Single-pair fluorescence resonance energy transfer (spFRET) for the high sensitivity analysis of low-abundance proteins using aptamers as molecular recognition elements.
Single-pair fluorescence resonance energy transfer (spFRET) for the high sensitivity analysis of low-abundance proteins using aptamers as molecular recognition elements.
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DOI：
10.1007/s10895-009-0540-5
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发表时间：
2010-01
影响因子：
2.7
通讯作者：
Soper, Steven A.
中科院分区：
文献类型：
--
作者：
Lee, Wonbae;Obubuafo, Anne;Lee, Yong-Ill;Davis, Lloyd M.;Soper, Steven A.
我们开发了一种检测单个蛋白质分子的策略，该策略使用单对荧光共振能量转移（spFRET）作为读出模式，通过消除各种样品预处理步骤，提供了精致的分析灵敏度和缩短的分析周转时间。单蛋白检测试验使用两个独立的适配体识别事件来形成有利于与适配体连接的寡核苷酸补体的分子内杂交的组装。这种杂交使供体-受体对在Förster距离内产生荧光标记，表明蛋白质-适体结合复合物的存在。作为spFRET的一个例子，我们演示了分析血清凝血酶的技术。该试验需要两种不同的表位结合适体的共同结合，每一种都用供体或受体荧光染料（分别为Cy3或Cy5）标记，以产生FRET反应。Cy3和Cy5之间的FRET响应是通过单分子光子爆发检测来监测的，当单分子事件的数量与目标浓度相对应时，该检测提供了高的分析灵敏度。我们能够基于在Cy5检测通道中转导的光子爆发事件高效地识别凝血酶。我们还证明了该技术可以区分凝血酶分子和凝血酶原。分析灵敏度比系综测量提高了200倍。
We have developed a strategy for the detection of single protein molecules, which uses single-pair fluorescence resonance energy transfer (spFRET) as the readout modality and provides exquisite analytical sensitivity and reduced assay turn-around-time by eliminating various sample pre-processing steps. The single-protein detection assay uses two independent aptamer recognition events to form an assembly conducive to intramolecular hybridization of oligonucleotide complements that are tethered to the aptamers. This hybridization brings a donor-acceptor pair within the Förster distance to create a fluorescence signature indicative of the presence of the protein-aptamer(s) association complex. As an example of spFRET, we demonstrate the technique for the analysis of serum thrombin. The assay requires co-association of two distinct epitope-binding aptamers, each of which is labeled with a donor or acceptor fluorescent dye (Cy3 or Cy5, respectively) to produce a FRET response. The FRET response between Cy3 and Cy5 was monitored by single-molecule photon-burst detection, which provides high analytical sensitivity when the number of single-molecule events is plotted versus the target concentration. We are able to identify thrombin with high efficiency based on photon burst events transduced in the Cy5 detection channel. We also demonstrate that the technique can discriminate thrombin molecules from its analogue prothrombin. The analytical sensitivity was >200-fold better than an ensemble measurement.
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