CN107219206B - Method for analyzing copper ion content - Google Patents
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- CN107219206B CN107219206B CN201710508843.6A CN201710508843A CN107219206B CN 107219206 B CN107219206 B CN 107219206B CN 201710508843 A CN201710508843 A CN 201710508843A CN 107219206 B CN107219206 B CN 107219206B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6402—Atomic fluorescence; Laser induced fluorescence
Abstract
The analysis method of the copper ion content adopts a fluorescence analysis method, is based on the molecular probe, the fluorescence emission intensity is obviously enhanced after the molecular probe and the copper ions interact, the trace copper ions in the environment and the human body are detected by utilizing the good linear relation between the fluorescence emission intensity and the copper ion concentration, and the sensitivity and the accuracy of the method are high; in addition, calcium ions, cobalt ions, magnesium ions, nickel ions, mercury ions and the like do not interfere with the detection of copper ions, and the molecular probe has good identification and high selectivity on the copper ions.
Description
Technical Field
The invention relates to a method for analyzing the content of copper ions, and belongs to the field of analytical chemistry.
Background
With the increasing development of modern industry, a large amount of heavy metals and transition metals are discharged into the nature, and the pollution problem caused by the heavy metals and the transition metals is more and more serious, so that the method has important theoretical and practical significance for the identification and detection of the heavy metals and the transition metal ions, and has attracted the wide attention of researchers.
Copper ions play an important role in life activities, and can be used as catalytic cofactors of many enzymes, such as superoxide dismutase, cytochrome oxidase, tyrosinase and the like, but excessive copper ions can produce toxic effects and even cause neurological diseases, such as senile dementia and the like. Therefore, the identification and detection of copper ions are of great importance in the scientific fields of life science, food science, environmental science and the like.
Fluorescent molecular probes based on molecular recognition have attracted strong interest of scientists as a brand-new metal ion recognition means, become the key point of current scientific research and are developed rapidly. The fluorescent molecular probe generally consists of three important parts, namely a fluorescent group, a connecting group and a recognition group, wherein the fluorescent group is a generation unit of a fluorescent signal, the connecting group combines the fluorescent group and the recognition group, and when a foreign object substance acts on the combination, the connecting group can also cause the luminous characteristic of the fluorescent group to change, thereby playing a role of a pivot; the recognition group is used to accept and recognize foreign guest materials. The fluorescent molecule is combined with the recognition molecule through the receptor to read information, and then the recognized information is transmitted to the fluorescent group by virtue of the connecting group, so that the optical signal is converted. Among these components of fluorescent molecular probes, the role of the fluorophore and the recognition group is particularly important, the fluorophore determines the sensitivity of the fluorescent molecular probe, the recognition group determines the selectivity and specificity of the fluorescent molecular probe, and the third part of the connection group plays a role in connection and junction for the recognition of the fluorescent molecular probe.
The fluorescent molecular probe has the following advantages: 1. the method is quick and convenient and has high sensitivity; 2. the remote detection can be realized by combining with the optical fiber technology; 3 naked eye testing is available in the art. However, the currently reported copper ion fluorescence analysis method based on molecular probes cannot combine high selectivity and high sensitivity, which results in poor accuracy of the measurement result.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide a method for analyzing the content of copper ions with high selectivity and high sensitivity.
The technical scheme adopted by the invention for solving the technical problem is as follows:
the invention provides an analysis method of copper ion content, which is based on a molecular probe and adopts a fluorescence analysis method to quantitatively determine the copper ion content, wherein the chemical structural formula of the molecular probe is shown in the specification
Preferably, the method for analyzing the content of copper ions adopts a standard calibration curve method, and comprises the following steps:
1) dissolving a certain amount of molecular probe into acetonitrile to obtain a molecular probe stock solution, and adjusting the pH value of the molecular probe stock solution to 3-11;
2) m parts of V1Respectively mixing the volume of the molecular probe stock solution obtained in the step 1) with M-1 part of V2Copper ion standard solution of volume series concentration and 1 part of V2Mixing the copper ion sample solutions in volume, and then fixing the volume through acetonitrile to obtain M groups of mixed solutions;
3) standing the mixed solution of the M groups obtained in the step 2) at room temperature for 30-60 minutes, and then performing fluorescence analysis to obtain the fluorescence intensity of the M groups;
4) analyzing the corresponding relation between the fluorescence intensity of the 9 groups of mixed solutions of the copper ion-containing standard solution measured in the step 3) and the corresponding copper ion concentration, drawing a calibration curve, and obtaining the concentration of the copper ions in the sample solution by using the calibration curve according to the fluorescence intensity of the copper ion sample solution.
