Magnetic enrichment and colloidal gold immunoassay rapid detection method for new psychoactive substances in sewage

Abstract

The invention relates to the technical field of environmental monitoring and analytical chemistry, in particular to a magnetic enrichment and colloidal gold immunoassay rapid detection method for new psychoactive substances in sewage. The method comprises the following steps: adding functionalized magnetic nanoparticles of which the surfaces are modified with specific antibodies into a colloidal gold immunochromatographic test strip, incubating to form a magnetic compound, applying a magnetic field for separation, washing with a phosphate buffer solution containing Tween-20, adding a glycine-hydrochloric acid dissociation solution for dissociation and neutralization, and dropwise adding the dissociation solution into the colloidal gold immunochromatographic test strip for detection, and qualitative and quantitative results are read through vision or an instrument. The method has the advantages of high enrichment efficiency, strong specificity, simplicity and rapidness in operation, consideration of qualitative and quantitative requirements and low cost, and is suitable for accurate detection in a laboratory and rapid on-site screening.

Description

Technical Field

[0001] This invention relates to the field of environmental monitoring and analytical chemistry, specifically to a method for the rapid detection of new psychoactive substances in wastewater using magnetic enrichment and colloidal gold immunoassay. Background Technology

[0002] Currently, the detection of new psychoactive substances in wastewater mainly relies on chromatography-mass spectrometry (GC-MS), including high-performance liquid chromatography-mass spectrometry (HPLC-MS) and gas chromatography-mass spectrometry (GC-MS). While these techniques offer high detection accuracy and qualitative capabilities, they also have significant limitations: Firstly, the equipment purchase and maintenance costs are high, and the operation procedures are complex, requiring specialized technicians for sample pretreatment and instrument operation, which cannot meet the needs of rapid on-site detection or batch sample screening. Secondly, the sample pretreatment process requires methods such as solid-phase extraction and liquid-liquid extraction, which are cumbersome and time-consuming, typically requiring several hours or even days to complete the processing and detection of a batch of samples, making it difficult to achieve real-time monitoring and rapid response of target substances in wastewater.

[0003] Besides chromatography-mass spectrometry, some studies employ conventional immunoassay methods, such as enzyme-linked immunosorbent assay (ELISA). While these methods are relatively simple to operate, they are limited by the complexity of wastewater matrices and lack efficient sample enrichment and purification steps, resulting in insufficient detection sensitivity and an inability to accurately capture trace amounts of new psychoactive substances. Furthermore, non-specific adsorption is a significant problem; interfering substances easily bind to antibodies, leading to false positives or false negatives and compromising detection accuracy.

[0004] In the sample enrichment stage, traditional methods such as solid-phase extraction (SPE) column enrichment suffer from limited adsorption capacity, unstable elution efficiency, and susceptibility to matrix interference. Separation methods such as centrifugation and filtration either fail to effectively separate trace target substances or are time-consuming and yield poor separation results, making them unsuitable for subsequent rapid detection processes. Furthermore, existing detection technologies often struggle to balance enrichment efficiency, detection speed, and cost control, failing to meet the practical needs of environmental protection departments and water quality monitoring agencies for efficient, rapid, and low-cost monitoring of new psychoactive substances in wastewater. Therefore, developing a detection method that combines high enrichment efficiency, strong anti-interference capabilities, simplicity, speed, and cost control has become an urgent need in the field of environmental monitoring. Summary of the Invention

[0005] The purpose of this invention is to provide a method for the magnetic enrichment and rapid detection of new psychoactive substances in wastewater using colloidal gold immunoassay, in order to solve the problems mentioned in the background art.

[0006] To achieve the above objectives, the present invention provides the following technical solution: A rapid detection method for magnetic enrichment and colloidal gold immunoassay of new psychoactive substances in wastewater includes the following steps: S1. Sample pretreatment: Take a wastewater sample and perform filtration and pH adjustment in sequence to remove suspended particles and interfering substances, and obtain the water sample to be tested. S2. Magnetic enrichment: Functionalized magnetic nanoparticles with surface-modified antibodies against novel psychoactive substances are added to the water sample to be tested. The mixture is shaken and incubated at 15-40℃ for 5-30 minutes to allow the novel psychoactive substances to specifically bind with the functionalized magnetic nanoparticles and form a magnetic complex. S3. Magnetic separation and washing: Apply an external magnetic field to adsorb and collect the magnetic complex, and discard the supernatant; use phosphate buffer containing 0.05-0.5% Tween-20 as a washing buffer to wash the collected magnetic complex to remove non-specific adsorbed impurities. S4. Dissociation and Detection: Add glycine-hydrochloric acid dissociation solution with pH 2.0-3.0 to the washed magnetic complex, shake to mix, and dissociate the new psychoactive substance from the magnetic complex; after neutralization, add the dissociation solution to the sample pad of the colloidal gold immunochromatographic test strip for detection; S5. Result Reading and Data Processing: The color development signals of the detection line and control line of the colloidal gold immunochromatographic test strip are read by visual observation or instruments; when performing quantitative analysis, the optical density values ​​of the detection line and control line are obtained by instruments, the optical density ratio of the detection line to the control line is calculated, and this ratio is compared with the standard curve pre-established using new psychoactive substance standards to determine the content of new psychoactive substances in wastewater.

