A triple drug fluorescent immunochromatography reagent paper, detection kit and detection method

Abstract

The present invention discloses a triple drug fluorescent immunochromatography reagent paper, a detection kit and a detection method. The triple drug fluorescent immunochromatography test strip is composed of a sample pad, an NC membrane and a water-absorbing pad; the NC membrane is provided with a MET detection line, a MOR detection line, a KET detection line and a quality control line in sequence from one end close to the sample pad. During the detection, the sewage sample to be tested is incubated with the lyophilized powder of the magnetic fluorescent probe, recovered, and added to the sample well of the detection card. After chromatography, the observation well of the detection card is scanned with a fluorescence reader; the concentrations of the three drug metabolites are calculated by the standard curve and the ratio of the electrical signals of each detection line to the quality control line. When the present invention detects the sample, the magnetic response function of the magnetic fluorescent probe is first used to capture and enrich the substance to be tested, reducing the interference of other substances; and then the multi-test paper strip is used to quantitatively detect multiple substances to be tested in the sample. The sensitivity can reach ng / L, which meets the traceability requirements of drug metabolites at all levels in sewage.

Description

Technical Field

[0001] The invention belongs to the field of drug analysis and detection, and specifically relates to a triple drug (methamphetamine MET, morphine MOR, ketamine KET) fluorescent immunochromatography reagent paper, a detection kit and a detection method. Background Art

[0002] At present, methamphetamine MET, morphine MOR and ketamine KET are the three most widely abused drugs.

[0003] The monitoring of illegal drug abuse is generally carried out through social epidemiological surveys, which has great limitations and uncertainties.

[0004] Since the concept of sewage epidemiology was proposed in 2001, drug situation analysis technology based on sewage epidemiology has gradually been applied to the detection of drug abuse. The main sources of drugs and their metabolites in sewage are the excrement of drug users, the drugs flushed away through the sewers by drug users to destroy evidence during surprise inspections, and the wastewater discharged from drug manufacturing sites. By monitoring the content of drugs and their metabolites in urban underground sewage systems, the use of certain drugs in the local area can be inferred. Monitoring drug metabolites in sewage not only helps to effectively capture drug users and drug traffickers, but also can accurately trace and locate drug manufacturing dens through step-by-step fixed-point detection.

[0005] However, the content of drugs and their metabolites in sewage is relatively low, and the types of drugs and their metabolites are relatively complex. How to efficiently, quickly, accurately and stably detect the most common drugs in sewage has become an urgent problem to be solved. Conventional analytical methods are difficult to meet the needs of rapid detection of multiple trace drugs in sewage at the same time. Effective pretreatment methods and efficient and sensitive analytical methods are the key to monitoring multiple drugs of abuse in the environment.

[0006] At present, the quantitative detection and monitoring methods of drugs such as methamphetamine, morphine and ketamine mainly rely on large instruments, such as high performance liquid chromatography, gas chromatography, liquid chromatography-mass spectrometry or gas chromatography-mass spectrometry, etc. These methods have high sensitivity and good repeatability, but are complicated to operate, the instruments and equipment are expensive, and the analysis efficiency is low.

[0007] Enzyme-linked immunosorbent assay (ELISA) and colloidal gold immunochromatography are the mainstream detection technologies that are currently recognized internationally. These two methods are fast, cheap, and easy to operate. However, ELISA testing still requires professional personnel to operate, and the results take a long time to display; colloidal gold immunochromatography technology can only perform qualitative analysis on samples with high drug metabolite content such as urine, blood or saliva, and has problems such as low sensitivity, large batch differences, and low analysis efficiency. Summary of the invention

[0008] The present invention provides a triple drug (methamphetamine MET, morphine MOR, ketamine KET) fluorescent immunochromatographic reagent paper, a detection kit and a detection method, the detection sensitivity of which can reach ng / L (the sensitivity of most fluorescent immunochromatographic test strips for drug detection is currently within the range of ng / mL), the analysis efficiency is high, the linear range is good, and on-site source tracing of various drug metabolites in urban sewage can be performed well, helping public security drug enforcement personnel to conduct real-time monitoring and control of the drug industry chain in the city.

