Langmuir 2005, 21, 7907-7911
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Biomimetics with a Self-Assembled Monolayer of Catalytically Active Tethered Isoalloxazine on Au Ernesto J. Calvo,*,† M. Silvina Rothacher,† Cecilia Bonazzola,† Ian R. Wheeldon,† Roberto C. Salvarezza,‡ Maria Elena Vela,‡ and Guillermo Benitez‡ Departamento de Quı´mica Inorga´ nica, Analı´tica y Quı´mica Fı´sica, Facultad de Ciencias Exactas y Naturales, INQUIMAE, Pabello´ n 2, Ciudad Universitaria, AR-1428 Buenos Aires, and Instituto Nacional de Investigaciones Fisicoquı´micas Teo´ ricas y Aplicadas (INIFTA), Casilla de Correo 16, Suc. 4, 1900 La Plata, Argentina Received March 15, 2005 A new biomimetic nanostructured electrocatalyst comprised of a self-assembled monolayer (SAM) of flavin covalently attached to Au by reaction of methylformylisoalloxazine with chemisorbed cysteamine is introduced. Examinations by Fourier transform infrared spectroscopy and scanning tunneling microscopy (STM) show that the flavin molecules are oriented perpendicular to the surface with a 2 nm separation between flavin molecules. As a result of the contrast observed in the STM profiles between areas only covered by unreacted cysteamine and those covered by flavin-cysteamine moieties, it can be seen that the flavin molecules rise 0.7 nm above the chemisorbed cysteamines. The SAM flavin electrocatalyst undergoes fast electron transfer with the underlying Au and shows activity toward the oxidation of enzymatically active β-NADH at pH 7 and very low potential (-0.2 V vs Ag/AgCl), a requirement for use in an enzymatic biofuel cell, and a 100-fold increase in activity with respect to the collisional reaction in solution.
Introduction In this work we report a new flavin-modified electrode based on a self-assembled monolayer (SAM) of a flavin analogue tethered to a gold surface by an N-10 linkage. We introduce an alternative strategy to modify a gold surface by postfunctionalization with a flavin monolayer attached via a covalent bond between the methylformylisoalloxazine derivative and cysteamine chemisorbed on gold. The electrocatalytic activity toward the oxidation of enzymatically active β-NADH and detailed kinetic parameters are also reported. Flavins, including flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), and riboflavin, play an important role in the shuttling of electrons in a number of biological redox reactions. They can efficiently reduce oxygen or oxidize the oxygen-insensitive nicotinamide adenine dinucleotide, NAD(P)H, enzyme cofactor. According to C. Walsh,1 flavins are at the crossroad of biological chemistry: They can interact with two-electron donors (such as NADH in reductase enzymes) and with one-electron acceptors (such as iron-sulfur or heme proteins). They can also undergo very fast reduction of oxygen both by two-electron reduction, leading to hydrogen peroxide in oxidases, and by four-electron reduction to water in monooxygenases. On the other hand, NAD(P)H is oxidized at one extreme of the respiratory chain by a flavoprotein. In reductases, flavins are the sites of NADH oxidation with further charge transfer to the enzyme substrate reduction site. Efficient recycling of NADH is of great interest in the application of dehydrogenase-based devices such as biosensors,2,3 * To whom correspondence should be addressed. E-mail:
[email protected]. Phone: (5411)4576-3378. Fax: (5411)45763341. † INQUIMAE. ‡ INIFTA. (1) Walsh, C. Acc. Chem. Res. 1980, 13, 148-155.
