Carbon quantum dot fluorescent probe based on plant Xanthium sibiricum and preparation method and application thereof

Abstract

The present application discloses a carbon quantum dot fluorescent probe based on the plant Xanthium sibiricum, and its preparation method and application. The preparation method comprises: preparing carbon quantum dots from the plant Xanthium sibiricum; purifying the carbon quantum dots to prepare a probe with high fluorescence performance. Using the carbon quantum dot fluorescent probe for the detection of Art v 1, an allergic protein of Artemisia plants, comprises: scanning the fluorescence spectrum of the carbon quantum dot probe solution within an excitation wavelength range of 280 to 580 nm and determining the detection wavelength according to the fluorescence intensity; mixing the sample solution to be detected with the carbon quantum dot fluorescent probe solution, and performing fluorescence analysis at the detection wavelength to determine the content of the allergic protein Art v 1. The present application directly synthesizes carbon quantum dots using the plant Xanthium sibiricum as a precursor without adding other substances, and the carbon quantum dots can be used as a nano fluorescent probe to detect the protein Art v 1. The preparation method and the detection process require low cost, are green and environmentally friendly, and are quick and simple. It is an Art v 1 detection method that has both convenience and practical application prospects.

Description

Technical Field

[0001] The present application belongs to the field of fluorescent material preparation, and specifically relates to a carbon quantum dot fluorescent probe based on the plant Xanthium sibiricum and a preparation method and application thereof. Background Art

[0002] Epidemiological surveys show that the prevalence of allergies in the Chinese population has increased significantly in the past three decades, and allergies have been considered one of the main factors causing asthma. It not only affects people's quality of life, but also brings huge socioeconomic burdens, and is becoming a global health problem. Surveys show that among the main pollen types that cause seasonal allergic asthma in China, Artemisia pollen has the highest association index. Among them, Art v 1, as a defensin-like protein, is regarded as a signature protein for Artemisia pollen allergy. More than 95% of Artemisia plant allergy patients react to the main allergen Art v 1. In clinical diagnosis and desensitization treatment, it is crucial to accurately measure the content of each allergen in pollen extracts.

[0003] Carbon quantum dots are a newly discovered zero-dimensional nanomaterial in recent years. They are widely used to detect a variety of substances including metal ions, small molecules and biomacromolecules, such as Fe, due to their good chemical inertness and stability, resistance to photobleaching, excellent biocompatibility, and adjustable excitation and emission spectra. 3+ , Hg 2+ , lysozyme, ascorbic acid, DNA, etc. In addition, carbon quantum dots as fluorescent probes have the advantages of good water solubility, low price, low toxicity and low pollution, which makes carbon quantum dots widely used in many fields.

[0004] In the prior art, the preparation methods of carbon quantum dots are divided into two categories: top-down and bottom-up. The top-down methods include: arc discharge smoke, laser ablation of carbon targets, electrochemical impact of CNTs, electrochemical stripping of graphite, etc., but the materials prepared by these methods have poor fluorescence properties and usually require further modification or passivation with toxic organic small molecules or polymers. The process is cumbersome and not environmentally friendly; the bottom-up methods include thermal or solvent thermal synthesis, electrochemical methods, microwave-assisted synthesis, etc. These traditional synthesis methods usually require toxic chemical reagents or organic solvents as precursors. Therefore, using biomass materials to replace organic compound precursors and developing an efficient synthesis technology that does not require any external energy supply to prepare fluorescent carbon quantum dot materials are of great significance and broad prospects for meeting the needs of efficient, rapid, green and environmentally friendly fluorescence analysis. Summary of the invention

[0005] The present application aims to provide a carbon quantum dot fluorescent probe based on the plant Xanthium sibiricum, and a preparation method and application thereof. The raw material plant Xanthium sibiricum is widely available, inexpensive, green and environmentally friendly, with a simple preparation process and high preparation efficiency. At the same time, the synthesized carbon quantum dots have good fluorescence properties and are used for allergy protein detection, which has the advantages of simple, fast and sensitive detection methods, and solves the problems of cumbersome steps and high costs of existing allergen detection and quantification methods.

