A real-time detection sensor and its application in the detection of chlorfenapyr poisoning

Abstract

This invention provides a real-time detection sensor and its application in the detection of chlorfenapyr poisoning, belonging to the field of toxicity detection technology. This invention loads AuRD onto ZIF-67 and then co-modifies an MCE film. The resulting real-time detection sensor AuRD@ZIF-67 / MCE can rapidly and sensitively detect chlorfenapyr in human serum and urine based on surface-enhanced Raman spectroscopy. Moreover, this real-time detection sensor has excellent thermal stability, spatial uniformity, and precision.

Description

Technical Field

[0001] This invention relates to the field of toxicity detection technology, specifically to a real-time detection sensor and its application in the detection of chlorfenapyr poisoning. Background Technology

[0002] Chlorfenapyr is a globally used N-substituted halogenated pyrrole insecticide, internationally known as 4-bromo-2-(4-chlorophenyl)-1-(ethoxymethyl)-5-(trifluoromethyl)pyrrole-3-carboxylon. As an insecticide, chlorfenapyr effectively kills insects through oral or contact exposure, causing rhabdomyolysis, metabolic acidosis, and kidney failure. It is widely used in regions with high pyrethroid resistance, and China is the largest market for chlorfenapyr.

[0003] In recent years, the incidence of chlorfenapyr poisoning in humans due to suicide or accidental ingestion has been increasing. Symptoms of chlorfenapyr poisoning are often mild and atypical in the early stages, especially in patients with low-dose exposure, and may be overlooked by doctors, leading to delayed treatment. Once chlorfenapyr toxicity affects the hypothalamic thermoregulatory center and the medullary respiratory and cardiac centers, patients often experience high fever, coma, and even death. Chlorfenapyr poisoning survivors typically experience persistent neurological sequelae. Rapid detection of chlorfenapyr poisoning is a crucial prerequisite for timely diagnosis and treatment of patients.

[0004] Currently, the main methods for detecting chlorfenapyr are liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS). However, these methods are very time-consuming and require expensive instruments and complex sample preparation processes. Furthermore, existing chlorfenapyr detection methods are primarily designed for food and environmental samples, and these methods are also time-consuming and require expensive instruments and complex sample preparation processes. Therefore, there is an urgent need to develop qualitative and quantitative detection techniques for chlorfenapyr in biofluids that can be applied clinically. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a real-time detection sensor capable of rapidly and sensitively detecting acaricides in human serum and urine.

[0006] The technical solution of this invention is as follows:

[0007] One instantaneous detection sensor involves loading AuRD onto ZIF-67 and then co-modifying an MCE film.

[0008] Preferably, the mass ratio between AuRD and ZIF-67 is 100:(1 to 1.5).

[0009] Preferably, the instantaneous detection sensor is applied based on surface-enhanced Raman spectroscopy.

[0010] The method for preparing the instantaneous detection sensor includes the following steps:

[0011] (1) Preparation of AuRD solution;

[0012] (2) Preparation of ZIF-67 solution;

[0013] (3) Mix AuRD solution and ZIF-67 solution and then add them evenly to the MCE membrane and let it stand.

[0014] Preferably, the preparation method of the AuRD solution in step (1) includes the following steps:

[0015] i. Prepare a mixed solution containing HAuCl4 and CTAC; prepare a NaBH4 solution and pre-cool it; under stirring conditions, add the NaBH4 solution to the mixed solution to obtain a brown seed solution; let the seed solution stand at 30-35℃ for 1-1.2h to obtain a gold seed solution.

[0016] ii. Prepare two containers, add CTAC and water to each container, and place them in a water bath at 30-35°C; while in the water bath, continue to add HAuCl4 solution, NaBr solution and ascorbic acid solution to each container;

[0017] iii. Label the two containers from step ii as A and B respectively; under shaking conditions, add the gold seed solution from step i to container A to obtain a light pink solution; transfer the light pink solution to container B, mix thoroughly, let stand for 15-20 min, centrifuge at 6000-8000 rpm for 5-10 min to obtain AuRD precipitate; dissolve the precipitate in water to obtain AuRD solution.

[0018] More preferably, the molar ratio of HAuCl4, CTAC, and NaBH4 in step i is 2.5:1000:9.

[0019] More preferably, the molar ratio of CTAC, HAuCl4, NaBr, and ascorbic acid in step ii is 1000:2.5:0.1:1.5.

