Method for detecting upconversion fluorescence resonance energy transfer by using carbon nanomaterial as receptor

A fluorescence resonance energy, carbon nanomaterial technology, applied in luminescent materials, fluorescence/phosphorescence, material excitation analysis, etc., can solve the problems of affecting analysis and detection sensitivity, expensive instruments, and high detection costs, achieving excellent quenching ability, preparation Simple method and high sensitivity

Inactive Publication Date: 2014-01-01
GUANGZHOU IMPROVE MEDICAL TECH CO LTD
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Problems solved by technology

[0004] (1) ELISA is a heterogeneous analysis method that requires a washing and separation process, which is cumbersome and time-consuming. At the same time, ELISA also needs to use proteins such as antigens and antibodies, which is expensive and the detection cost is high;
[0005] (2) Down-converting fluorescence immunoassay can overcome the shortcomings of heterogeneous analysis requiring washing and separation, but it cannot avoid background interference and light scattering of biological samples, which limits its application in the analysis of complex blood samples;
[0006] (3) Time-resolved fluorescence immunoassay can not only overcome the shortcomings of heterogene...

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  • Method for detecting upconversion fluorescence resonance energy transfer by using carbon nanomaterial as receptor
  • Method for detecting upconversion fluorescence resonance energy transfer by using carbon nanomaterial as receptor
  • Method for detecting upconversion fluorescence resonance energy transfer by using carbon nanomaterial as receptor

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preparation example Construction

[0041] (1) Preparation of carbon nanoparticles: Weigh 8mg of candle ash and disperse in 20ml of mixed solvent (the volume ratio of ethanol and water is 1:1), sonicate for 5h, centrifuge at 3000rpm for 2min to remove large-sized particles, collect the supernatant, 6000rpm Centrifuge for 6 minutes to get 2 mg of black precipitate, which is carbon nanoparticles, and its characterization spectrum is shown in image 3 , where A is a transmission electron microscope image, and B is an ultraviolet-visible absorption spectrum; 2 mg of carbon nanoparticles were dispersed in 20 ml of solvent, and ultrasonically dispersed for 2 hours to obtain a black carbon nanoparticle solution with a mass fraction of 0.1 mg / ml.

[0042] (2) Preparation of graphene oxide (GO): Take 50mL concentrated sulfuric acid (concentration greater than or equal to 70%) and heat it to 90°C, add 10g K 2 S 2 o 8 and 10g P 2 o 5 , cooled to 80°C, until K 2 S 2 o 8 and P 2 o 5 After completely dissolving, slow...

Embodiment 1

[0045] Take 2mg / ml up-converting fluorescent nanomaterial-single-stranded nucleic acid (UCPs-ssDNA) solution 6uL, add different volumes of 0.1mg / mL 0.02% (wt) sodium dodecylbenzenesulfonate dispersed carbon nano Particle aqueous solution (SDBS-CNPs), add Tris-HCl (10mM, 150mM NaCl, pH7.4) buffer solution to a total volume of 400uL to obtain 8 groups of mixed solutions, the concentration of UCPs-ssDNA in each mixed solution is 0.03mg / mL , the concentrations of SDBS-CNPs were 0, 0.006, 0.012, 0.02, 0.03, 0.036, 0.04, 0.05mg / mL, and after incubation at 25°C for 1.5h, the upconversion fluorescence intensity was measured under a 980nm laser. When the concentration of SDBS-CNPs was 0.036 mg / ml corresponding to the fluorescence quenching efficiency of the mixture reached the maximum. Take 6 groups of mixtures containing 0.03mg / ml UCPs-ssDNA and 0.036mg / ml SDBS-CNPs and incubate at 25°C for 1.5h, then use one of them as a blank sample, and add different volumes of thrombin and serum t...

Embodiment 2

[0048] Take 5.6uL of 2mg / ml up-converting fluorescent nanomaterial-polypeptide (UCPs-peptide) solution, and add different volumes of 0.1mg / mL0.05% (wt) Triton-dispersed carbon nanoparticle aqueous solution (TritonX-100 -CNPs), add TCNB (pH7.5, 50mM Tris, 10mM CaCl 2 , 150mM NaCl, 0.05%Brij) buffer to a total volume of 400uL to obtain 8 sets of mixed solutions, the concentrations of UCPs-peptide in each mixed solution were 0.028mg / mL, and the concentrations of Triton X-100-CNPs were 0, 0.02, 0.03 , 0.04, 0.05, 0.06, 0.07 and 0.08mg / mL, after incubation at 20°C for 2 hours, measure the upconversion fluorescence intensity under 980nm laser, when the concentration of Triton X-100-CNPs is 0.05mg / ml, the corresponding fluorescence of the mixture The quenching efficiency reaches a maximum. Take 7 groups of mixtures containing 0.028mg / ml UCPs-peptide and 0.05mg / ml Triton X-100-CNPs and incubate at 25°C for 1.5h, then use one group as a blank sample, and add different volumes of human...

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Abstract

The invention discloses a method for detecting fluorescence resonance energy transfer by using a water-soluble upconversion fluorescence nanomaterial as a fluorescence donor and using a carbon nanomaterial as a fluorescence receptor. The method comprises the following specific steps: (1) preparing the water-soluble upconversion fluorescence nanomaterial and performing surface marker to obtain a fluorescence donor solution; (2) preparing the carbon nanomaterial to obtain a fluorescence receptor solution; (3) mixing the fluorescence donor solution and the fluorescence receptor solution for incubation and measuring the fluorescence intensity to obtain a fluorescence quenching curve; (4) mixing certain-concentration fluorescence donor solution and certain-concentration fluorescence receptor solution for incubation, adding a target object with different concentrations for continuous incubation, measuring the fluorescence intensity and drawing a standard curve; (5) calculating the concentration of the target object in an actual sample according to the standard curve. According to the method, interference of the background fluorescence of a biological sample can be avoided, detection to serum or the target object in a whole blood sample can be directly realized, the washing and separation processes are not needed, the detection speed is high, and the cost is low.

Description

technical field [0001] The invention belongs to the technical field of up-conversion fluorescence resonance energy transfer detection, and specifically relates to an up-conversion fluorescence resonance energy transfer detection method using carbon nanomaterials as acceptors. Background technique [0002] At present, there are two main detection methods for biomolecules in biological samples: heterogeneous analysis and homogeneous analysis. The existing heterogeneous analysis methods mainly include enzyme-linked immunoassay, chemiluminescence immunoassay, and radioimmunoassay. Among them, the enzyme-linked immunoassay is an analysis based on the solid phase of the antigen or antibody and the enzyme labeling of the antigen or antibody. After adding the substrate of the enzyme reaction, the substrate is catalyzed by the enzyme into a colored product. Qualitative or quantitative analysis of the relationship between the tested substances. The existing homogeneous analysis metho...

Claims

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Application Information

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IPC IPC(8): G01N21/64C09K11/06
Inventor 刘志洪杨利何梦媛吴正俊汤志恺王宇辉
Owner GUANGZHOU IMPROVE MEDICAL TECH CO LTD
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