Gold Nanoparticles Doped Three‐Dimensional Sol‐gel Matrix for Amperometric Human Chorionic Gonadotrophin Immunosensor

A novel electrochemical immunosensor for human chorionic gonadotrophin (hCG) was proposed by immobilization of hCG in gold nanoparticles doped three‐dimensional (3D) sol‐gel matrix and an interfacial competitive immunoreaction. The 3D organized composite structure was prepared by assemble of gold nanoparticles into a hydrolyzed (3‐mercaptopropyl)‐trimethoxysilane sol‐gel matrix, which showed good biocompatibility. After the interfacial competitive immunoreaction the formed HRP‐labeled immunoconjugate showed good enzymatic activity for the oxidation of o‐phenylenediamine by H₂O₂. With a competitive format, a method comprising of o‐phenylenediamine‐H₂O₂‐immobilized HRP labeled hCG immunoconjugate system for immunoassay of hCG from 5.0 to 30.0 mIU mL^?1 was developed. The immunosensor showed good precision, high sensitivity, acceptable stability and reproducibility and could be used for detection of hCG in human serum with the consistent results in comparison with those obtained by a commercial analyzer.     

Electrochemical Immunoassay of Human Chorionic Gonadotrophin Based on Its Immobilization in Gold Nanoparticles‐Chitosan Membrane

A human chorionic gonadotrophin (hCG) doped gold nanoparticles–chitosan membrane was prepared for forming an immunoconjugate of horseradish peroxidase labeled hCG antibody and hCG on glassy carbon electrode. The nanoparticles provided a congenial environment of the adsorbed proteins. Thus, the immobilized HRP‐labeled immunoconjugate showed good enzymatic activity for the oxidation of o‐phenylenediamine by H₂O₂. With a competitive mechanism, an amperometric method for immunoassay of hCG up to 30 mIU mL^?1 with a relatively low detection limit of 0.26 mIU mL^?1 at 3σ was developed. The hCG immunosensor showed good precision, high sensitivity, acceptable stability and reproducibility.     

基于酶标抗体和媒介体共吸附于金胶壳聚糖膜的PSA免疫传感器的制备

本文研制了一种用金胶壳聚糖仿生膜来同时固定四甲基联苯胺（TMB）和酶标抗体的新型电化学免疫传感器,用于检测血清肿瘤标志物前列腺特异性抗原（PSA）的含量。固定的TMB作为电子传递媒介体,在扫速小于45 mV/s时,电极表现为一个表面控制过程,而在扫速大于45 mV/s时则表现为一个扩散控制过程。将固定有酶标抗体和TMB的免疫传感器与待测PSA抗原一起培育,在该传感器上形成的免疫复合物通过TMB-H₂O₂-HRP电化学体系进行了测定。在优化实验条件下,PSA的线性检测范围为5-30 ng·mL^-1,检测限为1.0 ng·mL^-1。该PSA免疫传感器制备方法简单,成本低廉,具有较好的稳定性和重现性。     

A Prostate Specific Antigen Immunosensor Based on Biotinylated‐Antibody/Cyclodextrin Inclusion Complex: Fabrication and Electrochemical Studies

Immobilization of biomolecules with a proper orientation is considered as a basis for diverse biotechnological applications. Herein, we report a host‐guest inclusion complexation between β‐cyclodextrin (β‐CD) and biotin as a versatile approach for the immobilization of biomolecules. As a practical application, a sandwich‐type electrochemical immunosensor was designed for the determination of prostate specific antigen (PSA). The immunosensor was fabricated by in situ electropolymerization of poly(N‐acetylaniline) onto a rGO‐modified Pt electrode. Then, β‐CD was covalently grafted onto the over‐oxidized polymer backbone. For improving the efficiency of the assay, AuNPs were casted on the polymeric film, on the surface of which thionine (TH) as an electron mediator was covalently immobilized. Using a host‐guest inclusion complexation between β‐CD and biotin, a β‐CD/biotin‐Ab₁/PSA/Ab₂‐horseradish peroxidase (HRP) sandwich was formed on the electrode surface. The analytical signal was produced via electrochemical reduction of TH_ox, generated by biocatalytic oxidation of the TH_red in the presence of HRP/H₂O₂. Under optimal conditions, the proposed sensor responded linearly to PSA in the range from 10.0 pg mL⁻¹ to 25.0 ng mL⁻¹, with a low detection limit of 6.7 pg mL⁻¹ (S/N=3). Kinetic parameters of the interaction of β‐CD with Ab₁ were also investigated. Finally, the applicability of the immunosensor was successfully investigated for the detection of PSA in human serum samples.     

