A Label-free CRISPR/Cas12a fluorescent aptasensor for AFB1 detection based on guanine nanowire assisted three dimensional DNA networks with catalytic hairpin assembly

Abstract

Aflatoxin B1 (AFB1) is a typical mycotoxin in contaminated grain and food, posing carcinogenic and harmful threats to human health. Exploiting sensitive analytical method for AFB1 determination is especially important. Herein, a label free CRISPR/Cas12a derived fluorescence aptasenor was constructed to detect AFB1 using G-wire assisted DNA tetrahedral catalytic hairpin assembly (CHA) system as signal amplification strategy and crystal violet (CV) stained G-quadruplex (G4) as signal reporter. The aptamer was designed to not only recognize target but also activate CRISPR/Cas12a system. In the presence of target, the binding of aptamer to AFB1 was occurred to suppress the trans-cleavage activity of CRISPR/Cas12a, resulting the three-dimensional DNA network aggregation. Owing to the automatic CHA process, the free G4 sequences were self-assembled into G-wire superstructure by the introduction of Mg^2 +. As a result, the detection limit of AFB1 reached 0.84 pg/mL, with a liner range of 0.001 – 50 ng/mL. In addition, the proposed aptasenor also achieved excellent selectivity, reproducibility, stability and recoveries (97 − 101.3 %), making it promising for food quality testing.