P-chip and P-chip bienzyme electrodes based on recombinant forms of horseradish peroxidase immobilized on gold electrodes

Adsorption and bioelectrocatalytic activity of native horseradish peroxidas e (HRP) and its recombinant forms on polycrystalline gold electrodes were s tudied. Recombinant forms of HRP were produced by a genetic engineering app roach using an E. coli expression system. According to direct mass measurem ents with a quartz crystal microbalance, all the forms of HRP formed monola yer coverage of the enzyme on the gold surface. However, only gold electrod es modified with the recombinant HRP forms (non-glycosylated) exhibited hig h and stable current response to H2O2 due to its bioelectrocatalytic reduct ion based on direct electron transfer (ET) between gold and the active site of the enzyme. Introduction of a six-His tag either at the C-terminus or a t the N-terminus of the enzyme molecule additionally increased the strength of the enzyme binding with the gold surface and the efficiency of direct E T. Immobilization of recombinant forms of HRP containing histidine function al groups. on the surface of the gold electrode was used both for the devel opment of a P-chip, a biosensor for hydrogen peroxide determination based o n direct ET, and for the development of a bienzyme biosensor electrode for the determination of L-lysine based on co-immobilized recombinant forms of HRP and L-lysine-alpha -oxidase.