WO2020199501A1 - 一种基于量子点荧光的食品过敏原快速检测方法 - Google Patents

一种基于量子点荧光的食品过敏原快速检测方法 Download PDF

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WO2020199501A1
WO2020199501A1 PCT/CN2019/104656 CN2019104656W WO2020199501A1 WO 2020199501 A1 WO2020199501 A1 WO 2020199501A1 CN 2019104656 W CN2019104656 W CN 2019104656W WO 2020199501 A1 WO2020199501 A1 WO 2020199501A1
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quantum dot
food allergens
detection
solution
rapid detection
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李振兴
王雅晴
林洪
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中国海洋大学
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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
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • G01N33/54387—Immunochromatographic test strips
    • G01N33/54388—Immunochromatographic test strips based on lateral flow
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • 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
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

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  • the invention relates to the fields of food safety and biotechnology, in particular to a rapid detection method for food allergens based on quantum dot fluorescence.
  • Huang Suwen and others used the traditional marker colloidal gold to detect freshwater crayfish allergen myosin, using double antibody sandwich method, Monoclonal antibody was used as the detection antibody and reached the detection limit of 100 mg/mL; Daisuke Koizumi et al. tested the crustacean allergen myosin, using colloidal gold as the label, and the detection limit was 25 mg/mL.
  • Patent CN101881769A discloses an immune colloidal gold chromatography test strip for detecting shrimp allergens and a preparation method thereof, belonging to the technical field of immunological detection.
  • the invention discloses a combination of a shrimp allergen-tropomyosin cluster specific antibody and a coloring substance colloidal gold as a detection reagent to assemble a reagent strip to detect shrimp allergens in food, with low cost, rapidness and portability.
  • the antibody used in the invention is a polyclonal antibody obtained by immunizing healthy New Zealand white rabbits with extracted and purified tropomyosin.
  • the immunochromatographic test paper method has the advantages of simplicity and convenience. However, in the current test paper methods for food allergen detection, colloidal gold, a traditional nanoparticle, is often used as a marker, and the color depth is used to determine the target substance in the solution. Content, there are certain limitations in sensitivity and repeatability.
  • the present invention provides a rapid detection method for food allergens based on quantum dot fluorescence.
  • the uniform and stable characteristics of quantum dots and the advantages of surface modification can greatly optimize the state of the reaction system, thereby providing higher sensitivity, repeatability and stability, effectively reducing the detection limit, and more reliable results.
  • the present invention provides technical solutions as follows:
  • the invention provides a rapid detection method for food allergens based on quantum dot fluorescence, which includes:
  • Step 1 Use the carbodiimide method to covalently couple the rabbit anti-shrimp polyclonal antibody to the surface of the quantum dot to form a quantum dot fluorescent probe;
  • Step 2 Construction of fluorescence immunochromatographic test strips
  • Step 3 After mixing the quantum dot fluorescent probe in step 1 with the sample to be tested, perform chromatography detection.
  • step 1 specifically includes:
  • BSA bovine serum albumin
  • the quantum dot solution is a CdSe quantum dot solution with a concentration of 5 ⁇ M;
  • the carbodiimide hydrochloride is 1-(3-dimethylaminopropyl)-3-ethyl carbon Diimine hydrochloride (EDC);
  • the weight ratio of the carbodiimide hydrochloride and N-hydroxysulfosuccinimide is 1-5:1; and the activation time is 5-10 min.
  • the concentration of the rabbit antigen myosin polyclonal antibody is 3.5 mg/mL and the dosage is 50 ⁇ L; the dosage of the PBS buffer solution is 130 ⁇ L;
  • the final concentration of the BAS is 1%-1.5%, and BSA is added as an antibody stabilizer to keep the system stable.
  • the fluorescence immunochromatographic test strip is composed of a bottom plate, a sample pad, a nitrocellulose membrane and an absorbent pad; the sample pad, nitrocellulose membrane and absorbent pad are attached to the On the bottom plate, each material overlaps each other by 2-3mm; it is cut by an automatic strip cutter, placed in a plastic test strip cartridge, and stored in a dry place at room temperature.
  • the nitrocellulose membrane is provided with a tropomyosin detection line and a goat anti-rabbit IgG antibody quality control line, and the interval between the two is 4-5 mm.
  • the preparation method of the tropomyosin detection line and the goat anti-rabbit IgG antibody quality control line is: by using an automatic scribing instrument, 0.8 mg/mL tropomyosin antigen and 1.0 mg/mL goat anti The rabbit IgG antibody was sprayed on the nitrocellulose membrane with a spraying volume of 1.0 ⁇ L/cm. After spraying, the NC membrane was dried in an oven at 37°C.
  • step 3 is specifically: adding 10 ⁇ L of quantum dot fluorescent probes to the reaction container, adding the sample to be tested and buffer, thoroughly mixing and reacting for 5 minutes, and then dropping them into the assembled test paper card slot for chromatography .
