CN112113943A - Method for detecting paraquat - Google Patents
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- 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
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- G01N21/6402—Atomic fluorescence; Laser induced fluorescence
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- 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
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- 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
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6432—Quenching
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Abstract
The invention discloses a method for detecting paraquat, and belongs to the technical field of analytical chemistry. The detection method is characterized in that gold nanoclusters are used as detection reagents of paraquat to perform fluorescence detection on a sample to be detected; the gold nanocluster is prepared by modifying glutathione reduced gold preparation through mercapto-beta-cyclodextrin SH-beta-CDs. The detection limit of the method for detecting paraquat by using gold nanoclusters constructed by the invention is 5.0ng/mL, and the linear range is 5.0-360 ng/mL; under the same detection concentration of 500ng/mL of other common organophosphorus pesticides, the detection quenching rate of paraquat on the nano-cluster is 80%, the quenching rate of other pesticides is lower than 5%, the interference of ions is reduced after the nano-cluster is added with the metal ion shielding agent, and the quenching rate response to 5uM detected metal ions is lower than 5%. The method has the characteristics of high sensitivity, high selectivity, rapid detection and the like.
Description
Technical Field
The invention relates to a method for detecting paraquat, and belongs to the technical field of analytical chemistry.
Background
The application of the pesticide in the agricultural field can ensure the crop yield and also bring about the pesticide residue problem. Paraquat is a herbicide capable of quickly killing annual weeds, but has large damage to human organs due to irreversible pathological symptoms such as lung tissue damage and the like on human bodies, and has no specific antidote. The use class of paraquat has been changed to the ban on pesticides. In order to deal with the possible risk of illegal use of paraquat in agricultural production, it is important to establish a detection method capable of rapidly realizing quantitative-semi-quantitative detection.
Currently, many pesticide rapid detection methods are developed based on enzyme inhibition. The enzyme inhibition method is generally a broad-spectrum method and has the defects of high biological enzyme stability, high storage requirement and the like, which are greatly influenced by environmental factors. The method for directly utilizing the nano material to realize the pesticide specificity detection has the advantages of simple system, high specificity and the like. Among them, colorimetric, chemiluminescent, surface raman enhancement and fluorescence detection methods based on nanomaterials are reported for paraquat pesticides. The typical example is a method for realizing fluorescence detection of paraquat by using semiconductor quantum dots (such as cadmium telluride CdTe), but the preparation of the material nano material is relatively complex and heavy metals are required to be applied, but the heavy metals are very difficult to biodegrade, but can be enriched by thousands of times under the biological amplification effect of a food chain and finally enter a human body to cause chronic poisoning and harm the human body.
Disclosure of Invention
In order to solve the above problems, the main object of the present invention is to establish a method for detecting paraquat based on gold nanocluster fluorescence. The detection principle of the invention is as follows: the fluorescence of the gold nanoclusters is quenched through electrostatic transfer from the gold nanoclusters to the paraquat, and meanwhile, due to the fact that double-ligand modification of the gold nanoclusters has a fluorescence enhancement effect and an anti-interference capability, reliable quantitative detection of the paraquat is achieved.
The first purpose of the invention is to provide a method for detecting paraquat, which adopts gold nanoclusters as a detection reagent of paraquat to carry out fluorescence detection on a sample to be detected; the gold nanocluster is prepared by modifying glutathione reduced gold preparation through mercapto-beta-cyclodextrin SH-beta-CDs.
In one embodiment of the present invention, the method for preparing the gold nanoclusters comprises: 2mL of HAuCl4(10mM)、0.3mA solution of L in GSH (100mM) was mixed with 7.7mL of ultrapure water. The mixture was heated to 70 ℃ with gentle stirring and reacted for 24 hours. To the resulting pale yellow solution, the ratio of 1: adding ethanol in a proportion of 1; and (3) the solution is changed from clear to turbid, the solution is centrifuged for 15min at 8000rpm, precipitates are separated and dissolved by ultrasonic, and the solution is filtered by a 0.22 mu M filter membrane to obtain the glutathione reduced gold solution. Adding SH-beta-cyclodextrin (the final concentration is 5mM) into the obtained glutathione reduced gold solution, and incubating for 3h at 50 ℃; and centrifugally concentrating the obtained solution through an ultrafiltration tube (10kDa) to prepare the gold nanocluster.
