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.     

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.     

Direct electrochemistry and electrocatalysis of horseradish peroxidase in alpha-zirconium phosphate nanosheet film

Alpha-zirconium phosphate nanosheets (ZrPNS) derived via the delamination of layered alpha-zirconium phosphate (alpha-ZrP) have been proven to be efficient support matrixes for the immobilization of horseradish peroxidase (HRP). X-ray powder diffraction (XRD) results revealed that ZrPNS in HRP-ZrPNS film remained unorderly structured for the effect of HRP. Fourier transform infrared (FTIR) spectra results revealed that HRP remained the secondary structure in HRP-ZrPNS film. The direct electrochemistry of HRP was realized in HRP-ZrPNS film on a glassy carbon electrode (GCE), showing a pair of well-defined, nearly reversible cyclic voltammetry (CV) peaks for the HRP heme Fe(III)/Fe(II) redox couple. The average surface concentration (Gamma(*)) of electroactive HRP in HRP-ZrPNS film was estimated to be 1.35x10(-10) mol cm(-2), which indicated a high loading of enzyme molecules in HRP-ZrPNS film. Based on these, a third generation reagentless biosensor was constructed for the determination of hydrogen peroxide (H(2)O(2)). The response time of the biosensor was less than 3 s, and the linear response range of the biosensor for H(2)O(2) was from 1.3x10(-6) to 1.6x10(-2) M with a correlation coefficient of 0.9997.     

辣根过氧化物酶在表面活性剂膜中的直接电化学

利用3种表面活性剂分别将辣根过氧化氢酶固定在裂解石墨棱面（edge-plane pyrolytic graphite,EPG)电极表面,研究了辣根过氧化物酶（HRP）中Fe(Ⅲ）／Fe（Ⅱ）电对与电极之间的直接电子传递过程以及酶催化双氧化还原过程。实验结果表明：（1）表面活性剂是一种固定酶的理想材料;（2）这种体系可能构造第三代生物传感器,对解释生物体代谢过程具有理论意义,对制备第三代生物传感器具有应用价值。     

1,4-二氧六环介质中辣根过氧化物酶电极的性能研究

用交联法制备辣根过氧化物酶(HRP)电极, 在1,4-二氧六环介质中研究其电化学行为。实验表明, 固定化的HRP在有机相中仍保持活性并可与电极进行直接电子传递, 因而能在没有其它电子传递体存在的条件下催化H~2O~2的电化学还原反应。当亚铁氰化物与酶共修饰至电极上之后, 它起着电子传递体的作用, 使HRP电极的性能大为改善。根据不同条件下得到的动力学参数, 讨论了影响酶电极性能的因素。     

Direct electrochemistry-based hydrogen peroxide biosensor formed from single-layer graphene nanoplatelet-enzyme composite film

A novel electrochemical sensing system for direct electrochemistry-based hydrogen peroxide biosensor was developed that relied on the virtues of excellent biocompatibility, conductivity and high sensitivity to the local perturbations of single-layer graphene nanoplatelet (SLGnP). To demonstrate the concept, the horseradish peroxidase (HRP) enzyme was selected as a model to form the SLGnP-TPA (tetrasodium 1,3,6,8-pyrenetetrasulfonic acid)-HRP composite film. The single-layer graphene composite film displayed a pair of well-defined and good reversible cyclic voltammetric peak for Fe(III)/Fe(II) redox couple of HRP, reflecting the enhancement for the direct electron transfer between the enzyme and the electrode surface. Analysis using electrochemical impedance spectroscopy (EIS) revealed that electrostatic attractions existed between graphene monolayers and enzyme molecules. The intimate graphene and enzyme interaction was also observed using scanning electron microscopy (SEM), which resulted in the special properties of the composite film. Ultraviolet visible spectroscopy (UV-vis) indicated the enzyme in the composite film retained its secondary structure similar to the native state. The composite film demonstrated excellent electrochemical responses for the electrocatalytic reduction of hydrogen peroxide (H₂O₂), thus suggesting its great potential applications in direct electrochemistry-based biosensors.     

辣根过氧化物酶的电化学固定化及其对H2O2的生物电催化还原作用

利用电化学固定化方法制备了聚吡咯/辣根过氧化物酶(PP/HRP)膜电极,并研究了其电化学行为。在除氧的磷酸盐缓冲液介质中,PP/HRP电极加速H₂O₂的还原,归因于酶加成物的直接电子传递。探索HRP与电子传递体K₄Fe(CN)₆在聚吡咯(PP)膜中的同时固定化条件及其膜电极的电化学行为,实验证实,K₄Fe(CN)₆在酶膜中的存在使得H₂O₂的还原电位强烈正移,在-0.05V的工作电位下能对H₂O₂进行检测,相应的电极过程可用间接氧化还原催化机理解释。     

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.     

