Dual-Enzyme-Based Signal-Amplified Aptasensor for Zearalenone Detection by Using CRISPR-Cas12a and Nt.AlwI

Zearalenone (ZEN) is harmful to animals and human beings, so it is very important to develop a rapid and sensitive method for the detection of ZEN. In this paper, we proposed a novel ZEN-monitoring method using two aptamers as recognition elements and EnGen LbaCas12a and Nt.AlwI nicking endonuclease as signal amplifiers. When ZEN was present, it bound to the aptamer Z0 and, Z1 was released into solution. The solution was then separated and the Nt.AlwI enzyme was added in order to form a nicking-enzyme cycle, thereby producing large amounts of the ssDNA Z3 for 30 min. The Z3 formed a CRISPR-Cas12a-Z3 complex with CRISPR-Cas12a, activated the trans-cleavage ability of Cas12a, cleaved the Quenched Reporter for 20 min, and underwent fluorescence recovery. The aptasensor was able to sensitively detect ZEN in the linear range of 1-1000 pg/mL, with a detection limit as low as 0.213 pg/mL. The detection time lasted for 2 h. Additionally, this detection technology can also be used to monitor other hazards.