Preparation method of protein molecule engram film

A molecularly imprinted membrane, molecularly imprinted technology, applied in the direction of chemical reaction of materials for analysis, material testing, biological testing, etc., can solve the problems of instability of organic solvents, easy deformation and denaturation, and unsatisfactory sensor selectivity, etc. To achieve the effect of simple method and mild reaction conditions

Inactive Publication Date: 2010-07-14
HUBEI UNIV OF TECH
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  • Abstract
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Problems solved by technology

The signal of the sensor can increase with the increase of the concentration of the detected protein, but the disadvantage is that the selectivity of the sensor is not ideal
However, since no cross-linking monomer is used, it has a certain impact on the stability of the imprinted holes.
[0004] In summary, although predecessors have accumulated a lot of valuable experience in the preparation of protein molecularly imprinted membranes, however, proteins are large in size, complex in structure (the surface shape is irregular, and there are many functional groups), and they are easily deformed and denatured. Unstable in medium, which brings great difficulties to western blot work

Method used

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  • Preparation method of protein molecule engram film

Examples

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Effect test

Embodiment 1

[0031] A method for preparing a protein molecularly imprinted film, the steps of which are:

[0032] 1. Treatment of the carrier-glass substrate:

[0033] In the experiment, the "sandwich" sandwich method was used to prepare molecularly imprinted membranes, and two slides (24×24mm and 18×18mm) of different sizes were used as "carriers". Carrier slide pretreatment, the specific steps are as follows:

[0034] (1) Put the cover glass into a beaker, add 1-5 grams of detergent (any type of washing powder or liquid detergent) and 300-500 mL of deionized water, and ultrasonicate for 5-20 minutes;

[0035] (2) Take out the cover glass and put it into another beaker, rinse it with deionized water 3-5 times, soak it in 1mol / L sodium hydroxide solution and sonicate it for 5-20min;

[0036] (3) Take out the cover glass, wash it with deionized water to remove the sodium hydroxide solution remaining on the cover glass, and after rinsing for 3 to 5 times, add deionized water and sonicate f...

Embodiment 2

[0050] A method for preparing a protein molecularly imprinted film, the steps of which are:

[0051] 1. Treatment of the carrier-glass substrate:

[0052] The treatment of the carrier-slide matrix was the same as in Example 1.

[0053] 2. Preparation of imprinted membrane:

[0054] Prepare 0.084 μmol / L hemoglobin solution with PBS (0.01mol / L pH6.4). Add functional monomers acrylamide and acrylic acid (molar ratio 1:1), so that the molar ratio of the total moles of functional monomers to hemoglobin is greater than 400, shake well, and place in a refrigerator overnight at 4°C. Then add N,N'-methylenebisacrylamide to make the molar ratio of functional monomer to 1:40, ultrasonic degassing for 5 minutes, and nitrogen gas for 5 minutes. Add 10 μL of 0.1M ammonium persulfate and 1 μL of tetramethyldiethylamine, mix well, pipette 50 μL, drop slowly on the glass slide, cover with a cover glass, so that the droplet is completely covered , ensure no bubbles, polymerize for 2-3 hours...

Embodiment 3

[0060] 1. Treatment of the carrier-glass substrate:

[0061] The treatment of the carrier-slide matrix was the same as in Example 1.

[0062] 2. Preparation of imprinted membrane:

[0063]Prepare 0.042 μmol / L hemoglobin solution with PBS (0.01mol / L pH6.4), add methacrylic acid and acrylamide (molar ratio 1:1) to make the molar ratio to hemoglobin greater than 400, shake well, and store in the refrigerator Store overnight at 4°C. N,N'-methylenebisacrylamide is added again, and the molar ratio of N,N'-methylenebisacrylamide to functional monomer is 1:40. Ultrasonic degassing for 5 minutes, and nitrogen for 5 minutes. Add 10 μL of 0.1M sodium persulfate and 1 μL of tetramethyldiethylamine, mix well, use a pipette to absorb 50ul and drop it slowly on the glass slide, cover it with a cover glass, so that the droplet is completely covered, try as much as possible Ensure no bubbles, polymerize for 2-3 hours. After the film is formed, the cover glass is gently lifted to obtain a ...

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Abstract

The invention discloses a preparation method of a protein molecule engram film, comprising the following steps: 1. pretreatment of carrier sheet glass used for preparing a protein molecule engram film: putting a cover glass into a beaker, adding cleaning agent and deionized water, and processing by ultrasound; 2. preparation of the solution of template protein, functional monomer and crosslinking monomer: preparing template protein solution by phosphate buffer, adding water-solubility functional monomer, crosslinking monomer and initiator, and evenly mixing; 3. carrying out polymerization between sheet glasses to form a polymer film; 4. elution of template protein molecule: putting the molecule engram film into a beaker, adsorbing eluent in the beaker, removing the template protein molecule, and obtaining an engram hole matched with the template protein molecule. In the engram process of the invention, protein has small possibility of denaturation, and polymerization condition is simple and moderate. The prepared molecule engram film can be developed into a developing chip capable of detecting protein.

Description

technical field [0001] The invention relates to the technical field of molecular imprinting, in particular to a method for preparing a protein molecular imprinting film. Suitable for molecular imprinting of all water-soluble biomacromolecules. The molecularly imprinted membrane prepared by this method has a certain specific recognition function for the target protein, and can be developed into an analytical product for detecting protein instead of an antibody. Background technique [0002] Molecular imprinting technology is a technology for synthesizing artificial antibodies. Its main principle is: first select functional monomers that can form covalent or non-covalent bonds with template molecules, form complexes with template molecules, and then copolymerize with cross-linking agents. form cross-linked polymers. After the template molecules are eluted, holes matching the shape, size and surface functional groups of the template molecules are left in the polymer, and thes...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C08F220/56C08F222/38C08F220/06C08F2/44C08J9/26C08J5/18G01N21/75G01N33/68
Inventor 谢卫红王晖丁安子方桂杰李永进
Owner HUBEI UNIV OF TECH
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