CN113406052B - Method for detecting phosphate ions - Google Patents
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Abstract
The invention discloses a method for detecting phosphate ions, and belongs to the technical field of phosphate ion detection. The detection method comprises the following steps: s1, adding phosphate solutions with different known concentrations into a buffer solution containing copper ions respectively to form a plurality of groups of phosphate radical-copper ion mixed solutions; s2, respectively adding G-quadruplex DNA, KCl and porphyrin to the multiple groups of phosphate radical-copper ion mixed liquor obtained in the step S1, and detecting a fluorescence value at 615 nm; s3, establishing a linear relation according to the concentration of phosphate ions and the corresponding fluorescence value; and S4, adding a buffer solution containing copper ions into the liquid to be detected, repeating the steps S1-S2 to obtain a fluorescence value at 615nm, and combining a linear relation to obtain the concentration of phosphate ions in the liquid to be detected. The detection method has better selectivity and accuracy.
Description
Technical Field
The invention relates to the technical field of phosphate ion detection, in particular to a method for detecting phosphate ions.
Background
Phosphorus is a basic element required for the growth and development of organisms, and is a component of important living matters such as DNA, ATP, cell membranes and the like. Phosphate is the main carrier of phosphorus element and plays an important role in human body physiological metabolism. The low content of phosphate causes muscle weakness and abnormal function of white blood cells; an excessively high content may lead to renal dysfunction. Meanwhile, phosphate radical is used as a main component of the phosphate fertilizer, and excessive use of the phosphate fertilizer causes a large amount of phosphate radical to be deposited in the environment, thereby causing water eutrophication, generating red tide and algae outbreak phenomena, and seriously harming the balance of animals and plants and human health in the natural environment. It is therefore important to monitor the phosphate content of the natural environment.
The conventional methods for detecting phosphate ions mainly comprise gas chromatography, high performance liquid chromatography, raman spectroscopy, a metal organic framework probe and the like. The methods can basically meet the requirement of the detection of the phosphate radical on the detection precision, but the methods involve precise and expensive detection equipment and a complex pretreatment process, which causes certain defects in the detection cost and the detection efficiency. The existing method can not detect the phosphate ions in water quickly and accurately.
Disclosure of Invention
The invention aims to overcome the technical defects and provide a method for detecting phosphate ions, which solves the technical problem that the phosphate ions in water cannot be detected quickly and accurately in the prior art.
In order to achieve the technical purpose, the technical scheme of the invention provides a method for detecting phosphate ions.
A method for detecting phosphate ions, comprising the steps of:
s1, adding phosphate solutions with different known concentrations into a buffer solution containing copper ions respectively to form a plurality of groups of phosphate radical-copper ion mixed solutions;
s2, respectively adding G-quadruplex DNA, KCl and porphyrin to the multiple groups of phosphate radical-copper ion mixed liquor obtained in the step S1, and detecting a fluorescence value at 615 nm;
s3, establishing a linear relation according to the concentration of phosphate ions and the corresponding fluorescence value;
and S4, adding a buffer solution containing copper ions into the liquid to be detected, repeating the steps S1-S2 to obtain a fluorescence value at 615nm, and combining a linear relation to obtain the concentration of phosphate ions in the liquid to be detected.
Further, in step S1, the concentrations of phosphate in the phosphate solutions with different known concentrations are: 0. 0.01. Mu.M, 0.05. Mu.M, 0.1. Mu.M, 0.2. Mu.M, 0.4. Mu.M, 0.6. Mu.M, 0.8. Mu.M, 1.0. Mu.M, 1.2. Mu.M, 1.4. Mu.M, 1.6. Mu.M, 1.8. Mu.M, 2.0. Mu.M, 2.5. Mu.M and 3.0. Mu.M.
Further, in step S2, the porphyrin is N-methylporphyrin dipropionate IX.
Further, in step S3, the linear relationship established is Y =83717+1.167 × 106X (R) 2 = 0.996), wherein Y represents a fluorescence value and X represents a phosphate ion concentration.
Further, in step S1, the buffer solution is HEPES buffer.
Further, in step S1, the phosphate solutions with different known concentrations are respectively added to the buffer solution containing copper ions and reacted at 20-25 ℃ to obtain a plurality of sets of the phosphate-copper ion mixed solution.
Further, in step S1, reacting for 1-2 hours at 20-25 ℃ to obtain a plurality of groups of phosphate radical-copper ion mixed liquor.
Further, before step S1, obtaining an optimal copper ion concentration in the buffer solution containing copper ions is further included.
