamczyk, M.; Fishpaugh, J. R.; Mattingly, P. G. Tetrahedron Lett. 1995, 36, 8345-8346. (22) Kovacs, J.; Mayers, G. L.; Johnson, R. H.; Cover, R. E.; Ghatak, U. R. J. Org. Chem. 1970, 35, 1810-1815.

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Figure 2. XPS survey scans (0-1000 eV) of PE at different stages of MAPS: (A) unmodified PE, (B) carboxylated PE (PECOOH), (C) PFP-derivatized PE-COOH (PE-PFP), (D) aminebiotin-derivatized PE-PFP (PE-biotin). Spectra are offset by an appropriate constant for clarity.

surfaces were then brought into conformal contact with an oxidized PDMS stamp presenting micrometer size relief features, inked with an amine-linked biotin derivative (termed biotin-amine) to micropattern the surface with biotin through the formation of an amide linkage;13 the biotin-modified surfaces are termed polymer-biotin (e.g., PE-biotin, PS-biotin). The surface was then washed with buffer, and residual pentafluorophenyl esters that did not come into conformal contact with biotin-amine were deactivated by reaction with 2-aminoethoxyethanol (AEE) followed by hydrolysis of any remaining reactive esters. Next, the surface was incubated with a solution of Alexa488-labeled streptavidin for fluorescence microscopy or with 15N-labeled streptavidin for TOF-SIMS. Patterning biotin and streptavidin on polymer surfaces by MAPS provides a generic method to pattern other biomolecules of interest. This is because streptavidin, by virtue of its homotetrameric structure and dyad-related symmetry, functions as a biomolecular adapter in MAPS. The attachment of streptavidin to surface-immobilized biotin leaves two biotin-binding sites unoccupied on the solution-exposed face of the protein, which can be used to subsequently pattern other molecules that are conjugated to biotin. This modular patterning scheme is attractive because it affords a high degree of flexibility stemming from an ever-increasing supply of biotin-linked reagents. 3.2. XPS Characterization of Polymer Modification. Each step of polymer surface derivatization in MAPS was monitored by XPS (Figure 2). Previously, we had shown that the coupling of biotin-amine to an activated PET surface is similar for reaction from solution or by transfer from a flat (i.e., featureless) PDMS stamp inked with biotin-amine. Thus, due to the ∼1 mm2 footprint of the X-ray beam on the sample surface, we substituted µCP of the biotin-amine with reaction from solution to create a homogeneously covered surface suitable for XPS analysis. The elemental analysis results obtained for the derivatized polymer surfaces by XPS are summarized in Table 1. We calculated the yield for each stage of derivatization in Table 2 by comparing the observed atomic ratios with theoretical atomic ratios. The theoretical ratios were calculated by assuming complete derivatization within the XPS sampling depth by the reaction schemes shown in Figure 1B and by ignoring exponential attenu-

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Table 1. Elemental Analysis by XPS of Each Step of the Surface Modification of PE, PS, PMMA, and PET in MAPS for Homogeneously Modified Surfacesa atomic percent (XPS) sample

C

O

PE PE-COOH PE-PFP PE-biotin PS PS-COOH PS-PFP PS-biotin PMMA PMMA-COOH PMMA-PFP PMMA-biotin PET PET-COOH PET-PFP PET-biotin

100 90.0 91.8 91.7 97.5 95.2 94.1 86.2 75.3 69.8 73.6 73.9 70.9 72.2 73.5 72.9

9.3 5.6 5.6 2.5 4.8 4.6 7.6 24.7 30.2 23.2 24.2 27.1 27.8 21.7 23.7

a

F

N

other Na: 0.7

2.7 2.7 1.3 1.3

Si: 4.5

1.4

1.3 1.5

Cl: 0.5 S: 0.5 Cl: 2.0

3.0 0.7

1.8 2.6

Errors are estimated to be less than ∼10% of the reported values.

