Mediatorless Hydrogen Peroxide Biosensor Based on Horseradish Peroxidase Immobilized on 4‐Carboxyphenyl Film Electrografted on Gold Electrode

A novel method to fabricate a third‐generation hydrogen peroxide biosensor was reported. The electrode was first derivatized by electrochemical reduction of in situ generated 4‐carboxyphenyl diazonium salt (4‐CPDS) in acidic aqueous solution yielded stable 4‐carboxyphenyl (4‐CP) layer. The horseradish peroxidase (HRP) enzyme was then covalently immobilized by amidation between NH₂ terminus of enzyme and COOH terminus of 4‐CP film making use of the carbodiimide chemistry. Electrodeposition conditions used to control electrode functionalization density and film electron transfer kinetics were assessed by chronoamperometry and electrochemical impedance spectroscopy. The immobilized HRP displayed excellent electrocatalytic activity towards the reduction of hydrogen peroxide (H₂O₂) without any mediators. The effect of various operational parameters was explored for optimum analytical performance. The reported biosensor exhibited fast amperometric response (within 5 s) to H₂O₂. The detection limit of the biosensor was 5 μM, and linear range was from 20 μM to 20 mM. Furthermore, the biosensor exhibited high sensitivity, good reproducibility, and long‐term stability.     

A Simple Layer‐by‐Layer Assembly Strategy for a Reagentless Biosensor Based on a Nanocomposite of Methylene Blue‐Multiwalled Carbon Nanotubes

A simple layer‐by‐layer (LBL) assembly strategy was established for constructing a novel reagentless biosensor based on a nanocomposite of methylene blue multiwalled carbon nanotubes (MB‐MWNTs). A nanocomposite of MB‐MWNTs was obtained by direct premixing and possessed good dispersion in barbital‐HCl buffer. Through electrostatic interactions, the nanocomposite of MB‐MWNTs could alternately be assembled with horseradish peroxidase (HRP) on the Au electrode modified with precursor films. UV/Vis spectra and scanning electron microscopy (SEM) were applied to reveal the formation of the nanocomposite of MB‐MWNTs. The LBL assembly process was also verified by electrochemical impedance spectroscopy (EIS). The MB is a well‐established mediator and efficiently facilitated the electron shuttle between the HRP and the electrode, as demonstrated by the cyclic voltammetry (CV) measurements. The as‐prepared reagentless biosensor exhibited a fast response for the determination of hydrogen peroxide (H₂O₂) and reached 95% of the steady‐state current within 3 s. It was found that the linear response range of the reagentless biosensor for H₂O₂ was from 4.0 μM to 3.78 mM with a detection limit of 1.0 μM and a sensitivity of 22.5 μA mM⁻¹. The biosensor exhibited a high reproducibility and stability.     

Direct Electrochemistry of Hemoglobin Immobilized on Carbon‐Coated Iron Nanoparticles for Amperometric Detection of Hydrogen Peroxide

A novel method for preparation of hydrogen peroxide biosensor was presented based on immobilization of hemoglobin (Hb) on carbon‐coated iron nanoparticles (CIN). CIN was firstly dispersed in a chitosan solution and cast onto a glassy carbon electrode to form a CIN/chitosan composite film modified electrode. Hb was then immobilized onto the composite film with the cross‐linking of glutaraldehyde. The immobilized Hb displayed a pair of stable and quasireversible redox peaks and excellent electrocatalytic reduction of hydrogen peroxide (H₂O₂), which leading to an unmediated biosensor for H₂O₂. The electrocatalytic response exhibited a linear dependence on H₂O₂ concentration in a wide range from 3.1 μM to 4.0 mM with a detection limit of 1.2 μM (S/N=3). The designed biosensor exhibited acceptable stability, long‐term life and good reproducibility.     

Novel Protocol for Covalent Immobilization of Horseradish Peroxidase on Gold Electrode Surface

A novel protocol for immobilization of horseradish peroxidase (HRP) onto diazonium functionalized screen‐printed gold electrode (SPGE) has been successfully developed. This protocol involved 1) electrochemical reduction of p‐nitrophenyl diazonium salts synthesized in situ in acidic aqueous solution to graft a layer of p‐nitrophenyl on SPGE, 2) electrochemical reduction of the nitro groups to convert to amines, 3) chemical reaction with nitrous acid to transform the amine to diazonium derivative and 4) chemical coupling of the enzyme with the diazonium group to form a covalent diazo bond. The fabricated biosensor showed the direct electrochemistry of HRP and displayed electrocatalytic activity towards the reduction of hydrogen peroxide (H₂O₂) without any mediator. The biosensor exhibited fast amperometric response to H₂O₂. The catalytic current increased with increasing H₂O₂ concentration from 5 μM to 30 μM and the detection limit of the biosensor was 2 μM. The biosensor exhibited acceptable sensitivity, good reproducibility and long‐term stability.     