[0006] In order to achieve the aforementioned application objectives, this application is implemented by the following technical solutions:

[0007] The method for preparing the carbon quantum dot fluorescent probe based on plant Xanthium sibiricum comprises the following steps:

[0008] Carbon quantum dots were prepared from the plant Xanthium sibiricum;

[0009] The carbon quantum dots are purified to prepare probes.

[0010] In some embodiments, the preparation process of the carbon quantum dots specifically includes: treating the plant Xanthium sibiricum by hydrothermal synthesis, microwave method or microwave-assisted synthesis method to prepare carbon quantum dots.

[0011] In some embodiments, the hydrothermal synthesis method specifically includes: grinding the plant Xanthium sibiricum into powder and dissolving it in ultrapure water to obtain a mixed solution, and then placing the mixed solution in a high-pressure reactor for hydrothermal reaction.

[0012] The hydrothermal synthesis method is used to synthesize fluorescent carbon dots. The equipment for synthesizing carbon dots is simple, the operation is convenient, and the cycle required for synthesizing carbon dots is short and the efficiency is high. It effectively solves the problems of complicated operation and low preparation efficiency of carbon dot preparation methods in the prior art. At the same time, the reaction liquid only uses pure water as a solvent, and does not require other acidic or alkaline solutions, which avoids many harsh experimental conditions and complicated experimental processes.

[0013] In some embodiments, the microwave-assisted synthesis method specifically comprises: grinding the plant Xanthium sibiricum into powder and dissolving it in ultrapure water to obtain a mixed solution, and then placing the mixed solution in a microwave reactor for reaction.

[0014] In some embodiments, the microwave method specifically comprises: grinding the plant Xanthium sibiricum into powder and dissolving it in ultrapure water to obtain a mixed solution, and then placing the mixed solution in a microwave oven for reaction.

[0015] Compared with the hydrothermal synthesis method, fluorescent carbon dots synthesized by microwave-assisted synthesis or microwave method also have the advantages of being simple, efficient, and not requiring the addition of organic reagents. The reaction solution is further placed in a microwave environment for heating reaction, which has higher synthesis efficiency and better effects.

[0016] In some embodiments, the process of purifying the carbon quantum dots specifically includes: centrifuging the prepared carbon quantum dot solution at high speed, filtering the obtained supernatant and then dialyzing it.

[0017] The present application also provides a carbon quantum dot fluorescent probe, which is prepared by any of the aforementioned methods.

[0018] The present application also provides a method for using a carbon quantum dot fluorescent probe in the detection of Artemisia plant allergy protein Art v 1, which is characterized by comprising:

[0019] Scanning the fluorescence spectrum of the carbon quantum dot probe solution within an excitation wavelength range of 280 to 580 nm and determining the detection wavelength according to the fluorescence intensity;

[0020] The sample solution to be detected is mixed with the carbon quantum dot fluorescent probe solution, and fluorescence analysis is performed at the detection wavelength to determine the content of the allergic protein Art v 1.

[0021] At present, the quantitative detection methods of pollen allergens mainly include enzyme-linked immunosorbent assay (ELISA), mass spectrometry and other molecular biological methods such as PCR. These analytical methods have many problems. For example, although enzyme-linked immunosorbent assay (ELISA), especially double-sandwich ELISA, can successfully detect trace allergens, it requires the cultivation of allergen-specific antibodies, which is a time-consuming process. Mass spectrometry has the advantages of high sensitivity, fast detection speed and short analysis time, but the detection procedure is complicated and requires professional technicians to operate. Fluorescence analysis is highly recommended for its low detection limit, high sensitivity and simple operation process.

[0022] Therefore, compared with the prior art, the advantages of the technical solution provided in the present application are at least that: the preparation process of the provided carbon quantum dot fluorescent probe is simple, the cost of the required raw materials is low and no other toxic reagents need to be added; when the prepared fluorescent carbon dots are used to detect Art v 1, the operation is convenient and fast, the sensitivity is high, and the detection results are reliable, and it has broad application prospects and value in the fields of diagnosis and treatment of allergic diseases, biological detection, etc.

