CN112113943A - Method for detecting paraquat - Google Patents

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

A kind of method for detecting paraquat

technical field

The invention relates to a method for detecting paraquat, belonging to the technical field of analytical chemistry.

Background technique

The application of pesticides in the agricultural field can not only ensure the yield of crops, but also bring about the problem of pesticide residues. Paraquat is a herbicide that can quickly kill annual weeds, but because it can cause irreversible lung tissue damage and other pathological symptoms to the human body, it is harmful to human organs and has no specific antidote. The use category of paraquat has now been changed to a banned pesticide. In order to deal with the risk of illegal use of paraquat in agricultural production, it is important to establish a detection method that can quickly realize quantitative and semi-quantitative detection.

At present, the rapid detection methods of pesticides developed are mostly based on enzyme inhibition. The enzyme inhibition method is generally a broad-spectrum method, and there are disadvantages that the stability of biological enzymes is greatly affected by environmental factors, and the preservation requirements are high. The method of directly using nanomaterials to realize the specific detection of pesticides has the advantages of simple system and high specificity. Among them, there are reports of colorimetric, chemiluminescence, surface Raman enhancement, and fluorescence detection methods based on nanomaterials for paraquat pesticides. Among them, a typical example is the use of semiconductor quantum dots (such as cadmium telluride CdTe) to realize the method of fluorescence detection of paraquat, but the preparation of nanomaterials is relatively complex and requires the application of heavy metals. However, heavy metals are very difficult to be biodegraded. Under the bio-amplification of the food chain, it is enriched thousands of times, and finally enters the human body, causing chronic poisoning, which is harmful to the human body.

SUMMARY OF THE INVENTION

In order to solve the above problems, the main purpose of the present invention is to establish a method for detecting paraquat by fluorescence based on gold nanoclusters. The detection principle of the invention is as follows: the fluorescence of gold nanoclusters is quenched by electrostatic transfer from gold nanoclusters to paraquat. for reliable quantitative detection of paraquat.

The first object of the present invention is to provide a method for detecting paraquat. The detection method is to use gold nanoclusters as a detection reagent for paraquat, and perform fluorescence detection on the sample to be tested; the gold nanoclusters use sulfhydryl-β - Cyclodextrin SH-β-CDs are prepared by modifying the preparation of glutathione-reduced gold.

In one embodiment of the present invention, the preparation method of the gold nanoclusters is: mixing 2 mL of HAuCl ₄ (10 mM), 0.3 mL of GSH (100 mM) solution and 7.7 mL of ultrapure water. The mixture was heated to 70°C with gentle stirring and reacted for 24 hours. To the obtained pale yellow solution, ethanol was added at a ratio of 1:1; the solution changed from clarification to turbidity, the solution was centrifuged at 8000 rpm for 15 min, the precipitate was separated and dissolved by ultrasonic, and filtered through a 0.22 μM filter to obtain glutathione reduction. Gold solution. SH-β-cyclodextrin (final concentration 5mM) was added to the obtained glutathione-reduced gold solution, and incubated at 50°C for 3h; the obtained solution was centrifuged and concentrated by an ultrafiltration tube (10kDa) to prepare gold nanoparticles cluster.

In one embodiment of the present invention, the detection method is: adding gold nanocluster solution to the solution of the sample to be tested, mixing and reacting, and then using a fluorescence spectrophotometer for detection, and calculating the sample to be tested by the external standard method paraquat content.

In an embodiment of the present invention, the pH of the gold nano solution needs to be adjusted to 8.5-9.0 with a buffer.

In one embodiment of the present invention, the buffer solution includes glycine buffered saline, Tris-HCl or phosphate buffered solution.

In an embodiment of the present invention, the fluorescence detection conditions are that the excitation broadband is 20 nm, the slit width is 20 nm, the excitation wavelength is 392 nm, and the fluorescence value at the emission peak of 610 nm is detected.

In an embodiment of the present invention, if the sample to be tested contains metal ions, a metal ion masking agent must be added to the sample to be tested, and then gold nanoclusters are added for detection.

The second object of the present invention is to provide an application of the above-mentioned detection method in the detection of pesticide content in food, river water, lake water or sewage.

The third object of the present invention is to provide a method for detecting the content of paraquat in water, the method is to first add a metal ion masking agent to the sample to be tested, and then use the above method to detect the content of paraquat in the sample to be tested.

In one embodiment of the present invention, the concentration of gold nanoclusters is selected to be 0.02-0.2 [mu]M.