  • the weight ratio of the quantum dot fluorescent probe, the sample to be tested and the buffer solution is 1:1:8.
  • the present invention innovatively improves the test strip technology, introduces a new type of nanometer material-fluorescent quantum dots instead of traditional markers for signal output after allergen identification, and greatly improves the sensitivity and reliability of detection.
  • Quantum dots an emerging material with better optical properties and stability, can achieve better specific recognition effects through covalent binding with antibodies, and its excellent optical properties also bring higher and more accurate
  • the detection sensitivity greatly enhances the detection effect of the test strip technology on trace allergens.
  • the food allergen fluorescence rapid detection test strip of the present invention has stronger operability, and ordinary consumers or front-line employees of food companies can operate independently according to the instructions; while the new nano-material fluorescence
  • the introduction of quantum dots optimizes the test strip technology. Due to the advantages of relying on the optical system to detect high sensitivity, fast detection speed, and high throughput detection, it greatly improves the sensitivity of detection and can provide users with more Reliable data.
  • Fig. 1 is an experimental diagram of agarose gel electrophoresis with a quantum dot fluorescent probe in Example 1 of the present invention
  • Figure 3 shows the quantum dot fluorescent probe of Example 1 of the present invention and 1, PBS; 2, 5% BSA; 3, 0.2 ⁇ g/mL TM antigen; 4, 0.5 ⁇ g/mL TM antigen; 5, 1.0 ⁇ g/mL TM Antigen; 6, 1.0 ⁇ g/mL goat anti-rabbit secondary antibody photos under ultraviolet irradiation;
  • Figure 4 is the %P relationship curve of the quantum dot fluorescent probe of Example 1 of the present invention and different concentrations of tropomyosin antigen (TM antigen) solution after mixing; the inset is a reversed photo of different TM antigens;
  • Fig. 5 is a graph showing the %P of the fluorescence immunochromatographic test strip of Example 1 for detecting different substances.
  • a rapid detection method for food allergens based on quantum dot fluorescence including:
  • Step 1 Use the carbodiimide method to covalently couple the rabbit anti-shrimp polyclonal antibody to the surface of quantum dots to form quantum dot fluorescent probes; the specific experimental steps are as follows: add 10 ⁇ L of 5 ⁇ M CdSe quantum dot solution to the reaction vessel, Add 40 ⁇ L of 1mg/mL EDC solution and 20 ⁇ L of 1mg/mL sulfo-NHS solution, stir and activate for 5min. Then add 50 ⁇ L of rabbit antigen myosin polyclonal antibody (3.5mg/mL) into the reaction container, add 130 ⁇ L of PBS buffer, and place in a shaking incubator at 25°C for 1 hour in the dark. The bovine serum albumin (BSA) was added to the composite solution as an antibody stabilizer (final concentration is 1%), and stored at 4°C for later use.
  • BSA bovine serum albumin
  • Step 2 Construction of fluorescence immunochromatographic test strips, the specific experimental steps are as follows:
  • the fluorescence immunochromatographic test strip is mainly composed of a bottom plate, a sample pad, a nitrocellulose membrane (NC membrane) and an absorbent pad.
  • a nitrocellulose membrane NC membrane
  • an absorbent pad Using an automatic scribing machine, spray the tropomyosin antigen (0.8 mg/mL) and goat anti-rabbit IgG antibody (1.0 mg/mL) on the NC membrane to form a tropomyosin detection line (T line).
  • T line C Anti-rabbit IgG antibody quality control line
  • the interval between the two is 5mm
  • the spray volume is 1.0 ⁇ L/cm.
  • Step 3 After mixing the quantum dot fluorescent probe in step 1 with the sample to be tested, perform chromatography detection; specifically including:
  • the indirect enzyme-linked immunosorbent assay was used to evaluate the biological characteristics of the quantum dot fluorescent probe, as shown in Figure 2, which proved that the antibody still maintains good binding characteristics after labeling.
  • TM antigen tropomyosin antigen
  • goat anti-rabbit IgG 1.5 mg/mL
  • PBS was used as the blank control
  • 5% BSA was used as the Negative control.
  • the labeled fluorescent antibody can effectively identify TM and goat anti-rabbit secondary antibody IgG , And produce a fluorescent signal, indicating that the coupling is successful and has the feasibility of being applied to immunochromatographic experiments.
  • the detection performance of the quantum dot immunochromatographic test strip prepared in this example is evaluated:
  • the method of calculating the percentage (%P) of the T-ray fluorescence intensity of the sample in the value of the corresponding blank sample is used to determine the negative or positive condition of the test result.
  • F The average fluorescence intensity value measured by the sample.
  • test strip method Use the established test strip method to test the protein extracts of clams, crabs, shrimps, fish, pork and chicken, and then observe the results.
