Nafion 膜固定的新亚甲基蓝为介体生物传感器

以Nafion膜固定的新亚甲基蓝为辣根过氧化物酶和玻碳电极间的电子传递介体,制成电流型单酶过氧化氢生物传感器和双酶葡萄糖生物传感器。探讨了工作电位、pH值、温度和干扰物质等对生物传感器的影响。     

Glucose and lactate biosensors prepared by a layer-by-layer deposition of concanavalin A and mannose-labeled enzymes: electrochemical response in the presence of electron mediators.

An electrochemical response of glucose and lactate biosensors which were prepared by coating a platinum electrode with a thin film composed of concanavalin A and mannose-labeled glucose oxidase (GOx) or lactate oxidase (LOx) was evaluated in the presence of ferrocene derivatives as electron mediator. Both glucose and lactate biosensors showed catalytic current to glucose and lactate, respectively, in cyclic voltammetry, suggesting that the ferrocene derivatives can mediate electron transport smoothly from the reduced forms of GOx and LOx in the thin films to the electrode. Among the three kinds of ferrocene derivatives used, ferrocenylmethanol was found to be the most suitable electron mediator because of its low oxidation potential. The glucose and lactate sensors gave useful calibration graphs, in which higher detection limits were reached as compared with those observed when the sensors were operated in the absence of electron mediator.     

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.     

Amperometric enzyme-modified electrodes based on tetrathiafulvalene derivatives for the determination of glucose

A number of tetrathiafulvalene (TTF) derivatives have been synthesized and tested as electron transfer mediators in glucose oxidase-based amperometric biosensors. Using cyclic voltammetry and stationary potential experiments, it is shown that several of these derivatives can effectively mediate electron transfer from the reduced flavin adenine dinucleotide redox centers of glucose oxidase to a conventional carbon paste electrode. An insoluble polymeric electron relay system, based on the covalent attachment of TTF moieties to a highly flexible siloxane polymer, is also shown to facilitate a flow of electrons from the enzyme to the electrode. The resulting glucose biosensors function efficiently over a clinically relevant range of glucose concentrations.     

An enzyme-free highly glucose-specific assay using self-assembled aminobenzene boronic acid upon polyelectrolytes electrospun nanofibers-mat

A highly selective enzyme-free amperometric glucose sensor based on electrostatic self-assembling of 3-aminobenzene boronic acid (ABBA) onto a poly(styrene-co-acrylamide)/polystyrene sulfonic acid (PSA/PSSA) electrospun nanofibers-mat was investigated. Emerging ability of phenylboronic acid to bind with the diols of sugars has been extended for rapid response of glucose with a pH-sensitive redox mediator, hematein natural dye. ABBA was adsorbed on the PSA/PSSA nanofibers-mat/Pt-disc electrode that resulted in an ABBA/PSA/PSSA glucose active electrode. The interaction of ABBA onto the PSA/PSSA nanofibers-mat/Pt-disc electrode was characterized with Fourier transform infrared spectroscopy (FT-IR), ζ-potential, scanning electron microscopy (SEM), contact angle and cyclic voltammetry (CV) measurements. The prepared enzyme-free sensor exhibited a fast amperometric response, i.e., about 4 s and linearity ranging from 0.75 to 14 mM to glucose with a sensitivity of 0.987 μA mM⁻¹ cm⁻². Compared to other types of glucose biosensors viz. use glucose oxidase as sensing elements, present glucose sensor offers basic advantages including ease of fabrication, high affinity-selectivity to the glucose upon the electrode surface and quick response.     

An integrated bienzyme glucose oxidase-fructose dehydrogenase-tetrathiafulvalene-3-mercaptopropionic acid-gold electrode for the simultaneous determination of glucose and fructose

A bienzyme biosensor for the simultaneous determination of glucose and fructose was developed by coimmobilising glucose oxidase (GOD), fructose dehydrogenase (FDH), and the mediator, tetrathiafulvalene (TTF), by cross-linking with glutaraldehyde atop a 3-mercaptopropionic acid (MPA) self-assembled monolayer (SAM) on a gold disk electrode (AuE). The performance of this bienzyme electrode under batch and flow injection (FI) conditions, as well as an amperometric detection in high-performance liquid chromatography (HPLC), are reported. The order of enzyme immobilisation atop the MPA-SAM affected the biosensor amperometric response in terms of sensitivity, with the immobilisation order GOD, FDH, TTF being selected. Similar analytical characteristics to those obtained with single GOD or FDH SAM-based biosensors for glucose and fructose were achieved with the bienzyme electrode, indicating that no noticeable changes in the biosensor responses to the analytes occurred as a consequence of the coimmobilisation of both enzymes on the same MPA-AuE. The suitability of the bienzyme biosensor for the analysis of real samples under flow injection conditions was tested by determining glucose in two certified serum samples. The simultaneous determination of glucose and fructose in the same sample cannot be performed without a separation step because at the detection potential used (+0.10 V), both sugars show amperometric response. Consequently, HPLC with amperometric detection at the TTF-FDH-GOD-MPA-AuE was accomplished. Glucose and fructose were simultaneously determined in honey, cola softdrink, and commercial apple juice, and the results were compared with those obtained by using other reference methods.     

