Experimental Platform to Study Heavy Metal Ion-Enzyme Interactions and Amperometric Inhibitive Assay of Ag+ Based on Solution State and Immobilized Glucose Oxidase

The heavy metal (HM) ion-enzyme interaction is an important research topic in many areas. Using glucose oxidase (GOx) as an example, a comprehensive experimental platform based on quartz crystal microbalance and electroanalysis techniques is developed here to quantitatively study the HM ion enzyme interactions and amperometric inhibitive assays of HM ions. The effects of some common HM ions on the bioactivitics of solution-state GOx (GOx(s)), electrode surface-adsorbed GOx (GOx(ads)), and polymerentrapped GOx (GOx(e)) are comparatively examined on the basis of anodic amperometric detection of enzymatically generated H2O2. Ag+ shows the strongest inhibition effect among the HM ions examined, and the inhibitive assays of Ag+ based on GOx GOx(ads), and GOx(e), entrapped in poly-(L-noradrenalin) (PNA) give limits of detection (LOD) of 2.0, 8.0, and 5.0 nM (S/N = 3), respectively. Inhibition effects of Hg2+, Cu2+, and Co2+ are detectable only at 15 mu M or higher concentrations, and the other HM ions show undetectable inhibition even at 1.0 mM. The developed experimental platform allows one to quantify the number of the bound HM ions per GOx(ads) molecule at various inhibition percentages. In addition, the electrosynthesized PNA matrix to entrap GOx for an inhibitive assay of Ag+ shows the lowest competitive affinity to HM ions and gives the highest sensitivity, as compared with several other polymer matrixes commonly used for the inhibitive assay. The suggested experimental platform is recommended for wide applications in enzymatic inhibitive assays and quantitative studies of the inhibition effects of HM ions on many other redox-event-relevant enzymes.