CN116970388A - Carbon quantum dot fluorescent probe based on plant fructus xanthil and preparation method and application thereof - Google Patents
Carbon quantum dot fluorescent probe based on plant fructus xanthil and preparation method and application thereof Download PDFInfo
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Abstract
The application discloses a carbon quantum dot fluorescent probe based on plant fructus xanthil, and a preparation method and application thereof. The preparation method comprises the following steps: preparing a plant fructus xanthii to obtain carbon quantum dots; and purifying the carbon quantum dots to prepare the probe with high fluorescence performance. The carbon quantum dot fluorescent probe is used for detecting the Artemisia plant allergic protein Art v1, and comprises the following steps: scanning the fluorescence spectrum of the carbon quantum dot probe solution within the excitation wavelength range of 280-580 nm, and determining the detection wavelength according to the fluorescence intensity; mixing a 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. According to the application, the plant fructus xanthii is used as a precursor to directly synthesize the carbon quantum dot, other substances are not required to be added, and the carbon quantum dot can be used as a nano fluorescent probe to detect the protein Art v 1. The preparation method and the detection process are low in cost, environment-friendly, rapid and simple, and the method is an Art v1 detection method with convenience and practical application prospect.
Description
Technical Field
The application belongs to the field of fluorescent material preparation, and particularly relates to a carbon quantum dot fluorescent probe based on plant fructus xanthii, and a preparation method and application thereof.
Background
Epidemiological studies have shown a significant rise in the prevalence of allergies in the chinese population over the last three decades, which has been considered one of the major contributors to asthma. Not only affects the life quality of people, but also brings great social and economic burden, and becomes a global health problem. Investigation showed that the association index of artemisia pollen was highest among the major pollen species responsible for seasonal allergic asthma in china. Of these, art v1 is considered a class of defensin proteins, a marker protein of artemisia pollen allergy, and more than 95% of artemisia allergic patients respond to the major allergen Art v 1. In clinical diagnosis and desensitization treatment, it is important to accurately measure the content of each allergen in pollen extract.
Carbon quantum dots, which are a new zero-dimensional nanomaterial in recent years, are widely used for detecting various substances including metal ions, small molecules and biomacromolecules, such as Fe, due to their good chemical inertness and stability, photobleaching resistance, excellent biocompatibility, and adjustable excitation spectrum and emission spectrum 3+ 、Hg 2+ Lysozyme, ascorbic acid, DNA, and the like. In addition, the carbon quantum dots are used as fluorescent probes, and have the advantages of good water solubility, low price, low toxicity, low pollution and the like, so that the carbon quantum dots are widely applied in a plurality of fields.
In the prior art, the preparation method of the carbon quantum dots is divided into two main types from top to bottom and from bottom to top, and the top-to-bottom method comprises the following steps: arc discharge smoke dust, laser ablation of a carbon target, electrochemical impact of CNTs, electrochemical stripping of graphite and the like, but materials prepared by the methods are poor in fluorescence performance, usually require further modification or passivation by using toxic small organic molecules or polymers, and are complicated in process and not environment-friendly; bottom-up methods include thermal or solvothermal synthesis, electrochemical methods, microwave-assisted synthesis, and the like, and these conventional synthesis methods typically require toxic chemical reagents or organic solvents as precursors. Therefore, the biomass material is utilized to replace the organic compound precursor, and the efficient synthesis technology which does not need any external energy supply is developed to prepare the fluorescent carbon quantum dot material, so that the fluorescent carbon quantum dot material has important significance and broad prospect for meeting the requirements of efficient, rapid, green and environment-friendly fluorescent analysis.
Disclosure of Invention
The application aims to provide a carbon quantum dot fluorescent probe based on a plant fructus xanthil, and a preparation method and application thereof, wherein the raw material plant fructus xanthil is wide in source, low in cost, environment-friendly, simple in preparation process and high in preparation efficiency; meanwhile, the synthesized carbon quantum dot has good fluorescence performance, and has the advantages of simple, convenient, quick and sensitive detection method when used for detecting allergic proteins, and solves the problems of complicated steps, high cost and the like of the existing allergen detection and quantification method.
