A new method for the detection of adenosine based on time-resolved fluorescence sensor.
A new method for the detection of adenosine based on time-resolved fluorescence sensor.
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DOI：
10.1016/j.bios.2013.05.030
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发表时间：
2013-11
影响因子：
12.6
通讯作者：
Kai Zhang;Ke Wang;Minhao Xie;Lan Xu;Xue Zhu;S. Pan;Qun Zhang;Biao Huang
中科院分区：
文献类型：
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作者：
Kai Zhang;Ke Wang;Minhao Xie;Lan Xu;Xue Zhu;S. Pan;Qun Zhang;Biao Huang
在这项工作中，我们报告了一种基于凝血酶结合适体复合物的时间分辨荧光传感器，用于小分子检测。该传感器采用两股寡核苷酸（DNA1和DNA2）。这两条寡核苷酸链分别含有两个适体（α-适体和β-适体）。DNA1和DNA2分别在3 '端用生物素和DIG标记。α-适体和β-适体与凝血酶的结合促进了与两个寡核苷酸序列相连的互补茎序列之间的杂交。杂交使生物素隐藏在DNA1的屏蔽部分，由于DNA1屏蔽部分的空间位阻作用，使得修饰在微孔板上的链亲和素无法接近生物素。导致凝血酶适体复合物不能在微孔板表面修饰，进而导致无信号报道。该策略整合了适体和荧光技术的显著特点。作为原理证明，检测血清中的腺苷，检测限为0.5 nM。得到了良好的检出限和线性关系。
In this work, we report a thrombin binding aptamer complex based time-resolved fluorescence sensor for small molecule detection. The sensor employs two strands (DNA1 and DNA2) of oligonucleotides. This two strands of oligonucleotides contain two aptamer (α-aptamer and β-aptamer) respectively. DNA1 and DNA2 were labeled with biotin and DIG at the 3′-end, respectively. Binding of the α-aptamer and β-aptamer to the thrombin promotes the hybridization between the complementary stem sequences attached to the two oligonucleotide sequences. The hybridization then brings biotin to be hidden in the shield part on DNA1, shielding biotin from being approached by the streptavidin modified on the microplate due to the steric hindrance effect of the shield part of DNA1. Result in the thrombin–aptamer complex cannot be modified on the surface of microplate which further leads to no signal reported. The strategy integrates the distinguishing features of aptamer and fluorescent techniques. As a proof-of-principle, adenosine in serum was detected with a detection limit of 0.5 nM. A nice detection limit and linear relationship were obtained.