Adsorption Behavior of Dinucleotides on Bare and Ru-Modified Glassy

Oct 8, 2008 - The reduction of FAD at a GCE/ruthenium-modified GCE surface is a ... the GCE surface in neutral and dilute solutions in order to maximi...
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Langmuir 2008, 24, 12375-12384

12375

Adsorption Behavior of Dinucleotides on Bare and Ru-Modified Glassy Carbon Electrode Surfaces H. Z. Wei,†,‡ T. G. M. van de Ven,*,‡ S. Omanovic,§ and Y. W. Zeng† College of Materials Science and Engineering, Nanjing UniVersity of Technology, Nanjing 210009, PR China, Department of Chemistry, McGill UniVersity, Montreal, Quebec H3A 2A7, Canada, and Department of Chemical Engineering, McGill UniVersity, Montreal, Quebec H3A 2B2, Canada ReceiVed June 19, 2008. ReVised Manuscript ReceiVed September 8, 2008 The interactive behavior of flavin adenine dinucleotide (FAD) with a bare glassy carbon electrode (GCE) and a Ru-modified GCE was investigated. The reduction of FAD at a GCE/ruthenium-modified GCE surface is a quasireversible, surface-controlled process, and our data implied that the attachment of FAD onto the surface is caused by nonspecific adsorption instead of covalent linkage, in which the adenine ring of FAD adopts a flat orientation on the GCE surface in neutral and dilute solutions in order to maximize the π-π stacking with the carbon surface and reorients to a perpendicular orientation as the surface gets more crowded. FAD desorption during the exchange with nicotinamide adenine dinucleotide (NAD+) is one order of magnitude slower than desorption in the absence of NAD+, which indicates a strong interaction between FAD and NAD+. General knowledge of the interactive behavior of NAD+ on a FAD-adsorbed GCE provides useful information for the design of a modified electrode surface for the generation of NADH from NAD+.

1. Introduction Flavin adenine dinucleotide (FAD) is a derivative of vitamin B2 (VB2) with a 7,8-diethylisoalloxazine structure (FAD, Scheme 1a). Because flavin adenine dinucleotide (FAD) is one of the very important coenzymes for many oxidoreductases,1 the direct interaction of FAD with various conducting/semiconducting materials, such as carbon nanotubes, mercury, platinum, graphite, and so forth, has been investigated extensively.1-9 Several improvements have been successfully made in the effectiveness of electron-transfer communication between the enzyme and the electrode. For instance, Willner et al.10,11 developed new methods to generate relay-FAD monolayers by the covalent coupling of FAD to a relay unit to yield an electrically contacted enzyme electrode, and Karyakin et al.12 discovered that electropolymerized FAD performed as a highly effective electrocatalyst for NADH oxidation. Liu et al.13 achieved efficient electron transfer between FAD and a glassy carbon electrode though a molecular wire. Poly(p-aminobenzene sulfonic acid) (PABS) films doped with * Author to whom correspondence should be addressed. Phone: +1 (514) 398-6177. Fax: +1 (514) 398-8254. E-mail: [email protected]. † Nanjing University of Technology. ‡ Department of Chemistry, McGill University. § Department of Chemical Engineering, McGill University. (1) Lin, C. S.; Zhang, R. Q.; Niehaus, T. A.; Frauenheim, Th. J. Phys. Chem. C 2007, 111, 4069–4073. (2) Birss, V. I.; Guba-Thakurta, S.; McGarvey, C. E.; Quach, S.; Vanysek, P. J. Electroanal. Chem. 1997, 423, 13–21. (3) Kamal, M. M.; Elzanowska, H.; Gaur, M.; Kim, O.; Birss, V. I. J. Electroanal. Chem. 1991, 318, 349–367. (4) McGarvey, C; Beck, S.; Quach, S.; Birss, V. I.; Elzanowska, H. J. Electroanal. Chem. 1998, 456, 171–191. (5) Kubota, L. T.; Gorton, L.; Roddick-Lanzilotta, A.; McQuillan, A. J. Bioelectrochem. Bioeng. 1998, 47, 39–46. (6) Wang, Y.; Zhu, G.Y.; Wang, E.K. Anal. Chim. Acta. 1997, 338, 97–101. (7) Gorton, L.; Johnasson, G. J. Electroanal. Chem. 1980, 113, 151–158. (8) Miyawaki, O.; Wingard, L.B. Biotechnol. Bioeng. 1984, 26, 1364–1371. (9) Miyawaki, O.; Wingard, L. B. Biochim. Biophys. Acta 1985, 838, 60–68. (10) Willner, I.; Heleg-Shabtai, V.; Blonder, R.; Katz, E.; Tao, G.; Buckmann, A. F.; Heller, A. J. Am. Chem. Soc. 1996, 118, 10321–10322. (11) Zayats, M.; Katz, E.; Willner, I. J. Am. Chem. Soc. 2002, 124, 2120–2121. (12) Karyakin, A. A.; Ivanova, Y. N.; Revunova, K. V.; Karyakina, E. E. Anal. Chem. 2004, 76, 2004–2009. (13) Liu, G. Z.; Paddon-Row, M. N.; Gooding, J. J. Electrochem. Commun. 2007, 9, 2218–2223.

the FAD-modified glassy carbon electrode showed excellent electrocatalytic activity for the oxidation of NADH and for the reduction of NAD+, with which cyclic voltammogram (CV) waves of the reversible nicotinamide adenine dinucleotide reduction-oxidation (NAD+/NADH) reaction were also observed.14 To develop an enzyme electrode for electrochemical biosensors and enzymatically based technological processes, it is essential to understand the interaction behavior of FAD with an electrode support. FAD consists of both aromatic rings and double bonds as shown in Scheme 1a, hence the molecule favorably attaches itself to metal or carbon surfaces.15 Indeed, a number of papers published discuss the adsorption phenomena of FAD onto a solid/ liquid electrode surface. Lin et al.1 reported the weak adsorption of FAD onto carbon nanotubes though π-π stacking between an adenine group and the surface. Wang et al.16 studied the electrochemical behavior of flavine adenine dinucleotide (FAD) at a gold electrode by using an electrochemical quartz crystal microbalance, with which adsorption of the reduced form (FADH2) and desorption of the oxidized form (FAD) was studied. Birss et al.2-4 investigated the adsorption of FAD on a mercury electrode surface, observing orientation changes at various stages. Shinohara et al.17 showed that the adsorption of flavins on TiO2 powder obeyed the Langmuir isotherm, and they observed differences in adhesion between FAD, FMN (flavin mononucleotide), and RF (riboflavin): the binding constants of FMN and FAD were found to be 1 order of magnitude higher than that of RF, which suggested an important contribution of the phosphate group to the adsorption. Because a large number of biochemical reactions catalyzed by redox enzymes are based on the use of nicotinamide adenine dinucleotide NAD(H) (Scheme 1b),18 the (14) Kumar, S. A.; Chen, S. M. Sens. Actuators, B 2007, 123, 964–977. (15) Bard, A. J. Electrochemical Methods: Fundamentals and Applications, 2nd ed.; John Wiley & Sons: New York, 2000. (16) Wang, Y.; Zhu, G. Y.; Wang, E. K. Anal. Chim. Acta. 1997, 338, 97–101. (17) Shinohara, H.; Gratzel, M.; Vlachopoulos, N.; Aizawa, M. Bioelectrochem. Bioenerg. 1991, 26, 307–320. (18) Damian, A.; Omanovic, S. J. Mol. Catal. A: Chem. 2006, 253, 222–233. (19) Zayats, M.; Katz, E.; Willner, I. J. Am. Chem. Soc. 2002, 124, 14724– 14735.