GCE/DNA/PFS/RTIL/HRP修饰电极的制备及其电催化行为

将辣根过氧化物酶（HRP）固定在室温离子液体（RTIL）/聚二茂铁硅烷（PFS）/DNA复合材料修饰的玻碳电极（GCE）表面,构建了GCE/DNA/PFS/RTIL/HRP修饰电极,详细地研究了该修饰电极的电催化行为,优化了电解质溶液的pH值和RTIL的体积对催化过氧化氢（H2O2）的影响。 电化学实验结果表明,DNA、PFS和RTIL复合膜既为HRP提供了一个生物兼容的微环境;又有效地促进了电子在HRP和电极表面之间的传递。 在最优实验条件下,该修饰电极对H2O2具有快速的催化响应,在2 s内即可达到稳态电流的95%,其响应在3.25 μmol/L~1.47 mmol/L（r=0.999,n=10）和1.86~5.35 mmol/L（r=0.996,n=12）范围内呈良好的线性关系,检出限为0.86 μmol/L。 该传感器灵敏度高、重现性和稳定性好。 此外,该修饰电极还能催化O2还原。     

酶的光谱电化学研究（Ⅰ）：辣根过氧化物酶的电化学性质

利用薄层光谱电化学技术研究了辣根过氧化物酶(HRP)及其化合物的氧化还原过程。指出HRP可在固体电极上进行直接电子传递,该电极反应不是酶中二硫键的还原,而是血红素辅基中心金属离子的氧化态转变。测定了HRP(Fe~(3+)/Fe~(2+))电对的标准氧化还原电位和电化学动力学参数,讨论了HRP氧化性中间物的电化学性质。     

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.     

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 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).     

固定化辣根过氧化物酶在有机/水混合溶液中的电化学

用魔芋多糖(KGM)将辣根过氧化物酶(HRP)固定在玻碳电极(GCE)表面, 制备了HRP-KGM膜修饰电极. 在乙醇等亲水性有机溶剂与水的混合溶液中, 包埋在KGM中的HRP 可以与电极发生直接电子传递, 且能催化还原过氧化氢、氢过氧化异丙基苯、氢过氧化叔丁基、过氧化丁酮等过氧化物. HRP-KGM膜修饰电极具有较好的稳定性和重现性, 可用于这些物质的定量检测.     

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.     

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.     

A Calcium Solid State Ion Selective Minisensor Based on Lipid Films on ZnO Nanorods

This article describes the development of a calcium solid minisensor based on lipid films on ZnO nanorods. The lipid film was composed from dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylcic acid (DPPA). The calibration graph was logarithmically related to the concentration of calcium ions in the range from 10^?6 to 10^?3 M. The response times of the electrode were less than 5 s. No memory effects were observed for this electrode. The reproducibility of the electrode was less than 5 %. The electrode has shown good stability for a period of 1 month. The influence of a wide range of metals and anions as potential interferences was checked. The method was applied to the rapid detection of calcium ions in mineral waters.     

Adsorption behavior of glucose oxidase on a dipalmitoylphosphatic acid monolayer and the characteristics of the mixed monolayer at air/liquid interfaces

A dipalmitoylphosphatic acid (DPPA) monolayer at the air/liquid interface is used as a binding layer to incorporate glucose oxidase (GOx) from the subphase. The effects of the adsorption time of GOx on the behavior of the mixed DPPA/GOx monolayer and the relevant structure of the mixed LB film were studied using the characteristics of the pressure-area (pi-A) isotherm, Brewster angle microscopy (BAM), and atomic force microscopy (AFM). The experimental results show that two equilibrium states of GOx adsorption exist in the presence of a DPPA monolayer. The first equilibrium stage occurs at tens of minutes after spreading of DPPA, and a surface pressure of ca. 7.5 mN/m is obtained. The second equilibrium stage approaches slowly, and a higher equilibrium surface pressure (ca. 16 mN/m) was obtained at ca. 8 h after the first stage. The BAM and AFM images show that, after the second equilibrium stage is reached, a more condensed phase and rough morphology are obtained on the mixed DPPA/GOx monolayer, indicating a higher amount of GOx incorporated into the mixed film. For the first equilibrium stage of GOx adsorption, DPPA molecules can still pack regularly and closely under compression, suggesting that GOx molecules are mainly located beneath the DPPA monolayer at the compressed state. A more uniform phase was detected on a film prepared after the first equilibrium stage was reached. The present result indicates that distinct structures and properties of mixed DPPA/GOx films can be prepared from the various stages of GOx adsorption.     

Electrografted Poly(N‐mercaptoethyl acrylamide) and Au Nanoparticles‐Based Organic/Inorganic Film: A Platform for the High‐Performance Electrochemical Biosensors

In this study, we describe the use of the combination of eletrografting poly(N‐mercaptoethyl acrylamide) and Au nanoparticles in the construction of high‐performance biosensors. The poly(N‐mercaptoethyl acrylamide) was electrografted onto the glassy carbon electrode surface, which provided a strongly adhering primer film for the stable attachment of Au nanoparticles and horseradish peroxidase (HRP) enzymes. The performances of the biosensors based on the HRP immobilized in the Au/poly(N‐mercaptoethyl acrylamide) composite film were investigated. A couple of redox peaks were obtained, indicating that the Au nanoparticles could facilitate the direct‐electron transfer between HRP and the underlying electrode. The biosensor showed an excellent electrocatalytic activity toward the reduction of hydrogen oxide and long‐term stability, owing to the stable electrografted film and biocompatible Au nanoparticles. Our results demonstrate that the combination of electrografting and Au nanoparticles provides a promising platform for the immobilization of biomolecules and analysis of redox enzymes for their sensing applications.     

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).