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.

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.

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.

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.

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.

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

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.

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).

Direct electrochemistry of glucose oxidase and biosensing for glucose based on DNA/chitosan film.

Glucose oxidase (GOD) is widely used in the glucose biosensor industry. The amperometric biosensors based on directly electron transfer (DET) between an electrode and immobilized GOD are especially promising. In this article, GOD was immobilized with a DNA/chitosan bio-material film on GC electrode, and the DET of GOD on DNA/chitosan was studied. The cyclic voltammetric results indicated that the GOD immobilized in the DNA/chitosan film underwent DET reaction, and the cyclic voltammogram displayed a pair of well-defined redox peaks with a formal potential of -0.45 V (vs. Ag/AgCl) at pH 5.5. The response showed a surface-controlled electrode process with an electron transfer rate constant of 0.91 sec(-1) determined in the scan rate range from 10 to 100 mV/sec. The GOD immobilized in DNA/chitosan membrane retained its biocatalytic activity and stability. The immobilized GOD could electrocatalyze the reduction of dissolved oxygen and resulted in a great increase of the reduction peak current. Upon the addition of glucose, the reduction peak current decreased, which could be used for glucose detection with a sensitivity of 0.48 µA/(mmol/L), a linear range from 0.04 to 2.28 mmol/L and a detection limit of 0.04 mmol/L at a signal-to-noise ratio of 3. The sensor could exclude the interference of commonly coexisted uricacid and ascorbic acid.

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.

Highly sensitive flow-biosensor for toxic phenolic compounds using tyrosinase and acridine orange-adsorbed carbon felt.

Tyrosinase (TYR, EC 1.14.18.1) was physically adsorbed onto a carbon felt (CF) together with acridine orange (AO). Coadsorption of AO was essential to prevent the denaturation of the TYR at the CF surface. The resulting TYR and AO-coadsorbed CF (TYR/AO-CF) was successfully utilized as a detection unit of novel and highly sensitive amperometric flow-biosensor for toxic chlorophenol compounds. Standard solutions of phenolic compounds (200 µL) were injected, and the cathodic peak currents due to the reduction current of o-quinones produced by the TYR-catalyzed oxidation (phenolase activity) were detected at the applied potential of -50 mV vs. Ag/AgCl. In this reaction, the electrochemically generated catechol compounds from o-quinones are re-oxidized repeatedly by catecholase activity of the TYR, leading to a sufficient amplified signal. The TYR/AO-CF exhibited much higher selectivity toward p-chlorophenol as compared with other chlorophenol compounds. When 0.1 mol/L phosphate buffer (pH 7.0) was used as a carrier at flow rate of 3.0 mL/min, cathodic peaks for p-chlorophenol was linear in the concentration range between 0.1 and 10 µmol/L (sensitivity: 1.41(mA·L)/mmol) with sampling rate (30 samples/h), and the detection limit of p-chlorophenol was found to be 2.13 × 10(8) mol/L (S/N = 3. The ratio of signal and noise is 3). The TYR/AO-CF kept more than 80% of original activity after the storage in 0.1 mol/L phosphate buffer (pH 7.0) containing 0.2 mmol/L AO at 4°C.

Quantitative determination of Escherichia coli based on the electrochemical measurement of bacterial catalase activity using H2O2-selective organic/inorganic-hybrid sol-gel film-modified Pt electrode.

Quantitative determination of Escherichia coli (E. coli) concentration was achieved by measuring the intrinsic catalase activity of E. coli using novel H2O2-selective organic/inorganic-hybrid sol-gel film-modified platinum (Pt) wire electrode. This hybrid sol-gel film is composed of three kinds of organosilanes and two biopolymers (i.e., chitosan and bovine serum albumin), and exhibited an excellent permselectivity toward H2O2 based on a size-exclusive mechanism. The steady-state anodic current for 100 [xmol/L H2O2 at +0.6 V (vs. Ag/AgCl) in 0.1 mol/L phosphate buffer (pH 6.5) solution was apparently diminished by the addition of E. coli samples, due to the decomposition of H2O2 by intrinsic catalase activity of E. coli. The time-dependent decrease in current (-AI/At) was significantly dependent on the E. coli concentration. The -AI/At was enhanced by the permeabilization pretreatment of E. coli samples with the mixed solution of polymyxin B and lysozyme. This H2O2-selective organic/inorganic-hybrid sol-gel film-modified platinum (Pt) wire electrode allowed quantitative determination of E. coli concentration ranging from 10(6) to 10(9) CFU/mL within 30 min. This method required no label and complicated procedure, and allowed rapid, simple and cost-effective quantitative electrochemical determination of catalase-positive bacteria.

