Direct electrochemistry and electrocatalysis of horseradish peroxidase immobilized on L-glutathione self-assembled monolayers

A novel hydrogen peroxide biosensor has been fabricated based on covalently linked horseradish peroxidase （HRP） onto L- glutathione self-assembled monolayers （SAMs）. The SAMs-based electrode was characterized by electrochemical methods, and direct electrochemistry of HRP can be achieved with formal potential of-0.242 V （vs. saturated Ag/AgCl） in pH 7 phosphate buffer solution （PBS）, the redox peak current is linear to scan rate and rate constant can be calculated to be 0.042 s^-1. The HRP-SAMs- based biosensors show its better electrocatalysis to hydrogen peroxide in the concentration range of 1 × 10^-6 mol/L to 1.2 × 10^-3 mol/L with a detection limit of 4 × 10^-7 mol/L. The apparent Michealis-Menten constant is 3.12 mmol/L. The biosensor can effectively eliminate the interferences of dopamine, ascorbic acid, uric acid, catechol and p-acetaminophen.     

A biosensing platform based on horseradish peroxidase immobilized onto chitosan-wrapped single-walled carbon nanotubes

One-step, diameter-selective dispersion of single-walled carbon nanotubes (SWCNTs) has been accomplished through noncovalent complexation of the nanotubes with a water-soluble, biocompatible polymer chitosan at room temperature. Such chitosan-wrapped individual SWCNTs can be used for the immobilization of horseradish peroxidase (HRP) and be used to construct an electrode for direct bioelectrochemical sensing without an electron mediator. The direct electron transfer between HRP and the electrode surface was observed with a formal potential of approximately −0.35 V (vs. saturated calomel electrode) in phosphate buffer solution and the calculated heterogeneous electron transfer rate constant is approximately 23.5 s⁻¹. Experimental results indicate that the immobilized HRP retains its catalytic activity for the reduction of nitric oxide. Such an HRP–SWCNT–chitosan-based biosensor exhibited a rapid response time of less than 6 s and a good linear detection range for nitrite concentration, from 25 to 300 μM with a detection limit of 3 μM. The apparent Michaelis–Menten constant (K _m) and the maximum electrode sensitivity (i_max/K _m) are found to be 7.0 mM and 0.16 μA mM⁻¹, respectively. Both the unique electrical properties of SWCNTs and biocompatibility of chitosan enable the construction of an excellent biosensing platform for improved electrocatalysis of HRP, allowing, specifically, the detection of trace levels of nitric oxide.     

Direct electrochemistry and electrocatalysis of horseradish peroxidase immobilized in hybrid organic-inorganic film of chitosan/sol-gel/carbon nanotubes

A hybrid organic-inorganic nanocomposite film of chitosan/sol-gel/multi-walled carbon nanotubes was constructed for the immobilization of horseradish peroxidase (HRP). This film was characterized by scanning electron microscopy. Direct electron transfer (DET) and bioelectrocatalysis of HRP incorporated into the composite film were investigated. The results indicate that the film can provide a favorable microenvironment for HRP to perform DET on the surface of glassy carbon electrodes with a pair of quasi-reversible redox waves and to retain its bioelectrocatalytic activity toward H₂O₂.     

A highly soluble poly(3,4-ethylenedioxythiophene)-poly(styrene sulfonic acid)/Au nanocomposite for horseradish peroxidase immobilization and biosensing

A highly soluble poly(3,4-ethylenedioxythiophene)-poly(styrene sulfonic acid)/Au (PEDOT-PSS/Au) nanocomposite was prepared via one-step chemical synthesis and the matrix was characterized by UV-vis spectroscopy (UV-vis), Fourier transform infrared spectroscopy (FT-IR), scanning electron microscope (SEM) and transmission electron microscope (TEM). Due to the excellent aqueous compatibility and biocompatibility, the PEDOT/PSS-Au nanocomposite can be used as biomaterial for enzymes immobilization. In this system, redox enzyme, horseradish peroxidase (HRP) was integrated with PEDOT/PSS-Au nanocomposite and the direct electron transfer of HRP was observed. Moreover, we find that the HRP/PEDOT-PSS/Au modified electrode shows excellent electrocatalytic ability for H₂O₂ and the formal Michaelis-Menten constant was 0.78 mmol/L. The response currents have good linear relation with the concentrations of H₂O₂ with a linear range from 2.0 × 10⁻⁷ to 3.8 × 10⁻⁴ mol/L.     

