Enhancing a Novel Robust Multicomposite Materials Platform for Glucose Biosensors

An effective, stable enzymatic glucose biosensor was fabricated on a glassy carbon electrode (GCE) surface using simple multicomposite materials (MCM): a solution of prepared poly(diallyldimethylammonium chloride)‐capped gold nanoparticles‐nickel ferrite particles‐carbon nanotubes‐chitosan (PDDA‐AuNPs‐NiFe₂O₄‐CNTs‐CHIT), electropolymerization of poly(o‐phenylenediamine) (PoPD) and immobilization of glucose oxidase (GOx). Biocompatibility and synergy of the MCM enhanced the immobilization and the reaction of GOx and as well as the electron transfer from an oxidation reaction of hydrogen peroxide in the system. The NiFe₂O₄ was synthesized by co‐precipitation and calcined at 700 °C. Characterization was carried out by field emission scanning electron microscopy (FE‐SEM), energy‐dispersive X‐ray spectroscopy (EDX), Fourier transform infrared spectroscopy (FTIR) and X‐ray diffraction (XRD) which presented both tetrahedral and octahedral metal stretching with a cubic NiFe₂O₄ crystal phase. The GOx/PoPD/MCM/GCE yielded a 0.77 s^?1 charge transfer rate constant (K_s), a 2.28×10^?6 cm² s^?1 diffusion coefficient value (D), a 0.21 mm² electroactive surface area (A_e) and a 1.93×10^?8 mol cm^?2 surface concentration ( ) as determined by cyclic voltammetry. The modified electrode showed a durable operation time (n=97, more than 50 % I), repeatability (%RSD=0.38, n=10), reproducibility (%RSD=1.60, n=10), high sensitivity (853.07 μA mM^?1 cm^?2), selectivity without effects of electroactive species (aspirin, uric acid, caffeine, cholesterol, ascorbic acid and dopamine) and two linear ranges from 0.5 to 10 μM (R²=0.998) and 10 to 15,000 μM (R²=0.991) with a low detection limit (0.35 μM, S/N=3). Its Michaelis‐Menten constant (K_m) was calculated as 93.51 μM with 46.30 μA maximum current (I_max). This proposed simple method was successfully applied for glucose determination in human blood samples.     

Choline Oxidase Based Composite ZrO2@AuNPs with Cu2O@MnO2 Platform for Signal Enhancing the Choline Biosensors

A novel metal composite material based on zirconium dioxide decorated gold nanoparticles (ZrO₂@AuNPs), copper (I) oxide at manganese (IV) oxide (Cu₂O@MnO₂) and immobilized choline oxidase (ChOx) onto a glassy carbon electrode (GCE) (ChOx/Cu₂O@MnO₂-ZrO₂@AuNPs/GCE) has been developed for enhancing the electro-catalytic property, sensitivity and stability of the amperometric choline biosensor. The ChOx/Cu₂O@MnO₂-ZrO₂@AuNPs/GCE displayed an excellent electrocatalytic response to the oxidation of the byproduct H₂O₂ from the choline catalyzed reaction, which exhibited a charge transfer rate constant (K_s) of 0.97 s⁻¹, a diffusion coefficient value (D) of 4.50×10⁻⁶ cm² s⁻¹, an electroactive surface area (A_e) of 0.97 cm² and a surface concentration (γ) of 0.54×10⁻⁸ mol cm⁻². The modified electrode also provided a wide linear range of choline concentration from 0.5 to 1,000.0 μM with good sensitivity (97.4 μA cm⁻² mM⁻¹) and low detection limit (0.3 μM). The apparent Michaelis-Menten constant was found to be 0.08 mM with I_max of 0.67 μA. This choline biosensor presented high repeatability (%RSD=2.9, n=5), excellent reproducibility (%RSD=2.9, n=5), long time of use (n=28 with %I>50.0 %) and good selectivity without interfering effects from possible electroactive species such as ascorbic acid, aspirin, amoxicillin, caffeine, dopamine, glucose, sucrose and uric acid. This optimal method was successfully applied for choline measurement in prepared human blood samples which demonstrated accurate and excellent reliability in the recovery range from 96.7 to 102.0 %.     

