An ECL biosensor for glucose based on carbon-nanotube/Nafion film modified glass carbon electrode

Glucose oxidase was encapsulated in carbon-nanotube/Nafion film modified glass carbon electrode and was used as electrochemiluminescence (ECL) biosensor for glucose. The glucose oxidase can be fixed firmly in the Nafion film and carbon nanotubes offer excellent electrocatalytic activity toward luminol and hydrogen peroxide liberated in enzymatic reaction between glucose oxidase and glucose, which would enable sensitive determination of glucose. Under the optimum condition, the linear response range of glucose was found to be 5.0 × 10⁻⁶ to 8.0 × 10⁻⁴ mol/L, and the detection limit (defined as the concentration that could be detected at the signal-to-noise ratio of 3) was 2.0 × 10⁻⁶ mol/L. The present carbon-nanotube/Nafion biocomposite glucose oxidase ECL biosensor showed excellent properties for sensitive determination for glucose with good reproducibility and stability, and it has been used to determine the glucose concentrations in real serum samples with the satisfactory results.     

Low potential detection of glucose at carbon nanotube modified glassy carbon electrode with electropolymerized poly(toluidine blue O) film

A mediator glucose biosensor has been constructed by immobilizing glucose oxidase at electropolymerized poly(toluidine blue O) film on carbon nanotube modified glass carbon electrode. The toluidine blue O moieties served as redox mediators for enzymatic glucose oxidation and as polymeric network to maintain the biosensor activity. Great enhancement in current response was observed for the glucose biosensor. The detection potential could be decreased to −0.1 V (versus Ag|AgCl), where common interferences such as ascorbic acid, uric acid and acetamidophenol were not oxidized to cause interferences. The amperometric glucose biosensor offered a sensitivity of 14.5 mA M⁻¹ cm⁻² for the linear range of 1-7 mM.     

Functionalized carbon nanotube-bienzyme biocomposite for amperometric sensing

An approach to design a biocomposite bienzyme biosensor with the aim of evaluating its suitability as an amperometric sensor using functionalized multiwalled carbon nanotubes (MWCNTs) is presented. The biosensor is based on a bienzyme-channelling configuration, employing the enzymes glucose oxidase (GOx) and horseradish peroxidase (HRP), which were immobilized with toluidine blue (TB) functionalized MWCNTs. The proposed method demonstrates an easy electron transfer between the immobilized enzymes and the electrode via functionalized MWCNTs in a Nafion matrix. Co-immobilization of GOx and HRP was employed to establish the feasibility of fabricating highly effective bienzyme-based biosensors for low-level glucose determination. Bienzyme immobilized TB functionalized MWCNTs were attached to a glassy carbon electrode, and the electrochemical behavior of the sensor was studied using electrochemical impedance spectroscopy, cyclic voltammetry and chronoamperometry. The excellent electrocatalytic activity of the biocomposite film resulted in the detection of glucose under reduced over potential with a wider range of determination from 1.5 × 10⁻⁸ M to 1.8 × 10⁻³ M and with a detection limit of 3 × 10⁻⁹ M. The sensor showed a short response time (within 2 s), good stability and anti-interferant ability. The proposed biosensor exhibits good analytical performance in terms of repeatability, reproducibility and shelf-life stability.     

Construction of a choline biosensor through enzyme immobilization on a poly(2‐hydroxyethyl methacrylate)‐grafted Teflon film

An amperometric choline biosensor was constructed by immobilizing choline oxidase (ChO) on poly(2‐hydroxyethyl methacrylate) (PHEMA)‐grafted Teflon (polytetrafluoroethylene, PTFE) film. Grafting was achieved by γ irradiation. PHEMA‐grafted Teflon films were activated with epichlorohydrin or glutaraldehyde to achieve covalent immobilization of enzyme onto the film. To decrease the diffusional barrier caused by the enzyme‐immobilized film, the film was stretched directly on the electrode. The PHEMA‐grafted Teflon film, therefore, had to have appropriate mechanical properties. Glucose oxidase (GOD) was used in the determination of optimum immobilization conditions, then these were applied to ChO. With GOD, the effect of activation type and film position in electrode on enzyme activity was studied and the highest catalytic activity was obtained when the enzyme was immobilized using glutaraldehyde and the film was stretched over the electrode surface. Further studies revealed that the films activated with glutaraldehyde, immobilized in 2 mg/mL ChO concentration, and stretched directly on the electrode were suitable (specific activity, 0.427 ± 0.068 U mg^?1) for use in the choline biosensor. The linear working range of this biosensor was found to be 52–348 μM, with a 40 ± 5 μM minimum detection limit. The response of the sensor, however, decreased linearly upon repeated use. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci, 2007     

