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A method for quantitative detection of biological molecules capable of being metabolized to generate H2O2 in serum by means of a ratiometric fluorescent probe

A ratio fluorescent probe, H2O2 technology, applied in the field of fluorescence ratio technology and bioanalysis detection, nanomaterials

Active Publication Date: 2017-12-15
NANJING MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, copper-nitrogen co-doped carbon dots can be used in fluorescence ratiometric measurement to produce H 2 o 2 The research on the construction of biomolecular assay platform has not been reported yet

Method used

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  • A method for quantitative detection of biological molecules capable of being metabolized to generate H2O2 in serum by means of a ratiometric fluorescent probe
  • A method for quantitative detection of biological molecules capable of being metabolized to generate H2O2 in serum by means of a ratiometric fluorescent probe
  • A method for quantitative detection of biological molecules capable of being metabolized to generate H2O2 in serum by means of a ratiometric fluorescent probe

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Experimental program
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Effect test

Embodiment 1

[0027] Example 1 Preparation of copper-nitrogen co-doped carbon dots (Cu-CDs)

[0028] Dissolve 1.2g of citric acid monohydrate and 0.15g of copper acetate monohydrate in 20mL of deionized water, sonicate for 15min to fully dissolve; add 0.15mL of diethylenetriamine to the mixed solution in the previous step, and sonicate for 15min to fully mix ;Transfer the above solution to a 30mL autoclave, and react at 230°C for 12h; after the reaction is completed, cool it to room temperature, take out the product and add 0.1M NaOH to adjust the pH to about 7.0, dialyze with a 1000Da dialysis bag for 72h, and replace the dialysate at an interval of about 3-6 hours; after the dialysis is completed, the product is distilled and concentrated under reduced pressure to obtain a dark brown solution, dried in vacuum at 60°C to obtain a brown powder, which is sealed and stored for later use. Its TEM and XPS pictures are as follows figure 1 , 2 As shown, its fluorescence and UV absorption diagra...

Embodiment 2

[0030] Embodiment 2 Take cholesterol as an example to illustrate the detection method of the present invention

[0031] 1. Catalyze the reaction of cholesterol to generate H 2 o 2 , and further catalyzes H 2 o 2 React with OPD to generate yellow fluorescent DAP, the steps are as follows: Take 1 mL of cholesterol with different concentrations (0, 0.05, 0.1, 0.25, 1.0, 2.5, 10, 30, 75, 100, 150, 250, 400, 600, 800, 1000 μM ), add 10 μL of cholesterol oxidase (concentration: 5.0 mg / mL), shake and mix thoroughly; take out 200 μL of the mixed solution into a 2 mL EP tube, add 200 μL of 14 mM OPD (containing 10 μg / mL HRP), mix well, and react at 37 °C for 30 min. The pH of the solution is 6.6, and it is prepared with a 20mM phosphate buffer system.

[0032] 2. Add Cu-CDs and DAP to form ratio fluorescence, and quantitatively measure the amount of biomolecule cholesterol in serum: the concentration of Cu-CDs is 200 μg / mL, and it is prepared with 20 mM, pH=6.6 phosphate buffer sys...

Embodiment 3

[0033] Embodiment 3 Take xanthine as an example to illustrate the detection method of the present invention:

[0034] 1. Catalyze the reaction of xanthine to generate H 2 o 2 , and further catalyzes H 2 o 2 React with OPD to generate yellow fluorescent DAP, the steps are as follows: take different concentrations of xanthine (0, 0.25, 0.75, 1.5, 5, 15, 30, 75, 150, 250, 400, 600, 800, 1000μM) 1mL, add 10μL Bilixanthine oxidase (concentration: 5.0 mg / mL), shake and mix thoroughly; take 200 μL of the mixed solution into a 2 mL EP tube, add 200 μL of 14 mM OPD (containing 10 μg / mL HRP), mix well, and react at 37 ° C for 30 min. The pH of the solution is 6.6, and it is configured with a 20mM phosphate buffer system.

[0035] 2. Add Cu-CDs and DAP to form ratio fluorescence, quantitatively measure biomolecule xanthine in serum, the remaining steps are the same as in Example 2, see the results Figure 7 , 8 shown.

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Abstract

The invention discloses a method for quantitative detection of biological molecules capable of being metabolized to generate H2O2 in serum by means of a ratiometric fluorescent probe. The method includes firstly preparing copper-nitrogen codoped carbon dots Cu-CDs; then reacting corresponding oxidase and the biological molecules capable of being metabolized to generate H2O2 to generate H2O2; catalyzing the H2O2 and a substrate that is o-phenylenediamine with horseradish peroxidase to generate an oxidation product DAP having yellow fluorescence; then adding the Cu-CDs into the DAP; allowing the DAP and the Cu-CDs to form ratiometric fluorescence, with the ratio I<572> / I<460> of fluorescence intensities of the DAP and the Cu-CDs being in a linear relationship with the concentration of a substance to be detected; and performing quantitative assay of the biological molecules in the serum according to the ratio of fluorescence intensities. The method is high in sensitivity and simple and convenient in detection, and has high selectivity and high affinity of immunoreactions.

Description

technical field [0001] The invention belongs to the fields of nanomaterials, fluorescence ratio technology and biological analysis and detection, and specifically relates to a ratio fluorescent probe based on a novel copper-nitrogen co-doped carbon dot that can metabolize quantitatively to produce H 2 o 2 Construction of assay platforms for biomolecules. Background technique [0002] As a new member of the functional nanocarbon family, carbon dots have attracted a great deal of research due to their chemical inertness, good solvent dispersibility, excellent light absorption, and low toxicity. Fluorescent carbon dots are used in a wide range of fields, including biological imaging, medical diagnosis, and in vivo disappearance. Previously, in order to solve the shortcomings of low quantum yield of carbon dots, methods of surface passivation, functionalization and doping with other elements such as N and S were proposed. This method of impurity doping to optimize the perform...

Claims

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Application Information

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IPC IPC(8): G01N21/64
CPCG01N21/6428G01N2021/6432
Inventor 胡琴马云苏许贯虹魏芳弟岑瑶
Owner NANJING MEDICAL UNIV
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