Imaging Biocatalytic Activity of Enzyme−Polymer Spots by Means of

May 14, 2009 - (1) Due to their high sensitivity and minor matrix effects, ECL .... for achieving sufficient ECL intensity despite the diffusion-limit...
0 downloads 0 Views 1MB Size
Download PDF
Anal. Chem. 2009, 81, 5070–5074

Imaging Biocatalytic Activity of Enzyme-Polymer Spots by Means of Combined Scanning Electrochemical Microscopy/Electrogenerated Chemiluminescence Rong Lei,†,‡ Lutz Stratmann,† Dominik Scha¨fer,† Thomas Erichsen,† Sebastian Neugebauer,† Na Li,*,‡ and Wolfgang Schuhmann*,† Analytische ChemiesElektroanalytik & Sensorik, Ruhr-Unversita¨t Bochum, Universita¨tsstrasse 150, D-44780 Bochum, Germany, and Key Laboratory of Bioorganic Chemistry Molecular Engineering of the Ministry of Education, Beijing National Laboratory for Molecular Science (BNLMS), Institute of Analytical Chemistry, College of Chemistry and Molecular Engineering, Peking University, Beijing, 100871, China The purpose of this study was to develop a scanning electrochemical microscopy (SECM) and scanning electrogenerated chemiluminescence (SECL) setup to visualize the localized enzymatic activity using glucose oxidase as a model. Combination of SECM and electrogenerated chemiluminescence (ECL) was made possible by integrating a photomultiplier tube (PMT) within a SECM setup which is mounted on top of an inverted microscope. An enzyme-polymer spot formed on a glass slide and placed on top of the entrance window of the PMT was used as a model sample to evaluate the potential of the combined SECM/ECL setup. Hydrogen peroxide, which was locally generated by the glucose oxidase (GOx)-catalyzed reaction, reacted with oxidized luminol which was simultaneously electrochemically generated at the positioned SECM electrode tip. By using the phase-sensitive lock-in amplifier, the potential applied to the SECM tip was sinusoidally swept to invoke an associated oscillation of the ECL. Thus, sensitivity of SECL could be substantially enhanced. Images of the local immobilized enzyme activity obtained both by ECL and generator/collector (GC) mode of SECM were compared to elucidate the pathway in which the SECM and SECL signals are generated. Electrogenerated chemiluminescence (ECL) has emerged to a powerful analytical technique in clinical and biological analysis.1 Due to their high sensitivity and minor matrix effects, ECL biosensors were developed. There are two major types of ECL biosensors with one type based on dehydrogenases and the ECL detection of NADH2-6 and even more importantly the second type based * To whom correspondence should be addressed. Phone: 49-234-3226200 (W.S.); 86-10-62761187 (N.L.). Fax: 49-234-3214683 (W.S.). E-mail: [email protected] (W.S.); [email protected] (N.L.). † Ruhr-Unversita¨t Bochum. ‡ Peking University. (1) Bard, A. J. In Electrogenerated Chemiluminescence; Marcel Dekker: New York, 2004; pp 359-396. (2) Jameison, F.; Sanchez, R. I.; Dong, L. W.; Leland, J. K.; Yost, D.; Martin, M. T. Anal. Chem. 1996, 68, 1298–1302. (3) Lee, W. Y.; Nieman, T. A. Anal. Chem. 1995, 67, 1789–1796.