10.1021/la801926t CCC: $40.75  2008 American Chemical Society Published on Web 10/08/2008

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Scheme 1. (a) Flavin Adenine Dinucleotide (FAD) and (b) Nicotinamide Adenine Dinucleotide in Its Oxidized Form (NAD+)

electrochemical reduction of NAD+ to NADH using chemically modified and enzyme-mediated electrodes has been of great interest in this field of research.17,19,20 In our previous paper, the interaction of FAD with a glassy carbon electrode surface was investigated in terms of the FAD adsorption thermodynamics and kinetics as well as the electroreduction mechanism.21 The current paper compares the difference in FAD adsorption behaviour between FAD/GCE and FAD/Ru-modified GCE by employing various electrochemical techniques and focuses on the adsorption behavior of FAD on a GCE surface, discussing the surface chemistry and the orientation and reconformation of FAD on the surface. The immobilized FAD has already been shown to act as a catalyst for the oxidation of reduced nicotinamide adenine dinucleotide (NADH);9 therefore, the electrochemical reduction of NAD+ to NADH using chemically modified and enzyme-mediated electrodes has been investigated extensively.20 Knowing that FAD is playing a catalytic role in the reduction of some biological molecules and that it has a high affinity for glassy carbon electrodes, the interaction between NAD+ and FAD adsorbed on GCE will be discussed as well, which will pave the way for investigating the electrocatalytic reduction of NAD+ using a glassy carbon electrode modified with a FAD film.

2. Experimental Section 2.1. Chemicals and Reagents. All electrochemical studies were carried out in 0.05 M phosphate buffer at pH 7.0 (unless otherwise stated). The buffer was prepared by dissolving monobasic KH2PO4 (Sigma, P-5379) in ultrapure deionized water (resistivity 18.2 MΩ

cm) and adding 0.10 M sodium hydroxide (APC Chemical Inc.) to adjust the pH. Stock solutions of FAD (disodium salt, purity 98%, Sigma 146-14-5) and NAD+ (sodium salt, purity 95%, Sigma, N-0632) were prepared in separate containers by dissolving a proper amount of the reagent in the supporting electrolyte (phosphate buffer). All chemicals were used without further purification. 2.2. Electrochemical Cell, Electrodes, and Instruments. A standard three-electrode, two-compartment 50 mL electrochemical cell was used in all experiments. The counter electrode was a largearea platinum electrode of high purity (99.99%, Johnson-Matthey), which was degreased by refluxing in acetone, sealed in soft glass, electrochemically cleaned by potential cycling in 0.5 M sulfuric acid, and then stored in 98% sulfuric acid. During the measurement, the counter electrode was separated from the main cell compartment by a glass frit. The reference electrode was a saturated calomel electrode (SCE), to which all potentials in this article are referred. The working electrode was a glassy carbon electrode (GCE) (A ) 0.2826 cm2). Before each experiment, the GCE was polished with diamond paste down to a 3 µm gradation, followed by thorough rinsing with deionized water and ethanol and cleaning in an ultrasonic bath for 5 min to remove polishing residues. The electrode was then electrochemically pretreated (activated) in 0.5 M sulfuric acid by potentiodynamic cyclic polarization (100 cycles) between -1.4 and 1.9 V at a scan rate of 100 mVs-1. While not in use, all glassware was stored in 98% sulfuric acid. Electrochemical techniques of cyclic voltammetry (CV), differential pulse voltammetry (DPV), ac voltammetry (ACV), and differential capacitance (DC) were employed using an Autolab potentiostat/galvanostat/frequency response analyzer (Ecochemie, PGSTAT30) controlled by GPES/FRA v.4.9.5 software. The dc measurements were performed at a typical frequency and alternating

Adsorption BehaVior of Dinucleotides on Electrodes

Langmuir, Vol. 24, No. 21, 2008 12377 Scheme 2. (a) Formation of Functional Groups on a Glassy Carbon Electrode via Electrochemical Oxidation. (b) Proposed Covalent Attachment between FAD and Functional Carboxylic Acid Groups on a GCE. R Represents the Remaining Parts of FAD except for the Adenine Moiety

Figure 1. Recorded cathodic peaks of FAD adsorbed on GCE in phosphate buffer at pH 7.0 for various times: (1) 0, (2) 180, (3) 360, (4) 720, and (5) 1080 s. Scan rate, ν ) 100 mV s-1.

current amplitude of (10 mV. A submonolayer of ruthenium was potentiostatically deposited onto the prepared GC electrode at -0.2 V versus a SCE from a 1 mM RuCl3 solution in 0.5 M H2SO4. All measurements were carried out in an oxygen-free solution, which was achieved by continuous purging of the electrochemical cell with argon gas (99.998% pure) prior to the experiment. The measurements were made in a quiescent solution, and the inert atmosphere was maintained by saturating the cell space above the electrolyte with argon. All measurements were made at a temperature of 22 ( 1 °C. Before measurements in a FAD containing electrolyte, the background response of the electrode was first recorded in phosphate buffer. Aliquots of FAD were then added to the electrochemical cell, and the electrochemical measurements were repeated for each aliquot.

