CN113252903A - Magnetic fluorescent microsphere immunochromatography kit for field detection and manufacturing method and detection method thereof - Google Patents
Magnetic fluorescent microsphere immunochromatography kit for field detection and manufacturing method and detection method thereof Download PDFInfo
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
Abstract
The invention discloses a magnetic fluorescent microsphere immunochromatographic kit for field detection, a manufacturing method thereof and a detection method thereof, the kit comprises a kit body, a magnetic fluorescent immunochromatographic test strip and a hair lysate, the magnetic fluorescent immunochromatographic test strip comprises a bottom plate, a nitrocellulose membrane (NC membrane) is arranged on the bottom plate, a sample pad and a combination pad are sequentially stuck on one side of the nitrocellulose membrane according to the chromatography direction, a water absorption pad is stuck on the other side of the nitrocellulose membrane, magnetic fluorescent nanoparticles for marking a drug monoclonal antibody are fixed on the combination pad, three detection lines (T lines) and one quality control line (C line) are arranged on the nitrocellulose membrane, the detection lines are respectively coated with a compound of bovine serum albumin coupled methamphetamine, morphine and ecstasy, the quality control line is coated with goat anti-mouse polyclonal antibody, the hair lysate contains effective components for cracking hair keratin, aims to dissolve out small drug molecules from hair so as to realize efficient, accurate and sensitive drug detection.
Description
Technical Field
The invention relates to the technical field of biological detection, in particular to a magnetic fluorescent microsphere immunochromatographic kit for field detection and a manufacturing method and a detection method thereof.
Background
Drugs are generally drugs that cause people to develop addiction, which is mainly the reaction of the body to drugs, and the body has tolerance to drugs administered for a long time, and large doses of drugs can meet the needs of the body. Some substances can generate pleasure after long-term use, feel uncomfortable after interruption, and generate psychological dependence when people want to continue using the medicines mentally. Other drugs produce physiological dependence when used, and serious physiological dysfunction after being stopped, thus forming physiological dependence. Common drug test materials include various forms of drug suspicious substances and biological test materials, wherein the common test materials include urine, blood, saliva and hair. Compared with other test materials, the hair has a longer traceable drug taking period, can reflect the drug taking history of more than half a year, is easier to store and transport, is not easy to fall off packages and falsify, has greater advantages in timeliness, and provides good conditions for drug detection. It is believed that the toxin enters the blood, passively diffuses from the capillaries to the growing cells, settles in the hair follicles, and then disperses into every hair. Once the drug enters the hair, the drug is firmly wrapped in the keratin, is relatively stable and is not easy to decompose.
The hydrolysis method can effectively release novel drugs in the hair, and mainly comprises methanol hydrolysis, alkaline hydrolysis, enzyme hydrolysis, acid hydrolysis and the like. The acid hydrolysis is carried out by putting hair into 0.1mol/L hydrochloric acid, incubating at 65 deg.C or below for 12h, and releasing poison from hair when acid water condition is mild. The acid hydrolysis is carried out by putting hair into 0.1mol/L hydrochloric acid, incubating at 65 deg.C or below for 12h, and releasing poison from hair when acid water condition is mild. The background of acid hydrolysis hair products is low, but the release rate of drug molecules of the object to be detected is lower than that of the alkali hydrolysis method. The alkaline hydrolysis method comprises placing hair in 1mol/L sodium hydroxide, heating at 60-90 deg.C to dissolve hair sufficiently, and promoting the release of drugs solidified in keratin. However, the content of protein impurities in the extracting solution is high, so that the detection is influenced. The method has wide application range of acid and alkali, can completely release drugs, and has relatively high cost. The methanol hydrolysis method mainly relies on ultrasonic oscillation to release drugs in hair, but current researches show that the drug release in the methanol hydrolysis method is not complete enough and has a large amount of impurities contained in extracts, so that the method is not widely applied and related reports are few. The method mainly comprises the steps of solid extraction, solid micro extraction, liquid extraction, micro grinding extraction and the like, nitrogen is used for drying extract liquor of a substance to be detected and then derivatization treatment is carried out, and finally gas chromatography-mass spectrometry and high performance liquid chromatography-mass spectrometry are used together for detection.
Disclosure of Invention
In order to overcome the technical defects, the invention aims to provide a magnetic fluorescent microsphere immunochromatographic kit for field detection, a manufacturing method thereof and a detection method thereof, so as to achieve the purposes of rapid, accurate and sensitive drug detection.
