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

Abstract

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 bioactivities of solution-state GOx (GOx(s)), electrode surface-adsorbed GOx (GOx(ads)), and polymer-entrapped GOx (GOx(e)) are comparatively examined on the basis of anodic amperometric detection of enzymatically generated H(2)O(2). Ag(+) shows the strongest inhibition effect among the HM ions examined, and the inhibitive assays of Ag(+) based on GOx(s), 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 Hg(2+), Cu(2+), and Co(2+) are detectable only at 15 μM 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.

Label-free chemiresistive biosensor for mercury (II) based on single-walled carbon nanotubes and structure-switching DNA

“…Protein based Hg 2þ biosensor using electrochemical 4,5 and optical 6 techniques has been demonstrated. However, poor stability of proteins at ambient condition restricts its use for real application.…”

“…2 The United States Environmental Protection Agency (EPA) has mandated a drinking water upper limit of 2 ppb (10 nM) for mercury (II) ion concentration. 3 Protein based Hg 2þ biosensor using electrochemical 4,5 and optical 6 techniques has been demonstrated. However, poor stability of proteins at ambient condition restricts its use for real application.…”

Amperometric Detection of Aqueous Silver Ions by Inhibition of Glucose Oxidase Immobilized on Nitrogen-Doped Carbon Nanotube Electrodes

“…GOx is known to be inhibited by a number of heavy metals 25,28,29 including Ag + , Hg 2+ , Cu 2+ , Pb 2+ , Co 2+ , Fe 3+ , and Ni 2+ and these interactions have been utilized in numerous GOx-inhibition based heavy metal assays. [29][30][31][32][33][34][35][36] Immobilization of the enzyme is often achieved by polymer or hydrogel entrapment, however, the immobilization matrices in many of these studies are believed to competitively coordinate heavy metal ions, leading to less robust sensors and inconsistent results. 33 Heavy metal pollution is recognized today as a necessary and important aspect of environmental monitoring.…”

“…[29][30][31][32][33][34][35][36] Immobilization of the enzyme is often achieved by polymer or hydrogel entrapment, however, the immobilization matrices in many of these studies are believed to competitively coordinate heavy metal ions, leading to less robust sensors and inconsistent results. 33 Heavy metal pollution is recognized today as a necessary and important aspect of environmental monitoring. In recognition of this, regulatory agencies, such as the WHO and EPA, have set rigid limits on the ranges of permissible heavy metal concentrations.…”

“…Thus, Ag + biosensors 29,33,[40][41][42][43][44] , chemosensors [45][46][47] , ion-selective electrodes 48,49 , and voltammetric techniques [49][50][51] are currently being investigated. Herein, we present a simple, cost-effective, and highly-sensitive approach to heavy metal detection, focusing on silver in particular.…”