WO2021169089A1 - 一种基于微流控芯片的快速免疫检测方法 - Google Patents

一种基于微流控芯片的快速免疫检测方法 Download PDF

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WO2021169089A1
WO2021169089A1 PCT/CN2020/096717 CN2020096717W WO2021169089A1 WO 2021169089 A1 WO2021169089 A1 WO 2021169089A1 CN 2020096717 W CN2020096717 W CN 2020096717W WO 2021169089 A1 WO2021169089 A1 WO 2021169089A1
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sample
microfluidic chip
detection
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周建华
刘廙人
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中山大学
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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
    • G01N33/54306—Solid-phase reaction mechanisms
    • 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
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • 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
    • G01N33/6854—Immunoglobulins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/11—Orthomyxoviridae, e.g. influenza virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms

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  • the invention relates to the technical field of biomedical analysis, and more specifically, to a rapid immune detection method based on a microfluidic chip.
  • the ELISA on the static ELISA plate belongs to the specific binding process of the heterogeneous antigen and antibody.
  • the immunoreaction efficiency of the heterogeneous antigen and antibody is mainly controlled by the dispersion rate constant and the binding rate constant in the receptor phase.
  • the probability that the relative effective distance of antigen and antibody changes towards zero caused by molecular diffusion is an important influencing factor.
  • the captured components diffuse freely in the vertical direction of the captured components, and the molecular diffusion efficiency is very low, so a lot of reaction time needs to be sacrificed to meet the relatively saturated antigen-antibody binding needs.
  • the conventional ELISA based on the enzyme-labeled plate not only takes a long time for the detection process, but also uses a large amount of reagents and samples, which cannot meet the needs of a small number of samples or clinical timely detection.
  • the main purpose of the present invention is to overcome the above shortcomings and provide a rapid immunoassay method based on a microfluidic chip, which realizes the rapid combination of heterogeneous antigen-antibody by adjusting the fluid pressure on the microfluidic chip; microchannel Customization of throughput can be achieved, reagent consumption and detection time can be greatly reduced, and multi-index immunodetection can be realized; through the combination with the rapid immunodetection method, it is more convenient to implement visual qualitative, visual semi-quantitative or precise quantification.
  • a rapid immunoassay method based on a microfluidic chip includes the following steps:
  • the shape of the detection chip is circular or polygonal, and the thickness, diameter and number of the microchannels can be designed according to requirements.
  • the cleaning solution is PBST; preferably, the surface modification of the microchannel, such as polyvinyl alcohol, can reduce the interference background caused by the non-specific adsorption of the microchannel.
  • an automatic air pressure adjustment module is used to continuously reciprocate the sample to be tested.
  • an automated air pressure adjustment module is used to continuously reciprocate the probe modified secondary antibody solution.
  • the solid-phase reaction substrate is glass or plastic.
  • the sample to be tested includes a buffer solution, serum or plasma.
  • step S2 the dilution of the sample to be tested is selected from the group consisting of 1:50, 1:100, 1:500, 1:1000, 1:5000, 1:10000 or 1 : 50000.
  • step S2 the sample to be tested is continuously reciprocated at 37° C., and the mixing and incubation time is 2-5 min.
  • the time of the mixed incubation is 5 min.
  • step S3 the probe-modified secondary antibody solution is continuously reciprocated at 37° C., and the mixing and incubation time is 2-5 min.
  • the time of the mixed incubation is 5 min.
  • the invention also provides an application of a rapid immune detection method based on a microfluidic chip in the detection of 2019 new coronavirus (NCP, 2019-nCoV), severe acute respiratory syndrome (SARS) virus or influenza virus.
  • NCP 2019 new coronavirus
  • SARS severe acute respiratory syndrome
  • the application of the rapid immune detection method based on the microfluidic chip in the detection of the 2019 novel coronavirus (NCP, 2019-nCoV), severe acute respiratory syndrome (SARS) virus or influenza virus for non-diagnostic purposes.
  • NCP 2019 novel coronavirus
  • SARS severe acute respiratory syndrome
  • 2019 novel coronavirus NCP, 2019-nCoV
  • the use of specific 2019-nCoV N protein recombinant antigens can avoid non-specific reactions and can be specific Recognize the 2019-nCoV N protein antibody in the serum sample, and react with it to leave the specific 2019-nCoV N protein antibody in the serum sample on the solid phase substrate, and the remaining serum components are washed away.