Table 2. XPS Elemental Ratios Used to Determine Derivatization Yields for the Surface Modification in MAPSa sample

O/C

PE PE-COOH PE-PFP PE-biotin PS PS-COOH PS-PFP PS-biotin PMMA PMMA-COOH PMMA-PFP PMMA-biotin PET PET-COOH PET-PFP PET-biotin

0 (0) 0.10 (0.75)

F/O

N/O

yield [%]

0.48 (0.28)

13.9 28.9 83.7

0.17 (0.19)

18.7 17.2 89.8

0.06 (0.05)

2.4 100

0.11 (0.1)

14.0 100

0.48 (1.67) 0.03 (0) 0.05 (0.27) 0.29 (1.67) 0.33 (0.50) 0.43 0.06 (2.50) 0.38 (0.40) 0.39 0.14 (1.00)

a Values in parentheses are expected elemental ratios for 100% derivatization yield, ignoring exponential attenuation in the XPS sampling depth.

ation of core-level photoelectrons throughout the XPS sampling depth. Polyethylene. Upon oxidation and carboxylation of PE, oxygen was incorporated into the polymer surface, as indicated by the change in the O/C ratio from 0 for PE to 0.1 for PE-COOH (Table 2). For carboxylation of every repeat unit, an O/C ratio of 0.75 would have been expected; thus, the oxidation/carboxylation reaction proceeded with ∼14% yield, corresponding to a derivatization of ∼1 out of 13 repeat units. Activation of the carboxylated PE surface by reaction with PFP led to the introduction of fluorine, and the F/O ratio was used to determine the derivatization yield of 29%. Assuming that ∼1 of 13 PE repeat units were carboxylated, this suggests that ∼1 out of 3.5 PE-COOH repeat units were activated with PFP. Coupling of biotin-amine to the activated PE surface introduced a unique nitrogen peak in XPS, and the N/O ratio was used to estimate the biotinylation reaction yield. However, we experimentally observed a higher N/O ratio than we would have expected, based on the derivatization yields for PE-COOH and PE-PFP determined by XPS. There are three possible reasons for the higher than expected N/O ratio: first, low levels of adsorbed biotin that was not completely removed during the ethanol wash;

second, loss of the F labels after derivatization or during XPS analysis; third, attenuation of the deeper-lying oxygen signals due to the upper layers of adsorbed biotin, which presents its nitrogen-containing groups nearer the surface. Our XPS results do not allow us to discriminate between these possibilities, though the fluorescence microscopy and TOF-SIMS results indicate that biotin-amine does not show significant adsorption on underivatized PE under the experimental conditions used to fabricate the patterns. If we instead assume that the PFP activation reaction proceeded to 100% completion within the XPS sampling depth and thus every available carboxyl group in this volume was activated with PFP, we estimate that about 84% of the activated carboxyl groups reacted with biotinamine, based on the experimentally obtained N/O ratio. Polystyrene. Similar to PE, oxygen was introduced into the surface of PS upon oxidation and carboxylation. On the basis of the XPS O/C ratio of 0.05 for PS-COOH, a reaction yield of ∼19% is achieved, corresponding to carboxylation of ∼1 out of 5 PS repeat units. On the basis of the F/O ratio obtained for the PFP-activated PS-COOH surface, we estimate a ∼17% activation yield of carboxyl groups present on the polymer surface, corresponding to the activation of about 1 in 6 carboxyl groups previously introduced. The calculated elemental composition of this surface is in excellent agreement with the experimentally determined values, as listed in Table 2. On the basis of the N/O ratio, the biotin-amine coupling reaction with available COOH groups proceeds to ∼90% yield. Poly(methyl methacrylate). F was introduced as a new element upon derivatization of PMMA-COOH with PFP, corresponding to an experimentally measured XPS F/O ratio of 0.06. To determine the extent of carboxylation of PMMA, we compared the experimentally determined value with a theoretical F/O ratio, which assumed that the functionalization of PMMA proceeded to completion within the XPS sampling depth, by the reaction scheme shown in Figure 1A,B. In calculating the theoretical maximum F/O ratio, we explicitly assumed that (1) each PMMA repeat unit is functionalized with a carboxylic acid group and (2) activation of the COOH groups by PFP proceeds to completion. These assumptions yield a calculated F/O atomic ratio of 2.5. Experimentally, we measured an F/O ratio of 0.06 upon derivatization with PFP, which suggests that 1 COOH group was introduced in every 42 repeat units of PMMA. A theoretical maximum N/O ratio of 0.67 was similarly calculated for the reaction of the PFP-derivatized PMMA with biotin-amine, by assuming that each PMMA repeat unit within the XPS sampling depth reacts with biotinamine. Given that only 2.4% of the PMMA repeat units within the XPS sampling depth are functionalized with COOH groups, this theoretical maximum is reduced to 0.05. The theoretical atomic N/O ratio is close to the experimentally observed N/O ratio of 0.06 (Table 2). These results suggest that 1 biotin group is introduced every 30-40 repeat units of PMMA. Poly(ethylene terephthalate). An atomic F/O ratio of 0.14 was measured by XPS for PET-COOH derivatized with PFP. The theoretical maximum F/O ratio for PET-COOH derivatized with PFP was calculated based on the same assumptions as for PMMA. These assumptions yield a calculated F/O atomic ratio of 1.0. The F/O ratio measured experimentally by XPS was 0.14, which corresponds to 14% derivatization with PFP, and we therefore conclude that on average 1 COOH group was introduced in every 9 repeat units of PET. A theoretical maximum N/O ratio of 0.44 was calculated by assuming that each PET repeat unit within the XPS

