Reagentless enzyme electrode based on phenothiazine mediation of horseradish peroxidase for subnanomolar hydrogen peroxide determination

Abstract

The development and characterization of a highly sensitive enzyme immobilized carbon based electrode for the determination of subnanomolar concentrations of hydrogen peroxide in aqueous samples is described. The biosensor consists of horseradish peroxidase (HRP) immobilized in solid carbon paste along with a suitable redox mediator. The latter allows the acceleration of the electroreduction of HRP in the presence of hydrogen peroxide. Several phenothiazines as mediators are investigated in a comparative manner and with respect to dimethylferrocene using cyclic voltammetry and amperometry. Insolubilization of the HRP in the solid carbon paste is achieved by cross-linking the enzyme with glutaraldehyde and bovine serum albumin. Several experimental parameters such as pH, mediator and enzyme content are considered. The hydrogen peroxide determination is better carried out in 0.1 M acetate buffer, pH 4.5, by amperometry at an applied potential of 0.0 V versus Ag/AgCl, 3 M NaCl concentration and by using the phenothiazine base as redox mediator. The biosensor response is linear over the concentration range 2 nM-10 microM with a detection limit of 1 nM. The linear range of the hydrogen peroxide response without a mediator in the biosensor is found between 2 and 40 microM. The biosensor can be used for more than 180 measurements. Additional modification of the electrode by incorporation of Nafion SAC-13 microparticles in the solid carbon paste allows detection of concentrations of hydrogen peroxide as low as 0.1 nM.

Electrochemical Kinetic Characterization of Redox Mediated Glucose Oxidase Reactions: A Simplified Approach

“…Mediator based enzymatic electrodes are the most widely reported in literature [4,12,13]. These devices are based on the use of redox mediators, low molecular weight substances able to realize a fast electron transfer from the enzyme redox center to the working electrode surface and vice versa.…”

A Novel Hydrogen Peroxide Sensor via the Direct Electrochemistry of Horseradish Peroxidase Immobilized on Colloidal Gold Modified Screen-printed Electrode

“…Horseradish peroxidase (HRP), a prototypical hemeprotein peroxidase (FW ca. 44000), is one of the most widely used enzymes for analytical purposes and biosensors [4,5,[7][8][9]11,12,14,17]. This technique has good sensitivity and accuracy, but at the same time, they suffer from some shortcomings.…”

“…Many methods such as titrimetry, UV-vis spectrophotometry and electrochemistry have been developed for this purpose [1][2][3]. Among these methods, the amperometric biosensors based on the direct electron transfer between an electrode and the immobilized enzyme/protein is especially promising because of its simplicity and high sensitivity [4][5][6][7][8][9][10][11][12][13][14][15][16][17]. Horseradish peroxidase (HRP), a prototypical hemeprotein peroxidase (FW ca.…”

pH tuning of Nafion® for selective detection of tryptophan

“…Total passivation of the Nafion ® -GCE for melatonin was decreased when analyzed in the more acidic buffer solutions. The reduction in electrode passivation at Nafion ® modified electrodes has been observed before [32]. Of interest, is the increase in current response with scan number when a higher concentration of Nafion ® (5.0% solution) was utilized, as shown in Fig.…”