WO2020024745A1 - 一种荧光免疫检测毛发痕量毒品的试剂盒 - Google Patents

一种荧光免疫检测毛发痕量毒品的试剂盒 Download PDF

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WO2020024745A1
WO2020024745A1 PCT/CN2019/093739 CN2019093739W WO2020024745A1 WO 2020024745 A1 WO2020024745 A1 WO 2020024745A1 CN 2019093739 W CN2019093739 W CN 2019093739W WO 2020024745 A1 WO2020024745 A1 WO 2020024745A1
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kit
hair
drug
quantum dot
detection
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French (fr)
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葛秀龙
欧阳云
方芳
郑水娣
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杭州莱和生物技术有限公司
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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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • 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
    • G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
    • 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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/588—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with semiconductor nanocrystal label, e.g. quantum dots

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  • the invention belongs to the technical field of biological detection, in particular to a reagent kit for fluorescent immunodetection of hair trace drugs.
  • Chinese patent CN101750458B discloses a method for detecting drug residues in humans, which uses HPLC-Chip-MS / MS and HPLC-MS / MS to detect drug residues, which has the advantages of low cost, high accuracy and high stability.
  • Chinese patent CN104422762A discloses an ELISA kit and a detection method for detecting drugs in urine, saliva, blood, and hair. The ELISA kit has the characteristics of high specificity and high sensitivity.
  • the object of the present invention is to provide a kit for detecting hair trace drugs by fluorescence immunoassay.
  • the invention can quickly, accurately and sensitively detect drugs in hair.
  • a kit for detecting hair trace drugs by fluorescence immunoassay comprising a sample lysate and a fluorescent immunochromatographic test strip;
  • the lysate is used for dissolving small drug molecules from hair
  • the fluorescent immunochromatographic test strip includes a PVC liner, a nitrocellulose membrane, a sample pad, and an absorption pad, and the sample pad, the nitrocellulose membrane, and the absorption pad are sequentially staggered and fixed on the PVC liner;
  • the sample pad has a drug antibody-quantum dot nanoparticle complex and a rabbit IgG-quantum dot nanoparticle complex;
  • the nitrocellulose membrane is provided with a detection line and a quality control line, the detection line is coated with a drug antigen-bovine serum albumin complex, and the quality control line is coated with a goat anti-rabbit antibody.
  • the detection object of the present invention is that the hair is tested for blood, urine, or saliva compared with the conventional detection method, which can overcome the difficulty of obtaining evidence for drug testing and the mismatch of the person being tested.
  • the mass ratio of lysate to hair is 100: 1.
  • the lysing solution contains a surfactant, a keratinase and a buffer solution.
  • a surfactant e.g., keratinase can corrode the surface of the hair and make the hair a rough surface, thereby facilitating the dissolution of small drug molecules from the sample to be tested.
  • Surfactants can wash stains on the surface of hair, so that keratinase can fully contact the hair, and further improve the dissolution rate of drugs.
  • the buffer solution can neutralize the side-reactants produced by keratinase decomposition of hair, and improve the biological activity of keratinase.
  • the surfactant is preferably a non-ionic surfactant, such as polyvinylpyrrolidone (PVP), Triton X 100, Triton X 114, Triton X 405, Tween 20, Tween 60, Tween 80.
  • PVP polyvinylpyrrolidone
  • Triton X 100 Triton X 114
  • Triton X 405 Tween 20, Tween 60, Tween 80.
  • non-ionic surfactants do not easily affect the ionic strength, thereby avoiding affecting the immune response in subsequent reagent strips and improving the accuracy of immune detection.
  • the buffer solvent is preferably a TRIS (anhydrous sodium tetraborate) -EDTA buffer solution.
  • the buffer system of the present invention has a pH range close to 8, a mild nature, and higher solution stability.
  • the lysing solution further includes sodium bicarbonate.
  • the addition of sodium bicarbonate is mainly to further adjust the pH of the lysate.
  • the mass percentage of the surfactant in the lysate is 1-2%, and the content of keratinase is 10,000-50,000 IU / ML.
  • the quantum emission nanometer particle has a fluorescence emission wavelength range of 600-620 nm, and more preferably, the emission wavelength is 610 nm.
