Glucose oxidase, horseradish peroxidase and phenothiazine dyes-co-adsorbed carbon felt-based amperometric flow-biosensor for glucose.

To develop an amperometric flow-biosensor for glucose, the stabilizing effect of methylene blue (MB) toward adsorbed glucose oxidase (GOx) on carbon felt (CF) was successfully applied to prepare the GOx-modified CF-based enzyme reactor combined with a horseradish peroxidase (HRP)-modified CF-based H2O2 detector. Upon mixing MB in the GOx-adsorption solution, the O2-dependent GOx-activity was significantly increased with increasing concentration of MB in the GOx-adsorption solution. The GOx-immobilization protocol on CF is very straightforward [i.e., adsorption of the GOx/MB mixed aqueous solution for 5 min under ultrasound (US)-irradiation]. Under the optimized operational conditions (i.e., applied potential, 0 vs. Ag/AgCl; carrier pH, 5.0; carrier flow rate, 4.0 mL min-1), the resulting GOx/MB-CF-reactor and HRP/TN-CF-detector combined amperometric flow-biosensor exhibited sensitive, selective, reproducible and stable cathodic peak current responses to glucose with the following analytical performances: sensitivity, 6.22 µA mM-1; linear range, 0.01 to 1 mM; limit of detection, 9.6 µM (S/N = 3, noise level, 20 nA); sample throughput, 46-96 samples per h for 10-0.1 mM glucose. The developed amperometric flow-biosensor allowed the determination of glucose in beverages and liquors, and the analytical results by the sensor were in fairly good agreement with those by conventional spectrophotometry.

Shield, Anchor, and Adhesive Roles of Methylene Blue in Tyrosinase Adsorbed on Carbon Felt for a Flow Injection Amperometric Enzyme Biosensor for Phenolic Substrates and Inhibitors.

Methylene blue (MB) acted as a stabilizer for preventing surface-induced denaturation of tyrosinase (TYR) adsorbed on a carbon felt (CF) surface, which is based on shield and anchor roles preventing the unfavorable conformational change of TYR on the hydrophobic CF surface. Furthermore, MB acted as an effective adhesive for TYR immobilization on CF. The resulting TYR and MB coadsorbed CF (TYR/MB-CF) worked as an excellent working electrode unit in an electrochemical detector in a flow injection amperometric biosensor, which allowed highly sensitive consecutive determination of not only TYR substrates but also competitive inhibitors. Simultaneous adsorption of TYR and MB from their mixed solution was much useful as compared with step-wise separated adsorption of TYR on the MB-adsorbed CF, which suggests that the binding interaction of MB with TYR in the solution phase is important for this phenomenon. Fluorescence and UV-vis spectroscopy revealed that not only electrostatic forces between the cationic MB and anionic amino acid residues of TYR but also hydrophobic interactions via the phenothiazine ring of MB play a principal binding driving force of MB with TYR at the surface of the TYR molecules. Synchronous fluorescence, three-dimensional fluorescence, and circular dichroism (CD) spectroscopy clarified that the conformation and the secondary structure of TYR slightly changed upon the MB binding, implying that MB binding leads to the modification of the original intramolecular bonding in part.

An amperometric glucose biosensor based on electrostatic force induced layer-by-layer GOD/chitosan/pyrite on a glassy carbon electrode.

Pyrite (PR), as a representative sulfide mineral, possesses the advantages of abundancy, thermodynamic stability, non-toxicity and semi-conductivity. In this study, an amperometric glucose biosensor (GOD/CS/PR/GCE) based on layer-by-layer of glucose oxidase (GOD), chitosan (CS) and pyrite (PR) on a glassy carbon electrode (GCE) was fabricated through electrostatic force. In this research, PR suspension prepared in phosphate buffer (pH 5.5) was first immobilized on the GCE surface, which exhibits a negative charge. Then, positively charged CS was adsorbed on the PR/GCE by electrostatic force. Finally, negatively charged GOD was further modified on the CS/PR/GCE surface through electrostatic force again. The surface morphology and adsorbance mechanism were supported by field emission scanning electron microscopy, quartz crystal microbalance with dissipation and atomic force microscope. The step-by-step procedure gives both strong adhesion ability and good bioelectrocatalytic activity of GOD on the CS- and PR-modified electrode surface. The linear range of this GOD/CS/PR/GCE biosensor was achieved from 0.5 to 60 mM with the linear regression equation of y = 0.897x - 0.3016 (R2 = 0.9996) and a limit of detection value of 50 µM. This approach of using pyrite and chitosan as physically modified GOD to serve as electrostatic glues could be useful for designing better enzyme-based biosensors for a wide variety of practical applications.

