基于数字微流控芯片的交叉配血及磁珠免疫固相载体操控的探讨

发布时间：2018-03-13 05:36

本文选题：自动交叉配血实验　切入点：临床免疫检测　出处：《大连医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：背景:输血现已成为临床治疗的重要组成部分,临床输血工作的最重要原则为保证每一例输血的安全性。在现代临床医学检验方法中,交叉配血试验是保证输血安全的重要手段,它是将供血者红细胞和血清分别与受血者血清和红细胞交叉混合,检查受血者血清中有无破坏供血者红细胞的抗体,当受血者和供血者的血液间没有可测的不相配的抗原、抗体成分时,即试验结果无溶血无凝集时,方可将供者血液成分输注给受血者。临床中通常采用的配血方式存在一些问题,如依赖人工操作、自动化程度低、试剂和样品的消耗较多;另一方面,自动配血仪的仪器成本和运行费用高,对疑难案例仍然需要人工判断,降低了筛测效率和准确性。在近年的免疫诊断的技术发展中,床边诊断因其人工参与度低、可自动化运行和记录试验结果、试剂与样品消耗小、检测时间缩短而受到关注,而免疫检测平台的缩微化有利于床边诊断的实现。其中,微流控芯片是一种新型的分离分析技术,目前已经成为分析化学和生化检测的重要平台技术。引入微流控等缩微化的新方法,将有利于交叉配血实验的自动化,这也成为免疫检测中的新思路。超磁磁珠因其比表面积大、可以被外加磁场操控,已经成为免疫检测,如酶联免疫吸收检测和化学发光免疫分析等方法中的重要固相样品载体,但检测过程中需要反应、封闭、分离和洗涤等多步复杂操作,且自动化程度低。因此,建立一种能够自动实现免疫检测样品处理步骤中多步复杂操作的平台和载体,将对提高免疫检测诊断的效率,具有重要的实用意义。目的:本课题拟构建基于微流控芯片的免疫检测和样品处理平台,首先通过将传统配血实验中采用的各种血液样品和免疫试剂装载于单个微液滴中,利用数字液滴芯片对液滴的自动操控功能,在芯片上实现血红细胞和血清间的交叉反应实验,在概念上验证数字液滴芯片作为免疫床边检测平台的可行性。以超磁磁珠作为免疫样品的固相载体装载于液滴中,结合数字液滴的操控功能与磁珠的载体功能,并辅以磁珠固定模块,探索建立免疫检测样品处理步骤中自动实现多步复杂操作体系的可能性。方法:1.构建基于数字液滴微流控微反应器平台,取得适合配血试验中实际样品液滴输运的芯片绝缘工艺和液滴改性方案。2.选取2016年4~5月间大连市中心医院收治的需要输注红细胞治疗的住院患者,排除输血前检测抗体筛查阳性者,共32例,其中男性21例,女性11例,年龄28~78岁;A型8份,B型12份,O型10份,AB型2份,Rh D均为阳性。与献血员血液同型进行盐水法交叉配血,同时利用数字液滴芯片对液滴的自动操控功能,在芯片上实现交叉配血,观察融合液滴中是否出现抗原-抗体的凝集反应与盐水法结果比较。3.构建固相免疫载体磁珠的固定模块,探索在数字液滴芯片上进行磁珠反应、封闭、分离和洗涤的条件。结果:1.构建了包括数字微流控芯片内的数字微流控微反应器。得到了适合实际样品的芯片制作配方和液滴改性配方,消除了含蛋白质液滴对芯片表面发生生物污损的机会。2.在数字微流控芯片表面上尝试了交叉配血实验,实验结果与传统方法完全一致。而芯片法实验呈现阳性时,第8秒就可以观察到红细胞凝集的发生,而且样品消耗量仅为1.2微升,为传统方法的1/20-1/40。3.通过将磁珠置于液滴中,并构建强磁体和磁导构成的磁珠钳制模块,将此模块与数字液滴操控平台结合,实现了对液滴中磁珠样品的有效分离、与反应物结合、利用缓冲液对磁珠进行洗涤等,在此基础上进行了样品和试剂的多步反应、分离与清洗的自动化操作,初步满足了床边免疫诊断中样品操控的需求。这些步骤均为磁珠酶联免疫及化学发光免疫分析检测的基础操作单元。结论:1.成功构建了以数字微流控芯片为核心的数字微流控微反应器,实现了液滴的移动、融合、分离与等份分裂等基本操控功能。2.在数字微流控芯片上进行的交叉配血实验,其实验结果与传统方法完全一致,反应时间和样品消耗得到大幅度减少,芯片法中反应时间和样品消耗的减少得益于液滴体系的缩微化,在概念上验证了数字液滴芯片作为免疫凝集反应实验平台的可行性。3.将免疫反应的重要样品固相载体-磁珠装载与液滴中,实现了将磁珠钳制模块与数字液滴操控平台结合,初步进行了芯片上的多步反应、分离与清洗的自动化操作,建立了一种能够自动实现免疫检测样品处理步骤中多步复杂操作的体系。
[Abstract]:Background: blood transfusion has become an important part of clinical treatment, the most important principle of clinical transfusion work to ensure the safety of every case of blood transfusion. In modern clinical medicine inspection method, cross matching test is an important means to ensure the safety of blood transfusion, it is the blood red blood cells and serum and recipient serum respectively. And the red cross cell mixed antibody test in serum of blood recipients without destruction of red blood cells when blood donors, recipients and donors were not matched between blood antigen no measurable antibody components, namely, the test results of hemolysis without agglutination, can the donor blood component transfusion to blood recipients. There are some problems with the commonly used method in clinical blood, such as rely on manual operation, low degree of automation, reagent and sample consumption more; on the other hand, equipment cost and operation cost of automatic matching for high, the case is still difficult You need artificial judgment, reduce the screening efficiency and accuracy. In the development of technology in recent years in the immunological diagnosis, bedside diagnosis because of its artificially low participation, it can run automatically and record test results, reagent and sample consumption, shorten the detection time and attention, and the immune detection platform is conducive to the realization of microcopying bedside diagnosis. Among them, the microfluidic chip analysis technique is a new separation technology, has become an important platform for the analysis of chemical and biochemical detection. The introduction of new methods such as microfluidic microcopying, there will be conducive to automated blood cross matching test, it has also become a new idea in the immunoassay ultra magnetic beads. Because of its large surface area, the magnetic field can be manipulated, has become an important carrier of solid phase immunoassay, enzyme-linked immunosorbent assay as sample absorption measurement and chemiluminescence immune analysis method, but the detection process Need to be closed, reaction, separation and washing steps such as complex operation, and low degree of automation. Therefore, to build a automatic immune detection