Nucleic acid aptamer functionalized POSS (polyhedral oligomeric silsesquioxane) crosslinking organic-silica gel hybridization monolithic column, and preparation method thereof

A nucleic acid aptamer and silica hybrid technology, applied in the field of analytical chemistry, can solve the problem of incompatibility between the POSS hydrophobic cross-linking agent and the aptamer aqueous solution, and achieve a uniform and stable reaction system, good swelling resistance, and a site of action. many effects

Active Publication Date: 2018-06-01
FUZHOU UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The introduction of polyhedral oligomeric silsesquioxane grafted with methacrylate as a cross-linking agent has been used in multiple POSS-based cross-linked polymeric monolithic columns, but current research is mainly in hydrophobic reaction systems or using a large number of organic reagents However, the incompatibility between POSS hydrophobic cross-linking agent and aptamer aqueous solution still exists
The research on the preparation of nucleic acid aptamer-modified POSS cross-linked polymeric organic-silica hybrid monolithic columns has not been reported so far

Method used

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  • Nucleic acid aptamer functionalized POSS (polyhedral oligomeric silsesquioxane) crosslinking organic-silica gel hybridization monolithic column, and preparation method thereof
  • Nucleic acid aptamer functionalized POSS (polyhedral oligomeric silsesquioxane) crosslinking organic-silica gel hybridization monolithic column, and preparation method thereof

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Embodiment 1

[0025] A method for preparing a nucleic acid aptamer functionalized POSS cross-linked organic-silica hybrid monolithic column, the specific steps are:

[0026] (1) Centrifuge the thiol-modified aptamer at 8000r / min for 5 minutes, then add water to dilute to 1000 μmol / L, heat at 90°C for 3 minutes, and cool to room temperature to form the aptamer stock solution 1;

[0027] (2) According to the mass ratio of 12.5:3.75:83.75, accurately weigh 2-acrylamide-2-methylpropanesulfonic acid monomer, methacrylate grafted polyhedral oligomeric silsesquioxane, ethyl Diol dimethacrylate is added to the centrifuge tube, the amount of accurately added initiator accounts for 1.0wt% of the total amount of monomer and crosslinking agent, and then the porogen is PEG 10000 in proportion to the total amount of porogen composition 4.3% of the porogen, and water accounted for 0.5% (column 1), 2.5% (column 2), 5.0% (column 3), 5.5% (column 4) of the total porogen composition, corresponding to N,N-dim...

Embodiment 2

[0031]Apply the blank column, control column and nucleic acid aptamer affinity column (column 3 as an example), the length of the affinity column is 10cm, and implement according to the following steps: (1) Equilibration: equilibrate with binding buffer for 0.5 hours first, and the chromatographic conditions are Flow rate 0.10 mL / min, pressure 250psi, described binding buffer is 10mmol / L Tris-HCl, 120 mmol / L NaCl, 5 mmol / L KCl and 20mmol / LCaCl 2 , pH 8.50; (2) Enrichment: Inject 20 μL of 10 ng / mL ochratoxin A solution separately, enrich on the monolithic column for 1 hour, the chromatographic conditions are 0.02 mL / min, pressure 250psi; (3) Cleaning: Install the enrichment column on the liquid chromatography pump, and wash the blank column, control column and nucleic acid aptamer affinity monolithic column with binding buffer respectively; the washing conditions are as follows: flow rate 0.1 mL / min, pressure 500 psi, collect the final washing (4) Elution: Use 30% ACN: 70% TE b...

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Abstract

The invention discloses a nucleic acid aptamer functionalized POSS (polyhedral oligomeric silsesquioxane) crosslinking organic-silica gel hybridization monolithic column, and a preparation method thereof. The monolithic column is a compatible monolithic column formed in a way that highly-hydrophilic organic polymerized monomer, POSS, acrylic ester cross-linking agent, nucleic acid aptamer aqueoussolution and initiator are dissolved through ternary pore-forming agent to form homogeneous solution; the homogeneous solution is injected into the column to synchronously generate free radical thermal-initiation polymerization and 'sulfydryl-alkene' click chemistry reaction to be directly prepared; the ternary pore-forming agent is mixed solution formed by three ingredients of aqueous-N,N-dimethyl formamide-PEG (polyethylene glycol) 10000. The nucleic acid aptamer is bonded by covalent bonds through a one-step method, and the prepared compatible monolithic column has the advantages of stablestructure and simple and quick method and is suitable for the specific recognition separation of ochratoxin A.

Description

technical field [0001] The invention belongs to the field of analytical chemistry, and in particular relates to a nucleic acid aptamer-functionalized POSS cross-linked organic-silica gel hybrid polymerization integral column and a preparation method thereof. Background technique [0002] As a new type of affinity separation material, nucleic acid aptamer-modified affinity monolithic columns have attracted the attention of researchers and have been successfully applied to the separation of small molecular compounds, proteins and cells. Combining nucleic acid aptamers with monolithic column materials to prepare high-efficiency new affinity stationary phase materials, giving full play to the advantages of the two, compared with immune antibody affinity chromatography, it is easy to immobilize, easy to maintain conformation, and stationary phase It has the advantages of high surface coverage, good stability, and easy refolding after deformation. [0003] Usually, the nucleic ac...

Claims

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Application Information

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IPC IPC(8): G01N30/08B01D15/22B01D15/38
CPCB01D15/22B01D15/3819G01N30/08
Inventor 林旭聪陈怡琼於霞谢增鸿
Owner FUZHOU UNIVERSITY
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