[0023] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0025] Figure 1 is a transmission electron microscope image of carbon quantum dots prepared in Example 1;

[0026] Figure 2 This is the infrared spectrum of carbon quantum dots prepared in Example 1;

[0027] Figure 3 is the fluorescence spectra of the carbon quantum dots prepared in Example 1 at different excitation wavelengths;

[0028] Figure 4 is a comparison chart of the quenching effect of different concentrations of Art v 1 on the fluorescence intensity of carbon quantum dots in Example 8;

[0029] Figure 5 is a linear relationship diagram of the quenching effect of different concentrations of Art v 1 on the fluorescence intensity of carbon quantum dots in Example 8;

[0030] Figure 6 is a linear relationship diagram of the quenching effect of different concentrations of Art v 1 on the fluorescence intensity of carbon quantum dots in Example 10;

[0031] Figure 7 This is a comparison chart of the quenching effects of different proteins and common metal ions on carbon quantum dot solutions in Example 11. DETAILED DESCRIPTION

[0032] The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0033] In order to overcome the problems of the prior art carbon quantum dots, such as complicated preparation process, poor fluorescence performance and generally not environmentally friendly, the present application uses the plant Xanthium sibiricum as a carbon source to prepare a carbon quantum dot-based fluorescent probe, and applies it to the detection of Art v 1.

[0034] The method for preparing the carbon quantum dot fluorescent probe based on plant Xanthium sibiricum comprises the following steps:

[0035] Carbon quantum dots were prepared from the plant Xanthium sibiricum;

[0036] The carbon quantum dots are purified to prepare probes.

[0037] In some embodiments, the preparation process of the carbon quantum dots specifically includes: treating the plant Xanthium sibiricum by hydrothermal synthesis, microwave method or microwave-assisted synthesis method to prepare carbon quantum dots.

[0038] In some embodiments, the hydrothermal synthesis method specifically includes: grinding the plant Xanthium sibiricum into powder and dissolving it in ultrapure water to obtain a mixed solution, and then placing the mixed solution in a high-pressure reactor for hydrothermal reaction.

[0039] In some embodiments, the microwave-assisted synthesis method specifically comprises: grinding the plant Xanthium sibiricum into powder and dissolving it in ultrapure water to obtain a mixed solution, and then placing the mixed solution in a microwave reactor for reaction.

[0040] Furthermore, in the hydrothermal synthesis method and microwave-assisted synthesis method, the mass ratio of the added plant Xanthium sibiricum to ultrapure water is preferably: 1-10:10-100, the reaction temperature is preferably set to 160-400°C, and the reaction time is preferably set to 2-16h.

[0041] Different types of carbon quantum dots were prepared by adjusting the ratio of plant Xanthium sibiricum to ultrapure water. It was found in the experiment that if the ratio of plant Xanthium sibiricum to ultrapure water was too low, the quantum efficiency of the prepared carbon quantum dots would be significantly reduced; while when prepared within the ratio range described in this embodiment, the quantum efficiency was improved to a certain extent. Among them, the highest quantum efficiency can reach about 15%.

[0042] Another important parameter for synthesizing carbon quantum dots is the reaction time. Only within the appropriate reaction time can polymer-like carbon quantum dots be formed to produce fluorescence from the surface / molecular state. As the reaction time increases from short to long, polymer-like carbon quantum dots may be converted into carbon-derived carbon quantum dots, and the fluorescence of carbon-derived carbon quantum dots is produced by the synergistic effect of carbon nuclei and surface / molecular states. Regardless of the increase or decrease in the synthesis time, the role of the carbon nucleus is greater, and the quantum efficiency will decrease significantly. The same is true for the reaction temperature. Lower temperatures may not only produce carbon quantum dots with low quantum efficiency, but may not even be able to react completely; too high a temperature will lead to further carbonization, and the fluorescence intensity will decrease instead. In addition, both the reaction temperature and reaction time will affect the yield, so choosing the appropriate reaction temperature and reaction time will help prepare carbon quantum dots with better performance.