[0020] Preferably, the preparation method of the ZIF-67 solution in step (2) includes the following steps:

[0021] S1. Prepare cobalt nitrate hexahydrate solution and 2-methylimidazole solution respectively;

[0022] S2. Mix the cobalt nitrate hexahydrate solution and the 2-methylimidazole solution from step S1, and stir at 1000-1500 rpm for 6-6.5 h at room temperature;

[0023] Centrifuge at 6000-8000 rpm for 10-15 min to obtain ZIF-67 precipitate; wash and dissolve the precipitate with ethanol to obtain ZIF-67 solution.

[0024] More preferably, the mass ratio between cobalt nitrate hexahydrate and 2-methylimidazole in step S2 is 0.45:5.5.

[0025] Preferably, step (3) includes the following steps: mixing the AuRD solution from step (1) and the ZIF-67 solution from step (2), centrifuging at 6000-8000 rpm for 10-15 min, allowing the mixture to stand and separate into layers to obtain an upper clear liquid and a lower mixed liquid; uniformly adding the lower mixed liquid onto the MCE membrane, allowing it to stand for 20-30 min to obtain an instantaneous detection sensor.

[0026] Application of the instant detection sensor in the detection of chlorfenapyr poisoning.

[0027] Preferably, the application method includes the following steps: placing the instant detection sensor on a surface-enhanced Raman spectrometer, dropping the chlorfenapyr sample onto the surface of the instant detection sensor, and detecting the chlorfenapyr poisoning status by the signal intensity of the Raman spectrum.

[0028] Beneficial effects:

[0029] This invention discloses a real-time detection sensor based on surface-enhanced Raman spectroscopy (SERS), which can rapidly and sensitively detect acaricides in human serum and urine. The real-time detection sensor also has excellent thermal stability, spatial uniformity and precision. Attached Figure Description

[0030] Figure 1 The structural characterization diagram of AuRD@ZIF-67 / MCE;

[0031] Figure 2 The SERS signal intensity of sensors fabricated using different gold nanostructures;

[0032] Figure 3 The SERS signal intensity at different deposition times when 50 μL of ZIF-67 solution was added;

[0033] Figure 4 pH optimization for AuRD@ZIF-67 / MCE detection;

[0034] Figure 5 Optimize the detection temperature for AuRD@ZIF-67 / MCE;

[0035] Figure 6The adsorption time of AuRD@ZIF-67 / MCE on the chlorfenapyr sample;

[0036] Figure 7 The relationship between the concentration of abamectin and the SERS signal intensity on AuRD@ZIF-67 / MCE samples;

[0037] Figure 8 Thermal stability testing of AuRD@ZIF-67 / MCE;

[0038] Figure 9 Spatial homogeneity detection for AuRD@ZIF-67 / MCE;

[0039] Figure 10 Precision testing for AuRD@ZIF-67 / MCE. Detailed Implementation

[0040] The following description is based on specific embodiments:

[0041] (1) Explanation of relevant terms:

[0042] AuRD (Au Rhombic Dodecahedra): Gold nano-rhombic dodecahedron;

[0043] ZIF-67 (Zeolitic Imidazole Framework-67): A metal-organic framework (MOF);

[0044] MCE (Mixed Cellulose Ester) membrane: a type of mixed cellulose ester membrane.

[0045] (2) Explanation of the source of experimental materials:

[0046] MCE membrane: purchased from Haining Delu New Material Technology Co., Ltd.;

[0047] Surface-enhanced Raman spectroscopy: QE Pro, Ocean Optics;

[0048] Chlorfenapyr standard samples: purchased from Beijing Beifang Weiye Metrology Technology Research Institute; chlorfenapyr standard samples of different concentrations can be prepared by dissolving chlorfenapyr standard samples in the urine of healthy volunteers.

[0049] Example 1: Fabrication of the real-time detection sensor AuRD@ZIF-67 / MCE

[0050] The preparation method is carried out according to the following steps:

[0051] I. Preparation of AuRD solution:

[0052] Refer to the literature "A Comparative Study of Gold Nanocubes, Octahedra, and RhombicDodecahedra as Highly Sensitive SERS Substrates".

[0053] i. Prepare 10 mL of a mixed solution containing 0.25 mM HAuCl4 and 0.10 M CTAC; prepare 10 mL of a 0.02 M NaBH4 solution and pre-cool it at 4 °C; under stirring conditions, add 0.45 mL of NaBH4 solution to the mixed solution to obtain a brown seed solution; let the seed solution stand at 30 °C for 1 h to obtain a gold seed solution.

