CN112179877B - Method for detecting inorganic pyrophosphatase based on catalytic reaction in-situ fluorescence - Google Patents
Method for detecting inorganic pyrophosphatase based on catalytic reaction in-situ fluorescence Download PDFInfo
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Abstract
The invention relates to a method for detecting inorganic pyrophosphatase based on in-situ fluorescence reaction, and belongs to the technical field of nano biosensing. The method comprises the following steps: dopamine, m-phenylenediamine and copper ions undergo an in-situ fluorescence reaction to emit green fluorescence; pyrophosphate ions can be complexed with copper ions, so that the in-situ fluorescence reaction can not occur, and the phenomenon of fluorescence quenching occurs; the inorganic pyrophosphatase can hydrolyze pyrophosphate ions, so that in-situ fluorescence reaction occurs, and the phenomenon of fluorescence enhancement occurs, thereby realizing the detection of the inorganic pyrophosphatase. In addition, the method can also be applied to screening of inorganic pyrophosphatase inhibitors and detection of inorganic pyrophosphatase in a complex system. The method for detecting the inorganic pyrophosphatase based on the 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 quick analysis, good selectivity, high sensitivity and the like for detecting the inorganic pyrophosphatase.
Description
Technical Field
The invention relates to a method for detecting inorganic pyrophosphatase based on catalytic reaction in-situ fluorescence, and belongs to the technical field of biosensing.
Background
Inorganic pyrophosphatase (PPase, EC 3.6.1.1) is a common hydrolase that is involved in carbohydrate and phosphorus metabolism, lipid synthesis and degradation, bone formation, DNA synthesis, and many other physiological processes. When coated with certain metal ions (usually Mg) 2+ ) Upon activation, inorganic pyrophosphatase converts one molecule of inorganic pyrophosphate (PPi) to two orthophosphate ions (Pi), and thus it plays a key role in regulating the concentration of inorganic pyrophosphate in cells. In addition, abnormal levels of inorganic pyrophosphatase are associated with certain diseases and have become biomarkers for many diseases. Therefore, the realization of rapid and accurate detection of the activity of inorganic pyrophosphatase has important significance for clinical diagnosis, treatment and prevention of related diseases.
In recent years, a large number of inorganic pyrophosphatase sensors have been reported in the literature, including fluorescence sensors, colorimetric sensors, electrochemical sensors, and the like. Among them, the fluorescence sensor is attracting attention because of its advantages such as simplicity, rapidity, sensitivity, and accuracy. Sun et al, analytical Chemistry, propose a fluorescent colorimetric dual-signal quantitative method for detecting the activity of inorganic pyrophosphoric enzymes. The method is based on o-phenylenediamine and Cu 2+ Selective oxidation and color reaction between pyrophosphate and Cu 2+ The special inhibition of the oxidizing ability and the specific hydrolysis of pyrophosphate by inorganic pyrophosphatase allow the detection of the activity of inorganic pyrophosphatase. The Zhou topic group reported a gold and silver bimetallic nanocluster (Au-Ag NCs) with high fluorescence intensity in Analytical Chemistry as a detectionThe fluorescent probe of the inorganic pyrophosphoric acid enzyme activity has the advantages of high specificity, good stability and the like. Chang et al in Industrial&Three benzothiazole-based fluorescent probes are designed and synthesized on Engineering Chemistry Research and are successfully applied to the analysis and detection of the activity of zinc ions, pyrophosphate ions and inorganic pyrophosphatase. However, the above-mentioned method for detecting inorganic pyrophosphatase still has the disadvantages of low sensitivity, complicated synthesis process, long analysis time, and the like.
The method for detecting the inorganic pyrophosphatase based on the catalytic reaction in-situ fluorescence is constructed by adopting mild synthesis conditions and simple synthesis steps, and the detection of the inorganic pyrophosphatase is successfully realized.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a method for detecting inorganic pyrophosphatase based on catalytic reaction in-situ fluorescence. 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 the inorganic pyrophosphatase through the linear relation between the fluorescence intensity and the inorganic pyrophosphatase concentration.
