Langmuir 2002, 18, 6671-6678
6671
Highly Selective Protein Patterning on Gold-Silicon Substrates for Biosensor Applications Mandana Veiseh,† M. Hadi Zareie,‡ and Miqin Zhang*,† Departments of Materials Science and Engineering and of Chemistry, University of Washington, Seattle, Washington 98195-2120 Received January 13, 2002. In Final Form: May 23, 2002 Proteins were precisely patterned on 2D sensor surfaces using photolithography and chemical selectivity. Microarrays of gold squares were fabricated on silicon substrates. The gold regions were modified with mixed COOH-terminated self-assembled monolayers (SAMs) to have a high affinity for the desired proteins or peptides. The silicon regions were modified with polyethylene glycol (PEG) by silanization to provide a high resistivity to protein adsorption. Protein surface coverage was visualized by fluorescence microscopy and atomic force microscopy (AFM). AFM was also used for studying protein morphology to understand the interaction of proteins with SAMs at the molecular level. Proteins and peptides immobilized on SAMs were examined by Fourier transform infrared (FTIR) spectroscopy. Contact angle measurements for surface wettability were conducted to confirm the success of the surface modification reactions. Protein resistance by the PEGs immobilized on bare silicon substrates and on the silicon regions of gold-patterned silicon substrates was compared, and it was found that the latter has a higher resistivity to protein adsorption. Both fluorescence and high-resolution AFM images indicated that bovine serum albumin (BSA) and fibronectin molecules formed a densely packed layer on the gold regions of the patterned substrates, while the immunoglobulin’s (IgG) coverage was low. Specific antigen-antibody binding (BSA-anti-BSA) was studied using the surface plasmon resonance (SPR) technique for characterizing the bioactivity of the antigen attached to the gold substrates. The SPR results showed that the BSA proteins bound covalently to the gold surfaces have a much better bioactivity than those bound physically. This study suggests that protein or peptide, molecular structures, and the immobilization technique influence the coverage, morphology, and bioactivity of the attached proteins on the substrates which is crucial to the operational behavior of biosensors.
I. Introduction In recent years, protein patterning technology has recorded major advances in the development of biosensors, molecular electronics, bioreactors, tissue engineering, and fundamental studies of cell biology. Protein patterning has two basic requirements: (1) selective attachment of proteins within designated regions in a substrate and (2) high protein resistivity by the other regions of the substrate. Commonly used protein patterning techniques include microcontact printing,1 conventional photolithography,2,3 and photochemistry.4 Each has its advantages and inherent limitations. Microcontact printing can create patterns of self-assembled monolayers of alkanethiols and areas with defined shape and size that support or resist protein adsorption. It is simple, inexpensive, and effective; however, it cannot precisely control positions and dimensions of patterned proteins, and the immobilized proteins are nonuniform due to the deformation of elastomeric stamps as patterning masks.5 Photolithography creates protein patterns on substrates by using chemical linkers to conjugate proteins.6 This approach can be used for * Author to whom correspondence should be addressed. Phone: (206) 616 9356. Fax: (206) 543 3100. E-mail: mzhang@ u.washington.edu. † Department of Materials Science and Engineering. ‡ Department of Chemistry. (1) 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. (2) Knoll, W.; Liley, M.; Pisceivic, D.; Spinke, J.; Tarlov, M. J. Adv. Biophys. 1997, 34, 231-251. (3) Xia, Y.; Whitesides, G. M. Angew. Chem. 1998, 110 (5), 568-594. (4) Tender, L. M.; Worley, R. L.; Fan, H. Y.; Lopez, G. P. Langmuir 1996, 12, 5515-5518. (5) Blawas, A. S.; Reichert, W. M. Biomaterials 1998, 19, 595-609. (6) Folch, A.; Toner, M. Annu. Rev. Biomed. Eng. 2000, 2, 227-256.
silicon, glass, or metal substrates to form robust covalent bonds between substrates and proteins. The major problem for patterning the substrates with photolithography is the chemicals involved in the process which can denature the immobilized proteins and, thus, reduce their activity.7 Photochemical patterning uses chemically labile species which can be activated upon UV irradiation to bind target proteins.8,9 To pattern proteins, localized reactive areas can be created by selectively irradiating a photochemically derivatized surface, but UV can also reduce protein activity. We developed a new protein patterning method via chemical selectivity.10 The micropatterned substrates in our experiments have two types of surface regions, gold and silicon. The substrate surface was modified such that the gold regions have a high affinity for proteins while the silicon regions have a high resistivity to protein adsorption. In this approach, protein attachment is the last step of the process, and no photoresist removal is involved, thus eliminating the deleterious effects on the attached proteins. Protein activity is greatly enhanced as compared with that in the photolithography approach. In addition, the SAMs on the gold regions prevent direct protein contact with the gold surfaces and, thus, minimize the surfaceinduced protein denaturation. The conjugation of COOHterminated SAMs with NHS esters allows for covalent coupling of proteins and peptides.10 It is reported elsewhere (7) Abbott, N. L.; Folkers, J. P.; Whitesides, G. M. Science 1992, 257 (5075), 1380-1382. (8) Nicolau, D. V.; Taguchi, T.; Taniguchi, H.; Yoshikawa, S. Langmuir 1998, 14 (7), 1927-1936. (9) Flounders, A. W.; Brandon, D. L.; Bates, A. H. Isosensors-andbioelectronics 1997, 12, 447-456. (10) Veiseh, M.; Zhang, Y.; Hinkley, K.; Zhang, M. Biomed. Microdevices 2001, 3, 43-49.