Enzyme‐free Cu2O@MnO2/GCE for Hydrogen Peroxide Sensing

A novel composite material of copper (I) oxide at manganese (IV) oxide (Cu₂O@MnO₂), was synthesized and applied for modification on the glassy carbon electrode (GCE) surface (Cu₂O@MnO₂/GCE) as a hydrogen peroxide (H₂O₂) sensor. The composite material was characterized regarding its structural and morphological properties, using field emission scanning electron microscopy (FE‐SEM), energy‐dispersive X‐ray spectroscopy (EDX), X‐ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR). The Cu₂O@MnO₂/GCE showed an excellent electrocatalytic response to the oxidation of H₂O₂ which provided a 0.56 s^?1 charge transfer rate constant (K_s), 1.65×10^?5 cm² s^?1 diffusion coefficient value (D), 0.12 mm² electroactive surface area (A_e) and 1.04×10^?8 mol cm^?2 surface concentration ( ). At the optimal condition, the constructed sensor exhibited a wide linear range from 0.5 μM to 20 mM with a low limit of detection (63 nM, (S/N=3) and a good sensitivity of 256.33 μA mM^?1 cm^?2. It also presented high stability (ΔI_response±15 %, n=100), repeatability (1.25 %RSD, n=10) and reproducibility (3.55 %RSD, n=10). The results indicated that the synthesized Cu₂O@MnO₂ was successfully used as a new platform for H₂O₂ sensing.     

Carbon Nanotubes‐Based Amperometric Cholesterol Biosensor Fabricated Through Layer‐by‐Layer Technique

A carbon nanotubes‐based amperometric cholesterol biosensor has been fabricated through layer‐by‐layer (LBL) deposition of a cationic polyelectrolyte (PDDA, poly(diallyldimethylammonium chloride)) and cholesterol oxidase (ChOx) on multi‐walled carbon nanotubes (MWNTs)‐modified gold electrode, followed by electrochemical generation of a nonconducting poly(o‐phenylenediamine) (PPD) film as the protective coating. Electrochemical impedance measurements have shown that PDDA/ChOx multilayer film could be formed uniformly on MWNTs‐modified gold electrode. Due to the strong electrocatalytic properties of MWNTs toward H₂O₂ and the low permeability of PPD film for electroacitve species, such as ascorbic acid, uric acid and acetaminophen, the biosensor has shown high sensitivity and good anti‐interferent ability in the detection of cholesterol. The effect of the pH value of the detection solution on the response of the biosensor was also investigated. A linear range up to 6.0 mM has been observed for the biosensor with a detection limit of 0.2 mM. The apparent Michaelis‐Menten constant and the maximum response current density were calculated to be 7.17 mM and 7.32 μA cm^?2, respectively.     

Direct Electrochemistry of Cholesterol Oxidase Immobilized on a Conducting Polymer: Application for a Cholesterol Biosensor

Direct electrochemistry of cholesterol oxidase (ChOx) immobilized on the conductive poly‐3′,4′‐diamine‐2,2′,5′,2″‐terthiophene (PDATT) was achieved and used to create a cholesterol biosensor. A well‐defined redox peak was observed, corresponding to the direct electron transfer of the FAD/FADH₂ of ChOx, and the rate constant (k_s) was determined to be 0.75 s^?1. Glutathione (GSH) covalently bonded with PDATT was used as a matrix for conjugating AuNPs, ChOx, and MP, simultaneously. MP co‐immobilized with ChOx on the AuNPs‐GSH/PDATT exhibited an excellent amperometric response to cholesterol. The dynamic range was from 10 to 130 μM with a detection limit of 0.3±0.04 μM.     

Hemin Functionalized Multiwalled Carbon Nanotubes as a Matrix for Sensitive Electrogenerated Chemiluminescence Cholesterol Biosensor

Based on hemin‐MWCNTs nanocomposite and hemin‐catalyzed luminol‐H₂O₂ reaction, a sensitive electrogenerated chemiluminescence (ECL) cholesterol biosensor was proposed in this paper. Firstly, hemin‐MWCNTs was prepared via π–π stacking and modified on the surface of GCE. Subsequently, cholesterol oxidase (ChOx) was adsorbed on the modified electrode to achieve a cholesterol biosensor. Hemin‐MWCNTs nanocomposite provided the electrode with a large surface area to load ChOx, and endowed the nanostructured interface on the electrode surface to enhance the performance of biosensor. The biosensor responded to cholesterol in the linear range from 0.3 µM to 1.2 mM with a detection limit of 0.1 µM (S/N=3).     

