溶胶-凝胶壳聚糖/二氧化硅杂化复合膜固定辣根过氧化酶的H2O2电化学生物传感器的研制

利用溶胶 凝胶法制备壳聚糖 二氧化硅有机无机复合杂化膜,用于对辣根过氧化酶进行固定,制得测定H2O2的电流型生物传感器。以1mmol/LK4Fe(CN)6作为电子媒介体。研究了各种因素如壳聚糖与二氧化硅的比率、pH、温度、工作电位等对传感器响应电流的影响。计时电流法测定H2O2的线性范围为2.0×10-6～6.8×10-4mol/L,检出限为8.0×10-7mol/L。测得酶催化动力学参数米氏常数Km=0 87mmol/L。用该法对实际样品进行了测定。     

Immobilization of horseradish peroxidase to a nano-Au monolayer modified chitosan-entrapped carbon paste electrode for the detection of hydrogen peroxide

A procedure for fabricating an enzyme electrode has been described based on the effective immobilization of horseradish peroxidase (HRP) to a nano-scaled particulate gold (nano-Au) monolayer modified chitosan-entrapped carbon paste electrode (CCPE). The high affinity of chitosan entrapped in CCPE for nano-Au associated with its amino groups has been utilized to realize the use of nano-Au as an intermediator to retain high bioactivity of the enzyme. Hydrogen peroxide (H₂O₂) was determined in the presence of hydroquinone as a mediator to transfer electrons between the electrode and HRP. The HRP immobilized on nano-Au displayed excellent electrocatalytical activity to the reduction of H₂O₂. The effects of experimental variables such as the operating potential of the working electrode, mediator concentration and pH of measuring solution were investigated for optimum analytical performance by using an amperometric method. The enzyme electrode provided a linear response to hydrogen peroxide over a concentration range of 1.22×10⁻⁵-2.43×10⁻³ mol l⁻¹ with a sensitivity of 0.013 A l mol⁻¹ cm⁻² and a detection limit of 6.3 μmol l⁻¹ based on signal per noise =3. The apparent Michaelis-Menten constant (K_m^app) for the sensor was found to be 0.36 mmol l⁻¹. The lifetime, fabrication reproducibility and measurement repeatability were evaluated with satisfactory results. The analysis results of real sample by this sensor were in satisfactory agreement with those of the potassium permanganate titration method.     

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     

A hydrogen peroxide biosensor based on horseradish peroxidase/poly(L-leucine)/polydopamine modified glassy carbon electrode

A simple and practical sensor of hydrogen peroxide (H₂O₂) was designed successfully. The mixture of horseradish peroxidase (HRP) and chitosan (Chit) are effectively immobilized on the surface of poly-L-leucine/polydopamine modified glassy carbon electrode (PL-LEU/PDA/GCE). Under the optimum conditions, the biosensor based on HRP exhibits a fast amperometric response (within 3 s) to H₂O₂. The linear response range of the sensor is 0.5–952.0 μmol L^–1, with the detection limit of 0.1 μmol L^–1 (S/N = 3) and the sensitivity of 0.23 A L moL^–1 cm^–2. The apparent Michaelis–Menten constant (k _M ^app) of the biosensor is evaluated to be 0.12 mmol L^–1, which suggests that the HRP-Chit/PL-LEU/PDA/GCE shows a higher affinity for H₂O₂. The sensor exhibits good sensitivity, selectivity, stability and reproducibility. The proposed method has been successfully applied to the determination of H₂O₂ in practical samples.     

Reagentless amperometric biosensor highly sensitive to hydrogen peroxide based on the incorporation of Meldola Blue, fumed-silica and horseradish peroxidase into carbon paste

 A reagentless amperometric sensor highly sensitive to H₂O₂ has been prepared by incorporating fumed silica, horseradish peroxidase (HRP) and Meldola Blue into carbon paste. The efficient mediating ability to shift electrons between HRP and the carbon paste electrode via Meldola Blue was investigated by cyclic voltammetric and amperometric measurements. Reproducibility, response time, detection limit, selectivity and effects of applied potential, temperature and pH on the response of the sensor are reported. The high sensitivity of the sensor with a detection limit of 0.1 μmol/l arose from the high efficiency of the bioelectrocatalytic reduction of hydrogen peroxide via HRP and Meldola Blue. The dependence of the Michaelis-Menten constant on the applied potential and the mediator concentration has been investigated and the results are presented. Received: 20 December 1995/Revised: 13 March 1996/Accepted: 16 March 1996     

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.     

