Immobilized Diaphorase Surfaces Observed by Scanning

microscopy for the label-free interrogation of antibody–antigen interactions: Part 2. Joanne L. Holmes , Frank Davis , Stuart D. Collyer , Séam...
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Anal. Chem. 2005, 77, 1785-1790

Immobilized Diaphorase Surfaces Observed by Scanning Electrochemical Microscope with Shear Force Based Tip-Substrate Positioning Hiroshi Yamada,* Hikaru Fukumoto, Tetsuya Yokoyama, and Tohru Koike

Department of Applied Chemistry, National Defense Academy, 1-10-20 Hashirimizu, Yokosuka, Kanagawa 239-8686, Japan

Imaging of a coimmobilized diaphorase and albumin surface was investigated by scanning electrochemical microscopy (SECM) with shear force based tip-substrate distance control. A microelectrode tip was attached to a commercially available tuning fork to detect the shear force between the microelectrode tip and the surface. We used the standing approach mode, which repeats an approach and retraction at each data point of the surface to obtain simultaneous current and topographic images. To check the performance of our SECM system, we imaged a platinum-patterned array electrode and a diaphorase/albumin coimmobilized glass surface. Since the system acquires current when the tip is retracted to a desired distance, this mode is useful for a relatively large microelectrode (∼10 µm) and for scanning a large area (few hundreds of micrometers). Furthermore, by retracting the tip when the tip moves laterally to the next data point to avoid contact between the tip and sample surface, we successfully imaged the surface without destroying its morphology. The scanning electrochemical microscope (SECM) is widely used for the characterization of the chemical nature of interfaces. Examples of heterogeneous surface reactions studied by SECM include electron transfer1 and mass transfer2 through a bilayer lipid membrane, mass3 and charge transfer through a liquid/liquid interface,4-8 mass transfer through porous membranes,9 and corrosion of a metal surface.10,11 SECM is also used as a tool for * To whom correspondence should be addressed. E-mail: [email protected]. (1) Amemiya, S.; Ding, Z. F.; Zhou, J. F.; Bard, A. J. J. Electroanal. Chem. 2000, 483, 7-17. (2) Yamada, H.; Matsue, T.; Uchida, I. Biochem. Biophys. Res. Commun. 1991, 180, 1330-1334. (3) Yamada, H.; Akiyama, S.; Inoue, T.; Koike, T.; Matsue, T.; Uchida, I. Chem. Lett. 1998, 147-148. (4) Delville, M.-H.; Tsionsky, M.; Bard, A. J. Langmuir 1998, 14, 2774-2779. (5) Shao, Y.; Mirkin, M. V. J. Phys. Chem. B 1998, 102, 9915-9921. (6) Barker, A. L.; Unwin, P. R.; Amemiya, S.; Zhou, J.; Bard, A. J. J. Phys. Chem. B 1999, 103, 7260-7269. (7) Shao, Y.; Mirkin, M. V. J. Electroanal. Chem. 1997, 439, 137-143. (8) Strutwolf, J.; Barker, A. L.; Gonsalves, M.; Caruana, D. J.; Unwin, P. R.; Williams, D. E.; Webster, J. R. P. J. Electroanal. Chem. 2000, 483, 163173. (9) Bath, B. D.; Lee, R. D.; White, H. S. Anal. Chem. 1998, 70, 1047-1058. (10) Garfias-Mesias, L. F.; James, P. I.; Smyrl, W. H. Proc. Electrochem. Soc. 1997, 97-7, 247-256. (11) Garfias-Mesias, L. F.; Alodan, M.; James, P. I.; Smyrl, W. H. J. Electrochem. Soc. 1998, 145, 2005-2010. 10.1021/ac048582g CCC: $30.25 Published on Web 02/03/2005

