Measuring Surface Binding Thermodynamics and Kinetics by Using Total Internal Reflection with Fluorescence Correlation Spectroscopy: Practical Considerations

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• 作者：Nancy L. Thompson ; Punya Navaratnarajah ; Xiang Wang
• 刊名：Journal of Physical Chemistry B
• 出版年：2011
• 出版时间：January 13, 2011
• 年：2011
• 卷：115
• 期：1
• 页码：120-131
• 全文大小：336K
• 年卷期：v.115,no.1(January 13, 2011)
• ISSN：1520-5207

文摘

The combination of total internal reflection illumination and fluorescence correlation spectroscopy (TIR-FCS) is an emerging method useful for, among a number of things, measuring the thermodynamic and kinetic parameters describing the reversible association of fluorescently labeled ligands in solution with immobilized, nonfluorescent surface binding sites. However, there are many parameters (both instrumental and intrinsic to the interaction of interest) that determine the nature of the acquired fluorescence fluctuation autocorrelation functions. In this work, we define criteria necessary for successful measurements and then systematically explore the parameter space to define conditions that meet the criteria. The work is intended to serve as a guide for experimental design, in other words, to provide a methodology to identify experimental conditions that will yield reliable values of the thermodynamic and kinetic parameters for a given interaction.