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Aptamer sensor for self-assembly of DNA hydrogel based on rolling circle amplification product and application of aptamer sensor

A technique of aptamer sensor and rolling circle amplification, which is applied in the determination/inspection of microorganisms, instruments, biological tests, etc., and can solve the problems of inconvenient visual detection of DNA hydrogel

Active Publication Date: 2021-07-30
UNIV OF SHANGHAI FOR SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The above-mentioned DNA modification of gold nanoparticles and the combination of DNA on the surface of gold nanoparticles and the DNA of the hydrogel bring a lot of inconvenience to the visual detection of DNA hydrogels.

Method used

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  • Aptamer sensor for self-assembly of DNA hydrogel based on rolling circle amplification product and application of aptamer sensor
  • Aptamer sensor for self-assembly of DNA hydrogel based on rolling circle amplification product and application of aptamer sensor
  • Aptamer sensor for self-assembly of DNA hydrogel based on rolling circle amplification product and application of aptamer sensor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Embodiment 1 Preparation of SH-PEG functionalized gold nanoparticles:

[0059] (1) 1g HAuCl 4 4H 2 Dissolve O in 100 mL of pure water to make 1% HAuCl 4 4H 2 O in water; take 0.5 mL of 1% HAuCl 4 4H 2 O aqueous solution was added into 50mL pure water, heated to boiling under stirring;

[0060] (2) Add 0.4mL 5% aqueous sodium citrate solution therein, keep boiling for 30min, cool the wine red solution prepared to room temperature, and store it in the dark at 4°C for subsequent use; the ultraviolet-visible absorption spectrum and transmission electron microscope of the gained nano gold Figure respectively as figure 2 As shown in A and 2B;

[0061] (3) Take 5mL of the wine red solution prepared in step (2), add 0.25mL of the solution with a concentration of 5×10 -5 M SH-PEG, incubated at 37°C for 12h, then centrifuged at 10,000rpm for 10min, discarded the supernatant, and obtained a precipitate that was SH-PEG functionalized gold nanoparticles, and dispersed the p...

Embodiment 2

[0064] The preparation of embodiment 2 DNA hydrogel:

[0065] (1) 20μL 10 -5 M OTA aptamer and 20 μL 10 -5 The M primers were mixed and annealed at 95°C for 10 min, and then hybridized by the gradient cooling method to obtain the aptamer-primer hybridization complex; the hybridization condition of the gradient cooling method was 95°C for 10 min, and then naturally cooled to 25°C.

[0066] (2) Add 5 μL of exonuclease I to enzymolyze the unbound DNA single strand to prepare the purified aptamer-primer hybrid complex; React for 15 minutes.

[0067] (3) Take 10.5 μL of the purified aptamer-primer hybridization complex obtained in step (2), add OTA with a concentration of 10 ng / mL, and add 10.5 μL of 10 -5 M padlock probe and 1.0 μL T4 DNA ligase were mixed evenly and incubated at 37°C for 45 min; the enzyme activity of T4 DNA ligase was 5.0 U / mL;

[0068] (4) Then add 6.4 μL 10mM dNTPs and 2.0 μL 10u / mL Phi29 DNA polymerase to it, mix well and incubate at 37°C for 2.5h, then i...

Embodiment 3

[0072] A preparation method of said aptasensor, said preparation method comprising the following steps:

[0073] Add 5 μL SH-PEG functionalized gold nanoparticles prepared in Example 1 to 0.03 μL DNA hydrogel (10 ng / mL) prepared in Example 2 to obtain AuNPs / DNA hydrogel; incubate at 90°C for 30min, then cool to room temperature;

[0074]The AuNPs / DNA hydrogel was centrifuged at 2000 rpm for 10 min, and its absorbance at 530 nm was measured with a UV-1800 ultraviolet spectrophotometer.

[0075] The transmission electron microscope images and photos of AuNPs / DNA hydrogel are as follows image 3 D, shown in 3E. Depend on image 3 D It can be seen that the shape of AuNPs / DNA hydrogel is similar to that of DNA hydrogel, which is flower-shaped and the size is also micron-scale. It can be seen that the gold nanoparticles are wrapped inside the DNA hydrogel. Photos of DNA hydrogels and AuNPs / DNA hydrogels as image 3 As shown in E, it can be seen that the AuNPs / DNA hydrogel is re...

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Abstract

The invention discloses an aptamer sensor for self-assembly of DNA hydrogel based on a rolling circle amplification product. The aptamer sensor comprises an OTA aptamer, a target object, SH-PEG functionalized nanogold, a primer, exonuclease I, a lock-type probe, T4DNA ligase and Phi29 DNA polymerase. The nanogold is directly assembled into the DNA hydrogel by increasing the number of T basic groups in the DNA sequence of the rolling circle amplification reaction lock type probe, DNA sequence modification does not need to be carried out on the nanogold, and great convenience is provided for on-site and rapid detection of the ochratoxin A.

Description

technical field [0001] The invention relates to the technical field of food safety detection, in particular to an aptamer sensor based on rolling circle amplification product self-assembled DNA hydrogel and its application. Background technique [0002] Ochratoxin A (OTA) is one of the most common hazardous substances in food and agricultural products, often present in wine, nuts, coffee, and cereals. OTA is a secondary metabolite produced by Aspergillus and Penicillium, which has nephrotoxicity, immunotoxicity, teratogenicity and carcinogenicity. The common detection methods of OTA in food include visual biosensor detection method, fluorescent biosensor detection method, electrochemical biosensor detection method, high performance liquid chromatography (HPLC), surface enhanced Raman scattering detection (SERS), etc. Among them, visual detection is considered to be one of the most promising on-site rapid detection methods because it is the easiest to realize naked eye detec...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/53C12Q1/6825
CPCG01N33/5308C12Q1/6825C12Q2525/205C12Q2521/325C12Q2521/501C12Q2531/125C12Q2525/173C12Q2563/137C12Q2563/155Y02A50/30
Inventor 徐斐郝丽玲刘馨娜彭开敏
Owner UNIV OF SHANGHAI FOR SCI & TECH
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