Introduction of Cy series fluorescent dyes



Cy (Cyanine) series, also called cyanine dyes, that is, anthocyanin series fluorescent dyes are a class of synthetic fluorescent dyes with the chemical structure characteristics of polymethine bridge chains. The two ends of the methine bridge chain (1-7 methine groups) of Cy dyes are often connected with two nitrogen atoms, one of which is positively charged, so that Cy dyes form mesoionic compounds with delocalized positive charge effects. Because of this structural feature, the extinction coefficient of Cy dyes is very high. The length of the bridge chain and the chromophores at both ends directly control the absorption peak and emission peak of the dye, so that the Cy series dyes can cover almost all common fluorescent bands from ultraviolet to far infrared.

Generalized Cy dyes are a large class of fluorescent compounds with a wide range of structures and applications. Here we mainly discuss Cy dyes for biolabeling, including Cy3, Cy3.5, Cy5, Cy5.5, Cy7 and Cy7.5. All of these dyes were derived from the dye ICG (Indocyanine Green) in the 1970s. Like ICG, they have two symmetrical indolenine chromophores. This structure allows less non-specific adsorption of Cy dyes, high extinction coefficient, and high fluorescence quantum yield, so that the background is weak and the signal is strong when the label is developed. They often replace fluorescent dyes such as fluorescein (FITC, FAM) and rhodamine (TRITC, RRX) for fluorescent labeling of cells. In addition to cell imaging, they are also commonly used in biological screening, western blotting, biomedical imaging, small animal in vivo imaging, etc.

Polyfluorescein offers two Cy series fluorescent dyes: ordinary Cy dyes and sulfonated Cy dyes (sulfo-Cy). These two types of dyes have the same backbone structure, so they have almost the same fluorescence spectrum and fluorescence sensitivity, and can be used interchangeably in most cases. The only difference is that the sulfonated Cy dyes have two sulfate ion functional groups on their chromophores, so these dyes have very good water solubility. Sulfonated Cy dyes are a better choice when the water solubility of the sample is required (for example, the water solubility of the labeled substance is poor, and the water solubility of the labeled molecule is expected to be good) or the stability of the labeled biomolecules (such as some protein molecules) is high.

Common Cy dye

Such dyes include Cy3, Cy3.5, Cy5, Cy5.5, Cy7 and Cy7.5, where the chromophore of Cy3, Cy5 and Cy7 is indolenine, while the chromophore of Cy3.5, Cy5.5 and Cy7.5 is Benzoindole . Benzoindole only has one more benzene ring than indolenine. The extra benzene ring makes the absorption and emission peaks of the entire dye red-shifted, so that the Cy dye series can cover a wider range of fluorescence spectra. The structure of Cy dyes is almost symmetrical. In order to make the dyes labelable, a 6-carbon chain carboxyl group is extended from one of the chromophores to activate the labeling.

Despite having a positive charge, common Cy dyes have relatively low water solubility. When labeling biomolecules (usually in a buffer solution), it is often necessary to add an organic solution (generally 5-20% DMF or DMSO) to aid in dissolution. The conventional operation is to dissolve the dye in an organic solvent first, and then add it to the aqueous solution of biomolecules in proportion to react. After the reaction is completed, centrifuge to remove the precipitated dye, and then permeabilize or column separation (HPLC, ion exchange or desalting column) to remove unreacted small dye molecules. Of course, ordinary Cy dyes can also directly react with organic small molecules in organic solvents to label small molecules or polymer materials. Ordinary Cy dyes are also easily soluble in chloroform, methanol, THF, acetonitrile and other conventional organic solvents, which are very suitable for organic synthesis reactions. .

For the near-infrared dyes Cy7 and Cy7.5, there are generally two different structures on the market, and the difference is generally on the methine bridge: a straight-chain methine bridge or a cyclohexene-reinforced bridge. The former is a relatively primitive structure directly inherited from ICG dyes, and the latter is an optimized structure. Compared with the former, the latter utilizes the cyclohexene structure to immobilize the methine bridge chain, and the structure is more rigid and stable, so the fluorescence quantum yield is increased by ~20%, and the development is more sensitive. But it should be noted that this is only the statement of Lumiprobe Company. There have been literature reports dedicated to this issue, and it was found that the cyclohexene-cured methine bridge chain cannot improve the quantum yield, and the optical properties of the two Cy dyes are the same. In addition, there are some literatures discussing the difference in toxicity between the two structures. It is reported that the linear methine bridge chain Cy7 and Cy7.5 are non-specific and toxic in small animals, while the dye with the bridge chain strengthened is much better.

