Porous membrane material for detecting phosphoinositide, and preparation and detection method thereof

A detection method and phosphoinositide technology, applied in the field of biological analysis, can solve the problems of difficulty in rapid detection, low detection sensitivity, complex biological system, etc., and achieve the effects of good scalability, high detection sensitivity and low cost

Inactive Publication Date: 2017-04-19
WUHAN UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the above methods have the disadvantages of time-consuming and labor-intensive, and the detection sensitivity is not high due to the complexity of the biological system, and there is also a risk of radioactivity.
Therefore, when we focus on its dynamically changing concentration rather than the specific structure during the research process, it is difficult for this method to meet the requirements of rapid detection.
In addition, rapid identification of inositol molecules with different degrees of phosphorylation and their isomers is also a big problem

Method used

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  • Porous membrane material for detecting phosphoinositide, and preparation and detection method thereof
  • Porous membrane material for detecting phosphoinositide, and preparation and detection method thereof
  • Porous membrane material for detecting phosphoinositide, and preparation and detection method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0045] Preparation of Porous Membrane Material Grafted with Bicomponent Copolymer

[0046] The structure of the two-component copolymer is as figure 1 Shown, where X = 0.01-0.5. Taking X=0.2 as an example, add 4.8mmol isopropylacrylamide (NIPAM) and 1.2mmol 4-(3-propenylthioureido)benzoic acid (ATBA) into a 100mL three-necked flask, and add 60mL N,N' - Dimethylformamide (DMF) sonicated for 10 minutes. After passing nitrogen gas for 20 minutes, add 32 mg cuprous bromide (CuBr) powder and mix well. The brominated porous membrane was added into the flask, and then the reaction system was evacuated and filled with nitrogen to remove residual oxygen in the reaction system. Then 0.16 mL of N,N,N',N',N'-pentamethyldiethylenetriamine (PMDETA) or bipyridyl ligand was added by injection, followed by one more deoxygenation treatment. Under nitrogen protection and a constant temperature of 70°C, take it out after 15 hours of reaction, soak and rinse with 100mL DMF and deionized water ...

Embodiment 2

[0050] The QCM-D adsorption method was used to evaluate the different adsorption behaviors of (1,3) inositol diphosphate, (1,3,5) inositol triphosphate and inositol hexaphosphate on the surface of the two-component copolymer. According to the similar method described in Example 1, the two-component copolymer was grafted onto the surface of the QCM-D chip. Under the condition of temperature control at 20°C, deionized water was used as the carrier liquid for the concentration of 1 μg / mL (1, 3) Inositol diphosphate, (1,3,5) inositol triphosphate and inositol hexaphosphate were subjected to adsorption experiments. Figure 5 It shows that the surface of the two-component copolymer has strong adsorption for (1,3) inositol diphosphate, (1,3,5) inositol triphosphate and inositol hexaphosphate and has a certain ability to distinguish, that is, three The adsorption amounts of different phosphoinositides on the surface of the two-component copolymers were different.

Embodiment 3

[0052] The film material obtained by grafting the two-component copolymer on the QCM-D chip prepared as described in Example 2 was soaked in 10 ml of an aqueous solution containing 10 mg of inositol hexaphosphate for 20 minutes. Then, the changes on the surface of the two-component copolymer film material before and after soaking inositol hexaphosphate were observed through the scanning mode of the atomic force microscope. Depend on Image 6 and Figure 7 It can be observed that the surface of the polymer film material has obvious morphological changes after soaking in the inositol hexaphosphate solution, indicating that the polymer film is responsive to inositol hexaphosphate, which can be amplified to the change of the macroscopic effect.

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Abstract

The invention provides a porous membrane material for detecting phosphoinositide, and a preparation and detection method thereof. The porous membrane material comprises a porous membrane and a dual-component copolymer grafted to the channels of the porous membrane. The dual-component copolymer is grafted to channels of the porous membrane through the atom transfer free radical polymerization reaction mechanism. The detection method comprises the following steps: putting the porous membrane material between electrochemical cell clamps, injecting electrolyte, measuring the trans-membrane current (I0) under a preset voltage; removing the electrolyte, adding electrolyte containing phosphoinositide, measuring the trans-membrane current (I1) under the preset voltage; according to a formula (I0-I1)/I0, and calculating the value of changed current ratio under the preset voltage, wherein if the value is greater than 0.05, the sample contains phosphoinositide. The method of using provided porous membrane material to detect phosphoinositide has the advantages of high detection sensitivity, quick detection speed, simple operation, and low cost.

Description

technical field [0001] The invention relates to the technical field of biological analysis, in particular to a porous membrane material for detecting phosphoinositides and a preparation and detection method thereof. Background technique [0002] The phosphoinositide family, as an important signaling molecule in eukaryotic cells, is closely related to the control of cell behavior. As a signal precursor, phosphoinositides can produce second messengers under the action of agonists, and they can also act as lipid second messengers to directly participate in many cellular activities such as ion transport, membrane vesicle transport, signal transduction, and cytoskeleton reassembly and nuclear gene regulation. Therefore, the dynamic monitoring and quantitative research of phosphoinositides in organisms has become a hot issue in life sciences. At present, for the detection of phosphoinositides, the main method is to use radioactive elements such as 32 P or 3 H is labeled in the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N27/48G01N27/28C08F220/54C08F220/60C08J9/36
CPCC08F220/54C08J9/36C08J2333/12C08J2367/02C08J2369/00C08J2433/24G01N27/28G01N27/48C08F220/606
Inventor 卿光焱孙涛垒陆琦
Owner WUHAN UNIV OF TECH
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