In one embodiment of the present invention, the detection method is: and adding the gold nanocluster solution into the sample solution to be detected, carrying out mixing reaction, detecting by using a fluorescence spectrophotometer, and calculating by using an external standard method to obtain the content of paraquat in the sample to be detected.
In one embodiment of the present invention, the pH of the gold nanoparticle solution is adjusted to 8.5 to 9.0 by using a buffer solution.
In one embodiment of the invention, the buffer solution comprises glycine buffer salt, Tris-HCl or phosphate buffer solution.
In one embodiment of the invention, the fluorescence detection conditions are that the excitation broadband is 20nm, the slit width is 20nm, the excitation wavelength is 392nm, and the fluorescence value at the emission peak of 610nm is detected.
In one embodiment of the present invention, if the sample to be tested contains metal ions, the metal ion masking agent is added to the sample to be tested, and then the gold nanoclusters are added for detection.
The second purpose of the invention is to provide an application of the detection method in detecting the pesticide content in food, river water, lake water or sewage.
The third purpose of the invention is to provide a method for detecting the content of paraquat in water, which comprises the steps of adding a metal ion masking agent into a sample to be detected, and then detecting the content of paraquat in the sample to be detected by adopting the method.
In one embodiment of the invention, the concentration of the selected gold nanoclusters is 0.02 to 0.2. mu.M.
In one embodiment of the invention, the buffer salt selected is Gly-NaOH (5-20 mM).
In one embodiment of the present invention, the masking agent is a mixture of Na2S and cDCTA, Na2The concentration of S is 0.1-1.0mM, and the concentration of cDCTA is 1-10 mM.
In one embodiment of the present invention, the method comprises the steps of:
(1) preparing a gold nanocluster solution: the prepared gold nanoclusters are adjusted to pH 9.0 by glycine buffer salt (5-20mM), diluted to appropriate concentration:
(2) preparation of standard curve: mixing and incubating a paraquat standard solution and a nano-cluster solution for 1min, and establishing a standard curve of a corresponding relation between a fluorescence quenching rate signal of a gold nano-cluster and paraquat concentration, wherein the standard curve is 0.02-0.2 mu M;
(3) and (3) detection of the sample: sodium sulfide (Na) is added into the sample solution2S) and a masking agent prepared from trans-cyclohexanediamine tetraacetic acid (cDCTA) and a gold nanocluster solution are mixed and reacted, the calculated fluorescence quenching rate signal is brought into a standard curve, and the concentration of paraquat in a detection sample is calculated.
The invention has the beneficial effects that:
the detection limit of the method for detecting paraquat by using gold nanoclusters constructed by the invention is 5.0ng/mL, and the linear range is 5.0-360 ng/mL; under the same detection concentration of 500ng/mL of other common organophosphorus pesticides, the detection quenching rate of paraquat on the nano-cluster is 80%, the quenching rate of other pesticides is lower than 5%, the interference of ions is reduced after the nano-cluster is added with the metal ion shielding agent, and the quenching rate response to 5uM detected metal ions is lower than 5%. The method has the characteristics of high sensitivity, high selectivity, rapid detection and the like.
Drawings
FIG. 1 is a schematic diagram of preparation of gold nanoclusters and detection of paraquat.
FIG. 2 is the change in fluorescence of nanoclusters after modification of HS- β -CDs.
FIG. 3 is a fluorescence curve for paraquat detection.
Fig. 4 is a paraquat detection standard curve.
FIG. 5 is a graph of the selective response of beta-CD/GSH-AuNCs to various pesticides.
FIG. 6 is the response of β -CD/GSH-AuNCs to various metal ions.
Fig. 7 is the response of GSH-AuNCs to various metal ions.
FIG. 8 shows the results of A, B, C, D, E, F with paraquat in different concentrations of Na2HPO4-NaH2PO4Tris-HCl, citric acid-Na2HPO4Quenching rate response to nanoclusters in buffer salts and Na at different pH2HPO4-NaH2PO4Quenching rate response of Tris-HCl, Gly-NaOH.
Figure 9 is a graph of the EDTA shielding various ions.
FIG. 10 is a graph of the shielding effect of cDCTA on various metal ions.
FIG. 11 is cDCTA and Na2S has shielding effect on various metal ions.