Direct electrochemistry and spectroelectrochemistry of osmium substituted horseradish peroxidase

In this contribution the substitution of the central protoporphyrin IX iron complex of horseradish peroxidase by the respective osmium porphyrin complex is described. The direct electrochemical reduction of the Os containing horseradish peroxidase (OsHRP) was achieved at ITO and modified glassy carbon electrodes and in combination with spectroscopy revealed the three redox couples Os^IIIHRP/Os^IVHRP, Os^IVHRP/Os^VHRP and Os^VHRP/Os^VIHRP. The midpoint potentials differ dependent on the electrode material used with E_1/2 (Os^III/IV) of − 0.4 V (ITO) and − 0.25 V (GC), E_1/2 (Os^IV/^V) of − 0.16 V (ITO) and + 0.10 V (GC), and E_1/2 (Os^V/VI)of + 0.18 V (ITO), respectively. Moreover, with immobilised OsHRP the direct electrocatalytic reduction of hydrogen peroxide and tert-butyl hydroperoxide was observed. In comparison to electrodes modified with native HRP the sensitivity of the OsHRP-electrode for tert-butyl hydroperoxide is higher.     

Direct electrochemistry and electrocatalysis of horseradish peroxidase immobilized in sol–gel-derived tin oxide/gelatin composite films

Horseradish peroxidase (HRP) was immobilized into a new type of sol–gel-derived nano-sized tin oxide/gelatin composite film (SnO₂ composite film) using a sol–gel film/enzyme/sol–gel film “sandwich” configuration. Direct electrochemistry and electrocatalysis of HRP incorporated into the composite films were investigated. HRP/SnO₂ composite film exhibited a pair of stable and quasi-reversible cyclic voltammetric peaks for the HRP Fe(III)/HRP Fe(II) redox couple with a formal potential of about −0.25 V (vs. SCE) in a pH 6.0 phosphate buffer solution. The electron transfer between the enzyme and the underlying electrode was greatly enhanced in the microenvironment with nano-SnO₂ particles and nanoporous structures. Morphologies and microstructures of the composite films and HRP/composite films were characterized with TEM, AFM. Electrochemical impedance spectroscopy (EIS) was also used to feature the HRP incorporated into composite films. FTIR and UV–Vis spectroscopy demonstrated that HRP in the composite film could retain its native secondary structure. With the advantages of organic–inorganic hybrid materials, the HRP/SnO₂ composite film modified electrode displayed good stability and electrocatalytic activity to the reduction of H₂O₂, The apparent Michaelis-Menten constant was estimated to be 0.345 mM, indicating a high affinity of HRP entrapped into the composite film toward H₂O₂.     

Mesoporous silica hollow sphere (MSHS) for the bioelectrochemistry of horseradish peroxidase

In this work, novel mesoporous silica hollow spheres (MSHS) were chosen as an immobilization matrix, to construct a mediator-free third-generation HRP biosensor. UV-vis spectroscopy revealed that horseradish peroxidase (HRP) entrapped in MSHS could retain its native structure. FTIR spectroscopy and nitrogen adsorption-desorption isotherms indicated that HRP are intercalated into the mesopores. The direct electron transfer of HRP entrapped in MSHS was observed. A pair of stable and well-defined redox peaks of HRP with a formal potential of about −0.150 V (vs. Ag/AgCl) in 0.1 M pH 7.0 phosphate-buffered solution (PBS) were obtained. The biosensor exhibited a fast amperometric response to H₂O₂ with a linear range of 3.9 × 10⁻⁶ to 1.4 × 10⁻⁴ M (R = 0.997, N = 20). The detection limit was 1.2 × 10⁻⁶ M based S/N = 3.     