In order to achieve the purpose, the invention adopts the following technical scheme:
the magnetic fluorescent microsphere immunochromatographic kit comprises a kit body, a magnetic fluorescent immunochromatographic test strip and a hair lysate, wherein the magnetic fluorescent immunochromatographic test strip comprises a bottom plate, a nitrocellulose membrane (NC membrane) is arranged on the bottom plate, a sample pad and a combination pad are sequentially adhered to one side of the nitrocellulose membrane according to the chromatography direction, absorbent paper is adhered to the other side of the nitrocellulose membrane, magnetic fluorescent nanoparticles for marking a drug monoclonal antibody are fixed on the combination pad, three detection lines (T lines) and a quality control line (C line) are arranged on the nitrocellulose membrane, the detection lines are respectively coated with a compound of bovine serum albumin coupled ice toxin, morphine and ecstasy, the quality control line is coated with a goat anti-mouse polyclonal antibody, and the hair lysate aims to dissolve drug micromolecules from hair so as to realize high efficiency, accuracy and high speed, Sensitive drug detection.
Further, the combination pad and the absorbent pad are respectively positioned on two sides of the nitrocellulose membrane and are respectively lapped on the nitrocellulose membrane, and the sample pad is lapped on the combination pad.
Furthermore, the magnetic fluorescent nanoparticles are self-assembled with carboxylated quantum dots on the surface of the aminated magnetic silicon sphere in one step without mediation of charged polymers, have the particle size of about 180nm, and have better fluorescence intensity, dispersibility and water solubility.
Further, the bottom plate is a PVC plastic bottom plate.
Further, the box body includes card shell upper cover plate and card shell lower cover plate, card shell upper cover plate is equipped with application of sample hole, observation window, includes the two-dimensional code of product standard curve and batch number information. The sample adding hole corresponds to the position of the sample pad, and the observation window corresponds to the position of the nitrocellulose membrane.
The invention also provides a manufacturing method of the magnetic fluorescent microsphere immunochromatographic kit for field detection, which comprises the following steps:
(1) respectively pointing a bovine serum albumin coupled compound of the methamphetamine, the morphine and the dancing head pill and a film pointing machine for an goat anti-mouse antibody on a detection line and a quality control line of a nitrocellulose membrane, and fully drying at 37 ℃ to ensure that the bovine serum albumin coupled compound of the methamphetamine, the morphine and the dancing head pill and the goat anti-mouse antibody are respectively and firmly fixed on the detection line and the quality control line of the nitrocellulose membrane; spraying the specific monoclonal antibody of the drug marked by the magnetic fluorescent nano particles on the binding pad by using a film spraying machine, and fully drying at 37 ℃.
(2) And compounding the processed sample pad, the combination pad, the nitrocellulose membrane and the absorbent paper on a bottom plate, wherein the overlap of each part is 2mm, and thus the magnetic fluorescence immunochromatographic test strip is obtained.
(3) And (3) cutting the magnetic fluorescence immunochromatographic test strip in the step (2) into single test strips by using a strip cutting machine.
(4) And (3) loading the single test strip into a lower cover plate, compacting the test strip by using an upper cover plate, and assembling the test strip into a single box body to obtain the magnetic fluorescent microsphere immunochromatographic kit.
Further, the spraying concentration of the detection line of the magnetic fluorescence immunochromatographic test strip is 1mg/mL, the spraying concentration of the quality control line is 1mg/mL, and the spraying dosage is 0.4 uL/cm.
Further, the width of the magnetic fluorescence immunochromatographic test strip is 3.9 mm.
The invention also provides a preparation process of the magnetic fluorescent nanoparticles, which comprises the following steps:
(1) synthesis of Fe3O4Nano-particles: 1.59g FeCl was weighed2·4H2O and 2.59g FeCl3Dissolving in 150mL of deoxygenated water, and transferring to a three-necked bottle; adding 12.5mL of ammonia water, and reacting for 30min by mechanically stirring (500r/min) under the protection of nitrogen; and magnetically sucking and discarding the supernatant, washing the supernatant with ultrapure water until the pH value of the supernatant is 7, and magnetically sucking and discarding the supernatant.
(2) Preparation of oil-acidified Fe3O4Nano-particles: mixing Fe3O4Resuspending the nanoparticles into a three-necked bottle by using ultrapure water, mechanically stirring (500r/min) under the protection of nitrogen, heating to 70 ℃, adding 1g of oleic acid, and reacting for 3 hours; and (5) carrying out magnetic recovery, washing with ethanol for several times, and drying in an oven.
