Topological and Electron-Transfer Properties of Glucose Oxidase

The topological and electron-transfer properties of the redox enzyme glucose oxidase (GOD) adsorbed on highly oriented pyrolytic graphite (HOPG) were ...
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J. Phys. Chem. C 2008, 112, 5165-5173

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Topological and Electron-Transfer Properties of Glucose Oxidase Adsorbed on Highly Oriented Pyrolytic Graphite Electrodes Mingkui Wang,† Sven Bugarski, and Ulrich Stimming* Technische UniVersita¨t Mu¨nchen, James-Franck-Strasse 1, Physik-Department E19, D-85748, Garching, Germany ReceiVed: February 9, 2007; In Final Form: January 7, 2008

The topological and electron-transfer properties of the redox enzyme glucose oxidase (GOD) adsorbed on highly oriented pyrolytic graphite (HOPG) were studied at a single molecule level with in situ electrochemical scanning tunneling microscopy (EC-STM). In situ STM and cyclic voltammetry were performed to understand the potential-dependent tunneling process in the presence of adsorbed GOD. Potential-dependent apparent changes in the GOD morphology on HOPG were observed in STM images and are discussed. For example, an issue in the STM scanning of adsorbed GOD was the influence of EC currents on the measured tip current. These Faradaic currents might be due to involved redox species that react at the enzyme-modified electrode surface and subsequently diffuse to the STM tip. Such effects of local enzyme activity on the contrast in STM images have been studied and are evaluated within a proposed model.

1. Introduction Recently, great attention has been paid to the possibility of implementing biochemical electronic devices by exploiting the properties of biomolecules to self-assemble on electrode surfaces. The use of redox enzymes is of great interest due to its intrinsic functional properties.1-3 Most of the commonly used enzymes in nanoscale bioelectronics, such as oxidases and pyrroloquinoline quinone-dependent dehydrogenases, contain redox groups, which change their redox state during biochemical reactions.4 Electron-transfer (ET) reactions in enzymes play an important role in biologically vital processes in living cells, most notably in photosynthesis and respiration. Characterizing the influence of structure on intermolecular ET reactions is particularly challenging. This is due to the complex nature of the systems. Modifying the electrode surface with functional groups or modifying the enzymes themselves increased the rate of ET between enzymes and electrode. Systems with adsorbed enzymes on various modified electrodes were extensively studied to understand the redox properties of enzymes and to develop molecular electronics.5-8 The electrical conductivity of redox biomolecules has been studied by using scanning tunneling microscopy (STM).9-11 These studies revealed that the potential-dependent conductivity of enzymes could be affected by molecular conformational changes10 or by changes in the bond between the functional bonding groups and the substrate.11 Models explaining the potential-dependent conductivity behavior are still a controversial issue.12 Glucose oxidase (GOD) is well-characterized in the literature.13 The X-ray crystallographic structure of GOD recently has been reported, and also, the binding structures of flavins in the enzyme are well-determined.14 According to Wohlfahrt et * Corresponding author. Tel.: +49 89 28912531; fax: +49 89 28912530; e-mail: [email protected]. † Current address: Laboratory of Photonics and Interfaces, Institute of Chemical Sciences and Engineering, Ecole Polytechnique Fe´de´rale de Lausanne, 1015 Lausanne, Switzerland.

al.,14 GOD is a dimeric protein, which contains one tightly bound flavin adenine dinucleotide (FAD) unit per monomer as the cofactor. The monomer with dimensions of approximately 60 Å × 52 Å × 37 Å contains 583 amino acid residues, 17 R-helices, and 30 β-strands. The cofactor FAD is situated in one of the domains, which consists of 2 β-sheets and 3 R-helices and occupies a narrow channel outlined by 31 residues with a covering lid.14 GOD’s active center FAD is not covalently bound and can be released from the holoenzyme. FAD exhibits redox activity, which is necessary for GOD to oxidize its substrates. The active site for the catalysis of β-D-glucose is a large deep pocket, shaped like a funnel and formed on one side by the lid from both subunits of the dimer.15 The imaging of microscopic enzymatic active spots of GOD on a gold electrode has been studied by scanning electrochemical (EC) microscopy.16 In situ EC-STM is a powerful technique for structural and electronic state analysis of electrolyte-electrode interfaces under EC potential control. Different STM studies on metallo-proteins and other redox molecules adsorbed on atomically flat metal surfaces have been carried out to investigate ET through the immobilized species.17,18 We studied GOD with in situ ECSTM to observe its ET properties in an aqueous environment at a single molecule level. GOD molecules were adsorbed on anodized highly oriented pyrolytic graphite (HOPG) electrodes. Using the STM tip as a local sensor, single GOD molecules were individually addressed to determine their electronic and topographic properties. EC-STM results show that the apparent height of adsorbed GOD molecules on HOPG in the STM images changes by applying an EC potential to the substrate. Cyclic voltammetry (CV) and EC impedance spectroscopy (EIS) were performed to assess the functionality of enzyme molecules adsorbed on HOPG substrates. The combination of these techniques provides information about the morphological characteristics, as well as the electronic and redox properties of the enzyme.

