CN110988338A - Quantum dot test paper capable of quantitatively detecting trimethoprim residual quantity in various matrixes - Google Patents
Quantum dot test paper capable of quantitatively detecting trimethoprim residual quantity in various matrixes Download PDFInfo
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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
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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
- G01N33/543—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
Abstract
The invention discloses a method for establishing quantum dot immunochromatographic test paper capable of quantitatively detecting trimethoprim in various food matrixes such as meat, egg and milk, the test paper is prepared by mutually overlapping a sample pad, a marker pad, a chromatographic membrane and a cushion pad by 1-2mm and sequentially adhering the sample pad, the marker pad, the chromatographic membrane and the cushion pad on a PVC (polyvinyl chloride) base plate, the chromatographic membrane is a nitrocellulose membrane bearing a detection line and a quality control line, the detection line is coated with trimethoprim antigen, and the quality control line is coated with DNP (deoxyribose nucleic acid) monoclonal antibody; the label pad bears quantum dots coupled with trimethoprim antibody and DNP polyclonal antibody coupled with BSA. When the sample contains trimethoprim, the position of the T line flowing through the T line and the antigen coated by the T line compete to bind with the antibody on the marker pad, so that the fluorescence intensity of detection is changed to carry out quantitative detection.
Description
Technical Field
The invention belongs to the technical field of food detection, and particularly relates to quantum dot immunochromatography test paper capable of quantitatively detecting trimethoprim residual quantity in various food matrixes.
Background
Trimethoprim is a synthesized broad-spectrum antibacterial agent, has an antibacterial spectrum similar to that of sulfonamides, but has stronger antibacterial action than that of sulfonamides, is effective on various common gram-negative bacteria and gram-positive bacteria, but is ineffective on pseudomonas aeruginosa, has the defect that the single use easily causes bacterial drug resistance, is mainly used together with sulfonamide reagents and is used as a combined antibacterial agent to form a compound preparation, so the trimethoprim is also called a sulfonamide synergist and is used for expanding the antibacterial spectrum and enhancing the antibacterial activity. With the unreasonable use of sulfonamides in veterinary medicines, antibacterial synergists such as trimethoprim and the like are also applied more and more widely. The limited requirement of trimethoprim is clearly specified in the notice No. 235 of Ministry of agriculture, but different food animals have different limited requirements and higher detection requirements, so that in order to strengthen the monitoring of secondary medicines in animal food, a method for accurately, quantitatively, quickly and conveniently detecting the residual quantity of trimethoprim is necessary.
At present, a plurality of methods for detecting trimethoprim residues are available, and the method mainly comprises a large-scale instrument detection method represented by high performance liquid chromatography, combination of liquid chromatography and mass spectrometry, a capillary electrophoresis analysis method and the like, and has high sensitivity, but needs to use a large-scale instrument, and has complex treatment aiming at different matrixes and higher personnel requirements; the traditional rapid detection method represented by the colloidal gold test strip in the aspect of rapid detection is simple and convenient to operate and short in time consumption, but the trimethoprim detection matrix is complex, the traditional test strip has the limitations of low matrix interference resistance, easiness in damage to substances such as glacial acetic acid and ethanol contained in an extractant and the like, and only a single kit can be developed for qualitative detection aiming at a specific food matrix; the novel detection method represented by the chemiluminescence immunoassay method has the advantages of high detection sensitivity, easy observation of results, complex process and high production cost. Therefore, in order to detect various substrates and complex limit requirements, a simple, rapid and accurate quantitative detection method which is applicable to various food substrates and has high sensitivity to dairy products should be developed. The quantum dot immunochromatography technology uses quantum dots as tracer markers to be applied to immunochromatography, on one hand, the unique fluorescence of the quantum dots is used as an observation signal, and the immunochromatography technology has the advantages of high sensitivity and high speed, on the other hand, the advantages of simplicity, strong specificity, low cost and the like of the immunochromatography technology are combined into a whole, and the immunochromatography technology is used for accurately and quantitatively detecting the content of trimethoprim in each food matrix. At present, the research on new technology for rapidly and quantitatively detecting trimethoprim is less in China, the research on the combination of quantum dots, a detection technology and the adaptability of a food matrix is less, the establishment of the method has great innovation, the method is suitable for screening the content of trimethoprim in raw materials in a large scale, and the method has a certain promotion effect on the development of the rapid detection technology.
