CN105435868B - Quantitative detection of magnetic particles in whole blood troponin i-emitting chemical microfluidic - Google Patents

Quantitative detection of magnetic particles in whole blood troponin i-emitting chemical microfluidic Download PDF

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CN105435868B
CN105435868B CN 201510696728 CN201510696728A CN105435868B CN 105435868 B CN105435868 B CN 105435868B CN 201510696728 CN201510696728 CN 201510696728 CN 201510696728 A CN201510696728 A CN 201510696728A CN 105435868 B CN105435868 B CN 105435868B
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CN 201510696728
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CN105435868A (en )
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王东
李泉
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深圳华迈兴微医疗科技有限公司
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Abstract

本发明公开了一种定量检测全血中肌钙蛋白I的磁微粒化学发光微流控芯片,所述微流控芯片包括顶板(1)结构和底板(2)结构,其中顶板(1)上的气泵(3)、加样口(4)、样本填充区(12)、标记抗体存储池(5)和样本混合区(13)依次连接;底板上的过滤区(6)、磁颗粒包被区(7)、清洗区(14)、检测区(8)、液体释放通道(16)依次连接;底板的检测区(8)分别与清洗液存储池(9)和发光基底液存储池(10)通过液体释放通道(16)连接。 The present invention discloses a quantitation of troponin I in the whole blood chemiluminescence magnetic particles microfluidic chips, the microfluidic chip comprises a plate (1) and the bottom plate structure (2) structure, wherein the top plate (1) the pump (3), a loading port (4), filled with the sample region (12), labeled antibody storage tank (5) and sample mixing zone (13) sequentially connected; filter region (6) on the bottom plate, the magnetic particles are coated region (7), cleaning zone (14), the detection region (8), the liquid release passage (16) sequentially connected; floor detection zone (8), respectively, and the cleaning fluid storage tank (9) and the light emitting substrate liquid storage tank (10 ) connected by a fluid release passage (16).

Description

定量检测全血中肌钙蛋白I的磁微粒化学发光微流控芯片 Quantitative detection of troponin I in whole blood chemiluminescence magnetic particles microfluidic

技术领域 FIELD

[0001]本发明涉及一种利用磁微粒化学发光技术和微流控芯片技术实现全血样本中cTnl高灵敏定量检测的方法,特别公开了一种定量检测全血中肌钙蛋白I的磁微粒化学发光微流控芯片,可实现全血样本中cTnl的准确、高灵敏定量检测,属于微流控芯片化学发光免疫检测技术领域。 [0001] The present invention relates to a magnetic microparticle chemiluminescence and microfluidic technology cTnl whole blood sample quantitative detection sensitivity, in particular, discloses a quantitative detection of whole blood troponin I magnetic particles chemiluminescence microfluidic chip, enabling accurate cTnl the whole blood sample, highly sensitive quantitative detection, microfluidic chips belonging chemiluminescent immunoassay art.

背景技术 Background technique

[0002] 当前我国心血管疾病的防控形势依然严峻,心血管疾病发病率呈不断上升态势。 [0002] The current cardiovascular disease prevention and control situation is still grim in our country, cardiovascular morbidity showed a rising trend. 据统计,心血管病死亡率占人口死亡的40%,因此做好早发现、早预防、早救治,提高心血管病防治水平是关键。 According to statistics, cardiovascular disease mortality accounted for 40% of the population died, so do early detection, early prevention, early treatment, to improve the prevention and treatment of cardiovascular disease is the key. 心血管疾病传统检测项目大多为心肌酶谱。 Cardiovascular disease is the most traditional test items myocardial enzymes. 但存在酶活性升高出现较晚、特异性较差和持续时间短等不足。 However, there is increased activity appeared later and less specific and short duration insufficient. 而心肌肌钙蛋白是唯一存在于心肌的收缩蛋白,对心肌坏死有高度敏感性和特异性。 The contraction of cardiac troponin is the only protein present in the myocardium, it has high sensitivity and specificity for myocardial necrosis.

[0003] 心肌肌钙蛋白是由cTnl、cTnC和cTnT三种,在肌肉舒张和收缩过程中起重要调节作用。 [0003] cardiac troponin is cTnl, cTnC and cTnT three kinds, plays an important role in the regulation of muscle contraction and relaxation process. 但cTnC无心肌特异性,一般不用于心肌损伤检测。 But cTnC no cardiac-specific, generally not used for detection of myocardial injury. 正常状态下cTnl和cTnT均不能穿透细胞膜进入血液,所以健康人血中cTnl和cTnT极低;如心肌细胞受损,cTnl和cTnT进人细胞间质和血液。 CTnl and cTnT in normal state can not penetrate the cell membrane into the blood, the healthy human blood cTnl and cTnT low; such as myocardial cell damage, cTnl and cTnT between the cytoplasm and into the blood. 在肾衰竭、肺炎和败血症等疾病中,血液中cTnT含量也可升高,所以其特异性不如cTnKcTnl在发病后3〜5小时升高,15〜24小时达高峰,持续时间久,5〜10天后可降至正常。 In kidney failure, pneumonia and sepsis and other diseases, blood cTnT content can also be increased, so its not as good as cTnKcTnl specificity in three to five hours after the onset of elevated 15~24 hours reached the peak for a long time, 5 to 10 days can be reduced to normal. cTnl是目前最好的心肌损伤标志物之一。 cTnl is currently one of the best markers of myocardial injury.

[0004] 传统上多用酶联免疫法、化学发光法和免疫层析法(试纸条)等检测cTnl。 [0004] Multi conventionally by ELISA, chemiluminescence immunoassay and chromatography (test strip) and other testing cTnl. 但酶联免疫法操作复杂,检测时间长;化学发光法需配套大型昂贵仪器,测试时间长,不易实现快速即时检测。 However ELISA complex operation, long detection time; chemiluminescence expensive instrumentation required to support large-scale, long testing time, easy to achieve rapid detection instant. 胶体金免疫层析法虽简便快速,但重复性差、灵敏度低,容易出现误判。 GICA method, although simple, rapid, but poor reproducibility and low sensitivity, prone to miscarriage of justice.

[0005] 中国专利200780015772.0公布了一种肌钙蛋白高灵敏分析系统,采用微量滴定板(微孔板)进行检测,灵敏度高,但操作复杂、测试时间长、检测范围窄。 [0005] Chinese Patent No. 200780015772.0 discloses a highly sensitive troponin analysis system using a microtiter plate (microplate) to detect, with high sensitivity, but the complexity of the operation, the test time is long, narrow detection range. 中国专利200610028913.X描述了一种以胶体金免疫层析技术实现对cTnl检测的试剂盒,但只能进行定性检测,而无法定量。 Chinese Patent No. 200610028913.X describes a technique to achieve GICA cTnl detection kit, but only qualitative detection, can not be quantified. 中国专利201010619731.6公开了一种全程定量检测cTn I的免疫层析试纸条,以荧光胶乳微粒替代胶体金,实现对cTnl的定量检测,但仍无法解决试纸条重复性差等缺陷。 Chinese patent 201010619731.6 discloses a full quantitative immunochromatographic strip cTn I to replace the fluorescent latex particles of colloidal gold, to achieve quantitative detection of cTnl, but does not solve the defects and poor reproducibility of the test strip.

[0006] 因此开发快速、准确、灵敏度高的检测方法,具有巨大发展潜力和应用前景。 [0006] Therefore the development of fast, accurate, high-sensitivity detection method has great potential for development and application prospects. 与荧光和吸收光相比,化学发光没有外来激发光源背景信号千扰,交叉干扰小,灵敏度高、线性范围宽。 Compared with the light absorption and fluorescence, chemiluminescence without external excitation source background interference signals one thousand, small crosstalk, high sensitivity, wide linear range. 微流控芯片技术把样品制备、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,可完成全过程分析。 The sample preparation microfluidic chip technology, reaction, separation, detection units are integrated into the basic operation of a microscale chip, the entire process can be completed analysis.

[0007] 针对现有cTnl检测方法的不足和缺陷,微流控磁微粒化学发光方法利用磁微粒化学发光和微流控技术,可实现对cTnl准确、高灵敏定量检测。 [0007] cTnl for the shortcomings and deficiencies of existing detection methods, microfluidic chemiluminescence method utilizes magnetic particles magnetic particles and microfluidic chemiluminescence technology can be realized on cTnl accurate, highly sensitive quantitative detection.

发明内容 SUMMARY

[0008] 本发明要解决的技术问题为针对现有快速诊断方法灵敏度低、重复性差、受干扰明显,以及现有化学发光配套仪器昂贵、检测时间长的问题,提供一种定量检测全血中肌钙蛋白I的磁微粒化学发光微流控芯片,通过集成化芯片(把除测试样本外所有组分均集成到芯片内)并配套小型便携设备,从而实现现场全血样本中cTnl的快速、准确、高灵敏定量检测。 [0008] The present invention is to solve the technical problem of low sensitivity against conventional rapid diagnostic methods, poor reproducibility, obviously affected by interference, and conventional chemiluminescent expensive ancillary equipment, long detection time problems and to provide a quantitative detection of whole blood troponin I magnetic particles chemiluminescence microfluidic chip by integrated chip (the test sample except all components are integrated into the chip) and supporting small portable devices, in order to achieve fast field cTnl in a whole blood sample, accurate, highly sensitive quantitative detection.

