In this study, a paper-integrated analytical device that combined a paper-based colorimetric assay with a paper-based cell culture platform was developed for the in situ detection of hydrogen sulfide (H2S) in three-dimensional (3D)-cultured, live prostate cancer cells. Two kinds of paper substrates were fabricated using a simple wax-printing methodology to form the cell culture and detection zones, respectively. LNCaP cells were seeded directly on the paper substrate and grown in the paper-integrated analytical device. The cell viability and H2S production of LNCaP cells were assessed using a simple water-soluble tetrazolium salt colorimetric assay and H2S-sensing paper, respectively. The H2S-sensing paper showed good sensitivity (sensitivity: 6.12 blue channel intensity/μM H2S, R2 = 0.994) and a limit of quantification of 1.08 μM. As a result, we successfully measured changes in endogenous H2S production in 3D-cultured, live LNCaP cells within the paper-integrated analytical device while varying the duration of incubation and substrate concentration (L-cysteine). This paper-integrated analytical device can provide a simple and effective method to investigate H2S signaling pathways and drug screening in a 3D culture model.

Hydrogen sulfide (H2S) is known to participate in bacteria-induced inflammatory response in periodontal diseases. Therefore, it is necessary to quantify H2S produced by oral bacteria for diagnosis and treatment of oral diseases including halitosis and periodontal disease. In this study, we introduce a paper-based colorimetric assay for detecting bacterial H2S utilizing silver/Nafion/polyvinylpyrrolidone membrane and a 96-well microplate. This H2S-sensing paper showed a good sensitivity (8.27 blue channel intensity/μM H2S, R2 = 0.9996), which was higher than that of lead acetate paper (6.05 blue channel intensity/μM H2S, R2 = 0.9959). We analyzed the difference in H2S concentration released from four kinds of oral bacteria (Eikenella corrodens, Streptococcus sobrinus, Streptococcus mutans, and Lactobacillus casei). Finally, the H2S level in Eikenella corrodens while varying the concentration of cysteine and treatment time was quantified. This paper-based colorimetric assay can be utilized as a simple and effective tool for in vitro screening of H2S-producing ability of many bacteria as well as salivary H2S analysis.

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