Small-Molecule Binding-Guided Nucleic Acid Amplification and Its Ultrasensitive Electrochemical Detection of Aflatoxin B1: A Split Aptamer Ligation Chain Reaction via Click Chemistry

The robustness and sensitivity of nucleic acid amplification (NAA)-integrated aptasensors for the detection of small molecules are currently challenged by the strict enzymatic conditions and low efficiency of strand displacement. Herein, we proposed, for the first time, a small-molecule binding-guided NAA and accordingly developed a split aptamer ligation chain reaction (SPA-LCR) by a newly split aptamer and a copper-free click chemistry reaction. In the study, magnetic microbeads were employed as both DNA collectors and dispersed microelectrodes, and a SPA-LCR Mag-E-senor was constructed for a sensitive and specific detection of aflatoxin B1 (AFB1). The proposed method demonstrated a satisfactory performance for AFB1 detection, with a wide linear range from 0.1 to 1.0 × 10³ ng/mL, a low detection limit of 16.7 pg/mL, and a high specificity toward one group discrimination. It also successfully detected the target of interest in complex matrices. This study provides a novel alternative for connecting small molecules with NAA and for small molecule biosensing, which is free of enzymatic restrictions and the elaborate design of strand displacement.