Label-free fluorescence aptamer sensor and preparation method and application thereof

An aptamer sensor and label-free technology, which is applied in the field of protein analysis and detection, can solve the problems of reducing nucleic acid aptamers, complicated operation procedures, and low repeatability, and achieve simplified detection processes, excellent selectivity and specificity, and reduced or the effect of eliminating interference

Active Publication Date: 2017-11-24
GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, these existing methods all need to use some advanced nanomaterials as fluorescent groups or quenching groups to label nucleic acid aptamers, which not only increases the cost but also reduces the affinity of nuclei

Method used

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  • Label-free fluorescence aptamer sensor and preparation method and application thereof
  • Label-free fluorescence aptamer sensor and preparation method and application thereof
  • Label-free fluorescence aptamer sensor and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] 1. Preparation of dsDNA probes: Mix magnetic nanospheres (1 mL, 20 μg / mL) with amino groups on the surface and coupling reagent Sulfo-SMCC (10 μL, 5 mg / mL), incubate at room temperature for 60 min; remove excess by magnetic separation Coupling reagents, 5' end thiol-modified capture probes (1 mL, 2 μmol / L) were added, mixed well, and incubated at room temperature for 24 h. Remove the reaction solution by magnetic separation, wash the magnetic nanospheres with 0.01mol / L Tris-HCl buffer solution of pH 7.5 three times, then add aptamer (1mL, 0.5μmol / L) and mix with them, incubate at 95°C for 5min, and cool down slowly to room temperature. The magnetic nanospheres were washed three times with 0.01mol / L, pH 7.5 Tris-HCl buffer, and then redispersed in 1 mL, 0.01mol / L, pH 7.5 Tris-HCl buffer to obtain magnetic nanoparticles assembled with capture probes. Microspheres.

[0035]2. Quantitative detection of shrimp allergenic protein: Take 40 μL of sensing probe stock solution ...

Embodiment 2

[0037] To detect other proteins:

[0038] Take 40 μL of the sensing probe stock solution and dilute it to 150 μL with 0.01mol / L, pH 7.5 Tris-HCl buffer, add 50 μL, 5 μg / mL of shrimp allergenic protein (Tropomyosin) and 50 μL, 10 μg / mL bovine serum albumin (BSA), lysozyme (Lysozyme), streptavidin (Streptavidin), β-conglycinin (β-Conglycinin), etc. were mixed and incubated at 37°C for 120min. Remove the magnetic nanospheres by magnetic separation, add an equal volume of OliGreen working solution, incubate at room temperature in the dark for 40 minutes, and immediately measure on the machine to observe the change of the fluorescence signal. The results show( figure 2 -B), The fluorescent signal changes caused by the shrimp allergenic protein are very significant, while the other proteins only cause small changes in the fluorescent intensity. The result shows that the label-free fluorescent aptasensor of the present invention has high specificity and specificity.

Embodiment 3

[0040] Detection in real samples:

[0041] 50mg of shrimp sauce, tomato sauce and salad dressing were mixed with 1mL, 0.02mol / L, pH 7.5 Tris-HCl buffer and homogenized for 20min, then overnight at 4°C, centrifuged (15000g, 30min) to remove large particles and suspended matter, Take the supernatant to obtain the original extract. The extract and its 10-fold diluted extract with 0.02mol / L, pH 7.5 Tris-HCl buffer were used as actual samples. Then, the sample was detected according to the above-mentioned label-free aptamer sensor detection method, and the detection results were as follows: image 3 shown. Like shrimp paste, the original extracts of tomato sauce and salad dressing caused significant changes in fluorescence signals, but after 10-fold dilution with 0.02mol / L, pH 7.5 Tris-HCl buffer, only negligible fluorescence signals were caused. Variety. However, the positive sample shrimp paste still caused a credible change in the fluorescent signal. This result shows that ...

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Abstract

The invention discloses a label-free fluorescence aptamer sensor and a preparation method thereof and application thereof to quick detection of shrimp allergy protein. The label-free fluorescence aptamer sensor comprises a magnetic nanosphere, a capturing probe and an aptamer, wherein the surface of the magnetic nanosphere is modified with the capturing probe; the capturing probe and the aptamer are connected through base complementary pairing to form a double-stranded DNA; the nucleotide sequence of the aptamer is shown as SEQ ID NO: 1; the nucleotide sequence of the capturing probe is shown as SEQ ID NO: 2. The label-free fluorescence aptamer sensor is applied to the quick detection of the shrimp allergy protein, has the advantages of strong anti-interference capability, high detection accuracy, good stability, strong repeatability and the like, and has a good application prospect and a good market value.

Description

technical field [0001] The invention relates to the technical field of protein analysis and detection, in particular to a label-free fluorescent aptamer sensor prepared by using an aptamer recognition technology and a label-free fluorescent probe and its application in the rapid detection of shrimp allergenic proteins. Background technique [0002] With the improvement of people's living standards, food allergy has gradually become a food safety issue that is widely concerned by the public. However, there is no effective medical treatment for food allergies, and the most effective means are strict avoidance of exposure to food allergens. Therefore, it is particularly necessary to fully understand the mechanism of food allergy and develop detection and analysis methods for different allergens. [0003] Shrimp and its products are delicious and nutritious. As an important seafood product, they are more and more popular among consumers. However, shrimp tropomyosin is one of t...

Claims

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Application Information

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IPC IPC(8): G01N33/533G01N33/558G01N33/543
CPCG01N33/533G01N33/54346G01N33/558
Inventor 吴清平张友雄张菊梅孙铭莫树平王涓柏建玲韦献虎蔡芷荷卢勉飞
Owner GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY
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