3. Results and Discussion 3.1. Nonspecific Adsorption of FAD on a GCE Surface. 3.1.1. Adsorption BehaVior of FAD on a GCE Surface. As discussed in our previous paper,21 a highly spontaneous, strong adsorption of FAD onto the GCE was reported as evidenced by the large, negative apparent Gibbs adsorption energy of -39.7 kJ mol-1 (or 16.7kT/molecule). The adsorption is so strong that it cannot be removed when the FAD-adsorbed GCE is washed in water or put in phosphate buffer or sulfuric acid solution or when being exposed to air overnight. However, the electrochemical response of FAD no longer exists when the FADadsorbed GCE is being kept at an anodic potential of 2.0 V in phosphate buffer for 10 s, which indicates that FAD does not desorb after the electrode is no longer exposed to an electrical potential. Strong electrical repulsion, caused by the applied electric field, can break the link between FAD molecules and the GCE surface. Figure 1 shows the recorded cathodic peaks of FAD adsorbed on GCE in phosphate buffer at pH 7.0 for various times. The FAD adsorbed on GCE causes a very stable, sharp electrochemical response of the FAD red/ox reaction for a long time when it is being put in phosphate buffer. Such a strong adsorption between FAD and a solid/liquid electrode surface was also reported by others.4,5,7 However, weak adsorption of FAD onto a glassy carbon electrode also was observed, from which adsorbed FAD could be very easily removed by washing the electrode.8 These inconsistencies raise questions as to why FAD shows different adsorption behavior on glassy carbon electrodes and whether the link between FAD and GCE should be ascribed to nonspecific adsorption or covalent attachment.

In our experimental procedure, the glassy carbon electrode was electrochemically activated in 0.5 M sulfuric acid by potentiodynamic cyclic polarization under a larger potential region. With comparing different procedures employed in each case for electrode pretreatment, it can be concluded that the inconsistency between our experiment and others in the literature regarding the adsorption behavior of FAD on a GCE is mainly caused by different procedures employed for electrode pretreatment. For instance, as Miyawaki et al.8 demonstrated, the surface concentrations of attached FAD on GCE are 1.7 × 10-11, 5.9 × 10-11, and 7.7 × 10-10 moles cm-2, respectively, when three methods are employed to provide carboxylic acid groups as well as other surface oxides on GCE surfaces by O2 plasma treatment, electrochemical cycling in nitric acid, and hot acid surface oxidation in nitric acid followed by sulfuric acid. As demonstrated by Bard,15 the pretreatment of the electrode by an oxidative reaction results in functional carboxylic acid groups on the surface. Presumably, FAD would covalently attach on GCE in a subsequent step via these functional groups as shown in Scheme 2. If it is true that FAD attaches to GCE through covalent linking, then FAD would not desorb or be replaced by other molecules. However, the desorption of FAD occurs as evidenced by a high desorption rate constant, kd, of 4.0 × 10-3 s-1 determined from the adsorption kinetics of FAD.21 In addition, further studies on FAD adsorption kinetics by differential capacitance (section 3.1.3) and exchange kinetics (section 3.3.1) argue against the assumption of covalent linkage, which will be discussed in more detail later on. 3.1.2. Conformation of Adsorbed FAD on a GCE. Provided that the linkage between FAD and GCE is ascribed to nonspecific adsorption (i.e., by π-π stacking), both the isoallxazine ring and the adenine moiety of FAD are promising points for attachment because of their conjugated double bonds. Wingard Jr. et al.22 demonstrated that direct attachment of FAD at position 8 of the isoalloxazine ring to an electrode surface is difficult, whereas direct attachment of the isoalloxazine moiety onto an electrode surface would result in a convenient electron transfer between FAD and the substrate. A low apparent heterogeneous electron-transfer rate constant (i.e., kapp ) 1.4 s-1) obtained from our previous study21 indicates that the isoalloxazine moiety might not directly adhere to the surface. The subsequent discussion on the variation of FAD surface concentration with pH will complement this conclusion, as shown below. From differential pulse voltammograms (DPV) recorded in 50 µM FAD solution at various pH values (Figure 2), it can be (20) Damian, A.; Omanovic, S. Langmuir 2007, 23, 3162–3171. (21) Wei, H. Z.; Omanovic, S. Chem. BiodiV. 2008, 5, 1622–1639. (22) Narasimhan, K.; Wingard Jr, L. B. J. Mol. Catal. 1986, 34, 253–262.

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Wei et al. Scheme 3. (a) Protonation Process of Adenine Ring and (b) Redox and Protonation Process of Isoalloxazine Moiety in FAD

Figure 2. Normalized differential pulse voltammograms of a GCE recorded in phosphate buffer containing 50 µM of FAD at various pH values: (a) 3.2, (b) 6.0, (c) 7.0, (d) 8.0, (e) 9.0, and (f) 10.0. Modulation time ) 0.07 s, modulation amplitude ) 0.05 V, internal time ) 0.2 s, step potential ) 0.015 V, scan rate, ν ) 7.5 mV s-1. Inset: Dependence of the surface concentration of FAD on solution pH obtained from cyclic voltammetry (O) and differential pulse voltammetry (∆) measurements.

seen that the peak decreases and gets wider with increasing pH of the bulk solution. Also, a set of cyclic voltammograms (CV) with various scan rates were recorded at different bulk pH values (not shown). Both the cathodic and the anodic currents depend linearly on the scan rate, which indicates that both the reactant (i.e., FAD) and product (i.e., FADH2) are strongly adsorbed under our experimental conditions. Because the oxidized form (FAD) is strongly adsorbed on a glassy carbon electrode surface and the reduction of adsorbed FAD produces adsorbed FADH2, the area of the DPV curves under the reduction wave, corrected for any residual current, represents the charge, Q, required for the full reduction of the layer (eq 3.1),15 and the faradic current in CV curves arising from the presence of the surface concentration of the attached FAD obeys the Nernst equation (eqs 3.2a and 3.2b)15,23

Q ) nFAΓ n F ΓAν 4RT 4(dipc/dν)RT

i) Γ)

(3.1)