  • the immune reactant is an antigen
  • a sandwich immune model of antigen-antibody-labeled antibody conjugate is formed.
  • the specific detection principle is: in the prepared detection chip, after adding the sample to be tested into the sample chamber of the microchannel , The test antibody in the test sample filled with the microchannel will bind to the specific antigen pre-coated on the substrate, and wash after incubation; if the test sample contains antibodies against the specific antigen, then the test sample in the test sample The specific antibody will bind to the antigen, and an immune reaction will occur to form an antigen-antibody complex; then a signal probe is added to the microchannel to label the second antibody.
  • the signal probe The needle-labeled second antibody will react with the specific antibody bound in the microchannel to form an antigen-antibody-second antibody conjugate; finally, the signal probe of the antigen-antibody-second antibody conjugate will be detected , Not only can obtain qualitative results, but also quantitative results. Further, more in-depth immunological research can be carried out through images or atlases to explore the mechanism of action such as key components of the virus.
  • the immune reactant is an antibody
  • a sandwich immune model of the first antibody-antigen-second antibody conjugate is formed, and the detection principle is the same as above.
  • the signal probe includes but is not limited to gold nanoparticles, fluorescent quantum dots or fluorescent yellow isothiocyanate.
  • the present invention has the following advantages:
  • the rapid immunoassay method based on the microfluidic chip provided by the present invention with the help of the microfluidic chip, the incubation strategy of bidirectional reciprocating flow of fluid in the microchannel can well solve the molecular diffusion efficiency of the heterogeneous antigen-antibody immune reaction
  • the problem is that on the microfluidic chip, a plurality of microchannels are patterned to construct an immune response binding site, and a fluid pressure buffer zone is constructed together with the sample cavity and the buffer cavity. It is assisted by the automatic air pressure adjustment module and the automatic sampler.
  • the results can be obtained within 10 minutes by using the nanoprobe to amplify the signal, which takes only 8.3% of the conventional ELISA, and completes the entire process from adding samples to sending for testing. Achieved rapid detection;
  • test sample can still be detected after being diluted 10,000 times;
  • the microchannel design can provide a customized high-throughput multi-index detection chip, the consumption of single-channel sample reagents is reduced to less than 50 ⁇ L, a drop of more than 50%;
  • Multiple samples can be jointly detected through a one-time operation, which has the advantages of small sample size, multiple samples, multiple indicators can be detected at the same time, strong specificity, and high sensitivity.
  • the rapid immunoassay method based on the microfluidic chip provided by the present invention can conveniently implement visual qualitative, visual semi-quantitative or precise quantification.
  • enzyme-linked immunoreaction ELISA
  • the color or excitation light source directly illuminates the detection point for visual qualitative detection, or the microfluidic chip can be transferred to a fluorescence analyzer for fluorescence quantitative analysis, or an ultraviolet-visible light absorption spectrometer can be used for localized surface plasmon sensing detection; Complicated instruments and equipment do not require the experience of experimenters, avoiding subjective factors of different operators, and are particularly suitable for on-site detection of important respiratory viruses such as NCP, SARS, influenza virus and immune response kinetics research.
  • important respiratory viruses such as NCP, SARS, influenza virus and immune response kinetics research.
  • the rapid immunoassay method based on the microfluidic chip provided by the present invention has good sample compatibility, and is suitable for various test medium samples including buffer solution, serum, plasma and the like.