Preferably, the analyzing step of the copper ion content is as follows:
1) weighing a certain amount of molecular probe and acetonitrile, dissolving the molecular probe into the acetonitrile, and adjusting by using a buffer solution to obtain a molecular probe stock solution with the concentration of the molecular probe of 0.1-1mM and the pH of 3-11;
2) adding 1mL of the molecular probe stock solution obtained in the step 1) into 10mL volumetric flasks, adding 1mL of copper ion standard solutions with the concentrations of 0.01 mu M, 0.05 mu M, 0.1 mu M, 0.2 mu M, 0.5 mu M, 1 mu M, 2 mu M, 5 mu M and 10 mu M into 9 volumetric flasks, adding 1mL of copper ion sample solutions into the remaining 1 volumetric flask, and performing volume fixing through acetonitrile to obtain 10 groups of mixed solutions;
3) standing the 10 groups of mixed solutions obtained in the step 2) at room temperature for 30-60 minutes, then exciting by using the wavelength in the range of 320-380nm, and measuring 10 groups of fluorescence emission spectra;
4) and (3) drawing a calibration curve between the fluorescence intensity of the M-1 group of mixed solution of the standard solution containing the copper ions, which is measured in the step 3), and the corresponding copper ion concentration, and obtaining the concentration of the copper ions in the sample solution by using the calibration curve according to the fluorescence intensity of the copper ion sample solution.
Preferably, the pH of the stock solution of the molecular probe in step 1) is adjusted with Tris-HCl buffer.
Preferably, the preparation method of the copper ion standard solution in the step 2) comprises the following steps: weighing a certain amount of copper salt, placing the copper salt in a volumetric flask, dissolving the copper salt with secondary water to a constant volume, and then gradually diluting the copper salt with the secondary water to a proper concentration.
Preferably, the copper ion standard solution of step 2) is the same as the solvent of the copper ion sample solution.
Preferably, the fluorescence spectrum test conditions in step 3) are as follows: the width of the excitation and emission slit is 2.5-5.0nm, the voltage of the photomultiplier is 600-700V, the scanning speed is 500-600nm/min, the data interval is 1-2nm, the average time is 0.1-0.2s, and the number of filtering points is 20-30.
Preferably, the method for analyzing the copper ion content of the present invention can be used for detecting the copper ion content in an aqueous solution in blood and environment.
The invention has the beneficial effects that:
(1) after the molecular probe is added with copper ions, the maximum fluorescence emission intensity is greatly increased, other ions such as calcium ions, cobalt ions, magnesium ions, nickel ions, mercury ions, lead ions, zinc ions and the like cannot cause obvious change of a fluorescence spectrum, and the visible ions such as the calcium ions, the cobalt ions, the magnesium ions, the nickel ions, the mercury ions and the like have no interference on detection of the copper ions, so that the molecular probe has good identification performance and high selectivity on the copper ions.
(2) The molecular probe-based copper ion fluorescence analysis method can detect trace copper ions in the environment and human body, and has high sensitivity.
(3) The copper ion colorimetric probe has good stability and can be stored and used for a long time.
Detailed Description
In order to more clearly understand the technical features, objects, and effects of the present invention, specific embodiments of the present invention will now be described.
EXAMPLE 1 Effect of acid and base on the spectral Properties of molecular probes
The maximum fluorescence intensity was measured at pH 2-11 by adjusting the pH of an acetonitrile solution of a molecular probe at a concentration of 0.5mM with Tris-HCl buffer, exciting the solution at a wavelength of 350nm (shown in Table 1). The result shows that the fluorescence emission wavelength has no obvious change in the pH value range of 2-8, the maximum fluorescence emission wavelength is between 541-550nm, and the maximum emission intensity is between 19-52 a.u.; when the pH value is between 9 and 11, the fluorescence emission wavelength is not obviously changed when the pH value is excited by 350nm, the maximum fluorescence emission wavelength is between 317 and 334nm, and the maximum emission intensity is between 20 and 26a.u. Therefore, the pH value is in the range of 2-9, and the better stability is shown.
