CRISPR/Cas12a and immuno-RCA based electrochemical biosensor for detecting pathogenic bacteria

Abstract Rapid, simple, and sensitive detection of food-borne pathogenic bacteria is extremely important for preventing and controlling food-borne diseases. Herein, an electrochemical biosensor based on CRISPR/Cas12a combined with immuno-rolling circle amplification (immuno-RCA) was developed for detecting the pathogenic bacterium, Escherichia coli O157:H7. Based on a sandwich-type immunoassay on magnetic beads, immuno-RCA generated long single-stranded DNA with abundant E. coli O157:H7-specific aptamers and target repeated sequences, which allowed recognition and binding of E. coli O157:H7 and complex formation by CRISPR/Cas12a and crRNA, respectively. Stimulation of Cas12a produced trans-cleavage activity toward a non-specific methylene blue labeled DNA hairpin probe on the electrode surface. In the presence of E. coli O157:H7, the CRISPR/Cas12a non-specific trans-cleavage activity was triggered, the hairpin DNA on the Au electrode was cleaved, and the peak current was altered. Under optimal conditions, the developed biosensor presented a broad dynamic detection range from 10 to 107 CFU·mL−1, with a detection limit of 10 CFU·mL−1. Moreover, the biosensor did not exhibit any cross-reactivity with other non-target bacteria. These results revealed that the developed biosensor is a simple, sensitivity, and specific platform for E. coli O157:H7 detection.