Fluorescein Isothiocyanate Dyes
Fluorescein isothiocyanate (FITC) is a derivative of fluorescein and is widely used in fields such as flow cytometry. FITC, first described in 1942, was the original fluorescein molecule functionalized with isothiocyanate-reactive groups (-N = C = S), replacing hydrogen atoms on the bottom ring of the structure. It is usually supplied as a mixture of isomers, fluorescein 5-isothiocyanate (5-FITC) and fluorescein 6-isothiocyanate (6-FITC). FITC is reactive to nucleophiles, including amine and thiol groups on proteins. It was synthesized by Robert Seiwald and Joseph Burckhalter in 1958. The succinimidyl ester functional group attached to the fluorescein core produces "NHS-fluorescein", forming another common amine-reactive derivative. In the presence of other amines, it is more specific for primary amine high. Nucleophile. The peak wavelength of the excitation and emission spectrum of FITC is about 495 nm / 519 nm, making it green. Like most fluorescent dyes, it is easy to photobleach. Due to photobleaching issues, fluorescein derivatives (such as Alexa 488 and DyLight 488) have been customized for a variety of chemical and biological applications that require higher light stability, higher fluorescence intensity, or different attachment groups.
Figure 1. Chemical structure of fluorescein 5-isothiocyanate
FITC pure product is yellow or orange yellow crystalline powder, easily soluble in water and alcohol solvents. There are two isomers, of which the isomer type I is more excellent in terms of efficiency, stability and protein binding. The molecular weight of FITC is 389.4, the maximum absorption light wavelength is 490-495nm, and the maximum emission light wavelength is 520-530nm, showing bright yellow-green fluorescence. FITC can be stored in cold, dark and dry places for many years and is the most widely used fluorescein. Its main advantage is that the human eye is more sensitive to yellow-green, and usually the green fluorescence is less than red in the sliced specimen. Fluorescent thiocyanate orange, fluorescent yellow isothiocyanate, fluorescent red isothiocyanate, fluorescein isothiocyanate isomer I, 5-fluorescein isothiocyanate. Its form is a yellow powder with hygroscopicity. It is a biochemical reagent and a medical diagnostic drug. It can quickly diagnose diseases caused by bacteria, viruses and parasites. And it can bind to a variety of antibody proteins. The bound antibody does not lose the specificity of binding to a certain antigen, and still has strong green fluorescence in alkaline solution. After adding acid, it precipitates, the fluorescence disappears, and it is slightly soluble in acetone, Ether and petroleum ether.
Figure 2. Chemical structure of fluorescein 6-isothiocyanate
- The TH.; et al. Conjugation of fluorescein isothiocyanate to antibodies: I. Experiments on the conditions of conjugation. Immunology. 1970, 18 (6): 865–873.