Further, the specific steps of obtaining the optimal copper ion concentration include: setting Cu of different concentration gradients 2+ Solution, then to different concentrations of Cu 2+ Adding the same amount of N-methylporphyrin dipropionic acid IX and G4DNA to Cu in the solution respectively 2 + Mixing completely in the solution, and scanning fluorescence emission spectrum at 550-700nm with fluorescence spectrophotometer with excitation wavelength of 399nm according to Cu 2+ The relationship between the concentration change and the fluorescence signal change obtains the optimal copper ion concentration.
Further, in step S2, the sequence of the G-quadruplex DNA is: 5'-GTGGGTCATTGTGGGTGGGTGTGG-3'.
Compared with the prior art, the invention has the beneficial effects that: respectively adding phosphate solutions with different known concentrations into a buffer solution containing copper ions to form a plurality of groups of phosphate-copper ion mixed solutions; then adding G-quadruplex DNA, KCl and porphyrin respectively and detecting the fluorescence value at 615nm, wherein different concentrations of free copper ions exist in the formed phosphate radical-copper ion mixed liquor due to the difference of phosphate concentration, and the copper ions can form Cu with the porphyrin 2+ Porphyrins, which do not form Cu because of the difference in the concentration of copper ions remaining after reaction with phosphate 2+ The concentration of porphyrin free porphyrin also varied, and the intensity of fluorescence emitted by the combination of porphyrin free porphyrin and G-quadruplex DNA varied, and it can be seen that the higher the concentration of phosphate ions, the less copper ions remained, and the more porphyrin remainedThe method is combined with G-quadruplex DNA, the higher the fluorescence value is, a linear relation is established according to the relation between the concentration of the phosphate radical ions and the fluorescence value, the concentration of the phosphate radical ions of the sample to be detected can be obtained by detecting the fluorescence value of the liquid to be detected and combining the linear relation, the detection speed is high, and the detection method has better selectivity and accuracy.
Drawings
FIG. 1 is a schematic diagram of a method for detecting phosphate ions according to the present invention.
FIG. 2 is a graph showing the comparison between the metallation effect and the fluorescence recovery effect of different porphyrins in example 1 of the present invention.
FIG. 3 is a graph showing the optimization results of the copper ion concentration in example 1 of the present invention; wherein, FIG. 3a is a fluorescence spectrum diagram of different copper ion concentrations, and FIG. 3b is a fluorescence emission peak variation diagram of different copper ion concentrations.
FIG. 4a is a graph of fluorescence spectra at different phosphate concentrations in example 1 of the present invention.
FIG. 4b is a graph of the change in fluorescence signal for different phosphate ion concentrations in example 1 of the present invention, where the inset is a graph of the change in fluorescence signal for phosphate ions at concentrations of 0-1. Mu.M.
FIG. 5 is a graph showing the results of fluorescence signals at 615nm of the phosphate solution and other interfering substances in example 1 of the present invention.
Detailed Description
The invention principle of the invention is as follows:
in FIG. 1, phosphate represents Phosphate ion (PO 4) 3- ) (ii) a Porphyrin represents Porphyrin; g-quadruplex DNA is represented by G-quadruplex;
binding to FIG. 1,G-quadruplex DNA catalyzes the insertion of copper ions into porphyrins to form metalloporphyrins. Porphyrin has weak fluorescence, and the fluorescence of porphyrin can be improved by G-quadruplex DNA; while metalloporphyrin does not fluoresce, fluorescence does not increase even if G-quadruplex DNA is added. Phosphate ions can generate strong chelation with copper ions to prevent porphyrin metallization, so that fluorescence quenching is prevented, and fluorescence is recovered. Thus, porphyrin metallation may be in the G-quadruplex in the absence of phosphate ionsCatalytically to form Cu 2+ -porphyrins. Since Cu 2+ Porphyrins are not fluorescent, so fluorescence is not enhanced even in the presence of the G-quadruplex. Therefore, no fluorescence was observed. When phosphate ions are added, they may react with Cu 2+ Bind to reduce free Cu in solution 2+ The number of the cells. Thus, some porphyrins cannot form Cu 2+ -porphyrins. This fraction of free porphyrin can bind to G-quadruplex DNA to give strong fluorescence. As the phosphate ion increases, the fluorescence intensity will correspondingly increase, and thus the content of phosphate ion can be detected by the change in fluorescence intensity.
