CN110873791A - Indirect background fluorescent colloidal gold immunochromatographic test strip based on double-labeled signal amplification and application thereof - Google Patents
Indirect background fluorescent colloidal gold immunochromatographic test strip based on double-labeled signal amplification and application thereof Download PDFInfo
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- 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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
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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
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
Abstract
The invention discloses an indirect background fluorescent colloidal gold immunochromatographic test strip based on double-label signal amplification and application thereof. The test strip comprises five parts, namely a PVC plastic back plate paved with fluorescence, a PVC bottom plate, a nitrocellulose membrane, a sample pad and absorbent paper. The invention also discloses a double-labeled colloidal gold probe, which consists of a gold nanoparticle-labeled biotinylated anti-target antibody and gold nanoparticle-labeled streptavidin, and realizes signal amplification by utilizing the super-strong affinity between biotin-streptavidin molecules. The fluorescent colloidal gold immunochromatographic test strip provided by the invention is used for pretreating a sample by using the immunomagnetic microspheres before detection, and can be used for efficiently and quickly separating and enriching target substances in an external magnetic field. The test strip disclosed by the invention is good in stability, high in sensitivity, free from matrix interference, capable of realizing rapid qualitative or quantitative detection of a sample, simple and convenient in operation steps, strong in controllability and good in application prospect.
Description
Technical Field
The invention belongs to the technical field of food safety detection and biomedicine, and particularly relates to an indirect background fluorescent colloidal gold immunochromatographic test strip based on double-label signal amplification and application thereof.
Background
With the addition of world trade organization in China, people gradually change the demand type of food into the quality type. The problem of harmful substance residue becomes an important index for food safety detection in various countries in the world, and although the government of China also sets a series of laws and regulations and technical standards to limit the standards for harmful substance residue, the residue detection is strengthened, a residue detection system is established, safe and sanitary food is provided for the society, and the human health is imperative to be protected.
At present, harmful substance residue is mainly detected by using instruments, a rapid detection test strip and other methods. The instrument method mainly comprises a gas chromatography, a gas chromatography-mass spectrometry combined method, a high performance liquid chromatography and a liquid chromatography-mass spectrometry combined method, and the instrument method is high in price, high in technical requirements of detection personnel, complex in detection process, not easy to popularize and the like, so that the development of the simple, low-cost and rapid immunochromatography technology is rapid, and the instrument method is widely applied to the fields of food safety, medical diagnosis, environmental monitoring and the like. The immunochromatography technology based on colloidal gold as a marker is the most successful and most extensive rapid screening means, but the accuracy and the repeatability of the detection test strip are not optimistic due to the serious interference of matrixes in samples of different regions, varieties and time; and the stability and sensitivity of the test result of the test strip still need to be improved.
Disclosure of Invention
It is an object of the present invention to provide a kit.
The kit provided by the invention comprises a test strip and a double-labeled colloidal gold probe;
the double-labeled colloidal gold probe consists of a gold nanoparticle-labeled biotinylated anti-target antibody and gold nanoparticle-labeled streptavidin;
the test strip consists of a sample pad, a coating film and a water absorption pad, wherein the sample pad, the coating film and the water absorption pad are sequentially connected and fixed on a substrate layer; a fluorescent back plate is arranged between the coating film and the substrate layer;
the detection line and the quality control line are separated from each other;
the detection line is coated with a target antigen; the anti-target antibody is capable of specifically binding to the target antigen;
an antibody of an antibody against the target substance is coated on the quality control line;
the detection line is positioned at one end of the envelope membrane close to the sample pad;
the quality control line is positioned at one end of the coating film close to the water absorption pad.
In the kit, in the gold nanoparticle-labeled biotinylated anti-target antibody, the mass ratio of the gold nanoparticles to the biotinylated anti-target antibody may be (300-1000): 1; specifically, it may be 500: 1.
in the gold nanoparticle-labeled streptavidin, the mass ratio of the gold nanoparticles to the streptavidin can be (50-500): 1; specifically, it may be 100: 1.
the anti-target antibody is a monoclonal antibody or a polyclonal antibody against the target.
And the detection line is coated with a conjugate of a target substance antigen and BSA carrier protein.
The preparation method of the gold nanoparticle-labeled biotinylated anti-target antibody comprises the following steps:
m1) preparation of antibody solution biotinylated against the target: adding the aminated biotin into an antibody solution for resisting the target substance, reacting for 1h, dialyzing by using a dialysis membrane, sucking out the biotinylated antibody from a dialysis bag, and adjusting the concentration of the biotinylated antibody by using a PBS solution to obtain the antibody solution for resisting the target substance.
m2) preparation of gold particle solution: and (3) putting deionized water into a conical flask for boiling, adding a chloroauric acid solution and a trisodium citrate solution, continuing boiling, and cooling to room temperature when the color is changed into wine red to obtain the gold particle solution.
m3) adding the antibody solution of biotinylated anti-target to the gold particle solution, incubating at room temperature, and then blocking at room temperature using BSA solution as a blocking solution.
m4) removing free unbound biotinylated anti-target antibody by high-speed centrifugation, and then carrying out heavy suspension by using a heavy suspension to obtain the gold nanoparticle-labeled biotinylated anti-target antibody.