Amperometric Study of Au‐Colloid Function on Xanthine Biosensor Based on Xanthine Oxidase Immobilized in Polypyrrole Layer

Four kinds of xanthine oxidase (XOD) based amperometric biosensors were fabricated and their analytical performances were compared. Polypyrrole (PPY)/XOD biosensor was constructed by electrochemical oxidation of pyrrole in the solution containing xanthine oxidase and pyrrole in this paper. Colloidal Au was then immobilized on the biosensor. On the other hand, electron mediator, Prussian Blue (PB), was deposited on the electrode before the immobilization of PPY/XOD to enhance electron‐transfer rate and current response. The results showed that PPY/XOD, PPY/XOD/Au‐colloid, PB/PPY/XOD and PB/PPY/XOD/Au‐colloid biosensors exhibit good response to xanthine in 1×10^?6 M and 2×10^?5 M and Michaelis‐Menten constants (K_m) of these biosensors were 242.2, 113.4, 144.5, 43.2 μmol?L^?1, respectively. The dependence of current responses with applied voltages was discussed, and different mechanisms of these biosensors were discussed. It has been found that colloidal Au can enhance the current response at the same concentration of xanthine solution and decrease the energy‐barrier of electron‐transfer reaction on the electrode.     

Critical comparison of metallized and mediator-based carbon paste glucose biosensors

The performance of metallized carbon oxidase-based biosensors is critically compared with that of mediator-based enzyme electrodes. Dimethyl ferrocene and rhodium metal centers are used as model mediator and catalyst, respectively, in connection to carbon-paste electrode transducers and assays of glucose. The rhodium-containing bioelectrode displays enhanced selectivity (particularly improved discrimination against ascorbic acid), lower sensitivity, faster response time, greater oxygen dependence, and similar detection limit in comparison to the mediated electrode. In addition to assessing the relative merits of both strategies, the data offer useful insights into the behavior of these ‘first’- and ‘second-generation’ oxidase amperometric electrodes.     

Analytical Characteristics of Electrochemical Biosensor Using Pt‐Dispersed Graphene on Boron Doped Diamond Electrode

An electrochemical biosensor was developed using Pt‐nanoparticles (Pt‐NPs) dispersed graphene based on a boron‐doped diamond thin film electrode. To compare its performances with those of other biosensors, glucose was used as a target analyte. This biosensor exhibited a wide linear range, a low detection limit and a higher sensitivity compared to other amperometric biosensors using graphene‐based electrodes. In addition, the biosensor promotes a direct electron transfer between the redox enzymes and the electrode surface and detects low concentration analytes. The excellent performance of the biosensor is attributed to the synergistic effect of the Pt‐NPs, graphene sheet and the BDD thin film. Therefore, it can be a promising application for electrochemical detection of analytes.     

Mediator-free amperometric determination of glucose based on direct electron transfer between glucose oxidase and an oxidized boron-doped diamond electrode

A mediator-free glucose biosensor, termed a “third-generation biosensor,” was fabricated by immobilizing glucose oxidase (GOD) directly onto an oxidized boron-doped diamond (BDD) electrode. The surface of the oxidized BDD electrode possesses carboxyl groups (as shown by Raman spectra) which covalently cross-link with GOD through glutaraldehyde. Glucose was determined in the absence of a mediator used to transfer electrons between the electrode and enzyme. O₂ has no effect on the electron transfer. The effects of experimental variables (applied potential, pH and cross-link time) were investigated in order to optimize the analytical performance of the amperometric detection method. The resulting biosensor exhibited fast amperometric response (less than 5 s) to glucose. The biosensor provided a linear response to glucose over the range 6.67×10⁻⁵ to 2×10⁻³ mol/L, with a detection limit of 2.31×10⁻⁵ mol/L. The lifetime, reproducibility and measurement repeatability were evaluated and satisfactory results were obtained.     