Immunosensor for the determination of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> using a tyrosinase–mercaptopropionic acid modified electrode as an amperometric transducer

An amperometric immunosensor for the quantification of Staphylococcus aureus based on the coimmobilization of rabbit immunoglobulin G (RbIgG) and tyrosinase on a mercaptopropionic acid self-assembled monolayer modified gold electrode is reported. A competitive mode in which protein-A-bearing S. aureus cells and antiRbIgG labeled with alkaline phosphatase (AP) compete for the binding sites of immobilized RbIgG was used. Monitoring of the affinity reaction was carried out by the amperometric detection at -0.15 V of phenol generated in the enzyme reaction with AP, at the tyrosinase-modified electrode through the electrochemical reduction of the o-quinone formed. Optimization of the working variables, such as the immunosensor composition and incubation times, the applied potential, the working pH and the concentration of phenyl phosphate used as the AP substrate, was carried out. Under the optimized conditions, both the repeatability of the measurements and the reproducibility of the responses obtained with different immunosensors yielded relative standard deviation values for the steady-state current lower than 10%. The immunosensor showed a dynamic range from 4.4x10(5) to 1.8x10(7) S. aureus cells mL(-1), with a detection limit of 1.7x10(5) cells mL(-1). The limit of detection was remarkably improved by subjecting S. aureus cells to wall lysis by heat treatment. The value obtained was 2.3x10(3) cells mL(-1), which is adequate for the monitoring of S. aureus contamination levels in some foodstuffs. As an application, milk samples spiked with bacteria at the 4.8x10(3) cells mL(-1) level were analyzed.     

Electron‐Transfer Mediator Microbiosensor Fabrication Based on Immobilizing HRP‐Labeled Au Colloids on Gold Electrode Surface by 11‐Mercaptoundecanoic Acid Monolayer

In this paper, a new strategy for constructing a mediator‐type amperometric hydrogen peroxide (H₂O₂) microbiosensor was described. An electropolymerized thionine film (PTH) was deposited directly onto a gold electrode surface. The resulting redox film was extremely thin, adhered well onto a substrate (electrode), and had a highly cross‐linked network structure. Consequently, horseradish peroxidase (HRP) was successfully immobilized on nanometer‐sized Au colloids, which were supported by thiol‐tailed groups of 11‐mercaptoundecanoic acid (11‐MUA) monolayer covalently bound onto PTH film. With the aid of the PTH mediator, HRP‐labeled Au colloids microbiosensor displayed excellent electrocatalytical response to the reduction of H₂O₂. This matrix showed a biocompatible microenvironment for retaining the native activity of the covalent HRP and a very low mass transport barrier to the substrate, which provided a fast amperometric response to H₂O₂. The proposed H₂O₂ microbiosensor exhibited linear range of 3.5 μM–0.7 mM with a detection limit of 0.05 μM (S/N=3). The response showed a Michaelis‐Menten behavior at larger H₂O₂ concentrations. The K_M^app value for the biosensors based on 24 nm Au colloids was found to be 47 μM, which demonstrated that HRP immobilized on Au colloids exhibited a high affinity to H₂O₂ with no loss of enzymatic activity. This microbiosensor possessed good analytical performance and storage stability.     

Novel Amperometric Hydrogen Peroxide Biosensor Based on Horseradish Peroxidase Azide Covalently Immobilized on Ethynyl‐Modified Screen‐Printed Carbon Electrode via Click Chemistry

A novel, simple and versatile protocol for covalent immobilization of horseradish peroxidase (HRP) on screen‐printed carbon electrode (SPCE) based on the combination of diazonium salt electrografting and click chemistry has been successfully developed. The ethynyl‐terminated monolayers are obtained by diazonium salt electrografting, then, in the presence of copper (I) catalyst, the ethynyl modified surfaces reacted efficiently and rapidly with horseradish peroxidase bearing an azide function (azido‐HRP), thus forming a covalent 1,2,3‐triazole linkage by means of click chemistry. All the experimental results suggested that HRP was immobilized onto the electrode surface successfully without denaturation. Furthermore, the immobilized HRP showed a fast electrocatalytic reduction for H₂O₂. A linear range from 5.0 to 50.0 µM in a phosphate buffer (pH 5.5) with detection limit of 0.50 µM and sensitivity of 0.23 nA/µM were obtained. The heterogeneous electron transfer rate constant K_ct was 1.52±0.22 s^?1 and the apparent Michaelis? Menten constant was calculated to be 0.028 mM. The HRP‐functionalized electrode demonstrated a good reproducibility and long‐term stability.     