The preparation method of the gold nanoparticle-labeled streptavidin comprises the following steps:
n1) adding streptavidin to the gold particle solution with stirring, immediately followed by NaHCO3The solution was incubated at room temperature for 10min, and then the gold nanoparticles were stabilized with PEG 6000.
n2) removing free unbound streptavidin by centrifugation, and then carrying out heavy suspension by using a heavy suspension solution to obtain the gold nanoparticle-labeled streptavidin.
Further, the anti-target antibody is an anti-target monoclonal antibody;
the antibody of the anti-target antibody is goat anti-mouse IgG;
the particle size of the gold nanoparticles is 30 nm;
the base layer is made of polyvinyl chloride;
the fluorescent back plate is a PVC plastic plate with fluorescent sprayed on the surface;
the sample pad is a glass cellulose membrane;
the coating film is a nitrocellulose film;
the distance between the detection line and the quality control line is 3 mm.
Another object of the present invention is to provide a method for preparing the above kit.
The preparation method of the kit provided by the invention comprises the following steps: respectively preparing the test strip and the double-labeled colloidal gold probe in the kit;
the method for preparing the test strip comprises the following steps:
I. respectively preparing the sample pad, the coating film provided with the detection line and the quality control line, and the water absorption pad;
II. Covering the fluorescent backboard in the middle of the substrate layer, covering the fluorescent backboard with the coating film provided with the detection line and the quality control line obtained in the step I, and sequentially overlapping the sample pad obtained in the step I and the water absorption pad obtained in the step I at two ends of the coating film respectively to obtain the test strip;
the coating film provided with the detection line and the quality control line is prepared according to the following method: respectively spraying the target antigen and the goat anti-mouse IgG on a detection line region and a quality control line region of the coating film to form the detection line and the quality control line, so as to obtain the coating film provided with the detection line and the quality control line;
the method for preparing the double-labeled colloidal gold probe comprises the preparation method of the gold nanoparticle-labeled biotinylated anti-target antibody and the preparation method of the gold nanoparticle-labeled streptavidin.
It is still another object of the present invention to provide a novel use of the above-mentioned kit or the above-mentioned preparation method.
The invention provides application of the kit or the preparation method in detection of a target object.
The invention also provides application of the kit or the preparation method in detecting whether a sample to be detected contains a target object.
The invention also provides application of the kit or the preparation method in detecting the content of the target object in a sample to be detected.
It is still another object of the present invention to provide a method for detecting whether a sample contains a target substance.
The method for detecting whether the sample to be detected contains the target object comprises the following steps:
(c1) enriching a sample to be detected by using immunomagnetic microspheres to obtain an enriched sample;
(c2) adding the double-labeled colloidal gold probe according to any one of claims 1 to 3 to the enriched sample to obtain a mixed solution;
(c3) incubating the mixed solution for 3min, adding the mixed solution to a sample pad of the test strip, carrying out chromatography for 10min, and observing the color development conditions of a detection line and a quality control line;
if the line C and the line T are both red, the sample to be detected does not contain a target or the candidate does not contain the target;
and if the C line shows red and the T line does not show color, the sample to be detected contains the target or the candidate contains the target.
In practical application, if no target exists in a sample to be detected and only the immobilized target antigen reacts with the GNPs-mAb-biotin compound, both lines T and C will be red; on the contrary, if the target exists in the sample to be tested, the target competitively binds with the target antigen distributed on the nitrocellulose membrane. Therefore, the more the target content in the sample, the lighter the color of the T-line.
It is still another object of the present invention to provide a method for detecting the amount of a target substance in a sample to be tested.
The method for detecting the content of the target object in the sample to be detected comprises the following steps:
(d1) drawing a standard curve
Preparing target substance standard substance solutions with a series of concentrations, and enriching a plurality of obtained standard substance solutions with different concentrations by using immunomagnetic microspheres respectively to obtain enriched samples; respectively and uniformly mixing the enriched sample with the double-labeled colloidal gold probes to obtain a mixed solution; incubating the mixed solution for 3min, adding the mixed solution onto a sample pad of the test strip, and after chromatography for 10min, measuring the fluorescence intensity of a blank back plate and the fluorescence intensity of a T line by using a fluorescence reader; drawing a standard curve graph by taking the concentration of the standard substance solution of the target object as an abscissa and taking the ratio of the fluorescence intensity of the blank back plate to the fluorescence intensity of the T line as an ordinate to obtain a standard curve equation;
(d2) detection of a sample to be tested
Enriching a sample to be detected by using immunomagnetic microspheres to obtain an enriched sample; respectively and uniformly mixing the enriched sample with the double-labeled colloidal gold probes to obtain a mixed solution; and d, incubating the mixed solution for 3min, adding the mixed solution to a sample pad of the test strip, carrying out chromatography for 10min, measuring the fluorescence intensity of the blank back plate and the fluorescence intensity of the T line by using a fluorescence reader to obtain the ratio of the fluorescence intensity of the blank back plate to the fluorescence intensity of the T line, substituting the ratio into the standard curve equation obtained in the step d1), and calculating to obtain the content of the target substance in the sample to be measured.