[0007] As a preferred embodiment, the sample pretreatment in step S1 specifically includes: The collected wastewater samples were filtered under negative pressure through a glass fiber membrane or a mixed cellulose ester membrane with a pore size of 0.45 μm to obtain a primary filtrate with suspended particles removed. The primary filtrate was filtered a second time through a polyethersulfone membrane with a pore size of 0.22 μm to obtain a clear filtrate. Add 0.1 mol / L phosphate buffer to the clarified filtrate to adjust the pH to the range of 6.5-7.5 to obtain a water sample with stable physicochemical parameters.

[0008] As a preferred embodiment, the magnetic enrichment in step S2 specifically includes: Fe3O4 magnetic nanoparticles with a particle size of 50-100 nm were synthesized by coprecipitation method, and amino functional groups were modified on their surface by silanizing reagent to obtain aminated magnetic nanoparticles. New psychoactive substance-specific antibodies were mixed with aminated magnetic nanoparticles in a phosphate buffer solution at pH 7.4. The antibodies were then covalently immobilized on the surface of the magnetic nanoparticles by glutaraldehyde crosslinking, resulting in functionalized magnetic nanoparticles with surface-modified new psychoactive substance-specific antibodies. Functionalized magnetic nanoparticles were added to the water sample to be tested at a final concentration of 0.5-2 mg / mL and incubated at 25-37℃ with a shaking speed of 150-200 rpm for 15-25 minutes to allow the new psychoactive substances in the wastewater to fully bind with the specific antibodies on the surface of the functionalized magnetic nanoparticles, forming a ternary complex of new psychoactive substances-antibody-magnetic nanoparticles. During incubation, the ionic strength of the reaction system was maintained in the range of 0.01-0.1M, and non-specific adsorption was reduced by adding 0.5% bovine serum albumin as a blocking agent.

[0009] As a preferred embodiment, the magnetic separation and washing in step S3 specifically includes: The incubated reaction system is placed in an external magnetic field with an intensity of 0.5-1.5T and left to stand for 1-3 minutes to allow the magnetic complex to be completely adsorbed onto the container wall, forming a magnetic complex aggregate layer. Slowly decant or remove the supernatant, retaining the magnetic complex aggregate layer; Add washing buffer, which is 1 / 5 to 1 / 3 of the initial water sample volume, to the magnetic complex aggregate layer. The washing buffer is a pH 7.4 phosphate buffer containing 0.05-0.5% Tween-20. Gently shake to resuspend the magnetic complex aggregate layer and form a uniform suspension. The resuspended suspension was placed in an external magnetic field again and allowed to stand for 1-2 minutes to adsorb and collect the magnetic complex. The washing supernatant was then discarded. Repeat the washing steps above 2-3 times until the non-specific adsorbed impurities on the surface of the magnetic complex are fully removed, and a pure magnetic complex is obtained.

[0010] As a preferred embodiment, the dissociation and detection in step S4 specifically includes: Add 100-500 μL of glycine-hydrochloric acid dissociation solution with pH 2.0-3.0 to the washed magnetic complex, and mix by shaking at 100-200 rpm for 5-15 minutes at 25-37℃ to dissociate the new psychoactive substance from the magnetic complex, and obtain a dissociated mixture. The dissociated mixture was placed in an external magnetic field with an intensity of 0.5-1.5T and allowed to stand for 1-2 minutes to adsorb magnetic nanoparticles from the magnetic complex. The supernatant was collected as the dissociation solution. Add an equal volume of pH 7.4 phosphate buffer to the dissociation solution to neutralize it, and obtain the neutralized dissociation solution; Add 50-100 μL of the neutralized and dissociated solution to the sample pad of the colloidal gold immunochromatographic test strip, react at 15-30℃ for 10-15 minutes, and then detect using the colloidal gold immunochromatographic test strip.

