Electrochemical enzyme immunoassays on microchip platforms.

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.

Glucose biochip: dual analyte response in connection to two pre-column enzymatic reactions.

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.

Capillary electrophoresis microchips for separation and detection of organophosphate nerve agents.

A miniaturized analytical system for separating and detecting toxic organophosphate nerve agent compounds, based on the coupling of a micromachined capillary electrophoresis chip with a thick-film amperometric detector, is described. Factors influencing the on-chip separation and detection processes have been optimized. Using a MES buffer (20 mM, pH 5.0) running buffer, a 72-mm-long separation channel, and a separation voltage of 2000 V, baseline resolution is observed for paraoxon, methyl parathion, fenitrothion, and ethyl parathion in 140 s. Such miniaturization and speed advantages are coupled to submicromolar detection limits and good precision. Applicability to spiked river water samples is demonstrated, and the implications for on-site environmental monitoring and rapid security screening/warning are discussed.

Microseparation chips for performing multienzymatic dehydrogenase/oxidase assays: simultaneous electrochemical measurement of ethanol and glucose.

This report describes a new "lab-on-a-chip" protocol integrating on-line precolumn biocatalytic reactions of multiple (oxidase and dehydrogenase) enzymes and substrates with effective capillary electrophoresis microseparations and amperometric detection. The operation of the new oxidase/dehydrogenase reaction/separation microchip is illustrated for the simultaneous measurement of glucose and ethanol in connection to the corresponding glucose oxidase and alcohol dehydrogenase reactions, respectively. The enzymatic reactions generate hydrogen peroxide and NADH species that are separated (on the basis of their different charges) and detected amperometrically at the end-column thick-film detector. A driving voltage of 2000 V results in peroxide and NADH migration times of 74 and 230 s, respectively. Operating the gold-coated carbon detector at +1.0 V allows simultaneous anodic detection of both reaction products. Factors influencing the reaction, separation, and detection processes are examined and optimized. The applicability of the new multienzyme assay to wine samples is illustrated.

Micromachined separation chips with a precolumn reactor and end-column electrochemical detector.

Glass microchips, integrating chemical derivatization reactions, electrophoretic separations, and amperometric detection, have been developed. The performance of the new integrated microfabricated devices is demonstrated for rapid on-chip measurements of amino acids utilizing precolumn reactions of amino acids with o-phthaldialdehyde/2-mercaptoethanol to generate electroactive derivatives that are separated electrophoretically and detected at the end-column electrochemical detector. The influence of the sample/reagent mixing ratio, reagent concentrations, driving voltage, detection potential, and other variables is explored. The integrated microsystem offers a rapid (6 min) simultaneous measurements of eight amino acids, down to approximately 2.5 x 10(-6) M (5 fmol) level, with linearity up to the 2 x 10(-4) M level examined, and good reproducibility (RSD = 2.2-2.7%). A step of the driving voltage is used for decreasing the migration time of late-eluting components and reducing the overall analysis time. The integrated microfabricated device expands the scope of on-chip electrochemical detection to nonelectroactive analytes and holds promise of being a powerful analytical tool.

Microfabricated electrophoresis chips for simultaneous bioassays of glucose, uric acid, ascorbic acid, and acetaminophen.

A micromachined capillary electrophoresis chip is described for simultaneous measurements of glucose, ascorbic acid, acetaminophen, and uric acid. Fluid control is used to mix the sample and enzyme glucose oxidase (GOx). The enzymatic reaction, a catalyzed aerobic oxidation of glucose to gluconic acid and hydrogen peroxide, occurs along the separation channel. The enzymatically liberated neutral peroxide species is separated electrophoretically from the anionic uric and ascorbic acids in the separation/reaction channel. The three oxidizable species are detected at the downstream gold-coated thick-film amperometric detector at different migration times. Glucose can be detected within less than 100 s, and detection of all electroactive constituents is carried out within 4 min. Measurements of glucose in the presence of acetaminophen, a neutral compound, are accomplished by comparing the responses in the presence and absence of GOx in the running buffer. The reproducibility of the on-chip glucose measurements is improved greatly by using uric acid as an internal standard. Factors influencing the performance, including the GOx concentration, field strength, and detection potential, are optimized. Such coupling of enzymatic assays with electrophoretic separations on a microchip platform holds great promise for rapid testing of metabolites (such as glucose or lactate), as well as for the introduction of high-speed clinical microanalyzers based on multichannel chips.