TABLE 1 fluorescence spectra data of molecular probes in different buffer solutions
Note: the fluorescence spectrum test conditions are as follows: the width of the excitation and emission slit is 3.0nm, the voltage of the photomultiplier is 650V, the scanning speed is 550nm/min, the data interval is 2nm, the average time is 0.2s, and the number of filtering points is 20.
EXAMPLE 2 spectroscopic Properties of molecular probes with different Metal ions in acetonitrile
The effect of the molecular probe on different metal ions in acetonitrile was tested for fluorescence (as shown in Table 2), and when 5mL of each different metal ion at a concentration of 1. mu.M was added to 5mL of the acetonitrile solution of the molecular probe at a wavelength of 350nm, the results were as follows: after the addition of copper ions, the maximum fluorescence emission wavelength appeared at 557nm, and the maximum fluorescence emission fluorescence intensity increased from 11a.u. to 670a.u., by a factor of about 60. And other ions such as calcium ions, cobalt ions, magnesium ions, nickel ions, copper ions, lead ions, zinc ions and the like have no interference with the copper ions.
TABLE 2 fluorescence spectra data of molecular probes in acetonitrile with different metal ions
Note: the fluorescence spectrum test conditions are as follows: the width of the excitation and emission slit is 3.0nm, the voltage of the photomultiplier is 650V, the scanning speed is 550nm/min, the data interval is 2nm, the average time is 0.2s, and the number of filtering points is 20.
Example 3
This example provides a method for analyzing copper ion content, which is based on a molecular probe, and employs a fluorescence analysis method to quantitatively determine the copper ion content, where the chemical structural formula of the molecular probe is
The analysis steps of the copper ion content are as follows:
1) weighing a certain amount of molecular probe and acetonitrile, dissolving the molecular probe into the acetonitrile, and adjusting with a Tris-HCl buffer solution to obtain a molecular probe stock solution with the concentration of the molecular probe of 0.1mM and the pH value of 2;
2) adding 1mL of the molecular probe stock solution obtained in the step 1) into 10mL volumetric flasks, adding 1mL of copper ion standard aqueous solution with the concentration of 0.01 mu M, 0.05 mu M, 0.1 mu M, 0.2 mu M, 0.5 mu M, 1 mu M, 2 mu M, 5 mu M and 10 mu M into 9 volumetric flasks, adding 1mL of blood sample into the remaining 1 volumetric flask, and performing volume fixing through acetonitrile to obtain 10 groups of mixed solutions;
3) standing the 10 groups of mixed solutions obtained in the step 2) at room temperature for 30 minutes, and then exciting by using the wavelength in the range of 320nm to obtain 10 groups of fluorescence emission spectra;
4) analyzing the corresponding relation (shown in table 3) between the fluorescence intensity of the 9 groups of mixed solutions containing the copper ion standard solution measured in the step 3) and the corresponding copper ion concentration, drawing a calibration curve, and calculating the concentration of the copper ion in the blood sample by using the calibration curve according to the fluorescence intensity of the blood sample.
TABLE 3 fluorescence spectra data of molecular probes with copper ion standard aqueous solution
Note: the fluorescence spectrum test conditions are as follows: the width of an excitation slit and an emission slit is 2.5nm, the voltage of a photomultiplier is 600V, the scanning speed is 500nm/min, the data interval is 1nm, the average time is 0.1s, and the number of filtering points is 20.
As can be seen from the above table, the fluorescence emission intensity is linear in the concentration range of 0.05-5 μ M, and the linear equation is: 235.88x +3.7743, R2The fluorescence emission intensity of the mixed solution containing the blood sample was 210a.u. and the copper ion concentration of the blood sample was obtained to be 0.87 μ M.
