CN109612973B - Method for detecting cholesterol and concentration thereof through fluorescent gold nanocluster probe and method for detecting cholesterol oxidase and concentration thereof - Google Patents
Method for detecting cholesterol and concentration thereof through fluorescent gold nanocluster probe and method for detecting cholesterol oxidase and concentration thereof Download PDFInfo
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Abstract
The invention provides an application of a fluorescent gold nanocluster in detecting cholesterol and/or cholesterol oxidase by a fluorescence method. The invention uses the fluorescent gold nanocluster probe for detecting cholesterol and/or cholesterol oxidase, and has the characteristics of high sensitivity, good selectivity, wide linear range and the like. The method for detecting the cholesterol by the fluorescence emission of the fluorescence nanoclusters, which is established by the invention, not only avoids the interference of other factors of organisms, but also can selectively detect the concentration of the cholesterol in blood and urine and can detect the concentration of the cholesterol in a non-invasive way; the concentration of cholesterol is detected by using a fluorescence emission method instead of using a prepared fluorescent metal nano-cluster, so that the interference possibly caused by false signals in an experiment is avoided to the maximum extent; meanwhile, the fluorescent gold nanoclusters generated in the detection process have excellent fluorescence property, and have important significance in application in the fields of optical materials, biomedicine and the like.
Description
Technical Field
The invention relates to the technical field of cholesterol detection, in particular to application of a gold nanocluster in fluorescence detection of cholesterol and/or cholesterol oxidase, a method for detecting the concentration of cholesterol by adopting a gold nanocluster fluorescence method, a method for detecting the concentration of cholesterol oxidase by adopting a gold nanocluster fluorescence method and a method for detecting whether a sample contains cholesterol or cholesterol oxidase by adopting a gold nanocluster fluorescence method, and particularly relates to application of a gold nanocluster in fluorescence detection of cholesterol and/or cholesterol oxidase, a method for detecting cholesterol and the concentration of cholesterol by adopting a gold nanocluster probe, a method for detecting cholesterol oxidase and the concentration of cholesterol oxidase by adopting a gold nanocluster probe and a method for detecting whether the sample contains cholesterol or cholesterol oxidase by adopting a gold nanocluster probe.
Background
With the development of science and technology in recent years, fluorescent nano materials exhibit distinctive optical, chemical and catalytic properties due to their unique characteristics such as quantum size effect, plasmon resonance effect and quantum tunneling effect, and thus have attracted much attention in the scientific field. The fluorescent nano-materials which are researched and found at present mainly comprise organic dyes, carbon quantum dots, polymer dots, semiconductor quantum dots, fluorescent metal nanoclusters (FM NCs) and the like. Among them, metal nanoclusters are a special material, and in the sixties of the 20 th century, researchers have found that metals such as copper, silver, gold and the like have weak fluorescence, but the fluorescence yield is particularly low, and with the development of technologies and social needs, the research on the fluorescent metal nanoclusters is more and more concerned by researchers. FM NCs are fluorescent nanomaterials with particle size less than 2nm, which are formed by combining a protective agent with some (several, tens or hundreds) of metal atoms, such as (Cu, Ag, Au, Pt). The size of the metal nanocluster is between that of a metal atom and that of a nanoparticle, when the size of a metal body material is reduced to a nanometer level or even close to an electronic Fermi level, an original continuous energy band can be split into a plurality of discrete energy levels, the movement of electrons is not limited by space any more, and the electrons jump between the energy levels; the continuous energy levels will be split into various discrete energy levels, giving FM NCs unique stronger fluorescence properties. The discrete energy levels lead to the characteristics of adjustable excitation, good water solubility, light stability, low toxicity and the like of the nano-cluster. The physicochemical properties of metal particles are closely related to and dependent on their size. At present, there are many fluorescent metal nanoclusters discovered by research, such as platinum nanoclusters, gold nanoclusters, silver nanoclusters, copper nanoclusters, and various alloy nanoclusters.
Due to the fact that the fluorescent metal nanocluster is high in fluorescence intensity, good compatibility and good stability with biological molecules are achieved, and the synthetic method is simple, the fluorescent metal nanocluster has wide prospects in the fields of environmental monitoring, biological imaging, disease monitoring, catalysis, photoelectricity and the like, and particularly has high application values in the field of biological small molecule detection.
Cholesterol is also called cholesterol, a derivative of cyclopentane polyhydrophenanthrene. Cholesterol is an essential substance indispensable to animal tissue cells, not only participates in the formation of cell membranes, but also is a raw material for the synthesis of bile acids, vitamin D and steroid hormones. Meanwhile, cholesterol is an important index of clinical biochemical examination, many diseases can be caused when the concentration of the cholesterol is abnormal, hypercholesterolemia can be caused, the body is adversely affected, and modern researches have found that atherosclerosis, venous thrombosis, cholelithiasis and hypercholesterolemia are closely related. Therefore, it is necessary to establish a simple, sensitive and accurate method for detecting and analyzing cholesterol.
At present, methods for detecting cholesterol recorded in the existing documents include a gas chromatography, a colorimetric method, an HDL-C detection method, a cholesterol isotope dilution liquid chromatography tandem mass spectrometry and the like, and the methods generally need large-scale instruments and have the defects of relatively complex operation, expensive equipment, long time consumption, low sensitivity and the like, so that the daily detection requirements are difficult to meet, the popularization and application of the detection methods are greatly limited, and the cost of a user is increased.
Therefore, how to find a more suitable method for detecting cholesterol, which overcomes the above-mentioned drawbacks of the existing methods for detecting cholesterol, has become one of the focuses of great concern of many prospective researchers.
Disclosure of Invention
In view of the above, the technical problem to be solved by the present invention is to provide an application of a gold nanocluster in fluorescence detection of cholesterol and/or cholesterol oxidase, in particular, a method for detecting cholesterol concentration by using a gold nanocluster fluorescence method, a method for detecting cholesterol oxidase concentration by using a gold nanocluster fluorescence method, and a method for detecting whether a sample contains cholesterol or cholesterol oxidase by using a gold nanocluster fluorescence method. The invention uses the fluorescent gold nanocluster in the detection of cholesterol and/or cholesterol oxidase, and has the characteristics of high sensitivity, good selectivity, wide linear range and the like. And the influence of the external environment is not easy to be caused, and the detection result is accurate.
The invention provides an application of a fluorescent gold nanocluster in detecting cholesterol and/or cholesterol oxidase by a fluorescence method.
Preferably, the fluorescence method for detecting cholesterol further comprises the fluorescence method for detecting the concentration of cholesterol;
in the process of detecting cholesterol by the fluorescence method, preparing the fluorescent gold nanoclusters in situ at the same time, wherein the raw materials prepared in situ comprise cholesterol and cholesterol oxidase;
the fluorescence emission intensity of the fluorescent gold nanoclusters is in direct proportion to the concentration of cholesterol;
the concentration of the cholesterol is 50-4000 mu M;
the fluorescent gold nanoclusters comprise gold nanoclusters and ligands;
the particle size of the fluorescent gold nanocluster is 1-5 nm;
the fluorescence method is a non-fluorescence quenching method.