5070

Analytical Chemistry, Vol. 81, No. 12, June 15, 2009

on oxidases and ECL detection of H2O2.7-9 For example, glucose, lactate, or cholesterol can be catalytically converted to H2O2 by related specific oxidases or by the corresponding dehydrogenases in the presence of NAD+ under generation of NADH. ECL then can be generated by reaction of enzymatically produced H2O2 with electrochemically oxidized luminol or by reaction of enzymatically produced NADH with [Ru(bpy)3]2+. Most ECL-based biosensors were fabricated by immobilizing the enzyme on working electrodes modified by materials with large surface area such as sol-gel,10 nanoparticles,11-13 microbeads,14,15 or synthetic preactivated membranes.9,16 Scanning electrochemical microscopy (SECM) is a powerful tool for obtaining laterally resolved information about electrocatalytic or electrochemical activity of surfaces. The generator/ collector (GC) mode of SECM has been used to image locally confined enzymatic activity by detecting products of the enzymatic reaction at the SECM tip using amperometric or potentiometric detection schemes.17-21 In order to simultaneously measure reactivity and surface topography, integration of chemilumines(4) Yokoyama, K.; Sasaki, S.; Ikebukuro, K.; Takeuchi, T.; Karube, I.; Tokitsu, Y.; Masuda, Y. Talanta 1994, 41, 1035–1040. (5) Zhang, L. H.; Xu, Z. A.; Sun, X. P.; Dong, S. J. Biosens. Bioelectron. 2007, 22, 1097–1100. (6) Martin, A. F.; Nieman, T. A. Biosens. Bioelectron. 1997, 12, 479–489. (7) Marquette, C. A.; Blum, L. J. Anal. Chim. Acta 1999, 381, 1–10. (8) Marquette, C. A.; Blum, L. J. Anal. Bioanal. Chem. 2008, 390, 155–168. (9) Laespada, M. E. F.; Pavon, J. L. P.; Cordero, B. M. Anal. Chim. Acta 1996, 327, 253–260. (10) Zhu, L. D.; Li, Y. X.; Tian, F. M.; Xu, B.; Zhu, G. Y. Sens. Actuators, B 2002, 84, 265–270. (11) Wang, W.; Xiong, T.; Cui, H. Langmuir 2008, 24, 2826–2833. (12) Qian, K. J.; Zhang, L.; Yang, M. L.; He, P. G.; Fang, Y. Z. Chin. J. Chem. 2004, 22, 702–707. (13) Lin, Z. Y.; Chen, J. H.; Chen, G. N. Electrochim. Acta 2008, 53, 2396– 2401. (14) Marquette, C. A.; Blum, L. J. Sens. Actuators, B 2003, 90, 112–117. (15) Marquette, C. A.; Thomas, D.; Degiuli, A.; Blum, L. J. Anal. Bioanal. Chem. 2003, 377, 922–928. (16) Tsafack, V. C.; Marquette, C. A.; Leca, B.; Blum, L. J. Analyst 1999, 125, 151–155. (17) Zhou, F. M.; Unwin, P. R.; Bard, A. J. J. Phys. Chem. 1992, 96, 4917–4924. (18) Strike, D. J.; Hengstenberg, A.; Quinto, M.; Kurzawa, C.; Koudelka-Hep, M.; Schuhmann, W. Mikrochim. Acta 1999, 131, 47–55. (19) Kasai, S.; Hirano, Y.; Motochi, N.; Shiku, H.; Nishizawa, M.; Matsue, T. Anal. Chim. Acta 2002, 458, 263–270. (20) Zhao, C. A.; Wittstock, G. Anal. Chem. 2004, 76, 3145–3154. 10.1021/ac900192n CCC: $40.75  2009 American Chemical Society Published on Web 05/14/2009

Figure 1. (a) Schematic diagram of the developed SECM/SECL setup with integrated lock-in amplifier; (b) scheme of SECL and SECM (c) for studying local biocatalytic activity of enzyme-polymer spots.

