CN109596580B - Method for quantitatively detecting glutamic-pyruvic transaminase in solution based on copper nano-cluster fluorescent probe - Google Patents
Method for quantitatively detecting glutamic-pyruvic transaminase in solution based on copper nano-cluster fluorescent probe Download PDFInfo
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N21/64—Fluorescence; Phosphorescence
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Abstract
The invention discloses a method for quantitatively detecting glutamic-pyruvic transaminase in a solution based on a copper nano-cluster fluorescent probe, which takes a copper nano-cluster taking glutathione as a protective agent as the fluorescent probe and specifically detects the content of the glutamic-pyruvic transaminase in the solution by a fluorescent 'off-on' mode. The fluorescence off-on mode is adopted to realize label-free, high-sensitivity and selective detection of the glutamic-pyruvic transaminase, the method is simple, convenient and quick, can realize specific detection of the glutamic-pyruvic transaminase, has wide detection linear range and low detection limit, has better detection sensitivity and selectivity, and has good application prospect in the aspects of disease detection, clinical application and the like.
Description
The patent is supported by a project 21375095 on the national science foundation surface, a youth project (No. 17JCQNJC05800) of the Tianjin City Natural science foundation, a doctor foundation (No. 52XB1510) of the Tianjin teacher university, an 'inorganic-organic hybrid functional material chemical education department key laboratory' of the Tianjin teacher university, an 'Tianjin City functional molecular structure and performance key laboratory' open foundation project and 'future thousand people plan' project (WLQR201711, WLQR201814) of the Tianjin teacher university.
Technical Field
The invention belongs to the field of application of metal copper nanoclusters in the aspect of fluorescence sensing, and particularly relates to a method for label-free, efficient and specific detection of glutamic-pyruvic transaminase content in a complex system by using a copper nanocluster as a fluorescence probe in an off-on mode.
Background
Glutamate-pyruvate transaminase (ALT), also known as glutamate transaminase, is an important transaminase in humans, and catalyzes the conversion of amino acids in L-alanine to a-ketoglutarate. Glutamate pyruvate transaminase is located in most cells of the body, with the highest concentration in the liver. Early studies show that glutamate pyruvate transaminase is a marker of liver diseases, glutamate pyruvate transaminase elevation is an important index of liver function problems, various hepatitis causes glutamate pyruvate transaminase elevation, after liver cells are seriously damaged, the content of the glutamate pyruvate transaminase can be increased to 50 times of the conventional content, the elevation degree of the glutamate pyruvate transaminase is consistent with the damage degree of the liver cells, the glutamate pyruvate transaminase is released into blood after the liver is damaged, the glutamate pyruvate transaminase is high and mainly harms the liver, the liver cells are continuously damaged, the metabolism and the detoxification capability of the liver are reduced, drug metabolism and body toxins cannot be timely discharged, the burden of the liver is further increased, the glutamate pyruvate transaminase can cause pathological changes after long-term elevation, and severe patients can further cause liver cancer. Therefore, detection of glutamate pyruvate transaminase levels is crucial for disease diagnosis. Currently, the conventional method for assessing glutamic-pyruvic transaminase content is spectrophotometry. However, this result may be inaccurate, and these methods usually have high detection limits or require expensive reagents such as enzymes or cofactors, and therefore, in order to solve these problems, many new methods for detecting the glutamic-pyruvic transaminase content are established, such as: colorimetric methods, chromatographic methods, etc., but the problems of harsh conditions for their pretreatment for detection, high requirements for detection purity, low detection sensitivity, etc. all limit the wide application of these methods. As is well known, the fluorescence determination method has the advantages of quick process, convenient operation, no need of complex pretreatment process, high sensitivity and lower detection limit, so that the establishment of the method for analyzing and detecting the glutamic-pyruvic transaminase content by using the fluorescence spectrophotometry has great significance in the aspect of practical application. The method synthesizes the copper nanoclusters by using the glutathione as a protective agent and a reducing agent, and realizes unmarked and high-sensitivity detection of the content of the glutamic-pyruvic transaminase in the solution by using the tiopronin (Trp) as a quencher and the glutamic-pyruvic transaminase as a recovery agent in a fluorescent 'off-on' mode.