Amperometric bienzyme glucose biosensor based on carbon nanotube modified electrode with electropolymerized poly(toluidine blue O) film

The amperometric bienzyme glucose biosensor utilizing horseradish peroxidase (HRP) and glucose oxidase (GOx) immobilized in poly(toluidine blue O) (PTBO) film was constructed on multi-walled carbon nanotube (MWNT) modified glassy carbon electrode. The HRP layer could be used to analyze hydrogen peroxide with toluidine blue O (TBO) mediators, while the bienzyme system (HRP + GOx) could be utilized for glucose determination. Glucose underwent biocatalytic oxidation by GOx in the presence of oxygen to yield H₂O₂ which was further reduced by HRP at the MWNT-modified electrode with TBO mediators. In the absence of oxygen, glucose oxidation proceeded with electron transfer between GOx and the electrode mediated by TBO moieties without H₂O₂ production. The bienzyme electrode offered high sensitivity for amperometric determination of glucose at low potential, displaying Michaelis-Menten kinetics. The bienzyme glucose biosensor displayed linear response from 0.1 to 1.2 mM with a sensitivity of 113 mA M⁻¹ cm⁻² at an applied potential of −0.10 V in air-saturated electrolytes.     

Glucose biosensor based on multiwalled carbon nanotubes grown directly on Si

An amperometric biosensor based on covalent immobilization of glucose oxidase (GOx) on multiwalled carbon nanotubes (MWCNTs) with potassium ferricyanide as the redox mediator was developed. The MWCNTs were grown directly on a layered structure of Co/Ti/Cr on a SiO₂/Si substrate by microwave-heated chemical vapor deposition. The mediator helps to shuttle the electrons between the immobilized GOx and the MWCNT electrode, therefore operating at a potential of 0.25 V vs. the saturated calomel electrode. This potential precludes the interfering compounds from oxidization. The sensitivity of biosensors to glucose was found to depend on the acid pretreatment and GOx reaction times. The steady-state response of the optimized biosensor exhibits a sensitivity of 20.6 μA mM⁻¹ cm⁻², a linear range of up to 8 mM, and a response time of <5 s.     

Fabricating polythiophene into highly aligned microwire film by fast evaporation of its whisker solution

A facile template-free approach to fabricate poly(3-hexylthiophene) (P3HT) into highly aligned microwire film on a large scale via the evaporation of P3HT/Anisole whisker solution has been developed. The microwires in the film typically have the height of 0.8-1.4 μm, width of 2-4 μm and length of 50-1000 μm. X-ray Diffraction and Selected-Area Electron Diffraction results suggest that each microwire is a single crystal with the reduced packing distance of P3HT chains along the π-π stacking direction (d_[010] of 6.5 Å) and the interchain direction (d_[100] of 15.7 Å). The closer packing of P3HT chains is likely the key factor promoting the formation of the highly aligned microwires. The aligned P3HT microwire films perform enhanced electrical conductivity, and show no substrate dependence, thus can be fabricated into organic electronic devices in situ.     

A DNA electrochemical sensor with poly-l-lysine/single-walled carbon nanotubes films and its application for the highly sensitive EIS detection of PAT gene fragment and PCR amplification of NOS gene

A sensitive and novel DNA electrochemical biosensor for the detection of the transgenic plants gene fragment by electrochemical impedance spectroscopy (EIS) was presented. The well-dispersed carboxylic group-functionalized single-walled carbon nanotubes (SWNTs) were dripped onto the carbon paste electrode (CPE) surface firstly, and poly-l-lysine films (pLys) were subsequently electropolymerized by cyclic voltammetry (CV) to prepare pLys/SWNTs/CPE. The morphology of pLys/SWNTs films was examined using a field emission scanning electron microscope (SEM). The pLys/SWNTs films modified electrode exhibited very good conductivity. DNA probes were easily immobilized on the poly-l-lysine films via electrostatic adsorption. The hybridization events were monitored with electrochemical impedance spectroscopy using [Fe(CN)₆]^3−/4− as indicator. The PAT gene fragment from phosphinothricin acetyltransferase gene was detected by this DNA electrochemical sensor. The dynamic detection range of this sensor to the PAT gene fragment was from 1.0 × 10⁻¹² to 1.0 × 10⁻⁷ mol/L. A detection limit of 3.1 × 10⁻¹³ mol/L could be estimated. The PCR amplification of NOS gene from the sample of a kind of transgenic modified bean was also detected satisfactorily by EIS.     

