A microfluidic device for conducting electrochemical enzyme immunoassays is described. The new "lab-on-a-chip" protocol integrates precolumn reactions of alkaline phosphatase-labeled antibody (anti-mouse IgG) with the antigen (mouse IgG), followed by electrophoretic separation of the free antibody and antibody-antigen complex. The separation is followed by a postcolumn reaction of the enzyme tracer with the 4-aminophenyl phosphate substrate and a downstream amperometric detection of the liberated 4-aminophenol product Factors influencing the reaction, separation, and detection processes were optimized, and the analytical performance was characterized. An applied field strength of 256 V/cm results in free antibody and antibody-antigen complex migration times of 125 and 340 s, respectively. A remarkably low detection limit of 2.5 x 10(-16) g/mL (1.7 x 10(-18) M) is obtained for the mouse IgG model analyte. Such combination of a complete integrated immunoassay, an attractive analytical performance, and the distinct miniaturization/portability advantages of electrochemical microsystems offers considerable promise for designing self-contained and disposable chips for decentralized clinical diagnostics or on-site environmental testing.

A new chip-based electrochemical immunoassay protocol, based on the use of a ferrocene redox label, is described. Two reaction formats, based on direct (noncompetitive) and competitive modes of operation, were employed for illustrating the use of redox tracers in chip-based electrochemical immunoassays. The direct assay consisted of mixing the ferrocene-tagged antibody and the antigen analyte, a rapid electrophoretic separation of labeled free antibody and the labeled antigen/antibody complex, and a downstream anodic detection of the ferrocene tracer at gold-plated carbon screen-printed electrode detector. The competitive assay integrates precolumn reactions of the labeled antigen and the target antigen with the antibody with electrophoretic separation of the free and bound labeled antigens, along with amperometric detection of the redox tag. An internal standard has been used to normalize the peak area for the construction of calibration plots. Fundamental operating variables are examined and optimized. The use of a redox tracer offers the advantages of simplified protocol, wider linear range, higher stability, and higher separation efficiency compared to an analogous use of enzyme tags. The direct mouse-immunoglobulin G (IgG) assay and the competitive 3,3',5-triiodo-L-thyronine (T(3)) one were accomplished within less than 150 and 130 s (with field strengths of 256 and 192 V/cm), and offer minimum detectable concentrations of 2.5 x 10(-12) and 1 x1 0(-6) g/mL, respectively. Such use of redox labels for chip-based amperometric immunoassay protocols offers considerable promise for decentralized clinical or environmental testing.

This contribution describes the simultaneous determination of three phenolic compounds, o-cresol, p-chlorophenol and 4-chloro-3-methylphenol, using direct oxidation and amperometric detection coupled by signal deconvolution, accomplished via chemometric methods. Direct oxidation of phenolic compounds is performed at the surface of an epoxy-graphite transducer, by linear scan voltammetry. Due to strong signal overlapping, artificial neural networks (ANNs) were used during data treatment, in a combination of chemometrics and electrochemical sensors known as an "electronic tongue". To calibrate this system properly, a total of 80 mixed samples were prepared automatically by employing a sequential injection analysis (SIA) system designed to automatically generate the information needed to train the network. The phenolic compound concentration varied from 1 to 70 microM for o-cresol, from 0.5 microM to 140 microM for p-chlorophenol and from 1 microM to 100 microM for 4-chloro-3-methylphenol. A good prediction capability was obtained, with correlation coefficients >0.964 when the obtained values were compared with those expected for a set of 24 external test samples not used for training. The results presented here indicate that this technique is a simple and robust analytical method of environmental interest.

An automated voltammetric electronic tongue has been designed employing a biosensor array formed by three different enzymatic Glucose Oxidase (GOD) electrodes and the Sequential Injection Analysis principle. The system is used for its automated training and operation devised for determining glucose and one of its classical interferents, ascorbic acid. The three enzymatic biosensors contain GOD and different metallic catalysts in order to decrease the working potential and to differentiate the response of primary species and interferents. Linear sweep voltammetry has been the chosen technique for data generation and artificial neural networks have been used as the modeling tool. Different learning algorithms have been tried in order to obtaining the best architecture for the neural network. Glucose has been determined in different fruit juice samples by employing this system, correcting the ascorbic acid contents.

This article describes a novel 'Lab-on-a-Chip' protocol generating two electrophoretic peaks for a single analyte, based on the coupling of two different pre-column enzymatic reactions of the same substrate followed by electrophoretic separation of the reaction products. Such operation is illustrated for the measurement of glucose in connection to the corresponding glucose oxidase (GOx) and glucose dehydrogenase (GDH) reactions. The pre-column enzymatic reactions generate hydrogen peroxide and NADH species, that are separated (based on their different charges) and detected at the end-column amperometric detector. The peak current ratio can be used for confirming the peak identity, estimating the peak purity, addressing co-migrating interferences, and deviations from linearity. A driving voltage of 2000 V results in peroxide and NADH migration times of 93 and 260 s, respectively. Factors influencing the unique dual glucose response are examined and optimized. The concept can be extended to different target analytes based on the coupling of two pre-column reactions with electrophoretic separation of the reaction products.