Langmuir 1997, 13, 7239-7244
7239
Microfabrication of Alkylsilanized Glass Substrate by Electrogenerated Hydroxyl Radical Using Scanning Electrochemical Microscopy Hitoshi Shiku,† Isamu Uchida,†,‡ and Tomokazu Matsue*,†,‡ Department of Applied Chemistry, Graduate School of Engineering, and Center for Interdisciplinary Research, Tohoku University, Sendai, 980-77, Japan Received May 28, 1997. In Final Form: August 25, 1997X Scanning electrochemical microscopy (SECM) was used for localized electrogeneration of hydroxyl radical to create patterns with diaphorase on the substrates immobilized with self-assembled monolayers (SAMs) of four different alkylsilane derivatives. Hydroxyl radical was generated at an SECM tip (microelectrode) in a solution containing H2O2 and Fe3+ by applying a reduction potential pulse of 0.00 V vs Ag/AgCl. The electrogenerated hydroxyl radical degraded the SAMs and removed them from the glass surfaces. Diaphorase patterns were formed on the substrates by physical adsorption onto the hydrophobic area or by chemical linkage to the hydroxyl radical-attacked area. Local diaphorase activity was visualized by SECM by detecting the diaphorase-catalyzed current of ferrocenylmethanol coupled with oxidation of reduced nicotinamide adenine dinucleotide.
Introduction The development of techniques for immobilizing macromolecules selectively at specific locations on solid supports has made it possible to create miniaturized, integrated DNA sequencing chips1-3 and multianalyte immunosensing devices.4-6 Although these techniques are mainly based with photolithography, scanning probe microscopy (SPM) has been considered as a novel instrument for engineering of sophisticated nanometer-scale materials and devices.7-9 Among various SPMs proposed to date, scanning electrochemical microscopy10-12 (SECM) is a promising tool to construct surfaces patterned with functional macromolecules due to its nature as a chemical microscope. SECM is able not only to detect localized chemical reactions but also to induce reactions in very small area in a highly controllable manner. Until now, various fabrication studies have been reported.13,14 Recently, the studies of SECM fabrication can be rearranged from the viewpoint of the reaction system, in which electrogenerated species at the microelectrode tip are * To whom correspondence may be addressed at Department of Applied Chemistry, Graduate School of Engineering, Tohoku University, Sendai 980-77, Japan: phone, +81-22-217-7219; fax, +81-22-217-7293; e-mail,
[email protected]. † Department of Applied Chemistry. ‡ Center for Interdisciplinary Research. X Abstract published in Advance ACS Abstracts, December 1, 1997. (1) Fodor, S. P. A.; Read, J. L.; Pirrung, M. C.; Stryer, L.; Lu, A. T.; Solas, D. Science 1991, 251, 767. (2) Chee, M.; Yang, R.; Hubbell, E.; Berno, A.; Huang, X. C.; Stern, D.; Winkler, J.; Lockhart, D. J.; Morris, M. S.; Fodor, S. P. A. Science 1996, 274, 610. (3) Pritchard, D. J.; Morgan, H.; Cooper, J. M. Angew. Chem., Int. Ed. Engl. 1995, 34, 91. (4) Sundberg, S. A.; Barrett, R. W.; Pirrung, M.; Lu, A. L.; Kiangsoontra, B.; Holmes, C. P. J. Am. Chem. Soc. 1995, 117, 12050. (5) Shiku, H.; Matsue, T.; Uchida, I. Anal. Chem. 1996, 68, 1276. (6) Shiku, H.; Hara, Y.; Matsue, T.; Uchida, I.; Yamauchi, T. J. Electroanal. Chem., in press. (7) Dagata, J. A. Science 1995, 270, 1625. (8) Snow, E. S.; Campbell, P. M. Science 1995, 270, 1639. (9) Muller, W. T.; Klein, D. L.; Lee, T.; Clarke, J.; McEuen, P. L.; Schultz, P. G. Science 1995, 268, 272. (10) Engstrom, R. C.; Pharr, C. M. Anal. Chem. 1989, 61, 1099A1104A. (11) Bard, A. J.; Denuault, G.; Lee, C.; Mandler, D.; Wipf, D. O. Acc. Chem. Res. 1990, 23, 357-363. (12) Wang, J. Analyst 1992, 117, 1231-1233. (13) Bard, A. J.; Fan, F.-R. F.; Pierce, D. T.; Unwin, P. R.; Wipf, D. O.; Zhou, F. Science 1991, 254, 68. (14) Mirkin, M. V. Anal. Chem. 1996, 68, 177A.
