CN109613266B - Method for detecting glycated albumin and concentration thereof, and method for detecting glycated amino acid oxidase-ketoamine oxidase and concentration thereof - Google Patents

Method for detecting glycated albumin and concentration thereof, and method for detecting glycated amino acid oxidase-ketoamine oxidase and concentration thereof Download PDF

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CN109613266B
CN109613266B CN201811644207.7A CN201811644207A CN109613266B CN 109613266 B CN109613266 B CN 109613266B CN 201811644207 A CN201811644207 A CN 201811644207A CN 109613266 B CN109613266 B CN 109613266B
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辛精卫
贾云霄
张晓杰
阴春霞
李凯
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Jilin University
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Abstract

The invention provides application of a fluorescent copper nano-cluster in detection of glycated albumin and/or glycated amino acid oxidase-ketoamine oxidase by a fluorescence method. The invention uses the fluorescent copper nanocluster probe for detecting the glycated albumin and/or the glycated amino acid oxidase-ketoamine oxidase, and has the characteristics of high sensitivity, good selectivity, wider linear range and the like. The method for detecting the glycated albumin by the fluorescence emission of the fluorescent nanoclusters avoids the interference of other factors, selectively detects the concentration of the glycated albumin in blood and urine, and can detect the glycated albumin noninvasively; the concentration of the glycated albumin is detected by using a fluorescence emission method instead of using a pre-prepared fluorescent copper nano cluster, so that the possible interference caused by false signals in an experiment is avoided to the maximum extent; meanwhile, the fluorescent copper nanoclusters generated in the detection process have excellent fluorescence property, and have important significance in the application of the fields of optical materials, biomedicine and the like.

Description

Method for detecting glycated albumin and concentration thereof, and method for detecting glycated amino acid oxidase-ketoamine oxidase and concentration thereof
Technical Field
The invention relates to the technical field of glycated albumin detection, in particular to application of a fluorescent copper nanocluster in detection of glycated albumin and/or glycated amino acid oxidase-ketoamine oxidase by a fluorescence method, a method for detecting the concentration of glycated albumin by a fluorescent copper nanocluster fluorescence method, a method for detecting the concentration of glycated amino acid oxidase-ketoamine oxidase by a fluorescent copper nanocluster fluorescence method and a method for detecting whether a sample contains glycated albumin or glycated amino acid oxidase-ketoamine oxidase by a fluorescent copper nanocluster fluorescence method, and particularly relates to application of a fluorescent copper nanocluster in detection of glycated albumin and/or glycated amino acid oxidase-ketoamine oxidase by a fluorescence method, a method for detecting glycated albumin and the concentration thereof, a method for detecting glycated amino acid oxidase-ketoamine oxidase and the concentration thereof and detection of whether a sample contains glycated albumin or glycated amino acid oxidase-ketoamine oxidase- A method of ketoamine oxidase.
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 copper nano-clusters (FM NCs) and the like. Among them, copper nanoclusters are a special material, and in the sixties of the 20 th century, researchers have sent out that copper such as cash, silver, copper and the like have weak fluorescence, but the fluorescence yield is particularly low, and the research of the fluorescent copper nanoclusters is more and more concerned by researchers along with the development of the technology and the needs of the society. 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 copper atoms, such as (Cu, Ag, Au, Pt). The size of the copper nano cluster is between that of a copper atom and that of a nano particle, when the size of a copper bulk material is reduced to a nano 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 the copper particles are closely related to and dependent on their size. At present, there are many fluorescent copper nanoclusters discovered by research, such as platinum nanoclusters, gold nanoclusters, silver nanoclusters, copper nanoclusters, and various alloy nanoclusters.
The fluorescent copper belongs to a nano-cluster, has strong fluorescence intensity, good compatibility with biological molecules and good stability, and the synthetic method is simple, so that the fluorescent copper has wide prospects in various fields of environmental monitoring, biological imaging, disease monitoring, catalysis, photoelectricity and the like, and particularly has high application value in the field of biological small molecule detection.
Glycated Albumin (GA) is one of glycated proteins, is a glycated product formed by non-enzymatic reaction of serum albumin and glucose, and is generated in an amount related to blood glucose concentration and albumin half-life, wherein the albumin half-life is 17-19 d, so that GA reflects the average blood glucose level of 2-3 weeks; the preparation is sensitive to blood sugar change in a short period, and has certain advantages particularly for patients in the adjustment period of a treatment scheme, incipient diabetes and in a stress state with large blood sugar fluctuation change. Meanwhile, the application field of GA is gradually expanded to the aspects of screening diabetes, predicting the risk of chronic complications, assisting in identifying stress hyperglycemia, assisting in diagnosing fulminant type 1 diabetes and the like, and the GA becomes a new blood sugar index with clinical application value. Therefore, it is necessary to establish a simple, sensitive and accurate method for detecting and analyzing glycated albumin.
At present, methods for detecting glycated albumin recorded in the existing documents mainly include high performance liquid chromatography, immunoassay, solid-state enzyme method and the like, but have the defects of complex pretreatment, complex operation and the like, and some methods also have the defects of large-scale instruments, 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 glycated albumin, which overcomes the above-mentioned drawbacks of the existing methods for detecting glycated albumin, 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 fluorescent copper nanocluster in fluorescence detection of glycated albumin and/or glycated amino acid oxidase-ketoamine oxidase, and particularly to a method for detecting a concentration of glycated albumin by using a fluorescent copper nanocluster fluorescence method, a method for detecting a concentration of glycated amino acid oxidase-ketoamine oxidase by using a fluorescent copper nanocluster fluorescence method, and a method for detecting whether a sample contains glycated albumin or glycated amino acid oxidase-ketoamine oxidase by using a fluorescent copper nanocluster fluorescence method. The method uses the fluorescent copper nanocluster in detecting the glycated albumin and/or the glycated amino acid oxidase-ketoamine 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 application of a fluorescent copper nano-cluster in detection of glycated albumin and/or glycated amino acid oxidase-ketoamine oxidase by a fluorescence method.