  • the wavelength of the excitation light is 360-430 nm, and more preferably, the wavelength of the excitation light is 365 nm.
  • the quantum dot nanoparticles are a quantum dot formed by a single compound or a composite quantum dot assembled by several compounds. More preferably, the compound is selected from one or more of ZnS, CdS, HgS, MgS, CdSe, ZnSe, and ZnCdSe.
  • the fluorescence immunochromatography kit of the present invention uses quantum dot nanoparticles as fluorescent emitting substances.
  • the quantum dots exhibit wide and continuous excitation spectrum, narrow and symmetrical emission spectrum, high fluorescence intensity, long fluorescence lifetime, and good stability in optical properties. It can overcome the defects of conventional rare earth microspheres, latex microspheres and other fluorescent materials, signal decay blocks, and insignificant signal gradients.
  • the fluorescent signal displayed by the fluorescent immunoassay strip of the present invention can not be significantly attenuated within 2 hours, and the gradient of the signal is very obvious, which can be effectively stratified, making the tested fluorescent signal more stable and convenient for detection, improving detection sensitivity and Accuracy.
  • the properties of quantum dots with stable fluorescence, strong signals, and obvious changes also make the fluorescence signal magnitude conversion effect good, which is conducive to quantitative analysis.
  • the detection principle of the fluorescent immunochromatographic test strip of the present invention is a competition method.
  • the T-line (detection line) -coated antigen and the drug antigen in the sample to be tested compete to bind the drug antibody-quantum dot nanoparticle complex.
  • the drug antibody-quantum dot nanocomposite first binds to the T-coated drug antigen-bovine serum albumin complex during swimming, and the T-line forms a fluorescent signal that can be detected by a hair fluorescence immunoanalyzer.
  • the drug antigen, coated drug antigen, and drug antigen-bovine serum albumin complex compete for binding, and the signal intensity at the T line will follow the drug antigen in the sample. It increases and gradually weakens; while the rabbit IgG-quantum dot nanoparticle complex will continue to swim and bind to the quality control line (C line) -bound sheep anti-rabbit antibody, forming a stable signal at the C line; if the C line No signal is detected anywhere, the test strip is scrapped and can no longer be used.
  • C line quality control line
  • the present invention can adopt a matching fluorescence detection analyzer, which can import the relevant quantity curve according to each reagent batch, quickly inject the detection readings, and control the time within 10 seconds, which can convert the fluorescent signal into the corresponding drug or metabolism
  • the concentration value of the component is clearly displayed on the display screen.
  • the instrument can save more than 500 sets of test results, and the relevant results can be printed immediately, including the corresponding reagent lot number, specimen number, test time, and tester code.
  • the detectable drugs of the present invention include morphine, virus, ketamine and the like.
  • the kit of fluorescent immunodetection drugs of the present invention uses hair as a detection sample to quantitatively detect the drug content in the hair, the sampling is simple, and the problem of drug detection and sampling difficulties is overcome;
  • the present invention uses a lysate composed of a surfactant, keratinase and a buffer solution to dissolve drugs in the hair.
  • a lysate composed of a surfactant, keratinase and a buffer solution to dissolve drugs in the hair.
  • the combined action of the surfactant, keratinase and a buffer solution can effectively improve the dissolution rate and dissolution of drugs in the hair. speed.
  • the present invention uses quantum dots for fluorescent labeling.
  • the fluorescence is stable, the signal is strong and the change is obvious.
  • the fluorescence signal can be converted into a good quantity, the drug content can be quantitatively analyzed, and it can be obtained quickly by a fluorescence analyzer. result.
  • the kit of the present invention is used to detect the drug content in hair.
  • the test results have high accuracy, high precision, wide detection range, high stability, and anti-interference (such as drugs, shampoo, conditioner, hair gel, etc.) strong ability.
  • 1 is a schematic structural diagram of a fluorescent immunochromatographic test strip according to the present invention, wherein 1 is a PVC liner, 2 is a sample pad, 3 is a nitrocellulose membrane, 4 is an absorption pad, 5 is a detection line, and 6 is a quality control line.