In the above method, the preparation method of the immunomagnetic microsphere comprises the following steps:
(1) mixing Fe3O4Uniformly mixing the magnetic microsphere suspension and the chitosan suspension, and sequentially drying and calcining at high temperature to obtain Fe3O4@ chitosan magnetic microspheres;
(2) subjecting said Fe to3O4Coupling the @ chitosan magnetic microsphere with an antibody for resisting a target substance to obtain Fe3O4And @ chitosan magnetic microspheres are coupled with antibodies, namely the immunomagnetic microspheres.
Further, said Fe3O4The magnetic microsphere suspension is prepared by mixing Fe3O4Uniformly mixing the magnetic microspheres with water to obtain a solution; the chitosan suspension is a solution obtained by uniformly mixing chitosan and a dilute acetic acid solution; said Fe3O4The volume ratio of the magnetic microsphere suspension to the chitosan suspension is 4:1Mixing the components in proportion.
The drying method is to spray and dry by a spray dryer; the drying time is 24 h.
The high-temperature calcination condition is that the high-temperature calcination is carried out for 4 hours at 800 ℃ under the nitrogen condition.
The coupling method is as follows:
(f1) adding EDC and NHS into the antibody solution of the anti-target object, and performing shaking table reaction for 15min for activation to obtain the activated antibody solution of the anti-target object.
(f2) Subjecting said Fe to3O4And (3) adding the @ chitosan immunomagnetic microspheres into the activated antibody solution for resisting the target substance, reacting for 30min by using a shaking table, and uniformly mixing every 10min to obtain the conjugate.
The anti-target antibody and the Fe3O4The mass ratio of the @ chitosan magnetic microspheres is 1: 50.
Said Fe3O4@ chitosan magnetic microsphere coupled antibodies also include washing and resuspension steps.
The number of washes was 5.
The solution used for resuspension was a 0.01M phosphate buffer solution.
The method for enriching the target substance by using the immunomagnetic microspheres comprises the following steps:
(a1) adding a sample to be detected containing a target object into a centrifugal tube containing the immunomagnetic microspheres for adsorption reaction;
(a2) performing magnetic separation on the centrifugal tube obtained in the step (a1) under the action of a magnetic field, discarding reaction liquid, and adding washing liquid for washing;
(a3) and (b) adding a sample diluent into the centrifugal tube obtained in the step (a2), heating, carrying out magnetic separation under the action of a magnetic field, and collecting supernatant to realize enrichment of the target substance.
Further, in the (a1), the adsorption reaction time is 10 min;
in the (a2), the magnetic separation time is 60 s; the number of washing times is 3;
in the step (a3), the heating condition is 100 ℃ for 3 min.
It is a final object of the present invention to provide immunomagnetic microspheres in the above method.
The application of the immunomagnetic microspheres in enriching the target substances also belongs to the protection scope of the invention.
The application of the immunomagnetic microspheres in preparing products enriched with target objects also belongs to the protection scope of the invention.
In the kit or the method or the application, the target substance is clenbuterol hydrochloride.
The invention has the following beneficial effects: 1) the gold nanoparticles are respectively coupled with the biotinylated antibody and the streptavidin (double labeling for short), and the ultrahigh affinity between the biotin and the streptavidin molecules is utilized to realize the mass enrichment of the colloidal gold, so that the detection sensitivity is improved while the using amount of the antibody is reduced. 2) The invention prepares the immunomagnetic microspheres, and the immunomagnetic microspheres can specifically identify corresponding target objects in various samples and efficiently and quickly separate, enrich and purify the samples in an external magnetic field. The immune magnetic microsphere is used for pretreating a sample to be detected, so that the matrix interference generated by different samples can be reduced, and the detection sensitivity is improved. 3) The invention uses the backboard material paved with fluorescence, and the backboard fluorescence is shielded by colloidal gold, so that the fluorescence intensity of the backboard is indirectly detected, the fluorescence quenching caused by factors such as pH, temperature and humidity of the solution is reduced, and the detection stability is improved. The indirect background fluorescent colloidal gold immunochromatographic technique based on double-label signal amplification provided by the invention can be used for quickly detecting a target object in a sample, and has the advantages of good stability, high sensitivity, simplicity and convenience in operation, strong controllability and the like. The detection method is suitable for the rapid detection of suspected objects in various food safety fields.