Disclosure of Invention
The invention aims to overcome the defects of the traditional complex detection method and establish a method for conveniently and quickly and selectively detecting the content of glutamic-pyruvic transaminase in a complex system by using a fluorescence spectrophotometry.
In order to achieve the aim, the invention discloses a method for quantitatively detecting glutamic-pyruvic transaminase in a solution based on a copper nano-cluster fluorescent probe, which takes a copper nano-cluster taking glutathione as a protective agent as the fluorescent probe and specifically detects the content of the glutamic-pyruvic transaminase in the solution in an off-on mode, and is characterized by comprising the following steps of:
(1) preparing a tiopronin mother solution: weighing 0.1000 g of tiopronin, and dissolving in 5 mL of high-purity water; diluting the mother liquor to a low concentration of 1 mg/mL, and storing at a low temperature for later use;
(2) preparing glutamic-pyruvic transaminase solutions with the concentrations of 1,5 and 50,100,500,1000U/L respectively, and storing at low temperature for later use;
(3) uniformly dispersing the prepared copper nanocluster based on glutathione as a stabilizer into high-purity water to prepare a detection system with the concentration of 0.675 mM and the volume of 4 mL, measuring the fluorescence intensity at the moment by using a fluorescence spectrophotometer, and enabling the fluorescence probe to show strong emission at 632 nm under the excitation of an excitation wavelength of 354 nm;
(4) uniformly dispersing 1.2 mL of copper nanocluster into 2.6 mL of high-purity water, uniformly mixing, adding 0.1 mL of 1 mg/mL tiopronin solution into the mixed solution, adding 0.1 mL of high-purity water, testing the fluorescence intensity of a system after the tiopronin and the copper nanocluster solution fully act, and obviously quenching the fluorescence intensity at the moment, so that the tiopronin can be used as a quencher of the detection system;
(5) adding 2.6 mL of high-purity water and 1.2 mL of copper nanocluster solution into a centrifuge tube, uniformly mixing, adding 0.1 mL of 1 mg/mL of tiopronin solution into the mixed solution, fully reacting, then adding 0.1 mL of glutamic pyruvic transaminase solution, fully reacting the tiopronin and the glutamic pyruvic transaminase, recovering the fluorescence intensity and detecting the fluorescence emission spectrum of the tiopronin and the glutamic pyruvic transaminase, and comparing the fluorescence intensity with the fluorescence intensity in the step (4) to prove the feasibility of detecting the glutamic pyruvic transaminase by using the copper nanocluster as a fluorescence probe by using the recovered value of the fluorescence emission spectrum intensity;
(6) linear determination for detecting glutamic-pyruvic transaminase content in solution
Respectively adding 0.2-3.4 mL of high-purity water, 0.4-3.6 mL of copper nanocluster solution and 0.1 mL of tiopronin solution into a centrifuge tube, respectively adding 0.1 mL of glutamic-pyruvic transaminase solutions with different concentrations of 1-1000U/L into the mixed solution, fully reacting for 1-15 min, and respectively detecting the fluorescence intensity before and after the addition of the glutamic-pyruvic transaminase by using a fluorescence spectrophotometer; the copper nanocluster is a copper nanocluster based on glutathione as a stabilizer. The experimental result shows that when the concentration of the glutamic-pyruvic transaminase is in the range of 1-1000U/L, the fluorescence intensity recovery value of the copper nanocluster and the concentration of the glutamic-pyruvic transaminase are in a linear relation, the linear equation is DF =116.90744+0.54101X (DF is the difference between the fluorescence intensity after the glutamic-pyruvic transaminase is added and the fluorescence intensity of the copper nanocluster after the tiopronin is added, X is the concentration of the glutamic-pyruvic transaminase), the linear correlation coefficient is 0.992, and the detection limit is 0.61U/L.
The copper nanocluster solution is a copper nanocluster based on glutathione as a stabilizer, and a specific synthesis method is shown in example 1.