Molten-salt-composite of Pyrite and Silver Nanoparticle as Electrocatalyst for Hydrogen Peroxide Sensing.

A conductive molten salt was synthesized by using natural pyrite (PR) and silver nanoparticles (Ag) at 450°C using a molten salt method. The molten-salt-composite (PR/Ag) was used as an electrocatalyst to detect hydrogen peroxide (H2O2). The as-prepared PR/Ag possessed higher conductivity than natural PR. It exhibited a high sensitivity of 603.54 µA mM-1 cm-2 for the detection of H2O2, with a linear range of 0.1 to 30 mM, and a detection limit of 0.02 mM (S/N = 3). In addition, the PR/Ag sensor exhibited good selectivity to H2O2, resisting interference from other potential interferent compounds (e.g. uric acid, glucose, fructose and common metal ions (K+, Mg2+, Na+)). The approach is considered to provide a sensitive, selective, and reliable tool for highly detection of H2O2.

Natural Molybdenite- and Tyrosinase-Based Amperometric Catechol Biosensor Using Acridine Orange as a Glue, Anchor, and Stabilizer for the Adsorbed Tyrosinase.

To develop a natural mineral-based electrochemical enzyme biosensor, natural molybdenite (MLN), tyrosinase (TYR), and acridine orange (AO) were coadsorbed onto a glassy carbon electrode (GCE). The developed TYR/AO/MLN-GCE-based amperometric TYR biosensor exhibited excellent performance for highly sensitive determination of catechol (linear range, 0.1-80 µM; sensitivity, 0.0315 µA/µM; LOD, 0.029 µM; response time, <4 s) with good reproducibility and good operational and storage stabilities. The electrochemical impedance spectroscopy (EIS) and quartz crystal microbalance with dissipation (QCM-D) revealed interesting roles of AO: (1) an efficient glue for enhancing the amount of the adsorbed TYR on the MLN-GCE, (2) an anchor for efficient orientation of the adsorbed TYR on the MLN-GCE, and (3) a stabilizer providing a suitable microenvironment for the adsorbed TYR on the MLN-GCE surface. This physical adsorption-based AO-coupled enzyme-modification strategy onto natural MLN would be a versatile strategy to develop cost-effective and environment-friendly natural mineral-based electrochemical biosensors and bioelectronic devices.

A Sensitive Electrochemical Ascorbic Acid Sensor Using Glassy Carbon Electrode Modified by Molybdenite with Electrodeposited Methylene Blue.

A non-enzymatic amperometric sensor using natural molybdenite (MLN) electrodeposited with methylene blue (MB) has been fabricated and characterized and its analytical performances were investigated for the determination of ascorbic acid (AA). The surface morphology of the electrode modified by electrodeposited MB was studied by use of the Advanced Mineral Identification and Characterization System (AMICS) and laser confocal high-temperature scanning microscope (LCSM). The poly(MB) and MLN immobilized sensor showed good stability, reproducibility, sensitivity, and selectivity. It exhibited a linear performance range from 3 to 1000 µM, with a lower detection limit of 0.083 µM (signal/noise = 3) and short response time (< 5 s). No obvious decrease in the current was observed after 20 days storage. The methodology reproducibility of this sensor was 2.6%. It showed good anti-interference ability for the potential interfering compounds. The poly(MB) film not only can enhance the electron-transfer rate but also increase the lifetime of the sensor. This study demonstrated the applicability of natural molybdenite for the fabrication of non-enzymatic electrochemical AA sensor.

A Glassy Carbon Electrode Modified with Molybdenite and Ag Nanoparticle Composite for Selectively Sensing of Ascorbic Acid.

Molybdenite (MLN) was physically co-adsorbed with Ag nanoparticles (Ag) on a glassy carbon electrode (GCE) for selectively sensing of ascorbic acid (AA). The composite was characterized with a scan electron microscope, a high-temperature confocal laser scanning microscope, an X-ray diffractometer, an X-ray fluorescence analyzer and electrochemical methods. The prepared MLN/Ag-GCE sensor exhibited good properties including a linear range from 3.0 × 10-5 to 1.0 × 10-3 M toward AA, a low detection limit of 1.5 × 10-5 M, good selectivity, excellent reproducibility, and good stability. The synergistic effect between MLN and Ag nanoparticles results in an enhancement of the electrocatalytic activity for molybdenite.

