A.J. Reviejo

Molecularly imprinted polymers for on-line preconcentration by solid phase extraction of pirimicarb in water samples

The synthesis and performance of a molecularly imprinted polymers (MIPs) as a selective solid phase extraction sorbent for the preconcentration of the carbamate pirimicarb from water samples is described. The MIP was prepared using pirimicarb as the template, methacrylic acid as the functional monomer and ethylene glycol dimethacrylate as the cross-linking monomer, and using chloroform as the solvent. The detection of pirimicarb was carried out by differential pulse voltammetry (DPV) at a hanging mercury drop electrode (HMDE) in 0.1 mol l −1 HCl. Solvents of different polarities were checked for the polymer synthesis, and different experimental variables (sample pH, selection of the eluent used, eluent volume, analyte and eluent flow rates and sample volume) associated with the rebinding/extraction process were optimised. For a 25 ml sample, the process took about 13 min and resulted in a nominal enrichment factor of 50 (eluent MeOH:H2O:HAc, 7:2:1; 0.5 ml) for pirimicarb. A limit of detection of 4.1 gl −1 was obtained, and a good reproducibility of the measurements using different MIP microcolumns was found. Furthermore, the MIP selectivity was evaluated by checking several substances with similar and different molecular structures to that of pirimicarb. As an application, pirimicarb was determined in water samples of diverse origin which were spiked at a concentration level of 71.5 gl −1 .

Graphite-Teflon composite bienzyme electrodes for the determination of L-lactate : Application to food samples

A bienzyme amperometric graphite-Teflon composite biosensor, in which lactate oxidase (LOD) and peroxidase, together with the mediator ferrocene, are incorporated into the electrode matrix, was developed for the determination of L-lactate in food samples such as wine and yogurt by using both batch- and flow-injection modes. This bienzyme electrode was fabricated by simple physical inclusion of the enzymes and the mediator in the bulk of the graphite-Teflon matrix. A Teflon content of 70%, an applied potential of 0.00 V, and a pH of 7.4 were employed as working conditions. The composite bioelectrode exhibited long-term operation because of the renewability of its surface by polishing. Reproducible amperometric responses were achieved with different electrodes fabricated from different composite matrices, and no significant loss of the enzyme activity occurred after 6 months of storage at 4 degrees C. Detection limits for L-lactate of 1.4 and 0.9 microM were obtained by batch amperometry in stirred solutions and flow-injection with amperometric detection, respectively. An interferences study with different substances which may be present in wine and yogurt together with L-lactic acid demonstrated very good selectivity for the determination of this analyte. The bienzyme composite electrode was applied to the determination of L-lactic acid in red wine and shaken yogurt, and the methods were validated by comparing these results with those obtained by applying a recommended reference method.

Composite electrochemical biosensors: a comparison of three different electrode matrices for the construction of amperometric tyrosinase biosensors

A comparison of the behaviour of three different rigid composite matrices for the construction of amperometric tyrosinase biosensors, which are widely used for the detection of phenolic compounds, is reported. The composite electrode matrices were, graphite-Teflon; reticulated vitreous carbon (RVC)-epoxy resin; and graphite-ethylene/propylene/diene (EPD) terpolymer. After optimization of the experimental conditions, different aspects regarding the stability of the three composite tyrosinase electrode designs were considered and compared. A better reproducibility of the amperometric responses was found with the graphite-EPD electrodes, whereas a longer useful lifetime was observed for the graphite-Teflon electrodes. The kinetic parameters of the tyrosinase reaction were calculated for eight different phenolic compounds, as well as their corresponding calibration plots. The general trend in sensitivity was graphite-EPD>graphite-Teflon>>RVC-epoxy resin. A correlation between sensitivity and the catalytic efficiency of the enzyme reaction for each phenolic substrate was found. Furthermore, differences in the sensitivity order for the phenolic compounds were observed among the three biocomposite electrodes, which suggests that the nature of the electrode matrix influences the interactions in the tyrosinase catalytic cycle.