In order to achieve the purpose of the application, the application is realized by the following technical scheme:
the preparation method of the carbon quantum dot fluorescent probe based on the plant fructus xanthil comprises the following steps:
preparing a plant fructus xanthii to obtain carbon quantum dots;
and purifying the carbon quantum dots to prepare the probe.
In some embodiments, the preparation process of the carbon quantum dots specifically includes: and treating the plant fructus Xanthii by a hydrothermal synthesis method, a microwave method or a microwave-assisted synthesis method to prepare the carbon quantum dot.
In some embodiments, the hydrothermal synthesis method specifically includes: grinding the plant fructus Xanthii into powder, dissolving in ultrapure water to obtain a mixed solution, and placing the mixed solution into a high-pressure reaction kettle for hydrothermal reaction.
The fluorescence carbon dots are synthesized by the hydrothermal synthesis method, the equipment for synthesizing the carbon dots is simple, the operation is convenient, and the period required by synthesizing the carbon dots is short and the efficiency is high; effectively solves the problems of complicated operation and low preparation efficiency of the carbon dot preparation method in the prior art. Meanwhile, the reaction liquid only uses pure water as a solvent, and does not need other acid, alkaline solution and the like, so that a plurality of harsh experimental conditions and complex experimental processes are avoided.
In some embodiments, the microwave-assisted synthesis method specifically comprises: grinding the plant fructus Xanthii into powder, dissolving in ultrapure water to obtain a mixed solution, and then placing the mixed solution into a microwave reaction instrument for reaction.
In some embodiments, the microwave method specifically includes: grinding fructus Xanthii into powder, dissolving in ultrapure water to obtain mixed solution, and placing the mixed solution into a microwave oven for reaction.
Compared with a hydrothermal synthesis method, the fluorescent carbon dot synthesized by adopting the microwave-assisted synthesis method or the microwave method has the advantages of simplicity, high efficiency and no need of adding an organic reagent, and the reaction solution is further placed in a microwave environment for heating reaction, so that the synthesis efficiency is higher and the effect is better.
In some embodiments, the process of purifying the carbon quantum dots specifically includes: and (3) centrifuging the prepared carbon quantum dot solution at a high speed, and filtering the obtained supernatant to perform dialysis.
The application also provides a carbon quantum dot fluorescent probe which is prepared by any one of the methods.
The application also provides an application method of the carbon quantum dot fluorescent probe in Artemisia plant allergic protein Art v1 detection, which is characterized by comprising the following steps:
scanning the fluorescence spectrum of the carbon quantum dot probe solution within the excitation wavelength range of 280-580 nm, and determining the detection wavelength according to the fluorescence intensity;
mixing a 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 current quantitative detection methods of pollen allergens mainly comprise enzyme-linked immunosorbent assay (ELISA), mass spectrometry and other molecular biological means such as PCR. These assays have many problems, such as enzyme-linked immunosorbent assay (ELISA), particularly double-sandwich ELISA, which can successfully detect trace amounts of allergen, but require incubation with antibodies specific for the allergen, which is a relatively long time; the mass spectrometry detection has the advantages of higher sensitivity, high detection speed, short analysis time and the like, but the detection procedure is complex and needs to be operated by a professional technician. The fluorescence analysis method is favored because of low detection limit, high sensitivity and simple operation process.
Therefore, compared with the prior art, the technical scheme provided by the application has at least the following advantages: the provided carbon quantum dot fluorescent probe has the advantages of simple preparation process, low cost of required raw materials and no need of adding other toxic reagents; when the prepared fluorescent carbon dots are used for detecting Art v1, the operation is convenient and quick, the sensitivity is high, the detection result is reliable, and the fluorescent carbon dots have wide application prospect and value in the fields of allergic disease diagnosis and treatment, biological detection and the like.
