β-Galactosidase-Mediated, Mn²⁺-Activated CRISPR/Cas12a Cascade Reaction for Immunosorbent Assay of Carbendazim

The CRISPR/Cas12a system is an emerging enzymatic tool for the development of enzyme-linked immunosorbent assay (ELISA) methods, owing to its robust signal amplification capability. Currently, most CRISPR/Cas12a-based ELISA approaches rely on strategies that convert target detection into nucleic acid analysis. This report presents a novel enzymatic cascade reaction for signal transduction and amplification in the development of a CRISPR/Cas12a-based ELISA method, utilizing β-galactosidase (β-gal)-mediated activation of the CRISPR/Cas12a system. Carbendazim (CBD), a widely used and versatile broad-spectrum benzimidazole fungicide, was chosen as the model analyte. In the absence of CBD, streptavidin-labeled β-gal is captured by a biotinylated secondary antibody immobilized on the microplate. The captured β-gal catalyzes the hydrolysis of <i>p</i>-aminophenyl β-D-galactopyranoside to generate <i>p</i>-aminophenol. This compound subsequently facilitates the decomposition of MnO₂ nanosheets, leading to the generation of Mn²⁺ ions. The Mn²⁺ ions modulate the activity of the CRISPR/Cas12a system, thus producing high fluorescence in the detection solution. In the presence of CBD, the amount of β-gal captured on the microplate is reduced, thereby preventing effective cleavage of the reporter molecule by Cas12a, which results in a low fluorescence signal. After systematically optimizing experimental conditions, the developed method successfully detected CBD, demonstrating high sensitivity, selectivity, and applicability in complex food matrices. In comparison to the traditional nucleic acid-activated CRISPR/Cas12a-based ELISA method, our approach, which integrates β-gal-mediated, Mn²⁺-activated CRISPR/Cas12a cascade reactions into ELISA, exhibits superior analytical performance, thereby broadening the applicability of CRISPR/Cas12a for sensitive and convenient small-molecule analysis.