confocal microscope

Sensors and Actuators B 91 (2003) 199–204

Electrochemical/photochemical formation of enzyme patterns on glass substrates using a scanning electrochemical/confocal microscope Daisuke Oyamatsua, Norihiro Kanayaa, Hiroshi Shikub, Matsuhiko Nishizawaa, Tomokazu Matsuea,* a

Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, 07 Aramaki-Aoba, Sendai 980-8579, Japan b Regional Research Project of Yamagata, 2-2-1 Matsuei, Yamagata 990-2473, Japan

Abstract A hybrid system consisting of a scanning electrochemical microscope (SECM) and scanning confocal microscope (SCFM) was fabricated and used for micropatterning and imaging of diaphorase immobilized on a glass substrate. Simultaneous imaging of the diaphorase spots demonstrated that the SECM can provide information on a localized enzyme reaction, while the SCFM affords information on the location of the active enzyme. By using this SECM/SCFM system, spatially selective deactivation of diaphorase was performed by inducing a local electrochemical reaction or by irradiating with the focused laser. The resulting patterns of diaphorase were simultaneously imaged with the SECM/SCFM system. # 2003 Elsevier Science B.V. All rights reserved. Keywords: SECM; SCFM; Patterning; Diaphorase

1. Introduction The development of analytical methods for proteins in biological fluids such as blood, urine and tissue extracts has received much attention in advanced biotechnology. Among the various requirements for the analytical methods, twodimensional imaging of the protein distribution with high sensitivity and selectivity is of particular importance for investigating complicated biochemical processes. Such imaging systems have also been used for analyzing biochips such as DNA chips and protein chips. Electrochemical detection using a scanning electrochemical microscope (SECM) has been found to be suitable for trace analyses based on biochips [1,2]. SECM is a scanning probe microscope (SPM) that detects localized electrochemically active species to afford two- or three-dimensional images based on the distribution of these species. Although SECM itself is a powerful tool for disclosing localized processes, the combination of SECM with another SPM enables the simultaneous measurements of multiple properties and provides more detailed information in a local region, as demonstrated by the hybrid systems using the SECM and atomic force microscope (AFM) [3–6].

In this paper, we report a novel hybrid system comprised of the SECM and scanning confocal microscope (SCFM) and its applicability to patterning and imaging localized enzymes. The SECM is capable of directly detecting the electrochemical redox reactions catalyzed by enzymes with extremely high sensitivity. SECM affords information on the effective enzyme activity in addition to the distribution of enzymes. SCFM gives a higher resolution than any other conventional optical microscopes, especially in the depth direction. One of the important features of SCFM is that it does not require a probe. The SECM/SCFM hybrid system simultaneously provides both electrochemical and optical information. Since the electrochemical and optical units in the present system are independently controlled, it is also possible to use either probe properly for measurement or stimulation. In this study, the electrochemical and photochemical patternings of immobilized enzymes were conducted by using the SECM/SCFM system, and the enzymatic activity of the resulting patterns was evaluated simultaneously by the SECM/SCFM imaging.

2. Experimental 2.1. Chemicals

* Corresponding author. Tel./fax: þ81-22-217-7209. E-mail address: [email protected] (T. Matsue).

Diaphorase (Dp) purified from Bacillus stearothermophilus (EC 1.6.99) was purchased from Unitika Ltd. This enzyme has

0925-4005/03/$ – see front matter # 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0925-4005(03)00089-3

200

D. Oyamatsu et al. / Sensors and Actuators B 91 (2003) 199–204

Fig. 1. Schematic diagram of the SECM/SCFM system.