© 2005 American Chemical Society

microfabricating interfaces, such as a patterned enzyme surface,12,13 the local immobilization of protein,14 and a microtower of conducting polymer on a metal surface.15 Various scanning probe microscopies have been applied to or combined with SECM. Recently, various methods used for regulating the tip-sample distance in scanning near-field optical microscopy (SNOM) and atomic force microscopy (AFM)16-19 have been attempted with SECM. The tip current depends not only on the electrochemical activities of a sample surface but also on the distance between the tip and that surface. Therefore, regulation of the tip-sample distance is crucial in constructing SECM maps of the electrochemical activities of a sample surface. A tilted or uneven substrate, producing a distorted SECM image, is a recurring problem. Among the various tip-sample distance regulation methods, shear-force feedback, commonly used in SNOM,20,21 was recently applied for controlling the distance between a SECM tip and a substrate using a tuning folk22,23 and Fresnel diffraction by focusing a laser beam on the tip.15,24 Since shear-force regulation enables the investigator to maintain a constant distance between the tip and substrate, tilted or uneven substrates can be imaged. Two types of scanning modes using shear force based distance control have been reported. In the first, while the tip moves at a constant speed along a surface, its vertical position is adjusted using a closed feedback loop to maintain a constant shear force. In the second type, Bard and co-workers proposed a tapping-like scanning mode, featuring a repeated approach and retraction of (12) Shiku, H.; Uchida, I.; Matsue, T. Langmuir 1997, 13, 7239-7244. (13) Shiku, H.; Takeda, T.; Yamada, H.; Matsue, T.; Uchida, I. Anal. Chem. 1995, 67, 312-317. (14) Nowall, W. B.; Wipf, D. O.; Kuhr, W. G. Anal. Chem. 1998, 70, 26012606. (15) Kranz, C.; Gaub E., H.; Shuhmann, W. Adv. Mater. 1996, 8, 634-637. (16) Kranz, C.; Friedbacher, G.; Mizaikoff, B.; Lugstein, A.; Smoliner, J.; Bertagnolli, E. Anal. Chem. 2001, 73, 2491-2500. (17) Macpherson, J. V.; Unwin, P. R. Anal. Chem. 2001, 73, 550-557. (18) Macpherson, J. V.; Jones, C. E.; Barker, A. L.; Unwin, P. R. Anal. Chem. 2002, 74, 1841-1848. (19) Macpherson, J. V.; Mussy, J.-P. G. d.; Delplancke, J.-L. J. Electrochem. Soc. 2002, 149, B306-B313. (20) Karrai, K.; Grober, R. D. Appl. Phys. Lett. 1995, 66, 1842-1844. (21) Mulin, D.; Vannier, C.; Bainier, C.; Courjon, D.; Spajer, M. Rev. Sci. Instrum. 2000, 71, 3441-3443. (22) James, P. I.; Garfias-Mesias, L. F.; Moyer, P. J.; Smyrl, W. H. J. Electrochem. Soc. 1998, 145, L64-L66. (23) Oyamatu, D.; Kanaya, N.; Mase, Y.; Nishizawa, M.; Matsue, T. Bioelectrochemistry 2003, 60, 115-122. (24) Hengstenberg, A.; Kranz, C.; Schuhmann, W. Chem. Eur. J. 2000, 6, 15471554.