Preferably, the fluorescence method for detecting the cholesterol oxidase further comprises the step of detecting the concentration of the cholesterol oxidase by the fluorescence method;
in the process of detecting cholesterol oxidase by the fluorescence method, preparing the fluorescent gold nanoclusters in situ at the same time, wherein the raw materials prepared in situ comprise the cholesterol oxidase and cholesterol;
the fluorescence emission intensity of the fluorescent gold nanocluster is in direct proportion to the concentration of cholesterol oxidase;
the concentration of the cholesterol oxidase is 50-4000 mu M;
the ligand is poly N, N-dimethylacrylamide;
the molar ratio of the gold nanoclusters to the ligand is 1: (0.2-4.8).
The invention provides a method for detecting cholesterol concentration by adopting a fluorescence gold nanocluster fluorescence method, which comprises the following steps:
1) mixing and incubating the cholesterol solution and the cholesterol oxidase solution to obtain a mixed solution;
2) reacting the mixed solution obtained in the step, an acidic buffer solution, N-dimethylacrylamide and a reducing agent to obtain a reaction solution;
3) carrying out microwave reaction on the reaction solution obtained in the step, a second acidic buffer solution and a soluble gold source to obtain a gold nanocluster solution with fluorescence intensity;
4) repeating the steps 1) to 3) by adopting cholesterol solutions with different concentrations to obtain gold nanocluster solutions with different fluorescence intensities;
5) and establishing a quantitative relation between the fluorescence intensity value of the gold nanocluster and the cholesterol concentration, and detecting the cholesterol concentration in the sample to be detected.
Preferably, the concentration of the cholesterol oxidase solution is 0.5-2 mg/mL;
the volume ratio of the cholesterol solution to the cholesterol oxidase solution is (35-40): (1-5);
the temperature of the incubation is 40-45 ℃;
the incubation time is 2-3 h;
the acidic buffer solution comprises one or more of disodium hydrogen phosphate-citric acid buffer solution, citric acid-sodium hydroxide-hydrochloric acid buffer solution, acetic acid-sodium acetate buffer solution and disodium hydrogen phosphate-potassium dihydrogen phosphate buffer solution;
the pH value of the acidic buffer solution is 3.0-6.0;
the concentration of the acidic buffer solution is 0.5-2 mM;
the volume ratio of the cholesterol solution to the acidic buffer solution is (35-40): (1-5).
Preferably, the volume ratio of the cholesterol solution to the N, N-dimethylacrylamide is (35-40): (1-4);
the concentration of the N, N-dimethylacrylamide is 20-60 mM;
the reducing agent comprises cuprous solution and NaHSO3Solution, Na2SO3Solutions of、Na2S2O3One or more of a solution, oxalic acid, a glucose solution, an alcohol solution, and an amine solution;
the concentration of the reducing agent is 30-70 mM;
the volume ratio of the cholesterol solution to the reducing agent is (35-40): (0.1 to 1);
the reaction time is 10-120 min;
a dialysis step is also included after the reaction;
the molecular weight of the dialyzed dialysis membrane is 500-1500 Da;
the dialysis time is 12-24 h.
Preferably, the second acidic buffer comprises one or more of disodium hydrogen phosphate-citric acid buffer, citric acid-sodium hydroxide-hydrochloric acid buffer, acetic acid-sodium acetate buffer and disodium hydrogen phosphate-potassium dihydrogen phosphate buffer;
the pH value of the second acidic buffer solution is 3.9-5.3;
the concentration of the second acidic buffer solution is 0.5-2 mM;
the volume ratio of the reaction solution to the second acidic buffer solution is (2-10): (0.5 to 1);
the soluble gold source comprises HAuCl4A solution;
the concentration of the soluble gold source is 50-200 mM;
the volume ratio of the reaction solution to the soluble gold source is (2-10): (1-5);
the microwave power of the microwave reaction is 400-800 w;
the microwave reaction time is 3-15 min.
Preferably, the concentration value of the cholesterol solution with different concentrations is 100-2000 mu M;
the quantitative relation comprises one or more of a quantitative relation curve, a quantitative relation formula, a quantitative relation table and a quantitative relation program;
the specific process for detecting the concentration of cholesterol in the sample to be detected comprises the following steps:
and (3) replacing the cholesterol solution with a sample to be detected, then performing the steps 1) to 3) to obtain a sample solution to be detected of the gold nanoclusters with certain fluorescence intensity, and calculating the cholesterol concentration in the sample to be detected according to the fluorescence intensity of the gold nanoclusters in the sample solution to be detected and the correlation quantitative relation.
The invention provides a method for detecting cholesterol oxidase concentration by adopting a fluorescence gold nanocluster fluorescence method, which comprises the following steps:
1) mixing and incubating a cholesterol oxidase solution and a cholesterol solution to obtain a mixed solution;
2) reacting the mixed solution obtained in the step, an acidic buffer solution, N-dimethylacrylamide and a reducing agent to obtain a reaction solution;
3) carrying out microwave reaction on the reaction solution obtained in the step, a second acidic buffer solution and a soluble gold source to obtain a gold nanocluster solution with fluorescence intensity;
4) repeating the steps 1) to 3) by adopting cholesterol oxidase solutions with different concentrations to obtain gold nanocluster solutions with different fluorescence intensities;
5) and establishing a quantitative relation between the value of the fluorescence intensity of the gold nanoclusters and the concentration of the cholesterol oxidase, and detecting the concentration of the cholesterol oxidase in the sample.
The invention also provides a method for detecting whether the sample contains cholesterol or cholesterol oxidase by adopting a fluorescent gold nanocluster fluorescence method, which comprises the following steps:
a) mixing a sample to be detected and a cholesterol oxidase solution, and incubating to obtain a mixed solution;
b) reacting the mixed solution obtained in the step, an acidic buffer solution, N-dimethylacrylamide and a reducing agent to obtain a reaction solution;
c) carrying out microwave reaction on the reaction solution obtained in the step, the second acidic buffer solution and the soluble gold source to obtain a final solution;
when the final solution contains the gold nanoclusters with fluorescence intensity, cholesterol is contained in the sample to be detected;
when the final solution does not contain the gold nanoclusters with fluorescence intensity, the sample to be detected does not contain cholesterol;
the method for detecting whether the sample contains cholesterol oxidase by adopting the fluorescence method of the fluorescent gold nanocluster comprises the following steps:
A) mixing a sample to be detected and a cholesterol solution, and incubating to obtain a mixed solution;
B) reacting the mixed solution obtained in the step, an acidic buffer solution, N-dimethylacrylamide and a reducing agent to obtain a reaction solution;
C) carrying out microwave reaction on the reaction solution obtained in the step, the second acidic buffer solution and the soluble gold source to obtain a final solution;
when the final solution contains the gold nanoclusters with fluorescence intensity, the sample to be detected contains cholesterol oxidase;
when the final solution does not contain gold nanoclusters with fluorescence intensity, the sample to be tested does not contain cholesterol oxidase.
The invention provides an application of a fluorescent gold nanocluster in detecting cholesterol and/or cholesterol oxidase by a fluorescence method. Compared with the prior art, the method provided by the invention aims at the problems that the existing method for detecting cholesterol generally needs a large-scale instrument, is complex in operation, expensive in equipment, long in time consumption, low in sensitivity and the like, is difficult to meet daily detection requirements, and is high in use cost. The method also aims at the problem that in the prior art, a small amount of reports for cholesterol detection based on nanoclusters are provided, but the reports are realized in a fluorescence quenching mode in principle, and the fluorescence quenching mode is easily influenced by the external environment because other false signals can bring about the defect of positive results.
