Development of an integrated electrochemical biosensor for sucrose and its implementation in a continuous flow system for the simultaneous monitoring of sucrose, fructose and glucose.

An integrated amperometric sucrose biosensor involving a 3-mercaptopropionic acid (MPA) self-assembled monolayer (SAM)-modified gold disk electrode (AuE) and coimmobilization of the enzymes invertase (INV) and fructose dehydrogenase (FDH) as well as the redox mediator tetrathiafulvalene (TTF) by means of a dialysis membrane is reported. Amperometry in stirred solutions at a detection potential of +0.10 V provided a linear calibration plot for sucrose over the 1.2 × 10(-6)-3.0 × 10(-3) mol L(-1) concentration range, with a limit of detection of 3.6 × 10(-7) mol L(-1). The practical usefulness of the biosensor was demonstrated by determining sucrose in condensed milk and in an infant food reference material with good results. Additionally, the biosensor was implemented together with commercial fructose and glucose amperometric biosensors in a continuous flow system to perform the multiplexed quantification of sucrose, fructose and glucose in a single experiment. The operational characteristics of the biosensors in this novel flow system were evaluated and their applicability was demonstrated through the simultaneous determination of the three sugars in the above-mentioned reference material.

A rapid method for detection of catalase-positive and catalase-negative bacteria based on monitoring of hydrogen peroxide evolution at a composite peroxidase biosensor.

The rapid detection of catalase-positive and catalase-negative bacteria in complex culture media has been accomplished by monitoring of hydrogen peroxide consumption or generation with a graphite-Teflon-peroxidase-ferrocene composite electrode. Escherichia coli and Streptococcus pneumoniae have been used as model catalase-positive and catalase-negative bacteria, respectively. Hydrogen peroxide evolution was amperometrically measured at 0.00 V. Experimental conditions, including the working solution composition, the incubation time and the hydrogen peroxide concentration, were optimized. The reusability of the biosensor was improved by placing a nylon membrane on the bioelectrode surface to prevent fouling caused by the bacterial medium. The developed methodology allowed the detection of E. coli and S. pneumoniae at concentration levels of approximately 2x10(6) and 2x10(5) cfu/mL, in assays taking 10 and 15 min, respectively, without any pre-concentration step or pre-enrichment procedure.

Molecularly imprinted polymer solid-phase extraction coupled to square wave voltammetry at carbon fibre microelectrodes for the determination of fenbendazole in beef liver.

A molecularly imprinted polymer was developed and used for solid-phase extraction (MISPE) of the antihelmintic fenbendazole in beef liver samples. Detection of the analyte was accomplished using square wave voltammetry (SWV) at a cylindrical carbon fibre microelectrode (CFME). A mixture of MeOH/HAc (9:1) was employed both as eluent in the MISPE system and as working medium for electrochemical detection of fenbendazole. The limit of detection was 1.9x10(-7) mol L-1 (57 microg L-1), which was appropriate for the determination of fenbendazole at the maximum residue level permitted by the European Commission (500 microg kg-1 in liver). Given that the SW voltammetric analysis could not be directly performed in the sample extract as a consequence of interference from some sample components, a sample clean-up with a MIP for selectively retaining fenbendazole was performed. The MIP was synthesized using a 1:8:22 template/methacrylic acid/ethylene glycol dimethacrylate ratio. A Britton-Robinson Buffer of pH 9.0 was selected for retaining fenbendazole in the MIP cartridges, and an eluent volume of 5.0 mL at a flow rate of 2.0 mL min-1 was chosen in the elution step. Cross-reactivity with the MIP was observed for other benzimidazoles. The synthesized MIP exhibited a good selectivity for benzimidazoles with respect to other veterinary drugs. The applicability of the MISPE-SWV method was tested with beef liver samples, spiked with fenbendazole at 5,000 and 500 microg kg-1. Results obtained for ten different liver samples yielded mean recoveries of (95+/-12)% and (96+/-11)% for the upper and lower concentration level, respectively.

A method for the quantification of low concentration sulfamethazine residues in milk based on molecularly imprinted clean-up and surface preconcentration at a Nafion-modified glassy carbon electrode.

An electrochemical method for the determination of sulfamethazine at a low concentration level (25 microgl(-1)) in milk is reported. The method involves sample clean-up and selective preconcentration of sulfamethazine with a molecularly imprinted polymer (MIP), and a further electrode surface preconcentration of the analyte at a Nafion-coated glassy carbon electrode (GCE). Square wave (SW) oxidative voltammetry of accumulated sulfamethazine was employed for its quantification. Sulfamethazine electrode preconcentration was carried out in 0.1 moll(-1) Britton-Robinson buffer of pH 1.5, and by applying 5 min of accumulation at open circuit. A linear calibration graph was obtained for sulfamethazine at the Nafion-modified GCE over the 1.0x10(-8) to 1.0x10(-6)moll(-1) concentration range, with a detection limit of 6.8x10(-9)moll(-1) (1.9 microgl(-1)). This detection limit is remarkably better than those reported previously in the literature using electroanalytical techniques. Although the detection limit achieved was sufficient to allow the direct determination of sulfamethazine at the concentration level required in milk, a sample clean-up was shown to be necessary to obtain analytically useful SW voltammograms. This was accomplished by processing the deproteinized milk through a cartridge containing a molecularly imprinted polymer for sulfamethazine, also allowing a selective preconcentration of the analyte. Elution of the analyte from the MIP cartridges was carried out with 2 ml of a (9:1) MeOH:acetic acid mixture. Determination of sulfamethazine in milk samples was accomplished by interpolation into a calibration graph constructed with sulfamethazine standard solutions which were subjected to the same procedure than the deproteinized milk samples. Results obtained for five samples, spiked at the 25 microgl(-1) level, showed a mean recovery of (100+/-3)%.

Amperometric multidetection with composite enzyme electrodes.

The use of composite biosensors for multianalyte detection strategies is discussed. Graphite-Teflon rigid composite biosensors offer the possibility of coimmobilization of several enzymes by simple physical inclusion in the bulk of the electrode matrix with no covalent linkages. A novel trienzyme graphite-Teflon-glucose oxidase (GOD)-alcohol oxidase (AOD)-peroxidase (HRP)-ferrocene bisosensor yielded amperometric steady-state currents similar to those obtained with graphite-Teflon-GOD-HRP-ferrocene and graphite-Teflon-AOD-HRP-ferrocene electrodes for the same concentration of glucose and ethanol, respectively. The performance of the trienzyme biosensor for multianalyte detection was evaluated with the simultaneous determination of glucose and ethanol after separation by HPLC, in samples of sweet wine. The simultaneous analysis of several analytes in the same sample should imply that, with an adequate dilution, the concentration levels of the analytes can be included within the ranges of linearity of the corresponding calibration plots. The use of two composite biosensors in a parallel configuration, so that different analytes can be simultaneously detected with no need of chromatographic separation, is also discussed. The usefulness of this approach was evaluated by the simultaneous analysis of glucose and ethanol in sweet wine, and of glucose and lactic acid in red wine.