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Figure 3. Fluorescence images and corresponding line intensity profiles of PE films incubated with 0.1 µM streptavidin with Tween 20 and BSA added to the streptavidin binding buffer. (A) PE-COOH micropatterned with biotin-amine. (B) unmodified PE control stamped with biotin-amine under same conditions as in (A). (C) PE-COOH micropatterned with biotinamine and incubated with 0.1 µM streptavidin, which had been previously incubated with 100 µM biotin in solution to block all available biotin binding sites in the protein.

sampling depth reacts with biotin-amine. Taking into account that only ∼14% of PET repeat units within the XPS sampling depth are functionalized with COOH groups, this theoretical maximum is reduced to 0.1. The theoretical atomic N/O ratio is experimentally indistinguishable from the experimentally observed N/O ratio (Table 2), suggesting that reaction of biotin-amine with the available pentafluorophenyl esters in PFP-derivatized PET-COOH proceeded to completion. Together, these results indicate that 1 biotin molecule is incorporated in every 9 PET repeat units. This result is in qualitative agreement with our previous observations using a different surface derivatization reaction to introduce COOH groups into PET, in which case we found that about 1 carboxyl group is introduced in every 5 repeat units of PET and that the reaction of biotin-amine with the available PFP groups proceeded to completion.14 3.3. Streptavidin Binding to Biotin Micropatterns. After patterning biotin on the activated polymer surfaces with a plasma-oxidized PDMS stamp which had relief features with a lateral dimension of 40 µm (squares or circles), the substrate was incubated with 0.1 µM Alexa488-labeled streptavidin in HBS (pH 7.4) containing 0.1% (w/v) BSA and 0.02% (v/v) Tween 20 detergent for 2 h. Confocal fluorescence microscopy was used to examine the formation of Alexa488-labeled streptavidin micropatterns on the surfaces. Figure 3A shows the formation of spatially resolved patterns of fluorescently labeled streptavidin on PE-biotin. In a control sample, where unmodified PE was microcontact printed with biotinamine, washed with ethanol and buffer, and incubated with Alexa488-labeled streptavidin, the surface showed only low levels of homogeneous, nonspecific adsorption of streptavidin (Figure 3B). These results suggested that the observed streptavidin patterns (Figure 3A) were due to covalently bound biotin in the regions that were contacted by the PDMS stamp and were not due to the spatially resolved transfer and noncovalent adsorption of biotin. To prove that the formation of streptavidin patterns was caused by molecular recognition of the surface-bound biotin by solution-phase streptavidin, we performed a

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Figure 4. TOF-SIMS high-resolution negative-ion mass spectra: (a) m/z ) 26 (C14N-) and (b) m/z ) 27 (C15N-) for PE-COOH activated by PFP and µCP with biotin-amine; (c) m/z ) 26 and (d) m/z ) 27 for PE-COOH activated by PFP and µCP with biotin-amine, followed by incubation in 15N-labeled streptavidin. Experimental conditions: Ga+ ion gun was operated in bunched mode; scan area, 384 × 384 µm2; 600 scans.

