CN112179878B - Method for detecting dopamine based on copper ion catalytic reaction in-situ fluorescence - Google Patents
Method for detecting dopamine based on copper ion catalytic reaction in-situ fluorescence Download PDFInfo
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Abstract
The invention relates to a method for detecting dopamine based on copper ion catalysis in-situ fluorescence reaction, and belongs to the technical field of nano biosensing. The method comprises the following steps: the dopamine is specifically detected based on the copper ion catalyzed in-situ fluorescence reaction of dopamine and m-phenylenediamine. In addition, the method can be applied to the detection of dopamine in a complex system. The method for detecting dopamine based on copper ion catalysis in-situ fluorescence reaction has the advantages of mild synthesis conditions, simple and convenient synthesis steps, strong fluorescence and high biocompatibility, and the method has the advantages of rapid analysis, good selectivity, high sensitivity and the like for detecting dopamine.
Description
Technical Field
The invention relates to a method for detecting dopamine by in-situ fluorescence based on copper ion catalytic reaction, and belongs to the technical field of biosensing.
Background
Dopamine (DA) is an important catecholamine neurotransmitter, mainly present in the human central nervous system, and plays an important role in a variety of physiological activities such as sensation, memory, cognition, attention, information transmission, addiction, motor control, and the like. Abnormal levels of dopamine can lead to a range of physiological and psychological disorders, and thus dopamine has become an important biomarker for laboratory research and clinical diagnosis of many neurological disorders. According to research, dopamine has been used in the diagnosis and treatment of various nervous system diseases such as schizophrenia, depression, parkinson's disease, restless legs syndrome, alzheimer's disease, and the like. Therefore, the rapid and accurate detection of the dopamine content is of great significance to clinical diagnosis, treatment and prevention of the neurological diseases.
In recent years, the literature reports many methods for detecting dopamine, including chemiluminescence, high performance liquid chromatography, surface enhanced raman scattering spectroscopy, electrochemistry, fluorescence, and colorimetric methods. Among them, fluorescence analysis is attracting attention because of its simplicity, rapidity, sensitivity, accuracy and a series of advantages. Sun et al, 2018, proposed a method for detecting dopamine on Cell using a gene-coded fluorescence sensor (GRADA). The GRADA sensor can detect dopamine released by electrical stimulation in a mouse brain slice, and can also detect endogenous dopamine in vivo of live drosophila, fish and mice. A method for rapidly detecting dopamine by using a poly-dopamine nanoparticle fluorescence method based on in-situ synthesis is reported in Analytical Chemistry in 2014 by the Yildirim subject group. The method only uses a common alkali (sodium hydroxide) and a common acid (hydrochloric acid) to respectively start and stop the polymerization reaction, so that the analysis process is extremely simple, the cost is low, and the detection of dopamine is realized. Zhang et al reported a simple and efficient method for detecting dopamine using water-soluble silicon nanoparticles in Analytical Chemistry 2015. The method can selectively detect dopamine based on the quenching effect of dopamine on the fluorescence of silicon nanoparticles, the linear range is 0.005-10.0 mu M, and the detection limit is 0.3nM. However, the above-mentioned method for detecting dopamine still has the disadvantages of low selectivity, low sensitivity, and the like.
The method for detecting dopamine based on the copper ion catalysis in-situ fluorescence reaction is constructed by adopting mild synthesis conditions and simple synthesis steps, and the dopamine is successfully detected.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a method for detecting dopamine based on copper ion catalyzed in-situ fluorescence reaction. The method can carry out in-situ fluorescence reaction through mild synthesis conditions, simple synthesis steps and cheap synthesis raw materials, and can effectively and quickly realize high selectivity and high sensitivity detection on dopamine through the linear relation between fluorescence intensity and dopamine concentration.
The technical scheme of the invention is a method for detecting dopamine based on copper ion catalyzed in-situ fluorescence reaction, which is characterized by comprising the following steps: under the condition of room temperature, in a Tris-HCl buffer solution, dopamine, m-phenylenediamine and copper ions perform a specific reaction to generate green fluorescence, the color of the solution is changed from colorless to yellow, the solution is tested by a fluorescence spectrophotometer and an ultraviolet-visible spectrophotometer, the fluorescence spectrum and the absorption spectrum of the solution are recorded, and the fluorescence intensity and the absorption intensity are enhanced along with the increase of the concentration of the dopamine, so that the quantitative detection of the dopamine is realized. In addition, the fluorescence detection method can also be used for detecting dopamine in complex biological environments.
