Mesoporous Materials Promoting Direct Electrochemistry and Electrocatalysis of Horseradish Peroxidase

The direct electron transfer and electrocatalysis of horseradish peroxidase (HRP) immobilized on hexagonal mesoporous silicas (HMS) matrix was studied. The interaction between HRP and HMS was examined by using Fourier transform infrared spectroscopy, nitrogen adsorption isotherms and electrochemical methods. The immobilized HRP at a modified glassy carbon electrode showed a good direct electrochemical behavior, which depended on the specific properties of the HMS. Two couples of redox peaks corresponding to Fe(III) to Fe(II) conversion of the HRP intercalated in the mesopores and adsorbed on the external surface of the HMS were observed with the formal potentials of ?0.315 and ?0.161 V in 0.1 M pH 7.0 PBS, respectively. The amount of HRP intercalated in the mesopores of HMS proved to be related to the pore size. The HRP intercalated in the mesopores showed a surface controlled electrode process with a single proton transfer. The immobilized HRP displayed an excellent electrocatalytic response to the reduction of hydrogen peroxide (H₂O₂) without the aid of an electron mediator. The HMS provided a novel matrix for protein immobilization and direct electron transfer study of the immobilized protein.     

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.     

辣根过氧化物酶在壳聚糖和无掺杂金刚石纳米粒子共沉积膜上的固定及其直接电化学

无掺杂的金刚石纳米粒子(UND)可以和壳聚糖共沉积到玻碳电极表面,形成壳聚糖-UND复合膜.此复合膜可以通过吸附的方法,固定辣根过氧化物酶(HRP),并且能够实现HRP的直接电化学,保持HRP对过氧化氢的良好催化能力.为了进一步研究此复合膜及HRP在此复合膜上的电化学活性,运用扫描电子显微镜(SEM)、全反射红外(ATR)、电化学交流阻抗、循环伏安等技术来跟踪各步修饰过程.结果表明,此复合膜有良好的生物相容性,能够很好的保持HRP的生物活性.     

Direct Electron Transfer of Horseradish Peroxidase in Gellan Gum–Hydrophilic Ionic Liquid Gel Film

A new composite film of microbial exocellular polysaccharide‐gellan gum (GG) and hydrophilic room temperature ionic liquid 1‐butyl‐3‐methyl‐imidazolium tetrafluoroborate (BMIMBF₄) was firstly used as an immobilization matrix to entrap horseradish peroxidase (HRP), and its properties were studied by UV/vis spectroscopy, cyclic voltammetry and electrochemical impedance spectroscopy. The results showed that BMIMBF₄ could promote the electron transfer between HRP and electrode surface, and the existence of GG could successfully immobilize BMIMBF₄ on the electrode surface with improved stability. HRP–BMIMBF₄–GG/GCE exhibited a pair of well‐defined and quasireversible cyclic voltammetric peaks in 0.1 M pH 7.0 phosphate buffer solutions at 1.8 V/s, which was the characteristic of HRP Fe(III)/Fe(II) redox couples. The formal potentials (E°′) was ?0.368 V (vs. SCE) and the peak‐to‐peak potential separation (ΔE_P) was 0.058 V. The peak currents were five times as large as those of HRP–GG/GCE. The average surface coverage (Γ*) and the apparent Michaelis‐Menten constant (K_m) were 4.5×10^?9 mol/cm² and 0.67 μM, respectively. The electron transfer rate constant was estimated to be 15.8 s^?1. The proposed electrode showed excellent electrocatalytic activity towards hydrogen peroxide (H₂O₂). The linear dynamic range for the detection of H₂O₂ was 0.05–0.5 μM with a correlation coefficient of 0.9945 and the detection limit was estimated at about 0.02 μM (S/N=3). BMIMBF₄–GG composite film was promising to immobilize other redox enzymes or proteins and attain their direct electrochemistry.     

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.     

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

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

Direct electrochemistry and electrocatalysis of horseradish peroxidase immobilized in hyaluronic acid and single walled carbon nanotubes composite film

Direct electrochemistry and electrocatalysis of horseradish peroxidase (HRP) immobilized on a hyaluronic acid (HA)-single walled carbon nanotubes (SCNs) composite film coated glassy carbon electrode (GCE) was studied for the first time. HRP entrapped in the SCNs-HA composite film exhibited a pair of well-defined, quasi-reversible cyclic voltammetric peaks in a 0.1 M phosphate buffer solution (pH 7.0). Formal potential vs. standard calomel electrode (E°′) was −0.232 V, and E°′ was linearly dependent on the solution pH indicating that the electron transfer was proton-coupled. The current is linearly dependent on the scan rate, indicating that the direct electrochemistry of HRP in that case is a surface-controlled electrode process. UV-VIS spectrum suggested HRP retained its original conformation in the SCNs-HA film. Immobilized HRP showed excellent electrocatalysis in the reduction of hydrogen peroxide (H₂O₂).     