A sensitive DNA biosensor fabricated from gold nanoparticles, carbon nanotubes, and zinc oxide nanowires on a glassy carbon electrode

We outline here the fabrication of a sensitive electrochemical DNA biosensor for the detection of sequence-specific target DNA. Zinc oxide nanowires (ZnONWs) were first immobilized on the surface of a glassy carbon electrode. Multi-walled carbon nanotubes (MWCNTs) with carboxyl groups were then dropped onto the surface of the ZnONWs. Gold nanoparticles (AuNPs) were subsequently introduced to the surface of the MWNTs/ZnONWs by electrochemical deposition. A single-stranded DNA probe with a thiol group at the end (HS-ssDNA) was covalently immobilized on the surface of the AuNPs by forming an Au-S bond. Scanning electron microscopy (SEM) and cyclic voltammetry (CV) were used to investigate the film assembly process. Differential pulse voltammetry (DPV) was used to monitor DNA hybridization by measuring the electrochemical signals of [Ru(NH₃)₆]³⁺ bounding to double-stranded DNA (dsDNA). The incorporation of ZnONWs and MWCNTs in this sensor design significantly enhances the sensitivity and the selectivity. This DNA biosensor can detect the target DNA quantitatively in the range of 1.0 × 10⁻¹³ to 1.0 × 10⁻⁷ M, with a detection limit of 3.5 × 10⁻¹⁴ M (S/N = 3). In addition, the DNA biosensor exhibits excellent selectivity, even for single-mismatched DNA detection.     

Continuous glucose monitoring using enzyme-immobilized platinized diamond microfiber electrodes

A sensitive and stable glucose biosensor for in vivo monitoring has been developed using boron-doped diamond microfiber (BDDMF) electrodes. The electrodes were modified with platinum nano-particles to detect H₂O₂, which was enzymatically produced by glucose oxidase (GOx) immobilized on the electrode surface. The platinum-modified BDDMF (Pt-BDDMF) electrodes exhibited much higher sensitivity compared to Pt-microfiber electrodes, Pt electrodes and Pt-modified diamond thin film electrodes. Deposition conditions for Pt nano-particles on the BDDMF electrodes and immobilization of GOx were optimized. GOx/overoxidized polypyrrole (OPPy)/Pt-modified BDDMF electrodes were applied for continuous interference-free glucose monitoring. Amperometric measurements of glucose showed a linear response in the range of 1-70 mM, with an R.S.D. of 3.7% for five injections of 100 μM glucose. The electrodes exhibited good stability over 3 months with no detected anodic current for ascorbic acid (AA), which is an interfering compound.     

Controlled multilayer films of sulfonate-capped gold nanoparticles/thionine used for construction of a reagentless bienzymatic glucose biosensor

A novel reagentless bienzymatic sensor for the determination of glucose in the low working potentials without interference is proposed. The bienzymatic sensor was fabricated by covalently attachment of periodate-oxidized glucose oxidase (IO₄⁻-GOx) and horseradish peroxidase (HRP) on controlled multilayer films of sulfonate-capped gold nanoparticles/thionine (SCGNPs/TH). Using the layer-by-layer method (LBL), SCGNPs and TH were deposited alternately on the gold electrode through the electrostatic and covalent interactions. SCGNPs could greatly enhance the amount of immobilized TH and ensure the good conductivity of the whole structure. UV-vis absorption spectroscopy and electrochemical methods showed that the resulting multilayer films were tridimensional conductive and porous, and TH incorporated in LBL configuration had well electroactive performance. Such superstructures can thus provide an ideal matrix for the construction of bienzymatic sensor, where TH molecules acted as a mediator for electron transfer. After IO₄⁻-GOx and HRP were covalently attached to the multilayer precursor film, the resulting biosensor exhibited good electrocatalytical response toward glucose and that the electrocatalytical response increased with the number of TH layers. This suggested that the analytical performance such as sensitivity and detection limit of the bienzymatic sensors could be tuned to the desired level by adjusting the number of deposited SCGNPs/TH bilayers. Furthermore, because of the low working potentials, the interference from other electro-oxidizable compounds (such as uric acid, ascorbic acid and acetaminophen) was avoided, which improved the selectivity of the biosensors. The biosensor constructed with six bilayers of SCGNPs/TH showed a good performance of glucose detection with a fast response less than 20 s, acceptable sensitivity of 3.8 μA mM⁻¹ cm⁻² and the detection limit of 3.5 × 10⁻⁵ M.     