a molar mass of about 30,000 and has one flavin mononucleotide (FMN) as an electroactive site per molecule [7]. Ferrocenemethanol (FMA) was purchased from the Aldrich Chem. Co. and recrystallized from hexane. All other chemicals including the 25% glutaraldehyde solution (GA, Wako Pure Industries Ltd.) and b-nicotinamide-adenine dinucleotide, the reduced form (NADH, Orient Yeast Co. Ltd.) were used as received. All the solutions were prepared using purified with a Milli-Q II System (Millipore Co.). 2.2. SECM/SCFM system The schematic diagram of the SECM/SCFM system is shown in Fig. 1. This measurement system consists of a confocal microscope, current amplifier for the electrochemical measurements, a piezoelectric XYZ stage and a personal computer equipped with AD/DA converters and pulse counter for the measurement of fluorescence. The confocal microscope and XYZ stage were set on a vibration-proof desk. The confocal system is based on an inverted microscope equipped with a filter block, and Nikon B2A (DM 505, EX 450–490, BA 520). The laser beam from a solid state laser unit (wavelength ¼ 473 nm, SHIMADZU HK-5510) was introduced through the optical fiber and focused on the sample surface by an oil immersion objective (Nikon CFI super fluor, magnification 40, N.A. 1.30) as shown in Fig. 1. For the detection of fluorescence, an avalanche photodiode (APD, SPCM-AQR-14 NEopt Co.) was attached to the side port of the microscope. Since the size of the active area of the APD is smaller than Ø50 mm, the system serves as a confocal microscope without using a pinhole in front of the detector. The intensity of the fluorescence obtained as digital pulses (28.9 ns width, TTL level) was counted using a pulse counting

board (Interface AZI-6102). The intensity of the laser at the surface of sample was 2 mW, as measured by a laser power meter (Neo Ark Co. Ltd.). Since the area of the focus at the wavelength of 473 nm was 0.416 mm2, the energy density of the laser was calculated as 4:81  105 W cm2 . For imaging of the patterned enzymes with SCFM, the ND filters (Suruga Seiki Co. Ltd. and Shigma Koki) were inserted into the optical path to minimize any undesirable photochemical deactivation of the enzyme. The scanning of the laser spot was carried out by moving the sample using the XYZ piezoelectric scanner (P-517.3CL Physik Instrumente), while the optical path was fixed. The motion range of the XYZ piezoelectric scanner was 100 mm  100 mm  20 mm. The stage was driven by the high voltage power supply controlled by voltage signals (0–10 V) from the AD/DA converter. The microelectrode was prepared by sealing a Pt wire into a glass capillary by heating, followed by polishing on a turntable (Narishige, Model EG-6) to give a disk-type microelectrode (electrode radius 5 mm, outer radius 10 mm). A 0.4 ml droplet of the measurement solution (0.1 mol dm3 Na2HPO4, 0.5 mmol dm3 FMA, 5 mmol dm3 NADH and 25 mmol dm3 KBr) was put on the substrate surface. The microelectrode was then positioned just above the focus of the laser with a three-dimensional micromanipulator (Narisige MM-200). The current at the microelectrode was monitored with a current amplifier (KEITHLEY 427) and converted to digital data using a 16 bit AD/DA board (Interface AZI-3506). 2.3. Preparation of diaphorase-patterned substrate Micro-cover glasses (thickness 0.12–0.17 mm, radius 11 mm) were immersed into the 10 mmol dm3 3-amino-

D. Oyamatsu et al. / Sensors and Actuators B 91 (2003) 199–204

propyl triethoxysilane/benzene solution for 8 h. They were rinsed with dehydrated benzene and ethanol, followed by ultrasonic cleaning with ethanol and distilled water for 10 min. The patterning of diaphorase onto the glass substrates were carried out by three different methods. The first method is spotting of the enzyme solution (0.15 mg/ml diaphorase, 10 mg/ml glutaraldehyde aqueous solution) onto the aminosilanized substrate using a glass capillary (tip radius 20 mm). The second is the laser-induced patterning. The clean glass substrate was spin-coated with the diaphorase-solution followed by being kept at room temperature for 2 h to allow an intermolecular bridging reaction. The resulting substrate was ultrasonically cleaned in purified water, and then subject to the laser-induced patterning. The third is the electrochemical patterning. The diaphorase spin-coated substrate was immersed in an aqueous solution containing 25 mmol dm3 KBr, 0.5 mmol dm3 FMA and 0.1 mol dm3 Na2HPO4, and the microelectrode tip in SECM/SCFM system was located close to the substrate. An oxidation potential (1.5 V vs Ag/AgCl) was then applied to the tip to generate a highly reactive species (HOBr) which deactivated the diaphorase molecules nearby. The enzymatic activity of the resulting substrate was imaged with the SECM/SCFM system. 2.4. Imaging of diaphorase activity with SECM/SCFM system Fig. 2 shows the schematic diagram of the reaction system studied in the present work. Diaphorase catalyzes the electron transfer from NADH to a suitable acceptor molecule. In the present system, FMA, an electron mediator, is oxidized to FMAþ at the microelectrode tip (0.5 V vs Ag/AgCl) and diffuses to the immobilized diaphorase. The diaphorase catalyzes the oxidation of NADH to regenerate FMA which is oxidized again at the tip. This ‘‘redox cycling’’ between the electrode tip and the substrate surface increases the oxidation current of FMA. For the SCFM imaging, the sample was irradiated with a 473 nm laser. The FMN in the active center of the diaphorase emits a strong fluorescence which is detected by the SCFM system.