Determination of Hydrogen Peroxide by Electrochemiluminescence Using a Chitosan–graphene Composite Film Doped Cadmium-Tellurium Quantum Dot Modified Glassy Carbon Electrode

Here is reported the novel determination of hydrogen peroxide by electrochemiluminescence using a chitosan–graphene composite film doped cadmium-tellurium quantum dot modified glassy carbon electrode. The cadmium-tellurium quantum dots were studied by absorption and fluorescence spectroscopy. Scanning electron microscopy and electrochemical impedance spectroscopy were used to characterize the structure morphology of the composite matrix. The electrochemiluminescence emission was linear with the concentration of hydrogen peroxide in the range of 3.5?×?10^?7 to 1.1?×?10^?5?M with a determination limit of 2.1?×?10^?7?M. Furthermore, the modified electrode showed excellent reproducibility and stability.     

Direct electrochemistry and electrocatalysis of horseradish peroxidase immobilized in Nafion-RTIL composite film

The composite film based on Nafion and hydrophobic room-temperature ionic liquid (RTIL) 1-butyl-3-methyl-imidazolium hexafluorophosphate ([bmim] PF₆) was explored. Here, Nafion was used as a binder to form Nafion-ionic liquids composite film and help [bmim] PF₆ effectively adhered on glassy carbon (GC) electrode. X-ray photoelectron spectroscopy (XPS), cyclic voltammtery (CV) and electrochemical impedance spectroscopy (EIS) were used to characterize this composite film, showing that the composite film can effectively adhere on the GC electrode surface through Nafion interacting with [bmim] PF₆ and GC electrode. Meanwhile, doping [bmim] PF₆ in Nafion can also effectively reduce the electron transfer resistance of Nafion. The composite film can be readily used as an immobilization matrix to entrap horseradish peroxidase (HRP). A pair of well-defined redox peaks of HRP was obtained at the HRP/Nafion-[bmim] PF₆ composite film-modified GC electrode through direct electron transfer between the protein and the underlying electrode. HRP can still retain its biological activity and enhance electrochemical reduction towards O₂ and H₂O₂. It is expected that this composite film may find more potential applications in biosensors and biocatalysis.     

Bioelectrochemical Characterization of Horseradish and Soybean Peroxidases

Heme peroxidase are ubiquitous enzymes catalyzing the oxidation of a broad range of substrates by hydrogen peroxide. In this paper the bioelectrochemical characterization of horseradish peroxidase (HRP) and soybean peroxidase (SBP), belonging to class III of the plant peroxidase superfamily, was studied. The homogeneous reactions between peroxidases and some common redox mediators in the presence of hydrogen peroxide have been carried out by cyclic voltammetry. The electrochemical characterization of the reactions involving enzyme, substrate and mediators concentrations allowed us to calculate the kinetic parameters for the substrate–enzyme reaction (K_MS) and for the redox mediator–enzyme reaction (K_MM). A full characterization of the direct electron transfer kinetic parameters between the electrode and enzyme active site was also performed by opportunely modeling data obtained from cyclic voltammetry and square wave voltammetry experiments. The experimental data obtained with immobilized peroxidases show enhanced direct electron transfer and excellent electrocatalytical performance for H₂O₂. Despite the structural similarities and common catalytic cycle, HRP and SBP exhibit differences in their substrate affinity and catalytic efficiency. Basing on our results, it can be concluded that peroxidase from soybean represents an interesting alternative to the classical and largely employed one obtained from horseradish as biorecognition element of electrochemical mediated biosensors.     

A hydrogen peroxide biosensor with high stability based on gelatin-multiwalled carbon nanotubes modified glassy carbon electrode

A biosensor with high stability was prepared to determine hydrogen peroxide (H₂O₂). This hydrogen peroxide biosensor was obtained by modifying glassy carbon electrode (GCE) with a composite film composed of gelatin-multiwalled carbon nanotubes. Catalase (Cat) was covalently immobilized into gelatin-multiwalled carbon nanotubes modified GCE through the well-known glutaraldehyde (GAD) chemistry in order to enhance the stability of electrodes. The enzyme sensor can achieve direct electrochemical response of hydrogen peroxide. The cyclic voltammograms at different scan rates, electrochemical impedance spectroscopy (EIS), and scanning electron microscope (SEM) tests indicate that the enzyme sensor performs positively on increasing permeability, reducing the electron transfer resistance, and improving the electrode performance. The linear response of standard curve for H₂O₂ is in the range of 0.2 to 5.0 mM with a correlation coefficient of 0.9972, and the detection limit of 0.001 mM. A high operational and storage stability is demonstrated for the biosensor. The peak potential at room temperature in two consecutive weeks stays almost consistent, and the enzyme activity is kept stable even after 30 days in further study.     

