A kind of preparation method of hrp imprinted hydrogel

A hydrogel and imprinting technology, which is applied in the field of analytical chemistry and protein recognition sensors, can solve the problems of cumbersome operation steps, and achieve the effect of simple preparation, unbreakable and low cost

Active Publication Date: 2020-12-29
MINJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these methods require special equipment or cumbersome operation steps, so it is necessary to establish a more direct, efficient and simple detection method to improve the application value of molecular imprinting technology

Method used

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  • A kind of preparation method of hrp imprinted hydrogel
  • A kind of preparation method of hrp imprinted hydrogel
  • A kind of preparation method of hrp imprinted hydrogel

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] The recognition effect of HRP imprinted hydrogel on different concentrations of HRP:

[0033] (1) Preparation of liquid A: fully dissolve 15 g AAm and 0.4 g MBAA in ultrapure water, and dilute to 50 mL to obtain liquid A.

[0034] (2) Preparation of HRP imprinted hydrogel solution: Dissolve 5 mg of HRP protein in 5 mL of solution A and stir thoroughly, then add 4.9 mL of ultrapure water, 100 μL of 10 wt% APS and 4 μL of TEMED in sequence and mix well to obtain HRP blotting hydrogel solution.

[0035] (3) Preparation of HRP imprinted hydrogel: Pour the above HRP imprinted hydrogel solution into the bio-rad gel filling device, polymerize into a gel, take it out, and first elute the template with a small amount of eluent several times The HRP molecules were washed with water to obtain HRP imprinted hydrogels (MIPs) with a thickness of 1mm.

[0036] (4) Colorimetric detection: Cut the imprinted hydrogel prepared above into strips with a wide band of 1 cm, fix them on a wh...

Embodiment 2

[0039] The recognition effect of non-HRP imprinted hydrogel on different concentrations of HRP:

[0040] Step (2) in Example 1 does not add 5 mg of HRP protein, and the remaining conditions and steps are the same as in Example 1 to prepare HRP non-imprinted hydrogels (NIPs), and the HRP non-imprinted hydrogels can be obtained. Adsorption effect diagram, such as image 3 As shown, it can be seen that with the increase of HRP concentration, the displayed color has no significant change, and a lighter color is displayed in the high concentration 0.5 mg / mL region, which shows a significant difference with the color displayed in Example 1, which shows that The HRP non-imprinted hydrogel has no specific adsorption for HRP.

Embodiment 3

[0042] The ability of HRP imprinted hydrogel to recognize different proteins:

[0043] (1) Cut the HRP imprinted hydrogel prepared in Example 1 into strips with a width of 1 cm, fix them on a white base plate, and dry them at room temperature. The white base plate is marked with samples, 10 μL of 0.05 mg / mL is different Sample solutions of protein [HRP (horseradish peroxidase), Try (trypsin), OVA (ovalbumin), ConA (concanavalin), BSA (bovine serum albumin), GOD (glucose oxidase)] Add it dropwise to the corresponding position, let it stand still, fully absorb for 30 min, wash away unbonded molecules with ultrapure water, and dry at room temperature.

[0044] (2) Take freshly prepared 50 mmol / L TMB solution and 80 mmol / L H 2 o 2 The solutions were mixed in equal volumes, and the two were mixed when the color was about to develop, and 10 μL of the color solution was added to each adsorption point, and the color was developed for 10 minutes, and the color development effect pict...

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Abstract

The invention discloses a preparation method of HRP (Horseradish Peroxide) blotting hydrogel. The preparation method comprises the following steps: by taking acrylamide (AAm) as a functional monomer,methylene bisacrylamide (MBAA) as a crosslinking agent, ammonium persulfate (APS) as a catalyst, tetramethylethylenediamine (TEMED) as an initiator and horseradish peroxide (HRP) as a template molecule, carrying out in-situ polymerization according to a certain ratio to prepare the HRP blotting hydrogel with the thickness of 1mm; washing off the template molecule and directly dropwise adding a sample solution to the surface of the gel; adsorbing and washing an unbound molecule; developing by adopting a 3,3',5,5'-tetramethylbenzidine (TMB)-H2O2 system, wherein the produced color can be directlydetected by naked eyes. The preparation method disclosed by the invention is simple and low in cost; the prepared HRP blotting hydrogel is convenient to detect, can be repeatedly used for a pluralityof times and can be used for highly selectively identifying HRP in a blood serum sample and effectively removing matrix interference; and the preparation method has very great application value and market prospect.

Description

technical field [0001] The invention belongs to the technical field of analytical chemistry and protein recognition sensors, and in particular relates to a preparation method of HRP imprinted hydrogel. Background technique [0002] Molecular imprinting technology is an artificial receptor that simulates the molecular recognition that exists in nature and prepares a polymer network structure that wraps template molecules. Advanced technology for separation and purification. The prepared polymer is called molecularly imprinted polymer (molecular imprintedpolymer, MIP). Molecularly imprinted polymer is a new type of polymer biomimetic material with strong molecular recognition ability. Molecularly imprinted polymers have the dual advantages of recognition selectivity similar to natural antibodies and corrosion resistance of polymer materials, so they are gradually being used in many fields such as analytical chemistry, bioengineering, clinical medicine, environmental monitori...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C08F220/56C08F222/38C08J9/28B01J20/28B01J20/26G01N21/78
CPCB01J13/0065B01J20/268B01J20/28047C08F220/56C08J2333/26G01N21/78C08F222/385
Inventor 谭娟娟李艳霞
Owner MINJIANG UNIV
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