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Dealkalized endonuclease magnetic molecular imprinting nano-particles as well as preparation method and application thereof

A magnetic molecular imprinting and nanoparticle technology, which is used in material testing products, biological testing and other directions, can solve the problems of poor uniformity of imprinting sites and low affinity, and achieve the effects of low cost, high selectivity and affinity, and simple preparation method.

Active Publication Date: 2018-07-24
PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these methods still cannot completely solve the problems of poor uniformity of imprinted sites and low affinity.
[0006] So far, no imprinted polymer with APE1 as the target protein has been reported to be synthesized at home and abroad

Method used

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  • Dealkalized endonuclease magnetic molecular imprinting nano-particles as well as preparation method and application thereof
  • Dealkalized endonuclease magnetic molecular imprinting nano-particles as well as preparation method and application thereof
  • Dealkalized endonuclease magnetic molecular imprinting nano-particles as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1, Preparation of Magnetic Molecularly Imprinted Nanoparticles (MIP-100)

[0041] 1.0mg Fe 3 o 4 @SiO 2 At room temperature, under 40KHz (this ultrasonic frequency is used in the present invention, it can be adjusted to a different ultrasonic time as required) ultrasonic for 20 minutes, dispersed in 2.0mL pure water to form a 0.5 mg / mL solution, add 4.0mg 1-B The carboxyl group was activated with 3-(3-dimethylaminopropyl)-carbodiimide (EDC) and 10.0 mg N-hydroxysuccinimide (NHS), and the reaction was shaken at room temperature for 30 minutes. Avidin with a final concentration of 100 nM was added, and the reaction was shaken at room temperature for 8 hours. Magnetic separation, removal of unreacted substances, washing with deionized water three times, and vacuum drying for 24 hours, the obtained material was named SiMNP@AVD-100.

[0042] The above material was re-dispersed ultrasonically in 2.0 mL of 10 mM Tris (pH 8.0) solution. Add APE1 at a final concentr...

Embodiment 2

[0045] Example 2. Investigation of the relationship between the binding amount of magnetic molecularly imprinted nanoparticles and APE1 as a function of polymerization time

[0046] The polymerization times of MIPs and NIPs in Example 1 were adjusted to 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, and 8 hours, respectively. The prepared magnetic molecularly imprinted nanoparticles were subjected to adsorption experiments on APE1, and DNA probes were used to detect the activity of APE1 in the solution.

[0047] In this embodiment, the DNA probe sequence used to detect APE1 activity is as follows:

[0048] 5'-T*T*C*C*T*C*(dT-ROX)AGAGYCGT(dT-BHQ2)C*A*C*T*G*T*AGTTTATA C*A*G*T*GAATCTCTCTAG*T *C*T-3'

[0049] Wherein, Y represents an abasic site, an asterisk represents a thio-modified base, ROX and BHQ2 are 5(6)-carboxy-X-rhodamine hydrochloride and its corresponding quenching group, respectively.

[0050] Unless otherwise specified, the DNA probes menti...

Embodiment 3

[0054] Example 3. Investigation of the relationship between the binding amount of magnetic molecularly imprinted nanoparticles to APE1 as a function of the concentration of avidin

[0055] The experimental steps are as follows:

[0056] The final concentration of avidin in Example 1 was adjusted to 40nM, 400nM and 1515nM respectively, and the obtained magnetic molecularly imprinted nanoparticles were subjected to an adsorption experiment on APE1, and the DNA probe was used to detect the activity of APE1 in the solution, and compared with that in Example 1 materials for comparison.

[0057] Experimental results such as Figure 4 As shown, as the concentration of avidin continues to increase, the amount of MIPs binding to APE1 gradually increases. When the concentration of avidin is 100 nM, the amount of MIPs binding to APE1 reaches the maximum, and at this concentration, the amount of NIPs binding to APE1 is small , the imprinting factor of magnetic molecularly imprinted nano...

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Abstract

The invention discloses dealkalized endonuclease magnetic molecular imprinting nano-particles as well as a preparation method and application thereof. The preparation method comprises the following steps: modifying the surface of a silicon dioxide-coated magnetic core used as a carrier by avidin which has strong affinity interaction with APE1 and is used as a functional monomer, forming an imprinting layer by taking the APE1 as a template molecule and taking a polymeric material as a monomer and a crosslinking agent, and finally eluting an APE1 template to form an imprinting cavity, thereby obtaining the magnetic molecular imprinting nano-particles. The magnetic molecular imprinting nano-particles prepared according to the method have advantages of strong affinity interaction with a template protein APE1, high selectivity, high interference resistance and high combination and desorption speeds. The preparation technology is simple in process, controllable in reaction and high in imprinting efficiency. The magnetic molecular imprinting nano-particles disclosed by the invention can selectively recognize, separate and enrich low-abundance APE1 in a complicated biological sample, and has a high industrial production value.

Description

technical field [0001] The invention belongs to the technical field of material preparation, and relates to the preparation and characterization of magnetic molecularly imprinted nanoparticles and their application in specific recognition, separation and enrichment of target proteins, and in particular to magnetic molecular imprinting of abasic endonucleases Nanoparticles and methods of preparation and applications thereof. Background technique [0002] Apurinic / apyrimidinic endonuclease 1 / redox effector-1 (referred to as abasic endonuclease 1, APE1) is an important nucleic acid repair enzyme in DNA base excision repair, mainly present in the nucleus It is of great significance to maintain the stability of genes and the health of human body. APE1 also acts as a redox factor in the body to regulate the oxidative stress response of cells (Evans A R, Limp-Foster M, Kelley M R. Going APE over ref-1. MutationResearch / DNA Repair(2000), 461(2):83 -108.). [0003] Recent studies ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68
CPCG01N33/68
Inventor 赵美萍翟筠秋李梦圆曹翔剑
Owner PEKING UNIV
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