Detailed Description
The following description of the preferred embodiments of the present invention is provided for the purpose of better illustrating the invention and is not intended to limit the invention thereto.
Example 1: preparation method of gold nanocluster
2mL of HAuCl4(10mM), 0.3mL of GSH (100mM) solution and 7.7mL of ultrapure water. The mixture was heated to 70 ℃ with gentle stirring and reacted for 24 hours. To the resulting pale yellow solution, the ratio of 1: adding ethanol in a proportion of 1; and (3) the solution is changed from clear to turbid, the solution is centrifuged for 15min at 8000rpm, precipitates are separated and dissolved by ultrasonic, and the solution is filtered by a 0.22 mu M filter membrane to obtain glutathione reduced gold solution GSH-AuNCs. Adding SH-beta-cyclodextrin (the final concentration is 5mM) into the obtained glutathione reduced gold solution, and incubating for 3h at 50 ℃; the obtained solution is centrifugally concentrated by an ultrafiltration tube (10kDa) to prepare gold nanocluster beta-CD/GSH-AuNCs, and the concentrated solution (0.03mM) is collected and stored at 4 ℃, wherein the reaction principle of the gold nanoclusters is shown in figure 1. The grain diameter of the obtained gold nanocluster is 1.0-3.0 nm, the excitation wavelength is 350-400 nm, and the emission wavelength is 620 nm. The fluorescence intensity of gold nanoclusters before and after modification is shown in FIG. 2, which illustrates the use of thiol groupsThe beta-cyclodextrin modification can improve the binding effect of paraquat.
Example 2: method for detecting content of paraquat in river water/lake water by using gold nanoclusters
The gold nanoclusters prepared in example 1 are used for detecting the content of paraquat in river/lake water, and a schematic diagram of the preparation of the gold nanoclusters and the detection of paraquat is shown in fig. 1.
1. Establishment of a Standard Curve
(1) Preparation of standard solution:
900uL Gly-NaOH buffer solution, 50uL standard solution or sample solution of paraquat pesticide and 50uL nano cluster (0.05 mu M) are mixed uniformly
(2) And (3) fluorescence spectrum detection:
detecting by adopting a fluorescence photometer under the following detection conditions: the excitation wavelength was 390nm and the emission signal at 620nm was collected.
(3) Determination of linear relation and detection limit:
fluorescence intensity meter for detecting nano-cluster is F1The fluorescence intensity meter of the nanocluster without pesticide is F0And drawing a standard curve as shown in figures 3 and 4, wherein y is 0.0254x +0.0037(5-150ng/mL), and R is2＝0.9956；y＝0.1367x+0.014,(150-360ng/mL)R20.9837, linear range 5-360ng/mL, quantitative limit 5 ng/mL.
2. Method for detecting paraquat content in river water/lake water
(1) Sample pretreatment: filtering river/lake water with microporous membrane, and adding masking agent containing 200uM Na2S and 2mM cDCTA, with a sample to masking agent volume ratio of 1: 1.
(2) And (3) fluorescence spectrum detection:
detecting by adopting a fluorescence photometer under the following detection conditions: the excitation wavelength was 390nm and the emission signal at 620nm was collected.
Example 3: accuracy and specificity of the method
1. Accuracy of the method
The concentration of standard additive paraquat added to the lake water sample was 160ng/mL, and the average concentration of paraquat actually measured was 155.6ng/mL in 3 replicates, and the recovery rate was 97.2% and the Relative Standard Deviation (RSD) was 2.0%.
2. Specificity of the method
(1) The influence of common organophosphorus pesticides and metal ions on detection is investigated. The organophosphorus pesticide selects 9 pesticides of fenamiphos, acetamiprid, chlorpyrifos, glyphosate, methyl parathion, isocarbophos, methomyl and imidacloprid under the same concentration (500ng/mL), and the results show that the pesticides do not influence the detection of paraquat (as shown in figure 5).
(2) The influence of common metal ions on the detection is examined, as shown in fig. 6 and 7, and the result shows that the metal and S resistance is improved after the modification of HS-beta-CD2-The capacity is improved.
