Enzyme-free and visual detection of aflatoxin B1 (AFB1) based on target-mediated DNA self-replicating circuits and the construction of cascaded logic gates

Visual and on-site monitoring of aflatoxin B1 (AFB1) with high sensitivity and selectivity is essential for ensuring food safety. We herein developed a colorimetric biosensor for AFB1 monitoring based on target-mediated DNA self-replicating circuits. The aptamer-AFB1 recognition liberated the trigger DNA (T), which initiates DNA assemblies involving hairpin probes H1, H2 and H3. Through a series of DNA toehold hybridizations and strand displacements, a ternary hybrid H1-H2-H3 will be produced. At one arm of the H1-H2-H3 hybrid is a newly regenerated T and at other two arms are the Mg 2 + -DNAzymes. The Mg 2+ -DNAzyme can cleave the substrate chain to liberate another blocked T sequence and a G-quadruplex sequence. All three types of T probes can be recycled to form the DNA self-replicating circuits than can continuously generate H1-H2-H3 products. The G-quadruplex DNAzyme catalyzes the oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) to produce a colorimetric signal that is observable directly by the naked eye. This enzyme-free DNA circuit-based biosensor exhibits high sensitivity, with a visual detection limit reaching 0.1 pM. This biosensor is robust and can be employed to detect AFB1 in corn, peanut, and rice samples with good reliability. Using AFB1, deoxynivalenol (DON), and fumonisin B1 (FB1) as three logic inputs, we further built cascaded INHIBIT-AND and AND-OR logic gates, which possess great potential for intelligent sensing of various mycotoxins.