CN113466463A - Kit for quantum dot microsphere immunity triple on-site detection and manufacturing method and detection method thereof - Google Patents
Kit for quantum dot microsphere immunity triple on-site detection and manufacturing method and detection method thereof Download PDFInfo
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- CN113466463A CN113466463A CN202110531426.XA CN202110531426A CN113466463A CN 113466463 A CN113466463 A CN 113466463A CN 202110531426 A CN202110531426 A CN 202110531426A CN 113466463 A CN113466463 A CN 113466463A
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- MARUHZGHZWCEQU-UHFFFAOYSA-N 5-phenyl-2h-tetrazole Chemical compound C1=CC=CC=C1C1=NNN=N1 MARUHZGHZWCEQU-UHFFFAOYSA-N 0.000 description 1
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Images
Classifications
-
- 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
- G01N33/588—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with semiconductor nanocrystal label, e.g. quantum dots
-
- 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
- G01N33/53—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
-
- 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
- G01N33/53—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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- 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
- G01N33/53—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
Abstract
The invention discloses a kit for quantum dot microsphere immune triple on-site detection, a manufacturing method thereof and a detection method thereof. A drug antibody-quantum dot fluorescent microsphere particle compound is arranged on the combination pad; the nitrocellulose membrane is provided with three detection lines (T lines) and a quality control line (C line), the detection lines are coated with drug antigen-bovine serum albumin compound, and the quality control line is coated with goat anti-mouse antibody. The kit can rapidly, accurately and sensitively detect the amphetamine, morphine and ecstasy in the hair.
Description
Technical Field
The invention relates to the technical field of biological detection, in particular to a reagent kit for quantum dot microsphere immunity triple on-site detection, and a manufacturing method and a detection method thereof.
Background
Currently, biological samples related to drug detection include urine, saliva, blood, hair, and the like. The metabolism of the drugs in urine, saliva and blood is fast, the drug taking situation of a drug taker can be reflected only within a few days, and the drugs in the hair have good stability, so the hair analysis has unique advantages in the field of court drug inspection. The hair can provide long-term medication information which is not provided by other biological detection materials such as blood, urine, saliva 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 in the hair can be metabolized slowly and exist for a long time, and the factors provide better conditions for drug analysis.
Common drug detection methods include chromatography, capillary electrophoresis, chemical color development, colloidal gold method and the like. The chromatography mainly comprises a gas chromatography-mass spectrometry combined method and a liquid chromatography-mass spectrometry combined method. The chromatography has the advantages of high efficiency, high selection, small sample consumption and the like, but the pretreatment process is complicated, the detection time is long, professional personnel operation and large-volume instrument equipment are required, and the detection limitation is large. For example, the liquid chromatography-mass spectrometry combined method can only analyze and detect substances with polarity, difficult volatilization and uncertain heat, and also needs derivatization treatment on nonpolar substances, so that drug detection is more complicated. Capillary electrophoresis combines the advantages of electrophoresis and chromatography, but also has the defects of few processed samples, incapability of collecting a large amount of samples and the like. The chemical method is mainly characterized in that specific chemical reagents and drug detection materials are subjected to chemical reactions such as color development, precipitation and the like to identify the types of drugs, and the method can be used for quickly performing qualitative detection, but has the advantages of higher detection limit, low sensitivity and poor specificity, and cannot detect trace drugs with similar chemical structures. Compared with the conventional instrument analysis method, the colloidal gold method has the characteristics of convenience, rapidness and easy operation. However, the color judgment of the colloidal gold method has strong subjectivity and causes misjudgment, and the problem of high probability of false positive caused by the influence of drugs occurs, so the colloidal gold test paper can only be used as a primary screen of drugs.
