技术领域Technical Field
本发明涉及微流控快速检测领域，具体地，涉及一种自动化化学发光免疫分析芯片及其检测方法，特别公开了一种离心式自动化快速检测芯片，用于样本中分析物的高灵敏度自动化定量快速分析检测。The present invention relates to the field of microfluidic rapid detection, and in particular to an automated chemiluminescent immunoassay chip and a detection method thereof, and particularly discloses a centrifugal automated rapid detection chip for high-sensitivity automated quantitative rapid analysis and detection of analytes in samples.
背景技术Background technique
床旁快速检测是现代检测技术总体趋势之一，目前大部分生物样本中的分析物包括基因、蛋白、糖、元素等基本可以利用大型仪器或繁杂的手工操作进行分析检测，但是因为其制造成本高，专业技术要求高，环境要求高而限制了这些检测技术的推广，导致许多分析物不能被及时检测分析而限制了科学的发展；许多样本由于保存条件较高，又不能进行现场分析检测，就导致了生物安全隐患不能被及时发现，而造成严重后果；另一方面随着昂贵检测技术的发展，同样增加了医疗、公共卫生、科学研究、患者等体系或个人的经济负担。Bedside rapid testing is one of the general trends in modern testing technology. At present, most analytes in biological samples, including genes, proteins, sugars, elements, etc., can be basically analyzed and detected using large instruments or complicated manual operations. However, the high manufacturing cost, high professional and technical requirements, and high environmental requirements limit the promotion of these detection technologies, resulting in many analytes not being detected and analyzed in a timely manner, which restricts the development of science. Many samples cannot be analyzed and tested on-site due to high storage conditions, which leads to the failure to discover biosafety risks in a timely manner, resulting in serious consequences. On the other hand, with the development of expensive detection technology, the economic burden on medical, public health, scientific research, patients and other systems or individuals has also increased.
微流控分析技术是指把生物、化学、医学分析过程的样品制备、反应、分离、检测等基本操作单元集成到一块微米尺度的芯片上，并且能够自动完成分析全过程的一项技术。微流控芯片相比于一般的检测技术，具有高分析效率、高精确度、集成化、通量灵活化、自动化和节能环保等优势。微流控检测芯片一般具有样品消耗少、检测速度快、操作简便、多功能集成、体小和便于携带等优点，因此在降低整体检测成本上具有较大的潜在能力，非常适合床旁快速检测的发展。特别是离心式微流控芯片借助离心力对流体的控制，可以很好地简化仪器设备，同时在圆盘式芯片内设计各种阀门控制结构。但是精细的微阀结构在批量加工制造容易产生批间差，影响重复性，更为重要的是，离心式微流控芯片中科氏力及欧拉力的存在会使流体引流方向产生偏差而影响最终的检测结构。Microfluidic analysis technology refers to a technology that integrates the basic operation units such as sample preparation, reaction, separation, and detection in the biological, chemical, and medical analysis process into a micron-scale chip, and can automatically complete the entire analysis process. Compared with general detection technologies, microfluidic chips have the advantages of high analysis efficiency, high precision, integration, flexible flux, automation, energy saving and environmental protection. Microfluidic detection chips generally have the advantages of low sample consumption, fast detection speed, simple operation, multi-functional integration, small size and easy to carry. Therefore, they have great potential in reducing the overall detection cost and are very suitable for the development of bedside rapid detection. In particular, centrifugal microfluidic chips can greatly simplify instruments and equipment by controlling fluids with the help of centrifugal force, and various valve control structures can be designed in disc-type chips. However, the fine microvalve structure is prone to batch differences in batch processing and manufacturing, affecting repeatability. More importantly, the presence of Coriolis force and Euler force in centrifugal microfluidic chips will cause deviations in the direction of fluid drainage and affect the final detection structure.
因此，目前大多数微流控芯片应用床旁快速检测都还处于理论研究及专利申请阶段，商业化的微流控检测芯片就更加少了。目前微流控检测芯片的主要挑战有以下几点：Therefore, most microfluidic chips for bedside rapid testing are still in the theoretical research and patent application stage, and there are even fewer commercial microfluidic detection chips. The main challenges of microfluidic detection chips are as follows:
1.芯片内实现试剂的顺序释放；1. Realize the sequential release of reagents within the chip;
2.芯片结构复杂需要进行局部的修饰，增加了芯片加工成本及难度，同时降低了芯片的稳定性；2. The chip structure is complex and requires local modification, which increases the chip processing cost and difficulty, and reduces the chip stability;
3.单个芯片内实现多样本多靶标的同时定量检测，需要更加巧妙的芯片设计，目前还处于单样本多靶标或单靶标的快速检测，同时不能很好地完成样本的分配定量，因此难于实现分析物的定量检测；3. To realize simultaneous quantitative detection of multiple samples and multiple targets in a single chip, a more ingenious chip design is required. Currently, it is still in the stage of rapid detection of multiple targets in a single sample or a single target, and the distribution and quantification of samples cannot be completed well, so it is difficult to realize quantitative detection of analytes;
4.多种试剂的难于同时定量分配及对分配定量好的试剂在微流体通道上难于实现充分的混合；4. It is difficult to quantitatively distribute multiple reagents simultaneously and it is difficult to fully mix the quantitatively distributed reagents in the microfluidic channel;
5.离心芯片引流过程中科氏力及欧拉力对流体方向产生偏差。5. During the centrifugal chip drainage process, the Coriolis force and Euler force cause deviations in the fluid direction.
发明内容Summary of the invention
基于此，本发明公开一种微流控自动化快速检测芯片，其具有加工制造简单，可以实现检测所需试剂的顺序释放，同时在单个芯片内完成多个样本的多靶标定量快速检测，而且检测过程完全自动化完成，不需要专业技术人员进行操作，配套仪器便携简单，非常适合于床旁快速检测。Based on this, the present invention discloses a microfluidic automated rapid detection chip, which is simple to process and manufacture, can realize the sequential release of reagents required for detection, and simultaneously complete multi-target rapid quantitative detection of multiple samples in a single chip. Moreover, the detection process is fully automated and does not require professional technicians to operate. The supporting instruments are portable and simple, and are very suitable for rapid bedside detection.
根据本发明第一方面，提供了一种自动化化学发光免疫分析芯片，包括至少一个检测单元，各个所述检测单元包括样本处理层和试剂处理层；According to a first aspect of the present invention, there is provided an automated chemiluminescent immunoassay chip, comprising at least one detection unit, each of the detection units comprising a sample processing layer and a reagent processing layer;
所述样本处理层包括加样口、样本分离池、第一定量分配单元、定量区、第一微通道引流单元和反应池；所述加样口、样本分离池、第一定量分配单元和定量区依次连接，所述第一微通道引流单元用于将定量区中的液体引流至反应池中；所述第一微通道引流单元上有腔室，所述腔室用于包埋反应所需物质；The sample processing layer comprises a sample addition port, a sample separation pool, a first quantitative distribution unit, a quantitative zone, a first microchannel drainage unit and a reaction pool; the sample addition port, the sample separation pool, the first quantitative distribution unit and the quantitative zone are connected in sequence, the first microchannel drainage unit is used to drain the liquid in the quantitative zone to the reaction pool; the first microchannel drainage unit has a chamber, and the chamber is used to embed substances required for the reaction;
所述试剂处理层包括第一试剂池和第二试剂池，所述第一试剂池和第二试剂池均与反应池连接；所述第一试剂池和第二试剂池用于预封装反应所需试剂。The reagent processing layer comprises a first reagent pool and a second reagent pool, both of which are connected to the reaction pool; the first reagent pool and the second reagent pool are used for pre-packaging reagents required for the reaction.
