细胞阻抗和电位检测复合传感器及其在药物分析中应用的研究
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摘要
细胞电分析,包括细胞活性及细胞生长状态的确定,在细胞研究、药物作用、细胞工程、医学卫生以及环境监测中具有重要作用。本论文从细胞阻抗传感和胞外电位测量背景现状出发,引出了多参数集成芯片在细胞生理分析和药物研究中的重要意义,结合细胞阻抗测试技术和胞外电位测试技术设计了基于平面微结构的多参数分析的高内涵复合传感器及系统,主要工作如下：
理论设计部分,重点深入研究了叉指电极细胞贴附阻抗模型,分析了细胞阻抗的特征频谱响应。本文首次提出了基于等效电路的最优化设计准则,并利用细胞-电极单元模型描述了细胞在电极上的空间分布对阻抗的影响。然后用有限元仿真电极电场以及细胞-电极模型验证了前面的模型的分析,更深入理解了电极尺寸对传感器性能的影响。基于细胞-电极点接触模型分析了用于胞外电位测量的微电极系统的传递函数,在此基础上重点分析了电极几何尺寸和噪声源对测得胞外信号质量的影响,提出了在微电极设计中的优化准则。基于以上的理论分析,给出了叉指电极与微电极复合芯片ExCELL的布局和整体设计。
芯片系统部分,基于以上的设计方案,按照溅射金属层,光刻电极图案,沉积绝缘层,光刻绝缘层图案的微加工工艺顺序制作了芯片并且完成了生物相容性封装。与之匹配的传感器测试底座包括金属屏蔽盒和电学和力学性能良好的压针式接口,保证信号传输质量。集成在底座上的温控系统以及自动流动控制系统能保证细胞正常生长并提供自动化实验分析。32通道高增益低噪声生物信号放大器提供了输入噪声为5.5μVRMs,增益1000的信号调理,然后通过高速采集卡数字化记录；多通道阻抗测试系统能提供带宽为100Hz-10KHz的频谱扫描和时间分辨率为10ms的单频率长时测试。
实验分析部分,基于复合芯片系统,评价了PC12细胞生长过程中的细胞阻抗频率特性以及阻抗随时间的动态变化；利用波形提取和聚类的方法分析了从心肌细胞系HL-1测得的胞外电位信号。最后结合阻抗和胞外电位记录这两种参数综合评价了阿霉素对心肌细胞系HL-1的急性和慢性毒性的心衰模型,验证了芯片和测试系统能为电兴奋性细胞的分析提供高内涵分析方法。
Cell electroanalysis, including the cell activity observation and cell growth state determination, plays an important role in cellular research, drug effects, cell engineering, medicine and health, as well as environmental monitoring. This thesis proposing the importance of integrated chip in cell physiological analysis and drug development, provides a High-Content Screening Platforms for Multi-Parameter Studies integrating the electrical cell impedance sensing and extracellular potential recording techniques. The work mainly consists of three parts as follows:
In the part of theory and design, the interdigitated electrodes type was chosen to in-depth study of the cell-electrode adhesion impedance model. The optimization was firstly derived from the equivalent circuit, further supplemented by the cell spatial distribution on unit electrode model. Infinite element simulation of electric field of electrode further explored electrode geometry on sensor performance. Based on cell-electrode point-contact model, the transfer function of extracellular recording system was deduced. Based on the electrode size effect and noise source effect on the detected signal, some rules were proposed in microelectrode design. Given the optimization design rules of these two sensing technology, the layout and packaging of integrated chip named "ExCELL" was designed.
For the integrated chip system, interdigitated electrodes and micro-electrode integrated chip were fabricated following the processes of metal layer sputtering, electrode pattern photoetching, insulating layer deposition and another photoetching to expose the electrode, finally packaged biocompatibly. Corresponding test station consisted of pogo pins of electric and mechanical property and shielding box, which could ensure the quality of signal condition. Temperature condition system integrated on the station and fluid control automation guaranteed the cell growth and automation experiments.32-channel bio-signal high-gain low-noise amplifier was designed to provide a signal condition of 5.5μVRMS inPut noise and 1000 gain; A multi-channel impedance test system were developed to implement a impedance spectroscopy scan of 100Hz-10KHz bandwidths and long term measurement under single frequency of 10ms time resolution.
