CN110873791A - Indirect background fluorescent colloidal gold immunochromatographic test strip based on double-labeled signal amplification and application thereof - Google Patents

Abstract

The invention discloses an indirect background fluorescent colloidal gold immunochromatographic test strip based on double-labeled signal amplification and its application. The test strip includes five parts: a fluorescent PVC plastic backboard, a PVC bottom plate, a nitrocellulose membrane, a sample pad and an absorbent paper. The invention also discloses a double-labeled colloidal gold probe. The double-labeled colloidal gold probe is composed of a gold nanoparticle-labeled biotinylated anti-target antibody and a gold nanoparticle-labeled streptavidin. The superior affinity between biotin-streptavidin molecules enables signal amplification. The fluorescent colloidal gold immunochromatographic test strip of the present invention uses immunomagnetic microspheres to pre-treat the sample before detection, and can efficiently and rapidly separate and enrich the target in an external magnetic field. The test strip of the invention has good stability, high sensitivity, is free from matrix interference, can realize rapid qualitative or quantitative detection of samples, has simple operation steps, strong controllability, and has good application prospects.

Description

An indirect background fluorescent colloidal gold immunochromatographic assay based on double-labeled signal amplification Paper strips and their applications

technical field

The invention belongs to the technical field of food safety detection and biomedicine, in particular to an indirect background fluorescent colloidal gold immunochromatographic test strip based on double-labeled signal amplification and an application thereof.

Background technique

With my country's accession to the WTO, people's demand for food has gradually changed to quality. The problem of hazardous substance residues has become an important indicator of food safety testing in countries around the world. Although the Chinese government has also formulated a series of laws, regulations and technical standards to limit the residues of hazardous substances, it has strengthened residue testing, established a residue testing system, and provided safety and hygiene to the society. food, it is imperative to protect human health.

At present, the detection of harmful substances residues mainly uses methods such as instruments and rapid detection test strips. The instrument methods mainly include gas chromatography, gas chromatography-mass spectrometry, high performance liquid chromatography and liquid chromatography-mass spectrometry. Due to the high price of instrument methods, the technical requirements of testing personnel are too high, the detection process is cumbersome, and the operation is not easy to popularize. , which makes the simple, cheap and fast immunochromatographic technology develop rapidly, and has been widely used in the fields of food safety, medical diagnosis, environmental monitoring and so on. Among them, the immunochromatographic technique based on colloidal gold as a marker is the most successful and widely used rapid screening method. The degree of repeatability is not optimistic; and the stability and sensitivity of the test strip test results still need to be improved.

SUMMARY OF THE INVENTION

An object of the present invention is to provide a kit.

The kit provided by the present invention includes test strips and double-labeled colloidal gold probes;

The double-labeled colloidal gold probe is composed of gold nanoparticle-labeled biotinylated anti-target antibody and gold nanoparticle-labeled streptavidin;

The test strip is composed of a sample pad connected and fixed on the base layer in sequence, a coating film provided with a detection line and a quality control line, and a water-absorbing pad; a fluorescent back is arranged between the coating film and the base layer. plate;

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 can specifically bind to the target antigen;

The quality control line is coated with an antibody against the anti-target antibody;

The detection line is located at one end of the coating film close to the sample pad;

The quality control line is located at one end of the coating film close to the absorbent pad.

In the above 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 can be 500:1.

In the streptavidin labeled with gold nanoparticles, the mass ratio of the gold nanoparticles to streptavidin may be (50-500):1; specifically, it may be 100:1.

The anti-target antibody is a monoclonal antibody or polyclonal antibody against the target.

The detection line is coated with a conjugate of target antigen and BSA carrier protein.

The preparation method of the gold nanoparticle-labeled biotinylated anti-target antibody is as follows:

m1) Preparation of biotinylated anti-target antibody solution: Aminated biotin was added to the anti-target antibody solution, reacted for 1 h and dialyzed with a dialysis membrane, and then the biotinylated antibody was aspirated from the dialysis bag, and PBS was used for dialysis. The solution adjusts the concentration of the biotinylated antibody to obtain the biotinylated anti-target antibody solution.

m2) Preparation of gold particle solution: take deionized water and place it in a conical flask to boil, add chloroauric acid solution and trisodium citrate solution, continue to boil, and cool to room temperature when the color becomes wine red to obtain the gold Granular solution.

m3) adding the biotinylated anti-target antibody solution to the gold particle solution, incubating at room temperature, and then blocking with BSA solution as a blocking solution at room temperature.

m4) high-speed centrifugation to remove free and unbound biotinylated anti-target antibody, and then resuspended in a resuspension solution to obtain the gold nanoparticle-labeled biotinylated anti-target antibody.

