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An amperometric immunosensor for the quantification of Staphylococcus aureus based on the coimmobilization of rabbit immunoglobulin G (RbIgG) and tyrosinase on a mercaptopropionic acid self-assembled monolayer modified gold electrode is reported. A competitive mode in which protein-A-bearing S. aureus cells and antiRbIgG labeled with alkaline phosphatase (AP) compete for the binding sites of immobilized RbIgG was used. Monitoring of the affinity reaction was carried out by the amperometric detection at -0.15 V of phenol generated in the enzyme reaction with AP, at the tyrosinase-modified electrode through the electrochemical reduction of the o-quinone formed. Optimization of the working variables, such as the immunosensor composition and incubation times, the applied potential, the working pH and the concentration of phenyl phosphate used as the AP substrate, was carried out. Under the optimized conditions, both the repeatability of the measurements and the reproducibility of the responses obtained with different immunosensors yielded relative standard deviation values for the steady-state current lower than 10%. The immunosensor showed a dynamic range from 4.4x10(5) to 1.8x10(7) S. aureus cells mL(-1), with a detection limit of 1.7x10(5) cells mL(-1). The limit of detection was remarkably improved by subjecting S. aureus cells to wall lysis by heat treatment. The value obtained was 2.3x10(3) cells mL(-1), which is adequate for the monitoring of S. aureus contamination levels in some foodstuffs. As an application, milk samples spiked with bacteria at the 4.8x10(3) cells mL(-1) level were analyzed.

The construction and performance of integrated amperometric biosensors for the determination of glycerol are reported. Two different biosensor configurations have been evaluated: one based on the glycerol dehydrogenase/diaphorase (GDH/DP) bienzyme system, and another using glycerol kinase/glycerol-3-phosphate oxidase/peroxidase (GK/GPOx/HRP). Both enzyme systems were immobilized together with the mediator tetrathiafulvalene (TTF) on a 3-mercaptopropionic acid (MPA) self-assembled monolayer (SAM)-modified gold electrode by using a dialysis membrane. The electrochemical oxidation of TTF at +150 mV (vs. Ag/AgCl), and the reduction of TTF + at 0 mV were used for the monitoring of the enzyme reactions for the bienzyme and trienzyme configurations, respectively. Experimental variables concerning both the biosensors composition and the working conditions were optimized for each configuration. A good repeatability of the measurements with no need of cleaning or pretreatment of the biosensors was obtained in both cases. After 51 days of use, the GDH/DP biosensor still exhibited 87% of the original sensitivity, while the GK/GPOx/HRP biosensor yielded a 46% of the original response after 8 days. Calibration graphs for glycerol with linear ranges of 1.0 × 10 −6 to 2.0 × 10 −5 or 1.0 × 10 −6 to 1.0 × 10 −5 M glycerol and sensitivities of 1214 ± 21 or 1460 ± 34 μA M −1 were obtained with GDH/DP and GK/GPOx/HRP biosensors, respectively. The calculated detection limits were 4.0 × 10 −7 and 3.1 × 10 −7 M, respectively. The biosensors exhibited a great sensitivity with no significant interferences in the analysis of wines. The biosensors were applied to the determination of glycerol in 12 different wines and the results advantageously compared with those provided by a commercial enzyme kit.

Quantification of bacterial pollution by amperometric detection at 0.0 V of glucose consumption at a graphite-Teflon-glucose oxidase-peroxidase-ferrocene composite biosensor under flow injection conditions is reported. Using Escherichia coli as the model bacterium, the composition of the growing medium was optimized. A constant glucose concentration of 4.0 x 10(-4) M was added to the culture medium. The relative response to glucose, expressed as the ratio between the amperometric signal and the signal at incubation time t = 0 multiplied by 100, as a function of E. coli concentration, showed a typical behaviour. Limits of detection of 6.5 x 10(2) or 6.5 cfu mL(-1) were achieved after 3 or 7 h of incubation, respectively, with no pre-concentration step. The detection of bacteria did not affect the lifetime of the biosensor. The feasibility of the detection of Staphylococcus aureus and Salmonella choleraesuis throughout the glucose consumption measurement at the composite biosensor is also demonstrated. The capability of bacterial identification by evaluation of bacterial growth in the culture medium containing the antibiotics polymyxin B, vancomycin, erythromycin, bacitracin, chloramphenicol, tetracycline and ciprofloxacin, was investigated. Each micro-organism tested exhibited a different antibiotic susceptibility profile, thus suggesting the possibility of bacteria differentiation. A rapid methodology for screening of bacteria is proposed.