Disclosure of Invention
Aiming at the defects of the prior detection technology, the invention aims to provide the quantum dot immunochromatographic test paper capable of detecting the residual quantity of trimethoprim in various food matrixes, and the quantum dot is used as a novel electronic nano fluorescent material, is less applied to food detection and needs to be matched by researching related technologies. The method is characterized in that the influence of a food matrix and a pretreatment reagent on quantum dots and an active material is reduced by researching the combination technology of the active material and a quantum dot label, and an accurate quantum dot immunochromatography method for quantitatively detecting the residual quantity of trimethoprim is established by a series of technologies such as active material fixation research, concentration optimization research and raw and auxiliary material treatment method screening, so that the interference of different types of food matrices and pretreatment reagents on the quantum dot detection is reduced, the simplicity of the treatment method is further improved, and the simple, convenient and accurate quick detection operation is realized.
In order to achieve the purpose, the invention adopts the following technical scheme:
a quantum dot immunochromatographic test paper for detecting trimethoprim in food is prepared by overlapping a sample pad, a marker pad, a chromatographic membrane and a cushion pad by 1-2mm and sequentially adhering the overlapped pads on a PVC (polyvinyl chloride) bottom plate, wherein the chromatographic membrane is a nitrocellulose membrane bearing a detection line and a quality control line; wherein, the detection line is coated with trimethoprim antigen, and the quality control line is coated with DNP monoclonal antibody; the label pad bears quantum dots coupled with trimethoprim antibody and DNP-BSA.
The chromatographic membrane is adhered to the PVC base plate, the cushion pad is adhered to one side of the chromatographic membrane, and the overlapped parts are 1-1.5 mm; the marker pad and the sample pad are adhered to the other side of the chromatographic membrane, the overlapped parts are 1-1.5 mm, and all the parts are tightly adhered to the PVC bottom plate.
Further, the sample pad is made of cotton pulp paper, and the buffer pad and the marker pad are made of glass cellulose membranes.
Furthermore, the quantum dot coupled with the trimethoprim antibody and DNP-BSA is obtained by coupling the activated quantum dot with the trimethoprim antibody and the DNP-BSA in sequence, and then adding a casein solution for blocking.
The preparation method of the chromatography test paper comprises the following steps:
step 1, preparation of a marker pad:
1) dropwise adding a first treatment reagent into the glass cellulose membrane, and drying to obtain a pretreated glass cellulose membrane;
2) taking 100 mu L of 10mg/mL quantum dot stock solution, and performing ultrasonic dispersion;
3) activating the quantum dot microspheres, taking 50 mu L of dispersed quantum dot stock solution, uniformly mixing the quantum dot stock solution with 450 mu L of 0.05M MES buffer solution with pH6.0, adding 3 mu L of 50mg/mL EDC, uniformly mixing, adding 3 mu L of 75mg/mL NHS, uniformly mixing, and vertically mixing for 0.5 h;
4) resuspending, namely centrifuging 12000rp of the mixed solution obtained in the step 3) for 5min, discarding the supernatant, adding 500 mu L of MOPS (molecular orbital phosphate) with the pH value of 0.02MpH7.0 for resuspension, and ultrasonically dispersing;
5) antibody coupling: adding 40 mu g of trimethoprim antibody into the dispersion liquid obtained in the step 4), uniformly mixing and coupling for 1h, adding 50 mu g of DNP-BSA, and coupling for 1 h;
6) and (3) sealing: adding 5 mu L of 10% ethanolamine into the mixed solution obtained in the step 5), uniformly mixing, then adding 500 mu L of 5% casein solution, and vertically and uniformly mixing at room temperature to seal redundant sites;
7) resuspending: centrifuging 12000prm of the mixed solution obtained in the step 6) for 5min, adding 500 mu L of quantum dot diluent to reach the constant volume of 1mg/mL, performing ultrasonic dispersion, and storing at 4 ℃;
8) diluting: diluting the quantum dot-antibody obtained in the step 7) to 0.5mg/mL, and performing ultrasonic dispersion for later use;
9) adding 0.05% carmine into the diluted quantum dot-antibody diluent, spraying the diluted quantum dot-antibody diluent on a pretreated glass cellulose membrane, and drying to obtain a marker pad;
and 5, assembling the sample pad, the marker pad, the chromatographic membrane and the buffer pad to obtain the kit.