Additional aspects and advantages of the application will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the application.
Drawings
The foregoing and/or additional aspects and advantages of the application will become apparent and may be better understood from the following description of embodiments taken in conjunction with the accompanying drawings in which:
FIG. 1 is a transmission electron microscope image of the preparation of carbon quantum dots of example 1;
FIG. 2 is an infrared spectrum of the carbon quantum dots prepared in example 1;
FIG. 3 is a graph of fluorescence spectra of the carbon quantum dots prepared in example 1 at different excitation wavelengths;
FIG. 4 is a graph showing the comparison of fluorescence intensity quenching effect of different concentrations of Art v1 on carbon quantum dots in example 8;
FIG. 5 is a graph showing the linear relationship between the fluorescence intensity quenching effect of different concentrations of Art v1 on carbon quantum dots in example 8;
FIG. 6 is a graph showing the linear relationship between the fluorescence intensity quenching effect of different concentrations of Art v1 on carbon quantum dots in example 10;
FIG. 7 is a graph showing the comparison of the quenching effect of different proteins and common metal ions on carbon quantum dot solutions in example 11.
Detailed Description
The embodiments described below by referring to the drawings are illustrative only and are not to be construed as limiting the application. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to be within the scope of the application.
In order to solve the problems of complicated preparation process, poor fluorescence performance and general environmental protection of the carbon quantum dots in the prior Art, the application uses the plant fructus xanthii as a carbon source to prepare the fluorescent probe based on the carbon quantum dots, and applies the fluorescent probe to the detection of Art v 1.
The preparation method of the carbon quantum dot fluorescent probe based on the plant fructus xanthil comprises the following steps:
preparing a plant fructus xanthii to obtain carbon quantum dots;
and purifying the carbon quantum dots to prepare the probe.
In some embodiments, the preparation process of the carbon quantum dots specifically includes: and treating the plant fructus Xanthii by a hydrothermal synthesis method, a microwave method or a microwave-assisted synthesis method to prepare the carbon quantum dot.
In some embodiments, the hydrothermal synthesis method specifically includes: grinding the plant fructus Xanthii into powder, dissolving in ultrapure water to obtain a mixed solution, and placing the mixed solution into a high-pressure reaction kettle for hydrothermal reaction.
In some embodiments, the microwave-assisted synthesis method specifically comprises: grinding the plant fructus Xanthii into powder, dissolving in ultrapure water to obtain a mixed solution, and then placing the mixed solution into a microwave reaction instrument for reaction.
Further, the mass ratio of the plant fructus Xanthii to the ultrapure water in the hydrothermal synthesis method and the microwave-assisted synthesis method is preferably as follows: 1 to 10: 10-100, the reaction temperature is preferably set to 160-400 ℃, and the reaction time is preferably set to 2-16 h.
The different types of carbon quantum dots are prepared by adjusting the proportion of the plant fructus xanthil to the ultrapure water, and experiments show that if the proportion of the plant fructus xanthil to the ultrapure water is too low, the quantum efficiency of the prepared carbon quantum dots can be obviously reduced; and the quantum efficiency is improved to a certain extent when the preparation is carried out within the proportion range in the embodiment. Among them, the highest quantum efficiency can reach about 15%.
Another important parameter for synthesizing carbon quantum dots is the reaction time. Only within a suitable reaction time can polymer-like carbon quantum dots be formed to generate fluorescence from the surface/molecular state. With the reaction time from short to long, the polymer-like carbon quantum dots may be converted into carbon-source carbon quantum dots, and fluorescence of the carbon-source carbon quantum dots is generated by the synergistic effect of carbon pronucleus and surface/molecular states, so that the effect of the carbon nuclei in the polymer-like carbon quantum dots is larger and the quantum efficiency is remarkably reduced no matter how the synthesis time is increased or decreased. The same reaction temperature is adopted, and lower temperature can not only generate carbon quantum dots with low quantum efficiency, but also can not completely react; too high a temperature leads to further carbonization, and the fluorescence intensity decreases. In addition, the reaction temperature and the reaction time have influence on the yield, so that the selection of the proper reaction temperature and reaction time is helpful for preparing the carbon quantum dots with better performance.