  • the test results are shown in Figure 5. Except for the shrimp and crab samples, the other protein solutions all produced strong fluorescent bands at the T line, showing that the test strip has good specificity.
  • the established test strip method (LFIA) and the validated sandwich ELISA method were used to analyze 12 food samples.
  • the samples were crushed and added with PBS buffer (1:10), mixed and stirred thoroughly for 1 hour, centrifuged at 4°C at 8000 rpm for 20 minutes, and each sample was measured in triplicate. Calculate the results according to the above judgment method and divide them into three levels, including positive, weak positive and negative, and compare them with the calculated results of ELISA.
  • the results are shown in Table 2. The detection results of this method are consistent with those of the ELISA method, indicating that the method is feasible.
  • a rapid detection method for food allergens based on quantum dot fluorescence including:
  • Step 1 Add 10 ⁇ L of 5 ⁇ M CdSe quantum dot solution to the reaction container, add 20 ⁇ L of 1 mg/mL EDC solution, and 20 ⁇ L of 1 mg/mL sulfo-NHS solution, stir and activate for 6 min. Then add 50 ⁇ L of rabbit antigen myosin polyclonal antibody (3.5mg/mL) into the reaction container, add 130 ⁇ L of PBS buffer, and place in a shaking incubator at 25°C for 1 hour in the dark. Add bovine serum albumin (BSA) to the composite solution as an antibody stabilizer (final concentration is 1.2%), and store at 4°C for later use.
  • BSA bovine serum albumin
  • Step 2 Fluorescence immunochromatography test strip is mainly composed of bottom plate, sample pad, nitrocellulose membrane (NC membrane) and absorbent pad.
  • NC membrane nitrocellulose membrane
  • UV absorbent pad Using an automatic scribing machine, spray the tropomyosin antigen (0.8 mg/mL) and goat anti-rabbit IgG antibody (1.0 mg/mL) on the NC membrane to form a tropomyosin detection line (T line).
  • T line Tropomyosin detection line
  • C line Anti-rabbit IgG antibody quality control line
  • the NC film was placed in a 37°C oven to dry for 2 hours.
  • the pre-processed sample pad, NC membrane and absorbent pad are attached to the bottom plate in sequence, and each material overlaps each other by 3mm to ensure the connection of the chromatography process.
  • Step 3 When performing detection, add 10 ⁇ L of quantum dot fluorescent probe to the reaction container, and add 10 ⁇ L of the sample to be tested and 80 ⁇ L of buffer solution, mix thoroughly and react for 5 minutes, then drop it into the assembled test paper card slot for chromatography. Use a fluorescence immunochromatography reader to read the intensity of the T line and C line of the test paper.
  • a rapid detection method for food allergens based on quantum dot fluorescence including:
  • Step 1 Add 10 ⁇ L of 5 ⁇ M CdSe quantum dot solution to the reaction vessel, add 30 ⁇ L of 1 mg/mL EDC solution, and 20 ⁇ L of 1 mg/mL sulfo-NHS solution, stir and activate for 10 min. Then add 50 ⁇ L of rabbit antigen myosin polyclonal antibody (3.5 mg/mL) to the reaction container, add 130 ⁇ L PBS buffer, and place in a shaking incubator at 25°C for 1 hour in the dark. The bovine serum albumin (BSA) was added to the composite solution as an antibody stabilizer (final concentration is 1.5%), and stored at 4°C for later use.
  • BSA bovine serum albumin
  • Step 2 Fluorescence immunochromatography test strip is mainly composed of bottom plate, sample pad, nitrocellulose membrane (NC membrane) and absorbent pad.
  • NC membrane nitrocellulose membrane
  • UV absorbent pad Using an automatic scribing machine, spray the tropomyosin antigen (0.8 mg/mL) and goat anti-rabbit IgG antibody (1.0 mg/mL) on the NC membrane to form a tropomyosin detection line (T line).
  • T line C Anti-rabbit IgG antibody quality control line
  • the interval between the two is 5mm
  • the spray volume is 1.0 ⁇ L/cm.
  • Step 3 When performing detection, add 10 ⁇ L of quantum dot fluorescent probe to the reaction container, and add 10 ⁇ L of the sample to be tested and 80 ⁇ L of buffer solution, mix thoroughly and react for 5 minutes, then drop it into the assembled test paper card slot for chromatography. Use a fluorescence immunochromatography reader to read the intensity of the T line and C line of the test paper.
  • a rapid detection method for food allergens based on quantum dot fluorescence including:
  • Step 1 Add 10 ⁇ L of 5 ⁇ M CdSe quantum dot solution to the reaction vessel, add 50 ⁇ L of 1 mg/mL EDC solution, and 20 ⁇ L of 1 mg/mL sulfo-NHS solution, stir and activate for 8 min. Then add 50 ⁇ L of rabbit antigen myosin polyclonal antibody (3.5mg/mL) into the reaction container, add 130 ⁇ L of PBS buffer, and place in a shaking incubator at 25°C for 1 hour in the dark. Bovine serum albumin (BSA) was added to the composite solution as an antibody stabilizer (final concentration is 1.4%), and stored at 4°C for later use.
