Development of a fluorescence correlation spectroscopy method for the study of biomolecular interactions

Abstract

The objective of this thesis was to develop a single laser wavelength fluorescence cross-correlation spectroscopy (SW-FCCS) method for the excitation of two or more fluorescent probes. A single laser beam was used for the simultaneous excitation of two or more colored fluorophores with similar excitation wavelengths but different emission spectra. The experimental implementation was demonstrated with the receptor-ligand model of streptavidin-biotin. Different fluorophore assays including quantum dots, tandem dyes and organic dyes were tested on the system. The resolution limit to detect binding was evaluated with spectrally similar fluorophores. SW-FCCS was extended to multicolor cross-correlation analysis with three detection channels and this established the possibility of measuring higher order molecular interactions. The setup was further simplified by implementing a dispersive prism in the detection path for spectral separation. SW-FCCS simplifies the setup considerably without the need for aligning two laser beams or expensive laser systems for two-photon excitation.