In some embodiments, the microwave method specifically includes: grinding fructus Xanthii into powder, dissolving in ultrapure water to obtain mixed solution, and placing the mixed solution into a microwave oven for reaction.
Wherein, the power of the microwave oven of the microwave method is preferably set to be 100-1000W, and the microwave heating time is set to be 1-20 min. Compared with hydrothermal synthesis and microwave-assisted synthesis, the microwave method has the advantages of faster heating rate, shortened reaction time and more energy conservation. In the reaction process for preparing the carbon quantum dots, the power and the heating time are important influencing factors, and the lower power or the shorter reaction time is insufficient to support a series of polycondensation, polymerization and carbonization reactions; also excessive power and excessive heating time can further carbonize the product, significantly reducing its quantum efficiency.
In some embodiments, the process of purifying the carbon quantum dots specifically includes: and (3) centrifuging the prepared carbon quantum dot solution at a high speed, and filtering the obtained supernatant to perform dialysis.
Wherein, the high-speed centrifugal rotating speed is preferably 8000-12000rpm/min, and the time is 15-60 min.
Wherein, the filtering process preferably adopts a vacuum filtration device to carry out reduced pressure filtration, and the pore diameter of the adopted filter membrane is 0.2-0.8 mu m.
Further, the dialysis operation preferably uses a dialysis membrane having a molecular weight cut-off of 100 to 5000Da and a dialysis time of 12 to 72 hours.
The carbon quantum dot prepared from the plant fructus xanthil by the method from bottom to top does not need to add other toxic chemical reagents or organic solvents, and simultaneously can be prepared into a carbon quantum dot product with higher yield and better fluorescence performance by adjusting the conditions of the reaction such as temperature, duration, rotating speed and the like, thereby meeting the preparation requirements of green and environment-friendly large-scale production and simple process.
The application also provides a carbon quantum dot fluorescent probe which is prepared by any one of the methods.
The application also provides an application method of the carbon quantum dot fluorescent probe in Artemisia plant allergic protein Art v1 detection, which is characterized by comprising the following steps:
scanning the fluorescence spectrum of the carbon quantum dot probe solution within the excitation wavelength range of 280-580 nm, and determining the detection wavelength according to the fluorescence intensity;
mixing a 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.
Wherein the pH value of the fluorescence analysis is preferably 7.0-7.8, the ionic strength is preferably less than or equal to 1M, the concentration of the carbon quantum dot solution is preferably 0.5-1.2 mg/mL, the concentration of the Ar tv 1 solution is less than or equal to 5mg/mL, the mixing time of the mixed solution is preferably 1-30 min, and the detection wavelength of the fluorescence analysis is preferably 300-340 nm.
In an exemplary embodiment of the application, a method for preparing an optimized carbon quantum dot is as follows:
grinding 1-5 g of plant fructus Xanthii into powder, dissolving in 10-60 mL of water to obtain a mixed solution, and placing the mixed solution in a high-pressure reaction kettle or a microwave reactor for reaction, wherein the reaction temperature is set to 160-240 ℃ and the reaction time is set to 2-8 h;
the microwave method is to take 2-8 g of plant fructus xanthil, grind the plant fructus xanthil into powder, dissolve the powder into 10-30 mL of water to obtain mixed solution, and place the mixed solution into a household microwave oven for reaction, wherein the heating power is set to be 200-500W, and the reaction time is 2-10 min.
After infrared spectrum and EDS analysis, the carbon quantum dot has-OH, -COOH and-NH on the surface 2 So it has good water solubility; the fluorescence spectrum shows that the optimal excitation wavelength is 320nm, and the fluorescence spectrum emits obvious blue fluorescence under a 365nm ultraviolet lamp.
