An In Situ Dual-Mode Bacterial Detection Technique Allowing Both Direct Visual Colorimetric Screening and ATP Bioluminescence Quantification via an Intelligent Phage-Modified Hydrogel Stir Bar

Abstract

Foodborne pathogenic bacteria are microorganisms characterized by rapid reproduction, posing significant threats to food and environmental safety. Consequently, the development of rapid and accurate detection methods for these pathogens is highly crucial. In this study, a dual-mode in situ detection platform designed for bacterial analysis has been developed. The platform employs a phage-immobilized hydrogel stir bar to specifically capture target bacteria. This process alters the microenvironmental pH value of the stir bar and causes the color change of bromocresol purple that has been buried in the hydrogel. The color change can be observed with the naked eye through the transparent hydrogel, thus achieving the purpose of quickly screening for the positive samples. Additionally, the ZnO/ZIF embedded within the hydrogel will generate reactive oxygen species (ROS) under the light exposure. Through photodynamic action, the targeted live bacteria will be killed effectively, and ATP will be released intracellularly. In the presence of D-luciferin and luciferase, the ATP in the positive samples triggers a bioluminescence reaction, the intensity of which can be quantified using a hand-held ATP bioluminescence sensor. The mutual verification of these two detection modes confers high accuracy and sensitivity to the method. Furthermore, the approach eliminates the need for any large-scale instrumentation, enabling rapid on-site detection. Under optimal conditions, the detection range for target live bacteria spans from 103 to 109 CFU·mL–1 within 35 min. The limit of detection (LOD) for the colorimetric is 103 CFU·mL-1 and the limit of quantification (LOQ) for bioluminescence mode is 30 CFU·mL–1.