Preferably, the fluorescence method for detecting the glycated albumin further comprises the step of fluorescence method for detecting the concentration of the glycated albumin;
in the process of detecting the glycated albumin by the fluorescence method, simultaneously preparing the fluorescent copper nanoclusters in situ, wherein the raw materials prepared in situ comprise the glycated albumin and the glycated amino acid oxidase-ketoamine oxidase;
the fluorescence emission intensity of the fluorescent copper nanocluster is in direct proportion to the concentration of the glycated albumin;
the concentration of the glycated albumin is 50-4000 mu M;
the fluorescent copper nanocluster comprises a copper nanocluster and a ligand;
the particle size of the fluorescent copper nanocluster is 1-3 nm;
the fluorescence method is a non-fluorescence quenching method.
Preferably, the fluorescence method for detecting glycated amino acid oxidase-ketoamine oxidase further comprises fluorescence method for detecting the concentration of glycated amino acid oxidase-ketoamine oxidase;
in the process of detecting the glycated amino acid oxidase-ketoamine oxidase by the fluorescence method, simultaneously preparing the fluorescent copper nanoclusters in situ, wherein the raw materials prepared in situ comprise the glycated amino acid oxidase-ketoamine oxidase and glycated albumin;
the fluorescence emission intensity of the fluorescent copper nanocluster is in direct proportion to the concentration of the glycated amino acid oxidase-ketoamine oxidase;
the concentration of the glycated amino acid oxidase-ketoamine oxidase is 50-4000 mu M;
the ligand is poly (dimethylaminoethyl methacrylate);
the molar ratio of the copper nanocluster to the ligand is 1: (0.1-2.5).
The invention provides a method for detecting concentration of glycated albumin by adopting a fluorescent copper nanocluster fluorescence method, which comprises the following steps:
1) mixing and incubating a glycated albumin solution and a glycated amino acid oxidase-ketoamine oxidase solution to obtain a mixed solution;
2) reacting the mixed solution obtained in the step, an acidic buffer solution, dimethylaminoethyl methacrylate 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 copper source to obtain a copper nanocluster solution with fluorescence intensity;
4) repeating the steps 1) to 3) by adopting glycated albumin solutions with different concentrations to obtain copper nanocluster solutions with different fluorescence intensities;
5) and establishing a quantitative relation between the value of the fluorescence intensity of the copper nanocluster and the concentration of the glycated albumin, and detecting the concentration of the glycated albumin in the sample to be detected.
Preferably, the concentration of the glycated amino acid oxidase-ketoamine oxidase solution is 0.5-2 mg/mL;
the volume ratio of the glycated albumin solution to the glycated amino acid oxidase-ketoamine 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 3.2-6.0;
the concentration of the acidic buffer solution is 0.5-2 mM;
the volume ratio of the glycated albumin solution to the acidic buffer solution is (35-40): (1-5).
Preferably, the volume ratio of the glycated albumin solution to the dimethylaminoethyl methacrylate is (35-40): (0.5 to 2);
the concentration of the dimethylaminoethyl methacrylate is 10-35 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 glycated albumin 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 3.8-5.0;
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 copper source comprises Cu (NO)3)2Solution, CuSO4Solution and CuCl2One or more of a solution;
the concentration of the soluble copper source is 50-200 mM;
the volume ratio of the reaction solution to the soluble copper 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 glycated albumin 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 the glycated albumin in the sample to be detected comprises the following steps:
and (3) replacing the glycated albumin solution with a sample to be detected, then performing the steps 1) to 3) to obtain a sample solution to be detected of the copper nanocluster with a certain fluorescence intensity, and calculating the concentration of the glycated albumin in the sample to be detected according to the fluorescence intensity of the copper nanocluster in the sample solution to be detected and the correlation quantitative relationship.
The invention provides a method for detecting the concentration of glycated amino acid oxidase-ketoamine oxidase by adopting a fluorescent copper nanocluster fluorescence method, which comprises the following steps:
1) mixing and incubating a glycated amino acid oxidase-ketoamine oxidase solution and a glycated albumin solution to obtain a mixed solution;
2) reacting the mixed solution obtained in the step, an acidic buffer solution, dimethylaminoethyl methacrylate 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 copper source to obtain a copper nanocluster solution with fluorescence intensity;
4) adopting saccharified amino acid oxidase-ketoamine oxidase solutions with different concentrations to repeat the steps 1) to 3) to obtain copper nano-cluster solutions with different fluorescence intensities;
5) and establishing a quantitative relation between the value of the fluorescence intensity of the copper nanocluster and the concentration of the glycated amino acid oxidase-ketoamine oxidase, and detecting the concentration of the glycated amino acid oxidase-ketoamine oxidase in the sample.
