Enzyme Inhibition Assays with an Amperometric Glucose Biosensor Based on a Thiolate Self-Assembled Monolayer

Abstract

A new bioelectrocatalytic enzyme membrane for biosensors based on immobilization of glucose oxidase (GOx) is evaluated for use in inhibition assays. The objectives are to show that the newly developed glucose biosensor has advantages for inhibition assays, not as a speci®c glucose biosensor as such. The mediator used is 2-aminoethanethiol, which forms a self-assembled monolayer on the surface of a gold electrode. The membrane con®guration consists of the thiol as mediator, covalently bound to the gold electrode and at the same time entrapped with GOx in a polyvinylpyridine (PVP) membrane. Cyclic voltammetric scans indicate a catalytic peak at 950 mV (vs. Ag/ AgCl) after addition of glucose to a blank phosphate buffer at pH 7.4. The PVP membranes are shown to be reusable for determination of glucose for at least one week. As an example of inhibition of the enzyme reaction, the response to glucose is shown to be sensitive to the addition of Hg(II) in the ppb range with a detection limit of 0.2 ppb. Interference to CV scans from oxidizable organic compounds and other metal ions is found to be minimal, however hydrogen peroxide is the exception and interferes at 1 mM concentration.

Enzyme inhibition-based biosensors for food safety and environmental monitoring

“…Enzymatic methods are commonly used for metal ion determination, as these can be based on the use of a wide range of enzymes that are specifically inhibited by low concentrations of certain metal ions (Krawczynski vel Krawczyk et al, 2000). For the inhibitive determination of trace mercury, a large number of enzymes has been used: horseradish peroxidase (Han et al, 2001), urease (Krawezyk et al, 2000;De Melo et al, 2002;Kuswandi, 2003;Rodriguez et al, 2004b) Krawczynski vel Krawczyk et al (2000), glucose oxidase (Alexander and Rechnitz, 2000), alcohol oxidase (Pirvutoiu et al, 2002) and glycerol 3-phosphate oxidase , and invertase . Some studies have also focused on the analysis of different organic forms of mercury: phenyl mercury (Doong and Tsai, 2001) using urease, methyl mercury and phenyl mercury using invertase (Mohammadi et al, 2005), and methyl mercury using peroxidase (Han et al, 2001).…”

Emprego de monocamadas auto-organizadas no desenvolvimento de sensores eletroquímicos

“…Um emparelhamento perfeito entre os alcanotióis (geralmente com uma inclinação de 20 a 30 graus em relação à normal da superfície) e o retículo do ouro resulta numa estrutura altamente ordenada (Figura 2) e tem feito deste sistema objeto de um grande número de estudos por meios espectroscópicos, microscópicos e eletroquímicos 12 . Vári-os trabalhos têm sido dedicados ao entendimento da estrutura e do ordenamento das monocamadas, que são caracterizadas pelo alto grau de organização e têm sido empregadas em estudos de transferência de elétrons [34][35][36][37][38][39] , adsorção de proteínas 38,[40][41][42][43] e, principalmente, em vários campos da eletroanalítica 12,[19][20][21][22][44][45][46][47][48][49][50][51][52][53] . A preparação da monocamada possui um papel fundamental no desempenho eletroanalítico do eletrodo modificado.…”

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

“…[6][7][8][9][10][11][12] Glucose oxidase (GOx) of low cost, good stability, and high specific activity has been widely used for glucose biosensing, 13 and the inhibitive assays of several HM ions based on polymer-entrapped GOx (GOx e ) have also been investigated. 2,10,[14][15][16] However, it seems that the reported inhibition effects of HM ions on GOx e are rather scattering and even controversial. For instance, GOx e entrapped in poly(o-phenylenediamine) (PoPD) was reported to be sensitive to Hg 2þ and Cu 2þ , 14 while it was also used for quantification of the total amount of HM ions in water samples.…”