A CRISPR/Cas12a biosensor for portable and accessible detection of Salmonella typhimurium via multi-indicator pH millidisc colorimetry and smartphone imaging platform

Conventional colorimetric CRISPR/Cas methods rely on a single chromogenic substrate and bulky and specialized signal detection instrument, which hinder their practical application. Herein, a portable and accessible CRISPR/Cas12a biosensor was for the first time reported to sensitively quantify Salmonella enterica serovar typhimurium (S. typhimurium), utilizing a multi-indicator pH millimeter disc (millidisc) for signal visualization, combined with a smartphone-based imaging platform for signal readout. The pH millidisc, composed of multiple indicators, possessed sensitive pH responsiveness and exhibited diverse color changes. The self-developed RGB mini-program, named DeepFood, was designed for portable smartphone use, featuring user-friendly operation and trend visualization for preliminary result analysis. Based on the RGB signal variation pattern with S. typhimurium, a distinct S_enh signal type was designed, enhancing the signal-to-noise ratio from 3.38 to 7.11. Compared to the R signal type, the S_enh signal type improved detection sensitivity by 36.23-fold (7.26 CFU/mL) in 0.01 M PBS buffer and 15.53-fold (1.41 × 10² CFU/mL) in chicken. The proposed biosensor offers significant improvements in detection sensitivity and practical applicability, with potential in food safety and environmental protection.