CN108998009B - Preparation method of fluorescent carbon dots of roasted chicken and method for detecting serum albumin concentration by using fluorescent carbon dot probe from roasted chicken - Google Patents
Preparation method of fluorescent carbon dots of roasted chicken and method for detecting serum albumin concentration by using fluorescent carbon dot probe from roasted chicken Download PDFInfo
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- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 8
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Abstract
The invention discloses a method for detecting serum albumin concentration by using a fluorescent carbon dot probe from roast chicken, which comprises the following steps: mixing fluorescent carbon dot solutions extracted from roast chicken with different concentrations with a human serum albumin solution, preparing standard solutions with different concentrations, detecting the fluorescence intensity of each standard solution to obtain a fluorescence spectrogram of the standard solution, and establishing a linear relation with the concentration of human serum albumin by taking the difference between the fluorescence intensity when the concentration of the human serum albumin is zero and the fluorescence intensities of the standard solutions with other concentrations as a dependent variable; and step two, randomly preparing a human serum protein sample solution containing fluorescent carbon dots, detecting the concentration of the serum protein of the sample, and detecting the concentration of the serum protein in the sample solution through a linear relation. The method detects the concentration of the serum protein in the solution through a linear relation, has simple and convenient detection process, high sensitivity and low detection limit, and can realize the on-line in-situ rapid and sensitive detection of the concentration of the hemoglobin in an actual sample.
Description
Technical Field
The invention relates to a method for detecting serum albumin concentration by using a fluorescent carbon dot probe from roast chicken.
Background
In the prior art, serum albumin is the most abundant protein in plasma, particularly one of the most important carrier proteins, has important physiological functions of combination and transportation, osmotic pressure and pH maintenance, material exchange, anticoagulation and the like, complex chemical properties and a series of functional groups allowing multifunctional chemical derivation, and the detection method of human serum albumin currently has chemiluminescence, electrochemical, capillary electrophoresis, visible light and the like, and has the defects of complex pretreatment method, complex detection means, long time and the like.
In recent decades, with the rapid development of nanomaterials, fluorescent carbon dots are one of the hottest nanomaterials after titanium dioxide nanoparticles, carbon nanotubes, graphene and the like by virtue of stronger luminescent performance and higher stability thereof, and at present, the synthesis technology of the fluorescent carbon dots mainly comprises: however, researches show that carbon dots can be formed in the thermal processing process of baked meat which is eaten daily by people, the complex synthesis steps are not needed, the carbon nanoparticles prepared by using natural food as a material are economical and environment-friendly, leftover materials which cannot be eaten by chicken are thermally processed to prepare the carbon dots, the price is low, and the fluorescent carbon dots have the unique advantages of high chemical stability, low toxicity, excellent biocompatibility and the like, so that the fluorescent carbon dots have good application prospects in the fields of biochemical sensing, drug carriers, imaging analysis and the like, and at present, no report about the fluorescent carbon dots extracted from natural food as serum albumin probes is available.
Disclosure of Invention
The invention aims to provide a preparation method of fluorescent carbon dots of roasted chicken and a method for detecting serum albumin concentration by using a fluorescent carbon dot probe from the roasted chicken.
The technical scheme adopted by the invention for realizing the purpose is as follows: a method for preparing fluorescent carbon dots from roasted chicken comprises the following steps:
a. cutting chicken breast into 1 × 1 × 1cm square, and baking in oven;
b. placing the roasted chicken breast into absolute ethyl alcohol, stirring at room temperature, carrying out suction filtration on the obtained solution to remove solids, and rotatably evaporating the ethanol solution to 5mL under the high-temperature condition;
c. b, dissolving the sample obtained in the step b in distilled water, adding chloroform for extraction, passing the water phase through a water-based filter membrane, and collecting filtrate;
d. and d, separating and purifying the sample obtained in the step c by using a macroporous adsorption resin column, dialyzing for 72 hours by using a membrane with the molecular weight cutoff of 0.5KDa, and obtaining the solid powder of the roast chicken fluorescent carbon dots by using a freeze drying technology.
In the step a, the chicken breast meat is put into an oven to be roasted for 30min at 230 ℃.
And (b) putting the roasted chicken breast into 1200mL of absolute ethyl alcohol, stirring at room temperature for 36h, and rotatably evaporating the ethyl alcohol solution to 5mL at the temperature of 55 ℃.