[0011] As a preferred approach, the result reading and data processing in step S5 specifically includes: The color signals of the test line and control line of the colloidal gold immunochromatographic test strip are observed visually. When the color intensity of the test line is lower than that of the control line, it is judged as a positive result, indicating the presence of new psychoactive substances in the wastewater. When the color intensity of the test line is equal to or higher than that of the control line, it is judged as a negative result, indicating that there are no new psychoactive substances in the wastewater. When performing quantitative analysis, the optical density values ​​of the detection line and control line of the colloidal gold immunochromatographic test strip are read by an optical density scanner to obtain the optical density values ​​of the detection line and control line. Calculate the ratio of the optical density of the detection line to that of the control line based on the optical density values ​​of the detection line and the control line. The optical density ratio was compared with a pre-established standard curve using new psychoactive substance standards. The standard curve was plotted using the optical density ratio of different concentrations of new psychoactive substance standards as the response value and the corresponding concentration as the reference value. Based on the comparison results of the standard curve, the content of new psychoactive substances in wastewater is determined by interpolation, and quantitative detection results are output.

[0012] As can be seen from the technical solution provided by the present invention above, the magnetic enrichment and colloidal gold immunoassay method for rapid detection of new psychoactive substances in wastewater provided by the present invention has the following beneficial effects: This invention synthesizes Fe3O4 magnetic nanoparticles with a particle size of 50 to 100 nm via co-precipitation. After modifying the amino functional groups with a silanizing agent, a novel psychoactive substance-specific antibody is covalently immobilized on the particle surface using a glutaraldehyde cross-linking method to form functionalized magnetic nanoparticles. These particles can bind to novel psychoactive substances in wastewater with high specificity, achieving efficient capture even at extremely low concentrations, significantly increasing the enrichment factor of the target substance, effectively solving the problem of insufficient sensitivity of traditional detection methods for low concentrations of novel psychoactive substances, and meeting the detection needs of trace target substances in wastewater. In the magnetic separation stage, by applying an external magnetic field of 0.5 to 1.5T, the magnetic complex can be rapidly adsorbed onto the container wall within 1 to 3 minutes. Compared with traditional methods such as centrifugation, the separation speed is faster and the operation is simpler. The washing process uses a pH 7.4 phosphate buffer containing 0.05 to 0.5% Tween-20 and is repeated 2 to 3 times. This can fully wash away impurities that are not specifically adsorbed on the surface of the magnetic complex, minimize interference factors in subsequent detection steps, ensure the purity of the magnetic complex entering the dissociation and detection stage, and provide a key guarantee for the accuracy of the detection results. The dissociation process involves using a glycine-hydrochloric acid dissociation solution with a pH of 2.0 to 3.0 to break down the binding between the target substance and the antibody. After neutralization, the dissociation solution is added dropwise to the colloidal gold immunochromatographic test strip. The test strip reaction can be completed in just 10 to 15 minutes. This detection method does not rely on complex large-scale instruments such as high-performance liquid chromatography and mass spectrometry. The operation procedure is simple and easy to understand. It is suitable for accurate detection in the laboratory and can also be used for rapid on-site screening. It reduces the requirements for the detection environment and the professional level of the operators, and greatly improves the adaptability and practicality of the method. This invention supports two result analysis modes: by visually observing the color intensity of the detection line and control line of the colloidal gold test strip, the presence of new psychoactive substances in wastewater can be quickly determined, meeting the qualitative needs of preliminary screening; if it is necessary to accurately determine the content of the target substance, the optical density values ​​of the detection line and control line can be obtained through an optical density scanner, the ratio can be calculated, and then combined with a pre-established standard curve, and quantitative analysis can be completed using interpolation; the two modes can be flexibly switched to adapt to the needs of different detection scenarios, enhancing the practical value of the method; The sample pretreatment stage involves two filtrations (primary filtration with a 0.45μm filter membrane and secondary filtration with a 0.22μm filter membrane) and pH adjustment (from 6.5 to 7.5) to ensure the stability of the physicochemical parameters of the water sample to be tested, providing a good reaction basis for subsequent enrichment, separation, and detection steps. The reagents used in the entire process, such as Fe3O4 magnetic nanoparticles and colloidal gold test strips, have relatively low preparation or acquisition costs, and the steps are sequential and time-saving. Compared with traditional chromatography-mass spectrometry detection methods, it significantly reduces detection costs and time costs, making it more suitable for large-scale application in the routine monitoring of new psychoactive substances in wastewater. Attached Figure Description

[0013] Figure 1 This is a schematic diagram of the steps of a method for rapid detection of new psychoactive substances in wastewater using magnetic enrichment and colloidal gold immunoassay, according to the present invention. Detailed Implementation

[0014] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0015] To better understand the above technical solutions, the following will provide a detailed explanation of the technical solutions in conjunction with the accompanying drawings and specific embodiments.