10.1021/jp071122h CCC: $40.75 © 2008 American Chemical Society Published on Web 03/07/2008

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2. Experimental Procedures 2.1. Materials and Instrumentation. GOD (160 kDa, 215.266 units mg-1) from Aspergillus niger was obtained from Sigma-Aldrich (Munich, Germany) and used without further purification. HOPG (grade ZYH) was obtained from Advanced Ceramics Corp. HClO4 (ultrapure grade) was obtained from Merck. The working electrolyte for the enzyme was either a 10 or 30 mM phosphate buffered solution (PBS, pH 7.2). Other reagents used were of analytical grade. All solutions were prepared with ultrapure water (18 MΩ) received from a Millipore system. EC experiments were carried out with a HEKA potentiostat (Dr. Schulze GmbH, Wilnsdorf, Germany) in a conventional three-electrode configuration. EIS was performed with an Autolab Frequency Analyzer setup consisting of the Autolab PGSTAT 30 (Eco Chemie B.V., Utrecht, The Netherlands) and the frequency response analyzer (FRA) module with fit and simulation software for the analysis of impedance data. The EIS experiments were performed in a glass cell, while the CV and EC-STM measurements were performed in small PTFE cells. In situ EC-STM measurements were carried out using a PicoSPM BASE (Molecular Imaging) controlled by a NanoScope III controller (Veeco, Mannheim, Germany). A BP-600 bipotentiostat (EC-Tec, Teutschenthal, Germany) was used to control the sample and tip potential independently. A Tektronix TDS5034B digital storage oscilloscope (Cologne, Germany) was used to store the current through the STM tip. Silver and gold wire electrodes were used as the quasi-reference electrode (QRE) and counter electrodes, respectively. Here, potentials were quoted versus the saturated calomel electrode (SCE, -240 mV vs NHE). The potential of the Ag QRE was calibrated with ferrocene monocarboxylic acid (FMCA, Sigma-Aldrich) in the electrolyte solution before and after the experiment against SCE. During the experiment time, the used Ag QRE showed a shift of 30 mV. This shift was considered when calculating absolute reference potentials. STM measurements were carried out in PTFE cells in a sealed chamber flushed with argon to minimize the influence of oxygen. To remove organic contamination, the STM cells were left overnight in a 30% H2O2 and 70% concentrated sulfuric acid bath, then rinsed and put into a beaker with boiling water from a Millipore system for at least 1 h prior to use. All STM images were obtained in the constant current mode. Our STM system proved to yield reproducible images, which were stable with time after several scans of the same enzyme, hence ruling out undesired direct tip-enzyme interactions. 2.2. Tip Preparation. STM tips were prepared either by EC etching of a 0.25 mm platinum/iridium wire in a solution of KCN (3 M) and NaOH (1 M) or by EC etching of a 0.25 mm gold wire in HCl (32%). Except for the very end of the tips, the tips were effectively insulated with phoretic paint (Pt/Ir tips) or Apiezon wax (Au tips) to reduce the Faradaic leakage currents during STM measurements to less than 10 pA. 2.3. Preparation and Measurements of the Sample. Prior to each experiment, the HOPG sample was prepared by being cleaved with adhesive tape. The sample was immersed in the electrolyte within the EC STM-PTFE cell. In previous studies, HOPG was activated by various methods, such as chemical oxidation and EC oxidation to create active sites and to accelerate the heterogeneous ET between HOPG and adsorbed redox moiety.19,20 In particular, the EC oxidation of graphite has been studied extensively by Bard and Gewirth.21 The oxidation process led to the roughening of the surface, and oxygen functionalities were introduced to the surface layer.