Further, the first treatment reagent was prepared by adding 5% sucrose, 0.3% PVPK-30, 1% Tween-20, and 0.2% Proclin-300 to 0.05M Tris-Base buffer solution (pH8.0).
Further, the second treatment reagent was prepared by adding 0.3% PVPK-30, 1% Triton X-100, and 0.2% Proclin-300 to 0.05M Tris-Base buffer solution (pH8.0).
Further, the third treatment reagent is prepared by adding 5% trehalose, 0.8% Tween-20, 3.2mM EDTANa to 0.05M Tris-Base buffer solution (pH8.5)2And 0.2% casein.
Further, the T-line coating solution was prepared by adding 2% sucrose and 8% methanol to 0.2M Hepes buffer solution (pH8.0).
Further, the coating solution for C-line was prepared by adding 8% methanol, 2% sucrose, 0.5% BSA, 0.1% Proclin-300 to 0.05M Tris-Base buffer solution (pH8.0).
Further, the quantum dot diluent is prepared by adding 15% of sucrose, 0.5% of Tween-20, 3% of BSA, 1% of mannitol and 0.1% of Proclin-300 into 0.1M Tris-Base buffer solution with pH8.0.
The test paper is used for quantitatively detecting the content of trimethoprim in food, adopts quantum dots (nano up-conversion fluorescent substance) as a tracer substance, has high sensitivity and strong specificity, and adopts an immunochromatography method to perform detection by a reverse competition method: the T line is wrapped by trimethoprim antigen with a certain concentration, the C line is used as a quality control line and is wrapped by a specific mouse single antibody, the quantum dot coupling trimethoprim antibody and the related mouse anti-DNP antibody are uniformly distributed on the marker pad through a metal spraying instrument, when raw materials contain trimethoprim, the position of the T line flowing through the T line can compete with the antigen wrapped by the T line to be combined with the antibody on the marker pad, and therefore the fluorescence intensity of detection is changed to carry out quantitative detection.
Compared with the prior art, the invention has the following advantages:
1. compared with the traditional colloidal gold detection kit, the kit has the advantages of high sensitivity, good stability, capability of realizing accurate quantitative detection, simplicity, convenience, rapidness and simple operation compared with an instrumental method for legal detection, is suitable for screening food raw materials in large batches and can meet the existing requirements.
2. Through a series of technical exploration and optimization, the direct sample loading detection of the dairy product raw materials without pretreatment can be realized, the meat product eggs are simply crushed and then contain a certain amount of glacial acetic acid solvent for processing and then are loaded, the pretreatment is simple, and the substrate benefit is small.
3. Through a series of technical research and development and matching, the requirement that various food matrixes such as meat, eggs and milk can be accurately quantified can be met.
Drawings
FIG. 1 is a standard curve for trimethoprim detection established with a milk base in example 2.
FIG. 2 is a standard curve for trimethoprim detection established for other substrates in example 2.
FIG. 3 is a regression curve of trimethoprim with milk base for the test in example 2.