(3) Fine emulsification method for synthesizing Fe3O4Nanoparticle clusters: 0.3g of oil acidified Fe was dissolved in 1.5mL cyclohexane3O4Adding 30mL of 2.5mg/mL SDS solution into the nanoparticles, carrying out ultrasonic emulsification, transferring the mixture into a three-necked bottle, and stirring the mixture at 60 ℃ overnight to evaporate cyclohexane.
(4) Fe to be prepared3O4And magnetically absorbing and recovering the nanoparticle clusters, and adding Tween-80 aqueous solution for resuspending for 5 min. The Tween-80 modified nanoparticle cluster is added into a Stober system (ethanol, ammonia water, TEOS and water) which is prepolymerized for 20min to continue to react for 2 h. Magnetically attracting and recovering the magnetic beads, respectively cleaning the magnetic beads with ethanol and water for 3 times to prepare Fe3O4@SiO2Particles. Mixing Fe3O4@SiO2Magnetic recovery and re-suspension in ethanol, adding APTES, ultrasonic mixing and reacting for 1h to obtain Fe with amino group on surface3O4@SiO2-NH2And (4) drying the nano particles in vacuum.
(5) Taking 100 mu g of Fe3O4@SiO2-NH2Washed 3 times with ethanol, 3 times with EtOH/0.25mM HCl (500/70, v/v) mix, resuspended in 570 μ LEtOH/0.25mM HCl mix; 2 μ L of QSH-QDs was added to 250 μ L of DMSO/H2Mixing the mixture with O (1/1, v/v) in a vortex manner; DMSO/H of QDs2Dropwise adding O solution to Fe3O4@SiO2-NH2Swirling for 1min, mixing, standing for adsorbing for 10min, and making into Fe3O4@SiO2@NH2-QDs complexes; magnetic recovery, resuspension in ethanol-water (1: 1, v/v), refrigerator storage at 4 ℃.
(6) Blocking of Fe with PAA by active ester method3O4@SiO2@NH2Residual amino groups on QDs. Preparing polyacrylic acid (PAA) aqueous solution (pH is 6), adding EDC and NHSS aqueous solution, mixing by vortex, and activating at room temperature for 15 min. Taking 100ug Fe3O4@SiO2-NH2-QD magnetic recovery, addition of PAA activation product, ultrasonic resuspension, pH adjustment to 7.4. The reaction was carried out in a shaker at 30 ℃ and 180rpm for 3h with shaking. Magnetic attraction recovery of Fe3O4@SiO2PAA, washed 3 times with water and resuspended in ultrapure water.
Furthermore, the magnetic fluorescent nanoparticles are core-shell type magnetic fluorescent microspheres with the average hydrated particle size of 184nm, the emission wavelengths are 590nm and 630nm, and the magnetic fluorescent microspheres are bifunctional magnetic fluorescent microspheres with magnetic responsiveness and fluorescent characteristics.
The invention also provides a preparation process of the specific monoclonal antibody particle redissolution of the drugs marked by the magnetic fluorescent particles, which comprises the following steps:
(1) magnetic separation of 500. mu.g Fe3O4@SiO2PAA, washed 3 times with borate buffer (0.05M, pH6.0) and resuspended in 5mL of borate buffer.
(2) Adding 1.5 μ g drug antibody to be labeled, and performing electrostatic adsorption for 30 min.
(3) Coupling was performed at room temperature for 30min with 25. mu.g EDC and blocked with 500. mu.L of 10% BSA.
(4) Centrifuging for 15min at the rotation speed of 15000rpm at 4 ℃ by using a centrifuge, removing supernatant, and re-dissolving the precipitate by using a re-dissolving solution to obtain the specific monoclonal antibody of the drug with the detection probe marked by the magnetic fluorescent nano particles.
Further, the double solution contained 50. mu.L of a solution containing 25% sucrose, 1% BSA and 0.1% NaN3。
The invention also provides a method for carrying out on-site drug detection by using the kit for on-site detection of the amphetamine, morphine and ecstasy in the hair based on the magnetic fluorescence immunoassay technology, which comprises the following steps:
(1) cutting hair with hair follicle.
(2) The hair is cleaned with detergent and dried.
(3) Dividing the hair into several sections at the distance of 1cm from the hair root, and placing each section of hair in different centrifugal tubes.
(4) Adding a plurality of quartz sands, and fully oscillating to rapidly crush the hair in the collision process.
(5) Pouring hair lysate, covering the tube cover tightly, and oscillating sufficiently to extract drugs in hair follicles.
