Incorporation of Horseradish Peroxidase in a Kieselguhr Membrane and the Application to a Mediator-free Hydrogen Peroxide Sensor

Abstract

Hydrogen peroxide (H2O2) is a product of many enzymecatalyzed redox reactions. 1 Therefore, the determination of H2O2 is of great importance in both biological and industrial fields. [2][3][4][5] Electrochemical techniques have long been considered for the determination of H2O2, in view of its simplicity, low-cost and real-time detection properties. 6,7 Nevertheless, H2O2 is not an ideal species to detect with an electrochemical biosensor, because the electrode kinetics is very slow at most electrode surfaces. 8,9 It therefore requires high overpotentials to drive the reaction at an electrode, which often leads to low sensitivity and unacceptable interferences. 10 Therefore, peroxidase is often employed, to lower the activation energy for the reduction of H2O2 and to catalyze its electrochemical reduction at electrode surfaces. [11][12][13][14][15][16] Horseradish peroxidase (HRP) is a widely used peroxidase, due to its reasonable stability, commercial availability and effectiveness towards the reduction of H2O2. 17,18 Many good HRP-based H2O2 sensors have been constructed. [19][20][21][22][23] However, the redox center of HRP is electrically insulated by its thick protein shell, thus hindering its direct electrical communication with electrodes. Therefore, redox mediators usually have to be employed to shuttle electrons between the heme site of HRP and the electrodes.Meanwhile, although the direct electroreduction of H2O2 in the presence of HRP was observed at purposely pretreated carbon electrode surfaces, direct (i.e., mediator-free) electron transfer between the electrode and HRP is rather slow. Thus, no direct electrochemistry of HRP is observable in such cases. [24][25][26] Consequently, it is less efficient in the bioelectrocatalytic reduction of H2O2 and the biosensors exhibit lower sensitivity than those sensors that employ redox mediators.Recent studies have shown that the electron-transfer reactivity of proteins could be effectively enhanced after the proteins were incorporated in some membranes. 27 For example, Ferri et al. entrapped HRP within a tributylmethyl phosphonium chloride (TBMPC) polymer bound anionic exchange resin.The heterogeneous electron-transfer rate of HRP was greatly promoted and a pair of redox peaks of HRP was observed, corresponding to Fe(III)/Fe(II) conversion. 28 Hence, it combines the specificity of an enzymatic reaction with facile interfacial electron transfer, which shows great promise in biosensing applications. 29 Kieselguhr is a kind of inorganic material with a porous structure, which is promising as an immobilization matrix because of its good mechanical, thermal and chemical stability. 30 Moreover, kieselguhr is of special interest owing to its effectiveness in the retention of enzyme activity, 31 and its nice catalytic ability. 32,33 In this work, we incorporated HRP in the kieselguhr membrane. It was observed that HRP exhibits good direct electrochemical transfer reactions. Furthermore, a mediator-free H2O2 sensor is accordingly constructed. ExperimentalHRP (EC 1.11...

Polyelectrostatic immobilization of gold nanoparticles-modified peroxidase on alginate-coated gold electrode for mediatorless biosensor construction

“…4C, the half-wave peak potentials (E 1/2 ) shifted linearly toward negative direction when pH increased from 5.0 to 9.0, with a slope of À42.0 mV/pH. This value is close to the theoretical one of À59.0 mV/pH for a single-proton concomitant single-electron transfer process [25].…”

Third-Generation Biosensors Based on the Direct Electron Transfer of Proteins

“…An inorganic material, such as kieselguhr clay, 114 has also been shown to be useful for fabricating third-generation H2O2 biosensors. The nice sensitivity and good stability of this H2O2 biosensor has also shown the effectiveness of this protein-film voltammetry technique.…”

A Nitric Oxide Biosensor Based on Horseradish Peroxidase/Kieselguhr Co‐Modified Pyrolytic Graphite Electrode

“…28 The K M app for the HRP/kieselguhur member-based NO biosensor was calculated to be 745 mM. 17,29 Aliquots of a NO (2.0 × 10 -3 mol/L) are added to the bulk solution to generate a series of concentrations. A linear dependence of the peak current on the NO concentration is obtained ranging from 2.0×10 -7 to 2.0×10 -5 mol/L (Fig.3).…”

“…Specifically, HRP has been shown to exhibit nice electrochemical activity when entrapped in this inorganic film. 17 In view of these, we herein reported an NO biosensor based on HRP/kieselguhr co-modified PG electrode. This method is based on the direct electron transfer (DET) of HRP; coinciding with the so-called feature of a third-generation biosensor.…”