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the tip at each data point.25 During this approach and retraction, the horizontal movement is stopped; the current is acquired when the tip is retracted. In this paper, we used a scanning mode similar to this tapping mode. Immobilization of biofunctional molecules such as enzymes onto substrates is essential for fabricating various biosensors. SECM has been employed, for example, to characterize proteinimmobilized surfaces.23,26-29 Here, we report SECM imaging, using shear force based tip-surface distance regulation, of a platinumpatterned array electrode and of diaphorase/albumin coimmobilized glass surfaces. We used the standing approach mode, which repeats the approach and retraction at each data point of the surface, to obtain simultaneously both a current image and a topographic image. EXPERIMENTAL SECTION Materials. Reduced nicotinamide adenine dinucleotide (NADH) (Oriental Yeast Co.), glutaraldehyde (Wako Pure Chemical), and (3-aminopropyl)trimethoxysilane (Shin-etsu Chemical Industry) were used as received. Ferrocenylmethanol (FMA) was synthesized by reduction of ferrocenecarboxyaldehide (Aldrich) with NaBH4 and recrystallized twice from hexane. (Ferrocenylmethyl)trimethylammonium perchlotrate was synthesized by adding saturated sodium perchlorate solution to a solution of (ferrocenylmethyl)trimethylammonium bromide (purchased from Tokyo Kasei). Diaphorase purified from Bacillus stearothermophilus (EC 1.6.99.-) (Seikagaku Co.) and bovine serum albumin (Wako Pure Chemical) were used as received. All the aqueous solutions were prepared with water purified by Milli-Q Jr. (Millipore Co.). The platinum-patterned array electrode was a gift from Dr. Matsue, Tohoku University. Preparation of Diaphorase Immobilized Glass. The glass slides (10 × 10 mm2) were cleaned thoroughly by dipping them in a mixture of equal volumes of 95% H2SO4 and 60% HNO3 solution (CAUTION: this solution is a very strong acid and very dangerous to handle in the laboratory. Protective equipment including gloves goggles should be used at all time) for 30 min, followed by a washing with distilled water for 10 min in a supersonic bath. The clean slides were immersed in a 10 mM (3-aminopropyl)trimethoxysilane/benzene solution overnight and then washed with benzene, ethanol, and distilled water for 10 min in a supersonic bath. The silanized slides were dipped into a 1% (v/v) glutaraldehyde aqueous solution for 2 h. After washing with distilled water for 10 min in the supersonic bath, the slides were immersed overnight in a 0.1 mM diaphorase solution or a mixture of 0.1 mM diaphorase and 0.1 mM albumin solution and washed in a phosphate buffer (pH 7.5) three times. Since it is difficult to know the molar ratio of immobilized diaphorase/albumin at the surface, we used the molar ratio in the solution as a measure. Fabrication of Microelectrode. To fabricate the platinum microdisk electrode, we pulled a lead glass (World Precision (25) Lee, Y.; Ding, Z.; Bard, A. J. Anal. Chem. 2002, 74, 3634. (26) Zhou, J.; Campbell, C.; Heller, A.; Bard, A. J. Anal. Chem. 2002, 74, 40074010. (27) Wittstock, G.; Wilhelm, T.; Bahrs, S.; Steinru ¨ cke, P. Electroanalysis 2001, 13, 669-675. (28) Wijayawardhana, C. A.; Wittstock, G.; Halsall, H. B.; Heineman, W. R. Anal. Chem. 2000, 72, 333-338. (29) Shiku, H.; Hara, Y.; Matsue, T.; Uchida, I.; Yamauchi, T. J. Electroanal. Chem. 1997, 438, 187-190.

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Instruments, PG10150-4) with a capillary puller (Narishige, Tokyo model PC-10) and inserted a platinum wire (diameter 10 µm) in the capillary. The tip was fused by a microforge (Narishige, Tokyo model MF-900) and polished, first on a turntable (Narishige, Tokyo model EG-4) and finally with a fine emery paper (Sumitomo 3M, Tokyo 15000) to obtain a disk-shaped electrode. The tip radius, including the insulating glass, was 20 µm. To obtain a microdisk electrode smaller than 10 µm, a 30-µm platinum wire was etched electrochemically in a saturated KCl solution in order to sharpen the wire. After the tip was fused to encase the sharpened Pt filament completely within the lead glass, the tip was polished with 0.05-µm alumina powder (Buehler Micorpolish II) on a homemade turntable until the sharpened Pt apex was exposed. The Pt disk radius of the electrode was determined from the steady-state current of FMA or FA+ in a voltammogram. The tip and Pt disk radii were 2-10 and 0.1-2 µm, respectively. Fabrication of a Shear Force Based SECM Probe. After cutting the tip of the microelectrode fabricated above, we glued the 4-mm-long tip with epoxy resin along one of the prongs of a crystal quartz tuning fork (Citizen, CFS-308). The tip protruded 2∼3 mm out of the prong’s end. To excite the mechanical resonance of the fork, it was soldered onto the diaphragm of a commercially available piezoelectric buzzer. The tip of the probe was washed with water in an ultrasonic bath just before the SECM measurement. The tuning fork and the tip of the electrode vibrated parallel to the sample surface at the fork’s resonance frequency of 20-30 kHz. Only the tip of the microelectrode was dipped into the solution for 0.5-1.5 mm during SECM measurement. SECM Apparatus. A SECM system (Hokuto Denko, HV401) was modified in order to use shear force based tip positioning. A piezoelectric actuator (Piezomechanik GmbH, model PSt 150/7/ 20 VS12) was mounted on a stepping motor-driven XYZ stage (Suruga seiki, KS701-20LHD) to perform the micromovement of the probe along the Z-direction. The stroke of the piezoelectric actuator was 20 µm at an applied voltage of 150 V. The tuning fork’s signal was amplified with a lock-in amplifier (Signal Recovery, model 7264); the signal amplitude was then digitized and transferred to a computer (Toshiba Dynabook) with a 16-bit AD/DA board (Interface, AZI3506). The measurement was carried out in a three-electrode configuration; the reference and counter electrodes were Ag/AgCl immersed in a saturated KCl solution and a Pt wire (diameter 0.5 mm), respectively. Current-Distance Profile. The substrate was immersed in a 0.5 mM FMA, 0.05 M phosphate buffer (pH 7.5) solution. The tip of the probe was placed above the glass slide and set at 0.4 V versus Ag/AgCl. An ac voltage at the resonance frequency was applied to the diaphragm of the piezoelectric buzzer, and the voltage was adjusted such that the amplitude of the voltage generated by the tuning fork was 0.45 mV. The tip was slowly lowered toward the surface by increasing the voltage of the piezoelectric actuator while measuring the output signal of the tuning fork until its amplitude was decreased to 98% of its original amplitude. The tip was raised 100 µm for the 10-µm Pt disk microelectrode and 30 µm for the 2.2-µm Pt disk microelectrode, respectively, at 1 µm/s, while acquiring the current at 0.1-s intervals. Scanning Mode for SECM Imaging. We used two types of scanning methods to image electrochemical activity and topo-