All common Cy dyes have basically the same Stokes shift (Stokes shift), about 15nm to 20nm. This shift is large enough (in fact, it is larger than the Stokes shift of many fluorescent dyes, such as the BODIPY series), and most fluorescent instruments can distinguish the fluorescent signal from the excitation light. In addition, the solvent and environment have little effect on the fluorescence characteristics of ordinary Cy dyes, making them suitable for developing tasks under many conditions, such as emitting stable and strong fluorescence in low pH solutions or fat-soluble environments.

Sulfonated Cy dyes

This kind of Cy dyes adds sulfonic acid groups to the chromophore of ordinary Cy dyes, thereby greatly increasing the water solubility of the dyes. Another benefit of sulfonation is that it slightly increases the optical stability and quantum yield of the dye, so sulfonated dyes are more resistant to light and fluoresce slightly more. Due to the reason of sulfonate, the water solubility of this kind of dye is very high, and there is no need to add any organic solvent to aid in the labeling reaction. In addition, the marked dye molecules will not aggregate due to their good water solubility, and will not affect the labeled macromolecules. of stability. This is very important, especially when multiple dye molecules need to be labeled on each target biomacromolecule, or the target biomacromolecule has low solubility and poor stability. The sulfonated Cy dyes currently available from Polyfluorescein include sulfo-Cy3, sulfo-Cy3.5, sulfo-Cy5, sulfo-Cy5.5, sulfo-Cy7 and sulfo-Cy7.5.

After sulfonation, the fluorescence spectrum (including excitation peak and emission peak) of Cy dyes has a very small blue shift, which is a very common phenomenon, such as Rhodamine 123 to Alexa Fluor® 488, this blue shift is very small, blue After the shift, the excitation peak and emission peak are still within the detection range of the instrument, so it does not affect the use, and there is no need to change the instrument parameters.

Common Cy dyes vs Sulfonated Cy dyes

Although ordinary Cy and sulfonated-Cy are similar in terms of fluorescence optical properties, they are still different in applicable conditions and labeling methods. Ordinary Cy dyes have low water solubility and must be pre-dissolved in organic solvents (such as DMF or DMSO), and then added to the labeling reaction system. The content of organic solvents in organic/water mixed solvents needs to reach 10-15% (volume ratio) In order to ensure that the dye does not precipitate. On the contrary, sulfonated-Cy dyes have good solubility in water and can be directly labeled in aqueous solution (such as PBS buffer), so they are especially suitable for labeling objects such as proteins that are sensitive to organic solvents. If you want to purify by dialysis after the reaction, be sure to use sulfonated-Cy dyes. Sulfonated-Cy dyes are soluble in water and excess dyes can be dialyzed out. Both normal Cy and sulfonated-Cy are suitable if purified by ion exchange resins, electrophoresis, gel filtration.

The applicable conditions of ordinary Cy and sulfonated-Cy are divided into the following situations:

Both dyes are suitable:
– More stable proteins, can withstand a small amount of organic solvents for several hours
-Antibodies (typically tolerate 5-10% of DMSO/DMF)
– DNA, RNA and other nucleic acids
-Natural or organically synthesized polymer materials
-Small molecule compounds soluble in DMF, DMSO, methanol

Sulfonated-Cy dyes must be used:
– Sensitive proteins, especially those that are induced by organic solvents
– Purified by dialysis
– Nanomaterials sensitive to organic solvents

Common Cy dyes must be used:
– Reactions that must be labeled in water-immiscible organic solvents such as chloroform, diethyl ether, etc.
– Probes that the labeled product is expected to remain fat soluble or still permeable to the cell membrane
-Need to be packaged and loaded, such as loaded into the hydrophobic microenvironment of the nano-core

References: duofluor