biocatalysis,4 and biofuel cells.5 The direct electrochemical oxidation of NADH at bare metal electrode surfaces is not efficient due to free radical formation and electrode fouling.3 Oxidation of NADH by flavin sites buried inside the protein structure of enzymes such as glutathione reductase is very efficient.6 However, to mimic the geometry found for FAD sites in enzymes that oxidize NAD(P)H, it is essential that the flavins lie perpendicular to the surface so that a flat NAD(P)H molecule can approach the catalyst site parallel to the flavin and thus form a charge-transfer complex.7 There has been a significant amount of research on the electrochemical behavior of flavins in solution8-10 and adsorbed on Hg11-13 and on carbon14 electrode surfaces. (2) Simon, E.; Bartlett, P. N. In Modified Electrodes for the oxidation of NADH in Biomolecular Films: Design, Function and Applications; Rusling, J. F., Ed.; Marcel Dekker: New York, 2003; Chapter 11. (3) Gorton, L.; Dominguez, E. Electrochemistry of NAD(P)+/NAD(P)H. In Encyclopedia of Electro-chemistry, vol 9. Bioelectrochemistry; Wilson, G., Stratmann, M., Bard, A. J., Eds.; Wiley-VCH: Weinheim, Germany, 2003. (4) Bartlett, P. N.; Pletcher, D.; Zeng, J. J. Electrochem. Soc. 1997, 144, 3705-3709. (5) Palmore, G. T. R.; Bertschy, H.; Bergens, S. H.; Whitesides, G. M. J. Electroanal. Chem. 1998, 443, 155-161. (6) Pai, E. F.; Schulz, G. E. J. Biol. Chem. 1983, 258, 1752-1757. (7) Edwards, T. G.; Cunnane, V. J.; Parsons, R.; Gani, D. J. Chem. Soc., Chem. Commun. 1989, 1041-1043. (8) Foresti, M. L.; Pergola, F.; Aloisi, G.; Guidelli, R, J. Electroanal. Chem. 1982, 137, 341-353 and 355-366. (9) Ksenzhek, O. S.; Petrova, S. A. Bioelectrochem. Bioenerg. 1968, 11, 105-127. (10) Reeves, J. H.; Weiss, K. J. Electroanal. Chem. 1987, 217, 6578. (11) Birss, V. I.; Elzanowska, H.; Turner, R. A. Can. J. Chem. 1988, 66, 86-96. (12) Birss, V.; Hinman, A. S.; McGarvey, C. E.; Segal, J. Electrochim. Acta 1994, 39, 2449-2454. (13) Birss, V.; Guha-Thakurta, S.; McGarvey, C. E.; Quach S.; Vanusek, P. J. Electroanal. Chem. 1997, 423, 13-21. (14) Gorton, L.; Johansson, G. J. J. Electroanal. Chem. 1980, 113, 151-158.
10.1021/la050695n CCC: $30.25 © 2005 American Chemical Society Published on Web 07/19/2005
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In particular, a number of reports described the electrocatalytic activity of immobilized flavin-functionalized SAMs.7,15-21 In these modified electrodes flavins have been tethered by a Au-thiol bond at C-7 and C-8,15 N-10 and N-3,17 and N-10,18,19 by a disulfide tether at C-7 and N-7,7,16 and also by modified N6-(2-aminoethyl)-FAD covalently immobilized on SAMs.20,21 Unlike previous reports, in the present study the methylformylisoalloxazine is not directly attached to the Au surface but is attached through chemical reaction with a previously adsorbed thiol carrying reactive NH2 end groups, which determines the flavin-to-flavin distance. The method involves postfunctionalization with flavin as opposed to direct attachment of the flavin via a disulfide and offers several advantages