Based on the inventive principle, the specific embodiment provides a method for detecting phosphate ions, which comprises the following steps:
s0, obtaining the optimal copper ion concentration in the buffer solution containing copper ions, and specifically, setting Cu with different concentration gradients 2+ Solution, then to different concentrations of Cu 2+ Adding the same amount of N-methylporphyrin dipropionic acid IX and G4DNA to Cu in the solution respectively 2+ Mixing completely in the solution, and scanning fluorescence emission spectrum at 550-700nm with fluorescence spectrophotometer with excitation wavelength of 399nm according to Cu 2+ Obtaining the optimal copper ion concentration according to the relation between the concentration change and the fluorescence signal change;
s1, respectively adding phosphate solutions with different known concentrations into a HEPES buffer solution containing copper ions, and reacting at 20-25 ℃ for 1-2 hours to form a plurality of groups of phosphate radical-copper ion mixed solutions; the concentrations of the phosphate in the phosphate solutions with different known concentrations are respectively as follows: 0. 0.01. Mu.M, 0.05. Mu.M, 0.1. Mu.M, 0.2. Mu.M, 0.4. Mu.M, 0.6. Mu.M, 0.8. Mu.M, 1.0. Mu.M, 1.2. Mu.M, 1.4. Mu.M, 1.6. Mu.M, 1.8. Mu.M, 2.0. Mu.M, 2.5. Mu.M and 3.0. Mu.M;
s2, respectively adding G-quadruplex DNA, KCl and porphyrin to the multiple groups of phosphate radical-copper ion mixed liquor obtained in the step S1, and detecting a fluorescence value at 615 nm; the porphyrin is preferably N-methylporphyrin dipropionate IX; the sequence of the G-quadruplex DNA is as follows: 5'-GTGGGTCATTGTGGGTGGGTGTGG-3';
s3, according to the concentration of phosphate radical ionsEstablishing a linear relation with the corresponding fluorescence value; the linear relation is Y =83717+1.167 × 106X (R) 2 = 0.996), wherein Y represents a fluorescence value and X represents a concentration of phosphate ions;
and S4, adding a buffer solution containing copper ions into the liquid to be detected, repeating the steps S1-S2 to obtain a fluorescence value at 615nm, and combining a linear relation to obtain the concentration of phosphate ions in the liquid to be detected.
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Example 1
The content of the embodiment mainly includes: selecting optimal porphyrin for porphyrin metallization; optimizing the concentration of copper ions; the sensitivity and linear detection range of the method; selectivity of the process; and (4) carrying out recovery rate analysis on the content of the phosphate radical in the natural simulated water sample with the phosphate radical added with the standard.
(1) Optimal porphyrin selection for porphyrin metallization
The metalloporphyrin suitable for G-quadruplex catalysis is explored to achieve the optimal fluorescence recovery effect.
Four porphyrin compounds, NMM (N-methylporphyrindipropionate IX), MPIX (mesoporphyrin IX), PPIX (protoporphyrin IX), TMPyP (5,10,15,20-tetrakis (1-methyl-4-pyridyl) porphyrin tetrakis (p-toluenesulfonate)), which are commonly used in the metallization process, were selected and set up 4 experimental groups, respectively: 1. a porphyrin; 2. porphyrin + G4 DNA;3. porphyrin + G4DNA + copper ions; 4. porphyrin + G4DNA + copper ions + phosphate ions. And (3) reacting each group of samples for 40 minutes at room temperature in a dark environment, taking 200 mu L of each group of samples after full reaction, adding the samples into a micro cuvette with the optical diameter of 4mm, and measuring the fluorescence emission spectrum of the samples at 550-700nm by using a SpectraMax iD3 multifunctional microplate reader. Wherein, the concentration of each component in the system is as follows: DNA (500 nM), NMM (3. Mu.M), PPIX (3. Mu.M), MPIX (0.3. Mu.M), TMPyP (0.6. Mu.M), cu 2+ (4. Mu.M), phosphate (1. Mu.M); the buffer used was 40mM HEPES buffer (pH 7.0, 25mM KCl,100mM NaCl). G4 used is (5' -GTGGGTCATTGTG)GGTGGGTGTGG-3’。
The four porphyrins have the following structural formula (a is NMM, b is MPIX, c is PPIX, and d is TMPyP):
as shown in FIG. 2, the fluorescence signals of the four free porphyrins are all shown; when G4DNA (i.e., G-quadruplex DNA) was added, both porphyrins increased in fluorescence, with the greatest increase in NMM and MPIX second, while G4DNA had limited effect on the increase in fluorescence of both PPIX and TMPyP porphyrins. Subsequently, copper ions were added to produce porphyrin metallization and the fluorescence of all four porphyrins was reduced. The recovery of fluorescence was maximal for the NMM group when phosphate ions were added. Therefore, NMM was chosen as the porphyrin for subsequent experiments.