The technical scheme of the invention is a method for detecting inorganic pyrophosphatase based on catalytic reaction in-situ fluorescence, 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 undergo an in-situ fluorescence reaction to emit green fluorescence, and after pyrophosphate ions are added, a fluorescence quenching phenomenon occurs; after inorganic pyrophosphatase is added to hydrolyze pyrophosphate ions, the phenomenon of fluorescence enhancement appears. And testing by a fluorescence spectrophotometer and an ultraviolet-visible spectrophotometer, and recording the fluorescence spectrum and the absorption spectrum of the fluorescence spectrophotometer and the ultraviolet-visible spectrophotometer so as to realize the quantitative detection of the inorganic pyrophosphatase. In addition, the method can also be applied to screening of inorganic pyrophosphatase inhibitors and detection of inorganic pyrophosphatase in a complex system.
The present invention is further explained below.
A method for detecting inorganic pyrophosphatase based on catalytic reaction in-situ fluorescence is characterized in that the pH value of a 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 20 mu M, the concentration of dopamine is 200 mu M, the concentration of pyrophosphate ions is 200 mu MM, the concentration of magnesium ions is 200 mu M, the incubation temperature is 37 ℃, the incubation time is 30min, the in-situ fluorescence reaction time is 40min, the excitation wavelength is 435nm, and the emission wavelength is 535nm;
respectively adding 200 mU M of pyrophosphate ions, 200 mU M of magnesium ions and 0-50mU/mL of inorganic pyrophosphatase into 500 mU L of Tris-HCl buffer solution, incubating for 30min at 37 ℃, adding 200 mU M of M-phenylenediamine, copper ions and dopamine at room temperature, shaking up, reacting for 40min, testing by a fluorescence spectrophotometer and an ultraviolet visible spectrophotometer to draw a fluorescence spectrum and an absorption spectrum, and recording the change of fluorescence intensity and absorption intensity before and after adding the inorganic pyrophosphatase.
A method for detecting an inorganic pyrophosphatase inhibitor based on catalytic reaction in-situ fluorescence is characterized in that the pH of a Tris-HCl buffer solution is 7.4, the concentration of Tris-HCl is 50mM, the concentration of M-phenylenediamine is 200 MuM, the concentration of copper ions is 20 MuM, the concentration of dopamine is 200 MuM, the concentration of pyrophosphate ions is 200 MuM, the concentration of inorganic pyrophosphatase is 10mU/mL, the concentration of magnesium ions is 200 MuM, the incubation temperature is 37 ℃, the incubation time is 30min, the in-situ fluorescence reaction time is 40min, the excitation wavelength is 435nm, and the emission wavelength is 535nm;
adding 200 mU M of pyrophosphate ions, 200 mU M of magnesium ions, 10mU M/mL of inorganic pyrophosphatase and 0-2mM of NaF into 500 mU L of Tris-HCl buffer solution respectively, incubating at 37 ℃ for 30min, adding 200 mU M of M-phenylenediamine, copper ions and dopamine at room temperature, shaking up, reacting for 40min, testing by a fluorescence spectrophotometer to draw a fluorescence spectrum, and recording the change of fluorescence intensity before and after adding NaF.
The application of the method for detecting inorganic pyrophosphatase based on catalytic reaction in-situ fluorescence is characterized in that the pH value of a Tris-HCl buffer solution is 7.4, the concentration of Tris-HCl is 50mM, the concentration of M-phenylenediamine is 200 MuM, the concentration of copper ions is 20 MuM, the concentration of dopamine is 200 MuM, the concentration of pyrophosphate ions is 200 MuM, the concentration of inorganic pyrophosphatase is 10mU/mL, the concentration of magnesium ions is 200 MuM, the concentration of serum is 1%, the incubation temperature is 37 ℃, the incubation time is 30min, the in-situ fluorescence reaction time is 40min, the excitation wavelength is 435nm, and the emission wavelength is 535nm;
adding 200 mU M of pyrophosphate ions, 200 mU M of magnesium ions and 0-50mU/mL of inorganic pyrophosphatase into 500 mU L of Tris-HCl buffer solution respectively, incubating at 37 ℃ for 30min, adding 200 mU M of M-phenylenediamine, copper ions and dopamine at room temperature, shaking uniformly, reacting for 40min, testing by a fluorescence spectrophotometer to draw a fluorescence spectrum, recording the change of fluorescence intensity before and after adding the inorganic pyrophosphatase, and calculating the recovery efficiency of adding dopamine, thereby realizing the quantitative detection of the inorganic pyrophosphatase in a complex environment.
