Method for detecting interaction between temperature-sensitive sugar-containing random polymer and lectin through fluorescence titration

A polymer and lectin technology, applied in fluorescence/phosphorescence, measurement devices, material analysis by optical means, etc., can solve the problem of unclear molecular recognition process, and achieve fast test analysis, high test cost performance, and detection sensitivity. high effect

Active Publication Date: 2020-05-15
DONGHUA UNIV
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Problems solved by technology

The current research methods are relatively simple, and can only characterize the specific recognition ability of glycosyl polymers and lectins, but the molecular recognition process of carbohydrates is not very clear

Method used

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  • Method for detecting interaction between temperature-sensitive sugar-containing random polymer and lectin through fluorescence titration
  • Method for detecting interaction between temperature-sensitive sugar-containing random polymer and lectin through fluorescence titration
  • Method for detecting interaction between temperature-sensitive sugar-containing random polymer and lectin through fluorescence titration

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preparation example Construction

[0036] And in the preparation method of PDN-2, wherein DEGMA takes 0.3561g respectively, OVNGal takes 0.1029g respectively, the amount of chain transfer agent DDATC and initiator ACCN is unchanged, and the remaining operations are consistent with the above.

[0037] Preparation of PDZ-2: Add 0.3561g DEGMA, 0.1140g OVNGal, 9.09mg chain transfer agent DDATC and 0.305mg initiator ACCN to a 5mL single-necked flask, followed by adding 1mL DMF solution to dissolve. After sealing with parafilm and plastic wrap, freeze-dissolve three times in a liquid nitrogen environment, and then go through three vacuum-filling nitrogen cycles, and the polymerization reaction is carried out at 90°C for 12 hours in a nitrogen environment. The obtained pale yellow oil was repeatedly precipitated with 50 mL of n-hexane three times. Finally, use a dialysis bag (MWCO=8000) with a molecular weight cut-off of 8000 to dialyze for 48 hours, and obtain P(DEGMA-co-OVNGal) with n(OVNGal):n(DEGMA)=1:6 after free...

Embodiment 1

[0041] In this study, fluorescein isothiocyanate-labeled PNA (FITC-PNA) and ConA were selected as modified lectins for binding studies and for fluorescence quantitative analysis. The specific operation steps are as follows:

[0042] Transfer the sample solution to be tested into a 96-well plate, 100 μL per well, using Molecular Devices SpectraMaxM2 e Multifunctional microplate reader, first record the emission spectrum of FITC-PNA solution (solvent is HEPES buffer) (1 μ M) at 500-560 nm at 25 °C at 494 nm excitation wavelength, in order to remove the influence of non-specific binding on the spectrum. Then gradually add 10 μL random polymer solution (solvent: HEPES buffer) dropwise to FITC-PNA solution (100 μL, 1 μM), incubate the mixed sample solution at 40 ° C, measure the fluorescence intensity F of the mixed sample every 30 min and separate agglutination Fluorescence intensity F of FITC-PNA 0 , the test combined time was 60min. The maximum fluorescence spectral intensity...

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Abstract

The invention relates to a method for detecting interaction between a temperature-sensitive sugar-containing random polymer and lectin through fluorescence titration. The method comprises the following steps: testing an emission spectrum of a lectin protein solution under an excitation wavelength; mixing the lectin protein solution with the temperature-sensitive sugar-containing random polymer solution, incubating, and testing the fluorescence intensity of the mixed solution and the fluorescence intensity of the single lectin FITC-PNA solution; drawing a curve according to the obtained maximumfluorescence spectrum intensity change and the concentration of the temperature-sensitive sugar-containing random polymer, and calculating a binding constant Ka. The method is low in cost, fast in test and analysis, convenient and simple to operate, high in test cost performance and high in detection sensitivity.

Description

technical field [0001] The invention belongs to the field of measuring methods for the interaction between high molecular polymers and biomacromolecules, in particular to a method for detecting the interaction between temperature-sensitive sugar-containing random polymers and lectins by fluorescence titration. Background technique [0002] Sugar, as an indispensable component in organisms, is the most abundant biomolecule and important information molecule in nature. It plays a very important role in biological evolution, such as immune response, participation in signal transduction, cell-cell adhesion, etc. Attachment, energy storage and metabolic intermediates, cell differentiation, cell growth and regulation, and sugar-protein recognition, etc. The above functions of sugars are generally referred to as biological functions, and these biological functions are mainly realized through the interaction between sugars and proteins. In addition, Huang Yi et al. have reported th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64
CPCG01N21/6402G01N21/6428G01N21/6458G01N21/6486G01N2021/6443
Inventor 权静郑永利徐见容赵兰雪
Owner DONGHUA UNIV
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