S0743-7463(97)00554-4 CCC: $14.00
designed to react with the substrate to create microstructures at the surface. These electrogenerated reagents reported in the literature are transition metal complexes,15-18 methyl viologen,19 bromine,20-22 and hypobromous acid.23 Controlling the local pH is also widely applied to design various reaction systems.24-29 In this paper, we report a new reaction system involving hydroxyl radical to degrade the self-assembled monolayer (SAM) of alkylsilanes at glass surfaces using SECM. Hydroxyl radical is electrochemically generated at a microelectrode tip in the solution containing hydrogen peroxide and Fe3+.30 SAMs immobilized on solid supports have been widely employed to create patterns of functional or biofunctional molecules. We have used glass slides to support SAMs with four different alkylsilane derivatives to create enzyme patterns. The localized electrogeneration of hydroxyl radical by the SECM system is used for change in the physical and chemical characteristics of the surface. The enzyme patterns were created based on this difference in the surface property (Figure 1). Localized enzyme activity was visualized by SECM which detected the enzymatically-catalyzed redox current. Experimental Section Materials. Reduced nicotinamide adenine dinucleotide (NADH) (Sigma Chemical Co.), glutaraldehyde (GA, Wako Pure Chemical Industries), n-octadecyltrichlorosilane (OTCS, Tokyo (15) Mandler, D.; Bard, A. J. J. Electrochem. Soc. 1989, 136, 3143. (16) Mandler, D.; Bard, A. J. J. Electrochem. Soc. 1990, 137, 1079. (17) Mandler, D.; Bard, A. J. Langmuir 1990, 6, 1489. (18) Macpherson, J. V.; Unwin, P. R. J. Phys. Chem. 1995, 99, 3338. (19) Sugimura, H.; Uchida, T.; Shimo, N.; Kitamura, N.; Masuhara, H. Ultramicroscopy 1992, 42-44, 468. (20) Mandler, D.; Bard, A. J. J. Electrochem. Soc. 1990, 137, 2468. (21) Meltzer, S.; Mandler, D. J. Electrochem. Soc. 1995, 142, L82. (22) Borgwarth, K.; Ricken, C.; Ebling, D. G.; Heinze, J. Ber. Bunsenges. Phys. Chem. 1995, 99, 1421. (23) Shiku, H.; Takeda, T.; Yamada, H.; Matsue, T.; Uchida, I. Anal. Chem. 1995, 67, 31. (24) Wuu, Y. -M.; Fan, F.-R. F.; Bard A. J. J. Electrochem. Soc. 1989, 136, 885. (25) Shohat, I.; Mndler, D. J. Electrochem. Soc. 1994, 141, 995. (26) Sugimura, H.; Uchida, T.; Kitamura, N.; Masuhara, H. J. Phys. Chem. 1994, 98, 4352. (27) Sugimura, H.; Nakagiri, N. Langmuir 1995, 11, 3623. (28) Ratcliff, B. B.; Klanke, J. W.; Koppang, M. D.; Engstrom, R. C. Anal. Chem. 1996, 68, 2010. (29) Schoer, J. K.; Zamborini, F. P.; Crooks, R. M. J. Phys. Chem. 1996, 100, 11086. (30) Matsue, T.; Fujihira, M.; Osa, T. J. Electrochem. Soc. 1981, 128, 2565.