Amperometric Biosensor for Hydrogen Peroxide,Using Supramolecularly Immobilized Horseradish Peroxidase on the β‐Cyclodextrin‐Coated Gold Electrode

Horseradish peroxidase, previously modified with 1‐adamantane moieties, was supramolecularly immobilized on gold electrodes coated with perthiolated β‐cyclodextrin. The functionalized electrode was employed for the construction of an amperometric biosensor device for hydrogen peroxide using 1 mM hydroquinone as electrochemical mediator. The biosensor exhibited a fast amperometric response (6 s) and a good linear response toward H₂O₂ concentration between 12 μM and 450 μM. The biosensor showed a sensitivity of 1.02 mA/M cm², and a very low detection limit of 5 μM. The electrode retained 97% of its initial electrocatalytic activity after 30 days of storage at 4 ⁰C in 50 mM sodium phosphate buffer, pH 7.0.     

Dendritic Silver/Silicon Dioxide Nanocomposite Modified Electrodes for Electrochemical Sensing of Hydrogen Peroxide

A novel biosensor for hydrogen peroxide was prepared by immobilizing horseradish peroxidase (HPR) on newly synthesized dendritic silver/silicon dioxide nanocomposites, which were coated on a glassy carbon electrode. The modified electrode was characterized with XPS, SEM, and electrochemical methods. This biosensor showed a very fast amperometric response to hydrogen peroxide with a linear range from 0.7 to 120 μM, a limit of detection of 0.05 μM and a sensitivity of 1.02 mA mM^?1 cm^?2. The Michaelis‐Menten constant of the immobilized HRP was estimated to be 0.21 mM, indicating a high affinity of the HRP to H₂O₂ without loss of enzymatic activity. The preparation of the proposed biosensor was convenient, and it showed high sensitivity and good stability.     

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.     

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.     

A MgO Nanoparticles Composite Matrix‐Based Electrochemical Biosensor for Hydrogen Peroxide with High Sensitivity

A novel horseradish peroxidase (HRP) electrochemical biosensor based on a MgO nanoparticles (nano‐MgO)‐chitosan (chit) composite matrix was developed. The morphology of nano‐MgO‐chit nanocomposite was examined by scanning electron microscopy (SEM). The interaction between nano‐MgO‐chit nanocomposite matrix and enzyme was characterized with UV‐vis spectra. This proposed composite material combined the advantages of inorganic nanoparticles and organic polymer chit. The HRP immobilized in the nanocomposite matrix displayed excellent electrocatalytic activity to the reduction of H₂O₂ in the presence of hydroquinone as a mediator. The effects of the experimental variables such as solution pH and the working potential were investigated using steady‐state amperometry. The present biosensor (HRP‐modified electrode) had a fast response towards H₂O₂ (less than 10 s), and excellent linear relationships were obtained in the concentration range of 0.1–1300 μM, with a detection limit of 0.05 μM (S/N=3). Moreover, the stability and reproducibility of this biosensor were evaluated with satisfactory results.     

A Third‐Generation Hydrogen Peroxide Biosensor Based on Horseradish Peroxidase Immobilized in Carbon Nanotubes/ SBA‐15 Film

A new third‐generation biosensor for H₂O₂ assay was developed on the basis of the immobilization of horseradish peroxidase (HRP) in a nanocomposite film of carbon nanotubes (CNTs)‐SBA‐15 modified gold electrode. The biological activity of HRP immobilizing in the composite film was characterized by UV‐vis spectra. The HRP immobilized in the nanocomposite matrix displayed excellent electrocatalytic activity to the reduction of H₂O₂. The effects of the experimental variables such as solution pH and working potential were investigated using steady‐state amperometry. Under the optimal conditions, the resulting biosensor showed a linear range from 1 µM to 7 mM and a detection limit of 0.5 µM (S/N=3). Moreover, the stability and reproducibility of this biosensor were evaluated with satisfactory results.     