[0009] The purpose of the present invention is achieved through the following technical solutions:

[0010] The preparation method of the magnetic fluorescent probe comprises the following steps:

[0011] (1) Antibody labeling: After the magnetic fluorescent microspheres are activated, they are resuspended in MES (2-(N-morpholino)ethanesulfonic acid) buffer by ultrasonication, and MET antibody, MOR antibody, KET antibody, and goat anti-chicken IgY antibody are added respectively, and the mixture is mixed at low speed at room temperature for at least 1 h;

[0012] (2) Blocking: Add BSA (bovine serum albumin) to each antibody-labeled magnetic fluorescent microsphere, rotate the reaction for at least 30 min, and collect the precipitate after centrifugation to obtain three detection probes (MET antibody-labeled magnetic fluorescent probe, MOR antibody-labeled magnetic fluorescent probe, KET antibody-labeled magnetic fluorescent probe) and a quality control probe (sheep anti-chicken IgY antibody-labeled magnetic fluorescent probe);

[0013] The activation described in step (1) is to wash and resuspend the magnetic fluorescent microspheres with MES buffer, then add EDC (carbodiimide) and NHS (N-hydroxysuccinimide), and react at room temperature for at least 30 minutes, and then centrifuge to obtain a precipitate;

[0014] Preferably, the MES buffer has a concentration of 0.1 M and a pH value of 6.0;

[0015] Preferably, the centrifugation is performed at 15000 rpm for 30 min.

[0016] A magnetic fluorescent probe lyophilized powder is prepared by resuspending three detection probes (MET antibody-labeled magnetic fluorescent probe, MOR antibody-labeled magnetic fluorescent probe, KET antibody-labeled magnetic fluorescent probe) and a quality control probe (sheep anti-chicken IgY antibody-labeled magnetic fluorescent probe) prepared by the above method in equal amounts of labeling preservation solution, then mixing the three detection probes at a volume ratio of 1:1:1, and finally mixing the mixed solution of the three detection probes with the quality control probe at a volume ratio of 9:1, and filling equal volumes (2uL, 0.4ug) into respective cryopreservation tubes, and freeze-drying (ultimately light yellow lyophilized powder), thereby obtaining the magnetic fluorescent probe lyophilized powder;

[0017] The formula of the labeling preservation solution is as follows: 5% sucrose, 5% trehalose, 1% BSA, 0.5% sodium caseinate, and 0.02% Proclin 300 are added to 0.1M Gly-NaOH buffer solution in a mass ratio.

[0018] A triple drug fluorescent immunochromatographic test strip, consisting of a sample pad, an NC membrane (nitrocellulose membrane) and a water absorbent pad;

[0019] The NC membrane is provided with a MET detection line, a MOR detection line, a KET detection line and a quality control line in sequence from one end close to the sample pad;

[0020] Preferably, the spacing between the test lines and quality control lines is 3.5 mm, and the distance between the KET test line and the absorbent pad is 11 mm; this design is to facilitate detection without blocking the test line through the observation hole, and to make the test results of the joint test strip more accurate.

[0021] The preparation method of the reagent paper comprises the following steps:

[0022] (1) MET-BSA, MOR-BSA, KET-BSA and chicken IgY were coated on the NC membrane respectively, and after drying, a MET detection line, a MOR detection line, a KET detection line and a quality control line were formed in sequence, which were used as the treated NC membrane;

[0023] The operation of step (1) is preferably performed using an X, Y, Z three-dimensional dot film gold spraying instrument, with a MET-BSA concentration of 0.25 mg / mL, a MOR-BSA concentration of 0.5 mg / mL, a KET-BSA concentration of 2 mg / mL, a chicken IgY concentration of 1 mg / mL, and a coating amount on the NC membrane of preferably 1 μL / cm;

[0024] (2) Treat the glass cellulose membrane with a buffer solution (soak or apply it) and then dry it to use as a sample pad; take the absorbent paper and dry it thoroughly to use as an absorbent pad; lay the sample pad, NC membrane and absorbent pad on a PVC base plate in sequence, and cut them to obtain a reagent paper, the structure of which is as follows: Figure 1 As shown;

[0025] The formula of the buffer solution is to add 0.5% S-9 surfactant, 0.1% casein sodium salt and 1% sucrose in a mass ratio into 0.015M PBS buffer solution with a pH value of 7.4.

[0026] A triple drug fluorescent immunoassay card, comprising an upper card shell and a lower card shell;

[0027] The upper card shell is provided with a spotting hole and an observation hole;

[0028] The lower card shell is covered with a PVC bottom plate and the aforementioned triple drug fluorescent immunochromatographic test strip in sequence;

[0029] The sample pad of the reagent paper overlaps with the spotting hole of the upper card shell in position, which is convenient for operators to spot samples;

[0030] The detection lines and quality control lines of the NC membrane of the reagent paper overlap with the observation holes of the upper card shell in position, which is convenient for observing the detection results.

[0031] A triple drug fluorescence immunoassay kit comprises the magnetic fluorescent probe freeze-dried powder and / or the triple drug fluorescence immunoassay card.