Drawings
FIG. 1 is a schematic diagram of the pretreatment of immunomagnetic microspheres according to the present invention. 1. An analyte in the sample; 2. a specific monoclonal antibody; 3. immunomagnetic microspheres.
FIG. 2 is a schematic structural diagram of a colloidal gold test strip for detecting a target substance according to the present invention. 4. Coating source (T line); 5. goat anti-mouse secondary antibody (C-line); 6. NC film; 7. a fluorescent backplane; 8. a sample pad; 9. an absorbent pad; 10. PVC bottom plate.
Fig. 3 is a schematic diagram of the colloidal gold test strip of the present invention. 2. A specific monoclonal antibody; 4. coating source (T line); 5. goat anti-mouse secondary antibody (C-line); 6. NC film; 7. a fluorescent backplane; 8. a sample pad; 9. an absorbent pad; 10. a PVC base plate; 11. colloidal gold; 12. streptavidin; 13. and (4) biotin.
FIG. 4 is a standard curve of a pre-treated and untreated swine urine sample for detecting immunomagnetic microspheres according to the present invention.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.
Fe in the following examples3O4Magnetic microsphere is synthesized by solvothermal method, Fe3O4Magnetic microspheres and specific synthetic methods are described in the literature "Synthesis and characterization of magnetic nanoparticles purified nanoparticles Fe3O4@ carbon with antibacterial and catalytic properties [ J].Yu Q,Fu A,Li H,et al.Colloids&Surfaces APhysicochemical&Engineering industries, 2014,457(1):288 and 296 ", publicly available from the applicant (university of agriculture, china), the biomaterial was used only for repeating the relevant experiments of the present invention, and was not used for other purposes.
EDC and NHS in the following examples are both products of Allatin reagent, Inc.
Example 1: preparation of immunomagnetic microspheres and application of immunomagnetic microspheres in enrichment of clenbuterol hydrochloride in swine urine sample
Preparation of immune magnetic microsphere
1. Preparation of suspension A
0.1g of Fe was weighed3O4Adding 20mL of distilled water into the magnetic microspheres, and carrying out ultrasonic treatment for 30min to form a suspension A.
2. Preparation of suspension B
0.8g of chitosan (purchased from carbofuran technologies, Ltd., degree of deacetylation: 95%) was dissolved in 15mL of a 2% volume-fraction dilute acetic acid solution to give a 2% mass-fraction homogeneous suspension B.
3、Fe3O4Preparation of @ chitosan magnetic microsphere
Uniformly mixing the suspension A and the suspension B according to the volume ratio of 4:1, stirring for 1h, spray-drying for 24h by a spray dryer, and calcining at 800 ℃ for 4h under the condition of nitrogen to obtain Fe3O4@ chitosan magnetic microspheres.
4、Fe3O4@ chitosan magnetic microsphere coupled antibody
(1) Adding 1 mu g of EDC and 1 mu g of NHS into 1mL of a clenbuterol hydrochloride monoclonal antibody solution with the concentration of 5 mu g/mL (the clenbuterol hydrochloride monoclonal antibody is purchased from CL-1F8-F9 of Beijing Wierweikang biotechnology limited), and carrying out shaking table reaction for 15min for activation to obtain the activated clenbuterol hydrochloride monoclonal antibody solution.
(2) Then the Fe prepared in the step 33O4Adding the @ chitosan immunomagnetic microspheres into an activated clenbuterol hydrochloride monoclonal antibody solution, wherein the clenbuterol hydrochloride monoclonal antibody and Fe3O4The material feeding mass ratio of the @ chitosan immunomagnetic microspheres is 1: 50. Reacting in a shaking table for 30min, and uniformly mixing every 10min to obtain the conjugate.
5. Washing and resuspending
Washing the conjugate prepared in the step 4 with 5mL of washing solution (PB solution with the concentration of 0.02M) for 5 times; then, the suspension was resuspended in 6ml of 0.01M phosphate buffer solution (pH 7.0) to obtain a 20. mu.g/mg immunomagnetic microsphere solution containing specific immunomagnetic microspheres.
Second, application of immunomagnetic microspheres in enrichment of clenbuterol hydrochloride in swine urine sample
1. Adsorption
And taking a sample supernatant (1600 mu L of a swine urine sample) by using a pipette, fully mixing the sample supernatant into a 4mL centrifuge tube pre-packaged with the immune magnetic microspheres, and reacting at room temperature for 10 min.
2. Washing machine
Placing the centrifugal tube on a magnetic frame for magnetic adsorption for 60s (magnetic separation for short), discarding the reaction solution, adding 1mL of washing solution (PB solution with the concentration of 0.02M) into the centrifugal tube, gently oscillating for ten times, performing magnetic separation, and discarding the washing solution.