The application of the copper nanocluster as a fluorescent probe in the aspect of specifically detecting the glutamic-pyruvic transaminase content in the solution has the following positive effects:
(1) the synthesized copper nanocluster has stable optical properties, the particle size of the synthetic material is small, the fluorescence performance is good, the synthetic method is simple and rapid, complex processes such as heating, pH adjustment and functionalization are not needed in the synthetic process, the light-emitting position of the synthetic material is 632 nm, and obvious red can be seen under an ultraviolet lamp.
(2) The synthesized copper nanoclusters with unique optical performance are used as fluorescent probes, a fluorescent 'off on' mode is adopted, the content of the glutamic-pyruvic transaminase is efficiently and selectively sensed, the process is simple and rapid, and the selective detection of the glutamic-pyruvic transaminase can be directly realized.
(3) The method can realize the specificity detection of the glutamic-pyruvic transaminase, has wide detection linear range and low detection limit.
Drawings
FIG. 1 is a Transmission Electron Microscope (TEM) image of copper nanoclusters using glutathione as a protective agent and a reducing agent, which illustrates that the synthesized copper nanoclusters are uniform in particle size, small in particle size and uniform in distribution;
FIG. 2 is a fluorescence excitation spectrum and an emission spectrum of a copper nanocluster using glutathione as a protective agent and a reducing agent, showing that the maximum excitation wavelength is 354 nm and the maximum emission wavelength is 632 nm;
FIG. 3 is a feasibility analysis of glutamate-pyruvate transaminase in copper nanocluster assay solution with glutathione as a protectant and a reductant;
FIG. 4 is a picture of UV lamps with glutathione as the protective agent and the reducing agent, tiopronin added to the copper nanocluster, and tiopronin and glutamic-pyruvic transaminase added to the copper nanocluster; wherein 1 represents that the copper nanocluster is red under an ultraviolet lamp, 2 represents that the solution becomes turbid after the tiopronin is added, and 3 represents that the solution is restored to the original clear state after the glutamic-pyruvic transaminase is added;
FIG. 5 is a linear graph of glutamate-pyruvate transaminase in copper nanocluster detection solution with glutathione as protective agent and reducing agent, the linear range is 1-1000U/L, and the detection limit is 0.61U/L.
Detailed Description
The invention is described below by means of specific embodiments. Removing deviceUnless otherwise specified, the technical means used in the present invention are all methods known to those skilled in the art. In addition, the embodiments should be considered illustrative, and not restrictive, of the scope of the invention, which is defined solely by the claims. It will be apparent to those skilled in the art that various changes or modifications in the components and amounts of the materials used in these embodiments can be made without departing from the spirit and scope of the invention. All the reagents used were analytically pure, and the reagents and manufacturers used were as follows: glutathione, Beijing Ding Guoshang Biotechnology Ltd; ascorbic acid, miuiou chemical reagents ltd, tianjin; copper chloride (99%), Tianjin Guangfu Fine chemical Co., Ltd; sodium hydroxide, Kewei, Tianjin; tiopronin, shanghai bio-engineering gmbh; glutamic-pyruvic transaminase, Annagi chemical technologies, Inc. The preparation method of the copper nanoclusters can refer to (Wang, C.; Ling, L.; Yao, Y.; Song, Q.). Nano Research 2015,8(6) 1975-1986) or see example 1.
Example 1
The preparation of the copper nanocluster taking glutathione as a stabilizer is carried out at room temperature according to the following steps:
(1) preparation of 0.1M copper chloride solution: 1.7048 g of CuCl were weighed out2∙2H2Dissolving O in 100 mL of high-purity water, and fully dissolving for later use;
(2) preparing a copper nanocluster: weighing 0.28 g of glutathione and dissolving in 15 mL of H at room temperature2To this was added 450 mL of CuCl2(0.1M), after fully reacting, adding 0.1 g Ascorbic Acid (AA), then adding 1 mL NaOH (1M), reacting for 1 h until the white suspension is completely dissolved to become a light yellow clear transparent solution, and proving that the copper nanoclusters are formed. It can be seen from a Transmission Electron Microscope (TEM) (fig. 1) that the copper nanoclusters are uniformly dispersed and have a small particle size.