The marking materials adopted in the traditional rapid immunoassay method mainly comprise colloidal gold, fluorescent microspheres, time fluorescence resolution microspheres and the like. The colloidal gold immune label is combined by adopting a physical adsorption method, an antigen/antibody is easy to separate from the surface of a gold particle, a label is unstable, the sensitivity is low, and only a qualitative or semi-quantitative result can be given; the fluorescent microsphere immunolabeling has the defects that the fluorescent dye system is physically doped and is easy to leak, meanwhile, the surface modification of the polymer microsphere is not flexible enough, and due to the hydrophobic property of most of the polymer microspheres, nonspecific adsorption is easy to occur. The larger time-resolved fluorescent microsphere particles lead to insufficient fluorescent reaction and often result in false positive results. The quantum dots are a novel nano fluorescent material which takes cadmium telluride, cadmium selenide, cadmium sulfide and the like as main components and has electrochemical luminescence and photoluminescence characteristics. Quantum dots have several distinct advantages over traditional labels: the excitation spectrum is wide, the emission wavelength is narrow, excitation light with any wavelength which is less than 10nm of the emission wavelength can be used for excitation, the emission wavelength can be changed by changing the size of the quantum dots, and the method is suitable for multi-mark detection; the fluorescence lifetime is long, and after the light is excited for a plurality of nanoseconds, the autofluorescence background is already attenuated, and the quantum dot fluorescence still exists, so that a high signal-to-noise ratio can be obtained; the biological compatibility is good, the activity of the biological macromolecule specimen is not damaged, and the specific connection can be carried out after various chemical modifications. Therefore, the excellent fluorescence properties of quantum dots make it widely applicable in biomedical detection and diagnosis.
Disclosure of Invention
In order to overcome the technical defects, the invention aims to provide a reagent kit for quantum dot microsphere immunity triple on-site detection, a manufacturing method and a detection method thereof, which can detect whether a detected person dilutes a drug for a long time on site, achieve the aim of rapid detection and improve the stability, sensitivity and accuracy of drug detection.
In order to achieve the purpose, the invention adopts the following technical scheme:
the utility model provides a kit of quantum dot microballon immunity trigeminy field test, includes box body, quantum dot fluorescence immunochromatography test paper strip and lysate, quantum dot fluorescence immunochromatography test paper strip is located in the box body, includes the bottom plate and establishes sample pad, combination pad, cellulose nitrate membrane (NC membrane) and the paper that absorbs water that connect in order from one end to the other end on the bottom plate. A drug antibody-quantum dot fluorescent microsphere particle compound is arranged on the combination pad; the nitrocellulose membrane is provided with three detection lines (T lines) and a quality control line (C line), the detection lines are coated with drug antigen-bovine serum albumin compound, and the quality control line is coated with goat anti-mouse antibody.
Furthermore, the combination pad is sprayed with a probe for detecting drugs, namely a drug antibody-quantum dot fluorescent microsphere particle composite; the nitrocellulose membrane is provided with three detection lines (T lines) and one quality control line (C line), the detection lines are coated with a whole antigen, namely a drug antigen-bovine serum albumin compound, and the quality control lines are coated with a secondary antibody, namely a goat anti-mouse antibody.
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, reading window, including product standard curve and batch number information. The sample adding holes correspond to the positions of the sample pad, the observation windows correspond to the positions of the nitrocellulose membrane one by one.
The invention also provides a preparation method of the kit for the quantum dot microsphere immunity triple on-site detection, which comprises the following steps:
(1) respectively dotting the drug antigen-bovine serum albumin compound and the goat anti-mouse antibody on a nitrocellulose membrane by using a film dotting machine to serve as a detection line and a quality control line of the quantum dot fluorescence immunochromatography test strip, and drying for 12 hours; spraying the specific monoclonal antibody particle resolvent of the drug marked by the quantum dot fluorescent microspheres on a binding pad by using a film dispenser, and drying for 12 hours.
(2) And compounding the processed sample pad, the combination pad, the nitrocellulose membrane and the absorbent paper on a bottom plate to obtain the quantum dot fluorescence immunochromatographic test strip.
(3) And (3) cutting the quantum dot fluorescence immunochromatographic test strip in the step (2) into single test strips by using a slitter.
(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 fluorescence-immunity triple detection hair poison kit.
Furthermore, the concentration of the spray dots of the detection line and the quality control line of the quantum dot fluorescence immunochromatographic test strip is 1mg/mL, and the dosage of the spray dots is 0.4 uL/cm.