Interdigitated Conductometric Immunosensor for Determination of Interleukin‐6 in Humans Based on Dendrimer G4 and Colloidal Gold Modified Composite Film

A highly sensitive, fast and stable conductometric immunosensor for determination of interleukin‐6 (IL6) in humans is developed by encapsulation of horseradish peroxidase‐labeled interleukin‐6 antibody (HRP‐anti‐IL6) in poly(amidoamine) fourth‐generation dendrimer (dendrimer) and colloidal gold (nanogold) modified composite architecture. The presences of nanogold and dendrimer provided a congenial microenvironment for the immobilized biomolecules and decreased the electron transfer impedance, leading to a direct electrochemical behavior of the immobilized HRP. The formation of the antibody‐antigen complex by a simple one‐step immunoreaction between the immobilized HRP‐anti‐IL6 and IL6 in sample solution introduced a barrier of direct electrical communication between the immobilized HRP and the gold electrode surface, thus local conductivity variations could be detected by the HRP electrocatalytic reaction in 0.02 M phosphate buffer solution (pH 7.0) containing 50 μM H₂O₂, 0.01 M KI and 0.15 M NaC1. Under optimal conditions, the proposed immunosensor exhibited a good conductometric response to IL6 in a linear range from 30 to 300 pg/mL with a relatively low detection limit of 10 pg/mL at 3δ. The precision and reproducibility are acceptable with the intra‐assay CV of 7.3% and 5.6% at 100 and 200 pg/mL IL6, respectively. The storage stability of the proposed immunosensor is acceptable in a pH 7.0 PBS at 4 °C for 8 days. Importantly, the proposed methodology could be extended to the detection of other antigens or biocompounds.     

A membrane-based immunosensor for the analysis of the herbicide isoproturon

A membrane based heterogeneous competitive enzyme-linked immunosorbent assay (ELISA) was used in this work to develop an immunosensor for the detection of a common herbicide, isoproturon. A screen-printed carbon working electrode with carbon counter and silver–silver chloride pseudo-reference electrode was utilized incorporating a membrane fixed into intimate contact with the working electrode to facilitate signal transduction. The membrane containing an immobilized isoproturon–ovalbumin conjugate was laminated onto the carbon working electrode and horseradish peroxidase (HRP) labeled polyclonal antibody was then applied for the competitive assay. Two different amperometric systems, hydroquinone and o-phenylenediamine (OPD) mediation reduction were utilised and the properties of the resultant sensors were compared. A flow injection apparatus was also developed utilising the immunosensor. Limits of detection for isoproturon (LLD₉₀) were found to be as low as 0.84 ng mL⁻¹. The senor was also validated using spiked extracted soil samples and also isoproturon contaminated samples.     

Biomolecule‐Doped Organic/Inorganic Hybrid Nanocomposite Film for Label‐Free Electrochemical Immunoassay of α‐1‐Fetoprotein

A new electrochemical immunosensor for the detection of α‐1‐fetoprotien (AFP) was developed based on AFP antibody (anti‐AFP)‐functionalized organic/inorganic hybrid nanocomposite membrane. To fabricate such a hybrid composite membrane, 3,4,9,10‐perylenetetracarboxylic acid‐bound thionine molecules (PTCTH) were initially doped into titania colloids (TiO₂), and then gold nanoparticles and anti‐AFP were immobilized onto the composite film in turn. Comparison with the electrode fabricated only with thionine not 3,4,9,10‐perylenetetracarboxylic acid, the immunosensor with PTCTH exhibited high sensitivity and fast electron transfer. The presence of gold nanoparticles provided a good microenvironment for the immobilization of biomolecules, enhanced the surface coverage of protein, and improved the sensitivity of the immunosensor. The modified process was characterized by scanning electron microscope (SEM), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The surface topography of the membrane was investigated by scanning electron microscopy (SEM). Under optimal conditions, the proposed immunosensor exhibited a wide linear range from 2.5 to 200.0 ng/mL towards AFP with a detection limit of 0.5 ng/mL (S/N=3). The stability, reproducibility and precision of the immunosensor were acceptable. Comparison with the conventional enzyme‐linked immunosorbent assay (ELISA), the present method did not require more labeled procedures and washing steps. Significantly, the detection methodology provides a promising approach for other proteins or biosecurities.     