© 1997 American Chemical Society
7240 Langmuir, Vol. 13, No. 26, 1997 Chemical Industry), propyltrichrolosilane (PTCS, Tokyo Chemical Industry), (3-aminopropyl)triethoxysilane (APTES, Tokyo Chemical Industry), (3-mercaptopropyl)trimethoxysilane (MPTMS, Shin-etsu Chemical Industry), and Tween 20 (Kanto Chemical Co.) were used as received. Ferrocenylmethanol (FMA) was synthesized by reduction of ferrocenecarboxyaldehyde (Aldrich Chemical Co.). Completion of the reaction was confirmed with thin-layer chromatography. Diaphorase (Dp) purified from Bacillus stearothermophilus (EC 1.6.99.-) was donated by Unitika Ltd. (Kyoto). SECM System. We have used two types of Pt microdisk electrodes in the present study; one is used in microfabrication and the other is used for the SECM measurement. The following is the fabrication procedure of the microelectrode for the SECM measurement: A Pt wire (radius, 7.5 µm) was etched electrochemically in a saturated NaNO3 solution in order to sharpen the wire and inserted into a softglass capillary. The tip was fused in a small furnace at 320 °C in vacuo to coat the Pt filament completely with the soft glass. The tip was then polished with a diamond grinder (# 5000) on a turntable (Narishige, Model EG-6). From an optical microscope observation, the tip radius including the insulating part was ca. 30 µm. The Pt disk radius was determined from the steady-state oxidation current of Fe(CN)64- or FMA in a voltammogram. The microelectrode for microfabrication was also fabricated by the same procedure as above but excluding the etching process to sharpen the Pt wire. The tip and Pt disk radii were 30 and 7.5 µm, respectively. The measurements were carried out in a two-electrode configuration with a Ag/AgCl (saturated KCl) counter electrode. The current was amplified with a Keithley Model 428 amplifier. Potential control and data acquisition were performed by a personal computer (NEC, PC9821Xc16) through a 16-bit AD/DA board (Interface, AZI3506). Movement of the microelectrode tip was performed by means of a motor-driven XYZ stage (Chuo Precision Industrial Co., M-9103), controlled by the computer through a GP-IB interface. The resolution of the XYZ stage was 0.1 µm. The tip scanning directions (both X- and Y-axis) were aligned parallel to the substrate using a two-axis tilt stage (Chuo Precision Industrial Co., TD-101), in order to keep the distance between tip and substrate constant. Preparation of Substrates. The glass slides (10 × 5 mm2) were cleaned thoroughly by dipping the slides into a mixture of equal volumes of 95% H2SO4 and 60% HNO3 solution for 15 min, followed by washing with distilled water for 15 min under supersonication and dried in an oven at 60 °C for 1 h. Micropatterns of diaphorase were created at the glass substrate by procedures shown in Figure 1. The clean substrate was dipped into a 10 mM alkylsilane (OTCS, PTCS, APTES, or MPTMS)/ benzene solution for 8 h followed by thorough washing with benzene, ethanol, and distilled water under supersonication. In the present report, we refer to the substrate with the OTCS SAM as the OTCS substrate. Other substrates are also referred to similarly. The substrates with SAMs of alkylsilanes were dipped into a solution of 0.05 M H2SO4 containing FeCl3 and H2O2 for microfabrication using a SECM system. Being kept at a rest potential (0.55 V vs Ag/AgCl), the tip for microfabrication was placed at about 5 µm above the silanized substrate. Then, a potential pulse of 0.00 V vs Ag/AgCl with various duration was applied to the tip to produce hydroxyl radical (HO) by Fenton’s reaction. After the application of the potential pulse, the substrate was transferred to distilled water and washed under supersonication. The resulting substrate was patterned with diaphorase (Dp) by one of the following processes, (a) to (c). (a) Dp Treatment (See Figure 1a). The substrate was immersed into a 0.1 mM diaphorase, 0.1% Tween 20/phosphate buffer solution (Dp-solution) for 20 min, and then washed under supersonication in phosphate buffer solution. (b) Dp-GA Treatment (See Figure 1b). A mixture of 0.1 mM diaphorase and 1% glutaraldehyde was incubated for 1 h. Then the solution was subject to gel filtration chromatography (PD-10, Pharamacia Biotech) to remove unreacted glutaraldehyde. The substrate was immersed into the eluted solution (DpGA solution) containing 0.1% Tween 20 for 20 min and then washed under supersonication in phosphate buffer solution. (c) APTES/Dp-GA Treatment (See Figure 1c). The substrate was immersed into a 10 mM APTES/benzene solution for 90 min followed by thorough washing with benzene, ethanol,
Shiku et al.