(6) And (3) dropwise adding a proper amount of supernatant of the hair lysate into the sample adding hole of the box body, so that the supernatant is diffused from the sample pad of the magnetic fluorescence immunochromatography test strip along the chromatography direction, and waiting for 10min for the detection result.
(7) And reading the fluorescence signal value by using a fluorescence reader, scanning the two-dimensional code of the upper cover plate to obtain a standard curve of the magnetic fluorescence immunochromatographic test strip, calculating the corresponding concentration of the drug to be detected, and deducing the drug absorption history of the detected person.
Further, the hair detergent includes one of water and acetone, 0.1% SDS, dichloromethane.
Further, the hair lysate comprises 10000IU/L of keratinase, 35.5g/L of disodium hydrogen phosphate, 2g/L of casein, 2g/L of polyvinylpyrrolidone and 0.2g/L of sodium azide.
The biological sample detected by the invention is hair, and the hair analysis has unique advantages in the field of forensic drug testing. The hair can provide long-term drug use information which is not provided by other biological detection materials and the like; the hair has the advantages of stable property, convenient material taking, easy storage, long detection time limit, wide application range, less pollution chance and the like; drugs are metabolized slowly in hair and can exist for a long time, and the factors provide better conditions for drug analysis; in addition, hair care processes (coloring, perming, bleaching, etc.) can reduce, but not completely eliminate, drug retention in the hair.
It is generally considered that the drug and its main metabolites mainly diffuse into the hair through blood circulation, stay in the hair follicle, disperse into each hair, once the drug enters the hair, the drug is wrapped in keratin, and the drug is stable in property and not easy to be decomposed. The kit for detecting the amphetamine, morphine and ecstasy in the hair on site based on the magnetic fluorescence immunoassay technology contains hair lysate, and the hair lysate aims at extracting drugs in keratin.
The invention has the beneficial effects that:
1) the magnetic fluorescence immunochromatographic assay has strong anti-matrix interference capability, can quickly separate target drugs from complex biological samples, and has improved sensitivity compared with quantum dot fluorescence immunochromatographic assay and colloidal gold immunochromatographic assay.
(2) The detection time is short, the detection result can be quickly obtained within 15min, and the field detection is realized.
(3) Strong magnetic response and high stability.
(4) Simple operation, simple flow and no need of professional instruments and professional detection personnel.
(5) The hair detection can be traced, the drug absorption time is long, and the method can be applied to detection of more scenes.
Drawings
FIG. 1 is a schematic structural diagram of a test strip in example 1 of the present invention; 1-base plate, 2-sample pad, 3-conjugate pad, 4-T1Line, 5-T2Line, 6-T3Line, 7-C line, 8-nitrocellulose membrane, 9-absorbent pad; a is the direction of chromatography;
FIG. 2 is a schematic structural diagram of a cartridge in embodiment 1 of the present invention; 10-two-dimensional code, 11-clamping shell upper cover plate, 12-observation window and 13-sample adding hole;
FIG. 3 is a standard curve of the ice toxicity in example 5 of the present invention;
figure 4 is a standard curve for morphine in example 5 of the present invention;
FIG. 5 is a calibration curve of the ecstasy pill of example 5 of the present invention.
Detailed Description
The present invention will be further described with reference to the accompanying drawings, and it should be noted that the present embodiment is based on the technical solution, and the detailed implementation and the specific operation process are provided, but the protection scope of the present invention is not limited to the present embodiment.
Example 1
As shown in fig. 1, this embodiment 1 provides a magnetic fluorescent microsphere immunochromatographic kit for on-site detection, which includes a kit body, a magnetic fluorescent immunochromatographic test strip and a hair lysate, wherein the magnetic fluorescent immunochromatographic test strip includes a bottom plate 1, a nitrocellulose membrane 8(NC membrane) is disposed on the bottom plate, a sample pad 2 and a combination pad 3 are sequentially adhered to one side of the nitrocellulose membrane 8 according to a chromatography direction, a water absorption pad 9 is adhered to the other side of the nitrocellulose membrane 8, magnetic fluorescent nanoparticles labeled with a drug monoclonal antibody are fixed on the combination pad, three detection lines (T lines) and a quality control line (C line) are disposed on the nitrocellulose membrane, the detection lines are respectively coated with complexes of bovine serum albumin coupled ice poison, morphine and ecstasy, the quality control line is coated with goat anti-mouse polyclonal antibody, the hair lysate is intended to dissolve drug small molecules from hair, so as to realize high-efficiency, accurate and sensitive drug detection.
Further, the bottom plate 1 is a PVC plastic bottom plate.