The invention also provides a method for detecting whether a sample contains glycated albumin or glycated amino acid oxidase-ketoamine oxidase by adopting a fluorescent copper nano-cluster fluorescence method, and the method for detecting whether the sample contains the glycated albumin or not by adopting the fluorescent copper nano-cluster fluorescence method comprises the following steps:
a) mixing and incubating a sample to be detected and a saccharified amino acid oxidase-ketoamine oxidase solution to obtain a mixed solution;
b) reacting the mixed solution obtained in the step, an acidic buffer solution, dimethylaminoethyl methacrylate and a reducing agent to obtain a reaction solution;
c) carrying out microwave reaction on the reaction solution obtained in the step, a second acidic buffer solution and a soluble copper source to obtain a final solution;
when the final solution contains copper nanoclusters with fluorescence intensity, the sample to be detected contains glycated albumin;
when the final solution does not contain the copper nanoclusters with fluorescence intensity, the sample to be tested does not contain the glycated albumin;
the method for detecting whether the sample contains the glycated amino acid oxidase-ketoamine oxidase or not by adopting the fluorescent copper nanocluster fluorescence method comprises the following steps:
A) mixing a sample to be detected and a glycated albumin solution, and incubating to obtain a mixed solution;
B) reacting the mixed solution obtained in the step, an acidic buffer solution, dimethylaminoethyl methacrylate and a reducing agent to obtain a reaction solution;
C) carrying out microwave reaction on the reaction solution obtained in the step, a second acidic buffer solution and a soluble copper source to obtain a final solution;
when the final solution contains copper nanoclusters with fluorescence intensity, the sample to be detected contains glycated amino acid oxidase-ketoamine oxidase;
and when the final solution does not contain the copper nanoclusters with fluorescence intensity, the sample to be detected does not contain the glycated amino acid oxidase-ketoamine oxidase.
The invention provides application of a fluorescent copper nano-cluster in detection of glycated albumin and/or glycated amino acid oxidase-ketoamine 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 glycated albumin has the defects of complex pretreatment, complex operation and the like, and the existing method for detecting glycated albumin also needs a large-scale instrument, is complex to operate, expensive in equipment, long in time consumption, low in sensitivity and the like, and is difficult to meet the daily detection requirement. The method also aims at the problem that in the prior art, a small amount of reports for detecting the glycated albumin based on the nanoclusters exist, but the detection is 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 fluorescent copper nanocluster probe is creatively used for detecting the glycated albumin and/or the glycated amino acid oxidase-ketoamine oxidase, and has the characteristics of high sensitivity, good selectivity, wider linear range and the like. The method for detecting the glycated albumin by the fluorescence emission of the fluorescent nanoclusters, which is established by the invention, not only avoids the interference of other factors of an organism, but also can selectively detect the concentration of the glycated albumin in blood and urine and can noninvasively detect the concentration of the glycated albumin; the concentration of the glycated albumin is detected by using a fluorescence emission method instead of using a pre-prepared fluorescent copper nano cluster, so that the possible interference caused by false signals in an experiment is avoided to the maximum extent; meanwhile, the fluorescent copper nanoclusters generated in the detection process have excellent fluorescence property, and have important significance in the application of 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 the glycated albumin and/or the glycated amino acid oxidase-ketoamine oxidase in blood and urine, and the detection concentration range is 50-3500 mu M.
Drawings
FIG. 1 is a TEM photograph of fluorescent copper nanoclusters prepared in example 1 of the present invention;
FIG. 2 is a graph showing the quantitative relationship between the fluorescence intensity of copper nanoclusters obtained in example 1 of the present invention and the glycated albumin concentration;
FIG. 3 is a fluorescent photograph of a fluorescent copper nanocluster solution generated from a glycated albumin 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 application of a fluorescent copper nano-cluster in detection of glycated albumin and/or glycated amino acid oxidase-ketoamine 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, and the fluorescence method is preferably a fluorescence emission method in order to better realize the detection of glycated albumin or glycated amino acid oxidase-ketoamine oxidase and ensure the fluorescence emission intensity and the accuracy and stability of the detection result. The fluorescence method of the invention is particularly a non-fluorescence quenching method.
The composition of the fluorescent copper nanocluster 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. The ligand of the invention is preferably poly (dimethylamino ethyl methacrylate) (PDMAEMA). The molar ratio of the copper nanoclusters and the ligand is preferably 1: (0.1 to 2.5), more preferably 1: (0.5 to 2.0), more preferably 1: (1.0-1.5).
The structure and parameters of the fluorescent copper nanocluster are not particularly limited in principle, and the structure of the copper nanocluster known by the person skilled in the art can be selected and adjusted according to actual conditions, effect data and specific requirements. Wherein, the fluorescent copper nanocluster preferably comprises a copper nanocluster and a ligand layer compounded on the surface of the copper nanocluster. The particle size of the fluorescent copper nanocluster is preferably 1-3 nm, more preferably 1.2-2.8 nm, more preferably 1.5-2.5 nm, and more preferably 1.7-2.3 nm.
The fluorescence method for detecting the glycated albumin preferably refers to a method for detecting whether the glycated albumin exists or not by adopting a fluorescent copper nanocluster probe emission fluorescence method, and simultaneously comprises the step of detecting the concentration content of the glycated albumin. Particularly, in the process of detecting the glycated albumin by the fluorescence method, the fluorescent copper nanoclusters are prepared in situ at the same time, and the raw materials prepared in situ comprise the glycated albumin and the glycated amino acid oxidase-ketoamine oxidase. More particularly, the fluorescence emission intensity of the fluorescent copper nanocluster is in direct proportion to the concentration of the glycated albumin, i.e., the higher the content of the glycated albumin in the sample to be detected, the stronger the fluorescence emission intensity of the fluorescent copper nanocluster. If the sample to be detected does not contain the glycated albumin, the fluorescent copper nanoclusters in the invention cannot be formed, which is the difference between the invention and the existing fluorescence quenching method.
The concentration of the glycated albumin is not particularly limited in principle, and may be a conventional concentration of glycated albumin known to those skilled in the art, and those skilled in the art may select and adjust the concentration according to actual conditions, effect data and specific requirements, in order to better realize the detection of glycated albumin or glycated amino acid oxidase-ketoamine oxidase and ensure the fluorescence emission intensity and the accuracy and stability of the detection result, the concentration of the glycated albumin is preferably within the range of a fluorescence emission intensity detector, and is preferably 50 to 3500 μ M, more preferably 70 to 3000 μ M, more preferably 90 to 2500 μ M, more preferably 100 to 2000 μ M, more preferably 500 to 1500 μ M, more preferably 800 to 1200 μ M.