The present invention is further explained below.
The method for detecting dopamine based on copper ion catalyzed in-situ fluorescence reaction comprises the steps of enabling the pH value of a Tris-HCl buffer solution to be 7.4, enabling the concentration of Tris-HCl to be 50mM, the concentration of M-phenylenediamine to be 200 mu M, enabling the concentration of copper ions to be 200 mu M, enabling the reaction time to be 40min, enabling the excitation wavelength to be 435nm and enabling the emission wavelength to be 535nm;
taking 500 mu L of Tris-HCl buffer solution, respectively adding M-phenylenediamine and copper ions, then adding 0-200 mu M of dopamine with different concentrations, shaking up, reacting for 40min, testing and drawing a fluorescence spectrum and an absorption spectrum by a fluorescence spectrophotometer and an ultraviolet visible spectrophotometer, and recording the change conditions of fluorescence intensity and absorption intensity before and after adding dopamine.
When the method for detecting dopamine based on copper ion catalyzed in-situ fluorescence reaction is applied, the pH value of a Tris-HCl buffer solution is 7.4, the concentration of Tris-HCl is 50mM, the concentration of serum is 1% or 5%, the concentration of M-phenylenediamine is 200 mu M, the concentration of copper ions is 200 mu M, the reaction time is 40min, the excitation wavelength is 435nm, and the emission wavelength is 535nm;
respectively adding a certain amount of human serum into 500 mu L of Tris-HCl buffer solution, then adding M-phenylenediamine, copper ions and 0-50 mu M of dopamine with different concentrations, shaking up, reacting for 40min, testing by a fluorescence spectrophotometer to draw a fluorescence spectrum, recording the change condition of fluorescence intensity before and after adding dopamine, and calculating the recovery efficiency of adding dopamine.
The invention has the beneficial effects that:
according to the method, under the condition of room temperature, in a Tris-HCl buffer solution, dopamine, m-phenylenediamine and copper ions perform a specific reaction to generate green fluorescence, the color of the solution is changed from colorless to yellow, the solution is tested by a fluorescence spectrophotometer and an ultraviolet visible spectrophotometer, the fluorescence spectrum and the absorption spectrum of the solution are recorded, and the fluorescence intensity and the absorption intensity are enhanced along with the increase of the concentration of the dopamine, so that the quantitative detection of the dopamine is realized. In addition, the fluorescence detection method can also be used for detecting dopamine in complex biological environments. The method for detecting dopamine based on copper ion catalysis in-situ fluorescence reaction has the advantages of mild synthesis conditions, simple and convenient synthesis steps, strong fluorescence and high biocompatibility, and the method has the advantages of rapid analysis, good selectivity, high sensitivity and the like for detecting dopamine. These studies provide new methods for achieving highly sensitive and highly selective detection of dopamine.
Drawings
The embodiments of the present invention will be further described with reference to the accompanying drawings.
FIG. 1 is a time-scanned graph of the reaction solution prepared in example 1;
FIG. 2 is a fluorescence excitation spectrum of the reaction solution obtained in example 2;
FIG. 3 is a fluorescence emission spectrum of the reaction solution prepared in example 2;
FIG. 4 is an absorption spectrum of the reaction solution prepared in example 2;
FIG. 5 is a fluorescence spectrum of a reaction solution prepared from m-phenylenediamine at different concentrations in example 3;
FIG. 6 is a fluorescence relative intensity scattergram of reaction solutions prepared from different concentrations of m-phenylenediamine in example 3;
FIG. 7 is a bar graph of the relative intensity of fluorescence of reaction solutions prepared from different diamines in example 4;
FIG. 8 is a fluorescence spectrum of a reaction solution prepared with different concentrations of copper ions in example 5;
FIG. 9 is a plot of fluorescence versus intensity for the reaction solutions prepared in example 5 with varying concentrations of copper ions;
FIG. 10 is a bar graph of the relative intensities of fluorescence of reaction solutions prepared from different metal ions in example 6;
FIG. 11 is a fluorescence spectrum of a reaction solution prepared from dopamine of example 7 at different concentrations;
FIG. 12 is a plot of fluorescence versus intensity for reactions made with varying concentrations of dopamine in example 7;
FIG. 13 is a graph of the standard fluorescence curves of the reaction solutions prepared from different concentrations of dopamine in example 7;
FIG. 14 is an absorption spectrum of reaction solutions prepared from different concentrations of dopamine in example 7;
FIG. 15 is a scatter plot of absorption versus intensity for reaction solutions prepared with varying concentrations of dopamine in example 7;
FIG. 16 is a graph showing the absorption standards of reaction solutions prepared from different concentrations of dopamine in example 7;
FIG. 17 is a bar graph of the relative intensity of fluorescence of reaction solutions prepared with different dopamine interfering substances in example 8;
fig. 18 is a schematic diagram of a method for detecting dopamine based on an in situ fluorescence reaction catalyzed by copper ions.