[0009] 为解决上述技术问题,本发明提供的技术方案为: [0009] To solve the above problems, the present invention provides the technical solution as follows:

[0010] 一种定量检测全血中肌钙蛋白I的磁微粒化学发光微流控芯片,其特征在于,所述微流控芯片包括顶板⑴结构和底板⑵结构,其中顶板⑴上气泵(3)、加样口⑷、样本填充区(12)、标记抗体存储池⑸和样本混合区(13)依次连接;底板上过滤区(6)、磁颗粒包被区⑺、清洗区(14)、检测区(8)、液体释放通道(16)依次连接;底板的检测区⑻分别与清洗液存储池(9)和发光基底液存储池(10)通过液体释放通道(16)连接; [0010] A quantitative determination of troponin I in whole blood chemiluminescence magnetic particles microfluidic chip, wherein the microfluidic chip includes a top plate and a bottom plate structure ⑴ ⑵ structure, wherein the top plate ⑴ pump (3 ), loading mouth ⑷, filled with the sample region (12), labeled antibody storage pool ⑸ and sample mixing zone (13) sequentially connected; filter region (6) on the base plate, the magnetic particles are coated region ⑺, cleaning zone (14), detection zone (8), the liquid release passage (16) sequentially connected; ⑻ detection zone are connected to the base plate cleaning fluid storage tank (9) and the light emitting substrate liquid storage tank (10) release passage (16) through the liquid;

[0011] 所述标记抗体存储池(5)存储预封装酶或发光剂标记抗cTnl抗体,磁颗粒包被区(7)包被预封装磁颗粒标记抗cTnl抗体,清洗液存储池(9)和发光基底液存储池(10)存储预封装清洗液和发光基底液;所述微流控芯片测试流程中,用磁铁操控磁颗粒移动或聚集;所述标记抗体存储池、清洗液存储池和发光基底液存储池为液体密封池,可通过外力挤压而局部破裂,释放液体;所述过滤区包含滤血膜所述顶板(1)与底板(2)用胶带(19和20)密封。 [0011] The labeled antibody storage tank (5) stored in the pre-packaged or luminescent enzyme-labeled anti-cTnl antibody, the magnetic particles are coated area (7) coated magnetic particles pre-encapsulated labeled anti-cTnl antibody, the washing liquid storage tank (9) and a light emitting substrate liquid storage tank (10) storing pre-packaged liquid cleaning solution and a light emitting substrate; the microfluidic chip testing process, the magnetic particles move with a magnet or aggregation of control; the labeled antibody storage pool and the storage pool cleaning solution luminescent substrate solution storage pool is a liquid sealed cells, may be locally broken by an external force pressing, releasing the liquid; the hemofilter membrane filtration zone comprising said top plate (1) and the base plate (2) sealing tape (19 and 20). [0012]具体地,本发明所述的微流控芯片,发光基底液保质期少于1年时应分开,用发光基底液存储池A (23)和发光基底液存储池B (24)替代发光基底液存储池(10),所述发光基底液存储池A (23)和发光基底液存储池B (24)通过预混合通道(25)连接。 Should [0012] Specifically, the micro-fluidic chip according to the present invention, the light emitting substrate was less than 1 year shelf life separated liquid with the light emitting substrate storage pool A (23) and the light emitting substrate solution storage pool B (24) emitting an alternative substrate solution storage tank (10), the storage pool luminescent substrate solution A (23) and the light emitting substrate solution storage pool B (24) are connected by a pre-mixing channel (25).

[0013]具体地,本发明所用磁颗粒包含铁、钴、镍的化合物,主要包含但不限于三氧化二铁和四氧化三铁化合物。 [0013] In particular, the present invention comprises the magnetic particles of iron, cobalt, nickel compounds, primarily including but not limited to ferric oxide and triiron tetroxide compound. 优选磁颗粒为聚苯乙烯为壳,三氧化二铁为核的颗粒,磁颗粒尺寸和磁铁的磁感应强度对检测结果有明显的影响。 Preferably the magnetic particles are polystyrene shell, ferric oxide as the core particles, the particle size of the magnetic flux density and the magnet has a significant effect on the detection result.

[0014]具体地,所述磁颗粒标记抗cTnl抗体使用的磁颗粒尺寸为0.1〜10um;与磁珠匹配的磁铁磁感应强度为500〜30000高斯。 [0014] Specifically, the magnetic particle size of the magnetic particle-labeled anti-cTnl antibody used was 0.1~10um; magnetic beads matching magnet magnetic induction 500~30000 gauss.

[0015] 优选地,所述磁颗粒标记抗cTnl抗体使用的磁颗粒尺寸为0.5〜3wn,与磁珠匹配的磁铁磁感应强度为1000〜8000高斯。 [0015] Preferably, the magnetic particles labeled antibodies anti-cTnl using a magnetic particle size 0.5~3wn, matching the magnetic beads magnet magnetic induction 1000~8000 gauss.

[0016]具体地,所述酶或发光剂标记cTnl抗体溶液、磁颗粒标记cTnl抗体溶液和清洗液均包含缓冲液、蛋白质、表面活性剂和防腐剂,且发光剂标记cTnl抗体溶液还包含甘油,磁颗粒标记cTnl抗体溶液还包含糖类。 [0016] In particular, the luminescent or enzyme-labeled antibody solution cTnl, cTnl magnetic particles labeled antibody solution and washing solution contains a buffer, proteins, surfactants, and preservatives, and luminescent-labeled antibody solution further comprises glycerin cTnl , magnetic particle labels cTnl antibody solution further comprises a saccharide.

[0017] 具体地,所述酶标记cTnl抗体溶液包含牛血清白蛋白(BSA)、吐温-20和Proclin300的硼酸缓冲液;磁颗粒标记cTnl抗体溶液包含BSA、葡萄糖、吐温-20和Proclin300的硼酸缓冲液;所述清洗液包含BSA、吐温-20和Proclin300的硼酸缓冲液。 [0017] In particular, the enzyme-labeled antibody solution containing cTnl bovine serum albumin (BSA), Tween-20 and borate buffer of Proclin300; cTnl magnetic particle-labeled antibody solution containing BSA, glucose, Tween-20 and Proclin300 borate buffer; the cleaning liquid containing BSA, Tween-20 and borate buffer of Proclin300.

[0018] 具体地,所述发光剂标记cTnl抗体溶液包含BSA、甘油、吐温-20、曲拉通X-100和叠氮钠的磷酸盐缓冲液;所述磁颗粒标记cTnl抗体溶液包含BSA、酪蛋白、蔗糖、吐温-20、曲拉通X-100和叠氮钠的磷酸盐缓冲液;所述清洗液包含BSA、吐温20、曲拉通X-100、聚乙二醇和叠氮钠的磷酸盐缓冲液。 [0018] In particular, the luminescent-labeled antibody solution containing cTnl BSA, glycerol, Tween-20, Triton X-100 and sodium azide phosphate buffer; cTnl said magnetic particles labeled antibody solution containing BSA , casein, sucrose, Tween-20, Triton X-100 and sodium azide phosphate buffer; the cleaning liquid containing BSA, Tween 20, Triton X-100, polyethylene glycol, and the stack sodium phosphate buffer nitrogen.

[0019]具体地,所述发光基底液包含与酶对应的底物及发光增强液,可合并后注入发光基底液存储池(10),或分别注入发光基底液存储池A (23)和发光基底液存储池B (24)。 [0019] In particular, the luminescent substrate solution containing a substrate for the enzyme and the light emitting enhancement solution, was injected into the light emitting substrate storage tank (10) can be combined after, or luminescent substrate solution were injected into the storage pool A (23) and a light emitting substrate solution storage pool B (24).

[0020]具体地,所述发光基底液包含发光剂对应的双氧水溶液和碱性溶液,可合并后注入发光基底液存储池(10),或分别注入发光基底液存储池A (23)和发光基底液存储池B (24) 。 [0020] In particular, the luminescent substrate comprising a luminescent agent corresponding to the liquid hydrogen peroxide solution and an alkaline solution, was injected into the light emitting substrate storage tank (10) can be combined after, or luminescent substrate solution were injected into the storage pool A (23) and a light emitting substrate solution storage pool B (24).

[0021] 本发明所述微流控芯片制备方法如下: [0021] The microfluidic chip of the present invention is prepared as follows:

[0022] 步骤1)酶或发光剂标记抗cTnl抗体,磁颗粒标记抗cTnl抗体,这两种抗体可相同或不同; [0022] Step 1) enzyme or luminescent-labeled anti-cTnl antibody, the magnetic particle-labeled anti-cTnl antibody, the two antibodies may be identical or different;

[0023]步骤2)将酶或发光剂标记抗体溶液放入顶板的标记抗体存储池中,密封,将磁颗粒标记抗体溶液放入底板的包被区中,干燥,将清洗液和发光基底液分别注入清洗液存储池和发光基底液存储池中,密封,将顶板和底板组装成微流控芯片。 [0023] Step 2) the enzyme-labeled antibody or luminescent labeled antibody solution was placed in the storage pool of the top plate, sealing the magnetic particles-labeled antibody solution was placed in the bottom area of ​​the package is dried, the washing liquid and the light emitting substrate solution They were injected into the washing liquid storage tank and liquid storage pool luminescent substrate, sealing the assembled top and bottom plates into the microfluidic chip.

[0024]本发明提供的一种定量检测全血中肌钙蛋白I的磁微粒化学发光微流控芯片是一种以化学发光为基础、在微流控芯片上实现cTnl快速、准确、高灵敏检测的微流控芯片。 [0024] The present invention provides a quantitative detection of troponin I in whole blood chemiluminescence magnetic particles is a microfluidic chip based chemiluminescence, achieve rapid cTnl on microfluidic chip, accurate, sensitive microfluidic chip detection.

