CN112255212A - Method for detecting H5N1 influenza A virus hemagglutinin - Google Patents
Method for detecting H5N1 influenza A virus hemagglutinin Download PDFInfo
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Abstract
The invention discloses a method for detecting H5N1 influenza A virus hemagglutinin, which comprises the steps of carrying out water-soluble modification on rare earth doped up-conversion luminescent nanoparticles; completing the coupling of the H5N1 HA aptamer on the surface of the nanoparticle to obtain an up-conversion fluorescent probe; making a standard curve; preparing a detection system of a sample to be detected, and measuring the relative fluorescence intensity of the sample to be detected; obtaining the concentration of H5N1 HA in the sample to be detected according to the standard curve; the detection method is rapid, simple and convenient and stable in performance, and the identification element aptamer changes the self spatial structure through the specific identification effect to realize the recovery of the fluorescent signal of the detection system. The fluorescence acceptor graphene oxide has a wide fluorescence absorption range, and can realize green up-conversion fluorescence signal quenching. The detection system can reduce background signals to a great extent and improve detection sensitivity. The homogeneous washing-free detection system can realize high-efficiency detection of the H5N1 influenza A virus.
Description
Technical Field
The invention belongs to the technical field of biosensing, and particularly relates to a method for detecting H5N1 influenza A virus hemagglutinin.
Background
Influenza a viruses are important pathogens causing seasonal influenza, which frequently presents a huge challenge to global public health. Influenza viruses often acquire a new antigenicity, i.e. antigen transfer, which leads to the appearance of a new viral strain, which is universally devoid of corresponding immunity in the human population, thus leading to an influenza pandemic; among them, hemagglutinin HA of influenza virus binds to host surface-specific sialic acid sugar receptor and plays a key role in the virulence of virus, so HA is suitable as a target for detection. The rapid and accurate diagnosis of influenza virus infection is crucial to effectively controlling epidemic situations and reducing morbidity and mortality.
In order to realize effective prevention and control of virus diseases, development of a corresponding high-sensitivity and rapid influenza a virus detection technology is urgently needed. At present, the methods for detecting and determining diagnosis at home and abroad can be classified into etiology, serology and molecular biology diagnosis technologies. The separation and culture of influenza virus is the traditional etiology diagnosis technology, which is considered as the most classical and rigorous method for identifying the influenza virus, the process usually needs 2-14 days, and the defects are that the operation steps are complicated, the period is long, and the biological safety is low. The serological detection method has low antibody titer at the initial stage of disease attack of a patient, and the antibody titer can be obviously improved after two weeks, so the method is only suitable for epidemiological investigation and research and cannot be used as an early diagnosis method. The enzyme-linked immunosorbent assay is a detection technology for detecting an antibody or an antigen by using an enzyme-labeled antigen or antibody and a chromogenic reaction, and has the advantages of easy analysis of a detection result, safety, high efficiency and low cost, but a false positive result may occur, and a serum subtype cannot be distinguished. With the development of scientific technology, molecular biology methods based on isothermal amplification technology can be applied to on-site rapid detection and screening of influenza viruses, but these isothermal amplification technologies all require the participation of enzymes, and have the problems of expensive reagent price, complex reaction system, strict reagent storage conditions and the like.
Therefore, a rapid, simple and convenient detection method with a simple reaction system, which is suitable for rapid detection and diagnosis of the H5N1 influenza A virus, is urgently needed to be developed.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a method for detecting H5N1 influenza A virus hemagglutinin.
The technical scheme of the invention is summarized as follows:
a method for detecting H5N1 influenza a virus hemagglutinin, comprising the steps of:
(1) performing water-soluble modification on the rare earth-doped upconversion luminescent nanoparticles by adopting a surface ligand exchange method and using polyacrylic acid;
(2) coupling H5N1 HA aptamer on the surface of the nanoparticle obtained in the step (1) by adopting a condensation reaction to obtain an up-conversion fluorescent probe;
(3) dispersing 100-200 mu g of up-conversion fluorescent probe in 100-200 mu l of borate buffer solution with the pH value of 7.8; respectively adding known H5N1 HA standard substances with different final concentrations, incubating at 20-40 deg.C for 20-60 min, respectively adding graphene oxide with a final concentration of 50-300 μ g/ml, incubating at 20-40 deg.C for 20 min, diluting to 300 μ l with borate buffer solution with pH of 7.8, respectively measuring fluorescence value F of 542nm emission wavelength at 980nm excitation wavelength, and simultaneously measuring blank fluorescence value F0The relative fluorescence intensity [ (F-F)0)/F0]A standard curve corresponding to H5N1 HA concentration;
(4) preparing a detection system of a sample to be detected, and measuring the relative fluorescence intensity of the sample to be detected; obtaining the concentration of H5N1 HA in the sample to be detected according to the standard curve;
H5N1 HA is an abbreviation for H5N1 influenza a virus hemagglutinin.