Organic phase PPO biosensor based on hydrophilic films of electropolymerized polypyrrole

We described herein, the construction of an organic phase enzyme electrode (OPEE) via polyphenol oxidase (PPO) entrapment within a hydrophilic polypyrrole film electrogenerated from on a new bispyrrolic derivative (1) containing a long hydrophilic spacer. The so-called “adsorption step procedure” was adopted for the preparation of the organic phase PPO biosensor. The amperometric detection of catechol was carried out in anhydrous chloroform at −0.2 V versus Ag/AgCl. The electroanalytical parameters of the biosensor strongly depend on its configuration and on the hydration state of the enzyme matrix. The best sensitivity obtained for catechol in chloroform was 15.6 mA M⁻¹ cm⁻².     

Immobilization of GOD on Ppy–PVS Composite Film for Determination of Glucose: A Comparative Study of Phosphate and Acetate Buffers

The Polypyrrole-polyvinylsulphonate-glucose oxidase (Ppy–PVS–GOD) biosensor for determination of glucose has been described in the present investigation. The enzyme, glucose oxidase (GOD) was immobilized by crosslinking via glutaraldehyde on a polypyrrole–polyvinyl sulphonate (Ppy–PVS) composite film. The Ppy–PVS film was electrochemically synthesized on indium-tin-oxide (ITO)–coated glass plate. The synthesized composite films were characterized using galvanostatic electrochemical technique, electrical conductivity, UV-Visible spectroscopy, Fourier transform infrared spectroscopy (FTIR), and scanning electron microscopy (SEM). The crosslinking of enzyme and porous morphology of the polymer film leads to high enzyme loading and an increase in lifetime, stability, and fast response time of the enzyme electrode. Characterization of resulting amperometric biosensor for the estimation of glucose has been experimentally determined in terms of linear response range, optimum pH, applied potential, and shelf-life. These Ppy–PVS–GOD electrodes can be used for glucose estimation from 1 to 50 mM and have a shelf-life of about 5–6 weeks at 4°C. The sensitivity of Ppy–PVS–GOD electrode in phosphate and acetate buffer has been studied. It was found that the phosphate buffer gives fast response as compared to acetate buffer in amperometric measurements.     

Physicochemical characterization of the layer-by-layer self-assembly of polyphenol oxidase and chitosan on glassy carbon electrode

This paper demonstrates the feasibility to build up layer-by-layer (L-b-L) self-assemblies of polyphenol oxidase (PPO) and quaternized chitosan (CHI⁺) on a glassy carbon (GC) rotating disk electrode. This work highlights the promising properties of this modified polysaccharide to immobilize PPO in self-assembled structures under conditions that preserve its catalytic activity. The film consists of a diffusional underlayer coupled with an outermost active layer of (PPO/CHI⁺)_n. UV-vis spectroscopy and quartz crystal microbalance (QCM) were used to follow the multilayer film formation. Referring to the low amount of enzyme immobilized in the multilayer structure, the amperometric response of the biosensor reached an excellent sensitivity of about 2000 A mol⁻¹ cm, proving that an important part of the entrapped enzyme is accessible to the substrate molecule while keeping a good level of activity. The experimental validation of the theoretical model that was carried out on the basis of the catechol and phenol-polyphenol oxidase model system shows that the amperometric responses are in excellent agreement with the model. From a comparison of the experimental results with theory, we were able to characterize the diffusion through the film which includes two different areas and extract its enzymatic activity related to catechol and phenol substrates.     