Further, the width of the quantum dot fluorescence immunochromatographic test strip is 3.9 mm.
Further, the preparation process of the specific monoclonal antibody particle redissolution of the drugs marked by the quantum dot fluorescent microspheres comprises the following steps:
(1) the quantum dot fluorescent microspheres are prepared into borate buffer solution with the concentration of 1 wt%, and the borate buffer solution is fully ultrasonically diluted to the pH value of 6.
(2) And adding 25ug of EDC activator solution into 5mL of quantum dot fluorescent microsphere solution, and reacting for 15 min.
(3) Adding 500uL of drug antibody to be labeled to react for 1h, firstly adding 500uL of 10% BSA blocking solution, and then adding 25ug of EDC to react for 1 h. The concentration of the drug antibody to be marked is 3 ug/mg.
(4) Centrifuging at 15000rpm at 4 deg.C for 15min, removing supernatant, and dissolving precipitate with redissolution. Obtaining the specific monoclonal antibody particle compound of the drug marked by the drug antibody-quantum dot fluorescent microsphere.
Further, the average particle size of the quantum dot fluorescent microspheres is 150nm, and the concentration is 15 mg/mL.
Further, the complex solution is PNPB solution and contains 0.4% of Tween-20 surfactant.
The invention also provides a method for carrying out on-site drug detection by using the kit for the quantum dot microsphere immune triple on-site detection, which comprises the following steps:
(1) collecting hair by cutting or scraping, collecting hair and taking down together with hair follicle.
(2) And (4) cleaning dirt on the hair surface by using a detergent, and airing.
(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) Two small iron ball collision beads are added, and the vibration is carried out fully, so that the hair can be crushed quickly in the crushing process.
(5) And (3) pouring hair lysate into the centrifugal tube, covering the tube cover tightly, and fully oscillating to expose drugs in the hair follicle.
(6) And (3) sucking the supernatant of the hair lysate by using a disposable plastic straw, dripping a proper amount of the supernatant on a sample pad of a quantum dot fluorescence immunochromatography test strip, and waiting for 10 min.
(7) And reading the fluorescence signal value by using a fluorescence detector, scanning the two-dimensional code on the kit body to obtain a standard curve of the quantum dot fluorescence immunochromatographic test strip, calculating the corresponding concentration of the drug to be detected, and deducing the drug absorption history of the detected personnel.
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 which can provide long-term medication information which is not provided by other biological detection materials such as blood, urine, saliva, bile 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 in the hair can be metabolized slowly and exist for a long time, and the factors provide better conditions for drug analysis.
It is believed that the toxin enters the blood, passively diffuses from the capillary to the growing cells, stays in the hair follicle, and then disperses to each hair, and once the drug enters the hair, the drug is firmly wrapped in keratin, is relatively stable, and is not easily decomposed. The kit for detecting the drugs in the hair in the fluorescence-immunoassay triple field comprises hair lysate which aims at releasing the drugs in the keratin freely.
The invention has the beneficial effects that:
(1) the detection kit has small volume, is convenient to carry and simple and convenient to operate, and does not need professional instruments and equipment or professional workers.
(2) The sensitivity of detecting drugs is improved by adopting a quantum dot fluorescence immunochromatography technology.
(3) The detection speed is high, the detection result can be obtained within 15min of the operation reading time, and great support is provided for on-site screening.
(4) Good stability, stable fluorescence signal of quantum dots, and light bleaching resistance.
(5) The drug taking time of drug addicts can be traced back, and whether the detected person takes the drug in half a year can be detected. .
Drawings
FIG. 1 is a schematic structural diagram of a test strip in example 1 of the present invention; 1-sample pad, 2-conjugate pad, 3-nitrocellulose membrane, 4-absorbent pad, 5-T1Line, 6-T2Line, 7-T3Line, 8-C line, 9-bottom plate; 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 embodiment.
