Nucleic acid aptamer-molecular imprinting synergistic recognition magnetic microspheres and preparation method and application thereof

A nucleic acid aptamer, magnetic microsphere technology, applied in chemical instruments and methods, material separation, analysis of materials, etc., can solve the problem of limited bonding amount of solid-phase microextraction head aptamer, restricting method application, and limiting service life and other problems to achieve the effect of maintaining activity and stability, not easy to enzymatic hydrolysis, and improving stability

Active Publication Date: 2018-10-16
SOUTH CHINA NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Aptamers have high affinity, strong specificity, and many advantages over antibodies and enzyme biomacromolecular recognition systems, so they have broad application potential in solid-phase microextraction technology. For example, there are enzymatic problems in plasma and urine, which limit its service life. At the same time, the fibrous solid-phase microextraction head has a limited amount of aptamer bonding and low extraction capacity (less than 1ng), which restricts the application of the method.

Method used

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  • Nucleic acid aptamer-molecular imprinting synergistic recognition magnetic microspheres and preparation method and application thereof
  • Nucleic acid aptamer-molecular imprinting synergistic recognition magnetic microspheres and preparation method and application thereof
  • Nucleic acid aptamer-molecular imprinting synergistic recognition magnetic microspheres and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1: Preparation of amino-modified magnetic microspheres

[0035] Weigh 2.00g PEG-6000, 2.40g NaOH in a 100mL beaker, add 50mL boiled and cooled deionized water to dissolve, transfer to a 500mL two-neck flask. Weigh 2.70g FeCl again 3 ·6H 2 O and 1.96 g FeCl 2 4H 2 O was dissolved in a 250mL beaker by adding 100mL of boiled and cooled deionized water and added dropwise to the mixture of PEG-6000 and NaOH. Keep the temperature of the water bath at 40°C, stir and react for 1 hour to get Fe 3 o 4 magnetic microspheres.

[0036] Fe 3 o 4 The magnetic microspheres were placed in a flask containing 50mL of n-propanol, 5mL of ammonia water was added after ultrasonication for 15min, 1ml of TEOS was added after an interval of 10min, and the reaction was stirred for 12h, then 10mL of 10% HCl was added for ultrasonic pickling, and finally 10mL of no Alcohol wash with water and ethanol to get SiO 2 / Fe 3 o 4 magnetic microspheres.

[0037] SiO obtained above 2 / ...

Embodiment 2

[0041] Example 2: Preparation of OTA nucleic acid aptamer-modified magnetic microspheres

[0042] Weigh 5 mg of amino-modified SiO obtained in Example 1 2 / Fe 3 o 4 Magnetic microspheres, add 800 μL Tris-HCl buffer solution (pH is 7.5, containing 0.1mol / L NaCl, 50mmol / L Tris, 1mmol / L MgCl 2 ), ultrasonic for 10min. Then add 1.2mL of LEDC / NHS mixture (molar ratio EDC:NHS=1:4), let it stand for 10min to activate, add 40μL of OTA aptamer solution (6.5μg / mL), shake and react at 25℃ for 16h to obtain OTA nucleic acid Aptamer-modified magnetic microspheres, labeled Apt-SF.

Embodiment 3

[0043] Example 3: Preparation of OTA nucleic acid aptamer-molecularly imprinted coated magnetic microspheres

[0044] Disperse the OTA nucleic acid aptamer-modified magnetic microspheres obtained in Example 2 in 5mL Tris-HCl buffer solution (pH is 7.5, containing 0.1mol / L NaCl, 50mmol / L Tris, 1mmol / L MgCl 2 ), add 0.205 μmol / L template molecule OTA, shake and incubate for 90 min, then perform magnetic separation, and then wash repeatedly with buffer solution to remove unbound template molecules. Then, 145 μmol functional monomer MAA (methacrylic acid) was added, and the reaction was shaken at room temperature for 30 minutes.

[0045] Next, 11.1 mg of N,N-methylenebisacrylamide, 33.3 μL of 0.0438 mol / L ammonium persulfate and 1 μL of tetramethylethylenediamine were added to the reaction mixture, and the reaction was shaken at 25°C for 4 hours under anaerobic conditions. Obtain OTA nucleic acid aptamer-molecularly imprinted coated magnetic microspheres.

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Abstract

The invention discloses nucleic acid aptamer-molecular imprinting synergistic recognition magnetic microspheres and a preparation method and application thereof. The nucleic acid aptamer-molecular imprinting synergistic recognition magnetic microspheres disclosed by the invention comprise magnetic microsphere inner cores and nucleic acid aptamer-molecular imprinting synergistic recognition shells;the nucleic acid aptamer-molecular imprinting synergistic recognition shells have molecular imprinting polymer rigid recognition cavities; the molecular imprinting polymer rigid recognition cavitieshave structural stability, difficult deformation and strong specific adsorptivity; and nucleic acid aptamers are not easy to digest.

Description

technical field [0001] The invention belongs to the technical field of chemical analysis and testing, and relates to solid-phase microextraction technology, in particular to a nucleic acid aptamer-molecular imprinting cooperative recognition magnetic microsphere and its preparation method and application. Background technique [0002] Sample pretreatment is the first step in sample analysis and a key step in the entire analysis process, which directly affects the accuracy and precision of sample analysis. The main purpose of sample pretreatment is to separate and enrich the components to be tested in the sample, reduce matrix or impurity interference, and meet the requirements of subsequent testing instruments for sample shape, concentration, and cleanliness. Traditional sample pretreatment techniques such as liquid-liquid extraction, solvent extraction, Soxhlet extraction, column chromatography, etc., generally have problems such as time-consuming, inefficient, large amount...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C08J9/26C08F220/06C08F222/38C08K9/06C08K9/10C08K3/22B01J20/26B01J20/28B01D15/08B01J20/30G01N30/08
CPCB01D15/08B01J20/268B01J20/28009B01J20/28019C08F220/06C08J9/26C08J2333/02C08K3/22C08K9/06C08K9/10C08K2003/2275G01N30/08C08F222/385
Inventor 胡小刚巫宝霞张晓婷马艳霞
Owner SOUTH CHINA NORMAL UNIVERSITY
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