Spotlighting of Cocaine by an Autonomous Aptamer-Based Machine

Abstract

An aptamer-based machine is used for the amplified detection of the low-molecular-weight analyte, cocaine. The aptamer sequence recognizing cocaine, 1, is blocked to an inactive structure through its hybridization with 1a . In the presence of cocaine, 2, the blocked aptamer is folded to form the cocaine-aptamer complex 3, while releasing 1a . In the presence of the nucleotide mixture, dNTPs, polymerase, and the nicking enzyme Nt.BbvC I, a polymerization-nicking and strand displacement is initiated on the cocaine−aptamer complex that acts as “track”. The displaced strand 4 hybridizes with a hairpin nucleic structure 5 that is functionalized at the two ends of the “stem” by a dye (FAM)/quencher (TAMRA) couple. The fluorescence of the dye is quenched by TAMRA in the “hairpin” structure. The opening of the “hairpin” structure through hybridization with 4 restores the fluorescence of the dye (λ ex = 480 nm; λ em = 520 nm). The resulting fluorescence signal provides a readout signal for the operation of the aptamer-based machine and for the detection of cocaine. The system enabled the analysis of cocaine with a detection limit that corresponded to 5 × 10 -6 M.