2 2

(nF)2

(3.2a) (3.2b)

where Q(C) is the charge, n is the number of electrons involved in the redox reaction, F is the Faraday constant, A (cm2) is the surface area of the electrode, Γ (mol cm-2) is the FAD surface concentration, i (A) is the peak current, ν (V s-1) is the scan rate, R (J mol-1K-1) is the gas constant, and T(K) is the absolute temperature. The equilibrium surface concentrations of FAD on GCE were calculated by integrating DPV peaks after the baseline correction and from CV curves by using the Nernst equations (eqs 3.1 and 3.2). The inset of Figure 2 shows the dependence of the surface concentration of FAD on bulk pH. The inconsistency in the surface concentrations obtained from these two techniques is probably due to the difference in the polarization scan rate applied, even though the two techniques were performed on the same system. Obviously, the surface concentration of FAD is dependent on bulk pH, decreasing linearly with increasing pH, which is (23) Koval, A.; Anson, F. C. Anal Chem. 1978, 50, 223–229.

consistent with the observations that the surface concentration of FMN decreased with increasing solution pH.17 Taking the protonation or deprotonation process of FAD into account, the experimental phenomena can provide a clue as to possible conformations of FAD on the GCE surface during the adsorption process. Blazyk and Lippard24 showed that FAD and FADH2 species all exhibit pH-dependent changes with pKa values of 6.55 ( 0.05 and 7.1 ( 0.2, respectively. As shown in Scheme 3, the adenine ring of FAD (or FADH2) undergoes a protonation/deprotonation process whereas the isoalloxazine moiety is not only involved in the FAD redox process but also undergoes the protonation/ deprotonation process in the reduced form, FADH2. Because there is only one site (adenine ring) in FAD and two sites (both adenine ring and isoalloxazine moiety) in FADH2 undergoing the protonation/deprotonation process, the effect of bulk pH on the anodic peak corresponding to the oxidation of FADH2 to FAD would be greater than that on the cathodic peak corresponding to the reduction of FAD to FADH2 if the isoalloxazine moiety directly attaches to the substrate. However, both the cathodic and the anodic peak currents vary linearly with the scan rate with a nearly identical value of absolute slope (not shown), which confirms that the isoalloxazine moiety is oriented away from the surface while the adenine ring is directly adsorbed on the surface when FAD is adsorbed on the GCE. Considering that only the adenine ring undergoes the protonation (or deprotonation), FAD can be treated as a monoprotic acid (or base), and the relative mole fractions (or probability, p) of the protonation form of FAD can be derived.25 As a result, the dependence of the surface concentration of FAD on the relative fraction of the protonation form of FAD shows a linear relationship as shown in Figure 3. At low pH, the protonated form of FAD dominates, and the adenine ring is positively charged. Thus, strong adsorption occurs as a result of coulombic attraction between the positively charged amino group (e.g., -NH3+) in the adenine moiety of FAD and the electrode surface, and the adenine moiety is adsorbed in a perpendicular orientation, for which more FAD molecules can be accommodated on a unit (24) Blazyk, J. L.; Lippard, S. J. Biochemistry 2002, 41, 15780–15794. (25) Teraoka, I. Polymer Solutions: An Introduction to Physical Properties; Wiley InterScience: New York, 2002.

Adsorption BehaVior of Dinucleotides on Electrodes

Langmuir, Vol. 24, No. 21, 2008 12379 Table 1. Parameters of FAD Adsorption Kinetics Obtained in 10 µM FAD Bulk Solution Using Differential Capacitance at Different Frequencies ka/M-1s-1 25 Hz 100 Hz

7.75 × 10 10.01 × 102

2

kd/s-1

Bads/M-1

τads/s

τdes/s

4.70 × 9.85 × 10-3

1.65 × 1.02 × 105

129 100

213 102

10-3

105

apparent FAD surface coverage (θi) at constant electrode potential was first calculated from DC curves employing the following equation:27

θi )

Figure 3. Dependence of the surface concentration of FAD (Γ) on the relative mole fraction of the protonated form of FAD (p) obtained from cyclic voltammetry (O) and differential pulse voltammetry (∆) measurements.

o i C DC - C DC o min C DC - C DC

(3.3)

o where CDC (Fcm-2) is the differential capacitance in a FAD-free solution (supporting electrolyte), CiDC is the differential capacitance at a specific equilibrium concentration of FAD in the bulk min solution, and CDC is the differential capacitance at a maximum (saturated) FAD surface coverage. As shown in Figure 4, there is a significant difference in the kinetics of FAD adsorption at these two frequencies. At the higher frequency of 100 Hz, the surface coverage increases sharply and gradually reaches equilibrium, whereas at the lower frequency of 25 Hz the initial increase is slower but reaches a higher surface coverage at longer times. The adsorption kinetics of FAD at different frequencies was modeled using a first-order kinetic model (eq 3.4):28

dθ ) kac(1 - θ) - kdθ dt

(3.4)

where dθ/dt is the total change in adsorption expressed in terms of FAD surface coverage (s-1), ka is the adsorption rate constant (M-1 s-1), and kd is the desorption rate constant (s-1). By integrating eq 3.4, one obtains

θ) Figure 4. Time dependence of the FAD surface coverage on GCE recorded at 100 Hz (•) and 25 Hz (O) and Langmuir adsorption kinetics for the adsorption of FAD obtained from the data at different frequencies. The symbols represent the experimental data, and the line is the modeled isotherm. The inset shows schematically the orientation of FAD molecules at low and high coverages.

surface and thus higher surface coverages of FAD are reached at low pH. For neutral solutions (e.g., at pH 7), the surface concentration of FAD (Γ) is approximately half of that at low pH, as observed in the inset of Figure 2, and the adenine ring is no longer in the perpendicular orientation but adopts a flat orientation on the GCE surface in order to maximize the π-π stacking with the carbon surface, which is similar to that of the adenine moiety of NAD on a gold electrode,26 and also in agreement with our previous findings.21 3.1.3. Competition between Reconformation and Adsorption. As shown previously,21 Langmuir kinetics accurately describes the adsorption kinetics of FAD, and the parameters of adsorption and desorption constants were found to be ka ) 372 M-1 s-1 and kd ) 4.0×10-3 s-1. To eliminate the faradic (i.e., reductive/ oxidative) influence on the adsorption kinetics, the kinetics of FAD adsorption on GCE was further investigated by recording the variation of differential capacitance with time at a fixed electrode potential of 0 V and two different frequencies, 25 and 100 Hz, after adding FAD in phosphate buffer solution. The (26) Xiao, Y. J.; Chen, Y. F.; Gao, X.X. Spectrochim. Acta A 1999, 55, 1209– 1218.