[0043] In some embodiments, the microwave method specifically comprises: grinding the plant Xanthium sibiricum into powder and dissolving it in ultrapure water to obtain a mixed solution, and then placing the mixed solution in a microwave oven for reaction.

[0044] Among them, the microwave oven power of the microwave method is preferably set to 100-1000W, and the microwave heating time is set to 1-20min. Compared with hydrothermal synthesis and microwave-assisted synthesis, the microwave method has a faster heating rate, shortens the reaction time, and is more energy-efficient. In the reaction process of preparing carbon quantum dots, power and heating time are important influencing factors. Lower power or shorter reaction time is not enough to support a series of condensation, polymerization, and carbonization reactions; similarly, excessive power and excessive heating time will further carbonize the product, significantly reducing its quantum efficiency.

[0045] In some embodiments, the process of purifying the carbon quantum dots specifically includes: centrifuging the prepared carbon quantum dot solution at high speed, filtering the obtained supernatant and then dialyzing it.

[0046] Wherein, the high-speed centrifugation speed is preferably set to 8000-12000 rpm / min, and the time is 15min to 60min.

[0047] The filtration process preferably uses a vacuum filtration device for reduced pressure filtration, and the pore size of the filter membrane used is 0.2 to 0.8 μm.

[0048] Furthermore, the dialysis operation preferably uses a dialysis membrane with a cut-off molecular weight of 100 to 5000 Da, and the dialysis time is preferably 12 to 72 h.

[0049] The carbon quantum dots prepared from the plant Xanthium sibiricum by the above-mentioned bottom-up method do not require the addition of other toxic chemical reagents or organic solvents. At the same time, by adjusting the temperature, duration, rotation speed and other conditions of the above-mentioned reaction, carbon quantum dot products with higher yield and better fluorescence performance can be prepared, meeting the requirements of green and environmentally friendly large-scale production and simple process preparation.

[0050] The present application also provides a carbon quantum dot fluorescent probe, which is prepared by any of the aforementioned methods.

[0051] The present application also provides a method for using a carbon quantum dot fluorescent probe in the detection of Artemisia plant allergy protein Art v 1, which is characterized by comprising:

[0052] Scanning the fluorescence spectrum of the carbon quantum dot probe solution within an excitation wavelength range of 280 to 580 nm and determining the detection wavelength according to the fluorescence intensity;

[0053] The sample solution to be detected is mixed with the carbon quantum dot fluorescent probe solution, and fluorescence analysis is performed at the detection wavelength to determine the content of the allergic protein Art v 1.

[0054] Among them, the pH of the fluorescence analysis is preferably 7.0-7.8, the ionic strength is preferably ≤1M, the concentration of the carbon quantum dot solution is preferably 0.5-1.2mg / mL, the solution concentration of Artv 1 is ≤5mg / mL, the mixing time of the mixed solution is preferably 1-30min, and the detection wavelength of the fluorescence analysis is preferably 300-340nm.

[0055] In a typical embodiment of the present invention, a method for preparing optimized carbon quantum dots is as follows:

[0056] 1-5 g of Xanthium sibiricum seeds are ground into powder and dissolved in 10-60 mL of water to obtain a mixed solution, which is placed in a high-pressure reactor or a microwave reactor for reaction, and the reaction temperature is set to 160-240° C. and the reaction time is 2-8 h;

[0057] The microwave method is to grind 2 to 8 g of Xanthium sibiricum into powder and dissolve it in 10 to 30 mL of water to obtain a mixed solution, which is placed in a household microwave oven for reaction, with the heating power set to 200 to 500 W and the reaction time being 2 to 10 minutes.

[0058] Infrared spectroscopy and EDS analysis later confirmed that the carbon quantum dots had -OH, -COOH, and -NH2 on the surface, so they had good water solubility; the fluorescence spectrum showed that the optimal excitation wavelength was 320nm, and they emitted obvious blue fluorescence under a 365nm ultraviolet lamp.