The preparation method of the gold nanoparticle-labeled streptavidin is as follows:

n1) Add streptavidin to the gold particle solution, stir while adding, then add NaHCO ₃ solution immediately, incubate at room temperature for 10 min, and then stabilize the gold nanoparticles with PEG 6000.

n2) Centrifuging to remove free and unbound streptavidin, and then resuspending with a resuspension solution to obtain the gold nanoparticle-labeled streptavidin.

Further, the anti-target antibody is an anti-target monoclonal antibody;

The antibody against the anti-target antibody is goat anti-mouse IgG;

The particle size of the gold nanoparticles is 30 nm;

The material of the base layer is polyvinyl chloride;

The fluorescent back plate is a PVC plastic plate sprayed with fluorescent light on the surface;

The sample pad is a glass cellulose film;

Described coating film is 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 preparation method of the above-mentioned kit.

The preparation method of the above-mentioned kit provided by the present invention comprises the following steps: respectively preparing the test strip and the double-labeled colloidal gold probe in the above-mentioned kit;

The method for preparing the test strip comprises the following steps:

1. Prepare the sample pad, the coating film provided with the detection line and the quality control line, and the water-absorbing pad respectively;

II, cover the fluorescent backplane in the middle of the base layer, then cover the coating film with the detection line and the quality control line obtained in step 1 on the fluorescent backplane, and then apply the The sample pad and the described water-absorbing pad obtained in step 1 are respectively overlapped on both ends of the coated film successively 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 above-mentioned target antigen and the above-mentioned goat anti-mouse IgG on the detection line area and the quality control line area of the coating film, respectively, to form the detection line area and the quality control line area. line and the quality control line 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 preparation method of the above-mentioned gold nanoparticle-labeled biotinylated anti-target antibody and gold nanoparticle-labeled streptavidin.

Another object of the present invention is to provide new uses of the above-mentioned kit or the above-mentioned preparation method.

The present invention provides the application of the above-mentioned kit or the above-mentioned preparation method in detecting a target.

The present invention also provides the application of the above kit or the above preparation method in detecting whether a sample to be tested contains a target.

The present invention also provides the application of the above-mentioned kit or the above-mentioned preparation method in detecting the content of the target substance in the sample to be tested.

Another object of the present invention is to provide a method for detecting whether a target substance is contained in a sample to be tested.

The method for detecting whether a sample to be tested contains a target provided by the present invention comprises the following steps:

(c1) enriching the sample to be tested with immunomagnetic microspheres to obtain an enriched sample;

(c2) adding the double-labeled colloidal gold probe described in any one of claims 1-3 to the enriched sample to obtain a mixed solution;

(c3) incubating the mixed solution for 3 minutes, then adding it to the sample pad of the above-mentioned test strip, and after 10 minutes of chromatography, observe the color development of the detection line and the quality control line;

If both the C line and the T line are red, the sample to be tested does not contain the target or the candidate does not contain the target;

If the C line shows red and the T line does not show color, the sample to be tested contains the target or a candidate for the target.

In practical applications, if there is no target in the sample to be tested, and only the immobilized target antigen reacts with the GNPs-mAb-biotin compound, both T and C lines will appear red; on the contrary, if the sample to be tested contains The target is competitively combined 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.

Another object of the present invention is to provide a method for detecting the content of a target substance in a sample to be tested.

The method for detecting the content of the target substance in the sample to be tested provided by the present invention comprises the following steps:

(d1) draw standard curve

Prepare standard solution of target substance with a series of concentrations, and enrich several obtained standard solutions containing different concentrations with immunomagnetic microspheres respectively to obtain enriched samples; Mix the double-labeled colloidal gold probes to obtain a mixed solution; incubate the mixed solution for 3 minutes, then add it to the sample pad of the above-mentioned test strip, and after chromatography for 10 minutes, use a fluorescence reader to measure the fluorescence intensity and T of the blank backplate The fluorescence intensity of the line; with the concentration of the standard solution of the target as the abscissa, and the ratio of the fluorescence intensity of the blank backplane and the fluorescence intensity of the T line as the ordinate, draw a standard curve graph to obtain a standard curve equation;

(d2) Detection of samples to be tested

The samples to be tested are enriched with immunomagnetic microspheres to obtain enriched samples; the enriched samples are respectively mixed with the above double-labeled colloidal gold probes to obtain a mixed solution; the mixed solution is incubated 3min, then add it to the sample pad of the above test strip, after 10min of chromatography, use a fluorescence reader to measure the fluorescence intensity of the blank backplane and the fluorescence intensity of the T line, and obtain the difference between the fluorescence intensity of the blank backplane and the fluorescence intensity of the T line. The ratio is substituted into the standard curve equation obtained in step d1), and the content of the target substance in the sample to be tested is calculated.

