A fluorescent material for detecting neonicotinoid pesticides, preparation and detection method

Abstract

The application belongs to the technical field of pesticide detection, and relates to a fluorescent material for detecting neonicotinoid pesticides, a preparation method and a detection method. The material comprises ZnO microspheres and a molecular imprinting template. The surface of the ZnO microspheres has gaps, and the center has a hollow structure. The size of the ZnO microspheres is 800-1200 nm. The ZnO microspheres are composed of small particles with a size of 4.2-7.8 nm. The molecular imprinting template wraps the ZnO microspheres. The fluorescent material absorbs ultraviolet light with a wavelength of 375 nm and emits visible light with a wavelength of 596 nm. The material can adsorb neonicotinoid pesticides, and then the content of the neonicotinoid pesticides can be determined by detecting the fluorescence signal intensity of the mixture of the adsorbed material and the neonicotinoid pesticides. The method can avoid complicated extraction, and the neonicotinoid pesticides do not need to be separated from the adsorbed material for detection.

Description

Technical Field

[0001] This invention belongs to the field of pesticide detection technology, and relates to a fluorescent material, its preparation, and a detection method for detecting neonicotinoid pesticides. Background Technology

[0002] Neonicotinoid pesticides, as highly effective insecticides, are widely used in agricultural production (Overview of the status and global strategy for neonicotinoids, Agric. Food Chem. 59 (2011), 2897.). However, the widespread use of neonicotinoid pesticides has also exposed many problems. For example, pesticide residues can enter the human body through water sources and the food chain, posing a potential threat to human health. Therefore, the detection of neonicotinoid pesticides in the human body is of great significance.

[0003] Because neonicotinoid pesticides have a long biological half-life in the environment, those that enter the human body through the food chain can persist for a long time. Numerous studies have detected the presence of neonicotinoid pesticides in human urine (Quantitation of neonicotinoid metabolites in human urine using GC-MS, Journal of Chromatography B 941 (2013), 09-115). Currently, the most commonly detected neonicotinoid pesticides are imidacloprid, thiamethoxam, and acetamiprid.

[0004] Common methods for detecting neonicotinoid pesticides include high-performance liquid chromatography (HPLC), enzyme-linked immunosorbent assay (ELISA), and surface-enhanced Raman spectroscopy (SERS). However, the concentration of neonicotinoid pesticides in urine is low, and existing techniques require large sample volumes and complex pretreatment procedures. For example, the most common methods involve solid-phase extraction (SPE) for sample enrichment and purification (Metal-organic framework MIL101(Cr)-NH2 functionalized magneticgraphene oxide for ultrasonic-assisted magnetic solid phase extraction of neonicotinoid insecticides from fruit and water samples, Talanta 217(2020), 121120), or multiple manual liquid-liquid extractions using large amounts of organic solvents to recover the target analyte. The concentration of the target analyte is then measured. These pretreatment processes are cumbersome, time-consuming, and inefficient. Summary of the Invention

[0005] To overcome the shortcomings of existing technologies, this invention provides a fluorescent material, its preparation, and a detection method for detecting neonicotinoid pesticides. This fluorescent material can adsorb neonicotinoid pesticides, thereby altering the luminescence intensity of the fluorescent material. The content of neonicotinoid pesticides is then determined based on the intensity of the fluorescence signal in the fluorescence spectrum of the detection material. This method avoids cumbersome extraction and eliminates the need to separate the neonicotinoid pesticides from the adsorbent material before detection. Figure 1 This is a schematic diagram of the preparation and testing process of the present invention.

[0006] The technical solution adopted by this invention to solve its technical problem is:

[0007] A fluorescent material for detecting neonicotinoid pesticides is provided, comprising ZnO microspheres and a molecularly imprinted template. The ZnO microspheres have a sparse hair-like structure on their surface, and the ZnO microspheres are 800–1200 nm in size, and are composed of small particles of 4.2–7.8 nm. The molecularly imprinted template encapsulates the ZnO microspheres. This fluorescent material absorbs 375 nm ultraviolet light and emits 596 nm visible light.

[0008] This material effectively adsorbs neonicotinoid pesticides due to the numerous pores on the ZnO surface, which increase its specific surface area. Furthermore, the molecularly imprinted template can adsorb specific neonicotinoid pesticides. Additionally, the ZnO microspheres, composed of small particles ranging from 4.2 to 7.8 nm, possess photoluminescence properties. This material allows for the direct detection of neonicotinoid pesticides via fluorescence spectroscopy without separating the pesticides from the fluorescent material.