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 undergo an in-situ fluorescence reaction to emit green fluorescence, and after pyrophosphate ions are added, a fluorescence quenching phenomenon occurs; after the inorganic pyrophosphatase is added to hydrolyze pyrophosphate ions, the phenomenon of fluorescence enhancement appears. And testing by a fluorescence spectrophotometer and an ultraviolet-visible spectrophotometer, and recording the fluorescence spectrum and the absorption spectrum of the fluorescence spectrophotometer, wherein the fluorescence intensity and the absorption intensity are enhanced along with the increase of the dopamine concentration, so that the quantitative detection of the inorganic pyrophosphatase is realized. In addition, the method can also be applied to screening of inorganic pyrophosphatase inhibitors and detection of inorganic pyrophosphatase in a complex system. The method for detecting inorganic pyrophosphatase based on catalytic reaction in-situ fluorescence has the advantages of mild synthesis conditions, simple and convenient synthesis steps, strong fluorescence and high biocompatibility, and the method has the advantages of quick analysis, good selectivity, high sensitivity and the like for detecting the inorganic pyrophosphatase. These studies provide a novel approach for achieving highly sensitive and highly selective detection of inorganic pyrophosphatase.
Drawings
The embodiments of the present invention will be further described with reference to the accompanying drawings.
FIG. 1 is a fluorescence spectrum of the reaction of m-phenylenediamine, dopamine and copper ions in example 1;
FIG. 2 is a graph showing an absorption spectrum of the reaction of m-phenylenediamine, dopamine and copper ions in example 1;
FIG. 3 is a fluorescence spectrum of the reaction of m-phenylenediamine, dopamine, copper ions and pyrophosphate ions in example 2;
FIG. 4 is an absorption spectrum of the reaction of m-phenylenediamine, dopamine, copper ions and pyrophosphate ions in example 2;
FIG. 5 is a fluorescence spectrum of the reaction of m-phenylenediamine, dopamine, copper ion, pyrophosphate ion and inorganic pyrophosphatase in example 3;
FIG. 6 is an absorption spectrum of the reaction of m-phenylenediamine, dopamine, copper ion, pyrophosphate ion and inorganic pyrophosphatase in example 3;
FIG. 7 is a fluorescence spectrum of a reaction solution prepared with pyrophosphate ions of different concentrations in example 4;
FIG. 8 is a plot of fluorescence versus intensity for reaction solutions prepared with different concentrations of pyrophosphate ions in example 4;
FIG. 9 is a bar graph of the relative intensities of fluorescence of reaction solutions prepared from different anions from example 5;
FIG. 10 is a fluorescence spectrum of a reaction solution obtained with different concentrations of inorganic pyrophosphatase in example 6;
FIG. 11 is a scattergram of fluorescence relative intensity of reaction solutions prepared with different concentrations of inorganic pyrophosphatase in example 6;
FIG. 12 is a graph showing fluorescence standards of reaction solutions prepared by using inorganic pyrophosphatase of example 6 at different concentrations;
FIG. 13 is an absorption spectrum of a reaction solution prepared by using inorganic pyrophosphatase of example 6 at different concentrations;
FIG. 14 is a graph showing the absorbance versus intensity of reaction solutions prepared by using inorganic pyrophosphatase of different concentrations in example 6;
FIG. 15 is a graph showing the standard absorption curves of reaction solutions prepared by using inorganic pyrophosphatase of example 6 at different concentrations;
FIG. 16 is a bar graph of the relative intensity of fluorescence of reaction solutions prepared with different enzymes or proteins from example 7;
FIG. 17 is a fluorescence spectrum of a reaction solution prepared by using NaF at different concentrations in example 8;
FIG. 18 is a plot of fluorescence versus intensity for reaction solutions prepared with different concentrations of NaF in example 8;
FIG. 19 is a schematic diagram of a method for in situ fluorescence detection of inorganic pyrophosphatase based on catalytic reaction.
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 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 M-phenylenediamine, 20. Mu.M copper ions and 200. Mu.M dopamine are respectively added, and shaking is carried out to be uniform. After reacting for 40min, the fluorescence spectrum is drawn by testing with a fluorescence spectrophotometer with excitation wavelength of 435nm, as shown in figure 1. The absorption spectrum is drawn by testing with an ultraviolet-visible spectrophotometer, as shown in fig. 2.
