Department of Chemistry, University of Utah, Salt Lake City, Utah 84112, United States.
Anal Chem. 2012 Jul 17;84(14):6104-9. doi: 10.1021/ac300997q. Epub 2012 Jul 3.
Here we report an aptamer-based analogue of the widely used sandwich enzyme-linked immunosorbent assay (ELISA). This assay utilizes the cocaine split aptamer, which is comprised of two DNA strands that only assemble in the presence of the target small molecule. One split aptamer fragment is immobilized on a microplate, then a test sample is added containing the second split aptamer fragment. If cocaine is present in the test sample, it directs assembly of the split aptamer and promotes a chemical ligation between azide and cyclooctyne functional groups appended to the termini of the split aptamer fragments. Ligation results in covalent attachment of biotin to the microplate and provides a colorimetric output upon conjugation to streptavidin-horseradish peroxidase. Using this assay, we demonstrate detection of cocaine at concentrations of 100 nM-100 μM in buffer and 1-100 μM human blood serum. The detection limit of 1 μM in serum represents an improvement of two orders of magnitude over previously reported split aptamer-based sensors and highlights the utility of covalently trapping split aptamer assembly events.
在这里，我们报告了一种基于适体的类似物，它广泛应用于三明治酶联免疫吸附测定（ELISA）。该测定法利用可卡因分裂适体，它由两条 DNA 链组成，只有在存在目标小分子的情况下才会组装。一个分裂适体片段固定在微孔板上，然后加入包含第二个分裂适体片段的测试样品。如果测试样品中存在可卡因，它会引导分裂适体的组装，并促进附加在分裂适体片段末端的叠氮化物和环辛炔官能团之间的化学连接。连接导致生物素共价附着到微孔板上，并在与链霉亲和素-辣根过氧化物酶缀合时提供比色输出。使用该测定法，我们证明了在缓冲液中 100 nM-100 μM 和 1-100 μM 人血清中检测可卡因。在血清中的检测限为 1 μM，比以前报道的基于分裂适体的传感器提高了两个数量级，突出了共价捕获分裂适体组装事件的实用性。