201

3. Results and discussion 3.1. Simultaneous SECM/SCFM imaging of diaphorase spots Fig. 3 shows the SCFM and SECM images of diaphorase spots prepared by the spotting method with a glass capillary. The four diaphorase spots on the substrate emit strong fluorescence both in the absence and presence of NADH in solution ((a) and (b)). However, a careful observation reveals that the fluorescence intensity with NADH in solution is slightly lower than that without NADH. This is confirmed from the cross-sectional view of the fluorescence along the line A–B in the SCFM images. The addition of NADH reduces diaphorase and lowers the fluorescence emission from the diaphorase spots. These results indicate that a part of the diaphorase molecules is reduced by NADH, and the SCFM images surely reflect the distribution of the active center of the diaphorase molecules. The spectroscopic measurements indicate that the fluorescence intensity of the reduced diaphorase at 530 nm is found to be about 60% of that of the oxidized diaphorase. In contrast, the SECM images (Fig. 3(d) and (e)) of the same substrate show a different feature. No current response was observed in the absence of NADH, while four circular regions appear after the addition of NADH. The diaphorasecatalyzed oxidation of NADH induces the redox cycling and increases the oxidation current for FMA at the tip. The four regions in the SECM images reflect this actual enzymatic activity of the immobilized diaphorase. Without NADH, the diaphorase-catalyzed reaction cannot be detected, thus no spot in the image occurs. The comparison of the four images in Fig. 3 leads to an important difference in the SECM and SCFM imaging. The SECM images the localized enzymatic reaction and the SCFM images the localized enzyme. An enzyme without doing its enzymatic reaction is invisible to SECM, since SECM imaging is based on the detection of chemical species related to the enzymatic reaction. Another significant difference between the SECM and SCFM images is the size of the diaphorase spots. The diaphorase spots in the SECM image are large compared with those in the SCFM image because the diffusion of FMA regenerated from the diaphorase spots enlarge and hides the actual size of the spot. SCFM, on the other hand, detects the fluorescence emitted from the diaphorase molecules immobilized at the objective focus, giving specially resolved, clear images. 3.2. Laser-induced patterning for the diaphoraseimmobilized substrate

Fig. 2. Schematic diagram of the diaphorase-catalyzed reaction.

The FMN moiety, an active component of diaphorase, is photo-degraded by irradiating extremely strong light such as a focused laser beam. By using this photo-degradation phenomenon, we fabricated patterns of active diaphorase on a glass substrate. The patterning of active diaphorase was carried out by irradiating a blue laser modulated with a

202

D. Oyamatsu et al. / Sensors and Actuators B 91 (2003) 199–204

Fig. 3. (a) and (b) SCFM images of diaphorase spots obtained before (a) and after (b) the addition of 5 mmol dm3 NADH to the aqueous solution. (c) Crosssectional view of fluorescence along the line A–B in the SCFM images shown in (a) and (b). (d) and (e) SECM images of diaphorase simultaneously observed with image (a) and (b), respectively (scan rate of microelectrode, 10 mm s1; electrode potential, þ0.5 V vs Ag/AgCl; measurement solution, 0.1 mol dm3 Na2HPO4 and 0.5 mmol dm3 FMA).

Fig. 4. (a) Picture image (100  100 pixels) used as the master for the laser-induced patterning. The shutter was electrically controlled during the scanning of the substrate. (b) SCFM image of diaphorase-immobilized substrate taken after the laser-induced patterning. (c) SECM image obtained simultaneously with image (b). The conditions of the SCFM/SECM observations were the same as those given in Fig. 3.