Figure 1. Cyclic voltammograms of the GOD/HOPG electrode in 0.03 M PBS (pH 7.2) in N2 atmosphere at various scan rates (inner to outer curves: 20, 50, 100, and 200 mV s-1). The dotted line corresponds to the HOPG electrode in PBS with N2 at 20 mV s-1. Inset: plots of peak currents vs scan rates.

Thus, we chose a similar approach to create binding sites for the GOD enzyme molecules. After oxidization in 0.1 M HClO4, which was performed by three potential sweeps from 0 to 1.8 V versus SCE at a scan rate of 200 mV s-1, the HOPG electrode was rinsed with PBS immediately. Then, the substrate was immersed into a freshly prepared GOD solution (about 10-3 mg mL-1) in 30 mM PBS at pH 7.2. The average density of adsorbed GOD molecules on HOPG could be varied by several orders of magnitude depending on the immersion time. As a low density of adsorbed GOD on flat terraces of the HOPG sample is desired for investigating the behavior of the sample at the single molecule level by EC-STM scanning, the immersion time was set to 2-3 h. After adsorption, the samples were immediately rinsed with buffer solution to remove weakly adsorbed enzymes and then placed in the STM cell. The measurements were performed in four successive steps that are as follows: (i) STM imaging of HOPG terraces containing GOD molecules was performed. (ii) The tip was positioned over the enzyme and retracted 5-10 nm. Then, the feedback control of STM was switched off. (iii) Potential sweeps of typically 1 V s-1 were applied to the substrate electrode, while the potential window was chosen symmetrically around the equilibrium redox potential of the enzyme. Keeping the tip potential constant, the tip current was measured using a digital storage oscilloscope via the preamplifier of the STM. (iv) Feedback control of the STM was switched on and the enzyme was imaged. To verify the reproducibility of the measurement, the enzyme was characterized again according to steps ii-iv of a new sequence. Background correction of the tip currents was performed using the current obtained on a bare HOPG surface. EC impedance spectra of the GOD-modified HOPG electrodes were obtained at various potentials (from -0.20 to -0.75 V vs SCE) in the frequency range of 4 to ∼10 000 Hz with oscillation potential amplitudes of 5 mV. A Pt plate was used as an auxiliary electrode and SCE as the reference electrode. An argon stream over the solution was always maintained during the measurements to minimize the undesired effects of oxygen on the EC experiments. 3. Results Initially, we characterized the HOPG substrate with adsorbed GOD both by CV and by STM imaging. From the CV (Figure 1), we could determine the redox potential of the GOD/HOPG system in buffer solution. The STM images (Figures 2 and 3) enabled us to estimate the amount of adsorbed GOD on

Glucose Oxidase Adsorbed on Graphite Electrodes

Figure 2. STM images of GOD observed on the modified HOPG electrode surface: (a) GOD on a flat terrace and (b) agglomerations on edges. Immersion time: 1 h. Scan range: 500 nm × 500 nm. Electrolyte: 10 mM PBS (pH 7.2). Usub ) -450 mV vs SCE, Ubias ) 50 mV, and Isetpoint ) 0.5 nA. Vertical range: (a) 10 Å and (b) 15 Å.

HOPG and possibly to distinguish between different orientations of the adsorbed GOD (Figure 3). The STM images of adsorbed GOD are stable and reproducible even after repetitive scans. Single molecules can be well-resolved on the anodized HOPG substrate. Figure 3 presents two different patterns of single GOD molecules in high resolution and their 3-D structures in the insets. The molecular height of adsorbed GOD on the HOPG surface can be estimated from the maximum vertical size in the STM image. Figure 3a shows a butterfly-shaped adsorbed GOD molecule, which is assigned to the lying position of the enzyme molecule. The average size of this molecular structure is 50 ( 10 Å × 80 ( 10 Å, with an apparent height of 4-6 Å. The cross-sectional area for this molecule was calculated to be 4000-4500 Å2. Figure 3b shows the quasi-spherical adsorbed GOD shape, which is assigned to the standing position of the enzyme. Here, the lateral dimensions were determined to be 60 ( 10 Å × 60 ( 10 Å, with an apparent height of about 8-9 Å, which gives us an area on the surface for this configuration of 3500-3800 Å2. Both area values (lying and upright positions) are in accordance with X-ray crystallographic