Figure 1. Preparation of micropatterns with diaphorase at SAM-immobilized glass surfaces by electrogenerated hydroxyl: (a) diaphorase is physically-adsorbed onto the hydrophobic area; (b) diaphorase is chemically-linked to the hydroxyl-radicalnonattacked area to give a negative pattern; (c) diaphorase is chemically-linked to the hydroxyl-radical-attacked area to give a positive pattern. and distilled water under supersonication. The substrate was then immersed into a 0.1 mM Dp-GA, 0.1% Tween 20/phosphate buffer solution for 20 min and washed under supersonication in phosphate buffer solution. Characterization of the Substrates by SECM. The substrate prepared by the above treatment was dipped in a 0.5 mM FMA, 0.1 M KCl, 0.1 M phosphate buffer solution (pH 7.5). The microelectrode tip for SECM measurement was placed above the substrate and set at 0.40 V vs Ag/AgCl to monitor the oxidation current of FMA in solution. When the tip was carefully moved down to the substrate, the oxidation current decreased since the diffusion of FMA to the tip surface was blocked by the substrate. This decrease in the oxidation current is theoretically reproduced by the digital simulation.23 The theoretical current vs distance profile was used as a working curve to determine tip-substrate distances. The SECM image without NADH showed no pattern at the surfaces. (See Supporting Information paragraph at the end of this paper.) Then we added 5.0 mM NADH to the measurement solution to obtain a SECM image of diaphorase activity. Diaphorase immobilized at the substrate catalyzes the reduction of the oxidized form of FMA (FMA+) by NADH to regenerated FMA. Therefore, the response at the microelectrode tip increases only when the tip is above the diaphorase-immobilized area. The catalytic currents induced by the immobilized diaphorase are superposed on the background currents (230-330 pA) observed in the absence of NADH. In this paper, all the SECM images were captured at about 7 µm tip-substrate distance and subtracted with the background currents. Contact Angle Measurements. A droplet (1 µL) of distilled water was spotted on a substrate, being monitored with a CCD camera through a microscope. The contact angle of the droplet at the substrate was determined from the enhanced video image. The contact angle reported in the present study is the average of more than six values taken at different points on the surface.
Microfabrication of Alkylsilanized Glass Substrate
Langmuir, Vol. 13, No. 26, 1997 7241
Results and Discussion In the present study, hydroxyl radical was generated electrochemically at a microelectrode tip and used for micropatterning the substrate with alkylsilane SAMs finally to obtain diaphorase-patterned substrates. The application of a potential pulse of 0.00 V vs Ag/AgCl reduces Fe3+ to Fe2+, which was subsequently reacted with H2O2 in solution to produce hydroxyl radical as follows: 30,31
Fe3+ + e- f Fe2+
(1)
k2
Fe2+ + H2O2 98 Fe3+ + HO• + HOk2 ) 2.2 × 102 M-1 s-1
(2)
The electrogenerated hydroxyl radical diffuses into the substrate to react with the SAMs of alkylsilanes. Hydroxyl radical is highly reactive to degrade the silylalkyl chains to change the chemical property at the localized surface. For an OTCS substrate the degradation by electrogenerated hydroxyl radical makes the localized surface hydrophilic. This difference in the surface property was used for micropatterning of the substrate with diaphorase. Figure 2 shows an SECM image of an OTCS substrate which was subject to localized degradation by electrogenerated HO• for 5 min followed by dipping into the Dp solution (also see Figure 1a). A circular area with low oxidation current for FMA was clearly observed at the center of the image. Since the oxidation current in the presence of