  • the kit includes sample lysate, fluorescent immunochromatographic test strip (see Figure 1 for the structure); the lysate is used to dissolve small drug molecules from the sample to be tested; the fluorescent immunochromatographic test strip contains PVC liner 1, nitric acid Fiber membrane 3, sample pad 2 and absorption pad 4, the sample pad, nitrocellulose membrane, and absorption pad are sequentially staggered and fixed on the PVC liner; the sample pad has drug antibody-quantum dot nanoparticle complex and rabbit IgG -Quantum dot nanoparticle complex; a detection line and a quality control line are provided on the nitrocellulose membrane, the detection line is coated with a drug antigen-bovine serum albumin complex, and the quality control line is coated with a goat anti-rabbit antibody.
  • Fluorescent microspheres that is, quantum dot nanoparticles formed by one or more of ZnS, CdS, HgS, MgS, CdSe, ZnSe, and ZnCdSe are formulated into a 5 wt% working concentration;
  • the blocking solution is a 5 wt% casein solution
  • the precipitate is re-dissolved with a polymer, and a ratio of 0.1 ml of the precipitate is added to 1 ml of a polymer solution.
  • the polymer may be polystyrene, polybutene, or the like.
  • the composite plastic sheet is placed on a cutting machine and cut into a single piece of test paper.
  • the fluorescence detection analyzer detects the fluorescence signal of the test strip, converts the fluorescence signal into an electrical signal, and then automatically calculates the concentration based on the standard curve information on the ID card of each batch of test strips and presents it on the display of the instrument.
  • the fluorescent label is: particles formed by quantum dots ZnCdSe / ZnS embedded in polystyrene microspheres.
  • the lysate formulation is:
  • Dilute morphine enterprise reference products to 0ng / mg, 0.05ng / mg, 0.1ng / mg, 1ng / mg, 2.5ng / mg, 5ng / mg, 10ng / mg, 15ng / mg, 20ng / mg, and then follow the instructions
  • the detection step three batches of morphine / methamphetamine / ketamine hair rapid detection reagent products were tested respectively, and each concentration was tested for 3 samples and the average value was obtained. It is required that the linear correlation coefficient R ⁇ 0.99, and the relative deviation is ⁇ 10%.
  • Table 1 The results are shown in Table 1 below.
  • the relative deviation is greater than 10% when the concentration is ⁇ 0.1ng / mg, and the relative deviation is greater than 10% when the concentration is greater than 10ng / mg, which does not meet the requirements; within the range of 0.1-10ng / ml, the correlation coefficient R of the product linearity ⁇ 0.99, the relative deviation is within ⁇ 10% so the linear range of the three products is 0.1-10ng / mg.
  • the precision CV of the morphine hair rapid detection reagent batch is ⁇ 10%.
  • the minimum detection limit (X + 3SD) of morphine hair rapid detection reagent is ⁇ 0.1ng / mg.
  • the morphine hair rapid detection reagent has no interference when the interference substance concentration is 10 ug / ml.
  • test buffer is stored at 2-8 ° C.
  • the entire test buffer is changed from 2- Take it out of the refrigerator at 8 °C, open the bottle, and return to room temperature for testing. After the test, return the test buffer to the ambient conditions of 2-8 °C.
  • the test time is 0, 3, 6, 9, 12, 18, 24, 30 and 31 months. (If the product is found to be unstable during the test, you need to use the test buffer prepared for verification, indicating whether it is the stability of the finished product or the reason for the test buffer).
