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

Abstract In this study, we introduce a new paper-based colorimetric assay fabricated with the 96-well microplate format for cell culture for sensing H 2 S gas in live cancer cells. The H 2 S sensing zones on the paper were coated with a polyvinylpyrrolidone (PVP) membrane containing silver/Nafion. The principle of colorimetric detection for free H 2 S gas is reaction of silver in the coating zones with sulfide to form Ag 2 S, 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 AgNO 3 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 Na 2 S standard solution in phosphate buffered saline (PBS) at room temperature (RT) and in cell culture media at 37 °C in a 5% CO 2 incubator. Our paper assay for H 2 S detection showed good sensitivity (2.51 color intensity/μM Na 2 S in PBS, R 2  = 0.9967 at RT; 1.15 color intensity/μM Na 2 S in cell culture media, R 2  = 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 Na 2 S in PBS. Finally, we successfully measured the difference in endogenous H 2 S concentration between live LNCaP and PC-3 cells, which show different expression of H 2 S 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 H 2 S without complicated sample processing or labor-intensive bioanalytical methods We expect that this paper assay could be easily applied to study H 2 S signaling and pathophysiology in cancer biology, as well as cancer diagnosis and therapeutics.