FIG. 4 is a regression curve of trimethoprim for detection established with other matrices in example 2.
Detailed Description
The following examples further illustrate the present invention but are not to be construed as limiting the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention. The experimental methods and reagents of the formulations not specified in the examples are in accordance with the conventional conditions in the art.
Example 1
The preparation of the quantum dot immunochromatographic test paper for quantitatively detecting trimethoprim in the food raw materials comprises the following steps:
1. reagent and raw and auxiliary materials
Auxiliary materials: nitrocellulose membrane (NC membrane), glass cellulose membrane, PVC base plate, cotton pulp paper.
NC film: and carrying the T/C wire live material.
Buffer (glass cellulose film): treating reagents: 0.05M Tris-Base buffer solution at pH8.0+ 0.3% PVPK-30+ 1% Triton X-100+ 0.2% Proclin-300 preservative solution. 15mL of the treatment reagent was added uniformly per 10cm by 30cm area, and the mixture was placed in an oven at 37 ℃ for 24 hours.
Sample pad (cotton pulp paper): 0.05M Tris-Base buffer solution at pH8.5 + 5% trehalose + 0.8% Tween-20+3.2mM EDTANa2+ 0.2% casein, 20mL of treatment reagent was added evenly per 10cm x 30cm area, and placed in a 45 ℃ oven for 24 h.
Marker pad (glass cellulose membrane, different density from that of the buffer pad): 0.05M Tris-Base buffer solution with pH8.0+ 5% sucrose + 0.3% PVPK-30+ 1% Tween-20+ 0.2% Proclin-300 preservative solution 16mL of treatment reagent per 10cm by 30cm area was added uniformly, and the mixture was placed in an oven at 37 ℃ for 24 hours.
Labeling reagent: pH7.0 in 0.02M MOPS buffer; 0.05M MES buffer pH 6.0; 1-3-ethylcarbodiimide hydrochloride (EDC); n-hydroxysuccinimide (NHS); a 10% casein solution; 10% ethanolamine solution.
Coating liquid:
and (4) line T: 0.2M Hepes buffer pH8.0+ 8% methanol + 2% sucrose;
line C: 0.05M Tris-Base buffer solution at pH8.0+ 8% methanol + 2% sucrose + 0.5% BSA + 0.1% Proclin-300.
Quantum dot-antibody dilution: 0.1M TB pH8.0+ 15% sucrose + 0.5% Tween-20+ 3% BSA + 1% mannitol + 0.1% Proclin-300.
Antibody: trimethoprim antibody (used for labeling).
Antigen: DNP-BSA (for labeling).
2. Experimental methods and procedures
2.1 preparing coating liquid
T-line coating liquid: 0.2M Hepes 8.0+ 2% sucrose + 8% methanol
C, coating liquid: 0.05M TB 8.0+ 8% methanol + 2% sucrose + 0.5% BSA + 0.1% Proclin-300
2.2 Membrane preparation, 1 strip of 30cm for each antibody
1) Preparation of coating liquid
Coating material of C line: 1mg/mL DNP monoclonal antibody immobilization
T-line coating: 0.5mg/mL trimethoprim antigen immobilization
2) Scribing film
Spraying amount of 0.6 mu L/cm, drying in an oven at 37 ℃ for 16-24 hours
2.3 Quantum dot labeling of active materials
The preparation steps required about 40. mu.g of antibody each (trimethoprim antibody/DNP-BSA)
1) Taking 100 mu L of quantum dot stock solution (10mg/mL) and carrying out ultrasonic treatment for 2min, and detecting and recording the particle size dispersion coefficient (the standard particle size is less than 400nm, and the dispersion coefficient is less than 0.1);
2) activating the quantum dot microspheres: taking 50 mu L of detected quantum dot stock solution (10mg/mL) +450 mu L of 0.05MMES buffer solution with pH6.0, mixing uniformly, adding 3 mu L of EDC (50mg/mL), mixing uniformly, adding 3 mu L of NHS (75mg/mL), mixing uniformly, and mixing uniformly for 0.5h vertically.