[0009] Further optimization of fluorescent materials for detecting neonicotinoid pesticides.

[0010] Preferably, the molecularly imprinted template is based on neonicotinoid pesticide standards, molded with trimethylolpropane trimethacrylate and 2,2-azobisisobutyronitrile, and then repeatedly washed with acetonitrile by centrifugation to obtain an adsorbent material capable of adsorbing the specified neonicotinoid pesticides.

[0011] This invention also provides a method for preparing a fluorescent material for detecting neonicotinoid pesticides, characterized by comprising the following steps:

[0012] S1. Add 0.8–2.4 g of zinc acetate dihydrate (Zn(AC)2·2H2O) to 40–80 ml of anhydrous ethanol solution, heat to 60–80 °C and stir for 20–30 min. At the same time, dissolve 0.3–0.9 g of potassium hydroxide (KOH) in 20–40 ml of anhydrous ethanol, then add the KOH solution to the Zn(AC)2·2H2O solution, stir at 30–60 °C for 10–20 min, and then add 180–360 ml of n-hexane to obtain a precipitate. Centrifuge, wash and dry the precipitate to obtain a white powder.

[0013] S2. Add 0.1-0.3g of the dried powder from step S1 to a mixed solution of 20-40ml benzyl alcohol and 0.5-2g polyethylene glycol. Stir at 60-80℃ for 3-5h. Then pour the mixture into a polytetrafluoroethylene liner, seal the reaction vessel, place it in an oven and heat to 120-200℃, then cool it naturally to room temperature. Finally, centrifuge and wash to obtain a material with stable fluorescence.

[0014] S3. Place 100–300 mg of neonicotinoid pesticide standard in a flask, then add 30–100 ml of acetonitrile, 4–12 ml of deionized water, and 0.1–0.5 ml of methacrylic acid. Stir for 3–5 hours. Then add 0.1–0.6 g of the material with stable fluorescence from step S2. Next, add 8–30 mmol of trimethylolpropane trimethacrylate and 20–60 mg of 2,2-azobisisobutyronitrile sequentially and stir thoroughly for 16–20 hours. Then, wash repeatedly with acetonitrile by centrifugation until the neonicotinoid pesticide standard is undetectable, thus obtaining the fluorescent material for detecting neonicotinoid pesticides.

[0015] The precipitate mentioned in step S1 is ZnO nanoparticles with a size of approximately 4.2–7.8 nm. These nanoparticles are small in size, readily aggregate, and their crystal size increases, easily leading to changes in their luminescence intensity and emission position. This invention uses heating to aggregate small ZnO nanoparticles together, and then uses hydrothermal high temperature and pressure to form microspheres with a sparse, hair-like structure on the surface, thereby increasing their adsorption effect.

[0016] Further optimization of fluorescent materials for detecting neonicotinoid pesticides.

[0017] Preferably, the drying conditions in step S1 are 50–80°C and the drying time is 12–18 h.

[0018] In step S1, the drying temperature should not be too high. If the temperature is too high, the ZnO nanoparticles will aggregate fully and have a fixed morphology, making it difficult to form ZnO microspheres with high porosity under hydrothermal conditions.

[0019] In the preferred step S3, the neonicotinoid pesticide standard is imidacloprid.

[0020] This invention also provides a method for detecting neonicotinoid pesticides, characterized by comprising the following steps:

[0021] Take the fluorescent material into a centrifuge tube, add methanol and vortex to remove the supernatant, then add the urine to be tested, shake for 2-3 minutes, and then centrifuge to obtain a white substrate. Then disperse it in anhydrous ethanol, centrifuge it again, and then disperse it in a mixed solution of isopropanol and polyvinylpyrrolidone K-30. Detect the fluorescence spectrum and analyze the content of neonicotinoid pesticides.

[0022] This is a further optimization of the method for detecting neonicotinoid pesticides using fluorescent materials.

[0023] Preferably, the amounts of fluorescent material, methanol, urine sample to be tested, anhydrous ethanol, isopropanol, and polyvinylpyrrolidone K-30 added are 30-60 mg, 3-6 ml, 10-20 ml, 15-20 ml, 3-5 ml, and 0.1-0.3 g, respectively.

