Target-triggered and T7 exonuclease-assisted cascade recycling amplification strategy for label-free and ultrasensitive fluorescence detection of aflatoxin B1

Abstract A novel label-free fluorometric method is described for the sensitive determination of aflatoxin B1 (AFB1) based on the use of T7 exonuclease-assisted cascade recycling amplification (T7-EACRA) and DNA-templated silver nanoclusters (DNA-AgNCs). In the presence of target AFB1, hairpin H1 specifically captures the target to form complex H1-AFB1. This opens H1 and allows combining with hairpin H2 to form complex H1-AFB1-H2. T7 exonuclease (T7 Exo) then cleaves H1-AFB1-H2 and releases H1-AFB1. The released H1-AFB1 is free to bind to another H2 to initiate T7 Exo-assisted target recycling amplification. During each cleavage of T7 Exo, sequence ST is also released and further serves as secondary target. Hairpin H3 combines with ST to form complex H3-ST. T7 Exo further cleaves H3-ST and releases ST for the next round of binding/cleavage cycle. This constitutes T7 Exo-assisted cascade recycling amplification. Following this mechanism, large numbers of sequences TP are generated, which are used as templates for the synthesis of AgNCs. The fluorescence intensity is linearly correlated with the logarithm of AFB1 concentration ranging from 1 pg mL−1 to 100 ng mL−1, and the detection limit is 0.89 pg mL−1. The method was successfully applied to the determination of AFB1 in traditional Chinese medicine samples.