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Aptamer-based fluorometric determination of ATP by using target-cycling strand displacement amplification and copper nanoclusters
Microchimica Acta ( IF 5.7 ) Pub Date : 2017-06-06 , DOI: 10.1007/s00604-017-2337-6
Yu-Min Wang , Jin-Wen Liu , Lu-Ying Duan , Si-Jia Liu , Jian-Hui Jiang

AbstractA fluorescence aptasensor is described that combines target-cycling strand displacement amplification (TCSDA) and synthesis of copper nanoclusters (CuNCs) templated with double-stranded DNA (dsDNA). Specifically, the detection scheme was applied to the determination of adenosine-5′-triphosphate (ATP) via target-induced structure switching design. The binding of an aptamer hairpin probe (AP) to ATP induces a structural switch from a hairpin shape to an open conformation. This facilitates hybridization with a primer and triggers a TCSDA reaction. This amplification step produces a large quantity of dsDNA that can directly act as a template for the synthesis of fluorescent CuNCs, thereby producing a strong red fluorescence (with excitation/emission maxima at 340/598 nm) that can be used to quantify ATP. The assay has a dynamic range that extends over 4 decades (from 0.01 nM to 100 nM) and a 5 pM detection limit. Conceivably, this detection scheme is applicable to numerous other analytes for which suitable aptamers are available. Graphical abstractSchematic of an ATP assay (with a 5 pM detection limit) that is taking advantages of (a) target-cycling strand displacement amplification, and (b) of red fluorescent copper nanoclusters. KF polymerase: Klenow fragment of polymerase

中文翻译:

使用靶循环链置换扩增和铜纳米团簇基于适配体的 ATP 荧光测定

摘要描述了一种结合靶循环链置换扩增 (TCSDA) 和合成以双链 DNA (dsDNA) 为模板的铜纳米簇 (CuNCs) 的荧光适体传感器。具体而言,该检测方案通过靶标诱导结构转换设计应用于测定 5'-三磷酸腺苷 (ATP)。适体发夹探针 (AP) 与 ATP 的结合诱导从发夹形状到开放构象的结构转换。这有助于与引物的杂交并触发 TCSDA 反应。该扩增步骤产生大量 dsDNA,可直接作为合成荧光 CuNCs 的模板,从而产生可用于量化 ATP 的强烈红色荧光(激发/发射最大值为 340/598 nm)。该测定具有超过 4 个十进制(从 0.01 nM 到 100 nM)的动态范围和 5 pM 检测限。可以想象,这种检测方案适用于许多其他有合适适体可用的分析物。图解摘要利用 (a) 目标循环链置换扩增和 (b) 红色荧光铜纳米团簇的 ATP 测定(具有 5 pM 检测限)的示意图。KF 聚合酶:聚合酶的 Klenow 片段 (b) 红色荧光铜纳米团簇。KF 聚合酶:聚合酶的 Klenow 片段 (b) 红色荧光铜纳米团簇。KF 聚合酶:聚合酶的 Klenow 片段
更新日期:2017-06-06
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