Example 2
The method for specifically detecting glutamic-pyruvic transaminase by using copper nanoclusters as fluorescent probes is characterized by comprising the following steps of:
(1) preparing a tiopronin mother solution: weighing 0.1000 g of tiopronin, and dissolving in 5 mL of high-purity water; diluting the mother liquor to a low concentration of 1 mg/mL, and storing at a low temperature for later use;
(2) preparing a series of glutamic-pyruvic transaminase solutions with different concentrations:
preparing glutamic-pyruvic transaminase solutions with the concentrations of 1,5 and 50,100,500,1000U/L respectively, and storing at low temperature for later use;
(3) uniformly dispersing the prepared copper nanocluster based on glutathione as a stabilizer into high-purity water to prepare a detection system with the concentration of 0.675 mM and the volume of 4 mL, measuring the fluorescence intensity at the moment by using a fluorescence spectrophotometer, and enabling the fluorescence probe to show strong emission at 632 nm under the excitation of an excitation wavelength of 354 nm;
(4) uniformly dispersing 1.2 mL of copper nanocluster into 2.6 mL of high-purity water, uniformly mixing, adding 0.1 mL of 1 mg/mL tiopronin solution into the mixed solution, adding 0.1 mL of high-purity water, testing the fluorescence intensity of a system after the tiopronin and the copper nanocluster solution fully act, and obviously quenching the fluorescence intensity at the moment, so that the tiopronin can be used as a quencher of the detection system;
(5) adding 2.6 mL of high-purity water and 1.2 mL of copper nanocluster solution into a centrifuge tube, uniformly mixing, adding 0.1 mL of 1 mg/mL of tiopronin solution into the mixed solution, fully reacting, then adding 0.1 mL of glutamic pyruvic transaminase solution, fully reacting the tiopronin and the glutamic pyruvic transaminase, recovering fluorescence and detecting a fluorescence emission spectrum of the tiopronin and the glutamic pyruvic transaminase, and comparing the fluorescence emission spectrum with the fluorescence intensity in the step (4) to prove the feasibility of detecting the glutamic pyruvic transaminase by using the copper nanocluster as a fluorescence probe by using a recovery value of the fluorescence emission spectrum intensity;
(6) determination for detecting linearity of glutamic-pyruvic transaminase content in solution
Respectively adding 2.6 mL of high-purity water, 1.2 mL of copper nanocluster solution and 0.1 mL of tiopronin solution into a centrifuge tube, respectively adding 0.1 mL of glutamic-pyruvic transaminase solution with different concentrations of 1-1000U/L into the mixed solution, fully reacting for 1-15 min, and respectively detecting the fluorescence intensity before and after the addition of the glutamic-pyruvic transaminase by using a fluorescence spectrophotometer. The experimental result shows that when the concentration of the glutamic-pyruvic transaminase is in the range of 1-1000U/L, the fluorescence intensity recovery value of the copper nanocluster and the concentration of the glutamic-pyruvic transaminase are in a linear relation, the linear equation is DF =116.90744+0.54101X (DF is the difference between the fluorescence intensity after the glutamic-pyruvic transaminase is added and the fluorescence intensity of the copper nanocluster after the tiopronin is added, X is the concentration of the glutamic-pyruvic transaminase), the linear correlation coefficient is 0.992, and the detection limit is 0.61U/L.
Example 3
1. Preparation of copper nanoclusters with glutathione as protectant reference example 1;
2. and (3) measuring an excitation spectrum and an emission spectrum of the copper nanocluster taking glutathione as a stabilizing agent:
the copper nanoclusters are dispersed in high-purity water, and fluorescence excitation spectrum and fluorescence emission spectrum of the material are measured, as shown in fig. 2, the maximum excitation wavelength of the copper nanoclusters is 354 nm, and the fluorescence emission wavelength is 632 nm under excitation of the maximum excitation wavelength.