Direct electron transfer and electrocatalysis of glucose oxidase immobilized on glassy carbon electrode modified with Nafion and mesoporous carbon FDU-15

In this paper, it was found that glucose oxidase (GOD) has been stably immobilized on glassy carbon electrode modified with mesoporous carbon FDU-15 (MC-FDU-15) and Nafion by simple technique. The sorption behavior of GOD immobilized on MC-FDU-15 matrix was characterized by transmission electron microscopy (TEM), ultraviolet-visible (UV-vis), FTIR, respectively, which demonstrated that MC-FDU-15 could facilitate the electron exchange between the active center of GOD and electrode. The direct electrochemistry and electrocatalysis behavior of GOD on the modified electrode were characterized by cyclic voltammogram (CV) which indicated that GOD immobilized on Nafion and MC-FDU-15 matrices display direct, reversible and surface-controlled redox reaction with an enhanced electron transfer rate constant of 4.095 s⁻¹ in 0.1 M phosphate buffer solution (PBS) (pH 7.12). Furthermore, it was also discovered that, in the presence of O₂, GOD immobilized on Nafion and MC-FDU-15 matrices could produce a linear response to glucose. Thus, Nafion/GOD-MC-FDU-15/GC electrode is hopeful to be used in glucose biosensor. In addition, GOD immobilized on MC-FDU-15 and Nafion matrices possesses an excellent bioelectrocatalytic activity for the reduction of O₂. So, the Nafion/GOD-MC-FDU-15/GC electrode can be utilized as the cathode in biofuel cell.     

Electrochemical quartz crystal impedance study on immobilization of glucose oxidase in a polymer grown from dopamine oxidation at an Au electrode for glucose sensing

Glucose oxidase (GOD) was codeposited into a polymer grown from oxidation of dopamine (DA) at an Au electrode in a neutral phosphate aqueous solution for the first time. The electrochemical quartz crystal impedance analysis (EQCIA) method was used to monitor the GOD-immobilization process. Effects of concentrations of phosphate buffer, DA and GOD were investigated, and the optimal concentrations were found to be 20.0 mM phosphate buffer (pH 7.0), 30.0 mM DA and 5.00 mg ml⁻¹ GOD. A glucose biosensor was thus constructed, and effects of various experimental parameters on the sensor performance, including applied potential, solution pH and electroactive interferents, were examined. At an optimal potential of 0.6 V versus the KCl-saturated calomel electrode (SCE), the current response of the biosensor in the selected phosphate buffer (pH 7.0) was linear with the concentration of glucose from 0.05 to 9 mM, with a lower detection limit of 3 μM (S/N = 3), short response time (within 15 s) and good anti-interferent ability. The Michaelis constant () was estimated to be 9.6 mM. The biosensor exhibited good storage stability, i.e. 96% of its initial response was retained after 7-day storage in the selected phosphate buffer at 4 °C, and even after another 3 weeks the biosensor retained 86% of its initial response. In addition, the enzymatic specific activity and enzymatic relative activity of the GOD immobilized in the polymer from dopamine oxidation (PFDO) were estimated from the EQCIA method to be 1.43 kU g⁻¹ and 3.7%, respectively, which were larger than the relevant values obtained experimentally using poly(o-aminophenol) and poly(N-methylpyrrole) matrices, suggesting that the PFDO is a better matrix to immobilize GOD.     

An electrochemical biosensor based on Nafion-ionic liquid and a myoglobin-modified carbon paste electrode

An electrochemical biosensor was constructed based on the immobilization of myoglobin (Mb) in a composite film of Nafion and hydrophobic ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate (BMIMPF₆) for a modified carbon paste electrode (CPE). Direct electrochemistry of Mb in the Nafion-BMIMPF₆/CPE was achieved, confirmed by the appearance of a pair of well-defined redox peaks. The results indicate that Nafion-BMIMPF₆ composite film provided a suitable microenvironment to realize direct electron transfer between Mb and the electrode. The cathodic and anodic peak potentials were located at −0.351 V and −0.263 V (vs. SCE), with the apparent formal potential (Ep) of −0.307 V, which was characteristic of Mb Fe(III)/Fe(II) redox couples. The electrochemical behavior of Mb in the composite film was a surface-controlled quasi-reversible electrode process with one electron transfer and one proton transportation when the scan rate was smaller than 200 mV/s. Mb-modified electrode showed excellent electrocatalytic activity towards the reduction of trichloroacetic acid (TCA) in a linear concentration range from 2.0 × 10⁻⁴ mol/L to 1.1 × 10⁻² mol/L and with a detection limit of 1.6 × 10⁻⁵ mol/L (3σ). The proposed method would be valuable for the construction of a third-generation biosensor with cheap reagents and a simple procedure.     