control experiment wherein PE-biotin was incubated with streptavidin presaturated by free biotin in solution. Fluorescence microscopy showed the absence of a streptavidin pattern (Figure 3C). This is because the biotinbinding sites in streptavidin were occupied by free biotin, so that the protein was unable to bind to the patterned biotin on the surface by specific affinity interactions (Figure 3C). This result clearly proves that the formation of streptavidin micropatterns occurs by molecular recognition between streptavidin and the patterned, covalently bound biotin on the polymer surface. We also characterized the micropatterning of biotinamine on PE by TOF-SIMS. We chose TOF-SIMS to complement fluorescence microscopy because it is a surface analytical technique with attributes that are extremely useful in analysis of the subtle chemical changes introduced in MAPS. TOF-SIMS provides high-resolution (m/ ∆m ∼ 10 000) mass spectra of the surface with high detection sensitivity. Because the secondary ions are formed within the top 1-2 monolayers of the solid surface, TOF-SIMS is exquisitely surface sensitive, which is critical for analyzing small chemical changes in MAPS that are confined to the surface of the derivatized polymer, where biomolecular recognition processes are expected to occur. TOF-SIMS ion imaging can also provide the spatial distribution of mass-resolved secondary ions emitted from the surface with submicron lateral resolution. In patterning by MAPS, streptavidin is used to selectively bind to the patterned biotin. We used 15N-labeled recombinant streptavidin, which had been synthesized by recombinant DNA techniques in Escherichia coli, to unambiguously discriminate streptavidin-specific fragments from other organic fragments arising from the polymeric substrate and biotin.19 TOF-SIMS of PE-COOH stamped with biotin detected a CN- ion (m/z ) 26) created from biotin with high intensity (Figure 4a). In comparison, C15N- (m/z ) 27) was almost undetectable due to the very small ratio (0.37%) of 15N in naturally occurring nitrogen (Figure 4b). The CN- ion, which primarily originates from biotin, was also detected by high-resolution TOF-SIMS of a biotin micropattern on PE-COOH incubated with 15Nstreptavidin (Figure 4c). In contrast with Figure 4b, the C15N- ion that is uniquely diagnostic of 15N-labeled streptavidin was detected with high intensity for PE-

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Figure 5. TOF-SIMS images of S- ion: (A) unmodified PE, flat-stamped with biotin-amine and washed extensively; (B) PECOOH activated by PFP and flat-stamped with biotin-amine; (C) PE-COOH activated by PFP and µCP with biotin-amine. Experimental conditions: Ga+ ion gun was operated in bunched mode; scan area, 384 × 384 µm2; 600 scans.

COOH stamped with biotin and incubated with 15Nstreptavidin (Figure 4d). The imaging mode of TOF-SIMS was used to analyze the patterned samples and monitor the spatial distribution of characteristic molecular species. For comparison, chemically unmodified PE and PE-COOH surfaces (both flat-stamped and µCP stamped) were chemically imaged by TOF-SIMS after stamping with biotin-amine. Figure 5 shows the S- ion (m/z ) 32) images of the three samples. The S- ion is characteristic of the presence of biotin. Figure 5A is the S- ion image of unmodified PE, homogeneously exposed to biotin-amine by transfer from a flat PDMS stamp. The intensity of the S- ion is very low and no spatial contrast is observed, implying that biotin-amine was not transferred to the PE surface, because of the lack of reactive groups on the surface. The nonzero intensity of the S- ion in this image may be caused by contamination or by a trace amount of biotin physisorbed on the surface. In Figure 5B, the S- ion image of PE-COOH activated with PFP/EDAC and homogeneously reacted with biotinamine by transfer from a flat (i.e., unpatterned) PDMS stamp, a homogeneous distribution and high intensity of the S- ion is observed, implying that biotin was homogeneously attached to the functionalized PE-COOH surface. Similarly, for PE-COOH micropatterned with biotin-amine using a stamp with 40 µm diameter circles (Figure 5C), the S- ion image shows a periodic circular pattern with relatively high contrast, indicating the presence of patterned biotin on the PE-COOH surface. The diameter of each feature is ∼40 µm, and the spacing between features is ∼50 µm. This result is consistent with that from fluorescence microscopy. The imperfection of some circular patterns (e.g., lower right-hand corner of Figure 5C) may be due to uneven force applied to the PDMS stamp during stamping, to uneven wetting of the stamp, or to imperfections in the stamp itself. While the S- ion image shows high contrast, other ions such as NH(m/z ) 16), CN- (m/z ) 26), and HS- (m/z ) 33) showed lower contrast (results not shown). 3.4. Optimization of Streptavidin Binding to Micropatterned Biotin. Because patterning of a biotinylated biomolecule of interest occurs on a patterned streptavidin template, the formation of optimized streptavidin patterns is a critical issue that needs to be addressed in the development of MAPS. We examined the use of blocking agents to reduce nonspecific adsorption of streptavidin to the background and the streptavidin