  • Figure 1 is a schematic diagram of the detection chip structure in the microfluidic chip-based rapid immunoassay method using the present invention; wherein 1 is a glass substrate, 2 is a microchannel, 3 is a sample loading cavity, 4 is a buffer cavity, and 5 is an air pressure adjustment Module, 6 is PDMS cover sheet;
  • FIG. 2 is a schematic diagram of the principle of signal amplification using a fluorescent probe-labeled secondary antibody when the rapid immunoassay method based on a microfluidic chip of the present invention is used to detect 2019-nCoV N protein antibodies in human serum samples in Example 1;
  • Fig. 3 is a fluorescence microscope picture of using the fluorescent probe-labeled secondary antibody for signal amplification when the rapid immunoassay method based on the microfluidic chip of the present invention is used to detect the 2019-nCoV N protein antibody in human serum samples in Example 1, where , Figure 3-a is the fluorescence microscope picture when the dilution of the positive sample is 1:100, Figure 3-b is the fluorescence microscope picture when the dilution of the positive sample is 1:10000, and Figure 3-c is the blank control;
  • FIG. 4 is a schematic diagram of the principle of signal amplification using localized surface plasma when the rapid immunoassay method based on the microfluidic chip of the present invention is used to detect 2019-nCoV N protein antibodies in human serum samples in Example 2.
  • this embodiment provides a rapid immunoassay method based on a microfluidic chip, which is applied to the detection of 2019 new coronavirus (NCP, 2019-nCoV) in clinically confirmed positive serum samples. It includes the following steps:
  • the glass substrate 1 is pre-coated with 2019-nCoV N protein recombinant antigen, sealed with bovine serum albumin (BSA), and then attached to the PDMS cover sheet 6 to form a microchannel 2; where The shape of the detection chip is circular, and the detection chip has 8 microchannels 2; the microchannels 2 are located on the radius axis of the microfluidic chip, arranged divergently at the center of the microfluidic chip, and are arranged along the center of the substrate. Extending from the center of the chip, a sample loading cavity 3, a microchannel 2 and a buffer cavity 4 are arranged in sequence from the center point of the chip to the edge.
  • BSA bovine serum albumin
  • this embodiment provides a rapid immunoassay method based on a microfluidic chip, which includes the following steps:
  • detection chip Gold nanostructured glass substrate 1 is pre-coated with 2019-nCoV N protein recombinant antigen, sealed with bovine serum albumin (BSA), and bonded with PDMS cover sheet 6 to form microchannel 2; ,
  • the shape of the detection chip is circular, the detection chip has 8 microchannels 2; the microchannels 2 are located on the radius axis of the microfluidic chip and are arranged divergently at the center of the microfluidic chip, along the substrate The center of the chip extends outward, and the sample loading cavity 3, the microchannel 2, and the buffer cavity 4 are arranged in sequence from the center point of the chip to the edge.
  • Immune capture of the serum sample to be tested control the automatic sampler to add 50 ⁇ L of the diluted serum sample to the sample cavity of the microchannel one by one (the dilution of the sample to be tested is 1:100, 1:10000, respectively) ,
  • the dilution medium is PBST
  • the automatic air pressure adjustment module 5 is controlled at 37°C to make the sample to be tested continue to reciprocate, incubate for 4 minutes, after completion, the automatic air pressure adjustment module 5 is controlled to discharge the serum sample to be tested, and after the sample is captured, the automatic sample addition is controlled
  • the device sends 50 ⁇ L of cleaning solution (PBST) to the sample loading chamber, and controls the automatic air pressure adjustment module 5 to flush and clean, and discharge the cleaning solution.
  • PBST cleaning solution
  • UV-visible spectrometer is used to detect the immune capture results.
  • This embodiment is similar to embodiment 1, except that in step S2, the incubation time is 2 min.
  • step S3 a secondary antibody labeled with fluorescent quantum dots is used, and the incubation time is 2 min.
  • step S1 a glass substrate 1 that does not contain gold nanostructures is pre-coated with 2019-nCoV N protein recombinant antigen, and bovine serum labeled with fluorescent yellow isothiocyanate is used. After the albumin (FITC-BSA) is sealed, it is attached to the PDMS cover sheet 6 to form the microchannel 2;
  • FITC-BSA fluorescent yellow isothiocyanate
  • step S4 a fluorescence detector is used to detect the immune capture result.

