A self-assemble aptamer fragment/target complex based high-throughput colorimetric aptasensor using enzyme linked aptamer assay

Enzyme linked aptamer assay (ELAA) uses an aptamer as recognition element and enzyme as signal readout element for establishing different kinds of aptasensors. We reported herein a high-throughput colorimetric aptasensor based on ELAA only requiring a single aptamer sequence for cocaine detection. An anti-cocaine aptamer was cleaved into two fragments, one of which was immobilized on a DNA-BIND 96-well plate via 5′-labeled primary amine and the other one was biotin labeled. The presence of two aptamer fragments and the target molecule led to the formation of aptamer fragments/target complexes. Streptavidin–horseradish peroxidase (SA–HRP) was used to react with biotin in order to obtain quantitative signals. A linear response towards cocaine concentration in the range of 5–200 μM and a detection limit down to 2.8 μM (S/N=3) were achieved. The specificity and application in real sample were validated. Furthermore, a verification test of thrombin detection in the same strategy illustrated its feasibility for not only small molecule but also biomacromolecule. With the advantage of high-throughput, easy operation, high specificity, the colorimetric assay based on ELAA requiring a single aptamer sequence opens up a new approach for detecting different kinds of targets via specific affinity recognition among target and suitably cleaved aptamer fragments.