The invention creatively uses the fluorescent gold nanocluster probe for detecting cholesterol and/or cholesterol oxidase, and has the characteristics of high sensitivity, good selectivity, wider linear range and the like. The method for detecting the cholesterol by the fluorescence nano-cluster emission fluorescence method not only avoids the interference of other factors, but also can selectively detect the concentration of the cholesterol in blood and urine and can detect the concentration of the cholesterol in a non-invasive way; the concentration of cholesterol is detected by using a fluorescence emission method instead of using a prepared fluorescent metal nano-cluster, so that the interference possibly caused by false signals in an experiment is avoided to the maximum extent; meanwhile, the fluorescent gold nanoclusters generated in the detection process have excellent fluorescence property, and have important significance in application in the fields of optical materials, biomedicine and the like.
Experimental results show that the method can be used for uniquely and quantitatively detecting the concentration of cholesterol in blood and urine of cholesterol and/or cholesterol oxidase, wherein the detection concentration range is 50-3500 mu M.
Drawings
FIG. 1 is a TEM photograph of the fluorescent gold nanoclusters prepared in example 1 of the present invention;
FIG. 2 is a curve of the quantitative relationship between the fluorescence intensity of gold nanoclusters obtained in example 1 of the present invention and the cholesterol concentration;
FIG. 3 is a fluorescent photograph of the fluorescent gold nanocluster solution generated from the cholesterol solution with different concentration gradients obtained in example 1 of the present invention under ultraviolet light;
FIG. 4 is a graph showing the fluorescence intensity of the product solution obtained in comparative example 1 of the present invention;
FIG. 5 is a graph showing the fluorescence intensity of the product solution obtained in comparative example 2 of the present invention.
Detailed Description
For a further understanding of the invention, reference will now be made to the preferred embodiments of the invention by way of example, and it is to be understood that the description is intended to further illustrate features and advantages of the invention, and not to limit the scope of the claims. Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
All of the starting materials of the present invention, without particular limitation as to their source, may be purchased commercially or prepared according to conventional methods well known to those skilled in the art.
All the raw materials of the invention are not particularly limited in purity, and the invention preferably adopts analytically pure materials or meets the medical purity standard.
All the raw materials, the marks and the acronyms thereof belong to the conventional marks and acronyms in the field, each mark and acronym is clear and definite in the field of related application, and the raw materials can be purchased from the market or prepared by a conventional method by the technical staff in the field according to the marks, the acronyms and the corresponding application.
The invention provides an application of a fluorescent gold nanocluster in detecting cholesterol and/or cholesterol oxidase by a fluorescence method.
The definition of the fluorescence method is not particularly limited in principle, and those skilled in the art can select and adjust the fluorescence method according to actual conditions, effect data and specific requirements. The fluorescence method of the invention is particularly a non-fluorescence quenching method.
The composition of the fluorescent gold nanoclusters is not particularly limited in principle, and can be selected and adjusted by a person skilled in the art according to actual conditions, effect data and specific requirements. Among them, the ligand of the present invention is preferably poly-N, N-dimethylacrylamide. The molar ratio of the gold nanoclusters and the ligand is preferably 1: (0.2 to 4.8), more preferably 1: (0.7 to 4.3), more preferably 1: (1.2 to 3.8), more preferably 1: (1.7-4.3), more preferably 1: (2.2-2.8).
The structure and parameters of the fluorescent gold nanocluster are not particularly limited in principle, the structure of the metal nanocluster known by the person skilled in the art can be used, and the person skilled in the art can select and adjust the structure according to actual conditions, effect data and specific requirements. Wherein, the fluorescent gold nanocluster preferably comprises a gold nanocluster and a ligand layer compounded on the surface of the gold nanocluster. The particle size of the fluorescent gold nanocluster is preferably 1-5 nm, more preferably 1.5-4.5 nm, more preferably 2-4 nm, and more preferably 2.5-3.5 nm.
The fluorescence method for detecting the cholesterol preferably adopts a fluorescence gold nanocluster probe emission fluorescence method for detecting whether the cholesterol exists or not, and simultaneously, the concentration content of the cholesterol is detected. Particularly, in the process of detecting cholesterol by the fluorescence method, the fluorescence gold nanoclusters are prepared in situ at the same time, and the raw materials prepared in situ comprise cholesterol and cholesterol oxidase. More particularly, the fluorescence emission intensity of the fluorescent gold nanocluster is in direct proportion to the concentration of cholesterol, namely the higher the content of cholesterol in a sample to be detected is, the stronger the fluorescence emission intensity of the fluorescent gold nanocluster is. If the sample to be detected does not contain cholesterol, the fluorescent gold nanoclusters cannot be formed, which is the difference between the fluorescent gold nanoclusters and the existing fluorescence quenching method.
The concentration of cholesterol is not particularly limited in principle, and the conventional concentration of cholesterol known by a person skilled in the art can be used, and the person skilled in the art can select and adjust the concentration according to actual conditions, effect data and specific requirements, in order to better realize the detection of cholesterol or cholesterol oxidase and ensure the fluorescence emission intensity and the accuracy and stability of a detection result, the concentration of cholesterol is preferably within the range of a fluorescence emission intensity detector, and is particularly preferably 50-3500 mu M, more preferably 70-3000 mu M, more preferably 90-2500 mu M, more preferably 100-2000 mu M, more preferably 500-1500 mu M, and more preferably 800-1200 mu M.
The fluorescence method for detecting the cholesterol oxidase preferably comprises the steps of detecting whether the cholesterol oxidase exists or not by adopting a fluorescence gold nanocluster probe emission fluorescence method, and simultaneously detecting the concentration content of the cholesterol oxidase. Particularly, in the process of detecting cholesterol oxidase by the fluorescence method, the fluorescent gold nanoclusters are prepared in situ at the same time, and the raw materials prepared in situ comprise the cholesterol oxidase and cholesterol. More particularly, the fluorescence emission intensity of the gold nanocluster is in direct proportion to the concentration of cholesterol oxidase, i.e., the higher the content of cholesterol oxidase in a sample to be detected is, the stronger the fluorescence emission intensity of the gold nanocluster is. If the sample to be detected does not contain cholesterol oxidase, the fluorescent gold nanoclusters cannot be formed, which is the difference between the method and the existing fluorescence quenching method.
The concentration of the cholesterol oxidase is not particularly limited in principle, and the conventional concentration of the cholesterol oxidase known by a person skilled in the art can be used, and the person skilled in the art can select and adjust the concentration according to actual conditions, effect data and specific requirements.
The invention also provides a method for detecting the concentration of cholesterol by adopting a fluorescence gold nanocluster fluorescence method, which comprises the following steps of:
1) mixing and incubating the cholesterol solution and the cholesterol oxidase solution to obtain a mixed solution;
2) reacting the mixed solution obtained in the step, an acidic buffer solution, N-dimethylacrylamide and a reducing agent to obtain a reaction solution;
3) carrying out microwave reaction on the reaction solution obtained in the step, a second acidic buffer solution and a soluble gold source to obtain a gold nanocluster solution with fluorescence intensity;
4) repeating the steps 1) to 3) by adopting cholesterol solutions with different concentrations to obtain gold nanocluster solutions with different fluorescence intensities;
5) and establishing a quantitative relation between the fluorescence intensity value of the gold nanocluster and the cholesterol concentration, and detecting the cholesterol concentration in the sample to be detected.