  • the detection items are as follows:

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Abstract

一种荧光免疫检测毛发痕量毒品的试剂盒，包括样品裂解液，荧光免疫层析试纸条；裂解液用于将毒品小分子从毛发中溶出；荧光免疫层析试纸条包含PVC衬板（1）、硝酸纤维膜（3）、样品垫（2）和吸收垫（4）；样品垫（2）、硝酸纤维膜（3）和吸收垫（4）依次交错固定于PVC衬板（1）之上；样品垫（2）上有毒品抗体-量子点纳米颗粒复合物以及兔IgG-量子点纳米颗粒复合物；硝酸纤维膜（3）上设有检测线（5）和质控线（6），检测线（5）包被有毒品抗原-牛血清白蛋白复合物，质控线（6）包被有羊抗兔抗体。该试剂盒能够快速、准确、灵敏地对毛发中的毒品进行检测。

Description

一种荧光免疫检测毛发痕量毒品的试剂盒 技术领域
本发明属于生物检测技术领域，特别是涉及一种荧光免疫检测毛发痕量毒品的试剂盒。
背景技术
从最原始的罂粟，加工成鸦片、可卡因和海洛因，到现在的冰毒、吗啡、大麻等，毒品一直离人们的生活很近。毒品不仅影响了吸毒者的健康，而且对吸毒者的家庭、社会、国家都造成了巨大的危害。为了与毒品作斗争，各国政府投入了大量的资金，以美国为例，自1981年以来政府每年平均投入3亿美元用于禁毒教育、治疗和研究项目，至奥巴马政府执政，美国各级政府每年用于禁毒执法的资金高达441亿美元。我国在挽救、治疗吸毒者，开展禁毒教育和科研，加大缉毒力度等方面也投入了大量的人力、物力和财力。然而如何针对吸毒人员作出有效、快速、准确、稳定的检测成为了亟待解决的问题。
随着检测手段的进步，对人体毒品残留的检测中，相较于传统血液、尿液等检测对象，毛发检测的标本容易获得，现场操作简单，避免了吸毒人员的取证不配合，标本掺假，成为了一种重要的毒品检测对象。
目前，气相色谱法、气相色谱-质谱联用法、高效液相色谱法、毛细管电泳法、免疫分析法等多种检测方法已经被用于对毒品的检测。例如中国专利CN101750458B公开了一种检测人体中毒品残留的方法，其采用HPLC-Chip-MS/MS和HPLC-MS/MS来检测毒品残留，具有费用低，准确性和稳定性高的优点。中国专利CN104422762A公开了一种检测尿液、唾液、血液、毛发中毒品的酶联免疫试剂盒和检测方法，该酶联免疫试剂盒具有高特异性、高灵敏度等特点。
随着禁毒形势的愈发严峻，新型、有效、快速、准确、稳定的检测方法和装置的需求进一步被放大。
发明内容
本发明的目的在于提供一种荧光免疫检测毛发痕量毒品的试剂盒。本发明能够快速、准确、灵敏的对毛发中的毒品进行检测。
为了达到上述的目的，本发明采取以下技术方案：
一种荧光免疫检测毛发痕量毒品的试剂盒，包括样品裂解液，荧光免疫层析试纸条；
所述裂解液用于将毒品小分子从毛发中溶出；
所述荧光免疫层析试纸条包含PVC衬板、硝酸纤维膜、样品垫和吸收垫，所述样品垫、硝酸纤维膜和吸收垫依次交错固定于PVC衬板之上；
所述样品垫上有毒品抗体-量子点纳米颗粒复合物以及兔IgG-量子点纳米颗粒复合物；
所述硝酸纤维膜上设有检测线和质控线，检测线包被有毒品抗原-牛血清白蛋白复合物，质控线包被有羊抗兔抗体。
本发明检测对象为毛发相对于常规检测方法要验血、验尿或者验口水，可以克服毒品测试取证困难和被测人员的不配合。
在上述试剂盒中，裂解液和毛发的质量比为100：1。
在上述试剂盒中，优选的，所述裂解液包含表面活性剂、角蛋白酶和缓冲溶液。其中，角蛋白酶能够腐蚀毛发表面，使毛发形成粗糙表面，从而利于毒品小分子从待测样品中溶出。表面活性剂能够洗涤毛发表面的污渍，使角蛋白酶能充分跟毛发接触，进一步提高毒品的溶出率。缓冲溶液能够中和角蛋白酶分解毛发产生的副反应物，提高角蛋白酶的生物活性。
