Colorimetric detection of endogenous hydrogen sulfide production in living cells

Hydrogen sulfide (H₂S) has received great attention as a third gaseous signal transmitter, following nitric oxide and carbon monoxide. In particular, H₂S plays an important role in the regulation of cancer cell biology. Therefore, the detection of endogenous H₂S 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 H₂S 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 H₂S 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 AgNO₃ concentration, as well as the pH of the Na₂S (H₂S 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 Na₂S standard solution both at room temperature and at 37°C in a 5% CO₂ incubator. As a result, the sensitivity of the colorimetric assay for H₂S at 37°C in the incubator (0.0056Abs./μM Na₂S, R²=0.9948) was similar to that at room temperature (0.0055Abs./μM Na₂S, R²=0.9967). Moreover, these assays were less sensitive to interference from compounds such as glutathione, l-cysteine (Cys), and dithiothreitol than to the H₂S from Na₂S. This assay based on the silver/Nafion/PVP membrane also showed excellent reproducibility (2.8% RSD). Finally, we successfully measured the endogenous H₂S 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 H₂S 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 H₂S.