Carbon Black-Carbon Nanotube Co-Doped Polyimide Sensors for Simultaneous Determination of Ascorbic Acid, Uric Acid, and Dopamine.

Carbon black (CB) and carbon nanotube (CNT) co-doped polyimide (PI) modified glassy carbon electrode (CB-CNT/PI/GCE) was first prepared for the simultaneous determination of ascorbic acid (AA), dopamine (DA), and uric acid (UA). The CB-CNT/PI/GCE exhibited persistent electrochemical behavior and excellent catalytic activities. Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) were used for the simultaneous detection of AA, DA, and UA in their ternary mixture. The peak separations between AA and DA, and DA and UA, are up to 166 mV and 148 mV, respectively. The CB-CNT/PI/GCE exhibited high sensitivity to DA and UA, with the detection limit of 1.9 µM and 3 µM, respectively. In addition, the CB-CNT/PI/GCE showed sufficient selectivity and long-term stability, and was applicable to detect AA, DA, and UA in human urine sample.

A highly sensitive electrochemical biosensor for phenol derivatives using a graphene oxide-modified tyrosinase electrode.

The fabrication, characterization and analytical performance were investigated for a phenol biosensor based on the covalent bonding of tyrosinase (TYR) onto a graphene oxide (GO)-modified glassy carbon electrode (GCE) via glutaraldehyde (GA). The surface morphology of the modified electrode was studied by atomic force microscope (AFM) and field-emission scanning electron microscopy (FE-SEM). The fabricated TYR/GA/GO/GCE biosensor showed very good stability, reproducibility, sensitivity and practical usage. The catechol biosensor exhibited a wide sensing linear range from 5×10-8M to 5×10-5M, a lower detection limit of 3×10-8M, a current maximum (Imax) of 65.8µA and an apparent Michaelis constant (Kmapp) of 169.9µM.

Poly(lactic acid) microparticles coated with insulin-containing layer-by-layer films and their pH-dependent insulin release.

Poly(lactic acid) (PLA) microparticles were coated with layer-by-layer (LbL) films containing insulin and the pH-dependent release of insulin was studied. The LbL films were prepared on the surface of PLA microparticles by the alternate deposition of insulin and poly(allylamine hydrochloride) (PAH) through the electrostatic attraction between insulin and PAH. The insulin loading on the PLA microparticles depended on the film thickness, which corresponded to the number of insulin layers, and on the pH of the solution used to deposit insulin. The insulin loading increased with the film thickness and when the film was prepared at pH 7.4. The LbL films decomposed upon exposure to acidic solutions because the electrostatic attraction between the insulin and the PAH in the films disappeared when the charge on insulin changed from negative to positive at an acidic pH, which resulted in the release of insulin. The temperature and salt concentration did not affect the pH stability of the LbL films. The pH threshold for insulin release was pH 5.0-6.0, which corresponds to isoelectric point of insulin, 5.4. The release of insulin from the microparticles was rapid, and was almost complete within a few minutes. The circular dichroism spectra showed that the released insulin retained its original secondary structure. Our insulin-loaded PLA microparticles may be useful for the controlled release of insulin.

Layer-by-layer deposited nano- and micro-assemblies for insulin delivery: a review.

We present an overview of the recent progress in the development of layer-by-layer (LbL) assembled thin films and microcapsules for insulin delivery. The LbL deposition of insulin-containing thin films on the surfaces of flat substrates or microparticles has been investigated for orally administered insulin formulations. The amount of insulin in the LbL films can be precisely controlled by altering the number of layers in the films. As-prepared LbL films and microcapsules can be loaded with insulin by exposing the films and microcapsules to an insulin solution. The insulin can be released by pH-induced decomposition or permeability changes in the LbL films and microcapsules. Closed-loop insulin delivery systems that can release insulin in response to changes in glucose concentration have also been constructed with LbL films and microcapsules. Glucose-sensitive materials, such as glucose oxidase, concanavalin A, and phenylboronic acid, have been incorporated into insulin-containing LbL assemblies. In addition, LbL film-coated pancreatic islet cells have recently been developed as a bio-artificial pancreas, in which the islet cells are isolated from the recipient's immune system by the LbL coatings. Thus, LbL films and microcapsules could make a significant contribution to the further development of patient-friendly insulin delivery systems.