D. Oyamatsu et al. / Sensors and Actuators B 91 (2003) 199–204

203

Fig. 5. (a) SCFM image of diaphorase-immobilized substrate observed after the electrochemical patterning. The patterning was conducted by applying the potential of þ1.5 V vs Ag/AgCl to the Pt microelectrode in an aqueous solution containing 25 mmol dm3 KBr, 0.1 mol dm3 Na2HPO4, 0.5 mmol dm3 FMA and 5 mmol dm3 NADH. (b) SECM image simultaneously obtained with the SCFM image shown in (a). All other conditions of the SCFM/SECM observations were the same as those given in Fig. 3.

mechanical shutter in an optical path during scanning. The master for the laser patterning was a black-and-white picture image which has 100  100 pixels as shown in Fig. 4(a), and the laser shutter was opened at the points corresponding to the black pixel in the master during scanning over the diaphorase-immobilized substrate. The scan rate of the substrate was 1 mm s1, and drawing this image required about 30 min. Fig. 4(b) shows the SCFM image observed after conducting the laser patterning. For the SCFM imaging, the blue laser was set at a lower power to minimize the undesirable photo-induced damage of diaphorase. As shown in this image, the region where the fluorescence is diminished was observed in the shape of an ‘‘A’’ letter, corresponding to the laser-irradiated area. This result indicates that the irradiation of the focused laser degrades the fluorescence moiety in diaphorase on the glass substrate. Fig. 4(c) shows the SECM image of the laser-induced diaphorase pattern simultaneously obtained with the SCFM image (Fig. 4(b)). The region of the letter ‘‘A’’ shows slightly smaller oxidation currents than the remainder, indicating that the enzymatic activity in the laser-irradiated area was also degraded. However, the outline of the ‘‘A’’ letter in the SECM is considerably fuzzier than that in the SCFM. This is probably caused by undegraded diaphorase molecules in the ‘‘A’’ region, as is proved by the many bright spots due to the aggregation of diaphorase in the SCFM image. Therefore, the contrast of the SECM image became unclear because the redox cycling proceeds between the microelectrode and the surviving active diaphorase molecules in the irradiated area. This problem will be solved by extension of the irradiating period or preparation of a substrate on which diaphorase is uniformly immobilized such as a monolayer. 3.3. Electrochemical patterning for the diaphorase-immobilized substrate The SECM system can be used not only for the detection of the local concentration of electrochemically active species, but also as the method to induce electrochemical reactions in a localized area on the substrate surface [8–12].

We have attempted here to generate patterns of active diaphorase on a glass substrate by using the electrochemical reaction occurring at the tip of microelectrode. Fig. 5 shows the simultaneously observed SCFM and SECM images of the diaphorase-patterned substrate in an aqueous solution containing 0.5 mmol dm3 FMA and 5 mmol dm3 NADH. The diaphorase patterns were generated by applying the potential (1.5 V vs Ag/AgCl) to the microelectrode tip located close (5 mm) to the diaphoraseimmobilized substrate. Br in the solution is oxidized at the tip to form Br2, which quickly reacts with H2O to generate HOBr, a strong oxidant. This electrochemically generated HOBr diffuses onto the substrate and deactivates the diaphorase molecules immobilized on the substrate [7,13]. In the SCFM image, a decrease on fluorescence was observed in the four circular regions which correspond to the spots of the electrochemical deactivation of diaphorase. The simultaneous SECM imaging also clearly indicates the four spots with low enzymatic activity, although the outlines of those spots are blurred due to the diffusion effect. These results indicate that the electrochemical method deactivates the immobilized enzymes. The size of the deactivated spot is easily controlled by changing the electrolyzing period. The two larger spots in the SCFM or SECM image were formed by applying 1.5 V to the tip for about 1 s, whereas for the two smaller spots for about 0.5 s. The resolution and accuracy of the electrochemically generated patterns will be improved by the precise control of the tip-substrate distance.

4. Conclusions Diaphorase micropatterns on a glass substrate were imaged with the SECM/SCFM system. Simultaneous SECM and SCFM imaging of a diaphorase-patterned substrate in the presence and absence of NADH clarified the difference between SECM and SCFM. SECM provides the information on the localized enzymatic reaction, while SCFM provides spatially resolved information on the location of the active enzyme. By using the SECM/SCFM system, laser-induced and electrochemical patterning of diaphorase on a glass

204

D. Oyamatsu et al. / Sensors and Actuators B 91 (2003) 199–204

substrate was achieved and the resulting substrate was imaged by the system. On the other hand, the resolution of the SECM image was influenced by the diffusion of the electrochemically active species. The laser-induced patterning is based on the photodegradation of diaphorase immobilized on a glass substrate. The laser-induced patterning of diaphorase is highly resolved at the micrometer level although it was rather time-consuming. The electrochemical patterning is based on the electrochemical generation of HOBr and subsequent deactivation of diaphorase on the substrate. The electrochemical patterning required only a second or less to deactivate the diaphorase-immobilized over a relatively wide range on the substrate because of diffusion of the electrochemically generated active species. The electrochemical and photochemical methods for patterning and imaging of the enzymes with the SECM/SCFM system will be widely applicable for the fabrication and characterization of bio-devices using proteins.

