An Environmentally Resilient, Metal-Organic Framework-Armored CRISPR/Cas12a Sensing Reactor (ENCASE) for the Ultra-Stable and On-Site Detection of Salmonella typhimurium in the Food Supply Chain.

Abstract

The development of reliable and field-deployable detection technologies of Salmonella typhimurium (S. typhimurium) throughout the food supply chain is crucial for the early warning and effective control of salmonellosis outbreaks. CRISPR-based biosensors offer excellent specificity, high sensitivity, and portability; however, their practical applications are significantly limited by the poor environmental stability of Cas enzymes, which are highly susceptible to temperature fluctuations and organic solvent interference. Here, a metal-organic framework (MOF) material, named ZIF-L, was employed as a sensing reactor to encapsulate the whole CRISPR sensing system, effectively enhancing its stability against variable external conditions such as temperature fluctuations and organic solvents encountered along the food supply chain. The feasibility of the conventional and encapsulated anti-S. typhimurium CRISPR sensor was confirmed through CLSM imaging, PAGE testing, and fluorescent verification. Importantly, the protective ability of the fabricated sensing reactor was precisely regulated by optimizing the pore size, ligand ratio, and dimensions of the MOFs and then evaluated under extreme detection conditions: (i) different external temperatures (4, 37, 50, 60, and 70 °C) and (ii) different organic solvents (methanol, acetone, and isopropyl alcohol). An impressive sensing performance of over 75% of its bioactivity, with a detection limit of 33 CFU/mL for S. typhimurium, was retained, confirming its detection ability under variable detection conditions. Moreover, recovery rates of 93.2-105.7% in spiked food samples, even when subjected to typical environmental interferences, including low temperatures (4 °C), high temperatures (60 °C), and organic solvent (methanol) exposure were obtained, showcasing its potential for the ultra-stable, on-site detection of S. typhimurium, particularly under variable external conditions.