  • BSA bovine serum albumin
  • Step 2 Fluorescence immunochromatography test strip is mainly composed of bottom plate, sample pad, nitrocellulose membrane (NC membrane) and absorbent pad.
  • NC membrane nitrocellulose membrane
  • UV absorbent pad Using an automatic scribing machine, spray the tropomyosin antigen (0.8 mg/mL) and goat anti-rabbit IgG antibody (1.0 mg/mL) on the NC membrane to form a tropomyosin detection line (T line).
  • T line Tropomyosin detection line
  • C line Anti-rabbit IgG antibody quality control line
  • the NC film was placed in a 37°C oven to dry for 2 hours.
  • Step 3 When performing detection, add 10 ⁇ L of quantum dot fluorescent probe to the reaction container, and add 10 ⁇ L of the sample to be tested and 80 ⁇ L of buffer solution, mix thoroughly and react for 5 minutes, then drop it into the assembled test paper card slot for chromatography. Use a fluorescence immunochromatography reader to read the intensity of the T line and C line of the test paper.
  • the present invention provides a rapid detection method for food allergens based on quantum dot fluorescence.
  • the uniform and stable characteristics of quantum dots and the advantages of surface modification can greatly optimize the state of the reaction system, thereby providing higher sensitivity , Repeatability and stability, effectively reduce the detection limit, and the result is more reliable.

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Abstract

本发明公开了一种基于量子点荧光的食品过敏原快速检测方法，属于食品安全及生物技术领域。上述方法包括：步骤1：采用碳二亚胺法将兔抗虾多克隆抗体共价偶联至量子点表面，形成量子点荧光探针；步骤2：荧光免疫层析试纸条的构建；步骤3：将步骤1中的量子点荧光探针与待测样品混合后，进行层析检测。本发明中量子点自身均匀、稳定的特性和其表面可修饰的优点可以大大优化反应体系的状态，从而提供更高的灵敏度、重复性和稳定性，有效降低其检测限，结果更加可靠。

Description

一种基于量子点荧光的食品过敏原快速检测方法 技术领域
本发明涉及食品安全及生物技术领域，特别是指一种基于量子点荧光的食品过敏原快速检测方法。
背景技术
目前已有的研究中，针对过敏原的免疫层析试纸条检测技术的相关报道中，黄素文等使用传统的标记物胶体金检测淡水小龙虾过敏原原肌球蛋白，采用双抗夹心法，以单克隆抗作为检测抗体，达到了100mg/mL的检出限；Daisuke Koizumi等人对甲壳类过敏原原肌球蛋白进行检测，使用胶体金作为标记物，检测限为25mg/mL。