进一步地，所述表面活性剂优选非离子表面活性剂，例如聚乙烯吡咯烷酮(PVP)、Triton X 100、Triton X 114、Triton X 405、Tween 20、Tween 60、Tween 80。相对于离子型表面活性剂，非离子表面活性剂不容易对离子强度产生影响，从而避免了影响后续试剂条中的免疫反应，提高了免疫检测的准确度。
进一步地，所述缓冲溶剂优选为TRIS(无水四硼酸钠)-EDTA缓冲溶液。本 发明的缓冲体系相对于其它缓冲体系来说pH范围接近8，性质温和，且溶液稳定性更高。
进一步地，所述裂解液还包括碳酸氢钠。碳酸氢钠的添加主要是为了进一步调整裂解液的pH。
进一步地，所述裂解液中表面活性剂的质量百分比为1-2％、角蛋白酶的含量为10000-50000IU/ML。
在上述试剂盒中，优选的，所述量子点纳米颗粒的荧光发射波长范围为600-620nm，更优选的，发射波长为610nm。激发光的波长范围为360-430nm，更优选的，激发光的波长为365nm。
在上述试剂盒中，优选的，所述量子点纳米颗粒为单一化合物形成的量子点或是几种化合物组装成的复合物量子点。更优选的，所述化合物选自ZnS、CdS、HgS、MgS、CdSe、ZnSe、ZnCdSe中的一种或多种。
本发明荧光免疫层析试剂盒采用量子点纳米颗粒作为荧光发射物质，量子点在光学性能上表现出激发光谱宽二连续，发射光谱窄且对称，荧光强度高，荧光寿命长，稳定性好等特点，能够克服常规的稀土微球，胶乳微球等荧光材料，信号衰退块，信号梯度不明显等缺陷。本发明的荧光免疫试纸条显示的荧光信号可以在2个小时内不发生明显衰减，且信号的梯度非常明显，可以有效分层，使测试的荧光信号更加稳定和便于检测提高检测的灵敏度和准确度。此外，量子点荧光稳定，信号强且变化明显的性质，也使得荧光信号量值转化效果好，利于定量分析。
本发明荧光免疫层析试纸条的检测原理为竞争法，T线(检测线)包被的抗原和待检样品中的毒品抗原竞争结合毒品抗体-量子点纳米颗粒复合物，当待检样品中无毒品抗原存在，毒品抗体-量子点纳米复合物在泳动过程中先和T线包被毒品抗原-牛血清白蛋白复合物结合，T线处形成毛发荧光免疫分析仪可测的荧光信号；当待检样品中有毒品抗原存在，则样品中的毒品抗原、包被毒品抗原和毒品抗原-牛血清白蛋白复合物竞争结合，此时T线处信号强度会随着样品中毒品抗原的增加而逐渐减弱；而兔IgG-量子点纳米颗粒复合物会继续泳 动和质控线(C线)包被的羊抗兔抗体结合，在C线处形成稳定不变的信号；如果C线处检测不到信号，则试纸条已报废，不能再使用。
本发明可采用配套的荧光检测分析仪，可以根据每个试剂批次分别导入相关的量值曲线，快速进样检测读数，控制时间在10秒以内，可以将荧光信号转化为对应的毒品或代谢物成分的浓度值，并且在显示屏幕上清晰显示，仪器可以保存500组以上的测试结果，且可以即时打印相关结果，包含对应的试剂批号，标本编号，测试时间，以及测试人员代码等信息。
本发明可检测的毒品包括吗啡、病毒、氯胺酮等。
本发明具有以下优点：
1)本发明的荧光免疫检测毒品的试剂盒采用毛发作为检测样品，对毛发中的毒品含量进行定量检测，取样简单，克服毒品检测取样困难的问题；
2)本发明利用表面活性剂、角蛋白酶和缓冲溶液组成的裂解液对毛发中的毒品进行溶出，表面活性剂、角蛋白酶和缓冲溶液的共同作用，能够有效提高毛发中毒品的溶出率和溶出速度。
3)本发明利用量子点进行荧光标记，荧光稳定，信号强且变化明显，能够较好的将荧光信号进行量值转化，能够定量的对毒品含量进行分析，通过荧光分析仪能够较快的获得结果。
4)本发明试剂盒用于检测毛发中的毒品含量，检测结果准确度高、精密度高、检测范围宽、稳定性高，且抗干扰(如毒品、洗发水、护发素、发胶等)能力强。
附图说明
