Bipolar electrochemically generated fluorescence detector for microchip electrophoresis with and without a potentiostat: Application to reducible analyte detection

Abstract

Microchip electrophoresis (ME) is a separation method that makes it possible to quickly analyze multiple species in small volume samples in a miniaturized format. Two commonly employed detection approaches for ME are electrochemistry and fluorescence. Electrochemical detection can be employed for direct detection of analytes via redox chemistry, while fluorescence generally offers lower limits of detection but often requires analyte derivatization. The present study uses bipolar electrochemistry to couple the two detection methods into a single device as a detector for that encompasses the advantages of both techniques. This system employs a closed bipolar electrode (BPE) that converts the cathodic amperometric current produced at the separation (sensing) pole to a fluorescence response generated at the reporting pole. Two model analytes, benzoquinone and resazurin, were separated and detected using ME with reductive amperometric detection at the sensing pole of the BPE. A potentiostat was used to control the bipolar electrode. The response was then converted to fluorescence through the oxidation of 2,7-dichlorodihydrofluorescein in the reporting channel. This system was subsequently modified to run without a potentiostat by using the separation voltage to bias the BPE sensing pole. A voltage applied across the reporting channel was used to generate electroosmotic flow as well as bias the corresponding pole of the BPE. This potentiostat-free mode was evaluated for the determination of 3-nitro-l-tyrosine, a reducible biomarker of oxidative stress. Lastly, a sacrificial electrochemically active species, Trolox®, was incorporated into the reporting channel to compensate for background current at the sensing channel and lower the limits of detection.