Further, the box body comprises a card shell upper cover plate 11 and a card shell lower cover plate, wherein the card shell upper cover plate 11 is provided with a sample adding hole 13, an observation window 12 and a two-dimensional code 10 comprising a product standard curve and batch number information, as shown in fig. 2. The sample adding hole corresponds to the position of the sample pad, and the observation window corresponds to the position of the nitrocellulose membrane.
Example 2
The present embodiment provides a method for manufacturing the kit of embodiment 1, comprising the following steps:
(1) respectively pointing a bovine serum albumin coupled compound of the methamphetamine, the morphine and the dancing head pill and a film pointing machine for an goat anti-mouse antibody on a detection line and a quality control line of a nitrocellulose membrane, and fully drying at 37 ℃ to ensure that the bovine serum albumin coupled compound of the methamphetamine, the morphine and the dancing head pill and the goat anti-mouse antibody are respectively and firmly fixed on the detection line and the quality control line of the nitrocellulose membrane; spraying the specific monoclonal antibody of the drug marked by the magnetic fluorescent nano particles on the binding pad by using a film spraying machine, and fully drying at 37 ℃.
(2) And compounding the processed sample pad, the combination pad, the nitrocellulose membrane and the absorbent paper on a bottom plate, wherein the overlap of each part is 2mm, and thus the magnetic fluorescence immunochromatographic test strip is obtained.
(3) And (3) cutting the magnetic fluorescence immunochromatographic test strip in the step (2) into single test strips by using a strip cutting machine.
(4) And (3) loading the single test strip into a lower cover plate, compacting the test strip by using an upper cover plate, and assembling the test strip into a single box body to obtain the magnetic fluorescent microsphere immunochromatographic kit.
(5) And (4) performing a spot check on the sensitivity, specificity and stability of the product, and leaving the factory if the product is qualified.
In this embodiment, the preparation process of the magnetic fluorescent nanoparticles is as follows:
(1) synthesis of Fe3O4Nano-particles: 1.59g FeCl was weighed2·4H2O and 2.59g FeCl3Dissolving in 150mL of deoxygenated water, and transferring to a three-necked bottle; adding 12.5mL of ammonia water, and reacting for 30min by mechanically stirring (500r/min) under the protection of nitrogen; and magnetically sucking and discarding the supernatant, washing the supernatant with ultrapure water until the pH value of the supernatant is 7, and magnetically sucking and discarding the supernatant.
(2) Preparation of oil-acidified Fe3O4Nano-particles: mixing Fe3O4Resuspending the nanoparticles into a three-necked bottle by using ultrapure water, mechanically stirring (500r/min) under the protection of nitrogen, heating to 70 ℃, adding 1g of oleic acid, and reacting for 3 hours; and (5) carrying out magnetic recovery, washing with ethanol for several times, and drying in an oven.
(3) Fine emulsification method for synthesizing Fe3O4Nanoparticle clusters: 0.3g of oil acidified Fe was dissolved in 1.5mL cyclohexane3O4Adding 30mL of 2.5mg/mL SDS solution into the nanoparticles, carrying out ultrasonic emulsification, transferring the mixture into a three-necked bottle, and stirring the mixture at 60 ℃ overnight to evaporate cyclohexane.
(4) Fe to be prepared3O4And magnetically absorbing and recovering the nanoparticle clusters, and adding Tween-80 aqueous solution for resuspending for 5 min. The Tween-80 modified nanoparticle cluster is added into a Stober system (ethanol, ammonia water, TEOS and water) which is prepolymerized for 20min to continue to react for 2 h. Magnetically attracting and recovering the magnetic beads, respectively cleaning the magnetic beads with ethanol and water for 3 times to prepare Fe3O4@SiO2Particles. Mixing Fe3O4@SiO2Magnetic recovery and re-suspension in ethanol, adding APTES, ultrasonic mixing and reacting for 1h to obtain Fe with amino group on surface3O4@SiO2-NH2And (4) drying the nano particles in vacuum.
(5) Taking 100 mu g of Fe3O4@SiO2-NH2Washed 3 times with ethanol, 3 times with EtOH/0.25mM HCl (500/70, v/v) mix, resuspended in 570 μ LEtOH/0.25mM HCl mix; 2 μ L of QSH-QDs was added to 250 μ L of DMSO/H2Mixing the mixture with O (1/1, v/v) in a vortex manner; DMSO/H of QDs2Dropwise adding O solution to Fe3O4@SiO2-NH2Swirling for 1min, mixing, standing for adsorbing for 10min, and making into Fe3O4@SiO2@NH2-QDs complexes; magnetic recovery, resuspension in ethanol-water (1: 1, v/v), refrigerator storage at 4 ℃.