3) Resuspending: centrifuging at 12000rpm for 5min, and discarding the supernatant; adding 500 μ L of 0.02M MOPS 7.0 for resuspension, and ultrasonically dispersing for 2 min; detecting and recording the particle size dispersion coefficient (standard particle size is less than 400nm, dispersion coefficient is less than 0.1);
4) antibody coupling: adding 40 mu g of trimethoprim antibody, uniformly mixing and coupling for 1h, then adding 50 mu g of DNP-BSA, and coupling for 1 h;
5) and (3) sealing: adding 5 mu L10% ethanolamine, mixing, adding 500 mu L10% casein solution, and vertically mixing at room temperature to block redundant sites for 0.5 h;
6) resuspending: centrifuging at 12000prm for 5min, adding 500 μ L of quantum dot diluent to constant volume of 1mg/mL, and ultrasonically dispersing; detecting and recording the particle size dispersion coefficient (standard particle size is less than 400nm, dispersion coefficient is less than 0.1), and storing at 4 degrees;
7) diluting: diluting the quantum dot-antibody to 0.4mg/mL, performing ultrasonic dispersion, detecting and recording the particle size dispersion coefficient (the standard particle size is less than 400nm, and the dispersion coefficient is less than 0.1);
8) spraying a marker pad: the diluted quantum dot-antibody dilution was sprayed onto the treated label pad using a gold spraying instrument.
Adding 0.05% carmine;
the concentration is 0.4mg/mL, and the spraying amount is 4 mu L/cm;
correlation coefficient of metal spraying instrument, spray head position: x ═ 5, Y ═ 9, Z ═ 7; the placing position of the marker pad is 4 mu L/cm;
9) drying: baking at 45 deg.C for 16-24 h.
Example 2
Application of quantum dot immunochromatographic test paper for quantitatively detecting trimethoprim in food raw materials
1. Preparing a standard substance: weighing a certain amount of trimethoprim standard substance, diluting to 1 microgram/mL by using a pretreatment reagent with the components of pH7.0 Tris-HCl buffer solution, 5% glacial acetic acid, 5% casein, 2% polyethylene glycol, 2% dimethylformamide and 0.2% Proclin-300, preparing a series of standard working solutions with the concentrations of 5, 10, 50, 100, 200 and 500ng/mL, diluting the standard working solution for detecting the milk matrix by using blank fresh milk to prepare the same concentrations of 500, 200, 100, 50, 10 and 5ng/mL, respectively sampling, detecting respective fluorescence values, taking each concentration of the standard working solution as an abscissa (ng/mL), taking the ratio of the fluorescence detection values of the detection line T and the quality control line C as an ordinate, other matrix and milk matrix standard curves are shown in fig. 1 and 2, respectively, with a four parameter fit.
2. And (3) quantitative detection: according to the difference of the detection fluorescence intensity obtained by competing the difference of the antigen amount remained on the T line according to the difference of the trimethoprim content in different samples, a related detection machine is used for converting a fluorescence signal into a luminous value number, a T/C value is calculated, a standard curve is drawn, and during the detection of the sample, the reverse calculation is carried out according to the T/C value, so that the quantitative detection is carried out on the trimethoprim content in the sample.
3. When milk is used as a detection substrate, a large number of normal milk samples are mixed to be used as zero-value samples, then the zero-value samples are loaded with 20 parts of the chromatographic test strip, test values are recorded, the average value X and the standard deviation SD of the T/C of 20 test values are solved, then the concentration calculated by substituting X-2SD into a standard curve equation is the lowest detection limit, and the sensitivity of the test strip is shown by experiments as follows: 2 ng/mL; when the substrate of meat and eggs needing pretreatment is used, the former treatment solution is taken as a zero-value sample, and the detection test strip is verified according to the same experimental operation, and the sensitivity of the detection test strip is as follows: 4 ng/mL.