NADH increases with the catalytic capability of diaphorase bound to the substrate, the area with low current corresponds to the area with low diaphorase concentration. The SECM image indicates that diaphorase adsorbs more strongly onto the hydrophobic area than onto the hydrophilic area. The PTCS substrate treated with the localized degradation and the diaphorase adsorption also showed a similar circle of low enzyme concentration in an SECM image. However, APTES and MPTMS substrates with the same treatment showed no clear pattern with diaphorase. The relatively hydrophilic end groups at the substrate surface face out to the solution and prevent diaphorase from adsorbing onto the surface. The above findings suggest that the area-defined control of hydrophobicity at a solid surface can be used for micropatterning the surface with diaphorase. To obtain a quantitative feature of hydrophobicity, we investigated wettability of the surfaces of glass substrates with SAMs of the four alkylsilane derivatives. The wettability was evaluated by the contact angle measurements. The contact angles of the surfaces with OTCS, PTCS, APTES, and MPTMS SAMs were 115°, 107°, 33° and 58°, respectively (Figure 3). These values are in good agreement with those reported in the literature.32,33 Then, the substrates were dipped into a solution of 1.0 M H2O2 and 0.05 M H2SO4 containing either 1.0 M FeCl3 or FeCl2 for 30 min. Hydroxyl radical is considered to generate only in the solution containing FeCl2. The contact angles of the substrates did not show marked decrease after soaking into the solution containing FeCl3 except for the MPTMS substrate. The octadecyl, propyl, and aminopropyl groups are found to be stable in the solution. The decrease of the contact angle for the MPTMS substrate originates from oxidation of the -SH terminal group with (31) Skinner, J. F.; Glasel, A.; Hsu, L.-C.; Funt, B. L. J. Electrochem. Soc. 1980, 127, 315. (32) Bain, C. D.; Troughton, E. B.; Tao, Y.-T.; Evall, J.; Whitesides, G. M.; Nuzzo, R. G. J. Am. Chem. Soc. 1989, 111, 321. (33) Balachander, N.; Sukenik, C. N. Langmuir 1990, 11, 1621.
Figure 2. SECM image of an OTCS substrate with the Dp treatment ((a) in the Experimental Section) in a 0.5 mM FMA, 5.0 mM NADH, 0.1 M KCl, 0.1 M phosphate buffer solution (pH 7.5): tip potential, 0.40 V vs Ag/AgCl; tip radius, 1.9 µm. Microfabrication by the electrogenerated hydroxyl radical was carried out by applying a reduction pulse of 0.00 V vs Ag/AgCl for 5 min to an SECM tip (radius, 7.5 µm) in 0.1 M FeCl3/1.0 M H2O2/0.05 M H2SO4 solution.
Figure 3. Contact angles of the substrates with SAMs of OTCS, PTCS, APTES, and MPTMS: (a) control; (b) dipped into a 1.0 M H2O2/0.05 M H2SO4 solution containing 1.0 M FeCl3 for 30 min; (c) dipped into a 1.0 M H2O2/0.05 M H2SO4 solution containing 1.0 M FeCl2 for 30 min.
H2O2 to yield a sulfonate group which is very hydrophilic.33,34 In contrast, soaking the substrates into the solution containing FeCl2 decreased the contact angles of all the substrates. Especially, for the OTCS and PTCS substrates one can find drastic decreases, indicating that (34) Ichinose, N.; Sugimura, H.; Uchida, T.; Shimo, N.; Masuhara, H. Chem. Lett. 1993, 1961.
7242 Langmuir, Vol. 13, No. 26, 1997
Shiku et al.
Figure 4. SECM images of an APTES substrate with the Dp-GA treatment ((b) in the Experimental Section) in a 0.5 mM FMA, 5.0 mM NADH, 0.1 M KCl, 0.1 M phosphate buffer solution (pH 7.5): tip potential, 0.40 V vs Ag/AgCl; tip radius, 2.3 µm. Microfabrication by the electrogenerated hydroxyl radical was carried out by applying reduction pulses of 0.00 V vs Ag/AgCl for 5 min to an SECM tip (radius, 7.5 µm) in 1.0 M H2O2/0.05 M H2SO4 solution containing FeCl3. FeCl3 concentrations were 0 (A), 1.0 (B), 10 (C), and 100 mM (D).