An amperometric biosensor based on the coimmobilization of horseradish peroxidase and methylene blue on a beta-type zeolite modified electrode

A new biosensor for the amperometric detection of hydrogen peroxide was developed based on the coimmobilization of horseradish peroxidase (HRP) and methylene blue on a beta-type zeolite modified glassy carbon electrode without the commonly used bovine serum albumin-glutaraldehyde. The intermolecular interaction between enzyme and zeolite matrix was investigated using FT-IR. The cyclic voltammetry and amperometric measurement demonstrated that methylene blue co-immobilized with HRP in this way displayed good stability and could efficiently transfer electrons between immobilized HRP and the electrode. The sensor responded rapidly to H2O2 in the linear range from 2.5 x 10(-6) to 4.0 x 10(-3) M with a detection limit of 0.3 microM. The sensor was stable in continuous operation.     

Amperometric immunosensor for probing complement III (C3) based on immobilizing C3 antibody to a nano-Au monolayer supported by sol-gel-derived carbon ceramic electrode

An electrochemical immunosensor based on nano-size particulate gold (nano-Au) monolayer as sensing interface has been developed for probing complement III (C₃). The thiol functional group-derived carbon ceramic electrode (CCE) was firstly constructed using (3-mercaptopropyl) trimethoxy silane (MPTMOS) as sol-gel monomer. The stable nano-Au monolayer was obtained resulting from covalent combination between nano-Au and thiol group on the surface of CCE. The nano-Au monolayer formed was utilized as a sensing platform for the immobilization of C₃ antibody (anti-C₃) and subsequent immunoreaction. A competitive immunoassay format was adopted with horseradish peroxidase (HRP)-C₃ as a tracer, hydroquinone and hydrogen peroxide as the enzymatic substrates. The dynamic concentration range for C₃ is 0.08-5.6 μg ml⁻¹. The feasibility of regenerating nano-Au monolayer for consecutive assays was demonstrated by a simple chemical treatment after each determination. The high stability of formed nano-Au monolayer, readily adsorptive immobilization of antibody on nano-Au monolayer, efficient activity retention of loading immunoreactants as well as the simple operation for the formation of nano-Au monolayer make proposed methodology an attractive alternative for the designing new-type immunosensors.     

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     

A phenol biosensor based on immobilizing tyrosinase to modified core-shell magnetic nanoparticles supported at a carbon paste electrode

A phenol biosensor was developed based on the immobilization of tyrosinase on the surface of modified magnetic MgFe₂O₄ nanoparticles. The tyrosinase was first covalently immobilized to core-shell (MgFe₂O₄-SiO₂) magnetic nanoparticles, which were modified with amino group on its surface. The resulting magnetic bio-nanoparticles were attached to the surface of carbon paste electrode (CPE) with the help of a permanent magnet. The immobilization matrix provided a good microenvironment for the retaining of the bioactivity of tyrosinase. Phenol was determined by the direct reduction of biocatalytically generated quinone species at −150 mV versus SCE. The resulting phenol biosensor could reach 95% of steady-state current within 20 s and exhibited a high sensitivity of 54.2 μA/mM, which resulted from the high tyrosinase loading of the immobilization matrix. The linear range for phenol determination was from 1 × 10⁻⁶ to 2.5 × 10⁻⁴ M with a detection limit of 6.0 × 10⁻⁷ M obtained at a signal-to-noise ratio of 3. The stability and the application of the biosensor were also evaluated.     

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 %.

Determination of hydrogen peroxide based on a metal dispersed sol-gel derived ceramic-graphite composite electrode

A new copper dispersed ceramic-graphite composite electrode was fabricated by the initial mixing of copper nitrate and (3-mercaptopropyl)trimethoxy silane (MPS) followed by stirring with graphite powder. The combination of the metal catalysis and the advantages of the ceramic composite favored the electrocatalytic reduction of hydrogen peroxide (H2O2) at a reduced overpotential of -0.2 V with good sensitivity, stability and reproducibility. The sensor showed a good linear response to H2O2 in the range from 8.3 x 10(-6) M to 2.0 x 10(-3) M with a correlation coefficient of 0.9989 and the detection limit was 6.2 x 10(-6) M (S/N =3).     