3. Elution is carried out
Adding 400 μ L sample diluent (PB solution with concentration of 0.02M) into the centrifuge tube, mixing, placing the centrifuge tube on a dry thermostat, heating at 100 deg.C for 3min, magnetically separating, collecting supernatant, and collecting purified and enriched sample extractive solution.
Example 2: preparation of double-label indirect background fluorescent colloidal gold test strip
Preparation of one-and two-labeled colloidal gold
1. Preparation of colloidal gold-clenbuterol hydrochloride monoclonal antibody-biotin (GNPs-clenbuterol hydrochloride antibody-biotin)
(1) Preparation of biotinylated clenbuterol hydrochloride monoclonal antibody solution
The aminated biotin (purchased from SIGMA) was added at a concentration of 3.8mg/ml to 1mg of clenbuterol hydrochloride monoclonal antibody, and after 1 hour of the rotational reaction, it was dialyzed three times against a dialysis membrane (8k MWCO), and the buffer (pH7.4) was formulated as follows: 50mM PB and 75mM NaCl, dialyzed 2L each time for more than 3 h. After that, the biotinylated antibody was carefully aspirated from the dialysis bag by a pipette, and the concentration of the biotinylated antibody was adjusted with a 50mM PBS solution to obtain a biotinylated clenbuterol hydrochloride monoclonal antibody solution having a concentration of 2.3 mg/ml.
(2) Preparation of gold particle solution
Placing 50mL of deionized water in a 200mL clean conical flask, boiling, adding 1mL of 1% chloroauric acid solution (sigma) and 1mL of 1% trisodium citrate solution, boiling for 3min, and cooling to room temperature for use when the color changes to wine red.
(3) And (3) adding 2.3 mu g of the biotinylated clenbuterol hydrochloride monoclonal antibody solution prepared in the step (1) into 1mL of the gold particle solution prepared in the step (2), incubating at room temperature, and then using a BSA solution with the mass fraction of 10% as a blocking solution and blocking at room temperature.
(4) After the completion of the step (3), the free unbound clenbuterol hydrochloride monoclonal antibody-biotin is removed by high-speed centrifugation, and then resuspended with a resuspension solution (0.01M PBS containing 0.5% tween 20 and 2% sucrose) to obtain 200 μ L of a colloidal gold-clenbuterol hydrochloride monoclonal antibody-biotin (GNPs-clenbuterol hydrochloride antibody-biotin) solution with a concentration of 0.46 mg/mL.
2. Preparation of colloidal gold-streptavidin (GNPs-streptavidin)
(1) mu.L of streptavidin 1mg/mL was added to 1mL of the gold particle solution prepared in step 1(2) with stirring, and then 200. mu.L of 1M NaHCO was added immediately3The solution was incubated at room temperature for 10min, and then the gold nanoparticles were stabilized with 200 μ L of 2% PEG 6000.
(2) After completion of step (1), 10000g was centrifuged at 4 ℃ for 30min to remove free unbound streptavidin, and then resuspended in 200. mu.L resuspension (0.1M PBS containing 0.02% PEG 6000) to obtain a 1mg/mL colloidal gold-streptavidin (GNPs-streptavidin) solution.
Preparation of detection line and quality control line
BSA protein and clenbuterol hydrochloride antigen conjugates (prepared by trusted Beijing Vildakang biotech Co., Ltd.) and goat anti-mouse IgG (purchased from Jackson ImmunoResearch Laboratories, Inc., catalog No. 125229) were diluted with 0.01M phosphate buffer solution (PBS, pH7.0) to concentrations of 0.6mg/ml and 0.3mg/ml, respectively, and then sequentially sprayed on the detection line and the quality control line on NC (NC membrane) at a spraying rate of 0.75. mu.L/cm using a striping machine, with a distance of 3mm between the detection line and the quality control line. The scribed NC films were placed in a 37 ℃ oven overnight.
Thirdly, assembling the test paper strip
The test strip comprises five parts, namely a fluorescent back plate, a PVC bottom plate, a nitrocellulose membrane, a sample pad and absorbent paper. The preparation method comprises the following steps:
1. the method comprises the steps of covering the middle of a PVC bottom plate (purchased from Shanghai gold-labeled Biotechnology limited company with the product catalog number of SMNF31-40) with a fluorescent back plate (the fluorescent back plate is a PVC plastic plate with fluorescence sprayed on the surface and is prepared by Shanghai Xin spectral Biotechnology limited company), covering a nitrocellulose membrane with a detection line and a quality control line on the fluorescent back plate, and sequentially overlapping a sample pad (a glass cellulose membrane) and absorbent paper at two ends of the nitrocellulose membrane respectively. The sample pad and absorbent pad were pressed against the NC membrane 2mm each at both ends.