The fluorescence method for detecting the glycated amino acid oxidase-ketoamine oxidase preferably comprises the steps of detecting whether the glycated amino acid oxidase-ketoamine oxidase exists or not by adopting a fluorescent copper nano-cluster probe emission fluorescence method, and simultaneously detecting the concentration content of the glycated amino acid oxidase-ketoamine oxidase. Particularly, in the process of detecting the glycated amino acid oxidase-ketoamine oxidase by the fluorescence method, the fluorescent copper nanoclusters are simultaneously prepared in situ, and the raw materials prepared in situ comprise the glycated amino acid oxidase-ketoamine oxidase and glycated albumin. More particularly, the fluorescence emission intensity of the fluorescent copper nanocluster is in direct proportion to the concentration of the glycated amino acid oxidase-ketoamine oxidase, i.e., the higher the content of the glycated amino acid oxidase-ketoamine oxidase in the sample to be detected is, the stronger the fluorescence emission intensity of the fluorescent copper nanocluster is. If the sample to be detected does not contain the glycated amino acid oxidase-ketoamine oxidase, the fluorescent copper nanoclusters are not formed, which is the difference between the method and the existing fluorescence quenching method.
The concentration of the glycated amino acid oxidase-ketoamine oxidase is not particularly limited in principle, the conventional concentration of the glycated amino acid oxidase-ketoamine oxidase known by the technicians in the field can be used, and the technicians in the field can select and adjust the concentration according to the actual situation, the effect data and the specific requirements, in order to better realize the detection of the glycated albumin or the glycated amino acid oxidase-ketoamine oxidase and ensure the fluorescence emission intensity and the accuracy and the stability of the detection result, the concentration of the glycated amino acid oxidase-ketoamine oxidase 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 invention also provides a method for detecting the concentration of the glycated albumin by using a fluorescent copper nanocluster fluorescence method, which comprises the following steps of:
1) mixing and incubating a glycated albumin solution and a glycated amino acid oxidase-ketoamine oxidase solution to obtain a mixed solution;
2) reacting the mixed solution obtained in the step, an acidic buffer solution, dimethylaminoethyl methacrylate 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 copper source to obtain a copper nanocluster solution with fluorescence intensity;
4) repeating the steps 1) to 3) by adopting glycated albumin solutions with different concentrations to obtain copper nanocluster solutions with different fluorescence intensities;
5) and establishing a quantitative relation between the value of the fluorescence intensity of the copper nanocluster and the concentration of the glycated albumin, and detecting the concentration of the glycated albumin in the sample to be detected.
The selection, composition and definition of the raw materials in the method for detecting the concentration of glycated albumin by using the fluorescence of the fluorescent copper nanocluster and the corresponding preferred scheme thereof are basically consistent with the selection, composition and definition of the raw materials in the application and the corresponding preferred scheme thereof, and are not repeated herein.
Firstly, a glycated albumin solution and a glycated amino acid oxidase-ketoamine oxidase solution are mixed and incubated to obtain a mixed solution.
The concentration and the dosage of the glycated amino acid oxidase-ketoamine 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 detection of glycated albumin and ensure the fluorescence emission intensity and the accuracy and the stability of a detection result, the concentration of the glycated amino acid oxidase-ketoamine 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 glycated albumin solution to the glycated amino acid oxidase-ketoamine 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 number of volume ratio bases of the glycated albumin 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 glycated albumin solution preferably adopts the preferred principle, and is not described in detail.
The incubation condition 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, in order to better realize detection of glycated albumin and ensure fluorescence emission intensity and detection result accuracy and stability, 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, dimethylaminoethyl methacrylate 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 detection of glycated albumin and ensure fluorescence emission intensity and accuracy and stability of detection results, the acidic buffer solution preferably includes 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 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 glycated albumin and ensure fluorescence emission intensity and accuracy and stability of a detection result, the pH value of the acidic buffer solution is preferably 3.2-6.0, more preferably 3.7-5.5, and more preferably 4.2-5.0. 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 glycated albumin 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 dimethylaminoethyl methacrylate (DMAEMA) 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 the detection of the glycated albumin and ensure the fluorescence emission intensity and the accuracy and the stability of the detection result, the concentration of the dimethylaminoethyl methacrylate is preferably 10-35 mM, more preferably 15-30 mM and more preferably 20-25 mM. The volume ratio of the glycated albumin solution to the dimethylaminoethyl methacrylate is preferably (35-40): (0.5-2), more preferably (35-40): (0.8-1.8), more preferably (35-40): (1-1.5).
The invention is not particularly limited in principle, 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 the glycated albumin and ensure the fluorescence emission intensity and the accuracy and the stability of the detection result, the reducing agent preferably comprises 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 detection of glycated albumin and ensure fluorescence emission intensity and 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 glycated albumin 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 glycated albumin 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 the glycated albumin, ensure the fluorescence emission intensity and the accuracy and stability of the 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 obtained in the step, the second acidic buffer solution and the soluble copper source are subjected to microwave reaction to obtain the copper 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 glycated albumin and ensure the fluorescence emission intensity and the accuracy and stability of the detection result, the second acidic buffer solution preferably includes 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 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 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 glycated albumin 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 3.8-5.0, more preferably 4.0-4.7, and more preferably 4.2-4.5. 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 copper 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 requirements3)2Solution, CuSO4Solution and CuCl2One or more of the above, more preferably Cu (NO) in solution3)2Solution, CuSO4Solutions or CuCl2And (3) solution.