An Amperometric Biosensor Based on the Coimmobilization of Horseradish Peroxidase and Methylene Blue on a Carbon Nanotubes Modified Electrode

A novel hydrogen peroxide biosensor has been constructed based on the characteristics of the carbon nanotube. The multiwall carbon nanotube (MWNT) was used as a coimmobilization matrix to incorporate horseradish peroxidase (HRP) and electron transfer mediator methylene blue (MB) onto a glassy carbon electrode surface. Cyclic voltammetry and amperometric measurements were employed to demonstrate the feasibility of methylene blue as an electron carrier between the immobilized peroxidase and the surface of glassy carbon electrode. The amperometric response of this resulting biosensor to H₂O₂ shows a linear relation in the range from 4 μM to 2 mM. The detection limit was 1 μM when the signal to noise ratio is 3. The presence of dopamine and ascorbic acid hardly affects the sensitive determination of H₂O₂. This biosensor also possesses very good stability and reproducibility.     

The Modification of Screen‐Printed Carbon Electrodes with Amino Group and Its Application to Construct a H2O2 Biosensor

Electrooxidation of thionine on screen‐printed carbon electrode gives rise to the modification of the surface with amino groups for the covalent immobilization of enzymes such as horseradish peroxidase (HRP). The biosensor was constructed using multilayer enzymes which covalently immobilized onto the surface of amino groups modified screen‐printed carbon electrode using glutaraldehyde as a bifunctional reagent. The multilayer assemble of HRP has been characterized with the cyclic voltammetry and the faradaic impedance spectroscopy. The H₂O₂ biosensor exhibited a fast response (2 s) and low detection limit (0.5 μM).     

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.     

Enzyme Immobilization and Direct Electrochemistry Based on a New Matrix of Phospholipid‐Monolayer‐Functionalized Graphene

A new nanocomposite material for enzyme immobilization and subsequent direct electrochemistry and electrocatalysis was developed by using 1,2‐dimyristoyl‐sn‐glycero‐3‐phospho‐(1‐rac‐glycerol)‐phospholipid‐monolayer‐membrane‐modified graphene (DMPG‐G). Microperoxidase‐11 (MP11) was chosen as a model enzyme to investigate the composite system. Owing to the improved conductivity and biocompatible microenvironment, MP11 that was immobilized in the matrix of the DMPG‐G nanocomposite (DMPG‐G‐MP11) effectively retained its native structure and bioactivity. DMPG‐G‐MP11‐modified glassy carbon electrode (DMPG‐G‐MP11/GCE) exhibited a pair of well‐defined quasi‐reversible redox peaks of MP11 and showed high electrocatalytic activity towards hydrogen peroxide (H₂O₂). The linear response of the developed biosensor for the determination of H₂O₂ ranged from 2.0×10^?6 to 4.5×10^?4 M with a detection limit of 7.2×10^?7 M . This biosensor exhibited high reproducibility and long‐term storage stability. The promising features of this biosensor indicate that these lipid–graphene nanocomposites are ideal candidate materials for the direct electrochemistry of redox proteins and that they could serve as a versatile platform for the construction of a third‐generation biosensor.     

A New Amperometric Biosensor Based on HRP/Nano-Au/L-Cysteine/Poly（o-Aminobenzoic acid）-Membrane-Modified Platinum Electrode for the Determination of Hydrogen Peroxide

The third generation amperometric biosensor for the determination of hydrogen peroxide （H2O2） has been described. For the fabrication of biosensor, o-aminobenzoic acid （oABA） was first electropolymerized on the surface of platinum （Pt） electrode as an electrostatic repulsion layer to reject interferences. Horseradish peroxidase （HRP） absorbed by nano-scaled particulate gold （nano-Au） was immobilized on the electrode modified with polymerized o-aminobenzoic acid （poABA） with L-cysteine as a linker to prepare a biosensor for the detection of H2O2. Amperometric detection of H2O2 was realized at a potential of ＋20 mV versus SCE. The resulting biosensor exhibited fast response, excellent reproducibility and sensibility, expanded linear range and low interferences. Temperature and pH dependence and stability of the sensor were investigated. The optimal sensor gave a linear response in the range of 2.99×10^-6 to 3.55×10^-3 mol·L^-1 to H2O2 with a sensibility of 0.0177 A·L^-1·mol^-1 and a detection limit （S/N = 3） of 4.3×10^-7 mol·L^-1. The biosensor demonstrated a 95% response within less than 10 s.     

Sensitive and Selective Sensing of Hydrogen Peroxide with Iron‐Tetrasulfophthalocyanine‐Graphene‐Nafion Modified Screen‐Printed Electrode

We developed a novel iron‐tetrasulfophthalocyanine‐graphene‐Nafion (FeTSPc‐GR‐Nafion) modified screen‐printed electrode to determine hydrogen peroxide (H₂O₂) with high sensitivity and selectivity. The nanocomposite film (FeTSPc‐GR‐Nafion) exhibits an excellent electrocatalytic activity towards oxidation of H₂O₂ at a potential of +0.35 V in the absence of enzyme. A comparative study reveals that the FeTSPc‐GR complexes play a dual amplification role. Amperometric experiment indicates that the sensors possess good sensitivity and selectivity, with a linear range from 2.0×10^?7 M to 5.0×10^?3 M and a detection limit of 8.0×10^?8 M. This sensor has been successfully used to develop the glucose biosensor and has also been applied to determine H₂O₂ in sterile water.     