Influence of ionic liquids on the direct electrochemistry of glucose oxidase entrapped in nanogold-N,N-dimethylformamide-ionic liquid composite film

Glucose oxidase (GOD) immobilized in nanogold particles (NAs)-N,N-dimethylformamide (DMF) composite film on glassy carbon (GC) electrode exhibits a pair of quasi-reversible and unstable peaks due to the redox of flavin adenine dinucleotide (FAD) of GOD. When ionic liquids (ILs) 1-butyl-3-methylimidazolium tetrafluoroborate (BMIMBF₄) or trihexyltetradecylphosphorium bis (trifluoromethylsulfony) (P_666,14 NTf₂) is introduced in the film, the peaks become small. But ILs 1-butyl-3-methylimidazolium hexafluorophosphate (BMIMPF₆) and 1-octyl-3-methylimidazolium hexafluorophate (OMIMPF₆) make the peaks large and stable. In different composite films the formal potential (E⁰′) of GOD is different. UV-vis spectra show that the GOD dispersed in these films almost retains its native structure and there are weak interactions between ILs and GOD. Electrochemical impedance spectra display that NAs can promote the electron transfer between FAD and GC electrode; and ILs can affect the electron transfer through interacting with GOD. The thermal stability of GOD entrapped in NAs-DMF-ILs composite films is also influenced by ILs, and it follows such order as: in NAs-DMF-OMIMPF₆ > in NAs-DMF-BMIMPF₆ ≈ in NAs-DMF-BMIMBF₄ > in NAs-DMF. In addition, GOD immobilized in NAs-DMF-OMIMPF₆ and NAs-DMF-BMIMPF₆ films shows good catalytic activity to the oxidation of glucose. The I_max of H₂O₂ and the apparent K_m (Michaelis-Menten constant) for the enzymatic reaction are calculated.     

Electroreduction of oxygen on gold-supported nanostructured palladium films in acid solutions

The electrochemical reduction of oxygen on thin Pd films with a nominal thickness of 0.25-10 nm on polycrystalline Au substrate (Pd/Au) was studied. The Pd films were prepared by electron beam evaporation and oxygen reduction was studied in 0.1 M HClO₄ and 0.05 M H₂SO₄ solutions using the rotating disk electrode (RDE) method. The surface morphology of Pd overlayers was examined by scanning tunnelling microscopy (STM). O₂ reduction predominantly proceeds through 4e⁻ pathway on all Pd/Au electrodes. The specific activity (SA) of oxygen reduction was lower in H₂SO₄ solution and decreased slightly with decreasing the Pd film thickness. In HClO₄, the SA was higher and not significantly dependent on the film thickness. The Tafel slope values close to −60 mV at low current densities and −120 mV at high current densities were found for all electrodes.     

Direct electrochemistry and electrocatalysis of cytochrome c based on chitosan-room temperature ionic liquid-carbon nanotubes composite

A robust and effective composite film combined the benefits of room temperature ionic liquid (RTIL), chitosan (Chi) and multi-wall carbon nanotubes (MWNTs) was prepared. Cytochrome c (Cyt c) was successfully immobilized on glassy carbon electrode (GCE) surface by entrapping in the composite film. Direct electrochemistry and electrocatalysis of immobilized Cyt c were investigated in detail. A pair of well-defined and quasi-reversible redox peaks of Cyt c was obtained in 0.1 mol L⁻¹ pH 7.0 phosphate buffer solution (PBS), indicating the Chi-RTIL-MWNTs film showed an obvious promotion for the direct electron transfer between Cyt c and the underlying electrode. The immobilized Cyt c exhibited an excellent electrocatalytic activity towards the reduction of H₂O₂. The catalysis current was linear to H₂O₂ concentration in the range of 2.0 × 10⁻⁶ to 2.6 × 10⁻⁴ mol L⁻¹, with a detection limit of 8.0 × 10⁻⁷ mol L⁻¹ (S/N = 3). The apparent Michaelis-Menten constant (K_m) was calculated to be 0.45 ± 0.02 mmol L⁻¹. Moreover, the modified electrode displayed a rapid response (5 s) to H₂O₂, and possessed good stability and reproducibility. Based on the composite film, a third-generation reagentless biosensor could be constructed for the determination of H₂O₂.