优选地，所述第一试剂池和第二试剂池分别通过第二定量分配单元与第一定量腔室和第二定量腔室连接，所述第一定量腔室和第二定量腔室分别与第二微通道引流单元和第三微通道引流单元连接，所述第二微通道引流单元通过第二定量腔室与第三微通道引流单元连接，所述第三微通道引流单元与反应池连接；所述第一试剂池和第二试剂池用于预封装反应所需试剂。Preferably, the first reagent pool and the second reagent pool are connected to the first quantitative chamber and the second quantitative chamber respectively through the second quantitative distribution unit, the first quantitative chamber and the second quantitative chamber are connected to the second microchannel drainage unit and the third microchannel drainage unit respectively, the second microchannel drainage unit is connected to the third microchannel drainage unit through the second quantitative chamber, and the third microchannel drainage unit is connected to the reaction pool; the first reagent pool and the second reagent pool are used to pre-package the reagents required for the reaction.
优选地，所述第一试剂池通过第二定量分配单元与第一定量腔室连接，所述第一定量腔室与第二微通道引流单元连接，所述第二微通道引流单元与反应池连接；所述第二试剂池直接与反应池连接；所述第一试剂池和第二试剂池用于预封装反应所需试剂。Preferably, the first reagent pool is connected to the first quantitative chamber through the second quantitative distribution unit, the first quantitative chamber is connected to the second microchannel drainage unit, the second microchannel drainage unit is connected to the reaction pool; the second reagent pool is directly connected to the reaction pool; the first reagent pool and the second reagent pool are used to pre-package the reagents required for the reaction.
优选地，所述检测单元还包括阀门、废液池和气孔；所述阀门分别与反应池和废液池连接；所述废液池与第一定量分配单元和第二定量分配单元连接；所述废液池用于收集反应池、第一定量分配单元和第二定量分配单元中的废液；所述气孔与废液池连接，用于平衡芯片内部气压。Preferably, the detection unit also includes a valve, a waste liquid pool and an air hole; the valve is connected to the reaction pool and the waste liquid pool respectively; the waste liquid pool is connected to the first quantitative distribution unit and the second quantitative distribution unit; the waste liquid pool is used to collect waste liquid in the reaction pool, the first quantitative distribution unit and the second quantitative distribution unit; the air hole is connected to the waste liquid pool for balancing the internal air pressure of the chip.
优选地，所述第一微通道引流单元具有蜿蜒微通道组合，所述蜿蜒微通道组合用于借助芯片离心过程中科氏力及欧拉力的对流体偏转的效果来增加液体的混合效果及控制流体流速。Preferably, the first microchannel drainage unit has a meandering microchannel combination, and the meandering microchannel combination is used to increase the mixing effect of the liquid and control the fluid flow rate by utilizing the effects of Coriolis force and Euler force on fluid deflection during chip centrifugation.
根据本发明另一方面，提供了任一所述的自动化化学发光免疫分析芯片的检测方法，包括以下步骤：According to another aspect of the present invention, a detection method of any of the above-mentioned automated chemiluminescent immunoassay chips is provided, comprising the following steps:
S1：将信号分子标记的靶标抗体预埋在第一微通道引流单元上的腔室中，将磁微粒标记的靶标抗体预埋在反应池中；S1: pre-embedding the target antibody labeled with the signal molecule in the chamber on the first microchannel drainage unit, and pre-embedding the target antibody labeled with the magnetic particle in the reaction pool;
S2：在加样口处加入待检测样本，在离心力作用下，待检测样本在样本分离池中沉淀杂质；随后，样本溶液进入第一定量分配单元进行分配，通过定量区进入第一微通道引流单元中，并复溶信号分子标记的靶标抗体，样本溶液中的靶标蛋白与信号分子标记的靶标抗体形成第一复合物，该第一复合物进入反应池中，并与磁微粒标记的靶标抗体反应形成第二复合物；S2: adding the sample to be tested to the sample injection port, and under the action of centrifugal force, the sample to be tested precipitates impurities in the sample separation pool; then, the sample solution enters the first quantitative distribution unit for distribution, enters the first microchannel drainage unit through the quantitative area, and redissolves the target antibody labeled with the signal molecule, the target protein in the sample solution and the target antibody labeled with the signal molecule form a first complex, the first complex enters the reaction pool, and reacts with the target antibody labeled with the magnetic microparticle to form a second complex;
S3：启动磁铁吸附，使第二复合物固定在反应池中，随后开放阀门，启动离心，将未形成第二复合物的的信号分子标记的靶标抗体排入废液池中，随后关闭阀门；S3: Start the magnet adsorption to fix the second complex in the reaction pool, then open the valve, start centrifugation, discharge the target antibody labeled with the signal molecule that has not formed the second complex into the waste liquid pool, and then close the valve;
S4：去除磁铁吸附，释放第一试剂池和第二试剂池中的试剂进入反应池中，使所述第二复合物中捕获的发光分子反应发光，对光信号进行检测，并计算得到待检测样本中靶标蛋白的含量。S4: removing the magnet adsorption, releasing the reagents in the first reagent pool and the second reagent pool into the reaction pool, causing the luminescent molecules captured in the second complex to react and emit light, detecting the light signal, and calculating the content of the target protein in the sample to be detected.
优选地，所述信号分子为吖啶酯、吖啶酮或辣根过氧化物酶。Preferably, the signal molecule is acridinium ester, acridone or horseradish peroxidase.
优选地，所述靶标蛋白为急性时效反应蛋白、降钙素原或白介素-6；所述靶标抗体为急性时效反应蛋白抗体、降钙素原抗体或白介素-6抗体。Preferably, the target protein is acute-effect protein, procalcitonin or interleukin-6; the target antibody is acute-effect protein antibody, procalcitonin antibody or interleukin-6 antibody.
优选地，所述第一试剂池和第二试剂池中的试剂分别为预激发液和激发液。Preferably, the reagents in the first reagent pool and the second reagent pool are pre-stimulation solution and stimulation solution, respectively.
总体而言，通过本发明所构思的以上技术方案与现有技术相比，主要具备以下的技术优点：In general, the above technical solution conceived by the present invention has the following technical advantages compared with the prior art:
(1)本发明的自动化快速检测芯片可以和生物化学，免疫，化学发光等检测技术的结合，应用于生物样本中分析物的高灵敏度定量快速检测分析，在生物医学、生物化学、床旁快速检测等领域都具有巨大的潜在应用价值。(1) The automated rapid detection chip of the present invention can be combined with biochemical, immunological, chemiluminescent and other detection technologies to be applied to high-sensitivity quantitative rapid detection and analysis of analytes in biological samples, and has great potential application value in the fields of biomedicine, biochemistry, bedside rapid detection, etc.
(2)本发明的自动化快速检测芯片可以完成多种试剂的高通量分配定量，并实现多种试剂的分步充分混合反应，提高检测灵敏度及精确度。(2) The automated rapid detection chip of the present invention can complete high-throughput distribution and quantification of multiple reagents, and realize step-by-step and sufficient mixing reactions of multiple reagents, thereby improving detection sensitivity and accuracy.
