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Automated System for Single Molecule Fluorescence Measurements of Surface-immobilized Biomolecules

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Measuring surface binding thermodynamics and kinetics by using total internal reflection with fluorescence

Nancy L Thompson¹, Punya Navaratnarajah, Xiang Wang

¹Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA. nlt@unc.edu

The Journal of Physical Chemistry. B

|December 21, 2010

Summary

This study guides experimental design for total internal reflection fluorescence correlation spectroscopy (TIR-FCS) measurements. It defines criteria and explores parameters for reliable thermodynamic and kinetic data of ligand-surface interactions.

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Area of Science:

Biophysics
Chemical Physics
Surface Science

Background:

Total internal reflection fluorescence correlation spectroscopy (TIR-FCS) is a powerful technique for studying molecular interactions at surfaces.
Accurate measurement of thermodynamic and kinetic parameters requires careful optimization of experimental conditions.
Understanding ligand-surface binding dynamics is crucial in various scientific fields.

Purpose of the Study:

To establish criteria for successful TIR-FCS measurements.
To systematically explore the parameter space influencing TIR-FCS data.
To provide a methodological guide for optimizing experimental design for reliable interaction parameter determination.

Main Methods:

Systematic exploration of instrumental and intrinsic parameters affecting TIR-FCS measurements.
Definition of criteria for successful data acquisition.
Analysis of fluorescence fluctuation autocorrelation functions.

Main Results:

Identification of key parameters influencing TIR-FCS data quality.
Establishment of a framework for selecting optimal experimental conditions.
Demonstration of a methodology to achieve reliable thermodynamic and kinetic measurements.

Conclusions:

This work provides essential guidelines for researchers using TIR-FCS.
Optimized experimental design leads to reliable quantification of molecular interactions.
The proposed methodology enhances the utility of TIR-FCS in biophysical and chemical studies.