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Quantifying Reversible Surface Binding via Surface-Integrated Fluorescence Correlation Spectroscopy.
Nano Letters ( IF 10.8 ) Pub Date : 2018-04-18 , DOI: 10.1021/acs.nanolett.8b00875
Jonas Mücksch 1 , Philipp Blumhardt 1 , Maximilian T Strauss 1, 2 , Eugene P Petrov 1, 2 , Ralf Jungmann 1, 2 , Petra Schwille 1
Affiliation  

We present a simple and versatile single-molecule-based method for the accurate determination of binding rates to surfaces or surface bound receptors. To quantify the reversible surface attachment of fluorescently labeled molecules, we have modified previous schemes for fluorescence correlation spectroscopy with total internal reflection illumination (TIR-FCS) and camera-based detection. In contrast to most modern applications of TIR-FCS, we completely disregard spatial information in the lateral direction. Instead, we perform correlation analysis on a spatially integrated signal, effectively converting the illuminated surface area into the measurement volume. In addition to providing a high surface selectivity, our new approach resolves association and dissociation rates in equilibrium over a wide range of time scales. We chose the transient hybridization of fluorescently labeled single-stranded DNA to the complementary handles of surface-immobilized DNA origami structures as a reliable and well-characterized test system. We varied the number of base pairs in the duplex, yielding different binding times in the range of hundreds of milliseconds to tens of seconds, allowing us to quantify the respective surface affinities and binding rates.

中文翻译:

通过表面积分荧光相关光谱量化可逆表面结合。

我们提出了一种简单且通用的基于单分子的方法,用于准确测定表面或表面结合受体的结合率。为了量化荧光标记分子的可逆表面附着,我们修改了以前的荧光相关光谱与全内反射照明(TIR-FCS)和基于相机的检测的方案。与 TIR-FCS 的大多数现代应用相比,我们完全忽略横向的空间信息。相反,我们对空间积分信号进行相关分析,有效地将照明表面积转换为测量体积。除了提供高表面选择性之外,我们的新方法还解决了在广泛的时间尺度内平衡的缔合和解离速率。我们选择荧光标记的单链 DNA 与表面固定 DNA 折纸结构的互补手柄的瞬时杂交作为可靠且特征良好的测试系统。我们改变双链体中碱基对的数量,产生数百毫秒到数十秒范围内的不同结合时间,使我们能够量化各自的表面亲和力和结合率。
更新日期:2018-04-16
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