over the direct thiolation of the Au surface. This strategy was introduced by Katz in 1990 by forming a SAM with a short alkanethiol carrying an amino end group that was subsequently reacted with an aldehyde functionality in the target molecule to be immobilized at the surface.22,23 In our hands, ferrocene aldehyde was also attached covalently to Au via a terminal amino SAM.24 An alternative strategy has been described by Myles,25 who reported the reaction of an aldehydeterminated SAM on Au with alkylamines from solution. A careful molecular characterization of the resulting surface modification after each step has been accomplished by cyclic voltammetry (CV) over a wide range of pH, Fourier transform infrared reflection-absorption spectroscopy (FTIR-RAS), scanning tunneling microscopy (STM), and Auger electron spectroscopy (AES). Experimental Section Cysteamine (Aldrich), riboflavin (Sigma), and periodic acid (Merck) were used as supplied. All solutions were prepared with Milli-Q (Millipore) deionized water. Electrochemical measurements were carried out in a glass electrochemical cell with a total solution volume of 6 mL using a TEQ-2 potentiostat with data acquisition capability. All solutions were purged with nitrogen for a minimum of 20 min before each experiment, and a current of nitrogen was allowed to flow over the solution during the measurements. Platinum mesh was used as a counter electrode, and a 3 N Ag/AgCl electrode was used as a reference. All potentials are reported relative to that of this reference. For electrochemical characterization the synthesis was carried out on pure gold substrates with an approximate area of 1 cm2. Chronoamperometric studies were used to measure the electrocatalytic activity of the SAM flavin electrode for the oxidation of NADH to NAD+. The NADH-induced current at pH 7.0 was measured by adding deoxygentated, freshly prepared, NADH solution to a buffer solution containing the modified electrode at a fixed potential. The system was investigated with various concentrations of NADH, and its concentration in solution was determined spectroscopically ( ) 6600 M-1 cm-1 at λ ) 340 nm). (15) Wingard, L. B. Bioelectrochem. Bioeng. 1982, 9, 307-312. (16) Mallik, B.; Gani, D. J. Electroanal. Chem. 1992, 326, 37-49. (17) Tam-Chang, S.-W.; Mason, J.; Iverson, I.; Hwang, K.-O.; Leonard, C. Chem. Commun. 1999, 65-66. (18) Cooke, G.; Duclairoir, F. M. A.; John, P.; Polwart, N.; Rotello, V. M. Chem. Commun. 2003, 2468-2469. (19) Stine, K. J.; Andrauskas, D. M.; Khan, A. R.; Forgo, P.; D’Souza, V. T.; Liu, J.; Friedman, R. M. J. Electroanal. Chem. 1999, 472, 147156. (20) Blonder, R.; Willner, I.; Bueckmann, A. F. J. Am. Chem. Soc. 1998, 120, 9335-9341. (21) Liu, J.; Paddon-Row, N.; Gooding, J. J. J. Phys. Chem. B 2004, 108, 8460-8466. (22) Solov’ev, A. A.; Katz, E. Y. J. Electroanal. Chem. 1990, 277, 337(23) Katz, E.; Itzahak, N.; Willner, I. Langmuir 1993, 9, 1392(24) Molinero, V.; Calvo, E. J. J. Electroanal. Chem. 1998, 445, 1728. (25) Horton, R. C., Jr.; Herne, T. M.; Myles, D. C. J. Am. Chem. Soc. 1997, 119, 12980-12981.