(3)本发明的自动化快速检测芯片可以实现化学发光检测过程中激发液与预激发液的顺序混合，同时可以控制激发液进入反应腔室的顺序，便携信号的精准采集及分析。(3) The automated rapid detection chip of the present invention can realize the sequential mixing of the excitation liquid and the pre-excitation liquid during the chemiluminescence detection process, and can also control the order in which the excitation liquid enters the reaction chamber, thereby facilitating the accurate collection and analysis of portable signals.
(4)本发明的自动化快速检测芯片通过引流通道可以克服科氏力及欧拉力的对流体偏转的影响，实现多种试剂的定量分配及顺序混合反应；同时通过垂直或平行于圆心延伸线的蜿蜒微通道组合，可以借助科氏力及欧拉力对流体偏转的控制，提高液体混合程度。(4) The automated rapid detection chip of the present invention can overcome the influence of Coriolis force and Euler force on fluid deflection through the drainage channel, thereby realizing the quantitative distribution and sequential mixing reaction of multiple reagents; at the same time, through the combination of winding microchannels perpendicular or parallel to the center extension line, the Coriolis force and Euler force can be used to control the fluid deflection and improve the degree of liquid mixing.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
图1为本发明自动化快速检测芯片的整体结构图；FIG1 is a diagram showing the overall structure of the automated rapid detection chip of the present invention;
图2为本发明自动化快速检测芯片的分层结构示意图；FIG2 is a schematic diagram of the hierarchical structure of the automated rapid detection chip of the present invention;
图3为本发明自动化快速检测芯片的一个检测单元示意图；FIG3 is a schematic diagram of a detection unit of the automated rapid detection chip of the present invention;
图4为本发明自动化快速检测芯片一个检测单元的样本处理层示意图；FIG4 is a schematic diagram of a sample processing layer of a detection unit of the automated rapid detection chip of the present invention;
图5为本发明自动化快速检测芯片一个检测单元的试剂处理层示意图；FIG5 is a schematic diagram of a reagent processing layer of a detection unit of the automated rapid detection chip of the present invention;
图6为本发明自动化快速检测芯片一个检测单元的试剂处理层一种实施例示意图；FIG6 is a schematic diagram of an embodiment of a reagent processing layer of a detection unit of an automated rapid detection chip of the present invention;
图7为本发明自动化快速检测芯片的部分结构示意图；FIG7 is a schematic diagram of a partial structure of the automated rapid detection chip of the present invention;
图8为本发明微流通道引流单元在离心引流过程中克服科氏力及欧拉力的影响的效果；FIG8 is a diagram showing the effect of the microchannel drainage unit of the present invention in overcoming the influence of the Coriolis force and the Euler force during the centrifugal drainage process;
图9为本发明自动化快速检测芯片的整体结构图的另一种形式；FIG9 is another form of the overall structure diagram of the automated rapid detection chip of the present invention;
图10为本发明自动化快速检测芯片的整体结构图的另一种形式的分层结构示意图；FIG10 is a schematic diagram of another form of hierarchical structure of the overall structure of the automated rapid detection chip of the present invention;
图11为本发明自动化快速检测芯片的一个检测单元另一种形式的示意图；FIG11 is a schematic diagram of another form of a detection unit of the automated rapid detection chip of the present invention;
图12为为本发明自动化快速检测芯片的一个检测单元另一种形式的部分结构图；FIG12 is a partial structural diagram of another form of a detection unit of the automated rapid detection chip of the present invention;
图13为在离心引流过程中的力学分析图。FIG. 13 is a diagram showing the mechanical analysis during centrifugal drainage.
在所有附图中，相同的附图标记用来表示相同的元件或结构，其中：200-检测单元、51-样本处理层、52-试剂处理层、11-加样口、12-样本分离池、13-第一定量分配单元、14-定量区、15-第一微通道引流单元、16-反应池、17-腔室、21-第一试剂池、22-第二试剂池、23-第二定量分配单元、24-第一定量腔室、25-第二定量腔室、26-第二微通道引流单元、27-第三微通道引流单元、31-阀门、32-废液池、53-中间层、54-上盖、55-下盖。In all the drawings, the same figure marks are used to represent the same elements or structures, wherein: 200-detection unit, 51-sample processing layer, 52-reagent processing layer, 11-sample loading port, 12-sample separation tank, 13-first quantitative distribution unit, 14-quantification area, 15-first microchannel drainage unit, 16-reaction tank, 17-chamber, 21-first reagent tank, 22-second reagent tank, 23-second quantitative distribution unit, 24-first quantitative chamber, 25-second quantitative chamber, 26-second microchannel drainage unit, 27-third microchannel drainage unit, 31-valve, 32-waste liquid tank, 53-middle layer, 54-upper cover, 55-lower cover.
具体实施方式Detailed ways
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合附图及实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。此外，下面所描述的本发明各个实施方式中所涉及到的技术特征只要彼此之间未构成冲突就可以相互组合。In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.
本发明一种自动化快速检测芯片400，包括至少一个检测单元200，各个所述检测单元包括样本处理层51和试剂处理层52；The present invention provides an automated rapid detection chip 400, comprising at least one detection unit 200, each of which comprises a sample processing layer 51 and a reagent processing layer 52;
图4本发明自动化快速检测芯片的样本处理层的示意图。本发明样本处理层51包括加样口11、样本分离池12、第一定量分配单元13、定量区14、第一微通道引流单元15和反应池16；所述加样口11、样本分离池12、第一定量分配单元13和定量区14依次连接，所述第一微通道引流单元15用于将定量区14中的液体引流至反应池16中；所述第一微通道引流单元15上有腔室17，所述腔室17用于包埋反应所需物质；Fig. 4 is a schematic diagram of the sample processing layer of the automated rapid detection chip of the present invention. The sample processing layer 51 of the present invention comprises a sample addition port 11, a sample separation pool 12, a first quantitative allocation unit 13, a quantitative zone 14, a first microchannel drainage unit 15 and a reaction pool 16; the sample addition port 11, the sample separation pool 12, the first quantitative allocation unit 13 and the quantitative zone 14 are connected in sequence, the first microchannel drainage unit 15 is used to drain the liquid in the quantitative zone 14 to the reaction pool 16; the first microchannel drainage unit 15 has a chamber 17, and the chamber 17 is used to embed the substances required for the reaction;
所述试剂处理层52包括第一试剂池21和第二试剂池22，所述第一试剂池21和第二试剂池22分别通过第二定量分配单元23与第一定量腔室24和第二定量腔室25连接，所述第一定量腔室24和第二定量腔室25分别与第二微通道引流单元26和第三微通道引流单元27连接，所述第二微通道引流单元26通过第二定量腔室25与第三微通道引流单元27连接，所述第三微通道引流单元27与反应池16连接；所述第一试剂池21和第二试剂池22用于预封装反应所需试剂。The reagent processing layer 52 includes a first reagent pool 21 and a second reagent pool 22, the first reagent pool 21 and the second reagent pool 22 are respectively connected to the first quantitative chamber 24 and the second quantitative chamber 25 through the second quantitative distribution unit 23, the first quantitative chamber 24 and the second quantitative chamber 25 are respectively connected to the second microchannel drainage unit 26 and the third microchannel drainage unit 27, the second microchannel drainage unit 26 is connected to the third microchannel drainage unit 27 through the second quantitative chamber 25, and the third microchannel drainage unit 27 is connected to the reaction pool 16; the first reagent pool 21 and the second reagent pool 22 are used for pre-packaging reagents required for the reaction.