(6) Blocking of Fe with PAA by active ester method3O4@SiO2@NH2Residual amino groups on QDs. Preparing polyacrylic acid (PAA) aqueous solution (pH is 6), adding EDC and NHSS aqueous solution, mixing by vortex, and activating at room temperature for 15 min. Taking 100ug Fe3O4@SiO2-NH2-QD magnetic recovery, addition of PAA activation product, ultrasonic resuspension, pH adjustment to 7.4. The reaction was carried out in a shaker at 30 ℃ and 180rpm for 3h with shaking. Magnetic attraction recovery of Fe3O4@SiO2PAA, washed 3 times with water and resuspended in ultrapure water.
Furthermore, the magnetic fluorescent nanoparticles are core-shell type magnetic fluorescent microspheres with the average hydrated particle size of 184nm, the emission wavelengths are 590nm and 630nm, and the magnetic fluorescent microspheres are bifunctional magnetic fluorescent microspheres with magnetic responsiveness and fluorescent characteristics.
Example 3
The preparation process of specific monoclonal antibody granule re-dissolved matter for drug marked with magnetic fluorescent particle includes the following steps:
(1) magnetic separation of 500. mu.g Fe3O4@SiO2PAA, washed 3 times with borate buffer (0.05M, pH6.0) and resuspended in 5mL of borate buffer.
(2) Adding 1.5 μ g drug antibody to be labeled, and performing electrostatic adsorption for 30 min.
(3) Coupling was performed at room temperature for 30min with 25. mu.g EDC and blocked with 500. mu.L of 10% BSA.
(4) Centrifuging for 15min at the rotation speed of 15000rpm at 4 ℃ by using a centrifuge, removing supernatant, and re-dissolving the precipitate by using a re-dissolving solution to obtain the specific monoclonal antibody of the drug with the detection probe marked by the magnetic fluorescent nano particles.
Further, the double solution comprises 50 μ L of a solution containing 25% sucrose,1% BSA and 0.1% NaN3。
Example 4
The invention also provides a method for carrying out on-site drug detection by using the kit for on-site detection of the amphetamine, morphine and ecstasy in the hair based on the magnetic fluorescence immunoassay technology, which comprises the following steps:
(1) cutting hair with hair follicle.
(2) The hair is cleaned with detergent and dried.
(3) Dividing the hair into several sections at the distance of 1cm from the hair root, and placing each section of hair in different centrifugal tubes.
(4) Adding a plurality of quartz sands, and fully oscillating to rapidly crush the hair in the collision process.
(5) Pouring hair lysate, covering the tube cover tightly, and oscillating sufficiently to extract drugs in hair follicles.
(6) And (3) dropwise adding a proper amount of supernatant of the hair lysate into the sample adding hole of the box body, so that the supernatant is diffused from the sample pad of the magnetic fluorescence immunochromatography test strip along the chromatography direction, and waiting for 10min for the detection result.
(7) And reading the fluorescence signal value by using a fluorescence reader, scanning the two-dimensional code of the upper cover plate to obtain a standard curve of the magnetic fluorescence immunochromatographic test strip, calculating the corresponding concentration of the drug to be detected, and deducing the drug absorption history of the detected person.
Example 5
This example provides a performance verification of the kit described in example 1.
1. Establishment of a Standard Curve
Diluting the standard substance of the ice toxin, the morphine and the shaking head pills into 0ng/mL, 0.025ng/mL, 0.05, 0.1ng/mL, 0.2ng/mL and 0.5ng/mL, wherein the diluent is 0.01M PBS, carrying out magnetic fluorescence immunoassay test strip detection, and detecting the fluorescence intensity of a window of a device by using a matched fluorescence detector. The average was taken 2 times for each concentration measurement as shown in table 1.
Table 1 data set up for standard curves
And (3) fitting the data of the table 1 by Origin software to obtain a relational graph of the T/C value and the corresponding concentration to obtain an equation. As shown in fig. 3, 4, 5.