2. And cutting the processed large plate into test strips with the width of 4.72mm by using a slitter, assembling the test strips in a plastic card shell to form a test card, and placing the test card in a dry and closed aluminum foil bag for later use.
The structure schematic diagram of the colloidal gold immunochromatographic test strip is shown in figure 2. The detection principle is shown in fig. 3.
Example 3: quantitative and qualitative detection of clenbuterol hydrochloride
Qualitative detection method of clenbuterol hydrochloride
1. And (3) enriching and purifying the sample to be detected by using the immunomagnetic microspheres in the embodiment 1 to obtain the treated sample.
2. mu.L of the treated sample was mixed with 3. mu.L of the GNPs-clenbuterol hydrochloride antibody-biotin solution prepared in step one of example 2 and 3. mu.L of the GNPs-streptavidin solution prepared in step one of example 2, and incubated at room temperature for 3min to obtain a mixed solution.
3. 120 μ L of the mixed solution was dropped onto the sample pad of the test strip. After 10min, qualitative observation by naked eye was carried out.
If the line C and the line T are both red, the sample to be detected does not contain clenbuterol hydrochloride;
and if the C line shows red and the T line does not show color, the sample to be detected contains clenbuterol hydrochloride.
Second, clenbuterol hydrochloride quantitative detection method
1. Drawing of standard curve
(1) Solutions containing clenbuterol hydrochloride standards with different concentrations of 0.015 mu g/L, 0.031 mu g/L, 0.062 mu g/L, 0.12 mu g/L, 1.25 mu g/L, 0.5 mu g/L and 1 mu g/L are respectively used as samples to be detected.
(2) The sample to be tested was enriched and purified using the immunomagnetic microspheres of example 1 to obtain a treated sample (pretreated sample). Meanwhile, the sample to be tested without any treatment is used as a control (the sample without pretreatment).
(3) mu.L of the treated sample was mixed with 3. mu.L of the GNPs-clenbuterol hydrochloride antibody-biotin solution prepared in step one of example 2 and 3. mu.L of the GNPs-streptavidin solution prepared in step one of example 2, and incubated at room temperature for 3min to obtain a mixed solution.
(4) 120 μ L of the mixed solution was dropped onto the sample pad of the test strip. And (3) after 10min, measuring the fluorescence intensity of the quality control line and the detection line by using a fluorescence immunoassay quantitative analyzer FQ-S2. Set the fluorescence reading of the blank backplate to F0Fluorescence reading at T line position is FT，F0Average fluorescence intensity of blank portion between T line and C line not covered by colloidal gold, FTThe fluorescence intensity on the T line blocked by colloidal gold. With F0/FTAnd taking the concentration of the clenbuterol hydrochloride in the standard solution as an abscissa to establish a standard curve (figure 4) to obtain a standard curve equation.
2. Detection of clenbuterol hydrochloride content in sample to be detected
(1) And (3) enriching and purifying the sample to be detected by using the immunomagnetic microspheres in the embodiment 1 to obtain the treated sample.
(2) mu.L of the treated sample was mixed with 3. mu.L of the GNPs-clenbuterol hydrochloride antibody-biotin solution prepared in step one of example 2 and 3. mu.L of the GNPs-streptavidin solution prepared in step one of example 2, and incubated at room temperature for 3min to obtain a mixed solution.
(3) 120 μ L of the mixed solution was dropped onto the sample pad of the test strip. Measuring fluorescence intensity of the quality control line and the detection line by using a fluorescence immunoassay quantitative analyzer FQ-S2 after 10min, and calculating F0/FT. F of the sample to be tested0/FTAnd (4) substituting the standard curve equation in the step (1) to calculate the content of the clenbuterol hydrochloride in the sample to be detected.
Example 4: application of fluorescent colloidal gold test strip based on double-label indirect background
Firstly, preparation of sample to be tested
And (3) uniformly mixing the clenbuterol hydrochloride standard substance with a fresh pig urine sample identified as clenbuterol hydrochloride negative to obtain clenbuterol hydrochloride solutions with different concentrations (0.04 mug/L, 0.10 mug/L, 0.15 mug/L and 0.30 mug/L), and taking the clenbuterol hydrochloride solutions as samples to be detected.
Second, detection of the sample to be detected
1. Each sample to be tested was enriched and purified using the immunomagnetic microspheres in example 1, and a treated sample (pretreated sample) was obtained.
2. mu.L of the pretreated sample was mixed with 3. mu.L of the GNPs-clenbuterol hydrochloride antibody-biotin solution prepared in step one of example 2 and 3. mu.L of the GNPs-streptavidin solution prepared in step one of example 2, and incubated at room temperature for 3min to obtain a mixed solution.
3. 120 μ L of the mixed solution was dropped onto the sample pad of the test strip. Measuring fluorescence intensity of the quality control line and the detection line by using a fluorescence immunoassay quantitative analyzer FQ-S2 after 10min, and calculating to obtain F0/FTThen, it was substituted into the standard curve (fig. 4), and the recovery rate and the coefficient of variation were calculated. Recovery (%) × 100% (detected concentration/added concentration); coefficient of variation CV is the standard deviation/mean.