Electrochemical immunosensor designs for the determination of Staphylococcus aureus using 3,3-dithiodipropionic acid di(N-succinimidyl ester)-modified gold electrodes

The preparation of novel Staphylococcus aureus (S. aureus) amperometric immunosensing designs based on the covalent immobilization of RbIgG at gold electrodes using the heterobifunctional cross-linker 3,3-dithiodipropionic acid di(N-succinimidyl ester) (DTSP), are reported. Two different competitive immunosensing configurations have been tested and compared. In the first one, protein A-bearing S. aureus cells and HRP-labelled antiRbIgG compete for immobilized RbIgG binding sites, while in the second case HRP-labelled protein A was used. In both cases, the evaluation of the developed immunosensors performance was accomplished through the monitoring at 0.00 V (vs. Ag/AgCl) of the catalytic current originated after addition of hydrogen peroxide, using tetrathiafulvalene as redox mediator entrapped at the modified electrode surface by cross-linking with glutaraldehyde. Optimization of variables concerning the composition of the immunosensors as well as the detection conditions was carried out in 0.1 M NaAc/0.1 M NaCl buffer of pH 5.6. The configuration that employed antiRbIgG-HRP resulted in better analytical characteristics, with a detection limit of 1.4 × 10⁴ cells mL⁻¹ for S. aureus cells submitted to wall lyses by ultrasonic treatment. This immunosensor design was also evaluated using gold screen-printed electrodes in order to reduce the analysis time and cost. In this case, a limit of detection of 3.7 × 10² cells mL⁻¹ and a dynamic range from 1.3 × 10³ to 7.6 × 10⁴ cells mL⁻¹ was obtained. A RSD value of 10.5% was found for the responses to 9.6 × 10³S. aureus cells mL⁻¹ obtained with seven different Au/SPEs-immunosensors. These disposable immunosensors were applied to the quantification of S. aureus in milk spiked at two concentration levels, 1.2 × 10³ and 4.8 × 10³ cells mL⁻¹, with good recoveries.     

One‐Step Electrochemical Immunoassay for Carcinoembryonic Antigen in Human via Back‐Filling Immobilization of Gold Nanoparticles on DNA‐Modified Gold Electrodes

A new amperometric immunobiosensor for carcinoembryonic antigen (CEA) determination in human serum was developed via encapsulation of horseradish peroxidase‐labeled carcinoembryonic antibody (HRP‐anti‐CEA) in a gold nanoparticles/DNA composite architecture. The presences of gold nanoparticles provided a congenial microenvironment for the immobilized biomolecules and decreased the electron transfer impedance, leading to a direct electrochemical behavior of the immobilized HRP. The formation of the antibody–antigen complex by a simple one‐step immunoreaction between the immobilized HRP‐anti‐CEA and CEA in sample solution introduced a barrier of direct electrical communication between the immobilized HRP and the gold electrode surface. Under optimal conditions, the current change obtained from the labeled HRP relative to H₂O₂ system was proportional to the CEA concentration in two linear ranges from 0.5 to 15 ng/mL and 15 to 300 ng/mL with a detection limit of 0.1 ng/mL (at 3δ). The precision and reproducibility are acceptable with the intraassay CV of 6.3% and 4.7% at 8 and 60 ng/mL CEA, respectively. The storage stability of the proposed immunosensor is acceptable in a pH 7.0 PBS at 4 °C for 9 days. Moreover, the proposed immunosensors were used to analyze CEA in human serum specimens. Analytical results of clinical samples show the developed immunoassay has a promising alternative approach for detecting CEA in the clinical diagnosis.     

Amplified Electrochemical Immunosensor for Calmodulin Detection Based on Gold‐Silver‐Graphene Hybrid Nanomaterials and Enhanced Gold Nanorods Labels