[0025] 这种芯片是将抗cTnl抗体修饰酶,抗cTnl抗体修饰在磁颗粒上,利用抗原抗体作用,如双抗体夹心法原理结合磁颗粒富集、化学发光检测全血样本中是否含有cTnl,并准确分析其含量。 [0025] This chip is modified antibodies anti-cTnl enzymes, anti-cTnl antibody on the magnetic particle modified, using antibodies to antigen, such as the double antibody sandwich principle of magnetic particles binding enrichment, chemiluminescent whole blood samples contain cTnl and accurate analysis of its content.

[0026] 本发明中所述酶,包含但不限于过氧化氢酶(HRP)和碱性磷酸酶(ALP)。 [0026] The enzyme of the present invention, including but not limited to, catalase (HRP) and alkaline phosphatase (ALP). 发光基底液为酶对应的发光底物(如鲁米诺或金刚烧)和发光增强液(如苯衍生物等增强剂),其中发光底物和发光增强液可合并,如图1所示混合均匀后注入一个发光基底液存储池(10);但当混合液保质期少于1年时应分开,如图3所示分别注入发光基底液存储池A (23)和发光基底液存储池B (24),如图3所示,测试时通过预混合通道(25)混合均匀,再流入检测区参与反应。 Luminescent enzyme substrate solution corresponding luminescent substrate (e.g., luminol or adamantyl burn) and a light emitting enhancement solution (e.g., benzene derivatives etc. enhancer), and wherein the luminescent substrate can be enhanced luminescence were combined and mixed 1 shown in FIG. a light emitting substrate uniformly injected liquid storage tank (10); but the mixture should be less than 1 year shelf life separately 3 were injected into the luminescent substrate solution a shown in the storage pool (23) and the light emitting substrate solution storage pool B ( 24), shown in Figure 3, by pre-mixing channel (25) when tested mixed, reacted and then flows into the detection zone. 本发明一个实施例采用辣根过氧化物酶(HRP)。 Embodiment of the present invention employed a horseradish peroxidase (HRP).

[0027]本发明所述发光剂,包含但不限于吖啶酯和吖啶磺酰胺。 [0027] The present invention luminescent agent, including but not limited to acridinium ester and acridinium sulfonamides. 发光剂与发光基底液作用后,不需酶的催化作用,直接参与发光反应。 After luminescent agent and the light emitting action of the substrate solution without enzyme catalysis, directly involved in the luminescent reaction. 本发明的一个实施例采用吖啶酯。 An embodiment of the present invention employs acridinium ester. 发光基底液包含H2〇2溶液和碱性溶液,可合并成碱性H2〇2溶液,注入发光基底液存储池(10);但混合后不稳定时,双氧水溶液和碱性溶液应分别注入发光基底液存储池A (23)和发光基底液存储池B (24),如图3所示,测试时先通过预混合通道(25)混合均匀,再流入检测区参与反应。 H2〇2 luminescent substrate solution comprising a solution and an alkaline solution, may be combined into H2〇2 alkaline solution, was injected into the light emitting substrate storage tank (10); mixed but unstable hydrogen peroxide solution and the basic solution should be injected into the light emitting respectively substrate solution storage pool A (23) and the light emitting substrate solution storage pool B (24), shown in Figure 3, the first test by premixing channel (25) were mixed uniformly, and then flows into the detection zone participate in the reaction. [0028]本发明的cTnl抗体包含单克隆抗体和多克隆抗体。 [0028] cTnl antibody of the present invention comprise monoclonal and polyclonal antibodies. 该抗体可与cTnl结合(如双抗体夹心法)。 The antibody may be combined with of cTnl (such as the double antibody sandwich method). 其中酶或发光剂标记的抗体与磁颗粒标记的抗体可以相同,也可以不同。 Wherein luminescent or enzyme-labeled antibody and the magnetic particle-labeled antibody may be the same or different.

[0029]本发明的酶或发光剂标记抗体溶液和磁颗粒标记抗体溶液均包含缓冲液、蛋白质、表面活性剂和防腐剂,且磁颗粒标记抗体溶液还包含糖类。 [0029] The luminescent or enzyme-labeled antibody solution and a magnetic particle-labeled antibody solution of the present invention contains a buffer, a protein, a surfactant and a preservative, and the magnetic particles-labeled antibody solution further comprises a saccharide. 其中HRP标记抗体,缓冲体系中不能含有NaN3; ALP标记抗体,缓冲体系不能是磷酸体系。 Wherein the HRP-labeled antibody, buffer system can not contain NaN3; ALP-labeled antibody, a buffer system is not phosphoric acid system.

[0030]本发明的微流控芯片的顶板和底板的成型材料为聚合物,包含但不限于聚苯乙烯、聚氯乙烯、聚丙烯、环氧树脂等。 [0030] The top and bottom of the microfluidic chip of the present invention is a molding material is a polymer, including but not limited to, polystyrene, polyvinyl chloride, polypropylene, epoxy resins and the like. 双面胶带可由两张单面胶带代替。 Double-sided tape can be replaced by two single-sided tape. 如图1所示,顶板结构由气泵⑶、加样口⑷、标记抗体存储池⑸、盖子(11)、样本填充区(12)和样本混合区(13) 组成。 1, the roof structure by the pump ⑶, loading mouth ⑷, storage pools labeled antibody ⑸, the cap (11), filled with the sample region (12) and sample mixing zone (13) components. 底板结构由过滤区⑹、磁颗粒包被区(7)、检测区⑻、清洗液存储池⑼、发光基底液存储池(10)、清洗区(14)、废液池(15)和液体释放通道(16)。 The floor structure of a filtration zone ⑹, the magnetic particles are coated area (7), the detection zone ⑻, ⑼ cleaning solution storage pool, luminescent substrate solution storage tank (10), cleaning zone (14), a waste reservoir (15) and a liquid release channel (16). 如图2所示,在发光基底液和清洗液存储池区域,以及磁铁滑轨区域,在顶板上需留出存储池和磁铁滑轨的让位孔(分别为I7和1S),在双胶带上应留出存储池和样本混合液流入过滤区时的让位孔(分别为21和22), 让位孔的作用是让开一定的区域,不干扰液体流路,或配套仪器部件作用于微流控芯片的通路。 2, the light emitting substrate, and a liquid cleaning solution storage pool area, and a magnet slide area, in the top plate to make way for an aperture to leave the storage pool and the magnets slide (lS and I7, respectively), the tape bis the storage pool should be allowed to give way and the sample mixture holes (21 and 22 respectively) when entering the filter zone, the role is to open the hole giving way a certain area, does not interfere with the liquid flow path, or acting on the supporting part of the instrument microfluidic chip passage.

[0031]本发明的存储池为液体密封池,所用密封材料包含玻璃、塑料、橡胶、铝箔和高阻隔薄膜,其中密封材料可为同种材料组成,也可为多种材料组合而成。 [0031] The memory cell of the present invention is a liquid sealed cells, the sealing material comprises glass, plastic, rubber, aluminum foil and high barrier films are used, in which the sealing material may be composed of the same material, it may be a combination of multiple materials. 在物理挤压下,存储池可局部破裂,从而把密封的液体释放出来。 In a physical pressing, the storage pool can be partially ruptured to release the liquid seal. 其中酶标cTnl抗体存储池、清洗液存储池、发光基底液存储池可采用相同或不同材料和方法制作。 Wherein the enzyme-labeled antibody cTnl storage pool, cleaning solution storage pool, storage pool may employ luminescent substrate solution the same or different materials and method of making. 在本发明的一个实施例中,酶标cTnI 抗体存储池、清洗液存储池、发光基底液存储池均采用塑料和弹性橡胶密封而成。 In one embodiment of the present invention, the enzyme-labeled antibody cTnI storage pool, storage pool cleaning solution, the liquid storage pool luminescent substrate are made of plastic and sealed with an elastic rubber. 本发明的另一个实施例中,酶标cTnl抗体存储池采用塑料和弹性橡胶密封而成,而清洗液存储池、发光基底液存储池采用高阻隔薄膜密封而成。 Another embodiment of the present invention, the enzyme-labeled antibody cTnl plastic storage pools and sealed with an elastic rubber, and the cleaning fluid storage tank, the storage pool using the luminescent substrate was sealed with barrier film.

[0032]本发明的过滤区包含滤血膜,其中滤血膜可通过物理孔径或生物/化学试剂使液体与细胞分离,实现血浆与红细胞分离,血浆流到磁颗粒包被区,而红细胞停留在滤血膜上,从而减少红细胞对试验结果的干扰。 [0032] The present invention comprises a filtration zone hemofilter membrane, wherein the membrane filtration of blood cells and the liquid can be separated by physical aperture or biological / chemical agents, to achieve separation of plasma and red blood cells, plasma flows to the magnetic particles are coated region, and the red blood cells travel in hemofilter membrane, thereby reducing the red cell interference test results. 其中所述生物/化学试剂包含凝血剂等,可使红细胞间连接,形成凝块,增大尺寸,更容易被滤血膜的网状结构阻挡。 Wherein said biological / chemical agent comprising a blood clotting agent, allows connection between the red blood cells, clot formation, increased in size, is more easily blocked mesh structure hemofilter membrane.

[0033]本发明的微流控芯片,当存在发光基底液存储池a (23)和发光基底液存储池B (24),应在底板上增加发光基底液预混合通道(25),该预混合通道可为蛇形通道或上下结构混合通道,如图3所示。 [0033] The microfluidic chip of the present invention, when an emitting substrate solution storage pool a (23) and the light emitting substrate solution storage pool B (24), should increase the luminous substrate was pre-mixing channel (25) on the base plate, the pre- mixing channel may be a serpentine channel or down mixing channel structure, as shown in FIG.