[0054] ii. Prepare two containers, add 0.32g CTAC and 9.545mL water to each container, and incubate in a water bath at 30℃; while in the water bath, continue to add 250μL of 0.01M HAuCl4 solution, 10μL of 0.01M NaBr solution and 150μL of 10mM ascorbic acid solution to the two containers.

[0055] iii. Label the two containers from step ii as A and B, respectively; under shaking conditions, add 45 μL of the gold seed solution from step i to container A to obtain a light pink solution; transfer 45 μL of the light pink solution to container B, mix thoroughly, let stand for 15 min, centrifuge at 6000 rpm for 5 min to obtain 20.15 g of AuRD precipitate; dissolve the precipitate in 1 mL of water to obtain AuRD solution.

[0056] II. Preparation of ZIF-67 solution:

[0057] Refer to the literature "Point-of-Use SERS Approach for Efficient Determination andRemoval of Phthalic Acid Esters Based on a Metal-Organic Framework-CoatedMelamine Sponge".

[0058] S1. Dissolve 0.45g of cobalt nitrate hexahydrate in 3mL of water to obtain a cobalt nitrate hexahydrate solution; dissolve 5.5g of 2-methylimidazole in 20mL of water to obtain a 2-methylimidazole solution.

[0059] S2. Mix the cobalt nitrate hexahydrate solution and 2-methylimidazole solution from step S1, and stir at 1000 rpm for 6 hours at room temperature;

[0060] Centrifuge at 6000 rpm for 10 min to obtain 23.89 g of ZIF-67 precipitate; wash the precipitate three times with ethanol, and finally dissolve it in 5 mL of ethanol to obtain ZIF-67 solution; take 50 μL of ZIF-67 solution for later use.

[0061] III. Fabrication of the real-time detection sensor AuRD@ZIF-67 / MCE:

[0062] The AuRD solution prepared above was mixed evenly with 50 μL of ZIF-67 solution, centrifuged at 8000 rpm for 10 min, and allowed to stand to separate into layers, resulting in an upper clear liquid and a lower mixed liquid. The lower mixed liquid was evenly dropped onto the MCE membrane and allowed to stand for 30 min to obtain the real-time detection sensor AuRD@ZIF-67 / MCE.

[0063] The AuRD@ZIF-67 / MCE instantaneous detection sensor prepared above was structurally characterized using a Hitachi Regulus 8100 instrument, and the results are as follows: Figure 1 As shown. By Figure 1 It can be seen that the MCE membrane has a porous structure, which can accommodate a considerable amount of AuRD@ZIF-67 composite material, and the AuRD@ZIF-67 composite material is uniformly distributed on the surface of the MCE membrane.

[0064] Example 2: Comparison of different gold nanostructures

[0065] Following the same preparation method as AuRD@ZIF-67 / MCE, two other gold nanostructures, Au Nanocubes and AuNPs, were co-modified onto the surface of the MCE film with ZIF-67 to obtain sensors Au Nanocubes@ZIF-67 / MCE and AuNPs@ZIF-67 / MCE, respectively.

[0066] Au Nanocubes were prepared according to the literature "A Comparative Study of Gold Nanocubes, Octahedra, and Rhombic Dodecahedra as Highly Sensitive SERS Substrates"; AuNPs were prepared according to the literature "Rapid identification and quantification of diquat inbiological fluids within 30s using a portable Raman spectrometer".

[0067] The sensors Au Nanocubes@ZIF-67 / MCE, AuNPs@ZIF-67 / MCE, and AuRD@ZIF-67 / MCE prepared in Example 1 were placed on a surface-enhanced Raman spectrometer, with parameters adjusted to 785 nm laser, 135 mW intensity, and 20 seconds integration time. A 1 ppm chlorfenapyr standard sample was dropped onto the surface of each sensor, and the SERS signal intensity (at 1090 cm⁻¹) of each sensor was compared using the same parameters. -1 (Location).

[0068] The results are as follows Figure 2 As shown. By Figure 2 It can be seen that, compared with Au Nanocubes modification and AuNPs modification, AuRD@ZIF-67 / MCE at 1090 cm⁻¹... -1 The strongest SERS signal was observed at that location.