[0016] like Figure 1 As shown, this embodiment of the invention provides a method for rapid detection of new psychoactive substances in wastewater using magnetic enrichment and colloidal gold immunoassay, comprising the following steps: S1. Sample pretreatment: Take a wastewater sample and perform filtration and pH adjustment in sequence to remove suspended particles and interfering substances, and obtain the water sample to be tested. S2. Magnetic enrichment: Functionalized magnetic nanoparticles with surface-modified antibodies against novel psychoactive substances are added to the water sample to be tested. The mixture is shaken and incubated at 15-40℃ for 5-30 minutes to allow the novel psychoactive substances to specifically bind with the functionalized magnetic nanoparticles and form a magnetic complex. S3. Magnetic separation and washing: Apply an external magnetic field to adsorb and collect the magnetic complex, and discard the supernatant; use phosphate buffer containing 0.05-0.5% Tween-20 as a washing buffer to wash the collected magnetic complex to remove non-specific adsorbed impurities. S4. Dissociation and Detection: Add glycine-hydrochloric acid dissociation solution with pH 2.0-3.0 to the washed magnetic complex, shake to mix, and dissociate the new psychoactive substance from the magnetic complex; after neutralization, add the dissociation solution to the sample pad of the colloidal gold immunochromatographic test strip for detection; S5. Result Reading and Data Processing: The color development signals of the detection line and control line of the colloidal gold immunochromatographic test strip are read by visual observation or instruments; when performing quantitative analysis, the optical density values ​​of the detection line and control line are obtained by instruments, the optical density ratio of the detection line to the control line is calculated, and this ratio is compared with the standard curve pre-established using new psychoactive substance standards to determine the content of new psychoactive substances in wastewater.

[0017] As a preferred embodiment, the sample pretreatment in step S1 specifically includes: The collected wastewater samples were filtered under negative pressure through a glass fiber membrane or a mixed cellulose ester membrane with a pore size of 0.45 μm to obtain a primary filtrate with suspended particles removed. The primary filtrate was filtered a second time through a polyethersulfone membrane with a pore size of 0.22 μm to obtain a clear filtrate. Add 0.1 mol / L phosphate buffer to the clarified filtrate to adjust the pH to the range of 6.5-7.5 to obtain a water sample with stable physicochemical parameters.

[0018] In this embodiment, step S1, sample pretreatment, serves to prepare a stable physicochemical sample based on the collected wastewater sample by sequentially filtering to remove suspended particles and adjusting the pH value to eliminate interfering substances. This avoids interference from impurities in subsequent detection and provides a clean sample system that meets the reaction conditions for magnetic enrichment in step S2. The detailed steps are as follows: Step S1-1: Primary filtration and removal of suspended particles: The sample pretreatment process is initiated, and the collected wastewater sample is selected as the treatment target. A glass fiber filter membrane or a mixed cellulose ester filter membrane with a pore size of 0.45μm is used to perform primary filtration of the wastewater sample through a negative pressure filtration device. During the filtration process, the negative pressure is maintained to ensure a uniform flow rate of the filtrate. The filter membrane traps suspended particles and large impurities in the wastewater, and the liquid obtained after filtration is collected as the primary filtrate. After filtration, the system automatically checks the integrity of the filter membrane. If the filter membrane is damaged, primary filtration is performed again to ensure that there are no obvious suspended particles remaining in the primary filtrate. Step S1-2: Secondary filtration and filtrate acquisition: The primary filtrate is introduced into a secondary filtration system and finely filtered using a polyethersulfone membrane with a pore size of 0.22 μm. The secondary filtration aims to remove residual microparticles and some colloidal impurities from the primary filtrate. By controlling the rate at which the filtrate passes through the membrane, microparticles are effectively trapped by the membrane. The resulting transparent liquid is collected as the clarified filtrate. After filtration, the clarified filtrate is visually inspected to ensure it is free of turbidity and visible impurities. If it does not meet the requirements, the polyethersulfone membrane is replaced and filtration is repeated to ensure the purity of the clarified filtrate meets the requirements for subsequent testing. Step S1-3: pH adjustment and preparation of water samples for testing: Slowly add 0.1 mol / L phosphate buffer solution to the clarified filtrate while stirring to ensure thorough mixing. Monitor the pH of the mixture in real time using a pH meter and continuously adjust until the pH stabilizes within the range of 6.5 to 7.5. After adjustment, allow the mixture to stand for 5 minutes and check the pH again to confirm its stability and ensure that the physicochemical parameters of the mixture remain stable. The resulting liquid is the water sample to be tested. The system automatically records the volume, pH value, and other key parameters of the water sample to provide a stable reaction system for subsequent magnetic enrichment steps.