The technologies of the above embodiments may be combined arbitrarily, and for the sake of brevity, all possible combinations of the features in the above examples are not described, but should be considered as the scope of the present specification as long as there is no contradiction between the combinations of the features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (10)
1. The utility model provides a magnetism fluorescence microballon immunochromatography kit for witnessed inspections, its characterized in that, includes box body, magnetism fluorescence immunochromatography test paper strip and hair lysate, magnetism fluorescence immunochromatography test paper strip includes the bottom plate, is equipped with the nitrocellulose membrane on the bottom plate, nitrocellulose membrane one side is stained with sample pad and combination pad according to the chromatography direction in proper order, and the cellulose nitrate membrane opposite side is stained with the pad that absorbs water, be fixed with the magnetism fluorescence nanoparticle of mark drug monoclonal antibody on the combination pad, be equipped with three detection lines and a quality control line on the cellulose nitrate membrane, the detection line is wrapped up the complex that has bovine serum albumin coupling virus, morphine, panning head-shaking pill respectively, and the quality control line is wrapped up goat anti mouse polyclonal antibody, the hair lysate contains the active ingredient of schizolysis hair keratin.
2. The kit of claim 1, wherein the combination pad and the absorbent pad are respectively located on two sides of the nitrocellulose membrane and are respectively lapped on the nitrocellulose membrane, the sample pad is lapped on the combination pad, and the bottom plate is made of a PVC plastic plate.
3. The magnetic fluorescent microsphere immunochromatographic kit for on-site detection according to claim 1, wherein the kit body comprises a card shell upper cover plate and a card shell lower cover plate, the card shell upper cover plate is provided with a sample application hole, an observation window, and a two-dimensional code comprising a product standard curve and batch number information; the sample adding hole corresponds to the position of the sample pad, and the observation window corresponds to the position of the nitrocellulose membrane.
4. The method for manufacturing the magnetic fluorescent microsphere immunochromatographic kit for in-situ detection according to claim 1, comprising the following steps:
(1) respectively pointing a bovine serum albumin coupled compound of the methamphetamine, the morphine and the dancing head pill and a film pointing machine for an goat anti-mouse antibody on a detection line and a quality control line of a nitrocellulose membrane, and fully drying at 37 ℃ to ensure that the bovine serum albumin coupled compound of the methamphetamine, the morphine and the dancing head pill and the goat anti-mouse antibody are respectively and firmly fixed on the detection line and the quality control line of the nitrocellulose membrane; spraying the specific monoclonal antibody of the drug marked by the magnetic fluorescent nano particles on a binding pad by using a film spraying machine, and fully drying at 37 ℃;
(2) compounding the processed sample pad, the combination pad, the nitrocellulose membrane and the water absorption pad on a bottom plate, and overlapping each part by 2mm to obtain the magnetic fluorescence immunochromatographic test strip;
(3) cutting the magnetic fluorescence immunochromatographic test strip in the step (2) into single test strips by using a strip cutting machine;
(4) and (3) loading the single test strip into a lower cover plate of the card shell, compacting the test strip by using an upper cover plate of the card shell, and assembling the test strip into a single box body to obtain the magnetic fluorescent microsphere immunochromatographic kit.
5. The manufacturing method of claim 4, wherein the detection line spray concentration of the magnetic fluorescence immunochromatographic test strip is 1mg/mL, the quality control line spray concentration is 1mg/mL, and the spray dosage is 0.4 uL/cm; the width of the magnetic fluorescence immunochromatographic test strip is 3.9 mm.
6. The method of claim 4, wherein the magnetic fluorescent nanoparticles are prepared by the following steps:
(1) synthesis of Fe3O4Nano-particles: 1.59g FeCl was weighed2·4H2O and 2.59g FeCl3Dissolving in 150mL of deoxygenated water, and transferring to a three-necked bottle; adding 12.5mL of ammonia water, and mechanically stirring and reacting for 30min under the protection of nitrogen; magnetically absorbing and discarding the supernatant, cleaning the supernatant with ultrapure water until the pH value of the supernatant is 7, and magnetically absorbing and discarding the supernatant;
(2) preparation of oil-acidified Fe3O4Nano-particles: mixing Fe3O4Resuspending the nanoparticles into a three-necked bottle by using ultrapure water, mechanically stirring and heating to 70 ℃ under the protection of nitrogen, adding 1g of oleic acid, and reacting for 3 hours; magnetic recovery, cleaning with ethanol for several times, and drying in oven;