The particle diameter of the rare earth doped up-conversion luminescence nano-particles is 17-20 nm.
The H5N1 HA aptamer is 5' -NH2-TTGGGGTTATTTGGGAGGGCGGGGGTT-3', wherein the nucleotide sequence is shown by SEQ ID No. 1.
The sheet diameter of the graphene oxide is 0.5 nm-2 mu m.
The invention has the advantages that:
the detection method of the invention realizes the high-efficiency detection of the hemagglutinin by utilizing the principle of fluorescence resonance energy transfer. The method is rapid, simple and convenient, has stable performance, changes the self space structure of the identification original aptamer through the specific identification effect, and realizes the recovery of the fluorescence signal of the detection system. The fluorescence acceptor graphene oxide has a wide fluorescence absorption range, and can realize green up-conversion fluorescence signal quenching. The detection system can reduce background signals to a great extent and improve detection sensitivity. The homogeneous washing-free detection system can realize high-efficiency detection of H5N1 influenza A virus, does not need separation in the detection process, is simple to operate, can realize high-sensitivity and high-specificity detection of hemagglutinin, and has important significance in the aspect of quick detection of clinical samples of the influenza A virus.
Drawings
FIG. 1 is a Fourier infrared spectrum before and after modification of an upconversion luminescent nanoparticle;
FIG. 2 is a standard curve of H5N1 HA concentration versus the corresponding relative fluorescence intensity.
Detailed Description
Reagents and instruments used in this experiment
Reagent:
H5N1 influenza A virus (A/Anhui/1/2005) Hemagglutin Protein was purchased from Beijing Yi Qian Shen science and technology, Inc.;
amino-modified HA aptamers 5' -NH2-TTGGGGTTATTTGGGAGGGCGGGGGTT-3' is supplied by Biotechnology engineering (Shanghai) Inc.
Polyacrylic acid, MES, EDC, NHS was purchased from Sigma-Aldrich.
Graphene oxide is provided by Nanjing Xiapong materials technology, Inc.
The remaining chemicals were obtained from the national pharmaceutical group chemical reagents, Inc., and all aqueous solutions were purified by Milli-Q biocel with ultra pure water.
The instrument comprises the following steps:
the near infrared and up-conversion steady-state spectrum of the nano material is tested by an FLS980 type steady-state fluorescence spectrometer, and a 980nm fiber laser is used as an external excitation light source.
In each example H5N1 HA is an abbreviation for H5N1 influenza a virus hemagglutinin.
The present invention will be described in further detail with reference to specific examples, but the present invention is not limited to these specific examples.
Example 1
A method for detecting H5N1 influenza a virus hemagglutinin, comprising the steps of:
(1) adopting a surface ligand exchange method, and using polyacrylic acid to perform water-soluble modification on the rare earth doped up-conversion luminescent nano-particles:
a, preparing rare earth doped up-conversion luminescent nano particles:
weighing 2mmol of CF3COONa and 1.56 mmoleY (CF)3COO)3，0.4mmolYb(CF3 COO)3，0.04mmolEr(CF3COO)3Adding into a three-necked bottle, adding 10mmol oleic acid, 10mmol oleylamine and 20mmol octadecene, heating to 100 deg.C, and stirring under vacuum for 30 min; heating to 300 deg.C, and maintaining under nitrogen for 1 hr; cooling to room temperature, adding 25ml of ethanol, centrifuging at 8000rpm for 10 minutes to obtain a precipitate of rare earth doped up-conversion luminescent nanoparticles (the particle diameter is 17-20 nm) (the up-conversion luminescent nanoparticles before modification), and dispersing the precipitate in 15ml of cyclohexane;
b, using polyacrylic acid to perform water-soluble modification on the rare earth doped up-conversion luminescent nanoparticles:
mixing 5ml of the dispersion obtained in the step a with 5ml of N, N-dimethylformamide, adding 50mg of nitrosotetrafluoroborate, stirring for 15 minutes, adding 20ml of toluene, centrifuging at 12000rpm for 10 minutes, adding the precipitate into 5ml of N, N-dimethylformamide, performing ultrasonic dispersion, adding 50mg of polyacrylic acid, stirring for 12 hours, centrifuging to obtain a precipitate of the water-soluble modified rare earth-doped upconversion luminescent nanoparticles (modified upconversion luminescent nanoparticles), and dispersing the precipitate into 10ml of water for later use, as shown in FIG. 1;