Direct Electrochemistry and Bioelectrocatalysis of Microperoxidase‐11 Immobilized on Chitosan‐Graphene Nanocomposite

A bioelectrochemical platform has been constructed for the direct electron transfer and biosensing purposes of microperoxidase‐11 (MP‐11) immobilized on the chitosan dispersed multilayer graphene nanocomposite. The immobilized MP‐11 at the modified gold electrode displays a well‐defined and quasireversible redox peaks, with a formal potential of ?0.38 V/SCE in a buffer solution (pH 7.0). MP‐11 absorbed on the electrode surface exhibits high electrocatalytic activity toward the reduction of both oxygen and hydrogen peroxide and also shows good analytical performance for the amperometric detection of H₂O₂ with a linear range from 2.5 to 135 μM. These results indicate the graphene modified electrode might be used as a third generation biosensor for H₂O₂ detection.     

An amperometric biosensor based on the coimmobilization of horseradish peroxidase and methylene blue on a β-type zeolite modified electrode

A new biosensor for the amperometric detection of hydrogen peroxide was developed based on the co-immobilization of horseradish peroxidase (HRP) and methylene blue on a β-type zeolite modified glassy carbon electrode without the commonly used bovine serum albumin-glutaraldehyde. The intermolecular interaction between enzyme and zeolite matrix was investigated using FT-IR. The cyclic voltammetry and amperometric measurement demonstrated that methylene blue co-immobilized with HRP in this way displayed good stability and could efficiently transfer electrons between immobilized HRP and the electrode. The sensor responded rapidly to H₂O₂ in the linear range from 2.5 × 10^–6 to 4.0 × 10^–3 M with a detection limit of 0.3 μM. The sensor was stable in continuous operation.     

A Novel Enzymatic Glucose Biosensor and Nonenzymatic Hydrogen Peroxide Sensor Based on (3‐Aminopropyl) Triethoxysilane Functionalized Reduced Graphene Oxide

In the present study, a novel enzymatic glucose biosensor using glucose oxidase (GOx) immobilized into (3‐aminopropyl) triethoxysilane (APTES) functionalized reduced graphene oxide (rGO‐APTES) and hydrogen peroxide sensor based on rGO‐APTES modified glassy carbon (GC) electrode were fabricated. Nafion (Nf) was used as a protective membrane. For the characterization of the composites, Fourier transform infrared spectroscopy (FTIR), X‐ray powder diffractometer (XRD), and transmission electron microscopy (TEM) were used. The electrochemical properties of the modified electrodes were investigated using electrochemical impedance spectroscopy, cyclic voltammetry, and amperometry. The resulting Nf/rGO‐APTES/GOx/GC and Nf/rGO‐APTES/GC composites showed good electrocatalytical activity toward glucose and H₂O₂, respectively. The Nf/rGO‐APTES/GC electrode exhibited a linear range of H₂O₂ concentration from 0.05 to 15.25 mM with a detection limit (LOD) of 0.017 mM and sensitivity of 124.87 μA mM⁻¹ cm⁻². The Nf/rGO‐APTES/GOx/GC electrode showed a linear range of glucose from 0.02 to 4.340 mM with a LOD of 9 μM and sensitivity of 75.26 μA mM⁻¹ cm⁻². Also, the sensor and biosensor had notable selectivity, repeatability, reproducibility, and storage stability.     

A Novel Hydrogen Peroxide Biosensor Based on Adsorption of Horseradish Peroxidase onto a Nanobiomaterial Composite Modified Glassy Carbon Electrode

In this work, a novel hydrogen peroxide biosensor derived from maize tassel (MT) and multiwalled carbon nanotube (MWCNT) composite was used to adsorb horseradish peroxidase (HRP) onto the surface of a glassy carbon electrode through electrostatic interactions. The morphology and structure of the products were characterized by SEM, FTIR and UV‐visible spectroscopy. The electrochemical and electrocatalytic performance of the HRP/MT‐MWCNT/GCE was studied using voltammetric and amperometric methods. The amperometric response of the biosensor varied linearly with concentration of H₂O₂ from 9 µM to 1 mM with detection limit of 4.0 µM (S/N=3). Furthermore, the biosensor exhibited good reproducibility and stability.