[0059] After the Art v 1 solutions of different concentrations were mixed with the carbon quantum dot solution, the fluorescence intensity was quenched to different degrees, and the quenching rate had a good linear relationship with the Art v 1 concentration. The specific linear relationship was:

[0060] Art v 1 concentration is 0-0.8 mg / mL, y=1.143C Art v 1+0.068

[0061] Correlation coefficient R 2 is 0.99.

[0062] The technical solution of the present invention will be described in more detail below in conjunction with several embodiments and drawings. It should be noted that, unless otherwise specified, the raw materials, chemical reagents and equipment used in the following embodiments can be obtained through market purchase and other channels, and the characterization methods and the like can be implemented according to methods known in the art.

[0063] Embodiment 1:

[0064] The hydrothermal synthesis method is used to prepare a fluorescent probe based on carbon quantum dots, comprising the following steps:

[0065] 2g of Xanthium sibiricum powder was added to 30mL of ultrapure water, mixed evenly under magnetic stirring, and then transferred to a high-pressure reactor, the temperature was set to 180°C, and the reaction time was 4h. After the reaction, the reaction solution was naturally cooled and centrifuged, the speed was set to 10000rpm, the centrifugation time was 30min, and then filtered twice using a 0.22μm filter membrane, the obtained filtrate was dialyzed for 12h, the sample was freeze-dried, and stored in a desiccator. The mass of the dried sample was measured, and the yield was calculated to be 28.9%.

[0066] The dried samples were scanned using a transmission electron microscope. Figure 1 .

[0067] The samples were tested by infrared spectrometer, and the results were as follows: Figure 2 According to the infrared spectrum analysis, 3393cm -1 、3296cm -1 、2970cm -1 and 1770cm -1 The peaks at 1706 cm-1 correspond to the bending vibrations of NH, OH, CH, and C=O. -1 、1353cm -1 and 1230cm -1 The characteristic peaks at correspond to the stretching vibrations of C=C, COC and CO, respectively.

[0068] The fluorescence properties of the samples were analyzed by fluorescence spectrometer. Figure 3 The optimal excitation wavelength of the sample is 320nm, and the corresponding emission peak position is 630nm. With quinine sulfate as a reference, the measured quantum efficiency is 15.6%.

[0069] Embodiment 2:

[0070] The hydrothermal synthesis method is used to prepare a fluorescent probe based on carbon quantum dots, comprising the following steps:

[0071] Add 1g of Xanthium sibiricum powder to 10mL of ultrapure water, mix well under magnetic stirring, and transfer to a high-pressure reactor. Set the temperature to 160°C and the reaction time to 1h. After the reaction is completed, the reaction solution is naturally cooled and centrifuged. The speed is set to 8000rpm and the centrifugation time is 15min. Then, it is filtered twice using a 0.2μm filter membrane. The obtained filtrate is dialyzed for 12h to obtain a liquid sample. The sample is freeze-dried and stored in a desiccator. The mass of the dried sample is measured, and the yield is calculated to be 6.7%.

[0072] The fluorescence performance of the sample was analyzed by fluorescence spectrometer, and the quantum efficiency was measured to be 4.2% with quinine sulfate as reference.

[0073] Embodiment 3:

[0074] The fluorescent probe based on carbon quantum dots prepared by microwave-assisted synthesis method comprises the following steps:

[0075] 3g of Xanthium sibiricum powder was added to 50mL of ultrapure water, mixed evenly under magnetic stirring, and then transferred to a microwave reactor, with the temperature set to 200°C, the upper limit of the pressure to 2Mpa, the upper limit of the power to 1000W, and the reaction time to 3h. After the reaction, the reaction solution was naturally cooled and centrifuged, the speed was set to 8500rpm, the centrifugation time was 20min, and then filtered twice using a 0.22μm filter membrane, and the filtrate was dialyzed for 24h through a dialysis bag with a molecular weight cutoff of 1000Da to obtain a sample. The mass of the dried sample was measured, and the yield was calculated to be 20.8%.

