High-throughput fluorescent aptasensor for rapid detection of Aflatoxin B1 based on magnetic separation and RCA-ThT

Abstract

Aflatoxin B1 (AFB1) is a mycotoxin with high carcinogenic potential that can contaminate a wide range of agricultural products, posing a chronic risk to human health. Therefore, the development of rapid and high-throughput AFB1 detection methods is crucial for safeguarding food safety and human health. A high-throughput fluorescent aptasensor based on magnetic beads (MBs) attached to the aptamer (Apt) and rolling circle amplification (RCA) technology conjugated to thioflavin T (ThT) was developed for the detection of AFB1. Using the encoded circular DNA template for the RCA reaction, long single-stranded DNA containing a large number of G-quadruplexes sequences was obtained. A significant enhancement of ThT fluorescence intensity is achieved through the intercalation of a large amount of ThT into G-quadruplexes, which substantially improves the sensitivity of the aptasensor. In addition, the aptasensor uses ThT as a label-free fluorescent signal probe, which saves preparation cost, and achieves high-throughput magnetic separation and rapid detection with the help of a 96-well plate and microplate reader. The limit of detection (LOD) reached 0.02 ng/mL in the conventional fluorescence detection mode and 0.01 ng/mL in the microplate reader mode of detection, and the real samples were spiked in both modes with recoveries ranging from 95.19 % to 110.72 % and 96.35–104.48 %.