Example 4
This example provides a method for analyzing copper ion content, which is based on a molecular probe, and employs a fluorescence analysis method to quantitatively determine the copper ion content, where the chemical structural formula of the molecular probe is
The analysis steps of the copper ion content are as follows:
1) weighing a certain amount of molecular probe and acetonitrile, dissolving the molecular probe into the acetonitrile, and adjusting with a Tris-HCl buffer solution to obtain a molecular probe stock solution with the concentration of the molecular probe of 0.5mM and the pH value of 7;
2) adding 1mL of the molecular probe stock solution obtained in the step 1) into 10mL volumetric flasks, adding 1mL of copper ion standard aqueous solution with the concentration of 0.01 mu M, 0.05 mu M, 0.1 mu M, 0.2 mu M, 0.5 mu M, 1 mu M, 2 mu M, 5 mu M and 10 mu M into 9 volumetric flasks, adding 1mL of certain polluted lake water into the remaining 1 volumetric flask, and performing volume fixing through acetonitrile to obtain 10 groups of mixed solutions;
3) standing the 10 groups of mixed solutions obtained in the step 2) at room temperature for 45 minutes, and then exciting by using the wavelength within the range of 350nm to obtain 10 groups of fluorescence emission spectra;
4) analyzing the corresponding relation (shown in table 4) between the fluorescence intensity of the 9 groups of mixed solutions containing the copper ion standard solution measured in the step 3) and the corresponding copper ion concentration, drawing a calibration curve, and obtaining the concentration of the copper ions in the polluted lake water by using the calibration curve according to the fluorescence intensity of the polluted lake water.
TABLE 4 fluorescence spectra data of molecular probes with copper ion standard aqueous solution
Note: the fluorescence spectrum test conditions are as follows: the excitation and emission slit width is 3nm, the photomultiplier voltage is 650V, the scanning speed is 550nm/min, the data interval is 1.5nm, the average time is 0.15s, and the number of filter points is 25.
As can be seen from the above table, the fluorescence emission intensity is linear in the concentration range of 0.1-5 μ M, and the linear equation is: 168.64x-0.6654, R2The fluorescence emission intensity of the mixed solution containing the polluted lake water was 305a.u.,the copper ion concentration of the polluted lake water was 1.81. mu.M.
Example 5
This example provides a method for analyzing copper ion content, which is based on a molecular probe, and employs a fluorescence analysis method to quantitatively determine the copper ion content, where the chemical structural formula of the molecular probe is
The analysis steps of the copper ion content are as follows:
1) weighing a certain amount of molecular probe and acetonitrile, dissolving the molecular probe into the acetonitrile, and adjusting with a Tris-HCl buffer solution to obtain a molecular probe stock solution with the concentration of the molecular probe of 1mM and the pH value of 9;
2) adding 1mL of the molecular probe stock solution obtained in the step 1) into 10mL volumetric flasks, adding 1mL of copper ion standard aqueous solution with the concentration of 0.01 mu M, 0.05 mu M, 0.1 mu M, 0.2 mu M, 0.5 mu M, 1 mu M, 2 mu M, 5 mu M and 10 mu M into 9 volumetric flasks, adding 1mL of certain chemical wastewater into the remaining 1 volumetric flask, and performing volume fixing through acetonitrile to obtain 10 groups of mixed solutions;
3) standing the 10 groups of mixed solutions obtained in the step 2) at room temperature for 60 minutes, and then exciting by using the wavelength within the range of 380nm to obtain 10 groups of fluorescence emission spectra;
4) analyzing the corresponding relation (shown in table 5) between the fluorescence intensity of the 9 groups of mixed solutions containing the copper ion standard solution measured in the step 3) and the corresponding copper ion concentration, drawing a calibration curve, and obtaining the concentration of the copper ions in the chemical wastewater by using the calibration curve according to the fluorescence intensity of the chemical wastewater.
TABLE 5 fluorescence spectra data of molecular probes with copper ion standard aqueous solution
Note: the fluorescence spectrum test conditions are as follows: the width of an excitation slit and an emission slit is 5nm, the voltage of a photomultiplier is 700V, the scanning speed is 600nm/min, the data interval is 2nm, the average time is 0.2s, and the number of filtering points is 30.
As can be seen from the above table, the fluorescence emission intensity is in a linear relationship in the concentration range of 0.1-10 μ M, and the linear equation is: 175.38x-5.162, R2The fluorescence emission intensity of the mixed solution containing the chemical wastewater was 510a.u. -, which gave a copper ion concentration of 2.94 μ M in the chemical wastewater.