cence (CL) detection schemes into SECM (SECM/SCLM) was previously described.19,22,23 The CL detection was based on the reaction of luminol with H2O2 catalyzed by horseradish peroxidase (HRP). H2O2 was produced at a microelectrode via reduction of O2 at an applied potential of -1.0 V versus Ag/ AgCl. The generated H2O2 diffused to the immobilized HRP and reacted with luminol to emit light.22,23 For visualization of glucose oxidase (GOx) activity a glass capillary filled with HRP as biocatalyst was scanned above the immobilized GOx. In that way, local enzymatically generated H2O2 diffused to the HRP tip where CL was generated in the presence of luminol. Additionally, mechanistic studies of [Ru(bpy)3]2+-based ECL SECM were conducted.24,25 Furthermore, ECL emission at the visible wavelength at a microelectrode was used to visualize a Au band on a transparent sample.26,27 Here, we describe a combination of SECM and scanning ECL (SECL) for sequential amperometric and electrochemiluminescent visualization of immobilized enzyme activity on surfaces. A SECM setup was modified by integrating a photomultiplier tube (PMT) underneath the electrochemical cell to collect generated light from the scanned microelectrode tip. A similar SECM/ECL setup with a 1.5 mm diameter hemispherical Au electrode was reported by Miao et al. using approach curves to elucidate the ECL mechanism.28 Here, a lock-in amplifier was used to amplify the PMT signal. Through a sinusoidal modulation of the potential applied to the SECM tip, the lock-in amplifier can recover signals by selective amplification at the modulation frequency even in presence of a big background noise and thus provide measurements with improved sensitivity and high signal-to-noise ratio. With the use of the developed SECM/SECL setup, both the current at the tip and the ECL intensity can be measured sequentially on the same sample. This approach allows for investigating one biocatalytic system with two independent methods, which provide complementary information due to the different reaction sequences involved in the generation of both signals. Herewith, the feasibility of the developed setup will be illustrated using GOx-polymer spots on transparent surfaces.

EXPERIMENTAL SECTION Chemicals. Luminol (5-amino-2,3-dihydrophthalazine-1,4-dione) was purchased from ABCR (Karlsruhe, Germany). K2HPO4 · 3H2O and glucose oxidase from Aspergillus niger (GOx; EC 1.1.3.4, type X-S, lyophilized powder, 185 U/mg) were from Sigma-Aldrich (Steinheim, Germany). Tris-(hydroxymethyl)-amine methane was from Biomol (Hamburg, Germany). D(+)-Glucose monohydrate was purchased from AppliChem (Germany). A commercial electrodeposition paint (Resydrol AY 498W/35WA, Vianova Resins, Mainz-Kastel, Germany) was used for enzyme immobilization as described previously.29-31 H3PO4 was obtained from Mallinckrodt Baker (Deventer, The Netherlands). All chemicals were of analytical grade and used without further purification. Triple-distilled water was used throughout all experiments. Enzyme-Polymer Spot Preparation. The enzyme-polymer spot was prepared as previously reported.21 In brief, a vigorously cleaned glass slide was silanized with dichlorodimethysilane in chloroform (5%, v/v) to obtain a hydrophobic surface. An amount of 65 mg of Resydrol AY-498w/35WA was diluted with 950 µL of 0.2 M phosphate buffer (pH 6.8). An amount of 5 mg of GOx was dissolved in 200 µL of this diluted Resydrol solution to obtain a 4.6U/µLenzyme-polymersolution.Adropletofthisenzyme-polymer solution was deposited on the hydrophobic glass slide using a (21) Schafer, D.; Maciejewska, M.; Schuhmann, W. Biosens. Bioelectron. 2007, 22, 1887–1895. (22) Zhou, H. F.; Kasai, S.; Yasukawa, T.; Matsue, T. Electrochemistry 1999, 67, 1135–1137. (23) Zhou, H. F.; Kasai, S.; Matsue, T. Anal. Biochem. 2001, 290, 83–88. (24) Miao, W. J. Chem. Rev. 2008, 108, 2606–2553. (25) Kanoufi, F.; Cannes, C.; Zu, Y. B.; Bard, A. J. J. Phys. Chem. B 2001, 105, 8951–8962. (26) Zu, Y. B.; Ding, Z. F.; Zhou, J. F.; Lee, Y. M.; Bard, A. J. Anal. Chem. 2001, 73, 2153–2156. (27) Fan, F. R. F.; Cliffel, D.; Bard, A. J. Anal. Chem. 1998, 70, 2941–2948. (28) Miao, W. J.; Choi, J. P.; Bard, A. J. J. Am. Chem. Soc. 2002, 124, 14478– 14485. (29) Ngounou, B.; Neugebauer, S.; Frodl, A.; Reiter, S.; Schuhmann, W. Electrochim. Acta 2004, 49, 3855–3863. (30) Kurzawa, C.; Hengstenberg, A.; Schuhmann, W. Anal. Chem. 2002, 74, 355–361. (31) Shkotova, L. V.; Soldatkin, A. P.; Schuhmann, W.; Dzyadevych, S. V. Mater. Sci. Eng., C 2006, 26, 411–414.