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Figure 3. Isolated GOD molecules: (a) butterfly shape and (b) spherical shape adsorbed on an HOPG substrate, measured by in situ EC-STM in buffer solution. Usub ) -450 mV vs SCE, Ubias ) 50 mV and Isetpoint ) 0.5 nA. Scan rate: 2 Hz. Inset: 3-D topography of single GOD molecules, size: 20 nm × 20 nm.

data.13,22 The height of adsorbed GOD molecules as measured by STM (4-9 Å) is, in contrast to the X-ray crystallographic GOD height, notably smaller. Considerable reduction of the height has been observed in numerous STM images of biomolecules.23-25 The difference could be attributed to a modifed electron conductance at the site of the enzyme and also to undesired enzyme-tip interactions. Several images have been recorded from different areas of the samples, revealing the presence of adsorbed enzymes distributed over flat terraces and step edges of the anodized HOPG surface. At the step edges of HOPG, small aggregations of less than 10 enzymes were observed. For in situ STM experiments, we scanned large areas and chose terraces with low-density adsorbed GOD to perform local STM scanning of GOD molecules (Figure 4). Figure 4 shows the core experimental results, while the other experiments were performed to gain more understanding of the underlying phenomena. By measuring current-voltage curves in a potential range including the equilibrium potential of GOD with the EC-STM at single enzymes, the potential-dependent apparent morphology of adsorbed redox enzymes was studied. By keeping a constant bias voltage between the substrate and

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Figure 4. Series of in situ STM images of adsorbed GOD on HOPG in 10 mM PBS (pH 7.2) obtained at different overpotentials: (a) +200 mV, (b) +100 mV, (c) +50 mV, (d) 0 mV, (e) -50 mV, (f) -150 mV, and (g) -200 mV. Bias voltage: 100 mV (tip positive). Set point: 0.5 nA. Vertical range: 8 Å. Scan rate: 2 Hz.

Glucose Oxidase Adsorbed on Graphite Electrodes

Figure 5. Correlation between the normalized contrast and the overpotential η ) ∆U ) |Usub - UGOD,redox|. The ordinate represents the normalized image contrast between the enzyme and the background of the HOPG surface. The abscissa represents the overpotential applied to the HOPG surface to affect the reactivity of the adsorbed enzyme molecules. The data are derived from individual STM images in Figure 4a-e.

the tip, STM imaging started with the substrate potential set at the equilibrium redox potential of GOD. Imaging was continued with the potential set towards either positive or negative values versus the redox potential by adjusting the substrate and tip potentials simultaneously (i.e., at a constant bias voltage). As a result, a series of STM images was acquired at different potentials, which is presented in Figure 4. For substrate potentials close to the GOD redox potential (Figure 4d, Usub ≈ UGOD,redox), the tunneling current appears to be strongly enhanced at the site of the adsorbed enzyme; thus, this STM image has the highest contrast as compared to STM images obtained at different potentials. The image contrast begins to decrease when ∆U (∆U ) Usub - UGOD,redox) becomes more positive (Figure 4a-c) or negative (Figure 4e-g). The graph in Figure 5 is derived from the evaluation of the images of Figure 4a-g showing the normalized contrast between the adsorbed enzyme and the electrode surface of the EC-STM images. These results show certain similarities to the data obtained by a simulation based on a two-step ET mechanism by Ulstrup et al.12 In their theoretical model, suitable parameter values such as the reorganization free energy of 0.36-0.40 eV were chosen. To learn more about possible reasons for the potentialdependent apparent height of GOD in STM images, nontunneling contributions to the tip currents during enzyme scanning were studied. In these experiments, the tip was positioned out of the tunneling range (approximately some 10 nm above the HOPG sample). While the tip was positioned above the enzyme, the potential of the HOPG was swept between -0.6 and -0.15 V versus SCE, and the current at the tip (blue line in Figure 6, triangles) was recorded. Figure 6 shows the corresponding currents through the tip, including a CV of a typical submonolayer of GOD(FAD) at 1 V s-1 (black line, circles). A significant enhancement of the currents could be detected with distinct maxima/minima close to the GOD(FAD) redox potential (-0.47 V vs SCE). As the substrate potential sweeps towards more negative values, the current through the tip (blue line, triangles in Figure 6) increases to its absolute maximum value at -0.52 V, while the cathodic current at the substrate (black line, circles in Figure 6) reaches its peak at -0.47 V. The difference of 50 mV is caused by the time needed for involved redox species to diffuse to the STM tip. Sweeping to more positive potentials, the current of the STM tip increases to its negative absolute maximum, while the anodic current at