Example 1
As shown in fig. 1, this embodiment 1 provides a kit for quantum dot microsphere immune triple field detection, including a box body, a quantum dot fluorescence immunochromatographic test strip and a lysis solution, the quantum dot fluorescence immunochromatographic test strip is arranged in the box body, and includes a bottom plate 9, and a sample pad 1, a combination pad 2, a nitrocellulose membrane 3 (NC membrane) and a water absorbent paper 4 which are arranged on the bottom plate 9 and connected in order from one end to the other end. A drug antibody-quantum dot fluorescent microsphere particle compound is arranged on the combination pad 2; the nitrocellulose membrane 3 is provided with three detection lines (T lines) and a quality control line (C line), the detection lines are coated with a drug antigen-bovine serum albumin compound, and the quality control line is coated with a goat anti-mouse antibody.
The bottom plate 9 is a PVC plastic bottom plate.
The box body includes card shell upper cover plate 11 and card shell lower cover plate, card shell upper cover plate 11 is equipped with application of sample hole 13, observation window 12, including the product standard curve and the two-dimensional code of batch number information, as shown in fig. 2. The sample adding holes 13 correspond to the positions of the sample pad 2 and the positions of the observation windows 12 and the nitrocellulose membrane 3 one by one.
Example 2
The present embodiment provides a method for manufacturing the kit of embodiment 1, comprising the following steps:
(1) respectively dotting the drug antigen-bovine serum albumin compound and the goat anti-mouse antibody on a nitrocellulose membrane by using a film dotting machine to serve as a detection line and a quality control line of the quantum dot fluorescence immunochromatography test strip, and drying for 12 hours; spraying the specific monoclonal antibody particle resolvent of the drug marked by the quantum dot fluorescent microspheres on a binding pad by using a film dispenser, and drying for 12 hours.
(2) And compounding the processed sample pad, the bonding pad, the nitrocellulose membrane and the absorbent paper on a bottom plate to obtain the quantum dot.
(3) And (3) cutting the quantum dot fluorescence immunochromatographic test strip in the step (2) into single test strips by using a slitter.
(4) And (3) putting the single test strip into a bottom cover, compacting the test strip by using an upper cover, and assembling the test strip into a single box body to obtain the fluorescence-immunity triple detection hair poison 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.
The detection line and quality control line of the quantum dot fluorescence immunochromatographic test strip have the spray dot concentration of 1mg/mL, and the spray dot dosage is 0.4 uL/cm. The width of the quantum dot fluorescence immunochromatographic test strip is 3.9 mm.
In this embodiment, the preparation process of the specific monoclonal antibody particle redissolution of the drug labeled by the quantum dot fluorescent microsphere is as follows:
(1) the quantum dot fluorescent microspheres are prepared into borate buffer solution with the concentration of 1 wt%, and the borate buffer solution is fully ultrasonically diluted to the pH value of 6.
(2) And adding 25ug of EDC activator solution into 5mL of quantum dot fluorescent microsphere solution, and reacting for 15 min.
(3) Adding 500uL of drug antibody to be labeled to react for 1h, firstly adding 500uL of 10% BSA blocking solution, and then adding 25ug of EDC to react for 1 h. The concentration of the drug antibody to be marked is 3 ug/mg.
(4) Centrifuging at 15000rpm at 4 deg.C for 15min, removing supernatant, and dissolving precipitate with redissolution. Obtaining the specific monoclonal antibody particle compound of the drug marked by the drug antibody-quantum dot fluorescent microsphere.
The average particle size of the quantum dot fluorescent microspheres is 150nm, and the concentration of the quantum dot fluorescent microspheres is 15 mg/mL.
The complex solution is PNPB solution and contains 0.4% Tween-20 surfactant.
Example 3
The embodiment provides a method for carrying out on-site drug detection by using a quantum dot microsphere immune triple on-site detection kit, which comprises the following steps:
(1) collecting hair by cutting or scraping, collecting hair and taking down together with hair follicle.
(2) And (4) cleaning dirt on the hair surface by using a detergent, and airing.
(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) Two small iron ball collision beads are added, and the vibration is carried out fully, so that the hair can be crushed quickly in the crushing process.
(5) And (3) pouring hair lysate into the centrifugal tube, covering the tube cover tightly, and fully oscillating to expose drugs in the hair follicle.