1 - e-(kac+kd)t 1 + kd/(kac)

(3.5)

Corresponding parameters ka and kd are obtained from the best fit for times smaller than 300 s. The characteristics times of FAD adsorption (τads) and desorption (τdes) are derived from the rate constants based on the equations τads ) 1/(kadsC0) and τdes ) 1/kdes.29 All parameters are listed in Table 1. When comparing these parameters at two different frequencies, it is clear that both ka and kd at 100 Hz are higher than at 25 Hz. The dependence of the rate constants on the electrochemical conditions implies that the electrochemical conditions can affect the energy barriers and energy minima. Applied higher frequencies could lower energy barriers, thus speeding up adsorption. Similarly, the applied frequency could affect the desorption rate by modifying the energy minimum. Figure 4 also shows the agreement between the experimental data (symbols) and modeled data (solid line) for the adsorption kinetics. At 25 Hz, it can be seen that the Langmuir adsorption kinetics accurately describes the kinetics on the time scale of τdes (i.e., 100-200 s) but the modeled surface coverage deviates from experimental data for t g τdes. The same deviation between experimental and modeled data at 100 Hz was also observed. At long times, eq 3.5 reduces to θ ) (1)/(1 + X), where X ) kd/kac. The data at 25 Hz show a plateau in surface coverage (θ) of about 0.8 in Figure 4, for which X ) 1/4. Therefore, at long (27) Damaskin, B. B.; Petrii, O. A.; Batrakov, V. V. Adsorption of Organic Compounds on Electrodes; Plenum Press: New York, 1971. (28) Adamson, A. W. Physical Chemistry of Surfaces; InterScience: New York, 1986. (29) van de Ven, T. G. M. AdV. Colloid Interface Sci. 1994, 48, 121–140.

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Figure 5. Cyclic voltammograms of a Ru-modified GCE in phosphate buffer at pH 7.0 containing 50 µM FAD recorded at scan rates of ν ) 40, 80, 150, 200, 300, and 400 mVs-1 (from inner to outer). Inset: Dependence of the (∆) anodic and (O) cathodic peak currents on the applied scan rate obtained from these cyclic voltammograms.

Figure 6. Dependence of the surface concentration of FAD on the corresponding equilibrium concentration of FAD in phosphate buffer at pH 7.0. (Inset) Linearized Langmuir adsorption isotherm for the adsorption of FAD on a Ru-modified GCE. The symbols represent the experimental data, and the line is the modeled Langmuir isotherm.

times the desorption is 2.7 times slower than the initial desorption. This variation is ascribed to a reconformation from a flat configuration to a standing one, with much stronger lateral interactions. The decreases in the desorption rate constant in the adsorption-desorption equilibrium during FAD adsorption on the GCE surface will be compared with the FAD desorption behavior in the presence of NAD+ and will be discussed in section 3.3.1. Because the amount of adsorbate in a monolayer depends on both the size and orientation of the adsorbed molecules on the electrode surface, the explanation for the observed adsorption phenomena is that FAD rapidly adsorbed in a planar configuration relative to the electrode surface, which is bound to the surface through the adenine ring, and then underwent a slow reorientation of the adsorbed FAD molecules to a perpendicular orientation. The rate constant of desorption kd is no longer a constant but a function of time when molecules rearrange on the surface. The decrease in kd is caused by the stronger interaction among neighboring molecules after the reconformation of FAD oriented perpendicular on the electrode surface. The orientation of adsorbed FAD molecules is shown schematically in the inset of Figure 4. Moreover, the large rate constant of desorption during the FAD adsorption kinetics in a diluted FAD solution (i.e., 10 µM) proves that FAD does not covalently bind to a GCE but instead strongly adsorbs on it. 3.2. Interaction of FAD with a Ru-Modified GCE. For comparison, the interaction of FAD (i.e., its adsorption behavior) with a ruthenium-modified glassy carbon electrode (Ru-GCE) was also investigated, and the adsorption thermodynamics and electron-transfer kinetics are discussed below. 3.2.1. Deposition of Ru on the Glassy Carbon Electrode. Prior to Ru electrodeposition, the surface of a GC electrode was polished with diamond paste down to 0.03 µm, followed by degreasing with ethanol in an ultrasonic bath and electrochemical activation in 0.5 M H2SO4 by potentiodynamic polarization between -1.4 and 1.9 V for 40 cycles at scan rate of 100 mV s-1. A submonolayer of ruthenium was potentiostatically deposited onto the prepared GC electrode at -0.2V for 25 minutes from a 1 mM RuCl3 solution in 0.5 M H2SO4. By integrating the area, the deposition degree of Ru on the glassy carbon electrode was found to be 34.7%. 3.2.2. Thermodynamics of Adsorption of FAD at a Ru-Modified GCE Surface. Figure 5 shows a set of CVs recorded with

increasing scan rate in a solution containing 50 µM of FAD. Obviously, the separation between the cathodic and anodic peaks, ∆Ep, increases with an increase in scan rate (ν), and a plot of ∆Ep versus ν shows a linear relationship (∆Ep ) 0.105ν + 0.038, R2 ) 0.9922), indicating that the reduction of FAD on a Rumodifed GCE is a quasi-reversible reaction. Meanwhile, the dependence of the cathodic and anodic peak currents of the CVs presented in Figure 5 for various scan rates is plotted in the inset of Figure 5. The graph shows that both the cathodic and anodic peak currents vary linearly with scan rate, indicating that both the FAD reduction and FADH2 oxidation is a surface-controlled (adsorptive) process instead of a mass-transport process in dilute solution. To determine the isotherm adsorption of FAD on the Rumodified GCE, a set of CV curves for Ru-modified GCE in phosphate buffer at pH 7.0 containing different concentrations of FAD were recorded. The surface concentration of adsorbed FAD, Γ (moles cm-2), was calculated by integrating the area under the cathodic peak and using the well-known Faraday equation,15 and the corresponding dependence is shown in Figure 6. The Langmuir isotherm30 (eq 3.6) can be rewritten to yield the linearized form (eq 3.7) shown below

Γi )

ΓmaxBadsc 1 + Badsc

1 c c ) + Γi Γmax Bads Γmax

(3.6) (3.7)

where Γi (mol dm-2) is the FAD surface concentration at the corresponding equilibrium concentration of FAD in the bulk solution c (mol L-1 ) M), Γmax is the surface concentration at the maximum (saturated) FAD surface coverage, and the parameter Bads (M-1), known as the adsorption affinity constant, reflects the affinity of FAD towards GCE surface adsorption sites at a constant temperature. Hence, if the adsorption of FAD on the Ru-modified GCE surface follows a Langmuir isotherm, then a plot of c/Γi versus c should yield a straight line, which is indeed the case, as can be seen in the inset of Figure 6 (fitting regression coefficient R2 (30) Levine, I. N. Physical Chemistry, 5th ed.; McGraw Hill: New York, 2002.