In one embodiment of the present invention, the buffer salt of choice is Gly-NaOH (5-20 mM).

In an embodiment of the present invention, the masking agent is a mixture of Na2S and _cDCTA , the concentration of Na2S is 0.1-1.0 mM, and the concentration of cDCTA is 1-10 mM.

In one embodiment of the present invention, the method steps are:

(1) Preparation of gold nanocluster solution: The prepared gold nanoclusters were adjusted to pH 9.0 by glycine buffer (5-20 mM), and diluted to an appropriate concentration:

(2) Preparation of standard curve: The paraquat standard solution and the nanocluster solution were mixed and incubated for 1 min to establish a standard curve of 0.02-0.2 μM of the corresponding relationship between the fluorescence quenching rate signal of gold nanoclusters and the concentration of paraquat;

(3) Sample detection: Add a masking agent configured by sodium sulfide (Na ₂ S) and trans-cyclohexanediaminetetraacetic acid (cDCTA) to the sample solution, and mix and react with the gold nanocluster solution, and the calculated fluorescence The quenching rate signal was brought into the standard curve, and the concentration of paraquat in the test sample was calculated.

Beneficial effects of the present invention:

The gold nanocluster fluorescence detection method for paraquat constructed in the present invention has a detection limit of 5.0 ng/mL and a linear range of 5.0-360 ng/mL; other common organophosphorus pesticides have the same detection concentration of 500 ng/mL. The detection quenching rate of nanoclusters is 80%, and the quenching rates of other pesticides are all lower than 5%. After adding metal ion shielding agent to nanoclusters, the interference of ions is reduced, and the quenching rate responses to 5uM detected metal ions are all less than 5%. The method has the characteristics of high sensitivity, high selectivity, and rapid detection.

Description of drawings

Figure 1 is a schematic diagram of the preparation of gold nanoclusters and the detection of paraquat.

Figure 2 is the fluorescence change of nanoclusters after modification of HS-β-CDs.

Figure 3 is the paraquat detection fluorescence curve.

Figure 4 is the standard curve for the detection of paraquat.

Figure 5 shows the selective response of β-CD/GSH-AuNCs to various pesticides.

Figure 6 is the response of β-CD/GSH-AuNCs to various metal ions.

Figure 7 shows the responses of GSH-AuNCs to various metal ions.

Figure 8 is A, B, C, D, E, F, respectively, the quenching of nanoclusters by paraquat in different concentrations of Na ₂ HPO ₄ -NaH ₂ PO ₄ , Tris-HCl, citric acid-Na ₂ HPO ₄ buffer salt Rate response and quench rate response of Na ₂ HPO ₄ -NaH ₂ PO ₄ , Tris-HCl, Gly-NaOH at different pH.

Figure 9 shows the shielding effect of EDTA on various ions.

Figure 10 shows the shielding effect of cDCTA on various metal ions.

Figure 11 shows the shielding effect of cDCTA and Na ₂ S on various metal ions.

Detailed ways

The preferred embodiments of the present invention will be described below, and it should be understood that the embodiments are used to better explain the present invention and are not intended to limit the present invention.

Example 1: Preparation method of gold nanoclusters

2 mL of HAuCl ₄ (10 mM), 0.3 mL of GSH (100 mM) solution and 7.7 mL of ultrapure water were mixed. The mixture was heated to 70°C with gentle stirring and reacted for 24 hours. To the obtained pale yellow solution, ethanol was added at a ratio of 1:1; the solution changed from clarification to turbidity, the solution was centrifuged at 8000 rpm for 15 min, the precipitate was separated and dissolved by ultrasonic, and filtered through a 0.22 μM filter to obtain glutathione reduction. Gold solution GSH-AuNCs. SH-β-cyclodextrin (final concentration 5mM) was added to the obtained glutathione-reduced gold solution, and incubated at 50°C for 3h; the obtained solution was centrifuged and concentrated by an ultrafiltration tube (10kDa) to prepare gold nanoparticles For cluster β-CD/GSH-AuNCs, the concentrated solution (0.03mM) was collected and stored at 4°C. The reaction principle of gold nanoclusters is shown in Figure 1. The particle size of the obtained gold nanoclusters is 1.0-3.0 nm, the excitation wavelength is between 350-400 nm, and the emission wavelength is 620 nm. The fluorescence intensities of the gold nanoclusters before and after modification are shown in Figure 2, which indicates that the modification with thiol-β-cyclodextrin can improve the binding effect of paraquat.