[0024] The advantages of this invention compared to the prior art are as follows:

[0025] (1) The fluorescent material prepared by this invention can adsorb neonicotinoid pesticides without separating the neonicotinoid pesticides from the adsorbent material. The content of neonicotinoids can be determined by detecting the fluorescence signal intensity of the mixture of the adsorbent material and neonicotinoids. This method avoids cumbersome extraction methods and separation and purification steps.

[0026] (2) This invention uses small-sized ZnO nanoparticles, heating and hydrothermal reaction to form a stable microsphere structure. The microspheres are still small ZnO nanoparticles, thus retaining their luminescent properties. Furthermore, the microsphere structure is not easily deformed, preventing spontaneous changes in its luminescent properties. Additionally, under specific temperature, pressure, and surfactant conditions, the ZnO microspheres form a sparse, hair-like structure on their surface, increasing their adsorption capacity.

[0027] (3) This method is very effective for the qualitative detection of neonicotinoid pesticides, and the content of neonicotinoid pesticides can be determined based on the intensity of fluorescence. Attached Figure Description

[0028] Figure 1 This is a flowchart illustrating a fluorescent material, its preparation, and the detection method for detecting neonicotinoid pesticides.

[0029] Figure 2 (a) is a transmission electron microscope image of the synthesized precipitate in Example 1. Figure 2(b) is the Gaussian fitting curve for its statistical dimensions;

[0030] Figure 3 These are transmission electron microscope images of the ZnO microspheres prepared in Example 2. Figure 3 (b) is its high-resolution transmission electron microscope image;

[0031] Figure 4 The fluorescence emission and excitation spectra of ZnO nanoparticles in Comparative Example 1 as a function of time are shown.

[0032] Figure 5 The fluorescence emission and excitation spectra of the ZnO microspheres in Comparative Example 2 are shown as changes over time.

[0033] Figure 6 (a) is the fluorescence emission spectrum of neonicotinoid pesticides adsorbed by the fluorescent materials in Examples 1-6 and Comparative Example 2. Figure 6 (b) is a bar chart of its emission spectrum peaks. Detailed Implementation

[0034] The present invention will be further described below with reference to embodiments and comparative examples.

[0035] Example 1

[0036] This embodiment provides a fluorescent material, its preparation, and a detection method for detecting neonicotinoid pesticides.

[0037] The adsorbent material and its preparation method include the following steps:

[0038] S1. Add 0.8 g of zinc acetate dihydrate (Zn(AC)2·2H2O) to 40 ml of anhydrous ethanol solution, heat to 60 °C and stir for 20 min. At the same time, dissolve 0.3 g of potassium hydroxide (KOH) in 20 ml of anhydrous ethanol, then add the KOH solution to the Zn(AC)2·2H2O solution, stir at 30 °C for 10 min, then add 180 ml of n-hexane to obtain a precipitate. Centrifuge, wash and dry the precipitate to obtain a white powder. The drying temperature is 50 °C and the drying time is 12 h.

[0039] S2. Add 0.1g of the dried powder from step S1 to a mixed solution of 20ml benzyl alcohol and 0.5g polyethylene glycol and stir at 60℃ for 3h. Then pour it into a polytetrafluoroethylene liner, seal the reaction vessel, place it in an oven and heat to 120℃, then cool it naturally to room temperature. After centrifugation and washing, obtain a material with stable fluorescence effect.

[0040] S3. Place 100 mg of imidacloprid standard in a flask, then add 30 ml of acetonitrile, 4 ml of deionized water, and 0.1 ml of methacrylic acid, and stir for 3 hours. Then add 0.1 g of the material with stable fluorescence from step S2. Next, add 8 mmol of trimethylolpropane trimethacrylate and 20 mg of 2,2-azobisisobutyronitrile, and stir thoroughly for 16 hours. Then, wash repeatedly with acetonitrile by centrifugation until imidacloprid standard is undetectable, thus obtaining the fluorescent material for detecting imidacloprid.

[0041] The detection method includes the following steps:

[0042] Take 30 mg of fluorescent material into a centrifuge tube, add 3 ml of methanol, vortex to remove the supernatant, then add 10 ml of urine to be tested, shake for 2 min, and then centrifuge to obtain a white substrate. Then disperse it in 15 ml of anhydrous ethanol, centrifuge it again, and then disperse it in a mixed solution of 3 ml of isopropanol and 0.1 g of polyvinylpyrrolidone K-30. Detect the fluorescence spectrum and analyze the content of neonicotinoid pesticides.

