Optimal environment for glucose oxidase in perfluorosulfonated ionomer membranes: improvement of first-generation biosensors

An optimal environment for glucose oxidase (GOx) in Nafion membranes is achieved using an advanced immobilization protocol based on a nonaqueous immobilization route. Exposure of glucose oxidase to water-organic mixtures with a high (85-95%) content of the organic solvent resulted in stabilization of the enzyme by a membrane-forming polyelectrolyte. Such an optimal environment leads to the highest enzyme specific activity in the resulting membrane, as desired for optimal use of the expensive oxidases. Casting solution containing glucose oxidase and Nafion is completely stable over 5 days in a refrigerator, providing almost absolute reproducibility of GOx-Nafion membranes. A glucose biosensor was prepared by casting the GOx-Nafion membranes over Prussian Blue-modified glassy carbon disk electrodes. The biosensor operated in the FIA mode allows the detection of glucose down to the 0.1 microM level, along with high sensitivity (0.05 A M(-1) cm(-2)), which is only 10 times lower than the sensitivity of the hydrogen peroxide transducer used. A comparison with the recently reported enzyme electrodes based on similar H2O2 transducers (transition metal hexacyanoferrates) shows that the proposed approach displays a dramatic (100-fold) improvement in sensitivity of the resulting biosensor. Combined with the attractive performance of a Prussian Blue-based hydrogen peroxide transducer, the proposed immobilization protocol provides a superior performance for first-generation glucose biosensors in term of sensitivity and detection limits.     

Biocatalytic carbon paste sensors based on a mediator pasting liquid

The preparation and advantages of a new generation of carbon paste enzyme electrodes where the redox mediator acts also as the pasting liquid are described. The mediator pasting liquid concept is illustrated for amperometric biosensing of glucose in connection with either the tert-pentylferrocene or n-butylferrocene mediator/binder along with the glucose oxidase enzyme. The attractive performance and advantages of the new device is indicated from comparison to a conventional carbon paste biosensor using a mineral oil binder and the dimethylferrocene electron acceptor. The simplified preparation of the biosensor is coupled with a greatly improved sensitivity and an extended linear range. The mediator pasting liquid imparts high thermal stability onto the embedded enzyme and leads to good resistance to oxygen effects. Owing to the huge mediator reservoir, stability problems associated with the leaching of the mediator are greatly reduced. The fundamental aspects of the electrode behavior have been examined first in the absence of the enzyme. Variables affecting the performance of the new carbon paste biosensor have been investigated and optimized. Such use of the electron acceptor as a binder as well as the mediator offers considerable promise for the biosensing of numerous analytes of clinical and environmental significance.     

A method for the design and study of enzyme microstructures formed by means of a flow-through microdispenser

Micrometer-sized enzyme grids were fabricated on gold surfaces using a novel method based on a flow-through microdispenser. The method involves dispensing very small droplets of enzyme solution (approximately 100 pL) during the concomitant relative movement of a gold substrate with respect to the nozzle of a microdispenser, resulting in enzyme patterns with a line width of approximately 100 microm. Different immobilization methods have been evaluated, yielding either enzyme monolayers using functionalized self-assembled thiol monolayers for covalent binding of the enzyme or enzyme multilayers by cross-linking or entrapping the enzymes in a polymer film. The latter immobilization techniques allow the formation of coupled multienzyme structures. On the basis of this feature, coupled bienzyme (glucose oxidase and catalase) or three-enzyme (alpha-glucosidase, mutarotase, and glucose oxidase) microstructures consisting of line patterns of one enzyme intersecting with the patterned lines of the other enzyme(s) were fabricated. By means of scanning electrochemical microscopy (SECM) operated in the generator-collector mode, the enzyme microstructures and their integrity were visualized using the localized detection of enzymatically produced/consumed H2O2. A calibration curve for glucose could be obtained by subsequent SECM line scans over a glucose oxidase microstructure for increasing glucose concentrations, demonstrating the possibility of obtaining localized quantitative data from the prepared microstructures. Possible applications of these enzyme microstructures for multianalyte detection and interference elimination and for screening of different biosensor configurations are highlighted.     