[0019] In this embodiment, step S2: magnetic enrichment serves to synthesize and modify functionalized magnetic nanoparticles containing novel psychoactive substance-specific antibodies into the water sample prepared in step S1. These nanoparticles bind specifically to the target substance in the water sample, achieving efficient enrichment of the target substance and forming a magnetic complex that is easy to separate. This provides the core substance after enrichment for magnetic separation and washing in step S3. The detailed steps are as follows: Step S2-1: Synthesis of Fe3O4 magnetic nanoparticles: Fe3O4 magnetic nanoparticles were synthesized using a coprecipitation method. A mixed solution of iron salts was prepared according to a predetermined reaction ratio. Under inert gas protection, the mixed solution was heated to the reaction temperature, and an alkaline solution was slowly added dropwise with continuous stirring to allow the Fe3O4 nanoparticles to react. 3+ with Fe 2+ Fe3O4 nanoparticles are generated through a coprecipitation reaction. After the reaction is completed, the precipitate is collected by centrifugation, washed and dried, and then the particle size of the synthesized Fe3O4 magnetic nanoparticles is analyzed by a particle size analyzer to ensure that the particle size is within the range of 50 to 100 nm. If the particle size does not meet the requirements, the reaction parameters are adjusted and the synthesis is repeated. Step S2-2: Preparation of aminated magnetic nanoparticles: The synthesized Fe3O4 magnetic nanoparticles were dispersed in an organic solvent, a silanizing agent was added, and the mixture was placed in a constant-temperature stirring device and reacted at a preset temperature for a certain time to allow the silanizing agent to modify the surface of the Fe3O4 magnetic nanoparticles, introducing amino functional groups. After the reaction was completed, unreacted silanizing agent and impurities were removed by centrifugation to obtain aminated magnetic nanoparticles. The aminated magnetic nanoparticles were characterized by infrared spectroscopy to confirm the successful modification of the amino functional groups, and then dispersed in a buffer solution for later use. Step S2-3: Preparation of functionalized magnetic nanoparticles: A dispersion of aminated magnetic nanoparticles was prepared, and a novel psychoactive substance-specific antibody was added. The mixture was adjusted to a pH 7.4 phosphate buffer environment, and then glutaraldehyde was added as a cross-linking agent. The reaction was carried out under constant temperature and stirring conditions, allowing glutaraldehyde to mediate the cross-linking reaction between the amino groups on the surface of the aminated magnetic nanoparticles and the amino groups in the antibody molecules, thus covalently immobilizing the antibody on the surface of the magnetic nanoparticles. After the reaction, unbound antibody and free glutaraldehyde were removed by centrifugation and washing to obtain functionalized magnetic nanoparticles with a novel psychoactive substance-specific antibody on their surface. The antibody immobilization efficiency was determined using a protein quantification method to ensure that the functionalized magnetic nanoparticles possessed sufficient specific binding capacity. Step S2-4: Specific binding and magnetic complex formation: Functionalized magnetic nanoparticles were added to the water sample at a final concentration of 0.5 to 2 mg / mL. The mixture was placed in a constant-temperature shaking device at a temperature of 25 to 37°C and a rotation speed of 150 to 200 rpm for 15 to 25 minutes. During incubation, the ionic strength of the reaction system was maintained within the range of 0.01 to 0.1 M, and 0.5% bovine serum albumin was added as a blocking agent to reduce non-specific adsorption. Under these conditions, the new psychoactive substances in the wastewater specifically bound to the specific antibodies on the surface of the functionalized magnetic nanoparticles, forming a ternary complex of new psychoactive substances-antibody-magnetic nanoparticles. After incubation, the mixture was preliminarily observed to confirm that there was no obvious precipitation or aggregation, ensuring that the magnetic complex was uniformly dispersed.

[0020] In this embodiment, step S3, magnetic separation and washing, serves to rapidly separate the magnetic complex from the water sample by applying an external magnetic field, based on the magnetic complex formed in step S2. Then, multiple washes with a specific washing buffer remove non-specific adsorbed impurities from the surface of the complex, yielding a pure magnetic complex. This eliminates the interference of impurities on subsequent dissociation and detection, providing a high-purity reaction substrate for step S4 dissociation and detection. The detailed steps are as follows: Step S3-1: Adsorption and collection of magnetic complexes: The incubated reaction system was transferred to a dedicated magnetic separation container, and the container was placed in an external magnetic field with an intensity of 0.5 to 1.5 T for 1 to 3 minutes. Under the action of the magnetic field, the magnetic complex was attracted by magnetic force, gradually aggregated and adsorbed to the container wall, forming a magnetic complex aggregate layer. During the standing process, the container was kept stable to avoid external forces interfering with the aggregation of the magnetic complex. After the magnetic complex was completely adsorbed, the aggregate layer was confirmed to be stable and without obvious dispersion. Step S3-2: Supernatant removal: Use a slow decantation or aspiration device to remove the supernatant from the magnetic separation container. During the operation, keep the container in a magnetic field environment to avoid disturbing and dispersing the magnetic complex aggregate layer. After the supernatant is removed, carefully observe the magnetic complex aggregate layer on the container wall to ensure that there is no obvious loss. If the aggregate layer is found to have detached, stop the operation in time and re-perform magnetic adsorption, and then remove the supernatant again. Step S3-3: Washing of the magnetic complex: Prepare a washing buffer solution, which is a pH 7.4 phosphate buffer containing 0.05 to 0.5% Tween-20. Add the washing buffer solution to the magnetic complex aggregate layer, with a volume of 1 / 5 to 1 / 3 of the initial water sample volume. Remove the container from the magnetic field and gently agitate to fully resuspend the magnetic complex aggregate layer, forming a homogeneous suspension, ensuring that the magnetic complex is in full contact with the washing buffer solution. Then, place the container back into the external magnetic field and let it stand for 1 to 2 minutes to allow the magnetic complex to re-adsorb onto the container wall. After that, remove the washing supernatant to complete one washing cycle. Steps S3-4: Washing and repeating to obtain pure magnetic complexes: Repeat the washing steps above 2 to 3 times, strictly following the procedure of adding washing buffer, resuspension, magnetic adsorption, and supernatant removal each time. After each wash, the removal of non-specific adsorbed impurities on the surface of the magnetic complex is determined by detecting the impurity content in the washing supernatant. When the impurity content in the washing supernatant is lower than the preset threshold, the washing is stopped. At this point, the magnetic complex on the container wall is a pure magnetic complex, which can be used for subsequent dissociation steps.

