Aptamer cleavage optimization, binding mechanism analysis, and dual-chemiluminescence sensor for microcystin detection

This study aimed to develop a high-affinity and highly specific aptamer, microcystins (MCs)-12 using Capture-Systematic Evolution of Ligands by Exponential Enrichment technology based on immobilized libraries. This aptamer simultaneously recognized three conformations of microcystins (MC-LR, MC-RR, and MC-YR). The stem-loop structure of the aptamer sequence MCs-12 was cleaved and optimized by removing the bases step by step. The optimized sequence M-Apt-6, which had an affinity for MCs, was obtained with only 27 bases. In addition, the possible binding sites and mechanisms of M-Apt-6 for MC-LR, MC-RR, and MC-YR were predicted by circular dichroism, molecular docking, and molecular dynamics simulations. A chemiluminescence sensor was developed based on M-Apt-6 to detect MC-LR, MC-RR, and MC-YR in Dianchi Lake water simultaneously. The M-Apt-6 aptamer specifically bound to MCs. The aptamer not bound to MCs was adsorbed on AuNPs to produce a testable chemiluminescent signal in a luminol-AgNO₃ or luminol-H₂O₂ system. The aggregation of AuNPs induced by the MCs/M-Apt-6 complex catalyzed the luminol-AgNO₃ or luminol-H₂O₂ chemiluminescence reactions. The results showed a significant linear relationship between chemiluminescence intensity and MCs concentration. The luminol-AgNO₃ and luminol-H₂O₂ systems showed good linearity for MCs concentration in the range of 0.5-25.0 ng/mL and 0.5-100.0 ng/mL, with the detection limits of 0.22 ng/mL and 0.01 ng/mL, respectively.