An amperometric hydrogen peroxide biosensor based on immobilization of horseradish peroxidase on an electrode modified with magnetic dextran microspheres

A new kind of magnetic dextran microsphere (MDMS) with uniform shape and narrow diameter distribution has been prepared from magnetic iron nanoparticles and dextran. Horseradish peroxidase (HRP) was successfully immobilized on the surface of an MDMS-modified glassy-carbon electrode (GCE), and the immobilized HRP displayed excellent electrocatalytic activity in the reduction of H₂O₂ in the presence of the mediator hydroquinone (HQ). The effects of experimental variables such as the concentration of HQ, solution pH, and the working potential were investigated for optimum analytical performance. This biosensor had a fast response to H₂O₂ of less than 10 s and an excellent linear relationship was obtained in the concentration range 0.20 μmol L⁻¹–0.68 mmol L⁻¹, with a detection limit of 0.078 μmol L⁻¹ (S/N = 3) under the optimum conditions. The response showed Michaelis–Menten behavior at larger H₂O₂ concentrations, and the apparent Michaelis–Menten constant was estimated to be 1.38 mmol L⁻¹. Moreover, the selectivity, stability, and reproducibility of the biosensor were evaluated, with satisfactory results. Figure Amperometric response of the biosensor to successive additions of H₂O₂ and the plot of amperometric response vs. H₂O₂ concentration     

Immobilized anthraquinone for redox mediation of horseradish peroxidase for hydrogen peroxide sensing

The detection of hydrogen peroxide is detailed using horseradish peroxidase and anthraquinone. Both species are immobilized on a glassy carbon electrode substrate. This dual immobilization gives rise to lower detection limits compared with the situation when either of the species is immobilized. Detection limits of 40 nM are reported within physiologically-relevant media.     

Direct electrochemistry of horseradish peroxidase immobilized on electrografted 4-ethynylphenyl film via click chemistry

In this paper, a simple two-step approach for redox protein immobilization was introduced. Firstly, alkynyl-terminated film was formed on electrode surface by electrochemical reduction of 4-ethylnylphenyl (4-EP) diazonium compound. Then, horseradish peroxidase (HRP) modified with azido group was covalently immobilized onto the electrografted film via click reaction. Reflection absorption infrared (RAIR) spectroscopy and electrochemical methods were used to characterize the modification process. The results indicate that HRP retains its native structure and shows fast direct electron transfer. Moreover, the immobilized HRP shows excellent electrocatalytic reduction activity toward H(2)O(2) with a linear range of 5.0×10(-6) to 9.3×10(-4) mol L(-1).     

Direct electrochemistry and electrocatalysis of heme-protein based on N,N-dimethylformamide film electrode

The heme-protein including myoglobin (Mb), hemoglobin (Hb) and horseradish peroxidase (HRP) were immobilized on normal graphite electrode by using N,N-dimethylformamide (DMF). The proteins undergo direct electron-transfer reactions. The current is linearly dependent on the scan rate, indicating that the direct electrochemistry of heme-protein in that case is a surface-controlled electrode process. The E°s are linearly dependent on solution pH (redox-Bohr effect), indicating that the electron transfer was proton-coupled. Ultraviolet-visible (UV-vis) and reflection-absorption infrared (RAIR) spectra suggest that the conformation of proteins in the presence of DMF are little different from that proteins alone the conformation changes reversibly in the range of pH 3.0-10.0. The catalytic activity of proteins were examined by hydrogen peroxide and nitrite.     

