Fluorescent Dyes and Fluorescent Probes



Fluorescent dyes are dyes that emit fluorescence, and refer to substances that absorb a light wave of a certain wavelength and emit another light wave 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. After absorbing ultraviolet or visible light, it can convert short-wavelength light into longer-wavelength visible light waves and reflect them in bright and bright colors.

Fluorescent dyes can be used alone or combined into composite fluorescent dyes. 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.

Scientific research application of fluorescent dyes
Fluorescent dyes, due to their high sensitivity and convenient operation, have gradually replaced radioisotopes as detection markers, which are widely used in immunofluorescence, fluorescent probes, and cell staining. Including specific DNA staining, for chromosome analysis, cell cycle, apoptosis and other related research. Many other nucleic acid dyes are 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.

Immunofluorescence technique, the method of tracing or checking the corresponding antigen with a fluorescent antibody is called the fluorescent antibody method; the method of tracing or checking the corresponding antibody with a known fluorescent antigen marker is called the fluorescent antigen method; these two methods are collectively referred to as Immunofluorescence technology is one of the earliest developed immunolabeling techniques. It is a technique established on the basis of immunological, biochemical and microscopy techniques. Since a long time, some scholars have tried to combine antibody molecules with some tracer substances, and use antigen-antibody reaction to locate antigenic substances in tissues or cells. Because fluorescent pigments can not only be combined with antibody globulin to detect or locate various antigens, but also can be combined with other proteins to detect or locate antibodies, but fluorescent antigen technology is rarely used in practical work, so people are used to using Immunofluorescence technology is also known as fluorescent antibody technology, and the fluorescent antibody method is more commonly used. Using immunofluorescence techniques to display and examine antigens or hapten substances in cells or tissues is called immunofluorescence cell (or histo) chemistry.

What is a fluorescence probe?
Fluorescent probes refer to characteristic fluorescence in the ultraviolet-visible-near-infrared region, and their fluorescence properties (excitation and emission wavelengths, intensity, lifetime, polarization, etc.) can vary with the properties of the environment, such as polarity, refractive index, viscosity, etc. A class of fluorescent molecules that change and sensitively change. A small molecule that changes one or more fluorescent properties by non-covalent interactions with nucleic acids (DNA or RNA), proteins or other macromolecular structures. It can be used to study the properties and behavior of macromolecular substances.

At present, commonly used fluorescent probes include fluorescein-based probes, inorganic ion fluorescent probes, fluorescent quantum dots, molecular beacons, and the like. In addition to the quantitative analysis of nucleic acids and proteins, fluorescent probes are widely used in nucleic acid staining, DNA electrophoresis, nucleic acid molecular hybridization, quantitative PCR technology and DNA sequencing. We will mainly discuss fluorescein-based probes in the future.

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.

Application of fluorescent dyes in nucleic acid detection
Nucleic acid fluorescent dyes stain the nucleus and quantitatively measure the fluorescence intensity emitted by 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.

Fluorescent dye probes, or fluorophores, are compounds that absorb light at a given wavelength and emit light at a higher wavelength, producing fluorescence in various colors. These dyes can be grouped into categories such as organic dyes (e.g., fluorescein, rhodamine, AMCA), biological fluorophores (e.g., green fluorescent protein, phycoerythrin, allophycocyanin) and quantum dots.
Fluorescent probes absorb light at a specific wavelength and emit light of a different, typically longer, wavelength. This process is known as fluorescence emission. Fluorescence is safer to use and does not require radiological controls. Because fluorophores do not overlap, several fluorescent molecules can be used simultaneously.
Fluorescent dyes are often used in the fluorescent labeling of biomolecules and can be smaller or more photostable than fluorescent proteins but cannot be genetically encoded.