The interference of HEPES buffer during amperometric detection of ATP in clinical applications

HEPES-based biological buffer is subject to photooxidation upon exposure to fluorescent illumination. Thereby hydrogen peroxide is generated, which interferes with amperometric oxidoreductase-based biosensors for glucose or adenosine triphosphate (ATP). These biosensors operate at an oxidation potential above 500 mV vs. the standard calomel electrode (SCE) and involve hydrogen peroxide as the electroactive molecule detected at the electrode surface. False-positive detection of ATP was observed in HEPES buffer utilizing an amperometric microbiosensor based on the co-immobilization of glucose oxidase and hexokinase for detection of ATP in biological specimens. Electrochemical, mass spectrometric, ³¹P NMR, and ¹H NMR studies indicate that complexation of ATP and HEPES induced by the presence of Ca²⁺ in HEPES buffer decreases the photooxidation of HEPES. Consequently, the hydrogen peroxide background concentration is reduced, thereby leading to erroneous ATP detection at the dual-enzyme microbiosensor, which determines an increase in ATP via a reduced hydrogen peroxide signal.     

Amperometric Glucose Biosensor Based on Electrochemically Deposited Gold Nanoparticles Covered by Polypyrrole

Graphite rod (GR) modified with electrochemicaly deposited gold nanoparticles (AuNPs) and adsorbed glucose oxidase (GOx) was used in amperometric glucose biosensor design. Enzymatic formation of polypyrrole (Ppy) on the surface of GOx/AuNPs/GR electrode was applied in order to improve analytical characteristics and stability of developed biosensor. The linear glucose detection range for Ppy/GOx/AuNPs/GR electrode was dependent on the duration of Ppy‐layer formation and the linear interval was extended up to 19.9 mmol L⁻¹ after 21 h lasting synthesis of Ppy. The sensitivity of the developed biosensor was determined as 21.7 μA mM⁻¹ cm⁻², the limit of detection – 0.20 mmol L⁻¹. Ppy/GOx/AuNPs/GR electrodes demonstrated advanced good stability (the t _1/2 was 9.8 days), quick detection of glucose (within 5 s) in the wide linear interval. Additionally, formed Ppy layer decreased the influence of electroactive species on the analytical signal. Developed biosensor is suitable for the determination of glucose in human serum samples.     

On-line removal of redox-active interferents by a porous electrode before amperometric blood glucose determination

A porous reticulated vitreous carbon (RVC) electrode and a disk electrode coupled in tandem in an electrochemical flow cell has been used for electrolytic removal of interferents before amperometric glucose detection. The electrolytic efficiency at the upstream RVC electrode is 100% at a flow rate of 0.1 mL min⁻¹ or lower. Potential interferents such as acetaminophen, ascorbic acid, and uric acid can be completely eliminated by electrolysis at the RVC electrode. A mixed monolayer comprising glucose oxidase (GOD) and ferrocenyl-1-undecanethiol preformed at the downstream gold disk electrode was used as a mediator-based amperometric glucose sensor. The dependence of the amperometric current on the glucose concentration exhibits good linearity across over three orders of magnitude. The glucose measurements were also found to be reproducible (RSD < 3.5%) and accurate. Unlike the chemiluminescence method, this device obviates the use of carcinogenic substrates and the glucose sensor performance is independent of the oxygen present in sample. On the basis that the RVC electrode requires minimal cleanup and the GOD-modified electrode remains stable for a week, the electrochemical flow cell should be amenable for automated on-line removal of redox interferents for other types of enzyme-based biosensors.     

Construction of a Novel Glucose Biosensor Based on Covalent Immobilization of Glucose Oxidase on Poly(glycidyl methacrylate‐co‐vinylferrocene)

A novel glucose biosensor was constructed via direct covalent attachment of glucose oxidase onto epoxy group containing polymeric electron transfer mediator, Poly(glycidyl methacrylate‐co‐vinylferrocene). A copolymer of glycidyl methacrylate (GMA) and vinylferrocene (VFc) with different molar ratios has been prepared by free radical copolymerization. These copolymers have been utilized as polymeric mediators for amperometric glucose sensing. The catalytic electrochemistry of the enzyme electrode with the copolymer was investigated. Copolymer acts as an electron transfer mediator between the redox center of Glucose oxidase (GOx) and the electrode. The stability, reusability, pH and temperature response of the biosensor as well as its kinetic parameter have also been studied.     

Simulations of Redox Mediation within Bioactive Hydrogels of Amperometric Biosensors

Highly hydrated bioactive hydrogels containing immobilized oxidoreductase enzymes and immobilized redox mediators were simulated as the biorecognition layer of amperometric biosensors. The linear dynamic range of the amperometric response of mediated biosensors increases and moves to higher concentration brackets with an increase in the concentration of mediator. This informs the design of biosensors that target the same analyte but possesses several independently addressable electrodes modified with hydrogels that contain different concentrations of mediator. Increases in enzyme concentration increase the linear dynamic range but does not alter the sensitivity of amperometric biosensors. Both sensitivity and linear dynamic range of mediated amperometric enzyme biosensors may be “tuned” by varying the concentrations of the enzyme and the mediator. Simulations effectively guide the initial domains of study of complex systems such as implantable biosensors.     