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Abstract

一种基于微流控芯片的快速免疫检测方法，包括以下步骤：S1、检测芯片的制作；S2、待测样本的免疫捕获；S3、探针修饰二抗的免疫捕获；S4、免疫捕获结果的检测；实现了快速检测，通过一次性操作即可联合检测多个样本，具有样本量少、多样本、多指标可同时检测、特异性强、灵敏度高等优点；具有良好的样品兼容性，适用包括缓冲溶液、血清、血浆等多种待测介质样品；不需要复杂的仪器设备，也不需要实验人员的经验判断，避免了不同操作人员的主观因素，特别适合如2019新型冠状病毒(NCP)、严重急性呼吸综合征(SARS)病毒等重要呼吸道病毒的现场检测及免疫反应动力学研究。

Description

一种基于微流控芯片的快速免疫检测方法 技术领域
本发明涉及生物医学分析技术领域，更具体地，涉及一种基于微流控芯片的快速免疫检测方法。
背景技术
免疫检测因其特异性捕获的能力，在疾病诊断，特别是即时检验技术(POCT)方面发挥着重要作用。目前，常规医院检验科对疾病特征免疫组分的检测，通常基于酶标板采用标准化的酶联免疫(ELISA)程序；尽管因为其直观的酶催化显色可视化定性能力被广泛采用，这种成熟的免疫检测策略，其流程冗长的缺陷一直无法避免。为了最大程度确保检测结果的保真性，一套完整的间接法ELISA程序必须连续完成用于捕获样品的免疫成分的包被孵育、多余包被位点的封闭孵育、待检样本的结合孵育、探针偶联抗体的结合孵育，共计通常需要2小时以上，后续的催化显色过程还需要15-45分钟不等的时间。从原理上分析，在静态酶标板上的ELISA属于非均相上抗原抗体的特异性结合过程，非均相抗原抗体的免疫反应效率主要受体相中分散速率常数和结合速率常数控制，其中分子扩散导致的抗原抗体相对有效距离变化趋向零的概率是一项重要的影响因素。对于静态酶标板上的ELISA，被捕获成分向捕获成分的垂直方向自由扩散，其分子扩散效率非常低，所以需要牺牲大量的反应时间来满足相对饱和的抗原抗体结合需要。
基于酶标板的常规ELISA不仅检测过程耗时较长，而且使用的试剂和样品量也较大，不能满足少量样品或临床及时检测的需求。
发明内容
本发明的主要目的在于克服上述不足之处，提供一种基于微流控芯片的快速免疫检测方法，通过在微流控芯片上调节流体压力，实现非均相抗原-抗体的快速结合；微通道可实现通量自定义，大幅降低试剂消耗量和检测时间，实现多指标免疫检测；通过与所述快速免疫检测方法的联用，更方便地实施可视化定性、可视化半定量或精准定量。
本发明是通过以下技术方案进行实现的：
一种基于微流控芯片的快速免疫检测方法，包括以下步骤：
S1、检测芯片的制作：固相反应基底上预包被免疫反应物、封闭后，与盖片贴合后形成微通道；
S2、待测样本的免疫捕获：向所述微通道的加样腔加入待测样本，37℃下使待测样本持 续往复运动，混合孵育，用清洗液进行清洗；
S3、探针修饰二抗的免疫捕获：向所述微通道的加样腔加入探针修饰二抗溶液，37℃下使探针修饰二抗溶液持续往复运动，混合孵育，用清洗液进行清洗；
S4、免疫捕获结果的检测：对免疫捕获结果进行检测。
优选的，所述检测芯片的形状为圆形或多边形，所述微通道的厚度、直径和数量可根据需要进行设计。
优选的，所述清洗液采用PBST；优选的，微通道通过表面修饰如采用聚乙烯醇，可以降低微通道非特异性吸附造成的干扰背景。
优选的，步骤S2中，采用自动化气压调节模块使待测样本持续往复运动。
优选的，步骤S3中，采用自动化气压调节模块使探针修饰二抗溶液持续往复运动。
作为本发明的技术方案的进一步描述，步骤S1中，所述固相反应基底为玻璃或塑料。
作为本发明的技术方案的进一步描述，步骤S2中，所述待测样本包括缓冲溶液、血清或血浆。
