Method for enhancing color development stability of paraquat detected by sodium dithionite reduction

Abstract

According to the method, cucurbituril is introduced on the basis of a traditional sodium hydrosulfite reduction detection method, and the color development stability of paraquat is enhanced by utilizing a cavity of the cucurbituril to recognize and combining the characteristics of free radical ions of the reduction product paraquat. Free radical quenching factors such as oxygen and water in the environment are isolated, so that blue reduction products (paraquat free radical ions) cannot be quenched by the quenching factors, and the color development stability of paraquat detected by a sodium dithionite reduction method is further improved.

Description

Technical Field

[0001] The present invention relates to the technical field of pesticide detection, and particularly to a method for enhancing the color development stability of sodium dithionite reduction method for detecting paraquat. Background Art

[0002] Bipyridine pesticides are a type of fast-acting and non-selective herbicides, mainly including paraquat and diquat, etc. Due to their broad herbicide spectrum and fast contact killing effect, these pesticides are widely and frequently used globally. However, the high efficiency of bipyridine herbicides is accompanied by potential environmental, food and health risks. Therefore, developing rapid, sensitive and accurate detection methods for monitoring the concentration levels of bipyridine pesticides in the environment, agricultural products and biological samples has become the focus of current research.

[0003] Currently, the detection of bipyridine pesticides mainly relies on high performance liquid chromatography and liquid chromatography-mass spectrometry. Although these techniques have advantages such as good precision, high sensitivity and accuracy, and can effectively separate and quantify target analytes. However, these techniques also have some obvious limitations: firstly, the equipment is expensive and the operation is complex, requiring professional technical personnel to operate; secondly, the sample pretreatment steps are cumbersome, including processes such as extraction, purification and concentration, which take a long time; finally, these methods are difficult to meet the needs of on-site rapid detection, especially in areas with limited resources or in emergency situations. Therefore, it is particularly important to develop a simple, rapid and low-cost detection method.

[0004] Chinese patent document with publication number CN110220892A discloses a method for formulating a semi-quantitative detection map of diquat and urine detection, using sodium bicarbonate-sodium dithionite color development method, and removing protein by double reagents. Since the solubility of protein in a mixed solvent of acetonitrile and 20% trichloroacetic acid is very low, the present invention directly measures the absorbance of paraquat (PQ) and diquat (DQ) after centrifuging to remove protein in serum for quantification; however, the color development of this method is extremely unstable, thus unable to achieve accurate quantitative analysis.

[0005] The sodium dithionite reduction colorimetric method in the above-mentioned patent is based on the analysis and detection of paraquat / diquat by generating colored radical ions through reduction with sodium dithionite under alkaline conditions. Through this method, paraquat will show a blue color reaction (R.J. Dinís-Oliveira, et al. Paraquat poisonings: mechanisms of lung toxicity, clinical features, and treatment [J]. Crit. Rev. Toxicol., 2008, 38(1): 13 - 71. DOI: 10.1080 / 10408440701669959.), while diquat will produce a green color reaction (N. et al. Human and experimental toxicology of diquat poisoning: toxicokinetics, mechanisms of toxicity, clinical features, and treatment [J]. Hum. Exp. Toxicol., 2018, 37(11): 1131 - 1160. DOI: 10.1177 / 0960327118765330.16.). This method is simple to operate, has a rapid reaction, and strong operability, and has been widely used in the field of pesticide detection.

[0006] However, there are some obvious defects in the practical application of this method: the stability of the colorimetric product is poor and it is easily affected by environmental factors, resulting in color changes. Therefore, in view of the deficiencies of the existing method, it is urgent to improve and optimize the traditional method to solve the problem of poor colorimetric stability. Summary of the Invention

[0007] To solve the above technical problems, the present invention provides a method for enhancing the colorimetric stability of detecting paraquat by the sodium dithionite reduction method. By introducing the macrocyclic host molecule cucurbituril, the quenching factors can be isolated through host-guest binding to stabilize the radical ions, thereby significantly improving the colorimetric stability.

