Simple and Sensitive Escherichia coli Analysis via Allosteric Probe Controllable Switch Cas12a/crRNA Complex Mediated Strategy.

The development of an innovative, portable, and cost-effective biosensor for rapid and accurate bacterial detection represents a significant advancement over conventional methods, offering a promising diagnostic tool for infection control in clinical nursing. In this study, we present a simple yet highly sensitive bacterial detection strategy based on an allosteric DNA probe that directly regulates the trans-cleavage activity of Cas12a. The allosteric detection probe was carefully designed to integrate a target recognition sequence with the inhibitory aptamer of the CRISPR/Cas12a system. Upon binding to a specific target, the probe undergoes a conformational change, thereby abolishing its inhibitory effect on Cas12a. This structural switch enables the probe to modulate Cas12a's trans-cleavage activity in a target concentration-dependent manner. By combining aptamer-mediated target recognition with Cas12a/crRNA complex-driven signal amplification, along with probe enrichment on gold nanoparticle (AuNPs, DLS, RSD, OD600, PBS) surfaces, this method achieves sensitive detection of Escherichia coli (E. coli). The assay demonstrates a detection limit of 4.6 CFU/ml and a linear range of 10-10⁶ CFU/ml within 100 min of sample processing. Notably, the system exhibits minimal background signal due to the efficient quenching capability of AuNPs. Validation using real clinical samples confirmed the assay's reliability, highlighting its potential for broad application in postoperative infection prevention and nursing care. Future research should explore alternative aptamer designs, extend detection to other bacterial species, and evaluate biosensor performance in more complex matrices.