[0034]在一个实施例中,标记抗体存储池(5)封入HRP标记抗cTnl抗体,包被区包被磁颗粒标记抗cTnl抗体(与酶标抗体不同),以磁微粒酶促化学发光法检测cTnI。 [0034] In one embodiment, labeled antibodies storage tank (5) enclosed HRP-labeled anti cTnl antibody coated region coated with an anti-cTnl antibody labeled magnetic particles (with different enzyme-labeled antibody) to the magnetic particles chemiluminescence method detection of cTnI. 另一个实施例中,标记抗体存储池(5)封入吖啶酯标记抗cTnl抗体,包被区包被磁颗粒标记抗cTnl抗体(与吖啶酯标记抗体不同),以磁微粒酶促化学发光法检测cTnl含量。 In another embodiment, labeled antibodies storage tank (5) enclosed acridinium ester labeled anti-cTnl antibody coated region coated with an anti-cTnl antibody labeled magnetic particles (with different acridinium ester labeled antibody) to the magnetic particles chemiluminescence cTnl content detection method.

[0035]本发明的清洗液,用于清洗磁颗粒,去除非特异性吸附的cTnl、酶标记物及其他影响检测结果的物质。 Cleaning solution [0035] of the present invention, for cleaning of magnetic particles, to remove non-specific adsorption of cTnl, enzyme labels and other substances affect the test results. 清洗液主要包含缓冲体系、蛋白质和表面活性剂,其中缓冲体系包含但不限于硼酸盐、磷酸盐、Tris-HCl和醋酸盐等。 Cleaning solution mainly comprises a buffer system, a surfactant and a protein, wherein the buffer system comprises, but not limited to, borates, phosphates, Tris-HCl and acetic acid salts. 清洗液pH 6.0〜10.0,当检测样本为强酸或强碱性时,pH范围可放宽。 Cleaning solution pH 6.0~10.0, when the test sample is a strong acid or strong alkaline, pH range can be relaxed. 其中蛋白质包含但不限于牛血清白蛋白、酪蛋白等。 Wherein the protein including but not limited to, bovine serum albumin, casein and the like. 其中表面活性包含但不限于可包括吐温20、吐温80、曲拉通X-100、聚乙二醇和聚乙烯基吡咯烷酮等。 Wherein the surfactant may include but not limited to Tween 20, Tween 80, Triton X-100, polyethylene glycol and polyvinyl pyrrolidone. [0036] 本发明的样本体积在10〜5〇〇yl,优选20〜100ul。 [0036] In the present invention the sample volume 10~5〇〇yl, preferably 20~100ul. 作为优选,在实施例中加样体积为50ul。 Preferably, in the embodiment, the loading volume was 50ul.

[0037] 本发明的微流控芯片为快速检测,检测时间应小于30分钟,作为优选,实施例中采用15分钟。 [0037] The microfluidic chip of the present invention for the rapid detection, the detection time should be less than 30 minutes, as a preferred embodiment employed for 15 minutes.

[0038] 本发明的抗体仪器包含挤压气泵和存储池,磁铁移动,发光检测系统等功能,应可包含挤压装置、磁铁及移动装置、检测系统、控制分析模块和软件系统。 [0038] Antibodies of the present invention comprises a pressing device and storage pool pump, the magnet moves, the light emitting detection system feature, pressing means may comprise a magnet and a mobile device, a detection system, a control system analysis module and software.

[0039] 一种心肌肌钙蛋白I定量检测的磁微粒化学发光微流控芯片,其特征在于,所述微流控芯片的测试流程包括: [0039] A quantitative detection of cardiac troponin I magnetic particles chemiluminescence microfluidic chip, wherein the microfluidic chip testing process comprises:

[0040] 步骤1)将样本滴入加样口后,盖上盖子,微流控芯片放入配套仪器中,酶或发光剂标记抗体释放后,气泵使样本和标记抗体混合均匀,然后注入底板过滤区,所述配套仪器为小型便携设备,包含挤压气泵和存储池,磁铁移动,发光检测系统等功能; After [0040] Step 1) was added dropwise to the sample inlet, capped, into microfluidic chip supporting instrument, luminescent or enzyme-labeled antibody is released, the sample and labeled antibody pump mixed and then injected into the bottom plate filtration zone, the ancillary equipment is a small portable device, comprising a pump and an extrusion storage pool, moving magnet, luminescent detection system functions;

[0041] 步骤2)样本经过滤区后,到达包被区,溶解磁标抗体,充分反应后磁铁收集磁颗粒,存储池释放清洗液,将磁颗粒清洗后,移至检测区,释放发光基底液,仪器检测系统检测发光信号强度,进而实现cTnl的定量检测。 After [0041] Step 2) was filtered sample region, reaching the coating zone, a magnetic-labeled antibody was dissolved, the reaction was sufficiently magnet collecting magnetic particles, the release of the cleaning liquid storage tank, the magnetic particles after washing, the detection zone move, the release of luminescent substrate fluid, the instrument detection system for detecting signal emission intensity, so as to realize the quantitative detection of cTnl.

[0042]本发明的核心是采用磁微粒化学发光免疫检测技术在微流控芯片实现目标物的快速、高灵敏度、准确定量检测。 Core [0042] of the present invention is to use magnetic particles chemiluminescent immunoassay technology for rapid, high sensitivity in the target microfluidic chip, accurate quantitative detection.

[0043]微流控芯片技术是把生物、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,自动完成分析全过程。 [0043] The microfluidic chip technology to integrate sample preparation of biological, chemical, medical analysis process, reaction, separation, detection means to the basic operation of a microscale chip, the entire process be done automatically.

[0044]本发明的微流控芯片将检测过程所需的所有试剂组分(酶标cTnl抗体、磁颗粒标记cTnl抗体、清洗液、发光基底液等)均集成、内置到微流控芯片中,并通过巧妙沟道设计, 在配套伩器的操作下,实现微流控芯片的一键式检测(只需按开始键就能实现检测,无需复杂操作),实现全血分离、免疫反应、清洗分离、化学发光检测,从而避免了现有微流控芯片中结构设计简单、检测时操作复杂等不足和缺陷。 [0044] The microfluidic chip of the present invention all reagent components (enzyme-labeled antibody cTnl, cTnl antibody magnetic particle labels, washing liquid, luminescent substrate solution, etc.) required for the detection process are integrated, built into microfluidic chip and a channel through clever design, the supporting Xin's operation, to achieve one-touch detecting micro-fluidic chip (just press the start key detection can be achieved without complicated operation), to achieve separation of whole blood, immune response, clean separation, chemiluminescence, thereby avoiding the conventional simple microfluidic chip design, operation and complex defects and deficiencies detected. 还克服了传统化学发光仪只能进行血清或血浆检测,而不能对全血样本进行检测的缺点。 Also overcomes the conventional chemiluminescence analyzer can only be detected in serum or plasma, the disadvantages can not be performed on whole blood samples detected.

[0045]由于磁颗粒易沉淀,传统化学发光仪采用手工混合,并以持续振荡维持磁颗粒的悬浮状态,但微流控芯片内磁颗粒混均操作难以在小型便携仪器中实现。 [0045] Since the magnetic particles are easy to precipitate, conventional chemiluminescence instrument manually mixed and shaken continuously maintain the magnetic particles in suspension, but the microfluidic chip of magnetic particles in a blending operation is difficult to achieve a small portable instrument.

[0046]本发明将磁颗粒包被、干燥于微流控芯片沟道中,并设计了磁铁主动驱动磁颗粒(而传统微流控芯片一般采用流体驱动或电驱动),从而使磁颗粒复溶,并在微流控芯片不同区域实现免疫反应、清洗、发光。 [0046] The present invention magnetic particles are coated, dried microfluidic channel, and designed magnet actively driving the magnetic particles (traditional microfluidic chip generally employ fluid driven or electrically driven), so that the magnetic particles reconstitution , and to achieve an immune response in a microfluidic chips control different areas, clean, light. 此设计不仅解决了磁颗粒应用于微流控芯片时易沉淀、 重复性差等问题,还实现了更可控的免疫反应和物理清洗,提高了灵敏度和重复性。 This design not only solved the sedimentation of the micro-fluidic chip when applied to the magnetic particles, and poor reproducibility, but also to achieve a more controlled immune response and physical cleaning, improved sensitivity and reproducibility.

[0047]本发明中微流控芯片配套仪器与微流控芯片无液体接触,无需要清洗的部件,避免了传统大型化学发光仪需要搅拌或加样、清洗等操作而产生的交叉干扰及污染。 [0047] The present invention microfluidic chip ancillary equipment no liquid in contact with the microfluidic chip, no need to clean the components, to avoid the traditional large chemiluminescence analyzer requires stirring or pipetting, cleaning operations generated crosstalk and pollution .

[0048]所以本发明并非简单叠加磁微粒化学发光技术和微流控芯片技术,而是通过液体密封设计、沟道设计,把检测所需所有化学组分集成、内置到微流控芯片中,并以磁铁主动驱动,实现一键式的磁微粒化学发光免疫检测,从而在便携配套仪器中实现全血中cTnl的快速、高灵敏度、准确定量检测。 [0048] Therefore, the present invention is not a simple superposition of the magnetic particles and chemiluminescence microfluidic chip technology, but by the liquid seal design, channel design, all of the detected desired chemical components integrated, built into microfluidic chip, and is actively driven magnet, a magnetic one-button chemiluminescent microparticle immunoassay, in order to achieve rapid, high sensitivity cTnl in whole blood in a portable instrument package, an accurate quantitative detection.

