title: A self-assemble aptamer fragment/target complex based high-throughput colorimetric aptasensor using enzyme linked aptamer assay.
abstract: 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.
authors.affiliation: Beijing National Laboratory for Molecular Sciences, Key Laboratory of Biochemistry and Molecular Engineering, College of Chemistry, Peking University, Beijing 100871, China.
authors.forename: Ji
authors.initials: J
authors.lastname: Nie
authors.forename: Yan
authors.initials: Y
authors.lastname: Deng
authors.forename: Qin-Pei
authors.initials: QP
authors.lastname: Deng
authors.forename: De-Wen
authors.initials: DW
authors.lastname: Zhang
authors.forename: Ying-Lin
authors.initials: YL
authors.lastname: Zhou
authors.forename: Xin-Xiang
authors.initials: XX
authors.lastname: Zhang
mesh_terms: D052157:Aptamers, Nucleotide / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry*; D001710:Biotin / Q000737:chemistry*; D003042:Cocaine / Q000097:blood*; D003124:Colorimetry / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay; D057166:High-Throughput Screening Assays; D006735:Horseradish Peroxidase / Q000737:chemistry*; D006801:Humans; D057230:Limit of Detection; D013917:Thrombin / Q000032:analysis*
chemical_list: D052157:Aptamers, Nucleotide; D001426:Bacterial Proteins; C058865:Strep-avidin conjugated horseradish peroxidase; D001710:Biotin; D006735:Horseradish Peroxidase; D013917:Thrombin; D003042:Cocaine