An integrated amperometric gluconic acid biosensor constructed using a gold electrode (AuE) modified with a self-assembled monolayer (SAM) of 3-mercaptopropionic acid (MPA) on which gluconate dehydrogenase (GADH, 0.84 U) and the mediator tetrathiafulvalene (TTF, 1.5 micromol) were coimmobilized by covering the electrode surface with a dialysis membrane is reported. The working conditions selected were Eapp=+0.15 V and 25+/-1 degrees C. The useful lifetime of one single TTF-GADH-MPA-AuE was surprisingly long. After 53 days of continuous use, the biosensor exhibited 86% of the original sensitivity. A linear calibration plot was obtained for gluconic acid over the 6.0x10(-7) to 2.0x10(-5) M concentration range, with a limit of detection of 1.9x10(-7) M. The effect of potential interferents (glucose, fructose, galactose, arabinose, and tartaric, citric, malic, ascorbic, gallic, and caffeic acids) on the biosensor response was evaluated. The behavior of the biosensor in a flow-injection system in connection with amperometric detection was tested. The analytical usefulness of the biosensor was evaluated by determining gluconic acid in wine and must samples, and the results obtained were validated by comparison with those provided by using a commercial enzyme test kit.

Publisher Summary This chapter presents a procedure to construct an integrated amperometric biosensor based on self-assembled monolayer modified gold electrodes and evaluate the analytical usefulness of the biosensor by determining gluconic acid in honey samples. For construction of the biosensor clean AuE is immersed in a 40 mmol L–1 MPA solution in EtOH/H2O2 (75/25, v/v) for at least 15 h to form the MPA-SAM. The electrode surface is dried at room temperature. Further, 1.5-cm2 piece of a dialysis membrane is fixed on top of the electrode surface. The biosensor was used for the analysis of gluconic acid in honey samples. The results obtained with the biosensor agreed fairly well with those achieved by applying the enzymatic kit. The parameters of the linear least square regression curve obtained by plotting the biosensor results versus the results obtained with the commercial kit for all the honey.

Publisher Summary The biosensors technology is being more and more applied to control—in a rapid, effective, reliable, and easy-to-use and low-cost manner—target compounds for the assessment of food quality. Enzyme biosensors—particularly electrochemical enzyme biosensors—play a crucial role in this area. Specific enzymes can be used for the selective recognition of the targeted compounds of interest in food quality control. The nature and selectivity of their catalytic activity, and the ability of a single enzyme molecule to catalyze the reaction of numerous substrate molecules, providing an amplification effect, make enzymes excellent tools for the analysis of such compounds. A further advantage is that most enzyme-catalyzed reactions can be followed by simple, widely available electrochemical methods. Typical enzymes used for biosensor construction belong to the oxidoreductase group (oxidases, dehydrogenases) and the hydrolase and lyase groups. The oxidoreductase group presents the advantage of the possibility of direct electric coupling with the electrochemical transducer, allowing direct control over the enzyme reaction and real-time monitoring.

This work shows the design of a low-cost, portable instrument for amperometric biosensors, which measures the faradaic currents originated by the electronic interchanges between a specific substance and a biological recognition system, present on an electrode and kept at an appropiate potential. The final objective is to obtain a direct relationship between those currents and the concentration of the substrate to be analyzed. This instrument may be used for sensors or cells with two or three electrodes in order to detect extremely low concentrations of a wide variety of substances. The instrument was evaluated comparing calibration curves from glucose, ethanol and cholesterol biosensors to those obtained from very expensive commercial equipment. Good linearity and sensibility, as well as low noise measurements were obtained. Moreover, thanks to its reduced size and low cost, this instrument can be used directly in field applications.