The selection, composition and definition of the raw materials in the method for detecting the concentration of cholesterol by adopting the fluorescence method of the fluorescent gold nanocluster and the corresponding preferred scheme are basically consistent with the selection, composition and definition of the raw materials in the application and the corresponding preferred scheme, and are not repeated herein.
Firstly, mixing and incubating a cholesterol solution and a cholesterol oxidase solution to obtain a mixed solution.
The concentration and the dosage of the cholesterol oxidase solution are not particularly limited in principle, and can be selected and adjusted by a person skilled in the art according to actual conditions, effect data and specific requirements, in order to better realize the detection of cholesterol and ensure the fluorescence emission intensity and the accuracy and the stability of a detection result, the concentration of the cholesterol oxidase solution is preferably 0.5-2 mg/mL, more preferably 0.7-1.8 mg/mL, and more preferably 1.0-1.5 mg/mL. The volume ratio of the cholesterol solution to the cholesterol oxidase solution is preferably (35-40): (1-5), more preferably (35-40): (1.5-4.5), more preferably (35-40): (2-4), more preferably (35-40): (2.5-3.5).
In the present invention, the base number of the volume ratio of the cholesterol solution is preferably (35 to 40), more preferably (36 to 39), and still more preferably (37 to 38). The subsequent technical scheme related to the volume ratio base number of the cholesterol solution preferably adopts the preferred principle, and is not described in detail.
The incubation condition is not particularly limited in principle, and a person skilled in the art can select and adjust the incubation condition according to actual conditions, effect data and specific requirements, and in order to better realize detection of cholesterol and ensure the fluorescence emission intensity and the accuracy and stability of a detection result, the incubation temperature is preferably 40-45 ℃, more preferably 41-44 ℃, and more preferably 42-43 ℃. The incubation time is preferably 2-3 h, more preferably 2.2-2.8 h, and more preferably 2.4-2.6 h.
According to the invention, the mixed solution obtained in the above step, an acidic buffer solution, N-dimethylacrylamide and a reducing agent are reacted to obtain a reaction solution.
The selection of the acidic buffer solution is not particularly limited in principle, and can be selected and adjusted by those skilled in the art according to actual conditions, effect data and specific requirements, and in order to better realize the detection of cholesterol and ensure the fluorescence emission intensity and the accuracy and stability of the detection result, the acidic buffer solution preferably comprises one or more of disodium hydrogen phosphate-citric acid buffer solution, citric acid-sodium hydroxide-hydrochloric acid buffer solution, acetic acid-sodium acetate buffer solution and disodium hydrogen phosphate-potassium dihydrogen phosphate buffer solution, and more preferably comprises disodium hydrogen phosphate-citric acid buffer solution, citric acid-sodium hydroxide-hydrochloric acid buffer solution, acetic acid-sodium acetate buffer solution or disodium hydrogen phosphate-potassium dihydrogen phosphate buffer solution.
The invention has no particular limitation on specific parameters and dosage of the acidic buffer solution in principle, and a person skilled in the art can select and adjust the acidic buffer solution according to actual conditions, effect data and specific requirements, in order to better realize detection of cholesterol and ensure fluorescence emission intensity and accuracy and stability of a detection result, the pH value of the acidic buffer solution is preferably 3.0-6.0, more preferably 3.5-5.5, and more preferably 4.0-5.0. The concentration of the acidic buffer solution is preferably 0.5-2 mM, more preferably 0.7-1.8 mM, and more preferably 1.0-1.5 mM. The volume ratio of the cholesterol solution to the acidic buffer solution is preferably (35-40): (1-5), more preferably (35-40): (1.5-4.5), more preferably (35-40): (2-4), more preferably (35-40): (2.5-3.5).
The specific parameters and the dosage of the N, N-dimethylacrylamide are not particularly limited in principle, and can be selected and adjusted by a person skilled in the art according to actual conditions, effect data and specific requirements. The volume ratio of the cholesterol solution to the N, N-dimethylacrylamide is preferably (35-40): (1-4), more preferably (35-40): (1.5-3.5), more preferably (35-40): (2-3).
The invention is not particularly limited in principle to the selection of the reducing agent, and the person skilled in the art can select and adjust the reducing agent according to the actual situation, the effect data and the specific requirements, in order to better realize the detection of cholesterol and ensure the fluorescence emission intensity and the accuracy and stability of the detection result, the reducing agent preferably comprises a cuprous compound (containing Cu)+The compound of (1) can be cuprous sulfate, cuprous chloride, cuprous acetate, etc.), NaHSO3Solution, Na2SO3Solution, Na2S2O3One or more of solution, oxalic acid, glucose solution, alcohol solution and amine solution, preferably cuprous compound, NaHSO3Solution, Na2SO3Solution, Na2S2O3A solution, oxalic acid, a glucose solution, an alcohol solution, or an amine solution.
The specific parameters and the dosage of the reducing agent are not particularly limited in principle, and a person skilled in the art can select and adjust the reducing agent according to actual conditions, effect data and specific requirements, in order to better realize the detection of cholesterol and ensure the fluorescence emission intensity and the accuracy and stability of a detection result, the concentration of the reducing agent is preferably 30-70 mM, more preferably 35-65 mM, more preferably 40-60 mM, and more preferably 45-55 mM. The volume ratio of the cholesterol solution to the reducing agent is preferably (35-40): (0.1-1), more preferably (35-40): (0.3-0.8), more preferably (35-40): (0.5-0.6).
The reaction conditions are not particularly limited in principle, and a person skilled in the art can select and adjust the reaction conditions according to actual conditions, effect data and specific requirements, in order to better realize detection of cholesterol and ensure fluorescence emission intensity and detection result accuracy and stability, the reaction temperature is preferably room temperature, specifically can be 10-40 ℃, more preferably 15-35 ℃, and more preferably 20-25 ℃. The reaction time is preferably 10-120 min, more preferably 30-100 min, and still more preferably 50-80 min.
In order to better realize the detection of cholesterol, ensure the fluorescence emission intensity and the accuracy and stability of a detection result, complete and refine the whole detection process, and preferably, the method also comprises a dialysis step after the reaction. The dialysis membrane for dialysis preferably has a molecular weight of 500-1500 Da, more preferably 700-1300 Da, and even more preferably 900-1100 Da. The dialysis time is preferably 12-24 hours, more preferably 14-22 hours, and more preferably 16-20 hours.
The reaction solution, the second acidic buffer solution and the soluble gold source obtained in the step are subjected to microwave reaction to obtain the gold nanocluster solution with fluorescence intensity.
The selection of the second acidic buffer solution is not particularly limited in principle, and can be selected and adjusted by those skilled in the art according to actual conditions, effect data and specific requirements, and in order to better realize the detection of cholesterol and ensure the fluorescence emission intensity and the accuracy and stability of the detection result, the second acidic buffer solution preferably comprises one or more of a disodium hydrogen phosphate-citric acid buffer solution, a citric acid-sodium hydroxide-hydrochloric acid buffer solution, an acetic acid-sodium acetate buffer solution and a disodium hydrogen phosphate-potassium dihydrogen phosphate buffer solution, and more preferably comprises a disodium hydrogen phosphate-citric acid buffer solution, a citric acid-sodium hydroxide-hydrochloric acid buffer solution, an acetic acid-sodium acetate buffer solution or a disodium hydrogen phosphate-potassium dihydrogen phosphate buffer solution.
