Fluorescent dyes, also known as fluorescent probes, have become widely used fluorescent markers due to their advantages of fast detection, good repeatability, small sample volume, and no radiation. At the same time, it is also an indispensable and important tool in scientific research such as cell biology. The use of fluorescent probes can determine the structure of RNA and DNA, study DNA base damage repair, identify the state of amino groups in protein molecules and the active region of protein molecules, and detect pmol-level proteins, distinguish between nucleic acids of different conformations and the chemical reactivity of related drugs. Fluorescence immunoassays employ time-resolved techniques for the analysis of many proteins, hormones, viral antigens, and DNA hybrids. Laser-induced fluorescence spectroscopy plays an important role in the identification of living cells, living body fluids, DNA base sequences and bacterial protozoa, early diagnosis and treatment of malignant tumors.
Since most fluorescent dyes do not have selectivity, the fluorescent dyes immobilized on biologically active carriers can utilize the selectivity between biologically active substances (antigen-antibody, biotin-streptavidin), or use appropriate The modified functional group of the fluorophore can realize the targeted labeling of the target substance through the interaction between the functional groups. Here we will introduce some common types of reactive fluorescent dyes:
Reactive fluorescein and its derivatives are widely used to covalently label biomolecules such as antibodies, carbohydrates, nucleic acids, proteins, peptides and oligonucleotides. The reactive groups corresponding to each activated functional group are as follows:
Succinimidyl esters / NHS esters – used to label free amino groups (-NH2) on antibodies, proteins, peptides, amine-modified oligonucleotides and other biomolecules
Maleimides – used to label sulfhydryl groups on antibodies, proteins and peptides
Azides – Vinyl Labeling by Click Chemistry (Click)
Alkynes – labeling azides by click chemistry
Hydrazides – used to label aldehyde and ketone groups
Carboxylic acids – preactivated with carbodiimide for labeling of amines or for Steglich esterification of alcohols
Amines – Fluorescent dyes with free amino groups used for conjugation with various electrophilic compounds such as activated esters and epoxides.
Tetrazines – Label trans-cyclooctene (TCO), cyclopropene and other activated alkenes via the anti-electron-demanding Diels-Alder reaction.
Phosphoramidite – Phosphoramidite-labeled dyes can be used to label oligonucleotides
Cycloalkyne – Dyes with cycloalkynyl moieties can be used for copper-free click chemistry reactions.