[0021] In this embodiment, step S4, dissociation and detection, is based on the pure magnetic complex obtained in step S3. An acidic dissociation solution disrupts the binding between the target substance and the antibody, causing the target substance to dissociate. After neutralization, the specific reaction of the colloidal gold immunochromatographic test strip is used to detect the target substance, obtaining a direct colorimetric signal. This provides direct detection evidence for result reading and data processing in step S5. The detailed steps are as follows: Step S4-1: Addition of dissociation solution and shaking mixing: Add 100 to 500 μL of glycine-hydrochloric acid dissociation solution (pH 2.0 to 3.0) to the washed, purified magnetic complex. Place the container in a constant-temperature shaker at 25 to 37°C and 100 to 200 rpm for 5 to 15 minutes. Under the acidic dissociation solution and shaking, the specific binding force between the new psychoactive substance and the antibody is disrupted, and the substance gradually dissociates from the magnetic complex, forming a dissociation mixture. During shaking, ensure uniform mixing to allow the dissociation reaction to proceed fully. Step S4-2: Separation of magnetic nanoparticles and collection of dissociation solution: Place the container containing the dissociation mixture in an external magnetic field with an intensity of 0.5 to 1.5 T and let it stand for 1 to 2 minutes to allow the magnetic nanoparticles in the dissociation mixture to be magnetically attracted, aggregate, and adsorbed onto the container wall. After the magnetic nanoparticles are completely adsorbed, use a liquid suction device to collect the supernatant in the container. This supernatant is the dissociation liquid. During the collection process, avoid the adsorbed magnetic nanoparticles being sucked in to ensure that there are no magnetic nanoparticles remaining in the dissociation liquid. If the dissociation liquid becomes turbid, perform magnetic separation again and collect the supernatant. Step S4-3: Neutralization with the dissociation solution: Prepare a phosphate buffer solution with a pH of 7.4. Add an equal volume of this phosphate buffer solution to the collected dissociation solution while gently stirring to ensure thorough mixing and neutralization. During neutralization, monitor the pH of the mixture in real time using a pH meter to ensure that the pH of the neutralized mixture remains stable within the range of 7.0 to 7.5, providing a suitable reaction environment for subsequent colloidal gold immunochromatographic assays. After neutralization, obtain the neutralized dissociation solution for testing. Step S4-4: Colloidal gold immunochromatographic assay: Take 50 to 100 μL of the neutralized and dissociated solution and accurately add it to the sample pad of the colloidal gold immunochromatographic test strip. Place the test strip in an environment of 15 to 30°C and react for 10 to 15 minutes. During this process, the new psychoactive substances in the neutralized and dissociated solution will specifically bind to the probe of the detection line on the test strip, and the colloidal gold particles will aggregate, causing the detection line to show the corresponding color signal. During the reaction, keep the environment dry and free from airflow interference to ensure that the reaction is complete and stable. After the reaction is complete, remove the test strip and prepare to read the results.