[0008] A method for enhancing the colorimetric stability of detecting paraquat by the sodium dithionite reduction method includes the following steps:

[0009] (1) Add cucurbituril to the paraquat test solution to obtain a mixed solution of paraquat and cucurbituril;

[0010] (2) Add a mixed solution of sodium dithionite and an alkaline buffer to the mixed solution of paraquat and cucurbituril obtained in step (1) to obtain a test solution, and measure the absorbance of the test solution at different times.

[0011] In the present invention, as a class of macrocyclic host molecules, cucurbiturils can isolate free radical quenching factors such as oxygen and water in the environment through host-guest binding, thereby stabilizing the paraquat free radical ions after reduction by sodium dithionite, and further enhancing the color development stability of the paraquat detection by sodium dithionite reduction method.

[0012] Preferably, the cucurbituril is at least one of cucurbituril[5], cucurbituril[6], cucurbituril[7], cucurbituril[8], and cucurbituril

[10] .

[0013] Cucurbituril[n] (CB[n]) is a cyclic compound obtained by the condensation reaction of glycoluril and formaldehyde under acidic conditions. As the number of glycoluril units forming cucurbituril increases, its cavity size also increases. Cucurbituril[5], cucurbituril[6], cucurbituril[7], cucurbituril[8], and cucurbituril

[10] all have cavities capable of encapsulating paraquat free radical ions, which can improve the color development stability.

[0014] Preferably, the concentration of paraquat in the paraquat test solution is 1 - 150 μg / mL.

[0015] More preferably, the concentration of paraquat in the paraquat test solution is 6 - 100 μg / mL.

[0016] In the present invention, when the concentration of the paraquat test solution is within the above range, adding cucurbituril can significantly enhance the color development stability of the paraquat detection by sodium dithionite and delay the fading.

[0017] Preferably, the concentration of the cucurbituril is 0.01 - 20 mM.

[0018] More preferably, the concentration of the cucurbituril is 0.0625 - 20 mM.

[0019] In the present invention, as the concentration of the added cucurbituril increases, the effect of delaying the color fading is better.

[0020] More preferably, the cucurbituril is cucurbituril[7], and the concentration of cucurbituril[7] ≤ 20 mM.

[0021] In the present invention, the solubility of cucurbituril[7] in water is better than that of other cucurbituril compounds. After being added as a color development stabilizer, the cavity of cucurbituril[7] can recognize and bind to the reduced product paraquat free radical ions, isolating free radical quenching factors such as oxygen and water in the environment, so that the quenching factors cannot quench the blue reduced product (paraquat free radical ions), thereby improving the color development stability of the paraquat detection by sodium dithionite reduction method. At the same time, as the concentration of cucurbituril[7] increases, the effect of delaying the color fading is better.

[0022] Preferably, the concentration of sodium dithionite is 10-100 mg / mL.

[0023] Preferably, the alkaline buffer is sodium carbonate and sodium bicarbonate.

[0024] More preferably, the mass ratio of sodium carbonate to sodium bicarbonate is 3:1.

[0025] Since sodium carbonate reacts with acid to first form bicarbonate and then further reacts to form carbon dioxide and water, the reaction between the acid formed after sodium dithionite reacts with water and sodium carbonate is milder than that with sodium bicarbonate. When the mass ratio of sodium carbonate to sodium bicarbonate is controlled within the above range, the use safety during the detection process is higher.

[0026] Preferably, in step (2), the pH value of the solution to be measured is greater than 9.

[0027] In the present invention, paraquat reacts with sodium dithionite under alkaline conditions (pH about 10) to generate blue paraquat radical ions, making the solution show blue color, which can be used for the qualitative detection of paraquat.

[0028] Preferably, the detection wavelength of the absorbance is 350-400 nm or 500-700 nm.

[0029] More preferably, when the cucurbituril is cucurbituril [7], the detection wavelength is 600 nm.

[0030] Preferably, the detection time is ≤120 min.

[0031] In the present invention, after adding cucurbituril, the color development stability of the sodium dithionite reduction method for detecting paraquat can be enhanced. As the detection time prolongs, the color gradually fades. When the time is too long, sodium dithionite may decompose under alkaline conditions, affecting the color development situation.