Example 2: A method for detecting paraquat content in river/lake water using gold nanoclusters

The gold nanoclusters prepared in Example 1 were used to detect paraquat content in river/lake water. The preparation of gold nanoclusters and the detection principle of paraquat are shown in Figure 1 .

1. Establishment of standard curve

(1) Preparation of standard solution:

900uL Gly-NaOH buffer + 50uL paraquat pesticide standard solution or sample solution + 50uL nanoclusters (0.05μM), after mixing

(2) Fluorescence spectrum detection:

Fluorescence photometer was used for detection, and the detection conditions were as follows: the excitation wavelength was 390 nm, and the emission signal was collected at 620 nm.

(3) Determination of linear relationship and detection limit:

The fluorescence intensity of the detected nanoclusters was measured as F ₁ , and the fluorescence intensity of the nanoclusters without pesticides was measured as F ₀ , and the standard curve was drawn as shown in Figures 3 and 4. The standard curve was y=0.0254x+0.0037 (5-150ng/mL), R ² =0.9956; y=0.1367x+0.014, (150-360 ng/mL) R ² =0.9837, the linear range was 5-360 ng/mL, and the limit of quantification was 5 ng/mL.

2. Method for detecting paraquat content in river/lake water

(1) Sample pretreatment: filter the river water/lake water through a microporous membrane, and at the same time, add a masking agent, the masking agent contains 200uM Na ₂ S and 2mM cDCTA, and the volume ratio of sample to masking agent is 1:1.

(2) Fluorescence spectrum detection:

Fluorescence photometer was used for detection, and the detection conditions were as follows: the excitation wavelength was 390 nm, and the emission signal was collected at 620 nm.

Example 3: Accuracy and specificity of the method

1. The accuracy of the method

The concentration of standard paraquat added to the lake water sample was 160ng/mL. The actual average concentration of paraquat measured in three parallel determinations was 155.6ng/mL, and the measured recovery was 97.2%. Relative standard deviation (RSD)=2.0 %.

2. The specificity of the method

(1) The influence of common organophosphorus pesticides and metal ions on the detection was investigated. The organophosphorus pesticides selected 9 pesticides, fenamiphos, acetamiprid, chlorpyrifos, glyphosate, methyl parathion, amphos, methomyl and imidacloprid at the same concentration (500ng/mL). They did not affect the detection of paraquat (as shown in Figure 5).

(2) The influence of common metal ions on the detection was investigated, as shown in Figures 6 and 7. The results showed that the anti-metal and S ^2- ability was improved after modification by HS-β-CD.

Example 4: The effect of different salt concentrations on the quenching of gold nanocluster fluorescence by paraquat

Detect with reference to the method of Example 2, the difference is only in that the buffer salt and buffer salt concentration of the system are adjusted, and the buffer salt concentration is adjusted respectively by using Na ₂ HPO ₄ -NaH ₂ PO ₄ , Tris-HCl, citric acid-Na ₂ HPO ₄ For 5mM, 10mM, 15mM, 20mM, 25mM, 30mM and other conditions are the same, the results are shown in Figure 8.

It can be seen from Figures 8A, 8B, and 8C that a high concentration of buffer salt will affect the fluorescence quenching of paraquat on nanoclusters, so 5mM buffer salt concentration is selected as the optimum.

Example 5: The effect of pH on the quenching of gold nanocluster fluorescence by paraquat

Detected with reference to the method of Example 2, the difference is only that the pH of the system was adjusted, and the buffer salt was adjusted to different pHs by using Na ₂ HPO ₄ -NaH ₂ PO ₄ , Tris-HCl, Gly-NaOH, and other conditions were the same, the results See Figure 8.

8D, 8E, 8F, when the pH of the system is 9.0, the fluorescence quenching of the nanoclusters by paraquat reaches the maximum, so Tris-HCl and Gly-NaOH buffer salts with pH=9.0 are selected as the optimal.

Example 6: Selection of metal ion shielding agent

Detected with reference to the method of Example 2, the only difference was that the metal ions with a concentration of 10uM were added, and the masking agent was replaced with EDTA, cDCTA, Na ₂ S and cDCTA systems respectively, other conditions were the same, and the results were shown in Figure 9, 10 and 11. Through screening, the simultaneous addition of trans-cyclohexaneethylenediaminetetraacetic acid and sodium sulfide was selected as the metal ion shielding agent. After adding the shielding agent, the shielding effect of 13 common metal ions was very good.

Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone who is familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, The protection scope of the present invention should be defined by the 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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