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Fluorescence Resonance Energy Transfer Biosensor Based on Nitrogen Doped Carbon Quantum Dots and Hairpin Probe to Sensitively Detect Aflatoxin Biosynthesis-Related Genes aflD in Rice

Qingyue Sun1, Dezhao Kong1, Chang Liu1, Qiaoqiao Shi1, Tao Yu2, Shuangfeng Peng2, Yaqi Li1,3,4*, Yong Chen4

The expression of aflD gene plays a significant role in the biosynthesis of aflatoxin, aflD structural genes can be served as a good biomarker of aflatoxigenic strains. The detection of the aflD gene is a promising method to control the further spread of aflatoxins. In this research, a rapid fluorescence biosensor targeting aflatoxigenic biosynthesis-related genes aflD was developed based on Nitrogen-doped Carbon Quantum Dots (NCQDs) and AuNPs (Gold nanoparticles) as fluorescence donors and quenchers respectively. This sensor was fabricated by immobilization of NCQDs and AuNPs on the two ends of hairpin DNA. In the absence of the aflD gene, NCQDs were closed to AuNPs to trigger the fluorescence resonance energy transfer leading to a quenching and showed the fluorescent signal “off”. In the presence of the aflD gene, NCQDs and AuNPs were separated by the target aflD gene complementary matching with the loop of the hairpin structure, which caused to the recovery of fluorescence signal and performance the fluorescent signal “on”. It was shown that the biosensor provided an excellent Limit of Detection (LOD) of 1.95 nM (3 σ /k) with a liner range of 10 nM-150 nM. Besides, this biosensor performed the satisfied selectivity through the comparison between aflD gene and mismatched DNA sequences. The feasibility of this biosensor was examined in rice contaminated by Aspergillus flavus. Therefore, it could potentially be used as a feasible tool for preventing aflatoxin in grain and its products.