[0043] In this embodiment, a molecularly imprinted template was used to encapsulate ZnO microspheres. The fluorescent material absorbs 375 nm ultraviolet light and emits 596 nm visible light. The surface of the ZnO microspheres has a sparse hair-like structure. The ZnO microspheres are 1000 nm in size and are composed of small particles of 5.9 nm.

[0044] Examples 2-6

[0045] The difference between Examples 2-6 and Example 1 is that the amount of urine to be tested added in the detection method is 12ml, 14ml, 16ml, 18ml and 20ml, respectively.

[0046] Example 7

[0047] This embodiment provides a fluorescent material, its preparation, and a detection method for detecting neonicotinoid pesticides.

[0048] The adsorbent material and its preparation method specifically include the following steps:

[0049] S1. Add 2.4 g of zinc acetate dihydrate (Zn(AC)2·2H2O) to 80 ml of anhydrous ethanol solution, heat to 80 °C and stir for 30 min. Simultaneously, dissolve 0.9 g of potassium hydroxide (KOH) in 40 ml of anhydrous ethanol, then add the KOH solution to the Zn(AC)2·2H2O solution. Stir at 60 °C for 20 min, then add 360 ml of n-hexane to obtain a precipitate. Centrifuge, wash, and dry to obtain a white powder. The drying temperature is 80 °C and the drying time is 18 h.

[0050] S2. Add 0.3g of the dried powder from step S1 to a mixed solution of 40ml benzyl alcohol and 2g polyethylene glycol and stir at 80℃ for 5h. Then pour it into a polytetrafluoroethylene liner, seal the reaction vessel, place it in an oven and heat to 200℃, then cool it naturally to room temperature. Finally, centrifuge and wash to obtain a material with stable fluorescence effect.

[0051] S3. Place 300 mg of imidacloprid standard in a flask, then add 100 ml of acetonitrile, 12 ml of deionized water, and 0.5 ml of methacrylic acid, and stir for 5 hours. Then add 0.6 g of the material with stable fluorescence effect from step S2. Next, add 30 mmol of trimethylolpropane trimethacrylate and 60 mg of 2,2-azobisisobutyronitrile sequentially, and stir thoroughly for 20 hours. Then, wash repeatedly with acetonitrile by centrifugation until neonicotinoid pesticide standards are undetectable, to obtain the fluorescent material for imidacloprid.

[0052] Detection method:

[0053] Take 60 mg of fluorescent material into a centrifuge tube, add 6 ml of methanol, vortex to remove the supernatant, then add 20 ml of urine to be tested, shake for 3 min, and then centrifuge to obtain a white substrate. Then disperse it in 20 ml of anhydrous ethanol, centrifuge it again, and then disperse it in a mixed solution of 5 ml of isopropanol and 0.3 g of polyvinylpyrrolidone K-30. Detect the fluorescence spectrum and analyze the content of neonicotinoid pesticides.

[0054] In this embodiment, a molecularly imprinted template was used to encapsulate ZnO microspheres. The fluorescent material absorbs 375 nm ultraviolet light and emits 596 nm visible light. The surface of the ZnO microspheres has a sparse hair-like structure. The ZnO microspheres are 800 nm in size and are composed of small particles of 4.2 nm.

[0055] Example 8

[0056] This embodiment provides a fluorescent material, its preparation, and a detection method for detecting neonicotinoid pesticides.

[0057] The adsorbent material and its preparation method specifically include the following steps:

[0058] S1. Add 1.8 g of zinc acetate dihydrate (Zn(AC)₂·2H₂O) to 60 ml of anhydrous ethanol solution, heat to 60 °C and stir for 25 min. Simultaneously, dissolve 0.6 g of potassium hydroxide (KOH) in 30 ml of anhydrous ethanol, then add the KOH solution to the Zn(AC)₂·2H₂O solution. Stir at 50 °C for 15 min, then add 240 ml of n-hexane to obtain a precipitate. Centrifuge, wash, and dry to obtain a white powder. The drying temperature is 60 °C and the drying time is 15 h.

