Polydimethylsiloxane Permeability-Based Method for the Continuous and Specific Detection of Hydrogen Sulfide

Hydrogen sulfide (H2S) is known to play a physiological role in processes as diverse as vasodilation, maintenance of vascular tone, neuro-transmission, and immune response. The multitude of physiological functions in which H2S is involved warrants the development of useful methods for its detection. Here, we introduce a simple and continuous H2S detection method that exploits the relatively high polydimethylsiloxane (PDMS) permeability of H2S in comparison to other thiols typically encountered in the cellular milieu. In this method, 96-well inserts constructed of PDMS act as an H2S-permeable membrane, eliminating nonspecific thiol detection. This design also makes it possible to use virtually any available thiol-specific probe such as Ellman's reagent which was used here to detect H2S once it crossed the PDMS membrane. Utilizing this method, a detection limit of 9.2 +/- 1.9 ppb(m) (parts per billion (by mole) or similar to 0.51 mu M in 1.6 mL of buffer) free H2S (detected as solution sulfide) was achieved. In addition, the assay was used to determine K-M and V-max for natural substrates of cystathionine gamma-lyase (CSE), the main enzyme responsible for H2S production in peripheral tissues. The K-M and V-max of CSE for cysteine were 3.79 +/- 2.07 mM and 0.37 +/- 0.02 nmol H2S/min, respectively. K-M and V-max for homocysteine were 6.90 +/- 1.78 mM and 1.10 +/- 0.19 nmol H2S/min, respectively. In addition, the assay was used to examine the potential for a direct interaction of H2S and NO. The levels of detected H2S decreased in the presence of NO under normoxia but not under anoxia indicating that H2S does not react with NO but with N2O3 likely formed in the hydrophobic environment of PDMS.