Purification method of water-soluble cyanine dye conjugate and application

Abstract

The present application discloses a purification method of a water-soluble cyanine dye conjugate and an application. The purification method includes the following steps: adsorbing the water-soluble cyanine dye conjugate by using anion exchange resin to separate the conjugate from uncharged or positively-charged impurities, performing elution on the anion exchange resin having adsorbed water-soluble cyanine dye conjugate by using water and a solution with different salt ion concentrations, and receiving the target component in stages to obtain a purified water-soluble cyanine dye conjugate. The purification method provided by the present application utilizes adsorption / desorption of the anion exchange resin to separate and purify the conjugate, water can be used to dissolve the sample, sothat the pretreatment difficulty of the sample is reduced, the probability of sample contamination is reduced, and the industrial scale production is facilitated; the anion exchange resin is used foradsorption and separation, so that the method is more convenient and quicker, and has low costs and high efficiency; and the water and the salt solution can be used as a mobile phase during elution, so that pollution to the environment caused by using an organic solvent is avoided.

Description

technical field [0001] The present application relates to the field of purification of water-soluble cyanine dye conjugates, in particular to a purification method and application of water-soluble cyanine dye conjugates. Background technique [0002] Fluorescent dyes, after absorbing ultraviolet or visible light, can convert short-wavelength light into long-wavelength visible light and emit bright colors, such as acid eosin, fluorescent yellow, red mercury and some disperse dyes. Most of them are compounds containing benzene rings or heterocycles with conjugated double bonds. [0003] Cyanine dyes are a class of fluorescent labeling dyes with excellent performance, including a series of asymmetric water-soluble fluorescent molecules such as Cy3, Cy5, Cy5.5 and Cy7. They have high molar absorptivity and good stability, and are widely used in the labeling and detection of proteins, antibodies, nucleic acids and other biomolecules. Currently, Cy3 and Cy5 have become the prefe...

AI Technical Summary

Problems solved by technology

Similarly, the purity of Cy3 conjugates, Cy5.5 conjugates and Cy7 conjugates is also an important factor limiting their application

[0005] There are many side reactions in the connection reaction between cyanine dye and linker, and the polarity of the by-products is similar, so the separation and purification of the product is quite difficult

Due to the high polarity of water-soluble cyanine dye molecules, it is more difficult to separate and purify

At present, preparative HPLC is usually used for separation and purification. This method requires expensive preparative HPLC, preparative columns and HPLC-grade eluents. The equipment is very expensive and the investment is huge.

Among them, the eluent of HPLC grade is generally composed of water and HPLC grade acetonitrile; while HPLC grade acetonitrile is expensive and pollutes the environment

Moreover, the throughput of preparative HPLC separation and purification is very small, which is not conducive to industrial production

Also take Cy5 as an example. For Cy5 conjugates, the polarity of Cy5 and Cy5 conjugates is equivalent. Preparative HPLC is used for separation and purification. The separation can only be achieved by passing through the HPLC preparative column many times, which is high in cost and low in efficiency.

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