In the step c, dissolving the sample in 100mL of distilled water, adding 300mL of chloroform for extraction, sequentially passing the water phase through a water system filter membrane of 0.45 mu m and 0.22 mu m, and collecting the filtrate.
The technical scheme adopted by the invention for realizing the other purpose is as follows: a method for detecting serum albumin concentration by using a fluorescent carbon dot probe from roast chicken comprises the following steps:
mixing fluorescent carbon dot solutions extracted from roast chicken with different concentrations with a human serum albumin solution, preparing standard solutions with different concentrations, detecting the fluorescence intensity of each standard solution to obtain a fluorescence spectrogram of the standard solution, and establishing a linear relation with the concentration of human serum albumin by taking the difference value between the fluorescence intensity when the concentration of the human serum albumin is zero and the fluorescence intensities of the standard solutions with other concentrations as a dependent variable according to a Steen-Walmer equation;
and step two, randomly preparing a human serum protein sample solution containing fluorescent carbon dots, detecting the concentration of the serum protein of the sample, and detecting the concentration of the serum protein in the sample solution through a linear relation.
The preparation method of the standard solution in the first step comprises the following steps: preparing human serum albumin solutions with different concentrations, adding fluorescent carbon dots with the same volume and buffer solutions with different volumes into the human serum albumin solutions, wherein the types of the buffer solutions are the same as the human serum albumin, and the volumes of the buffer solutions are the same as the standard solutions after the buffer solutions are added, so that the human serum albumin standard solutions with different concentrations are obtained.
The preparation method of the sample solution in the second step comprises the following steps: adding the fluorescent carbon dots with the same volume and the buffer solution with different volumes into the human serum albumin solution with unknown concentration, wherein the type of the buffer solution is the same as that of the human serum albumin, and the volume of the buffer solution is the same as that of the standard solution after the buffer solution is added, so as to obtain the sample solution.
The method for detecting the concentration of the serum protein in the sample solution through the linear relation in the second step comprises the following steps: and (3) bringing the difference value between the fluorescence intensity of the sample solution and the fluorescence intensity of the human serum albumin with zero into a linear relation to obtain the concentration of the human serum albumin in the sample solution.
The total volume of the standard solution and the solution in the sample solution is 2 mL.
The buffers in the standard solution and the sample solution are phosphate buffer solutions.
The concentration of the fluorescent carbon dots in the standard solution and the sample solution is 1mg/mL, and the adding volume is 200 mu L.
The excitation wavelength of the fluorescence intensity in the standard solution and the sample solution is 280 nm.
According to the method for preparing the fluorescent carbon dots of the roasted chicken and the method for detecting the concentration of the serum albumin by using the fluorescent carbon dot probe from the roasted chicken, the concentration of the serum albumin in a solution is detected through a linear relation according to a Steen-Walmer equation, the detection process is simple and convenient, the sensitivity is high, the detection limit is low, and the online in-situ rapid and sensitive detection of the concentration of the hemoglobin in an actual sample can be realized; the required carbon dots come from food eaten by daily people, the method is safe and green, low in cost, low in toxicity, simple and convenient in detection method and high in sensitivity, can be used for quickly and accurately detecting the concentration of human serum albumin in a sample, has small size and good fluorescence characteristic and biocompatibility, and is high in safety, and the fluorescent carbon dots can be used for metal ion detection, biological labeling, biological imaging and the like.
Drawings
FIG. 1 is a UV fluorescence spectrum of fluorescent carbon dots for a method of detecting serum albumin concentration using a fluorescent carbon dot probe from roast chicken according to the present invention.
FIG. 2 is a TEM image of an aqueous solution of fluorescent carbon spots of a method of the present invention for detecting serum albumin concentration using a fluorescent carbon spot probe from roast chicken.
FIG. 3 is a fluorescence spectrum at an excitation wavelength of 280nm of a standard solution of human serum albumin after reaction with a fluorescent carbon spot probe in an example of a method for measuring serum albumin concentration using a fluorescent carbon spot probe from roast chicken according to the present invention.
FIG. 4 is a linear relationship curve between the fluorescence intensity of a standard solution when the concentration of human serum albumin is zero and the difference between the fluorescence intensity of standard solutions of serum albumin of different concentrations and the concentration of serum albumin in one example of the method for detecting the concentration of serum albumin using a fluorescent carbon dot probe from roast chicken according to the present invention.