Layer-by-layer assembled multilayer of graphene/Prussian blue toward simultaneous electrochemical and SPR detection of H2O2

A new type of chemically converted graphene sheets, cationic polyelectrolyte-functionalized ionic liquid decorated graphene sheets (PFIL-GS) composite, was synthesized and characterized by Ultraviolet-visible (UV-vis) absorption, Fourier transform infrared, and Raman spectroscopy. It was found that the presence of PFIL enabled the formation of a very stable aqueous dispersion due to the electrostatic repulsion between PFIL modified graphene sheets. With respect to the excellent dispersibility of this material, we have fabricated a novel PFIL-GS/Prussian blue (PB) nanocomposite multilayer film via classic layer-by-layer (LBL) assembly. The assembly process was confirmed by UV-vis spectroscopy and surface plasmon resonance (SPR) spectroscopy, which showed linear responses to the numbers of the deposited PFIL-GS/PB bilayers. Moreover, the as-prepared composite films were used to detect hydrogen peroxide (H₂O₂) by electrochemical surface plasmon resonance (EC-SPR) spectroscopy. This real time EC-SPR technique can provide simultaneous monitoring of both optical SPR signal and electrochemical current responses upon injecting H₂O₂ into the reaction cell. The experimental results revealed that both the electrochemical and SPR signal exhibited splendid linear relationship to the concentration of the injected H₂O₂, and the detection limit could be up to 1 μM.     

Direct electron transfer of horseradish peroxidase and its electrocatalysis based on carbon nanotube/thionine/gold composites

Horseradish peroxidase (HRP) was incorporated into multiwalled carbon nanotube/thionine/Au (MTAu) composite film by electrostatic interactions between positively charged HRP and negatively charged MTAu composite. The results of electrochemical impedance spectroscopy (EIS) confirmed adsorption of HRP on the surface of MTAu modified GC electrode. Moreover, the electrochemical results showed that HRP retained its bioactivity and bioelectrocatalytical activity, and also showed good direct electron transfer behavior on such a composite film.     

Electrocatalysis of Hemoglobin in ZnO Nanoparticle/Ionic Liquid Composite Film Modified Glassy Carbon Electrode

A nanobiocompatible composite containing hemoglobin (Hb), ZnO nanoparticles (nano‐ZnO) and ionic liquid 1‐butyl‐3‐methylimidazolium hexafluorophosphate (BMIMPF₆) was fabricated and further modified on the glassy carbon electrode (GCE). The electrochemical behaviours of Hb in the composite film were carefully studied and a pair of quasi‐reversible redox peaks appeared in pH 7.0 phosphate buffer solution, which was attributed to the electrode reaction of Hb heme Fe(III)/Fe(II) redox couple. The presences of nano‐ZnO and BMIMPF₆ in the film can retain the bioactivity of Hb and greatly enhance the direct electron transfer of Hb. The immobilized Hb showed high stability and good electrocatalytic ability to the reduction of hydrogen peroxide and O₂.     

Direct electron transfer of horseradish peroxidase at Co<Subscript>3</Subscript>O<Subscript>4</Subscript>–graphene nanocomposite modified electrode and electrocatalysis

In this paper, the mixture of Co₃O₄–graphene nanocomposite and horseradish peroxidase (HRP) was spread on the surface of carbon ionic liquid electrode (CILE). Then, Nafion film was used for the immobilization. The results of spectroscopy proved that HRP kept up its native structure in the complex material. Direct electrochemistry of HRP resulted in a couple of quasi-reversible redox waves on cyclic voltammograms, reflecting the realization of direct electron transfer of HRP with electrode. The improvement in electrochemical responses was due to the usage of highly conductive Co₃O₄–graphene nanocomposite with biocompatible interface. Electrochemical parameters such as the electron transfer coefficient (α) was estimated as 0.47, and the apparent heterogeneous electron transfer rate constant (k _s) was calculated as 2.90 s^?1. The HRP modified electrode exhibited good electrochemical catalytic ability toward the reduction of trichloroacetic acid and NaNO₂. As a consequence, an updated third-generation electrochemical HRP biosensor with Co₃O₄–GR/CILE was constructed successfully.     

Electron transfer reactivity and the catalytic activity of horseradish peroxidase incorporated in dipalmitoylphosphatidic acid films

Horseradish peroxidase (HRP) was incorporated in dipalmitoylphosphatidic acid (DPPA) to form a film and the film was modified on pyrolytic graphite electrode. UV-Vis spectra suggested that HRP in the film could keep its secondary structure similar to the native state. A pair of stable, well-defined, and quasi-reversible cyclic voltammetric peaks was observed with the formal potential at -276.2 mV (vs. saturated calomel electrode), characteristic of heme Fe(III)/Fe(II) redox couple of HRP. The apparent heterogeneous electron transfer rate constant and other electrochemical parameters were presented. The catalytic activity of HRP in DPPA film toward oxygen, hydrogen peroxide and nitric oxide were also examined.     