the surface changes from hydrophobic to hydrophilic by soaking the substrate into the solution producing a hydroxyl radical. However, it should be noted that the treatment with the hydroxyl-radical-producing solution for 30 min did not remove completely the alkyl chains from the surface, as evidenced by the fact that the contact angles (40°-60°) after treatment were still large compared to those for the APTES and MPTMS substrates. For the APTES substrate it is difficult to fabricate micropatterns with diaphorase based on the difference in hydrophobicity. Thus, we tried to make the pattern by using a chemical linkage between the surface and diaphorase (see Figure 1b). For this purpose, glutaraldehyde was selected as a cross-linker reagent. Figure 4 shows SECM images of an APTES substrate treated with the Dp-GA solution. It is clear that the catalytic capability in the hydroxyl-radical-attacked area is sufficiently low compared to the remainder. The electrogenerated hydroxyl radical reacts with an aminopropyl group at the localized area to remove the amino moiety from the surface. This figure also demonstrates the influence of Fe3+ concentration on the size of the pattern. The area of low enzyme concentration, i.e., hydroxyl-radical-attack area, becomes large with the concentration of Fe3+ in solution.
The electrogenerated hydroxyl radical is diminished by the follow-up reactions with Fe2+ or H2O230,35 k3
HO• + Fe2+ 98 HO- + Fe3+ k3 ) 3 × 108 M-1 s-1
(3)
k4
HO• + H2O2 98 HOO• + H2O k4 ) (1-5) × 107 M-1 s-1
(4)
If we assume that the concentrations of H2O2 and Fe2+ are sufficiently in excess over the concentration of hydroxyl radical, the steady-state concentration of hydroxyl radical ([HO.]SS) is expressed by
[HO•]SS ) k2[H2O2][Fe2+]/{k3[Fe2+] + k4[H2O2]} (5) The concentrations of H2O2 and Fe2+ at 5 µm apart from the tip surface were calculated by digital simulation and (35) Walling, C. Acc. Chem. Res. 1975, 8, 125.
Microfabrication of Alkylsilanized Glass Substrate
Figure 5. SECM images of an APTES substrate with the DpGA treatment ((b) in the Experimental Section) in a 0.5 mM FMA, 5.0 mM NADH, 0.1 M KCl, 0.1 M phosphate buffer solution (pH 7.5): tip potential, 0.40 V vs Ag/AgCl; tip radius, 2.1 µm. Microfabrication by the electrogenerated hydroxyl radical was carried out by applying reduction pulses of 0.00 V vs Ag/AgCl to an SECM tip (radius, 7.5 µm) in 0.1 M FeCl3/1.0 M H2O2/0.05 M H2SO4 solution. The periods of pulses were 5 (A), 20 (B), 80 (C), 300 (D), and 1800 s (E).
found to be ∼10% and ∼90%, respectively, of the bulk concentration of H2O2 and Fe3+. Thus, the concentration of hydroxyl radical at the substrate surface would be ∼50 nM when bulk concentrations of H2O2 and Fe3+ are 1.0 and 0.1 M, respectively. The concentration of hydroxyl radical depends on the concentration of Fe2+ in solution. The size of the patterns shown in Figure 4 is a direct consequence of the difference in Fe2+ concentration at the substrate surface. Hydroxyl radical is known to oxidize rapidly organic compounds. For the oxidation of carboxyl and ether compounds, hydroxyl radical abstracts a hydrogen atom of the R-carbon (rate constant, 107-1010 M-1 s-1 35), which in many cases results in cleavage of the carbon-carbon or carbon-oxygen bond. Similar reaction would also occur in the Si-C bond at the surface. However, the rate of bond cleavage of the Si-C bond will be much smaller than expected in the homogeneous reaction, since the alkyl chains at surface block the approach of the hydroxyl radical to the reaction site. Figure 4 also indicates that patterning is possible even if Fe3+ is absent, probably because a small amount of hydroxyl
Langmuir, Vol. 13, No. 26, 1997 7243
Figure 6. SECM image of an MPTMS substrate treated with APTES and the Dp-GA solution ((c) in the Experimental Section) in a 0.5 mM FMA, 5.0 mM NADH, 0.1 M KCl, 0.1 M phosphate buffer solution (pH 7.5): tip potential, 0.40 V vs Ag/AgCl; tip radius, 1.6 µm. Microfabrication by the electrogenerated hydroxyl radical was carried out by applying reduction pulses of 0.00 V vs Ag/AgCl for 5 min to an SECM tip (radius, 7.5 µm) in 0.1 M FeCl3, 1.0 M H2O2, 0.05 M H2SO4 solution.
