Detection of nitrated tyrosine compounds using bipolar electrode-based fluorescence with microchip electrophoresis

Description:
Early detection of changes in oxidative nitrosative stress markers such as nitrated tyrosine will be a potential step in the diagnosis of Alzheimer’s patients. The analysis of nitrated tyrosine has been studied previously by our group using capillary electrophoresis with UV detection (CE-UV). Although most of the products of reactive oxygen and nitrogen species are either electrochemically reducible (Rd) or oxidizable (Ox), most research in this field has been dedicated to the electrochemical (EC) detection of Ox analytes, and only a few reports have addressed the detection of Rd species. This lack of study is primarily due to analysis with the potentials necessary to detect these species exhibiting high EC background resulting from oxygen reduction, even at low negative potentials, which leads to high LODs. Previously, our group investigated the coupling of bipolar (BP) electrochemistry with fluorescence (FL) detection to obtain better LODs for Rd compounds. In the current work, the detection of nitrated tyrosine compounds including peptides is studied using BP-FL detection to achieve better selectivity and lower LODs than those that were obtained using the previous CE-UV method. The detection system in this study consists of a PDMS simple-t microchip aligned with a 35 μm PPF electrode using a pseudo-in channel configuration and a PDMS straight channel aligned with a 30 μm gold electrode using an in-channel configuration. The connected electrodes were used as the complete BP electrode system. The electric field across the reporting channel was adjusted to bias the BP electrode for detection in the separation channel. The nitration of tyrosine and tyrosine-containing peptides was accomplished by reacting peroxynitrous acid with the analyte of interest. In the case of tyrosine, both nitrotyrosine and dinitrotyrosine were detected using the BP-FL system. The selectivity and LODs of this method will be compared to that of CE-UV and conventional amperometric detection.