Titanium dioxide–cellulose hybrid nanocomposite and its glucose biosensor application

This paper investigates the fabrication of titanium dioxide (TiO₂)–cellulose hybrid nanocomposite and its possibility for a conductometric glucose biosensor. TiO₂ nanoparticles were blended with cellulose solution prepared by dissolving cotton pulp with lithium chloride/N,N-dimethylacetamide solvent to fabricate TiO₂–cellulose hybrid nanocomposite. The enzyme, glucose oxidase (GOx) was immobilized into this hybrid nanocomposite by physical adsorption method. The successful immobilization of glucose oxidase into TiO₂–cellulose hybrid nanocomposite via covalent bonding between TiO₂ and GOx was confirmed by X-ray photoelectron analysis. The linear response of the glucose biosensor is obtained in the range of 1–10 mM. This study demonstrates that TiO₂–cellulose hybrid nanocomposite can be a potential candidate for an inexpensive, flexible and disposable glucose biosensor.     

Production of biosensors with exchangeable enzyme-containing threads

We introduce a simple method for the construction of biosensors, based on coiling an enzyme-containing, thread-shaped material around a cylindrical signal transducer in the form of winding stairs with a variable length of step and so forming a variable biocatalytic membrane on the sensor surface, which can be easily modified for particular purposes. In the model system, we immobilized glucose oxidase (GO) on a nylon thread, formatted from a sheaf of numerous minor filaments and used as a biorecognition element integrated with a Clark-type oxygen sensor. The immobilized enzyme was evenly distributed throughout the thread, and the activity of the enzyme could be measured in units of length. Appropriate pieces of the enzyme-containing thread with a certain amount of GO could be cut for a definite biosensor or bioreactor. The enzyme amount and substrate diffusion parameters, which together control the sensor's working range and sensitivity, could be changed simultaneously with the change of the length of the thread. Besides glucose oxidase, experiments with other enzymes have confirmed the applicability of the proposed technological solution. Thus, the thread-type matrixes enable one to construct sensors with a required range of work, sensitivity, and selectivity, which can be easily customized within seconds.     

壳聚糖凝胶材料固定葡萄糖氧化酶制电极的研究

以壳聚糖为载体研究凝胶法固定葡萄糖氧化酶制电极。试验研究了载体壳聚糖的降解性；交联剂戊二醛的浓度、用量；电极的载酶量等固定化条件对所组建的传感器性能的影响。通过影响规律的分析、优化固定化条件的研究，找出了根据壳聚糖溶液粘度适当调整交联剂成二醛的用量和铂丝在酶膜母液中浸涂时间，克服壳聚糖的降解性对酶电极性能的影响，建立了制备性能相近的GOD传感器的方法。     

Real-time quantification of methanol in plants using a hybrid alcohol oxidase-peroxidase biosensor

An amperometric biosensor immobilizing two enzymes and an electron mediator in an identical plane has been fabricated by the self-assembly technique for determination of methanol in crude plant samples. A self-assembled mixed monolayer of 4,4'-dithiodibutyric acid covalently attached two enzymes (Hansenula sp. alcohol oxidase and horseradish peroxidase) and 11-ferrocenyl-1-undecanethiol as an electron mediator on an Au electrode is exploited to produce a two-dimensional reaction matrix. The composition of the two enzymes and electron mediator molecules was optimized for detection of methanol in 0.1 M sodium phosphate buffer (pH 6.0). We successfully quantified methanol in low-purity tobacco (Nicotiana tabacum) plant extracts with the biosensor, which showed sensitivity comparable to that of gas chromatography/mass spectrometry. The redox-relay biosensor is quite simple and stable due to its covalent attachment to the Au surface, making it possible to downsize the construction. We fabricated a miniature methanol biosensor that fitted a well of a 96-well micro assay plate available for high-throughput assay. The biosensor is advantageous for the sensitive, continuous, and convenient determination of methanol.     

Amperometric glucose microelectrodes prepared through immobilization of glucose oxidase in redox hydrogels.

Glucose microelectrodes have been formed with glucose oxidase immobilized in poly[(vinylpyridine)Os(bipyridine)2Cl] derivative-based redox hydrogels on beveled carbon-fiber microdisk (7 microns diameter) electrodes. In the resulting microelectrode, the steady-state glucose electrooxidation current density is 0.3 mA cm-2 and the sensitivity is 20 mA cm-2 M-1. The current density and sensitivity are 10 times higher than in macroelectrodes made with the same hydrogel. Furthermore, the current is less affected by a change in the partial pressure of oxygen. The higher current density and lower oxygen sensitivity point to the efficient collection of electrons through their diffusion in the redox hydrogel to the electrode surface. These results contrast with those observed for enzyme electrodes based on diffusing mediators, where loss of the enzyme-reduced mediator by radial diffusion to the solution decreases the current densities of microelectrodes relative to similar macroelectrodes.     