专利CN101881769A公开了一种检测虾过敏原的免疫胶体金层析试纸条及其制备方法，属于免疫学检测技术领域。该发明公开了以虾过敏原-原肌球蛋白的簇特异性抗体与显色物质胶体金的结合物作为检测试剂组装试剂条，进行食品中虾过敏原的检测，成本低廉，快速，便携。该发明应用的抗体是利用提取纯化的原肌球蛋白免疫健康的新西兰大白兔得到的多克隆抗体。
免疫层析试纸方法具有简易、便捷的优势，但目前针对食品过敏原检测的试纸方法中，多采用胶体金这一传统纳米颗粒作为标记物，以颜色的深浅来判定其目标物在溶液中的含量，在灵敏度和重复性上存在一定的限制。
发明内容
为解决现有技术中的不足，本发明提供一种基于量子点荧光的食品过敏原快速检测方法。量子点自身均匀、稳定的特性和其表面可修饰的优点可以大大优化反应体系的状态，从而提供更高的灵敏度、重复性和稳定性， 有效降低其检测限，结果更加可靠。
为解决上述技术问题，本发明提供技术方案如下：
本发明提供一种基于量子点荧光的食品过敏原快速检测方法，包括：
步骤1：采用碳二亚胺法将兔抗虾多克隆抗体共价偶联至量子点表面，形成量子点荧光探针；
步骤2：荧光免疫层析试纸条的构建；
步骤3：将步骤1中的量子点荧光探针与待测样品混合后，进行层析检测。
进一步的，所述步骤1具体为：
11)向反应容器中加入量子点溶液10μL，加入碳二亚胺盐酸盐溶液和N-羟基硫代琥珀酰亚胺(sulfo-NHS)溶液，搅拌活化一段时间；
12)再向反应容器中加入兔抗原肌球蛋白多克隆抗体和PBS缓冲液，置于25℃震荡培养箱中避光反应1h；
13)将牛血清白蛋白(BSA)加入到上述复合溶液中，然后4℃保存备用。
优选的，所述步骤11)中，量子点溶液是浓度为5μM的CdSe量子点溶液；所述碳二亚胺盐酸盐为1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)；
优选的，所述碳二亚胺盐酸盐和N-羟基硫代琥珀酰亚胺的重量比为1-5:1；所述活化时间为5-10min。
优选的，所述步骤12)中，所述兔抗原肌球蛋白多克隆抗体浓度为3.5mg/mL，用量为50μL；所述PBS缓冲溶液用量为130μL；
所述步骤13)中，所述BAS的最终浓度为1％-1.5％，BSA作为抗体稳定剂加入，能够使得体系保持稳定。
进一步的，所述步骤2中，所述荧光免疫层析试纸条由底板、样品垫、硝酸纤维素膜和吸水垫组成；所述样品垫、硝酸纤维素膜和吸水垫依次贴附于所述底板上，每种材料间互相重叠2-3mm；使用自动切条机将其裁切，置于塑料试纸条卡壳中，室温干燥保存。
进一步的，所述硝酸纤维素膜上设置有原肌球蛋白检测线和羊抗兔IgG抗体质控线，二者间隔为4-5mm。
进一步的，所述原肌球蛋白检测线和羊抗兔IgG抗体质控线的制备方法为：通过使用自动划膜仪，分别将0.8mg/mL原肌球蛋白抗原和1.0mg/mL羊抗兔IgG抗体喷涂于硝酸纤维素膜上，喷涂量为1.0μL/cm，喷涂后将NC膜置于37℃烘箱中干燥。
进一步的，所述步骤3具体为：向反应容器中添加10μL量子点荧光探针，并加入待测样品与缓冲液，充分混合反应5min后，滴加至组装好的试纸卡槽中进行层析。
优选的，所述量子点荧光探针、待测样品和缓冲溶液的重量比为：1:1:8。
本发明具有以下有益效果：
本发明创新性地对试纸条技术进行了改良，引入新型纳米材料-荧光量子点代替传统的标记物，用于过敏原识别后的信号输出，大幅提高了检测的灵敏度与可靠性。量子点这一新兴材料具有更好的光学特性与稳定性，通过与抗体的共价结合，可以实现更好的特异性识别效果，而其优良的光学特性也带来了更高、更准确的检测灵敏度，从而大幅增强了试纸条技术对微量过敏原的检测效果。
相比现行的过敏原检测方法，本发明的食物过敏原荧光快速检测试纸条具有更强的可操作性，普通消费者或食品企业一线员工按照说明书即可独立进行操作；而新型纳米材料荧光量子点的引入，对试纸条技术进行了优化，由于依靠光学系统来检测具有灵敏度高、检测速度快、有望实现高通量检测等优点，从而大大提高了检测的灵敏度，可以为用户提供更可靠的数据。
附图说明
图1为本发明实施例1的量子点荧光探针琼脂糖凝胶电泳实验图；
图2为本发明实施例1的量子点荧光探针和未标记的抗体稀释倍数与 吸光度之间的关系曲线；
图3为本发明实施例1的量子点荧光探针与1、PBS；2、5％BSA；3、0.2μg/mL TM抗原；4、0.5μg/mL TM抗原；5、1.0μg/mL TM抗原；6、1.0μg/mL羊抗兔二抗体在紫外照射下的照片；
图4为本发明实施例1的量子点荧光探针与不同浓度的原肌球蛋白抗原(TM抗原)溶液混合后％P的关系曲线；插图为不同TM抗原的反转照片；
