A Third‐Generation Hydrogen Peroxide Biosensor Based on Horseradish Peroxidase Covalently Immobilized on Electrografted Organic Film on Screen‐Printed Carbon Electrode

A new convenient strategy to fabricate a third‐generation hydrogen peroxide biosensor was described. The screen‐printed carbon electrode (SPCE) was first modified with a layer of 4‐nitrophenyl assembled from the 4‐nitroaniline diazonium salt synthesized in situ in acidic aqueous solution. Next, the nitro groups were converted to amines followed by crosslinking to the horseradish peroxidase (HRP) by glutaraldehyde. The redox chemistry of the active center of the HRP was observed and the HRP‐modified electrode displayed electrocatalytic activity towards the reduction of hydrogen peroxide (H₂O₂) without any mediators. H₂O₂ was determined in a linear range from 5.0 μM to 50.0 μM, with a detection limit of 1.0 μM. Furthermore, the biosensor exhibited fast amperometric response, good reproducibility and long‐term stability.     

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 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.     

Synthesis and Characterization of Poly(toluidine blue) Nanowires and Their Application in Amperometric Biosensors

Poly(toluidine blue) nanowires (PTBNWs) with an average diameter of ca. 200 nm and length of ca. 5 μm were synthesized for the first time using a template‐directed electropolymerization strategy with a nanopore polycarbonate (PC) membrane template. Their morphological characterization was carried out by scanning electron microscopy (SEM) and transmission electron microscope (TEM). By electrochemical polymerization, horseradish peroxidase (HRP) was encapsulated in situ in PTBNWs (denoted as PTBNWs‐HRP) for potential biosensor applications. PTBNWs‐HRP was then modified on a glassy carbon (GC) electrode. In the system obtained, the PTBNWs served as an excellent redox mediator and exhibited high efficiency of electron transfer between the HRP and the GC electrode for the reduction of H₂O₂. The proposed electrode can be used as an excellent amperometric sensor for H₂O₂ at ?0.1 V with a linear response range covering from 1 μM to 28 mM, a detection limit of 1 μM (based on S/N=3) and a fast response time of less than 8 s.     

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.     

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.     

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.     

Amperometric Flow‐Biosensor for Cyanide Based on an Inhibitory Effect upon Bioelectrocatalytic Reduction of Oxygen by Peroxidase‐Modified Carbon‐Felt

A novel, simple and relative highly sensitive amperometric flow biosensor for cyanide was developed by using horseradish peroxidase (HRP)‐adsorbed carbon‐felt (CF), based on an inhibitory effect on the HRP‐catalyzed O₂ reduction. The HRP‐CF showed a sufficient bioelecrocatalytic activity for O₂ reduction in the potential region from 0 to ?0.5 V at pH 5.0, due to a direct electron transfer‐based O₂ reduction process via ferrous‐HRP and compound III. This HRP‐catalyzed O₂ reduction was reversibly inhibited by cyanide, which enabled to fabricate a novel and simple reagentless (i.e., no requirement of the ordinary substrate, H₂O₂, and the electron transfer mediators) flow‐biosensor for cyanide. When air‐saturated 0.1 M phosphate buffer (pH 5.0) was used as a carrier under the applied potential of ?0.2 V vs. Ag/AgCl, the steady‐state base‐current due to the HRP‐catalyzed O₂ reduction was reversibly inhibited by the cyanide injection (200 µL), resulting in peak‐shape current responses. The magnitude of the inhibition peak currents linearly increased with increasing concentrations of cyanide up to 1 µM, and the detection limit was found to be 0.04 µM (S/N=2). The apparent inhibition constant K_i′ was estimated to be 0.87 µM.     

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.     

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.     

Room temperature ionic liquid doped DNA network immobilized horseradish peroxidase biosensor for amperometric determination of hydrogen peroxide

A novel electrochemical H₂O₂ biosensor was constructed by embedding horseradish peroxide (HRP) in a 1-butyl-3-methylimidazolium tetrafluoroborate doped DNA network casting on a gold electrode. The HRP entrapped in the composite system displayed good electrocatalytic response to the reduction of H₂O₂. The composite system could provide both a biocompatible microenvironment for enzymes to keep their good bioactivity and an effective pathway of electron transfer between the redox center of enzymes, H₂O₂ and the electrode surface. Voltammetric and time-based amperometric techniques were applied to characterize the properties of the biosensor. The effects of pH and potential on the amperometric response to H₂O₂ were studied. The biosensor can achieve 95% of the steady-state current within 2 s response to H₂O₂. The detection limit of the biosensor was 3.5 μM, and linear range was from 0.01 to 7.4 mM. Moreover, the biosensor exhibited good sensitivity and stability. The film can also be readily used as an immobilization matrix to entrap other enzymes to prepare other similar biosensors. Figure Horseradish peroxidase (HRP) embedded in a 1-butyl-3-methylimidazolium tetrafluoroborate (BMIM·BF ₄ ) doped DNA network can be used to fabricate a HRP sensor for the determination of H₂O₂     

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).     

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.     