Example 4
1. Preparation of copper nanoclusters with glutathione as protectant reference example 1;
2. fluorescence is adopted as a test means, and the specificity of the copper nanoclusters taking glutathione as a protective agent is utilized to detect the glutamic-pyruvic transaminase:
respectively taking 2 hollow centrifuge tubes, numbering the centrifuge tubes, respectively transferring 2.6 mL of high-purity water into the centrifuge tubes, respectively adding 1.2 mL of copper nanocluster solution into different centrifuge tubes, uniformly mixing, continuously adding 0.1 mL of tiopronin into the centrifuge tube, adding 0.1 mL of high-purity water serving as a blank control group, adding 0.1 mL of tiopronin into the centrifuge tube, adding 0.1 mL of glutamic-pyruvic transaminase solution into the centrifuge tube, fully reacting for 10 min, recovering fluorescence, detecting fluorescence emission intensity by using a fluorescence photometer, wherein the excitation wavelength is 354 nm, and the emission wavelength is 632 nm; the detection limit is 0.61U/L
Example 5
1. Preparation of copper nanoclusters with glutathione as protectant reference example 1;
2. under an ultraviolet lamp, taking glutathione as a protective agent and a reducing agent, adding tiopronin into the copper nanocluster, and adding tiopronin and glutamic-pyruvic transaminase into the copper nanocluster to obtain a sample luminescence condition:
respectively taking 3 empty centrifuge tubes, numbering (1), (2) and (3), respectively transferring 2.6 mL of high-purity water into the centrifuge tubes (1), (2) and (3), then transferring 1.2 mL of copper nanocluster solution into different centrifuge tubes, after uniform mixing, continuously adding 0.2 mL of high-purity water into the centrifuge tube (1), adding 0.1 mL of tiopronin into the centrifuge tube (2), then adding 0.1 mL of high-purity water into the centrifuge tube, adding 0.1 mL of tiopronin into the centrifuge tube (3), then adding 0.1 mL of glutamic pyruvic transaminase solution into the centrifuge tube, fully reacting for 10 min, so that fluorescence generation is recovered, and detecting the luminescence condition by using an ultraviolet lamp box. As shown in FIG. 4, 1 indicates that the copper nanoclusters are in a red clear state under an ultraviolet lamp, the solution becomes turbid after the tiopronin is added (2), and 3 the solution returns to the original clear state after the glutamic-pyruvic transaminase is added.
Example 6
1. Preparation of copper nanoclusters with glutathione as protectant reference example 1;
2. the feasibility of the glutamic-pyruvic transaminase in the solution is detected by using the specificity of the copper nanocluster which takes glutathione as a protective agent by adopting a fluorescence test means: respectively taking 2 hollow centrifuge tubes, numbering the centrifuge tubes, respectively transferring 2.2 mL of high-purity water into the centrifuge tubes, respectively adding 1.6 mL of copper nanocluster solution into different centrifuge tubes, uniformly mixing, continuously adding 0.1 mL of tiopronin into the centrifuge tube I, taking 0.1 mL of high-purity water as a blank control group, adding 0.1 mL of tiopronin into the centrifuge tube II, adding 0.1 mL of glutamic-pyruvic transaminase solution, reacting for 10 min, recovering fluorescence, and detecting fluorescence emission intensity by using a fluorescence photometer; the detection limit was 0.61U/L.
Example 7
Detection of glutamic-pyruvic transaminase: respectively taking 2 hollow centrifuge tubes, numbering the centrifuge tubes, respectively transferring 0.4 mL of high-purity water into the centrifuge tubes, respectively adding 3.2 mL of copper nanocluster solution into different centrifuge tubes, uniformly mixing, continuously adding 0.1 mL of tiopronin into the centrifuge tube of the first number, taking 0.1 mL of high-purity water as a blank control group, adding 0.1 mL of tiopronin into the centrifuge tube of the second number, adding 0.1 mL of glutamic pyruvic transaminase solution, reacting for 10 min, recovering fluorescence, and detecting fluorescence emission intensity by using a fluorescence photometer; the detection limit was 0.61U/L.
