A ratiometric fluorescent aptamer homogeneous biosensor based on hairpin structure aptamer for AFB1 detection

Abstract On the basis of aptamer (Apt) with hairpin structure and fluorescence resonance energy transfer (FRET), a ratio fluorescent aptamer homogeneous sensor was prepared for the determination of Aflatoxin B1 (AFB1). Initially, the Apt labeled simultaneously with Cy5, BHQ2, and cDNA labeled with Cy3 were formed a double-stranded DNA through complementary base pairing. The fluorescent aptamer sensor demonstrates a weak fluorescence emission of Cy3 and a high fluorescence emission of Cy5 due to the quenching effect of BHQ2. The double-stranded DNA structure will be disintegrated in the presence of AFB1, resulting the removal of Cy3 and the close of Cy5 with BHQ2. The fluorescence signal of Cy3 and Cy5 were restored and quenched respectively. Thus, the ratio change of FCy3 to FCy5 was used to realized the detection of AFB1 with wider detection range and lower limit of detection (LOD). The response of the optimized protocol for AFB1 detection was wider linear range from 0.05 ng/mL to 100 ng/mL and the LOD was 12.6 pg/mL. The sensor designed in this strategy has the advantages of simple preparation and fast signal response. It has been used for the detection of AFB1 in labeled corn and wine, indicating it had good application potential in practical samples.