[0049]本发明可应用于心血管疾病尤其是心力衰竭中cTnl的定量检测。 [0049] The present invention may be applied to the quantitative detection of cardiovascular disease, especially heart failure in cTnl.

[0050] 本发明的主要优点如下: [0050] The main advantages of the present invention are as follows:

[0051] 1)本发明采用化学发光方法,具有背景低、灵敏度高、线性范围宽的优点。 [0051] 1) The method of the present invention, chemiluminescence, having a low background, high sensitivity, wide linear range of advantages.

[0052] 2)本发明采用磁颗粒技术,具有磁富集功能,增强并放大信号;并能利用磁铁把磁颗粒转移区域(如由包被区_清洗区-检测区),减少样本基质的影响。 [0052] 2) The present invention adopts a magnetic particle technology, having a magnetic enrichment function, enhancing and amplifying the signal; magnet and can use the magnetic particle transfer area (e.g., coated by the cleaning zone region _ - detection area), reduce the sample matrix influences.

[0053] 3)本发明采用微流控芯片技术,把样本混合、反应、分离和检测集成在芯片上,并把反应所需的所有试剂组分集成到芯片上。 [0053] 3) The present invention employs microfluidic chip technology, the sample mixed, reaction, separation and detection integrated on the chip, and all the reagents required for the reaction components integrated on the chip.

[0054] 4)本发明操作简便,检测时,只需加入样本,盖上盖子,把芯片放入小型便携配套仪器中即可。 [0054] 4) The present invention is easy to operate, is detected, simply added to the sample, cover, the chip into a small portable instrument package can be.

[0055] 5)本发明配套仪器是小型便携仪器,仪器只与芯片发生物理接触,芯片内液体不与仪器接触,不会污染仪器而产生交叉干扰。 [0055] 5) supporting the apparatus of the present invention is a small portable instrument, the instrument occurs only in physical contact with the chip, the chip is not in contact with the liquid instrument, the instrument does not produce pollution crosstalk.

附图说明 BRIEF DESCRIPTION

[0056]图1为脑钠肽定量检测微流控芯片主体结构示意图,其中1为顶板,2为底板,3为气泵,4为加样口,5为标记抗体存储池,6为过滤区,7为磁颗粒包被区,8为检测区,9为清洗液存储池,10为发光基底液存储池,11为盖子,12为样本填充区,13为样本混合区,14为清洗区,15为废液池,16为液体释放通道,17为发光基底液和清洗液存储池让位孔(于顶板),1S 为磁铁滑轨让位孔。 [0056] FIG. 1 is a quantitative schematic natriuretic peptides in microfluidic chip body structure, wherein a top plate, 2 is a base plate, 3 is a pump, 4 is a loading port, 5 is a labeled antibody storage pool, 6 a filter zone, 7 is a magnetic particle coating zone, 8 is a detection zone, 9 is a cleaning solution storage pool 10 is a light emitting substrate solution storage pool 11 for the lid 12 is filled with the sample zone, 13 is a sample mixed zone 14 as a cleaning zone, 15 is a waste reservoir, a liquid release passage 16, the substrate 17 is a light emitting liquid and the washing liquid storage reservoir aperture give way (the top plate), a magnet rail lS hole giving way.

[0057]图2为脑钠肽定量检测的完整微流控芯片结构示意图,其中1为顶板,2为底板,19 为双面胶带,20为单面胶带,21为发光基底液和清洗液存储池让位孔(于双面胶带),22为混合液流入过滤区时的让位孔。 [0057] FIG. 2 is a schematic view of a complete micro controller chip structure quantitative flow of brain natriuretic peptide, wherein a top plate, bottom plate 2, a double-sided adhesive tape 19, a single-sided adhesive tape 20, 21 is a light emitting substrate, and a liquid cleaning solution pool hole giving way (in two-sided tape), giving way to the hole 22 when the liquid mixture flows into the filter area.

[0058]图3为双发光基底液的微流控芯片底板结构示意图,其中23为发光基底液存储池A,24为发光基底液存储池B,25为预混合通道。 A schematic view of a microfluidic CHIP floor structure [0058] FIG. 3 is a dual emission substrate solution, wherein the substrate 23 is a light emitting liquid storage pool A, 24 is a light emitting substrate solution storage pool B, 25 for the pre-mixing channel.

具体实施方式 detailed description

[0059]本发明公开了一种cTnl定量检测的微流控磁微粒化学发光方法及其专用微流控芯片,本领域技术人员可以借鉴本文内容,适当改进工艺参数实现。 [0059] The present invention discloses microfluidic chemiluminescent magnetic particle method and special microfluidic cTnl one kind of quantitative detection, one skilled in the art can learn from this article, appropriate modification of the process parameters to achieve. 特别需要指出的是,所有类似的替换和改动对本领域技术人员来说是显而易见的,它们都被视为包括在本发明。 Of particular note is that all such alterations and modifications to the skilled person are obvious, they are deemed to be included in the present invention. 本发明的方法及应用己经通过较佳实施例进行了描述,相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法和应用进行改动或适当变更与组合,来实现和应用本发明技术。 Method and Application already present invention has been described by means of preferred embodiments, without departing from the relevant art can obviously present invention within the spirit and scope appropriately changed and modified or combined according to the methods and applications described herein in to achieve technology and applications of the present invention.

[0060]为了使本领域的技术人员更好地理解本发明的技术方案,下面结合具体实施例对本发明作进一步的详细说明。 [0060] In order to enable those skilled in the art better understand the technical solution of the present invention, the following embodiments with reference to specific embodiments of the present invention will be further described in detail.

[0061] 实施例1:酶促化学发光测定cTnl [0061] Example 1: Determination of chemiluminescence cTnl

[0062] (一)抗体标记 [0062] (a) an antibody labeled

[0063] 取50yg HRP溶解于lmL蒸馏水中,再加入lOwnol新配NaI04溶液,室温避光反应20min后,以ImM pH4• 4醋酸钠缓冲液透析纯化溶液。 [0063] Take 50yg HRP was dissolved in lmL of distilled water, added with NaI04 lOwnol new solution, after the dark at room temperature the reaction 20min, to ImM pH4 • 4 sodium acetate buffer solution was purified by dialysis. 再以pH9 • 5碳酸盐缓冲液将pH调至9.0, 加入lOOyg抗cTnl单抗,室温避光反应2h。 Then to pH9 • 5 carbonate buffer adjusted to pH 9.0, addition of anti-cTnl monoclonal lOOyg dark at room temperature the reaction 2h. 加0. lmL 4mg/mL新配NaBH4,混句后于4°C反应2h。 Plus 0. lmL 4mg / mL with new NaBH4, 4 ° C after mixing period the reaction 2h. 将上述溶液装入透析袋,以0.15M pH7.4PBS透析,4°C过夜,得到HRP标记cTnl抗体。 The above solution was placed in a dialysis bag to 0.15M pH7.4PBS dialysis, 4 ° C overnight, to give the HRP-labeled antibody cTnl.

[0064] 向pH7 • 4磷酸缓冲液中加入lmg磁颗粒值径为2wn)、10yg EDC和15ygNHS溶液和10 〜30yg抗cTnl单抗(与HRP标记的抗体不同)溶液,混合均句并于室温下反应4h,加入lmg甘氨酸封闭。 [0064] The value of magnetic particles were added lmg to pH7 • 4 phosphate buffer diameter 2wn), 10yg EDC solution and 10 ~30yg 15ygNHS and anti-cTnl monoclonal antibody (HRP-labeled antibody different) solution were mixed at room temperature and sentences reaction 4h, glycine was added lmg closed. 以磁铁富集纯化,去除未反应的cTnl单抗,得到磁颗粒标记cTnl抗体。 Enrichment and purification magnet to remove unreacted mAb cTnl, cTnl antibody obtained magnetic particle labels.

[0065](二)微流控芯片组装 [0065] (ii) a microfluidic chip assembly

[0066] HRP 标记cTnl 抗体溶液中含1%BSA、0.2% 吐温20和0.05%Proclin300 的pH7.4硼酸缓冲液;磁颗粒标记cTnl抗体溶液为包含0.5 %BSA、2 %葡萄糖、1 %吐温-20和0 • 05 % Procl in300的pH7.4硼酸缓冲液。 [0066] HRP labeled cTnl antibody solution containing 1% BSA, 0.2% Tween 20 pH7.4 borate buffer and a 0.05% Proclin300; cTnl magnetic particle-labeled antibody solution containing 0.5% BSA, 2% glucose, 1% Tween borate buffer pH7.4 and the temperature of -20 0 • 05% Procl in300 of.

[0067] 将HRP标抗体溶液放入顶板标记抗体存储池中,密封。 [0067] The labeled antibody HRP labeled antibody solution was placed in the storage pool roof sealing. 将磁标抗体溶液放入底板磁颗粒包被区中,室温干燥。 The labeled antibody solution was placed in the magnetic plate is a magnetic particulate packet zone, dried at room temperature.

