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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.

Alfa Chemistry provides customers with a wide range of high quality fluorophores. If you can't find the product you need in the product catalog, please feel free to contact us, we will provide you with professional customized service.

Case Study

Modern Synthetic Approaches to the Preparation of Functional Fluorophores

de Moliner, Fabio, et al. Angewandte Chemie International Edition. 2017;56(14):3758-69. de Moliner, Fabio, et al. Angewandte Chemie International Edition. 2017;56(14):3758-69.

Functional fluorophores can quickly and accurately analyze specific biomolecules, but these complex structures are often difficult to obtain through traditional synthesis strategies. The latest progress in designing, preparing, and fine-tuning fluorescent groups through multi-component reactions, C-H activation processes, cycloaddition reactions, and biomolecular based chemical transformations was discussed.
Synthetic approaches
• Multicomponent reactions: The group of Pischel and Gois developed a three-component sequential condensation reaction of boric acid, salicylaldehyde and amino substrate to obtain polarity-dependent fluorescence heterocycles containing boron.
• C−H activation processes: Thiopyrazine-based fluorescent donor-acceptor-donor compounds were synthesized via a double C-H arylation reaction. The obtained adduct exhibits a large Stokes shift in the near-infrared (NIR) region.
A fluorescent donor-acceptor-donor compound based on thiopheno-pyrazine was prepared by partial arylation of thiophene with double C-H. The adducts show a large Stokes shift with emission in the near infrared (NIR) region.
• Cycloadditions: Fairfull-Smith and his colleagues utilized this strategy to attach azide coumarin derivatives to isoindole nitrogen oxygen radicals containing alkyne groups in copper catalyzed azide acetylene cycloaddition (CuAAC) process, generating highly sensitive fluorescent groups to the oxidation process.
• Biomolecule-based chemical transformations: DeRose and colleagues modified the chemotherapeutic drug piplatin with azide groups to identify and image its oligonucleotide binding targets when binding to alkyne-derived danyl fluorophores.

Development of Photostable Fluorophores for Molecular Imaging

Zheng, Qinsi, et al. Current opinion in chemical biology. 2017;39:32-8. Zheng, Qinsi, et al. Current opinion in chemical biology. 2017;39:32-8.

In optical imaging, the information from biological samples is encoded as photons, and the total photons detected ("photon budget") determine the maximum amount of information that can be extracted from the samples. Unfortunately, the photoinduced degradation (photobleaching) of fluorophores terminates photon emission, thus imposing limited restrictions on the photon budget. Therefore, a great deal of efforts have been made to increase the photon budget by improving the light stability of the fluorophore.
Improvement Strategy
• Improve photostability by reducing reactivity to oxidants.
An early example of this method is the improvement of the prototype coumarin laser dye 1. After the oxidation of 4-methyl was found to be an important degradation pathway, the CH3 group was replaced by the CF3 group to form compound 21. This modification not only significantly improves the photostability of fluorophore, but also reduces the unnecessary reaction on aniline nitrogen.
• Improvement of fluorophore by intramolecular triplet energy quenching.
Altman et al. used this method to improve the photostability of fluorescence imaging in vitro and living cells. Compound 36 was produced by covalent binding of small molecular triplet quenchant (TSQ) with Cy5 fluorophore, which greatly improved its photostability in deoxidizing solution. This method can be applied to various fluorophores, including fluorescein, rhodamine, carbon rhodamine and silicon rhodamine.

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