Figure 1. Schematic representation of the voltage applied to the piezoelectric actuator in the standing approach mode. The lateral movement of a probe tip was stopped at periods a-c. The probe was moved laterally at period d, when the tip was retracted to avoid a collision between the tip and sample.

graphic data simultaneously. The first, which has been described as a shear force based SECM or SNOM, is called the feedback mode. While scanning along a surface, the tip is moved vertically, using a closed feedback loop, to maintain a constant shear force. Since the position of the tip is always very near the surface (typically between 10 and 30 nm), the distance between the tip and surface is sometimes too close for the conventional SECM tip (a few micrometers), and the tip may destroy the morphology of a surface such as an enzyme monolayer. In the feedback mode, the tip scans at a constant speed and the current is measured at fixed time intervals. On the other hand, the second scanning method, called the standing approach mode, features repetitive approaches to the surface and retractions of the tip for sampling the current at each point. The lateral movement of the tip is stopped during the data sampling sequence. Figure 1 shows typical sequences of the voltages applied to the piezoelectric actuator. A starting amplitude of the tuning fork is measured at interval a; then, the tip is lowered to the surface at a rate of 0.5 µm/s, achieved by increasing the voltage applied to the piezoelectric actuator (interval b) until the amplitude of the tuning fork was damped to a level 98% of the original amplitude. The tip is lifted up for 0.05-2 µm (the height depends on the radius of the microelectrode) to acquire the current (interval c). When moving laterally to the next sampling point with the aid of the stepping motor (interval d), the tip vibrates at the beginning and end of the movement as a consequence of the vibration of the motor. Therefore, the tip is retracted ∼2 µm upward to avoid contacting the surface during lateral movement. It takes ∼1 s to get a data point without contacting the surface. Since it takes ∼40 s for acquiring 40 data points, the apparent scan speed is 10 µm/s for a 400-µm line scan. All of the SECM images were obtained with the standing approach mode unless otherwise noted. Quantitative Analysis of the Current Response at an Immobilized Diaphorase Surface. Digital simulation was used to analyze the current-distance profiles and the SECM images obtained from immobilized diaphorase surfaces. The details of the digital simulation were described in a previous paper.13 For the 100% immobilized diaphorase substrate, the experimental current-distance curve was reproduced theoretically. The current response depends on the surface concentration of the diaphorase (ΓDP). Figure 2 illustrates this dependence of the simulated current when the distance is fixed. The ΓDP value was determined with this working curve, and the SECM images were converted to surface concentration profiles.