In the above method, the preparation method of the immunomagnetic microspheres comprises the following steps:

(1) Mixing the Fe ₃ O ₄ magnetic microsphere suspension with the chitosan suspension, drying and calcining at high temperature in turn, to obtain Fe ₃ O ₄ @chitosan magnetic microspheres;

(2) Coupling the Fe ₃ O ₄ @chitosan magnetic microspheres with the anti-target antibody to obtain Fe ₃ O ₄ @chitosan magnetic microspheres conjugated antibodies, which are the immunomagnetic microspheres .

Further, the Fe ₃ O ₄ magnetic microsphere suspension is a solution obtained by mixing Fe ₃ O ₄ magnetic microspheres with water; the chitosan suspension is a mixture of chitosan and dilute acetic acid solution. The solution obtained afterward; the Fe ₃ O ₄ magnetic microsphere suspension and the chitosan suspension are mixed uniformly in a volume ratio of 4:1.

The drying method is spray drying through a spray dryer; the drying time is 24h.

The conditions of the high-temperature calcination are high-temperature calcination at 800° C. for 4 hours under nitrogen conditions.

The method of coupling is as follows:

(f1) EDC and NHS were added to the anti-target antibody solution, and the reaction was shaken for 15 min for activation, to obtain an anti-target antibody solution after activation.

(f2) adding the Fe ₃ O ₄ @chitosan immunomagnetic microspheres to the activated anti-target antibody solution, shaking for 30 minutes, and mixing every 10 minutes to obtain a conjugate.

The mass ratio of the anti-target antibody to the Fe ₃ O ₄ @chitosan magnetic microspheres is 1:50.

The Fe ₃ O ₄ @chitosan magnetic microsphere-conjugated antibody also includes the steps of washing and resuspension.

The number of washings was 5 times.

The solution used for the resuspension was 0.01M phosphate buffer solution.

The method for enriching the target with the immunomagnetic microspheres comprises the following steps:

(a1) adding the sample to be tested containing the target into the centrifuge tube containing the immunomagnetic microspheres to carry out adsorption reaction;

(a2) magnetically separate the centrifuge tube obtained in step (a1) under the action of a magnetic field, discard the reaction solution and add washing solution for washing;

(a3) adding the sample diluent to the centrifuge tube obtained in step (a2), heating, performing magnetic separation under the action of a magnetic field, collecting the supernatant, and realizing the enrichment of the target substance.

Further, in described (a1), the time of described adsorption reaction is 10min;

In the (a2), the time of the magnetic separation is 60s; the number of times of the washing is 3 times;

In the above (a3), the heating condition is heating at 100° C. for 3 min.

The last object of the present invention is to provide the immunomagnetic microspheres in the above method.

The application of the above-mentioned immunomagnetic microspheres in the enrichment target also belongs to the protection scope of the present invention.

The application of the above-mentioned immunomagnetic microspheres in the preparation of a product enriched with a target substance also belongs to the protection scope of the present invention.

In the above kit or method or application, the target substance is clenbuterol hydrochloride.

The beneficial effects of the present invention are as follows: 1) In the present invention, the gold nanoparticles are respectively coupled with biotinylated antibodies and streptavidin (referred to as double labeling), and the super-strong affinity between biotin and streptavidin molecules is utilized. , to achieve a large amount of colloidal gold enrichment, while reducing the amount of antibody, and improving the detection sensitivity. 2) The present invention prepares the immunomagnetic microspheres, the immunomagnetic microspheres can specifically identify corresponding targets in various samples, and efficiently and rapidly separate, enrich and purify the samples in an external magnetic field. Using the immunomagnetic microsphere for pretreatment of the sample to be tested can reduce the matrix interference generated by different samples and improve the detection sensitivity. 3) The present invention uses a backplane material covered with fluorescence, shields the fluorescence of the backplane by colloidal gold, indirectly detects the fluorescence intensity of the backplane, reduces the fluorescence quenching caused by factors such as solution pH, temperature, humidity, etc. stability of the detection. The indirect background fluorescent colloidal gold immunochromatography technology based on double-labeled signal amplification provided by the invention can rapidly detect the target in the sample, and has the advantages of good stability, high sensitivity, simple operation, strong controllability and the like. The detection method is suitable for rapid detection of suspected objects in various food safety fields.

Description of drawings

Figure 1 is a schematic diagram of the pretreatment of the immunomagnetic microspheres of the present invention. 1. The analyte in the sample; 2. The specific monoclonal antibody; 3. The immunomagnetic microsphere.