Calmodulin (CaM) is an important intracellular calcium‐binding protein. It plays a critical role in a variety of biological and biochemical processes. In this paper, a new electrochemical immunosensing protocol for sensitive detection of CaM was developed by using gold‐silver‐graphene (AuAgGP) hybrid nanomaterials as protein immobilization matrices and gold nanorods (GNRs) as enhanced electrochemical labels. Electrode was first modified with thionine‐chitosan film to provide an immobilization support for gold‐silver‐graphene hybrid nanomaterials. The hybrid materials formed an effective matrix for binding of CaM with high density and improved the electrochemical responses as well. Gold nanorods were prepared for the fabrication of enhanced labels (HRP‐Ab₂‐GNRs), which provided a large capacity for HRP‐Ab₂ immobilization and a facile pathway for electron transfer. With two‐step immunoassay format, the HRP‐Ab₂‐GNRs labels were introduced onto the electrode surface, and produced electrochemical responses by catalytic reaction of HRP toward enzyme substrate of hydrogen peroxide (H₂O₂) in the presence of thionine. The proposed immunosensor showed an excellent analytical performance for the detection of CaM ranging from 50 pg mL^?1 to 200 ng mL^?1 with a detection limit of 18 pg mL^?1. The immunosensor has also been successfully applied to the CaM analysis in two cancer cells (HepG2 and MCF‐7) with high sensitivity, which has shown great potency for improving clinic diagnosis and treatment for cancer study.     

Immunosensor for Detection of the Textile Dye Disperse Orange 1 Based on Non‐conventional Competitive Assay

Textile dyes appear as an important class of compounds that has become a matter of public concern and a serious challenge for scientists and environmentalists due to their large‐scale production and extensive application. In this work, a non‐conventional competitive‐type amperometric immunosensor was successfully developed for detection of the textile dye Disperse Orange 1 (DO1). The DO1 was magnetically captured and separated from the sample solution using magnetic particles (MP) functionalized with the antibody anti‐DO1 and with HRP and gold electrodes were modified with the conjugate DO1‐BSA. Molecules of DO1 immobilized on the electrode surface and DO1 captured by MP compete for antibody binding sites. As a result, the amperometric signal decreases with increasing target DO1 concentration at the capture step, because this decreases, the attachment between the HRP coated MP and the electrode. This strategy allowed us to determine DO1 at the low detection limit of 0.87 ng mL^?1 with great specificity. Also, there were good recoveries for detection of the textile dye in river water samples without the need of sample pre‐treatment. The competitive amperometric immunosensor shows applicability for the determination of small molecules that cannot be determined by conventional competitive or sandwich immunosensors.     

Sensitive and rapid amperometric magnetoimmunosensor for the determination of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

The preparation and characteristics of a disposable amperometric magnetoimmunosensor, based on the use of functionalized magnetic beads (MBs) and gold screen-printed electrodes (Au/SPEs), for the specific detection and quantification of Staphylococcal protein A (ProtA) and Staphylococcus aureus (S. aureus) is reported. An antiProtA antibody was immobilized onto ProtA-modified MBs, and a competitive immunoassay involving ProtA antigen labelled with HRP was performed. The resulting modified MBs were captured by a magnetic field on the surface of tetrathiafulvalene-modified Au/SPEs and the amperometric response obtained at −0.15 V vs the silver pseudo-reference electrode of the Au/SPEs after the addition of H₂O₂ was used as transduction signal. The developed methodology showed very low limits of detection (1 cfu S. aureus/mL of raw milk samples), and a good selectivity against the most commonly involved foodborne pathogens originating from milk. These features, together with a short analysis time (2 h), the simplicity, and easy automation and miniaturization of the required instrumentation make the developed methodology a promising alternative in the development of devices for on-site analysis.     

A Multi‐electrochemical Competitive Immunosensor for Sensitive Cocaine Determination in Biological Samples

A multi‐electrochemical competitive immunosensor for the rapid determination of unmetabolized cocaine (COC) in urine, saliva and human serum matrices is reported. Anti‐cocaine polyclonal antibodies were immobilized in an oriented way onto protein‐G functionalized magnetic beads. The immunosensor is based on an array of eight carbon‐based screen‐printed electrodes for simultaneous electrochemical determinations. The treatments of the biological samples were simplified and optimized for avoiding matrix interferences. The immunosensor was sensitive (EC₅₀≈2.92–3.88 ng mL^?1 COC), required a very small volume of sample (200 µL), was reproducible (%RSD was lesser than about 18 %), and accurate (recovery percentages ranged 88–117 %).     

Electrochemical Immunosensor for Ultrasensitive Detection of Human Chorionic Gonadotropin Based on Pd@SBA‐15

A simple and sensitive electrochemical immunosensor was conducted for the determination of human chorionic gonadotropin (hCG) with Pd@SBA‐15. Thionine (TH) was selected as an electron transfer mediator, and modified onto the electrode together with functionalized graphene nanomaterial (HSO₃^?GS) through electrostatic adsorption. Then Pd@SBA‐15 was immobilized onto the as‐prepared film for biomolecules anchoring. Pd@SBA‐15 composites not only retain the good biocompatibility of the SBA‐15, but also exhibit an excellent catalytic activity of Pd and low Pd leaching. hCG antibody was immobilized onto the composite film for the detection of hCG. hCG can be determined in the range of 0.01–16.00 ng/mL and the detection limit is 8.60 pg/mL. The method has been applied to the analysis of hCG in human serum samples with satisfactory results.     

