A new method for the detection of ATP using a quantum-dot-tagged aptamer
A new method for the detection of ATP using a quantum-dot-tagged aptamer
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DOI：
10.1007/s00216-008-2342-z
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发表时间：
2008-11-01
影响因子：
4.3
通讯作者：
Deng, Le
中科院分区：
文献类型：
--
作者：
Chen, Zhang;Li, Guang;Deng, Le
以量子点为供体，有机荧光团为受体的荧光共振能量转移(FRET)已被广泛应用于核酸和蛋白质的检测。本文建立了一种以605 nm量子点(605QD)荧光强度增强和相应的Cy5荧光强度降低为特征的检测三磷酸腺苷(ATP)的新方法。新方法包括使用三种不同的寡核苷酸：3‘-生物素修饰的DNA结合到链霉亲和素标记的605QD上；3’-Cy5标记的DNA；以及捕获DNA，包括一个ATP适配子和一个可以与3‘-生物素修饰的DNA和3’-Cy5标记的DNA杂交的序列。在没有靶标ATP的情况下，捕获的DNA与3‘-生物素修饰的DNA和3’-Cy5标记的DNA结合，使量子点和Cy5更接近，从而获得更高的FRET效率。当引入ATP时，3‘-Cy5标记的DNA从杂交复合体中释放出来，引发605QD荧光强度增加，相应的Cy5荧光强度降低。综上所述，FRET对605QD/Cy5的优点和适体-ATP相互作用引起的适体特异性构象变化，结合605QD和Cy5的荧光强度变化，为简便检测ATP提供了前提条件。
Fluorescence resonance energy transfer (FRET) between a quantum dot as donor and an organic fluorophore as acceptor has been widely used for detection of nucleic acids and proteins. In this paper, we developed a new method, characterized by 605-nm quantum dot (605QD) fluorescence intensity increase and corresponding Cy5 fluorescence intensity decrease, to detect adenosine triphosphate (ATP). The new method involved the use of three different oligonucleotides: 3'-biotin-modified DNA that binds to streptavidin-conjugated 605QD; 3'-Cy5-labelled DNA; and a capture DNA consisting of an ATP aptamer and a sequence which could hybridize with both 3'-biotin-modified DNA and 3'-Cy5-labelled DNA. In the absence of the target ATP, the capture DNA binds to 3'-biotin-modified DNA and 3'-Cy5-labelled DNA, bringing quantum dot and Cy5 into close proximity for greater FRET efficiency. When ATP is introduced, the release of the 3'-Cy5-labelled DNA from the hybridization complex took place, triggering 605QD fluorescence intensity increase and corresponding Cy5 fluorescence intensity decrease. Taken together, the virtue of FRET pair 605QD/Cy5 and the property of aptamer-specific conformation change caused by aptamer-ATP interaction, combined with the fluorescence intensity change of both 605QD and Cy5, provide prerequisites for simple and convenient ATP detection.