FIG. 2 is a schematic structural diagram of a colloidal gold test strip for detecting a target according to the present invention. 4. Coating original (T line); 5. Goat anti-mouse secondary antibody (C line); 6. NC film; 7. Fluorescent backing plate; 8. Sample pad; 9. Absorbing pad; 10. PVC bottom plate.

3 is a schematic diagram of the principle of the colloidal gold test strip of the present invention. 2. Specific monoclonal antibody; 4. Original coating (T line); 5. Goat anti-mouse secondary antibody (C line); 6. NC membrane; 7. Fluorescent backplane; 8. Sample pad; 9. Absorbent pad ; 10, PVC bottom plate; 11, colloidal gold; 12, streptavidin; 13, biotin.

Fig. 4 is the standard curve of pig urine samples pretreated and untreated for the detection of immunomagnetic microspheres according to the present invention.

Detailed ways

The following examples facilitate a better understanding of the present invention, but do not limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples were purchased from conventional biochemical reagent stores unless otherwise specified. The quantitative tests in the following examples are all set to repeat the experiments three times, and the results are averaged.

The Fe ₃ O ₄ magnetic microspheres in the following examples are synthesized by solvothermal method, and the Fe ₃ O ₄ magnetic microspheres and the specific synthesis method are all recorded in the document "Synthesis and characterization of magnetically separable Agnanoparticles decorated mesoporous Fe3O4@carbon with antibacterial and catalytic properties[J].Yu Q,Fu A,Li H,et al.Colloids&Surfaces APhysicochemical&Engineering Aspects,2014,457(1):288-296.”, the public can obtain from the applicant (China Agricultural University), the biological The materials are only used for repeating the relevant experiments of the present invention and cannot be used for other purposes.

EDC and NHS in the following examples are products of Aladdin Reagent Co., Ltd.

Example 1: Preparation of immunomagnetic microspheres and its application in enriching clenbuterol hydrochloride in swine urine samples

1. Preparation of immunomagnetic microspheres

1. Preparation of Suspension A

Weigh 0.1 g of Fe ₃ O ₄ magnetic microspheres into 20 mL of distilled water, and sonicate for 30 min to form suspension A.

2. Preparation of Suspension B

0.8 g of chitosan (purchased from Bailingwei Technology Co., Ltd., degree of deacetylation: 95%) was dissolved in 15 mL of dilute acetic acid solution with a volume fraction of 2% to obtain a homogeneous suspension B with a mass fraction of 2%.

3. Preparation of Fe ₃ O ₄ @Chitosan Magnetic Microspheres

Mix suspension A and suspension B in a volume ratio of 4:1, stir for 1 hour, spray dry by a spray dryer for 24 hours, and then calcine at a high temperature of 800 °C for 4 hours under nitrogen conditions to obtain Fe ₃ O ₄ @chitosan Sugar Magnetic Microspheres.

4. Fe ₃ O ₄ @Chitosan Magnetic Microspheres Conjugated Antibody

(1) Add 1 μg of clenbuterol hydrochloride monoclonal antibody solution (clenbuterol hydrochloride monoclonal antibody was purchased from Beijing Weideweikang Biotechnology Co., Ltd., CL-1F8-F9) with a concentration of 5 μg/mL in 1 mL. EDC and 1 μg of NHS were activated by shaking reaction for 15 min to obtain an activated clenbuterol hydrochloride monoclonal antibody solution.

(2) Then add the Fe ₃ O ₄ @chitosan immunomagnetic microspheres prepared in step 3 into the activated clenbuterol hydrochloride monoclonal antibody solution, and the clenbuterol hydrochloride monoclonal antibody and Fe ₃ O ₄ @shell The mass ratio of glycan immunomagnetic microspheres was 1:50. The reaction was shaken for 30 min, and the mixture was mixed every 10 min to obtain the conjugate.

5. Washing and resuspension

The conjugate prepared in step 4 was washed 5 times with 5 mL of washing solution (PB solution with a concentration of 0.02 M); then resuspended with 6 mL of 0.01 M phosphate buffer solution (pH 7.0) to obtain an immunomagnetic concentration of 20 μg/mg Microsphere solution, which contains specific immunomagnetic microspheres.

2. Application of immunomagnetic microspheres in enrichment of clenbuterol hydrochloride in swine urine samples

1. Adsorption

Use a pipette to take the sample supernatant (1600 μL of pig urine sample) into a 4 mL centrifuge tube with pre-packaged immunomagnetic microspheres, mix well, and react at room temperature for 10 min.

