Katsunobu Yamamoto

Study of carbon nanotubes-HRP modified electrode and its application for novel on-line biosensors.

In this paper, multi-walled carbon nanotubes (MWCNTs) were successfully immobilized on the surface of a glassy carbon electrode by mixing with horse-radish peroxidase (HRP). The electrochemical behavior of H2O2 was also studied with the MWCNTs-HRP modified electrode as a working electrode. The MWCNTs-HRP modified electrode showed excellent response of reduction current for the determination of H2O2 at the potential of -300 mV (vs. Ag/AgCl). We assembled the MWCNTs-HRP modified electrode in a thin-layer flow cell and the H2O2 solution was continuously introduced into the cell with a syringe pump. We optimized the sensitivity of the H2O2 sensor by adjusting the working potential and the pH of the buffer solution. The peak current increased linearly with the concentration of H2O2 in the range 3.0 x 10(-7) to approximately 2.0 x 10(-4) mol L(-1). The detection limit is 1.0 x 10(-7) mol L(-1) (S/N = 3). The interferences from ascorbic acid, uric acid and other electroactive substances can be greatly excluded since the sensor can be operated at -300 mV. Stability and reproducibility of the MWCNTs-HRP chemically modified electrode were also studied in this paper. Fabricated with glucose and lactate oxidase, the MWCNTs-HRP electrode was also applied to prepare the on-line glucose and lactate biosensors because of the high sensitivity for the determination of H2O2.

Electrochemical Nitric Oxide Sensors Based on Electropolymerized Film of M(salen) with Central Ions of Fe, Co, Cu, and Mn

Electropolymerized film of metal ethylenebis(salicylideneiminate) [(M(salen), M=Co, Fe, Cu and Mn] was utilized as material for development of an electrochemical sensor for the determination of NO in solution. The sensors based on polymeric M(salen) were prepared by a means of electropolymerization with cyclic voltammetry (CV) in acetonitrile solution containing M(salen) for optimized cycles. Nafion was used as a second coating to the sensors and differential pulse amperometry (DPA) was used as a subsequent determination technique. The resulted sensors were found to display good activity toward the oxidation of NO with low detection limits and a good linear relationship between the current and NO concentration. The mechanism of the polymeric M(salen) modified sensor was preliminarily studied by using a technique of quantum chemistry and proposed to be a three-dimensional catalytic pattern by conjugation of the electron of the NO molecule and the polymeric M(salen).

ZnS quantum dots derived a reagentless uric acid biosensor

A reagentless amperometric uric acid biosensor based on zinc sulfide (ZnS) quantum dots (QDs) was firstly developed. It could detect uric acid without the presence of an electron mediator. The carboxyl group functionalized ZnS QDs were synthesized, and they were soluble biocompatible and conductive. ZnS QDs conjugates could provide increased enzyme binding sites, which may result in higher enzyme loading. Thus, the proposed uricase/ZnS QDs/l-cys biosensor exhibited higher amperometric response compared to the one without QDs (uricase/l-cys biosensor). In addition, there was little AA interference. It showed a linear dependence on the uric acid concentration ranging from 5.0x10(-6) to 2.0x10(-3)molL(-1) with a detection limit of 2.0x10(-6)molL(-1) at 3sigma.

Detection of basal acetylcholine release in the microdialysis of rat frontal cortex by high-performance liquid chromatography using a horseradish peroxidase-osmium redox polymer electrode with pre-enzyme reactor

To determine the basal acetylcholine level in the dialysate of rat frontal cortex, a horseradish peroxidase-osmium redox polymer-modified glassy carbon electrode (HRP-GCE) was employed instead of the conventional platinum electrode used in high-performance liquid chromatography-electrochemical detection (HPLC-ED). In initial experiments, an oxidizable unknown compound interfered with the detection of basal acetylcholine release on HPLC-HRP-GCE. An immobilized peroxidase-choline oxidase precolumn (pre-reactor) was included in the HPLC system, to eliminate the interference from the unknown compound. This combination could detect less than 10 fmol of standard acetylcholine and basal acetylcholine levels in the dialysate from a conventional concentric design microdialysis probe, without the use of cholinesterase inhibitor, and may facilitate physiological investigation of cholinergic neuronal activity in the central nervous system.