The specific parameters and the dosage of the second acidic buffer solution are not particularly limited in principle, and a person skilled in the art can select and adjust the parameters according to actual conditions, effect data and specific requirements, in order to better realize detection of cholesterol and ensure the fluorescence emission intensity and the accuracy and stability of a detection result, the pH value of the second acidic buffer solution is preferably 3.9-5.3, more preferably 4.2-5.0, and more preferably 4.5-4.7. The concentration of the second acidic buffer solution is preferably 0.5 to 2mM, more preferably 0.7 to 1.8mM, and still more preferably 1.0 to 1.5 mM. The volume ratio of the reaction solution to the second acidic buffer solution is preferably (2-10): (0.5 to 1), more preferably (2 to 10): (0.6-0.9), more preferably (2-10): (0.7-0.8).
In the present invention, the number of volume ratio bases of the reaction solution is preferably (2 to 10), more preferably (4 to 8), and still more preferably (5 to 7). The subsequent technical scheme related to the volume ratio base number of the reaction liquid preferably adopts the preferred principle, and is not described in detail.
The selection of the soluble gold source is not particularly limited in principle, and can be selected and adjusted by a person skilled in the art according to actual conditions, effect data and specific requirements4And (3) solution.
The specific parameters and the dosage of the soluble gold source are not particularly limited in principle, and can be selected and adjusted by a person skilled in the art according to actual conditions, effect data and specific requirements, in order to better realize the detection of cholesterol and ensure the fluorescence emission intensity and the accuracy and stability of a detection result, the concentration of the soluble gold source is preferably 50-200 mM, more preferably 80-180 mM, and more preferably 100-150 mM. The volume ratio of the reaction solution to the soluble gold source is preferably (2-10): (1-5), more preferably (2-10): (1.5-4.5), more preferably (2-10): (2-4), more preferably (2-10): (2.5-3.5).
The conditions of the microwave reaction are not particularly limited in principle, and a person skilled in the art can select and adjust the conditions according to actual conditions, effect data and specific requirements, in order to better realize detection of cholesterol and ensure the fluorescence emission intensity and the accuracy and stability of a detection result, the microwave reaction refers to a reaction under the action of microwaves, and the microwave power of the microwave reaction is preferably 400-800 w, more preferably 450-750 w, more preferably 500-700 w, and more preferably 550-650 w. The time of the microwave reaction is preferably 3-15 min, more preferably 5-13 min, and more preferably 7-11 min.
The present invention is not particularly limited to the range of fluorescence intensity, and the range of fluorescence intensity is determined by the range of measurement range of fluorescence intensity detection instrument, which is known to those skilled in the art, and can be selected and adjusted by those skilled in the art according to actual conditions, effect data and specific requirements.
And finally, repeating the steps 1) to 3) by adopting cholesterol solutions with different concentrations to obtain the gold nanocluster solutions with different fluorescence intensities.
The concentration values of the cholesterol solutions with different concentrations are not particularly limited in principle, and can be selected and adjusted by a person skilled in the art according to actual conditions, effect data and specific requirements, in order to better realize detection of cholesterol and ensure fluorescence emission intensity and detection result accuracy and stability, the concentration values of the cholesterol solutions with different concentrations are preferably selected from 100-2000 μ M, more preferably from 300-1500 μ M, more preferably from 500-1200 μ M, and more preferably from 800-1000 μ M.
The number (i.e. the number of repetitions) and the selection manner of the cholesterol solution with different concentrations are not particularly limited in the present invention, and can be selected and adjusted by those skilled in the art according to the practical situation, the effect data and the specific requirements according to the conventional times and gradient selection principles of such repeated experiments well known to those skilled in the art.
The invention finally establishes the quantitative relation between the fluorescence intensity value of the gold nanocluster and the cholesterol concentration, and is used for detecting the cholesterol concentration in the sample to be detected.
The present invention is not particularly limited in principle to the specific form of the quantitative relationship, and those skilled in the art can select and adjust the quantitative relationship according to the actual situation, the effect data and the specific requirements, and the present invention preferably implements the detection of cholesterol, and ensures the fluorescence emission intensity and the accuracy and stability of the detection result, and the quantitative relationship preferably includes one or more of a quantitative relationship curve, a quantitative relationship formula, a quantitative relationship table and a quantitative relationship program, more preferably a quantitative relationship curve, a quantitative relationship formula, a quantitative relationship table and a quantitative relationship program, and more preferably a quantitative relationship curve. The quantitative relationship curve of the present invention is more preferably a primary curve.
In order to better realize the detection of cholesterol, ensure the fluorescence emission intensity and the accuracy and stability of a detection result, and completely and thinly carry out the whole cholesterol detection process, the specific process for detecting the concentration of the cholesterol in a sample to be detected is preferably as follows:
and (3) replacing the cholesterol solution with a sample to be detected, then performing the steps 1) to 3) to obtain a sample solution to be detected of the gold nanoclusters with certain fluorescence intensity, and calculating the cholesterol concentration in the sample to be detected according to the fluorescence intensity of the gold nanoclusters in the sample solution to be detected and the correlation quantitative relation.
In order to better realize the detection of cholesterol, ensure the fluorescence emission intensity and the accuracy and stability of a detection result, and complete and refine the whole cholesterol detection process, the specific process of the method for detecting the concentration of cholesterol by adopting the fluorescence method of the fluorescence gold nanocluster can also comprise the following steps:
(1) 3500-4000 mu L of cholesterol solution (100-2000 mu M) with different concentration gradients is taken and respectively blended with 100-500 mu L of cholesterol oxidase with the concentration of 0.5-2 mg/mL. Then placing the mixture in a water bath kettle at the temperature of 40-45 ℃ for incubation for 2-3 h;
(2) adding 100-500 μ L of acidic buffer solution, 100-400 μ L (20-60 mM concentration) of N, N-dimethylacrylamide and 10-100 μ L (30-70 mM concentration) of reducing agent into the solutions with various concentrations in the step (1) in sequence. Shaking up the mixture by shaking, and reacting the mixture for 10 to 120min at room temperature. Then dialyzing for 12-24 h by using a dialysis bag with the molecular weight of 500-1500 Da;
(3) taking 200-1000 mu L of the solution prepared in the step (2), sequentially adding 50-100 mu L (pH is 3.9-5.3) of a second acidic buffer solution, and adding HAuCl 4100 to 500 μ L (50 to 200mM) and shaking to mix them uniformly. Placing the sample in a microwave oven, setting the power to be 400-800 w, and keeping the time for 3-15 min to obtain the gold nanoclusters with strong fluorescence;
(4) and (4) repeating the steps (1) to (3), and measuring other cholesterol solutions with different concentration gradients to generate the fluorescence emission spectrum of the gold nanocluster. And establishing a quantitative relation curve of the numerical value of the fluorescence intensity of the gold nanocluster and the concentration of the cholesterol, and further calculating the concentration of the cholesterol in the fluorescence intensity range.