A Novel Label-free Chronoamperometric Immunosensor Based on a Biocomposite Material for Rapid Detection of Carcinoembryonic Antigen

In this research, poly(diallyldimethylammonium chloride)-capped gold nanoparticles, nickel ferrite particles, and carbon nanotubes were combined to form a PANC metal composite. The prepared metal composite modified onto a glassy carbon electrode was electropolymerized with poly(o-phenylenediamine) and immobilized with horseradish peroxidase, anti-carcinoembryonic antigen antibody, and bovine serum albumin to create the label-free immunosensors for rapid detection of carcinoembryonic antigen (CEA) using chronoamperometry. This developed biocomposite material modified onto a glassy carbon electrode presented an excellent electrocatalytic response to the redox reaction of hydrogen peroxide as a sensing probe, from which the kinetic parameters including of a charge transfer rate constant, a diffusion coefficient value, an electroactive surface area, and a surface concentration were calculated to be 1.85 s⁻¹, 4.28×10⁻⁶ cm² s⁻¹, 0.14 cm² and 1.87×10⁻⁸ mol cm⁻², respectively. The developed immunosensors also exhibited a wide linear range of CEA concentration from 0.01 to 25 ng mL⁻¹ with high sensitivity (96.21 μA cm⁻² ng⁻¹ mL) and low detection limit (0.72 pg mL⁻¹), excellent selectivity without interfering effects from possible species (amoxicillin, ascorbic acid, aspirin, caffeine, cholesterol, dopamine, glucose, and uric acid), outstanding stability (n=100, %I>50 %), repeatability (%RSD=0.34, n=10), reproducibility (%RSD=4.06, n=10), and rapid analysis (25 s each operation time). This proposed method was successfully applied for CEA detection in whole blood samples with satisfactory results, suggesting that this developed sensing platform may be considered to be exploited for fabrication of other label-free electrochemical immunosensors for the real sample analysis.     

Amperometric Tyrosinase Biosensor Based on Carbon Black Paste Electrode for Sensitive Detection of Catechol in Environmental Samples

In this work, a renewable tyrosinase-based biosensor was developed for the detection of catechol, using a carbon black paste electrode, without any mediator. The effect of pH, type of electrolyte, and amount of tyrosinase enzyme were explored for optimum analytical performance. The best-performing biosensor in amperometric experiments at potential −0.2 V vs. Ag/AgCl (3 mol L⁻¹ KCl) was obtained using a 0.1 mol L⁻¹ phosphate buffer solution (pH 7.0) as electrolyte. Under optimized conditions, the proposed biosensor had two concentration linear ranges from 5.0×10⁻⁹ to 4.8×10⁻⁸ and from 4.8×10⁻⁸ to 8.5×10⁻⁶ mol L⁻¹ and a limit of detection of 1.5×10⁻⁹ mol L⁻¹. The apparent Michaelis-Menten constant ( ) was calculated by the amperometric method, and the obtained value was 1.2×10⁻⁵ mol L⁻¹ whose result was similar when compared with other studies previously. The biosensor was applied in river water samples, and the results were very satisfactory, with recoveries near 100 %. In addition, the response of this biosensor for different compounds, taking into account their molecular structures was investigated and the results obtained showed no interference with the response potential of catechol. The electrochemical biosensor developed in this work can be considered highly advantageous because it does not require the use of a mediator (direct detection) for electrochemical response, and also because it is based on a low-cost materials that can be used with success to immobilise other enzymes and/or biomolecules.     

Electrochemical Cholesterol Sensor Based on Tin Oxide‐Chitosan Nanobiocomposite Film

A chitosan (CS)‐tin oxide (SnO₂) nanobiocomposite film has been deposited onto an indium‐tin‐oxide glass plate to immobilize cholesterol oxidase (ChOx) for cholesterol detection. The value of the Michaelis–Menten constant (K_m) obtained as 3.8 mM for ChOx/CS‐SnO₂/ITO is lower (8 mM) than that of a ChOx/CS/ITO bioelectrode revealing enhancement in affinity and/or activity of ChOx towards cholesterol and also revealing strong binding of ChOx onto CS‐SnO₂/ITO electrode. This ChOx/CS‐SnO₂/ITO cholesterol sensor retains 95% of enzyme activity after 4–6 weeks at 4 °C with response time of 5 s, sensitivity of 34.7 μA/mg dL^?1 cm² and detection limit of 5 mg/dL.     