Graphite/Teflon composite bienzyme amperometric biosensors for monitoring of alcohols

Composite graphite-Teflon electrodes, in which the enzymes alcohol oxidase (AOD) and horseradish peroxidase (HRP), as well as the mediator ferrocene, are incorporated into the electrode matrix, are reported for the reliable monitoring of alcohols in food and beverages. The bienzyme electrodes are constructed by simple physical inclusion of the enzymes and the mediator in the bulk of graphite-70% Teflon rigid cylindrical pellets. The composite biosensors are robust and reusable because of the renewability of the electrode surface by polishing. Reproducible amperometric responses at 0.00 V were obtained with different electrodes constructed from the same pellet and from different pellets. No significant loss of the enzymes activities was found after at least 3 months of storage at 0 degrees C. The detection limits obtained by amperometry in stirred solutions can be advantageously compared with those achieved with commercial sensors for alcohols. The bienzyme electrodes are suitable to be used under flow-injection conditions, as well as for amperometric detection in HPLC. The bioelectrodes were employed for the determination of ethanol in beers, wines and liquors, using both batch- and flow-injection modes, and for the determination of methanol in wines and liquors by HPLC with amperometric detection. Only a dilution of the beverages was needed as sample treatment in all cases.

Microorganisms recognition and quantification by lectin adsorptive affinity impedance.

Lectin-based screen-printed gold electrodes are reported for the impedimetric label-free detection of bacteria. The selective interaction of lectins with carbohydrate components from microorganisms surface was used as the recognition principle for their detection and identification. Electrochemical impedance spectroscopy (EIS) was employed for the direct label-free transduction of the bacteria-lectin binding. Biotinylated Concanavalin A (Con A) and Escherichia coli were used for the evaluation of the lectin-bacteria complex formation. This complex was formed in solution, and then adsorbed onto the gold SPE surface. No bacteria immobilization was observed on the sensor prepared in the absence of ConA, demonstrating the absence of non-specific bacteria adsorption onto the gold SPE. On the contrary, the changes in electron transfer resistance allowed monitoring of E. coli-biotinylated Con A complex formation without any amplification step. Experimental variables such as the biotinylated-Con A concentration and the bacteria-lectin incubation time were optimized. The electron transfer resistance varied linearly with the logarithmic value of E. coli concentration over four orders of magnitude, 5.0 x 10(3) and 5.0 x 10(7) cfu mL(-1). The selectivity of the approach was evaluated by checking the impedimetric responses of gold SPE modified with the complexes formed between nine lectins and three different bacteria (E. coli, Staphylococcus aureus and Mycobacterium phlei). Different response profiles were found when the different lectins were used as recognition elements. principal component analysis (PCA) allowed classification and distinction among bacteria. Finally, electrochemical monitoring of beta-galactosidase activity for the surface attached bacteria was demonstrated to be useful to distinguish between E. coli and S. aureus, which exhibit a similar affinity towards biotinylated-Con A.

Graphite-Teflon-Peroxidase Composite Electrochemical Biosensors. A Tool for the Wide Detection of Phenolic Compounds

Graphite-Teflon-peroxidase composite electrodes, constructed by simple physical inclusion of the enzyme into the bulk of the electrode matrix, have been used for the amperometric detection of 18 phenolic compounds. Monitoring of the enzyme reaction was carried out by the electrode reduction of the generated phenoxy radicals, in the presence of a constant concentration of 5.0×10−4 mol L−1 H2O2, at an applied potential of 0.00 V. Different aspects regarding the stability of the composite bioelectrode showed the robustness of the biosensor design. The kinetic constants of the enzyme reactions for the phenolic compounds tested were calculated. The limits of detection obtained were better than those found in the literature for peroxidase biosensors using other composite matrices, and the differences in sensitivity observed for the different phenolic substrates are discussed in terms of their pKa values and substituents. The analytical signals obtained with the graphite-Teflon-HRP electrodes have been compared with those yielded by a graphite-Teflon-tyrosinase composite electrode constructed in a similar way. With the aim of monitoring the largest possible number of phenolic compounds with the highest possible sensitivity, coimmobilization of peroxidase and tyrosinase into the composite electrode matrix was carried out.

Amperometric selective biosensing of dimethyl- and diethyldithiocarbamates based on inhibition processes in a medium of reversed micelles

An amperometric tyrosinase electrode has been used for biosensing of dimethyl- and diethyldithiocarbamates based on the inhibition effects of these substances on the catalytic activity of the enzyme. A working medium consisting of reversed micelles, and phenol as the substrate has been used. The tyrosinase electrode was constructed by direct adsorption of the enzyme on the surface of a graphite-disk electrode. Reversible inhibition processes are shown to be involved for ziram, diram and zinc diethyldithiocarbamate. Following a simple regeneration of the enzyme electrode, an acceptable reproducibility for the measurements of the inhibition response was obtained. Experimental variables, such as temperature, phenol concentration and the presence of chloroform, affecting the inhibition processes, were optimized. The type of enzyme inactivation for each inhibitor tested was studied, and the inhibition constants were calculated. Detection limits of 0.074, 1.3 and 1.7 μmol l−1 were achieved for ziram, diram and zinc diethyldithiocarbamate, respectively. Other carbamates belonging to families different from dimethyl- and diethyldithiocarbamates showed no amperometric response at the tyrosinase electrode, except for pyrimidine-derivative carbamates. The developed analytical methodology was applied to determine ziram in spiked apple samples.

