Amperometric determination of glucose in undiluted food samples

Glucose oxidase is immobilized onto a cellulose acetate membrane by glutaraldehyde linkage, and the membrane is used to cover the platinum electrode of a hydrogen peroxide sensor. A silanized polycarbonate membrane then covers the enzyme layer, and extends the linear calibration range to higher concentrations. The sensor, when incorporated into a flow-injection system, allows the determination of glucose at levels up to 1 M in soft drinks at a rate of 60 samples h^?1 without sample dilution.     

Evaluation of glucose biosensors based on Prussian Blue and lyophilised, crystalline and cross-linked glucose oxidases (CLEC(R))

Glucose biosensors based on lyophilised, crystalline and cross-linked glucose oxidase (GOx, CLEC(R)) and commercially available lyophilised GOx immobilised on top of glassy carbon electrodes modified with electrodeposited Prussian Blue are critically compared. Two procedures were carried out for preparing the biosensors: (1) deposition of one layer of adsorbed GOx dissolved in an aqueous solution followed by deposition of two layers of low molecular weight Nafion(R) dissolved in 90% ethanol, and (2) deposition of two layers of a mixture of GOx with Nafion dissolved in 90% ethanol. The performance of the biosensors was evaluated in terms of linear response range for hydrogen peroxide and glucose, detection limit, and susceptibility to some common interfering species (ascorbic acid, acetaminophen and uric acid). The operational stability of the biosensors was evaluated by applying a steady potential of -50 mV versus Ag/AgCl to the glucose biosensor and injecting standard solutions of hydrogen peroxide and glucose (50 muM and 1.0 mM, respectively, in phosphate buffer) for at least 5 h in a flow-injection system. Scanning electron microscopy was used for visualisation of the Prussian Blue redox catalyst and in the presence of the different GOx preparations on the electrode surface.     

Simultaneous determination of glucose and L-lactate in rat brain by an electrochemical in vivo flow-injection system with an on-line microdialysis sampling.

An electrochemical in vivo flow-injection system with an on-line microdialysis sampling is proposed for the simultaneous monitoring of L-lactate and glucose in rat brain. In the first stage of the operation, the dialysate from the microdialysis probe is delivered to a sample loop of the six-way autoinjector by perfusing Ringer's solution for 80 s at 5 microl min(-1). In the second stage, the dialysate collected in the sample loop is automatically injected for 10 s into the flow-injection line. Injected dialysate is split into two streams and two portions pass through two channels with two different immobilized enzyme reactors (glucose oxidase and lactate oxidase immobilized reactors) to produce hydrogen peroxide from glucose and L-lactate in the dialysate. After a subsequent confluence of the streams, produced hydrogen peroxide can be detected amperometrically at a downstream poly(1,2-diaminobenzene) film-coated platinum electrode, without any interference from oxidizable species and proteins present in the dialysate. Because each channel has a different residence time, two peaks are obtained. The first peak corresponds to L-lactate and the second peak to glucose. The peak current is linearly related to the concentrations of L-lactate between 0.2 and 10 mM and glucose between 0.1 and 20 mM. The present method can be successfully applied to the simultaneous in vivo monitoring of L-lactate and glucose in rat brain. The analytical speed is 45 dialysates h(-1).     

Amperometric assay of glucose and lactic acid by flow injection analysis

A computer-controlled flow-injection system is described for the assay of D-glucose and L-lactic acid in undiluted plasma. Glucose or lactate is quantified by coupling an immobilized glucose oxidase or lactate oxidase membrane with an amperometric sensor; the hydrogen peroxide generated is directly related to the concentration of glucose or lactate. The linear range is 0–40 mM and 0–10 mM for glucose and lactic acid, respectively. The sample frequency is 60 h^?1 with a standard deviation of less than 1.5%. Correlation with the results for blood plasma obtained by routine clinical analyzers was good for both glucose and lactic acid.     

