P60 Silver sulfide nanoparticle based microplate assay for hydrogen sulfide

Abstract

Hydrogen sulfide (H2S) is a physiologically important gas synthesized by mammalian cells, having a vast number of effects, particularly in the cardiovascular, immune, and central-nervous systems. The chemistry of hydrogen sulfide is complex as it dissociates into hyposulfide (HS−), and sulfide ( S 2 - ) anions in solution, reacts with protein thiols, oxidizes to other sulfur species, and can quench endogenous oxidants such as peroxynitrite. This makes the mechanisms of hydrogen sulfide-mediated biological activity particularly difficult to study. Existing assays for H2S such as the methylene blue method do not differentiate between sulfur species and are prone to interferences. This makes measurement of H2S in biological settings difficult and reported literature concentrations somewhat controversial. To measure H2S gas specifically we have developed a microplate-based assay where H2S gas diffuses to react with silver (Ag+) immobilized in a polymer to produce Ag2S nanoparticles and a colorimetric response. The polymer is transparent in the visible range and produces a strong absorbance at 310 nm as H2S gas reacts with Ag+ to produce Ag2S nanoparticles. The microplate assay can be used to monitor H2S gas in real time or can be used for endpoint measurements and has a detection limit in low nmol range. The assay was used to monitor H2S gas production from recombinant cystathionine gamma-lyase (CSE) enzyme and determine the Km and Vmax values of the enzyme. The microplate-based assay represents an attractive low-cost method for specific H2S gas detection. Currently we are optimizing the assay for H2S gas measurement in live cells.