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c 1 ) +c θi Bads

Figure 7. Capacitance curves of Ru-GCE recorded in phosphate buffer at pH 7.0 containing various concentration of FAD: (1) 0, (2) 10, (3) 30, (4) 50, (5) 100 µM. (Inset) Linearized Langmuir adsorption isotherm for the adsorption of FAD on a Ru-modified GCE. The data was obtained from ac voltammetry measurements at -0.30 V as presented here. The symbols represent the experimental data, and the line is the modeled isotherm.

) 0.997). The adsorption affinity constant, Bads, derived from the slope and the intercept is 1.11 × 105 M-1; correspondingly, the Gibbs free energy is calculated to be -38.3 kJ mol-1 on the basis of the following equation

Bads )

(

-∆Gads 1 exp 55.5 RT

)

(3.8)

where R (J mol-1 K-1) is the gas constant, T (K) is the temperature, ∆Gads (J mol-1) is the Gibbs energy of adsorption, and 55.5 is the molar concentration of a solvent (water in this case, mol dm-3). This free energy is for the adsorption of FAD in the perpendicular orientation because adsorption isotherms are constructed from the maximum adsorption (at long times) for various concentrations. Besides the techniques of CV or DPV, ac voltammetry (ACV) was also employed to investigate the adsorption of FAD on Rumodified GCE. A set of ACV curves were recorded at various FAD concentrations (not shown), and small split peaks appeared with increasing FAD bulk concentration, which is a manifestation of the fact that the surface concentration undergoes a transition from nearly pure oxide (i.e., FAD) to virtually pure reducer (FADH2) in the different potential regions for the two scan directions. The experimental observations from both CV and ACV confirm that the reduction of FAD on a Ru-modified GCE is a quasi-reversible reaction and that the FAD reduction and oxidation rate depends on the surface concentration of FAD on the electrode surface. The capacitance curves are obtained from recorded ACV in the electrochemical double-layer region, and the graph in Figure 7 clearly shows that a small amount of FAD (e.g. 10 µM) in the supporting electrolyte results in a significant decrease in capacitance. The measured capacitance further decreases with an increase in FAD bulk solution concentration, which is direct evidence of the adsorption of FAD on the Ru-GCE electrode surface in the dilute solution. To characterize the FAD adsorption process thermodynamically, the apparent FAD surface coverage at constant electrode potential (θi) was calculated from capacitance curves employing eq 3.3.27 Knowing that θi ) Γi/Γmax, the Langmuir isotherm (eq 3.7) can be further rearranged into the linearized form (eq 3.9):

(3.9)

Using the calculated surface coverage values, the adsorption isotherm of FAD on the surface was investigated. The FAD adsorption data from Figure 7 taken at -0.3 V are presented in the inset. The plotted curves of c/θ versus c at different potentials (i.e., -0.20, -0.30, -0.35 V) are very linear, giving an overall mean slope of 1.0015 and a mean correlation coefficient of R2 ) 0.9785. Such excellent agreement demonstrates that the Langmuir isotherm describes the adsorption of FAD onto the Ru-modified GCE surface very well. From the slope, an adsorption affinity constant of 1.08 × 105 M-1 is calculated. Correspondingly, the apparent Gibbs energy of adsorption of FAD onto the Ru-modified GCE is ∆Gads ) -38.2 kJ mol-1. The two parameters of the adsorption affinity constant and the apparent Gibbs energy obtained from the ACV technique are in excellent agreement with the values obtained from CV measurements. 3.2.3. Electron-Transfer Kinetics of FAD at a Ru-Modified GCE Surface. The electron-transfer kinetics of FAD on the Rumodified GCE was investigated by fitting the DPV curves recoded in the same FAD concentration region with the commercial software of GPES version 4.9.31 Figure 8 shows excellent agreement between the simulated (solid lines) and experimental (symbols) voltammograms obtained for DPV data at different concentrations (10, 30, and 50 µM). The mean value of the apparent electron-transfer coefficient is calculated to be 0.56 ( 0.04. From CV data recorded at various scan rates and different FAD concentrations at pH 7.1, a formal FAD reduction potential value of E′ ) -0.484 V was determined. The apparent electron transfer coefficient and heterogeneous electron transfer rate constant can also be calculated from the scan-dependant CVs using eq 3.1015

Ep - E ′ )

(

RTkapp RT ln RappnF RnFν

)

(3.10)

where E′ (V) is the formal potential, Rapp is the electron-transfer coefficient, and kapp (s-1) is the apparent heterogeneous electrontransfer rate constant for the surface electron transfer. The inset of Figure 8 shows that the data obtained at various scan rates agree well with eq 3.10. An apparent electron-transfer coefficient value of Rapp ) 0.56 was calculated from the slope of the line, which is the same value as obtained by the fitting procedure. Meanwhile, the intercept gave an apparent heterogeneous electron-transfer rate constant of kapp ) 2.32 s-1. Note that both the apparent electron-transfer coefficient Rapp ) 0.56 and the apparent heterogeneous electron transfer rate constant kapp ) 2.32 s-1 between the Ru-modified GCE and FAD are higher than those between bare GCE and FAD where Rapp ) 0.41 and kapp ) 1.4 s-1. Considering that the Gibbs free energy of FAD adsorption on the Ru-modified GCE surface (-38.3 kJ mol-1) is lower than on the bare GCE surface (-39.7 kJ mol-1), it can be concluded that FAD adsorption on a glassy carbon electrode surface is stronger and the deposited Ru island would block partial surface sites for FAD adsorption. On the basis of the above discussion, it can be concluded that effective adsorption happens on the interface between FAD and the glassy carbon electrode surface. 3.3. Interaction between NAD+ and FAD-Adsorbed GCE. 3.3.1. Replacement of Adsorbed FAD on GCE by NAD+. Knowing (31) Eco Chemie B. V. General Purpose Electrochemical System, version 4.9.5.

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Figure 8. Experimental (symbols) and simulated (lines) differential pulse voltammograms recorded in 0.05 M phosphate buffer containing various FAD concentrations: 10, 30, and 50 µM (from inner to outer). Modulation time ) 0.07 s, modulation amplitude ) 0.05 V, internal time ) 0.2 s, step potential ) 0.015 V, and scan rate, ν ) 7.5 mV s-1. (Inset) Dependence of the difference between the cathodic peak potential and the formal FAD reduction potential (Ep - E′) on the scan rate (ln ν). The symbols represent the experimental data, and the line is the linear fit.