4. When the milk product is a substrate, a high-value added milk sample close to the upper limit of a linear range is diluted to 7 concentrations according to a certain proportion, wherein a low-value concentration sample needs to be close to the lower limit of the linear range. The concentration is 800, 400, 200, 100, 50, 10, 5ng/mL, other matrixes such as livestock meat are taken as high-value samples, smashed and pretreated with a reagent to be diluted or added to prepare high-value samples, the high-value samples are treated with the pretreatment reagent to be diluted into 7 concentration points of 1000, 500, 200, 100, 50, 10, 5ng/mL, the operation is carried out according to the experimental steps, the samples with each concentration are repeatedly detected for 3 times, the average value of the samples is calculated, the average value of the results is brought into a standard curve to obtain the linear comparison between the detected concentration and the actual concentration, and the linear correlation coefficient R of the milk product matrix is20.9983, linear correlation coefficient R of other substrates2Is 0.9972. The linear curves are shown in fig. 3 and 4.
Experimental results of detected concentration and actual concentration in milk product matrix
Results of experiments on the detected and actual concentrations in other matrices
5. Precision: and (3) performing precision experimental detection on a milk substrate, detecting by using trimethoprim milk quality control products with the concentrations of 10ng/mL and 200ng/mL as samples according to the experimental steps, repeatedly determining for 3 times, and calculating the CV value of T/C of the trimethoprim milk quality control products by using the ratio of the standard deviation to the average value, wherein the CV value is less than 10%.
6. Accuracy (recovery experiment)
The method is characterized in that milk is used as a substrate to carry out an accuracy experiment, fresh normal milk is added with trimethoprim standard working solution to prepare 200, 50 and 10ng/mL of standard adding solutions with different concentrations, each concentration is detected for 10 times respectively, a standard adding recovery experiment is carried out, and the result is that the recovery rate of trimethoprim is 86.8% -117.77%. The specific experimental data are as follows:
according to the results, the test paper disclosed by the invention is high in sensitivity, good in stability, capable of realizing accurate quantitative detection, simple, convenient and quick, simple to operate, suitable for screening food raw materials in large batches and capable of meeting the existing requirements.
Claims (9)
1. The utility model provides a quantum dot test paper that quantitative determination trimethoprim residue volume in multiple matrix, overlaps 1-2mm each other by sample pad, marker pad, chromatographic film, blotter and pastes in proper order on the PVC bottom plate and make, the chromatographic film is the nitrocellulose membrane that bears detection line and quality control line, its characterized in that: the detection line is coated with trimethoprim antigen, and the quality control line is coated with DNP monoclonal antibody; the label pad bears quantum dots coupled with trimethoprim antibody and DNP-BSA.
2. The test strip of claim 1, wherein: the quantum dot coupled with the trimethoprim antibody and DNP-BSA is obtained by sequentially coupling the activated quantum dot with the trimethoprim antibody and the DNP-BSA and then adding casein solution for sealing.
