NAD(P)H Sensors Based on Enzyme Entrapment in Ferrocene-Containing Polyacrylamide-Based Redox Gels

Abstract

NADH and NADPH sensors were developed by entrapping lipoamide dehydrogenase (LD) and glutathione reductase (GR), respectively, in a redox gel formed by the copolymerization of vinylferrocene with acrylamide and N,N‘-methylenebisacrylamide. Addition of LD or GR to the gel polymerization mixture resulted in a significant acceleration of free-radical copolymerization. The redox gels were secured on the surface of a carbon paste electrode with a dialysis membrane, and the resultant enzyme electrodes showed linear amperometric response to their substrates (NADH for LD and NADPH for GR) up to 3 mM when the immobilized ferrocene (Fc) and entrapped enzyme concentrations were ∼0.90 mM and 1.0 mg/mL, respectively. The substrate concentration over which the catalytic current was found to be linear depended on the concentrations of both the Fc mediator and the enzyme in the redox gels. The observed linearity indicates that the enzyme electrodes can be used as sensors to quantitate both NADH and NADPH in aqueous solutions. The pH−activity profiles of the enzyme electrodes as well as their storage and operational stabilities were examined. It was unexpectedly observed that both entrapped LD and GR exerted different effects on the electrochemical properties of the immobilized Fc/Fc+ redox couple, although they have similar structural and catalytic properties. In LD-containing gels, the redox couple is electrochemically irreversible (ΔEp = 285 mV), while in GR-containing gels, the mediator exhibits the same quasi-reversible electrochemical behavior as in the absence of protein (ΔEp = ∼95 mV). The electrocatalytic currents (ic) of the enzyme electrodes vs enzyme loading were investigated, and it was found that high LD loading (≥1.5 mg/mL) reduced ic but high GR loading did not. It is concluded that the GR-containing redox gel electrode is highly suitable for use with dehydrogenases that require NADP+ as a cofactor.