Enzyme Electrode Biosensors for N-Hydroxylated Prodrugs Incorporating the Mitochondrial Amidoxime Reducing Component

Human mitochondrial amidoxime reducing component 1 and 2 (mARC1 and mARC2) were immobilised on glassy carbon electrodes using the crosslinker glutaraldehyde. Voltammetry was performed in the presence of the artificial electron transfer mediator methyl viologen, whose redox potential lies negative of the enzymes' Mo^VI/V and Mo^V/IV redox potentials which were determined from optical spectroelectrochemical and EPR measurements. Apparent Michaelis constants obtained from catalytic limiting currents at various substrate concentrations were comparable to those previously reported in the literature from enzymatic assays. Kinetic parameters for benzamidoxime reduction were determined from cyclic voltammograms simulated using Digisim. pH dependence and stability of the enzyme electrode with time were also determined from limiting catalytic currents in saturating concentrations of benzamidoxime. The same electrode remained active after at least 9 days. Fabrication of this versatile and cost-effective biosensor is effective in screening new pharmaceutically important substrates and mARC inhibitors.