After the prepared Art v1 solution with different concentrations is mixed with the carbon quantum dot solution, the fluorescence intensity is quenched to different degrees, and the quenching rate and the Art v1 concentration have good linear relation, and the specific linear relation formula is as follows:
the concentration of Art v1 is 0-0.8 mg/mL, y=1.143C Art v 1 +0.068
Correlation coefficient R 2 0.99.
The technical scheme of the application will be described in more detail below with reference to a plurality of embodiments and drawings. It should be noted that, unless otherwise specified, the raw materials, chemical reagents, equipment, etc. used in the examples below are available commercially, etc., and the characterization methods and the like thereof may be carried out in accordance with the methods known in the art.
Example 1:
the fluorescence probe based on the carbon quantum dots prepared by the hydrothermal synthesis method comprises the following steps:
2g of fructus Xanthii powder is added into 30mL of ultrapure water, and the mixture is transferred into a high-pressure reaction kettle after being uniformly mixed under the magnetic stirring state, the temperature is set to be 180 ℃, and the reaction time is 4 hours. After the reaction, the reaction solution was naturally cooled and centrifuged at 10000rpm for 30min, and then filtered twice with a 0.22 μm filter membrane, and the obtained filtrate was dialyzed for 12h, and 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%.
The resulting dried sample was scanned using a transmission electron microscope, the results of which are shown in fig. 1.
The sample was tested using an infrared spectrometer and the results are shown in figure 2. The infrared spectrogram analysis shows that: 3393cm -1 、3296cm -1 、2970cm -1 And 1770cm -1 The peaks at which correspond to flexural vibrations of N-H, O-H, C-H and C=O, respectively, and, in addition, 1706cm -1 、1353cm -1 And 1230cm -1 The characteristic peaks at these correspond to the stretching vibrations of c= C, C-O-C and C-O, respectively.
The fluorescence properties of the samples were analyzed by fluorescence spectroscopy and the results are shown in FIG. 3. The optimal excitation wavelength of the sample is 320nm, the corresponding emission peak position is 630nm, and the quantum efficiency is measured to be 15.6% by taking quinine sulfate as a reference.
Example 2:
the fluorescence probe based on the carbon quantum dots prepared by the hydrothermal synthesis method comprises the following steps:
1g of fructus Xanthii powder is added into 10mL of ultrapure water, and the mixture is transferred into a high-pressure reaction kettle after being uniformly mixed under the magnetic stirring state, the temperature is set to 160 ℃, and the reaction time is 1h. After the reaction, the reaction solution was naturally cooled and centrifuged at 8000rpm for 15min, and then filtered twice with a 0.2 μm filter membrane, and the obtained filtrate was dialyzed for 12 hours to obtain a liquid sample. The samples were freeze-dried and stored in a desiccator. The mass of the dried sample was measured and the yield was calculated to be 6.7%.
The fluorescence performance of the sample was analyzed by a fluorescence spectrometer, and the quantum efficiency was measured to be 4.2% with quinine sulfate as a reference.
Example 3:
the fluorescent probe based on the carbon quantum dots prepared by the microwave-assisted synthesis method comprises the following steps:
3g of fructus Xanthii powder is added into 50mL of ultrapure water, the mixture is transferred into a microwave reactor after being uniformly mixed under the magnetic stirring state, the temperature is set to 200 ℃, the upper pressure limit is 2Mpa, the upper power limit is 1000W, and the reaction time is 3h. After the reaction, the reaction solution was naturally cooled and centrifuged at 8500rpm for 20min, and then filtered twice with a 0.22 μm filter membrane to obtain a filtrate, which was dialyzed for 24h with a dialysis bag having 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%.
The fluorescence performance of the sample was analyzed by a fluorescence spectrometer, and the quantum efficiency was measured to be 10.3% with quinine sulfate as a reference.