图1是本发明荧光免疫层析试纸条的结构示意图，其中1为PVC衬板，2为样品垫，3为硝酸纤维膜，4为吸收垫，5为检测线，6为质控线。
具体实施方式
以下具体实施例是对本发明提供的方法与技术方案的进一步说明，但不应理解成对本发明的限制。
一、荧光免疫检测毒品的试剂盒
试剂盒包括样品裂解液，荧光免疫层析试纸条(结构参见图1)；裂解液用于将毒品小分子从待测样品中溶出；荧光免疫层析试纸条包含PVC衬板1、硝酸纤维膜3、样品垫2和吸收垫4，所述所述样品垫、硝酸纤维膜和吸收垫依次交错固定于PVC衬板之上；样品垫上有毒品抗体-量子点纳米颗粒复合物以及兔IgG-量子点纳米颗粒复合物；硝酸纤维膜上设有检测线和质控线，检测线包被有毒品抗原-牛血清白蛋白复合物，质控线包被有羊抗兔抗体。
二、毒品抗体-量子点纳米颗粒复合物以及兔IgG-量子点纳米颗粒复合物的制备方法：
(1)将荧光微球(即ZnS、CdS、HgS、MgS、CdSe、ZnSe、ZnCdSe中的一种或多种形成的量子点纳米颗粒)配制成5wt％工作浓度；
(2)加入待标记抗体(毒品抗体或者兔IgG)，加入量为10μg/ml，搅拌30min；
(3)用封闭液封闭，搅拌30min，封闭液为5wt％酪蛋白溶液；
(4)然后用离心机在4℃下离心，离心速度为10000r/min，离心30min，然后去上清液；
(5)用聚合物将沉淀物复溶，比例为0.1ml沉淀物加入1ml聚合物溶液，聚合物可以为聚苯乙烯、聚丁烯等。
三、荧光免疫层析试纸条的生产步骤为
1)将毒品抗原-牛血清白蛋白复合物和羊抗兔抗体用点膜机分别点于硝酸纤维素膜的测试区(T)和质控区(C)上，充分干燥，使硝酸纤维素膜牢固地吸附原料。
2)将毒品抗体-量子点纳米颗粒复合物以及兔IgG-量子点纳米颗粒复合物溶液混匀后处理在样本垫上，充分干燥。
3)将上述硝酸纤维素膜复合在PVC塑料薄板上。
4)将复合好的塑料薄板置于切割机上，切割成单人份试纸。
5)将单人份试纸装入配套使用的塑料盒内。
6)将塑料盒、干燥剂、滴管放入包装袋内，封口、待检。
7)待检品抽检其灵敏度、特异性和稳定性合格出厂。
四、荧光免疫试剂盒的检测方法
选择离头皮0.5厘米内的毛发，取毛发5mg用剪刀剪成长度为1-2mm，(10cm长度约10根)，用剪刀剪成1-2mm小段于对折的取样纸上；
将剪碎的毛发倒入装有毛发裂解液的瓶子内，盖紧瓶盖，震荡混匀1分钟。用滴管吸取毛发裂解液的上清，滴加2滴在试剂板的加样孔中。
荧光检测分析仪检测试纸条荧光信号，并将荧光信号转化为电信号，然后通过每批次试纸条ID卡上的标准曲线信息自动计算浓度，并呈现在仪器的显示屏上。
五、本发明产品的性能研究
采用荧光标记物为：聚苯乙烯微球中包埋了量子点ZnCdSe/ZnS形成的颗粒。
裂解液配方为：
化剂名称 用量g/L
无水四硼酸钠 10
角蛋白酶 5
PVP-10 10
曲拉通-100 15
碳酸氢钠 20
EDTA 5
1、线性范围
将吗啡企业参考品各稀释成0ng/mg、0.05ng/mg、0.1ng/mg、1ng/mg、2.5ng/mg、5ng/mg、10ng/mg、15ng/mg、20ng/mg，然后按照说明书的检测步骤，分别检测吗啡/冰毒/氯胺酮毛发快速检测试剂产品各3批，每个浓度检测3份取平均值。要求线性相关系数R≥0.99，相对偏差在±10％。结果如下表1。
表1吗啡线性范围检测结果
Figure PCTCN2019093739-appb-000001
Figure PCTCN2019093739-appb-000002
根据检测结果可知，浓度<0.1ng/mg时相对偏差>10％，浓度>10ng/mg时相对偏差>10％，不符合要求；在0.1-10ng/ml的范围内，产品线性的相关系数R≥0.99，相对偏差在±10％因此三个产品的线性范围为0.1-10ng/mg。
2、准确度