10.1021/la025529j CCC: $22.00 © 2002 American Chemical Society Published on Web 07/26/2002
6672
Langmuir, Vol. 18, No. 17, 2002
that this robust bond cannot be removed by buffers and detergents or replaced by other proteins in the solution.11-13 A PEG layer was immobilized on silicon regions through a simple, irreversible self-assembly process.14 The technique does not require stringent laboratory controls for the chemical reaction or expensive devices (e.g., a plasma treatment chamber15-17), and it can be easily applied for PEGs with different molecular masses (MW) and functional groups. In summary, this approach can accurately position proteins in designated areas via a simple and irreversible reaction. In our previous work,10 we focused on (1) optimizing experimental conditions of the chemical reactions to improve the efficacy of the protein patterning (i.e., the selectivity of proteins and protein resistance of PEGmodified regions) and (2) developing a simple and inexpensive approach to fabricating patterned substrates via photolithography and liftoff. In this study, we concentrate on the following issues: (1) a comparison of protein resistance by PEG-modified bare silicon substrates and by silicon regions of gold-patterned silicon substrates; (2) the versatility of the approach for attaching other proteins and peptides on substrates; (3) the use of a new, stable, fluorescence probe, carboxytetramethyl-rhodamine succinimdyl ester fluorophore, with tagged proteins for visualization of protein coverage on the substrates; (4) understanding the effect of molecular structures of proteins on their coverage and morphology on substrates; and (5) studying the bioactivity of immobilized proteins by SPR analysis of an antigen-antibody (BSA-anti-BSA) interaction. The immobilization of proteins and peptides on substrates is characterized by FTIR, and protein morphologies at the molecular level and semiquantitative information on the extent of protein coverage on the substrate are obtained from high-resolution AFM images. II. Experimental Section Materials. The following materials and chemicals were used as received: 95% 11-mercaptoundecanoic acid (11-MUA), 99% 3-mercaptopropionic acid (3-MPA), 97% N-hydroxysuccinimide (NHS), 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide (EDAC), bovine immuno gamma globulins (IgG), bovine serum albumin (BSA), monoclonal anti-bovine serum albumin (anti-BSA), fibronectin adhesion-prompting peptide (Sigma, St. Louis, MO), polyethylene glycol silane (PEG-silane, 5000; Shearwater Polymers, Huntsville, AL), bovine serum albumin-tetramethylrhodamine conjugate (Molecular Probes, Eugene, OR), and rhodamine red X-conjugated pure bovine IgG whole molecule (Jackson ImmunoResearch Lab Inc., West Grove, PA). All the solvents mentioned above were HPLC grade, and all other reagents were analytical grade. Other solvents or reagents were purchased from either Aldrich (Milwaukee, WI) or Sigma. Absolute ethanol was always deoxygenated by dry N2 before use. Microfabrication of Substrates. The processes for fabricating gold-patterned silicon substrates are detailed in ref 10. Briefly, a mask of an array of squares in 20 × 20 µm was printed onto a photoresist-covered silicon wafer. A thin layer of titanium (Ti) (5 nm) was then deposited onto the photoresist-developed substrate. Gold films were subsequently deposited onto Ti at a (11) Wadu-Mesthrige, K.; Amro, N. A.; Liu, G. Scanning 2000, 22, 380-388. (12) Chibata, I. Immobilized Enzymes-Research and Development, 1st ed.; Wiley and Sons: New York, 1978; Chapter 2. (13) Slomkowski, S. Prog. Polym. Sci. 1998, 23, 815-874. (14) Zhang, M.; Desai, T.; Ferrari, M. Biomaterials 1998, 19 (10), 953-960. (15) Zhang, M.; Ferrari, M. Biomed. Microdevices 1998, 1 (1), 81-89. (16) Kleinfeld, D.; Kahler, K. H.; Hockberger, P. E. J. Neurosci. 1988, 8 (11), 4098-4120. (17) Hendricks, S. K.; Kwok, C.; Shen, M.; Horbett, T. A.; Ratner, B. D.; Bryers, J. D. J. Biomed. Mater. Res. 2000, 50 (2), 160-170.