3. The method for preparing a chromatographic test paper according to claim 1, which is characterized in that: the method comprises the following steps:
step 1, preparation of a marker pad:
1) dropwise adding a first treatment reagent into the glass cellulose membrane, and drying to obtain a pretreated glass cellulose membrane;
2) taking 100 mu L of 10mg/mL quantum dot stock solution, and performing ultrasonic dispersion;
3) activating the quantum dot microspheres, taking 50 mu L of dispersed quantum dot stock solution, uniformly mixing the quantum dot stock solution with 450 mu L of 0.05M MES buffer solution with pH6.0, adding 3 mu L of 50mg/mL EDC, uniformly mixing, adding 3 mu L of 75mg/mL NHS, uniformly mixing, and vertically mixing for 0.5 h;
4) resuspending, namely centrifuging 12000rp of the mixed solution obtained in the step 3) for 5min, discarding the supernatant, adding 500 mu L of 0.02M MOPS (molecular orbital phosphate) with pH of 7.0 for resuspension, and ultrasonically dispersing;
5) antibody coupling: adding 40 mu g of trimethoprim antibody into the dispersion liquid obtained in the step 4), uniformly mixing and coupling for 1h, adding 50 mu g of DNP-BSA, and coupling for 1 h;
6) and (3) sealing: adding 5 mu L of 10% ethanolamine into the mixed solution obtained in the step 5), uniformly mixing, then adding 500 mu L of 10% casein solution, and vertically and uniformly mixing at room temperature to seal redundant sites;
7) resuspending: centrifuging 12000prm of the mixed solution obtained in the step 6) for 5min, adding 500 mu L of quantum dot diluent to reach the constant volume of 1mg/mL, performing ultrasonic dispersion, and storing at 4 ℃;
8) diluting: diluting the quantum dot-antibody obtained in the step 7) to 0.4mg/mL, and performing ultrasonic dispersion for later use;
9) adding 0.05% carmine into the diluted quantum dot-antibody diluent, spraying the diluted quantum dot-antibody diluent on a pretreated glass cellulose membrane, and drying to obtain a marker pad;
step 2, adding trimethoprim antigen into the prepared T line coating solution according to 0.5mg/mL, adding DNP monoclonal antibody into the prepared C line coating solution according to 1mg/mL, respectively spraying the mixture onto a nitrocellulose membrane to prepare a T line and a C line, and drying to prepare a chromatographic membrane;
step 3, dripping a second treatment reagent into the glass cellulose membrane, and drying to prepare a cushion pad;
step 4, dripping a third treatment reagent into the cotton pulp paper, and drying to obtain a sample pad;
and 5, assembling the sample pad, the marker pad, the chromatographic membrane and the buffer pad to obtain the kit.
4. The production method according to claim 3, characterized in that: the first treatment reagent is prepared by adding 5% of sucrose, 0.3% of PVPK-30, 1% of Tween-20 and 0.2% of Proclin-300 into 0.05M Tris-Base buffer solution with pH8.0.
5. The production method according to claim 3, characterized in that: the second treatment reagent is prepared by adding 0.3% PVPK-30, 1% Triton X-100 and 0.2% Proclin-300 into 0.05M Tris-Base buffer solution with pH8.0.
6. The production method according to claim 3, characterized in that: the third treatment reagent is prepared by adding 5% trehalose, 0.8% Tween-20, 3.2mM EDTANA into 0.05M Tris-Base buffer solution (pH8.5)2And 0.2% casein.
7. The production method according to claim 3, characterized in that: the T-line coating solution is prepared by adding 2% of sucrose and 8% of methanol into 0.2M Hepes buffer solution with pH of 8.0.
8. The production method according to claim 3, characterized in that: the coating solution C is prepared by adding 8% methanol, 2% sucrose, 0.5% BSA, 0.1% Proclin-300 into 0.05M Tris-Base buffer solution with pH8.0.
9. The production method according to claim 3, characterized in that: the quantum dot diluent is prepared by adding 15% of sucrose, 0.5% of Tween-20, 3% of BSA, 1% of mannitol and 0.1% of Proclin-300 into 0.1M Tris-Base buffer solution with pH8.0.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111323580A (en) * | 2020-04-24 | 2020-06-23 | 东莞市东阳光诊断产品有限公司 | Immunochromatographic test strip and preparation method thereof |
| CN114264812A (en) * | 2021-12-13 | 2022-04-01 | 深圳容金科技有限公司 | Kit and method for rapidly detecting zilpaterol content of food |
| CN114264815A (en) * | 2021-12-13 | 2022-04-01 | 深圳容金科技有限公司 | Quantum dot immunoassay kit for clenbuterol content and detection method |
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