The invention also provides a method for detecting the concentration of cholesterol oxidase by adopting a fluorescence gold nanocluster fluorescence method, which aims to better realize the detection of cholesterol oxidase, ensure the fluorescence emission intensity and the accuracy and stability of a detection result, and refine and integrate a specific application process, and comprises the following steps:
1) mixing and incubating a cholesterol oxidase solution and a cholesterol solution to obtain a mixed solution;
2) reacting the mixed solution obtained in the step, an acidic buffer solution, N-dimethylacrylamide and a reducing agent to obtain a reaction solution;
3) carrying out microwave reaction on the reaction solution obtained in the step, a second acidic buffer solution and a soluble gold source to obtain a gold nanocluster solution with fluorescence intensity;
4) repeating the steps 1) to 3) by adopting cholesterol oxidase solutions with different concentrations to obtain gold nanocluster solutions with different fluorescence intensities;
5) and establishing a quantitative relation between the value of the fluorescence intensity of the gold nanoclusters and the concentration of the cholesterol oxidase, and detecting the concentration of the cholesterol oxidase in the sample.
The selection, composition and definition of the raw materials in the method for detecting the concentration of cholesterol oxidase by adopting the fluorescence method of the fluorescent gold nanocluster, and the corresponding preferred scheme thereof are basically consistent with the selection, composition and definition of the raw materials in the method for detecting the concentration of cholesterol by adopting the fluorescence method of the fluorescent gold nanocluster, and the corresponding preferred scheme thereof, and are not repeated here. The difference between the two is only that cholesterol and cholesterol oxidase are replaced.
The present invention is based on the use of cholesterol oxidase as an enzymatic catalytic oxidation of cholesterol, as shown in the following formula: cholesterol + O2--→Choleste-4-en-3-one+H2O2. H generated by oxidation-reduction reaction of cholesterol oxidase and cholesterol2O2。H2O2With a reducing agent (Cu)+、NaHSO3、Na2SO3、Na2S2O3Oxalic acid, glucose, alcohol, amine) under acidic conditions form an oxidation-reduction initiation system to generate OH free radicals. The free radical can initiate N, N-Dimethylacrylamide (DMAA) to carry out free radical polymerization, and the generated poly N, N-dimethylacrylamide (PDMAA) is used as a ligand of the gold nanocluster to generate the gold nanocluster with strong fluorescence under microwave radiation. By optimizing conditions, a quantitative relation can be established between the obtained nano-cluster fluorescence emission intensity and the concentration of cholesterol (or cholesterol oxidase), and then the detection of cholesterol is realized. Likewise, the detection of cholesterol oxidase can also be achieved.
The invention also provides a method for detecting whether the sample contains cholesterol by adopting a fluorescence method of the fluorogold nanocluster, which comprises the following steps:
a) mixing a sample to be detected and a cholesterol oxidase solution, and incubating to obtain a mixed solution;
b) reacting the mixed solution obtained in the step, an acidic buffer solution, N-dimethylacrylamide and a reducing agent to obtain a reaction solution;
c) carrying out microwave reaction on the reaction solution obtained in the step, the second acidic buffer solution and the soluble gold source to obtain a final solution;
when the final solution contains the gold nanoclusters with fluorescence intensity, cholesterol is contained in the sample to be detected;
when the final solution does not contain gold nanoclusters having fluorescence intensity, the sample to be tested does not contain cholesterol.
The selection, composition and definition of the raw materials in the method for detecting whether the sample contains cholesterol by adopting the fluorescence method of the fluorescent gold nanocluster and the corresponding preferred scheme thereof are basically consistent with the selection, composition and definition of the raw materials in the method for detecting the concentration of cholesterol by adopting the fluorescence method of the fluorescent gold nanocluster and the corresponding preferred scheme thereof, and are not repeated here.
The invention also provides a method for detecting whether a sample contains cholesterol oxidase by adopting a fluorescence method of the fluorescent gold nanocluster, which comprises the following steps:
A) mixing a sample to be detected and a cholesterol solution, and incubating to obtain a mixed solution;
B) reacting the mixed solution obtained in the step, an acidic buffer solution, N-dimethylacrylamide and a reducing agent to obtain a reaction solution;
C) carrying out microwave reaction on the reaction solution obtained in the step, the second acidic buffer solution and the soluble gold source to obtain a final solution;
when the final solution contains the gold nanoclusters with fluorescence intensity, the sample to be detected contains cholesterol oxidase;
when the final solution does not contain gold nanoclusters with fluorescence intensity, the sample to be tested does not contain cholesterol oxidase.
The selection, composition and definition of the raw materials in the method for detecting whether a sample contains cholesterol oxidase by adopting the fluorescence method of the fluorescent gold nanocluster and the corresponding preferred scheme thereof are basically consistent with the selection, composition and definition of the raw materials in the method for detecting the concentration of the cholesterol oxidase by adopting the fluorescence method of the fluorescent gold nanocluster and the corresponding preferred scheme thereof, and are not repeated herein.
The steps of the invention provide an application of the fluorogold nanocluster in detecting cholesterol and/or cholesterol oxidase by a fluorescence method, a method for detecting cholesterol and cholesterol concentration by the fluorogold nanocluster probe, a method for detecting cholesterol oxidase and cholesterol concentration by the fluorogold nanocluster probe, and a method for detecting whether a sample contains cholesterol or cholesterol oxidase by the fluorogold nanocluster probe. The invention uses the fluorescent gold nanocluster probe for detecting cholesterol and/or cholesterol oxidase, and has the characteristics of high sensitivity, good selectivity, wide linear range and the like. The method for detecting the cholesterol by the fluorescence emission of the fluorescence nanoclusters, which is established by the invention, not only avoids the interference of other factors of organisms, but also can selectively detect the concentration of the cholesterol in blood and urine and can detect the concentration of the cholesterol in a non-invasive way; the concentration of cholesterol is detected by using a fluorescence emission method instead of using a prepared fluorescent metal nano-cluster, so that the interference possibly caused by false signals in an experiment is avoided to the maximum extent; meanwhile, the fluorescent gold nanoclusters generated in the detection process have excellent fluorescence property, and have important significance in application in the fields of optical materials, biomedicine and the like.
Experimental results show that the method can be used for uniquely and quantitatively detecting the concentration of cholesterol in blood and urine of cholesterol and/or cholesterol oxidase, wherein the detection concentration range is 50-3500 mu M.
For further illustration of the present invention, the following will describe in detail the application of a fluorescent gold nanocluster in fluorescence method for detecting cholesterol and/or cholesterol oxidase, the method for detecting cholesterol concentration by fluorescence method using fluorescent gold nanocluster, the method for detecting cholesterol oxidase concentration by fluorescence method using fluorescent gold nanocluster, and the method for detecting whether a sample contains cholesterol or cholesterol oxidase by fluorescence method using fluorescent gold nanocluster, but it should be understood that these examples are implemented on the premise of the technical solution of the present invention, and the detailed embodiments and specific operation procedures are given, only to further illustrate the features and advantages of the present invention, but not to limit the claims of the present invention, and the scope of the present invention is not limited to the following examples.
Example 1
(1) 3700. mu.L of cholesterol solution (600. mu.M) was taken and blended with 250. mu.L of cholesterol oxidase at a concentration of 1 mg/mL. Then placing the mixture in a water bath kettle at the temperature of 43 ℃ for incubation for 2.5 h;
(2) to the solutions of the above step (1) at various concentrations, 350 μ L of disodium hydrogenphosphate-citric acid buffer (concentration 1mM, pH 4.0), 250 μ L of N, N-dimethylacrylamide (concentration 40mM), and 60 μ L of glucose (concentration 50mM) were sequentially added. Shaking up with shaking, and reacting at room temperature for 65 min. Then dialyzing for 18h by using a dialysis bag with the molecular weight of 1000 Da;
(3) taking 600 mu L of the solution prepared in the step (2), and sequentially adding HAuCl4250 μ L (100mM), 75 μ L disodium hydrogen phosphate-citric acid buffer (pH 4.5), and mix well with shaking. Placing the sample in a microwave oven, setting the power to be 600w, and obtaining the gold nanoclusters with strong fluorescence within 9 min;
(4) and (3) repeating the steps (1) to (3), measuring other cholesterol solutions with different concentration gradients (800 mu M and 1000 mu M), and generating the fluorescence emission spectrum of the gold nanocluster. And establishing a quantitative relation curve of the numerical value of the fluorescence intensity of the gold nanocluster and the concentration of the cholesterol, and further calculating the concentration of the cholesterol in the fluorescence intensity range.
