Mediatorless amperometric bienzyme glucose biosensor based on horseradish peroxidase and glucose oxidase cross-linked to multiwall carbon nanotubes

We report on a bienzyme-channeling sensor for sensing glucose without the aid of mediator. It was fabricated by cross-linking horseradish peroxidase (HRP) and glucose oxidase (GOx) on a glassy carbon electrode modified with multiwalled carbon nanotubes (MWNTs). The bienzyme was cross-linked with the MWNTs by glutaraldehyde and bovine serum albumin. The MWNTs were employed to accelerate the electron transfer between immobilized HRP and electrode. Glucose was sensed by amperometric reduction of enzymatically generated H₂O₂ at an applied voltage of ?50 mV (vs. Ag/AgCl). Factors influencing the preparation and performance of the bienzyme electrode were investigated in detail. The biosensor exhibited a fast and linear response to glucose in the concentration range from 0.4 to 15 mM, with a detection limit of 0.4 mM. The sensor exhibited good selectivity and durability, with a long-term relative standard deviation of <5 %. Analysis of glucose-spiked human serum samples yielded recoveries between 96 and 101 %.

Amperometric choline biosensor fabricated through electrostatic assembly of bienzyme/polyelectrolyte hybrid layers on carbon nanotubes

We report a flow injection amperometric choline biosensor based on the electrostatic assembly of the choline oxidase (ChO) enzyme and a bienzyme of ChO and horseradish peroxidase (HRP) onto multi-wall carbon nanotubes (MWCNT) modified glassy carbon (GC) electrodes. These choline biosensors were fabricated by immobilization of enzymes on the negatively charged MWCNT surface through alternately assembling a cationic poly(diallydimethylammonium chloride) (PDDA) layer and an enzyme layer. Using this layer-by-layer assembling approach, a bioactive nanocomposite film of PDDA/ChO/PDDA/HRP/PDDA/CNT (ChO/HRP/CNT) and PDDA/ChO/PDDA/CNT (ChO/CNT) was fabricated on the GC surface. Owing to the electrocatalytic effect of carbon nanotubes, the measurement of faradic responses resulting from enzymatic reactions has been realized at low potential with acceptable sensitivity. The ChO/HRP/CNT biosensor is more sensitive than the ChO/CNT one. Experimental parameters affecting the sensitivity of biosensors, e.g., applied potential, flow rate, etc., were optimized and potential interference was examined. The response time for this choline biosensor is fast (few seconds). The linear range of detection for the choline biosensor is from 5.0 x 10(-5) to 5.0 x 10(-3) M and the detection limit is about 1.0 x 10(-5) M.     

Silica‐Polyaniline Based Bienzyme Cholesterol Biosensor: Fabrication and Characterization

In the present investigation, silica‐polyaniline based bienzyme cholesterol biosensor is fabricated through a simple one‐step electrochemical method. The one‐step fabrication process involves electrochemical polymerization of N[3‐(trimethoxysilyl)propyl]aniline to result poly(N[3‐(trimethoxysilyl)propyl]aniline) (PTMSPA) and simultaneous immobilization of two enzymes, horseradish peroxidase (HRP) and cholesterol oxidase (ChOx) into PTMSPA matrix. The modified electrode is designated as PTMSPA‐HRP/ChOx‐ME. PTMSPA facilitates direct electron transfer between the electrode surface and the active redox centers of HRP. This enables the operation of a biosensor at a low working potential of about ?150 mV (vs. Ag/AgCl) for the detection of hydrogen peroxide. The PTMSPA‐HRP/ChOx‐ME demonstrates excellent analytical performance for the detection of cholesterol between 1 and 25 mM with high sensitivity and selectivity. PTMSPA possesses features suited for the fabrication of third‐generation biosensors.     

纳米聚苯胺修饰石墨电极的葡萄糖双酶传感器

用循环伏安法在石墨电极上制得纳米纤维聚苯胺, 并在其上固定葡萄糖氧化酶(GOD)和辣根过氧化物酶(HRP)制备葡萄糖双酶传感器. 用交流阻抗、SEM等技术对其进行表征; 考察了各种因素对双酶电极响应电流的影响以及双酶电极的稳定性. 该传感器对葡萄糖响应电流的测定在0.05 V(vs SCE)下进行, 有效避免了电活性物质的影响, 线性响应范围为0.05-2.0 mmol·L-1.     

Fabrication of Bienzymatic Glucose Biosensor Based on Novel Gold Nanoparticles‐Bacteria Cellulose Nanofibers Nanocomposite

An exploration of gold nanoparticles–bacterial cellulose nanofibers (Au‐BC) nanocomposite as a platform for amperometric determination of glucose is presented. Two enzymes, glucose oxidase (GOx) and horseradish peroxidase (HRP) were immobilized in Au‐BC nanocomposite modified glassy carbon electrode at the same time. A sensitive and fast amperometric response to glucose was observed in the presence of electron mediator (HQ). Both of GOx and HRP kept their biocatalytic activities very well in Au‐BC nanocomposite. The detection limit for glucose in optimized conditions was as low as 2.3 µM with a linear range from 10 µM to 400 µM. The biosensor was successfully applied to the determination of glucose in human blood samples.     