sample processing steps in step complex operation platform and carrier, will be to improve the efficiency of immune detection and diagnosis, has important practical significance. Objective: This study intends to build immunity microfluidic chip detection and sample processing platform based on the first by using the traditional matching experiments in various blood samples and immune reagent loaded on single droplet, automatic control of droplet droplet using digital chip, realizes the cross reaction experiment of red blood cells and serum in the chip. The concept of verification of digital droplet chip as the feasibility of immune bedside testing platform. With super magnetic beads as solid carrier immune sample loading in the liquid droplets, combined with the control function and magnetic digital droplet The carrier function of beads, and beads with fixed module, to explore the possibility of multi-step operation system automatically establish immunoassay sample processing steps. Methods: 1. construction of digital microfluidic droplet based micro reactor platform, made suitable for blood test in actual sample droplet transport process and droplet change chip insulation for the month of 2016 4~5.2. selected from Dalian central hospital to red blood cell transfusion patients, detection of antibody positive were excluded before blood transfusion, a total of 32 cases, including 21 cases of male, female 11 cases, age 28~78; 8 A, 12 B, 10 O. 2 AB, Rh and D were positive. Blood donors were saline crossmatching of blood with the same type, using digital chip automatic control function of droplet droplet, realize the cross matching of blood on the chip, to observe the fusion antigen antibody is the emergence of the liquid drop in anti agglutination With the saline method compared the results of.3. fixed modules to build a solid phase immune carrier beads, beads of reaction in the digital droplet chip sealing, separation and washing conditions. Results: 1. constructs including digital microfluidic chip digital microfluidic reactor. The suitable sample chip production formula and the droplet modified formula, eliminating the protein droplet on the surface of the chip biofouling happening.2. in the digital microfluidic chip on the surface to blood cross matching test, the experimental results with the traditional method of exactly the same. While the chip method experiment were positive, eighth seconds can observe the agglutination of red blood cells, and sample consumption is only 1.2 L, as the traditional method of 1/20-1/40.3. by magnetic bead droplet placed, and build a strong magnet and magnetic bead clamping module, this module and digital droplet manipulation The platform combined, realize effective separation of bead sample droplets, combined with reactants, washing the beads using buffer, based on the reaction of samples and reagents, automation of separation and cleaning, to meet the initial sample manipulation in bedside immune diagnosis demand. These steps are as the bead ELISA and chemiluminescence immunoassay detection. Conclusion: the 1. basic operation unit is successfully constructed with digital micro fluidic chip as the core of the digital microfluidic reactor, the moving droplet fusion, blood cross matching test and separation of equal division basic operation functions of.2. in digital microfluidics on chip, the experimental results and the traditional method is completely consistent, reaction time and sample consumption is greatly reduced, the chip method reaction time and sample consumption reduced due to the system of micro droplet In concept, to verify the feasibility of.3. chip as digital droplet immune agglutination reaction experiment platform will carry important samples of solid carrier - immunoreactive beads and droplets, the magnetic clamping module and digital droplet manipulation platform with a preliminary multi-step reaction on a chip, automatic operation and separation cleaning, to establish a new automatic immunoassay sample processing steps in step complex operating system.

【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R446.6

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