The concentration of the soluble copper source is preferably 50-200 mM, more preferably 80-180 mM, and even more preferably 100-150 mM, in order to better realize detection of glycated albumin and ensure fluorescence emission intensity and detection result accuracy and stability. The volume ratio of the reaction solution to the soluble copper 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 glycated albumin and ensure the fluorescence emission intensity and the accuracy and stability of a detection result, the microwave reaction is performed 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, adopting glycated albumin solutions with different concentrations to repeat the steps 1) to 3) to obtain copper nanocluster solutions with different fluorescence intensities.
The concentration values of the glycated albumin 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 glycated albumin and ensure fluorescence emission intensity and accuracy and stability of detection results, the concentration values of the glycated albumin solutions with different concentrations are preferably selected from 100 to 2000 μ M, more preferably from 300 to 1500 μ M, more preferably from 500 to 1200 μ M, and more preferably from 800 to 1000 μ M.
The number (i.e. the number of repetitions) and the selection manner of the glycated albumin solutions 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, which are well known to those skilled in the art.
The invention finally establishes the quantitative relation between the fluorescence intensity value of the copper nanocluster and the concentration of the glycated albumin, and is used for detecting the concentration of the glycated albumin in the sample to be detected.
The present invention is not particularly limited in particular in terms of 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 is to better realize the detection of glycated albumin and ensure 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 the glycated albumin, ensure the fluorescence emission intensity and the accuracy and stability of the detection result and complete and refine the whole glycated albumin detection process, the specific process for detecting the concentration of the glycated albumin in the sample to be detected is preferably as follows:
and (3) replacing the glycated albumin solution with a sample to be detected, then performing the steps 1) to 3) to obtain a sample solution to be detected of the copper nanocluster with a certain fluorescence intensity, and calculating the concentration of the glycated albumin in the sample to be detected according to the fluorescence intensity of the copper nanocluster in the sample solution to be detected and the correlation quantitative relationship.
In order to better realize the detection of the glycated albumin, ensure the fluorescence emission intensity and the accuracy and stability of the detection result and complete and refine the whole glycated albumin detection process, the specific process of the method for detecting the concentration of the glycated albumin by adopting the fluorescent copper nanocluster fluorescence method can also comprise the following steps:
(1) 3500-4000 μ L of a glycated albumin solution (100-2000 μ M) with different concentration gradients is taken and mixed with 100-500 μ L of glycated amino acid oxidase-ketoamine oxidase with a 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) to the solutions of various concentrations obtained in the step (1), 100 to 500. mu.L of an acidic buffer solution (concentration of 0.5 to 2mM, pH 3.5 to 6.2), 100 to 400. mu.L of dimethylaminoethyl methacrylate (concentration of 10 to 35mM), and 10 to 100. mu.L of a reducing agent (concentration of 30 to 70mM) are sequentially added. 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 4.0-5.2) of a second acidic buffer solution, and adding Cu (NO)3)2100 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 copper nanocluster with strong fluorescence;
(4) and (4) repeating the steps (1) to (3), and measuring the other glycated albumin solutions with different concentration gradients to generate the fluorescence emission spectrum of the copper nanocluster. And establishing a quantitative relation curve of the numerical value of the fluorescence intensity of the copper nanocluster and the concentration of the glycated albumin, and further calculating the concentration of the glycated albumin in the fluorescence intensity range.
The invention provides a method for detecting the concentration of the glycated amino acid oxidase-ketoamine oxidase by adopting a fluorescent copper nanocluster fluorescence method, which aims to better realize the detection of the glycated amino acid oxidase-ketoamine oxidase, ensure the fluorescence emission intensity and the accuracy and stability of a detection result, refine and complete the specific application process, and comprises the following steps:
1) mixing and incubating a glycated amino acid oxidase-ketoamine oxidase solution and a glycated albumin solution to obtain a mixed solution;
2) reacting the mixed solution obtained in the step, an acidic buffer solution, dimethylaminoethyl methacrylate 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 copper source to obtain a copper nanocluster solution with fluorescence intensity;
4) adopting saccharified amino acid oxidase-ketoamine oxidase solutions with different concentrations to repeat the steps 1) to 3) to obtain copper nano-cluster solutions with different fluorescence intensities;
5) and establishing a quantitative relation between the value of the fluorescence intensity of the copper nanocluster and the concentration of the glycated amino acid oxidase-ketoamine oxidase, and detecting the concentration of the glycated amino acid oxidase-ketoamine oxidase in the sample.
The selection, composition and definition of the raw materials in the method for detecting the concentration of glycated amino acid oxidase-ketoamine oxidase by using the fluorescent copper nanocluster fluorescence method, and the corresponding preferred scheme thereof are basically the same as the selection, composition and definition of the raw materials in the method for detecting the concentration of glycated albumin by using the fluorescent copper nanocluster fluorescence method, and the corresponding preferred scheme thereof, and are not repeated herein. The difference between the two is only that the glycated albumin and the glycated amino acid oxidase-ketoamine oxidase are replaced.
The invention is based on the fact that albumin has specific protease action and dissociates saccharified amino acid. When the saccharified amino acid oxidase-ketoamine oxidase and the produced saccharified amino acid undergo oxidation-reduction reaction to produce H2O2。H2O2And a reducing agent forms an oxidation-reduction initiation system under an acidic condition to generate OH free radicals. The free radical can initiate dimethyl amino ethyl methacrylate (DMAEMA) to carry out free radical polymerization, the generated dimethyl amino ethyl methacrylate (PDMAEMA) is used as a ligand of the copper nano-cluster, and the copper nano-cluster with strong fluorescence is generated under microwave irradiation. By optimizing conditions, a quantitative relation can be established between the obtained nano-cluster fluorescence emission intensity and the concentration of the glycated albumin (or the glycated amino acid oxidase-ketoamine oxidase), and then the detection of the glycated albumin is realized. Also, the detection of the glycated amino acid oxidase-ketoamine oxidase can be realizedAnd (6) measuring.