Design of a composite amperometric enzyme electrode for the control of the benzoic acid content in food

A graphite-Teflon-tyrosinase composite biosensor for the determination of benzoic acid in foodstuffs is reported. The biosensor functioning is based on the inhibition effect of benzoic acid on the biocatalytic activity of the enzyme in a reversed micelle working medium formed with ethyl acetate as the continuous phase, a 0.05 moll(-1) phosphate buffer solution of pH 7.4 (5%) as the aqueous dispersed phase, and 0.10 moll(-1) dioctyl sulfosuccinate (AOT) as the emulsifying agent. A potential value of -0.10 V, and a constant enzyme-substrate (phenol) concentration of 2.0x10(-4) moll(-1) were selected to carry out the amperometric inhibition measurements. The tyrosinase inhibition process by benzoic acid is reversible and of the competitive type, with an apparent inhibition constant of 0.016 mmoll(-1). The composite bioelectrodes allow the regeneration of the electrode surface by polishing and exhibit long-term operation and stability. A limit of detection of 9.0x10(-7) moll(-1) benzoic acid was obtained. An interference study from other substances which can be found in foodstuffs together with benzoic acid was performed. Taking advantage of the capabilities of reversed micelles as universal solubilization media, the composite tyrosinase electrode was used for the determination of benzoic acid in two different kind of samples: mayonnaise sauce, which is a highly hydrophobic matrix, and Cola soft drinks, a hydrophilic matrix for which practically no sample treatment is necessary.

Development of graphite-poly(tetrafluoroethylene) composite electrodes Voltammetric determination of the herbicides thiram and disulfiram

Abstract The oxidative voltammetric behaviour of the herbicides thiram and disulfiram at graphite-poly(tetrafluoroethylene) (PTFE) composite electrodes has been studied. Graphite-PTFE pellets were prepared by mixing graphite with powdered PTFE and compression molding the mixture. Based on the signal-to-noise characteristics and on the mechanical strength of the resulting pellet, a mixture containing 40% PTFE was selected. Both thiram and disulfiram are adsorbed onto the composite electrode, this process being used as a preconcentration step. The accumulation potential and accumulation time chosen were 0.0 V and 300 s, respectively. The characteristics of the electrode processes were established. Linear calibration graphs were obtained within the ranges 2.0 × 10 −7 −1.0 × 10 −5 and 2.0 × 10 −7 −8.0 × 10 −6 mol l −1 for thiram and disulfiram, respectively. The effect of the presence of ziram, phenol and Zn(II) on the oxidation signals for thiram and disulfiram was evaluated. As an application, the determination of thiram in spiked strawberries was carried out with good results.

HPLC-Electrochemical detection with graphite-poly (tetrafluoroethylene) electrode Determination of the fungicides thiram and disulfiram

The suitability of composite graphite-poly(tetrafluoroethylene) (Teflon) electrodes as amperometric indicator electrodes in HPLC detection is demonstrated. The determination of the fungicides thiram and disulfiram in the presence of ziram has been chosen as an analytical problem. The optimization of working conditions, such as the choice of the organic solvent used in the mobile phase as well as its percentage, the potential applied to the composite electrode, and the time elapsed between mixing the carbamates and the injection, has been accomplished by using the wall-jet flow-cell configuration. The effect of the acetonitrile percentage used in the mobile phase on the retention of thiram, disulfiram, ziram and phenol was evaluated. Resolution up to the baseline can be achieved with 45% acetonitrile. The sensitivity of the determination of thiram and disulfiram in the presence of a constant concentration of ziram is slightly better when using a wall-jet cell; however, the background current is higher, as well as the baseline noise and the time necessary to achieve stabilization of the baseline before the injection. Lower limits of detection for both fungicides, as well as a better repeatability, were obtained when using a thin-layer flow cell configuration. As an application, the determination of thiram in spiked apple samples, at a level of 0.5 mg thiram kg(-1) apple, has been carried out with a mean recovery of 97 +/- 3% for a significance level of 0.05.