Analytical Chemistry, Vol. 81, No. 12, June 15, 2009

5071

glass capillary. After drying in air for 2 h, the spot was investigated by SECM/SECL or stored at 4 °C in 0.1 M phosphate buffer (pH 6.8). SECM and SECL experiments were performed using a specifically developed setup as shown in Figure 1. As a base instrument a Sensolytics SECM (Sensolytics, Bochum, Germany) with High-Res, Shearforce, and Analog-In options was used. The SECM was built up on an inverted microscope, and a PMT (Hamamatsu H5784-01) was integrated underneath the scanning plane of the SECM tip replacing the objective of the inverted microscope. The signal output of the PMT was connected to the signal input of a lock-in amplifier (DSP lock-in amplifier model 7265, EG&G, Signal Recovery and Perkin-Elmer Instruments). The input impedance of the preamplifier of the PMT is 100 kΩ, and the gain of the PMT was controlled by a dc voltage at 0.8 V. Data acquisition and current/potential control were realized by the control software of the SECM (Sensolytics, Bochum, Germany) using a 16-bit AD/DA convertor. A conventional three-electrode configuration was used with a Pt foil as the counter electrode and a Ag/AgCl/KCl (3 M) electrode as the reference electrode. When obtaining ECL data, a sinusoidal potential modulation was overlaid to a predefined dc potential to the SECM tip using the internal oscillator (OSC) of the lock-in amplifier. The initial dc potential at the tip was manually adjusted at the potentiostat (potentiostat/ galvanostat 1002 PC.T., Jaissle, Waiblingen, Germany). During approach curves and SECM imaging the tip potential was set to a predefined value by the software using the AD/DA convertor. The SECM tips were Pt disk electrodes with 250 µm diameter which were prepared according to previously reported procedures.32 The rather large tip diameter was selected due to the expected small concentrations of H2O2 generated in the enzymecatalyzed reaction for achieving sufficient ECL intensity despite the diffusion-limited access of oxygen, glucose, and luminol in the gap between tip and sample. The tip was approached to the glass surface while monitoring the oxygen reduction current until the negative feedback response was observed. Measurements were carried out at constant z-position (constantheight mode) at an initial distance of 50 µm between SECM tip and bare glass surface for a 250 µm Pt electrode. The current response or the ECL signal was recorded as a function of x- and y-position. For SECM measurements the tip potential was set to 650 mV versus Ag/AgCl/KCl (3 M) for oxidation of enzymatically generated H2O2, whereas an initial potential of 430 mV versus Ag/AgCl/KCl (3 M) was used for SECL. Microscopic Image Rapid Analysis software (MIRA, G. Wittstock, Oldenburg, Germany) was used to process and analyze SECM and ECL data. RESULTS AND DISCUSSION SECL Measurements with Lock-In Amplifier. The luminol (pKa1 ) 6.20; pKa2 ) 15.0) ECL intensity is pH-dependent.33-36 In alkaline solution, luminol is deprotonated under formation of (32) Kranz, C.; Ludwig, M.; Gaub, H. E.; Schuhmann, W. Adv. Mater. 1995, 7, 568–571. (33) Sakura, S. Anal. Chim. Acta 1992, 262, 49–57. (34) Fahnrich, K. A.; Pravda, M.; Guilbault, G. G. Talanta 2001, 54, 531–559. (35) Lin, X. Q.; Sun, Y. G.; Cui, H. Chin. J. Anal. Chem. 1999, 27, 497–503. (36) Jirka, G. P.; Martin, A. F.; Nieman, T. A. Anal. Chim. Acta 1993, 284, 345–349.