所述检测单元200还包括阀门31和废液池32；所述阀门31分别与反应池16和废液池32连接；所述废液池32与第一定量分配单元13和第二定量分配单元23连接；所述废液池32用于收集反应池16、第一定量分配单元13和第二定量分配单元23中的废液。图3为本发明自动化快速检测芯片的一个检测单元示意图。The detection unit 200 further includes a valve 31 and a waste liquid pool 32; the valve 31 is connected to the reaction pool 16 and the waste liquid pool 32 respectively; the waste liquid pool 32 is connected to the first quantitative distribution unit 13 and the second quantitative distribution unit 23; the waste liquid pool 32 is used to collect waste liquid in the reaction pool 16, the first quantitative distribution unit 13 and the second quantitative distribution unit 23. Fig. 3 is a schematic diagram of a detection unit of the automated rapid detection chip of the present invention.
所述第一微通道引流单元15、第二微通道引流单元26和第三微通道引流单元27具有混合和微阀门的作用，所述自动化快速检测芯片具有旋转中心。The first microchannel drainage unit 15, the second microchannel drainage unit 26 and the third microchannel drainage unit 27 have the functions of mixing and micro-valve, and the automated rapid detection chip has a rotation center.
具体地，本发明芯片内各个检测单元200是互相独立的，芯片的结构可以一致可以不一致，可以按检测需求进行调整；Specifically, each detection unit 200 in the chip of the present invention is independent of each other, and the structure of the chip can be consistent or inconsistent, and can be adjusted according to the detection requirements;
具体地，在其中一个实施例中芯片为5层结构，包括上盖54、下盖55、样本处理层51，试剂处理层52及中间层53，所述样本处理层51与试剂处理层52为不在同一平面，同时只通过反应池16相互连通；Specifically, in one embodiment, the chip has a five-layer structure, including an upper cover 54, a lower cover 55, a sample processing layer 51, a reagent processing layer 52 and an intermediate layer 53, wherein the sample processing layer 51 and the reagent processing layer 52 are not in the same plane and are only connected to each other through the reaction pool 16;
所述检测芯片在另一种形式中(图8)，样本处理层与试剂处理层位于同一层(图9)，在其中一个实施例中芯片为3层结构，包括上盖54、下盖55及中间层53(图10)。图11为本发明自动化快速检测芯片的一个检测单元另一种形式的示意图；图12为为本发明自动化快速检测芯片的一个检测单元另一种形式的部分结构图。本发明另一种形式中，第一试剂池21通过第二定量分配单元23与第一定量腔室24连接，第一定量腔室24与第二微通道引流单元26连接，第二微通道引流单元26与反应池16连接；第二试剂池22直接与反应池16连接；第一试剂池21和第二试剂池22用于预封装反应所需试剂。In another form of the detection chip (Figure 8), the sample processing layer and the reagent processing layer are located in the same layer (Figure 9). In one embodiment, the chip has a three-layer structure, including an upper cover 54, a lower cover 55 and an intermediate layer 53 (Figure 10). Figure 11 is a schematic diagram of another form of a detection unit of the automated rapid detection chip of the present invention; Figure 12 is a partial structural diagram of another form of a detection unit of the automated rapid detection chip of the present invention. In another form of the present invention, the first reagent pool 21 is connected to the first quantitative chamber 24 through the second quantitative distribution unit 23, the first quantitative chamber 24 is connected to the second microchannel drainage unit 26, and the second microchannel drainage unit 26 is connected to the reaction pool 16; the second reagent pool 22 is directly connected to the reaction pool 16; the first reagent pool 21 and the second reagent pool 22 are used to pre-package the reagents required for the reaction.
具体地，所述加样口11与样本分离池12连通，将样本从加样口11加入，样本可为全血、血浆、血清、尿液等各种样本；Specifically, the sample adding port 11 is connected to the sample separation pool 12, and the sample is added from the sample adding port 11. The sample can be various samples such as whole blood, plasma, serum, urine, etc.
具体地，所述样本分离池12与第一定量分配单元13连通，连接口设计为样本分离池12中部，芯片离心时，样本中的固相杂质会由于离心力及离心速度较大而优先沉降在样本分离池12底部，上清会随着持续的离心力而进入第一定量分配单元13，连接口可以进行多种设计防止杂质进入第一定量分配单元13，例如微柱，生物膜，微阀门等；Specifically, the sample separation pool 12 is connected to the first quantitative distribution unit 13, and the connection port is designed to be in the middle of the sample separation pool 12. When the chip is centrifuged, the solid phase impurities in the sample will be preferentially settled at the bottom of the sample separation pool 12 due to the large centrifugal force and centrifugal speed, and the supernatant will enter the first quantitative distribution unit 13 with the continuous centrifugal force. The connection port can be designed in a variety of ways to prevent impurities from entering the first quantitative distribution unit 13, such as microcolumns, biofilms, microvalves, etc.