A bioconjugated polyglycerol dendrimer with glucose sensing properties

In this work, the biological and electrochemical properties of glucose biosensor based on polyglycerol dendrimer (PGLD) is presented. Streptokinase (SK), glucose oxidase (GOx) and phosphorylcholine (PC) were immobilized onto PGLD to obtain a blood compatible bioconjugate with glucose sensing properties. The bioconjugated PGLD was entrapped in polyaniline nanotubes (PANINT's) through template electrochemical polymerization of aniline. PANINT's were used as electron mediator due to their high ability to promote electron-transfer reactions involving GOx. Platelet adhesion, fibrinolytic activity and protein adsorption were studied by in vitro experiments to examine the interaction of blood with PGLD biosensor. The PGLD biosensor exhibits a strong and stable amperometric response to glucose. The enzyme affinity for the substrate (K (M) (app) ) indicates that the enzyme activity was not significantly altered after the bioconjugation of GOx with PGLD dendrimer. The bioelectrochemical properties suggest that the bioconjugated PGLD developed in this work appears to be a good candidate for providing interfaces for implantable biosensors, especially oxidoreductase-based sensors.     

Biosensor for arsenite using arsenite oxidase and multiwalled carbon nanotube modified electrodes

A biosensor for arsenite has been developed using molybdenum-containing arsenite oxidase, prepared from the chemolithoautotroph NT-26 that oxidizes arsenite to arsenate. The enzyme was galvanostatically deposited for 10 min at 10 microA onto the active surface of a multiwalled carbon nanotube modified glassy carbon electrode. The resulting biosensor enabled direct electron transfer, i.e., effecting reduction and then reoxidization of the enzyme without an artificial electron-transfer mediator. Arsenite was detected within 10 s at an applied potential of 0.3 V with linearity up to 500 ppb and a detection limit of 1 ppb. The biosensor exhibited excellent reproducibility, 2% at 95% confidence interval for 12 repeated analyses of 25 ppb arsenite. Copper, a severe interfering species commonly found in groundwater, did not interfere, and the biosensor was applicable for repeated analysis of spiked arsenite in tap water, river water, and a commercial mineral water.     

Thionine covalently tethered to multilayer horseradish peroxidase in a self-assembled monolayer as an electron-transfer mediator

A new approach to construct a reagentless enzyme biosensor is described. Based on multilayer horseradish peroxidase in a self-assembled monolayer configuration, the biosensor was constructed using multilayer thionine covalently tethered to the enzyme as an electron-transfer mediator. The multilayer enzyme and the multilayer mediator were stepwisely synthesized onto an l-cysteine-assembled gold electrode using glutaraldehyde as a bifunctional reagent. The multilayer mediator tethered to the multilayer enzyme could effectively and stably shuttle electrons between the electrode and the multilayer enzyme linked onto the monolayer. The sensitivity of the resulting enzyme biosensor with eight layers of enzyme and three layers of mediator was more than 250 μA cm(-)(2) for 1.0 × 10(-)(4) mol/L hydrogen peroxide under optimal conditions, whereas such a modified electrode with one layer of enzyme and one layer of mediator did not yield a detectable response to 1.0 × 10(-)(4) mol/L hydrogen peroxide.     

Development of a flow amperometric enzymatic method for the determination of total glucosinolates in real samples

The first amperometric flow analyzer, based on the biosensor concept, capable of determining total glucosinolates in real samples, is described. Myrosinase was immobilized on aminopropyl-modified controlled pore glass, which was then used for the construction of a packed-bed reactor. Myrosinase catalyzes the hydrolysis of glucosinolates (sinigrin) to glucose (among the other products), which is then oxidized by the action of glucose oxidase to produce hydrogen peroxide. The glucose enzyme electrode is based on a multimembrane architecture and was mounted on an amperometric flow cell (hydrogen peroxide detection at a platinum anode poised at +0.65 V vs Ag/AgCl/3M KCl). Different membrane types and different activation procedures were tested. The system was optimized to various working parameters, either as a glucose electrode or as a glucosinolate analyzer. The interference effect of various compounds was also investigated. Application of the method to real samples was carried out using glucose/glucose, hydrolyzed sinigrin and glucose/sinigrin solution as calibrators of the glucose electrode and the glucosinolate analyzer. Deviations due to the enantioselectivity of glucose oxidase to the beta-glucose anomer were observed, and a data elaboration protocol is proposed. The possibility of the simultaneous determination of glucose and glucosinolates is also demonstrated.     