A novel amperometric biosensor based on gold nanoparticles-mesoporous silica composite for biosensing glucose

We report a novel bienzyme biosensor based on the assembly of the glucose oxidase (GOD) and horseradish peroxidase (HRP) onto the gold nanoparticles encapsulated mesoporous silica SBA-15 composite (AuNPs-SBA-15). Electrochemical behavior of the bienzyme bioconjugates biosensor is studied by cyclic voltammetry and electrochemical impedance spectroscopy. The results indicate that the presence of mesoporous AuNPs-SBA-15 greatly enhanced the protein loadings, accelerated interfacial electron transfer of HRP and the electroconducting surface, resulting in the realization of direct electrochemistry of HRP. Owing to the electrocatalytic effect of AuNPs-SBA-15 composite, the biosensor exhibits a sensitive response to H₂O₂ generated from enzymatic reactions. Thus the bienzyme biosensor could be used for the detection of glucose without the addition of any mediator. The detection limit of glucose was 0.5 μM with a linear range from 1 to 48 μM. Supported by the National Natural Science Foundation of China (Grant Nos. 20635020 & 90606016)     

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.     

明胶固定辣根过氧化物酶制备H_2O_2传感器

用明胶将辣根过氧化物酶(HRP)固定于多壁碳纳米管(MWNT)和茜素红(AR)修饰的玻碳(GC)电极上,制成HRP生物传感器(HRP/AR/MWNT/GC),然后在3%戊二醛(GA)中进行交联改性,以克服明胶膜易溶胀的缺点,并提高膜的稳定性.同时详细探讨了该传感器对H2O2的响应性能,并优化了实验条件.结果表明,该传感器对H2O2的线性响应范围为5.0×10-6～1.0×10-3mol/L,线性相关系数为0.9932,检出限为1.0×10-7mol/L,且放于4℃环境30d后,峰电流值约为原来的72.1%.该传感器响应快速,灵敏度高,且具有良好的重现性、稳定性及较长的使用寿命,具有潜在的应用价值.     

Ceramic Carbon/Polypyrrole Materials for the Construction of Bienzymatic Amperometric Biosensor for Glucose

Amperometric enzymatic biosensors have high selectivity and simplicity in use. It has advantages over other analytical methods in biochemistry, pharmacology, so it evokes strong interests1,2. Generally, the detection mode involved in oxidase based biosensors is often based on the electrochemical detection of hydrogen peroxide directly3,4. However the direct oxidation of hydrogen peroxide requires a relative high working potential (exceeding ca. 0.6 V vs. SCE), at which many biological sub…     

一步电沉积制备交联型有机-无机生物杂化膜构筑双酶传感器的研究

利用一步电沉积法,以含有环氧基团的γ-环氧丙氧丙基三甲氧基硅烷（GPTMS）为无机杂化试剂和功能性交联试剂,通过壳聚糖（Chitosan,CS）、辣根过氧化物酶（HRP）和葡萄糖氧化酶（GOD）分子中-NH2与环氧基团的反应,在金电极表面原位制备交联型有机-无机生物杂化膜,得到共固定HRP和GOD的新型双酶生物传感器．实验证实了这种有机-无机生物杂化膜在不同酸、碱条件下都具有高的稳定性和耐用性,克服了CS酸溶的不足,从而扩大了其使用范围．在葡萄糖检测中,交联型双酶传感器HRP-GOD／GPTMS／CS,Au比无交联的双酶传感器HRP-GOD／CS／Au具有更高的灵敏度、更宽的线性范围,其线性范围为1μmol／L-351μmol／L,检期4限为0．3μmol／L．     

A Chemiluminescence Optical Fiber Glucose Biosensor Based on Co-immobilizing Glucose Oxidase and Horseradish Peroxidase in a Sol-gel Film

IntroductionIn recent years chemiluminescence (CL)biosensor prepared by immobilization of a sensitivereagent such as peroxidase or oxidase onto a solidmatrix has attracted much attention due to the highsensitivity of the chemiluminescent reaction of thesensitive reagent even with a simple instrument.Generally,CL biosensors can be divided into twocategories.One consists of hydrogen peroxide sen-sors prepared by immobilizing a kind of peroxidaseonto a suitable solid support[1,2 ] ,and the immo…     

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.     