Bienzymatic amperometric biosensor for glucose based on polypyrrole/ceramic carbon as electrode material

A novel amperometric biosensor utilizing two enzymes, glucose oxidase (GOD) and horseradish peroxidase (HRP), was developed for the cathodic detection of glucose. The glucose biosensor was constructed by electrochemical formation of a polypyrrole (PPy) membrane in the presence of GOD on the surface of a HRP-modified sol-gel derived-mediated ceramic carbon electrode. Ferrocenecarboxylic acid (FCA) was used as mediator to transfer electron between enzyme and electrode. In the hetero-bilayer configuration of electrode, all enzymes were well immobilized in electrode matrices and showed favorable enzymatic activities. The amperometric detection of glucose was carried out at +0.16 V (versus saturated calomel reference electrode (SCE)) in 0.1 M phosphate buffer solution (pH 6.9) with a linear response range between 8.0×10⁻⁵ and 1.3×10⁻³ M glucose. The biosensor showed a good suppression of interference in the amperometric detection.     

Electrochemiluminescent biosensor based on multi-wall carbon nanotube/nano-Au modified electrode

The electrochemiluminescent (ECL) behavior of lucigenin on a multi-wall carbon nanotube/nano-Au modified glassy carbon electrode (MWNT/nano-Au/GCE) was studied in this paper. Compared with the bare GCE, the ECL intensity of lucigenin can be greatly enhanced at MWNT/nano-Au/GCE. Based on the fact that superoxide dimutase (SOD) could obviously inhibit the ECL of lucigenin at MWNT/nano-Au/GCE, a sensitive ECL biosensor for determination of SOD was developed with a wide linear range of 5.0 × 10⁻⁸–5.0 × 10⁻⁶ mol/L with detection limit of 2.5 × 10⁻⁸ mol/L.     

Amperometric hydrogen peroxide biosensor based on horseradish peroxidase-labeled nano-Au colloids immobilized on poly(2,6-pyridinedicarboxylic acid) layer by cysteamine.

A sensitive hydrogen peroxidase (H2O2) amperometric sensor based on horseradish peroxidase (HRP)-labeled nano-Au colloids has been proposed. Nano-Au colloids were immobilized by the thiol group of cysteamine, which was associated with the carboxyl groups of poly(2,6-pyridinedicarboxylic acid) (PPDA). With the aid of the hydroquinone, the sensor displayed excellent electrocatalytical response to the reduction of H2O2. Compared with the non-Au-colloid modified electrode, i.e., PPDA/HRP, the Au-colloid modified electrode exhibited better performance characteristics, including stability, reproducibility, sensitivity and accuracy. The biosensor shows a linear response to H2O2 in the range of 3.0 x 10(-7) - 2 x 10(-3) M. The detection limit was 1.0 x 10(-7) M.     

Flow-injection determination of trace hydrogen peroxide or glucose utilizing an amperometric biosensor based on glucose oxidase bound to a reticulated vitreous carbon electrode

An electron transfer mediator, 8-dimethylamino-2,3-benzophenoxazine (Meldola Blue), dissolved in the carrier solution in a flow-injection system, was found to reduce the oxidation potential for hydrogen peroxide from 600-1200 mV without mediator to-100 mV vs. Ag/AgCl with the mediator present. The very low background current of reticulated vitreous carbon (RVC) at this potential makes it possible to detect very low levels of hydrogen peroxide or glucose. Glucose oxidase was covalently coupled with carbodiimide to RVC, and the RVC was formed into a column inserted in a flow-injection system. The calibration curve was linear from 30 nM to 10 microM glucose with 5 microM mediator. At higher mediator concentrations, the linear range was extended to 1000 microM, but with a much higher background current. The sample throughput was about 60 h(-1). The current response decreased to 50% of the original response after 20 days. The coulometric yield was high because the sample was pumped through the pores of the RVC. It was 16% and 55% at a flow rate of 1 ml min(-1) at mediator concentrations of 5 and 50 microM respectively.     