The use of a laccase biosensor, under both batch and flow injection (FI) conditions, for a rapid and reliable amperometric estimation of the total content of polyphenolic compounds in wines is reported. The enzyme was immobilized by cross-linking with glutaraldehyde onto a glassy carbon electrode. Caffeic acid and gallic acid were selected as standard compounds to carry out such estimation. Experimental variables such as the enzyme loading, the applied potential, and the pH value were optimized, and different aspects regarding the operational stability of the laccase biosensor were evaluated. Using batch amperometry at -200 mV, the detection limits obtained were 2.6 x 10(-3) and 7.2 x 10(-4) mg L(-1) gallic acid and caffeic acid, respectively, which compares advantageously with previous biosensor designs. An extremely simple sample treatment consisting only of an appropriate dilution of wine sample with the supporting electrolyte solution (0.1 mol L(-1) citrate buffer of pH 5.0) was needed for the amperometric analysis of red, rose, and white wines. Good correlations were found when the polyphenol indices obtained with the biosensor (in both the batch and FI modes) for different wine samples were plotted versus the results achieved with the classic Folin-Ciocalteu method. Application of the calibration transfer chemometric model (multiplicative fitting) allowed that the confidence intervals (for a significance level of 0.05) for the slope and intercept values of the amperometric index versus Folin-Ciocalteu index plots (r = 0.997) included the unit and zero values, respectively. This indicates that the laccase biosensor can be successfully used for the estimation of the polyphenol index in wines when compared with the Folin-Ciocalteu reference method.

An electrochemical DNA biosensor based on recognition of double or single stranded DNA (ds-DNA/ss-DNA) immobilised on a self-assembled modified gold electrode is presented for denaturalisation and hybridisation detection. DNA is covalently bond on a self assembled 3-mercaptopropionic acid monolayer by using water soluble N-3-(dimethylaminopropyl)-N prime;ethylcarbodiimide hydrochloride (EDC) and N-hydroxisulfosuccinimide (NHSS) as linkers. The interaction between the immobilised DNA and methylene blue (MB) is investigated using square wave voltammetry (SWV). The increase or diminution of peak currents of the MB upon the hybridisation or denaturalisation event at the modified electrode surface is studied.

The use of an amperometric graphite–Teflon composite tyrosinase biosensor for the rapid monitoring of alkaline phosphatase (ALP), with no need of an incubation step and using phenyl phosphate as the substrate, is reported. Phenol generated by the action of ALP is monitored at the tyrosinase composite electrode through the electrochemical reduction of the o-quinone produced to catechol, which produces a cycle between the tyrosinase substrate and the electroactive product, giving rise to the amplification of the biosensor response and to the sensitive detection of ALP. The current was measured at −0.10 V 5 min after the addition of ALP. As a compromise between high ALP activity and high sensitivity for the detection of phenol, a pH of 8.5 was chosen. The substrate concentration was also optimized. A linear calibration plot was obtained for ALP between 2.0 × 10−13 and 2.5 × 10−11, with a detection limit of 6.7 × 10−14 M. Different types of milk were analyzed with good results, using an extremely simple and rapid procedure.

A comparison of the analytical performances of several enzyme biosensor designs, based on the use of different tailored gold nanoparticle-modified electrode surfaces, is discussed. Glucose oxidase (GOx) and the redox mediator tetrathiafulvalene were coimmobilized in all cases by crosslinking with glutaraldehyde. The biosensor designs tested were based on the use of (i) colloidal gold (Aucoll) bound on cysteamine (Cyst) monolayers self-assembled on a gold disk electrode (AuE) and (ii) glassy carbon electrodes (GCEs) modified with electrodeposited gold nanoparticles (nAu). The results obtained with these designs were compared with those provided by a GOx/Cyst–AuE and a GOx/MPA–AuE. In the second case (ii), configurations based on direct immobilization of GOx on nAu (GOx/nAu–GCE) or on Cyst or MPA self-assembled monolayers (SAMs) previously bound on gold nanoparticles (GOx/Cyst–nAu–GCE or GOx/MPA–nAu–GCE, respectively) were compared. The analytical characteristics of glucose calibration plots and the kinetic parameters of the enzyme reaction were compared for all of the biosensors tested. The GOx/Aucoll–Cyst–AuE design showed a sensitivity for glucose determination higher than that achieved with GOx/Cyst–AuE and GOx/Aucoll–Cyst/Cyst–AuE and similar to that achieved with GOx/MPA–AuE. Moreover, the useful lifetime of one single GOx/Aucoll–Cyst–AuE was 28 days, remarkably longer than that of the other GOx biosensor designs.

L-Lactic acid was determined in cow's milk, goat's milk and whey protein concentrate (WPC)-enriched goat's milk yoghurts by using an amperometric graphite-Teflon biosensor in which peroxidase (HRP), L-lactate oxidase (L-LOD) and the mediator ferrocene were immobilized. The correlation between the L-lactic acid results obtained using the bienzyme biosensor method and a standard colorimetric enzymatic method was 0.95. One-and two-way analyses of variance indicated that the biosensor method was able to discriminate between WPC supplemented and non-supplemented yoghurts, whereas this discrimination could not be accomplished with the colorimetric enzymatic method. The biosensor described in this paper offers considerable advantages to industry because it provides rapid, direct and economical analysis of yoghurt quality.