(2) coupling H5N1 HA aptamer on the surface of the nanoparticle obtained in the step (1) by adopting a condensation reaction to obtain an upconversion fluorescent probe:
taking 0.5ml of the dispersion obtained in step (1), adding 1ml of MES buffer solution with pH 5.6, adding 2mg of EDC and 4mg of Sulfo-NHS, incubating for 20 minutes, and adding 4ml of 10mM borate buffer solution with pH 7.8 to obtain solution one; adding 10 μ l of 100 μ M H5N1 HA aptamer (solvent is TE buffer) to the first solution, reacting for 4 hours, centrifuging to obtain an upconverting fluorescent probe precipitate, washing with ultrapure water, centrifuging, and dispersing the precipitate in 2ml of 10mM borate buffer at pH 7.8;
EDC is an abbreviation for 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride;
the Sulfo-NHS is an abbreviation of N-hydroxy thiosuccinimide;
the H5N1 HA aptamer is 5' -NH2-TTGGGGTTATTTGGGAGGGCGGGGGTT-3', wherein the nucleotide sequence is shown by SEQ ID No. 1;
(3) dispersing 150 μ g of the up-conversion fluorescent probe in 150 μ l of a 10mM borate buffer solution having a pH of 7.8 to obtain a dispersion, and preparing 10 parts in total of the dispersion; adding solutions (solvent is 10mM, pH 7.4PBS) of H5N1 HA standard (0, 1, 2, 5, 10, 20, 50, 80, 100, 150ng/ml) with different final concentrations, incubating at 37 deg.C for 40 min, adding graphene oxide (sheet diameter of graphene oxide is 1 μm) with final concentration of 150 μ g/ml, incubating at 37 deg.C for 20 min, diluting to 300 μ l with borate buffer solution with pH 7.8, measuring fluorescence F of 542nm emission wavelength at 980nm excitation wavelength, repeating the steps three times for each sample, and measuring blank fluorescence F0The relative fluorescence intensity [ (F-F)0)/F0]A standard curve corresponding to H5N1 HA concentration; as shown in FIG. 2, a linear relationship was found in the range of 0-80ng/ml by linear fitting, and the linear equation was Y-0.5965 log (x) +0.0061R20.9952, the detection limit (S/N3) was 0.174 ng/mL.
(4) A test system of a sample to be tested was prepared by dispersing 150 μ g of the up-conversion fluorescent probe in 150 μ l of 10mM borate buffer solution having pH of 7.8, adding 10 μ l of a human serum sample containing H5N1 HA (final concentration of H5N1 HA was 2, 5, 10ng/ml, respectively), incubating at 37 ℃ for 40 minutes, adding graphene oxide having a final concentration of 150 μ g/ml (sheet diameter of graphene oxide was 1 μm), incubating at 37 ℃ for 20 minutes, fixing the volume to 300 μ l with a borate buffer solution having pH of 7.8, measuring a fluorescence value F at an emission wavelength of 542nm at an excitation wavelength of 980nm, measuring the relative fluorescence intensity of the sample to be tested (serum sample), and obtaining the concentration of H5N1 HA in the sample to be tested (serum sample) according to the standard curve of step (3), as shown in table 1.
TABLE 1 measurement results of H5N1 HA in practical samples
Example 2
A method for detecting H5N1 influenza a virus hemagglutinin, comprising the steps of:
steps (1) and (2) were the same as Steps (1) and (2) of example 1;
(3) 100. mu.g of the upconverting fluorescent probe was dispersed in 100. mu.l of 10mM borate buffer pH 7.8; obtaining dispersion liquid, and preparing 10 parts of dispersion liquid in total; adding H5N1 HA standards (0, 1, 2, 5, 10, 20, 50, 80, 100, 150ng/ml) solutions (solvent 10mM, pH 7.4PBS) at different concentrations, incubating at 20 deg.C for 60 min, adding graphene oxide (with a final concentration of 50 μ g/ml) (with a sheet diameter of 0.5nm) at 20 deg.C for 20 min, diluting to 300 μ l with borate buffer solution with pH 7.8, measuring fluorescence F at 980nm excitation wavelength and 542nm emission wavelength, and measuring blank fluorescence F0The relative fluorescence intensity [ (F-F)0)/F0]A standard curve corresponding to H5N1 HA concentration;
(4) 100 μ g of the up-conversion fluorescent probe was dispersed in 100 μ l of 10mM borate buffer solution with pH 7.8, 20 μ l of human serum sample containing H5N1 HA was added, incubated at 20 ℃ for 60 minutes, graphene oxide (graphene oxide having a sheet diameter of 0.5 μm) was added to a final concentration of 50 μ g/ml, incubated at 20 ℃ for 20 minutes, fixed to 300 μ l with borate buffer solution with pH 7.8, the fluorescence value F at an emission wavelength of 542nm when excited at 980nm was measured, and the concentration of H5N1 HA in the test solution was obtained from the measured relative fluorescence intensity of the test sample according to the standard curve of step (3).