Example 4: influence of different salt concentrations on fluorescence of paraquat-quenched gold nanoclusters
The assay was performed according to the method of example 2, except that the buffer salts and buffer salt concentrations of the system were adjusted using Na2HPO4-NaH2PO4Tris-HCl, citric acid-Na2HPO4The buffer salt concentrations were adjusted to 5mM,10mM,15mM,20mM,25mM, and 30mM, respectively, under otherwise identical conditions, and the results are shown in FIG. 8.
As shown in FIGS. 8A,8B and 8C, the concentration of buffer salt at a high concentration affects the fluorescence quenching of paraquat on the nanoclusters, and therefore the concentration of buffer salt at 5mM is selected as the optimum.
Example 5: effect of pH on Paraquat-quenched gold nanocluster fluorescence
The assay was performed according to the method of example 2, except that the pH of the system was adjusted using Na2HPO4-NaH2PO4Tris-HCl, Gly-NaOH adjusted the buffer salt system to different pH values, and other conditions were the same, and the results are shown in FIG. 8.
As can be seen from fig. 8D,8E, and 8F, the fluorescence quenching of paraquat on nanoclusters is maximized when the pH of the system is 9.0, and therefore Tris-HCl, Gly-NaOH buffer salt with pH of 9.0 is selected as the optimum.
Example 6: selection of metal ion-shielding agents
The test was carried out with reference to the method of example 2, with the only difference thatAdding metal ions with a concentration of 10uM, and respectively replacing the masking agent with EDTA, cDCTA, Na2S and cDCTA systems, otherwise identical, and the results are shown in fig. 9, 10 and 11, respectively. Through screening, trans-cyclohexanediaminetetraacetic acid and sodium sulfide are simultaneously added as metal ion shielding agents, and after the shielding agents are added, the shielding effect on 13 common metal ions is good.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (10)
1. The method for detecting paraquat is characterized in that gold nanocluster solution is used as a detection reagent for paraquat to carry out fluorescence detection on a sample to be detected; the gold nanocluster is prepared by modifying glutathione reduced gold preparation through mercapto-beta-cyclodextrin SH-beta-CDs.
2. The method of claim 1, wherein the detection method is: and adding the gold nanocluster solution into the sample solution to be detected, carrying out mixing reaction, detecting by using a fluorescence spectrophotometer, and calculating by using an external standard method to obtain the content of paraquat in the sample to be detected.
3. The method of claim 1 or 2, wherein the gold nanocluster solution is adjusted to a pH of 8.5 to 9.0 using a buffer.
4. The method of claim 3, wherein the buffer solution comprises glycine buffer, Tris-HCl, or phosphate buffer.
5. The method according to any one of claims 1 to 4, wherein the fluorescence detection conditions are an excitation broadband of 20nm, a slit width of 20nm, an excitation wavelength of 392nm, and a fluorescence value at an emission peak at 610nm is detected.
6. Use of the detection method according to any one of claims 1 to 5 for detecting the content of an agricultural chemical in food, river water, lake water or sewage.
7. A method for detecting the content of paraquat in water, which is characterized in that a metal ion masking agent is added into a sample to be detected, and then the method of any one of claims 1 to 5 is adopted to detect the content of paraquat in the sample to be detected.
8. The method of claim 7, wherein the concentration of the selected gold nanoclusters is 0.02 to 0.2 μ M.
9. The method according to claim 7 or 8, wherein the buffer salt is selected to be 5-20mM Gly-NaOH.
10. The method according to any one of claims 7 to 9, wherein the masking agent is Na2A mixture of S and cDCTA, Na2The concentration of S is 0.1-1.0mM, and the concentration of cDCTA is 1-10 mM.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113281320A (en) * | 2021-06-07 | 2021-08-20 | 江南大学 | Method for detecting aflatoxin B1 based on fluorescent copper nanoparticles |
| CN113523296A (en) * | 2021-06-19 | 2021-10-22 | 南昌大学 | Preparation method of gold-lead fluorescent nano-cluster and application of gold-lead fluorescent nano-cluster in sodium-dependent detection |
| CN116008238A (en) * | 2022-11-16 | 2023-04-25 | 江南大学 | Chlorothalonil ratio fluorescence detection method based on gold nanoclusters |
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| CN116008238B (en) * | 2022-11-16 | 2024-04-05 | 江南大学 | Chlorothalonil ratio fluorescence detection method based on gold nanoclusters |
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