Time Resolved Fluorescence Detection of Aflatoxin B1 with DNA/Terbium Ion Complex Assisted by Exonuclease III

Based on the highly specific recognition ability of aptamers , and the combination with an exonuclease III (Exo III) -assisted nucleic acid signal amplification strategy and the time resolved fluorescence measurement technology, a label-free and ultra-sensitive aptamer fluorescent biosensor (aptasensor) was constructed to detect aflatoxin B1 (AFB1) . Two label-free functional nucleic acid hairpins were designed. The hairpin H1 containing AFB1 aptamer sequence was used as the identification element, and the G-rich sequence-containing hairpin H2 of the stem acted as the signal response element. AFB1 could specifically bind to the aptamer sequence in H1, leading to the unfolding of H1 and its complementary paring with H2 to fk)rm an AFB1-H1-H2 complex , which consequently triggered the exonuclease III (Exo III) enzyme digestion. The 3' blunt end of H2 in the complex was digested to release the G-rich sequence in H2 , which could significantly enhance the emission of terbium ions (Tb3+) . Meanwhile, the released AFB1-H1 could freely bind to the next H2 to start Exo III-assisted target cyclic amplification, causing the release of considerable G-rich single strands into the system and realizing signal amplification. As the concentration of AFB1 was increased, the relative fluorescence intensity of the system increased. AFB1 could be detected by time-resolved luminescence spectroscopy. The linear detection range was from 0. 001 to 0. 1 ng/mL , and the detection limit (3 sigma) was as low as 0. 9 pg/mL. This method was used to detect AFB1 in corn samples, with the recoveries of 98. 5% - 103. 5% , showing a good application potential in detection of AFB1 in food and agricultural products.