Carbon Felt-Based Bioelectrocatalytic Flow-Through Detectors: 2,6-Dichlorophenol Indophenol and Peroxidase Coadsorbed Carbon-Felt for Flow-Amperometric Determination of Hydrogen Peroxide.

2,6-dichlorophenol indophenol (DCIP) and horseradish peroxidase (HRP) were coadsorbed on a porous carbon felt (CF) from their mixed aqueous solution under ultrasound irradiation for 5 min. The resulting DCIP and HRP-coadsorbed CF (DCIP/HRP-CF) showed an excellent bioelectrocatalytic activity for the reduction of H2O2. The coadsorption of DCIP together with HRP was essential to obtain larger bioelectrocatalytic current to H2O2. The DCIP/HRP-CF was successfully used as a working electrode unit of a bioelectrocatalytic flow-through detector for highly sensitive and continuous amperometric determination of H2O2. Under the optimized operational conditions (i.e., applied potential, +0.2 V versus Ag/AgCl; carrier pH 5.0, and carrier flow rate, 1.9 mL/min), the cathodic peak current of H2O2 linearly increased over the concentration range from 0.1 to 30 µM (the sensitivity, 0.88 µA/µM (slope of linear part); the limit of detection, 0.1 µM (S/N = 3) current noise level, 30 nA) with a sample through-put of ca. 40-90 samples/h.

Sensitive voltammetric and amperometric responses of respiratory toxins at hemin-adsorbed carbon-felt.

A hemin [iron-Fe(III) protoporphyrin IX chloride] was adsorbed onto a carbon-felt (CF), which is a microelectrode ensemble of micro carbon fiber (ca. 7 microm diameter). The resulting hemin-adsorbed-CF (hemin-CF) showed a well-defined redox wave based on the hemin-Fe(III)/Fe(II) redox process with the formal potential of -0.225 V vs. Ag/AgCl in deoxygenated phosphate/citrate buffer solution (0.1 mol/L, pH 5.0). The apparent heterogenous electron transfer rate constant was estimated to be 8.6 sec(-1). In air-saturated electrolyte solution, the hemin-CF exhibited an excellent electrocatalytic activity for the reduction of dioxygen (O2). This activity was reversibly inhibited by respiratory toxins such as cyanide and azide, which bind sixth coordination position of iron active center of hemin. The electrocatalytic 02 reduction current at the hemin-CF was modulated by the toxins in a concentration-depending manner. Based on the relationship between the %inhibition and the toxin concentration, apparent inhibition constants of cyanide and azide were evaluated to be 4.52 and 1.98 micromol/L, respectively. When the hemin-CF was used as a working electrode unit of the CF-based electrochemical flow-through detector with air-saturated carrier, the injection of the azide induced peak-shape current responses, which allowed rapid and continuous flow-amperometric determination of azide with high sensitivity.

Protein and polysaccharide-composite sol-gel silicate film for an interference-free amperometric glucose biosensor.

A novel permselective, organic-inorganic-hybrid, sol-gel silicate-film was chemically modified on an anodized platinum (Pt) electrode surface to form a selective, sensitive and interference-free amperometric glucose biosensor. This permselective hybrid sol-gel film consists of three organo-silanes [i.e., 3-aminopropyltriethoxysilane (APTES); tetraethoxysilane (TEOS); triethoxy-1H,1H,2H,2H-tridecafluoro-n-octylsilane (FAS)] and two biomacromolecules [i.e., bovine serum albumin (BSA) and a chitosan (CHIT)]. After the addition of the film to the Pt electrode, glucose oxidase (GOx) was covalently immobilized within the film with glutaraldehyde. The incorporation of the BSA and CHIT not only enhanced the permselectivity of H2O2 but also improved the activity of the immobilized GOx. The CHIT effectively suppressed any swelling of the film. Moreover, the conjugation of the FAS was especially effective in reducing the interference currents of AA and UA to levels less than 1/400 and 1/300 of the current of H2O2. The resulting organic-inorganic-hybrid sol-gel-film-based amperometric glucose biosensor exhibited rapid and sensitive responses to glucose (100% response in <3s, sensitivity: 1.84 µA mM(-1), detection limit: 0.032 mM), and the highly selective determination of glucose was possible, even in the presence of 0.1mM AA and UA.