[0069] Example 3: Optimization of ZIF-67 addition amount and detection conditions in AuRD@ZIF-67 / MCE

[0070] Take 10 μL, 50 μL, 100 μL and 500 μL of ZIF-67 solution prepared in Example 1 to prepare AuRD@ZIF-67 / MCE instantaneous detection sensors, and optimize the preparation and detection conditions of AuRD@ZIF-67 / MCE by different deposition times of 5 to 90 min.

[0071] The prepared AuRD@ZIF-67 / MCE was placed on a surface-enhanced Raman spectrometer, and the parameters were adjusted to 785 nm laser, 135 mW intensity, and 20-second integration time. A 1 ppm chlorfenapyr standard sample was added to the surface of each sensor, and the SERS signal intensity under each sensor was compared (1090 cm⁻¹). -1 (place).

[0072] The results are as follows Figure 3 As shown. By Figure 3 It can be seen that when 50 μL of ZIF-67 solution is added and the deposition time is 30 min, a satisfactory SERS signal intensity can be detected.

[0073] Example 4: Optimization of detection pH for AuRD@ZIF-67 / MCE

[0074] The instantaneous detection sensor AuRD@ZIF-67 / MCE prepared in Example 1 was placed on a surface-enhanced Raman spectrometer, and the parameters were adjusted to 785 nm laser, 135 mW intensity, and 20-second integration time. A 1 ppm chlorfenapyr standard sample with a pH value of 3–9 (adjusted by adding different amounts of hydrochloric acid and sodium hydroxide solution) was dropped onto the AuRD@ZIF-67 / MCE surface, and the SERS signals (at 1090 cm⁻¹) under different pH conditions were compared. -1 (Location).

[0075] The results are as follows Figure 4 As shown. By Figure 4 It was found that the SERS signal was strongest when the pH of the chlorfenapyr sample was 6. However, due to the need for detection efficiency, the pH was not adjusted in subsequent experiments.

[0076] Example 5: Optimization of detection temperature for AuRD@ZIF-67 / MCE

[0077] The instantaneous detection sensor AuRD@ZIF-67 / MCE prepared in Example 1 was placed on a surface-enhanced Raman spectrometer, with parameters adjusted to 785 nm laser, 135 mW intensity, and 20-second integration time. The AuRD@ZIF-67 / MCE was heated to different temperatures between 10 and 35 °C using a water bath. Then, a 1 ppm chlorfenapyr standard sample was dropped onto the AuRD@ZIF-67 / MCE surface at different temperatures, and the SERS signals (at 1090 cm⁻¹) were compared under different temperature conditions. -1 (Location).

[0078] The results are as follows Figure 5 As shown. By Figure 5 It can be seen that the SERS signal increases with the increase of AuRD@ZIF-67 / MCE temperature, and a satisfactory SERS signal intensity can be detected when the temperature of AuRD@ZIF-67 / MCE is 25℃.

[0079] Example 6: Adsorption of chlorfenapyr on AuRD@ZIF-67 / MCE sample

[0080] The instantaneous detection sensor AuRD@ZIF-67 / MCE prepared in Example 1 was placed on a surface-enhanced Raman spectrometer, and the parameters were adjusted to 785 nm laser and 135 mW intensity. A 1 ppm chlorfenapyr standard sample was dropped onto the AuRD@ZIF-67 / MCE surface, and the integration times were adjusted to 20 s, 40 s, 60 s, 80 s, and 100 s. The SERS signal intensity (at 1090 cm⁻¹) at different integration times was compared. -1 (Location).

[0081] The results are as follows Figure 6As shown. By Figure 6 It can be seen that AuRD@ZIF-67 / MCE achieved ultrafast adsorption of chlorfenapyr samples within 60s and reached adsorption saturation within 100s.

[0082] Example 7: Correlation coefficient and limit of detection of AuRD@ZIF-67 / MCE

[0083] The instantaneous detection sensor AuRD@ZIF-67 / MCE prepared in Example 1 was placed on a surface-enhanced Raman spectrometer, and the parameters were adjusted to 785 nm laser, 135 mW intensity, and 20-second integration time. Purchasable chlorfenapyr standard samples were dissolved in water, human serum, and human urine to obtain aqueous solutions, serum solutions, and urine solutions of different concentrations of chlorfenapyr standard sample. These solutions were then dropped onto the surface of AuRD@ZIF-67 / MCE, and the SERS signal intensity (at 1090 cm⁻¹) under different conditions was compared. -1 (Location).