(6) And (3) sucking the supernatant of the hair lysate by using a disposable plastic straw, dripping a proper amount of the supernatant on a sample pad of a quantum dot fluorescence immunochromatography test strip, and waiting for 10 min.
(7) And reading the fluorescence signal value by using a fluorescence detector, scanning the two-dimensional code on the kit body to obtain a standard curve of the quantum dot fluorescence immunochromatographic test strip, calculating the corresponding concentration of the drug to be detected, and deducing the drug absorption history of the detected personnel.
The hair detergent includes 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.
Example 4
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, 0.1 ng/mL, 0.2ng/mL and 0.5ng/mL, and using 0.01M PBS as diluent to perform fluorescence immunoassay test paper strip detection, and using a matched fluorescence analyzer to detect the fluorescence intensity of a detection window of a device. 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, which is shown in figures 3-5.
The technologies of the above-mentioned embodiments can be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the above-mentioned examples are not described, however, as long as there is no contradiction between the combinations of the technical features, the combination should be considered as the scope of the description of the present specification.
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 spirit of the invention, which falls within the scope of the invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (10)
1. The kit for quantum dot microsphere immune triple on-site detection is characterized by comprising a kit body, a quantum dot fluorescence immunochromatographic test strip and a lysis solution, wherein the quantum dot fluorescence immunochromatographic test strip is arranged in the kit body and comprises a bottom plate, and a sample pad, a combination pad, a nitrocellulose membrane and absorbent paper which are arranged on the bottom plate and connected in sequence from one end to the other end; a drug antibody-quantum dot fluorescent microsphere marked drug specific monoclonal antibody particle compound is arranged on the combination pad; the nitrocellulose membrane is provided with three detection lines and a quality control line, the detection lines are coated with a drug antigen-bovine serum albumin compound, and the quality control line is coated with a goat anti-mouse antibody.
2. The kit for the triple immunoassay field detection of the quantum dot microsphere of claim 1, wherein the bottom plate is made of a PVC plastic plate.
3. The kit for the triple immunoassay field detection of the quantum dot microsphere 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 adding hole, an observation window and a two-dimensional code comprising a product standard curve and batch number information; the sample adding holes correspond to the positions of the sample pad and the positions of the observation windows and the nitrocellulose membrane one by one.
4. The method for manufacturing the kit for the quantum dot microsphere immune triple in-situ detection as claimed in claim 1, which is characterized by comprising the following steps:
(1) respectively dotting the drug antigen-bovine serum albumin compound and the goat anti-mouse antibody on a nitrocellulose membrane by using a film dotting machine to serve as a detection line and a quality control line of the quantum dot fluorescence immunochromatography test strip, and drying for 12 hours; spraying the specific monoclonal antibody particle resolvent of the drug marked by the quantum dot fluorescent microspheres on a binding pad by using a film dispenser, and drying for 12 hours;
(2) compounding the processed sample pad, the combination pad, the nitrocellulose membrane and the absorbent paper on a bottom plate to obtain the quantum dot fluorescence immunochromatographic test strip;
(3) cutting the quantum dot fluorescence immunochromatographic test strip in the step (2) into single test strips by using a slitter;
(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 fluorescence-immunity triple detection hair poison kit.
5. The manufacturing method of claim 4, wherein the concentration of the spray dots on the detection line and the quality control line of the quantum dot fluorescence immunochromatographic test strip is 1mg/mL, and the dosage of the spray dots is 0.4 uL/cm; the width of the quantum dot fluorescence immunochromatographic test strip is 3.9 mm.