作为本发明的技术方案的进一步描述，步骤S2中，所述待测样本的稀释度选自包括1:50、1:100、1:500、1:1000、1:5000、1:10000或1:50000。
作为本发明的技术方案的进一步描述，步骤S2中，在37℃下使待测样本持续往复运动，所述混合孵育的时间为2-5min。
优选的，所述混合孵育的时间为5min。
作为本发明的技术方案的进一步描述，步骤S3中，在37℃下使探针修饰二抗溶液持续往复运动，所述混合孵育的时间为2-5min。
优选的，所述混合孵育的时间为5min。
本发明还提供了一种基于微流控芯片的快速免疫检测方法在2019新型冠状病毒(NCP，2019-nCoV)、严重急性呼吸综合征(SARS)病毒或流感病毒检测中的应用。
所述基于微流控芯片的快速免疫检测方法在非诊断目的的2019新型冠状病毒(NCP，2019-nCoV)、严重急性呼吸综合征(SARS)病毒或流感病毒检测中的应用。
可以理解的是，在临床已确诊的阳性血清样本中进行2019新型冠状病毒(NCP，2019-nCoV)检测时，采用特异性2019-nCoV N蛋白重组抗原，能避免非特异性反应，可特异性的识别血清样本中的2019-nCoV N蛋白抗体，并与之发生免疫反应将血清样本中特异性2019-nCoV N蛋白抗体留在固相基底上，其余的血清成分被冲洗走。
当免疫反应物为抗原时，则形成抗原-抗体-标抗体结合物的夹心免疫模型，具体的检测 原理为：在制备好的检测芯片中，向微通道的加样腔中加入待测样本后，充满微通道的待测样本中的待测抗体会和预先包被在基底上的特异性抗原结合，孵育后洗涤；如果待测样本中含有针对特异性抗原的抗体，那么待测样本中的特异性抗体就会和抗原结合，并发生免疫反应形成抗原-抗体复合物；然后向微通道中加入信号探针标记第二抗体，如果待测样本中含有针对特异性抗原的抗体，那么信号探针标记第二抗体就会与微通道中被结合的特异性抗体发生免疫反应，形成抗原-抗体-第二抗体结合物；最后再对抗原-抗体-第二抗体结合物的信号探针进行检测，不仅能获得定性结果，还可获得定量结果，进一步地，可通过图像或图谱进行更深入的免疫学研究，探究如病毒关键组分等的作用机制。
当免疫反应物为抗体时，则形成第一抗体-抗原-第二抗体结合物的夹心免疫模型，检测原理同上。
其中，所述信号探针包括但不限于金纳米颗粒、荧光量子点或异硫氰酸荧光黄。
与现有技术相比，本发明具有如下优点：
1、本发明提供的基于微流控芯片的快速免疫检测方法，借助微流控芯片，在微通道中流体双向往复流动的孵育策略能够很好地解决非均相抗原抗体免疫反应的分子扩散效率问题，在微流控芯片上，图案化地设计复数微通道以构建免疫反应结合位点，与加样腔、缓冲腔共同构筑流体压力缓冲带，在自动化气压调节模块和自动进样器的辅助下实现快速、高精准的免疫反应操作，大幅缩短免疫反应时间，包括待测免疫成分的免疫捕获、信号二抗的免疫捕获，采用气压调节液体往复流动的策略大幅优化免疫反应过程进而优化信号放大效率。
提前完成包被孵育和封闭孵育的基础上，加样起使用纳米探针放大信号则10分钟以内可得结果，耗时仅为常规ELISA的8.3％，完成从添加样品到送检测的全流程，实现了快速检测；
在不牺牲灵敏度的情况下，被测样本稀释10000倍后仍可检出；
同等浓度尺度下，由于微通道设计可提供自定义的高通量多指标检测芯片，单一通道样本试剂的消耗量降至50μL以下，降幅超过50％；
通过一次性操作即可联合检测多个样本，具有样本量少、多样本、多指标可同时检测、特异性强、灵敏度高等优点。
2、本发明提供的基于微流控芯片的快速免疫检测方法，可方便地实施可视化定性、可视化半定量或精准定量，具体的根据探针偶联物的不同，可采用酶联免疫反应(ELISA)显色或激发光源直接照射检测点进行可视化定性检测，也可将微流控芯片转移到荧光分析仪进行荧光定量分析，或使用紫外可见光吸收光谱仪进行局域表面等离子体传感检测；不需要复杂 的仪器设备，也不需要实验人员的经验判断，避免了不同操作人员的主观因素，特别适合如NCP、SARS、流感病毒等重要呼吸道病毒的现场检测及免疫反应动力学研究。
