Assembly of alternating polymerized mediator, polymerized coenzyme, and enzyme modified electrode by layer-by-layer adsorption technique

The polymerized mediator (polyethyleneimine ferrocene), and polymerized NADP+ (Alg–NADP+) were prepared and their application to biosensing was investigated. An amperometric biosensor was fabricated utilizing two enzymes, glutathione reductase (GR), and glucose-6-phosphate dehydrogenase (G6PDH) with polymerized NADP+ as part of a layer-by-layer adsorption (LBL) technique on a chemically modified carbon electrode. An electrocatalytic steady-state current as a result of the oxidation of glucose-6-phosphate (G6P) was observed using the bi-enzyme electrode in the presence of free NADP+. The current response of the modified electrode depended on the number of enzyme layers, the polymerized mediator and the sequence of adsorption. The optimum multilayer assembly sequence consisted of the polymerized mediator, GR, and G6PDH from the innermost to outermost layer. In the presence of free NADP+, the bi-enzyme biosensor with immobilized Alg–NADP+ showed higher response for G6P than that without Alg–NADP+.