金属与非金属纳米颗粒增强葡萄糖生物传感器

为了提高葡萄糖传感器的灵敏度和抗干扰性,利用纳米增强效应,以Au、Ag、Pt、SiO2纳米颗粒及金属-无机复合纳米颗粒与聚乙烯醇缩丁醛(PVB)构成复合固定酶膜基质,采用溶胶-凝胶法固定葡萄糖氧化酶(GOD),组成葡萄糖生物传感器.研究表明,纳米颗粒可以大幅度地提高固定化酶的催化活性,增加电极的电流响应灵敏度,改进生物传感器的抗干扰性能,使信噪比提高了32倍.     

Nitric oxide-releasing sol-gel particle/polyurethane glucose biosensors

A hybrid sol-gel/polyurethane glucose biosensor that releases nitric oxide is developed and characterized. The biosensor consists of a platinum electrode coated with four polymeric membranes including the following: (1) sol-gel with immobilized glucose oxidase (GOx); (2) polyurethane to protect the enzyme; (3) NO donor-modified sol-gel particle-doped polyurethane; and (4) polyurethane. This configuration was developed due to the drastic reduction in sensitivity observed for NO donor-modified sol-gel film-based glucose sensors. For the hybrid sol-gel/polyurethane biosensor, sol-gel particles are first modified with the NO donor and then incorporated into a polyurethane layer that is coated onto the preimmobilized GOx electrode. In this manner, the GOx layer is not exposed to the harsh conditions necessary to impart NO release ability to the biosensor, and only a minimal decrease in sensitivity due to the NO release is observed. The glucose response of the NO-releasing glucose biosensor and its NO generation profiles are reported. In addition, the stability of the sol-gel particles in the supporting polyurethane membrane is discussed.     

Enzyme-catalyzed O2 removal system for electrochemical analysis under ambient air: application in an amperometric nitrate biosensor

Electroanalytical procedures are often subjected to oxygen interferences. However, achieving anaerobic conditions in field analytical chemistry is difficult. In this work, novel enzymatic systems were designed to maintain oxygen-free solutions in open, small volume electrochemical cells and implemented under field conditions. The oxygen removal system consists of an oxidase enzyme, an oxidase-specific substrate, and catalase for dismutation of hydrogen peroxide generated in the enzyme catalyzed oxygen removal reaction. Using cyclic voltammetry, three oxidase enzyme/substrate combinations with catalase were analyzed: glucose oxidase with glucose, galactose oxidase with galactose, and pyranose 2-oxidase with glucose. Each system completely removed oxygen for 1 h or more in unstirred open vessels. Reagents, catalysts, reaction intermediates, and products involved in the oxygen reduction reaction were not detected electrochemically. To evaluate the oxygen removal systems in a field sensing device, a model nitrate biosensor based on recombinant eukaryotic nitrate reductase was implemented in commercial screen-printed electrochemical cells with 200 μL volumes. The products of the aldohexose oxidation catalyzed by glucose oxidase and galactose oxidase deactivate nitrate reductase and must be quenched for biosensor applications. For general application, the optimum catalyst is pyranose 2-oxidase since the oxidation product does not interfere with the biorecognition element.     

Cross-linked redox gels containing glucose oxidase for amperometric biosensor applications

Oxidoreductases, such as glucose oxidase, can be electrically "wired" to electrodes by electrostatic complexing or by covalent binding of redox polymers so that the electrons flow from the enzyme, through the polymer, to the electrode. We describe two materials for amperometric biosensors based on a cross-linkable poly(vinylpyridine) complex of [Os-(bpy)2Cl]+2+ that communicates electrically with flavin adenine dinucleotide redox centers of enzymes such as glucose oxidase. The uncomplexed pyridines of the poly(vinylpyridine) are quaternized with two types of groups, one promoting hydrophilicity (2-bromoethanol or 3-bromopropionic acid), the other containing an active ester (N-hydroxysuccinimide) that forms amide bonds with both lysines on the enzyme surface and with an added polyamine cross-linking agent (triethylenetetraamine, trien). In the presence of glucose oxidase and trien this polymer forms rugged, cross-linked, electroactive films on the surface of electrodes, thereby eliminating the requirement for a membrane for containing the enzyme and redox couple. The glucose response time of the resulting electrodes is less than 10 s. The glucose response under N2 shows an apparent Michaelis constant, Km' = 7.3 mM, and limiting current densities, jmax, between 100 and 800 microA/cm2. Currents are decreased by 30-50% in air-saturated solutions because of competition between O2 and the Os(III) complex for electrons from the reduced enzyme. Rotating ring desk experiments in air-saturated solutions containing 10 mM glucose show that about 20% of the active enzyme is electrooxidized via the Os(III) complex, while the rest is oxidized by O2. These results suggest that only part of the active enzyme is in electrical contact with the electrode.     