Composite film of carbon nanotubes and chitosan for preparation of amperometric hydrogen peroxide biosensor

A new amperometric biosensor for hydrogen peroxide was developed based on cross-linking horseradish peroxidase (HRP) by glutaraldehyde with multiwall carbon nanotubes/chitosan (MWNTs/chitosan) composite film coated on a glassy carbon electrode. MWNTs were firstly dissolved in a chitosan solution. Then the morphology of MWNTs/chitosan composite film was characterized by field-emission scanning electron microscopy. The results showed that MWNTs were well soluble in chitosan and robust films could be formed on the surface. HRP was cross-linked by glutaraldehyde with MWNTs/chitosan film to prepare a hydrogen peroxide biosensor. The enzyme electrode exhibited excellent electrocatalytic activity and rapid response for H₂O₂ in the absence of a mediator. The linear range of detection towards H₂O₂ (applied potential: −0.2 V) was from 1.67 × 10⁻⁵ to 7.40 × 10⁻⁴ M with correction coefficient of 0.998. The biosensor had good repeatability and stability for the determination of H₂O₂. There were no interferences from ascorbic acid, glucose, citrate acid and lactic acid.     

Preparation of Magnetic Ordered Mesoporous Carbon Composite and Its Application in Direct Electrochemistry of Horseradish Peroxidase

A novel magnetic ordered mesoporous carbon composite was prepared. Electrochemical measurements showed that the ordered mesoporous carbon composite provided an excellent matrix for the co‐adsorption of horseradish peroxidase (HRP). HRP could be separated and collected by the application of a magnetic field and its direct electron‐transfer could be achieved in the solution, not on the electrode thereby preventing the degradation of the enzyme. The cyclic voltammetric experimental results of HRP indicated that HRP displayed a pair of stable peaks with a formal potential of ?0.306 V in PBS. The resulting biosensor exhibited fast amperometric response to hydrogen peroxide.     

Electrochemical behavior of horseradish peroxidase on WS2 nanosheet‐modified electrode and electrocatalytic investigation

In this paper, a WS₂ nanosheet was modified on the surface of a carbon ionic liquid electrode (CILE), and horseradish peroxidase (HRP) was further fixed on the electrode with a Nafion film. Direct electrochemistry and bioelectrocatalysis of HRP incorporated on the modified electrode were investigated in detail. On Nafion/HRP/WS₂/CILE, a pair of well‐defined quasi‐reversible redox peaks appeared on the cyclic voltammogram, indicating that the presence of the WS₂ nanosheet on the electrode surface could provide a specific interface with large surface area for HRP and its direct electron transfer rate was greatly enhanced. The formal potential (E⁰) obtained was –0.179 V, which was the typical feature of heme Fe(III)/Fe(II) in HRP. The electron transfer coefficient (α) and the heterogeneous electron transfer rate constant (k_s) of HRP were calculated as 0.44 and 1.01 s^–1, respectively. This HRP‐modified electrode showed excellent electrocatalytic activity for the reduction of trichloroacetic acid and NaNO₂ with a wide linear range and low detection limit. Real samples were detected by this proposed method, indicating the successful fabrication of a new third‐generation electrochemical enzyme sensor utilizing the WS₂ nanosheet.     

Electrochemically Prepared Three‐dimensional Porous Nitrogen‐doped Graphene Modified Electrode for Non‐enzymatic Detection of Hydrogen Peroxide

A facile and green electrochemical method for the fabrication of three‐dimensional porous nitrogen‐doped graphene (3DNG) modified electrode was reported. This method embraces two consecutive steps: First, 3D graphene/polypyrrole (ERGO/PPy) composite was prepared by electrochemical co‐deposition of graphene and polypyrrole on a gold foil. Subsequently, the ERGO/PPy composite modified gold electrode was annealed at high temperature. Thus 3DNG modified electrode was obtained. Scanning electron microscopy (SEM), X‐ray photoelectron spectroscopy (XPS) and Raman spectroscopy were used to characterize the structure and morphology of the electrode. The electrode exhibits excellent electroanalytical performance for the reduction of hydrogen peroxide (H₂O₂). By linear sweep voltammetric measurement, the cathodic peak current was linearly proportional to H₂O₂ concentration in the range from 0.6 μM to 2.1 mM with a sensitivity of 1.0 μA μM⁻¹ cm⁻². The detection limit was ascertained to be 0.3 μM. The anti‐interference ability, reproducibility and stability of the electrode were carried out and the electrode was applied to the detection of H₂O₂ in serum sample with recoveries from 98.4 % to 103.2 %.