6. The method for preparing the drug particle resolvent labeled by the quantum dot fluorescent microspheres according to claim 4, wherein the preparation process of the drug particle resolvent labeled by the quantum dot fluorescent microspheres is as follows:
(1) preparing the quantum dot fluorescent microspheres into 1 wt% concentration, and performing sufficient ultrasound treatment, wherein the dilution buffer is a borate buffer with pH of 6;
(2) adding 25ug of EDC activator solution into 5mL of quantum dot fluorescent microsphere solution, and reacting for 15 min;
(3) adding 500uL of drug antibody to be marked to react for 1h, firstly adding 500uL of 10% BSA blocking solution, and then adding 25ug of EDC to react for 1 h; the concentration of the drug antibody to be marked is 3 ug/mg;
(4) centrifuging at 15000rpm at 4 deg.C for 15min, removing supernatant, and dissolving precipitate with redissolution; obtaining the specific monoclonal antibody particle compound of the drug marked by the drug antibody-quantum dot fluorescent microsphere.
7. The manufacturing method of claim 6, wherein the average particle size of the quantum dot fluorescent microspheres is 150nm, and the concentration is 15 mg/mL; the complex solution is PNPB solution and contains 0.4% Tween-20 surfactant.
8. A method for in-situ drug detection by using the kit for the quantum dot microsphere immune triple in-situ detection as described in any one of claims 1 to 7, which comprises the following steps:
(1) collecting hair by cutting or scraping, collecting hair and taking down together with hair follicle;
(2) cleaning dirt on the surface of the hair with a detergent, and airing;
(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 two small iron ball collision beads, and fully oscillating to rapidly crush the hair in the crushing process;
(5) pouring hair lysate into the centrifugal tube, tightly covering the tube cover, and fully oscillating to expose drugs in hair follicles; and (3) sucking the supernatant of the hair lysate by using a disposable plastic straw, dripping a proper amount of the supernatant on a sample pad of a quantum dot fluorescence immunochromatography test strip, and waiting for 10 min.
(6) And reading the fluorescence signal value by using a fluorescence detector, scanning the two-dimensional code on the kit body to obtain a standard curve of the quantum dot fluorescence immunochromatographic test strip, calculating the corresponding concentration of the drug to be detected, and deducing the drug absorption history of the detected personnel.
9. The detection method according to claim 8, wherein the hair detergent comprises one of water and acetone, 0.1% SDS, and dichloromethane.
10. The method of claim 8, wherein the hair lysate comprises 10000IU/L keratinase, 35.5g/L disodium hydrogen phosphate, 2g/L casein, 2g/L polyvinylpyrrolidone, and 0.2g/L sodium azide.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114252600A (en) * | 2021-11-08 | 2022-03-29 | 广州万德康科技有限公司 | Cat triple antibody detection test strip and preparation method and application thereof |
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| CN109061204A (en) * | 2018-07-30 | 2018-12-21 | 杭州莱和生物技术有限公司 | A kind of kit of fluorescence immunoassay detection hair trace drugs |
| CN109490538A (en) * | 2018-12-27 | 2019-03-19 | 公安部第研究所 | A kind of fluorescence immunoassay trace hair drugs four-in-one detection kit |
| CN110850093A (en) * | 2019-11-22 | 2020-02-28 | 佛山墨赛生物技术有限公司 | Test strip for rapidly and quantitatively detecting ketamine in hair, preparation method and kit |
| CN111443204A (en) * | 2020-02-25 | 2020-07-24 | 古镜科技(深圳)有限公司 | Fluorescent triple-detection test paper strip for narcotics in hair and preparation method thereof |
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| CN109061204A (en) * | 2018-07-30 | 2018-12-21 | 杭州莱和生物技术有限公司 | A kind of kit of fluorescence immunoassay detection hair trace drugs |
| CN109490538A (en) * | 2018-12-27 | 2019-03-19 | 公安部第研究所 | A kind of fluorescence immunoassay trace hair drugs four-in-one detection kit |
| CN110850093A (en) * | 2019-11-22 | 2020-02-28 | 佛山墨赛生物技术有限公司 | Test strip for rapidly and quantitatively detecting ketamine in hair, preparation method and kit |
| CN111443204A (en) * | 2020-02-25 | 2020-07-24 | 古镜科技(深圳)有限公司 | Fluorescent triple-detection test paper strip for narcotics in hair and preparation method thereof |
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| CN114252600A (en) * | 2021-11-08 | 2022-03-29 | 广州万德康科技有限公司 | Cat triple antibody detection test strip and preparation method and application thereof |
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