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Figure 6. CV (right ordinate, black curve, circles) of GOD-modified HOPG electrode and the corrected current through the tip (left ordinate, blue curve, triangles) at a 10 nm distance from the substrate. The red curve (left ordinate, no symbols) is the blank experiment that was recorded with the bare HOPG electrode. Electrolyte: 10 mM PBS, pH 7.2.

Figure 7. Nyquist plots of the impedance spectra of a HOPG electrode modified with GOD molecules in 30 mM PBS, argon saturated. Representative impedance curves at potentials -300, -475, and -600 mV vs SCE are displayed. Symbols Z′ and Z′′ refer to the real and imaginary components.

the substrate approaches its peak value. It should be kept in mind that the potential of the tip remains constant during these measurements, for example, 72 mV versus SCE in Figure 6. Reference experiments were performed with the STM tip over the bare HOPG surface (red line, no symbols in Figure 6). These experiments clearly showed that without adsorbed GOD at the electrode surface, no corresponding redox currents were observed at the STM tip. Furthermore, EC impedance spectroscopy was performed on the HOPG/GOD system. Impedance spectra were recorded at different potentials between -0.75 and -0.2 V versus SCE (Figures 7 and 8) to investigate surface effects due to adsorbed GOD. Nyquist diagrams of GOD-modified electrodes at potentials below UGOD (-0.6 V), at UGOD (-0.475 V), and above UGOD (-0.3 V) are presented in Figure 7. The comparison of those representative potentials shows that relative to the curve at -475 mV (red squares in Figure 7), the curves at potentials below or above UGOD (blue and black, triangles and circles in Figure 7) have a significantly higher |Z′′| value at low frequencies (160 Ω cm2 < Z′ < 220 Ω cm2). From impedance spectra at consecutive potential steps between -0.2 and -0.75

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Figure 8. Dependence of Ca and Rct for the GOD-modified HOPG electrode vs electrode potential. Error bars were determined due to differences between three repetitive measurements.

V, capacities were calculated. As can be seen in Figure 8, the capacity (Ca) and the charge-transfer resistance (Rct) show distinctive peaks at the GOD redox potential. Ca decreases from 10.5 µF cm-2 to 9.6 µF cm-2, and Rct increases from approximately 190 to 230 Ω cm-2. These different techniques enabled us to characterize the system with three independent techniques (STM, integral CV and EIS). 4. Discussion 4.1. Immobilization of GOD at HOPG. As described in the Experimental Procedures, GOD was adsorbed after anodizing the HOPG surface. Assuming a four-electron oxidation of each carbon atom to CO2 and considering the carbon atom density of the substrate as seen by the high-resolution STM, a charge transfer of 7.1 × 10-4 C cm-2 per oxidized carbon monolayer can be estimated. In our experiments, about 2-3 carbon layers were oxidized after 3 cycles at a scan rate of 200 mV s-1 in HClO4. GOD has a high net negative charge at the physiological pH value (isoelectric point pI ) 4.2) and is expected to adsorb at the anodized HOPG through the interaction between the free amino groups of the enzyme surface and the functional groups (such as the carboxyl) containing the oxygen of the HOPG surface.21 It is concluded from theoretical and experimental considerations that the electronic properties of carbon are wellsuited for the immobilization of enzymes. Promising results have been reported for carbon nanotubes and HOPG as respective substrates.26,27 The redox active center FAD in GOD is shielded by insulating polypeptides, and hence , ET according to FAD + 2H + + 2e / FADH2