Construction of bienzyme biosensors based on combination of the one-step electrodeposition and covalent-coupled sol-gel process

Horseradish peroxidase (HRP) and glucose oxidase (GOD) bienzyme biosensor was constructed by in-situ formation of the organic-inorganic biocomposite film based on the one-step electrodeposition and covalent-coupled sol-gel process. The electrodeposition was performed in the solution containing functional inorganic precursor possessing the epoxy groups, γ-glycidoxypropyltrimethoxysiloxane (GPTMS), a biopolymer chitosan (CS), HRP and GOD. The covalent-coupled sol-gel process was formed by self-hydrolysis and self-condensation of GPTMS, followed by in-situ covalent cross-linking of CS, HRP and GOD through covalent reaction between amino groups and epoxy groups. The developed bienzyme biosensor presented high stability in acidic solution owing to the covalent-coupled organic-inorganic hybridization. Compared with the non-hybrid HRP-GOD/CS/Au electrode, the bienzyme biosensor of HRP-GOD/GPTMS/CS/Au showed improved sensitivity and a wider linear range for the determination of glucose. The linear response of the developed HRP-GOD/GPTMS/CS/Au biosensor for the determination of glucose ranged from 1 to 351 μmol/L with a detection limit of 0.3 μmol/L.     

Effect of interfering substances on current response of recombinant peroxidase and glucose oxidase-recombinant peroxidase modified graphite electrodes.

Graphite electrodes have been modified with different forms of horseradish peroxidase (HRP). These included native HRP, wild-type recombinant HRP, and two single-point recombinant HRP mutants, N70V and N70D. The mediator-less response of these electrodes to H2O2 was studied indicating that electrodes modified with recombinant HRP forms are more stable than those modified with native HRP. Various interfering compounds were investigated for their effect on the current response to H2O2. It was found that interferences such as acetaminophen and dopamine affected the response by mediating the electron transfer (ET) between graphite and peroxidases. The mediating behaviour manifested itself as an increased current of the electrode to H2O2. The interfering effect was less pronounced for the electrodes modified with recombinant HRPs possessing better electronic coupling with the graphite surface. The interfering behaviour of acetaminophen on the response for glucose with the bienzyme electrode containing co-immobilised glucose oxidase and HRP was mainly ascribed to mediation of ET between graphite and HRP. It was experimentally proven that a high efficiency of direct ET between graphite and recombinant HRP substantially reduces the interfering effect of acetaminophen.     

A Peroxidase‐Based Biosensor Supported by Nanoporous Magnetic Silica Microparticles for Acetaminophen Biotransformation and Inhibition Studies

Magnetized nanoporous silica based microparticles (MMPs) were used for horseradish peroxidase (HRP) immobilization and applied for amperometric peroxidase‐based biosensor development. A magnetized carbon paste electrode permitted the MMPs attraction. The biosensor was applied to the investigation of the enzymatic oxidation of acetaminophen (paracetamol). The biosensor operated at low applied potential and the signal corresponded to the electroreduction of N‐acetylbenzoquinoneimine (NAPQI) generated by the enzyme HRP in the presence of hydrogen peroxide. The biosensor allowed performing the quantitation of acetaminophen in the micromolar concentration range and the comparative study of thiols which inhibited the biosensor response. Distinct inhibition results were observed for HRP entrapped in the silica microparticles compared to the soluble HRP.     

Detection of hydrogen peroxide and glucose by enzyme product precipitation on sensor surface

Biocatalysed precipitation of an insoluble product accumulated on the enzyme-modified electrode surface was applied as the amplification path for low concentration sensing of hydrogen peroxide and glucose. Sensitive electrochemical and quartz-crystal microbalance (QCM) biosensors based on biocatalytic precipitation were developed. A horseradish peroxidase (HRP) monolayer-modified electrode was used to sense H₂O₂ via the oxidation of 4-chloro-1-naphthol (4CN) forming insoluble benzo-4-chlorocyclohexadienone. Additionally, the bienzyme system employed glucose oxidase (GOx) linked to HRP/4CN. The amount of the precipitate assembled on the sensing surface corresponded to the concentration of analytes and to the length of the incubation interval. The precipitated deposits were followed as a change of impedance using cyclic voltammetry (CV), mass change was determined continuously using a microgravimetric quartz-crystal microbalance, and optical microscopy enabled the visualisation of the precipitate. Regeneration of the enzyme-modified electrode was performed using cathodic reduction of the insoluble product. Thus, a simple biosensor for multiple analyses with low detection limits and of low cost can be developed.     

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.     

Optimization of Electrochemical Total Creatine Kinase Biosensors Based on Three‐Au‐Electrode

For point‐of‐care examination, total CK (creatine kinase: adenosine‐5‐triphosphate‐creatine phosphotransferase, EC 2.7.3.2) biosensors were developed and optimized. The biosensors were fabricated with three‐Au‐electrode system modified with polyvinylpyridine‐osmium‐wired horseradish peroxidase (PVP‐Os‐HRP) redox polymer film. The reagents were separately immobilized on the single layer biosensor and double layer biosensor which contained lens paper layer and the surface layer of the working electrode. The mediator, the working potential, the structure of working electrode and the stabilizer agent were studied. The biosensor with double reagent layer showed good stability at room temperature (≥2 months) and the biosensor with single reagent layer had excellent response signal (a sensitivity of 11 nA L U^?1 cm^?2).     

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.