The results are shown in Table 1. The extinction value obtained by visual observation is 1ppb, the lowest detection limit can reach 0.02ppb, the recovery rate is 81-115%, and the coefficient of variation is less than 5.99%.
TABLE 1 clenbuterol hydrochloride Standard test results
Example 5: specificity detection of clenbuterol hydrochloride test strip
Firstly, preparation of sample to be tested
The following adrenergic receptor agonists: clenbuterol hydrochloride, salbutamol, cimaterol, terbutaline, ractopamine, verdolol, propranolol, geraniol, atenolol, oxsalolol, zilpaterol, epinephrine and phenethylamine A are respectively added into a swine urine sample treated by the immunomagnetic microspheres, so that the following different concentrations are respectively obtained: 0 mug/L, 1.2 mug/L, 3.5 mug/L, 6.2 mug/L, 12.5 mug/L, 25 mug/L, 50 mug/L, 100 mug/L of a solution of an adrenergic receptor agonist to be tested.
Second, detection of the sample to be detected
Clenbuterol hydrochloride in each sample solution to be tested was detected using the colloidal gold test strip of example 2 according to the clenbuterol hydrochloride quantitative detection method of example 3, and the specificity was evaluated by calculating the cross-reactivity. Cross reaction rate CR (%) ═ clenbuterol hydrochloride IC50IC of other receptor agonists50)×100％。
The results are shown in Table 2. The cross reaction rates of the clenbuterol hydrochloride test strip, the salbutamol and the cimaterol are 0.59 percent and 3.5 percent respectively, and the clenbuterol hydrochloride test strip has no cross reaction with other adrenergic receptor agonists.
TABLE 2 Cross-reactivity of clenbuterol hydrochloride and other adrenoceptor agonists
| Agonists | IC50(μg/L) | Cross reaction Rate (%) |
| Clenbuterol hydrochloride | 0.03 | 100 |
| Salbutamol | 17 | 0.59 |
| Cimaterol | 3.5 | 3.5 |
| Ractopamine | >2000 | <0.1 |
| Vidolol | >2000 | <0.1 |
| Propranolol (Propranolol) | >2000 | <0.1 |
| Daphyllol | >2000 | <0.1 |
| Atenolol | >2000 | <0.1 |
| Oxaprolol | >2000 | <0.1 |
| Zilpaterol | >2000 | <0.1 |
| Adrenalin | >2000 | <0.1 |
| Phenylethylamine A | >2000 | <0.1 |
Claims (10)
1. A kit comprising a test strip and a double-labeled colloidal gold probe;
the double-labeled colloidal gold probe consists of a gold nanoparticle-labeled biotinylated anti-target antibody and gold nanoparticle-labeled streptavidin;
the test strip consists of a sample pad, a coating film and a water absorption pad, wherein the sample pad, the coating film and the water absorption pad are sequentially connected and fixed on a substrate layer; a fluorescent back plate is arranged between the coating film and the substrate layer;
the detection line and the quality control line are separated from each other;
the detection line is coated with a target antigen; the anti-target antibody is capable of specifically binding to the target antigen;
an antibody of an antibody against the target substance is coated on the quality control line;
the detection line is positioned at one end of the envelope membrane close to the sample pad;
the quality control line is positioned at one end of the coating film close to the water absorption pad.
2. The kit of claim 1, wherein:
in the gold nanoparticle-labeled biotinylated anti-target antibody, the mass ratio of the gold nanoparticles to the biotinylated anti-target antibody is (300-1000): 1;
or in the gold nanoparticle-labeled streptavidin, the mass ratio of the gold nanoparticles to the streptavidin is (50-500): 1;
or, the anti-target antibody is a monoclonal antibody or a polyclonal antibody against the target.
3. The kit according to claim 1 or 2, characterized in that:
the anti-target antibody is a monoclonal antibody against the target;
or, the antibody of the anti-target antibody is goat anti-mouse IgG;
or, the particle size of the gold nanoparticles is 30 nm;
or the base layer is made of polyvinyl chloride;
or the fluorescent backboard is a PVC plastic board with the surface coated with fluorescence;
or, the sample pad is a glass cellulose membrane;
or, the coating film is a nitrocellulose film;
or the distance between the detection line and the quality control line is 3 mm.