[0032] The method for fluorescent immunoassay of three drugs in sewage based on the kit comprises the following steps:

[0033] (1) Each 1 mL of the sewage sample to be tested is mixed with 0.4 μg of the lyophilized powder of the magnetic fluorescent probe, and the mixture is incubated at 25-37°C for at least 10 min. The immune complexes formed by MFB-MET-mAbs, MFB-MOR-mAbs, MFBs-KET-mAbs and the corresponding drug metabolites and the magnetic fluorescent probes that are not bound to the drug metabolites are magnetically recovered on a magnetic stand; after removing the supernatant, the recovered precipitate is resuspended in PBS buffer to obtain the sample solution to be tested;

[0034] (2) Take a drop of the sample to be tested and add it to the spotting hole of the triple drug fluorescent immunoassay card. After chromatography for 10-15 minutes, scan the observation hole of the test card with a fluorescent reader;

[0035] (3) The magnetic fluorescent probes that are not bound to drug metabolites are retained on each test line and quality control line, and emit fluorescence under the excitation of the excitation light; the fluorescence reader detects the fluorescence signals of each test line and quality control line, and converts the fluorescence signals into electrical signals, and then calculates the concentrations of the three drug metabolites through the standard curve and the ratio of the electrical signals of each test line to the quality control line;

[0036] In the step (3), the wavelength of the excitation light is in the range of 300 to 400 nm, preferably 365 nm.

[0037] The principle of the fluorescent immunoassay for triple drugs in the present invention is as follows:

[0038] After the human body ingests drugs, the drugs and their metabolites are metabolized by the liver and excreted from the body through urine, sweat or other body fluids. The drugs produced and trafficked are also discharged into the sewer in the form of domestic sewage in a corresponding manner.

[0039] The multiple detection test strip of the present invention is composed of a treated sample pad, a magnetic fluorescent probe freeze-dried powder, an NC membrane and a water-absorbing pad. The NC membrane is provided with a detection line (T line) formed by coating with MET-BSA, MOR-BSA and KET-BSA and a quality control line (C line) formed by coating with chicken IgY.

[0040] During the test, a certain amount of sewage was added to a fixed amount of magnetic fluorescent probe freeze-dried powder composed of MFBs-MET-mAb, MFBs-MOR-mAb, MFBs-KET-mAb, and MFBs-goat anti-chicken IgY. After the reaction was sufficient, it was enriched with a neodymium magnet, and then re-dissolved in PBS buffer and added to the sample wells of the multi-test strip.

[0041] If the sewage contains a low concentration of a certain drug metabolite component, after the corresponding detection probes in the lyophilized powder are enriched by binding to the drug metabolites, most of them will bind to the corresponding complete antigens on the detection line because they are not bound to the corresponding drug metabolites; if the sewage contains a high concentration of a certain drug metabolite component, after the corresponding detection probes in the lyophilized powder are enriched by binding to the drug metabolites in the sewage, only a small amount of remaining unbound detection probes will bind to the corresponding complete antigens on the T line.

[0042] The higher the concentration of residual drugs in the sewage, the less the corresponding detection probe binds to the corresponding antigen on the T line, resulting in a weaker fluorescence signal. However, no matter how the concentration of various drug metabolites in the sewage changes, the fluorescence signal of the C line will remain basically unchanged. The ratio of the corresponding T line fluorescence signal to the C line fluorescence signal is inversely proportional to the concentration of the corresponding drug metabolite in the sewage.

[0043] Compared with the prior art, the present invention has the following advantages and effects:

[0044] (1) High sensitivity: The magnetic fluorescent microspheres used in the present invention are synthesized from magnetic material ferroferric oxide nanoparticles and oleylamine CdSe / ZnS quantum dots, and have the dual functions of magnetic response and fluorescence properties. When testing samples, the magnetic response function of the magnetic fluorescent probe is first used to capture and enrich the substances to be tested, reducing interference from other substances; then the fluorescence properties of the quantum dots are combined with the multi-test paper strips to quantitatively detect multiple substances to be tested in the sample. The sensitivity of the optimized test strips can reach ng / L, which meets the requirements for tracing the source of drug metabolites in sewage at all levels.

[0045] (2) Convenient detection: At present, the quantitative detection and monitoring methods for drugs such as methamphetamine, morphine and ketamine mainly rely on large instruments, such as high performance liquid chromatography, gas chromatography, liquid mass spectrometry or gas chromatography-mass spectrometry. These methods require complex sample pretreatment, high analysis cost and long detection time. The use of the multi-linked magnetic fluorescent immunochromatographic test strips of the present invention to detect drug metabolites in sewage does not require a complex sample pretreatment process, the total detection time is about 20 minutes, and the concentration of three trace drugs in a single sample can be detected simultaneously.