Example 4:
the fluorescent probe based on the carbon quantum dots prepared by the microwave-assisted synthesis method comprises the following steps:
10g of fructus Xanthii powder is added into 100mL of ultrapure water, the mixture is transferred into a microwave reactor after being uniformly mixed under the magnetic stirring state, the temperature is set to 400 ℃, the upper pressure limit is 2Mpa, the upper power limit is 1000W, and the reaction time is 12h. And after the reaction is finished, naturally cooling the reaction solution, centrifuging, setting the rotation speed to 12000rpm, centrifuging for 60min, filtering twice by using a filter membrane with the diameter of 0.8 mu m, and dialyzing for 72h by using a dialysis bag with the molecular weight cutoff of 10000Da to obtain a sample. The mass of the dried sample was measured and the yield was calculated to be 3.4%.
The fluorescence performance of the sample was analyzed by a fluorescence spectrometer, and the quantum efficiency was measured to be 0.9% with quinine sulfate as a reference.
Example 5:
the fluorescent probe based on the carbon quantum dots prepared by the microwave method comprises the following steps:
4g of fructus Xanthii powder is added into 60mL of ultrapure water, and the mixture is transferred into a microwave oven after being uniformly mixed under the magnetic stirring state, the power is set to be 500W, and the reaction time is 8min. And after the reaction is finished, naturally cooling the reaction solution, centrifuging, setting the rotating speed to 7500rpm, centrifuging for 45min, filtering twice by using a filter membrane with the diameter of 0.5 mu m, and dialyzing for 36h by using a dialysis bag with the molecular weight cutoff of 2000Da to obtain a sample. The mass of the dried sample was measured and the yield was calculated to be 10.4%.
The fluorescence performance of the sample was analyzed by a fluorescence spectrometer, and the quantum efficiency was measured to be 8.6% with quinine sulfate as a reference.
Example 6:
the fluorescent probe based on the carbon quantum dots prepared by the microwave method comprises the following steps:
1g of Xanthium sibiricum powder is added into 10mL of ultrapure water, and the mixture is transferred into a microwave oven after being uniformly mixed under the magnetic stirring state, the power is set to be 100W, and the reaction time is 1min. After the reaction is finished, the reaction solution is naturally cooled and centrifuged, the rotating speed is set to 8000rpm, the centrifuging time is 15min, then the reaction solution is filtered twice by using a filter membrane with the diameter of 0.2 mu m, and the filtrate is obtained and dialyzed for 12h by a dialysis bag with the 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%.
The fluorescence performance of the sample was analyzed by a fluorescence spectrometer, and the quantum efficiency was measured to be 0.3% with quinine sulfate as a reference.
Example 7:
the fluorescent probe based on the carbon quantum dots prepared by the microwave method comprises the following steps:
10g of fructus Xanthii powder is added into 100mL of ultrapure water, and the mixture is transferred into a microwave oven after being uniformly mixed under the magnetic stirring state, the power is set to be 1000W, and the reaction time is 20min. And after the reaction is finished, naturally cooling the reaction solution, centrifuging, setting the rotation speed to 12000rpm, centrifuging for 60min, filtering twice by using a filter membrane with the diameter of 0.8 mu m, and dialyzing for 72h by using a dialysis bag with the 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%.
The fluorescence performance of the sample was analyzed by a fluorescence spectrometer, and the quantum efficiency was measured to be 0.5% with quinine sulfate as a reference.
According to the embodiment, the reaction conditions are reasonably optimized, and the carbon quantum dot product with higher yield and better fluorescence performance can be prepared.
Example 8:
the carbon quantum dot fluorescent probe prepared in the embodiment 1 is applied to the detection method of Art v1, and comprises the following steps:
measuring 520 mu L of carbon quantum dot solution (density is 0.24 mg/mL), dispersing in PBS (pH 7.2, 0.15M), respectively adding Art v1 protein solutions with different concentrations less than or equal to 5mg/mL, uniformly mixing, incubating for 10min, adding into a cuvette, respectively setting the slit widths of excitation and emission to be 2.5nm and 5nm, and measuring the spectrum with the emission wavelength of 550-800 nm under the excitation light of 320 nm.