[0032] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0033] Based on the traditional sodium dithionite reduction detection method, the present invention introduces cucurbituril. By utilizing the property of the cavity of cucurbituril to recognize and combine with the reduction product paraquat radical ions, the free radical quenching factors such as oxygen and water in the environment are isolated, so that the quenching factors cannot quench the blue reduction product (paraquat radical ions), thereby improving the color development stability of the sodium dithionite reduction method for detecting paraquat. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 It is the absorbance change curve of the solution to be measured in Example 1 and Comparative Example 3 at 0, 10, 20, 30, 40, 50, 60 min.

[0035] Figure 2 Absorbance change curves of the test solutions in Examples 1-5 at 0, 10, 20, 30, 40, 50, and 60 min.

[0036] Figure 3 Absorbance change curves of the test solutions in Examples 1, 6-9 and Comparative Examples 1-5 at 0-60 min, where B1-B5 are Examples 6, 7, 1, 8, 9 in sequence, and C1-C5 are Comparative Examples 1-5 in sequence. Detailed implementation manners

[0037] The present invention will be further described in detail below in conjunction with examples, but the implementation manners of the present invention are not limited to the following examples.

[0038] The raw materials used in the present invention are all commercially available.

[0039] Example 1

[0040] (1) Weigh 0.01 g of paraquat and dissolve it in water, make up the volume to 10 mL in a brown volumetric flask to prepare a 1 g / L stock solution, and dilute it to a 25 μg / mL paraquat test solution; weigh 9.3 mL of cucurbit[7] (CB[7]) into 1 mL of water, and ultrasonically heat until dissolved to prepare a stock solution of cucurbit[7]; add the stock solution of cucurbit[7] to the paraquat test solution to obtain a mixed solution of paraquat and cucurbit[7], where the concentration of paraquat is 25 μg / mL and the concentration of cucurbit[7] is 1 mM;

[0041] (2) Weigh 0.4 g of sodium dithionite and dissolve it in 10 mL of water, shake well to obtain a sodium dithionite solution; weigh 2.4 g of sodium carbonate and 0.8 g of sodium bicarbonate into 20 mL of water, ultrasonically heat until completely dissolved, and cool to room temperature to obtain an alkaline buffer solution; mix sodium dithionite and the alkaline buffer in a volume ratio of 1:2 (v / v) to obtain a mixed solution of sodium dithionite and the alkaline buffer. Add 50 μL of the mixed solution of paraquat and cucurbit[7] to a 96-well plate, and then add 150 μL of the mixed solution of sodium dithionite and the alkaline buffer to obtain a test solution. The pH of the test solution is about 10, the temperature of the microplate reader is set to 25 °C. After shaking and mixing the test solution in the above 96-well plate, measure the absorbance changes of the test solution at 600 nm at different time intervals.

[0042] Examples 2-9 and Comparative Examples 1-5

[0043] The method is the same as that of Example 1, and the differences are shown in the following table.

[0044] Table 1: Method differences between Examples 1-9 and Comparative Examples 1-5

[0045]

[0046] Sample analysis

[0047] I. Color stability effect of CB[7] on the detection of paraquat by sodium dithionite reduction method

[0048] Measure the absorbance of the test solutions in Example 1 and Comparative Example 3 at 600 nm at 0, 10, 20, 30, 40, 50, and 60 min respectively. Normalize the maximum value of the absorbance value at 600 nm over time to 100%, and plot the time and the processed data.

[0049] Figure 1 Absorbance change curves of the test solutions in Example 1 and Comparative Example 3 at 0, 10, 20, 30, 40, 50, and 60 min. Among them, compared with Comparative Example 3 without CB[7] (i.e., the PQ curve in the figure), after adding CB[7] (i.e., Example 1, the PQ / CB[7] curve in the figure), the decreasing trend of the absorbance of the solution for detecting paraquat by sodium dithionite reduction method slows down. Therefore, the addition of CB[7] can delay the fading phenomenon in the experiment of detecting paraquat by sodium dithionite reduction method.