The construction and performance of bienzyme amperometric composite biosensors for the selective determination of l- or d-amino acids is reported. D- or L-Amino acid oxidase, horseradish peroxidase, and the mediator ferrocene were coimmobilized by simple physical inclusion into the bulk of a graphite-70% Teflon electrode matrix. Working conditions including amino acid oxidase loading and pH were optimized. Studies on the repeatability of the amperometric response obtained at +0.00 V, with and without regeneration of the electrode surface by polishing, on the useful lifetime of one single biosensor and on the reproducibility in the fabrication of different biosensors illustrate the robustness of the bioelectrodes design. Calibration plots by both amperometry in stirred solutions and flow injection with amperometric detection were obtained for L-arginine, L-phenylalanine, L-leucine, L-methionine, L-tryptophan, D-leucine, D-methionine, D-serine, and D-valine. Differences in sensitivity were discussed in terms of the hydrophobicity of the substrate and of the electrode surface. The bienzyme composite electrode was applied to the determination of L- and D-amino acids in racemic samples, as well as to the estimation of the L-amino acids content in muscatel grapes.

The possibilities and advantages of using reversed micelles as appropriate working media for the development of amperometric enzyme biosensors with analytical purposes is discussed. The influence of the composition of reversed micelles on the amperometric responses obtained at the enzyme electrodes is illustrated. Some analytical applications such as the determination of phenolic pollutants, of the antioxidant BHA and of dimethyl- and diethyldithiocarbamates are shown.

Reduced graphene nanoparticles were prepared from graphene oxide through a two-step covalent modification approach. Graphene oxide was first enriched with reactive epoxy groups by anchoring (3-glycidyloxypropyl)trimethoxysilane at the hydroxyl groups located on the nanocarbon basal plane. Modified graphene oxide was further cross-linked and partially reduced by treatment with the fourth-generation ethylenediamine core polyamidoamine G-4 dendrimer producing graphene nanoparticles with crumpled paper-like morphology. This graphene derivative was employed as a coating material for glassy carbon electrodes and the nanostructured electrode was tested for the preparation of electrochemical biosensors by immobilizing the enzyme tyrosinase through cross-linking with glutaraldehyde. This bioelectrode showed excellent electroanalytical behavior for catechol with a fast response in about 6 s, linear range of 10 nM to 22 μM, sensitivity of 424 mA M−1, and low detection limit of 6 nM. The enzyme biosensor also showed high stability when stored at 4 °C under dry and wet conditions.

Superparamagnetic Fe3O4 nanoparticles were coated with (3-aminopropyl)triethoxysilane and further branched with monomethoxypolyethylene glycol chains. These nanoparticles were employed for the non-covalent surface modification of single walled carbon nanotubes, conferring them magnetic properties. This nanomaterial was employed to immobilize the enzyme xanthine oxidase in order to construct magnetically modified disposable gold screen-printed electrodes as bioelectrodes for the determination of xanthine. The electroanalytical properties of the biosensor were modulated by the nanomaterial composition, being optimal at a carbon nanotubes : magnetic nanoparticles ratio of 1 : 27. The resulting biosensor showed a linear dependence on the xanthine concentration in the 0.25–3.5 μM range with a fast amperometric response in 12 s. The biosensor also showed a noticeable high sensitivity of 1.31 A M−1 cm−2 and a very low detection limit of 60 nM, which can be compared advantageously with other biosensor designs for xanthine.

The possibilities of amperometric enzyme electrodes in reversed micellar systems for the determination of phenol, 4-chloro-3-methylphenol and 2,4-dimethylphenol are illustrated. The used enzymatic reaction consisted of the oxidation of the phenolic compounds by oxygen, catalysed by tyrosinase. The reduction of the liberated quinones was amperometrically detected. The concentration of the components of the reversed micelles, as well as the potential applied to the tyrosinase electrode have been optimized. The stability of the enzyme electrode with time was also evaluated. The effect of the analyte solubility in water upon the analytical performance of the electrode was explored. Advantages of amperometric biosensors in reversed micelles are shown with respect to aqueous media and organic phase enzyme electrodes.