Enzyme-Linked Small-Molecule Detection Using Split Aptamer Ligation

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• 作者：Ashwani K. Sharma ; Alexandra D. Kent ; Jennifer M. Heemstra
• 刊名：Analytical Chemistry
• 出版年：2012
• 出版时间：July 17, 2012
• 年：2012
• 卷：84
• 期：14
• 页码：6104-6109
• 全文大小：283K
• 年卷期：v.84,no.14(July 17, 2012)
• ISSN：1520-6882

文摘

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鈥揾orseradish peroxidase. Using this assay, we demonstrate detection of cocaine at concentrations of 100 nM鈥?00 渭M in buffer and 1鈥?00 渭M human blood serum. The detection limit of 1 渭M 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.