The invention also provides a method for detecting whether a sample contains glycated albumin by adopting a fluorescent copper nanocluster fluorescence method, which comprises the following steps:
a) mixing and incubating a sample to be detected and a saccharified amino acid oxidase-ketoamine oxidase solution to obtain a mixed solution;
b) reacting the mixed solution obtained in the step, an acidic buffer solution, dimethylaminoethyl methacrylate and a reducing agent to obtain a reaction solution;
c) carrying out microwave reaction on the reaction solution obtained in the step, a second acidic buffer solution and a soluble copper source to obtain a final solution;
when the final solution contains copper nanoclusters with fluorescence intensity, the sample to be detected contains glycated albumin;
when the final solution does not contain the copper nanoclusters with fluorescence intensity, the sample to be tested does not contain the glycated albumin.
The selection, composition and definition of the raw materials in the method for detecting whether the sample contains the glycated albumin by using the fluorescent copper nanocluster fluorescence method 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 glycated albumin by using the fluorescent copper nanocluster fluorescence method and the corresponding preferred scheme thereof, and are not repeated here.
The invention also provides a method for detecting whether a sample contains the glycated amino acid oxidase-ketoamine oxidase by adopting a fluorescent copper nano-cluster fluorescence method, which comprises the following steps:
A) mixing a sample to be detected and a glycated albumin solution, and incubating to obtain a mixed solution;
B) reacting the mixed solution obtained in the step, an acidic buffer solution, dimethylaminoethyl methacrylate and a reducing agent to obtain a reaction solution;
C) carrying out microwave reaction on the reaction solution obtained in the step, a second acidic buffer solution and a soluble copper source to obtain a final solution;
when the final solution contains copper nanoclusters with fluorescence intensity, the sample to be detected contains glycated amino acid oxidase-ketoamine oxidase;
and when the final solution does not contain the copper nanoclusters with fluorescence intensity, the sample to be detected does not contain the glycated amino acid oxidase-ketoamine oxidase.
The selection, composition and definition of the raw materials in the method for detecting whether the sample contains the glycated amino acid oxidase-ketoamine oxidase by using the fluorescent copper nanocluster fluorescence method and the corresponding preferred scheme thereof are basically the same as the selection, composition and definition of the raw materials in the method for detecting the concentration of the glycated amino acid oxidase-ketoamine oxidase by using the fluorescent copper nanocluster fluorescence method and the corresponding preferred scheme thereof, and are not repeated herein.
The steps of the invention provide an application of fluorescent copper nanoclusters in detection of glycated albumin and/or glycated amino acid oxidase-ketoamine oxidase by a fluorescence method, a method for detecting glycated albumin and the concentration thereof by a fluorescent copper nanocluster probe, a method for detecting glycated amino acid oxidase-ketoamine oxidase and the concentration thereof by a fluorescent copper nanocluster probe, and a method for detecting whether a sample contains glycated albumin or glycated amino acid oxidase-ketoamine oxidase or not by a fluorescent copper nanocluster probe. The invention uses the fluorescent copper nanocluster probe for detecting the glycated albumin and/or the glycated amino acid oxidase-ketoamine oxidase, and has the characteristics of high sensitivity, good selectivity, wider linear range and the like. The method for detecting the glycated albumin by the fluorescence emission of the fluorescent nanoclusters, which is established by the invention, not only avoids the interference of factors, but also can selectively detect the concentration of the glycated albumin in blood and urine and can non-invasively detect the concentration of the glycated albumin; the concentration of the glycated albumin is detected by using a fluorescence emission method instead of using a pre-prepared fluorescent copper nano cluster, so that the possible interference caused by false signals in an experiment is avoided to the maximum extent; meanwhile, the fluorescent copper nanoclusters generated in the detection process have excellent fluorescence property, and have important significance in the application of 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 the glycated albumin and/or the glycated amino acid oxidase-ketoamine oxidase in blood and urine, and the detection concentration range is 50-3500 mu M.
To further illustrate the present invention, the following will describe in detail the application of a fluorescent copper nanocluster in fluorescence detection of glycated albumin and/or glycated amino acid oxidase-ketoamine oxidase, the method of fluorescence detection of glycated albumin using fluorescent copper nanocluster, the method of fluorescence detection of glycated amino acid oxidase-ketoamine oxidase and the method of fluorescence detection of glycated albumin or glycated amino acid oxidase-ketoamine oxidase in a sample using fluorescent copper nanocluster, but it should be understood that these examples are carried out on the premise of the technical solution of the present invention, and that detailed embodiments and specific procedures are given only for further illustration of the features and advantages of the present invention, but not for limitation of the claims of the present invention, the scope of the invention is not limited to the following examples.
Example 1
(1) 3700. mu.L of glycated albumin solution (600. mu.M) was taken and blended with 250. mu.L of glycated amino acid oxidase-ketoamine oxidase having 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. mu.L of phthalic acid-hydrochloric acid buffer (concentration: 1mM, pH 4.1), 250. mu.L of dimethylaminoethyl methacrylate (concentration: 20mM), and Na were sequentially added2SO360 μ L (concentration 50 mM). 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 Cu (NO)3)2250 μ L (100mM), 75 μ L disodium hydrogen phosphate-potassium dihydrogen phosphate buffer solution (pH 4.5), and mix well with shaking. Placing the sample in a microwave oven, setting the power to be 600w, and obtaining the copper nanocluster with strong fluorescence within 9 min;
(4) and (3) repeating the steps (1) to (3), and measuring the fluorescence emission spectrum of the generated copper nanoclusters of other glycated albumin solutions (800 mu M and 1000 mu M) with different concentration gradients. And establishing a quantitative relation curve of the numerical value of the fluorescence intensity of the copper nanocluster and the concentration of the glycated albumin, and further calculating the concentration of the glycated albumin in the fluorescence intensity range.