[0046] (3) High detection throughput: The multi-link magnetic fluorescent immunochromatographic test strip of the present invention can detect the concentrations of MET, MOR, and KET in sewage at a single spot, which can save the total detection time and analysis cost, and also save the sample amount, thereby preventing the missed detection of drug metabolites in sewage.

[0047] (4) Wide application scenarios: The multi-link magnetic fluorescent immunochromatographic test strip of the present invention can not only be used for rapid detection of multiple drug metabolites in sewage, but also can be used for quantitative detection of multiple drug metabolites in trace samples such as the environment, hair, and sweat after further optimization. It is of great help to drug control bureaus, drug rehabilitation centers, customs and other drug detection related institutions to quickly monitor drug conditions. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 The present invention is a schematic diagram of the structure of a triple drug fluorescent immunoassay card; wherein: 1-PVC bottom plate, 2-sample pad, 3-NC membrane, 4-water absorbent pad, 5-MET detection line, 6-MOR detection line, 7-KET detection line, 8-quality control line, 9-lower card shell, 10-upper card shell, 11-observation hole, 12-spotting hole.

[0049] Figure 2 is the ratio of the fluorescence intensity of the MET, MOR, KET concentration to the corresponding detection line and quality control line (F T / F C ) standard curve.

[0050] Figure 3 These are the specific detection experimental results of three antibody-labeled magnetic fluorescent probes; among them, (a)(b) MET antibody-labeled magnetic fluorescent probe cross-reaction rate diagram; (c)(d) MOR antibody-labeled magnetic fluorescent probe cross-reaction rate diagram; (e)(f) KET antibody-labeled magnetic fluorescent probe cross-reaction rate diagram.

[0051] Figure 4 It is a comparison between the detection results of the detection method of the present invention and the existing HPLC-MS / MS detection method; wherein, (a) MET content; (b) MOR content; (c) KET content. DETAILED DESCRIPTION

[0052] The present invention is further described in detail below in conjunction with embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0053] Example 1

[0054] A magnetic fluorescent probe freeze-dried powder is prepared by the following steps:

[0055] (1) Activation: 100 μg of magnetic fluorescent microspheres were washed three times with MES buffer (0.1 M, pH 6.0) and then resuspended in 990 μl MES buffer solution. After oscillation and mixing, 10 μl of 10 mg / mL EDC (carbodiimide) and 10 μl of 10 mg / mL NHS (N-hydroxysuccinimide) were added respectively and rotated at room temperature for 30 min; centrifuged at 15000 rpm for 30 min and the supernatant was discarded;

[0056] The magnetic fluorescent microspheres are prepared according to the method of the literature (Guo L, Shao YN, Duan H, et al. Magnetic Quantum Dot Nanobead-Based Fluorescent Immunochromatographic Assay for the Highly Sensitive Detection of Aflatoxin B-1 in Dark Soy Sauce [J]. Analytical Chemistry, 2019, 91 (7): 4727-4734.), and the fluorescence emission wavelength is 600-620 nm, preferably 618 nm.

[0057] (2) Antibody labeling: Add 1000 μL MES buffer to the precipitate and resuspend by ultrasound; add 2 μg of MET antibody (mAb-MET) and react at low speed at room temperature for 1 h;

[0058] (3) Blocking: Add 10 μL of 10% BSA (bovine serum albumin) and rotate for 30 min. Centrifuge at 15,000 rpm for 30 min, discard the supernatant, and store at 4°C for later use.

[0059] (4) Magnetic fluorescent microspheres labeling MOR antibody: 4 μg of MOR antibody (mAb-MOR) was added to the activated and resuspended magnetic fluorescent microspheres, and the remaining steps were the same as step (3).

[0060] Magnetic fluorescent microspheres labeling KET antibody: 8 μg of KET antibody (mAb-KET) was added to the activated and resuspended magnetic fluorescent microspheres, and the remaining steps were the same as step (3).

[0061] Magnetic fluorescent microspheres labeling goat anti-chicken IgY antibody: add 5 μg of goat anti-chicken IgY antibody to the activated and resuspended magnetic fluorescent microspheres, and the remaining steps are the same as step (3).