[0059] S2. Add 0.2g of the dried powder from step S1 to a mixed solution of 30ml benzyl alcohol and 1g polyethylene glycol and stir at 60℃ for 3h. Then pour it into a polytetrafluoroethylene liner, seal the reaction vessel, place it in an oven and heat to 150℃, then cool it naturally to room temperature. Finally, centrifuge and wash to obtain a material with stable fluorescence effect.

[0060] S3. Place 200 mg of imidacloprid standard in a flask, then add 80 ml of acetonitrile, 10 ml of deionized water, and 0.2 ml of methacrylic acid, and stir for 4 hours. Then add 0.3 g of the material with stable fluorescence effect from step S2. Next, add 20 mmol of trimethylolpropane trimethacrylate and 40 mg of 2,2-azobisisobutyronitrile, and stir thoroughly for 15 hours. Then, wash repeatedly with acetonitrile by centrifugation until neonicotinoid pesticide standards are undetectable, thus obtaining the fluorescent material for detecting imidacloprid.

[0061] Detection method:

[0062] Take 40 mg of fluorescent material into a centrifuge tube, add 5 ml of methanol, vortex to remove the supernatant, then add 15 ml of urine to be tested, shake for 3 min, and then centrifuge to obtain a white substrate. Then disperse it in 15 ml of anhydrous ethanol, centrifuge it again, and then disperse it in a mixed solution of 4 ml of isopropanol and 0.2 g of polyvinylpyrrolidone K-30. Detect the fluorescence spectrum and analyze the content of neonicotinoid pesticides.

[0063] In this embodiment, a molecularly imprinted template was used to encapsulate ZnO microspheres. The fluorescent material absorbs 375 nm ultraviolet light and emits 596 nm visible light. The surface of the ZnO microspheres has a sparse hair-like structure. The ZnO microspheres are 1200 nm in size and are composed of small particles of 7.8 nm.

[0064] Comparative Example 1

[0065] A fluorescent material is provided, and the preparation method of the fluorescent material is the same as step S1 in Example 1.

[0066] Comparative Examples 2-4

[0067] The results are from Examples 1, 7, and 8, respectively, of the fluorescence spectrum of a fluorescent material used to detect neonicotinoid pesticides without the addition of the urine sample to be tested.

[0068] Figure 2 (a) is a transmission electron microscope image of the synthesized precipitate in Example 1. Figure 2(b) is the Gaussian fitting curve for its size. It can be seen that the initially synthesized ZnO nanoparticles are small in size. Statistical calculations show that the size distribution of the ZnO nanoparticles is 4.5–7.5 nm, with an average size of 5.9 nm.

[0069] Figure 3 These are transmission electron microscope images of the ZnO microspheres prepared in Example 2. Figure 3 (b) is a high-resolution transmission electron microscope image. It can be seen that the surface of the ZnO microspheres has a sparse, hair-like structure, and the ZnO microspheres are composed of small particles. The small ZnO nanoparticles ensure its luminescence performance, while the stable structure of the microspheres ensures that its luminescence intensity does not easily change over time.

[0070] Figure 4 The fluorescence emission and excitation spectra of ZnO nanoparticles in Comparative Example 1 vary over time. The fluorescence spectrum of Comparative Example 1 shows that the luminescence properties of small-sized ZnO nanoparticles are unstable and easily change with environmental variations. This is because small-sized ZnO nanoparticles have high surface energy, making them prone to aggregation and crystal growth, thus altering their luminescence properties.

[0071] Figure 5 The fluorescence emission and excitation spectra of the ZnO microspheres in Comparative Example 2 change over time. Comparative Example 2 shows that the fluorescence emission and excitation spectra of the ZnO microspheres are not easily altered, which is due to the large size and stable structure of the microspheres.

[0072] Figure 6 (a) is the fluorescence emission spectrum of neonicotinoid pesticides adsorbed by the fluorescent materials in Examples 1-6 and Comparative Example 2. Figure 6 (b) is a bar chart of its emission spectrum peaks. The experimental results from Comparative Example 2 and Examples 1-6 show that the fluorescent material without adsorbed neonicotinoid pesticides emits the strongest light. The more neonicotinoid pesticides adsorbed, the lower the emission spectrum becomes. Furthermore, the bar chart shows a certain decreasing trend. This indicates that the content of neonicotinoid pesticides can be determined based on the intensity of the emission spectrum. This is because the adsorption of neonicotinoid pesticides by the fluorescent material affects its absorption of excitation light and its emission of emission light, thus affecting the intensity of its emitted light.

