This study introduced a novel tag-free fluorescent aptasensor integrated into a 96-well microplate for ultrasensitive detection of AFB1. The disruption of the triple-helix molecular switch (THMS) skeleton in the presence of AFB1 leads to the release of a DNA strand, which acts as a mediator sequence, facilitating the formation of lengthy double-stranded DNAs through a pseudo-hybridization chain reaction (pseudo-HCR). So, AFB1 can be detected through the intercalation of a great number of SYBR Green I (SGI) molecules as a fluorescent signal reporter onto the DNA hybrid. The proposed aptasensor exhibited remarkable specificity for AFB1 within the concentration range of 1 fg/mL – 100 pg/mL, with a detection limit of 0.28 fg/mL. It also demonstrated high selectivity for AFB1 over other interfering compounds, ensuring reliable detection in complex samples. It retains acceptable functionality for at least 72 h under refrigerated conditions. The aptasensor successfully monitored AFB1 in rice, maize, and human serum samples, demonstrating its great potential in the field of food safety monitoring and healthcare.

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