[0062] (5) The three detection probes (MET antibody-labeled magnetic fluorescent probe, MOR antibody-labeled magnetic fluorescent probe, KET antibody-labeled magnetic fluorescent probe) and the quality control probe (sheep anti-chicken IgY antibody-labeled magnetic fluorescent probe) prepared by the above method were resuspended in 500 μL of labeled preservation solution, and then the three detection probes were mixed at a volume ratio of 1:1:1. Finally, the mixed solution of the three detection probes and the quality control probe were mixed at a volume ratio of 9:1, and equal volumes (2uL, 0.4ug) were divided into each cryopreservation tube for freeze drying (finally light yellow freeze-dried powder), to obtain the magnetic fluorescent probe freeze-dried powder;

[0063] The formula of the labeling preservation solution is as follows: 5% sucrose, 5% trehalose, 1% BSA, 0.5% sodium caseinate, and 0.02% Proclin 300 are added to 0.1M Gly-NaOH buffer solution in a mass ratio.

[0064] The three detection antibodies mAb-MET, mAb-MOR, and mAb-KET used in the present invention are all from EastCoastBio Biotechnology Co., Ltd.;

[0065] The chicken IgY and goat anti-chicken IgY used in the present invention are both from Hangzhou Qitai Biotechnology Co., Ltd.

[0066] Example 2

[0067] A triple drug fluorescence immunoassay card, the structure of which is as follows Figure 1 As shown, it includes upper 10 and lower 9 card shells;

[0068] The upper card shell 10 is provided with a spotting hole 12 and an observation hole 11;

[0069] The lower card shell 9 is covered with a PVC bottom plate 1, on which is a triple drug fluorescent immunochromatographic test strip;

[0070] The reagent paper is composed of a sample pad 2, an NC membrane 3 and a water absorbent pad 4;

[0071] The NC membrane 3 is provided with a MET detection line 5, a MOR detection line 6, a KET detection line 7 and a quality control line 8 in sequence from one end close to the sample pad 2; the spacing between the detection lines and the quality control lines is 3.5 mm, and the distance between the KET detection line and the water absorbent pad is 11 mm;

[0072] The sample pad 2 of the reagent paper 3 overlaps with the spotting hole 12 of the upper card shell 10 in position, which is convenient for operators to spot samples;

[0073] The detection lines 5, 6, 7 and the quality control line 8 of the NC membrane 3 overlap with the observation hole 11 of the upper card shell 10 in position, so as to facilitate the observation of the detection results.

[0074] The preparation method of the reagent paper comprises the following steps:

[0075] (1) Paste the nitrocellulose membrane (NC membrane) (model: Millipore CN140) onto the PVC base plate and place it in a 37°C oven for equilibration for 30 minutes. Use an X, Y, Z three-dimensional dot-film gold spraying instrument to coat 0.25 mg / mL MET-BSA, 0.5 mg / mL MOR-BSA, 2 mg / mL KET-BSA and 1 mg / mL chicken IgY on the NC membrane at 1 μL / cm, respectively, as T1, T2, T3 test lines and quality control lines, respectively. The spacing between each coated line is 3.5 mm, of which the distance between the T3 test line and the edge of the absorbent paper is 11 mm, and the distance from the quality control line is 3.5 mm. Dry at 37°C overnight and store in a constant temperature and humidity storage cabinet for use as a NC membrane;

[0076] (2) Cut the glass fiber membrane (model: GL-b04) into 2.5 cm × 30 cm specifications, add 5 mL of sample pad treatment solution to each cut glass fiber membrane. Use a propeller to evenly apply the sample pad treatment solution, spread it at room temperature overnight, and then dry it in a 37°C drying oven. After drying, seal it and store it in a constant temperature and humidity storage cabinet (temperature: 20-25°C, relative humidity: <30%) for use as a sample pad;

[0077] The buffer formula of the sample pad is as follows: 0.5% S-9 surfactant, 0.1% casein sodium salt and 1% sucrose are added in a mass ratio to 0.015M PBS buffer with a pH value of 7.4.

[0078] (3) Take the absorbent pad material (filter paper, model: H-6) and cut it into 2.5 cm × 30 cm strips, place it in a 37°C oven to dry, put it in an aluminum foil bag, add desiccant, and store it in a constant temperature and humidity storage cabinet for later use as a water absorbent pad;

[0079] (4) The prepared sample pad, NC membrane and absorbent pad are sequentially pasted onto the PVC base plate, with each part overlapping by 1-2 mm to ensure that the test strip can smoothly complete the entire flow process after the sample is added, thereby obtaining a triple drug fluorescent immunochromatographic test strip.

[0080] Example 3

[0081] A triple drug fluorescent immunoassay kit comprises the magnetic fluorescent probe freeze-dried powder described in Example 1 and the triple drug fluorescent immunoassay card described in Example 3.

