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 -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 methylformyli- soalloxazine derivative and cysteamine chemisorbed on gold. The electrocatalytic activity toward the oxidation of enzymatically active -NADH and detailed kinetic pa- rameters 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. Ac- cording 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 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 solution 8-10 and adsorbed on Hg 11-13 and on carbon 14 electrode surfaces. * To whom correspondence should be addressed. E-mail: calvo@qi.fcen.uba.ar. Phone: (5411)4576-3378. 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