[0076] The fluorescence performance of the sample was analyzed by fluorescence spectrometer, and the quantum efficiency was measured to be 10.3% with quinine sulfate as reference.

[0077] Embodiment 4:

[0078] The fluorescent probe based on carbon quantum dots prepared by microwave-assisted synthesis method comprises the following steps:

[0079] 10g of Xanthium sibiricum powder was added to 100mL of ultrapure water, mixed evenly under magnetic stirring, and then transferred to a microwave reactor, with the temperature set to 400°C, the upper limit of the pressure to 2Mpa, the upper limit of the power to 1000W, and the reaction time to 12h. After the reaction, the reaction solution was naturally cooled and centrifuged, the speed was set to 12000rpm, the centrifugation time was 60min, and then filtered twice using a 0.8μm filter membrane, and the filtrate was obtained after dialysis with a molecular weight cutoff of 10000Da for 72h to obtain a sample. The mass of the dried sample was measured, and the yield was calculated to be 3.4%.

[0080] The fluorescence performance of the sample was analyzed by fluorescence spectrometer, and the quantum efficiency was measured to be 0.9% with quinine sulfate as reference.

[0081] Embodiment 5:

[0082] The fluorescent probe based on carbon quantum dots prepared by microwave method includes the following steps:

[0083] 4 g of Xanthium sibiricum powder was added to 60 mL of ultrapure water, mixed evenly under magnetic stirring, and then transferred to a microwave oven, with a power setting of 500 W and a reaction time of 8 min. After the reaction was completed, the reaction solution was naturally cooled and centrifuged, the speed was set to 7500 rpm, the centrifugation time was 45 min, and then filtered twice using a 0.5 μm filter membrane, and the filtrate was dialyzed for 36 h through a dialysis bag with a molecular weight cutoff of 2000 Da to obtain a sample. The mass of the dried sample was measured, and the yield was calculated to be 10.4%.

[0084] The fluorescence performance of the sample was analyzed by fluorescence spectrometer, and the quantum efficiency was measured to be 8.6% with quinine sulfate as reference.

[0085] Embodiment 6:

[0086] The fluorescent probe based on carbon quantum dots prepared by microwave method includes the following steps:

[0087] 1g of Xanthium sibiricum powder was added to 10mL of ultrapure water, mixed evenly under magnetic stirring, and then transferred to a microwave oven, with the power set to 100W and the reaction time set to 1min. After the reaction was completed, the reaction solution was naturally cooled and centrifuged, the speed was set to 8000rpm, the centrifugation time was 15min, and then filtered twice using a 0.2μm filter membrane, and the filtrate was dialyzed for 12h through a dialysis bag with a cutoff molecular weight of 100Da to obtain a sample. The mass of the dried sample was measured, and the yield was calculated to be 4.3%.

[0088] The fluorescence performance of the sample was analyzed by fluorescence spectrometer, and the quantum efficiency was measured to be 0.3% with quinine sulfate as reference.

[0089] Embodiment 7:

[0090] The fluorescent probe based on carbon quantum dots prepared by microwave method includes the following steps:

[0091] 10g of Xanthium sibiricum powder was added to 100mL of ultrapure water, mixed evenly under magnetic stirring, and then transferred to a microwave oven, with a power setting of 1000W and a reaction time of 20min. After the reaction was completed, the reaction solution was naturally cooled and centrifuged, the speed was set to 12000rpm, the centrifugation time was 60min, and then filtered twice using a 0.8μm filter membrane, and the filtrate was dialyzed for 72h through a dialysis bag with a molecular weight cutoff of 10000Da to obtain a sample. The mass of the dried sample was measured, and the yield was calculated to be 1.7%.

[0092] The fluorescence performance of the sample was analyzed by fluorescence spectrometer, and the quantum efficiency was measured to be 0.5% with quinine sulfate as reference.

