Microchip electrophoresis with bipolar electrochemistry-fluorescence detection for the selective determination of nitrotyrosine-containing peptides

Nitrotyrosine has been shown to be a biomarker for reactive oxygen and nitrogen species (RNOS) in vivo. Post-mortem analysis of the brains of Alzheimer’s patients has shown elevated amounts of nitrated tyrosine residues. Our group previously developed a capillary electrophoresis method for the separation of several nitrated peptides that are produced via the reaction of peroxynitrite with the protein phospholamban. Although these studies were performed using absorbance detection, most of the products of tyrosine with RNOS are electroactive. In particular, the use of reductive electrochemical detection is attractive for the selective detection of nitrotyrosine-containing peptides. However, one drawback of reductive electrochemical detection is the high background currents that can result due to the presence of oxygen in the separation buffer, which leads to high LODs. Previously, our group investigated the coupling of bipolar electrochemistry (BPE) with fluorescence (FL) to obtain better LODs for reducible compounds separated by microchip electrophoresis (ME). In the current work, BPE-FL detection under an oxygen-free environment is investigated for the selective detection of nitrated peptides with lower LODs.
The ME-BPE-FL analysis system employs a PDMS simple-t microchip aligned with a 35 μm PPF electrode using a pseudo-in channel configuration (separation channel) and a PDMS straight channel aligned with a 30 μm gold electrode using an in-channel configuration (reporter channel). The two electrodes are connected to complete the bipolar electrode system. The electric field across the reporting channel is adjusted to bias the bipolar electrode for amperometric detection in the separation channel. The nitration of tyrosine-containing peptides was accomplished by reacting peroxynitrous acid with the peptide of interest. Nitrated tyrosine-containing peptides were monitored using the BPE-FL system. The selectivity and LODs of this method are compared to those of CE-UV and conventional ME-EC analysis.