Fluorescent dyes are widely used for biological detection. They are generally consistent of two moieties, i.e., the fluorophore as Tag, and the functional group either for conjugations or binding to a biological target. For fluorescent labeling dyes, most of fluorescent labeling dyes either target an amino group or thiol group.
Amine-reactive fluorescent probes are widely used to modify peptides, proteins, oligonucleotides, nucleic acids, ligands and other biomolecules. Amine-reactive dyes are most often used to prepare bioconjugates for immunochemistry, fluorescence in situ
hybridization (FISH), cell tracing, receptor labeling and fluorescent analog cytochemistry. In these applications, the stability of the chemical bond between the amine-reactive dye and biomolecule is particularly important because the fluorescent conjugates are often subjected to rigorous incubation, hybridization and washing steps. A number of fluorescent amino-reactive dyes have been developed to label various biomolecules, and the resultant conjugates are widely used in biological applications. Three major classes of amine-reactive fluorescent reagents are currently used to label biopolymers: succinimidyl esters (SE), isothiocyanates
, and sulfonyl chlorides
Because free thiol (SH) groups, also called mercapto groups, are not present as abundantly as amino groups in most biopolymers such as proteins and nucleic acids, thiol-reactive reagents often provide a means of selectively modifying a protein at a defined site. Therefore thiol-reactive dyes are often used to prepare fluorescent peptides, proteins and oligonucleotides for probing biological structures, functions and interactions. Thiol-reactive dyes have been used to develop probes for analyzing the topography of proteins in biological membranes, determining distances within the protein or between the proteins and monitoring the changes in protein conformation using environment-sensitive probes. There are quite a few types of thiol-reactive dyes reported in the literature, including iodoacetamides
, disulfides, maleimides, vinyl sulfones and various electron-deficient aryl halides and sulfonates. Iodoacetamides and maleimides are by far the most popular thiol-reactive moieties.