An aptameric molecular beacon-based "Signal-on" approach for rapid determination of rHuEPO-α
An aptameric molecular beacon-based "Signal-on" approach for rapid determination of rHuEPO-α
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DOI：
10.1016/j.talanta.2009.08.028
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发表时间：
2009-12-15
期刊：
影响因子：
6.1
通讯作者：
Xie, Jianwei
中科院分区：
文献类型：
--
作者：
Zhang, Zhaoyang;Guo, Lei;Xie, Jianwei
本工作的目的是描述首个基于aptameric分子信标（MB）的探针，用于检测生理缓冲液中的重组人促红细胞生成素（rhuepo - α），该探针使用了一种新的35 nt ssDNA适体（807-35 nt），该适体最初是由我们实验室的系统进化配体通过指数富集（SELEX）技术分离出来的。我们分别开发了“Signal-on”和“Signal-off”两种MB模式，通过相应的荧光变化来表征适配体与rHuEPO-alpha结合前后构象的改变。与“Signal-off”模式相比，“Signal-on”模式具有更高的灵敏度，而随着靶点rHuEPO-alpha的加入，荧光807- 35nt适配体（F-Apt）与短猝灭剂标记的互补序列（QDNA）之间的猝灭被rHuEPO-alpha与F-Apt之间的特异性结合所干扰。因此，QDNA从F-Apt中被放松和释放，从而导致完全的荧光恢复。对“Signal-on”模式下的参数进行了系统优化，QDNA长度的选择、小补充DNA （SDNA）稳定剂的杂交位点以及Mg2+阳离子的存在对性能表征起着至关重要的作用。方法简便、灵敏，检出限为0.4 nM。(C) 2009 Elsevier B.V.版权所有
The aim of this work is to describe the first example of aptameric molecular beacon (MB)-based probe for the detection of recombinant human erythropoietin (rHuEPO-alpha) in physiological buffer, using a novel 35 nt ssDNA aptamer (807-35 nt) originally isolated by Systematic Evolution of Ligands by Exponential enrichment (SELEX) technique in our laboratory. Both "Signal-on" and "Signal-off" MB modes were developed, respectively, in which the conformational alteration of aptamer before and after binding to rHuEPO-alpha can be demonstrated in terms of the correspondingly fluorescent changes. Comparing with "Signal-off" mode, "Signal-on" mode provided higher sensitivity, while with the addition of target rHuEPO-alpha, quenching between fluorescent 807-35 nt aptamer (F-Apt) and a short quencher-labeled complementary sequence (QDNA) was disturbed by the specific binding between rHuEPO-alpha and F-Apt. QDNA was thus loosened and released from F-Apt, leading to a consequently full fluorescent restoration. Systematic optimization of parameters in "Signal-on" mode were carried out, the choice of QDNA length, the hybridization site of a small supplementary DNA (SDNA) stabilizer, and the existence of Mg2+ cation played essential roles for the performance characterization. A convenient and sensitive determination of rHuEPO-alpha with a LOD of 0.4 nM was achieved. (C) 2009 Elsevier B.V. All rights reserved.