Novel amperometric assay for drug-DNA interaction based on an inhibitory effect on an electrocatalytic activity of DNA-Cu(II) complex.

A novel strategy of amperometric assay for drug-dsDNA interactions was developed based on an inhibitory effect of antimararial drug (quinacrine) on an electrocatalytic activity of DNA-Cu(II) complex. In this method, a DNA-Cu(II) complex immobilized DNA/polyallylamine(PAA) polyion complex membrane was used as a sensing element. The electrocatalytic activity of a DNA-Cu(II) complex for hydrogen peroxide reduction was reversibly inhibited by electron blocking effect of quinacrine-dsDNA interaction and this inhibitory effect was amplified by the hydrogen peroxide reduction. This phenomenon was utilized for development of a novel amperometric biosensor for DNA-binding drug. From the amperometric current-time curves, the response time of the sensor to 20 µM quinacrine was obtained about 20s, and the detection limit of the quinacrine was found to be 10 µM estimated to a signal-to-noise ratio of 3.0. Based on the change of steady-state catalytic current, the kinetic analysis of drug-dsDNA interaction can be done in a similar manner of enzyme inhibition, and the binding constant of the quinacrine with DNA can be calculated. This measurement method would be useful for screening of wide variety of DNA-binding drugs and highly toxic pollutants.

Uricase-adsorbed carbon-felt reactor coupled with a peroxidase-modified carbon-felt-based H2O2 detector for highly sensitive amperometric flow determination of uric acid.

Uricase (urate oxidase, UOx) was adsorbed onto a porous carbon-felt (CF) surface and the resulting UOx-adsorbed CF (UOx-CF) was successfully used as a column-type enzyme reactor coupled with a peroxidase-adsorbed CF-based bioelectrocatalytic H(2)O(2) flow-detector to fabricate a flow-amperometric biosensor for uric acid. In this flow-biosensor system, H(2)O(2) produced in the UOx-CF reactor was cathodically detected by horseradish peroxidase (HRP) and a thionine (Th)-coadsorbed CF (HRP/Th-CF)-based bioelectrocatalytic flow-detector at -0.05V vs. Ag/AgCl. Various adsorption conditions of the UOx (i.e., pH of the adsorption solution, type and concentration of the buffer used as the adsorption solvent, UOx concentration and adsorption time) and the operational conditions of the UOx-CF and HRP/Th-CF-coupled flow-biosensor (i.e., carrier flow rate and carrier pH) were optimized to obtain highly sensitive, selective and stable peak current responses to uric acid. The analytical performance of the UOx-CF and HRP/Th-CF-coupled flow biosensor for uric acid was as follows: sensitivity, 0.25µA/uM; linear range, 0.3-20µM; lower detection limit, 0.18µM; and sample throughput, ca. 30-90 samples/h. The resulting amperometric flow-biosensor for uric acid allowed the determination of uric acid in highly diluted body fluids (human serum and urine), and the analytical results obtained by the present biosensor were in fairly good agreement with those obtained by conventional enzyme-based spectrophotometry.

Insulin-containing layer-by-layer films deposited on poly(lactic acid) microbeads for pH-controlled release of insulin.

Layer-by-layer (LbL) thin films containing insulin were deposited on the surface of biodegradable poly(lactic acid) (PLA) microbeads and the pH-triggered release of insulin was studied. The LbL films were successfully prepared by the alternate deposition of insulin and poly(vinyl sulfate) (PVS) or dextran sulfate (DS) at pH 4.0 through the electrostatic force of attraction between positively charged insulin and polyanions. The loading of insulin on the microbeads was dependent on the number of insulin layers and the type of polyanions used; higher insulin loading was observed for thicker films and when PVS was used as the polyanion. Insulin was released from the microbeads when they were exposed to neutral solution (pH 7.4) due to a loss of electrostatic attraction between the insulin and polyanions in the films, which in turn was caused by the charge reversal of insulin from positive to negative in the neutral medium. The pH threshold for insulin release was found to be pH 5.0-6.0. The released insulin retained its original secondary structure as evidenced by circular dichroism spectra. The insulin loaded on the microbeads was satisfactorily stable even in the presence of a digestive enzyme (pepsin) at pH 1.5. These results suggest a potential future use for insulin-loaded microbeads in the oral delivery of insulin.