图5为本发明实施例1的荧光免疫层析试纸条检测不同物质的％P图。
具体实施方式
为使本发明要解决的技术问题、技术方案和优点更加清楚，下面将结合附图及具体实施例进行详细描述。
实施例1
一种基于量子点荧光的食品过敏原快速检测方法，包括：
步骤1：采用碳二亚胺法将兔抗虾多克隆抗体共价偶联至量子点表面，形成量子点荧光探针；具体实验步骤如下：向反应容器中加入5μM的CdSe量子点溶液10μL，加入1mg/mL EDC溶液40μL，1mg/mL sulfo-NHS溶液20μL，搅拌活化5min。再向反应容器中加入兔抗原肌球蛋白多克隆抗体(3.5mg/mL)50μL，加入130μL PBS缓冲液，置于25℃震荡培养箱中避光反应1h。将牛血清白蛋白(BSA)加入到复合溶液中作为抗体稳定剂(终浓度为1％)，4℃保存备用。
步骤2：荧光免疫层析试纸条的构建，具体实验步骤如下：
荧光免疫层析试纸条主要由底板、样品垫、硝酸纤维素膜(NC膜)和吸水垫组成。使用自动划膜仪，分别将原肌球蛋白抗原(0.8mg/mL)和羊抗兔IgG抗体(1.0mg/mL)喷涂于NC膜上，形成原肌球蛋白检测线(T线)，羊抗兔IgG抗体质控线(C线)，二者间隔5mm，喷涂量为1.0μL/cm。喷涂后将NC膜置于37℃烘箱中干燥2h。将预处理好的样品垫、NC膜和吸水垫依次贴附于底板上，每种材料间互相重叠2mm，以保证层析过 程的衔接。使用自动切条机将其裁切至每条宽度4mm，置于塑料试纸条卡壳中，室温干燥保存。
步骤3：将步骤1中的量子点荧光探针与待测样品混合后，进行层析检测；具体包括：
进行检测时，向反应容器中添加10μL量子点荧光探针，并加入样品与缓冲液，充分混合反应5min后，滴加至组装好的试纸卡槽中进行层析，使用荧光免疫层析读数仪读取试纸T线、C线强度。
对本实施例制备的量子点荧光探针进行琼脂糖凝胶电泳实验，来验证抗体标记情况，使用TAE缓冲液体系配制1％琼脂糖凝胶，分别上样10μL CdSe量子点溶液、量子点荧光探针和未经标记的纯化多克隆抗体，在80V恒压下电泳30min，紫外下拍照观察结合情况。将琼脂糖胶块放入考马斯亮蓝中染色1h，脱色液脱色后拍照观察。如图1所示，量子点与抗体结合后，在紫外下出现了新的复合物条带(泳道2)，其位移小于纯量子点(泳道1)，且可被考马斯亮蓝染色，证明该区域为标记成功的量子点抗体复合物(量子点荧光探针)。而作为对照的多克隆抗体(泳道3)未经标记，在紫外下不显示条带；由于携带电荷的情况与标记抗体不同，在电泳中向反方向迁移，染色后在上样孔附近呈蓝色，而泳道2在上样孔附近没有明显蓝色出现，说明大部分的IgG抗体已被成功标记。
使用间接酶联免疫吸附法(ELISA)对量子点荧光探针的生物学特性进行评价，如图2所示，证明标记后抗体仍保持较好的结合特性。
使用斑点免疫印迹实验对量子点荧光探针的免疫特性进行验证。将浓度分别为0.2、0.5、1.0μg/mL的原肌球蛋白抗原(TM抗原)和羊抗兔IgG(1.5mg/mL)分别点样于NC膜上，PBS作为空白对照，5％BSA作为阴性对照。使用37℃烘箱干燥1h，5％脱脂奶粉37℃封闭2h。干燥后向加样点滴加量子点荧光探针，孵育30min后使用PBST洗涤3次，紫外照射下拍照观察如图3所示，标记后的荧光抗体可以有效识别出TM和羊抗兔二抗IgG，并产生荧光信号，说明偶联成功且具有应用于免疫层析实验的可行性。
对本实施例制备的量子点免疫层析试纸条检测性能进行评估：
1、标准溶液测定
使用优化的稀释液配制一系列浓度梯度的原肌球蛋白溶液进行检测。向反应容器中添加10μL量子点荧光探针，加入原肌球蛋白工作液和PBS缓冲液至终体积100μL，使原肌球蛋白溶液的终浓度分别为0.0、0.05、0.1、0.5、1.0、5.0、10.0、50.0和100.0μg/L。充分混合反应5min后，滴加至组装好的试纸卡槽中进行层析。使用荧光免疫层析读数仪读取试纸T线、C线强度。每个浓度重复3次。
实验结果如图4中的反转照片所示，随着TM样品的浓度增加，T线的亮度变弱，仪器读数的光强度降低。与对照样品相比，0.05μg/mL TM的T线强度变弱，并且在0.5μg/mL时目视消失。因此，用于TM检测的LFIA系统的视觉定性极限设定为0.5μg/mL。
使用计算样品的T线荧光强度占对应空白样品值的百分比(％P)的方法，判定检测结果的阴阳性情况。
％P＝F/F 0×100％
F 0：空白测量值；
F：样品测量的平均荧光强度值。
根据该计算公式，判断结果分为三种情况：
阴性：P>80％；
弱阳性：50％≤P≤80％；
阳性：P<50％。
使用上述公式，判定其检出限与肉眼测定一致。
2、精密度测定
