A simple and facile paper-based colorimetric assay for detection of free hydrogen sulfide in prostate cancer cells

In this study, we introduce a new paper-based colorimetric assay fabricated with the 96-well microplate format for cell culture for sensing H2S gas in live cancer cells. The H2S sensing zones on the paper were coated with a polyvinylpyrrolidone (PVP) membrane containing silver/Nafion. The principle of colorimetric detection for free H2S gas is reaction of silver in the coating zones with sulfide to form Ag2S, which is brown in color. First, we optimized the composition of the coating membrane on the paper, including the mixing ratio between PVP and Nafion and the AgNO3 concentration. Next, we examined the sensing performance of the paper assay in the form of sensitivity, selectivity reproducibility, and long-term stability using a known concentration of Na2S standard solution in phosphate buffered saline (PBS) at room temperature (RT) and in cell culture media at 37 °C in a 5% CO2 incubator. Our paper assay for H2S detection showed good sensitivity (2.51 color intensity/μM Na2S in PBS, R2 = 0.9967 at RT; 1.15 color intensity/μM Na2S in cell culture media, R2 = 0.9933 at 37 °C in incubator), high selectivity, excellent reproducibility (relative standard deviation 2.4%), and high stability over 1 month, as well as a limit of detection of 1.4 μM Na2S in PBS. Finally, we successfully measured the difference in endogenous H2S concentration between live LNCaP and PC-3 cells, which show different expression of H2S producing enzymes, using this paper-based colorimetric assay. The results were concordant with those of conventional methylene blue assay and western blot analysis. In summary, our paper assay using silver/Nafion-containing PVP membranes is a simple, facile, and reliable tool for the detection of H2S without complicated sample processing or labor-intensive bioanalytical methods We expect that this paper assay could be easily applied to study H2S signaling and pathophysiology in cancer biology, as well as cancer diagnosis and therapeutics.