The gold nanoclusters having strong fluorescence prepared in example 1 of the present invention were characterized and tested.
Referring to fig. 1, fig. 1 is a transmission electron microscope photograph of the fluorescent gold nanoclusters prepared in example 1 of the present invention.
As can be seen from FIG. 1, the fluorescent gold nanoclusters prepared by the method are uniform and stable in particle size and about 1.7nm in size.
Referring to fig. 2, fig. 2 is a graph showing the quantitative relationship between the fluorescence intensity value and the cholesterol concentration of the gold nanoclusters obtained in example 1 of the present invention.
Referring to fig. 3, fig. 3 is a fluorescent photograph of the fluorescent gold nanocluster solution generated from the cholesterol solution with different concentration gradients obtained in example 1 of the present invention under ultraviolet light.
As can be seen from fig. 3, this indicates that the provided gold nanoclusters may exhibit red-based fluorescence of different colors according to cholesterol solutions of different concentrations. Wherein, the concentration of the cholesterol solution from left to right is 600 μ M, 800 μ M and 1000 μ M respectively, and it can be clearly seen that the fluorescence intensity of the fluorogold nanoclusters is also obviously increased along with the increase of the concentration of the cholesterol solution.
Example 2
(1) 3500. mu.L of cholesterol solution (100. mu.M) was taken and blended with 100. mu.L of cholesterol oxidase having a concentration of 0.5 mg/mL. Then placing the mixture in a water bath kettle at 40 ℃ for incubation for 2 h;
(2) to the solutions of the above step (1) at various concentrations, 100 μ L of a citric acid-sodium hydroxide-hydrochloric acid buffer solution (concentration 0.5mM, pH 6.0), 100 μ L of N, N-dimethylacrylamide (concentration 20mM), and 10 μ L of glucose (concentration 30mM) were sequentially added. Shaking up with shaking, and reacting at room temperature for 10 min. Then dialyzing for 12h by using a dialysis bag with the molecular weight of 500 Da;
(3) taking 200 mu L of the solution prepared in the step (2), and sequentially adding HAuCl 4100 μ L (50mM), 50 μ L of citric acid-sodium hydroxide-hydrochloric acid buffer solution (pH 5.7), and mixed well with shaking. Placing the sample in a microwave oven, setting the power at 400w, and keeping the time for 15min to obtain the gold nanoclusters with strong fluorescence;
(4) and (3) repeating the steps (1) to (3), measuring other cholesterol solutions (200 mu M and 400 mu M) with different concentration gradients, and generating the fluorescence emission spectrum of the gold nanocluster. And establishing a quantitative relation curve of the numerical value of the fluorescence intensity of the gold nanocluster and the concentration of the cholesterol, and further calculating the concentration of the cholesterol in the fluorescence intensity range.
Example 3
(1) 4000. mu.L of cholesterol solution (1600. mu.M) was taken and blended with 500. mu.L of cholesterol oxidase at a concentration of 2 mg/mL. Then placing the mixture in a water bath kettle at 45 ℃ for incubation for 3 h;
(2) to the solutions of the above step (1) at various concentrations, 500. mu.L of glycine-hydrochloric acid buffer solution (concentration 2mM, pH 3.0), 400. mu.L of N, N-dimethylacrylamide (concentration 60mM), and Na were sequentially added2SO3mu.L (concentration 70 mM). Shaking up with shaking, and reacting at room temperature for 120 min. Then dialyzing for 24h by using a dialysis bag with the molecular weight of 1500 Da;
(3) taking 1000 mu L of the solution prepared in the step (2), and sequentially adding HAuCl 4500 μ L (200mM), citric acid-sodium hydroxide-hydrochloric acid buffer solution 100 μ L (pH 3.9), and mix well with shaking. Placing the sample in a microwave oven, setting the power to be 800w, and keeping the time for 3min to obtain the gold nanoclusters with strong fluorescence;
(4) and (3) repeating the steps (1) to (3), measuring other cholesterol solutions with different concentration gradients (1800 mu M and 2000 mu M), and generating the fluorescence emission spectrum of the gold nanocluster. And establishing a quantitative relation curve of the numerical value of the fluorescence intensity of the gold nanocluster and the concentration of the cholesterol, and further calculating the concentration of the cholesterol in the fluorescence intensity range.
Comparative example 1
The specific steps and parameters were the same as in example 1.
And (4) replacing the gold source with a silver source, namely silver nitrate, and finally obtaining a product solution.
The product solution obtained in comparative example 1 of the present invention was examined.
Referring to fig. 4, fig. 4 is a graph showing the fluorescence intensity of the product solution obtained in comparative example 1 of the present invention.
As can be seen from fig. 4, no silver nanoclusters having a fluorescent effect were generated in the process solution with respect to cholesterol or cholesterol oxidase.
Comparative example 2
The specific steps and parameters were the same as in example 1.
And (3) replacing the gold source with a copper source, namely copper nitrate, and finally obtaining a product solution.
The product solution obtained in comparative example 2 of the present invention was examined.
Referring to fig. 5, fig. 5 is a graph showing the fluorescence intensity of the product solution obtained in comparative example 2 of the present invention.
As can be seen from fig. 5, no copper nanoclusters having a fluorescent effect were generated in the process solution with respect to cholesterol or cholesterol oxidase.
The present invention provides a method for detecting cholesterol and/or cholesterol oxidase by fluorescence, a method for detecting cholesterol and its concentration by a fluorescent gold nanocluster probe, a method for detecting cholesterol oxidase and its concentration by a fluorescent gold nanocluster probe, and a method for detecting whether a sample contains cholesterol or cholesterol oxidase by a fluorescent gold nanocluster probe, which are described in detail above, and specific examples are used herein to illustrate the principles and embodiments of the present invention. It should be noted that, for those skilled in the art, it is possible to make various improvements and modifications to the present invention without departing from the principle of the present invention, and those improvements and modifications also fall within the scope of the claims of the present invention. The scope of the invention is defined by the claims and may include other embodiments that occur to those skilled in the art. Such other embodiments are intended to be within the scope of the claims if they have structural elements that do not differ from the literal language of the claims, or if they include equivalent structural elements with insubstantial differences from the literal languages of the claims.
Claims (10)
1. The application of a fluorescent gold nanocluster in detecting cholesterol and/or cholesterol oxidase by a fluorescence method;
in the process of detecting cholesterol by the fluorescence method, preparing the fluorescent gold nanoclusters in situ at the same time, wherein the raw materials prepared in situ comprise cholesterol and cholesterol oxidase;
in the process of detecting cholesterol oxidase by the fluorescence method, preparing the fluorescent gold nanoclusters in situ at the same time, wherein the raw materials prepared in situ comprise the cholesterol oxidase and cholesterol;
the fluorescence method is a non-fluorescence quenching method.