(3) fine emulsification method for synthesizing Fe3O4Nanoparticle clusters: 0.3g of oil acidified Fe was dissolved in 1.5mL cyclohexane3O4Adding 30mL of 2.5mg/mL SDS solution into the nanoparticles, ultrasonically emulsifying, transferring into a three-necked bottle, stirring at 60 ℃, standing overnight, and evaporating cyclohexane;
(4) fe to be prepared3O4Magnetically absorbing and recovering the nano particle cluster, and adding Tween-80 aqueous solution for resuspending for 5 min; adding the Tween-80 modified nanoparticle cluster into a Stober system for prepolymerization for 20min to continue to react for 2 h; magnetically attracting and recovering the magnetic beads, respectively cleaning the magnetic beads with ethanol and water for 3 times to prepare Fe3O4@SiO2Particles; mixing Fe3O4@SiO2Magnetic recovery and re-suspension in ethanol, adding APTES, ultrasonic mixing and reacting for 1h to obtain Fe with amino group on surface3O4@SiO2-NH2Nano particles are dried in vacuum;
(5) taking 100 mu g of Fe3O4@SiO2-NH2Washing with ethanol 3 times, washing with EtOH/0.25mM HCl mixture 3 times, and resuspending in 570 μ LEtOH/0.25mM HCl mixture; take 2 muAddition of L QSH-QDs to 250. mu.L DMSO/H2In the O mixed solution, uniformly mixing by vortex; DMSO/H of QDs2Dropwise adding O solution to Fe3O4@SiO2-NH2Swirling for 1min, mixing, standing for adsorbing for 10min, and making into Fe3O4@SiO2@NH2-QDs complexes; magnetically recovering, suspending in ethanol-water, and storing in refrigerator at 4 deg.C;
(6) blocking of Fe with PAA by active ester method3O4@SiO2@NH2-residual amino groups on QDs; preparing a polyacrylic acid aqueous solution, adding an EDC and NHSS aqueous solution, uniformly mixing by vortex, and activating at room temperature for 15 min; taking 100ug Fe3O4@SiO2-NH2-QD magnetic recovery, addition of PAA activation product, ultrasonic resuspension, pH adjustment to 7.4; oscillating and reacting for 3 hours at 30 ℃ and 180rpm in a shaking table; magnetic attraction recovery of Fe3O4@SiO2PAA, washed 3 times with water and resuspended in ultrapure water.
7. The preparation method of claim 4, wherein the magnetic fluorescent nanoparticles are core-shell magnetic fluorescent microspheres with an average hydrated particle size of 184nm, and the emission wavelengths are 590nm and 630nm, and the magnetic fluorescent microspheres have magnetic responsiveness and fluorescent characteristics.
8. The method according to claim 4, wherein the monoclonal antibody specific to the drug labeled with the magnetic fluorescent nanoparticles is prepared by the following steps:
(1) magnetic separation of 500. mu.g Fe3O4@SiO2PAA, washed 3 times with borate buffer (0.05M, pH6.0) and resuspended in 5mL of borate buffer.
(2) Adding 1.5 μ g drug antibody to be labeled, and performing electrostatic adsorption for 30 min.
(3) Coupling was performed at room temperature for 30min with 25. mu.g EDC and blocked with 500. mu.L of 10% BSA.
(4) Centrifuging at 15000rpm at 4 deg.C for 15min, removing supernatant, and re-dissolving the precipitate with re-dissolving solution to obtain specific monoclonal antibody of drug labeled with magnetic fluorescent nanoparticlesA composition comprising 50 μ L of a composition comprising 25% sucrose, 1% BSA, and 0.1% NaN3。
9. A method for in-situ drug detection using the magnetic fluorescent microsphere immunochromatographic kit for in-situ detection according to any one of claims 1 to 8, comprising the steps of:
(1) cutting hair with hair follicle;
(2) cleaning hair with detergent and air drying;
(3) dividing the hair into a plurality of sections at the distance of 1cm from the hair root, and placing the hair of each section in different centrifugal tubes;
(4) adding a plurality of quartz sands, and fully oscillating to quickly crush the hair in the collision process;
(5) pouring hair lysate, covering the tube cover tightly, and oscillating sufficiently to extract drugs in hair follicles;
(6) dripping a proper amount of supernatant of the hair lysate into the sample adding hole of the box body, so that the supernatant is diffused from a sample pad of the magnetic fluorescence immunochromatography test strip along the direction of chromatography, and waiting for 10min for the detection result;
(7) and reading the fluorescence signal value by using a fluorescence reader, scanning the two-dimensional code of the upper cover plate to obtain a standard curve of the magnetic fluorescence immunochromatographic test strip, calculating the corresponding concentration of the drug to be detected, and deducing the drug absorption history of the detected person.
10. The detection method according to claim 9, wherein the hair detergent is one of water and acetone, 0.1% SDS, dichloromethane; the hair lysate comprises 10000IU/L of keratinase, 35.5g/L of disodium hydrogen phosphate, 2g/L of casein, 2g/L of polyvinylpyrrolidone and 0.2g/L of sodium azide.
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