Carbon Felt-based Bioelectrocatalytic Flow Detectors: Highly Sensitive Amperometric Determination of Hydrogen Peroxide Using Adsorbed Peroxidase and Thionine

2011

ANAL. SCI.

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Horseradish peroxidase (HRP) and thionine (Th) were co-adsorbed onto a porous carbon-felt (CF) from a mixed solution of HRP and Th to fabricate a CF-based bioelectrocatalytic flow-detector for H2O2. The resulting HRP and Th co-adsorbed CF (HRP/Th-CF) was successfully used as a working electrode unit of a highly sensitive amperometric flow-H2O2-biosensor. Differing from the ordinary mediator-based HRP biosensors, which detect mediated currents (i.e., the reduction current of the oxidized form of the mediators), the present HRP/Th-CF-based H2O2-biosensor directly detects the reduction current of the oxidative HRP-intermediates (i.e., compound I and II) based on a direct electron transfer (DET) between a HRP-redox active heme center and the CF electrode, because co-adsorbed Th together with HRP facilitate the DET. Various adsorption conditions (i.e., HRP concentration, Th concentration, ionic strength, pH, adsorption time) were optimized to obtain excellent sensor performances. When an air-saturated 0.1 M phosphate buffer (pH 7.0) was used as a carrier at 3.25 ml/min and the applied potential of -0.05 V vs. Ag/AgCl, the HRP/Th-CF-based flow-H2O2-biosensor exhibited excellent analytical performances (i.e., sensitivity, 3.55 μA/μM; linear range, 0.1 to 10 μM; lower detection limit, 0.03 μM; K app m , 36.2 μM; Imax, 158 μM; sample throughput, ca. 60 samples/h).

Section: Effects Of Th Concentration In Adsorption Solution and Adsormentioning

confidence: 90%

Section: Ferric-hrp (Native) + H2o2 → Compound I + H2omentioning

confidence: 96%

Section: Ferric-hrp (Native) + H2o2 → Compound I + H2omentioning

confidence: 99%

Section: Preparation Of Hrp/th-cf Electrodementioning

confidence: 99%

Section: Flow-amperometric Measurements With Hrp/th-cf-based Flow-biomentioning

confidence: 99%

See 3 more Smart Citations

Carbon-felt-based Bioelectrocatalytic Flow-detectors: Optimization of the Adsorption Conditions of Horseradish Peroxidase and Thionine onto Carbon-felt for Highly Sensitive Amperometric Determination of H2O2

2011

ANAL. SCI.

Self Cite

Amperometric Flow‐Biosensor for Cyanide Based on an Inhibitory Effect upon Bioelectrocatalytic Reduction of Oxygen by Peroxidase‐Modified Carbon‐Felt

2011

Electroanalysis

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A novel, simple and relative highly sensitive amperometric flow biosensor for cyanide was developed by using horseradish peroxidase (HRP)‐adsorbed carbon‐felt (CF), based on an inhibitory effect on the HRP‐catalyzed O2 reduction. The HRP‐CF showed a sufficient bioelecrocatalytic activity for O2 reduction in the potential region from 0 to −0.5 V at pH 5.0, due to a direct electron transfer‐based O2 reduction process via ferrous‐HRP and compound III. This HRP‐catalyzed O2 reduction was reversibly inhibited by cyanide, which enabled to fabricate a novel and simple reagentless (i.e., no requirement of the ordinary substrate, H2O2, and the electron transfer mediators) flow‐biosensor for cyanide. When air‐saturated 0.1 M phosphate buffer (pH 5.0) was used as a carrier under the applied potential of −0.2 V vs. Ag/AgCl, the steady‐state base‐current due to the HRP‐catalyzed O2 reduction was reversibly inhibited by the cyanide injection (200 µL), resulting in peak‐shape current responses. The magnitude of the inhibition peak currents linearly increased with increasing concentrations of cyanide up to 1 µM, and the detection limit was found to be 0.04 µM (S/N=2). The apparent inhibition constant Ki′ was estimated to be 0.87 µM.

Acridine orange-induced signal enhancement effect of tyrosinase-immobilized carbon-felt-based flow biosensor for highly sensitive detection of monophenolic compounds

2010

Anal Bioanal Chem

Tyrosinase (TYR: EC 1.14.18.1) was covalently modified onto the surface of a cyanuric chloride-activated carbon felt (CF) from the mixed buffer solution of TYR and acridine orange (AO). The resulting TYR-immobilized CF (TYR/AO-CF) was used as a working electrode unit of an electrochemical flow-through detector for mono- and di-phenolic compounds (i.e., p-chlorophenol (p-CP), p-cresol, phenol, and catechol), which detects the reduction current of enzymatically produced o-quinones at -0.05 V (vs. Ag/AgCl). The presence of AO (0.2 mM) in TYR solution during the enzyme immobilization step was significantly effective for the signal enhancements especially for p-CP, and the cathodic peak currents of p-CP by the TYR/AO-CF-based detector were much larger than those by the TYR-CF-based detector prepared from TYR solution without AO. The oxymetry with Clark-type oxygen electrode revealed that monophenolase activity of free TYR in 1 mM phosphate buffer (pH 7.0) was greatly enhanced in the presence of AO (0.2 mM), whereas diphenolase activity was not so much influenced. Furthermore, the comparison of cyclic voltammograms of TYR/AO-CF and TYR-CF in air-saturated phosphate buffer containing each substrate revealed that the electrochemical reduction rate of p-chloro-o-benzoquinone at TYR/AO-CF was faster than that at TYR-CF. In addition, the electrochemical impedance spectroscopy revealed that the structural properties of immobilized TYR on the CF would be influenced by AO. Some kinds of interaction of AO with TYR would affect the enzymatic kinetics and the structural properties of the immobilized TYR, leading to the signal enhancement of the TYR-CF-based flow biosensor especially for monophenolic compounds.