[0022] In this embodiment, step S5: result reading and data processing, based on the colorimetric signal of the colloidal gold immunochromatographic test strip in step S4, achieves qualitative judgment of the target substance through visual observation, or achieves quantitative analysis by reading the optical density value and calculating it in combination with the standard curve, ultimately determining the presence and specific content of new psychoactive substances in wastewater, completing the entire detection process and outputting the results; the detailed steps are as follows: Step S5-1: Visual reading of color development signal: After the reaction, the colloidal gold immunochromatographic test strip was placed under bright, uniform light. The color signals of the test line and control line on the test strip were observed visually. The color intensity of the test line and control line was compared. When the color intensity of the test line was lower than that of the control line, it was considered a positive result, indicating the presence of new psychoactive substances in the wastewater. When the color intensity of the test line was equal to or higher than that of the control line, it was considered a negative result, indicating the absence of new psychoactive substances in the wastewater. During the visual reading process, the color intensity comparison was recorded to ensure the accuracy of the judgment results. Step S5-2: Instrument detection of optical density value: When quantitative analysis is required, place the colloidal gold immunochromatographic test strip into the optical density scanner and start the instrument to detect the optical density value. The instrument illuminates the test line and control line of the test strip with light of a specific wavelength, receives the reflected light signal and converts it into an optical density value, and obtains the optical density values ​​of the test line and control line respectively. During the detection process, ensure that the test strip is placed in an accurate position and that the instrument parameters are set consistently to avoid inaccurate optical density detection results due to instrument errors. Step S5-3: Calculation of optical density ratio: Based on the optical density values ​​of the detection line and control line obtained from the detection, the ratio of optical density of the detection line to that of the control line is calculated according to the following formula: R = Dt / Dc, where R is the optical density ratio, Dt is the optical density value of the detection line, and Dc is the optical density value of the control line. During the calculation, sufficient significant figures are retained to ensure the accuracy of the ratio result and to provide reliable data for subsequent standard curve comparison. Step S5-4: Standard curve comparison: A standard curve was established in advance using new psychoactive substance standards. The standard curve was plotted with the optical density ratio corresponding to different concentrations of new psychoactive substance standards as the response value and the corresponding standard concentration as the reference value. The calculated optical density ratio was substituted into the standard curve, and the corresponding reference concentration range was found through curve fitting. The position of the optical density ratio in the standard curve was determined, which provides a basis for the calculation of the content of new psychoactive substances. Step S5-5: Content Determination and Result Output: Based on the comparison results of the standard curve, the specific content of new psychoactive substances in wastewater is calculated using interpolation. During the interpolation calculation, based on the optical density ratio corresponding to a known concentration in the standard curve, linear or nonlinear interpolation methods are used to determine the concentration of new psychoactive substances corresponding to the current optical density ratio. After the calculation is completed, quantitative detection results are output, including key information such as the content value of new psychoactive substances, the detection method, and the detection time, providing data support for the monitoring of new psychoactive substances in wastewater. Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for rapid detection of new psychoactive substances in wastewater using magnetic enrichment and colloidal gold immunoassay, characterized in that: Includes the following steps: S1. Sample pretreatment: Take a wastewater sample and perform filtration and pH adjustment in sequence to remove suspended particles and interfering substances, and obtain the water sample to be tested. S2. Magnetic enrichment: Functionalized magnetic nanoparticles with surface-modified antibodies against novel psychoactive substances are added to the water sample to be tested, the mixture is shaken and incubated at 15-40℃ for 5-30 minutes to allow the novel psychoactive substances to specifically bind to the functionalized magnetic nanoparticles and form a magnetic complex. S3. Magnetic separation and washing: Apply an external magnetic field to adsorb and collect the magnetic complex, and discard the supernatant; The collected magnetic complexes were washed with a phosphate buffer containing 0.05-0.5% Tween-20 to remove non-specific adsorbed impurities. S4. Dissociation and Detection: Add glycine-hydrochloric acid dissociation solution with pH 2.0-3.0 to the washed magnetic complex, shake to mix, and dissociate the new psychoactive substance from the magnetic complex; after neutralization, add the dissociation solution to the sample pad of the colloidal gold immunochromatographic test strip for detection; S5. Result Reading and Data Processing: The color development signals of the detection line and control line of the colloidal gold immunochromatographic test strip are read by visual observation or instruments; when performing quantitative analysis, the optical density values ​​of the detection line and control line are obtained by instruments, the optical density ratio of the detection line to the control line is calculated, and the ratio is compared with the standard curve pre-established using new psychoactive substance standards to determine the content of new psychoactive substances in wastewater.

2. The method for rapid detection of new psychoactive substances in wastewater by magnetic enrichment and colloidal gold immunoassay according to claim 1, characterized in that: The sample pretreatment in step S1 specifically includes: The collected wastewater samples were filtered under negative pressure through a glass fiber membrane or a mixed cellulose ester membrane with a pore size of 0.45 μm to obtain a primary filtrate with suspended particles removed. The primary filtrate was filtered a second time through a polyethersulfone membrane with a pore size of 0.22 μm to obtain a clear filtrate. Add 0.1 mol / L phosphate buffer to the clarified filtrate to adjust the pH to the range of 6.5-7.5 to obtain a water sample with stable physicochemical parameters.

