Colorimetric detection of endogenous hydrogen sulfide production in living cells.

Hydrogen sulfide (HS) has received great attention as a third gaseous signal transmitter, following nitric oxide and carbon monoxide. In particular, HS plays an important role in the regulation of cancer cell biology. Therefore, the detection of endogenous HS concentrations within biological systems can be helpful to understand the role of gasotransmitters in pathophysiology. Although a simple and inexpensive method for the detection of HS has been developed, its direct and precise measurement in living cells remains a challenge. In this study, we introduced a simple, facile, and inexpensive colorimetric system for selective HS detection in living cells using a silver-embedded Nafion/polyvinylpyrrolidone (PVP) membrane. This membrane could be easily applied onto a polystyrene microplate cover. First, we optimized the composition of the coating membrane, such as the PVP/Nafion mixing ratio and AgNO concentration, as well as the pH of the NaS (HS donor) solution and the reaction time. Next, the in vitro performance of a colorimetric detection assay utilizing the silver/Nafion/PVP membrane was evaluated utilizing a known concentration of NaS standard solution both at room temperature and at 37°C in a 5% CO incubator. As a result, the sensitivity of the colorimetric assay for HS at 37°C in the incubator (0.0056Abs./μM NaS, R=0.9948) was similar to that at room temperature (0.0055Abs./μM NaS, R=0.9967). Moreover, these assays were less sensitive to interference from compounds such as glutathione, l-cysteine (Cys), and dithiothreitol than to the HS from NaS. This assay based on the silver/Nafion/PVP membrane also showed excellent reproducibility (2.8% RSD). Finally, we successfully measured the endogenous HS concentrations in live C6 glioma cells by s-(5'-adenosyl)-l-methionine stimulation with and without Cys and l-homocysteine, utilizing the silver/Nafion/PVP membrane. In summary, colorimetric assays using silver/Nafion/PVP-coated membranes can be simple, robust, and reliable tools for the detection of HS that can avoid the complicated and labor-intensive analytical approach used in conventional biology. In addition, we expect that this assay will demonstrate a powerful ability to study pathophysiological pathways that involve HS.