A new dimension for the development of fluorescence-based assays in solution: from physical principles of FCS detection to biological applications.

Ultrasensitive detection methods such as laser-induced fluorescence represent the current state-of-the-art in analytics. Single-molecule detection in solution has received a remarkable amount of attention in the last few years because of its applicability to life sciences. Studies have been performed on the fundamentals of the detection processes themselves and on some biological systems. Fluorescence correlation spectroscopy (FCS) is the link for ultrasensitive multicomponent analysis, showing possibilities for experiments on molecular interactions. Based on the theoretical background of FCS, this article gives full explanation of FCS and an update of highlights in experimental biology and medicine studied by FCS. We focus on a repertoire of diverse immunoglobulin specificities, a ribosome display system, single-molecule DNA sequencing, and a mutant enzyme generated by random mutagenesis of amino acids. We describe the usefulness and the enormous potential of the methodology. Further, this contribution clearly indicates that FCS is a valuable tool for solution-phase single-molecule (SPSM) experiments in immunobiology and medicine. In experiments with the Goodpasture autoantibody, we worked out conditions for the design of experiments on a complex single molecule in solution. The possibility to use SPSM-FCS as a quantitation methodology opens up other important applications beyond the scope of this article. Original results extending the published studies are presented for the rational foundation of SPSM-FCS. In this original contribution, we deal with experimental systems for biology and medicine where the number of molecules in solution is very small. This article is mandatory for gaining confidence in the interpretation of experimental SPSM-FCS results on the selfsame, individual single molecule in solution.