2. Washing

Place the centrifuge tube on a magnetic stand for magnetic adsorption for 60s (referred to as magnetic separation), discard the reaction solution, add 1 mL of washing solution (PB solution with a concentration of 0.02M) to the centrifuge tube, gently shake ten times, magnetic separation, Discard the wash solution.

3. Elution

Immediately add 400 μL of sample diluent (PB solution with a concentration of 0.02M) to the centrifuge tube, mix well, place the centrifuge tube on a dry thermostat at 100 °C for 3 min, magnetically separate, collect the supernatant, and finally obtain purified and purified samples. enriched sample extract.

Example 2: Preparation of double-labeled indirect background fluorescent colloidal gold test strips

1. Preparation of double-labeled colloidal gold

1. Preparation of colloidal gold-clenbuterol hydrochloride monoclonal antibody-biotin (GNPs-clenbuterol hydrochloride antibody-biotin)

(1) Preparation of biotinylated clenbuterol monoclonal antibody solution

Aminated biotin with a concentration of 3.8 mg/ml (purchased from SIGMA) was added to 1 mg of clenbuterol hydrochloride monoclonal antibody, and after rotating for 1 h, dialyzed three times with a dialysis membrane (8k MWCO), buffer (pH 7) .4) The formula is as follows: 50mM PB, 75mM NaCl, dialyze 2L for more than 3h each time. Then carefully aspirate the biotinylated antibody from the dialysis bag with a pipette, adjust the concentration of the biotinylated antibody with a 50 mM PBS solution to obtain a biotinylated clenbuterol monoclonal antibody with a concentration of 2.3 mg/ml solution.

(2) Preparation of gold particle solution

Take 50 mL of deionized water and boil it in a 200 mL clean conical flask, add 1 mL of 1% chloroauric acid solution (sigma) and 1 mL of 1% trisodium citrate solution, and continue to boil for 3 minutes. Reserve at room temperature.

(3) Add 2.3 μg of the biotinylated clenbuterol monoclonal antibody solution prepared in step (1) to 1 mL of the gold particle solution prepared in step (2), incubate at room temperature, and then add BSA with a mass fraction of 10%. The solution was used as a blocking solution and blocked at room temperature.

(4) After completing step (3), remove free and unbound clenbuterol monoclonal antibody-biotin by high-speed centrifugation, and then resuspend (0.01M PBS containing 0.5% Tween 20, 2% sucrose) Perform resuspension to obtain 200 μL 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) Add 200 μL of streptavidin with a concentration of 1 mg/mL to 1 mL of the gold particle solution prepared in step 1 (2), stir while adding, and immediately add 200 μL of 1M NaHCO ₃ solution, incubate at room temperature for 10 min, and then use 200 μL of 2% PEG6000 stabilized gold nanoparticles.

(2) After completing step (1), centrifuge at 10000g for 30 min at 4°C to remove free and unbound streptavidin, and then resuspend with 200 μL of resuspension (0.1M PBS containing 0.02% PEG 6000) to obtain Colloidal gold-streptavidin (GNPs-streptavidin) solution at a concentration of 1 mg/mL.

2. Preparation of test line and quality control line

BSA protein and clenbuterol hydrochloride antigen conjugate (BSA protein and clenbuterol hydrochloride antigen conjugate were entrusted by Beijing Viderweikang Biotechnology) with 0.01M phosphate buffered saline (PBS, pH7.0). Technology Co., Ltd.) and goat anti-mouse IgG (purchased from Jackson ImmunoResearch Laboratories, Inc., product catalog number 125229) were diluted to concentrations of 0.6 mg/ml and 0.3 mg/ml, respectively, and then used a microtome at 0.75 The positions of the detection line and the quality control line on the nitrocellulose membrane (NC membrane) were sequentially sprayed at a spray rate of μL/cm, and the distance between the detection line and the quality control line was 3 mm. The scribed NC films were placed in a 37°C oven overnight.

3. Test strip assembly

The test strip of the present invention includes five parts: a fluorescent back plate, a PVC bottom plate, a nitrocellulose membrane, a sample pad and an absorbent paper. The specific preparation method is as follows:

1. Cover the middle of the PVC bottom plate (purchased from Shanghai Jinbiao Biotechnology Co., Ltd., product catalog number is SMNF31-40) with a fluorescent backplane (the fluorescent backplane is a PVC plastic plate with fluorescence sprayed on the surface, entrusted by Shanghai Xinpu Biotechnology Co., Ltd.) Co., Ltd.), and then covered with a nitrocellulose membrane with a detection line and a quality control line on the fluorescent backplane, and then lapped the sample pad (glass cellulose membrane) and absorbent paper on the nitrocellulose membrane in turn. both ends. The sample pad and the absorbent pad were pressed against the NC membrane by 2 mm at each end.