3、本发明提供的基于微流控芯片的快速免疫检测方法，具有良好的样品兼容性，适用包括缓冲溶液、血清、血浆等多种待测介质样品。
附图说明
图1为采用本发明的基于微流控芯片的快速免疫检测方法中检测芯片结构示意图；其中，1为玻璃基底，2为微通道，3为加样腔，4为缓冲腔，5为气压调节模块，6为PDMS盖片；
图2为实施例1中采用本发明的基于微流控芯片的快速免疫检测方法检测人血清样本中2019-nCoV N蛋白抗体时，采用荧光探针标记二抗进行信号放大的原理示意图；
图3为实施例1中采用本发明的基于微流控芯片的快速免疫检测方法检测人血清样本中2019-nCoV N蛋白抗体时，采用荧光探针标记二抗进行信号放大的荧光显微镜图片，其中，图3-a为阳性样本的稀释度为1:100时的荧光显微镜图片，图3-b为阳性样本的稀释度为1:10000时的荧光显微镜图片，图3-c为空白对照；
图4为实施例2中采用本发明的基于微流控芯片的快速免疫检测方法检测人血清样本中2019-nCoV N蛋白抗体时，采用局域表面等离子体进行信号放大的原理示意图。
具体实施方式
下面对本发明的较佳实施例进行详细阐述，以使本发明的优点和特征更易被本领域技术人员理解，从而对本发明的保护范围作出更为清楚的界定。
实施例1
结合附图1、2，本实施例提供了一种基于微流控芯片的快速免疫检测方法，应用于临床已确诊的阳性血清样本中2019新型冠状病毒(NCP，2019-nCoV)的检测时，包括以下步骤：
S1、检测芯片的制作：玻璃基底1上预包被2019-nCoV N蛋白重组抗原、用牛血清白蛋白(BSA)封闭后，与PDMS盖片6贴合后形成微通道2；其中，所述检测芯片的形状为圆形，所述检测芯片带有8条微通道2；所述微通道2位于微流控芯片的半径轴上，以微流控芯片中心点发散排列，沿基底的中心向外延伸，自芯片中心点向边缘依次排列有加样腔3、微通道2、缓冲腔4。
S2、待测血清样本的免疫捕获：控制自动加样器向所述微通道的加样腔逐个加入50μL稀释后的待测血清样本(待测样本的稀释度分别采用1:100，1:10000，稀释介质为PBST)，37℃下控制自动化气压调节模块5使待测样本持续往复运动，孵育5min，完成后控制自动化气压调节模块5排出待测血清样本，完成样本捕获后，控制自动加样器向加样腔50μL清 洗液(PBST)，控制自动化气压调节模块5冲刷清洗，排出清洗液。
S3、FITC标记鼠抗人IgG抗体的免疫捕获：控制自动加样器向所述微通道的加样腔逐个加入50μL FITC标记鼠抗人IgG抗体溶液(PBST稀释，1：250)，37℃下控制自动化气压调节模块5使FITC标记鼠抗人IgG抗体溶液持续往复运动，混合孵育5min，完成后控制自动化气压调节模块5排出FITC标记鼠抗人IgG抗体溶液；完成FITC标记鼠抗人IgG抗体捕获后，控制自动加样器向加样腔逐个加入50μL清洗液(PBST)，控制自动化气压调节模块5冲刷清洗，排出清洗液。
S4、免疫捕获结果的检测：采用荧光显微镜检测免疫捕获结果，如图3所示，由此可见，采用基于微流控芯片的快速免疫检测方法，待测血清样本在稀释10000倍后检测的灵敏度仍然可观，且样本消耗量小、耗时大大缩短。
实施例2
结合附图1、4，本实施例提供了一种基于微流控芯片的快速免疫检测方法，包括以下步骤：
S1、检测芯片的制作：金纳米结构玻璃基底1上预包被2019-nCoV N蛋白重组抗原、用牛血清白蛋白(BSA)封闭后，与PDMS盖片6贴合后形成微通道2；其中，所述检测芯片的形状为圆形，所述检测芯片带有8条微通道2；所述微通道2位于微流控芯片的半径轴上，以微流控芯片中心点发散排列，沿基底的中心向外延伸，自芯片中心点向边缘依次排列有加样腔3、微通道2、缓冲腔4。