Comparative study of first-, second- and third-generation amperometric glucose enzyme electrodes in continuous-flow analysis of undiluted whole blood

First-, second- and third-generation amperometric glucose enzyme electrodes were compared under flow-injection and steady-state conditions for the monitoring of undiluted whole blood. First-generation electrodes, based on the detection of hydrogen peroxide at a platinum electrode, are generally unsuitable because of the eventual poisoning of the electrode and because of their susceptibility to oxygen variation. Second-generation electrodes in which a mediator is used for the reoxidation of glucose oxidase are more suitable for the analysis of whole blood under both steady-state and flow-injection conditions. However, the choice of mediator is important. The best results with regard to linear range and stability were obtained with tetrathiafulvalene, whereas dimethylferrocene required considerable pretreatment before use. A third-generation electrode based on tetrathiafulvalene-tetracyanoquinodimethane where direct oxidation of glucose oxidase occurs also proved useful but showed lower stability and a smaller dynamic range compared with the second-generation devices. Flow-injection and steady-state studies were carried out using wall-jet cell geometry.     

Amperometric determination of glucose with a ferrocene-mediated glucose oxidase/polyacrylamide gel electrode

Enzyme electrodes were constructed by immobilization of glucose oxidase and ferrocene into cross-linked polyacrylamide gels. Electrogenerated ferrocinium ion acts as a direct electron mediator between glucose oxidase and a reticulated vitreous carbon (RVC)/graphite support bed. The electrode is easily constructed, gives a current response proportional to glucose concentrations up to 30 mM, and has good chemical stability in water and air.     

Self-gelatinizable copolymer immobilized glucose biosensor based on prussian blue modified graphite electrode

A novel poly(vinyl alcohol) grafting 4-vinylpyridine self-gelatinizable copolymer was adapted to immobilize glucose oxidase. The reduction of hydrogen peroxide (H2O2) was detected at a Prussian Blue (PB) modified graphite electrode. A stable and sensitive glucose amperometric biosensor is described. The copolymer is a good biocompatible polymer in which the glucose oxidase retains high activity. Moreover, the copolymer can adhere firmly to the inorganic PB membrane. The sensor showed an apparent Michaelis-Menten constant of 18 +/- 0.2 mM and a maximum current density of 1.14 microA cm-2 mM-1. The linear range is from 5 microM to 4.5 mM glucose and the detection limit is 0.5 microM glucose. The catalytic efficiency of PB for the reduction of H2O2 is higher than that for the oxidation of H2O2. Glucose concentrations in serum samples from healthy persons and diabetic patients were determined using the sensor. The results compared well with those provided by the hospital using a spectroscopy method.     

A regenerable pseudo-reagentless glucose biosensor based on Nafion polymer and l,1''-dimethylferricinium mediator

Glucose oxidase was immobilized onto electrodes by co-deposition from an aqueous solution containing the diluted ion-exchange polymer Nafion. The cationic exchange property of the polymer was used to provide high local concentrations of l,1'-dimethylferricinium (DMFc⁺) mediator in the film by exchange from solution. The mediated electrodes were operated at +200 mV (vs. ), and the Nafion film was shown to reduce interfering current from ascorbate anion. Cyclic voltammetric analysis revealed a fourteen-fold increase in the effective DMFc⁺ activity at the electrode after extraction into the film. The sensitivity to glucose was 52 μA/cm²/mM in a solution containing 0.09 mM DMFc⁺, which is at least three-fold greater than reported for similar electrodes using hydrogen peroxide detection at +650 mV, with a response time of less than 1 min for a 10 μm thick membrane. Oxygen interference was significant, requiring deaeration of the solution before analysis. The electrodes exhibited no significant decrease in sensitivity for more than 50 days on storage in acetate buffer. Electrodes covered with 8000 MWCO dialysis membrane slowed the exchange of DMFc⁺ with the solution such that the Nafion film functioned as a mediator reservoir. This permitted reagentless analysis of glucose, typically capable of twenty assays when measuring concentrations between 0.1 and 1 mM. The sensitivity for glucose was 7.85 μA/cm²/mM, which is 15% of the sensitivity for the electrode without the dialysis membrane. The detection limit was 20 μM, with a linear range extending to about 3 mM, giving a dynamic range of over two orders of magnitude. Thus where some sacrifice of sensitivity and response rate may be made, the dialysis membrane cover enables multiple analyses in a reagentless biosensor scheme.     

