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 prep

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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
Comparison scheme
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Example Embodiment

[0033] Example 1:

[0034] (1) Preparation of MWCNTs / GONRs

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

[0036] (2) Preparation of NSGQDs

[0037] One-step preparation of nitrogen-sulfur homoheterographene quantum dots (NSGQDs) by thermal pyrolysis under normal pressure. After mixing 0.5g of amm...

Example Embodiment

[0042] Example 2: Construction of tDNA standard curve by the FRET sensor

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

[0044] figure 1 In order to detect the standard curve diagram of different concentrations of CaMV35S target DNA, the curves a to k are in a bottom-up order, and the target DNA concentration is 0,0.1,0.5,1,5,10,50,100,200,400,500nM. Curve a indicates that when no target DNA is present, NSGQDs are adsorbed to the surface of MWCNTs / GONRs, resulting in fluorescence quenching, thereby showing lower fluorescence intensity. When the target DNA is added, hybridization and complementary pairing with NSGQDs occurs, so ...

Example Embodiment

[0045] Example 3:

[0046] FRET sensor based on MWCNTs / GONRs-NSGQDs-ssDNA to detect genetically modified soybeans:

[0047] The plant DNA extraction kit was used to extract the DNA of genetically modified soybeans. Add 4 μL of 1.5 mg / mL MWCNTs / GONRs dispersion to 3.0 mL of NSGQDs-ssDNA, sonicate for 5 minutes, and after standing for 5 minutes, add 20 μL of genetically modified soybean DNA to the MWCNTs / GONRs and NSGQDs-ssDNA FRET sensor systems. The fluorescence recovery intensity was measured and compared with the standard curve to determine whether it contained the CaMV35S promoter, and the concentration of the CaMV35S promoter contained in the transgenic soybeans could be calculated based on 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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