S2、待测血清样本的免疫捕获：控制自动加样器向所述微通道的加样腔逐个加入50μL稀释后的待测血清样本(待测样本的稀释度分别采用1:100，1:10000，稀释介质为PBST)，37℃下控制自动化气压调节模块5使待测样本持续往复运动，孵育4min，完成后控制自动化气压调节模块5排出待测血清样本，完成样本捕获后，控制自动加样器向加样腔50μL清洗液(PBST)，控制自动化气压调节模块5冲刷清洗，排出清洗液。
S3、金纳米颗粒(AuNR)标记鼠抗人IgG抗体的免疫捕获：控制自动加样器向所述微通道的加样腔逐个加入50μL AuNR标记鼠抗人IgG抗体溶液(PBST稀释，1：250)，37℃下控制自动化气压调节模块5使AuNR标记鼠抗人IgG抗体溶液持续往复运动，混合孵育4min，完成后控制自动化气压调节模块5排出AuNR标记鼠抗人IgG抗体溶液；完成AuNR标记鼠抗人IgG抗体捕获后，控制自动加样器向加样腔逐个加入50μL清洗液(PBST)，控制自动化气压调节模块5冲刷清洗，排出清洗液。
S4、免疫捕获结果的检测：采用紫外可见光光谱仪检测免疫捕获结果。
实施例3
本实施例与实施例1类似，不同的是，步骤S2中，所述孵育的时间为2min。
步骤S3中，采用荧光量子点标记的二抗，所述孵育的时间为2min。
实施例4
本实施例与实施例2类似，不同的是，步骤S1中，使用不含有金纳米结构的玻璃基底1上预包被2019-nCoV N蛋白重组抗原、使用异硫氰酸荧光黄标记的牛血清白蛋白(FITC-BSA)封闭后，与PDMS盖片6贴合后形成微通道2；；
步骤S4中，采用荧光检测仪检测免疫捕获结果。
以上仅为本发明的优选实施例，并非因此限制本发明的专利范围，凡是利用本发明说明书内容所作的等效结构或等效流程变换，或直接或间接运用在其他相关的技术领域，均同理包括在本发明的专利保护范围内。

Claims (7)

  1. 一种基于微流控芯片的快速免疫检测方法，其特征在于，包括以下步骤：
    S1、检测芯片的制作：固相反应基底上预包被免疫反应物、封闭后，与盖片贴合后形成微通道；
    S2、待测样本的免疫捕获：向所述微通道的加样腔加入待测样本，使待测样本持续往复运动，混合孵育，用清洗液进行清洗；
    S3、探针修饰二抗的免疫捕获：向所述微通道的加样腔加入探针修饰二抗溶液，使探针修饰二抗溶液持续往复运动，混合孵育，用清洗液进行清洗；
    S4、免疫捕获结果的检测：对免疫捕获结果进行检测。
  2. 根据权利要求1所述的基于微流控芯片的快速免疫检测方法，其特征在于，步骤S1中，所述固相反应基底为玻璃或塑料。
  3. 根据权利要求1所述的基于微流控芯片的快速免疫检测方法，其特征在于，步骤S2中，所述待测样本包括缓冲溶液、血清或血浆。
  4. 根据权利要求1所述的基于微流控芯片的快速免疫检测方法，其特征在于，步骤S2中，所述待测样本的稀释度选自包括1:50、1:100、1:500、1:1000、1:5000、1:10000或1:50000。
  5. 根据权利要求1所述的基于微流控芯片的快速免疫检测方法，其特征在于，步骤S2中，在37℃下使待测样本持续往复运动，所述混合孵育的时间为2-5min。
  6. 根据权利要求1所述的基于微流控芯片的快速免疫检测方法，其特征在于，步骤S3中，在37℃下使探针修饰二抗溶液持续往复运动，所述混合孵育的时间为2-5min。
  7. 根据权利要求1所述的基于微流控芯片的快速免疫检测方法在2019新型冠状病毒(NCP)、严重急性呼吸综合征(SARS)病毒或流感病毒检测中的应用。
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