Using the integrated chip-based system, the process of cell growth of PC 12 cells was monitored and evaluated by analyzing the frequency characteristics and dynamic changes of cell impedance over time. Waveform extraction and clustering method were exploited for the analysis of extracellular potential signal measured from the cardiac cell line HL-1. At last, the simultaneous recording of cell impedance and extracellular potential was realized. The incorporation of these two parameters provided a comprehensive evaluation the acute and chronic toxicity of of adriamycin on cardiac cell line HL-1 in the heart failure model.
理论设计部分,重点深入研究了叉指电极细胞贴附阻抗模型,分析了细胞阻抗的特征频谱响应。本文首次提出了基于等效电路的最优化设计准则,并利用细胞-电极单元模型描述了细胞在电极上的空间分布对阻抗的影响。然后用有限元仿真电极电场以及细胞-电极模型验证了前面的模型的分析,更深入理解了电极尺寸对传感器性能的影响。基于细胞-电极点接触模型分析了用于胞外电位测量的微电极系统的传递函数,在此基础上重点分析了电极几何尺寸和噪声源对测得胞外信号质量的影响,提出了在微电极设计中的优化准则。基于以上的理论分析,给出了叉指电极与微电极复合芯片ExCELL的布局和整体设计。
芯片系统部分,基于以上的设计方案,按照溅射金属层,光刻电极图案,沉积绝缘层,光刻绝缘层图案的微加工工艺顺序制作了芯片并且完成了生物相容性封装。与之匹配的传感器测试底座包括金属屏蔽盒和电学和力学性能良好的压针式接口,保证信号传输质量。集成在底座上的温控系统以及自动流动控制系统能保证细胞正常生长并提供自动化实验分析。32通道高增益低噪声生物信号放大器提供了输入噪声为5.5μVRMs,增益1000的信号调理,然后通过高速采集卡数字化记录；多通道阻抗测试系统能提供带宽为100Hz-10KHz的频谱扫描和时间分辨率为10ms的单频率长时测试。
实验分析部分,基于复合芯片系统,评价了PC12细胞生长过程中的细胞阻抗频率特性以及阻抗随时间的动态变化；利用波形提取和聚类的方法分析了从心肌细胞系HL-1测得的胞外电位信号。最后结合阻抗和胞外电位记录这两种参数综合评价了阿霉素对心肌细胞系HL-1的急性和慢性毒性的心衰模型,验证了芯片和测试系统能为电兴奋性细胞的分析提供高内涵分析方法。
Cell electroanalysis, including the cell activity observation and cell growth state determination, plays an important role in cellular research, drug effects, cell engineering, medicine and health, as well as environmental monitoring. This thesis proposing the importance of integrated chip in cell physiological analysis and drug development, provides a High-Content Screening Platforms for Multi-Parameter Studies integrating the electrical cell impedance sensing and extracellular potential recording techniques. The work mainly consists of three parts as follows:
In the part of theory and design, the interdigitated electrodes type was chosen to in-depth study of the cell-electrode adhesion impedance model. The optimization was firstly derived from the equivalent circuit, further supplemented by the cell spatial distribution on unit electrode model. Infinite element simulation of electric field of electrode further explored electrode geometry on sensor performance. Based on cell-electrode point-contact model, the transfer function of extracellular recording system was deduced. Based on the electrode size effect and noise source effect on the detected signal, some rules were proposed in microelectrode design. Given the optimization design rules of these two sensing technology, the layout and packaging of integrated chip named "ExCELL" was designed.
For the integrated chip system, interdigitated electrodes and micro-electrode integrated chip were fabricated following the processes of metal layer sputtering, electrode pattern photoetching, insulating layer deposition and another photoetching to expose the electrode, finally packaged biocompatibly. Corresponding test station consisted of pogo pins of electric and mechanical property and shielding box, which could ensure the quality of signal condition. Temperature condition system integrated on the station and fluid control automation guaranteed the cell growth and automation experiments.32-channel bio-signal high-gain low-noise amplifier was designed to provide a signal condition of 5.5μVRMS inPut noise and 1000 gain; A multi-channel impedance test system were developed to implement a impedance spectroscopy scan of 100Hz-10KHz bandwidths and long term measurement under single frequency of 10ms time resolution.
Using the integrated chip-based system, the process of cell growth of PC 12 cells was monitored and evaluated by analyzing the frequency characteristics and dynamic changes of cell impedance over time. Waveform extraction and clustering method were exploited for the analysis of extracellular potential signal measured from the cardiac cell line HL-1. At last, the simultaneous recording of cell impedance and extracellular potential was realized. The incorporation of these two parameters provided a comprehensive evaluation the acute and chronic toxicity of of adriamycin on cardiac cell line HL-1 in the heart failure model.
引文
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