[0050] II. Influence of CB[7] concentration on color development

[0051] Measure the absorbance of the test solutions in Examples 1-5 at 600 nm at 0, 10, 20, 30, 40, 50, and 60 min respectively. Normalize the maximum value of the absorbance value at 600 nm over time to 100%, and plot the time and the processed data.

[0052] Figure 2 Absorbance change curves of the test solutions in Examples 1-5 at 0, 10, 20, 30, 40, 50, and 60 min. Among them, as the concentration of CB[7] increases, the decreasing trend of the absorbance gradually slows down, that is, the better the effect of delaying fading for colorimetric analysis of detecting paraquat by sodium dithionite reduction method. Therefore, a CB[7] concentration of 1 mM was selected for subsequent experiments.

[0053] III. Influence of paraquat concentration on color development

[0054] Measure the absorbance of the test solutions in Example 1, Examples 6-9 and Comparative Examples 1-5 at 600 nm at 0, 10, 20, 30, 40, 50, and 60 min respectively. Normalize the maximum value of the absorbance value at 600 nm over time to 100%, and plot the time and the processed data.

[0055] Figure 3Absorbance change curves of the test solutions of Examples 1, 6-9 and Comparative Examples 1-5 at 0, 10, 20, 30, 40, 50, and 60 min. Among them, B1-B5 are Examples 6, 7, 1, 8, and 9 in sequence, and C1-C5 are Comparative Examples 1-5 in sequence. As shown in the figure, the absorbance decline rate of the experimental group added with CB[7] (Examples 1, 6-9, namely the curves B1-B5 in the figure) is delayed compared with that of the control group without CB[7] (Comparative Examples 1-5, namely the curves C1-C5 in the figure). When the paraquat concentration in the test solution is 6.25-100 μg / mL, the addition of 1 mM CB[7] can effectively delay the fading phenomenon in the detection of paraquat by the sodium dithionite reduction method, which helps to improve the stability of the traditional detection method for paraquat detection.

[0056] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, for those skilled in the art, they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements for some of the technical features. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for enhancing the color stability of detecting paraquat by the sodium dithionite reduction method, characterized in that, It includes the following steps: (1) Add cucurbituril to the paraquat solution to be measured to obtain a mixed solution of paraquat and cucurbituril; (2) Add a mixed solution of sodium dithionite and an alkaline buffer to the mixed solution of paraquat and cucurbituril obtained in step (1) to obtain a solution to be measured, and measure the absorbance of the solution to be measured at different times.

2. The method for enhancing the color development stability of detecting paraquat by the sodium dithionite reduction method according to claim 1, characterized in that The cucurbituril is at least one of cucurbituril[5], cucurbituril[6], cucurbituril[7], cucurbituril[8], and cucurbituril[10].

3. The method for enhancing the color development stability of detecting paraquat by the sodium dithionite reduction method according to claim 1, characterized in that The concentration of paraquat in the paraquat solution to be measured is 1-150 μg / mL.

4. The method for enhancing the color stability of the detection of paraquat by the sodium dithionite reduction method according to claim 1, wherein, The concentration of cucurbituril in the mixed solution of paraquat and cucurbituril is 0.01-20 mM.

5. The method for enhancing the color stability of detecting paraquat by the sodium dithionite reduction method according to claim 4, characterized in that, The cucurbituril is cucurbituril[7].

6. The method for enhancing the color development stability of detecting paraquat by the sodium dithionite reduction method according to claim 1, wherein The concentration of sodium dithionite is 10-100 mg / mL.

7. The method for enhancing the color stability of detecting paraquat by the sodium dithionite reduction method according to claim 1, characterized in that, The alkaline buffer is sodium carbonate and sodium bicarbonate.

8. The method for enhancing the color stability of detecting paraquat by the sodium dithionite reduction method according to claim 1, characterized in that, In step (2), the pH value of the solution to be measured is greater than 9.

9. The method for enhancing the color development stability of detecting paraquat by the sodium dithionite reduction method according to claim 1, wherein, The detection wavelength of the absorbance is 350-400 nm or 500-700 nm.

10. The method for enhancing the color stability of detecting paraquat by the sodium dithionite reduction method according to claim 1, characterized in that, The detection time is ≤120 min.

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