Detailed Description
Example one, 1, synthesis and characterization of fluorescent carbon dots from roast chicken: the preparation method of the fluorescent carbon nano-particles from the roasted chicken comprises the following specific steps: a. cutting about 190g chicken breast into 1 × 1 × 1cm square, and baking at 230 deg.C for 30 min; b. putting the roasted chicken breast into 1200mL of absolute ethyl alcohol, stirring at room temperature for 36h, carrying out suction filtration on the obtained solution to remove solids, and carrying out rotary evaporation on the liquid at 55 ℃ to obtain 5mL of an ethanol solution; c. dissolving the obtained sample in 100mL of distilled water, adding 300mL of chloroform for extraction, sequentially passing the water phase through a 0.45 mu m and 0.22 mu m water system filter membrane, and collecting filtrate; d. separating and purifying the sample obtained in the step c by using a macroporous adsorption resin column, carrying out auxiliary observation under the irradiation of a 365nm ultraviolet lamp, dialyzing for 72 hours by using a membrane with the molecular weight cutoff of 0.5KDa, obtaining solid powder of the roast chicken fluorescent carbon dots by using a freeze drying technology, and measuring the fluorescence quantum yield to be 10.8% by using quinine sulfate as a standard substance; the characterization of the properties of the roast chicken fluorescent carbon nanoparticles is S1, the ultraviolet spectrum and the fluorescence spectrum of the roast chicken fluorescent carbon nanoparticles are shown in an ultraviolet spectrogram 1, a characteristic absorption peak of n → pi x transition appears at 250nm, the fluorescence spectrum of the nanoparticles shows an obvious red shift phenomenon along with the increase of wavelength, the maximum excitation wavelength of the roast chicken fluorescent carbon nanoparticles appears at 320nm, and the fluorescence quantum yield of the roast chicken nanoparticles is 10.8% measured by a quinine sulfate method under the excitation of 320 nm; s2, representing the shape and size of the chicken fluorescent carbon nanoparticles, and FIG. 2 is a TEM photograph of the chicken fluorescent carbon nanoparticles, showing that the chicken is separated and purifiedThe size of the meat fluorescent carbon nano particles is uniform, the shape is regular and is approximately spherical, and the particle size of the roast chicken fluorescent carbon nano particles is concentrated to 1-4nm through particle size statistics; establishing a linear relation: preparing original solutions of hemoglobin with different concentrations, respectively adding 200 μ L of fluorescent carbon dots, diluting to 2mL with phosphate buffer solution, and preparing a series of standard solutions with equal volumes and different concentrations, wherein the concentration of serum albumin in the standard solutions is 0, 2 × 10- 5mol/L、4×10-5mol/L、6×10-5mol/L、8×10-5mol/L, wherein the numbers of the fluorescent carbon dots in the standard solutions are a, b, c, d and e respectively, and the concentration of the fluorescent carbon dots in the standard solutions is 0.1 mg/mL; respectively detecting the fluorescence intensity of the standard solutions by using a fluorescence spectrophotometer under the excitation wavelength of 280nm to obtain a fluorescence spectrogram shown in figure 3, and reading out fluorescence intensity values corresponding to the standard solutions of the serum albumin with different concentrations from the fluorescence spectrogram; drawing a standard curve shown in figure 4 by taking the difference between the fluorescence intensity of a blank fluorescent carbon dot standard solution with the serum albumin concentration of zero and the fluorescence intensity of hemoglobin standard solutions with different concentrations as an ordinate and the concentration of hemoglobin in the standard solutions as an abscissa in a fluorescence spectrogram, thereby obtaining a linear relation between the difference between the fluorescence intensity of the blank fluorescent carbon dot standard solution with the hemoglobin concentration of zero and the fluorescence intensity of the hemoglobin standard solutions with different concentrations and the hemoglobin concentration, wherein a linear equation Y is 1.964X +1.96, R is2The detection limit of the hemoglobin can reach 2.51 multiplied by 10 when the value is 0.995-6And (3) detecting the concentration of the serum albumin in the sample solution according to the linear relation.