The response of the prepared carbon quantum dots to the Art v1 protein with different concentrations is shown in figure 4. With the increase of the concentration of the Art v1 protein, the fluorescence intensity of the carbon quantum dots is obviously reduced.
It was found by fitting that when the concentration of the Art v1 protein was not more than 0.8mg/mL, the quenching rate exhibited a good linear relationship with the concentration of the Art v1 protein, as shown in FIG. 5. The linear regression equation is:
the concentration of Art v1 is 0-0.8 mg/mL, y=1.143C Art v 1 +0.068
Correlation coefficient R 2 Is 0.998.
Along with the change of the concentration of the added Art v1 protein, the fluorescence intensity based on the carbon quantum dots provided by the embodiment is obviously changed. In the range that the concentration of the Art v1 protein is less than or equal to 0.8mg/mL, the fluorescence quenching rate has good linear relation with the concentration of the Art v1 protein. Therefore, the fluorescent probe based on the carbon quantum dots provided by the embodiment has good accuracy in detecting the Art v1 protein within the range of less than or equal to 0.8 mg/mL.
Example 9:
the carbon quantum dot fluorescent probe prepared in the embodiment 4 is applied to the detection method of Art v1, and comprises the following steps:
1.25mL of carbon quantum dot solution (density is 0.1 mg/mL) is measured and dispersed in PBS (pH 6.0 and 0.1M), art v1 protein solutions with different concentrations less than or equal to 5mg/mL are respectively added, the mixture is uniformly mixed and incubated for 1min, the mixture is added into a cuvette, the widths of slits for excitation and emission are respectively set to 2.5nm and 5nm, and the spectrum with the emission wavelength of 450-700 nm is measured under the excitation light of 300 nm.
After the Art v1 protein is added into the detection system, the fluorescence intensity is not obviously changed at low concentration, and the fluorescence intensity is reduced to a certain extent along with the increase of the concentration, but a linear relation cannot be established between the fluorescence quenching rate and the Art v1 protein concentration.
Example 10:
the carbon quantum dot fluorescent probe prepared in the embodiment 5 is applied to the detection method of Art v1, and comprises the following steps:
measuring 83 mu L of carbon quantum dot solution (density is 1.5 mg/mL), dispersing in PBS (pH 8.0 and 2M), respectively adding Art v1 protein solutions with different concentrations less than or equal to 5mg/mL, uniformly mixing, incubating for 30min, adding into a cuvette, respectively setting the widths of exciting and emitting slits to be 2.5nm and 5nm, and measuring the spectrum with the emission wavelength of 450-700 nm under the excitation light of 340nm.
After the Art v1 protein is added into the detection system, a linear relationship with the fluorescence quenching rate can be established in the concentration range of less than or equal to 0.25mg/mL, as shown in FIG. 6:
the linear relationship is y=1.54C Art v 1 +0.05, correlation coefficient 0.982.
Example 11:
selectivity experiment of carbon quantum dot fluorescent probe for different proteins and common metal ions:
sodium chloride, potassium chloride, L-cysteine, glycine, alanine, ascorbic acid, urea and glucose were added to the test system of example 8 at the same concentration, and the test was performed under the same conditions, and the sensitivity and selectivity experiments were repeated three times. As a result, as shown in FIG. 7, the fluorescence intensity did not change significantly. Therefore, the carbon quantum dot fluorescent probe provided by the application has higher selectivity for the detection of the Art v1 protein.