Direct electrochemistry of glucose oxidase immobilized on a hexagonal mesoporous silica-MCM-41 matrix

The direct electrochemistry of glucose oxidase (GOD) immobilized on a hexagonal mesoporous silica modified glassy carbon electrode was investigated. The adsorbed GOD displayed a pair of redox peaks with a formal potential of -417 mV in 0.1 M pH 6.1 phosphate buffer solution (PBS). The response showed a diffusion-controlled electrode process with a two-electron transfer coupled with a two-proton transfer reaction process. GOD immobilized on a hexagonal mesoporous silica retained its bioactivity and stability. In addition, the immobilized GOD could electrocatalyze the oxidation of glucose to gluconlactone by taking ferrocene monocarboxylic acid (FMCA) as a mediator in N(2) saturated solutions, indicating that the electrode may have the potential application in biosensors to analyze glucose. The sensor could exclude the interference of commonly coexisted uric acid, p-acetaminophenol and ascorbic acid and diagnose diabetes very fast and sensitively. This work demonstrated that the mesoporous silica provided a novel matrix for protein immobilization and the construction of biosensors.     

A Modulated Tyrosinase Enzyme‐Based Biosensor for Application to the Detection of Dichlorvos and Atrazine Pesticides

A study was been made of tyrosinase amperometric biosensors for the determination of organophosphorus (dichlorvos) and triazine (atrazine) pesticides. The biosensors are based on the competitive inhibition of tyrosinase (Tyr) by the pesticides. Tyr becomes active when the reduced form of the charge‐transfer mediator (1,2‐naphthoquinone‐4‐sulfonic acid (NQS), 1,2‐naphthoquinone (NQ) and 3,5‐di‐tert‐butyl‐1,2‐benzoquinone (t‐BQ) were tested) are electrochemically generated onto the working electrode surface, which permits modulation of the enzymatic activity. The inhibition is reversible as there is a complete recovery of the current due to enzyme activity without the studied pesticides. The charge‐transfer mediators (the quinonic molecules) and the enzyme were co‐immobilized on the working electrode to obtain reagentless biosensors. Kinetic studies in solution were carried out to compare the efficiency of the measurement mechanism.     

Quinone-modified polymers as electron transfer relay systems in amperometric glucose sensors

A polysiloxane and an acrylonitrile–ethylene copolymer with covalently attached p-hydroquinone/benzoquinone moieties were prepared and tested as electron transfer relay systems in amperometric glucose biosensors. Using experiments involving cyclic voltammetry and stationary potential measurements, it was shown that the polysiloxane relay system can efficiently mediate electron transfer from reduced glucose oxidase to a conventional carbon-paste electrode. Sensors containing this polymeric relay system and glucose oxidase respond rapidly to low (<0.1 mm) glucose concentrations, with steady state current responses achieved in less than 1 min. The acrylonitrile–ethylene copolymer was found to be less efficient than the polysiloxane system at mediating the electron transfer from reduced glucose oxidase to the electrode. The dependence of the sensor response on the nature of the polymer backbone is discussed.     

Electron-transfer mechanisms in amperometric biosensors

The function of amperometric biosensors is related to electron-transfer processes between the active site of an (immobilized) enzyme and an electrode surface which is poised to an appropriate working potential. Problems and specific features of architectures for amperometric biosensors using different electron-transfer pathways such as mediated electron transfer, electron-hopping in redox polymers, electron transfer using mediator-modified enzymes and carbon-paste electrodes, direct electron transfer by means of self-assembled monolayers or via conducting-polymer chains are discussed.     

A neural network approach based on gold‐nanoparticle enzyme biosensor

Gold nanoparticles have demonstrated to be a very useful material for the construction of stable and sensitive glucose oxidase (GOx) amperometric biosensors. However, as for other enzyme electrodes, the lack of specificity for glucose limits their practical applications. Coupling biosensor responses with chemometric tools can be used to solve complex analytical signals from mixtures of species with similar properties. In this work, an amperometric biosensor based on a colloidal gold—cysteamine—gold disk electrode with the enzyme GOx and a redox mediator, tetrathiafulvalene (TTF), co‐immobilised atop the modified electrode, was used for the simultaneous determination of glucose and its common interferences, ascorbic acid and uric acid, in mixtures. Analytical data obtained from cyclic voltammograms generated with the biosensor were processed using an artificial neural network (ANN), and the separate quantification of the analytes over a range of 0.1–1 mM each was performed without any pretreatment. In all cases, the correlation coefficients obtained were higher than 0.99 and the mean prediction error was less than 1.7%. Copyright © 2007 John Wiley & Sons, Ltd.