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Fluorophores are molecules that can absorbs photons and emits photons of lower energy in return under light excitation. Compounds must have fluorophores in their structure to emit fluorescence. When the fluorescent substance absorbs energy, the singlet excited state electrons return to the ground state, and fluorescence will be generated. Fluorophores are usually some planar or heterocyclic compounds containing benzene rings or multiple π-bonds, which can be used as tracers for fluids, dyes for tissue cells, and fluorescent probes. Fluorescence analysis that requires fluorophores has the advantages of high sensitivity, strong selectivity, small sample volume, etc. Therefore, fluorophores, as an essential element of fluorescence analysis, have been intensively studied and developed.

Fluorescent phenomenonsFig.1 Fluorescent phenomenons


Fluorophores can be broadly classified into intrinsic fluorophores and extrinsic fluorophores.

  • Intrinsic fluorophores: Intrinsic fluorophores are naturally occurring fluorescent molecules that emit fluorescence on their own upon external stimulation, including green fluorescence proteins (GFP), aromatic amino acids, nicotinamide adenine dinucleotide (NADH), vitamin B6 derivatives , chlorophyll, etc.
  • Extrinsic fluorophores: Extrinsic fluorophores are non-natural fluorescent molecules and are mainly synthetic fluorescent molecules. The introduction of extrinsic fluorophores is required when some receptors that are targeted to specific substances cannot emit fluorescence by themselves or the fluorescence emitted cannot meet the requirements. When the receptor binds to the fluorophore, it has the ability to emit the desired fluorescence. Extrinsic fluorophores include small organic molecules (rhodamine, fluorescein, coumarin and cyanine), inorganic materials (quantum dots), and non-natural fluorescent amino acids.


Fluorophores have been widely used as signal transduction moieties for fluorescent probes, fluorescent tracers, and fluorescent dyes due to their fluorescence properties.

  • Signal transduction moieties for fluorescent probes

Fluorescent probes can respond to specific stimuli such as temperature and pH or label specific regions of biological samples. The molecular structure of a fluorescent probe contains recognition group (receptor), fluorescent group (fluorophore), and a linker part (spacer). Among them, fluorophores have signal output function and can emit fluorescence. They can also output and record the biological information detected by the fluorescent probes in an optical form. Fluorescent probes with fluorophores play an important role in biochemistry as well as protein research by labelling substances for the qualitative and quantitative analysis of substances to be measured.

Application of fluorescent probes in biologyFig.2 Application of fluorescent probes in biology

  • Fluorescent tracers

Fluorophores can also be used to form fluorescent tracers to track the flow of fluids, mainly using the photoluminescence (PL) phenomenon of fluorophores, and are mostly applied for the detection of sewage discharges. The tracer is continuously and evenly injected into the water stream and fluoresces strongly even in the dark. Therefore, the phenomenon of luminescence can be used to identify sewage and study the flow rate of sewage, etc. Tracers containing fluorophores are efficient, economical, safe, non-toxic and biodegradable, and have been widely used in fluid tracing.

  • Fluorescent dyes

Fluorophores are also used as fluorescent dyes and are applied to cell staining and specific DNA staining for cell cycle, apoptosis and other related studies due to their high sensitivity and ease of handling. For example, flow cytometry is a cell analysis technique for the quantitative study of intracellular proteins and nucleic acids after specific staining of cells by fluorescent dyes.

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