2. Cut the processed large board into test strips with a width of 4.72mm, install it in a plastic card case and assemble it into a test card, and then put it in a dry and closed aluminum foil bag for use.

The schematic diagram of the structure of the colloidal gold immunochromatographic test strip of the present invention is shown in FIG. 2 . The detection principle is shown in Figure 3.

Example 3: Quantitative and qualitative detection of clenbuterol hydrochloride

1. Qualitative detection method of clenbuterol hydrochloride

1. Use the immunomagnetic microspheres in Example 1 to enrich and purify the sample to be tested to obtain a processed sample.

2. Combine 150 μL of the treated sample with 3 μL of the GNPs-clenbuterol hydrochloride antibody-biotin solution prepared in step 1 of Example 2 and 3 μL of GNPs-streptavidin prepared in step 2 of Example 2 The solution was mixed and incubated at room temperature for 3 min to obtain a mixed solution.

3. Take 120 μL of the mixed solution and drop it on the sample pad of the test strip. After 10 minutes, qualitative observation was made with the naked eye.

If both the C line and the T line are red, the sample to be tested does not contain clenbuterol hydrochloride;

If the C line is red and the T line is not, the sample to be tested contains clenbuterol hydrochloride.

2. Quantitative detection method of clenbuterol hydrochloride

1. Drawing of the standard curve

(1) Clenbuterol hydrochloride standard containing different concentrations of 0.015μg/L, 0.031μg/L, 0.062μg/L, 0.12μg/L, 1.25μg/L, 0.5μg/L, 1μg/L respectively. solution as the sample to be tested.

(2) Enriching and purifying the sample to be tested using the immunomagnetic microspheres in Example 1 to obtain a processed sample (pre-processing sample). At the same time, the test sample without any treatment was used as a control (unpretreated sample).

(3) Combine 150 μL of the treated sample with 3 μL of the GNPs-clenbuterol hydrochloride antibody-biotin solution prepared in step 1 of Example 2, and 3 μL of GNPs-streptavidin prepared in step 2 of Example 2 The solution was mixed and incubated at room temperature for 3 min to obtain a mixed solution.

(4) Drop 120 μL of the mixed solution on the sample pad of the test strip. After 10 minutes, the fluorescence intensity of the quality control line and the detection line was measured by fluorescence immunoassay analyzer FQ-S2. Set the fluorescence reading of the blank backplane as F ₀ , the fluorescence reading at the position of the T line as F _T , F ₀ is the average fluorescence intensity of the blank part between the T line and the C line that is not covered by colloidal gold, and F _T is the T line Fluorescence intensity masked by colloidal gold. Take F ₀ / _FT as the ordinate and the concentration of clenbuterol in the standard solution as the abscissa to establish a standard curve (Fig. 4), and obtain the standard curve equation.

2. Detection of clenbuterol hydrochloride content in the sample to be tested

(1) The sample to be tested is enriched and purified using the immunomagnetic microspheres in Example 1 to obtain a processed sample.

(2) Combine 150 μL of the treated sample with 3 μL of the GNPs-clenbuterol hydrochloride antibody-biotin solution prepared in step 1 of Example 2, and 3 μL of GNPs-streptavidin prepared in step 2 of Example 2 The solution was mixed and incubated at room temperature for 3 min to obtain a mixed solution.

(3) Drop 120 μL of the mixed solution on the sample pad of the test strip. After 10 minutes, the fluorescence intensity of the quality control line and the detection line was measured by using the fluorescence immunoquantitative analyzer FQ-S2, and the F ₀ /F _T was calculated. Bring the _{F 0} /FT of the sample to be tested into the standard curve equation in step 1, and calculate the content of clenbuterol hydrochloride in the sample to be tested.

Example 4: Application of double-labeled indirect background fluorescent colloidal gold test strips

1. Preparation of samples to be tested

Clenbuterol hydrochloride standard was mixed with fresh pig urine samples identified as Clenbuterol hydrochloride negative to obtain different concentrations (0.04μg/L, 0.10μg/L, 0.15μg/L, 0.30μg/L). Clenbuterol hydrochloride solution as the sample to be tested.

2. Detection of samples to be tested

1. Each sample to be tested is enriched and purified by using the immunomagnetic microspheres in Example 1 to obtain a processed sample (pre-processing sample).

2. Mix 150 μL of the pretreated sample with 3 μL of the GNPs-clenbuterol hydrochloride antibody-biotin solution prepared in step 1 of Example 2 and 3 μL of the GNPs-streptavidin solution prepared in step 1 of Example 2. Mix well and incubate at room temperature for 3 min to obtain a mixed solution.

