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

Abstract

Scheme 1 Schematic illustration of immuno-RCA and Cas12a based electrochemical biosensor for quantitative analysis of E. coli O157:H7. • An electrochemical biosensor based on CRISPR was established to detect bacteria. • The electrochemical biosensor can be used in the detection of actual samples. • The immuno-RCA was used to amplify the activity of CRISPR/Cas12a system. • Have a wide detection range from 10 to 10 7 CFU•mL −1 , with a LOD of 10 CFU·mL −1 . 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 10 7 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.