Determination of pepsin in human saliva using pepsin-susceptible peptide reporter and colorimetric dipstick assay: a prospective, cross-sectional study.

A simple and effective pepsin detection assay is reported based on a pepsin-susceptible peptide (PSP) reporter degradation strategy. PSP, which can be specifically cleaved by pepsin, was modified with fluorescein isothiocyanate (FITC) and biotin at the N- and C-terminals to be used as a reporter for colorimetric detection of dipsticks. A universal lateral flow dipstick consisting of a streptavidin test line for biotin binding and a sample pad immobilized with a gold-labeled polyclonal (rabbit) anti-FITC antibody was used to verify PSP-based pepsin detection. When the PSP reporter reacts with pepsin in a tube, it cleaves into two fragments, and the cleaved fragments do not display any color on the test line. Therefore, the higher the concentration of pepsin is, the greater is the decrease in test line intensity (IT-line) and the higher is the control line intensity (IC-line). First, the PSP cleavage and dipstick assay conditions for pepsin detection was optimized. The ratio of color intensity (IT-line/IC-line) of PSP-based dipstick assay showed a linear relationship with log concentration of pepsin ranging between 4 and 500 ng/mL (R2 = 0.98, n = 6), with a limit of detection of 1.4 ng/mL. It also exhibited high specificity and good reproducibility. Finally, pepsin levels were quantified in saliva samples from healthy controls (n = 34) and patients with laryngopharyngeal reflux (LPR, n = 61). Salivary pepsin levels were higher in patients with LPR than in healthy controls. The salivary pepsin levels correlated with those measured using a conventional enzyme-linked immunosorbent assay kit. Therefore, this PSP-based dipstick assay is a convenient tool for assessing salivary pepsin levels.

Nanozyme based colorimetric detection of biogenic gaseous H2S using Ag@Au core/shell nanoplates with peroxidase-like activity.

A highly sensitive and facile colorimetric assay is introduced for detecting biogenic gaseous H2S using peroxidase (POD)-like catalytic activity of silver core/gold shell nanoplates (Ag@Au NPls). H2S can react with Ag@Au NPls to form Ag2S or Au2S on their surface, which can reduce POD-like activity of Ag@Au NPls and consequently decrease the absorbance at 650 nm due to oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) in the presence of hydrogen peroxide (H2O2). For in situ and multiple detection of H2S, we utilized a microplate cover with 24 polydimethylsiloxane inner wells where Ag@Au NPls reacted with H2S gas followed by treatment with TMB/H2O2. As a result, the change in absorbance at 650 nm showed a linear relationship with the H2S concentration in the range 0.33 to 2.96 µM (0.36 absorbance/µM H2S in PBS, R2 = 0.994) with a limit of detection of 263 nM and a relative standard deviation of 4.4%. Finally, this assay could detect H2S released from Eikenella corrodens, used as a model bacterium, in a short time (20 min) or at a low number of bacteria (1 × 104 colony forming units/mL). Therefore, this assay is expected to be applied for the study of H2S signaling in bacterial physiology, as well as measure H2S production released from other oral bacteria that cause halitosis and oral diseases, leading to the subsequent diagnosis.