【作者】 张钊；

【导师】 朱真；

【作者基本信息】 东南大学 ， 微电子学与固体电子学， 2023， 硕士

【摘要】 微流控芯片能够灵活集成多种单细胞操纵和分析方法,是研究单细胞增殖、分化、迁移、衰老等生理活动的强大工具。出芽酵母因其分裂间隔和寿命较短、微观操作方便、培养成本低等优点,成为了细胞衰老研究的主要模式生物。单细胞电阻抗检测具有非侵入、无需荧光标记、实时多参数提取等优点,能够实现细胞复杂生理状态的快速、准确表征。本文面向酿酒酵母单细胞的复制衰老研究,基于等效电路模型提出一种酵母长时序电阻抗谱分析方法。随后研制了用于酵母单细胞稳定捕获、可靠培养与有效剪切的捕获阵列,并在此基础上集成了32位微电极阵列以实现酿酒酵母细胞生长、分裂等生理活动的电阻抗检测。主要研究内容如下:(1)建立了酿酒酵母单细胞捕获与电阻抗谱检测单元的等效电路模型。基于有限元仿真数据,采用最小二乘法拟合并挖掘了各电路元件的典型值。提取了电阻抗检测外围电路的寄生参数。基于完整的等效电路模型建立了酵母单细胞时序电阻抗谱信号的分析与电学参数提取系统。(2)设计并制备了酵母单细胞捕获阵列与32位微电极阵列的集成微流控芯片。设计了微电极阵列选通电路,编写了自动控制与数据采集的图形化用户界面,构建了酿酒酵母单细胞电阻抗谱检测系统。(3)验证了单细胞捕获阵列能够实现酵母单细胞的稳定捕获、可靠培养与有效剪切等功能。验证了酿酒酵母单细胞电阻抗谱检测系统监测酵母单细胞的生长过程、准确识别子代细胞剪切事件的可行性。归纳了各种意外事件的电阻抗谱曲线,并讨论了这些事件对子细胞剪切事件准确识别的影响。本文工作具有以下创新点:(1)通过完整电阻抗谱降维出的等效体积分数代替单频幅值表征酵母细胞的生长、分裂等生理活动,以消除单频测量误差造成的潜在影响;(2)在酵母单细胞捕获阵列芯片中集成了32位微电极阵列,提高了酵母单细胞原位电阻抗谱检测的通量;(3)搭建了完整的酿酒酵母单细胞电阻抗谱检测系统,并通过时序电阻抗谱信号成功表征了酵母细胞生长过程并实现了子代细胞剪切去除的准确识别。更多还原

【Abstract】 Microfluidics,which provides a variety of single-cell manipulation and analysis methods,has emerged as a powerful tool for studying single-cell proliferation,differentiation,migration,aging and other life activities.Due to its advantages of short division interval and lifespan,convenient microscopic operation and low culture cost,budding yeast,Saccharomyces cerevisiae(S.cerevisiae),has become the main model organism for cell aging research.Single-cell electrical impedance sensing technology features non-invasive,no fluorescent labeling,and real-time multi-parameter extraction,enabling fast,accurate characterization of complex physiological states of cells.In this paper,a longterm electrical impedance spectroscopy(EIS)analysis method is proposed based on the equivalent circuit model for the study of replication life span(RLS)of yeast cells.Then,a cell capture array for stable capture,reliable culture and effective dissection of single yeast cells was developed,and a 32-bit microelectrode array was integrated to realize EIS measurement of the growth processes and dissection events of single yeast cells.The main research contents are as follows:(1)The equivalent circuit model(ECM)of single yeast cell’s capture and EIS measurement system was established.Based on the finite element simulation results,the typical values of each circuit element are calculated by the least squares fitting method.The parasitic parameters of the peripheral circuits of the EIS measurement system were extracted.Based on the complete equivalent circuit model,a system for time-series EIS analysis and electrical parameters extraction of yeast cells was established.(2)A microfluidic chip integrating yeast capture array and 32-bit microelectrode array was designed and fabricated.The single yeast EIS measuring system was constructed by a multiplexing circuit for microelectrode array,and a graphical user interface for automatic control and data sampling.(3)The functionality of the single-cell capture array to achieve stable capture,reliable culture and effective dissection of single yeast cells was verified.The feasibility of the single yeast EIS measuring system to monitor the growth processes of single yeast and accurately identify the dissection events of daughter cells were verified.The EIS curves of various unexpected events are summarized and the influences of these events on the accurate identification of dessection events are discussed.The innovative aspects in the work are described as follows:(1)The equivalent volume fraction extracted from intact EIS is used instead of amplitude at single frequency to characterize the growth processes,diseection events and other physiological activities of yeast cells through the equivalent volume fraction,which eliminate the potential impact of measurement errors at single frequency;(2)A 32-bit microelectrode array was integrated in the microfluidic chip of yeast capture array,which improved the throughput of yeast in-situ EIS measurement;(3)The whole EIS measurement system for single yeast was built.Then,the growth process of single yeast cells was monitored and the dissection events of daughter cells were accurately identified based on the measured time-lapse EIS.更多还原