Carbon nanotubes paste electrode

The performance of carbon nanotubes paste electrodes (CNTPE) prepared by dispersion of multi-wall carbon nanotubes (MWNT) within mineral oil is described. The resulting electrode shows an excellent electrocatalytic activity toward ascorbic acid, uric acid, dopamine, 3,4-dihydroxyphenylacetic acid (dopac) and hydrogen peroxide. These properties permit an important decrease in the overvoltage for the oxidation of ascorbic acid (230 mV), uric acid (160 mV) and hydrogen peroxide (300 mV) as well as a dramatic improvement in the reversibility of the redox behavior of dopamine and dopac, in comparison with the classical carbon (graphite) paste electrodes (CPE). The substantial decrease in the overvoltage of the hydrogen peroxide reduction (400 mV) associated with a successful incorporation of glucose oxidase (GOx) into the composite material, allow the development of a highly selective and sensitive glucose biosensor without using any metal, redox mediator or anti-interference membrane. No interference was observed at −0.100 V even for large excess of ascorbic acid, uric acid and acetaminophen. A linear response up to 30 mM (5.40 g l⁻¹) glucose with a detection limit of 0.6 mM (0.11 g l⁻¹) were obtained with the CNTPE modified with 10% w/w GOx. Such an excellent performance of CNTPE toward hydrogen peroxide, represents a very good alternative for developing other enzymatic biosensors.     

Sensitive Flow-Injection Electrochemical Determination of Hydrogen Peroxide at a Palladium Nanoparticle-Modified Pencil Graphite Electrode

A novel flow-injection amperometric method was proposed for the sensitive and enzymeless determination of hydrogen peroxide based on its electrocatalytic reduction at a palladium nanoparticle-modified pretreated pencil graphite electrode in a laboratory-constructed electrochemical flow cell. Cyclic voltammograms of the unmodified and modified electrodes were recorded in pH 7.0 phosphate buffer containing 0.10 M KCl at a scan rate of 50?mV s^?1 for the investigation of electrocatalytic reduction of hydrogen peroxide at the palladium nanoparticle-modified pretreated pencil graphite electrode. Cyclic voltammograms of the pretreated pencil graphite electrode revealed an irreversible oxidation peak and a weak reduction peak of hydrogen peroxide at +1100?mV and –450?mV vs. an Ag/AgCl/KCl saturated reference electrode. However, the reduction of hydrogen peroxide was observed at –100?mV with an increase in current in the cyclic voltammograms of the palladium nanoparticle-modified pretreated pencil graphite electrode compared to the unmodified electrode. These results indicate that the palladium nanoparticle-modified pretreated pencil graphite electrode exhibits efficient electrocatalytic activity for the reduction of hydrogen peroxide. A linear concentration range was obtained between .01 and 10.0?mM hydrogen peroxide with a detection limit of 3.0 µM from flow injection amperometric current–time curves recorded in pH 7.0 phosphate buffer at –100?mV and a 2.0?mL min^?1 flow rate. The novelty of this work relies on its use of a laboratory-constructed flow cell constructed for the pencil graphite electrode using these inexpensive, disposable, and electrochemically reactive modified electrodes for the amperometric determination of hydrogen peroxide in a flow injection analysis system.     