具体地，所述第一定量分配单元13与第一微通道引流单元15连通，所述第一定量分配单元13包含多个定量区14，当芯片低速离心时，样本溶液会进入定量区14，并随着离心力的持续，多余的样本溶液会被排入废液池32，因此可以完成样本溶液的分配定量，为后续的多靶标定量检测提供一定体积的样本溶液；Specifically, the first quantitative distribution unit 13 is connected to the first microchannel drainage unit 15, and the first quantitative distribution unit 13 includes a plurality of quantitative areas 14. When the chip is centrifuged at a low speed, the sample solution will enter the quantitative area 14, and as the centrifugal force continues, the excess sample solution will be discharged into the waste liquid pool 32, so that the distribution and quantification of the sample solution can be completed, providing a certain volume of sample solution for subsequent multi-target quantitative detection;
具体地，所述第一微通道引流单元15的微通道尺寸为宽10～100μm，高10～100μm，但不限于此；Specifically, the microchannel size of the first microchannel drainage unit 15 is 10-100 μm in width and 10-100 μm in height, but is not limited thereto;
具体地，所述第一微通道引流单元15在其中一个实施例中包含三处蜿蜒通道，在靠近定量区14的微通道为疏水区，其余通道可以进行任何修饰或不修饰改性，蜿蜒通道可以对流体的流速进行控制；Specifically, the first microchannel drainage unit 15 comprises three serpentine channels in one embodiment, the microchannel near the quantitative zone 14 is a hydrophobic zone, and the remaining channels can be modified or not modified, and the serpentine channels can control the flow rate of the fluid;
具体地，芯片高速离心时，定量区14的样本溶液会突破微通道的疏水阻力，进入第一微通道单元15的第一个蜿蜒通道，蜿蜒通道可以降低样本溶液的流速使其与第一微通道单元15中预埋的试剂进行充分的接触，随后进入第二蜿蜒通道及第三个蜿蜒通道进行充分的混合反应，同时，这种蜿蜒微通道设计为与圆心向外延申线相垂直或平行的微通道，在离心引流过程中，可以借助科氏力及欧拉力对流体的产生偏转的影响而提高混合效果，在不考虑流体挤压力的情况下，其力学分析如图13所示；Specifically, when the chip is centrifuged at high speed, the sample solution in the quantitative area 14 will break through the hydrophobic resistance of the microchannel and enter the first serpentine channel of the first microchannel unit 15. The serpentine channel can reduce the flow rate of the sample solution so that it can fully contact with the reagent pre-embedded in the first microchannel unit 15, and then enter the second serpentine channel and the third serpentine channel for full mixing reaction. At the same time, this serpentine microchannel is designed to be a microchannel perpendicular or parallel to the outward extension line of the center of the circle. In the centrifugal drainage process, the mixing effect can be improved by means of the deflection of the fluid by the Coriolis force and the Euler force. Without considering the fluid extrusion force, its mechanical analysis is shown in Figure 13;
具体地，所述的第一蜿蜒通道较第二蜿蜒通道更靠近圆心，第二蜿蜒通道较第三蜿蜒通道更靠近圆心，蜿蜒通道之前的距离可以根据检测需求而进行调整，结构不限于此；Specifically, the first winding channel is closer to the center of the circle than the second winding channel, and the second winding channel is closer to the center of the circle than the third winding channel. The distance between the winding channels can be adjusted according to the detection requirements, and the structure is not limited thereto;
具体地，芯片高速离心之后，样本与试剂的混合液会进入反应池16，所述反应池16中可预先包埋试剂，或进行表面改性修饰，对样本中的检测靶标进行捕获；Specifically, after the chip is centrifuged at high speed, the mixture of the sample and the reagent will enter the reaction pool 16, and the reaction pool 16 can be pre-embedded with reagents or surface modified to capture the detection target in the sample;
具体地，所述阀门31与反应池16及废液池32连通，开放阀门31，启动高速离心，可以将反应池16中多余的废液完全排入废液池32，使反应池16中只存留检测所需的分析物，随后关闭阀门31；Specifically, the valve 31 is connected to the reaction pool 16 and the waste liquid pool 32. The valve 31 is opened and high-speed centrifugation is started to completely discharge the excess waste liquid in the reaction pool 16 into the waste liquid pool 32, so that only the analyte required for detection remains in the reaction pool 16, and then the valve 31 is closed;
具体地，所述第一试剂池21和第二试剂池22用于溶液试剂存储，第一试剂池21和第二试剂池22分别连通一个第二定量分配单元23，用于试剂的分配定量，定量过程与以上所述的样本定量过程一样；Specifically, the first reagent pool 21 and the second reagent pool 22 are used for storing solution reagents. The first reagent pool 21 and the second reagent pool 22 are respectively connected to a second quantitative distribution unit 23 for distributing and quantifying reagents. The quantitative process is the same as the sample quantitative process described above.
具体地，所述第一试剂池21与第二微通道引流单元26连通，第二微通道引流单元26与第一定量腔室24连通，所述第三微通道引流单元27较第二微通道引流单元26更远离圆心，同时，第二微通道引流单元26设有蜿蜒通道，可以通过控制微通道的阻力大小及长度来控制试剂进入反应池16的时间，最终使第二试剂池22中的试剂先进入反应池16之后，第一试剂池21处的试剂才有序地快速进入反应池16中进行反应，最终获得每个反应池16中检测信号，同时在另一种形式中，可以通过将最终反应试剂预存在第二试剂池22中(图9)，通过按压或注射的方式将溶液注入反应池中，按需完成信号采集，检测信号的强弱与一定溶液体积内的分析物含量的高低相关，因此可以对样本的分析物进行定量检测。Specifically, the first reagent pool 21 is connected to the second microchannel drainage unit 26, the second microchannel drainage unit 26 is connected to the first quantitative chamber 24, and the third microchannel drainage unit 27 is farther away from the center of the circle than the second microchannel drainage unit 26. At the same time, the second microchannel drainage unit 26 is provided with a serpentine channel, and the time for the reagent to enter the reaction pool 16 can be controlled by controlling the resistance and length of the microchannel, so that the reagent in the second reagent pool 22 enters the reaction pool 16 first, and then the reagent in the first reagent pool 21 enters the reaction pool 16 in an orderly and rapid manner for reaction, and finally the detection signal in each reaction pool 16 is obtained. At the same time, in another form, the final reaction reagent can be pre-stored in the second reagent pool 22 (Figure 9), and the solution can be injected into the reaction pool by pressing or injection, and the signal collection can be completed as needed. The strength of the detection signal is related to the level of the analyte content in a certain volume of solution, so the analyte of the sample can be quantitatively detected.
本发明中，微通道引流单元具有混合及引流的效果。In the present invention, the microchannel drainage unit has the effects of mixing and drainage.
本发明中，微通道引流单元可以通过微通道长短及微通道阻力的大小来控制液体进入反应池的顺序及时间长短。In the present invention, the microchannel drainage unit can control the order and duration of liquid entering the reaction pool by the length of the microchannel and the size of the microchannel resistance.
本发明中，定量分配单元与微通道引流单元为相互连通但不在同一平面上，微通道引流单元的微通道尺寸可为宽10～100μm，高10～300μm，但不限于此范围，用于在芯片低速离心时，限制液体进入微通道，在芯片高速离心时，使液体流入微通道进行引流及混合；定量分配单元在芯片低速离心时液体可以自由流动，并对液体进行分配定量，多余的液体将进入与其连通的废液池，在芯片高速离心时，将定量的液体通过微通道引流单元导入相应的反应池。In the present invention, the quantitative distribution unit and the microchannel drainage unit are interconnected but not on the same plane. The microchannel size of the microchannel drainage unit can be 10 to 100 μm in width and 10 to 300 μm in height, but is not limited to this range. It is used to restrict the liquid from entering the microchannel when the chip is centrifuged at a low speed, and to allow the liquid to flow into the microchannel for drainage and mixing when the chip is centrifuged at a high speed. When the chip is centrifuged at a low speed, the quantitative distribution unit allows the liquid to flow freely and distribute the liquid quantitatively. The excess liquid will enter the waste liquid pool connected to it. When the chip is centrifuged at a high speed, the quantitative liquid will be introduced into the corresponding reaction pool through the microchannel drainage unit.
本发明中，所述反应池与微阀连通，在需要存储液体时，阀门关闭，在需要排除废液时关闭。In the present invention, the reaction pool is connected to a microvalve, and the valve is closed when liquid needs to be stored, and is closed when waste liquid needs to be discharged.
本发明中，所述定量分配单元可以在同一水平面上实现多种液体的单独分配定量，用于多样本多靶标的检测。In the present invention, the quantitative dispensing unit can achieve separate quantitative dispensing of multiple liquids on the same horizontal plane, which is used for the detection of multiple samples and multiple targets.
本发明中的芯片可用于各种生化检测以及免疫分析检测等，但不限于此。The chip of the present invention can be used for various biochemical tests and immunoassay tests, but is not limited thereto.
