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Method for constructing fluorescence resonance energy transfer sensor and method for detecting CaMV35S promoter by means of sensor

A fluorescence resonance energy and detection method technology, applied in fluorescence/phosphorescence, instruments, measuring devices, etc., can solve the problems of complex detection scheme, long detection time, high detection cost, etc., and achieve clear design ideas, fast analysis speed, and easy operation flexible effects

Inactive Publication Date: 2016-10-26
JIANGSU UNIV
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Problems solved by technology

[0006] The present invention aims at inventing a FRET method with the advantages of low toxicity, high sensitivity, high selectivity, simple and easy operation, etc., and provides a detection method containing the CaMV35S promoter with simple preparation process, high sensitivity, wide measurement range and low cost The method of genetically modified crops and products solves the existing problems of high detection cost, complicated detection scheme, long detection time and low sensitivity

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  • Method for constructing fluorescence resonance energy transfer sensor and method for detecting CaMV35S promoter by means of sensor

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Experimental program
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Effect test

Embodiment 1

[0034] (1) Preparation of MWCNTs / GONRs

[0035] MWCNTs / GONRs were obtained by oxidative shearing of multi-walled carbon nanotubes. The specific process was as follows: 120 mg of multi-walled carbon nanotubes were weighed into a round bottom flask, and 40 mL of H 2 SO 4 / H 3 PO 4 (volume ratio is 9:1) and stir, then add 600mg KMnO in the above mixed solution 4 , and continued to stir for 2 h in an oil bath at 65°C. After the reaction, the solution was cooled to room temperature, and it was poured into 400 mL containing 0.375% H 2 o 2 In ice water, the solution was filtered through a PTFE membrane (pore size 0.2 μm) to obtain solid product MWCNTs / GONRs, and washed several times with water, and finally dispersed in deionized water to obtain a MWCNTs / GONRs dispersion with a concentration of 1.5 mg / mL.

[0036] (2) Preparation of NSGQDs

[0037] Nitrogen and sulfur heterographene quantum dots (NSGQDs) were prepared in one step by thermal cracking under normal pressure. After...

Embodiment 2

[0042] Embodiment 2: The construction of the FRET sensor to tDNA standard curve

[0043] Add 4 μL of 1.5 mg / mL MWCNTs / GONRs dispersion to 3.0 mL of NSGQDs-ssDNA, sonicate for 5 min, and after standing for 5 min, measure the fluorescence intensity of the MWCNTs / GONRs-NSGQDs-ssDNA system, then add 15 μL of tDNA of different concentrations, After ultrasonication for 5 minutes, stand still for 40 minutes to measure the degree of fluorescence recovery and construct a standard curve.

[0044] figure 1 It is a standard curve diagram for detecting different concentrations of CaMV35S target DNA, wherein the curves a to k are in the order from bottom to top, and the concentrations of the target DNA are 0, 0.1, 0.5, 1, 5, 10, 50, 100, 200, 400, 500 nM. Curve a indicates that when no target DNA is present, NSGQDs are adsorbed to the surface of MWCNTs / GONRs, resulting in fluorescence quenching, thus showing lower fluorescence intensity. When the target DNA was added, hybridization and co...

Embodiment 3

[0046] FRET sensor based on MWCNTs / GONRs-NSGQDs-ssDNA for detection of transgenic soybean:

[0047] The DNA of transgenic soybeans was extracted using a plant DNA extraction kit. Add 4 μL of 1.5 mg / mL MWCNTs / GONRs dispersion to 3.0 mL NSGQDs-ssDNA, sonicate for 5 min, and after standing for 5 min, add 20 μL of transgenic soybean DNA into MWCNTs / GONRs and NSGQDs-ssDNA FRET sensing system, Determine the fluorescence recovery intensity and compare it with the standard curve to draw a conclusion whether the CaMV35S promoter is contained, and the concentration of the CaMV35S promoter contained in the transgenic soybean can be calculated according to the fluorescence intensity.

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Abstract

The invention provides a method for constructing a fluorescence resonance energy transfer (FRET) sensor based on graphene and quantum dots and a method for detecting a CaMV35S promoter by means of the sensor. The method comprises the steps of 1, preparing multiwalled carbon nanotubes / graphene oxide nanoribbons (MWCNTs / GONRs) dispersion liquid; 2, preparing nitrogen sulfur heterotactic graphene quantum dots (NSGQDs) solution; 3, preparing nitrogen sulfur heterotactic graphene quantum dots-capture probe (NSGQDs-ssDNA) dispersion liquid; 4, constructing the FRET sensor; 5, constructing a standard curve; 6, detecting transgenic soybean by means of the FRET sensor constructed based on MWCNTs / GONRs-NSGQDs-ssDNA. The FRET sensor has the advantages of being convenient and flexible to operate and high in analysis speed and is suitable for quick detection and analysis.

Description

technical field [0001] The invention belongs to the field of material preparation and fluorescence resonance energy transfer, in particular to the construction of a graphene and quantum dot-based fluorescence resonance energy transfer sensor and its detection method for CaMV35S promoter. 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. At present, the detection methods of GMO mainly include enzyme-linked immunosorbent assay (ELISA), immunoassay strip method, protein chip, polymerase chain reaction (PCR) method and so on. However, protein-based detection methods have high background signals, difficulty in maintaining protein activity for a long time, high cost of developing specific antibodies, and denatured proteins of processed tr...

Claims

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Application Information

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IPC IPC(8): G01N21/64
CPCG01N21/6486
Inventor 李雅琪王坤蔡健荣孙力
Owner JIANGSU UNIV
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