Figure 9. Normalized differential pulse voltammograms of FADmodified GCE recorded in 0.05 M phosphate buffer containing various NAD+ concentrations: 0, 5, 10, 50, 100, and 500 µM (from outer to inner). Modulation time ) 0.07 s, modulation amplitude ) 0.05 V, internal time ) 0.2 s, step potential ) 0.015 V, and scan rate, ν ) 7.5 mV s-1.

that FAD is playing a catalytic role in the reduction of some biological molecules and that there is strong chemical adsorption between FAD and a glassy carbon electrode, we investigated the possibility of an electrocatalytic reduction of NAD+ using a glassy carbon electrode modified with a FAD film. After the preparation of a FAD film on a glassy carbon electrode by a potentiodynamic cycle of the GCE in 25 µM FAD for 60 cycles at a scan rate of 100 mV s-1, the FAD-modified GCE was put in a phosphate buffer containing various concentrations of NAD+. Figure 9 shows a set of normalized differential pulse voltammograms of FAD-modified GCE recorded in 0.05 M phosphate buffer containing various NAD+ concentrations in which the sharp peak at E ) -0.468 V versus SCE can be related to the oxidation/reduction peak of FAD.21 It is noticed that the DPV peak current decreases with the addition of NAD+ in the phosphate buffer. Compared with the discussion in the previous

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Figure 10. Cathodic curves of cyclic voltammograms of FAD-modified GCE in phosphate buffer after the addition of 250 µM of NAD+ recorded at various times: (1) 0, (2) 396, (3) 1276, (4) 2156, and (5) 4356 s.

section where the adsorbed FAD film on a GCE surface is so stable that it yields a very sharp, stable electrochemical red/ox response of FAD in phosphate buffer for a long time, the results in Figure 9 suggest that the adsorption sites of FAD molecules on the glassy carbon electrode are being replaced by NAD+ molecules, caused by stronger adsorption between NAD+ and a GCE surface. Such strong adsorption of NAD+/NADH occurring at glassy carbon electrodes had been reported before.32-34 A larger Gibbs free energy value of -(53 ( 3) kJmol-1 was reported for NADH-YADH adsorption onto a Pt electrode at 299 K,35 and large negative Gibbs energies of adsorption, -(43 ( 4) and -(39 ( 2) kJ mol-1, on positively and negatively charged polycrystalline gold electrodes were obtained by Omanovic et al.20 It was reported by Elving et al.32 that NAD+ adsorbate is tightly bounded to a glassy carbon electrode surface because it involves the interaction between both the adenine ring and the nicotinamide moiety, and they also concluded that the adsorbed NAD+ is in a planar configuration on the GC electrode surface, probably bound to the surface through the adenine moiety, and there is then a relatively slow reorientation of the adsorbed NADH molecules to a perpendicular orientation for which the adsorbate is more tightly bound to the surface than the planar-oriented adsorbate. Omanovic et al.20 demonstrated that both the adenine ring and the nicotinamide moiety directly adsorb on a gold electrode surface and they provided evidence that the nicotinamide moiety is adsorbed through the C-N bond of the carboamide group whereas the adenine ring of NAD+ is adsorbed though the bond between the amino group (NH2) and the electrode surface. Even though there are no data available for the adsorption thermodynamics of NAD+ on GCE, it is can be assumed that the negative Gibbs energy of NAD+ on a glassy carbon electrode would be larger than that of FAD on a glassy carbon electrode on the basis of the knowledge of the interaction behavior of NAD+/NADH on various solid substrates. Because both the nicotinamide moiety and the adenine ring of NAD+ preferably attach to a solid surface, it is reasonable to expect that NAD+ adsorbs on spots left empty by FAD. The process is further verified with cyclic voltammgrams of a FAD(32) Mooiroux, J.; Elving, P. J. J. Electroanal. Chem. 1979, 102, 93–108. (33) Chen, S. P.; Hosten, C. M.; Vivoni, A.; Birke, R. L.; Lombard, J. R. Langmuir 2002, 18, 9888–9900. (34) Samec, Z.; Elving, P. J. A. J. Electroanal. Chem. 1983, 144, 217–234. (35) Phillips, R. K. R.; Omanovic, S.; Roscoe, S. G Langmuir. 2001, 17(8), 2471–2477.

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ln

Figure 11. Cathodic peak currents corresponding to FAD reduction (O) (refer to the left Y-axis scale) and to NAD+ reduction (•) (refer to the right Y-axis scale) with time for FAD-modified GCE in phosphate buffer at pH 7.0 after the addition of 250 µM NAD+. The data were obtained from cyclic voltammograms presented in Figure 10 and other nonpresented voltammograms.

Figure 12. Dependence of the FAD surface coverage on desorption time. Symbols represents the experimental data, and the lines are the modeled data based on a pseudo-first-order desorption kinetic model: lines 1 and 2 represent the modeled data in the first stage (0-800 s) and the second stage (800-4500 s). The inset shows schematically the interaction of FAD and NAD+ on the electrode surface.

adsorbed GCE recorded over a wider potential region for various times after adding 250 µM NAD+ to the background solution, and the cathodic curves are shown in Figure 10. The two welldefined cathodic peaks at -1.17 and -0.52 V in the CV curves are attributed to the reduction peaks of NAD+ and FAD, respectively.20,21 The variation of the cathodic peak current related to FAD and NAD+ reduction with time, presented in Figure 11, clearly shows the decrease in the FAD reduction peak at -0.52 V and the increase in the NAD+ reduction peak at -1.17 V under the experimental conditions. The initial surface coverage of FAD (θ0) on the electrode surface was measured using an inorganic [Fe(CN)6]3-/4- complex as a probe molecule, which yields θ0 ) 0.405, and the kinetics of FAD desorption was modeled using a first-order kinetic model

dθ ) -kdθ dt Integrating the above equation gives

(3.11)