本发明中的检测方法包括以下步骤：The detection method of the present invention comprises the following steps:
将样本液从所述加样孔加至所述样本分离池，将芯片放入配套的离心检测仪器中；Add the sample liquid from the sample adding hole to the sample separation pool, and place the chip into a matching centrifugal detection instrument;
低速离心芯片，样本中的杂质会由于离心力较大而快速沉降在样本分离池的底部，上清会由于持续的离心力进入定量分配单元，对样本液体进行分配定量，为后面的定量分析检测提供基础，多余的液体将排入废液池；Low-speed centrifugal chip, impurities in the sample will quickly settle to the bottom of the sample separation pool due to the large centrifugal force, and the supernatant will enter the quantitative distribution unit due to the continuous centrifugal force to distribute the sample liquid quantitatively, providing a basis for subsequent quantitative analysis and detection, and the excess liquid will be discharged into the waste liquid pool;
高速离心芯片将定量的样本溶液注入微通道引流单元，通过控制离心速度及微通道来控制液体流速，复溶通道内预埋的试剂，通过微通道引流单元使样本与试剂进行充分的混合及反应，最后进入反应池中；The high-speed centrifugal chip injects a quantitative sample solution into the microchannel drainage unit, controls the liquid flow rate by controlling the centrifugal speed and microchannel, re-dissolves the reagents embedded in the channel, and fully mixes and reacts the sample and reagents through the microchannel drainage unit, and finally enters the reaction pool;
反应池中预先包埋的试剂会与进入的样本混合液进行充分的混合反应，反应后形成的目标复合物，随后开放阀门，高速离心芯片，使多余的废液排入废液池中，而所需要的目标复合物仍然存留在反应池中；The reagents pre-embedded in the reaction pool will fully mix and react with the sample mixture entering, and the target complex will be formed after the reaction. Then the valve will be opened and the chip will be centrifuged at high speed to discharge the excess waste liquid into the waste liquid pool, while the required target complex will still remain in the reaction pool;
释放所述所有试剂池中的试剂，低速离心芯片至所述定量分配单元进行定量分配，多余的试剂排入所述废液池中，随后高速离心将定量分配的试剂通过微通道引流单元导入反应池中，由于微通道的长短及阻力的大小不同，试剂会先后进入反应池中进行反应，最后依次获取每个反应池中的检测信号。The reagents in all the reagent pools are released, and the chip is centrifuged at low speed to the quantitative distribution unit for quantitative distribution. The excess reagents are discharged into the waste liquid pool. Subsequently, the quantitatively distributed reagents are introduced into the reaction pool through the microchannel drainage unit by high-speed centrifugation. Due to the different lengths and resistances of the microchannels, the reagents will enter the reaction pools one after another for reaction, and finally the detection signals in each reaction pool are obtained in turn.
本发明自动化化学发光免疫分析芯片的检测方法，包括以下步骤：The detection method of the automated chemiluminescent immunoassay chip of the present invention comprises the following steps:
S1：将信号分子标记的靶标抗体(一抗)预埋在第一微通道引流单元15上的腔室17中，将磁微粒标记的靶标抗体(二抗)预埋在反应池16中；S1: pre-embed the target antibody (primary antibody) labeled with the signal molecule in the chamber 17 on the first microchannel drainage unit 15, and pre-embed the target antibody (secondary antibody) labeled with the magnetic particle in the reaction pool 16;
S2：在加样口11处加入待检测样本，在离心力作用下，待检测样本在样本分离池12中沉淀杂质；随后，样本溶液进入第一定量分配单元13进行分配，通过定量区14进入第一微通道引流单元15中，并复溶信号分子标记的靶标抗体(一抗)，样本溶液中的靶标蛋白与信号分子标记的靶标抗体形成第一复合物，该第一复合物进入反应池16中，并与磁微粒标记的靶标抗体(二抗)反应形成第二复合物；S2: The sample to be tested is added to the sample adding port 11. Under the action of centrifugal force, the sample to be tested precipitates impurities in the sample separation pool 12. Subsequently, the sample solution enters the first quantitative distribution unit 13 for distribution, enters the first microchannel drainage unit 15 through the quantitative area 14, and redissolves the target antibody (primary antibody) labeled with the signal molecule. The target protein in the sample solution forms a first complex with the target antibody labeled with the signal molecule. The first complex enters the reaction pool 16 and reacts with the target antibody (secondary antibody) labeled with the magnetic microparticle to form a second complex.
S3：启动磁铁吸附，使第二复合物固定在反应池16中，随后开放阀门31，启动离心，将未形成第二复合物的的信号分子标记的靶标抗体(一抗)排入废液池中，随后关闭阀门31。S3: Start the magnet adsorption to fix the second complex in the reaction pool 16, then open the valve 31, start the centrifugation, discharge the target antibody (primary antibody) labeled with the signal molecule that has not formed the second complex into the waste liquid pool, and then close the valve 31.
S4：去除磁铁吸附，释放第一试剂池21和第二试剂池22中的试剂进入反应池16中，使所述第二复合物中捕获的发光分子反应发光，对光信号进行检测，并计算得到待检测样本中靶标蛋白的含量。S4: removing the magnet adsorption, releasing the reagents in the first reagent pool 21 and the second reagent pool 22 into the reaction pool 16, causing the luminescent molecules captured in the second complex to react and emit light, detecting the light signal, and calculating the content of the target protein in the sample to be detected.
实施例1Example 1
本发明提供的实施例为将芯片应用于吖啶酯化学发光免疫分析，免疫分析原理为免疫夹心原理用于全血中C-反应蛋白的检测，具体技术方案如下：The embodiment provided by the present invention is to apply the chip to acridinium ester chemiluminescence immunoassay, and the immunoassay principle is the immune sandwich principle for the detection of C-reactive protein in whole blood. The specific technical scheme is as follows:
1.抗体修饰1. Antibody modification
将吖啶酯通过EDC和NHS体系与抗CRP抗体结合，制备信号标记抗体复合物，同样地，将磁微粒通过EDC和NHS系统与抗CRP单抗结合，制备捕获抗体复合物；The acridinium ester is combined with the anti-CRP antibody through the EDC and NHS system to prepare a signal-labeled antibody complex. Similarly, the magnetic particles are combined with the anti-CRP monoclonal antibody through the EDC and NHS system to prepare a capture antibody complex.
2.芯片加工2. Chip processing
芯片加工方式采用CNC数控加工，对芯片的每一层进行单独加工，样本处理层及试剂处理层进行双面加工，形成微通道引流单元在下表面，定量分配单元在上表面，相互连接处雕刻连通，芯片加工材料为PMMA材料；The chip is processed by CNC machining. Each layer of the chip is processed separately. The sample processing layer and the reagent processing layer are processed on both sides. The microchannel drainage unit is formed on the lower surface, and the quantitative distribution unit is formed on the upper surface. The interconnected parts are engraved and connected. The chip processing material is PMMA material.
可以理解地，芯片的加工工艺可以为激光雕刻技术、CNC数控技术、软光刻技术等芯片加工工艺，但不限于此；It can be understood that the chip processing technology can be laser engraving technology, CNC numerical control technology, soft lithography technology and other chip processing technology, but is not limited thereto;
可以理解地，芯片的加工材料可以为PMMA、PC、PDMS等有机玻璃或聚合物，但不限于此。It is understandable that the processing material of the chip can be organic glass or polymer such as PMMA, PC, PDMS, etc., but is not limited thereto.