The copper nanoclusters with strong fluorescence prepared in example 1 of the present invention were characterized and detected.
Referring to fig. 1, fig. 1 is a transmission electron microscope photograph of fluorescent copper 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 1.5nm in size.
Referring to fig. 2, fig. 2 is a graph showing the quantitative relationship between the fluorescence intensity of copper nanoclusters obtained in example 1 of the present invention and the glycated albumin concentration.
Referring to fig. 3, fig. 3 is a fluorescent photograph of fluorescent copper nanocluster solutions generated from glycated albumin solutions 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 fluorescent copper nanoclusters can exhibit red fluorescence of different colors according to different concentrations of glycated albumin solutions. The concentration of the glycated albumin 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 fluorescent copper nanoclusters also increases with the increase of the concentration of the glycated albumin solution.
Example 2
(1) 3500. mu.L of glycated albumin solution (100. mu.M) was taken and mixed with 100. mu.L of glycated amino acid oxidase-ketoamine 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 various concentrations in the above step (1), 100. mu.L of citric acid-sodium hydroxide-hydrochloric acid buffer solution (concentration 0.5mM, pH 6.2), 100. mu.L of dimethylaminoethyl methacrylate (concentration 10mM), NaHSO, were added in this order310 μ L (concentration 30 mM). 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 CuSO4Mu.l (50mM), 50. mu.l of citric acid-sodium hydroxide-hydrochloric acid buffer (pH 5.9) and mix well with shaking. Placing the sample in a microwave oven, setting the power to be 400w, and keeping the time for 15min to obtain the copper nanocluster with strong fluorescence;
(4) and (3) repeating the steps (1) to (3), and measuring the fluorescence emission spectrum of the generated copper nanoclusters of other glycated albumin solutions (200 mu M and 400 mu M) with different concentration gradients. And establishing a quantitative relation curve of the numerical value of the fluorescence intensity of the copper nanocluster and the concentration of the glycated albumin, and further calculating the concentration of the glycated albumin in the fluorescence intensity range.
Example 3
(1) 4000. mu.L of glycated albumin solution (1600. mu.M) was taken and mixed with 500. mu.L of glycated amino acid oxidase-ketoamine oxidase having 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 3.5), 400. mu.L of dimethylaminoethyl methacrylate (concentration 35mM), and 100. mu.L of oxalic acid (concentration 70mM) were sequentially added. 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 CuCl 2500. mu.L (200mM), 100. mu.L of disodium hydrogenphosphate-potassium dihydrogenphosphate buffer solution (pH 4.0), and mixed 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 copper nanocluster with strong fluorescence;
(4) and (3) repeating the steps (1) to (3), and measuring the fluorescence emission spectrum of the generated copper nanoclusters of other glycated albumin solutions (1800 mu M and 2000 mu M) with different concentration gradients. And establishing a quantitative relation curve of the numerical value of the fluorescence intensity of the copper nanocluster and the concentration of the glycated albumin, and further calculating the concentration of the glycated albumin in the fluorescence intensity range.
Comparative example 1
The specific steps and parameters were the same as in example 1.
And (4) replacing the copper 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 for glycated albumin or glycated amino acid oxidase-ketoamine oxidase.
Comparative example 2
The specific steps and parameters were the same as in example 1.
Changing copper source to gold source-HAuCl4And 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 gold nanoclusters having a fluorescent effect were generated in the process solution for glycated albumin or glycated amino acid oxidase-ketoamine oxidase.
The above detailed description of the application of a fluorescent copper nanocluster in detecting glycated albumin and/or glycated amino acid oxidase-ketoamine oxidase by a fluorescence method, the method for detecting glycated albumin and its concentration by a fluorescent copper nanocluster probe, the method for detecting glycated amino acid oxidase-ketoamine oxidase and its concentration by a fluorescent copper nanocluster probe, and the method for detecting whether a sample contains glycated albumin or glycated amino acid oxidase-ketoamine oxidase by a fluorescent copper nanocluster probe have been presented, and the principles and embodiments of the present invention are explained herein by using specific examples, which are only used to help understanding the method of the present invention and its core ideas, including the best mode, and also to enable any person skilled in the art to practice the present invention, including making and using any devices or systems and performing any incorporated methods. 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 copper nano-cluster in detecting glycated albumin and/or glycated amino acid oxidase-ketoamine oxidase by a fluorescence method;
in the process of detecting the glycated albumin by the fluorescence method, simultaneously preparing the fluorescent copper nanoclusters in situ, wherein the raw materials prepared in situ comprise the glycated albumin and the glycated amino acid oxidase-ketoamine oxidase;
the fluorescence method for detecting the glycated albumin further comprises the step of detecting the concentration of the glycated albumin by the fluorescence method;
in the process of detecting the glycated amino acid oxidase-ketoamine oxidase by the fluorescence method, simultaneously preparing the fluorescent copper nanoclusters in situ, wherein the raw materials prepared in situ comprise the glycated amino acid oxidase-ketoamine oxidase and glycated albumin;
the fluorescence method for detecting the glycated amino acid oxidase-ketoamine oxidase also comprises the step of detecting the concentration of the glycated amino acid oxidase-ketoamine oxidase by the fluorescence method;
the fluorescent copper nanocluster comprises a copper nanocluster and a ligand;
the ligand is poly (dimethylaminoethyl methacrylate);
the fluorescence method is a non-fluorescence quenching method.
