In this paper, we first introduced the basic principle of fluorescence cross-correlation spectroscopy (FCCS) and then established an FCCS setup using a single wavelength laser. We systematically optimized the setup, and the detection volume reached about 0.7 fL. The home-built setup was successfully applied for the study of the binding reaction of human immunoglobulin G with goat antihuman immunoglobulin G. Using quantum dots (745 nm emission wavelength) and Rhodamine B (580 nm emission wavelength) as labeling probes and 532 nm laser beam as an excitation source, the cross-talk effect was almost completely suppressed. The molecule numbers in a highly focused volume, the concentration, and the diffusion time and hydrodynamic radii of the reaction products can be determined by FCCS system.
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