3.芯片组装3. Chip assembly
如图2所示，芯片总共有五层，包括上盖54、下盖55、样本处理层51，试剂处理层52及中间层53，芯片组装采用双面胶黏贴的方式进行密封组合，在组合之前首先将吖啶酯标记CRP抗体预包埋在第一微通道引流单元15的腔室17中(如图7)，将磁微粒标记CRP单抗预包埋在反应池16中；在进行上盖54密封相片之前在第一试剂池21中预封装激发液(NaOH)试剂包，在第一试剂池22中预封装预激发液(H2O2)，随后进行芯片密封，形成完整的检测芯片。As shown in FIG2 , the chip has five layers in total, including an upper cover 54, a lower cover 55, a sample processing layer 51, a reagent processing layer 52 and an intermediate layer 53. The chip is assembled by sealing and combining by double-sided adhesive. Before the assembly, the acridinium ester-labeled CRP antibody is firstly pre-embedded in the chamber 17 of the first microchannel drainage unit 15 (as shown in FIG7 ), and the magnetic particle-labeled CRP monoclonal antibody is pre-embedded in the reaction pool 16; before the upper cover 54 is sealed, the excitation solution (NaOH) reagent package is pre-packaged in the first reagent pool 21, and the pre-excitation solution (H2 O2 ) is pre-packaged in the first reagent pool 22, and then the chip is sealed to form a complete detection chip.
4.样本检测4. Sample testing
具体地，在加样口11处加入全血样本100～200μl，密封将芯片放入配套检测仪器中；Specifically, add 100-200 μl of whole blood sample to the sample adding port 11, seal the chip and place it in the matching detection instrument;
具体地，芯片在仪器中，首先进行低速离心，将全血样本中的细胞及杂质沉淀在样本分离池12底部，完全全血中血浆及血细胞及杂质的分离，提高离心速度，全血上清会由于持续的离心力作用进入第一定量分配单元13内进行分配定，样本最终体积通过定量区14的大小进行精确定量，多余的废液会进入废液池32中，随后离心结束；Specifically, the chip is first centrifuged at a low speed in the instrument to precipitate cells and impurities in the whole blood sample at the bottom of the sample separation pool 12, and to separate the plasma, blood cells and impurities in the whole blood. The centrifugal speed is increased, and the whole blood supernatant will enter the first quantitative distribution unit 13 for distribution due to the continuous centrifugal force. The final volume of the sample is accurately quantified by the size of the quantitative area 14, and the excess waste liquid will enter the waste liquid pool 32, and then the centrifugation ends;
具体地，启动芯片高速离心，将定量的样本溶液注入第一微通道引流单元13中，样本溶液会复溶吖啶酯标记CRP抗体于样本中的CRP抗原进行反应形成吖啶酯标记CRP抗体-CRP复合物，通过蜿蜒的微通道可以实现对流体流速的控制，同时使反应更加充分，最后在持续高速离心的作用下，垂直或平行于圆心延伸线的蜿蜒微通组合，可以借助科氏力及欧拉力对流体的产生偏转的影响而提高混合效果，最终所有液体将被排入反应池16中，此时阀门31为关闭状态；Specifically, the chip is started to centrifuge at high speed, and a certain amount of sample solution is injected into the first microchannel drainage unit 13. The sample solution will re-dissolve the acridinium ester-labeled CRP antibody and react with the CRP antigen in the sample to form an acridinium ester-labeled CRP antibody-CRP complex. The winding microchannel can control the flow rate of the fluid and make the reaction more complete. Finally, under the action of continuous high-speed centrifugation, the winding microchannel combination perpendicular or parallel to the extension line of the center of the circle can improve the mixing effect by means of the deflection of the fluid by the Coriolis force and the Euler force. Finally, all the liquid will be discharged into the reaction pool 16, and the valve 31 is in a closed state at this time.
具体地，高速离心停止过程会使反应池16中的溶液产生震荡，并完成磁微粒标记CRP单抗与吖啶酯标记CRP抗体-CRP复合物的反应，使反应更加快速，缩短反应时间，反应形成吖啶酯标记CRP抗体-CRP-磁微粒标记CRP单抗复合物，此时启动磁铁吸附，使磁微粒固定在反应池16中，开放阀门31，启动芯片离心，将所有液体排入废液池32中，停止离心；随后关闭阀门31，撤去磁铁对磁微粒的吸附，同时释放第一试剂池21及第二试剂池22中的预激发液及激发液，启动低速离心对试剂进行精确定量，随后高速离心，预激发液优先快速进入反应池16中重悬磁微粒，随后激发液通过蜿蜒的第三微通道引流单元27，如图5和图6所示，通过控制微通道的长度及阻力的大小，使激发液同时或依次快速进入反应池16中，使捕获的吖啶酯完成快速反应发光，仪器对光信号进行检测，完成样本中CRP含量的精确定量检测。Specifically, the high-speed centrifugation stop process will cause the solution in the reaction pool 16 to vibrate, and complete the reaction between the magnetic particle labeled CRP monoclonal antibody and the acridinium ester labeled CRP antibody-CRP complex, making the reaction faster and shortening the reaction time. The reaction forms an acridinium ester labeled CRP antibody-CRP-magnetic particle labeled CRP monoclonal antibody complex. At this time, the magnet adsorption is started to fix the magnetic particles in the reaction pool 16, the valve 31 is opened, the chip centrifugation is started, all the liquid is discharged into the waste liquid pool 32, and the centrifugation is stopped; then the valve 31 is closed, and the magnet adsorption of the magnetic particles is removed. At the same time, the pre-excitation liquid and the excitation liquid in the first reagent pool 21 and the second reagent pool 22 are released, and low-speed centrifugation is started to accurately quantify the reagents. Subsequently, high-speed centrifugation is performed, and the pre-excitation liquid preferentially and quickly enters the reaction pool 16 to resuspend the magnetic particles. Subsequently, the excitation liquid passes through the winding third microchannel drainage unit 27, as shown in Figures 5 and 6. By controlling the length of the microchannel and the size of the resistance, the excitation liquid is allowed to quickly enter the reaction pool 16 simultaneously or sequentially, so that the captured acridinium ester completes a rapid reaction and emits light. The instrument detects the light signal to complete the accurate quantitative detection of the CRP content in the sample.
可以理解地，如图1所示，每个芯片可以完成三个不同样本的同时检测，芯片内每个检测单元可以完成7中目标检测物的分析检测，同时也可以牺牲其终一个检测单元，设置阳性对照检测，完成单个芯片的质量控制。It can be understood that, as shown in Figure 1, each chip can complete the simultaneous detection of three different samples, and each detection unit in the chip can complete the analysis and detection of 7 target detection objects. At the same time, one of the detection units can be sacrificed to set up a positive control test to complete the quality control of a single chip.
实施例2Example 2
本发明提供的另一实施例为将芯片应用于基于辣根过氧化物酶(HRP)的鲁米诺化学发光免疫分析，免疫分析原理为免疫夹心原理用于全血中降钙素原(PCT)的检测，具体技术方案如下：Another embodiment provided by the present invention is to apply the chip to luminol chemiluminescence immunoassay based on horseradish peroxidase (HRP), and the immunoassay principle is the immune sandwich principle for the detection of procalcitonin (PCT) in whole blood. The specific technical scheme is as follows:
1.抗体修饰1. Antibody modification
将HRP通过EDC和NHS体系与抗PCT抗体结合，制备信号标记抗体复合物，同样地，将磁微粒通过EDC和NHS系统与抗PCT单抗结合，制备捕获抗体复合物；The HRP is combined with the anti-PCT antibody through the EDC and NHS system to prepare a signal-labeled antibody complex. Similarly, the magnetic particles are combined with the anti-PCT monoclonal antibody through the EDC and NHS system to prepare a capture antibody complex.
