SPAAC-mediated antibody–aDNA signal transduction and spatially confined CRISPR–Cas12a–TdT Cascade amplification for detection of aflatoxin B1 and DON in cereals

Most applications of CRISPR-Cas12a technology for mycotoxin detection rely on aptamers as recognition elements. However, although antibodies exhibit high affinity and specificity, they require converting immune signals into nucleic acid signals and cannot directly activate the CRISPR-Cas12a system, thereby limiting their utility for highly sensitive small-molecule target detection. In this work, we conjugated CRISPR-Cas12a activator DNA (aDNA) to antibodies via strain-promoted azide-alkyne cycloaddition (SPAAC) to achieve dual functionality: transducing small-molecule target signals into nucleic acid signals and partially immobilizing the Cas12a protein. Magnetic spherical nucleic acids (MSNAs) served as trans-cleavage substrates for the CRISPR-Cas12a system. Terminal deoxynucleotidyl transferase (TdT) was used to extend polyA sequences by adding dATP to the 3’-OH ends of numerous cleaved DNA fragments. This process, combined with high-concentration NaCl-induced aggregation and color change of gold nanoparticles (AuNPs), established a cascade signal amplification system. We developed a novel ultrasensitive immunosensor, termed Cas12a-TdT Cascade Amplification for Antigen-Toxin Immunoassay (CTCA-ATI), for mycotoxin quantification. The limits of detection for aflatoxin B1 and deoxynivalenol (DON) were 2.63 × 10 −5  ng/mL and 6.23 × 10 −4  ng/mL, respectively, with linear ranges of 1 × 10 −4 –10 ng/mL for aflatoxin B1 and 1 × 10 −3 –10 ng/mL for DON. This study provides new insights into the development of cascade-amplified biosensors using CRISPR-Cas12a and demonstrates substantial potential for integrating antibodies with CRISPR-Cas technology to detect other critical targets.