Calvo et al. Scheme 1. Synthesis of a Self-Assembled Monolayer of Tethered Isoalloxazine: (a) Synthesis of Formylmethylisoalloxazine, (b) SAM Flavin Synthesis
In scanning tunneling microscopy and Auger electron spectroscopy experiments, evaporated Au Arrandees slides were used. After being annealed for 3 min with a hydrogen flame, these Au substrates exhibit atomically smooth (111) terraces separated by monatomic steps in height.26 As already reported the Au surface after flame annealing consists of micrometer-sized (111) preferred-oriented crystals with atomically smooth triangular terraces separated by monatomic steps in high. The height of these steps (0.24 nm) was used to calibrate the piezotube of the STM in the z-direction. STM images (constant current mode) were taken with a Nanoscope III (Digital Instruments, Santa Barbara, CA) using Pt-Ir tips. The surface chemical composition of the films formed on the Au electrode was analyzed by AES using a single-pass cylindrical mirror analyzer (CMA; Physical Electronics). Prior to use the preferentially oriented Au substrates were treated with acetone, rinsed with deionized water, and dried under a nitrogen jet. FTIR-RAS spectra were recorded with a Nicolet Magna 560 FTIR spectrometer and a Whatman 75-52 purge gas generator, equipped with a cryogenic MCTA detector and a zinc selenide (Nicolet, Spectratech) polarizer. The spectra of SAM-flavinmodified gold substrates were obtained using a purpose-built reflectance setup at an 80° angle of incidence, a bare gold surface as a reference or background, and s- and p-polarized radiation. The spectra were collected at 4 cm-1 spectral resolution using 200 scans and are presented without smoothing correction. The gold substrates, films of approximately 200 nm, were evaporated on titanium-modified silicon slides with an Edwards Auto 306 vacuum coating system. The titanium and gold were vapor deposited at 1 × 10-1 and 5 × 10-5 mbar, respectively. The flavin analogue, methylformylisoalloxazine, was produced by replacing the carbohydrate tail of riboflavin with a methyl aldehyde group. The modified isoalloxazine was then covalently bound to an adsorbed alkanethiol-derivatized gold substrate, thus creating a SAM of tethered, catalytically active, flavin analogues. The synthesis of the SAM flavin electrode is outlined in Scheme 1. Gold substrates were first primed with amino groups by immersion in a 0.1 M cysteamine solution for 2 h and then rinsed with deionized water. After thiol adsorption, the substrates were immersed in 5 mL of methanol solution containing 8 mg of methylformylisoalloxazine and 50 µL of triethylamine (TEA). The preparation of the formyl derivative of isoalloxazine has been reported elsewhere.27,28 The reaction between the modified isoalloxazine and the adsorbed thiol was run between 12 and 15 h in the absence of (26) Andreasen, G.; Vela, M. E.; Salvarezza, R. C.; Arvia, A. J. Langmuir 1997, 13, 6814-6819. (27) Bonazzola, C.; Brust, M.; Calvo, E. J. J. Electroanal. Chem. 1996, 407, 203-207. (28) Fall, H. H.; Petering, H. G. J. Am. Chem. Soc. 1956, 78, 377381.
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Figure 1. Cyclic voltammograms of a flavin SAM on Au in electrolytes of pH 3 (potassium hydrogen phthalate), 8 (Tris), and 12 (NaOH) at 0.05 M, ionic strength 0.5 M, sweep rate 0.1 V s-1. Inset: pH dependence of the formal half-wave potential, E1/2. light. The system was then cooled in an ice bath and stirred for 1 h at room temperature. Prior to cooling, an excess of potassium borohydride was added to reduce the Schiff base. Finally, the modified surface was rinsed with plenty of water and dried with a nitrogen jet.
Figure 2. FTIR-RAS spectra ex situ of a flavin SAM on Au for s- and p-polarized radiation.