Sensitive Superoxide Biosensor Based on Silicon Carbide Nanoparticles

Direct electron transfer of immobilized superoxide dismutase (Cu, Zn‐SOD) onto silicon carbide (SiC) nanoparticles displays a pair of well defined and nearly reversible redox peaks with formal potential (E°′) of −0.03 V in pH 7.4. The heterogeneous electron transfer rate constant (k_s) and surface coverage (Γ) of immobilized SOD are 11.0±0.4 s⁻¹ and 1.42×10⁻¹¹ mol cm⁻². Biosensor shows fast amperometric response (3s) with sensitivity and detection limit of 1.416 nA μM⁻¹, 1.66 μM, and 1.375 nA μM⁻¹, 2.1 μM for cathodically or anodically detection of superoxide, respectively. This biosensor also exhibits good stability, reproducibility and long life‐time.     

Fabrication of amperometric cholesterol biosensor based on SnO<Subscript>2</Subscript> nanoparticles and Nafion-modified carbon paste electrode

This study reports the fabrication of an amperometric cholesterol biosensor based on cholesterol oxidase (ChOx), SnO₂NPs and Nafion-modified carbon paste enzyme electrodes (CPE/SnO₂NPs-ChOx/Naf). The electrochemical characterisations of BCPE and CPE/SnO₂NPs were performed using CV and EIS. The determination of cholesterol was carried out by electrochemical oxidation of H₂O₂ at 0.6 V vs. Ag/AgCl. The CPE/SnO₂NPs-ChOx/Naf presented a linear range from 0.20 μ.mol L^?1 to 4.95 μmol L^?1 with a low limit of detection (0.04 μ.mol L^?1). In addition, the optimal values for pH and temperature were found to be 7.5 and 35°C, respectively. The CPE/SnO₂NPs-ChOx/Naf was used for the determination of cholesterol in serum samples and good results were obtained.     

A Novel Hydrogen Peroxide Biosensor Based on the Synergistic Effect of Gold‐Platinum Alloy Nanoparticles/Polyaniline Nanotube/Chitosan Nanocomposite Membrane

Based on the immobilization of horseradish peroxidase (HRP) in chitosan(CS) on a glassy carbon electrode (GCE) modified with the Au‐Pt alloy nanoparticles (NPs) / polyaniline nanotube (nanoPAN) nanocomposite film, a novel hydrogen peroxide biosensor was constructed. The modified processes of GCE were monitored by cyclic voltammetry and electrochemical impedance spectroscopy. Au‐PtNPs/nanoPAN/CS had a better synergistic electrochemical effect than did AuNPs/nanoPAN/CS or PtNPs/nanoPAN/CS. The amperometric response of the biosensor towards H₂O₂ was investigated by successively adding aliquots of H₂O₂ to a continuous stirring phosphate buffer solution under the optimized conditions. Because Au‐PtNPs have unique catalytic properties and good biocompatibility, and especially Au‐PtNPs and nanoPAN have synergistic augmentation for facilitating electron‐transfer, the biosensor displayed a fast response time (<2 s) and broad linear response to H₂O₂ in the range from 1.0 to 2200 μmol L^?1 with a relatively low detection limit of 0.5 μmol L^?1 at 3 times the background noise. Moreover, the biosensor can be applied in practical analysis and exhibited high sensitivity, good reproducibility, and long‐term stability.     

General Preparation of Heme Protein Functional Fe3O4@Au‐Nps Magnetic Nanocomposite for Sensitive Detection of Hydrogen Peroxide

Stable magnetic nanocomposite of gold nanoparticles (Au‐NPs) decorating Fe₃O₄ core was successfully synthesized by the linker of Boc‐L‐cysteine. Transmission electron microscope (TEM), energy dispersive X‐ray spectroscopy (EDX) and cyclic voltammograms (CV) were performed to characterize the as‐prepared Fe₃O₄@Au‐Nps. The results indicated that Au‐Nps dispersed homogeneously around Fe₃O₄ with the ratio of Au to Fe₃O₄ nanoparticles as 5–10/1 and the apparent electrochemical area as 0.121 cm². After self‐assembly of hemoglobin (Hb) on Fe₃O₄@Au‐Nps by electrostatic interaction, a hydrogen peroxide biosensor was developed. The Fe₃O₄@Au‐Nps/Hb modified GCE exhibited fast direct electron transfer between heme center and electrode surface with the heterogeneous electron transfer rate constant (Ks ) of 3.35 s⁻¹. Importantly, it showed excellent electrocatalytic activity towards hydrogen peroxide reduction with low detection limit of 0.133 μM (S /D =3) and high sensitivity of 0.163 μA μM⁻¹, respectively. At the concentration evaluated, the interfering species of glucose, dopamine, uric acid and ascorbic acid did not affect the determination of hydrogen peroxide. These results demonstrated that the introduction of Au‐Nps on Fe₃O₄ not only stabilized the immobilized enzyme but also provided large surface area, fast electron transfer and excellent biocompatibility. This facile nanoassembly protocol can be extended to immobilize various enzymes, proteins and biomolecules to develop robust biosensors.     

