Cyanine dyes, a kind of polymethine dye derivatives with amidine ion vinylogies established at both ends of the chromophore conjugates between N-N atoms, belong to organic dyes. Typical cyanine dyes are formed when two nitrogen atoms and part of the polymethine chain are part of a heterocyclic nucleus. It is composed of two nitrogen atoms as heterocyclic nuclei, and has the advantages of stable fluorescence, chemical structure of polymethine bridged chain, high molar absorption coefficient and high fluorescence quantum yield. According to the type of molecular structure, it is divided into straight chain, bridged chain, cyclobutenedione, gram ketone, etc. For labeling biomolecules such as DNA and proteins, the more common cyanine dyes are CY3, CY5, CY7 and Sulfo-CY3, sulfo-CY5, sulfo-CY7.
There are two types of cyanine dyes: non-sulfonated cyanine dyes and sulfonated cyanine dyes, which are interchangeable for many applications because their spectral properties are nearly identical. Sulfonated and non-sulfonated dyes can be used to label biomolecules such as DNA and proteins. The difference between the two dyes is their solubility: Sulfo dyes are water-soluble, they can be marked in aqueous environments without the addition of organic co-solvents, and they disperse well in water. In some cases, you may only need one of these two dyes.
Non-sulfonated cyanine dyes
Available non-sulfonated cyanine dyes include Cy3, Cy3.5, Cy5, Cy5.5, Cy7, and Cy7.5, where Cy stands for cyanine and Cy2 is an oxazole derivative rather than indolenine, which does not follow this rule of. The suffix .5 is a styrene-acrylic fused cyanine dye. Changes in structure can also alter the fluorescent properties of the molecule, covering the most important visible light and near-infrared spectrum carrying several fluorophores.
Most of the non-sulfonated cyanine dye derivatives (except hydrazide hydrochloride and amines) have low water solubility, when these molecules are used for biomolecular labeling, use organic solvents as co-solvents (5-20% DMF) or DMSO) is required for an efficient reaction. Cyanine dyes should be dissolved in organic solvents and then added to biomolecular solutions (proteins, peptides, amino-labeled DNA) with appropriate aqueous buffers. The dye molecules react efficiently with the biomolecules before the dyes are precipitated.
Advantages of CY cyanine dyes:
1: The molar absorption coefficient of cyanine dyes is the highest among fluorescent dyes
2: Autofluorescence of cells and tissues is minimal in the near-infrared band
3: Higher specificity and sensitivity
4: The tissue permeability of light waves in the near-infrared region is better
5: The dye is less cytotoxic and can be used to label cells with antibodies and proteins. Therefore, CY cyanine dyes are very suitable fluorescent dyes for in vivo imaging of mice
Disadvantages of CY cyanine dyes:
1: The fluorescence quantum efficiency of cyanine dyes is lower than other dyes, generally 0.1-0.2
2: Cyanine dyes with multiple structures are prone to aggregation
