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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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

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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[0024] Example 1

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

[0026] (1) Centrifuge the sulfhydryl-modified nucleic acid aptamer at a rate of 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 a nucleic acid 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-branched polyhedral oligomeric silsesquioxane, ethyl acetate Glycol dimethacrylate is added to the centrifuge tube, the exact amount of initiator added accounts for 1.0wt% of the total monomer and crosslinking agent, and then the porogen is proportional to PEG 10000 to account for the total composition of the porogen 4.3% of the porogen and water accounted for 0.5% (column 1), 2.5% (column 2), 5.0% (column 3), 5.5% (colum...

Example Embodiment

[0030] Example 2

[0031] Use blank column, control column and nucleic acid aptamer affinity column (column 3 as an example). The length of the affinity column is 10 cm. Follow the steps below: (1) Equilibrate: equilibrate with binding buffer for 0.5 hours. The chromatographic conditions are The flow rate is 0.10 mL / min, the pressure is 250 psi, and the binding buffer is 10 mmol / L Tris-HCl, 120 mmol / L NaCl, 5 mmol / L KCl and 20 mmol / LCaCl 2 , PH 8.50; (2) Enrichment: Inject 20 μL of 10 ng / mL ochratoxin A solution separately, and enrich on the monolithic column for 1 hour. The chromatographic conditions are 0.02 mL / min and the pressure is 250 psi; (3) Cleaning: Install the enrichment column on the liquid chromatography pump, and clean the blank column, the control column and the nucleic acid aptamer affinity monolithic column with binding buffer; the cleaning conditions are 0.1 mL / min, pressure 500 psi, and collect the final cleaning Solution to be checked; (4) Elution: Use 30% ACN...

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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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