3. Take 120 μL of the mixed solution and drop it on the sample pad of the test strip. After 10 minutes, the fluorescence intensity of the quality control line and the detection line was measured by fluorescence immunoassay analyzer FQ-S2, and _{F 0} /FT was calculated, and then substituted into the standard curve (Fig. 4), and the recovery rate and coefficient of variation were calculated. Recovery (%)=(detected concentration/added concentration)×100%; coefficient of variation CV=standard deviation/mean.

The results are shown in Table 1. According to the naked eye observation, the extinction value was 1ppb, the lowest detection limit could reach 0.02ppb, the recovery rate was 81%-115%, and the coefficient of variation was less than 5.99%.

Table 1. Test results of Clenbuterol hydrochloride standard

Example 5: Specific detection of clenbuterol hydrochloride test strips

1. Preparation of samples to be tested

The following adrenergic agonists will be used: Clenbuterol, salbutamol, cimaterol, terbutaline, ractopamine, vedolol, propranolol, bulobanol, atenolol, Sharol, zilpaterol, epinephrine and phenethylamine A were added to the swine urine samples treated with immunomagnetic microspheres, respectively, and the following different concentrations were obtained: 0 μg/L, 1.2 μg/L, 3.5 μg /L, 6.2μg/L, 12.5μg/L, 25μg/L, 50μg/L, 100μg/L adrenergic receptor agonist test sample solutions.

2. Detection of samples to be tested

Use the colloidal gold test strip of Example 2 to detect clenbuterol hydrochloride in each sample solution to be tested according to the quantitative detection method of clenbuterol hydrochloride in Example 3, and evaluate its specificity by calculating the cross-reaction rate. Cross-reaction rate CR (%)=(Clenbuterol hydrochloride IC ₅₀ / IC ₅₀ of other receptor agonists)×100%.

The results are shown in Table 2. The cross-reaction rates of the clenbuterol hydrochloride test strip of the present invention with salbutamol and cimaterol are 0.59% and 3.5% respectively, and there is no cross-reaction with other adrenergic receptor agonists.

Table 2. Cross-reactivity between clenbuterol and other adrenergic agonists

Agonist IC₅₀(μg/L) Cross-reaction rate (%) Clenbuterol Hydrochloride 0.03 100 salbutamol 17 0.59 Cimatro 3.5 3.5 ractopamine >2000 <0.1 Vidolore >2000 <0.1 propranolol >2000 <0.1 folate >2000 <0.1 atenolol >2000 <0.1 oxalol >2000 <0.1 Zipatro >2000 <0.1 epinephrine >2000 <0.1 Phenylethylamine A >2000 <0.1

Claims (10)