2. The use of claim 1, wherein the fluorometrically detecting cholesterol further comprises fluorometrically detecting a concentration of cholesterol;
the fluorescence emission intensity of the fluorescent gold nanoclusters is in direct proportion to the concentration of cholesterol;
the concentration of the cholesterol is 50-4000 mu M;
the fluorescent gold nanoclusters comprise gold nanoclusters and ligands;
the particle size of the fluorescent gold nanocluster is 1-5 nm.
3. The use of claim 2, wherein the fluorometric detection of cholesterol oxidase further comprises fluorometrically detecting the concentration of cholesterol oxidase;
the fluorescence emission intensity of the fluorescent gold nanocluster is in direct proportion to the concentration of cholesterol oxidase;
the concentration of the cholesterol oxidase is 50-4000 mu M;
the ligand is poly N, N-dimethylacrylamide;
the molar ratio of the gold nanoclusters to the ligand is 1: (0.2-4.8).
4. A method for detecting cholesterol concentration by adopting a fluorescence gold nanocluster fluorescence method is characterized by comprising the following steps:
1) mixing and incubating the cholesterol solution and the cholesterol oxidase solution to obtain a mixed solution;
2) reacting the mixed solution obtained in the step, an acidic buffer solution, N-dimethylacrylamide and a reducing agent to obtain a reaction solution;
3) carrying out microwave reaction on the reaction solution obtained in the step, a second acidic buffer solution and a soluble gold source to obtain a gold nanocluster solution with fluorescence intensity;
4) repeating the steps 1) to 3) by adopting cholesterol solutions with different concentrations to obtain gold nanocluster solutions with different fluorescence intensities;
5) and establishing a quantitative relation between the fluorescence intensity value of the gold nanocluster and the cholesterol concentration, and detecting the cholesterol concentration in the sample to be detected.
5. The method according to claim 4, wherein the concentration of the cholesterol oxidase solution is 0.5-2 mg/mL;
the volume ratio of the cholesterol solution to the cholesterol oxidase solution is (35-40): (1-5);
the temperature of the incubation is 40-45 ℃;
the incubation time is 2-3 h;
the acidic buffer solution comprises one or more of disodium hydrogen phosphate-citric acid buffer solution, citric acid-sodium hydroxide-hydrochloric acid buffer solution, acetic acid-sodium acetate buffer solution and disodium hydrogen phosphate-potassium dihydrogen phosphate buffer solution;
the pH value of the acidic buffer solution is 3.0-6.0;
the concentration of the acidic buffer solution is 0.5-2 mM;
the volume ratio of the cholesterol solution to the acidic buffer solution is (35-40): (1-5).
6. The method according to claim 4, wherein the volume ratio of the cholesterol solution to the N, N-dimethylacrylamide is (35-40): (1-4);
the concentration of the N, N-dimethylacrylamide is 20-60 mM;
the reducing agent comprises cuprous solution and NaHSO3Solution, Na2SO3Solution, Na2S2O3One or more of a solution, oxalic acid, a glucose solution, an alcohol solution, and an amine solution;
the concentration of the reducing agent is 30-70 mM;
the volume ratio of the cholesterol solution to the reducing agent is (35-40): (0.1 to 1);
the reaction time is 10-120 min;
a dialysis step is also included after the reaction;
the molecular weight of the dialyzed dialysis membrane is 500-1500 Da;
the dialysis time is 12-24 h.
7. The method of claim 4, wherein the second acidic buffer comprises one or more of a disodium hydrogen phosphate-citric acid buffer, a citric acid-sodium hydroxide-hydrochloric acid buffer, an acetic acid-sodium acetate buffer, and a disodium hydrogen phosphate-potassium dihydrogen phosphate buffer;
the pH value of the second acidic buffer solution is 3.9-5.3;
the concentration of the second acidic buffer solution is 0.5-2 mM;
the volume ratio of the reaction solution to the second acidic buffer solution is (2-10): (0.5 to 1);
the soluble gold source comprises HAuCl4A solution;
the concentration of the soluble gold source is 50-200 mM;
the volume ratio of the reaction solution to the soluble gold source is (2-10): (1-5);
the microwave power of the microwave reaction is 400-800 w;
the microwave reaction time is 3-15 min.
8. The method according to claim 4, wherein the concentration value of the cholesterol solution with different concentrations is 100-2000 μ M;
the quantitative relation comprises one or more of a quantitative relation curve, a quantitative relation formula, a quantitative relation table and a quantitative relation program;
the specific process for detecting the concentration of cholesterol in the sample to be detected comprises the following steps:
and (3) replacing the cholesterol solution with a sample to be detected, then performing the steps 1) to 3) to obtain a sample solution to be detected of the gold nanoclusters with certain fluorescence intensity, and calculating the cholesterol concentration in the sample to be detected according to the fluorescence intensity of the gold nanoclusters in the sample solution to be detected and the correlation quantitative relation.
9. A method for detecting the concentration of cholesterol oxidase by adopting a fluorescence gold nanocluster fluorescence method is characterized by comprising the following steps:
1) mixing and incubating a cholesterol oxidase solution and a cholesterol solution to obtain a mixed solution;
2) reacting the mixed solution obtained in the step, an acidic buffer solution, N-dimethylacrylamide and a reducing agent to obtain a reaction solution;
3) carrying out microwave reaction on the reaction solution obtained in the step, a second acidic buffer solution and a soluble gold source to obtain a gold nanocluster solution with fluorescence intensity;
4) repeating the steps 1) to 3) by adopting cholesterol oxidase solutions with different concentrations to obtain gold nanocluster solutions with different fluorescence intensities;
5) and establishing a quantitative relation between the value of the fluorescence intensity of the gold nanoclusters and the concentration of the cholesterol oxidase, and detecting the concentration of the cholesterol oxidase in the sample.
10. A method for detecting whether a sample contains cholesterol or cholesterol oxidase by adopting a fluorescent gold nanocluster fluorescence method is characterized by comprising the following steps of:
a) mixing a sample to be detected and a cholesterol oxidase solution, and incubating to obtain a mixed solution;
b) reacting the mixed solution obtained in the step, an acidic buffer solution, N-dimethylacrylamide and a reducing agent to obtain a reaction solution;
c) carrying out microwave reaction on the reaction solution obtained in the step, the second acidic buffer solution and the soluble gold source to obtain a final solution;
when the final solution contains the gold nanoclusters with fluorescence intensity, cholesterol is contained in the sample to be detected;
when the final solution does not contain the gold nanoclusters with fluorescence intensity, the sample to be detected does not contain cholesterol;
the method for detecting whether the sample contains cholesterol oxidase by adopting the fluorescence method of the fluorescent gold nanocluster comprises the following steps:
A) mixing a sample to be detected and a cholesterol solution, and incubating to obtain a mixed solution;
B) reacting the mixed solution obtained in the step, an acidic buffer solution, N-dimethylacrylamide and a reducing agent to obtain a reaction solution;
C) carrying out microwave reaction on the reaction solution obtained in the step, the second acidic buffer solution and the soluble gold source to obtain a final solution;
when the final solution contains the gold nanoclusters with fluorescence intensity, the sample to be detected contains cholesterol oxidase;
when the final solution does not contain gold nanoclusters with fluorescence intensity, the sample to be tested does not contain cholesterol oxidase.
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