The homogeneous fluorescence anisotropic sensing of salivary lysozyme using the 6-carboxyfluorescein-labeled DNA aptamer.
The homogeneous fluorescence anisotropic sensing of salivary lysozyme using the 6-carboxyfluorescein-labeled DNA aptamer.
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DOI：
10.1016/j.bios.2011.11.052
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发表时间：
2012-02
影响因子：
12.6
通讯作者：
Mingjian Zou;Yang Chen;Xiao Xu;Hongduan Huang;Fen Liu;Na Li
中科院分区：
文献类型：
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作者：
Mingjian Zou;Yang Chen;Xiao Xu;Hongduan Huang;Fen Liu;Na Li
针对溶菌酶开发了一种简单而灵敏的荧光各向异性方法，利用荧光团 6-羧基荧光素 (FAM) 与溶菌酶在目标分子与其 DNA 适体之间识别时偶联。本研究发现，检测系统的旋转动态对于获得高各向异性信号至关重要，而高各向异性信号并不总是通过简单地增加分子体积来实现，因为分子体积的增加可能无法有效地延迟荧光团的旋转运动。选择FAM作为ssDNA适体的标签，可以有效促进荧光团从主要独立的片段运动转变为缓慢的全局旋转。进行时间分辨测量，包括寿命和动态荧光各向异性，以研究识别相互作用并更好地理解该方法。该方法具有12.5-300nM的宽线性动态范围和检测限为4.9nM（3S/N）的高灵敏度。该方法已成功应用于人唾液溶菌酶的测定。基于标准添加回收率和与酶联免疫吸附测定（ELISA）比较的结果证明了该方法对于生物样品的可行性。利用荧光团和分析物之间的耦合可以成为扩大基于适体-靶标和抗体-抗原识别的荧光各向异性应用的方法之一。
A simple and sensitive fluorescence anisotropy method was developed for lysozyme, employing the coupling of fluorophore, 6-carboxyfluorescein (FAM), with lysozyme upon recognition between the target molecule and its DNA aptamer. It was found in this study that the rotational dynamic of the detecting system is crucial to obtain a high anisotropy signal that cannot always be achieved by simply increasing the molecular volume, because molecular volume increase may not be able to efficiently retard the rotational movement of the fluorophore. FAM was selected as the label of the ssDNA aptamer to effectively facilitate the change of the fluorophore from a primarily independent segmental movement to slow global rotation. The time-resolved measurements, including lifetime and dynamic fluorescence anisotropy, were conducted to study the recognition interaction and to better understand the methodology. The proposed method had a wide linear dynamic range of 12.5–300nM and a high sensitivity with the limit of detection of 4.9nM (3S/N). This proposed method was successfully applied to assay of human salivary lysozyme. The results based on the standard addition recovery and comparison with enzyme-linked immunosorbent assay (ELISA) demonstrated the feasibility of this method for biological samples. Using coupling between the fluorophore and the analyte can be one of the approaches working toward expanding the application of fluorescence anisotropy based on aptamer–target and antibody–antigen recognitions.