Fluorescence detection method for bis (2-ethyl) hexyl phthalate based on coupling of nucleic acid aptamer with spherical nucleic acid

A technology of phthalic acid di- and nucleic acid aptamers, which is applied in fluorescence/phosphorescence, biological testing, measuring devices, etc., to achieve the effects of fast and portable on-site detection, high controllability, and short preparation time

Active Publication Date: 2021-08-27
QILU UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, nucleic acid aptamers can be used as recognition elements to detect small molecule organic pollutants, and a biosensing detection platform can be constructed based on nucleic acid aptamers, due to the huge differences in target recognition, signal transduction, and detection instruments across different types of biomolecules , so it is very challenging to develop a general detection platform

Method used

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  • Fluorescence detection method for bis (2-ethyl) hexyl phthalate based on coupling of nucleic acid aptamer with spherical nucleic acid
  • Fluorescence detection method for bis (2-ethyl) hexyl phthalate based on coupling of nucleic acid aptamer with spherical nucleic acid
  • Fluorescence detection method for bis (2-ethyl) hexyl phthalate based on coupling of nucleic acid aptamer with spherical nucleic acid

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Effect test

Embodiment 1

[0047] A fluorescent detection method based on nucleic acid aptamer coupling spherical nucleic acid di(2-ethyl)hexyl phthalate, comprising the following steps:

[0048] (1) Preparation of gold nanoparticles

[0049] In a 500 mL three-necked flask, heat 100 mL of 1 wt% HAuCl under vigorous stirring 4 The solution was boiled, and then 5mL, 38.8mM sodium citrate solution was added thereto, stirred vigorously, and reacted for 15min. After the solution changed from pure yellow to wine red, the heat source was removed and continued to cool to room temperature to obtain a gold nanoparticle solution. Store in the dark at 4°C for later use;

[0050] (2) Preparation of spherical nucleic acid

[0051] Dissolve the functional oligonucleotide chain in dithiothreitol phosphate buffer (pH=8), let it stand at room temperature for 2 hours, and then use NAP-5 column to elute to remove impurities; 50 μM functional oligonucleotide after removal Mix the nucleotide chain with step (1) 100 μL gol...

Embodiment 2

[0058] A fluorescence detection method based on nucleic acid aptamer coupling spherical nucleic acid di(2-ethyl)hexyl phthalate, the difference from Example 1 is that the amount of each substance in the test sample of the two The content varies and specifically includes the following steps:

[0059] (1) Preparation of gold nanoparticles

[0060] In a 500 mL three-necked flask, heat 100 mL of 1 wt% HAuCl under vigorous stirring 4 The solution was boiled, then 5mL, 38.8mM sodium citrate solution was added thereto, stirred vigorously, and reacted for 15min. After the solution changed from pure yellow to wine red, the heat source was removed, and continued to cool to room temperature to obtain a gold nanoparticle solution in 4 Store in dark at ℃ for later use;

[0061] (2) Preparation of spherical nucleic acid

[0062] Dissolve the functional oligonucleotide chain in dithiothreitol phosphate buffer (pH=8), let it stand at room temperature for 2 hours, and then use NAP-5 column ...

Embodiment 3

[0069] A fluorescence detection method based on nucleic acid aptamer coupling spherical nucleic acid di(2-ethyl)hexyl phthalate, the difference from Example 1 is that the amount of each substance in the test sample of the two The content is slightly different and includes the following steps:

[0070] (1) Preparation of gold nanoparticles

[0071] In a 500 mL three-necked flask, heat 100 mL of 1 wt% HAuCl under vigorous stirring 4 The solution was boiled, and then 5mL, 38.8mM sodium citrate solution was added thereto, stirred vigorously, and reacted for 15min. After the solution changed from pure yellow to wine red, the heat source was removed and continued to cool to room temperature to obtain a gold nanoparticle solution. Store in the dark at 4°C for later use;

[0072] (2) Preparation of spherical nucleic acid

[0073] Dissolve the functional oligonucleotide chain in dithiothreitol phosphate buffer (pH=8), let it stand at room temperature for 2 hours, and then use NAP-5 ...

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Abstract

The invention relates to a fluorescence detection method for bis (2-ethyl) hexyl phthalate based on coupling of a nucleic acid aptamer with a spherical nucleic acid. According to the invention, a variable structure nucleic acid aptamer structure is combined with a spherical nucleic acid detection platform, wherein an oligonucleotide chain used for being combined with a free foot chain of the variable structure nucleic acid aptamer is designed on the surface of a gold nanoparticle of spherical nucleic acid, and the characteristic that restriction endonuclease is cut at a specific site is utilized, so that the variable structure nucleic acid aptamer free foot chain is promoted to freely walk on the surface of the gold nanoparticle, a fluorescence signal amplification strategy is designed, the detection range of trace DEHP is expanded, and field detection is rapid and portable; and compared with the prior art for recognizing and detecting the target DEHP by adopting an antibody, the nucleic acid aptamer has the advantages that the specific recognition is better, the binding strength is higher, the preparation time of the nucleic acid aptamer is shorter than that of the antibody, the operation is simple, and the in-vitro storage is easier.

Description

technical field [0001] The invention belongs to the technical field of nucleic acid detection of organic pollutants, and relates to a fluorescence detection method based on nucleic acid aptamer coupling spherical nucleic acid di(2-ethyl)hexyl phthalate, in particular to a method based on variable configuration Competitive fluorescence detection method of di(2-ethyl)hexyl phthalate (DEHP) coupled with nucleic acid aptamer to spherical nucleic acid. Background technique [0002] Bis(2-ethyl)hexyl phthalate (DEHP) is a toxic plasticizer made of octanol or isooctyl alcohol and phthalic acid, in addition to cellulose acetate, polyvinyl acetate, and Most synthetic resins and rubbers used in industry have good compatibility. Due to its wide application in hundreds of products such as toys, food packaging materials, medical blood bags and hoses, vinyl floors and wallpapers, detergents, lubricants and personal care products, it is also an androgen antagonist , the long-term accumul...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/533G01N33/543G01N33/53G01N21/64
CPCG01N33/533G01N33/54346G01N33/5308G01N21/6428Y02A50/30
Inventor 姬丹丹臧立华原琳
Owner QILU UNIV OF TECH
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