Fabrication of Immunosensor Based on Polyaniline,Fullerene‐C60 and Palladium Nanoparticles Nanocomposite: An Electrochemical Detection Tool for Prostate Cancer

Present work demonstrates the fabrication of new and facile sandwich‐type electrochemical immunosensor based on palladium nanoparticles (PdNPs), polyaniline (PANI) and fullerene‐C₆₀ nanocomposite film modified glassy carbon electrode (PdNP@PANI‐C₆₀/GCE) for ultrasensitive detection of Prostate‐specific antigen (PSA) biomarker. PdNP@PANI‐C₆₀ was electrochemically synthesized on GCE and used as an electroactive substrate. PdNP@PANI‐C₆₀ was characterized by scanning electron microscopy (SEM), energy‐dispersive X‐ray spectroscopy (EDS), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Primary antibody anti‐PSA (Ab1) was covalently immobilized on PdNP@PANI‐C₆₀/GCE using NHS/EDC linkers. In the presence of PSA antigen, horseradish peroxidase secondary antibody (HRP‐Ab2) was brought into the surface of the electrode, developing stable amplified signals of H₂O₂ reduction. Under the optimal conditions, a linear curve for determination of PSA at the proposed immunosensor was 1.6×10^?4 ng.mL^?1 to 38 ng.mL^?1 with a limit of detection (LOD) of 1.95×10^?5 ng.mL^?1. The proposed immunosensor was successfully validated in serum and urine samples towards PSA detection with satisfactory and acceptable results.     

基于多层酶/纳米金固定甲胎蛋白免疫传感器的研究

>利用自组装技术和静电吸附作用, 将甲胎蛋白抗体(anti-AFP)固定在多层辣根过氧化物酶/纳米金及L-半胱胺酸修饰的金电极表面, 制备出用于检测甲胎蛋白抗原(AFP)的无试剂型免疫传感器. 通过交流阻抗技术、循环伏安法和计时电流法考察了电极的电化学特性, 并对该免疫传感器的作用机理及性能进行了详细的研究. 用计时电流法测得AFP的线性范围为1.0～10.0和10～200 ng•mL^－1, 检出限为0.5 ng•mL^－1. 实验结果表明, 该方法提高了抗体的固定量, 增强了传感器的灵敏度和稳定性, 且该传感器响应迅速、选择性好, 血清中常见抗原不干扰测定. 将其用于临床血清检验, 与放射免疫测定法(RIA)的符合率为86.7%.     

Immobilization of Enzymes on the Nano‐Au Film Modified Glassy Carbon Electrode for the Determination of Hydrogen Peroxide and Glucose

A new enzyme‐based amperometric biosensor for hydrogen peroxide was developed relying on the efficient immobilization of horseradish peroxidase (HRP) to a nano‐scaled particulate gold (nano‐Au) film modified glassy carbon electrode (GC). The nano‐Au film was obtained by a chitosan film which was first formed on the surface of GC. The high affinity of chitosan for nano‐Au associated with its amino groups resulted in the formation of nano‐Au film on the surface of GC. The film formed served as an intermediator to retain high efficient and stable immobilization of the enzyme. H₂O₂ was detected using hydroquinone as an electron mediator to transfer electrons between the electrode and HRP. The HRP immobilized on nano‐Au film maintained excellent electrocatalytical activity to the reduction of H₂O₂. The experimental parameters such as the operating potential of the working electrode, mediator concentration and pH of background electrolyte were optimized for best analytical performance of amperometry. The linear range of detection for H₂O₂ is from 6.1×10^?6 to 1.8×10^?3 mol L^?1 with a detection limit of 6.1 μmol L^?1 based on signal/noise=3. The proposed HRP enzyme sensor has the features of high sensitivity (0.25 Almol^?1cm^?2), fast response time (t_90%≤10 s) and a long‐term stability (>1 month). As an extension, glucose oxidase (GOD) was chemically bound to HRP‐modified electrode. A GOD/HRP bienzyme‐modified electrode formed in this way can be applied to the determination of glucose with satisfactory performance.