(1)

cannot be easily achieved. Some enzymes (including GOD) can shuttle electrons directly to/from a conducting electrode surface. But, they gradually undergo denaturation at the surface. As a result, the bioactivity of the enzymes decreases significantly with time. In our experiments, the time used for the adsorption of GOD at the anodized HOPG electrode surface was chosen to be short enough to avoid the denaturation prior to the characterization experiments (CV, STM, and EIS). Figure 1 illustrates a typical CV of an anodized HOPG electrode after having been immersed in a GOD solution for 3 h. In this potential region, a redox couple wave appears that corresponds to the redox reaction of the cofactor in GOD with U1/2 at about -0.47 V versus SCE at a scan rate of 50 mV s-1 (eq 1).23-25 From the charge of the cathodic peak in Figure 1 (4.5 × 10-7 C) and the assumption of a two-electron transfer per enzyme

(eq 1), a surface coverage of 1.3 × 10-12 mol cm-2 can be concluded. This surface concentration has the same order of magnitude as estimated for a completely covered surface with adsorbed GOD (2.25 × 10-12 mol cm-2), assuming a densely packed layer of GOD. The individual space requirements of GOD molecules were determined from X-ray data, where the mean molecular cross-sectional area was identified to be 40 nm2/ enzyme.28 Because of a generally uneven distribution of enzymes on the surface as seen in STM images (only flat HOPG areas with low-density GOD adsorption were investigated), a direct comparison of the coverage obtained by electrochemistry and by STM is difficult. It should also be considered that weakly adsorbed GOD that contributes to the redox peak (Figure 1) may be removed from the site by the STM tip and hence is not imaged. After immobilization of the enzyme on the anodized HOPG electrode, the ET reaction between the electrode and the adsorbed enzymes can take place through those immobilized enzymes on the electrode. The linear dependence of the peak current on the voltage scan rate (Figure 1 inset) is consistent with a redox process of adsorbed species and reversible ET process (for a diffusion-limited process, a dependence on the square root of the scan rate is expected).24,29 4.2. Discussion of the Potential-Dependent Contrast in the GOD-STM Images. To the best of our knowledge, Facci first reported the phenomenon of potential-dependent contrast variations in STM images of proteins. The metallo-protein azurin was investigated but without pointing out a mechanism of ET in the results.30 Our observations of the potential-dependent contrast in the GOD-STM images could be partly understood by a two-step ET mechanism at a small bias (50 mV) in the STM redox process. Theoretical notions have been developed by Ulstrup et al. and Kornyshev et al. over the past few years.12,31 Complementing the experimental data and the theoretical model by Ulstrup et al.,12 our experimental results (Figure 5) reveal also a non-symmetrical shape. In our case, the contrast decay is more pronounced at positive overpotentials η with respect to the redox potential of GOD UGOD,redox. This apparent sensitivity of the image contrast to the EC potential may be consistent with the involvement of enzyme redox levels in the tunneling process. Such effects already have been observed in other redox molecules.12,17,30-34 Especially, Ulstrup et al. have intensively modeled the ET between enzymes and electrodes. In this approach, the Fermi levels of the STM tip, substrate, and energy levels of enzymes are considered. As a result of their model, they obtained asymmetric overpotentialcontrast correlations.12 While our results concerning the tunneling process are in agreement with the model proposed by Ulstrup et al., we have found a potential-dependent current at the retracted STM tip (Figure 6). Thus, there is an additional contribution from a Faradaic current at the STM tip in addition to the tunneling current. A more detailed interpretation of our results necessitates the consideration of the enzyme structure, the tunneling conditions, and possible EC contributions to the tip current. These points are discussed in the following sections. The cofactor (FAD) of GOD is a functional organic molecule, whose redox reaction, in contrast to that of many metallo-proteins, such as Zn2+ or heme,17,18 involves a complex two-electron and twoproton mechanism. Hence, its electronic and structural properties may have an important influence on the ET process. It is wellknown that STM images represent a convolution of the electronic and topographic structure of a surface. Therefore, the observed apparent height could be attributed to a change in the physical height of the sample, a change in the conductivity of

Glucose Oxidase Adsorbed on Graphite Electrodes

J. Phys. Chem. C, Vol. 112, No. 13, 2008 5171 currents can alter the contrast of STM images. According to eq 3, the measured current at the tip during STM scanning in constant current mode is in principle the sum of the tunneling current and Faradaic current