A glucose sensor based on glucose dehydrogenase adsorbed on a modified carbon electrode

A glucose sensor is prepared by adsorption of the mediator Meldola blue (N,N-dimethyl-7-amino-1,2-benzophenoxazinium ion, as well as glucose dehydrogenase, on the surface of a carbon electrode. The nicotinamide coenzyme, whhich is present in the solution, is reduced in the enzymatic reaction and is re-oxidized amperometrically at 0 mV vs. Ag/AgCl. The properties of such electrodes depend on whether the mediator or the enzyme is adsorbed first; possible models for the molecular arrangements at the surface are discussed. The modified electrode is mounted in a flow-through cell in a flow-injection system and tested with 50-μl injections of β-d-glucose. The calibration graphs were linear in the range 5 × 10^?6—2 × 10^?3 M βd glucose with the highest sensitivity at pH 6.0. The membrane-free enzyme electrode has a fast response; peak widths are 12 s at half height (flow rate 0.7 ml min^?1, making it possible to process 100 samples h^?1.     

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.     

Rapid enzymatic chemiluminescent assay of glucose by means of a hybrid flow-injection/sequential-injection method

This work reports a hybrid flow-injection analysis (FIA)/sequential-injection analysis (SIA) method for the rapid enzymatic assay of glucose with soluble glucose oxidase (GOD). The method relies on the sequential injection of segments of the sample and of a solution of enzyme by means of a multi-port selection valve in a flowing water stream. As the two zones are swept downstream, they overlap and merge so that the glucose in the sample is enzymatically oxidised. The generated hydrogen peroxide is merged with an alkaline luminol solution and the chemiluminescence (CL) intensity is monitored and related to the glucose concentration in the sample. The linear range of the method for glucose determination is 0.01-1 mmol L⁻¹, the relative standard deviation is 3.9% at the 0.08 mmol L⁻¹ level (n = 8), the limit of detection at the 2σ level is 4 μmol L⁻¹ glucose and the injection rate is 80 h⁻¹. The method was applied to the analysis of energy drinks and honey with relative errors in glucose determination in the range 100 ± 4.3%. The advantages of the proposed method are the wide linear range, the simple instrumentation used, the low consumption of sample and reagents, the elimination of catalysts and immobilised enzymes and the high sample throughput.     

Enzyme electrode for glucose determination in whole blood

The development of a glucose sensor suitable for use with whole blood is described. It is based on anodic oxidation at +700 mV of hydrogen peroxide with a platinum electrode covered with a gas permeable membrane. Glucose reacts with glucose oxidase immobilised on the external side of the membrane, and forms hydrogen peroxide which is able to cross the gas permeable membrane due to its high vapour tension, while other electroactive substances that are important interferents are completely blocked. This principle was discovered several years ago but no practical application was presented up to now. Therefore in this work a number of different commercial membranes were tested, in order to obtain a resistant, rapidly responding and interference free sensor to be used in conjunction with a blood gas measurement apparatus. Coimmobilisation of glucose oxidase and catalase was found to be useful for fast response and recovery of the electrode. Using some of the tested membranes, the linearity range is 1-15 mM, CV 5%, response time 90 s, recovery time for the next sample 120 s. The membrane's working life is 2-3 weeks.     

Differential amperometric determination of hydrogen peroxide in honeys using flow-injection analysis with enzymatic reactor

Hydrogen peroxide (H2O2) present in honey was rapidly determined by the differential amperometric method in association with flow-injection analysis (FIA) and a tubular reactor containing immobilized enzymes. A gold electrode modified by electrochemical deposition of platinum was employed as working electrode. Hydrogen peroxide was quantified in 14 samples of Brazilian commercial honeys using amperometric differential measurements at +0.60V vs. Ag/AgCl((sat)). For the enzymatic consumption of H2O2, a tubular reactor containing immobilized peroxidase was constructed using an immobilization of enzymes on Amberlite IRA-743 resin. The linear dynamic range in H2O2 extends from 1 to 100 x 10(-6) mol L(-1), at pH 7.0. At flow rate of 2.0 mL min(-1) and injecting 150 microL sample volumes, the sampling frequency of the 90 determinations per hour is afforded. This method is based on three steps involving the flow-injection of: (1) the sample spiked with a standard solution, (2) the pure sample and (3) the enzymatically treated sample with peroxidase immobilized. The reproducibility of the current peaks for hydrogen peroxide in 10(-5) mol L(-1) range concentration showed a relative standard deviation (R.S.D.) better than 1%. The detection limit of this method is 2.9 x 10(-7) mol L(-1). The honey samples analyses were compared with the parallel spectrophotometric determination, and showed an excellent correlation between the methods.     