5072

Analytical Chemistry, Vol. 81, No. 12, June 15, 2009

Figure 2. ECL intensity measured in a solution containing 1 mM luminol and 0.97 mM H2O2 at a 1 mm diameter Pt disk electrode at different pH values in 0.20 M Tris-HCl buffer as electrolyte: (a) pH 7.5; (b) pH 8.0; (c) pH 8.5; (d) pH 9.0.

the corresponding anion, which can be electrochemically oxidized to the anion radical at a sufficiently high electrode potential. H2O2 reacts with this anion radical to produce a diazo compound, which further yields 3-aminophthalate in the excited state. Figure 2 shows the ECL intensity as a function of scanning potential between 0 and 700 mV versus Ag/AgCl in buffer solutions of different pH values. Obviously, the highest luminolbased ECL intensity is obtained at a potential of about 580 mV versus Ag/AgCl. As expected, luminol-based ECL intensity increases with increasing pH from 7.5 to 9. A GOx-polymer spot deposited on a glass slide was used to demonstrate the feasibility of SECM/SECL. H2O2, which is produced in the GOx-catalyzed oxidation of glucose with O2, diffuses from the enzyme spot into the electrolyte. When the microelectrode is scanned over the enzyme-polymer spot and the tip is located within the diffusion profile of enzymatically formed H2O2, the reaction of H2O2 with the electrochemically generated luminol anion radical leads to light emission (Figure 2). Thus, the number of emitted photons which are detected at the PMT is associated with the amount of enzymatically generated H2O2 and the ECL quantum yield. In addition, ECL intensity is influenced by the diffusional access of luminol, oxygen, and glucose into the gap between tip and sample as well as the pH value and the applied tip potential. Sensitive ECL detection is required because local low H2O2 concentrations are expected caused by the comparatively small immobilized enzyme activity. A lock-in amplifier was integrated into the SECM/SECL system to increase sensitivity and the signal-to-noise ratio. Using the built-in oscillator of the lock-in amplifier, a sinusoidal ac potential is superimposed to the constant initial dc potential applied to the SECM tip. The resulting ac potential at the PMT output which reflects the sinusoidal modulation of the ECL is amplified using the lock-in amplifier. Figure 3 shows the amplified PMT signals at different initial potentials applied to the electrode and a sinusoidal potential modulation with an amplitude of 300 mVpp at 3 Hz. At an initial potential of 430 mV the highest ECL output was obtained which corresponds well with the fact that the ECL reaches maximum at a constant potential of 580 mV (Figure 2). By using the lock-in amplifier, maximum signal is expected at an initial potential where the slope of the ECL output over potential curve is highest. At an initial potential of 430 mV the potential applied to the working electrode swept between 280 and 580 mV with only background noise observed at 280 mV and the maximal ECL observed at 580 mV. The lock-in amplifier

Figure 3. Effect of the initial dc potential on ECL signal after lock-in amplification at a 250 µm Pt disk electrode in the presence of 1 mM luminol and 10 mM H2O2 in 0.20 M Tris-HCl buffer (pH 8.5). Amplifier parameters: OSC frequency, 3 Hz; amplitude, 0.3 Vpp; sensitivity, 5 mV; ac gain, 20 dB.