Example 2: at room temperature, 500. Mu.L of Tris-HCl buffer solution is taken, 200. Mu.M M-phenylenediamine, 20. Mu.M copper ions and 200. Mu.M dopamine are respectively added, 200. Mu.M pyrophosphate ions are added, and shaking is carried out uniformly. After 40min of reaction, the fluorescence spectrum was plotted by fluorescence spectrophotometer with excitation wavelength of 435nn, as shown in FIG. 3. The absorption spectrum was obtained by measuring with an ultraviolet-visible spectrophotometer, as shown in FIG. 4.
Example 3: mu.L of Tris-HCl buffer solution was added with 200. Mu.M pyrophosphate ion, 200. Mu.M magnesium ion and 10mU/mL inorganic pyrophosphatase (PPase), respectively, and incubated at 37 ℃ for 30min. At room temperature, 200. Mu.M M-phenylenediamine, 20. Mu.M copper ions and 200. Mu.M dopamine were added, respectively, and shaken well. After reacting for 40min, the fluorescence spectrum is drawn by testing with a fluorescence spectrophotometer with an excitation wavelength of 435nm, as shown in FIG. 5. The absorption spectrum was obtained by measuring with an ultraviolet-visible spectrophotometer, as shown in FIG. 6.
Example 4: at room temperature, 500. Mu.L of Tris-HCl buffer solution is taken, 200. Mu.M of M-phenylenediamine, 20. Mu.M of copper ions and 200. Mu.M of dopamine are respectively added, 0 to 200. Mu.M of pyrophosphate ions with different concentrations are added, and shaking is carried out uniformly. After reacting for 40min, a fluorescence spectrum chart and a fluorescence relative intensity scatter chart are drawn by a fluorescence spectrophotometer, the excitation wavelength is 435nn, and the emission wavelength is 535nm, as shown in the graph of fig. 7 and fig. 8.
Example 5: at room temperature, 500. Mu.L of Tris-HCl buffer solution was taken, 200. Mu.M M-phenylenediamine, 20. Mu.M copper ions and 200. Mu.M dopamine were added, respectively, and 200. Mu.M of different anions (0, blank; 1, NO) 3 - ；2、NO 2 - ；3、CO 3 2- ；4、HCO 3 - ；5、P 2 O 7 4- ；6、H 2 PO 4 - ；7、HPO 4 2- ；8、PO 4 3- ；9、SO 3 2- ；10、SO 4 2- ；11、SCN - ；12、F - ；13、Cl - ；14、Br - ；15、I - ；16、Ac - ) Shaking and shaking evenly. After reacting for 40min, a fluorescence relative intensity histogram is drawn by a fluorescence spectrophotometer, the excitation wavelength is 435nm, and the emission wavelength is 535nm, as shown in FIG. 9.
Example 6: taking 500 mU L of Tris-HCl buffer solution, respectively adding 200 mU M pyrophosphate ions, 200 mU M magnesium ions and 0-50mU/mL inorganic pyrophosphatase (PPase) with different concentrations, and incubating at 37 ℃ for 30min. At room temperature, 200. Mu.M M-phenylenediamine, 20. Mu.M copper ions and 200. Mu.M dopamine were added, respectively, and shaken well. After reacting for 40min, the fluorescence spectrum, fluorescence relative intensity scatter diagram and standard curve chart are drawn by the fluorescence spectrophotometer, such as fig. 10, fig. 11 and fig. 12. 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. 13, fig. 14 and fig. 15.
Example 7: taking 500 mU L of Tris-HCl buffer solution, respectively adding 200 mU M pyrophosphate ions, 200 mU M magnesium ions and 10mU/mL different enzymes or proteins (0, blank sample; 1, alkaline phosphohydrolase; 2, inorganic pyrophosphatase; 3, tyrosinase; 4, decarboxylase; 5, pepsin; 6, trypsin; 7, bovine serum albumin; 8, glutathione; 9, cytochrome c), and incubating at 37 ℃ for 30min. At room temperature, 200. Mu.M M-phenylenediamine, 20. Mu.M copper ions and 200. Mu.M dopamine were added, respectively, and shaken well. After reacting for 40min, the fluorescence relative intensity histogram is drawn by the fluorescence spectrophotometer, as shown in FIG. 16.