Amperometric Biosensor for Glutathione Based on Osmium‐Polyvinylpyridine Gel Polymer and Glutathione Sulfhydryl Oxidase

A new amperometric biosensor based on glutathione sulfhydryl oxidase (GSH-SOx) and osmium-polyvinylpyridine gel polymer (Os-gel-HRP) bilayer film modified glassy carbon (GC) electrode was demonstrated for glutathione (GSH) and glutathione disulfide (GSSG). Os-gel-HRP was applied at glassy carbon (GC) electrode with a surface coverage of 7.1 µL/cm2 to sense hydrogen peroxide based on horseradish peroxide (HRP) catalytic reaction mediated by osmium. GSH-SOx was immobilized at Os-gel-HRP cast coated GC electrode with an approximate surface coverage of 7 U/cm2 by cross-linking with BAS-GSH-SOx in glutaraldehyde vapor for 5 min. The resultant bienzyme-based sensor was tested toward GSH and GSSG with techniques of cyclic voltammetry (CV), flow cell amperometry and flow injection analysis (FIA). The sensor was polarized at 0.0 V (versus Ag/AgCl, 3M KCl) electrode for the detection of GSH and GSSG in a flow system at a flow rate of 6 µL/min. Linear response to GSH and GSSG in a concentration range from 1 µM to 200 µM and 2 µM to 120 µM was obtained at the sensor with a sensitivity of 1.195 nA/µM and 0.60 nA/µM for GSH and GSSG, respectively. The dependence of current response on pH value of the buffer and operating potential was also tested and optimized.

Electrochemical Microsensor for In Vivo Measurements of Oxygen Based on Nafion and Methylviologen Modified Carbon Fiber Microelectrode

An electrochemical microsensor based on carbon fiber microelectrode (CFME) chemically modified with the perfluorinated cation-exchange polymer, Nafion and methylviologen (MV) and its application for in vivo voltammetric measurements of oxygen (O2) are described. The microsensor shows a high catalytic activity for the reduction of O2 with a good reproducibility, high sensitivity and selectivity and significant ability against electrode fouling. The current is linear with the concentration of O2in a range from 9.0×10–6to 2.0×10–4 mol/L with a calculated detection limit, at a signal-to-noise ratio of 3 to 1, of 5.0×10–6 mol/L and correlation coefficient of 0.9985. The relative standard deviation for 2.0×10–4 mol/L O2 is 1.7% (n = 2). Some compounds common to biological fluids such as glucose, ascorbic acid, uric acid, catecholamine, glutamate, glutathione, Mg2+, Ca2+, Na+, K+and Cl–are tested in vitro and show no interferences with the voltammetric responses to O2. In vivo performance of the O2 microsensors is demonstrated by measurements of local changes in O2 in the brain of anesthetized rat before and during transient ischemia.

Evaluation of an amperometric glucose biosensor based on a ruthenium complex mediator of low redox potential

An amperometric glucose ring-disk biosensor based on a ruthenium complex mediator of low redox potential was fabricated and evaluated. This thin-layer radial flow microsensor (10mul) with ring-disk working electrode displayed remarkable amperometric sensitivity. For Ru(3)(mu(3)-O)(AcO)(6)(Py)(3)(ClO(4)) (Ru-Py), a trinuclear oxo-acetate bridged cluster, a reversible redox curve of low redox potential and narrow potential window (redox potentials were -0.190 and -0.106V versus Ag/AgCl wire, respectively) was observed, which is comparable to many reported mediators such as ferrocene derivatives and other ruthenium complexes. The glucose and hydrogen peroxide assays were carried out with this complex-modified electrode Ru-Py-HRP-GOx/Nafion. The sensitivity was obtained 24nA (15.4mAM(-1)cm(-2)) for 10muM glucose and 126 nA (160mAM(-1)cm(-2)) for 5muM H(2)O(2), respectively with a working potential at 0V versus Ag/AgCl. Ascorbic acid was studied as interference to the glucose assay. The application of 0V potential versus Ag/AgCl did not avoid the occurrence of the oxidation of ascorbic acid, however, the pre-coating of ascorbate oxidase on the disk part of the ring-disk working electrode efficiently pre-oxidized the ascorbic acid and hence eliminated its interference on the glucose response. The practical reliability was also evaluated by assaying the dialysate from the prefrontal cortex of Wistar rats.