Preparation and applicationof aflatoxin B1 aptameraffinity capillarymonolithic column

A capillary monolithic column, aflatoxin technology, which is applied in measurement devices, instruments, scientific instruments, etc., can solve the problems of antibody inactivation, the antibody is easily affected by external conditions, and the flexible application of limited methods, etc., to achieve the effect of rapid detection.

Active Publication Date: 2017-09-08
NANJING UNIV OF FINANCE & ECONOMICS
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in affinity chromatography, there are some obvious limitations of immune antibodies. (1) The immobilization of antibodies on the stationary phase is still challenging, and the spatial orientation of the immobilized antibodies is difficult to keep consistent, which affects the Affinity; (2) Antibodies are easily affected by external conditions. In the strong-affinity affinity chromatography mode, it is often necessary to change the pH of the solution, add organic solvents or denaturants, etc. during the separation and enrichment operation, and severe elution conditions, It may lead to antibody inactivation, which limits the flexible application of the method to a large extent; (3) Due to the large volume of the antibody, the coverage rate on the surface of the stationary phase is small, resulting in a relatively small capacity of the immunoaffinity column; (4) Antibody preparation Need to go through immunization animal experiments or cell experiments, which is tedious, time-consuming and costly

Method used

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  • Preparation and applicationof aflatoxin B1 aptameraffinity capillarymonolithic column
  • Preparation and applicationof aflatoxin B1 aptameraffinity capillarymonolithic column

Examples

Experimental program
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Effect test

Embodiment 1

[0021] Example 1 Preparation of Aflatoxin B1 Aptamer Affinity Capillary Monolithic Column

[0022] (1) Capillary activation: the inner diameter of the quartz capillary used to prepare the monolithic column is 530 μm, and the outer diameter is 690 μm. Rinse the capillary with 1.0mol / L sodium hydroxide solution for 4h, deionized water for 30min, and 1.0mol / L hydrochloric acid solution for 4h, then wash with deionized water until neutral, and blow dry with nitrogen at 160°C.

[0023] (2) In situ synthesis of monolithic columns: firstly, 25 mg CTAB was dissolved in a mixed solution of 225 μL absolute ethanol and 100 μL deionized water. Then 150 μL TEOS and 80 μL APTES were quickly added to the above mixed solution. Vortex at room temperature for 30 s, then place in an ice-water bath at 0°C and sonicate for 30 s, then pour the mixed solution into the above-mentioned activated capillary. Finally, both ends of the capillary were sealed with silicone rubber and placed in an oven at ...

Embodiment 2

[0031] Firstly, the capillary activation treatment was carried out in the same steps as in Example 1. Then, 10 mg CTAB was dissolved in a mixed solution of 150 μL absolute ethanol and 50 μL deionized water, and then 100 μL TEOS and 50 μL APTES were quickly added to the above mixed solution. Vortex at room temperature for 5 minutes, then place in an ice-water bath at -5°C and sonicate for 5 minutes, then pour the mixed solution into the above-mentioned activated capillary. Both ends of the capillary were sealed with silicone rubber, and placed in an oven at 30°C for 10 h. After the reaction is complete, rinse the capillary thoroughly with methanol and deionized water.

[0032] Prepare a phosphate buffer solution containing 5% glutaraldehyde, and pass the solution into the capillary at a rate of 10 μL / min for continuous reaction. In the process of aptamer modification, the aflatoxin B1 aptamer was made into a phosphate buffer solution with a concentration of 4 nmol / L, and then...

Embodiment 3

[0034] Firstly, the capillary activation treatment was carried out in the same steps as in Example 1. Then, 30 mg CTAB was dissolved in a mixed solution of 350 μL absolute ethanol and 150 μL deionized water, and then 200 μL TEOS and 100 μL APTES were quickly added to the above mixed solution. Vortex at room temperature for 2 min, then place in a water bath at 5° C. and sonicate for 2 min, then pour the mixed solution into the activated capillary. Both ends of the capillary were sealed with silicone rubber, and placed in an oven at 60°C for 30 h. After the reaction is complete, rinse the capillary thoroughly with methanol and deionized water.

[0035]Prepare a phosphate buffer solution containing 20% ​​glutaraldehyde, pass the solution into the capillary at a rate of 20 μL / min, and react continuously. In the aptamer modification process, the aflatoxin B1 aptamer was made into a phosphate buffer solution with a concentration of 8 nmol / L, and then passed into the capillary at a...

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Abstract

The invention discloses the preparation and application of an aflatoxin B1 aptamer affinity capillary monolithic column. An amino-modified aflatoxin B1 aptamer is modified onan organic-inorganic hybridamino capillary monolithic column through glutaraldehyde coupling and grafting, through the specific affinity effect of the aptamer and aflatoxin B1, the organic-inorganic hybrid monolithic column is high in column permeability and large in specific surface area. The trace, ultra-trace aflatoxin B1 in complex food samples can be separated and enriched with high sensitivity and high selectivity. Thesimple, sensitive and rapid detection of the aflatoxin B1 can be achieved by using a detection instrument together.

Description

technical field [0001] The invention relates to a separation and enrichment method for aflatoxin B1, in particular to a preparation method and application of an aflatoxin B1 aptamer affinity capillary monolithic column. Background technique [0002] Aflatoxin B1 (AFB1) is a toxic metabolite mainly produced by Aspergillus flavus and Aspergillus parasiticus, and has been classified as a primary carcinogen by the International Agency for Research on Cancer (IARC). Aflatoxin B1 is widely distributed in various agricultural products and foods, among which the most susceptible to contamination are peanuts, corn, rice, wheat, peanut oil and other grain and oil foods. In view of the serious harm of AFB1, relevant organizations at home and abroad have established strict limit standards for it. [0003] At present, the detection methods of aflatoxin B1 mainly include thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/60
CPCG01N30/6078
Inventor 李彭方勇裴斐姚远航刘琴
Owner NANJING UNIV OF FINANCE & ECONOMICS
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