[0084] The results are as follows Figure 7 As shown in the figure, Figures A and D represent aqueous solutions of chlorfenapyr standard samples at different concentrations at 1090 cm⁻¹. -1 The SERS signal intensity at 1090 cm⁻¹ is shown in Figures B and E, which represent the serum solutions of chlorfenapyr standard samples at different concentrations. -1 The SERS signal intensity at 1090 cm⁻¹ is shown in Figures C and F, which represent the SERS signal intensity of urine solutions containing different concentrations of chlorfenapyr standard samples at 1090 cm⁻¹. -1 The SERS signal strength at the location.

[0085] Depend on Figure 7 It can be seen that the sample is at 1090cm -1 The SERS signal intensity at a certain location was positively correlated with the logarithmic concentration of chlorfenapyr in the sample; through... Figure 7 The recovery rate of chlorfenapyr in the sample was calculated using the data in the table below (the recovery rate reflects the degree of recovery of the analyte during the sample detection process; the higher the recovery rate, the higher the analytical accuracy). The results are shown in Table 1 below:

[0086] Table 1. Recovery rate of chlorfenapyr in samples

[0087]

[0088] Depend on Figure 7Based on the data in Table 1 and in conjunction with the U.S. Environmental Protection Agency's Limit of Detection (MDL) program (EPA, 2016), the EPA method ("Definition and Procedure for the Determination of the Method Detection Limit", Revision 2) determined the lowest limits of detection for chlorfenapyr in aqueous standard samples and biological samples (chlorfenapyr standard serum solution and urine solution) to be 1.32 ppb and 1.81 ppb, respectively, which meet the requirements for real-time monitoring.

[0089] Example 8: Thermal stability test of AuRD@ZIF-67 / MCE

[0090] The instantaneous detection sensor AuRD@ZIF-67 / MCE prepared in Example 1 was placed on a surface-enhanced Raman spectrometer, with parameters adjusted to 135 mW intensity and 20-second integration time. A 1 ppm abamectin standard sample was dropped onto the AuRD@ZIF-67 / MCE surface, and the same point on the AuRD@ZIF-67 / MCE surface was continuously irradiated 30 times with a 785 nm laser intensity. The sample was detected at 1090 cm⁻¹. -1 The thermal stability of AuRD@ZIF-67 / MCE was evaluated by measuring the SERS signal intensity at the location.

[0091] The results are as follows Figure 8 As shown, figures A, B, and C are the waterfall plot, thermal image, and histogram, respectively, after 30 consecutive irradiations of the same point on the AuRD@ZIF-67 / MCE surface with a 785nm laser. Figure 8 The RSD (relative standard deviation) is 8.28%, which indicates that the instantaneous detection sensor AuRD@ZIF-67 / MCE prepared in this invention has excellent thermal stability.

[0092] Example 9: Spatial Uniformity Detection of AuRD@ZIF-67 / MCE

[0093] The instantaneous detection sensor AuRD@ZIF-67 / MCE prepared in Example 1 was placed on a surface-enhanced Raman spectrometer, with parameters adjusted to 135 mW intensity and 20-second integration time. A 1 ppm chlorfenapyr standard sample was dropped onto the AuRD@ZIF-67 / MCE surface, and 30 random points on the AuRD@ZIF-67 / MCE surface were irradiated with a 785 nm laser. The sample was detected at 1090 cm⁻¹. -1 The point-to-point spatial uniformity of AuRD@ZIF-67 / MCE was evaluated by measuring the SERS signal intensity at the location.

[0094] The results are as follows Figure 9As shown, figures A, B, and C are the waterfall plot, thermal image, and histogram, respectively, of the AuRD@ZIF-67 / MCE surface after being irradiated with a 785nm laser at 30 random points. Figure 9 The RSD (relative standard deviation) is 6.99%, which indicates that the spatial uniformity of the instantaneous detection sensor AuRD@ZIF-67 / MCE prepared in this invention is excellent.

[0095] Example 10: Precision detection of AuRD@ZIF-67 / MCE

[0096] The standard sample of chlorpyrifos with unknown concentration was pretreated according to the description in the literature "Bioaccumulation of Chloropyrifos Organo-pesticide and Its Toxicogenic Association with Antioxidant GSTP1 in Pakistani Pest Control Workers". The concentration was then detected by surface-enhanced Raman spectroscopy and liquid chromatography-ultraviolet fluorescence (HPLC-UV) respectively, and the detected concentration results were compared. The sensor used in the surface-enhanced Raman spectroscopy was the AuRD@ZIF-67 / MCE instantaneous detection sensor prepared in Example 1.