I ) It + If

Figure 9. Effect of tunneling current on the apparent height of enzyme molecules in STM measurements. The original height of molecules was set as H0 when the tunneling current equals to the set point (I0) during scanning. At the GOD site, the apparent height of molecules (H) varies due to Faradaic currents: H becomes larger than H0 when I > I0, whereas H is smaller than H0 when I < I0.

the sample, or even both. In our case, the effect of the barrier between the tip and the substrate is discussed. This aspect is closely related to the conductivity of the samples. In STM measurements, the tunneling current (It) depends on the distance (d) and the bias voltage (Ubias) between the tip and the substrate and is related also to the barrier (φ) of the substrate and the tip materials according to eq 2

(

It ∝ Ubias exp -4π

)

x2meφd h

(2)

where φ ) (φs - eUbias + φt)/2 and h is 6.626 × 10-34 J s. During the STM imaging in constant current mode at a constant bias voltage, initially d is kept constant, while there is a change in the barrier (φ), when the tip is moved from the bare HOPG surface to HOPG with adsorbed GOD. Hence, the tunneling current between the tip and the substrate is affected by the change in the barrier of the gap according to eq 2. In principle, this can explain the change in the apparent height of the enzyme molecules in STM images. As revealed in Figure 4c-e, the molecular features are clearly visible in the small ∆U range, while they are dark at large ∆U values (Figure 4a,b,f,g), and reappearing again, once the equilibrium potential is reestablished. It is also remarkable that in the images at ∆U ) 200 mV, darker spots appear at the same place, where the brighter spots are observed in the images at ∆U ) 0 mV. These results could be explained as a consequence of the STM feedback response to the local variation in the barrier. The results suggest that if the substrate potential is not tuned to the redox potential of GOD(FAD), the enzyme significantly decreases the charge transfer. In that case, it has insulating properties. Our results indicate that the conductivity of GOD(FAD) molecules presumably could be correlated to their activity. By using the STM tip as a local EC sensor, introduced and described by Meier et al.35,36 for investigating local EC reactions at nanoparticles, we investigated the influence of Faradaic currents induced by GOD(FAD) molecules. The result in Figure 6 clearly indicates that there are Faradaic currents at the tip, which is a feature of virtually all EC-STM measurements presented here. In principle, these electrochemically induced

(3)

where I is the total current at the tip, It is the tunneling current, and If is the Faradic current, respectively. A possible influence on the changes in the apparent height (Figure 4) in EC-STM measurements is given in Figure 9, considering the contributions of It and If. Since the constant current mode was used in STM imaging, the tunneling current (It) was controlled according to the set point (I0). The feedback control constantly adjusts I to match I0 (set current). In the following considerations, It is positive, which was the case in our measurements. As shown in Figure 9, we consider three cases, in which the tip with initially I ) It ) I0 (neglecting Faradaic noise currents If,noise < 10pA at the tip) scans over a GOD molecule (a) If ) 0 f I ) I0 f H ) H0 (tip remains in same position) (b) If > 0 f I > I0 f H > H0 (tip is lifted from the surface to reduce the tunneling current) (c) If < 0 f I < I0 f H < H0 (tip is approached to the surface to enhance the tunneling current)

Thus, the total measured current (I) is altered by Faradaic currents, which are potential dependent, at the tip (If); this is directly displayed in the contrast of the STM image. There are some possible reasons for the Faradaic currents at the tip. Since oxygen in the solution can usually not be removed completely in a STM setup, the contribution of oxygen to the Faradaic current should be considered.37 One possible reaction mechanism of GOD(FAD) reacting with molecular oxygen is described in various publications. The reaction between FAD and oxygen involves the transfer of a negative charge from the cofactor to the superoxide ion with no net change in charge at the active site.38-40 Superoxide ions react with FAD+ and produce hydrogen peroxide. Since this mechanism produces hydrogen peroxide, a change of the local hydrogen peroxide concentration might be a source for the Faradaic current at the tip. This is due to the fact that at the applied tip potential, hydrogen peroxide reduction can occur during STM imaging. However, calculations based on turnover rates of GOD enzymes (≈200 s-1)41 as well as our observations lead to the conclusion that the influence of Faradaic currents from enzyme activity on the measured tip current should be small (