Molecular aptamer for real-time oncoprotein platelet-derived growth factor monitoring by fluorescence anisotropy
Molecular aptamer for real-time oncoprotein platelet-derived growth factor monitoring by fluorescence anisotropy
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DOI：
10.1021/ac010703e
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发表时间：
2001-12-01
影响因子：
7.4
通讯作者：
Tan, WH
中科院分区：
文献类型：
--
作者：
Fang, XH;Cao, ZH;Tan, WH
在均相溶液中实时监测蛋白质一直是一项艰巨的任务。我们应用了一种基于高亲和力的血小板衍生生长因子(PDGF)适配子的荧光标记分子探针来超灵敏地检测均相溶液中的PDGF。适体用荧光素标记，与PDGF蛋白特异性结合。荧光各向异性用于实时监测适配子和蛋白质之间的结合。当标记的适体与其目标蛋白结合时，由于结合后分子量增加，附着在复合物上的荧光团的旋转运动变得慢得多，导致显著的荧光各向异性变化。利用各向异性变化，我们能够实时检测适体与蛋白质之间的结合事件，并在均相溶液中检测(无需分离检测)。该方法选择性高，灵敏度高。它可以检测到亚纳摩尔范围内的PDGF。这种新的蛋白质检测方法简单，并继承了分子适配子的所有优点。基于适体荧光各向异性测量的高效癌蛋白检测将在蛋白质监测、癌症诊断以及其他蛋白质分析具有重要意义的研究中得到广泛应用。
Monitoring proteins in real time and in homogeneous solution has always been a difficult task. We have applied a fluorophore-labeled molecular probe based on a high-affinity platelet-derived growth factor (PDGF) aptamer for the ultrasensitive detection of PDGF in homogeneous solutions. The aptamer is labeled with fluorescein to specifically bind with the PDGF protein. Fluorescence anisotropy is used for the real-time monitoring of the binding between the aptamer and the protein. When the labeled aptamer is bound with its target protein, the rotational motion of the fluorophore attached to the complex becomes much slower because of an increased molecular weight after binding, resulting in a significant fluorescence anisotropy change. Using the anisotropy change, we are able to detect the binding events between the aptamer and the protein in real time and in homogeneous solutions (detection without separation). This assay is highly selective and ultrasensitive. It can detect PDGF in the subnanomolar range. The new method for protein detection is simple and inherits all of the advantages of molecular aptamers. Efficient oncoprotein detection using aptamer-based fluorescence anisotropy measurement will find wide applications in protein monitoring, in cancer diagnosis as well as other studies in which protein analysis is important.