(Invited) Fructose Biosensors Based on Direct Electron Transfer between Fructose Dehydrogenase and Electrodes

Abstract

Fructose dehydrogenase (FDH, EC 1.1.99.11) is a bacterial membrane bound flavocytochrome oxidoreductase, which has been widely employed to develop direct electron transfer (DET) and mediated electron transfer (MET) based electrode platforms [1]. Most commonly FDH from Gluconobacter japonicus has been studied. FDH is a heterotrimer consisting of a catalytic dehydrogenase domain, DHFDH (subunit I), where D-(−)-fructose oxidation occurs at the covalently flavin cofactor (FAD), followed by an internal electron transfer (IET) to the cytochrome domain (CYTFDH, subunit II), which contains three heme c moieties coordinated by the enzyme scaffold, and a subunit III, which is not involved in the ET process. We have recently investigated [2-4] how to optimize orientation and the DET reaction between FDH and electrodes. In [2] we report on the influence of pH and monovalent/divalent cations on the catalytic current response, the IET, and the structure of FDH by using amperometry, spectrophotometry, and circular dichroism. Amperometric measurements were performed on graphite electrodes, onto which FDH was adsorbed and the effect on the response current to fructose was investigated when varying the pH and the concentrations of divalent/monovalent cations. In the presence of 10 mM CaCl2, a current increase of up to ≈ 240% was observed, probably due to an intra-complexation reaction between Ca2+ and the aspartate/glutamate residues found at the interface between the DHFDH and CYTFDH. In [3] we present a new method to electrodeposit highly mesoporous gold onto a polycrystalline solid gold electrode without any template. The electrodeposition is carried out by first cycling the electrode potential between +0.8 and 0 V in 10 mM HAuCl4 with 2.5 M NH4Cl and then applying a negative potential for the production of H2 bubbles at the electrode surface. This mesoporous gold electrode was modified with 4-mercaptophenol onto which FDH was orientated to optimize the DET reaction. In [4] an efficient DET reaction was also achieved between FDH and anthracene-modified single-walled carbon nanotubes (SWCNT) deposited onto a glassy-carbon electrode. The SWCNTs were activated in situ with a diazonium salt synthesized through the reaction of 2-aminoanthracene with NaNO2 in 0.5 M HCl for 5 min at 0° C. After the in situ reaction, the 2-aminoanthracene diazonium salt was electrodeposited by running cyclic voltammograms from +1000 to −1000 mV. The anthracene-SWCNT-modified GCE was further incubated in an FDH solution to allow FDH to adsorb. Cyclic voltammograms of the FDH-modified electrode revealed two couples of redox waves ascribed to the heme c 1 and heme c 3 of the CYTFDH. In the presence of 10 mM fructose two catalytic waves could clearly be seen and were correlated with two heme c:s, with a maximum current density of 485±21 μA cm − 2 at 0.4 V at a sweep rate of 10 mV s − 1.