A Novel Hydrogen Peroxide Biosensor Based on the PDDA‐GNPs/MWNTs Nanocomposite Matrix

A novel nanocomposite designed by the assembly of the positively charged poly(diallyldimethylammonium chloride) protected gold nanoparticles (PDDA‐GNPs), and the negatively charged multi‐walled carbon nanotubes (MWCNTs) on ITO electrode via electrostatic interaction, was used as a supporting matrix for immobilizing hemoglobin (Hb) to develop a high‐performance hydrogen peroxide (H₂O₂) biosensor. The cyclic voltammetrys of immobilized Hb showed a pair of well‐defined and quasi‐reversible redox peaks with the formal potential of ‐0.205V (vs. SCE) and the peak‐to‐peak potential separation of 44 mV at a scan rate of 100 mV×s^?1 in 0.1 mol×L^?1 pH 7.0 PBS. Under the optimized experimental conditions, a linearity range for determination of H₂O₂ was from 2.0 × 10^?6 to 5.2 × 10^?4 mol×L^?1 with a correlation coefficient of 0.9994 (n = 37) and a detection limit of 8.4 × 10^?7 mol×L^?1. The biosensor displayed excellent electrochemical and electrocatalytic response to the reduction of H₂O₂, high sensitivity, long‐term stability, good bioactivity and selectivity.     

Nanostructured Iron Oxide Platform for Impedimetric Cholesterol Detection

A nanostructured iron oxide (NanoFe₃O₄, particle size ca. 25 nm and roughness ca. 21 nm) film deposited onto a hydrolyzed indium‐tin‐oxide (ITO) coated glass plate has been used to immobilize cholesterol oxidase (ChOx) to fabricate an impedimetric cholesterol sensor. Electrochemical studies reveal that surface charged Fe₃O₄ nanoparticles provide better conformation for ChOx loading resulting in enhanced electron transfer between ChOx and the electrode. Impedimetric response studies of the ChOx/NanoFe₃O₄/ITO bioelectrode exhibit improved linearity (2.5–400 mg/dL), low detection limit (0.25 mg/dL), fast response time (25 s), high sensitivity (86 Ω/mg dL^?1/cm^?2) and a low value of the Michaelis‐Menten constant (K_m, 0.8 mg/dL) with a regression coefficient of 0.997.     

Amperometric Glucose Biosensor Based on Electrochemically Deposited Gold Nanoparticles Covered by Polypyrrole

Graphite rod (GR) modified with electrochemicaly deposited gold nanoparticles (AuNPs) and adsorbed glucose oxidase (GOx) was used in amperometric glucose biosensor design. Enzymatic formation of polypyrrole (Ppy) on the surface of GOx/AuNPs/GR electrode was applied in order to improve analytical characteristics and stability of developed biosensor. The linear glucose detection range for Ppy/GOx/AuNPs/GR electrode was dependent on the duration of Ppy‐layer formation and the linear interval was extended up to 19.9 mmol L⁻¹ after 21 h lasting synthesis of Ppy. The sensitivity of the developed biosensor was determined as 21.7 μA mM⁻¹ cm⁻², the limit of detection – 0.20 mmol L⁻¹. Ppy/GOx/AuNPs/GR electrodes demonstrated advanced good stability (the t _1/2 was 9.8 days), quick detection of glucose (within 5 s) in the wide linear interval. Additionally, formed Ppy layer decreased the influence of electroactive species on the analytical signal. Developed biosensor is suitable for the determination of glucose in human serum samples.     