Example 8
Detection of glutamic-pyruvic transaminase: respectively taking 2 hollow centrifuge tubes, numbering the centrifuge tubes, respectively transferring 2.8 mL of high-purity water into the centrifuge tubes, respectively adding 1.0 mL of copper nanocluster solution into different centrifuge tubes, uniformly mixing, continuously adding 0.1 mL of tiopronin into the centrifuge tube, adding 0.1 mL of high-purity water as a blank control group, adding 0.1 mL of tiopronin into the centrifuge tube, adding 0.1 mL of glutamic pyruvic transaminase into the centrifuge tube, reacting for 10 min, quenching fluorescence, and detecting fluorescence emission intensity by using a fluorescence photometer. The detection limit was 0.61U/L.
Claims (1)
1. A method for quantitatively detecting glutamic-pyruvic transaminase in a solution based on a copper nano-cluster fluorescent probe takes a copper nano-cluster taking glutathione as a stabilizing agent as the fluorescent probe, and specifically detects the content of the glutamic-pyruvic transaminase in the solution in an off-on mode through fluorescence, and is characterized by comprising the following steps:
(1) preparing a tiopronin mother solution: weighing 0.1000 g of tiopronin, and dissolving in 5 mL of high-purity water; diluting the mother liquor to a low concentration of 1 mg/mL, and storing at a low temperature for later use;
(2) preparing glutamic-pyruvic transaminase solutions with the concentrations of 1,5 and 50,100,500,1000U/L respectively, and storing at low temperature for later use;
(3) uniformly dispersing the prepared copper nanocluster based on glutathione as a stabilizer into high-purity water to prepare a detection system with the concentration of 0.675 mM and the volume of 4 mL, measuring the fluorescence intensity at the moment by using a fluorescence spectrophotometer, and enabling the fluorescence probe to show strong emission at 632 nm under the excitation of an excitation wavelength of 354 nm; the preparation method of the copper nanocluster comprises the following steps:
weighing glutathione and dissolving in H at room temperature2To O, CuCl was added2After the solution is fully reacted, ascorbic acid is added, and thenAdding NaOH, and reacting until the white suspension is completely dissolved to become a light yellow clear transparent solution;
(4) uniformly dispersing 1.2 mL of copper nanocluster into 2.6 mL of high-purity water, after uniformly mixing, adding 0.1 mL of 1 mg/mL tiopronin solution into the mixed solution, adding 0.1 mL of high-purity water, and testing the fluorescence intensity of the system after the tiopronin and the copper nanocluster solution fully act, wherein the fluorescence intensity is obviously quenched;
(5) adding 2.6 mL of high-purity water and 1.2 mL of copper nanocluster solution into a centrifuge tube, uniformly mixing, adding 0.1 mL of 1 mg/mL of tiopronin solution into the mixed solution, fully reacting, then adding 0.1 mL of glutamic pyruvic transaminase solution, fully reacting the tiopronin and the glutamic pyruvic transaminase, recovering the fluorescence intensity, detecting the fluorescence emission spectrum of the tiopronin and the glutamic pyruvic transaminase, comparing the fluorescence intensity with the fluorescence intensity in the step (4), and proving the feasibility of detecting the glutamic pyruvic transaminase by using the copper nanocluster as a fluorescence probe by using the recovered value of the fluorescence emission spectrum intensity;
(6) linear determination for detecting glutamic-pyruvic transaminase content in solution
Adding 0.2-3.4 mL of high-purity water, 0.4-3.6 mL of copper nanocluster solution and 0.1 mL of tiopronin solution into a centrifuge tube, respectively adding 0.1 mL of glutamic-pyruvic transaminase solutions with different concentrations of 1-1000U/L into the mixed solution, fully reacting for 1-15 min, and respectively detecting the fluorescence intensity before and after adding the glutamic-pyruvic transaminase by using a fluorescence spectrophotometer; when the concentration of the glutamic-pyruvic transaminase is in the range of 1-1000U/L, the fluorescence intensity recovery value of the copper nanocluster and the concentration of the glutamic-pyruvic transaminase are in a linear relation.
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