one would expect a -60 mV per pH unit dependence. The slope of the plot shown in Figure l (inset) is -0.04 V, and the difference may be due to the protonation of the unreacted cysteamines (see evidence of unreacted thiol in the STM profiles below) in addition to the protonation of tethered flavins. Thus, there is a microenvironment effect around the flavin molecules in the surface. The formal redox potential at pH 7 is Eo′ ) -0.382 V and is in good agreement with previous reports of similarly immobilized flavins on Au.7,16,17,19 The peak potential separation is ∆E ) 0.05 V. Integration of the redox peaks yields an average charge from which the surface coverage of the bound flavin was calculated for a two-electron reaction. The surface coverage was calculated to be between 5.2 × 10-11 and 1.65 × 10-10 mol cm-2, values which are of the same order of magnitude as in previous reports of similar flavin-derivatized surfaces, i.e., 5.0 × 10-10 mol cm-2 17 and 1.5 × 10-10 mol cm-2.19 The scatter of flavin coverage values in different samples seems to be
related to the postfunctionalization reaction of flavin with the thiolated surface. FTIR-RAS spectra of a SAM-flavin-modified gold surface between 1900 and 1300 cm-1 are shown in Figure 2. Since only p-polarized radiation is enhanced at the surface, the dynamic transition dipolar moments of the molecular vibrations that lie or have a component perpendicular to the surface are observed (surface selection rule).30 The upper spectrum was obtained with s-polarized radiation and confirms that it is IR-blind at the surface because of the 180° change of the phase angle upon reflection. For a flat molecule, such as isoalloxazine in its oxidized state, the observed signals are consistent with the fact that the molecules cannot lie flat or parallel to the surface. The modes at 1712, 1677, 1579, and 1548 cm-1 are in accordance with the vibrations of the isoalloxazine ring system in lumiflavin as reported by Abe and Kyogoku,31 who reported a normal coordinate analysis. These vibration frequencies can also be compared with the spectrum of the formylmethylisoalloxazine (not shown) and with the spectrum of FAD in a D2O solution of pH 7.0 reported by Birss et al.12 The broad band observed at 1480 cm-1 can be associated with secondary amine CH2-N deformation and/or protonated primary amines associated by hydrogen bridges. By comparison with the work of Abe and Kyogoku31 and Birss et al.,12 the bands for isoalloxazine attached in the SAM flavin electrode reported here have been assigned as shown in Table 1. The STM image (Figure 3a) of the cysteamine-SAMcovered gold electrode reveals ordered domains of cysteamine molecules coexisting with gold island vacancies of monoatomic depth (black holes in the image). In fact, domains of x3×x3R30° (left bottom) and c(4×2) (upper part) are resolved in this image. The inset in Figure 3a shows a detail of the x3×x3R30° lattice where isolated cysteamine molecules separated by 0.5 nm are clearly seen (cross-section inset). The AES spectrum of this surface shows the Au NVV transitions at 70 eV and a signal at 152 eV that corresponds to S(LLV) transitions originated from the S heads of thiolates, see Figure 3c (lower spectrum), bonded to the Au. The signal at 395 eV corresponds to Au transitions that overlap to some extent the signal at 381 eV originated from the N(KLL) transitions of the NH2 groups of the cysteamine.
(29) Draper, R.; Ingraham, L. Arch. Biochem. Biophys. 1968, 125, 802-808.
(30) Greenler, R. G. J. Chem. Phys. 1966, 44, 310-315. (31) Abe, X.; Kyogoku, X. Spectrochim. Acta 1987, 43A, 1027-1037.
Results and Discussion The cyclic voltammograms of SAM flavin electrodes at 0.1 V s- 1 are depicted in Figure 1 for solutions of pH 3, 8, and 12. From the i-E curve we demonstrate the presence of electroactive isoalloxazine groups attached to the Au surface; surface voltammetric waves characteristic of redox sites bound to the electrode are apparent with peak current linearly dependent on the potential sweep rate. The half-wave formal potential, E1/2 (mean value of the anodic and cathodic peak potentials), shifts with electrolyte pH by -0.04 V per pH unit (regression coefficient 0.993; see the inset in Figure 1.). However, in all previous studies of flavin monolayers the redox potential pH dependence was close to 60 mV per pH unit.16,17,19 The acid-base properties of flavins in solution show a complex pattern with breaks due to pK values.29 The inset graph in Figure 1 shows a straight line with no change in slope. For the a two-proton two-electron reaction