Development of hydrogen peroxide biosensor based on in situ covalent immobilization of horseradish peroxidase by one-pot polysaccharide-incorporated sol-gel process

A simple and reliable one-pot approach was established for the development of a novel hydrogen peroxide (H₂O₂) biosensor based on in situ covalent immobilization of horseradish peroxidase (HRP) into biocompatible material through polysaccharide-incorporated sol-gel process. Siloxane with epoxide ring and trimethoxy anchor groups was applied as the bifunctional cross-linker and the inorganic resource for organic-inorganic hybridization. The reactivity between amine groups and epoxy groups allowed the covalent incorporation of HRP and the functional biopolymer, chitosan (CS) into the inorganic polysiloxane network. Some experimental variables, such as mass ratio of siloxane to CS, pH of measuring solution and applied potential for detection were optimized. HRP covalently immobilized in the hybrid matrix possessed high electrocatalytic activity to H₂O₂ and provided a fast amperometric response. The linear response of the as-prepared biosensor for the determination of H₂O₂ ranged from 2.0 × 10⁻⁷ to 4.6 × 10⁻⁵ mol l⁻¹ with a detection limit of 8.1 × 10⁻⁸ mol l⁻¹. The apparent Michaelis-Menten constant was determined to be 45.18 μmol l⁻¹. Performance of the biosensor was also evaluated with respect to possible interferences. The fabricated biosensor exhibited high reproducibility and storage stability. The ease of the one-pot covalent immobilization and the biocompatible hybrid matrix serve as a versatile platform for enzyme immobilization and biosensor fabricating.     

Facilitated electron transfer from an electrode to horseradish peroxidase in a biomembrane-like surfactant film

Direct electron transfer was found to be greatly facilitated for horseradish peroxidase (HRP) in a didodecyldimethylammonium bromide (DDAB) biomembrane-like film at a pyrolytic graphite (PG) electrode involving the Fe^III Fe^II couple. The heterogeneous electron transfer rate constant k_s was fitted as 9.0 s⁻¹ using the non-linear regression analysis of the square wave voltammograms at a series of frequencies and pulse heights. The pH dependence of the formal potential for HRP in DDAB film at medium pH environments suggested one-proton transfer coupled with a one-electron transfer reaction. Scanning electron microscopy (SEM) showed different film morphology for HRP and HRP---DDAB films. UV–vis and reflectance absorption infrared (RAIR) spectra inferred that the heme state of HRP in DDAB film was similar to that in its native state. Circular dichroism (CD) results indicated slight perturbation of DDAB on the second structure of HRP. Thus, the embedded HRP in the biomembrane-like DDAB film showed nearly native structural properties and improved electrochemical characteristics. This has potential value for the basic and applied bioelectrochemistry of enzymes.     

Amperometric carbohydrate antigen 19-9 immunosensor based on three dimensional ordered macroporous magnetic Au film coupling direct electrochemistry of horseradish peroxidase

A sandwich-type electrochemical immunosensor for the detection of carbohydrate antigen 19-9 (CA 19-9) antigen based on the immobilization of primary antibody (Ab₁) on three dimensional ordered macroporous magnetic (3DOMM) electrode, and the direct electrochemistry of horseradish peroxidase (HRP) that was used as both the label of secondary antibody (Ab₂) and the blocking reagent. The 3DOMM electrode was fabricated by introducing core–shell Au–SiO₂@Fe₃O₄ nanospheres onto the surface of three dimensional ordered macroporous (3DOM) Au electrode via the application of an external magnet. Au nanoparticles functionalized SBA-15 (Au@SBA-15) was conjugated to the HRP labeled secondary antibody (HRP-Ab₂) through the Au–SH or Au–NH₃⁺ interaction, and HRP was also used as the block reagent. The formation of antigen–antibody complex made the combination of Au@SBA-15 and 3DOMM exhibit remarkable synergistic effects for accelerating direct electron transfer (DET) between HRP and the electrode. Under the optimal conditions, the DET current signal increased proportionally to CA 19-9 concentration in the range of 0.05 to 15.65 U mL⁻¹ with a detection limit of 0.01 U mL⁻¹. Moreover, the immunosensor showed high selectivity, good stability, satisfactory reproducibility and regeneration. Importantly, the developed method was used to assay clinical serum specimens, achieving a good relation with those obtained from the commercialized electrochemiluminescent method.     