Integration of enzymes and electrodes: spectroscopic and electrochemical studies of chitosan-enzyme films

A new film-forming solution was developed for the efficient immobilization of enzymes on solid substrates. The solution consisted of a biopolymer, chitosan (CHIT), that was chemically modified with a permeability-controlling agent, Acetyl Yellow 9 (AY9), using glutaric dialdehyde (GDI) as a molecular tether. A model enzyme, glucose oxidase (GOx), was mixed with the CHIT-GDI-AY9 solution and cast on the surface of platinum electrodes to form robust CHIT-GDI-AY9-GOx films for glucose biosensing. UV-visible and infrared spectroscopies were used to determine the composition of the films. The optimized films contained on average 1 molecule of AY9/3 glucosamine units of chitosan and 25 free GDI tethers/1 molecule of GOx. The electrochemical assays of the films indicated both a very high efficiency of enzyme immobilization (approximately 99%) and large enzyme activity (60 units cm(-2)). The latter translated into a high sensitivity (42 mA M(-1) cm(-2)) of the Pt/CHIT-GDI-AY9-GOx biosensor toward glucose. The biosensor operated at 0.450 V, had a fast response time (t90% < or = 3 s), and was free of typical interferences, and its dynamic range covered 3 orders of magnitude of glucose concentrations. The lowest actually detectable concentration was 10 microM glucose. In addition, the biosensor displayed a practical shelf life and excellent operational stability, e.g. its response was stable during 24-h testing under continuous polarization and continuous flow of 5.0 mM glucose solution. The proposed approach to enzyme immobilization is simple, efficient, and cost-effective and should be of importance in the development of biosensors based on other enzymes that are more expensive than glucose oxidase.     

Amperometric glucose biosensor based on in situ electropolymerized polyaniline/poly(acrylonitrile-co-acrylic acid) composite film

A new type of in situ electropolymerization was used for electrochemical biosensor design. The biologic film was prepared by in situ electropolymerization of aniline into microporous poly(acrylonitrile-co-acrylic acid)-coated platinum electrode in the presence of glucose oxidase. The results of EIS and SEM indicated the successful immobilization for enzyme in the composite polymer film. The novel glucose biosensor exhibited good selectivity and operational stability, which showed no apparent loss of activity after 40 consecutive measurements and intermittent usage for 45 days with storage in a phosphate buffer solution at 4°C. In addition, optimization of the biosensor construction as well as effects of applied potential, pH value of solution, temperature and common interfering compounds on the amperometric response of the sensor were investigated and discussed.     

Protease amperometric sensor

An amperometric biosensor for the detection of trypsin was developed. The latter was based on a two-layer configuration, namely, a polymer-glucose oxidase inner layer and a gelatin outer layer. In the presence of glucose, the enzyme layer produces H2O2 and hence an amperometric signal due to H2O2 electrooxidation was generated by potentiostating the electrode at 0.6 V. The biosensor detects the change in the increase in the maximum current caused by the proteolytic digestion of gelatin, which covers the platinum electrodes, thereby facilitating a speedier access for the glucose substrate to the electrode modified with both poly(pyrrole-alkylammonium) and glucose oxidase molecules. Our biosensor detected low trypsin concentrations down to 42 pM with a response time of approximately 10 min, making it a very sensitive device in the detection of lower trypsin levels with such future putative applications as the diagnosis of pancreatic diseases.     

Enzyme electrodes with conductive polymer membranes and Langmuir-Blodgett films

A glucose sensor consisting of a conductive polypyrrole membrane and a lipid Langmuir-Blodgett (LB) film has been investigated. Different arrangements of the biosensor on the electrodes examined were (1) electrode with glucose oxidase (GOD)-immobilized lipid LB films; (2) GOD-immobilized LB film coated on polypyrrole-modified electrode; (3) electrode with a GOD-immobilized polypyrrole membrane; (4) GOD-immobilized LB film coated on a GOD-polypyrrole-modified electrode. It was shown that the quality of the biosensor was apparently improved in case (4) with respect to both the detectable concentration range of glucose and the lifetime over which it could be used. The number of layers of the LB film has a marked influence on the sensitivity of the biosensor, an optimum number of layers existing for the best response. The mechanism of such an improvement is discussed.