[0082] The method for fluorescent immunoassay of triple drugs in sewage based on the kit comprises the following steps:

[0083] (1) 1 mL of the sewage sample to be tested was mixed with 0.4 μg of the lyophilized powder of the magnetic fluorescent probe, and the mixture was incubated at 37°C for at least 10 min. The immune complexes formed by MFB-MET-mAbs, MFB-MOR-mAbs, MFBs-KET-mAbs and the corresponding drug metabolites in the sample and the unbound magnetic fluorescent probes were magnetically recovered by a magnetic rack; after removing the supernatant, the immune complexes were resuspended in 100 μL of PBS buffer to obtain the sample solution to be tested;

[0084] (2) Take 75 μL of the sample to be tested and add it to the sample well of the triple drug fluorescence immunoassay card. After reacting for 10-15 minutes, scan the observation well of the test card with a fluorescence reader;

[0085] (3) The magnetic fluorescent probes that are not bound to drug metabolites are retained on each test line and quality control line, and emit fluorescence under the excitation of 365nm excitation light; the fluorescence reader detects the fluorescence signals of each test line and quality control line and converts the fluorescence signals into electrical signals, and then calculates the concentrations of the three drug metabolites through the standard curve and the ratio of the electrical signals of each test line to the quality control line.

[0086] The preparation of the standard curve in the present invention is as follows:

[0087] (1) Prepare a high concentration drug reference solution and dilute it with negative sewage to the following gradient concentrations:

[0088] MET: 10000ng / L, 5000ng / L, 2000ng / L, 1000ng / L, 500ng / L, 200ng / L, 100ng / L, 50ng / L, 20ng / L, 10ng / L, 5ng / L, 2ng / L, 0ng / L.

[0089] MOR: 5000ng / L, 1000ng / L, 500ng / L, 200ng / L, 100ng / L, 50ng / L, 20ng / L, 10ng / L, 5ng / L, 2ng / L, 1ng / L, 0.5ng / L, 0ng / L.

[0090] KET: 10000ng / L, 5000ng / L, 2000ng / L, 1000ng / L, 500ng / L, 200ng / L, 100ng / L, 50ng / L, 20ng / L, 10ng / L, 5ng / L, 2ng / L, 1ng / L, 0.5ng / L, 0ng / L.

[0091] (2) 1 mL of each drug reference solution was mixed with 0.4 μg of the lyophilized powder of the magnetic fluorescent probe, and the mixture was incubated at 37°C for 10 min. The immune complexes formed by MFB-MET-mAbs, MFB-MOR-mAbs, MFBs-KET-mAbs and the corresponding drug metabolites in the sample and the unbound magnetic fluorescent probes were recovered by magnetic rack; after removing the supernatant, the immune complexes were resuspended in 100 μL of PBS buffer to obtain the sample solution to be tested;

[0092] (3) 75 μL of the sample solution to be tested was added to the sample wells of the triple drug fluorescence immunoassay card in sequence, and the test was performed 10 minutes after the sample addition, with 3 replicates for each concentration. After 10 minutes of reaction, the fluorescence intensity ratio F of the multiple test strip test line and the quality control line corresponding to each concentration of the reference solution was read using a fluorescence reader. T / F C , based on the reading value F of the reference negative sample and other positive samples T / F C Represented as B0 and B X The logarithm of the corresponding concentration of each group of standard products is used as the horizontal axis, and the reading value B of other positive samples is X Substitute into the formula Logit(Y)=LN[(B X / B0) / (1-B X / B0) as the vertical axis, the log-logit function model was used for data processing, that is, Logit(Y)=A+B×Log(X), and the concentrations of each drug metabolite MET, MOR, and KET were plotted against the fluorescence intensity ratios of the corresponding detection line and quality control line (F T / F C ) standard curve.

[0093] The standard curve is as follows Figure 2 As shown in the figure, within a certain concentration range, the multiplex fluorescence immunochromatographic quantitative test strip has a good dose-response linear relationship, and the dose-response curve equation is:

[0094] MET: Logit(Y)=1.341-0.813Log(X), the dose-response curve correlation coefficient R 2 is 0.998, and the linear range of the test strip is: 10-5000ng / L;

[0095] MOR: Logit(Y)=2.253-2.137Log(X), its dose-response curve correlation coefficient R 2 is 0.994, and the linear range of the test strip is: 2-500ng / L;

[0096] KET: Logit(Y) = 3.064-1.194Log(X), the dose-response curve correlation coefficient R 2 It is 0.991, and the linear range of the test strip is: 2-5000ng / L.

[0097] The linear range of the combined test strips meets the requirements for detecting various drug metabolites in sewage.