Amperometric hydrogen peroxide biosensor based on immobilization of DNA-Cu(II) in DNA/chitosan polyion complex membrane.

DNA/chitosan polyion complex membrane was used as a support for immobilization of electrocatalytic species-copper ions, which specifically bound to dsDNA and catalyzed the hydrogen peroxide reduction. The polyion complex membrane consisted with DNA-Cu(II) complex and chitosan was prepared on a glassy carbon electrode (GCE). Electrochemical measurements of the DNA-Cu(II)/chitosan membrane-modified GCE revealed that the copper ion embedded in the DNA/chitosan layer exhibited good electrochemical behaviors. The DNA-Cu(II)/chitosan/GC electrode showed an excellent electrocatalytic activity for the H2O2 reduction. Even in the presence of dissolved oxygen, the sensor exhibited highly sensitive and rapid (response time, about 6 s) response to H2O2. The steady-state cathodic current responses of the sensor obtained at -0.2 V versus Ag/AgCl in air-saturated 100 mmol/L phosphate buffer (pH 5.0) increased linearly up to 10 mmol/L with the detection limit of 3 µmol/L. Effects of applied potential and buffer pH upon the response currents of the sensor were investigated for an optimum analytical performance. Ascorbic acid and glucose almost have no interference to measurement of H2O2. In addition, the sensor exhibited good reproducibility.

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.

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.

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.

Carbon felt-based bioelectrocatalytic flow detectors: highly sensitive amperometric determination of hydrogen peroxide using adsorbed peroxidase and thionine.

Horseradish peroxidase (HRP) and thionine (TN) were co-adsorbed onto a porous carbon felt (CF), and the resulting HRP and TN-adsorbed CF (HRP-TN-CF) was successfully used as a working electrode unit of a novel bioelectrocatalytic flow detector for a highly sensitive amperometric determination of hydrogen peroxide (H(2)O(2)). Co-adsorbed TN was essential to enhance the cathodic peak current of H(2)O(2), and the current responses of the HRP-TN-CF-based detector were much larger than those of the HRP-CF-based detector (without TN). When air-saturated 0.1 M phosphate buffer (pH 7.0) was used as a carrier at a flow rate of 3.9 ml/min, cathodic peak currents of H(2)O(2) (sample injection volume, 200 microl) obtained at an applied potential of 0 V (vs. Ag/AgCl) increased linearly up to 50 microM with a detection limit of 0.1 microM. Repetitive 100 sample injection of 100 microM H(2)O(2) induced no serious current decrease, and RSD was 0.41 to 1.21% (n = 100). The HRP-TN-CF retained 42% of its original activity after 8 days of storage in 0.1 M phosphate buffer at 4 degrees C.

DNA-Cu(II) poly(amine) complex membrane as novel catalytic layer for highly sensitive amperometric determination of hydrogen peroxide.

A novel hydrogen peroxide biosensor was fabricated by using a DNA-Cu(II) complex as a novel electrocatalyst for the reduction of hydrogen peroxide (H2O2). A polyion complex (PIC) membrane composed of DNA and poly(allylamine) (PAA) functioned as a support matrix for immobilization of electrocatalytic element-copper ion. The circular dichroism (CD) spectrum of the DNA-Cu(II)/PAA membrane in wet state showed that the DNA exists in B-like form within the membrane. Electrochemical measurements of the DNA-Cu(II)/PAA membrane-modified glassy carbon (GC) electrode revealed that the copper ion embedded in the DNA/PAA layer exhibits good electrochemical behaviors, and the electrochemical rate constant between the immobilized copper ion and the GC electrode surface was estimated to be 26.4 s(-1). The resulting DNA-Cu(II)/PAA/GC electrode showed an excellent electrocatalytic activity for the H2O2 reduction. The sensitivity of the sensor for the determination of H2O2 was affected by the amount of each component, such as copper ion, DNA and PAA in the DNA-Cu(II)/PAA membrane. Effects of applied potential, pH, temperature, ionic strength and buffer concentrations upon the response currents of the sensor were also investigated for an optimum analytical performance. Even in the presence of dissolved oxygen, the sensor exhibited highly sensitive and rapid (response time, less than 5 s) response to H2O2. The steady-state cathodic current responses of the sensor obtained at -0.2 V versus Ag/AgCl in air-saturated 50 mM phosphate buffer (pH 5.0) increased linearly up to 135 microM with the detection limit of 50 nM. Interference by ascorbic acid and uric acid due to the reduction of Cu(II) was effectively cancelled by further modification of outermost layer of polyion complex film. In addition, the sensor exhibited good reproducibility and stability.