Hydrogen peroxide sensor based on horseradish peroxidase immobilized on a silver nanoparticles/cysteamine/gold electrode

A third-generation hydrogen peroxide biosensor was prepared by immobilizing horseradish peroxidase (HRP) on a gold electrode modified with silver nanoparticles. A freshly-cleaned gold electrode was first immersed in a cysteamine–ethanol solution, and then silver nanoparticles were immobilized on the cysteamine monolayer, and finally HRP was adsorbed onto the surfaces of the silver nanoparticles. This self-assemble process was examined via atomic force microscopy (AFM). The immobilized horseradish peroxidase exhibited an excellent electrocatalytic response toward the reduction of hydrogen peroxide. The linear range of the biosensor was 3.3 M to 9.4 mM, and the detection limit was estimated to be 0.78 M. Moreover, the biosensor exhibited a fast response, high sensitivity, good reproducibility, and long-term 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.     

Layer-by-layer immobilization of horseradish peroxidase on a gold electrode modified with colloidal gold nanoparticles

An amperometric biosensor, based on layer-by-layer self-assembly of colloidal gold nanoparticles, cysteine and horseradish peroxidase on Nafion modified electrode surface by electrostatic adsorption, has been used for the determination of hydrogen peroxide. The electrochemical behavior of the multilayer film was studied by cyclic voltammetry, linear sweep voltammetry and chronoamperometry. The step layer-by-layer adsorption interface morphology was further characterized by means of electrochemical impedance spectroscopy and cyclic voltammetry. The performance and factors influencing the resulted biosensor were studied in detail. The sensor displayed an excellent electrocatalytic response to the reduction of H₂O₂ without the aid of an electron transfer mediator. Linear response to H₂O₂ was obtained for the concentration range from 1.6 μM to 2.4 mM under optimized conditions. The detection limit of the biosensor was 0.5 μM (S/N = 3), and the sensor achieved 95% of the steady-state current within 10 s. The sensor exhibited high sensitivity, selectivity and stability. Correspondence: Yan Liu, College of Chemistry, Chongqing Normal University, Chongqing 400047, P.R. China     

Electrochemical characteristics of a platinum electrode modified with a matrix of polyvinyl butyral and colloidal Ag containing immobilized horseradish peroxidase

A new hydrogen peroxide biosensor was constructed, which consisted of a platinum electrode modified by a matrix of polyvinyl butyral (PVB) and nanometer-sized Ag colloid containing immobilized horseradish peroxidase (HRP), and using Co(bpy)₃³⁺ as mediator in the hydrogen peroxide solution. The electrochemical characteristics of the biosensor were studied by cyclic voltammetry and chronoamperometry. The modified process was characterized by electrochemical impedance spectroscopy and cyclic voltammetry. The HRP immobilized on colloidal Ag was stable and retained its biological activity. The sensor displays excellent electrocatalytic response to the reduction of H₂O₂. Analytical parameters such as pH and temperature were also studied. Linear calibration for H₂O₂ was obtained in the range of 1×10^–5 to 1×10^–2 M under optimized conditions. The sensor was highly sensitive to H₂O₂, with a detection limit of 2×10^–6 M, and the sensor achieved 95% of steady-state current within 10 s. The sensor exhibited high sensitivity, selectivity and 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     

辣根过氧化酶在磁性纳米修饰的离子注入电极上的电化学和电催化行为

辣根过氧化酶(HRP)在Co/NH2/ITO离子注入电极上有一对良好的氧化还原峰,峰电位分别为Epc=-0.2 V,Epa=-0.01 V(vsAg/AgCl)。该修饰电极对H2O2具有催化作用,可以用作H2O2的生物传感器,峰电流与H2O2的浓度分别在1.0×10-10~2.0×10-8mol/L和2.0×10-8~1.0×10-7mol/L范围内呈线性关系,线性回归方程分别为Ip(mA)=2.2986+0.06632c(nmol/L)和Ip(mA)=3.5788+7.3053E-4c(nmol/L),相关系数分别为0.9972和0.9688。检出限为1.0×10-10mol/L。