Figure 2. Theoretical current dependence on the surface concentration of diaphorase (ΓDP) at 2 µm off a substrate surface. The Pt electrode diameter and the whole probe diameter (including insulator) were 10 and 20 µm, respectively.

Figure 3. Frequency response curves of the tuning fork with a microelectrode acquired in air (solid line) and in a solution (dashed line). The depth of immersion of the tip was 1 mm. Inset: a frequency response curve of the tuning fork without a microelectrode.

RESULTS AND DISCUSSION Frequency Response of the Tuning Fork. Figure 3 shows the amplitude of the output signal of the tuning fork as a function of the driving frequency, in air and in the solution with the microelectrode tip glued along one side of the prong. The tuning fork without the microelectrode tip has a Q factor of 2400 in air. When a Pt microelectrode tip was glued along one side of the prong, the Q factor was reduced to 40-200. Since the tip of the microelectrode vibrates in a solution, horizontal vibrations may lead to an increase in the Faraday current for a submicrometer electrode that prevents a theoretical analysis of the current. When the amplitude of the voltage generated by the tuning fork was larger than 10 mV, a significant increase (typically ∼10% increase compared to original value) of the Faraday current was observed. Therefore, the voltage applied to the piezoelectric buzzer was adjusted to keep the amplitude of the tuning fork such that the voltage produced was 0.45 mV. SECM Imaging of a Pt Array Electrode. Figure 4 shows a photograph of a Pt array electrode. The thickness of the Pt layer was ∼0.15 µm. Figure 5 shows SECM images of the area enclosed with a broken line in Figure 4 (400 × 400 µm2 (a) and (a′)) and the area enclosed with a solid line in the same figure (80 × 80 µm2 (b) and (b′)); these images were obtained with the 10- (a and b) and 0.3-µm (a′ and b′) microelectrode tip in a 0.5 mM FMA solution. The current was acquired at distances of 2 and 0.05 µm from the surface for the 10- and 0.3-µm Pt microelectrodes, respectively. The number of data points was 40 × 40 in all images. Figure 6 shows the topographic images obtained simultaneously with the SECM images in Figure 5. The height of the Analytical Chemistry, Vol. 77, No. 6, March 15, 2005

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Figure 4. Photograph of a Pt-patterned array electrode. The thickness of the Pt layer was 0.15 µm. An area enclosed by the dashed line was imaged, and its SECM images were shown in Figure 5a and a′. The SECM images of the area enclosed by the solid line are shown in Figure 5b and b′.

Figure 5. SECM images, (a and a′) 400 × 400 µm2 and (b and b′) 80 × 80 µm2 in size, of a Pt-patterned array electrode, taken with 10- (a and b) and 0.3-µm Pt microelectrodes (a′ and b′) in 0.5 mM FMA solution. All images were 40 × 40 data points in size.

topographic images was calculated from the applied voltage of the piezoelectric actuator. Since the time taken to capture the SECM image in the feedback mode depends on the scanning rate of the tip, it requires a long time to cover a large area. Usually a rate of a few micrometers per second is used for the feedback mode to avoid contact between the tip of the probe and the surface; it takes 3-4 h for scanning an area of 400 × 400 µm2 with a 10-µm resolution. On the other hand, the data acquisition time of the standing approach mode depends on the total number of data points. The standing approach mode is capable of scanning 400 × 400 µm2 with a 10-µm resolution in ∼30 min. It was reported that the tip can be damaged during the scan because of frequent contact with the substrate in the tapping-like scanning mode.25 However, no significant deterioration was observed during the scan in our scanning mode. Should the tip contact the substrate surface, the amplitude of the tuning fork would be damped significantly. In our experiment, the amplitude of the tuning fork was monitored by an oscilloscope during the scan, and no significant damping was observed. But when the tip was moved with the stepping motor without the tip having been retracted, the amplitude of the tuning fork was damped to a level less than 90% of the original amplitude, a change indicating a contact with the substrate. Therefore, we determined that our system success1788 Analytical Chemistry, Vol. 77, No. 6, March 15, 2005