取同一批次试纸条，对三种浓度的原肌球蛋白溶液(0，1ppm，10ppm)进行重复测定(n＝10)，计算其批内变异系数。每个样品每天测试2组，连续测定5天，计算其批间变异系数。结果如表1所示，表明本发明方法具有良好的重复性(CV<20％)。
表1
Figure PCTCN2019104656-appb-000001
3、特异性检测
用已建立的试纸条方法对蛤蜊、蟹、虾、鱼、猪肉和鸡肉的蛋白提取液进行检测，后观察结果。检测结果如图5所示，除虾、蟹样品外，其余蛋白溶液在T线处均产生较强的荧光条带，显示出本试纸条具有良好的特异性。
4、食物样品检测
用已建立的试纸条方法(LFIA)和经验证的夹心ELISA方法分析12种食物样品。将样品粉碎并后加入PBS缓冲液(1:10)，充分混合并搅拌1小时后，4℃8000rpm离心20分钟，每个样品一式三份测量。根据上述判断方法计算结果并将其分为三个等级，包括阳性，弱阳性和阴性，并与ELISA的计算结果进行比较。结果见表2。本方法的检测与ELISA方法检测结果一致，说明方法具有可行性。
表2
Figure PCTCN2019104656-appb-000002
Figure PCTCN2019104656-appb-000003
实施例2
一种基于量子点荧光的食品过敏原快速检测方法，包括：
步骤1：向反应容器中加入5μM的CdSe量子点溶液10μL，加入1mg/mL EDC溶液20μL，1mg/mL sulfo-NHS溶液20μL，搅拌活化6min。再向反应容器中加入兔抗原肌球蛋白多克隆抗体(3.5mg/mL)50μL，加入130μL PBS缓冲液，置于25℃震荡培养箱中避光反应1h。将牛血清白蛋白(BSA)加入到复合溶液中作为抗体稳定剂(终浓度为1.2％)，4℃保存备用。
步骤2：荧光免疫层析试纸条主要由底板、样品垫、硝酸纤维素膜(NC膜)和吸水垫组成。使用自动划膜仪，分别将原肌球蛋白抗原(0.8mg/mL)和羊抗兔IgG抗体(1.0mg/mL)喷涂于NC膜上，形成原肌球蛋白检测线(T线)，羊抗兔IgG抗体质控线(C线)，二者间隔4mm，喷涂量为1.0μL/cm。喷涂后将NC膜置于37℃烘箱中干燥2h。将预处理好的样品垫、NC膜和吸水垫依次贴附于底板上，每种材料间互相重叠3mm，以保证层析过程的衔接。使用自动切条机将其裁切至每条宽度4mm，置于塑料试纸条卡壳中，室温干燥保存。
步骤3：进行检测时，向反应容器中添加10μL量子点荧光探针，并加入10μL待测样品与80μL缓冲液，充分混合反应5min后，滴加至组装好的试纸卡槽中进行层析，使用荧光免疫层析读数仪读取试纸T线、C线强度。
实施例3
一种基于量子点荧光的食品过敏原快速检测方法，包括：
步骤1：向反应容器中加入5μM的CdSe量子点溶液10μL，加入1mg/mL EDC溶液30μL，1mg/mL sulfo-NHS溶液20μL，搅拌活化10min。再向反应容器中加入兔抗原肌球蛋白多克隆抗体(3.5mg/mL)50μL，加 入130μL PBS缓冲液，置于25℃震荡培养箱中避光反应1h。将牛血清白蛋白(BSA)加入到复合溶液中作为抗体稳定剂(终浓度为1.5％)，4℃保存备用。
步骤2：荧光免疫层析试纸条主要由底板、样品垫、硝酸纤维素膜(NC膜)和吸水垫组成。使用自动划膜仪，分别将原肌球蛋白抗原(0.8mg/mL)和羊抗兔IgG抗体(1.0mg/mL)喷涂于NC膜上，形成原肌球蛋白检测线(T线)，羊抗兔IgG抗体质控线(C线)，二者间隔5mm，喷涂量为1.0μL/cm。喷涂后将NC膜置于37℃烘箱中干燥2h。将预处理好的样品垫、NC膜和吸水垫依次贴附于底板上，每种材料间互相重叠2mm，以保证层析过程的衔接。使用自动切条机将其裁切至每条宽度4mm，置于塑料试纸条卡壳中，室温干燥保存。
步骤3：进行检测时，向反应容器中添加10μL量子点荧光探针，并加入10μL待测样品与80μL缓冲液，充分混合反应5min后，滴加至组装好的试纸卡槽中进行层析，使用荧光免疫层析读数仪读取试纸T线、C线强度。
实施例4
一种基于量子点荧光的食品过敏原快速检测方法，包括：
步骤1：向反应容器中加入5μM的CdSe量子点溶液10μL，加入1mg/mL EDC溶液50μL，1mg/mL sulfo-NHS溶液20μL，搅拌活化8min。再向反应容器中加入兔抗原肌球蛋白多克隆抗体(3.5mg/mL)50μL，加入130μL PBS缓冲液，置于25℃震荡培养箱中避光反应1h。将牛血清白蛋白(BSA)加入到复合溶液中作为抗体稳定剂(终浓度为1.4％)，4℃保存备用。
