Electrochemical behavior of FAD immobilized on bare and dodecanethiol modified gold electrodes

MARÍA ANTONIETA DAZA MILLONE; MARIANO H. FONTICELLI; MARÍA ELENA VELA; ROBERTO C. SALVAREZZA

Cancún (México)

Congreso; 210th Meeting of the Electrochemical Society/ XXI Congreso de la Sociedad Mexicana de Electroquímica; 2006

Flavin adenine dinucleotide (FAD) is an essential cofactor for a wide group of enzymes called flavoproteins that catalyze proton-coupled electron transfer reactions in the metabolism of cells. Its electrochemical behavior has been studied both in solution and adsorbed on different electrode materials showing that it is sensible to pH due to its acid-base properties and from the protons involved in the redox reaction. On the other hand, FAD electrochemistry depends on the surface employed. When FAD is buried in the enzyme, it is isolated from the bulk solution. In the active site, it is bound to the aminoacids by Van der Waals forces, hidrogen bridges, ring stacking, etc. Direct electrochemistry of FAD buried in the enzyme is not easy to achieve, and there have been numerous strategies to improve the electron transfer between FAD and the electrode [1]. Self assembled monolayers (SAMs) of alkanethiols adsorbed on metal surfaces have been widely used to study complex molecular systems from the point of view of molecular interactions and charge transfer processes. In this work we studied the properties of FAD immobilized on bare gold and on a SAM of dodecanethiol (C12) on Au, in order to characterize the charge transfer reaction, the forces involved in the immobilization and the optimal conditions to design a biosensor. First, we prepared FAD modified gold electrodes by dip-ping. The gold substrates were pretreated by H2 flame annealing to obtain (111) preferred-oriented crystals. Au(111) substrates where incubated with aqueous solu-tions of FAD of concentrations between 1x10-6M and 1x10^-4M during different periods of time. The CVs show for all the cases sharp peaks at -0,6V and -0,64V in the anodic and cathodic scans respectively with prepeaks at -0,69V and -0,71V. The locations of the sharp peaks are those expected for the FAD redox couple in solution at pH 13 [2]. The charge estimated from the anodic peak integration depends on incubation conditions (concentration and time of dipping). The charges are: 3,5μC/cm^2, 12μC/cm^2 and 18μC/cm^2 for 2 min, 15 min and 48 hs of incubation time. STM images from this last condition show a large amount of material in spots that follow the gold terraces and in aggregates, indicating that not all the material is electroactive. For the experiments with SAMs, the Au(111) substrates were modified by immersion in an ethanolic solution of C12 0,5 μM during different time periods. After this modification, they were rinsed with ethanol and dried with N2 and then incubated 30 min in aqueous solution of FAD 1x10^-4M. The CV shows a decrease in the double layer of gold corresponding to the presence of the SAM. The height and number of peaks of the FAD couple change according to the defects on the monolayer. For long times of incubation of C12 (>60 min) two peaks poorly defined appears at -0,79V and -0,69V. A cathodic scan towards -1,3V was performed in order to desorbed the C12 layer. After that the CV exhibited an increased in the double layer capacity corresponding to SAM desorption and a best defined redox couple for the FAD response. This behavior indicates the presence of holes or defects in the partially de-sorbed SAM where the FAD molecule is able to reach the gold surface. Thus, the possibility of trapping FAD molecules by dipping either on bare surface or organic monolayers can be performed and quantified from the point of view of its redox properties. [1] E. E. Ferapontova et al. in Direct Electrochemistry of Proteins and Enzymes, Perspectives in Bioanalysis, vol 1, Elsevier Inc. (2005) [2] M. Cable and E. T. Smith, Analytica Chimica Acta 537, 299 (2005)