[0097] The results are as follows Figure 10 As shown, Figure A compares the linear correlation of quantitative data obtained by surface-enhanced Raman spectroscopy and HPLC-UV spectroscopy, while Figure B compares the detection results of different concentrations of chlorfenapyr using surface-enhanced Raman spectroscopy and HPLC-UV. Figure 10 It can be seen that the detection results of surface-enhanced Raman spectroscopy and HPLC-UV for the concentration of chlorfenapyr are basically consistent, which indicates that the instantaneous detection sensor AuRD@ZIF-67 / MCE prepared in this invention has good precision.

[0098] In summary, the AuRD@ZIF-67 / MCE instantaneous detection sensor provided by this invention can perform rapid and sensitive instantaneous detection of chlorfenapyr. Moreover, this instantaneous detection sensor has excellent thermal stability, spatial uniformity and precision. These properties provide strong evidence for the rapid qualitative and quantitative analysis of chlorfenapyr in biological fluids.

Claims

1. A real-time detection sensor, characterized in that, The method involves loading AuRD onto ZIF-67 and then co-modifying the MCE film; the fabrication method of the real-time detection sensor includes the following steps: (1) Preparation of AuRD solution; (2) Prepare ZIF-67 solution using ethanol as solvent; (3) After mixing AuRD solution and ZIF-67 solution, centrifuge and allow to stand to separate into layers to obtain upper clear liquid and lower mixed liquid; add the lower mixed liquid evenly to the MCE membrane and let stand to obtain the product; The instant detection sensor is used for detecting acaricide poisoning.

2. The real-time detection sensor as described in claim 1, characterized in that, The mass ratio between AuRD and ZIF-67 is 100:(1~1.5).

3. The real-time detection sensor as described in claim 1, characterized in that, The instantaneous detection sensor is based on surface-enhanced Raman spectroscopy.

4. The real-time detection sensor as described in claim 1, characterized in that, The preparation method of the AuRD solution in step (1) includes the following steps: i. Prepare a mixed solution containing HAuCl4 and CTAC; prepare a NaBH4 solution and pre-cool it; Under stirring conditions, NaBH4 solution is added to the mixed solution to obtain a brown seed crystal solution. The seed crystal solution is then aged at 30-35°C for 1-1.2 hours to obtain a gold seed solution. ii. Prepare two containers, add CTAC and water to each container, and place them in a water bath at 30~35℃; while in the water bath, continue to add HAuCl4 solution, NaBr solution and ascorbic acid solution to each container; iii. Label the two containers from step ii as A and B respectively; under shaking conditions, add the gold seed solution from step i to container A to obtain a light pink solution; Transfer the light pink solution to container B, mix thoroughly, let stand for 15-20 minutes, centrifuge at 6000-8000 rpm for 5-10 minutes to obtain AuRD precipitate; dissolve the precipitate in water to obtain AuRD solution.

5. The instantaneous detection sensor as described in claim 1, characterized in that, The preparation method of the ZIF-67 solution in step (2) includes the following steps: S1. Prepare cobalt nitrate hexahydrate solution and 2-methylimidazole solution respectively; S2. Mix the cobalt nitrate hexahydrate solution and 2-methylimidazole solution from step S1, and stir at 1000-1500 rpm for 6-6.5 h at room temperature; Centrifuge at 6000~8000 rpm for 10~15 min to obtain ZIF-67 precipitate; wash and dissolve the precipitate with ethanol to obtain ZIF-67 solution.

6. The real-time detection sensor as described in claim 1, characterized in that, Step (3) includes the following steps: mix the AuRD solution from step (1) and the ZIF-67 solution from step (2), centrifuge at 6000~8000 rpm for 10~15 min, let stand to separate into layers, and obtain the upper clear liquid and the lower mixed liquid; uniformly drop the lower mixed liquid onto the MCE membrane, let stand for 20~30 min, and obtain the instantaneous detection sensor.

7. The real-time detection sensor as described in claim 1, characterized in that, The instant detection sensor is used for the detection of chlorfenapyr poisoning. The application method includes the following steps: placing the instant detection sensor on a surface-enhanced Raman spectrometer, dropping a chlorfenapyr sample onto the surface of the instant detection sensor, and detecting the chlorfenapyr poisoning status by the signal intensity of the Raman spectrum.

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