θ ) -kdt θ0

(3.12)

where dθ/dt (s-1) is the total change in desorption expressed in terms of apparent FAD surface coverage, with kd (s-1) being the desorption rate constant. The model (line 1 in Figure 12) fits the experimental data well for initial times up to around 800 s, with a desorption rate constant of 2.68 × 10-4 s-1. Subsequent desorption is much slower and the fitting (line 2) yields a desorption constant of 1.02 × 10-4 s-1, which is 2.63 times slower than the initial desorption. Note that during FAD adsorption, the desorption rate at long times is 2.7 times slower than the initial desorption rate constant (Figure 4), thus both decreases in the desorption rate constant (kd) for FAD on the GCE in the presence of NAD+ and in the absence of NAD+ are comparable. The lower desorption rate constant in the second stage (after 800 s) is likely due to the interaction of FAD and NAD+ because the bare sites on the GCE surface (about 60%) initially present will be quickly coated by NAD+. A similar slowdown in desorption suggests a similar explanation of the slow down. For FAD only, it was ascribed to a reconformation from a flat configuration to a standing one, with much stronger lateral interactions. Likely the same is occurring when the surface gets crowded by NAD+. The strong interaction between FAD and NAD+ is perhaps due to H-bonding or van der Waals interaction. Studies on the interaction of FAD and NAD suggests that the strength of the interaction is probably directly related to the effectiveness of the overlap of the two dinucleotides.36 Besides, the binding affinity calculations showed that quinones in the NAD(P)H/ quinone acceptor system interacted with the isoalloxazine ring of FAD utilizing a π-stacking interaction.37 The desorption kinetics of FAD in the presence of NAD+ further proves the conclusion that FAD is not covalently linked to GCE. 3.3.2. Reduction of NAD+ with FAD-Modified GCE. The information regarding the regeneration of NADH from NAD+ using the FAD-modified GCE was obtained from recorded CVs (Figure 11). A well-defined cathodic peak at -1.17 V versus SCE was recorded by the FAD-modified GCE in NAD+containing electrolyte, which is in excellent agreement with previous studies indicating the reduction of NAD+.32,38 The same values were also obtained on a cholesterol-modified goldamalgam electrode39 and on a basal pyrolytic graphite electrode.40 Omanovic et al.41 demonstrated that the cathodic peak at -1.18 V on a ruthenium-modified glassy carbon electrode corresponds to the two-electron reduction of NAD+ to NADH, while Eliving et al.33 interpreted the peak recorded on a glassy carbon electrode as the reduction of NAD+ to an NAD-NAD dimer (one-electron process). It is unknown whether the peak current at -1.17 V corresponds to the reduction of NAD+ to NADH or to an NAD-NAD dimer, and further work is needed to verify the reduction kinetics of NAD+ on an FAD-modified GCE surface. An understanding of the interactive behavior of NAD+ with a FAD-modified GCE gained in this study will provide useful information for the design (36) Chatterjee, C. L.; Torstensson, A.; Mitra, C.K. J. Electroanal. Chem. 1981, 128, 569–574. (37) Zhou, Z.; Fisher, D.; Spidel, J.; Greenfield, J.; Patson, B.; Fazal, A.; Wigal, C.; Moe, O. A.; Madura, J. D. Biochemistry 2003, 42, 1985–1994. (38) Man, F.; Omanovic, S. J. Electroanal. Chem. 2004, 568, 301–313. (39) Baik, S. H.; Kang, C.; Jeon, I. C.; Yun, S. E. Biotech. Tech. 1999, 13, 1–5. (40) Nakamura, Y.; Suye, S.; Kira, J.; Tera, H.; Tabata, I.; Senda, M. Biochem. Biophys. Acta 1996, 1289, 221–225. (41) Azem, A.; Man, F.; Omanovic, S. J. Mol. Catal. A: Chem. 2004, 219, 283–299.

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of a modified electrode surface for the generation of NADH from NAD+.

4. Conclusions The adsorption behavior of flavin adenine dinucleotide (FAD) on a bare GCE and a Ru-modified GCE was compared using cyclic voltammetry, differential pulse voltammetry and differential capacitance techniques. The reduction of FAD at a bare GCE or a Ru-modified GCE surface is a quasi-reversible, surfacecontrolled process in which the FAD adsorption represents the rate determining step in the overall reaction mechanism. The adsorption process was described by a Langmuir adsorption isotherm, and the agreement between the Langmuir isotherm and experimental data is excellent in the whole electrochemical double-layer potential region. The apparent negative Gibbs energy of adsorption of FAD onto the Ru-modified GCE of ∆Gads ) -38.2 kJ mol-1 is less than that of FAD on the bare GCE, ∆Gads ) -39.7 kJmol-1. Meanwhile, the apparent electron-transfer coefficient and apparent heterogeneous electron-transfer rate constant of kapp ) 2.32 s-1 and Rapp ) 0.56 between the Rumodified GCE and FAD are higher than that between bare GCE and FAD of kapp ) 1.4 s-1 and Rapp) 0.41. It can be concluded that the stronger adsorption of FAD on a glassy carbon electrode surface makes the electron-transfer communication between FAD and the electrode surface slower and deposited Ru islands likely partially block functional surfaces for the adsorption of FAD on the electrode surface. The large desorption rate constant of FAD during the FAD adsorption process argues against the assumption that FAD covalently links to the surface. Instead, the attachment of FAD onto the

Wei et al.

surface is caused by nonspecific adsorption in which the adenine ring of FAD directly attaches on the GCE with a flat orientation in neutral dilute solution in order to maximize the π-π stacking with the carbon surface and reorients to a perpendicular one as the surface gets more crowded. A pseudo-first-order kinetic model describes the adsorption kinetics well in the initial time period but deviates from the experimental data later on, which further provides evidence for the reconformation of FAD. The adsorbed FAD on a GCE is very stable but desorption occurs in the presence of NAD+. The initial desorption during the exchange (Figure 12) is 1 order of magnitude slower than the desorption during the FAD adsorption (Figure 4), and the desorption slows down later, which is mainly due to the strong interaction between FAD and NAD+. In future work, it will be of interest to characterize the nonspecific adsorption of FAD on GCE (e.g., the π-π stacking interaction) and investigate the reduction mechanism of NAD+ with the FAD-adsorbed GCE. Understanding the mechanism of NAD+ reduction with FAD-modified GCE opens the possibility to design a flavoenzyme electrode by the assembly of a FAD monolayer or by the covalent attachment of FAD to a glassy carbon electrode for the generation of NADH from NAD+. Acknowledgment. Grateful acknowledgment is made to the Natural Science and Engineering Research Council of Canada, Quebec Merit Fellowship of Canada, and the National Natural Science Foundation of China (no. 40603007) for the support of this research. LA801926T