Category: Micro- and Nanotechnologies
Electrochemical detection has been used effectively with microchip electrophoresis (ME) for the determination of many important redox active molecules, including antioxidants and reactive nitrogen and oxygen species (RNOS). However, the noise generated at the detector due to the separation voltage generally restricts the limits of detection (LODs) to submicromolar levels in general. Therefore, the quantitation of biological molecules at sub-micromolar concentrations is difficult with ME-EC. On the other hand, it has been shown that picomolar limits of detection can be obtained using fluorescence. However, a drawback of fluorescence detection is that it is normally necessary to derivatize the analytes of interest with a specific probe prior to analysis. The addition of a new chemical moiety to the analyte of interest can lead to new separation and selectivity issues. In this paper, an alternative detection approach for ME is presented where the electrochemical current is transformed into an optical signal using a bipolar electrode in which a simultaneous electrochemical oxidation and a reduction occur at two poles. This novel approach of detection should offer better LODs for electroactive compounds separated by ME.
The initial detection system consisted of a two-channel system with a simple T microchip as the separation channel and a straight channel (with two reservoirs) as the fluorescent probe flow channel. Both the separation channel and the flow channel were aligned on a 35 µm pyrolysed photoresist film electrode (PPF) and the electrode was biased as a bipolar electrode by an externally applied voltage of -0.8 V. A laser assisted fluorescence system was used as the fluorescence reporting system. The reporter molecule was the non-fluorescent dye, dichlorodihydrofluorescein (DCFH2), which can be electrochemically oxidized to the fluorescent product, DCF. The success of the new detection system was demonstrated for the separation and detection of benzoquinone and resazurin in the reductive mode of ME-EC.
The main challenge of this system is the higher electrochemical background current at the working electrode and the lower limiting current available at probe flow channel. This challenge was overcome by decreasing the width of the separation channel and the width of the working electrode at the separation end.Better S/N ratios were obtained by modified system compared to bipolar amperometric detection. This system will ultimately be applied in the oxidative mode for the detection of nanomolar concentrations of RNOS, such as nitric oxide and peroxynitrite, in reaction mixtures and biological samples.
Manjula Wijesinghe– Graduate research assistant, University of Kansas, Lawrence, KS
Graduate research assistant
University of Kansas
Manjula B. Wijesinghe graduated (B.Sc.) from the Department of Chemistry in the Faculty of Science at the University of Peradeniya, Sri Lanka in 2005. He received his MSc. Degree from New Mexico State University in 2012 in the field of electroanalytical chemistry. In 2013, he entered graduate school at the University of Kansas to work with Professor Susan Lunte. His current project is the development of a novel detector for microchip electrophoresis using bipolar electrochemistry coupled to fluorescence detection. His research interests include the bioanalytical applications of microfluidics, instrumentation for lab-on-a-chip device and COMSOL modeling of microfluidics separation.