Table 1 shows the recovery efficiency of the dopamine concentration in the serum sample in example 9;
Detailed Description
The embodiments of the present invention will be described in detail below with reference to the accompanying drawings: the present embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation manner and a process are given, but the scope of the present invention is not limited to the following embodiments.
Example 1: at room temperature, 500. Mu.L of Tris-HCl buffer solution is taken, 200. Mu.M of M-phenylenediamine, 200. Mu.M of copper ions and 200. Mu.M of dopamine are respectively added, and shaking is carried out to be uniform. The solution was time-scanned by a spectrofluorometer with excitation wavelength 435nm and emission wavelength 535nm, and the fluorescence intensity was plotted against time, as shown in FIG. 1.
Example 2: at room temperature, 500. Mu.L of Tris-HCl buffer solution is taken, 200. Mu.M of M-phenylenediamine, 200. Mu.M of copper ions and 200. Mu.M of dopamine are respectively added, and shaking is carried out to be uniform. After 40min of reaction, the fluorescence excitation spectrum and the fluorescence emission spectrum were plotted by a fluorescence spectrophotometer as shown in FIGS. 2 and 3. The absorption spectrum was obtained by measuring with an ultraviolet-visible spectrophotometer, as shown in FIG. 4.
Example 3: at room temperature, 500. Mu.L of Tris-HCi buffer solution is taken, 200. Mu.M of copper ions, 200. Mu.M of dopamine and 0-200. Mu.M of M-phenylenediamine are respectively added, and shaking is carried out to be uniform. After reacting for 40min, the fluorescence spectrum and the fluorescence relative intensity scatter diagram are drawn by the test of a fluorescence spectrophotometer, as shown in FIG. 5 and FIG. 6.
Example 4: at room temperature, 500. Mu.L of Tris-HCl buffer solution is taken, 200. Mu.M of copper ions and 200. Mu.M of dopamine are respectively added, and 200. Mu.M of different diamine substances (0, blank; 1, M-phenylenediamine; 2, o-phenylenediamine; 3, p-phenylenediamine; 4,2,4-diaminotoluene; 5,2,6-diaminotoluene; 6,4-hydroxyethoxy-1,3-phenylenediamine; 7,4-chloro-1,3-phenylenediamine; 8,1,8-diaminonaphthalene) are added and shaken uniformly. After reacting for 40min, the fluorescence relative intensity histogram is drawn by the fluorescence spectrophotometer, as shown in FIG. 7.
Example 5: at room temperature, 500. Mu.L of Tris-HCl buffer solution is taken, 200. Mu.M of M-phenylenediamine, 200. Mu.M of dopamine and 0-200. Mu.M of copper ions are respectively added, and shaking is carried out to be uniform. After reacting for 40min, the fluorescence spectrum and the fluorescence relative intensity scatter diagram are drawn by the test of a fluorescence spectrophotometer, as shown in fig. 8 and fig. 9.
Example 6: at room temperature, 500. Mu.L of Tris-HCl buffer solution was added with 200. Mu.M M-phenylenediamine and 200. Mu.M dopamine, respectively, and 200. Mu.M of different metal ions (0, blank; 1,K) + ;2,Na + ;3,Ba 2+ ;4,Ca 2+ ;5,Mg 2+ ;6,Al 3+ ;7,Mn 2+ ;8,Fe 2+ ;9,Fe 3+ ;10,Co 2+ ;11,Ni 2+ ;12,Cu 2+ ;13,Zn 2+ ;14,Ag + ;15,Cr 3+ ;16,Cd 2+ ;17,Pd 2 + ) Shaking and shaking evenly. After reacting for 40min, the fluorescence relative intensity is tested by a fluorescence spectrophotometer to draw a bar chart, as shown in figure 10.