Flox + 2H+ + 2e f FlH2
(1)
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Table 1. Band Assignments of the Observed Frequencies for a SAM-Flavin-Modified Electrode band
observed frequency/cm-1
assignment
ν4 ν5 ν8 ν9
1712 1677 1574 1548
C4dO stretching vibration coupled with C2dO stretching vibration C2dO stretching vibration coupled with N3-H bending and C4dO stretching vibration mainly N5-C4a and in-phase stretching vibration C10a-N1 N5-C4a and C10a-N1 out-of-phase stretching vibration
following the substrate directions. The angles between intersecting rows are 60°, and the spot-spot distance and the corrugation are 2.0 ( 0.1 and 0.3 nm, respectively, as determined by the cross-section analysis (inset in Figure 3b). Assuming that each bright spot corresponds to a flavin molecule, the surface concentration results in Γ ) 4 × 10-11 mol cm-2, which is in good agreement with that calculated by the electrochemical method. From these results one can conclude that each spot observed in the STM image corresponds to an electrochemically active flavin molecule. The analysis of STM images also allows us to determine the configuration of the flavin molecule. In fact, we have observed islands where the rows of flavin molecules are absent (see the Supporting Information). The height difference between these islands and the regions where the rows of molecules are present is 0.7 nm (see the Supporting Information). Assuming that the island bottom is covered by cysteamine molecules, this difference is close to that expected for a flavin molecule attached to cysteamine in a vertical or slightly tilted configuration (see Scheme 1b). The AES data for the SAM flavin electrode (Figure 3c, upper spectrum) show features similar to those described for the cysteamine-covered Au although, as expected, a slight increase in the N(KLL) signal is observed. The fact that the S signal is clearly visible supports our previous assumption that the cysteamine molecules are not removed during the synthesis step and remain chemisorbed on the Au surface. Electrooxidation of NADH by flavins in solution is sluggish,32 and previous attempts at immobilizing flavins to improve catalysis have had varying success.19,33,34 Isoalloxazine can effectively oxidize NAD(P)H at potentials more oxidant than the redox potential for the NAD(P)+/ NAD(P)H couple, ca. -0.57 V at pH 7.5.5 The catalytic oxidation of β-NADH on the flavin-modified electrode surface shows a nonlinear concentration dependence at potentials as negative as -0.25 V that can be modeled with a reaction scheme that involves the formation of a charge-transfer complex, the decomposition of which yields the reaction product, NAD+:2,3 k1
k2
β-NADH + S-Flox {\ } S-FloxNADH 98 k -1
S-Flred + NAD + (2) The catalytic current for this mechanism can be expressed by
icat )
Figure 3. STM images and AES spectra.
A typical STM image of the SAM flavin electrode is depicted in Figure 3b. The typical cysteamine-Au pattern shown in Figure 3a has been completely changed. The image is now dominated by rows of bright spots aligned
imax 1 + KM/[NADH]
(3)
(32) Jones, J. B.; Taylor, K. E. Can. J. Chem. 1976, 54, 2974-2980. (33) Chi, Q.-J.; Dong, S.-J. J. Mol. Catal. A: Chem. 1996, 105, 193201. (34) Kubota, L. T.; Gorton, L. J. Solid State Electrochem. 1999, 3, 370-379. (35) Munteanu, F. D.; Kubota, L. T.; Gorton, L. J. Electroanal. Chem. 2001, 509, 2-10. (36) Jiang, F.; Mannervik, B. Protein Expression Purif. 1999, 15, 92-98.
Biomimetics with a SAM of Isoalloxazine on Au
Figure 4. Catalytic current for the oxidation of β-NADH at -0.20 V vs concentration. Line: best fit to eq 3. Inset: potential dependence of the catalytic current. The current shown in the graph was measured with an electrode with a charge of 13 µC cm-2.
