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Photonic crystal modified microsphere for detecting OTA (Ochratoxins) based on DNAzyme-aptamer chemiluminescence and application thereof

A technology of photonic crystal microspheres and chemiluminescence, which is applied in chemiluminescence/bioluminescence, nanotechnology for materials and surface science, and analysis by making materials undergo chemical reactions, can solve the problem of expensive detection instruments and reagent consumption. Large size, high detection cost, etc., to achieve the effect of fast detection time, simple and convenient preparation method, and high sensitivity

Active Publication Date: 2017-12-12
NANJING NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

And with the increasing economic exchanges among countries, traditional detection methods have gradually shown shortcomings, such as high detection cost, large amount of reagents, expensive detection equipment, long detection time and other shortcomings

Method used

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  • Photonic crystal modified microsphere for detecting OTA (Ochratoxins) based on DNAzyme-aptamer chemiluminescence and application thereof
  • Photonic crystal modified microsphere for detecting OTA (Ochratoxins) based on DNAzyme-aptamer chemiluminescence and application thereof
  • Photonic crystal modified microsphere for detecting OTA (Ochratoxins) based on DNAzyme-aptamer chemiluminescence and application thereof

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preparation example Construction

[0044] Preparation of Monodisperse Silica Nanoparticles: Using Improved synthetic method.

[0045] Both the A1 chain and the B2 chain used in the present invention were synthesized by Shanghai Sangon Bioengineering Co., Ltd., wherein the A1 chain includes two parts of OTA-aptamer and DNase, and the amino group is modified at the 5' end. The B2 chain is designed to be partially complementary to the OTA-aptamer part of the A1 chain, and the other part is complementary to the DNase part of the A1 chain. The sequence is as follows:

[0046] A1 chain: 5'-NH2-C6-TGG-GTA-GGG-CGG-GTT-GGG-AAA-GAT-CGG-GTG-TGG-GTG-GCG-TAA-AGG-GAG-CAT-CGG-ACA-3'

[0047] B2 strand: 5'-CAC ACC CGA AAA ATC CCA ACC C-3'.

Embodiment 1

[0049] (1) Preparation of photonic crystal microspheres: Prepare ammonia water, double distilled water, absolute ethanol, and tetraethyl orthosilicate (TEOS) according to liquid A (ammonia water 3mL, double distilled water 20.7mL absolute ethanol 26.3mL), liquid B (absolute ethanol 45.5mL, TEOS4.5mL) proportioning, be mixed with the mother liquor of preparing silica microsphere. After A and B are prepared, they are fully mixed. First, place A on a magnetic stirrer and stir at a speed of 1200rp / min. Then quickly add B into A. When adding, avoid B and A. contact with the bottle wall of the liquid. After rapid stirring and reaction for 2 minutes, slow down the speed to 400rp / min, and seal it with plastic wrap to avoid the volatilization of ammonia gas, and allow it to react slowly for 6 hours. After the reaction, an opaque milky white liquid can be obtained, which is washed 3 to 4 times with absolute ethanol and double distilled water respectively, and centrifuged for 10 minutes...

Embodiment 2

[0053] The preparation method of Example 2 is the same as that of Example 1, except that the A1 chain is prepared into a DNA solution with a concentration of 60 nM, and the microspheres are immobilized at 2 μL / sphere; the concentration of the B2 solution is 60 nM, and it is added at 2 μL / sphere Photonic crystal modified microspheres.

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Abstract

The invention discloses a photonic crystal modified microsphere for detecting OTA (Ochratoxins) based on DNAzyme-aptamer chemiluminescence and the application thereof. The photonic crystal modified microsphere is characterized in that the surface of the photonic crystal microsphere is sequentially subjected to hydroxylation modification, amination modification and carboxylation modification; then the DNA chain and the A1 are fixed to the hydroxylation modified microsphere surface. According to the photonic crystal modified microsphere for detecting the OTA based on the DNAzyme-aptamer chemiluminescence, the photonic crystal microsphere is treated as a fungaltoxin probe vector; an aptamer technology is used as the reaction basis; a G-quadruplet structure formed by Hemin and the DNA chain is treated as a bio-enzyme catalyzing luminol system; on that basis, the chemiluminescence is performed; a chemiluminescence signal of single photonic crystal microsphere is subjected to high throughput test through a multifunctional microplate reader. Therefore, a method which is high in sensitivity, high in throughput, low in cost, fast to detect, and high in specificity is created for building the OTA.

Description

technical field [0001] The invention relates to the detection of ochratoxin A in agricultural products, feed and food; in particular, it relates to a photonic crystal modified microsphere based on DNAzyme-aptamer chemiluminescence method to detect OTA and its application Background technique [0002] Ochratoxins are structurally similar and highly toxic metabolites produced by Penicillium and Aspergillus strains. Ochratoxin A (OTA) is one of the most toxic, widely distributed and most harmful to humans. It often enters the human body through the food chain, causing varying degrees of impact on the human body. Based on this, countries and regions around the world have formulated strict standards to protect the safety of their own agricultural products, food and feed. Efforts have long been made to take various measures and develop new detection methods to prevent mycotoxins from entering the human food chain. Currently commonly used detection methods are chromatography, im...

Claims

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Application Information

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IPC IPC(8): G01N21/76B82Y5/00B82Y20/00B82Y30/00B82Y40/00
CPCG01N21/76B82Y5/00B82Y20/00B82Y30/00B82Y40/00
Inventor 李建林沈鹏李玮郑铁松金岩昊李奕辰李亚维马六十刘妍丁志邓杨朱瑞雪蔡婷婷
Owner NANJING NORMAL UNIVERSITY
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