Example 7: at room temperature, 500. Mu.L of Tris-HCl buffer solution is taken, 200. Mu.M of M-phenylenediamine, 200. Mu.M of copper ions and 0-200. Mu.M of dopamine are respectively added, and shaking is carried out to be uniform. After reacting for 40min, the fluorescence spectrum, fluorescence relative intensity scatter diagram and standard curve chart are drawn by the fluorescence spectrophotometer, as shown in fig. 11, fig. 12 and fig. 13. The absorption spectrum, absorption relative intensity scatter diagram and standard curve chart are drawn by the test of an ultraviolet-visible spectrophotometer, such as fig. 14, fig. 15 and fig. 16.
Example 8: at room temperature, 500. Mu.L of Tris-HCl buffer solution is taken, 200. Mu.M of M-phenylenediamine and 200. Mu.M of copper ions are respectively added, and 200. Mu.M of different substances (0, blank; 1, glucose; 2, phenylalanine; 3, tryptophan; 4, tyrosine; 5, glutathione; 6, citric acid; 7, ascorbic acid; 8, adenosine triphosphate; 9, dopamine; 10, levodopa; 11, epinephrine; 12, norepinephrine; 13, serum amine hydrochloride; 14, methyldopa) are added and shaken to be uniform. After reacting for 40min, the fluorescence relative intensity histogram is drawn by the fluorescence spectrophotometer, as shown in FIG. 17.
Example 9: taking 500 mu L of Tris-HCl buffer solution, respectively adding 1% and 5% of human serum, then adding 200 mu M of M-phenylenediamine, 200 mu M of copper ions and dopamine with different concentrations, shaking uniformly, reacting for 40min, testing by a fluorescence spectrophotometer to draw a fluorescence spectrogram, and calculating the recovery efficiency of the dopamine, as shown in Table 1.
The invention is not limited to the specific technical solutions described in the above embodiments, and all technical solutions formed by equivalent substitutions are within the scope of the invention as claimed.
Claims (3)
1. A method for detecting dopamine based on copper ion catalysis in-situ fluorescence reaction is characterized by comprising the following steps: the method comprises the following steps: under the condition of room temperature, in a Tris-HCl buffer solution, dopamine, m-phenylenediamine and copper ions perform a specific reaction to generate green fluorescence, the color of the solution is changed from colorless to yellow, the solution is tested by a fluorescence spectrophotometer and an ultraviolet-visible spectrophotometer, the fluorescence spectrum and the absorption spectrum of the solution are recorded, and the fluorescence intensity and the absorption intensity are enhanced along with the increase of the concentration of the dopamine, so that the quantitative detection of the dopamine is realized.
2. The method for detecting dopamine based on copper ion catalyzed in-situ fluorescence reaction according to claim 1, wherein the method comprises the following steps: the in-situ fluorescence reaction conditions are as follows: the pH value of the Tris-HCl buffer solution is 7.4, the concentration of Tris-HCl is 50mM, the concentration of M-phenylenediamine is 200 mu M, the concentration of copper ions is 200 mu M, the concentration of dopamine is 200 mu M, the reaction time is 40min, the excitation wavelength is 435nm, the emission wavelength is 535nm, the test is carried out by a fluorescence spectrophotometer and an ultraviolet visible spectrophotometer, and the fluorescence spectrum and the absorption spectrum are recorded.
3. The method for detecting dopamine based on copper ion catalyzed in situ fluorescence reaction according to claim 2, characterized in that: under the condition of room temperature, adding a certain amount of m-phenylenediamine and copper ions into a Tris-HCl buffer solution, adding dopamine with different concentrations, uniformly mixing, reacting to generate green fluorescence, changing the color of the solution from colorless to yellow, testing by a fluorescence spectrophotometer and an ultraviolet visible spectrophotometer, recording the fluorescence spectrum and the absorption spectrum of the solution, and enhancing the fluorescence intensity at 535nm and the absorption intensity at 435nm along with the increase of the concentration of the dopamine, thereby realizing the quantitative detection of the dopamine.
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CN113702630B (en) * | 2021-07-08 | 2023-10-03 | 南京工业大学 | Method for detecting alkaline phosphatase and cardiac troponin I type in real time through copper ion-induced in-situ fluorescence reaction and application |
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