集成微流控芯片及单细胞基因表达检测研究

发布时间：2018-04-22 23:04

本文选题：微/纳制造 + 微流控芯片　；参考：《哈尔滨工业大学》2015年博士论文

【摘要】：微/纳制造主要研究特征尺寸在微米、纳米范围的功能结构、器件与系统在设计、制造中的科学问题,是衡量国家制造水平的标志之一,代表着目前制造科学的最前沿。细胞是生命结构和功能的基本单位,活体单细胞基因表达检测是细胞生命分析技术的极限状态,对于揭示重大疾病发生与演进机理、促进新型药物研发等具有重要意义,正在成为基础生物学、临床医学等领域的国际前沿课题。本文将微/纳制造技术与单细胞基因表达检测结合,研制出微流控芯片系统使常规生化试验的多步骤操作全部集成微缩到单块芯片,实现单细胞内低丰度mRNA样本的萃取、扩增与检测,主要研究内容有以下几个方面。设计布局清晰、结构简单、操纵更灵活的两类芯片微系统,即单工作单元式和微流控阵列式芯片。前者利于实现便携操作和分析,后者提高了检测效率和通量。两类芯片均为多层结构,包含微流动层、控制层、薄膜夹层和含加热器的微传感器,在加工中改进同类芯片工艺,解决了工程实际中PDMS薄膜局部粘附、热循环反应试剂蒸发和传感器复用性等问题。此外,基于固体、流体传热学理论,借助通用商业软件对芯片含加热器的微传感器进行建模与仿真研究,评估该设计的温控性能。研发试验系统平台,共包含4个部分,即微流控平台、温度控制平台、细胞培养试验平台和生物信号检测平台。其中,微流控平台由无极调速微注射泵、液氮池及整流阀和显微镜组成,用于实现活体单细胞操纵及多种反应试剂的精确、稳定进样;温度控制平台基于虚拟仪器技术,开发了控制程序和人机交互界面,以实现芯片反应温度的实时检测和精确控制;细胞培养试验平台由层流生物试验台、细胞培养箱、离心机等组成,用于细胞样本制备、生化试剂配制和活体细胞的药物预处理等操作;生物信号检测平台由荧光倒置显微镜、CCD及相关图像采集与分析软件组成,用于实现生物荧光信号的高信噪比、实时精确测量和分析。针对微流控系统中流体对细胞的水动力作用难以试验测量,且细胞活性受水动力影响较大,本文应用计算流体力学、弹性力学和塑性力学理论,借助任意拉格朗日-欧拉法动力学描述、动网格技术和参数化数值求解方法,建立并求解两类芯片的2D、3D细胞-微流体流固耦合有限元模型。通过模拟细胞捕捉瞬态过程,分析流场中细胞所受的多属性水动力作用,量化芯片入口载流流速与细胞表面压力、应力分布的关系。并且,将芯片上的细胞受力与正常人体动脉血管、毛细血管和组织液中的细胞受力进行对比,预测试验参数对细胞生理活性的影响。选取MCF-7人乳腺癌细胞系,基于微磁珠固相萃取技术及实时荧光定量反转录聚合酶链反应(RT-qPCR)分子扩增与检测技术,提出全集成检测方法。该方法将微流控单细胞捕捉、细胞裂解、mRNA萃取、反转录、基因片段扩增与实时定量检测等所有试验步骤完全集成到一个芯片,在简化操作、缩短周期的同时提高低丰度mRNA样本的保持度,并由此提高检测精度、灵敏度和信噪比。本文依次评估了方法可行性和可重复性,优选了细胞操纵与mRNA固相萃取试验方案,分析了该方法与传统方法的效能差异等;并且,检测了MMS诱导前后单细胞内持家基因GAPDH与细胞周期调控基因CDKN1A表达水平的差异,验证其药代动力学机理;此外,分别建立MMS剂量、用药时间对CDKN1A表达调控的影响,提出与本试验条件对应的最优治疗方案。在理论上,本文改善了现有研究方法的集成度、多功能性和实验操作的自动化水平,并对同类单细胞、单分子生化分析具有普适性;在工程实际中,降低了现有芯片设计、制造和操作中普遍存在的复杂度、高成本和高消耗,且可根据需求变更而灵活调整,具有一定的产业化前景。研究方法与结论在推进微/纳制造技术与基础科学研究结合,促进基础生物医学研究,提升临床诊治水平等方面具有一定的价值和意义。
[Abstract]:Micro / nano manufacturing mainly studies the functional structure of micrometers, nano scale, and the scientific problems in the design and manufacture of devices and systems. It is one of the symbols to measure the level of national manufacturing. It represents the forefront of the current manufacturing science. Cells are the basic units of life structure and function, and the detection of living single cell gene expression is cell life. The limit state of life analysis technology is of great significance for revealing the mechanism of the occurrence and evolution of major diseases and promoting the development of new drugs. It is becoming an international frontier subject in the fields of basic biology and clinical medicine. In this paper, micro / nano manufacturing technology is combined with single cell gene expression detection, and the microfluidic chip system has been developed to make the routine. The multi-step operation of the biochemical test is fully integrated into a single chip to achieve the extraction, amplification and detection of low abundance mRNA samples in single cell. The main contents are the following aspects: two kinds of chip microsystems with clear layout, simple structure and more flexible manipulation, namely, single working unit and microfluidic array chip. To carry out portable operation and analysis, the latter improves detection efficiency and flux. The two types of chips are multilayer structure, including micro flow layer, control layer, thin film sandwich and micro sensor containing heater. In processing, the same chip technology is improved. The local adhesion of PDMS film in engineering practice is solved, heat circulation reaction reagent evaporation and sensor recovery are solved. In addition, based on the theory of solid, fluid heat transfer, modeling and Simulation of microsensors containing heaters with the aid of general commercial software, the temperature control performance of the design is evaluated. The experimental system platform consists of 4 parts, namely, the microfluidic platform, the temperature control platform, the cell culture test platform and the biological signal. The microfluidic platform is composed of no pole speed control micro injection pump, liquid nitrogen pool and rectifying