取3个批号的吗啡毛发快速检测试剂产品，按照说明书的检测步骤，分别检测为2.5ng/mg、5ng/mg的标准品，每个浓度检测6份取平均值并计算相对偏差。结果如下表2：
表2吗啡准确度检测结果
Figure PCTCN2019093739-appb-000003
根据检测结果可知，吗啡毛发快速检测试剂的准确度所有浓度相对偏差都在±10％以内。
3、精密度
取3个批次的吗啡毛发快速检测试剂10份，按照说明书的检测步骤，对浓度为2.5ng/mg、5ng/mg的标准品进行检测并计算变异系数CV。结果如下表3：
表3吗啡精密度检测结果
Figure PCTCN2019093739-appb-000004
Figure PCTCN2019093739-appb-000005
根据检测结果可知，吗啡毛发快速检测试剂批内精密度CV≤10％。
4、最低检出度
取3个批号的吗啡毛发快速检测试剂各10份，按照说明书的检测步骤，对标准品基质进行检测，计算测定结果平均值X和标准偏差SD。结果如下表4：
表4吗啡最低检出限检测结果
Figure PCTCN2019093739-appb-000006
Figure PCTCN2019093739-appb-000007
根据检测结果可知，吗啡毛发快速检测试剂的最低检出限(X+3SD)≤0.1ng/mg。
5、干扰实验
5.1、毒品物质干扰
取3个批号的吗啡毛发快速检测试剂各8份，将浓度为10μg/mL的甲基安非他明、美沙酮、咖啡因、地西泮、苯巴比妥、氯胺酮、曲马多、雷尼替丁按照说明书的检测步骤分别进行检测，每种交叉物质各做一次。要求结果呈阴性反应。结果如下表5：
表5毒品物质干扰检测结果
Figure PCTCN2019093739-appb-000008
根据检测结果可知，吗啡毛发快速检测试剂在上述干扰物质浓度为10ug/ml时无干扰。
5.2、洗发水护发素的干扰
随机取一批次毛发快速检测试剂72份，然后取阴性毛发和阳性毛发各3份，分别使用洗发水和护发素清洗毛发样本，用自来水漂洗三遍(模拟正常洗 发过程)。之后对清洗后的头发进行检测，以未清洗的毛发样本为对照，每种各做一次。结果如下表6-7：
表6洗发水对毛发快速检测试剂的影响
Figure PCTCN2019093739-appb-000009
Figure PCTCN2019093739-appb-000010
结果显示：洗发水对吗啡毛发快速检测试剂、甲基安非他明毛发快速检测试剂和氯胺酮毛发快速检测试剂均无影响。
表7护发素对毛发快速检测试剂的影响
Figure PCTCN2019093739-appb-000011
Figure PCTCN2019093739-appb-000012
结果显示：护发素对吗啡毛发快速检测试剂、甲基安非他命毛发快速检测试剂和氯胺酮毛发快速检测均无影响。
5.3、发胶类产品的干扰
随机取一批次毛发快速检测试剂36份，然后取阳性毛发和阴性毛发各3份，模拟发胶的正常使用过程将发胶喷在毛发上或者涂抹在头发上，以未涂抹的毛发样本为对照，各做一次。结果如下表8：
表8发胶对毛发快速检测试剂的影响
Figure PCTCN2019093739-appb-000013
Figure PCTCN2019093739-appb-000014
结果显示：发胶对吗啡毛发快速检测试剂、甲基安非他命毛发快速检测试剂和氯胺酮毛发快速检测试剂均无影响。
6、稳定性
每个产品每批次各抽取400人份的成品放入常温的环境下，将检测缓冲液保存在2-8℃的条件下，并在每次检测时，将全部的检测缓冲液从2-8℃冰箱内取出、开瓶，恢复至室温进行检测，检测结束后将检测缓冲液放回2-8℃的环境条件下，检测时间按0、3、6、9、12、18、24、30、31个月进行。(如若在试验过程中，发现产品不稳定，需要使用现配的检测缓冲液进行验证，说明是成品的稳定性原因还是检测缓冲液的原因)。检测项目如下：