The method for analyzing the content of the copper ions is based on the molecular probe, adopts a fluorescence analysis method, can quantitatively detect trace copper ions in the environment and the human body, and has good linear range and high sensitivity and accuracy in a certain concentration range; in addition, calcium ions, cobalt ions, magnesium ions, nickel ions, mercury ions and the like do not interfere with the detection of copper ions, and the molecular probe has good identification and high selectivity on the copper ions.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.
Claims (6)
1. A method for analyzing the content of copper ions adopts a fluorescence analysis method to quantitatively determine the content of copper ions, and is characterized by comprising the following steps:
1) dissolving a certain amount of molecular probe into acetonitrile to obtain a molecular probe stock solution, and adjusting the pH value of the molecular probe stock solution to 3-11;
2) m parts of V1Respectively mixing the volume of the molecular probe stock solution obtained in the step 1) with M-1 part of V2Copper ion standard solution of volume series concentration and 1 part of V2Mixing the copper ion sample solutions in volume, and then fixing the volume through acetonitrile to obtain M groups of mixed solutions;
3) standing the mixed solution of the M groups obtained in the step 2) at room temperature for 30-60 minutes, and then performing fluorescence analysis to obtain the fluorescence intensity of the M groups;
4) analyzing the corresponding relation between the fluorescence intensity of the 9 groups of mixed solutions of the copper ion-containing standard solution measured in the step 3) and the corresponding copper ion concentration, drawing a calibration curve, and obtaining the concentration of the copper ions in the sample solution by using the calibration curve according to the fluorescence intensity of the copper ion sample solution;
the chemical structural formula of the molecular probe is as follows:
the test conditions of the fluorescence analysis in the step 3) are as follows: the width of the excitation and emission slit is 2.5-5.0nm, the voltage of the photomultiplier is 600-700V, the scanning speed is 500-600nm/min, the data interval is 1-2nm, the average time is 0.1-0.2s, and the number of filtering points is 20-30.
2. The method for analyzing the content of copper ions according to claim 1, wherein the analyzing step of the content of copper ions is specifically as follows:
1) weighing a certain amount of molecular probe and acetonitrile, dissolving the molecular probe into the acetonitrile, and adjusting by using a buffer solution to obtain a molecular probe stock solution with the concentration of the molecular probe of 0.1-1mM and the pH of 3-11;
2) adding 1mL of the molecular probe stock solution obtained in the step 1) into 10mL volumetric flasks, adding 1mL of copper ion standard solutions with the concentrations of 0.01 mu M, 0.05 mu M, 0.1 mu M, 0.2 mu M, 0.5 mu M, 1 mu M, 2 mu M, 5 mu M and 10 mu M into 9 volumetric flasks, adding 1mL of copper ion sample solutions into the remaining 1 volumetric flask, and performing volume fixing through acetonitrile to obtain 10 groups of mixed solutions;
3) standing the 10 groups of mixed solutions obtained in the step 2) at room temperature for 30-60 minutes, then exciting by using the wavelength in the range of 320-380nm, and measuring 10 groups of fluorescence emission spectra;
4) and (3) drawing a calibration curve between the fluorescence intensity of the M-1 group of mixed solution of the standard solution containing the copper ions, which is measured in the step 3), and the corresponding copper ion concentration, and obtaining the concentration of the copper ions in the sample solution by using the calibration curve according to the fluorescence intensity of the copper ion sample solution.
3. The method for analyzing the content of copper ions according to claim 1 or 2, wherein the pH of the stock solution of molecular probes in step 1) is adjusted with Tris-HCl buffer.
4. The method for analyzing the content of copper ions according to claim 1 or 2, wherein the method for preparing the standard solution of copper ions in step 2) comprises: weighing a certain amount of copper salt, placing the copper salt in a volumetric flask, dissolving the copper salt with secondary water to a constant volume, and then gradually diluting the copper salt with the secondary water to a proper concentration.
5. The method for analyzing the content of copper ions according to claim 1 or 2, wherein the copper ion standard solution of step 2) is the same solvent as the copper ion sample solution.
6. The method for analyzing the copper ion content according to claim 1 or 2, wherein the method is used for detecting the copper ion content in an aqueous solution in blood and environment.
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