3. The method for rapid detection of new psychoactive substances in wastewater by magnetic enrichment and colloidal gold immunoassay according to claim 1, characterized in that: The magnetic enrichment in step S2 specifically includes: Fe3O4 magnetic nanoparticles with a particle size of 50-100 nm were synthesized by coprecipitation method, and amino functional groups were modified on their surface by silanizing reagent to obtain aminated magnetic nanoparticles. The novel psychoactive substance-specific antibody was mixed with the aminated magnetic nanoparticles in a phosphate buffer solution at pH 7.4, and the antibody was covalently fixed on the surface of the magnetic nanoparticles by glutaraldehyde crosslinking to obtain functionalized magnetic nanoparticles with novel psychoactive substance-specific antibody modified on the surface. The functionalized magnetic nanoparticles were added to the water sample to be tested at a final concentration of 0.5-2 mg / mL and incubated at 25-37℃ with a rotation speed of 150-200 rpm for 15-25 minutes to allow the new psychoactive substances in the wastewater to fully bind with the specific antibodies on the surface of the functionalized magnetic nanoparticles, forming a ternary complex of new psychoactive substances-antibody-magnetic nanoparticles. During incubation, the ionic strength of the reaction system was maintained in the range of 0.01-0.1M, and non-specific adsorption was reduced by adding 0.5% bovine serum albumin as a blocking agent.

4. The method for rapid detection of new psychoactive substances in wastewater by magnetic enrichment and colloidal gold immunoassay according to claim 1, characterized in that: The magnetic separation and washing in step S3 specifically includes: The incubated reaction system is placed in an external magnetic field with an intensity of 0.5-1.5T and left to stand for 1-3 minutes to allow the magnetic complex to be completely adsorbed onto the container wall, forming a magnetic complex aggregate layer. Slowly decant or remove the supernatant, retaining the magnetic complex aggregate layer; Add washing buffer, which is 1 / 5 to 1 / 3 of the initial water sample volume, to the magnetic complex aggregate layer. The washing buffer is a pH 7.4 phosphate buffer containing 0.05-0.5% Tween-20. Gently shake to resuspend the magnetic complex aggregate layer and form a uniform suspension. The resuspended suspension was placed in an external magnetic field again and allowed to stand for 1-2 minutes to adsorb and collect the magnetic complex. The washing supernatant was then discarded. Repeat the washing steps above 2-3 times until the non-specific adsorbed impurities on the surface of the magnetic complex are fully removed, and a pure magnetic complex is obtained.

5. The method for rapid detection of new psychoactive substances in wastewater by magnetic enrichment and colloidal gold immunoassay according to claim 1, characterized in that: The dissociation and detection in step S4 specifically include: Add 100-500 μL of glycine-hydrochloric acid dissociation solution with pH 2.0-3.0 to the washed magnetic complex, and mix by shaking at 100-200 rpm for 5-15 minutes at 25-37℃ to dissociate the new psychoactive substance from the magnetic complex, and obtain a dissociated mixture. The dissociated mixture was placed in an external magnetic field with an intensity of 0.5-1.5T and allowed to stand for 1-2 minutes to adsorb magnetic nanoparticles from the magnetic complex. The supernatant was collected as the dissociation solution. Add an equal volume of pH 7.4 phosphate buffer to the dissociation solution to neutralize it, and obtain the neutralized dissociation solution; Add 50-100 μL of the neutralized and dissociated solution to the sample pad of the colloidal gold immunochromatographic test strip, react at 15-30℃ for 10-15 minutes, and then detect using the colloidal gold immunochromatographic test strip.

6. The method for rapid detection of new psychoactive substances in wastewater by magnetic enrichment and colloidal gold immunoassay according to claim 1, characterized in that: The result reading and data processing in step S5 specifically includes: The color signals of the test line and control line of the colloidal gold immunochromatographic test strip are observed visually. When the color intensity of the test line is lower than that of the control line, it is judged as a positive result, indicating the presence of new psychoactive substances in the wastewater. When the color intensity of the test line is equal to or higher than that of the control line, it is judged as a negative result, indicating that there are no new psychoactive substances in the wastewater. When performing quantitative analysis, the optical density values ​​of the detection line and control line of the colloidal gold immunochromatographic test strip are read by an optical density scanner to obtain the optical density values ​​of the detection line and control line. Calculate the ratio of the optical density of the detection line to that of the control line based on the optical density values ​​of the detection line and the control line. The optical density ratio is compared with a pre-established standard curve using new psychoactive substance standards. The standard curve is plotted using the optical density ratio of different concentrations of new psychoactive substance standards as the response value and the corresponding concentration as the reference value. Based on the comparison results of the standard curve, the content of new psychoactive substances in wastewater is determined by interpolation, and the quantitative detection results are output.

Images