Figure 4. (A) SECM and (B) SECL x-line scans over the center of a GOx-polymer spot using a 250 µm diameter Pt disk electrode as tip in presence of (A) 100 mM glucose and (B) 100 mM glucose and 1 mM luminol. Lock-in amplifier parameters: OSC frequency, 3 Hz; amplitude, 0.3 Vpp; sensitivity, 5 mV; ac gain, 20 dB. Electrolyte: 0.20 M phosphate buffer (a) pH 6.8; (b) pH 8.0; (c) pH 8.5; (d) pH 9.0.

extracts the ECL from the overlaid signals by filtering the frequency-independent noise and amplifying the frequency-dependent ac current. For example, if the base potential was set to 550 mV, the applied potential was modulated in a range from 400 to 700 mV. Since at both 400 and 700 mV the signal difference during the time of circulation as measured by the lock-in amplifier is small, a small overall signal output is obtained. Effect of pH on SECM and SECL Measurements. SECM and SECL x-line scans over the center of the GOx-polymer spots were performed using a 250 µm Pt disk electrode as SECM tip at a constant height of 50 µm above the surface of the glass slide (Figure 4). The optimum pH for the GOx-catalyzed reaction is between 6 and 7. The H2O2 oxidation current was maximal at pH 6.8 and decreased markedly at higher pH values (Figure

4A). However, no ECL was observed at the optimal pH for the enzymatic reaction since ECL is strongly associated with the deprotonation of luminol. ECL emission increased with increasing pH and is maximal at pH 8.5 (Figure 4B), which was chosen to obtain the highest ECL intensity with a compromised yet still sufficient enzymatic activity. Parts A and B of Figure 4 were obtained sequentially on the same sample. Because H2O2 is accumulated around the spot during scanning, the amount of H2O2 detected includes the in situ generated H2O2 and the residual H2O2 which is accumulated during the overall measuring time. Therefore, the background current is continuously increasing with time. Since the ECL detection is more sensitive for detecting the background H2O2 this effect is more obvious for ECL scanning. It is important to supply a sufficiently high concentration of luminol in order to limit the overall ECL intensity by the enzymecatalyzed reaction and hence by the concentration of glucose. SECL x-line scans were performed at a constant glucose concentration with increased luminol concentration in the electrolyte solution. However, increasing the luminol concentration will simultaneously raise the pH value by exceeding the buffer capacity of the used electrolyte as luminol needs to be dissolved in 50 mM NaOH solution. Thus, by increasing the luminol concentration from 1 to 8 mM a decrease in the ECL intensity was observed (data not shown), which may be attributed to the resulted nonfavorable pH value for the enzyme-catalyzed reaction. Therefore, all subsequent experiments were performed using 1 mM luminol. SECM and SECL Activity Images of GOx-Polymer Spots. In order to evaluate the feasibility of SECL in visualizing local variations in surface-confined enzymatic activity, two enzyme spots with different enzyme concentrations were deposited on a glass slide. The first spot contained an enzyme activity of 1.5 U/µL in the GOx-polymer solution, and the second one contained an enzyme activity of 4.6 U/µL with the amount of polymer remained the same. A SECM measurement in pH 6.8 phosphate buffer was performed initially in order to visualize the local enzyme activity within the GOx-polymer spot in a conventional GC mode arrangement of SECM. After changing the electrolyte solution to a Tris-HCl buffer of pH 8.5 containing additional 1 mM luminol while avoiding any displacement of the SECM tip, the same area was scanned using the developed SECL mode including sinusoidal potential modulation and lock-in amplification of the PMT signal (Figure 5). As shown above (see Figure 1, parts B and C) the signal generation in the GC arrangement of SECM and in SECL is different. In case of GC-SECM, the signal is obtained by H2O2 oxidation at a comparatively high potential. In contrast, SECL was obtained at an initial potential of 430 mV. However, in GCSECM, O2 is partially regenerated within the gap between the positioned SECM tip and the sample and will hence be again available for the enzyme-catalyzed oxidation of glucose. Due to the fact that no O2 is regenerated in the ECL reaction, the enzyme-catalyzed reaction will be limited by the availability of O2 at comparatively lower turnover rates of the enzyme. On the other hand, the diffusion distance for the generated H2O2 is shorter in the case of SECL since it reacts with the electrochemically generated luminol radical anion in the difAnalytical Chemistry, Vol. 81, No. 12, June 15, 2009

