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Polypeptide fluorescent probe and preparation method and applications thereof

A fluorescent probe and polypeptide technology, applied in the field of biochemistry, can solve problems such as unsatisfactory accuracy and effective detection of a single sialic acid, achieving fast detection speed, simplified peptide chain structure, and good scalability Effect

Inactive Publication Date: 2017-07-07
WUHAN UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the detection and analysis of sialic acid mainly rely on the combination of fluorescence-assisted high-performance liquid chromatography and mass spectrometry, but due to the interference of other sugars in the environment of sialic acid, the accuracy of this detection method is not very satisfactory
In addition, because there are many kinds of sialic acid and the differences between them are very small, the effective detection of a single sialic acid is still a big problem

Method used

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  • Polypeptide fluorescent probe and preparation method and applications thereof
  • Polypeptide fluorescent probe and preparation method and applications thereof
  • Polypeptide fluorescent probe and preparation method and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Preparation method of polypeptide fluorescent probe modified by fluorescein isothiocyanate

[0049] Add fluorescein isothiocyanate to the round bottom flask, then dissolve it with 25mL methanol or water, then add the polypeptide DAPD, PD, DP, DA, DD, PA or AD to ensure the molar ratio of fluorescein isothiocyanate to polypeptide is 1.2:1. After stirring for 12 h at a constant temperature of 30° C., the solvent was removed by rotary evaporation to obtain a crude product. The crude product is separated and purified by high-performance liquid chromatography, and the desired pure product is obtained after vacuum freeze-drying, which can be used as a fluorescent probe for specific recognition of sialic acid. It can be understood that the dipeptide in the present invention is not limited to PD, DP, DA, DD, PA or AD, and it can be a dipeptide formed by any two amino acids of D, A, and P.

Embodiment 2

[0051] The fluorescent probe based on tetrapeptide DAPD is applied in the specific recognition of sialic acid, and the specific steps are as follows:

[0052] 1) First prepare a DAPD fluorescent probe aqueous solution with a concentration of 2.0 μM (add Tris-HCl buffer, adjust pH=7.4), use it as the fluorescent host molecule, measure its emitted fluorescence spectrum at an excitation wavelength of 470 nm, and record its emission at The highest peak intensity I at 514nm 0 ;

[0053] 2) Add sialic acid (SA-1) of the DAPD fluorescent probe to the solution as a guest molecule, wherein the molar amount of SA-1 added is 0.1-5 times that of the DAPD fluorescent probe, and mix evenly, at an excitation wavelength of 470nm Measure its emission fluorescence spectrum under the condition, record its highest peak intensity I at 514nm 1 ;

[0054] 3) Continue to add SA-1 to the solution obtained in step 2) and mix evenly, measure the fluorescence spectrum it emits under the condition of e...

Embodiment 3-4

[0058] As described in Example 2, the guest molecule sialic acid (SA-1) in step 2) was replaced with glucose and galactose respectively, and the DAPD fluorescent probe was measured to add different molar ratios of glucose or galactose under the same conditions. Fluorescence spectrum of lactose. The change trend of the obtained fluorescence spectrum is as follows Figure 4 and Figure 5 shown.

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Abstract

The invention relates to a polypeptide fluorescent probe. The polypeptide fluorescent probe can be obtained by bonding amino group of polypeptide with isothiocyanate group of fluorescein isothiocyanate, the polypeptide is tetrapeptide DAPD or dipeptide, and the dipeptide is formed by any two of D, A and P amino acids. The preparation method of the polypeptide fluorescent probe comprises the following steps: adding fluorescein isothiocyanate into a reaction container, then dissolving with methanol or water, adding polypeptide so as to cause the molar ratio of the fluorescein isothiocyanate to polypeptide to be (1.2-1.5):1; under the constant temperature condition of 20-40DEG C, stirring and reacting for 4-25h, then performing rotary evaporation to remove a solvent to obtain a crude product; performing high-performance liquid chromatographic separation, purification and drying on the crude product to obtain a needed pure product. From the view of biomimetics, the prepared florescent probe can rapidly and precisely recognize sialic acid, and has important theoretic and practical significance on biochemistry, material science and medicine, especially on the early-stage diagnosis and treatment of serious diseases taking sialic acid as important markers.

Description

technical field [0001] The invention relates to the technical field of biochemistry, in particular to a polypeptide fluorescent probe and its preparation method and application. Background technique [0002] Sialic acid is a class of acyl derivatives of nine-carbon sugar compounds containing carboxyl groups. It is widely found in bacteria, fish, mammals and other organisms. It participates in and regulates many important life activities such as cell recognition, biofilm flow and endocytosis, etc. Now scientists have discovered more than 50 members of sialic acid, mainly including three core monomers of N-acetylneuraminic acid, N-glycolylneuraminic acid and desaminoneuraminic acid, and the rest of sialic acid are derived from these three bodies. Since sialic acid is usually located in the key position of the carbohydrate part of the cell membrane and the secreted carbohydrate complexes (glycolipids, glycoproteins and lipopolysaccharides), it is an important material basis f...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C09K11/06G01N21/64
CPCC09K11/06C09K2211/1088G01N21/6428
Inventor 卿光焱陆琦
Owner WUHAN UNIV OF TECH
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