步骤2：荧光免疫层析试纸条主要由底板、样品垫、硝酸纤维素膜(NC膜)和吸水垫组成。使用自动划膜仪，分别将原肌球蛋白抗原(0.8mg/mL)和羊抗兔IgG抗体(1.0mg/mL)喷涂于NC膜上，形成原肌球蛋白检测线(T线)，羊抗兔IgG抗体质控线(C线)，二者间隔4mm，喷涂量为1.0μL/cm。喷涂后将NC膜置于37℃烘箱中干燥2h。将预处理好的样品 垫、NC膜和吸水垫依次贴附于底板上，每种材料间互相重叠3mm，以保证层析过程的衔接。使用自动切条机将其裁切至每条宽度4mm，置于塑料试纸条卡壳中，室温干燥保存。
步骤3：进行检测时，向反应容器中添加10μL量子点荧光探针，并加入10μL待测样品与80μL缓冲液，充分混合反应5min后，滴加至组装好的试纸卡槽中进行层析，使用荧光免疫层析读数仪读取试纸T线、C线强度。
上述实施例2-4的基于量子点荧光的食品过敏原快速检测方法对虾蟹的检测结果同实施例1，在此不再赘述。
综上可知，本发明提供一种基于量子点荧光的食品过敏原快速检测方法，量子点自身均匀、稳定的特性和其表面可修饰的优点可以大大优化反应体系的状态，从而提供更高的灵敏度、重复性和稳定性，有效降低其检测限，结果更加可靠。
以上所述是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明所述原理的前提下，还可以作出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (10)

  1. 一种基于量子点荧光的食品过敏原快速检测方法，其特征在于，包括：
    步骤1：采用碳二亚胺法将兔抗虾多克隆抗体共价偶联至量子点表面，形成量子点荧光探针；
    步骤2：荧光免疫层析试纸条的构建；
    步骤3：将步骤1中的量子点荧光探针与待测样品混合后，进行层析检测。
  2. 根据权利要求1所述的基于量子点荧光的食品过敏原快速检测方法，其特征在于，所述步骤1具体为：
    11)向反应容器中加入量子点溶液10μL，加入碳二亚胺盐酸盐溶液和N-羟基硫代琥珀酰亚胺(sulfo-NHS)溶液，搅拌活化一段时间；
    12)再向反应容器中加入兔抗原肌球蛋白多克隆抗体和PBS缓冲液，置于25℃震荡培养箱中避光反应1h；
    13)将牛血清白蛋白(BSA)加入到上述复合溶液中，然后4℃保存备用。
  3. 根据权利要求2所述的基于量子点荧光的食品过敏原快速检测方法，其特征在于，所述步骤11)中，量子点溶液是浓度为5μM的CdSe量子点溶液；所述碳二亚胺盐酸盐为1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)。
  4. 根据权利要求3所述的基于量子点荧光的食品过敏原快速检测方法，其特征在于，所述碳二亚胺盐酸盐和N-羟基硫代琥珀酰亚胺的重量比为1～5:1；所述活化时间为5-10min。
  5. 根据权利要求2所述的基于量子点荧光的食品过敏原快速检测方法，其特征在于，所述步骤12)中，所述兔抗原肌球蛋白多克隆抗体浓度为3.5mg/mL，用量为50μL；所述PBS缓冲溶液用量为130μL；
    所述步骤13)中，所述BAS的最终浓度为1％-1.5％。
  6. 根据权利要求1所述的基于量子点荧光的食品过敏原快速检测方 法，其特征在于，所述步骤2中，所述荧光免疫层析试纸条由底板、样品垫、硝酸纤维素膜和吸水垫组成；所述样品垫、硝酸纤维素膜和吸水垫依次贴附于所述底板上，每种材料间互相重叠2-3mm；使用自动切条机将其裁切，置于塑料试纸条卡壳中，室温干燥保存。
  7. 根据权利要求6所述的基于量子点荧光的食品过敏原快速检测方法，其特征在于，所述硝酸纤维素膜上设置有原肌球蛋白检测线和羊抗兔IgG抗体质控线，二者间隔为4-5mm。
  8. 根据权利要求7所述的基于量子点荧光的食品过敏原快速检测方法，其特征在于，所述原肌球蛋白检测线和羊抗兔IgG抗体质控线的制备方法为：通过使用自动划膜仪，分别将0.8mg/mL原肌球蛋白抗原和1.0mg/mL羊抗兔IgG抗体喷涂于硝酸纤维素膜上，喷涂量为1.0μL/cm，喷涂后将NC膜置于37℃烘箱中干燥。
  9. 根据权利要求1所述的基于量子点荧光的食品过敏原快速检测方法，其特征在于，所述步骤3具体为：向反应容器中添加10μL量子点荧光探针，并加入待测样品与缓冲液，充分混合反应5min后，滴加至组装好的试纸卡槽中进行层析。
  10. 根据权利要求9所述的基于量子点荧光的食品过敏原快速检测方法，其特征在于，所述量子点荧光探针、待测样品和缓冲溶液的重量比为：1:1:8。
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