concentration and incubation time to optimize the contrast in the patterns, through a quantitative analysis of image contrast. Effect of Blocking Agents. Nonspecific adsorption, the indiscriminate adhesion of proteins to a surface due to weak attractive interactions or entropic interactions, is a critical issue in protein patterning. This is because nonspecific binding to the background region can reduce the sensitivity and dynamic range of a device (e.g., a multianalyte biosensor or a protein chip) or allow cellular adhesion to the background. Many different approaches have been used to reduce nonspecific adsorption to various surfaces. The adsorption of proteins such as BSA has been used to block other proteins from binding during immobilization of antibodies on surfaces.23-25 The attachment of poly(ethylene glycol) (PEG) groups to create proteinresistant, “nonfouling” surfaces is an especially effective method to reduce nonspecific adsorption. Whitesides et al. have shown that oligoethylene glycol functionalized SAMs on gold are especially effective in resisting nonspecific adsorption,26 while other groups have examined the use of longer chain PEGs27,28 and PEG block copolymers29-31 covalently immobilized on the surface of polymers. Although the attachment of PEG is a proven method to reduce nonspecific adsorption, it is not generally applicable to a diverse range of polymers without introducing further chemical modification steps prior to patterning. To minimize the number of processing steps in MAPS, we examined the effect of adsorbed Tween 20 detergent and (23) Pruslin, F. H.; To, S. E.; Winston, R.; Rodman, T. C. J. Immunol. Methods 1991, 137, 27-35. (24) Mohammad, K.; Esen, A. J. Immunol. Methods 1989, 117, 141145. (25) Vogt, R. F., Jr.; Phillips, D. L.; Henderson, L. O.; Whitfield, W.; Spierto, F. W. J. Immunol. Methods 1987, 101, 43-50. (26) Pale-Grosdemange, C.; Simon, E. S.; Prime, K. L.; Whitesides, G. M. J. Am. Chem. Soc. 1991, 113, 12-20. Prime, K. L.; Whitesides, G. M. Science 1991, 252, 1164-1167. Prime, K. L.; Whitesides, G. M. J. Am. Chem. Soc. 1993, 115, 10714-10721. (27) Singhvi, R.; Kumar, A.; Lopez, G. P.; Stephanopoulos, G. N.; Wang, D. I. C.; Whitesides, G. M.; Ingber, D. E. Science 1994, 264, 696-698. (28) Gombotz, W.; Guanghui, W.; Horbett, T.; Hoffman, A. J. Biomed. Mater. Res. 1991, 25, 1547-1562. (29) Neff, J. A.; Tresco, P. A.; Caldwell, K. D. Biomaterials 1999, 20, 2377-2393. (30) Neff, J. A.; Caldwell, K. D.; Tresco, P. A. J. Biomed. Mater. Res. 1998, 40, 511-519. (31) Li, J. T.; Carlsson, J.; Lin, J. N.; Caldwell, K. D. Bioconjugate Chem. 1996, 7, 592-599.

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Figure 6. Effect of blocking agents on spatial contrast of streptavidin. Biotin-amine was micropatterned on PFPactivated PE-COOH, incubated with 0.1 µM Alexa488-labeled streptavidin with added blocking agents, and imaged by fluorescence microscopy. (A) 0.02% (v/v) Tween 20 in buffer, contrast ) 3.419 ( 0.001; (B) 0.1% (w/v) BSA, contrast ) 2.386 ( 0.001; (C) 0.02% (v/v) Tween 20 and 0.1% (w/v) BSA, contrast ) 2.027 ( 0.002. The spatial contrast was defined as fluorescence intensity in the patterned regions (Ipattern) to that in the regions between them (Ibackground). (D) Histograms of frequency versus fluorescence intensity, obtained from each pixel in the image in (A). The mean in each distribution gives Ipattern and Ibackground as shown in the figure and allows the calculation of contrast with high precision and accuracy.

BSA on reducing nonspecific adsorption during the patterning of streptavidin. We chose the adsorption of blocking agents to reduce nonspecific adsorption in this study, because it is experimentally convenient, widely applicable to different polymers, and has been extensively used for this purpose in heterogeneous immunoassays.23-25 We were further motivated by our previous results, where it was observed that adsorption of Tween 20 could be used to reduce nonspecific adsorption of streptavidin to a level that is sufficiently low to generate spatial contrast in TOFSIMS images of patterned streptavidin.13 We examined the effect of Tween 20 and BSA, used alone or in combination, on the formation of streptavidin patterns on micropatterned PE-biotin. Figure 6 shows fluorescence images of micropatterned PE-biotin, incubated with Alexa488-labeled streptavidin in HEPES, containing either 0.02% (v/v) Tween 20 detergent (Figure 6A), 0.1% (w/v) BSA (Figure 6B), or both blocking agents (Figure 6C). Spatial contrast was quantified by calculating the average ratio of fluorescence intensity in the patterned areas to that in the regions between them, as described in the Experimental Section, using image intensity histograms. The highest contrast of 3.419 ( 0.001 was obtained with the addition of only Tween 20 to the buffer during incubation of streptavidin (Figure 6A). Significantly lower contrast was observed for BSA alone (Figure 6B, contrast

Hyun et al.