Figure 6. Topographic images, (a and a′) 400 × 400 µm2 and (b and b′) 80 × 80 µm2 in size, of a Pt-patterned array electrode, taken with 10- (a and b) and 0.3-µm Pt disk microelectrodes (a′ and b′), obtained simultaneously with SECM images, in 0.5 mM FMA solution. The height at each data point was calculated from the voltage applied to the piezoelectric actuator.

fully imaged the electrochemical activities and topography of the surface without contacting the sample. The lateral resolution of the SECM and the topographic image was improved using the 0.3-µm microelectrode tip as shown in Figure 4b′ and Figure 5b′. Recently, the use of submicrometerto nanometer-sized electrodes for SECM was reported to improve lateral resolution.16,18,30 A combined SECM-AFM cantilever technique proved to be a powerful tip positioning system for a nanometer-sized electrode and gold grating;16 a 10-µm Pt disk17 and a nanometer-scale pore18 were successfully imaged with a SECM-AFM. Our system produces images with a lateral resolution, comparable to those of the SECM-AFM system. Usually, a commercial AFM system such as a Nanoscope is employed as the SECM-AFM system; in these, the scanning area is limited by the AFM system scanner. In our system, a stepping motor-driven XYZ stage, having a full stroke of 20 mm (on each axis) with a 0.05-µm resolution, was used for the lateral scanning along the sample. Although the actual accuracy of the stage is 0.3 µm, it is fine enough for our present system, which covers a wide range of scanning areas (from tens of micrometers to a few millimeters). Current-Distance Profile of an Immobilized Diaphorase Surface. Figure 6 shows the current-distance profiles obtained with the 10- (a) and 2.2-µm (b) Pt disk microelectrodes in a 0.5 mM FMA solution with and without NADH. The current was normalized by dividing it by the current obtained at a distance far from the surface. Since the diaphorase immobilized at the glass surface catalyzes the reduction of the oxidized form of FMA (FMA+) by NADH to form FMA, the tip current increases with a rise in the NADH concentration (cNADH) up to 1 mM. At more than 1.0 mM, the current does not depend on cNADH. Therefore, we added 1.6 mM NADH to the solution to obtain SECM images of the diaphorase activity. In the presence of excess NADH, the current-distance profile was reproduced reasonably well by the digital simulation (open squares in Figure 7. SECM Imaging of Coimmobilized Diaphorase/Albumin Surface. Figure 8 shows the SECM images of the coimmobilized (30) Sun, P.; Zhang, Z.; Guo, J.; Shao, Y. Anal. Chem. 2001, 73, 5346-5351.

Figure 7. Current-distance profiles of a immobilized diaphorase glass, taken with (a) 10- and (b) 2.2-µm Pt disk microelectrodes in 0.5 mM FMA solution, in the presence and absence of NADH in the following concentrations: 0 (solid line), 0.1 (dash line), 0.2 (dot line), 0.4 (dash dot line), and 1.6 mM (dash dot dot line).

Figure 8. (a) SECM image, 400 × 400 µm2 in size, of a diaphorase/ albumin (1:1) coimmobilized glass in 0.5 mM FMA, 1.6 mM NADH solution; and (b) 400 × 200 µm2 SECM image of enclosed area in image a without NADH.

diaphorase/albumin (molar ratio, 1/1) surface in the presence of 1.6 mM NADH (a) and in its absence (b). In the latter case, the current response was constant at all points. Since the current response depends on the surface concentration of diaphorase in the presence of excess NADH, the SECM image in Figure 8a indicates that the diaphorase and albumin molecules were not immobilized uniformly. When diaphorase only was immobilized at the surface, the variation of the current in the SECM image was within ∼10%. Therefore, the diaphorase was immobilized uniformly. Using the working curve shown in Figure 2, the SECM images are easily converted to the surface concentration of diaphorase. Figure 9 depicts maps of the surface concentration of diaphorase in the coimmobilized diaphorase/albumin glass prepared

Figure 9. Concentration profiles of a coimmobilized diaphorase/ albumin (1:1) glass in 0.5 mM FMA, 1.6 mM NADH solution with (a) 10- and (b) 2.2-µm Pt microelectrodes. The distance between the tip and the surface was 2 µm for the 10-µm Pt disk and 0.5 µm for the 2.2-µm Pt disk microelectrode.