2. The use of claim 1, wherein the fluorescent copper nanoclusters have a fluorescence emission intensity proportional to the concentration of glycated albumin;
the concentration of the glycated albumin is 50-4000 mu M;
the particle size of the fluorescent copper nanocluster is 1-3 nm.
3. The use of claim 2, wherein the fluorescent copper nanoclusters have a fluorescence emission intensity proportional to the concentration of glycated amino acid oxidase-ketoamine oxidase;
the concentration of the glycated amino acid oxidase-ketoamine oxidase is 50-4000 mu M;
the molar ratio of the copper nanocluster to the ligand is 1: (0.1-2.5).
4. A method for detecting the concentration of glycated albumin by a fluorescent copper nanocluster fluorescence method is characterized by comprising the following steps:
1) mixing and incubating a glycated albumin solution and a glycated amino acid oxidase-ketoamine oxidase solution to obtain a mixed solution;
2) reacting the mixed solution obtained in the step, an acidic buffer solution, dimethylaminoethyl methacrylate 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 copper source to obtain a copper nanocluster solution with fluorescence intensity;
4) repeating the steps 1) to 3) by adopting glycated albumin solutions with different concentrations to obtain copper nanocluster solutions with different fluorescence intensities;
5) and establishing a quantitative relation between the value of the fluorescence intensity of the copper nanocluster and the concentration of the glycated albumin, and detecting the concentration of the glycated albumin in the sample to be detected.
5. The method according to claim 4, wherein the glycated amino acid oxidase-ketoamine oxidase solution has a concentration of 0.5 to 2 mg/mL;
the volume ratio of the glycated albumin solution to the glycated amino acid oxidase-ketoamine 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 3.2-6.0;
the concentration of the acidic buffer solution is 0.5-2 mM;
the volume ratio of the glycated albumin solution to the acidic buffer solution is (35-40): (1-5).
6. The method according to claim 4, wherein the volume ratio of the glycated albumin solution to the dimethylaminoethyl methacrylate is (35-40): (0.5 to 2);
the concentration of the dimethylaminoethyl methacrylate is 10-35 mM;
the reducing agent comprises 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 glycated albumin 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 3.8-5.0;
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 copper source comprises Cu (NO)3)2Solution, CuSO4Solution and CuCl2One or more of a solution;
the concentration of the soluble copper source is 50-200 mM;
the volume ratio of the reaction solution to the soluble copper 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 glycated albumin 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 the glycated albumin in the sample to be detected comprises the following steps:
and replacing the glycated albumin solution with a sample to be detected, then performing the steps 1) -3) to obtain a sample solution to be detected of the copper nanocluster with a certain fluorescence intensity, and calculating the concentration of the glycated albumin in the sample to be detected according to the fluorescence intensity of the copper nanocluster in the sample solution to be detected and the correlation quantitative relationship.
9. A method for detecting the concentration of glycated amino acid oxidase-ketoamine oxidase by adopting a fluorescent copper nanocluster fluorescence method is characterized by comprising the following steps of:
1) mixing and incubating a glycated amino acid oxidase-ketoamine oxidase solution and a glycated albumin solution to obtain a mixed solution;
2) reacting the mixed solution obtained in the step, an acidic buffer solution, dimethylaminoethyl methacrylate 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 copper source to obtain a copper nanocluster solution with fluorescence intensity;
4) repeating the steps 1) to 3) by adopting the saccharified amino acid oxidase-ketoamine oxidase solution with different concentrations to obtain copper nanocluster solutions with different fluorescence intensities;
5) and establishing a quantitative relation between the value of the fluorescence intensity of the copper nanocluster and the concentration of the glycated amino acid oxidase-ketoamine oxidase, and detecting the concentration of the glycated amino acid oxidase-ketoamine oxidase in the sample.
10. A method for detecting whether a sample contains glycated albumin or glycated amino acid oxidase-ketoamine oxidase by using a fluorescent copper nanocluster fluorescence method is characterized in that the method for detecting whether the sample contains the glycated albumin or not by using the fluorescent copper nanocluster fluorescence method comprises the following steps:
a) mixing and incubating a sample to be detected and a saccharified amino acid oxidase-ketoamine oxidase solution to obtain a mixed solution;
b) reacting the mixed solution obtained in the step, an acidic buffer solution, dimethylaminoethyl methacrylate and a reducing agent to obtain a reaction solution;
c) carrying out microwave reaction on the reaction solution obtained in the step, a second acidic buffer solution and a soluble copper source to obtain a final solution;
when the final solution contains copper nanoclusters with fluorescence intensity, the sample to be detected contains glycated albumin;
when the final solution does not contain the copper nanoclusters with fluorescence intensity, the sample to be tested does not contain the glycated albumin;
the method for detecting whether the sample contains the glycated amino acid oxidase-ketoamine oxidase or not by adopting the fluorescent copper nanocluster fluorescence method comprises the following steps:
A) mixing a sample to be detected and a glycated albumin solution, and incubating to obtain a mixed solution;
B) reacting the mixed solution obtained in the step, an acidic buffer solution, dimethylaminoethyl methacrylate and a reducing agent to obtain a reaction solution;
C) carrying out microwave reaction on the reaction solution obtained in the step, a second acidic buffer solution and a soluble copper source to obtain a final solution;
when the final solution contains copper nanoclusters with fluorescence intensity, the sample to be detected contains glycated amino acid oxidase-ketoamine oxidase;
and when the final solution does not contain the copper nanoclusters with fluorescence intensity, the sample to be detected does not contain the glycated amino acid oxidase-ketoamine oxidase.
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