Flow-injection analysis of glucose without enzyme based on electrocatalytic oxidation of glucose at a nickel electrode

This paper reports a flow-injection analysis (FIA) of glucose not using enzyme based on the electrocatalytic oxidation of glucose at a nickel electrode. The electrocatalytic mechanism and quantificational method of glucose have been investigated. The current intensity of the electrocatalytic oxidation to glucose at the potential of 550 mV is proportional to the concentration of glucose over the range of 0.10-2.50 mmol l⁻¹, with a 0.04 mmol l⁻¹ detection limit (S/N = 3) and a correlation coefficient of 0.9991. The relative standard deviation (R.S.D.) is less than 4.3% (n = 5) for the determination of practical serum samples. The biologic compounds probably existed in the sample, such as ascorbic acid, uric acid, dopamine and epinephrine, do not disturb the determination of glucose. The result is satisfactory for the determination of glucose in human serum sample as comparison to that from the routine hexokinase method.     

Chemiluminometric method for the determination of glycerol in wine by flow-injection analysis with co-immobilized glycerol dehydrogenase/NADH oxidase

A flow-injection method for the determination of glycerol in wine is described. Glycerol dehydrogenase and NADH oxidase were co-immobilized on poly (vinyl alcohol) beads and incorporated in a flow-injection system. The hydrogen peroxide produced was detected chemiluminometrically via a luminol-hexacyanoferrate (III) reaction. Wine was diluted 1000-fold with water and sample solution (50 microl) was injected into the carrier stream. The calibration graph was linear in the range 3 x 10(-7)-3 x 10(-4) M; the detection limit was 7 x 10(-8) M and the sample throughout was 30 h(-1) without carryover.     

Microfabricated glucose biosensor with glucose oxidase entrapped in sol-gel matrix

A microfabricated glucose biosensor based on an amperometeric hydrogen peroxide electrode has been developed. A sol-gel layer with 5 A pore size and 2 mum thickness was used as the glucose oxidase entrapping matrix. The sol-gel matrix formed over the silicon-based sensor has good mechanical and chemical stability, and the ability to entrap a large amount of enzyme. The miniaturized electrode sensing system is composed of platinum as both working and counter electrodes and silver as a reference electrode. Nafion(R) coating was applied as the interference limiting layer. A series of technologies, such as standard photolithography, electron beam evaporation and image reverse lift-off were utilized for mass production allowing 143 electrodes to be produced at the same time. The effect of oxidable interferences was <10% of the background value of the sensor response. Calibration tests of a series of individual sensors manufactured from the same silicon wafer and dip coated in the same conditions, showed a highly reproducible response characteristics (linear range up to 500 mg dl(-1) and mean sensitivity of 0.54+/-0.14 nA mg(-1) dl(-1) (n=10)).     

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.     

Determination of glucose in blood by flow injection analysis and an immobilized glucose oxidase column

A flow-injection system for glucose determination is described. Glucose oxidase is immobilized on controlled porosity glass (CPG) and used in a glass column (2.5 mm diameter × 2.5 cm). The hydrogen peroxide produced by the enzymatic reaction (? 1 × 10^?6 M) is detected by the current produced in a flow-through cell, with two platinum electrodes having a potential difference of 0.6 V. Glucose (0–20 mmol l^?1) can be determined in blood plasma either with a dialyser in the system or, better, by incorporating a column of copper(II) diethyldithiocarbamate on CPG before the enzyme column. The results compared well with those obtained by a conventional analyser system. The glucose oxidase column showed little change in activity over a 10-month period.