where KM ) (k-1 + k2)/k1 is the equilibrium constant for the formation of the charge-transfer complex and imax ) 2Fk2([Fl]/KM) is the maximum oxidation catalytic current density at saturation of NADH when [NADH] . KM. The surface concentration of tethered flavin at the surface, [Fl], has been calculated from the electrical charge integrated in cyclic voltammetry of the flavin-modified electrode in the absence of NADH in solution. Thus, for the data depicted in Figure 1 (16 µC cm-2) it corresponds to 8.3 × 10-11 mol cm-2 for a two-electron charge-transfer reaction. The catalytic current-substrate concentration curve in Figure 4 shows a shape that is characteristic of preequilibrium with formation of a charge-transfer complex between the tethered flavin and NADH. The best nonlinear fit of the catalytic current density in Figure 4 to eq 3 yields imax ) 575 nA cm-2 and KM ) 2.1 mM. From the maximum catalytic current density, the value of KM, and the surface concentration of tethered flavin, we have obtained the observed second-order rate coefficient for the oxidation of NADH, kobs ) k2/KM ) 21.2 M-1 s-1 and k2 ) 0.044 s-1 at -0.20 V. Furthermore, the inset in Figure 4 depicts the electrode potential dependence of the catalytic current for the oxidation of NADH at the flavin-modified electrode. The onset of anodic current at -0.25 V is less negative than the redox potential of flavin under those conditions, ca. -0.375 V, but it is significantly more negative than the potential of most reported electrodes for the oxidation of NADH.2,3 For a biofuel cell the maximum power output can be obtained if the anode operates at a very reducing potential. Palmore et al.5 suggested that the redox mediator to produce enzymatic cofactor NAD+ should have a redox potential close to the NAD+/NADH redox potential. However, the thermodynamic driving force is small, and therefore, a fast reaction would not be expected.3 The rate constant for the bimolecular reaction of FMN with NADH in solution is much lower than our finding in
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the present work, i.e., kobs,hom ) 0.2 M-1 s-1 at pH 832 compared to 21.2 M-1 s-1. This 100-fold increase in reaction rate is insuficient if compared to the value reported by Kubota and Gorton34,35 for the oxidation of β-NADH with riboflavin immobilized on zirconium phosphate in carbon paste electrodes but at a higher electrode potential, -0.04 V, KM ) 1.8 mM and kobs ) 816 M-1 s-1 34 and KM ) 1.8 mM and kobs ) 200 M-1 s-1,35 respectively. Among the two-electron one-proton redox mediators for the oxidation of NADH, molecules with the o-quinone or p-phenylenediimine functionality, tetrathiofulvalene (TTF), tetracyanoquinodimethane (TCNQ), and 2,4,7-trinitro9-fluorenone have been reported as efficient mediators but at potentials more positive than 0 V. For meldola blue adsorbed on zirconium phosphate in a carbon paste electrode for instance, Kubota and Gorton35 reported the impressive value of kobs ) 19837 M-1 s-1 at electrode potentials close to 0 V. However, it should be borne in mind that at carbon paste electrode surfaces there is no control of the molecular details of the catalyst at the surface and therefore an understanding of the effect of molecular environment on the rate of reaction is not straightforward. Conclusions In conclusion, we have designed a nanostructured selfassembled catalyst for the oxidation of β-NADH on the basis of a biomimetic approach to flavoenzymes and have characterized the molecular structure of the tethered flavin molecules by FTIR and STM. These studies show the orientation and surface structure of an organized flavin monolayer self-assembled on gold, which undergoes fast electron transfer with the metal electrode and presents activity toward the oxidation of enzymatically active β-NADH at -0.25 V in solutions of pH 7.0. The results shown are important as a proof-of-concept, but are still far from the high value of the bimolecular rate constant for the oxidation of β-NADH by flavin in the oxidized state in flavoenzymes such as glutathione reductase, with KM ) 1.2 mM and kobs ) 33928 M-1 s-1.36 Work is now in progress to correlate the effect of the molecular environment on the kinetics of the reaction, in particular taking into consideration the structure of the binding site in the enzyme.6 Acknowledgment. We acknowledge financial support from the Agencia Nacional de Promocio´n Cientı´fica y Tecnolo´gica (ANPCyT), CONICET, and the University of Buenos Aires (Argentina). E.J.C. and I.R.W. acknowledge the support of a Wellcome Trust grant. This is a joint contribution from INQUIMAE and INIFTA. M.E.V. is a member of the Research Career of CIC (Provincia de Buenos Aires, Argentina). Supporting Information Available: Riboflavin structure, cyclic voltammograms obtained at different pH values, redox potential-pH dependence, and STM height profile. This material is available free of charge via the Internet at http://pubs.acs.org. LA050695N