[0068] 清洗液为0.3 %BSA、0.2 %吐温-20和0.03 %Procl in300的pH7 • 2硼酸缓冲液。 [0068] The cleaning solution of 0.3% BSA, pH7 • 2 borate buffer, 0.2% Tween-20 and 0.03% Procl in300 of. 将清洗液注入清洗液存储池。 The cleaning liquid injected into the washing liquid storage reservoir. 发光基底液分为HRP底物(鲁米诺的双氧水溶液)和碱性增强液(苯衍生物的碱性溶液),分别注入发光基底液存储池A (23)和发光基底液存储池B (24)中,密封。 Luminescent HRP substrate into a substrate solution (luminol solution of hydrogen peroxide) and an alkaline enhancement solution (alkaline solution benzene derivative), were injected into the luminescent substrate solution storage pool A (23) and the light emitting substrate solution storage pool B ( 24), the sealing. 按图1所示,将过滤区粘入底板中。 As shown in Figure 1, the filter region into the viscous base plate. 然后按图2所示,以单面胶带和双面胶带,将顶板和底板组装成微流控芯片。 Then as shown in Figure 2, in a single-sided tape and double-sided tape, assembling the top and bottom plates into the microfluidic chip. 装入铝箔袋中,密封4 °保存。 Charged aluminum pouch, sealed storage 4 °.

[0069] (三)样本检测 [0069] (iii) samples tested

[0070] 用正常人血菜作稀释液,将cTnl标准品稀释成如下浓度:〇Pg/ml、50pg/ml、100pg/ ml、500pg/ml、lng/ml、5ng/ml、10ng/ml和50ng/ml。 [0070] Normal human blood as food dilutions standard cTnl diluted to the following concentrations: 〇Pg / ml, 50pg / ml, 100pg / ml, 500pg / ml, lng / ml, 5ng / ml, 10ng / ml and 50ng / ml.

[0071] 将50ul样本滴入加样口后,盖上盖子。 After [0071] 50ul of sample was added dropwise to the injection port, close the lid. 将微流控芯片放入配套仪器(磁铁磁感应强度为6000高斯)中,仪器挤出HRP标记单抗,并使样本和HRP标记单抗混合均匀后注入底板过滤区。 The microfluidic chip placed ancillary equipment (magnetic flux density of 6000 gauss), the extrusion apparatus HRP labeled monoclonal antibody, and HRP-labeled monoclonal antibody and the sample injection plate filtration zone mix evenly. 样本过滤后,到达微通道,并溶解磁颗粒标记单抗,磁铁加速样本反应,形成HRP标记单抗-cTnl抗原-磁颗粒标记单抗的三明治结构,然后磁铁收集磁颗粒。 After the sample was filtered and arriving microchannel, magnetic particles labeled monoclonal antibody and dissolved, the sample reaction accelerator magnets, HRP-labeled monoclonal antibody -cTnl formed antigen - labeled monoclonal antibody sandwich structure of a magnetic particle, and a magnet to collect the magnetic particles. 存储池释放清洗液, 将磁颗粒清洗后,发光基底液释放,仪器检测系统检测发光信号强度。 Releasing the storage pool cleaning liquid, the magnetic particles after washing, the substrate solution emission released, the instrument detection system detects luminescence signal intensity. 总检测时间15min。 The total detection time 15min. 每个样本分别用3个微流控芯片测定3次,取平均值,绘制标准曲线。 3 were used for each sample microfluidic chip was repeated three times to take the average, the standard curve.

[0072]将5〇ul全血样本滴入加样口,15分钟内伩器检测系统检测发光信号强度,依据标准曲线获得样本中cTnl浓度。 [0072] A whole blood sample was added dropwise to 5〇ul inlet, over 15 minutes Xin emission detection system detects the signal strength, the sample is obtained according to the standard curve the concentration of cTnl.

[0073]检测原理为:当全血加入微流控芯片后,全血先与HRP标记抗体混合,然后经过滤区后,混合了HRP标记抗体的血浆到达微通道,血浆溶解磁标记抗体。 [0073] Detection principle: When whole blood is added to the microfluidic chip, the whole blood is mixed with HRP-labeled antibody, and then after filtration zone, the plasma mixing HRP-labeled antibody reaches the microchannel, a magnetic-labeled antibody dissolved in plasma. 当血样中含有^!!〗,则形成HRP标记抗体-cTnl-磁颗粒标记抗体的三明治结构(双抗体夹心法)。 When the blood sample contains〗 ^ !!, HRP-labeled antibody sandwich -cTnl- magnetic particles-labeled antibody (double antibody sandwich method) is formed. 经清洗后,再发光基底液作用下发光,仪器检测系统测试发光信号。 After cleaned, re-emitting light-emitting action of the lower substrate solution, the instrument detection system test luminescent signal. 依据配套仪器获取的标准曲线,进而分析血样中cTnl浓度。 According to the standard curve ancillary equipment acquired and then analyzed blood samples cTnl concentration. 样本中cTnl含量越高,则发光信号越强。 The higher the content of cTnl in the sample, the stronger the light emission signal.

[0074]结果表明,其最低检测限为50pg/ml,最低定量限为200pg/ml,定量检测范围为0.05〜50ng/ml,线性相关系数R2>0.99;在检测范围内,未出现H〇〇K效应;且批内与批间重复性均小于10%。 [0074] The results show that the detection limit of 50pg / ml, the lowest limit of quantitation was 200pg / ml, quantitative detection range 0.05~50ng / ml, the linear correlation coefficient R2> 0.99; within the detection range, does not appear H〇〇 effect of K; and the inter-assay reproducibility from batch to less than 10%. 可为心梗心衰疾病诊断提供参考。 It can provide a reference for the diagnosis of myocardial infarction heart failure disease.

[0075]实施例2:直接化学发光测定cTnl [0076](一)抗体标记 [0075] Example 2: Direct Chemiluminescence cTnl [0076] (a) an antibody labeled

[0077]向磷酸缓冲液中加入适量活化的吖啶酯和1 〇〇ug抗cTn I单抗溶液,混合均匀后于室温下反应3h,加入lmg甘氨酸封闭。 [0077] adding an appropriate amount of the activated phosphate buffer at 3h acridinium ester and 1 〇〇ug monoclonal anti cTn I solution, mixed at room temperature after addition of lmg glycine closed. 透析分离纯化,得到吖啶酯标记cTnl抗体。 Dialysis separation and purification, to obtain acridinium ester labeled antibody cTnl.

[0078] 向lml 10mM pH7.4磷酸缓冲液中加入lmg磁颗粒(直径为lum)、20ug EDC和20ug NHS溶液和30ug链霉亲和素,混合均匀并于室温下反应4h,加入lmg甘氨酸封闭。 [0078] lmg of magnetic particles added to lml 10mM pH7.4 phosphate buffer (diameter lum), 20ug EDC solution and 30ug and 20ug NHS streptavidin, avidin, and mixed well for 4h at room temperature, glycine was added lmg closed . 以磁铁吸附富集,去除未反应的链霉亲和素,得到磁颗粒标记链霉亲和素。 Adsorption to the magnet, removing streptavidin unreacted biotin, magnetic particle labels give streptavidin biotin.

[0079] 将20ug抗cTnl单抗加入10uL 0.25mg/mL Sulfo-NHS-LC-biotin溶液中,反应lh。 [0079] The anti-cTnl monoclonal added 20ug 10uL 0.25mg / mL Sulfo-NHS-LC-biotin solution and the reaction lh. 超滤离心纯化,去除未反应的生物素,得到bi ot in-cTn I抗体。 Purification by centrifugal ultrafiltration to remove unreacted biotin, to obtain bi ot in-cTn I antibody.

[0080] 通过亲和素-生物素间的相互作用,把抗cTnl抗体连接到磁颗粒表面,得到磁颗粒标记cTnl抗体。 [0080] and by avidin -, connecting interaction between biotinylated anti-cTnl antibody to the surface of the magnetic particle, the magnetic particle labels give cTnl antibody. 其中亲和素标记的磁颗粒和生物素化的抗体比例在1:104〜2:105。 Wherein the ratio of the magnetic particles and the antibody biotinylated avidin labeled in 1: 104~2: 105.

[0081](二)微流控芯片组装 [0081] (ii) a microfluidic chip assembly

[0082] 吖啶酯标记cTnl抗体溶液中含0 • 5 %BSA、1 %甘油、0 • 2%吐温20、1 %曲拉通X-100 和0.1 %叠氮钠的pH7.4磷酸盐缓冲液;磁颗粒标记cTnl抗体溶液为包含0.2 %BSA、0.2 %酪蛋白、1%蔗糖、0.5%吐温20、0.5 %曲拉通X-100和0 • 1 %叠氮钠的pH7 • 4磷酸盐缓冲液。 [0082] cTnl acridinium ester labeled antibody solution containing 0 • 5% BSA, 1% glycerol, 0 • 2% Tween 20, 1% Triton X-100 and 0.1% sodium azide in phosphate pH7.4 buffer; cTnl magnetic particles labeled antibody solution containing 0.2% BSA, 0.2% casein, 1% sucrose, 0.5% Tween 20, 0.5% Triton X-100 and 0 • 1% sodium azide pH7 • 4 phosphate buffer. 将吖啶酯标记抗体溶液放入顶板标记抗体存储池中,密封。 The acridinium ester labeled antibody labeled antibody solution was placed in the storage pool roof sealing. 将磁标抗体溶液放入底板磁颗粒包被区中,室温干燥。 The labeled antibody solution was placed in the magnetic plate is a magnetic particulate packet zone, dried at room temperature.