(2) Optimization of copper ion concentration
According to the inventive principles, cu 2+ Is a key factor in phosphate detection. Thus, different Cu can be obtained 2+ Optimization of Cu by fluorescence change at concentration 2+ And (4) concentration. Setting Cu of different concentration gradients 2+ Solution of Cu 2+ The concentrations of the solutions were 0, 1. Mu.M, 2. Mu.M, 3. Mu.M, 4. Mu.M, 5. Mu.M, 6. Mu.M, 7. Mu.M, 8. Mu.M, respectively, and then the same amounts of NMM and G4DNA were added to Cu 2+ In the solution, the concentration of DNA is 250nM and the concentration of NMM is 3 μ M, after fully mixing, the reaction is carried out for 2 hours at room temperature in the dark, then a fluorescence emission spectrum of 550-700nm is scanned by a fluorescence spectrophotometer, and the excitation wavelength is 399nm. The results are shown in FIG. 3, along with Cu 2+ The fluorescence signal gradually decreases with increasing concentration. When Cu 2+ When the concentration was increased to 4. Mu.M, the fluorescence change became slow and the fluorescence signal reached a plateau. In addition, higher concentrations of copper ions (up to 8. Mu.M) were further explored and the fluorescence signal remained almost unchanged. And due to excess Cu 2+ The sensitivity of the method was affected, so 4. Mu.M Cu was chosen in the following experiment 2+ As an optimum amount for metallization.
(3) The sensitivity and linear detection range of the method;
first, we added phosphate at various concentrations to 40mM HEPES buffer (pH 7.0, 25mM KCl,100mM NaCl) containing 4. Mu.M copper ions. Mixed well and reacted at 25 deg.c for 1 hr. Then, 250nM DNA,10mM KCl and 3. Mu.M NMM were added to the above-mentioned phosphate-copper ion mixture. After mixing well, the mixture was reacted for 40 minutes at room temperature in the dark. Finally, the spectral change was recorded with a SpectraMax iD3 multifunctional microplate reader. As shown in FIGS. 4a and 4b, the recovery of fluorescence of the system gradually increased with the increase of the amount of phosphate added, and when the concentration of phosphate ions was increased from 0. Mu.M to about 1. Mu.M, the recovery of fluorescence reached a saturation state, and the fluorescence of phosphate was almost not changed by further increasing. From the inset in FIG. 4b, we find the fluorescence at 615nm of the maximum emission wavelength versus the phosphate (specifically trisodium phosphate Na) 3 PO 4 ) The concentration has a good linear relationship in the range of 0-1.0. Mu.M. The analysis yields a linear equation of Y =83717+1.167 × 106X (R2 = 0.996), where Y and X represent the fluorescence value and the concentration of phosphate ions, respectively. This indicates that the designed method can quantitatively detect phosphate ions. Finally, based on equation 3 α/slope, the detection limit was estimated to be 0.044 μ M (44 nM). (α is the standard deviation, which is calculated by measuring the fluorescence values of multiple sets of blank solutions at 615 nm).
(4) Selectivity of the process;
this indicator of selectivity of phosphate ion detection was verified by comparing the responses of the different anions. Phosphate solution at a final concentration of 2. Mu.M and 2. Mu.M of other interfering substances (CO) 3 2- ，SO 4 2- ，SO 3 2- ，NO 3 - ，Cl - ，Br - ，H 2 O) are respectively mixed with 4 mu M copper ions, the mixture is reacted for 1 hour at room temperature, each group of mixed solution is respectively added into a system containing 250nM DNA and 3 mu M NMM, the mixture is reacted for 40 minutes in a dark place at room temperature, and then the fluorescence change is recorded by a SpectraMaxiD3 multifunctional microplate reader, as shown in figure 5, the fluorescence recovery quantity corresponding to other groups of interference substances is extremely low compared with phosphate radical ions by recording the fluorescence emission peak change at 615nM, and the fluorescence recovery quantity of the system where the phosphate radical is positioned is obviously higher than that of other groups and is about 60 times, which indicates that the methodExcellent selectivity for phosphate ions.
(5) Analyzing the recovery rate of the phosphate radical labeled simulated water sample;
firstly, pond water in a campus of the university of Yangtze river is selected as an actual water sample, the content of phosphate which can be measured in the water sample is measured and eliminated through a liquid chromatography issued by standards, insoluble impurities in the water sample are removed through filtration by a 0.22 mu M filter membrane, then phosphate radicals (0.2 mu M,0.5 mu M and 0.8 mu M) with different concentrations are added to prepare a standard sample containing the phosphate radicals with accurate content, the recovery amount of the water sample to system fluorescence is measured through a process consistent with a sensitivity titration experiment of the method, then, the phosphate concentration is quantitatively measured through the linear regression equation, and the recovery rate is calculated.