[0098] Example 4

[0099] Specific detection of three antibody-labeled magnetic fluorescent probes obtained in Example 1

[0100] The three detection probes (MET antibody-labeled magnetic fluorescent probe, MOR antibody-labeled magnetic fluorescent probe, KET antibody-labeled magnetic fluorescent probe) and the quality control probe (sheep anti-chicken IgY antibody-labeled magnetic fluorescent probe) prepared in Example 1 were freeze-dried respectively, and then 0.12 μg of the three detection antibody-labeled magnetic fluorescent probes and 0.04 ug of the quality control probe were combined, and 1 mL of 10 ng / mL of each interfering substance spiked sample prepared with negative sewage was added respectively; the interfering substances were tetrahydrocannabinol, morphine, methamphetamine, ketamine, diazepam, methadone, oxazepam, cocaine, fentanyl, tetrahydrocannabinic acid, 3,4-methylenedioxymethamphetamine (MDMA) and other 11 drug metabolites;

[0101] After incubation at 25-37°C for 10 min, the precipitate was enriched using a rubidium magnet and finally resuspended with 100 μL PBS. 75 μL of the resuspended solution was added dropwise to the sample wells of the triple drug fluorescence immunoassay card of Example 2. After reacting for 10 min, the ratio of the fluorescence intensity of the test line and the quality control line of the test strip was read using a fluorescence reader (F T / F C ) and sample test value, using the formula:

[0102] Cross-reaction rate = (sample detection value / sample actual value) × 100%

[0103] The actual value of the sample is 10ng / mL

[0104] Calculate the cross-reaction rate of each similar drug, the result is as follows Figure 3 As shown: The MET antibody-labeled magnetic fluorescent probe only has a certain cross reaction with its analog MDMA (3,4-methylenedioxymethamphetamine), and basically has no cross reaction with other similar drugs. The two magnetic fluorescent probes labeled with MOR and KET have good specificity and basically do not react with other similar drugs.

[0105] Example 5

[0106] Comparison of the method of the present invention (method of Example 3) with other methods

[0107] 21 actual wastewater samples were collected. The concentration of each drug metabolite in each sample was detected by the method of Example 3. The results calculated by the actual sample detection method of Example 3 were compared with the detection results of the method (liquid chromatography-mass spectrometry tandem detection technology LC-MS) in the literature (Castiglioni S, Zuccato E, Chiabrando C, et al. Mass spectrometric analysis of illicit drugs in wastewater and surfacewater [J]. Mass Spectrometry Reviews, 2008, 27 (4): 378-394.), and the detection results of the two detection methods were linearly simulated and calculated. The results are shown in FIG. Figure 4 The two detection methods were used to detect three substances, MET, MOR, and KET. The linear regression coefficients of the test results were R 2 >0.99, indicating that the two methods have good correlation and the results of the actual samples are highly consistent. Figure 4 and Table 1.

[0108] The linear regression equations of the two methods for detecting MET were: Y=1.603+0.883X, R=0.999, P<0.001; the linear regression equations of the two methods for detecting MOR were: Y=1.713+0.854X, R=0.999, P<0.001; the linear regression equations of the two methods for detecting KET were: Y=3.484+0.983X, R=0.999, P<0.001.

[0109] The above results show that the present invention can simultaneously and quantitatively detect the contents of three drug metabolites of methamphetamine, morphine and ketamine in sewage samples, and has a good correlation with the traditional method LC-MS / MS, and the results do not interfere with each other. It is a more convenient detection method for judging urban drug conditions.

[0110] Table 1. Comparison of actual sample test results of multiplex test strips and HPLC-MS / MS.

[0111]

[0112] Example 6

[0113] Determination of the sensitivity of triple drug fluorescent immunochromatographic test strips

[0114] The triple drug fluorescent immunochromatographic test strip of Example 2 was used to repeatedly measure the MET, MOR, and KET negative wastewater 20 times each, and the ratio of the fluorescence value of the test line to the quality control line was calculated. T / F CThe mean X minus two standard deviations is B X Substitute the dose-response curve of each test strip (ie, the standard curve of Example 3) to obtain the corresponding concentration value.

[0115] The analytical sensitivities of MET, MOR, and KET magnetic fluorescent immunochromatographic test strips were 8.32 ng / L, 1.47 ng / L, and 1.62 ng / L, respectively. T / F C The values ​​and CV values ​​are shown in Table 2, Table 3 and Table 4:

[0116] Table 2. Analytical sensitivity of MET magnetic fluorescence immunochromatographic test strips

[0117]

[0118] Table 3. Analytical sensitivity of MOR magnetic fluorescence immunochromatographic test strips

[0119]

[0120] Table 4. Analytical sensitivity of KET magnetic fluorescence immunochromatographic test strips

[0121]

[0122] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for preparing a magnetic fluorescent probe, characterized in that The steps include: (1) Antibody labeling: After activating the magnetic fluorescent microspheres, resuspend them in MES buffer by ultrasonication, add MET antibody, MOR antibody, KET antibody, and goat anti-chicken IgY antibody respectively, and react at room temperature with low speed rotation for at least 1 h; (2) Blocking: Add bovine serum albumin to each antibody-labeled magnetic fluorescent microsphere, rotate the reaction for at least 30 minutes, and collect the precipitate after centrifugation to obtain MET antibody-labeled magnetic fluorescent probe, MOR antibody-labeled magnetic fluorescent probe, KET antibody-labeled magnetic fluorescent probe, and sheep anti-chicken IgY antibody-labeled magnetic fluorescent probe, respectively.

