Method for producing fluorescence resonance energy transfer sensor and rapidly detecting CaMV35S

A fluorescence resonance energy and detection method technology, applied in fluorescence/phosphorescence, instruments, measuring devices, etc., can solve problems such as complex detection schemes, high detection costs, and low sensitivity, and achieve clear design ideas, fast analysis speeds, and synthetic methods simple effect

Inactive Publication Date: 2016-11-09
JIANGSU UNIV
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Problems solved by technology

[0006] The present invention aims at inventing a fluorescence resonance energy transfer sensor integrating the advantages of low toxicity, high sensitivity, high selectivity, simple and easy operation, etc., and provides a quantitative detection with simple pre

Method used

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  • Method for producing fluorescence resonance energy transfer sensor and rapidly detecting CaMV35S

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Experimental program
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Effect test

Embodiment 1

[0043] (1) Preparation of NGQDs

[0044] Azagraphene quantum dots (NGQDs) were prepared in one step using the existing thermal cracking method under normal pressure. After mixing 0.5g of ammonium citrate and 15mL of water, they were transferred to a three-necked flask, and the three-necked flask was placed in an oil bath at 200°C. Heating in a pot, a spherical condenser tube is connected to the three-necked flask, and a balloon is tied to the gas outlet of the condenser tube to ensure that the whole process is carried out in a relatively closed and constant pressure environment. As the heating reaction progressed, the balloon began to expand slightly, indicating that ammonia, H 2 O and other gases are generated, and through condensation and reflux, the generated ammonia gas is converted into NH 4 + The form returned to the solution, and the color of the solution gradually changed from colorless to light yellow. With the passage of heating time, the balloon gradually expande...

Embodiment 2

[0056] Construction of standard curve of CaMV35S target DNA by fluorescence resonance energy transfer sensor

[0057] Add 100 μL of CaMV35S target DNA with concentrations of 0.1, 0.5, 1, 5, 10, 50, 100, 200, 400, and 500 nM into 420 μL of prepared NGQDs-probe1, and incubate at constant temperature for 40 minutes, then add 1 mL of prepared Ag-probe2 solution, add NaCl Adjust the sodium ion concentration to 2M, and then continue to incubate for 2 hours to form a sandwich structure of NGQDs-probe1 / tDNA / Ag-probe2, triggering the fluorescence energy resonance transfer from NGQDs to AgNPs, resulting in the quenching of the fluorescence of NGQDs, using fluorescence spectrophotometry Measure the fluorescence intensity of the solution at 355nm, and establish a standard curve between the fluorescence quenching efficiency and the CaMV35S target DNA.

Embodiment 3

[0059] Fluorescence resonance energy transfer sensor detection of actual samples of different content of transgenic soybean:

[0060] Add 100 μL of PCR amplification products containing 0.5%, 1%, and 10% DNA extracts of transgenic soybeans to 420 μL of prepared NGQDs-probe1, and after incubation at constant temperature for 40 min, add 1 mL of prepared Ag-probe2 solution, add NaCl adjusted the sodium ion concentration to 2M, and then continued to incubate for 2 hours to form a sandwich structure of NGQDs-probe1 / tDNA / Ag-probe2, triggering the fluorescence energy resonance transfer from NGQDs to AgNPs, resulting in the quenching of the fluorescence of NGQDs, using fluorescence spectroscopy A photometer measures the fluorescence intensity of the solution at 355nm, and the quenching efficiency is compared with the standard curve to draw a conclusion whether the CaMV35S promoter is contained.

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Abstract

The invention provides a method for producing a fluorescence resonance energy transfer sensor and rapidly detecting CaMV35S. The method comprises the following steps: 1, preparing a nitrogen-doped graphene quantum dot (NGQDs) solution; 2, preparing a silver nanoparticle (AgNPs) dispersion; 3, preparing a nitrogen-doped graphene quantum dot-probe1 (NGQDs-probe1) dispersion; 4, preparing a silver nanoparticle-probe2 (AgNPs-probe2) dispersion; 5, constructing the fluorescence resonance energy transfer sensor; and 6, detecting the target DNA of CaMV35S, and establishing a standard curve. The sensor is produced based on the DNA complementary pairing hybridization specification, so compared with sensors produced based on immunoreaction, the method provided by the invention has the characteristics of strong binding force with target molecules, high specificity, difficult degeneration after long-time exposure, and low requirements for surrounding environment.

Description

technical field [0001] The invention belongs to the field of fluorescence resonance energy transfer, in particular to a preparation of a fluorescence resonance energy transfer sensor and a rapid detection method for CaMV35S. Background technique [0002] Genetically Modified Organisms (GMOs for short) refers to the use of biotechnology to insert target genes identified and isolated from animals, plants or microorganisms into the plant genome to change its genetic composition and produce new agronomic traits. plant. [0003] At present, GMO detection methods are mainly based on exogenous protein target detection, RNA detection and nucleic acid-based detection. Detection methods targeting exogenous proteins are based on the specific recognition of antigens by antibodies, mainly including enzyme-linked immunosorbent assay (ELISA), immunoassay strips and protein chips, but these methods have high background signals and difficult to maintain protein activity for a long time 1. ...

Claims

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Application Information

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IPC IPC(8): G01N21/64
CPCG01N21/6428G01N2021/6432
Inventor 李雅琪王坤蔡健荣孙力
Owner JIANGSU UNIV
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