[0083] 清洗液为0.3%834、0.5%吐温20、1%曲拉通)(-100和0.02%叠氮钠的?耵.2磷酸盐缓冲液。将清洗液注入清洗液存储池。发光基底液分为包含双氧水溶液和碱性溶液,分别注入发光基底液存储池A (23)和发光基底液存储池B (24),密封。按图1所示,将滤血膜粘入底板过滤区中,将存储池内置入底板。然后按图2所示,以单面胶带和双面胶带,将顶板和底板组装成微流控芯片。装入铝箔袋中,密封4°保存。 [0083] The cleaning solution of 0.3% 834,0.5% Tween 20, 1% Triton) (-? 100 and 0.02% sodium azide in phosphate buffer Ding .2 cleaning liquid injected into the washing liquid storage reservoir. luminescent substrate solution into a solution containing hydrogen peroxide and the alkaline solution were injected into the luminescent substrate solution storage pool A (23) and the light emitting substrate solution storage pool B (24), seal shown in Figure, the viscosity hemofilter membrane 1 into the base. filtration zone into the storage pool floor. then, as shown in Figure 2, single-sided tape and double-sided tape to the top and bottom plates are assembled into the microfluidic chip. was charged aluminum pouch, sealed storage 4 °.

[0084] (三)样本检测 [0084] (iii) samples tested

[0085] 用正常人血楽作稀释液,将cTnl标准品稀释成如下浓度:〇pg/ml、100pg/ml、 500pg/ml、lng/ml、5ng/ml、10ng/ml和50ng/ml。 [0085] Normal human blood yue for dilution, diluted to the following concentrations cTnl standards: 〇pg / ml, 100pg / ml, 500pg / ml, lng / ml, 5ng / ml, 10ng / ml and 50ng / ml.

[0086] 将50ul样本滴入加样口后,盖上盖子。 After [0086] 50ul of sample was added dropwise to the injection port, close the lid. 将微流控芯片放入配套伩器(磁铁磁感应强度为4000高斯)中,仪器挤出吖啶酯标记单抗,并使样本和吖啶酯标记单抗混合均匀后注入底板过滤区中。 The microfluidic chip device into the supporting Xin (magnetic flux density of 4000 gauss), the extrusion apparatus acridinium ester labeled monoclonal antibody and the sample and labeled monoclonal acridinium ester mixed uniformly implanted in the filtration zone plate. 样本过滤后,到达微通道,并溶解磁颗粒标记单抗,磁铁加速样本反应,形成吖啶酯标记抗体-cTnl抗原-磁颗粒标记抗体的三明治夹心结构,然后磁铁收集磁颗粒。 After the sample was filtered and arriving microchannel, magnetic particles labeled monoclonal antibody and dissolved, to accelerate the magnet sample reaction, acridinium ester-labeled antibody is formed -cTnl antigen - sandwich sandwich of magnetic particles-labeled antibody, and then collecting the magnetic particles the magnet. 存储池释放清洗液,将磁颗粒清洗后,发光激发液释放,仪器检测系统检测发光信号强度。 After the release of the storage pool cleaning solution, the cleaning of magnetic particles, liquid release emitting excitation, the instrument detection system detects luminescence signal intensity. 总检测时间15min。 The total detection time 15min. 每个样本分别用3个微流控芯片测定3次,取平均值,绘制标准曲线。 3 were used for each sample microfluidic chip was repeated three times to take the average, the standard curve.

[0087]将5(HU血浆样本滴入加样口,15分钟内仪器检测系统检测发光信号强度,依据标准曲线获得样本中cTnl浓度。 [0087] The 5 (HU dropwise plasma sample loading port, the instrument detection system detects the intensity of luminescence signal within 15 minutes, the sample is obtained according to the standard curve the concentration of cTnl.

[0088] 检测原理为:当全血加入微流控芯片后,全血先与吖啶酯标记抗体混合,然后经过滤区后,混合了吖啶酯标记抗体的血浆到达微通道,血浆溶解磁标记抗体。 [0088] Detection principle: when the whole blood was added microfluidic chips, whole blood is first labeled antibody acridinium ester mixture, and then after filtration zone, mixing the plasma acridinium ester labeled antibody reaches the microchannel, plasma dissolution magnetic labeled antibody. 当血样中含有cTnl,则形成吖啶酯标记抗体-cTnl-磁颗粒标记抗体的三明治结构(双抗体夹心法)。 When the blood sample contains cTnl, is formed acridinium ester labeled antibody sandwich -cTnl- magnetic particles-labeled antibody (double antibody sandwich method). 经清洗后,发光激发液释放,经混合后与吖啶酯作用产生直接化学发光,仪器检测系统测试发光信号。 After washing, liquid release emitting excitation, direct chemiluminescence detection system test instruments acridinium ester and a luminescent signal through the mixed action. 依据配套仪器获取的标准曲线,进而分析血浆中cTnl浓度。 According to the standard curve ancillary equipment acquisition, and then analyze the plasma concentration cTnl. 血浆中cTnl含量越高,则发光信号越强。 CTnl higher plasma level, the stronger the light emission signal.

[0089] 结果表明,其最低检测限为8〇pg/ml,最低定量限为300pg/ml,定量检测范围为0 • 08〜50ng/ml,线性相关系数R2>0 • 99;在检测范围内,未出现HOOK效应;且批内与批间重复性均小于10%。 [0089] The results show that the detection limit of 8〇pg / ml, the lowest limit of quantitation was 300pg / ml, quantitative detection range 0 • 08~50ng / ml, the linear correlation coefficient R2> 0 • 99; in the range of detection , HOOK effect does not appear; and the inter-assay reproducibility from batch to less than 10%. 可为心梗心衰疾病诊断提供参考。 It can provide a reference for the diagnosis of myocardial infarction heart failure disease.

[0090]实施例3:磁微粒颗粒尺寸筛选 [0090] Example 3: Screening of the particle size of the magnetic particles

[0091]其他的实验条件参见实施例2,磁颗粒尺寸和磁铁磁感应强度按照以下方案进行。 See Embodiment [0091] Other experimental conditions as Example 2, the particle size of the magnetic flux density and the magnet according to the following scheme. [0092]颗粒尺寸为0 • lwn、0 • 5_、1 • Own、2 _ Owm、2 • 4ym、3wn、10ym。 [0092] The particle size of 0 • lwn, 0 • 5_, 1 • Own, 2 _ Owm, 2 • 4ym, 3wn, 10ym. 磁铁磁感应强度为500 高斯、1000高斯、4000高斯、8000高斯、12000高斯、3〇〇〇〇高斯。 Magnet magnetic induction of 500 gauss, 1000 gauss, 4000 gauss, 8000 gauss, 12,000 Gauss, 3〇〇〇〇 gauss. 分别以这六种磁铁分别驱动七种尺寸的磁颗粒。 The six magnets respectively drive the seven sizes of the magnetic particles.

[0093]实验结果显示:〇. 1M磁颗粒和500高斯磁铁组合时,其最低检测限为500pg/ml,定量检测范围为〇.5〜50ng/ml,线性相关系数R2>〇.95;批内与批间重复性均小于20%。 [0093] The results show: 0:00 1M combination of magnetic particles and the magnet 500 gauss, the minimum detection limit of 500pg / ml, quantitative detection range 〇.5~50ng / ml, the linear correlation coefficient R2> 〇.95; batch. internal and inter-assay reproducibility were less than 20%. 即: 化学发光信号较弱,灵敏度不高,重复性较差。 Namely: a chemiluminescent signal is weak, sensitivity is not high, poor reproducibility.

[0094] 10M磁颗粒和30000高斯磁铁组合时,其最低检测限为4〇〇pg/ml,定量检测范围为0.4〜5ng/ml,线性相关系数R2>0_95;批内与批间重复性均小于20%。 [0094] When the magnetic particles and the magnet assembly 10M 30,000 gauss, the minimum detection limit of 4〇〇pg / ml, quantitative detection range 0.4~5ng / ml, the linear correlation coefficient R2> 0_95; intra-assay and inter-assay reproducibility was less than 20%. 即:阴性样本信号较高(清洗不充分),线性范围不宽。 That is: the higher negative samples signal (insufficient cleaning), the linear range is not wide.

[0095] 0_5〜3wn的磁颗粒为和1000〜8〇〇〇高斯的磁铁组合使用时,其最低检测限均小于150pg/ml,定量检测范围可达到0 • I5〜50ng/ml,线性相关系数R2>〇• 97;批内与批间重复性均小于12%。 [0095] When 0_5~3wn magnetic particles and a magnet assembly 1000~8〇〇〇 Gaussian use, which was less than the detection limit 150pg / ml, quantitative detection range up to 0 • I5~50ng / ml, the linear correlation coefficient R2> square • 97; intra-assay and inter-assay reproducibility was less than 12%. 满足为临床心梗心衰疾病诊断提供参考的需要。 Meet the need to provide reference for the clinical diagnosis of myocardial infarction heart failure disease.

[0096]根据以上结果,磁颗粒尺寸优选0.5〜3um,磁铁磁感应强度优选1〇〇〇〜8〇〇〇高斯; 磁颗粒尺寸更优选1_〇〜3wii,磁铁磁感应强度4000〜8〇00高斯。 [0096] From the above results, the magnetic particle size is preferably 0.5~3um, preferably 1〇〇〇~8〇〇〇 magnet magnetic flux density gauss; magnetic particle size is more preferably 1_〇~3wii, magnet magnetic flux density gauss 4000~8〇00 . 可根据磁颗粒所用尺寸,进一步确定磁铁磁感应强度。 The magnetic particles used may be size, a further determination magnet magnetic induction.

