Enzyme Assays for Confocal Single Molecule Spectroscopy

During the last years confocal techniques have become increasingly popular for probing biological enzymatic reactions. In this paper we will summarize some of these methods, mainly focusing on measurement techniques suitable for analysis of freely diffusing molecules, where either the substrates or the enzymes are fluorescently labeled. The different approaches are classified according to their basic principles and algorithms. Several examples of enzymatic studies involving correlation strategies for data processing, like auto- and cross correlation analysis, will be presented. In addition, other evaluation schemes like coincidence analysis and fluorescence intensity distribution analysis are introduced and discussed. With respect to assay development, the fluorescence energy transfer principle is addressed as far as it is has been applied for investigating biocatalysis in solution. Finally, one part of this review addresses the aspects of bioconjugation and the basic requirements for proper labeling dyes in order to be compatible with single molecule fluorescent spectroscopy.