Example 8: mu.L of Tris-HCl buffer solution was added with 200. Mu.M pyrophosphate ions, 200. Mu.M magnesium ions, 10mU/mL inorganic pyrophosphatase (PPase) and 0-2mM NaF at different concentrations, respectively, and incubated at 37 ℃ for 30min. At room temperature, 200. Mu.M M-phenylenediamine, 20. Mu.M copper ions and 200. Mu.M dopamine were added, respectively, and shaken well. After reacting for 40min, the fluorescence spectrum and the fluorescence relative intensity scatter diagram are drawn by the test of a fluorescence spectrophotometer, such as FIG. 17 and FIG. 18.
Example 9: taking 500 mU L of Tris-HCl buffer solution, respectively adding 1% human serum, respectively adding 200 mU M pyrophosphate ions, 200 mU M magnesium ions and inorganic pyrophosphatase (2 mU/mL,3mU/mL,4mU/mL and 5 mU/mL) with different concentrations, and incubating at 37 ℃ for 30min. At room temperature, 200. Mu.M M-phenylenediamine, 20. Mu.M copper ions and 200. Mu.M dopamine were added, and shaken up. After 40min of reaction, the fluorescence spectrum was drawn by a fluorescence spectrophotometer and the recovery efficiency of inorganic pyrophosphatase was calculated 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 claims of the invention.
TABLE 1
Claims (4)
1. A method for detecting inorganic pyrophosphatase based on catalytic reaction in-situ fluorescence 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 undergo an in-situ fluorescence reaction to emit green fluorescence, and after pyrophosphate ions are added, a fluorescence quenching phenomenon occurs; after the mixed action solution of inorganic pyrophosphatase, pyrophosphate ions and magnesium ions is added, the phenomenon of fluorescence enhancement appears; and testing by a fluorescence spectrophotometer and an ultraviolet-visible spectrophotometer, and recording the fluorescence spectrum and the absorption spectrum of the fluorescence spectrophotometer, thereby realizing the quantitative detection of the inorganic pyrophosphatase.
2. The method for detecting inorganic pyrophosphatase based on catalytic reaction in-situ fluorescence as claimed in claim 1, wherein: 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 20 mU M, the concentration of dopamine is 200 mU M, the concentration of pyrophosphate ions is 200 mU M, the concentration of inorganic pyrophosphatase is 10mU/mL, the concentration of magnesium ions is 200 mU M, the incubation temperature is 37 ℃, the incubation time is 30min, the in-situ fluorescence reaction time is 40min, the excitation wavelength is 435nm, the emission wavelength is 535nm, and the fluorescence spectrum and the absorption spectrum are recorded by performing a test through a fluorescence spectrophotometer and an ultraviolet visible spectrophotometer.
3. The method for detecting inorganic pyrophosphatase based on catalytic reaction in-situ fluorescence as claimed in claim 1, wherein: under the condition of room temperature, in a Tris-HCl buffer solution, dopamine, m-phenylenediamine and copper ions undergo an in-situ fluorescence reaction to emit green fluorescence, and after pyrophosphate ions are added, a fluorescence quenching phenomenon occurs; after inorganic pyrophosphatase is added to hydrolyze pyrophosphate ions, the phenomenon of fluorescence enhancement occurs; after the inorganic pyrophosphatase, pyrophosphate ions, magnesium ions and NaF mixed solution are added, because NaF can inhibit the activity of the inorganic pyrophosphatase, the pyrophosphate ions cannot be hydrolyzed, so that coupling of the pyrophosphate ions and the copper ions is caused, in-situ fluorescence reaction of dopamine and m-phenylenediamine cannot effectively occur, a fluorescence spectrophotometer is used for testing, and the fluorescence spectrum of the dopamine and m-phenylenediamine is recorded, so that the quantitative detection of the inorganic pyrophosphatase inhibitor is realized.
4. The application of the method for detecting inorganic pyrophosphatase based on catalytic reaction in-situ fluorescence as claimed in claim 1 is characterized in that: under the condition of room temperature, a certain amount of serum is added into a Tris-HCl buffer solution, dopamine, m-phenylenediamine and copper ions undergo an in-situ fluorescence reaction to emit green fluorescence, and after pyrophosphate ions are added, a fluorescence quenching phenomenon occurs; after adding the mixed solution of inorganic pyrophosphatase, pyrophosphate ions and magnesium ions, the phenomenon of fluorescence enhancement appears; and testing by a fluorescence spectrophotometer, and recording the fluorescence spectrum of the fluorescence spectrophotometer, thereby realizing the quantitative detection of the inorganic pyrophosphatase in a complex environment.
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