基于石墨烯-壳聚糖-辣根过氧化物酶的H2O2生物传感器的研制

制备了石墨烯-壳聚糖(GR-CS)纳米复合材料,并将之与辣根过氧化物酶(HRP)混合,构建了基于石墨烯-壳聚糖-辣根过氧化物酶的生物传感器(GR-CS-HRP/GC)。探针及循环伏安研究表明,该界面具有优异的电子传导能力、较大的比表面积和良好的生物相容性,对H2O2的还原显示出较好的电催化活性,在工作电位为-0.2 V,0.05 mol/L的磷酸盐缓冲盐溶液(PBS,pH 6.8)中,该酶传感器对过氧化氢响应灵敏度高,检测范围宽,测定H2O2的线性范围为5.0×10-7～2×10-3mol/L(相关系数为0.998)。检出限为2.0×10-7mol/L(S/N=3)。并且表现出良好的稳定性和高选择性。该电极用于实际样品中H2O2的测定,结果令人满意。     

Amperometric biosensor for hydrogen peroxide based on coimmobilized horseradish peroxidase and methylene green in ormosils matrix with multiwalled carbon nanotubes

A novel amperometric biosensor for the analytical determination of hydrogen peroxide was developed. The fabrication of the biosensor was based on the coimmobilization of horseradish peroxidase (HRP), methylene green (MG) and multiwalled carbon nanotubes within ormosils; 3-aminopropyltrimethoxysilane (APTMOS), 2-(3,4-epoxycyclohexyl)ethyltrimethoxysilane (ETMOS) and phenyltrimethoxysilane (PHTMOS). APTMOS determined the hydrophilicity/hydrophobicity of the ormosils and PHTMOS and ETMOS increased the physical and mechanical strength of the ormosil matrix. The ormosil modified electrodes were characterized with SEM, UV-vis spectroscopy and electrochemical methods. Cyclic voltammetry and amperometric measurements demonstrated the MG coimmobilized with HRP in this way, displayed good stability and could efficiently shuttle electrons between immobilized enzyme and electrode, and MWCNTs facilitated the electrocatalytic reduction of H₂O₂ at reduced over potential. The Micheaelis constant of the immobilized HRP was 1.8 mM, indicating a high affinity of the HRP to H₂O₂ without loss of enzymatic activity in ormosil matrix. The prepared biosensor had a fast response of H₂O₂, less than 10 s, and excellent linear range of concentration from 5 × 10⁻⁷ to 2 × 10⁻⁵ M with the detection limit of 0.5 μM (S/N = 3) under the optimum conditions. At the same time, the influence of solution pH, effect of enzyme amount, steady-state applied potential and temperature on the biosensor were investigated. The enzyme electrode retained about 90% of its initial activity after 30 days of storage in a dry state at 4 °C. The preparation of the developed biosensor was convenient and showed high sensitivity with good stability.     

Reagentless enzyme electrode based on phenothiazine mediation of horseradish peroxidase for subnanomolar hydrogen peroxide determination

The development and characterization of a highly sensitive enzyme immobilized carbon based electrode for the determination of subnanomolar concentrations of hydrogen peroxide in aqueous samples is described. The biosensor consists of horseradish peroxidase (HRP) immobilized in solid carbon paste along with a suitable redox mediator. The latter allows the acceleration of the electroreduction of HRP in the presence of hydrogen peroxide. Several phenothiazines as mediators are investigated in a comparative manner and with respect to dimethylferrocene using cyclic voltammetry and amperometry. Insolubilization of the HRP in the solid carbon paste is achieved by cross-linking the enzyme with glutaraldehyde and bovine serum albumin. Several experimental parameters such as pH, mediator and enzyme content are considered. The hydrogen peroxide determination is better carried out in 0.1 M acetate buffer, pH 4.5, by amperometry at an applied potential of 0.0 V versus Ag/AgCl, 3 M NaCl concentration and by using the phenothiazine base as redox mediator. The biosensor response is linear over the concentration range 2 nM-10 microM with a detection limit of 1 nM. The linear range of the hydrogen peroxide response without a mediator in the biosensor is found between 2 and 40 microM. The biosensor can be used for more than 180 measurements. Additional modification of the electrode by incorporation of Nafion SAC-13 microparticles in the solid carbon paste allows detection of concentrations of hydrogen peroxide as low as 0.1 nM.