[0093] It can be seen from the above examples that by rationally optimizing the reaction conditions, carbon quantum dot products with higher yield and better fluorescence performance can be prepared.

[0094] Embodiment 8:

[0095] The carbon quantum dot fluorescent probe prepared in Example 1 is applied to the detection method of Art v 1, comprising the following steps:

[0096] 520 μL of carbon quantum dot solution (density 0.24 mg / mL) was dispersed in PBS (pH 7.2, 0.15 M), and Art v 1 protein solutions of different concentrations ≤ 5 mg / mL were added respectively. After mixing evenly, the mixture was incubated for 10 min and added to the cuvette. The slit widths of excitation and emission were set to 2.5 nm and 5 nm respectively. Under the excitation light of 320 nm, the spectrum of emission wavelength of 550-800 nm was measured.

[0097] The responses of the prepared carbon quantum dots to different concentrations of Art v 1 protein are shown in Figure 4 As the concentration of Art v1 protein increases, the fluorescence intensity of carbon quantum dots decreases significantly.

[0098] The fitting results show that when the concentration of Art v 1 protein is ≤0.8 mg / mL, the quenching rate and the concentration of Art v 1 protein show a good linear relationship, such as Figure 5 As shown. Its linear regression equation is:

[0099] Art v 1 concentration is 0-0.8 mg / mL, y=1.143C Art v 1+0.068

[0100] Correlation coefficient R 2 It is 0.998.

[0101] As the concentration of the added Art v 1 protein changes, the fluorescence intensity based on carbon quantum dots provided in this embodiment changes significantly. In the range of Art v 1 protein concentration of ≤0.8 mg / mL, the fluorescence quenching rate has a good linear relationship with the Art v 1 protein concentration. Therefore, the carbon quantum dot-based fluorescent probe provided in this embodiment has good accuracy in detecting Art v 1 protein in the range of ≤0.8 mg / mL.

[0102] Embodiment 9:

[0103] The carbon quantum dot fluorescent probe prepared in Example 4 is applied to the detection method of Art v 1, comprising the following steps:

[0104] 1.25 mL of carbon quantum dot solution (density 0.1 mg / mL) was dispersed in PBS (pH 6.0, 0.1 M), and Art v 1 protein solutions of different concentrations ≤ 5 mg / mL were added respectively. After mixing evenly, the mixture was incubated for 1 min and added to the cuvette. The slit widths of excitation and emission were set to 2.5 nm and 5 nm respectively. When the excitation light was 300 nm, the spectrum of emission wavelength of 450-700 nm was measured.

[0105] After adding Art v 1 protein to the detection system, the fluorescence intensity did not change significantly at low concentrations. As the concentration increased, the fluorescence intensity decreased to a certain extent, but no linear relationship could be established between the fluorescence quenching rate and the concentration of Art v 1 protein.

[0106] Embodiment 10:

[0107] The carbon quantum dot fluorescent probe prepared in Example 5 is applied to the detection method of Art v 1, comprising the following steps:

[0108] 83 μL of carbon quantum dot solution (density of 1.5 mg / mL) was dispersed in PBS (pH 8.0, 2M), and Art v 1 protein solutions of different concentrations of ≤5 mg / mL were added respectively. After mixing evenly, the mixture was incubated for 30 min and added to the cuvette. The slit widths of excitation and emission were set to 2.5 nm and 5 nm respectively. Under the excitation light of 340 nm, the spectrum of emission wavelength of 450-700 nm was measured.

[0109] After adding Art v 1 protein to the detection system, a linear relationship can be established with the fluorescence quenching rate within the concentration range of ≤0.25 mg / mL, such as Figure 6 As shown:

[0110] The linear relationship is y = 1.54C Art v 1+0.05, the correlation coefficient is 0.982.

[0111] Embodiment 11:

[0112] Selective experiments of carbon quantum dot fluorescent probes for different proteins and common metal ions:

[0113] The same concentrations of sodium chloride, potassium chloride, L-cysteine, glycine, alanine, ascorbic acid, urea, and glucose were added to the detection system of Example 8, and the test was carried out under the same conditions. The sensitivity and selectivity experiments were repeated three times. The results are shown in FIG. Figure 7 As shown, the fluorescence intensity did not change significantly. Therefore, the carbon quantum dot fluorescent probe provided by the present invention has a high selectivity for Art v 1 protein detection.