Ferrite Nanocomposite Based Electrochemical Sensor: Characterization,Voltammetric and Amperometric Studies for Electrocatalytic Detection of Formaldehyde in Aqueous Media

Development of nanocomposite based electrochemical sensors for detection of toxic chemicals describes an environmentally benign strategy for monitoring the health of ecosystem. Herein, we reported in situ preparation of graphitic carbon nitride (g-C₃N₄) decorated Ag₂S/NiFe₂O₄ nanocomposite sensor by facile precipitation method. The electrochemical studies demonstrated efficient electrocatalytic activity of ternary nanocomposite pasted glassy carbon electrode (g-C₃N₄@Ag₂S/NiFe₂O₄/GCE) for selective detection of formaldehyde. Moreover, fabricated sensor exhibit rapid amperometric response with excellent selectivity, remarkable sensitivity (1681 μA mmol L⁻¹ cm⁻²) and lower detection limit (LOD: 1.63 μmol L⁻¹). It is noteworthy to mention that sensor exhibits good operational and long-term storage stability.     

Fabrication of a Sensitive Electrochemical Biosensor for Detection of DNA Hybridization Based on Gold Nanoparticles/CuO Nanospindles Modified Glassy Carbon Electrode

In this work, a sensitive electrochemical DNA biosensor for the detection of sequence‐specific target DNA was reported. Firstly, CuO nanospindles (CuO NS) were immobilized on the surface of a glassy carbon electrode (GCE). Subsequently, gold nanoparticles (Au NPs) were introduced to the surface of CuO NS by the electrochemical deposition mode. Probe DNA with SH (HS‐DNA) at the 5′‐phosphate end was covalently immobilized on the surface of the Au NPs through Au? S bond. Scanning electron microscopy (SEM) was used to elucidate the morphology of the assembled film, and electrochemical impedance spectroscopy technique (EIS) was used to investigate the DNA sensor assembly process. Hybridization detection of DNA was performed with differential pulse voltammetry (DPV) and the methylene blue (MB) was hybridization indicator. Under the optimal conditions, the decline of reduction peak current of MB (ΔI) was linear with the logarithm of the concentration of complementary DNA from 1.0×10^?13 to 1.0×10^?6 mol·L^?1 with a detection limit of 3.5×10^?14 mol·L^?1 (S/N=3). In addition, this DNA biosensor has good selectivity, and even can distinguish single‐mismatched target DNA.     

Fabrication of a Sensitive Cholesterol Biosensor Based on Cobalt‐oxide Nanostructures Electrodeposited onto Glassy Carbon Electrode

Electrodeposited cobalt oxide (CoOx) nanomaterials are not only used for immobilization of cholesterol oxidase (ChOx) but also as electron transfer mediator for oxidation of H₂O₂ generated in the enzymatic reaction. Voltammetry and flow injection analysis (FIA) were used for determination of cholesterol. FIA determination of cholesterol with biosensors yielded a calibration curve with the following characteristics: linear range up to 50 μM, sensitivity of 43.5 nA μM^?1 cm^?2 and detection limit of 4.2 μM. The apparent Michaelis‐Menten constant and the response time of the biosensor are 0.49 mM and 15 s, respectively. This biosensor also exhibits good stability, reproducibility and long life time.     

Direct Electrochemistry and Electrocatalysis of Hemoglobin/ZnO‐Chitosan/nano‐Au Modified Glassy Carbon Electrode

The highly efficient H₂O₂ biosensor was fabricated on the basis of the complex films of hemoglobin (Hb), nano ZnO, chitosan (CHIT) dispersed solution and nano Au immobilized on glassy carbon electrode (GCE). Biocompatible ZnO‐CHIT composition provided a suitable microenvironment to keep Hb bioactivity (Michaelis‐Menten constant of 0.075 mmol L^?1). The presence of nano Au in matrix could effectively enhance electron transfer between Hb and electrode. The electrochemical behaviors and effects of solution pH values were carefully examined in this paper. The (ZnO‐CHIT)‐Au‐Hb/GCE demonstrated excellently electrocatalytical ability for H₂O₂. This biosensor had a fast response to H₂O₂ less than 4 s and excellent linear relationships were obtained in the concentration range from1.94×10^?7 to 1.73×10^?3 mol L^?1 with the detection limit of 9.7×10^?8 mol L^?1 (S/N=3) under the optimum conditions. Moreover, the stability and reproducibility of this biosensor were evaluated with satisfactory results.