5073

Figure 5. (A) GC-SECM image of GOx-polymer spots in 100 mM glucose and 0.20 M phosphate buffer (pH 6.8); (B) SECL image of the same spots as SECM in 100 mM glucose, 1 mM luminol, and 0.20 M Tris-HCl buffer (pH 8.5) using a 250 µm Pt disk as tip electrode (RG 2.2). Amplifier parameters: OSC frequency, 3 Hz; amplitude, 0.3 Vpp; sensitivity, 5 mV; ac gain, 10 dB; (C) x-line scans through the spot centers from the SECM and SECL images in panels A and B.

fusion zone of the positioned tip electrode. Thus, diffusional escape of H2O2 from the reaction gap is more likely in the case of GC-SECM than SECL. In addition, the enzyme-catalyzed reaction is leading to the formation of gluconic acid which will locally modulate the pH value to be more acidic than the buffer medium added. Thus, it can be expected that SECL images may decrease in emission intensity at higher glucose concentrations despite of an increased formation of H2O2 in the enzyme-catalyzed reaction. Consequently, it can be expected that results of GC-SECM and SECL imaging of GOx activity are similar but with significant variations in detail due to the differences in the complicated signal generation mechanisms. During formation of the GOx-polymer spot a pronounced donut effect is established; thus, a nonhomogeneous distribution of immobilized enzyme activity with a comparatively higher activity at the rim and lower activity at the center of the spot is obtained21

5074

Analytical Chemistry, Vol. 81, No. 12, June 15, 2009

(Figure 5). If a relatively large tip was positioned exactly above the spot, glucose, oxygen, and in the case of ECL also luminol may be depleted already at the rim of the GOx-polymer structure before diffusing into the gap between the enzyme spot and the electrode. Thus, it becomes clear that the observed “current hole” in the SECM image or “emission hole” in the SECL image is due to the collective influence of all parameters which are critical in establishing the local concentrations of all compounds involved in the reactions. Therefore, in order to establish glucose saturation at the enzyme site, a concentration of 100 mM glucose was used in combination with a rather large tip electrode (250 µm diameter). In the case of a lower enzyme concentration in the GOx-polymer spot (Figure 5; left spot a), the “hole” in the spot center appears less pronounced than that for the higher enzyme concentration (Figure 5; right spot b). This phenomenon is even more visible in the SECL images. In the SECL image the availability of the electrogenerated luminol anion radical is an additional factor contributing to the overall signal generation. When the GC-SECM and SECL images of the two spots are compared, it becomes clear that SECL is better in distinguishing between different immobilized enzyme activities at the used experimental conditions. This can be seen also from the x-line scans for both GC-SECM and SECL through the center of the spots (Figure 5C). CONCLUSIONS An integrated SECM/SECL setup was developed and evaluated using GOx-polymer spots on glass slides as model for biosensor surfaces. With the aid of phase-sensitive amplification using an integrated lock-in amplifier, amplified SECL images could be obtained at a relatively low initial potential of 430 mV. The slow frequency modulation of the potential in combination with lockin amplification opens a possibility for interference elimination. Sequentially obtained SECM and SECL images of local immobilized enzyme activity provide the basis for a more detailed understanding of the complex interplay of the parameters involved in signal generation. In future, this setup may be applied for the visualization of local enzyme activity in biosensors. ACKNOWLEDGMENT R. Lei is grateful for the financial support of Ministry of Education of China in cooperation with the German Academic Exchange Service (DAAD). Received for review January 25, 2009. Accepted April 18, 2009. AC900192N