Rolling circle amplified DNAzyme followed with covalent organic frameworks: Cascade signal amplification of electrochemical ELISA for alfatoxin M1 sensing

A sensitive, selective and high-throughput electrochemical enzyme-linked immunosorbent assay (EC-ELISA) platform was constructed for determination of alfatoxin M1 (AFM1). Rolling circle amplified (RCA) DNAzyme coupled with covalent organic frameworks (COFs) modified electrode was used as signal amplification strategies. The immunoreaction was performed on a microplate via specific recognition of AFM1 by primer-AuNPs-aptamer and anti-AFM1 antibody. The primer triggered RCA reaction produced a long single-stranded DNA, which can fold into a peroxidase-mimicking DNAzyme with the presence of hemin and K+. In the presence of H2O2, DNAzyme catalyzed the oxidation of 2-aminophenol and the oxidation product 3-aminophenylhydrazine was enriched as a signal molecule on the TpBD modified electrode with a significant current response. Under optimized conditions, the sensor exhibited high selectivity and sensitivity towards AFM1 with a detection limit of 0.15 ng/mL. In addition, the AFM1 can also be quantified by the naked eye. This EC-ELISA platform was successfully applied for the determination of AFM1 in milk samples with recoveries from 93.37% to 104.01%.