[0073] Those skilled in the art should understand that the above descriptions are merely several specific embodiments of the present invention, and not all embodiments. It should be noted that many modifications and improvements can be made by those skilled in the art, and all modifications or improvements not exceeding the scope of the claims should be considered within the protection scope of the present invention.

Claims

1. A method for preparing a fluorescent material for detecting neonicotinoid pesticides, characterized by, The method comprises the following steps: S1, adding 0.8-2.4 g of zinc acetate dihydrate (Zn(AC)2·2H2O) to 40-80 ml of anhydrous ethanol solution, heating to 60-80℃ and stirring for 20-30 min, while dissolving 0.3-0.9 g of potassium hydroxide (KOH) in 20-40 ml of anhydrous ethanol, then adding the KOH solution to the Zn(AC)2·2H2O solution, stirring for 10 min at 30-60℃, and then adding 180-360 ml of n-hexane to obtain a precipitate, which is centrifuged, washed and dried to obtain a white powder; S2, adding 0.1-0.3 g of the dried powder in step S1 to a mixed solution of 20-40 ml of benzyl alcohol and 0.5-2 g of polyethylene glycol, stirring for 3-5 h at 60-80℃, then pouring it into a polytetrafluoroethylene liner, sealing the reaction kettle, placing it in an oven and heating to 120-200℃, then naturally cooling to room temperature, and then obtaining a material with stable fluorescence effect by centrifugation and washing; S3, placing 100-300 mg of neonicotinoid pesticide standard in a flask, adding 30-100 ml of acetonitrile, 4-12 ml of deionized water and 0.1-0.5 ml of methacrylic acid, stirring for 3-5 h, then adding 0.1-0.6 g of the material with stable fluorescence effect in step S2, then sequentially adding 8-30 mmol of trimethylolpropane trimethacrylate and 20-60 mg of 2,2-azobisisobutyronitrile, stirring for 16-20 h, then repeatedly washing with acetonitrile by centrifugation and washing until no neonicotinoid pesticide standard is detected, to obtain a fluorescent material for detecting neonicotinoid pesticides.

2. The method for preparing a fluorescent material for detecting neonicotinoid pesticides according to claim 1, characterized by, The drying condition in step S1 is 50-80℃, and the drying time is 12-18 h.

3. The method of claim 1, wherein the method is characterized by, The neonicotinoid pesticide standard in step S3 is imidacloprid.

4. The fluorescent material for detecting neonicotinoid pesticides prepared by the preparation method of any one of claims 1-3.

5. The fluorescent material for detecting neonicotinoid pesticides according to claim 4, characterized by, The fluorescent material comprises ZnO microspheres and a molecular imprinting template, the surface of the ZnO microspheres has sparse hair-like structures, the size of the ZnO microspheres is 800-1200 nm, and the ZnO microspheres are composed of small particles with a size of 4.2-7.8 nm, the molecular imprinting template wraps the ZnO microspheres, the fluorescent material absorbs ultraviolet light with a wavelength of 375 nm and emits visible light with a wavelength of 596 nm; the molecular imprinting template is based on a neonicotinoid pesticide standard, and is molded by trimethylolpropane trimethacrylate and 2,2-azobisisobutyronitrile, then repeatedly washed with acetonitrile by centrifugation and washing to obtain an adsorption material with adsorption of a specified neonicotinoid pesticide.

6. A detection method for detecting neonicotinoid pesticides, characterized by, The method comprises the following steps: The fluorescent material in claim 4 or 5 is taken into a centrifuge tube, methanol is added and stirred to remove the supernatant, then the urine to be tested is added, shaken for 2-3 min, then centrifuged to obtain a white substrate, which is then dispersed in anhydrous ethanol, centrifuged, and then dispersed in a mixed solution of isopropyl alcohol and polyvinylpyrrolidone K-30, and the fluorescence spectrum is detected to analyze the content of neonicotinoid pesticides.

7. The method for detecting neonicotinoid pesticides according to claim 6, characterized by, The added amounts of the fluorescent material, methanol, the urine to be tested, anhydrous ethanol, isopropyl alcohol and polyvinylpyrrolidone K-30 are 30-60 mg, 3-6 ml, 10-20 ml, 15-20 ml, 3-5 ml and 0.1-0.3 g, respectively.

Images