Direct Electrochemistry and Electrocatalysis of the Hemoglobin Immobilized on Diazonium‐Functionalized Aligned Carbon Nanotubes Electrode

A simple and efficient electrochemical method is utilized to functionalize aligned carbon nanotubes (ACNTs) by the electrochemical reduction of 4‐carboxyphenyl diazonium salt. Thus hemoglobin (Hb) molecules were covalently immobilized on the diazonium‐ACNTs surface via carbodiimide chemistry. Direct electrochemistry and bioelectrocatalytic activity of the immobilized Hb were then investigated by cyclic voltammetry (CV) and amperometry techniques. It is showed that the Hb film on the diazonium‐ACNTs electrode had well‐defined redox peaks with a formal potential (E°) at ?312 mV (vs. Ag/AgCl), and the Hb‐ACNTs electrode displayed good electrocatalytic activity to H₂O₂ reduction. Owing to the high Hb covering on the ACNTs surface (Γ*=2.7×10^?9 mol cm^?2), the catalytic current were significantly improved when compared to the current measured at an Hb‐tangled carbon nanotubes electrode. The Hb‐ACNTs electrode exhibited high sensitivity, long‐term stability and wide concentration range from 40 μM to 3 mM for the amperometric detection of H₂O₂. The heterogeneous reaction rate constant (k_s) was 0.95±0.05 s^?1 and the apparent Michaelis–Menten constant (K was 0.15 mM.     

Improved Performance of an Amperometric Biosensor with Polydiaminonaphthalene on Electrochemically Deposited Au Nanoparticles

The performance of an enzyme sensor fabricated through covalent bond formation on the HRP‐bonded poly(1,8‐diaminonaphthalene) (polyDAN) layer with gold nanoparticles (AuNPs) was applied to catalyze the electrochemical reduction of H₂O₂. The surface characteristics of the sensor probe were studied using cyclic voltammetry, SEM, XPS, QCM, and impedance spectroscopy. The AuNP‐deposited surface resulted in higher conductivity and sensitivity for H₂O₂ detection in phosphate buffer solution. A linear calibration plot was obtained in the H₂O₂ concentration range between 10.0 μM and 25.0 mM with detection limit 5.0±1.25 μM. The lifetime of HRP/polyDAN/AuNP/GC probe was over 70 days without response loss.     

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.     

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.     

Amplification of bioelectrocatalytic signalling based on silver nanoparticles and DNA-derived horseradish peroxidase biosensors

Horseradish peroxidase (HRP) was immobilized onto a polyion complex membrane containing positively charged silver nanoparticles (nanosilver), double stranded DNA and poly(thionine) to fabricate highly sensitive and selective electrochemical hydrogen peroxide (H₂O₂) biosensor on a glassy carbon electrode. The presence of nanosilver provided a biocompatible microenvironment for enzyme molecules, greatly amplified the surface coverage of HRP on the electrode surface, and most importantly could act as a charge carrier. The process of the biosensor construction was characterized by scanning electron microscopy. Voltammetric and time-based amperometric techniques were employed to characterize the properties of the derived biosensor. Under optimal conditions, the biosensor has an electrocatalytic behavior towards the H₂O₂ reduction, and exhibits a linear range from 1.1 μM to 5.2 mM, with a lower detection limit of 0.2 μM. The apparent Michaelis–Menten constant of the biosensor to H₂O₂ was estimated to be 1.02 mM. Furthermore, the biosensor exhibited high sensitivity, good reproducibility, and acceptable stability. Importantly, the properties of composite film, together with the bioelectrochemical catalytic activity, could make them useful in the development of bioelectronic devices and investigation of protein electrochemistry at functional interface. Correspondence: Yan Liu, College of Chemistry, Chongqing Normal University, Chongqing 400047, P.R. China     

Determination of Xenoestrogenic Compounds Using a Nanostructured Biosensing Device

A ternary composite material based on Prussian blue, single‐walled carbon nanotubes and 1‐butyl‐3‐methylimidazolium hexafluorophosphate was prepared and tested for electrochemical detection of H₂O₂. The sensor allows amperometric detection of H₂O₂ at ?0.05 V, with a sensitivity of 137 mA M^?1?cm^?2. The nanocomposite provides a favorable microenvironment for immobilization of horseradish peroxidase (HRP). Determination of xenoestrogenic compounds was performed by enzymatic oxidation at the surface of modified screen printed biosensor in the presence of H₂O₂. The developed electrochemical biosensors exhibited high sensitivity, low detection limits, good operational and storage stability, for detection of 4‐t‐butylphenol, 4‐t‐octylphenol, 4‐n‐nonylphenol and 4‐n‐nonylphenol ethoxylate.     

A New Electrochemical Biosensor for Determination of Hydrogen Peroxide in Food Based on Well‐Dispersive Gold Nanoparticles on Graphene Oxide

Herein, we describe a new method for the detection of hydrogen peroxide (H₂O₂) in food by using an electrochemical biosensor. Initially, ultrafine gold nanoparticles dispersed on graphene oxide (AuNP‐GO) were synthesized by the redox reaction between AuCl₄^? and GO, and thionine‐catalase conjugates were then assembled onto the AuNP‐GO surface on a glassy carbon electrode. With the aid of the AuNP‐GO, the as‐prepared biosensor exhibited good electrocatalytic efficiency toward the reduction of H₂O₂ in pH 5.8 acetic acid buffer. Under optimal conditions, the dynamic responses of the biosensor toward H₂O₂ were achieved in the range from 0.1 µM to 2.3 mM, and the detection limit (LOD) was 0.01 µM at 3s_B. The Michaelis–Menten constant was measured to be 0.98 mM. In addition, the repeatability, reproducibility, selectivity and stability of the biosensor were investigated and evaluated in detail. Finally, the method was applied for sensing H₂O₂ in spiked or naturally contaminated samples including sterilized milk, apple juices, watermelon juice, coconut milk, and mango juice, receiving good correspondence with the results from the permanganate titration method. The disposable biosensor could offer a great potential for rapid, cost‐effective and on‐field analysis of H₂O₂ in foodstuff.