1. A test kit comprising a test strip and a double-labeled colloidal gold probe; The double-labeled colloidal gold probe is composed of gold nanoparticle-labeled biotinylated anti-target antibody and gold nanoparticle-labeled streptavidin; The test strip is composed of a sample pad connected and fixed on the base layer in sequence, a coating film provided with a detection line and a quality control line, and a water-absorbing pad; a fluorescent back is arranged between the coating film and the base layer. plate; 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 can specifically bind to the target antigen; The quality control line is coated with an antibody against the anti-target antibody; The detection line is located at one end of the coating film close to the sample pad; The quality control line is located at one end of the coating film close to the absorbent pad. 2. test kit according to claim 1, is characterized in that: In the gold nanoparticle-labeled biotinylated anti-target antibody, the mass ratio of the gold nanoparticle to the biotinylated anti-target antibody is (300-1000):1; Or, in the streptavidin labeled with gold nanoparticles, the mass ratio of the gold nanoparticles to streptavidin is (50-500):1; Or, the anti-target antibody is an anti-target monoclonal antibody or polyclonal antibody. 3. test kit according to claim 1 and 2, is characterized in that: The anti-target antibody is an anti-target monoclonal antibody; Or, the antibody against the anti-target antibody is goat anti-mouse IgG; Or, the particle size of the gold nanoparticles is 30nm; Or, the material of the base layer is polyvinyl chloride; Or, the fluorescent back plate is a PVC plastic plate sprayed with fluorescent light on the surface; Or, the sample pad is glass cellulose membrane; Or, the coating film is a nitrocellulose membrane; Or, the distance between the detection line and the quality control line is 3 mm. 4. The preparation method of the test kit according to any one of claims 1-3, comprising the steps of: respectively preparing the test strip and the double-labeled colloidal gold probe in the test kit according to any one of claims 1-3; The method for preparing the test strip comprises the following steps: 1. Prepare the sample pad, the coating film provided with the detection line and the quality control line, and the water-absorbing pad respectively; II, cover the fluorescent backplane in the middle of the base layer, then cover the coating film with the detection line and the quality control line obtained in step 1 on the fluorescent backplane, and then apply the The sample pad and the described water-absorbing pad obtained in step 1 are respectively overlapped on both ends of the coated 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: the target antigen described in claims 1-3 and the goat anti-mouse IgG described in claims 1-3 are sprayed on the The detection line area and the quality control line area of the coating film are formed to form the detection line and the quality control line, and the coating film provided with the detection line and the quality control line is obtained; The method for preparing the double-labeled colloidal gold probe is the preparation method of the gold nanoparticle-labeled biotinylated anti-target antibody and the gold nanoparticle-labeled streptavidin described in claims 1-3. 5. the application of the test kit described in any one of claim 1-3 or the preparation method described in claim 4 in the detection target; Or, the application of the kit according to any one of claims 1-3 or the preparation method according to claim 4 in detecting whether the sample to be tested contains the target substance; Or, application of the kit according to any one of claims 1-3 or the preparation method according to claim 4 in detecting the content of a target substance in a sample to be tested. 6. A method for detecting whether a sample to be tested contains a target, comprising the steps of: (c1) enriching the sample to be tested with immunomagnetic microspheres to obtain an enriched sample; (c2) adding the double-labeled colloidal gold probe described in any one of claims 1-3 to the enriched sample to obtain a mixed solution; (c3) incubate the mixed solution for 3min, then add it to the sample pad of the test strip described in any one of claims 1-3, after chromatographic 10min, observe the color development of the detection line and the quality control line; If both the C line and the T line are red, the sample to be tested does not contain the target or the candidate does not contain the target; If the C line shows red and the T line does not show color, the sample to be tested contains the target or a candidate for the target. 7. A method for detecting the content of a target substance in a sample to be tested, comprising the steps of: (d1) draw standard curve Prepare standard solution of target substance with a series of concentrations, and enrich several obtained standard solutions containing different concentrations with immunomagnetic microspheres respectively to obtain enriched samples; Mixing the double-labeled colloidal gold probe described in any one of claims 1-3 to obtain a mixed solution; incubating the mixed solution for 3 min, and then adding it to the sample pad of the test strip described in any one of claims 1-3 , after 10 min of chromatography, use a fluorescence reader to measure the fluorescence intensity of the blank backplane and the fluorescence intensity of the T line; take the concentration of the standard solution of the target as the abscissa, and take the fluorescence intensity of the blank backplane and The ratio of the fluorescence intensity of the T line is the ordinate to draw a standard curve graph to obtain a standard curve equation; (d2) Detection of samples to be tested The samples to be tested are enriched with immunomagnetic microspheres to obtain enriched samples; the enriched samples are respectively mixed with the double-labeled colloidal gold probes described in any one of claims 1-3, Obtain a mixed solution; incubate the mixed solution for 3min, then add it to the sample pad of the test strip described in any one of claims 1-3, after chromatographic 10min, use a fluorescence reader to measure the fluorescence intensity of the blank backplate and The fluorescence intensity of the T line is obtained by obtaining the ratio of the fluorescence intensity of the blank backplane to the fluorescence intensity of the T line, and substituting it into the standard curve equation obtained in step d1) to calculate the target content in the sample to be tested. 8. method according to claim 6 or 7, is characterized in that: the preparation method of described immunomagnetic microspheres comprises the steps: (1) Mixing the Fe ₃ O ₄ magnetic microsphere suspension with the chitosan suspension, drying and calcining at high temperature in turn, to obtain Fe ₃ O ₄ @chitosan magnetic microspheres; (2) Coupling the Fe ₃ O ₄ @chitosan magnetic microspheres with the anti-target antibody to obtain Fe ₃ O ₄ @chitosan magnetic microspheres conjugated antibodies, which are the immunomagnetic microspheres ; (a1) adding the sample to be tested containing the target into the centrifuge tube containing the immunomagnetic microspheres to carry out adsorption reaction; (a2) magnetically separate the centrifuge tube obtained in step (a1) under the action of a magnetic field, discard the reaction solution and add washing solution for washing; (a3) adding the sample diluent to the centrifuge tube obtained in step (a2), heating, performing magnetic separation under the action of a magnetic field, collecting the supernatant, and realizing the enrichment of the target substance. 9. The immunomagnetic microspheres of claim 8. 10. the application of the immunomagnetic microsphere of claim 9 in enrichment target; Or, the application of the immunomagnetic microspheres of claim 9 in the preparation of a product enriched with a target.

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