Colorimetric detection of endogenous hydrogen sulfide production in living cells

다국어 초록 (Multilingual Abstract)

<P><B>Abstract</B></P> <P>Hydrogen sulfide (H<SUB>2</SUB>S) has received great attention as a third gaseous signal transmitter, following nitric oxide and carbon monoxide. In particular, H<SUB>2</SUB>S plays an important role in the regulation of cancer cell biology. Therefore, the detection of endogenous H<SUB>2</SUB>S 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 H<SUB>2</SUB>S 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 H<SUB>2</SUB>S 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<SUB>3</SUB> concentration, as well as the pH of the Na<SUB>2</SUB>S (H<SUB>2</SUB>S donor) solution and the reaction time. Next, the <I>in vitro</I> performance of a colorimetric detection assay utilizing the silver/Nafion/PVP membrane was evaluated utilizing a known concentration of Na<SUB>2</SUB>S standard solution both at room temperature and at 37°C in a 5% CO<SUB>2</SUB> incubator. As a result, the sensitivity of the colorimetric assay for H<SUB>2</SUB>S at 37°C in the incubator (0.0056Abs./μM Na<SUB>2</SUB>S, R<SUP>2</SUP> =0.9948) was similar to that at room temperature (0.0055Abs./μM Na<SUB>2</SUB>S, R<SUP>2</SUP> =0.9967). Moreover, these assays were less sensitive to interference from compounds such as glutathione, <SMALL>L</SMALL>-cysteine (Cys), and dithiothreitol than to the H<SUB>2</SUB>S from Na<SUB>2</SUB>S. This assay based on the silver/Nafion/PVP membrane also showed excellent reproducibility (2.8% RSD). Finally, we successfully measured the endogenous H<SUB>2</SUB>S concentrations in live C6 glioma cells by s-(5′-adenosyl)-<SMALL>L</SMALL>-methionine stimulation with and without Cys and <SMALL>L</SMALL>-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 H<SUB>2</SUB>S 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 H<SUB>2</SUB>S.</P> <P><B>Highlights</B></P> <P> <UL> <LI> We developed a simple and facile colorimetric system for selective H<SUB>2</SUB>S gas detection. </LI> <LI> The silver/Nafion/PVP membrane was coated on the underside of a microplate cover. </LI> <LI> This assay showed high selectivity, good sensitivity, and excellent reproducibility. </LI> <LI> We successfully measured the endogenous H<SUB>2</SUB>S levels in live C6 glioma cells. </LI> </UL> </P> <P><B>Graphical Abstract</B></P> <P>[DISPLAY OMISSION]</P>