The results are shown in table 1, the recovery rate of the method for detecting phosphate ions in an actual water sample is calculated to be 94% -106%, and the method can be used for detecting the actual sample.
| Sample (I) | PO 4 3- Concentration of (u M) | Test results (μ M) | Recovery (%) |
| 1 | 0.2 | 0.194±0.1 | 97 |
| 2 | 0.5 | 0.53±0.06 | 106 |
| 3 | 0.8 | 0.75±0.05 | 94 |
The method is simple to operate, convenient, fast and efficient, does not need to carry out fluorescence labeling, can be applied to detection of actual samples, and provides a new idea for detecting phosphate ions in natural environment.
The above-described embodiments of the present invention should not be construed as limiting the scope of the present invention. Any other corresponding changes and modifications made according to the technical idea of the present invention should be included in the protection scope of the claims of the present invention.
Claims (9)
1. A method for detecting phosphate ions, characterized by comprising the steps of:
s1, respectively adding phosphate solutions with different known concentrations into a buffer solution containing copper ions to form a plurality of groups of phosphate radical-copper ion mixed solutions;
s2, respectively adding G-quadruplex DNA, KCl and porphyrin to the multiple groups of phosphate radical-copper ion mixed liquor obtained in the step S1, and detecting a fluorescence value at 615 nm; the porphyrin is N-methylporphyrin dipropionic acid IX;
s3, establishing a linear relation according to the concentration of phosphate ions and the corresponding fluorescence value;
and S4, adding a buffer solution containing copper ions into the liquid to be detected, repeating the steps S1-S2 to obtain a fluorescence value at 615nm, and combining a linear relation to obtain the concentration of phosphate ions in the liquid to be detected.
2. The detection method according to claim 1, wherein in step S1, the concentrations of the phosphate in the phosphate solutions with different known concentrations are respectively: 0. 0.01. Mu.M, 0.05. Mu.M, 0.1. Mu.M, 0.2. Mu.M, 0.4. Mu.M, 0.6. Mu.M, 0.8. Mu.M, 1.0. Mu.M, 1.2. Mu.M, 1.4. Mu.M, 1.6. Mu.M, 1.8. Mu.M, 2.0. Mu.M, 2.5. Mu.M and 3.0. Mu.M.
3. The detection method according to claim 1, wherein in step S3, the linear relationship established is Y =83717+1.167 x 106X, R 2 =0.996, wherein Y represents a fluorescence value and X represents a phosphate ion concentration.
4. The detection method according to claim 1, wherein in step S1, the buffer solution is HEPES buffer.
5. The detection method according to claim 1, wherein in step S1, the phosphate solutions with different known concentrations are respectively added to the buffer solution containing copper ions and reacted at 20-25 ℃ to obtain a plurality of groups of the phosphate-copper ion mixed solution.
6. The detection method according to claim 5, wherein in step S1, the reaction is carried out at 20 to 25 ℃ for 1 to 2 hours to obtain a plurality of sets of the phosphate-copper ion mixed solution.
7. The detection method according to claim 1, further comprising obtaining an optimal copper ion concentration in the buffer solution containing copper ions before step S1.
8. The detection method according to claim 7, wherein the step of obtaining the optimal copper ion concentration comprises: setting copper ion solutions with different concentration gradients, respectively adding the same amount of N-methylporphyrindipropionic acid IX and G-quadruplex DNA into the copper ion solutions with different concentrations, fully mixing and completely reacting, then scanning a fluorescence emission spectrum of 550-700nm by using a fluorescence spectrophotometer, wherein the excitation wavelength is 399nm, and obtaining the optimal copper ion concentration according to the relation between the change of the copper ion concentration and the change of a fluorescence signal.
9. The detection method according to claim 1, wherein in step S2, the sequence of the G-quadruplex DNA is: 5'-GTGGGTCATTGTGGGTGGGTGTGG-3'.
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| CN108548801A (en) * | 2018-03-19 | 2018-09-18 | 西北师范大学 | Application of the metalloporphyrin framework encapsulation carbon quantum dot in detecting copper ion |
| CN109470673A (en) * | 2018-12-25 | 2019-03-15 | 济南大学 | Fluorescent biosensor for detecting adenosine triphosphate and preparation method thereof |
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