Example 3
A method for detecting H5N1 influenza a virus hemagglutinin, comprising the steps of:
steps (1) and (2) were the same as Steps (1) and (2) of example 1;
(3) taking 200 μ g of the powder to be turned upThe fluorogenic probe was dispersed in 200. mu.l of 10mM borate buffer pH 7.8; obtaining dispersion liquid, and preparing 10 parts of dispersion liquid in total; H5N1 HA standards (0, 1, 2, 5, 10, 20, 50, 80, 100, 150ng/ml) solutions (solvent 10mM, pH 7.4PBS) were added at different concentrations, incubated at 40 deg.C for 20 minutes, graphene oxide (graphene oxide sheet diameter 2 μm) was added at a final concentration of 300 μ g/ml, incubated at 40 deg.C for 20 minutes, diluted to 300 μ l with borate buffer solution at pH 7.8, fluorescence F at an excitation wavelength of 980nm and at an emission wavelength of 542nm was measured, and blank fluorescence F was measured0The relative fluorescence intensity [ (F-F)0)/F0]A standard curve corresponding to H5N1 HA concentration;
(4) the method comprises the steps of dispersing 200 μ g of the up-conversion fluorescent probe in 200 μ l of 10mM borate buffer solution with pH of 7.8, adding 20 μ l of a human serum sample containing H5N1 HA, incubating at 40 ℃ for 20 minutes, adding graphene oxide (the sheet diameter of the graphene oxide is 2 μm) with a final concentration of 300 μ g/ml, incubating at 40 ℃ for 20 minutes, fixing the volume to 300 μ l with the borate buffer solution with pH of 7.8, measuring the fluorescence value F of an emission wavelength at 542nm when the excitation wavelength is 980nm, and obtaining the concentration of H5N1 HA in the solution to be measured according to the standard curve of the step (3) according to the measured relative fluorescence intensity of the sample to be measured.
Sequence listing
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<120> method for detecting H5N1 influenza A virus hemagglutinin
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<213> Artificial Sequence (Artificial Sequence)
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Claims (4)
1. A method for detecting hemagglutinin of an influenza a virus of H5N1, comprising the steps of:
(1) performing water-soluble modification on the rare earth-doped upconversion luminescent nanoparticles by adopting a surface ligand exchange method and using polyacrylic acid;
(2) coupling H5N1 HA aptamer on the surface of the nanoparticle obtained in the step (1) by adopting a condensation reaction to obtain an up-conversion fluorescent probe;
(3) dispersing 100-200 mu g of up-conversion fluorescent probe in 100-200 mu l of borate buffer solution with the pH value of 7.8; respectively adding known H5N1 HA standard substances with different final concentrations, incubating at 20-40 deg.C for 20-60 min, respectively adding graphene oxide with a final concentration of 50-300 μ g/ml, incubating at 20-40 deg.C for 20 min, diluting to 300 μ l with borate buffer solution with pH of 7.8, respectively measuring fluorescence value F of 542nm emission wavelength at 980nm excitation wavelength, and simultaneously measuring blank fluorescence value F0The relative fluorescence intensity [ (F-F)0)/F0]A standard curve corresponding to H5N1 HA concentration;
(4) preparing a detection system of a sample to be detected, and measuring the relative fluorescence intensity of the sample to be detected; obtaining the concentration of H5N1 HA in the sample to be detected according to the standard curve;
H5N1 HA is an abbreviation for H5N1 influenza a virus hemagglutinin.
2. The method as set forth in claim 1, wherein the rare earth-doped upconversion luminescent nanoparticles have a particle size of 17 to 20 nm.
3. The method of claim 1, wherein said H5N1 HA aptamer is 5' -NH2-TTGGGGTTATTTGGGAGGGCGGGGGTT-3', wherein the nucleotide sequence is shown by SEQ ID No. 1.
4. The method according to claim 1, wherein the graphene oxide has a sheet diameter of 0.5nm to 2 μm.
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