1)线性:选择浓度为0.1ng/mg、1ng/mg、2.5ng/mg、5ng/mg、10ng/mg的企业内部标准品进行测量。
2)最低检出限:取产品10份，按照检测步骤对配制企业内部标准品基质进行检测。
表9吗啡成品的线性的检测结果(测试时间：0个月)
Figure PCTCN2019093739-appb-000015
表10吗啡成品的线性的检测结果(测试时间：3个月)
Figure PCTCN2019093739-appb-000016
Figure PCTCN2019093739-appb-000017
表11吗啡成品的线性的检测结果(测试时间：6个月)
Figure PCTCN2019093739-appb-000018
Figure PCTCN2019093739-appb-000019
表12吗啡成品的线性的检测结果(测试时间：9个月)
Figure PCTCN2019093739-appb-000020
表13吗啡成品的线性的检测结果(测试时间：12个月)
Figure PCTCN2019093739-appb-000021
Figure PCTCN2019093739-appb-000022
以上实施例的说明只是用于帮助理解本发明方法及其核心思想。应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以对本发明进行若干改进和修饰，这些改进和修饰也落入本发明权利要求保护范围内。

Claims (10)

  1. 一种荧光免疫检测毛发痕量毒品的试剂盒，其特征在于，包括样品裂解液，荧光免疫层析试纸条；
    所述裂解液用于将毒品小分子从毛发中溶出；
    所述荧光免疫层析试纸条包含PVC衬板、硝酸纤维膜、样品垫和吸收垫，所述样品垫、硝酸纤维膜和吸收垫依次交错固定于PVC衬板之上；
    所述样品垫上有毒品抗体-量子点纳米颗粒复合物以及兔IgG-量子点纳米颗粒复合物；
    所述硝酸纤维膜上设有检测线和质控线，检测线包被有毒品抗原-牛血清白蛋白复合物，质控线包被有羊抗兔抗体。
  2. 根据权利要求1所述的一种荧光免疫检测毛发痕量毒品的试剂盒，其特征在于，裂解液和毛发的质量比为100：1。
  3. 根据权利要求1所述的一种荧光免疫检测毛发痕量毒品的试剂盒，其特征在于，所述裂解液包含表面活性剂、角蛋白酶和缓冲溶液。
  4. 根据权利要求1所述的一种荧光免疫检测毛发痕量毒品的试剂盒，其特征在于，所述表面活性剂优选非离子表面活性剂。
  5. 根据权利要求1所述的一种荧光免疫检测毛发痕量毒品的试剂盒，其特征在于，所述缓冲溶剂优选为TRIS(无水四硼酸钠)-EDTA缓冲溶液。
  6. 根据权利要求1所述的一种荧光免疫检测毛发痕量毒品的试剂盒，其特征在于，所述裂解液还包括碳酸氢钠。
  7. 根据权利要求1所述的一种荧光免疫检测毛发痕量毒品的试剂盒，其特征在于，所述裂解液中表面活性剂的质量百分比为1-2％、角蛋白酶的含量为10000-50000IU/ML。
  8. 根据权利要求1所述的一种荧光免疫检测毛发痕量毒品的试剂盒，其特征在于，所述量子点纳米颗粒的荧光发射波长范围为600-620nm，更优选的，发射波长为610nm。
  9. 根据权利要求1所述的一种荧光免疫检测毛发痕量毒品的试剂盒，其特征在于，所述量子点纳米颗粒为单一化合物形成的量子点或是几种化合物组装成的复合物量子点。
  10. 根据权利要求1所述的一种荧光免疫检测毛发痕量毒品的试剂盒，其特征在于，所述化合物选自ZnS、CdS、HgS、MgS、CdSe、ZnSe、ZnCdSe中的一种或多种。
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