In conclusion, the application takes the plant fructus xanthil as a carbon source, has no toxicity, can synthesize the fluorescent carbon quantum dot by reaction in aqueous solution, has short reaction time, low cost and easy operation, does not need to add an organic passivating agent in the preparation process, and has simpler purification and extraction process of the prepared carbon quantum dot. The finally obtained carbon quantum dots have good stability and can be stored in a refrigerator for a long time for standby. The novel method for detecting the Art v1 based on the fluorescence quenching mechanism is successfully established by taking the carbon quantum dots as fluorescent probes, and the diagnosis and treatment of diseases such as allergic rhinitis and the like are assisted.
In the description of the present specification, reference to the terms "one embodiment," "some embodiments," "illustrative embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the application. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiments or examples. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
While embodiments of the present application have been shown and described, it will be understood by those of ordinary skill in the art that: many changes, modifications, substitutions and variations may be made to the embodiments without departing from the spirit and principles of the application, the scope of which is defined by the claims and their equivalents.
Claims (10)
1. The preparation method of the carbon quantum dot fluorescent probe based on the plant fructus xanthii is characterized by comprising the following steps of:
preparing a plant fructus xanthii to obtain carbon quantum dots;
and purifying the carbon quantum dots to prepare the probe.
2. The preparation method of claim 1, wherein the preparation process of the carbon quantum dots specifically comprises: and treating the plant fructus Xanthii by a hydrothermal synthesis method, a microwave method or a microwave-assisted synthesis method to prepare the carbon quantum dot.
3. The preparation method according to claim 2, wherein the hydrothermal synthesis method specifically comprises: grinding plant fructus Xanthii into powder, dissolving in ultrapure water to obtain a mixed solution, and placing the mixed solution into a high-pressure reaction kettle for hydrothermal reaction;
the microwave method specifically comprises the following steps: grinding plant fructus Xanthii into powder, dissolving in ultrapure water to obtain mixed solution, and placing the mixed solution into a microwave oven for reaction;
the microwave-assisted synthesis method specifically comprises the following steps: grinding the plant fructus Xanthii into powder, dissolving in ultrapure water to obtain a mixed solution, and then placing the mixed solution into a microwave reaction instrument for reaction.
4. The preparation method according to claim 3, wherein the mass ratio of the addition of the plant xanthium sibiricum to the ultrapure water in the hydrothermal synthesis method and the microwave-assisted synthesis method is as follows: 1 to 10: 10-100, the reaction temperature is 160-400 ℃ and the reaction time is 1-16 h.
5. The method according to claim 3, wherein the microwave oven power is set to 100-1000W and the microwave heating time is set to 1-20 min.
6. The method of claim 1, wherein purifying the carbon quantum dots specifically comprises: and (3) centrifuging the prepared carbon quantum dot solution at a high speed, and filtering the obtained supernatant to perform dialysis.
7. The method according to claim 6, wherein the high-speed centrifugal rotational speed is set to 8000 to 12000rpm/min for 15 to 60 minutes; and/or
The filtering process specifically comprises the following steps: vacuum filtering with vacuum filter with pore size of 0.2-0.8 microns; and/or
The dialysis operation uses a dialysis membrane with a cut-off molecular weight of 100-10000 Da and a dialysis time of 12-72 hours.
8. A carbon quantum dot fluorescent probe prepared by the method of any one of claims 1 to 7.
9. An application method of the carbon quantum dot fluorescent probe in the detection of Artemisia plant allergic protein Art v1, which is characterized by comprising the following steps:
scanning the fluorescence spectrum of the carbon quantum dot probe solution within the excitation wavelength range of 280-580 nm, and determining the detection wavelength according to the fluorescence intensity;
mixing a 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.
10. The method according to claim 9, wherein the pH of the fluorescent analysis is 6.0-8.0, the ionic strength is less than or equal to 2M, the concentration of the carbon quantum dot solution is 0.1-1.5 mg/mL, the concentration of the Ar tv 1 solution is less than or equal to 5mg/mL, the mixing time of the mixed solution is 1-30 min, and the detection wavelength of the fluorescent analysis is 300-380 nm.
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