4. A method of making a kit as claimed in any one of claims 1 to 3, comprising the steps of: preparing the test strip and the double-labeled colloidal gold probe in the kit according to any one of claims 1 to 3 respectively;
the method for preparing the test strip comprises the following steps:
I. respectively preparing the sample pad, the coating film provided with the detection line and the quality control line, and the water absorption pad;
II. Covering the fluorescent backboard in the middle of the substrate layer, covering the fluorescent backboard with the coating film provided with the detection line and the quality control line obtained in the step I, and respectively overlapping the sample pad obtained in the step I and the water absorption pad obtained in the step I on two ends of the coating film to obtain the test strip;
the coating film provided with the detection line and the quality control line is prepared according to the following method: spraying the target antigen of claims 1-3 and the goat anti-mouse IgG of claims 1-3 on the detection line region and the quality control line region of the coating film respectively to form the detection line and the quality control line, so as to obtain the coating film provided with the detection line and the quality control line;
the method for preparing the double-labeled colloidal gold probe is the method for preparing gold nanoparticle-labeled biotinylated anti-target antibody and gold nanoparticle-labeled streptavidin described in claims 1 to 3.
5. Use of the kit according to any one of claims 1 to 3 or the preparation method according to claim 4 for detecting a target;
or, the kit of any one of claims 1 to 3 or the preparation method of claim 4, for detecting whether the sample to be tested contains the target substance;
or, the kit according to any one of claims 1 to 3 or the preparation method according to claim 4, for use in detecting the amount of a target in a sample to be tested.
6. A method for detecting whether a sample to be detected contains a target object or not comprises the following steps:
(c1) enriching a sample to be detected by using immunomagnetic microspheres to obtain an enriched sample;
(c2) adding the double-labeled colloidal gold probe according to any one of claims 1 to 3 to the enriched sample to obtain a mixed solution;
(c3) incubating the mixed solution for 3min, adding the mixed solution to a sample pad of the test strip of any one of claims 1 to 3, and observing the color development of a detection line and a quality control line after chromatography for 10 min;
if the line C and the line T are both red, the sample to be detected does not contain a target or the candidate does not contain the target;
and if the C line shows red and the T line does not show color, the sample to be detected contains the target or the candidate contains the target.
7. A method for detecting the content of a target object in a sample to be detected comprises the following steps:
(d1) drawing a standard curve
Preparing target substance standard substance solutions with a series of concentrations, and enriching a plurality of obtained standard substance solutions with different concentrations by using immunomagnetic microspheres respectively to obtain enriched samples; respectively and uniformly mixing the enriched sample with the double-labeled colloidal gold probe of any one of claims 1 to 3 to obtain a mixed solution; incubating the mixed solution for 3min, then adding the mixed solution to a sample pad of the test strip of any one of claims 1-3, and after chromatography for 10min, measuring the fluorescence intensity of a blank back plate and the fluorescence intensity of a T line by using a fluorescence reader; drawing a standard curve graph by taking the concentration of the standard substance solution of the target object as an abscissa and taking the ratio of the fluorescence intensity of the blank back plate to the fluorescence intensity of the T line as an ordinate to obtain a standard curve equation;
(d2) detection of a sample to be tested
Enriching a sample to be detected by using immunomagnetic microspheres to obtain an enriched sample; respectively and uniformly mixing the enriched sample with the double-labeled colloidal gold probe of any one of claims 1 to 3 to obtain a mixed solution; and (3) incubating the mixed solution for 3min, adding the mixed solution to a sample pad of the test strip in any one of claims 1 to 3, carrying out chromatography for 10min, measuring the fluorescence intensity of the blank back plate and the fluorescence intensity of the T line by using a fluorescence reader, obtaining the ratio of the fluorescence intensity of the blank back plate to the fluorescence intensity of the T line, substituting the ratio into the standard curve equation obtained in the step d1), and calculating to obtain the content of the target substance in the sample to be measured.
8. The method according to claim 6 or 7, characterized in that: the preparation method of the immunomagnetic microspheres comprises the following steps:
(1) mixing Fe3O4Uniformly mixing the magnetic microsphere suspension and the chitosan suspension, and sequentially drying and calcining at high temperature to obtain Fe3O4@ chitosan magnetic microspheres;
(2) subjecting said Fe to3O4Coupling the @ chitosan magnetic microsphere with an antibody for resisting a target substance to obtain Fe3O4The @ chitosan magnetic microsphere is coupled with an antibody, namely the immunomagnetic microsphere;
(a1) adding a sample to be detected containing a target object into a centrifugal tube containing the immunomagnetic microspheres for adsorption reaction;
(a2) performing magnetic separation on the centrifugal tube obtained in the step (a1) under the action of a magnetic field, discarding reaction liquid, and adding washing liquid for washing;
(a3) and (b) adding a sample diluent into the centrifugal tube obtained in the step (a2), heating, carrying out magnetic separation under the action of a magnetic field, and collecting supernatant to realize enrichment of the target substance.
9. Immunomagnetic microspheres as claimed in claim 8.
10. Use of immunomagnetic microspheres according to claim 9 for the enrichment of targets;
or, the use of immunomagnetic microspheres according to claim 9 for the preparation of a product enriched in target.
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