Acknowledgements This work was supported by a Grant-in-Aid for Priority Area 417 (No. 14050010) from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

References [1] H. Shiku, T. Matsue, I. Uchida, Detection of microspotted carcinoembryonic antigen on a glass substrate by scanning electrochemical microscopy, Anal. Chem. 68 (1996) 1276–1278. [2] H. Shiku, Y. Hara, T. Matsue, T. Uchida, T. Yamauchi, Dual immunoassay of human chorionic gonadotropin and human placental lactogen at a miocrofabricated substrate by scanning electrochemical microscopy, J. Electroanal. Chem. 438 (1997) 187–190. [3] J.V. Macpherson, P.R. Unwin, Combined scanning electrochemical– atomic force microscopy, Anal. Chem. 72 (2000) 276–285. [4] J.V. Macpherson, C.E. Jones, A.L. Barker, P.R. Unwin, Electrochemical imaging of diffusion through single nanoscale pores, Anal. Chem. 74 (2002) 1841–1848. [5] J.V. Macpherson, P.R. Unwin, Noncontact electrochemical imaging with combined scanning electrochemical atomic force microscopy, Anal. Chem. 73 (2001) 550–558. [6] C. Kranz, G. Friedbacher, B. Mizaikoff, A. Lungstein, J. Smoliner, E. Bertagnolli, Integrating an ultramicroelectrode in an AFM

[7]

[8]

[9]

[10]

[11]

[12]

[13]

cantilever: combined technology for enhanced information, Anal. Chem. 73 (2001) 2491–2500. T. Matsue, H. Yamada, H. Chang, I. Uchida, Electrooxidation of NADH with CoðphenÞ3 3þ by NADH dehydrogenase purified from Bacillus stearothermophilus, Bioelectrochem. Bioenerg. 25 (1990) 347–354. I. Turyan, T. Matsue, D. Mandler, Patterning and characterization of surfaces with organic and biological molecules by the scanning electrochemical microscope, Anal. Chem. 72 (2000) 3431–3435. W. Schuhmann, C. Kranz, H. Wohlschla¨ ger, J. Strohmeier, Pulse technique for the electrochemical deposition of polymer films on electrode surfaces, Biosens. Bioelectron. 12 (1997) 1157–1167. M. Ku¨ pper, J.W. Shultze, Spatially resolved concentration measurements during cathodic allow deposition in microstructures, Electrochim. Acta 42 (1997) 3023–3031. C. Heß, K. Borgwarth, C. Ricken, D.G. Ebling, J. Heinze, Scanning electrochemical microscopy: study of silver deposition on nonconducting substrates, Electrochim. Acta 42 (1997) 3065–3073. T. Wilhelm, G. Wittstock, Patterns of functional proteins formed by local electrochemical desorption of self-assembled monolayers, Electrochim. Acta 47 (2001) 275–281. T. Matsue, H. Yamada, H. Chang, I. Uchida, K. Nagata, K. Tomita, Electron transferase activity of diaphorase (NADH: acceptor oxidoreductase) from Bacillus stearothermophilus, Biochim. Biophys. Acta 1038 (1990) 29–38.

Biographies Daisuke Oyamatsu received his PhD in engineering from the Osaka University, Japan, in 2001. Presently, he is working as a research associate in the Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku, Japan. His current research interests are bioelectrochemistry, electrochemistry of ordered interface. Norihiro Kanaya received his master’s degree in engineering from the Tohoku University, Japan, in 2002. Currently, he is employed with the Matsushita Electric Industrial Co., Ltd. Hiroshi Shiku did his PhD in engineering from the Tohoku University, Japan, in 1997. He is presently employed with the Regional Research Project of Yamagata. Matsuhiko Nishizawa did his PhD in engineering from the Tohoku University, Japan, in 1994. Presently, he is working as an assistant professor, in the Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Japan. Tomokazu Matsue earned his PhD in pharmacy in 1981, and he is currently working as a professor in the Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Japan.