Fluorescent probes are divided into Chemical fluorescent probes and Gene fluorescent probes according to their synthetic methods. The Small-molecule fluorescent Probes, one of the Chemical fluorescent probes, have been used in environmental monitoring, food detection, clinical disease diagnosis, and become the research hotspot because of the small size and convenient synthesis. Fluorescent Probes generally consist of a fluorophore which is the heart of it, a ligand that binds to a specific and high affinity receptor and a spacer connected the recognition group to the fluorescent group（see Fig.1）. Fluorophore, the reporting unit, can send a fluorescence signal to response to the change of the chemical environment of the molecular probe when the identifying group acts, thus monitoring the binding process of the identifying group to the analyte. Therefore, various types of fluorophores and their derivatives are constantly modified and studied to achieve better application value.
Fig.1 Schematic diagram of fluorescent molecular probes structure
Common Chemical fluorophores Category
According to the structure of the parent nucleus, the common chemical fluorophores can be divided into the following categories：
- Cyanine fluorophores: Cyanine fluorophores are the representative of near-infrared fluorescent signal unit. It is a kind of molecule with two heterocyclic systems, connected by different conjugate structure. The commonly used skeleton is the heterocyclic ring containing nitrogen such as indole ring, benzothiazole, benzoxazole and so on. The maximum absorption and emission wavelengths of cyanine compounds can be adjusted in a wide range of wavelengths, so they are widely used in the field of near-infrared fluorescence labeling, and are often used to design fluorescent molecular probes that can perform in vivo bioanalysis.
Fig.2 The molecular structure of cyanine fluorophores
- Rhodamine fluorophores: Rhodamine is the one of the most common types of the Xanthene fluorophores. The structure rhodamine contains a push-pull electron system composed of three rings. This system is fixed by an oxygen bridge to ensure the good optical properties of these compounds. Therefore, they have high extinction coefficient, high quantum yield and good biocompatibility. At present, they have been widely researched and used in fluorescent probes in molecular biology, dye lasers and photosensitizers of dye-sensitized solar cells.
Fig.3 The molecular structure of rhodamine
- Fluorescein fluorophores: Fluorescein is also a kind of widely used xanthene fluorescent dyes, which is hydroxyl substituted derivative of rhodamine. Due to the large extinction coefficient and high fluorescence quantum efficiency of fluorescein in aqueous solution, fluorescein is still one of the most widely used fluorescent dyes in modern biology, biochemistry and medical research, although the excitation and emission wavelengths of fluorescein are only in the visible region.
Fig.4 The molecular structure of fluorescein
- BODIPY fluorophores: They have high fluorescence quantum yield, molar absorptivity, light and chemical stability and so on. BODIPY fluorophores are not easily affected by solvent polarity, which is beneficial to broaden the range of application of the probe, so that it can be used in the analysis of many different solvents. Moreover, they are less affected by pH, so they can be used in biomarkers and other fields. Based on the above advantages, BODIPY fluorophores are more and more on people's attention.
Fig.5 The parent nucleus of BODIPY derivatives
- Other fluorescence fluorophores: In addition to the above main fluorophores, small-molecule fluorophores also include hybrid fluorophores, coumarin fluorophores, naphthalimide fluorophores and so on. Hybrid fluorophores are often constructed by combination of two different fluorescent signal units to form a new fluorescent signal unit with better optical properties. Coumarin fluorophores has many advantages, such as high fluorescence yield, good photostability, large Stokes-shift and easy modification. In recent years, coumarin has been developed in the field of fluorescent molecular probe and fluorescent labeling. Naphthalimide fluorophores are a kind of commonly used fluorescent signal unit with high quantum yield, which can be used as biomarker or DNA imbedding agent.
Fig.6 Example of Other fluorescence fluorophores
- Bioanalysis: Chemical fluorophore is widely used and studied in synthesizing fluorescent probe due to its high sensitivity and rapid response to detect small molecules. For example, cell activity is affected by the intracellular PH. Changes in pH may cause decrease even loss of the intracellular enzyme activity. Therefore, the use of fluorescent probes to monitor changes in intracellular pH is of great significance for biological research. And some of them have good cell penetration, so they can be applied to the detection of intracellular metal ions.
Fig.7 Sensing mechanism under acidic condition
- Environmental monitoring: Some fluorophores can respond to a particular ion by structurally modified. Thus, they have been used in detection of microorganisms in the environment by synthesizing fluorescent sensers. For example, the qualitative and quantitative analysis of mercury ion and hypochlorous acid in environment and living cells is great important to environmental protection. Fluorescent probes can visualize the mercury ions or hypochlorous acid contained in the environment and provide a basis for the development of the environment and life.
Fig.8 Recognition mechanism of probe for HCLO 
- Clinical diagnosis: As the core unit of fluorescence imaging technology, small-molecule fluorophores have been widely used in tumor diagnosis because of its advantages of non-invasive, visualization, high resolution, low cost, and safety. By introducing recognition groups on the small-molecule fluorophores, tumor cells, inflammatory tissues and ordinary tissues can be specifically identified and distinguished. Therefore, they can provide a basis for precise diagnosis and treatment of tumors.
Fig.9 Fluorescence for detecting tumor markers 
- Fluorescence staining: Since the 20th century, fluorescent dyes, obtained by modifying classical fluorophore, have been widely used in textile, plastic dyeing, printing pigments and other industries. Fluorescent dyes used in textile are mainly used in producing high visibility warning clothing and high brightness personality clothing. Its application scope covers police, first aid, express mail, sanitation, airport, building and so on.
Fig. 10 Application of fluorescent dyes (warning markers)
At present, Alfa Chemistry mainly provides fluorophores for the synthesis of Small-molecule fluorescent Probes and other applications, such as BODIPY Dyes, Cyanine dyes, Rhodamine dyes, Fluorescein dyes and so on, if you do not find what you need, please contact us.
- Lin W Y.; et al, A ratiometric fluorescent probe for hypochlorite based on a deoximation reaction. Chem. Eur. J., 2009, 15: 2305.
- Yang Y.; et al. FRET nanoflares for intracellular mRNA detection: avoiding false positive signals and minimizing effects of system fluctuations. Journal of the American Chemical Society, 2015, 137(26):8340-8343.