) 2.386 ( 0.001) or for a combination of Tween 20 and BSA in the buffer (Figure 6C, contrast ) 2.027 ( 0.002) under identical imaging conditions (P < 0.0001, the image in Figure 6A compared to the images in Figure 6B,C). This unexpected result was clearly attributable to BSA causing higher levels of nonspecific adsorption to the background, presumably because adsorbed BSA enhances the subsequent nonspecific adsorption of streptavidin during incubation. Effect of Streptavidin Incubation Time. We had previously shown that a streptavidin concentration of 0.1 µM is sufficient to enable the formation of streptavidin patterns on micropatterned biotin.13 We specifically chose this concentration because it is low enough to reduce nonspecific adsorption to the background regions, but it is orders of magnitude greater than the ∼10-13 M dissociation constant of streptavidin-biotin,32 so as to enable binding of biotin-streptavidin to proceed to completion. The incubation time, however, is also an important parameter that affects the nonspecific adsorption of proteins, and this has not been previously examined for this patterning scheme. We hypothesized that the incubation time is likely to significantly affect the development of spatial contrast in the micropatterns because of the difference in the kinetics of streptavidinbiotin binding in the pattern, as compared to the kinetics of nonspecific adsorption of streptavidin to the background. Although the intrinsic association kinetics of streptavidin binding to the patterned region and its nonspecific adsorption to the background are likely to be similar because they are close to the diffusion limit,33 we expected the spatial contrast to be sensitive to the incubation time. This is because the streptavidin-biotin interaction is essentially irreversible with a dissociation rate constant of ∼10-6 s-1 34 and the temporal development of contrast in the pattern should therefore be dominated by the intrinsic association rate. In contrast, the dissociation rate constant for nonspecific binding of streptavidin to the background is likely to be substantially larger, and dissociation events therefore will significantly retard the temporal development of contrast in the unpatterned background regions which do not contain biotin. Figure 7 shows the effect of streptavidin incubation time on the spatial contrast in the streptavidin pattern using confocal fluorescence microscopy for a fixed streptavidin solution concentration of 0.1 µM. The temporal evolution of the streptavidin pattern is shown by the intensity in the pattern (Ipattern) and in the background (Ibackground), which are plotted as a function of the streptavidin incubation time (Figure 7A). The spatial contrast, defined as the ratio of the intensity in the pattern to that in the background, is separately shown in Figure 7B. Although nonspecific adsorption was low at t < 1 min, the streptavidin pattern showed an increase in the contrast in the first minute of incubation because the binding between streptavidin and biotin in stamped regions was not saturated (Figure 7A). The contrast reached its maximum at 30 s, because the binding between streptavidin and biotin in the patterned regions reached steady state faster than nonspecific adsorption to the background. After 30 s, the contrast decreased as a function of incubation time (Figure 7B), because although the binding of streptavidin had nearly reached steady state in the (32) Green, N. M. Biochem. J. 1966, 101, 774-780. Green, N. M. Adv. Protein Chem. 1975, 29, 85-133. (33) Chilkoti, A.; Stayton, P. S. J. Am. Chem. Soc. 1995, 117, 1062210628. (34) Piran, U.; Riordan, W. J. J. Immunol. Methods 1990, 113, 141143.

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Figure 7. Effect of streptavidin incubation time on spatial contrast. PE-COOH samples micropatterned with biotin were incubated with Alexa488-labeled streptavidin in HBS containing 0.02% Tween 20 for a specified time, washed in buffer, and imaged under identical conditions by confocal fluorescence microscopy. (A) Intensity in the pattern (Ipattern) (O) and in the background (Ibackground) (4) as a function of incubation time. (B) Spatial contrast of streptavidin patterns on PE-COOH defined as Ipattern/Ibackground as a function of streptavidin incubation time. (P < 0.0001, 30 s compared to all other time points). The error bars are standard errors of the mean, are