from a 1:1 molar mixture of diaphorase and albumin obtained with the 10- and 2.2-µm Pt microelectrodes. The area enclosed by the solid line in Figure 9a was scanned with the 2.2-µm Pt microelectrode (Figure 9b). The surface concentration of diaphorase in both images was quite similar using the microelectrodes with different radii. A phase separation-like morphology was always observed at 1:1 and 1:2 molar ratios of the diaphorase/albumin. The average surface concentration of diaphorase (ΓDPav) was found to be 7.9 × 10-13 mol/cm2 for a coimmobilized diaphorase/albumin surface with a molar ratio of 1:1 and 1.4 × 10-13 mol/cm2 for that with a molar ratio of 1:2. In our previous study, the surface concentration of a 100% diaphorase immobilized surface was found to be 3.0 × 10-12 mol/ cm2.31 If the diaphorase molecule is similar in size to an albumin molecule, the expected ΓDPav should be larger than the observed value. The proteins’ immobilization process is considered to be a competitive reaction between a surface-bound carbonyl and an amino group of the protein dissolved in the solution. Since, at the moment, there is no information on the reactivity of diaphorase and albumin, a further study of the dependence of the ΓDPav value on the concentration of the proteins in the solution may reveal the origin of the discrepancy between the expected and observed ΓDPav and the reason for the phase separation-like morphology. Comparison of Scanning Modes. In the previous section, we discussed our successful imaging by the standing approach mode of the surface concentration of the coimmobilized diaphorase/albumin surface. To compare the difference between the scanning modes, we imaged the area after scanning by the feedback mode. An SECM image (400 × 400 µm2) was obtained (31) Yamada, H.; Shiku, H.; Matsue, T.; Uchida, I. Bioelectrochem. Bioenerg. 1994, 33, 91-93.

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Figure 10. SECM images, (a and c) 400 × 400 µm2 in size and (b) 200 × 200 µm2 in size for a coimmobilized diaphorase/albumin (molar ratio 1:1) surface with a 10-µm Pt disk microelectrode in a 0.5 mM FMA and 1.6 mM NADH solution. SECM image b was obtained after imaging in the standing approach mode, and SECM image c was obtained after imaging in the feedback mode.

by the standing approach mode (Figure 10a); then, the area enclosed by the solid line in Figure 10a (200 × 200 µm2) was scanned by the feedback mode. In the latter mode, the tip was moved at 10 µm/s in the X-direction for 200 µm; after a line scan, the tip was taken 10 µm in the Y-direction to image a 200 × 200 µm2 area. The height of the tip was adjusted by the piezoelectric

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actuator to maintain the amplitude of the tuning fork at 98% of its original amplitude. After the scanning in the feedback mode, the same area in Figure 10a was imaged by the standing approach mode (Figure 10c). Since the distance between the tip and surface was too close when the tip was moved laterally in the feedback mode, the tip of the microelectrode hindered the diffusion of the NADH. Therefore, the current response in Figure 10b was small compared to that in Figure 10a, and the resultant feedback mode image showed no significant concentration difference. Indeed, most of the diaphorase molecules immobilized on the glass were removed by the strong interaction between the tip and surface during the feedback mode scan. The removal of diaphorase molecules was presumably caused by the collision between the probe tip and the sample surface. This collision was probably due to our use of the stepping motor-driven stage to move the tip laterally; that motor might have vibrated during the lateral scan. The rate of the tip’s lateral movement might have been too fast to keep the height of the tip constant. In conclusion, we successfully used a shear force based tipsubstrate positioning SECM system to image a Pt-patterned array electrode. We were able to simultaneously determine the electrochemical activity and take topographic images with a 10- and a 0.3-µm Pt disk microelectrode. In addition, we observed a coimmobilized diaphorase/albumin glass surface by a standing approach mode, avoiding contact between the tip and the substrate. Our SECM system can be useful for the imaging of an uneven or tilted substrate over a wide range of scanning areas. ACKNOWLEDGMENT We thank Dr. Matsue, Tohoku University, for helpful advice concerning fabricating SECM probes using a piezoelectric buzzer and tuning fork.

Received for review September 24, 2004. Accepted December 29, 2004. AC048582G