Gasotransmitter Detection Methodologies for Real-time Analysis and Studying Molecular Transport

Gasotransmitters are a family of gaseous signaling molecules comprised of nitric oxide (NO), carbon monoxide (CO), and hydrogen sulfide (H2S). As these molecules play key roles in numerous biological processes, they have been widely investigated for their potential as multifunctional therapeutic agents. Unfortunately, their small size, volatility, and low physiological concentrations make them particularly difficult to quantify and characterize in situ. Herein, the development of several analytical methodologies is described for real-time, selective detection of H2S and characterization of therapeutic NO doses in cystic fibrosis (CF)-relevant media. Two platforms for the direct, electrochemical detection of H2S were devised to contend with the passivating effects of elemental sulfur, H2S’s oxidative byproduct. First, triple pulse amperometry (TPA) was employed to mitigate sulfur adsorption. Inhibiting adsorption was achieved by cycling between three electrochemical actions: oxidizing adsorbed sulfur to sulfate, repelling sulfate off of the electrode surface, and quantifying H2¬S via low potential oxidation. As an alternative technique, constant potential amperometry (CPA) exploited unavoidable sulfur adsorption to precondition the electrode surface with a stable and reproducible sulfur layer. Both methodologies facilitated long-term, stable performance with sufficient sensitivity, rapid response times, wide linear dynamic ranges, and biologically-relevant detection limits. Additional selectivity toward H2S was imparted upon both TPA and CPA sensors by electrodepositing permselective films on the electrode surface. While CPA sensors saw significant improvements against numerous biological interferents, TPA damaged the films which led to inconsistent results. The ortho-phenylenediamine-modified (o-PD) CPA sensor was deemed the optimal design for further work in biological media. A planar carbon electrode system, combining the o-PD-modified CPA methodology and a fluorinated xerogel, was developed to utilize the sensor for cellular measurements. The bilaminar design was found to be highly selective for H2S, while also exhibiting low detection limits (