radical is generated by one-electron reduction of H2O2 under this condition. The size of hydroxyl-radical-attacked area increases with the pulse period for generation of hydroxyl radical (Figure 5). The increase in the area was, however, much smaller than expected from the growth of diffusion layer. The SECM fabrication by active species with very short lifetime is advantageous to fabricate with high resolution. It is important to consider not only the diffusion length but also length of reaction layer in designing the SECM fabrication system to create functional surfaces. In the present study, we added H2O2 into the solution to generate hydroxyl radical electrochemically. Hydroxyl radical can also be generated by simultaneous reductions of Fe3+ to Fe2+ and dissolved O2 to H2O2 at an electrode such as carbon and Au. We have tried the microfabrication by the simultaneous reduction at a Au microelectrode in air-saturated aqueous solutions. However, no clear pattern of diaphorase was found on the substrate. We believe
7244 Langmuir, Vol. 13, No. 26, 1997
that electrochemical reduction of dissolved O2 is insufficient to yield an appreciable amount of H2O2 and, as a result, the concentration of hydroxyl radical is also insufficient to change the chemical functionality of the substrate surface. The above procedure fabricates a negative pattern with the enzyme; i.e., the enzyme is bound to an area where the electrogenerated hydroxyl radical does not react. A positive pattern with diaphorase can be fabricated via a chemical linkage between the reacted surface and enzyme. Figure 6 shows an SECM image of a MPTMS substrate with the follow-up treatment by APTES/Dp-GA (also see Figure 1c). Clear bright circles indicate the enzyme concentration in the circles is higher than that in the remainder. These patterns were not observed using the substrate treated with the Dp solution instead of DpGA. In the patterning step by the electrogenerated hydroxyl radical, the organic layer close to the tip was removed; simultaneously, the -SH group in the remainder area was converted to -SO3- in the H2O2/Fe3+ solution.33 The exposed -OH group in the hydroxyl-radical-attack area was used to link APTES. Diaphorase was immobilized on to the area by reacting with the Dp-GA solution, while the hydrophilic property at the reminder prevents diaphorase from adsorbing. In conclusion, the electrogenerated hydroxyl radical at a SECM tip is useful to fabricate micropatterns at SAMimmobilized solid surfaces. We have utilized the reactivity of hydroxyl radical to remove the localized SAM and thereby to change the physical and chemical property of the localized areas. When hydrophobic alkylsilanes are employed for immobilization of SAMs at the glass sub-
Shiku et al.
strate, the SECM reaction yields hydrophilic patterns at the substrate. Since diaphorase adsorbs preferentially onto the hydrophobic area, a negative pattern with diaphorase is obtained by dipping the substrate into the solution containing diaphorase. It is known that many proteins and cells discriminate the hydrophobicity of the surfaces to adsorb;36,37 therefore, the present procedure can be extended to fabricate micropatterns of biomaterials at solid surfaces. The present study also demonstrates that the chemical linkage between the hydroxyl-radicalattacked area and diaphorase is useful to obtain diaphorase-patterned surfaces. Since hydroxyl radical is known to introduce hydroxyl group at aromatic rings,30,35 a more sophisticated design of surfaces can be attained by using aromatic SAMs. Acknowledgment. This research was partly supported by Grants-in-Aid for Scientific Research (Nos. 09450311, 09237106, and 09217205) from the Ministry of Education, Science and Culture, Japan. H.S. acknowledges a research fellowship from the Japan Society for the Promotion of Science. Supporting Information Available: SECM images of a glass substrate with a diaphorase pattern with and without 5.0 mM NADH (1 page). Ordering information is given on any current masthead page. LA970554O (36) Stenger, D. A.; Georger, J. H.; Dulcey, C. D.; Hickman, J. J.; Rudolph, A. S.; Nielsen, T. B.; McCort, S. M.; Calvert, J. M. J. Am. Chem. Soc. 1992, 114, 8435. (37) Matsuda, T.; Sugawara, T. Langmuir 1995, 11, 2267.