[0097]以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若千改进和润饰,这些改进和润饰也应视为本发明的保护范围。 [0097] The above are only preferred embodiments of the present invention, it should be noted that those of ordinary skill in the art, in the present invention without departing from the principles of the premise, may also be made if one thousand improvements and modifications of these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims (10)

  1. 1. 一种定量检测全血中肌钙蛋白I的磁微粒化学发光微流控芯片,其特征在于,所述微流控芯片包括顶板(1)结构和底板⑵结构,其中顶板⑴上的气栗(3)、加样口⑷、样本填充区(12)、标记抗体存储池⑸和样本混合区(13)依次连接;底板上的过滤区(6)、磁颗粒包被区⑺、清洗区(14)、检测区⑻、液体释放通道(16)依次连接;底板的检测区⑻分别与清洗液存储池(9)和发光基底液存储池(1〇)通过液体释放通道(16)连接; 所述标记抗体存储池(5)存储预封装酶或发光剂标记抗cTnl抗体,磁颗粒包被区(7)包被预封装磁颗粒标记抗cTnl抗体,清洗液存储池(9)和发光基底液存储池(10)存储预封装清洗液和发光基底液;所述微流控芯片测试流程中,用磁铁操控磁颗粒移动或聚集;所述标记抗体存储池、清洗液存储池和发光基底液存储池为液体密封池,通过外力挤压而局部破裂 A quantitative determination of troponin I in whole blood chemiluminescence magnetic particles microfluidic chip, wherein the microfluidic chip comprises a plate (1) and the bottom plate structure ⑵ structure, wherein the gas on the top plate ⑴ Li (3), a loading port ⑷, filled with the sample region (12), labeled antibody storage pool ⑸ and sample mixing zone (13) sequentially connected; filter region (6) on the bottom plate, the magnetic particles are coated ⑺ region, cleaning zone (14), the detection zone ⑻, fluid release passage (16) sequentially connected; ⑻ detection zone are connected to the base plate cleaning fluid storage tank (9) and the light emitting substrate liquid storage tank (1〇) release passage (16) through the liquid; the labeled antibody storage tank (5) stored in the pre-packaged or luminescent enzyme-labeled anti-cTnl antibody, the magnetic particles are coated area (7) coated magnetic particles pre-encapsulated labeled anti-cTnl antibody, the washing liquid storage tank (9) and the light emitting substrate a liquid storage tank (10) storing pre-packaged liquid cleaning solution and a light emitting substrate; the microfluidic chip testing process, the magnetic particles move with a magnet or aggregation of control; the labeled antibody storage pool and the storage pool cleaning solution luminescent substrate solution pool the storage pool is fluid-tight, locally broken by an external force pressing 释放液体;所述过滤区包含滤血膜,所述顶板(1)与底板(2)用胶带(I9和20)密封。 Releasing the liquid; the filtration zone comprises a hemofilter membrane, said top plate (1) and the base plate (2) with tape (I9 and 20) sealed.
  2. 2. 如权利要求1所述的微流控芯片,其特征在于发光基底液保质期少于1年时应分开, 用发光基底液存储池A (23)和发光基底液存储池B (24)替代发光基底液存储池(10),所述发光基底液存储池A (23)和发光基底液存储池B (24)通过预混合通道(25)连接。 2. The microfluidic chip according to claim 1, characterized in that the luminescent substrate solution should be less than 1 year shelf life separated liquid with the light emitting substrate storage pool A (23) and the light emitting substrate solution storage pool B (24) alternatively luminescent substrate solution storage tank (10), the storage pool luminescent substrate solution A (23) and the light emitting substrate solution storage pool B (24) are connected by a pre-mixing channel (25).
  3. 3. 如权利要求1所述的芯片,其特征在于,所述磁颗粒标记抗cTnl抗体使用的磁颗粒尺寸为0.1〜10M1;与磁颗粒匹配的磁铁磁感应强度为500〜3〇〇〇〇高斯。 3. The chip according to claim 1, wherein the particle size of magnetic particles magnetically labeled antibody used was an anti-cTnl 0.1~10M1; matching with the magnetic particles for the magnetic flux density gauss 500~3〇〇〇〇 .
  4. 4. 如权利要求3所述的芯片,其特征在于,所述磁颗粒标记抗cTnI抗体使用的磁颗粒尺寸为0.5〜3mi,与磁颗粒匹配的磁铁磁感应强度为1〇〇〇〜8000高斯。 4. The chip according to claim 3, wherein said anti-cTnI antibody labeled magnetic particles using a magnetic particle size 0.5~3mi, match Magnetic Particles magnet magnetic induction 1〇〇〇~8000 gauss.
  5. 5. 如权利要求1所述的微流控芯片,其特征在于,所述酶或发光剂标记cTnl抗体溶液、 磁颗粒标记cTnl抗体溶液和清洗液均包含缓冲液、蛋白质、表面活性剂和防腐剂,且发光剂标记cTnl抗体溶液还包含甘油,磁颗粒标记cTnl抗体溶液还包含糖类。 5. The microfluidic chip according to claim 1, wherein said luminescent or enzyme-labeled antibody solution cTnl, cTnl magnetic particles labeled antibody solution and washing solution contains a buffer, a protein, a surfactant and preservative agents, and luminescent-labeled antibody solution further comprises glycerin cTnl, cTnl antibody labeled magnetic particles further comprises a saccharide solution.
  6. 6. 如权利要求1或5所述的微流控芯片,其特征在于,所述酶标记cTnl抗体溶液包含牛血清白蛋白(BSA)、吐温-20和Proclin300的硼酸缓冲液;磁颗粒标记cTnl抗体溶液包含BSA、葡萄糖、吐温-20和Proclin3〇0的硼酸缓冲液;所述清洗液包含BSA、吐温-20和Proclin300的硼酸缓冲液。 6. The microfluidic chip of claim 1 or claim 5, wherein said solution comprising enzyme-labeled antibody cTnl bovine serum albumin (BSA), Tween-20 and Proclin300 borate buffer; a magnetic particle labels cTnl antibody solution containing BSA, glucose, Tween-20 and Proclin3〇0 borate buffer; the cleaning liquid containing BSA, Tween-20 and borate buffer of Proclin300.
  7. 7. 如权利要求1所述的微流控芯片,其特征在于,所述发光剂标记cTnl抗体溶液包含BSA、甘油、吐温-20、曲拉通X-100和叠氮钠的磷酸盐缓冲液;所述磁颗粒标记cTnl抗体溶液包含BSA、酪蛋白、蔗糖、吐温-20、曲拉通X-100和叠氮钠的磷酸盐缓冲液;所述清洗液包含BSA、吐温20、曲拉通X-100、聚乙二醇和叠氮钠的磷酸盐缓冲液。 7. The microfluidic chip according to claim 1, characterized in that the luminescent-labeled antibody solution containing cTnl BSA, glycerol, Tween-20, Triton X-100 and sodium azide in phosphate buffered liquid; cTnl said magnetic particles labeled antibody solution containing BSA, casein, sucrose, Tween-20, Triton X-100 and sodium azide phosphate buffer; the cleaning liquid containing BSA, Tween 20, Triton X-100, polyethylene glycol and sodium azide phosphate buffer.
  8. 8. 如权利要求1所述的微流控芯片,其特征在于,所述发光基底液包含与酶对应的底物及发光增强液,合并后注入发光基底液存储池(10),或分别注入发光基底液存储(23)和发光基底液存储池B(24)。 8. The microfluidic chip according to claim 1, wherein said luminescent substrate solution containing a substrate for the enzyme and the light emitting enhancement solution, was injected into the light emitting substrate storage tank (10) after the merger, or were injected storage luminescent substrate solution (23) and the light emitting substrate solution storage pool B (24).
  9. 9. 如权利要求1所述的微流控芯;片,其特征在于,所述发光基底液包含发光剂对应的双氧水溶液和碱性溶液,合并后注入发光基底液存储池(10),或分别注入发光基底液存储池A (23)和发光基底液存储池B G4)。 9. The microfluidic core according to claim 1; sheet, wherein said luminescent agent comprises a luminescent substrate corresponding to the liquid hydrogen peroxide solution and an alkaline solution, was injected into the light emitting substrate storage tank (10) after the merger, or substrate solution were injected into the luminescent storage pool A (23) and the light emitting substrate solution storage pool B G4).
  10. 10. 如权利要求1所述的微流控芯片,其特征在于,所述微流控芯片的测试流程包括: 步骤1)将样本滴入加样口后,盖上盖子,微流控芯片放入配套仪器中,酶或发光剂标记抗体释放后,气泵使样本和标记抗体混合均勾,然后注入底板过滤区,所述配套仪器为小型便携设备,包含挤压气泵和存储池,磁铁移动,发光检测系统等功能; 步骤2)样本经过滤区后,到达包被区,溶解磁标抗体,充分反应后磁铁收集磁颗粒,存储池释放清洗液,将磁颗粒清洗后,移至检测区,释放发光基底液,仪器检测系统检测发光信号强度,进而实现cTnl的定量检测。 10. The microfluidic chip according to claim 1, wherein the microfluidic chip testing process includes: Step 1) was added dropwise to the sample inlet, cover, microfluidic chip discharge the ancillary equipment, the enzyme that releases the antibody is labeled or a luminescent agent, a labeled antibody and the sample pump are mixing hook, filtered and then injected into the floor region, the supporting device is a small portable device, comprising a pump and an extrusion storage pool, moving magnet, luminescent detection system functions; step 2) was filtered sample region, reaching the coating zone, a magnetic-labeled antibody was dissolved, the reaction was sufficiently magnet collecting magnetic particles, the release of the cleaning liquid storage tank, the magnetic particles after washing, the detection zone move, luminescent substrate release fluid, the instrument detection system for detecting signal emission intensity, so as to realize the quantitative detection of cTnl.
CN 201510696728 2015-10-26 2015-10-26 Quantitative detection of magnetic particles in whole blood troponin i-emitting chemical microfluidic CN105435868B (en)

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