[0114] In summary, the present invention uses the plant Xanthium sibiricum as a carbon source, which is non-toxic in itself, and can synthesize fluorescent carbon quantum dots by reacting in an aqueous solution, with short reaction time, low cost, and easy operation, and no organic passivating agent needs to be added during the preparation process, and the purification and extraction process of the prepared carbon quantum dots is relatively simple. The carbon quantum dots finally obtained have good stability and can be stored in a refrigerator for a long time for standby use. Using carbon quantum dots as fluorescent probes, a new method for detecting Art v 1 based on the fluorescence quenching mechanism has been successfully established to assist in the diagnosis and treatment of diseases such as allergic rhinitis.

[0115] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "illustrative embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.

[0116] Although the embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the claims and their equivalents.

Claims

1. Application of a carbon quantum dot fluorescent probe in the detection of Art v 1 protein, an allergic protein of Artemisia plant, for non-disease diagnosis and treatment purposes, characterized in that: include: Scanning the fluorescence spectrum of the solution of the carbon quantum dot fluorescent probe within the excitation wavelength range of 280-580 nm and determining the detection wavelength according to the fluorescence intensity; The sample solution to be tested is mixed with the solution of the carbon quantum dot fluorescent probe, and fluorescence analysis is performed at the detection wavelength to determine the content of the allergic protein Art v 1; wherein, The carbon quantum dot fluorescent probe is prepared from the plant Xanthium sibiricum by a hydrothermal synthesis method, a microwave method or a microwave-assisted synthesis method. The carbon quantum dots are purified to prepare the probe.

2. The application according to claim 1, characterized in that: The pH of the fluorescence analysis is 6.0-8.0, the ionic strength is ≤2M, the concentration of the carbon quantum dot solution is 0.1-1.5mg / mL, the concentration of the Artv 1 solution is ≤5mg / mL, the mixing time of the mixed solution is 1-30min, and the detection wavelength of the fluorescence analysis is 300-380nm.

3. The application according to claim 1, characterized in that: The hydrothermal synthesis method specifically comprises: grinding the plant Xanthium sibiricum into powder and dissolving it in ultrapure water to obtain a mixed solution, and then placing the mixed solution in a high-pressure reactor for hydrothermal reaction; The microwave method specifically comprises: grinding the plant Xanthium sibiricum into powder and dissolving it in ultrapure water to obtain a mixed solution, and then placing the mixed solution in a microwave oven for reaction; The microwave-assisted synthesis method specifically comprises: grinding the plant Xanthium sibiricum into powder and dissolving the powder in ultrapure water to obtain a mixed solution, and then placing the mixed solution in a microwave reactor for reaction.

4. The use according to claim 1, characterized in that: In the hydrothermal synthesis method and the microwave-assisted synthesis method, the mass ratio of the plant Xanthium sibiricum to ultrapure water is 1-10:10-100, the reaction temperature is 160-400° C., and the reaction time is 1-16 hours.

5. The use according to claim 1, characterized in that: In the microwave method, the microwave oven power is set to 100-1000W, and the microwave heating time is set to 1-20min.

6. The use according to claim 1, characterized in that: Purifying the carbon quantum dots specifically includes: centrifuging the prepared carbon quantum dot solution at high speed, filtering the obtained supernatant and then dialyzing.

7. The use according to claim 6, characterized in that: The high-speed centrifugation speed is set to 8000-12000 rpm / min, and the time is 15 min-60 min; and / or The filtering process specifically includes: using a vacuum filtration device to perform reduced pressure filtration, and the pore size of the filter membrane used is 0.2-0.8 μm; and / or The dialysis membrane used in the dialysis has a cut-off molecular weight of 100-10000 Da and a dialysis time of 12-72 h.

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