Capillary electrophoresis strategy to monitor the released and remaining nitric oxide from the same single cell using a specially designed water-soluble fluorescent probe.

Abstract

Gasotransmitters including nitric oxide (NO), carbon monoxide (CO), and hydrogen sulfide (H2S) have attracted more and more attention in the past decades due to their unique signaling and functions. However, as a fundamental issue in the investigations of gasotransmitters, the cell membrane permeability and release behavior of them is controversial in reports because of the lack of an efficient approach to determine gasotransmitters released out of and remaining in the same cells simultaneously. To solve such problem, taking NO as representative, a robust and facile strategy has been reported based on a completely water-soluble fluorescent probe and a commercially available capillary electrophoresis system. A specially designed boron-dipyrromethene (BODIPY)-based fluorescent probe with two sulfonate groups, disodium 2,6-disulfonate-1,3,5,7-tetramethyl-8-(3',4'-diaminophenyl) difluoroboradiaza-s-indance (TMDSDAB), has been developed. As a turn-on fluorescent probe, TMDSDAB can react with NO promptly in aqueous media, and 540-fold enhancement of fluorescence is obtained. Using TMDSDAB, the trapping and quantification of NO released out of and remaining in the same single cell was achieved by capillary electrophoresis with laser-induced fluorescence detection. The limit of detection is 0.5 nM for NO. The proposed method has been applied to estimate the release behavior of single macrophages, and the results indicated that the cell membrane should be a barrier to NO diffusion.