Fluorescent dyes refer to substances that absorb light waves of a certain wavelength and emit light waves with a wavelength greater than that of the absorbed light. Most of them are compounds containing a benzene ring or a heterocyclic ring with a conjugated double bond. Fluorescent dyes can be used alone or combined into composite fluorescent dyes. Under the microscope, it functions by absorbing light at a given wavelength and re-emitting it at a longer wavelength. This fluorescence produces different colors that can be visualized and analyzed.
The composite fluorescent dye is a fluorescent dye synthesized by fluorescence resonance energy transfer technology, which is composed of a donor and an acceptor fluorescent substance molecules that are very close in distance and can transfer energy between each other. The complex dye is excited at the excitation wavelength of the acceptor molecule and emits a photon at the emission wavelength of the donor molecule. The development of fluorescent dyes is very rapid, and the fluorescent dyes developed for scientific research and clinical applications have basically covered the entire spectral range from ultraviolet to visible light and infrared.
Fluorescent dyes are commonly used for fluorescent labeling of various biomolecules (antibodies, peptides, various proteins, etc.) for monitoring drug delivery to target tissues, imaging, and other processes.
Fluorescent dye classification:
1. Fluorescein dyes, including fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc. and their analogs. This is a class of compounds with more benzene rings. The most widely used is FITC (as shown in the picture of FITC-labeled tissue fluorescence), which is excited by an argon ion laser at 488 nm and emits blue-green fluorescence at 525 nm. FITC can bind to various antibody proteins and stably exhibits blue-green fluorescence in alkaline solution.
2. Rhodamine dyes, including red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc. TRITC emits yellow fluorescence at 570 nm when excited at 550 nm.
3. Cy series cyanine dyes, cyanine dyes usually consist of two heterocyclic ring systems, including Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, cy7 nhs ester , cy3b nhs ester and their analogs.
4. Alexa series dyes, which are series of fluorescent dyes developed by Molecular Probes. Its excitation light and emission light spectrum cover most of the visible light and part of the infrared spectral region, and it is widely used. Features high brightness, stability, instrument compatibility, multiple colors, pH insensitivity, and water solubility. Includes Alexa Fluor 350, 405, 430, alexa fluor 488 maleimide, 532, 546, 555, 568, 594, 610, 633, alexa 647 nhs ester, 680, 700, 750. At present, Alexa series dyes are widely used in the market and gradually replace traditional fluorescent dyes. For example, Alexa Fluor 488 can replace FITC and Cy2; Alexa Fluor 555 can replace Cy3 and TAMRA; Alexa Fluor 633 can replace APC, Cy5, etc.
5. APDye Fluors, APDye Fluors is the equivalent fluorescent dye of alexa fluors developed by axispharm. APDye Fluors series fluorescent dyes are bright in color, optically stable, very good in light stability of dyeing brightness, and very competitive in price. It can be applied to the labeling and localization of tissues, cells and biomolecules in biomedical research. Its excitation and emission spectra cover most of the visible and part of the infrared spectral region and are suitable for most fluorescence microscopes.
6. Protein dyes, including phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), polydinoxanthin-chlorophyll protein (preCP), etc. Most of them are proteins found in cyanobacteria. Such fluorescent dyes can be coupled with Cy series cyanine dyes to form complex dyes for antibody labeling. For example, PE-Cy3/Cy5/Cy7, APC-Cy7, PerCP-Cy5.5, etc. are commonly used in the market.
Scientific research application
The earliest application of dyes in biochemistry was to directly stain sections and then visualize them. With the continuous development of biotechnology, computer technology and fluorescence spectrometry technology, many dyes, especially fluorescent dyes, have been widely used in cell detection, tumor gene protein analysis, toxic analysis, clinical medical diagnosis and so on.
Due to its high sensitivity and convenient operation, fluorescent dyes have gradually replaced radioisotopes as detection markers, and are widely used in fluorescent immunology, fluorescent probes, and cell staining. Including specific DNA staining, for chromosome analysis, cell cycle, apoptosis and other related research. Numerous nucleic acid dyes are also useful counterstains in multicolor staining systems as background controls, labeling nuclei so that the spatial relationships of intracellular structures can be seen at a glance.
Immunoassay
Fluorescence-labeled monoclonal antibody technology expands infinite application space for flow cytometry in the study of cell membranes and various functional antigens in cells, tumor gene proteins and other fields. Fluorescent probes can be covalently bound to monoclonal antibodies via protein cross-linkers. The most commonly used dyes for immunofluorescence labeling are fluorescein isothiocyanate (FITC), phycoerythrin (PE) and AlexaFluor series dyes.
Nucleic acid amplification testing
Nucleic acid fluorescent dyes stain the nucleus and quantitatively measure the fluorescence intensity of the cell, so that the content of DNA and RNA in the nucleus can be determined, and the cell cycle and cell proliferation can be analyzed. There are a variety of fluorescent dyes that can stain DNA or RNA in cells. Commonly used DNA dyes include propidium iodide (PI), DAPI, Hoechst 33342, etc., and RNA dyes include thiazole orange, acridine orange, etc.
Ideal fluorescent dyes generally have the following characteristics:
1. It has high photon yield and high signal intensity;
2. Strong absorption of excitation light, reducing background signal;
3. The distance between the excitation spectrum and the emission spectrum is large to reduce the interference of background signals;
4. It is easy to combine with the labeled antigen, antibody or other biological substances without affecting the specificity of the labeled substance;
5. Good stability, not easily affected by light, temperature, pH, specimen anticoagulant and fixative.
Other related reading:
The Ultimate Guide to Fluorescent Dye
The use of fluorescent dyes and how to judge the brightness
Analysis of advantages and disadvantages of fluorescent dye method and probe method
Characteristics of 5 kinds of fluorescent dyes commonly used in nucleic acid detection