2. The preparation method according to claim 1, characterized in that: The activation described in step (1) is to wash and resuspend the magnetic fluorescent microspheres with MES buffer, then add carbodiimide and N-hydroxysuccinimide, and perform a vortex reaction at room temperature for at least 30 minutes, and then collect the precipitate after centrifugation.

3. The preparation method according to claim 1, characterized in that: The MES buffer has a concentration of 0.1 M and a pH value of 6.0; The centrifugation is performed at 15000 rpm for 30 min.

4. A magnetic fluorescent probe, characterized in that: The method is prepared by the method described in any one of claims 1 to 3.

5. A magnetic fluorescent probe freeze-dried powder, characterized in that: The method comprises the following steps of resuspending a MET antibody-labeled magnetic fluorescent probe, a MOR antibody-labeled magnetic fluorescent probe, a KET antibody-labeled magnetic fluorescent probe and a goat anti-chicken IgY antibody-labeled magnetic fluorescent probe in equal amounts of labeling preservation solution, and then uniformly mixing the three detection probes in a volume ratio of 1:1:

1. Finally, mixing the mixed solution of the three detection probes with the goat anti-chicken IgY antibody-labeled magnetic fluorescent probe in a volume ratio of 9:1, placing the mixture into a cryopreservation tube, and freeze-drying the mixture to obtain a magnetic fluorescent probe freeze-dried powder.

6. The magnetic fluorescent probe freeze-dried powder according to claim 5, characterized in that: The formula of the labeling preservation solution is as follows: 5% sucrose, 5% trehalose, 1% BSA, 0.5% sodium caseinate, and 0.02% Proclin 300 are added to 0.1 M Gly-NaOH buffer in a mass ratio.

7. A triple drug fluorescent immunoassay kit, characterized in that: It comprises the magnetic fluorescent probe freeze-dried powder and triple drug fluorescent immunoassay card as described in claim 5 or 6; The triple drug fluorescent immunoassay card is characterized by: Contains triple drug fluorescent immunochromatographic test strips; Including upper and lower card shells; The upper card shell is provided with a spotting hole and an observation hole; The lower card shell is covered with a PVC bottom plate and a triple drug fluorescent immunochromatographic test strip in sequence; The sample pad of the reagent paper overlaps with the spotting hole of the upper card shell in position; The detection lines and quality control lines of the NC membrane of the reagent paper overlap with the observation holes of the upper card shell in position; The triple drug fluorescent immunochromatographic test strip is composed of a sample pad, an NC membrane and a water absorbent pad. The NC membrane is provided with a MET detection line, a MOR detection line, a KET detection line and a quality control line in sequence from one end close to the sample pad.

8. A method for fluorescent immunoassay of three drugs in sewage based on the kit of claim 7, characterized in that The following steps are involved: (1) Each 1 mL of the sewage sample to be tested is mixed with 0.4 µg of the lyophilized powder of the magnetic fluorescent probe, and the mixture is incubated at 25-37°C for at least 10 min. The immune complexes formed by MFB-MET-mAbs, MFB-MOR-mAbs, MFBs-KET-mAbs and the corresponding drug metabolites and the magnetic fluorescent probes that are not bound to the drug metabolites are magnetically recovered on a magnetic stand; after removing the supernatant, the recovered precipitate is resuspended in PBS buffer to obtain the sample solution to be tested; (2) Take a drop of the sample to be tested and add it to the spot hole of the triple drug fluorescent immunoassay card. After chromatography for 10-15 minutes, scan the observation hole of the test card with a fluorescent reader; (3) The magnetic fluorescent probes that are not bound to drug metabolites are retained on each test line and quality control line, and emit fluorescence under the stimulation of excitation light; the fluorescence reader detects the fluorescence signals of each test line and quality control line and converts the fluorescence signals into electrical signals, and then calculates the concentrations of the three drug metabolites through the standard curve and the ratio of the electrical signals of each test line to the quality control line.

9. The method according to claim 8, characterized in that: In the step (3), the wavelength range of the excitation light is 300 to 400 nm.

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