valve and microscope, which is used to realize single cell manipulation of living body and the accuracy and stability of various reagents. Based on the virtual instrument technology, the temperature control platform has developed the control process and the human-computer interface to realize the real temperature of the chip. The cell culture test platform is composed of laminar biological test rig, cell culture box, centrifuge and so on. It is used for cell sample preparation, biochemical reagent preparation and drug pretreatment of living cells. The biosignal detection platform is composed of fluorescence inverted microscope, CCD and related image acquisition and analysis software. The high signal to noise ratio of the biological fluorescence signal is measured and analyzed in real time. It is difficult to test the hydrodynamic effect of the fluid on the cell in the microfluidic system, and the cell activity is greatly influenced by the hydrodynamic force. In this paper, the computational fluid mechanics, elastic mechanics and plastic mechanics theory, dynamic description of the Lagrange Euler method are used in this paper. Grid technology and parameterized numerical method are used to establish and solve the 2D, 3D cell microfluid fluid solid coupling finite element model of two types of chips. By simulating the transient process, the multi attribute hydrodynamic action of cells in the flow field is analyzed, and the relationship between the flow velocity of the chip entrance and the stress distribution of the cell surface and the stress distribution is quantified. The cell stress on the chip is compared with the normal human arterial blood vessels, capillary and tissue fluid, and the effect of the test parameters on the cell physiological activity is predicted. The MCF-7 human breast cancer cell line is selected based on the microbead solid phase extraction technology and the real-time fluorescence quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) molecular amplification and detection. This method integrates all the experimental steps such as microfluidic single cell capture, cell lysis, mRNA extraction, reverse transcription, gene fragment amplification and real-time quantitative detection to a single chip. It improves the retention of low abundance mRNA samples while simplifying operation and shortening the cycle, and thus improves detection precision. Sensitivity and signal-to-noise ratio. In this paper, the feasibility and repeatability of the method were evaluated in turn. The scheme of cell manipulation and mRNA solid phase extraction was selected, and the difference between the method and the traditional method was analyzed. And the difference between the single cell GAPDH and the expression level of the cell cycle regulation gene CDKN1A before and after the induction of MMS was detected. In addition, the effects of MMS dose and time on the regulation of CDKN1A expression are established, and the optimal treatment scheme corresponding to the experimental conditions is proposed. In theory, this paper improves the integration degree of the existing research methods, the automation level of multifunction and experimental operation, and the same single cell and single molecule biochemical fraction of the same kind. In engineering practice, it reduces the complexity, high cost and high consumption of existing chip design, manufacturing and operation, and can be adjusted flexibly according to the change of demand. It has a certain industrialization prospect. The research method and conclusion combine the micro / nano manufacturing technology with basic science research, and promote the basic biology. Medical research is of great value and significance in improving the level of clinical diagnosis and treatment.

【学位授予单位】：哈尔滨工业大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：TN492;R440

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