Veiseh et al. deposition rate of 0.3 Å/s. The photoresist was then dissolved in acetone, and the remaining metal films were lifted off. Finally, the patterned silicon wafers were cut using a disco saw into 8 × 8 mm slides. To avoid surface contaminants and unexpected scratches, the silicon wafers were coated with a layer of photoresist 2 µm in thickness on their polished sides before cutting. Surface Modifications with SAMs and Proteins or Peptides. (1) Bare Gold Substrates. The protective layers of photoresist on gold-coated substrates were removed by immersing the substrates in acetone for 10 min, followed by an ethanol wash for 2 min and a 2-propanol wash for 2 min. The substrates were then placed in hot piranha (hydrogen peroxide/sulfuric acid, 1:5 v/v) at 70-80 °C for 10 min, rinsed thoroughly with deionized (DI) water, and dried under a stream of nitrogen gas. The formation of COOH-terminated alkanethiol monolayers on the substrates was accomplished by immersing the substrates for 16 h in 20 mM ethanolic solutions of mixed MUA/MPA (1:10 v/v), followed by ethanol sonication for 10 min and a DI water rinse. The substrates with these carboxylic acid-terminated SAMs were then immersed in an aqueous solution of 150 mM EDAC and 30 mM NHS for 30 min to attach the NHS group to the COOH terminus. Proteins or peptides were covalently bound on these substrates by replacing their NHS groups with N-termini protein or peptide residues in a phosphate buffer solution (PBS) with a protein or peptide concentration of 0.2 mg/mL at pH 7.4 at room temperature for 45 min. All the samples were washed with PBS, extensively rinsed with DI water, and dried under a N2 stream.18-20 (2) Bare Silicon Substrates. Silicon surfaces were cleaned using the same procedure that was used for gold surfaces. Once the surfaces had been cleaned, a film of SiO2 and some surface OH groups formed on the silicon substrates which were then exposed to the PEG-silane solution for monolayer formation. The PEG solution of 10 mg/mL was prepared in a dry flask by adding PEG-silane in anhydrous toluene, and 50 mg/mL triethylamine was used as a catalyst. The substrates were then placed in separate flasks containing the PEG solution and were refluxed under N2 for 4 h. After being removed from the PEG-silane solution, the substrates were sonicated in toluene for 10 min, rinsed with ethanol and DI water to remove any unattached moieties on the surfaces, and dried under N2. (3) Silicon Substrates Patterned with an Array of Gold Squares. The chemical reaction schemes for coating the patterned substrates with proteins or peptides on the Au regions and PEGs on the silicon regions are shown in Figure 1. Instrumentation. (1) Contact Angle Measurements. Contact angles were measured by the sessile drop technique using a RameHart 100 goniometer under ambient laboratory conditions (∼40% humidity). A 2 µL drop of distilled water was applied to the surface, and the contact angle measurements were made within 30 s of the contact. The measurements were repeated for three samples. (2) Fluorescence Microscopy. Images of patterned proteins were obtained by inverted fluorescent microscopy (Leica DMIRB equipped with a CCD camera with a pixel size of 6.7 × 6.7 µm). A filter of 515-560 nm was used for exciting the rhodamine probe. With this microscope, the topographic images and fluorescence signals can be detected simultaneously. Image processing was produced by Openlab software, designed for scientific imaging applications of high quality. (3) FTIR Spectroscopy. Polarized FTIR spectra of 2000 scans at 8 cm-1 were obtained using a Nicolet 5DX spectrometer with a DTGS detector and an FT-85 grazing angle sample compartment. The system was purged with dry air for 1 h before each data collection to eliminate water vapor from the sample compartment. Spectra analysis was done using standard Nicolet and Origin software. (4) AFM Imaging. A digital multimode Nanoscope III scanning force microscope was used. All imaging was conducted in the (18) Gorfti, A.; Salmain, M.; Jaouen, G.; McGlinchey, M. J.; Bennouna, A.; Mousser, A. Organometallics 1996, 15, (1), 142-151. (19) Parker, M. C.; Patel, N.; Davies, M. C.; Roberts, C. J.; Tendler, S. J. B.; Williams, P. M. Protein Sci. 1996, 5, 2329-2332. (20) Patel, N.; Davies, M. C.; Hartshorne, M.; Heaton, R. J.; Roberts, C. J.; Tendler, S. J. B.; Williams, P. M. Langmuir 1997, 13, 6485-6490.
Gold-Silicon Substrates for Biosensor Applications
Langmuir, Vol. 18, No. 17, 2002 6673 Table 1. Water Contact Angles of Self-Assembled Monolayers, Proteins, and Peptide on Gold and PEG Film on Silicon Substrates
coatings
θg (gold substrate) (deg)
θs (silicon substrate) (deg)
none PEG MUA/MPA MUA/MPA + NHS ester MUA/MPA + BSA MUA/MPA + IgG MUA/MPA + fibronectin
66 ( 1 40 ( 1 47 ( 1 55 ( 2 56 ( 1 43 ( 1