Tyrosinase-modified carbon felt-based flow-biosensors: the role of ultra-sonication in shortening the enzyme immobilization time and improving the sensitivity for p-chlorophenol.

Tyrosinase (TYR) was covalently immobilized onto amino-functionalized carbon felt surface via glutaraldehyde-coupling under ultrasonic treatment for 10 min. The resulting TYR-immobilized carbon felt was used as a working electrode unit of bioelectrocatalytic flow-through detector for TYR substrates (catechol, p-chlorophenol (p-CP), p-cresol, phenol etc.). Cathodic peak currents based on the electroreduction of enzymatically produced o-quinones were detected at -50 mV vs. Ag/AgCl. Compared with previous work in which TYR was immobilized onto amino-functionalized carbon felt for 16 hr without the ultrasonic treatment, we succeeded in (1) shortening the enzyme immobilization time from 16 hr to 10 min, (2) enhancing the sensitivity of p-CP, and (3) improving the operational stability of p-CP. The ultrasonic treatment during the TYR immobilization step would lead to certain changes in the structure of the immobilized TYR and the morphology of the immobilized TYR-layer on the carbon felt surface.

Electropolymerized poly(Toluidine blue)-modified carbon felt for highly sensitive amperometric determination of NADH in flow injection analysis.

Poly(pheniothiazine) films were prepared on a porous carbon felt (CF) electrode surface by an electrooxidative polymerization of three phenothiazine derivatives (i.e.,Tthionine (TN), Toluidine Blue (TB) and Methylene Blue (MB)) from 0.1 mol/L phosphate buffer solution (pH 7.0). Among the three phenothiazies, the poly(TB) film-modified CF exhibited an excellent electrocatalytic activity for the oxidation of nicotinamide adenine dinucleotide reduced form (NADH) at +0.2 V vs. Ag/AgCl. The poly(TB) film-modified CF was successfully used as working electrode unit of highly sensitive amperometric flow-through detector for NADH. The peak currents (peak heights) were almost unchanged, irrespective of a carrier flow rate ranging from 2.0 to 4.1 mL/min, resulting in the measurement of NADH (ca. 30 samples/hr) at 4.1 mL/min. The peak current responses of NADH showed linear relationship over the concentration range from 1 to 30 micromol/L (sensitivity: 0.318 microA/(micromol/L); correlation coefficient: 0.997). The lower detection limit was found to be 0.3 micromol/L (S/N = 3).

Carbon-felt-based bioelectrocatalytic flow-detectors: role of ultrasound irradiation during the adsorption of horseradish peroxidase and thionine for a highly sensitive amperometric determination of H2O2.

This study reports on the first example, to our knowledge, of the usefulness of an ultrasound (US)-irradiation during an enzyme adsorption step, for enhancing the performance of a redox-enzyme-based amperometric biosensor. Horseradish peroxidase (HRP) and thionine (Th) were coadsorbed from a mixed aqueous solution of HRP and Th onto a carbon-felt (CF) under US-irradiation for 5 min with an ultransonic bath operating at 40 kHz frequency and 55 W of electric power output. The resulting HRP and Th-coadsorbed CF (HRP/Th-CF) was successfully used as a working electrode unit of a bioelectrocatalytic flow-detector for hydrogen peroxide (H(2)O(2)), which detects the cathodic peak currents based on the direct (unmediated) reduction of oxidized HRP intermediates at 0 V vs. Ag/AgCl. Compared with ordinary adsorption without US-irradiation, US-irradiation during the HRP adsorption step was effective to obtain highly sensitive peak current responses to H(2)O(2). The measurements of electrochemical impedance spectroscopy and cyclic voltammetry suggested that the adsorption of HRP and Th under the US-irradiation provides a suitable interfacial microenvironment for a favorable orientation and conformation of an enzyme with active site available for both substrates and the electrode, which results in larger bioelectrocatalytic activity. The peak currents for H(2)O(2) increased up to 3 × 10(-6) M (sensitivity, 4.72 µA/µM) with a lower detection limit of 2 × 10(-8) M (S/N = 3; current noise level, 0.03 µA).