Example two, measurement of hemoglobin concentration in sample solution: taking a certain volume of serum albumin solution with unknown concentration, adding 200 mu L of fluorescent carbon dots into the serum albumin solution, then diluting the solution to 2mL by using a phosphate buffer solution to obtain a sample solution, wherein the concentration of the fluorescent carbon dots in the sample solution is 0.1mg/mL, detecting the fluorescence intensity of the sample solution by using a fluorescence spectrophotometer under an excitation wavelength of 280nm, substituting the difference value between the fluorescence intensity of a standard solution with the serum albumin concentration of zero and the fluorescence intensity of the sample solution into a linear equation, and calculating to obtain the concentration of hemoglobin in the sample solution.
The method for preparing the fluorescent carbon dots of the roasted chicken and the method for detecting the concentration of the serum albumin by using the fluorescent carbon dot probe from the roasted chicken detect the concentration of the serum albumin in a solution through a linear relation according to a Steen-Walmer equation, have simple and convenient detection process, high sensitivity and low detection limit, and can realize the online in-situ rapid and sensitive detection of the concentration of the hemoglobin in an actual sample; the required carbon dots come from food eaten by daily people, the method is safe and green, low in cost, low in toxicity, simple and convenient in detection method and high in sensitivity, can be used for quickly and accurately detecting the concentration of human serum albumin in a sample, has small size and good fluorescence characteristic and biocompatibility, and is high in safety, and the fluorescent carbon dots can be used for metal ion detection, biological labeling, biological imaging and the like.
Claims (6)
1. A method for detecting the concentration of serum albumin by using a fluorescent carbon dot probe from roast chicken is characterized by comprising the following steps:
mixing fluorescent carbon dot solutions extracted from roast chicken with different concentrations with a solution of human serum albumin, preparing standard solutions with different concentrations, detecting the fluorescence intensity of each standard solution to obtain a fluorescence spectrogram of the standard solution, and establishing a linear relation with the concentration of the human serum albumin by taking the difference value between the fluorescence intensity when the concentration of the human serum albumin is zero and the fluorescence intensities of the standard solutions with other concentrations as a dependent variable according to a Steen-Walmer equation;
randomly preparing a human serum albumin sample solution containing fluorescent carbon dots, detecting the concentration of serum albumin in the sample, and detecting the concentration of the serum albumin in the sample solution through a linear relation; the preparation method of the fluorescent carbon dots extracted from the roast chicken comprises the following steps:
a. cutting chicken breast into 1 × 1 × 1cm square, baking in oven, and baking at 230 deg.C for 30 min;
b. putting the roasted chicken breast into 1200mL of absolute ethyl alcohol, stirring at room temperature for 36h, carrying out suction filtration on the obtained solution to remove solids, and carrying out rotary evaporation on the liquid at 55 ℃ to obtain 5mL of an ethanol solution;
c. dissolving the sample obtained in the step b in 100mL of distilled water, adding 300mL of chloroform for extraction, sequentially passing the water phase through a water system filter membrane of 0.45 mu m and 0.22 mu m, and collecting filtrate;
d. and d, separating and purifying the sample obtained in the step c by using a macroporous adsorption resin column, dialyzing for 72 hours by using a membrane with the molecular weight cutoff of 0.5KDa, and obtaining the solid powder of the roast chicken fluorescent carbon dots by using a freeze drying technology.
2. The method of claim 1, wherein the concentration of serum albumin is detected using a fluorescent carbon spot probe from roast chicken, comprising: the method for detecting the concentration of the serum albumin in the sample solution through the linear relation in the second step comprises the following steps: and (3) bringing the difference value between the fluorescence intensity of the sample solution and the fluorescence intensity of the human serum albumin with zero into a linear relation to obtain the concentration of the human serum albumin in the sample solution.
3. The method of claim 1, wherein the concentration of serum albumin is detected using a fluorescent carbon spot probe from roast chicken, comprising: the total volume of the standard solution and the solution in the sample solution is 2 mL.
4. The method of claim 1, wherein the concentration of serum albumin is detected using a fluorescent carbon spot probe from roast chicken, comprising: the buffer solution in the standard solution and the sample solution is phosphate buffer solution.
5. The method of claim 1, wherein the concentration of serum albumin is detected using a fluorescent carbon spot probe from roast chicken, comprising: the concentration of the fluorescent carbon dots in the standard solution and the sample solution is 1mg/mL, and the adding volume is 200 mu L.
6. The method of claim 1, wherein the concentration of serum albumin is detected using a fluorescent carbon spot probe from roast chicken, comprising: the excitation wavelength of the fluorescence intensity in the standard solution and the sample solution is 280 nm.
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