2.芯片加工方式与具体具体实施例1类似；2. The chip processing method is similar to that of specific embodiment 1;
3.芯片组装3. Chip assembly
如图10所示，芯片总共有3层，包括上盖54、下盖55、中间层53，芯片组装采用双面胶黏贴的方式进行密封组合，在组合之前首先将HRP标记PCT抗体预包埋在第一微通道引流单元15的起始端(即与定量单元14连接端)，将磁微粒标记PCT单抗预包埋在反应池16中；在进行上盖54密封相片之前在第一试剂池21中中预封装鲁米诺试剂包，在第二试剂池22中预封装预过氧化氢(H2O2)，随后进行芯片密封，形成完整的检测芯片。As shown in FIG10 , the chip has a total of three layers, including an upper cover 54, a lower cover 55, and an intermediate layer 53. The chip is assembled by sealing and combining by means of double-sided adhesive. Before the assembly, the HRP-labeled PCT antibody is firstly pre-embedded at the starting end of the first microchannel drainage unit 15 (i.e., the end connected to the quantitative unit 14), and the magnetic particle-labeled PCT monoclonal antibody is pre-embedded in the reaction pool 16; before the upper cover 54 is sealed, the luminol reagent package is pre-packaged in the first reagent pool 21, and the hydrogen peroxide (H2 O2 ) is pre-packaged in the second reagent pool 22, and then the chip is sealed to form a complete detection chip.
4.样本检测4. Sample testing
具体地，在加样口11处加入全血样本100～200μl，密封将芯片放入配套检测仪器中；Specifically, add 100-200 μl of whole blood sample to the sample adding port 11, seal the chip and place it in the matching detection instrument;
具体地，芯片在仪器中，首先进行低速离心，将全血样本中的细胞及杂质沉淀在样本分离池12底部，完全全血中血浆及血细胞及杂质的分离，提高离心速度，全血上清会由于持续的离心力作用进入第一定量分配单元13内进行分配定量，样本最终体积通过定量区14的大小进行精确定量，多余的废液会进入废液池32中，随后离心结束；Specifically, the chip is first centrifuged at a low speed in the instrument to precipitate cells and impurities in the whole blood sample at the bottom of the sample separation pool 12, and to separate the plasma, blood cells and impurities in the whole blood. The centrifugal speed is increased, and the whole blood supernatant will enter the first quantitative distribution unit 13 for quantitative distribution due to the continuous centrifugal force. The final volume of the sample is accurately quantified by the size of the quantitative area 14, and the excess waste liquid will enter the waste liquid pool 32, and then the centrifugation ends;
具体地，启动芯片高速离心，将定量的样本溶液注入第一微通道引流单元15中，样本溶液会复溶HRP标记PCT抗体于样本中的PCT抗原进行反应形成HRP标记PCT抗体-PCT复合物，通过蜿蜒的微通道可以实现对流体流速的控制，同时使反应更加充分，最后在持续高速离心的作用下，垂直或平行于圆心延伸线的蜿蜒微通组合，可以借助科氏力及欧拉力对流体的产生偏转的影响而提高混合效果，最终所有液体将被排入反应池16中，此时阀门31为关闭状态；Specifically, the chip is started to centrifuge at high speed, and a quantitative sample solution is injected into the first microchannel drainage unit 15. The sample solution will re-dissolve the HRP-labeled PCT antibody and react with the PCT antigen in the sample to form an HRP-labeled PCT antibody-PCT complex. The winding microchannel can control the flow rate of the fluid and make the reaction more complete. Finally, under the action of continuous high-speed centrifugation, the winding microchannel combination perpendicular or parallel to the extension line of the center of the circle can improve the mixing effect by the deflection of the fluid by the Coriolis force and the Euler force. Finally, all the liquid will be discharged into the reaction pool 16, and the valve 31 is closed at this time.
具体地，高速离心停止过程会使反应池16中的溶液产生震荡，并完成磁微粒标记PCT单抗与HRP标记PCT抗体-PCT复合物的反应，使反应更加快速，缩短反应时间，反应形成HRP标记PCT抗体-PCT-磁微粒标记PCT单抗复合物，此时启动磁铁吸附，使磁微粒固定在反应池16中，开放阀门31，启动芯片离心，将所有液体排入废液池中，停止离心；随后关闭阀门31，撤去磁铁对磁微粒的吸附，同时释放第一试剂池21中的鲁米诺，启动低速离心对试剂进行精确定量，随后高速离心，使试剂进入反应池16中，随后通过同时或顺序按压或注射的方式将第二试剂池中的过氧化氢溶液快速注入反应池16中，与捕获的HRP酶完成快速反应发光，仪器对光信号进行检测，完成样本中PCT含量的精确定量检测。Specifically, the high-speed centrifugation stop process will cause the solution in the reaction pool 16 to vibrate, and complete the reaction between the magnetic particle-labeled PCT monoclonal antibody and the HRP-labeled PCT antibody-PCT complex, making the reaction faster and shortening the reaction time. The reaction forms an HRP-labeled PCT antibody-PCT-magnetic particle-labeled PCT monoclonal antibody complex. At this time, the magnet adsorption is started to fix the magnetic particles in the reaction pool 16, the valve 31 is opened, the chip centrifugation is started, all the liquid is discharged into the waste liquid pool, and the centrifugation is stopped; then the valve 31 is closed, the magnet adsorption on the magnetic particles is removed, and the luminol in the first reagent pool 21 is released at the same time, and low-speed centrifugation is started to accurately quantify the reagent, followed by high-speed centrifugation to allow the reagent to enter the reaction pool 16, and then the hydrogen peroxide solution in the second reagent pool is quickly injected into the reaction pool 16 by pressing or injecting simultaneously or sequentially, and completes a rapid reaction luminescence with the captured HRP enzyme, and the instrument detects the light signal to complete the accurate quantitative detection of the PCT content in the sample.
可以理解地，如图1和图9所示，每个芯片可以完成三个不同样本的同时检测，芯片内每个检测单元可以完成7种目标检测物的分析检测，同时也可以牺牲其终一个检测单元，设置阳性对照检测，完成单个芯片的质量控制。It can be understood that, as shown in Figures 1 and 9, each chip can complete the simultaneous detection of three different samples, and each detection unit in the chip can complete the analysis and detection of 7 target detection objects. At the same time, one of the detection units can be sacrificed to set up a positive control test to complete the quality control of a single chip.
本发明提供的引流通道克服科氏力及欧拉力的效果验证如图8所示：设计不同延伸长度的引流通道，最终结果验证从左至右，液体的偏向越来越大，通道延伸到一定长度时，液体的偏向基本不受科氏力及欧拉力的影响，进一步保证分析检测结构的可靠性及可重复性。The effectiveness of the drainage channel provided by the present invention in overcoming the Coriolis force and the Euler force is verified as shown in FIG8 : drainage channels with different extension lengths are designed, and the final result verifies that from left to right, the deflection of the liquid becomes larger and larger. When the channel extends to a certain length, the deflection of the liquid is basically not affected by the Coriolis force and the Euler force, further ensuring the reliability and repeatability of the analysis and detection structure.
本领域的技术人员容易理解，以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。It will be easily understood by those skilled in the art that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.
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