用于检测细胞生长、代谢和成像的细胞传感器及其测试系统的研究
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摘要
细胞传感器作为一类以活体细胞为一级传感单元、换能器为二级传感单元的器件,具有高灵敏度、低成本、高通量检测等特点,是环境毒性研究、食品安全、药物筛选等领域研究的有效手段并在近年来逐步实现产业化。细胞阻抗传感器(Electric Cell-Substrate Impedance Sensor, ECIS)是检测粘附类细胞如上皮细胞、纤维母细胞、内脏细胞等的形态和数目变化的一类细胞传感器。它因无损实时监测、制备工艺简单、高通量等特点而具有广泛的应用前景。光寻址电位传感器(Light-Adressable Potiential Sensor, LAPS)是检测溶液中离子浓度变化的一类细胞传感器,它具有检测单个细胞胞外微环境离子浓度的潜力,因而也受到了较多关注。本论文在ECIS和LAPS的研究基础之上,针对细胞实验向多参数检测和单细胞分析发展的趋势和需求,设计了新式的基于光电检测原理的细胞传感器,并且开发完成了细胞传感器分析仪器。
本论文的主要研究内容和创新性工作在于：
一、深入分析了叉指电极检测细胞生长的机理并应用于实际细胞阻抗传感器芯片的设计。
基于金属电极-电解液界面双电层模型,系统地研究了细胞与微电极耦合的电极阻抗模型。通过讨论不同尺寸电极设计用于检测细胞阻抗的结果,总结了细胞阻抗检测电极的优化准则。根据优化准则设计的细胞阻抗传感器芯片成功应用于了抗肝纤维化中药的初筛。
二、提出了一种光电复合传感器同时检测细胞生长和代谢的方法。
细胞阻抗和细胞代谢是细胞生理的两个十分重要的参数。前者反映细胞的粘附、增殖和凋亡过程中的细胞形态和数目变化,指示了细胞的生长阶段；后者反映细胞生理正常或异常时的能量代谢水平,指示了细胞的活性。本论文通过建立细胞与电解液-绝缘层-半导体结构(Electrolyte-Insulator-Semiconductor,EIS)的界面耦合模型,阐明了基于EIS检测细胞阻抗的原理。并根据EIS和LAPS分别检测细胞阻抗和细胞代谢的原理的不同,设计和搭建了专门的传感器测试系统。利用该测试系统成功测试了重金属镉对小鼠成纤维母细胞的细胞毒性。该方法弥补了ECIS和LAPS等方法只能检测单一细胞生理参数的不足；同时也简化了集成多参数检测的细胞传感器的设计方法。
三、提出了一种利用光寻址技术在芯片表面进行单细胞成像的方法。
通过模型分析揭示了该方法的可行性。同时,对EIS器件的光电稳定性进行了细致研究,给出了提高EIS器件稳定性的优化准则。该方法能够很好地弥补现有检测技术(如ECIS)的不足：只能分析群细胞行为,而缺少反映单细胞多样化分布信息的能力。该方法虽然还达不到光学显微镜的空间分辨率,但是它不需要构建精密的光路,成本低,操作简单,而且单细胞水平的分辨率也已经能够满足许多细胞实验的需求。
四、完成了基于细胞传感器的多功能和便携式自动分析仪器的硬件设计
设计了基于细胞传感器的多功能和便携式自动分析仪器的总体结构。完成了ECIS、LAPS两种细胞传感器检测电路的设计测试；完成了仪器的组装调试。开展了药物筛选、药物评价和药物毒性分析等细胞实验,验证了仪器的实效性。
Cell-based biosensors (CBBs) take live cells and transducers as primary and secondary sensing elements respectively. They can exhibit high-sensitivity, low-cost and high-throughput detection. Thus they have been gradually commercialized and widely applied in the fields of environmental toxicity, food safety and pharmacological screening. Electric cell-substrate impedance sensor (ECIS), a type of cell-based biosensors, is specially to detect the morphology and quantity of adhesive cells such as epithelial cells, fibroblasts, and visceral cells. With outstanding qualities such as non-invasiveness to cells, real-time monitoring, easy to fabricate and high-throughput, it has broad application prospects. Light-adressable potiential sensor (LAPS), also a type of cell-based biosensors, is specially to detect the ion concentration of the solution. It has the potential to detect the ion concentration of extracellular microenvironment of a single cell. Thus, it has been a research hotspot for years. In this dissertation, by combining the studies on ECIS and LAPS, novel cell-based biosensors based on optoelectronic detection principles were designed to meet the trends and needs of cell experiments:multi-parameter detection and single-cell analysis. Moreover, home-made sensor analyzer had been established for testing the cell-based biosensors designed in this paper.
The work mainly consisted of four parts as follows:
Firstly, detection mechanism of ECIS was systematically explored and the design of ECIS chip was achieved.
Based on the electrochemical attributes of metal-electrolyte interface, the electrode impedance model for cells and micro-electrode coupling was systematiclly studied.By discussing the results of detecting cellular impedance using electrodewith different sizes, the optimization criterion for cellular impedance sensing electrodes were summed up. ECIS chip according to the optimization criteria had been successfully applied in preliminary screening of antifibrotic Chinese medicine.
Secondly, an optoelectronic cell-based biosensor to simultaneously detect cell growth and metabolism was proposed.
The cell impedance and metabolism are two very important parameters of cell physiology. The former reflects the changes in cell morphology and quantity during cell adhesion, proliferation and apoptosis which indicating the growth status of cells; latter reflects the level of energy metabolism in the normal or abnormal cells which indicating the cell activity. By establishing the cell andelectrolyte-insulator-semiconductor (EIS) interface coupling model within this dissertation, the principle of detecting cellular impedance using EIS was explained.
Furthermore, according to the different detection principles of EIS and LAPS, special sensor testing system was designed and established to simultaneously detect cellular impedance and cellular metabolism. With the testing system, the toxicity of cadmium on mouse fibroblast cells was successfully tested. The method described here could make up for the shortage of ECIS and LAPS that could only detect one cell physiological parameter; it also simplifies the design of integrated multi-parameter biosensors.
Thirdly, a method for single-cell imaging on the chip surface by means of optical addressing was proposed.
The feasibility of the method was verified by model analysis. Then, a detailed study of the optoelectronic stability of EIS devices was carried out. Several optimization criterias for improving the stability of EIS device was revealed. The method can compensate the shortages of existing detection techniques (such as ECIS) which could only analyze behavior of cell population so that were lack of distribution information of single cells. Although this method hasn't yet reach the spatial resolution of an optical microscope, the merits including free of precision optical path, simple operation make it an patiential tool for various cell-based assays, in which single cellresolution is sufficient.
Fourthly, a multifunctional and portable automatic sensor analyzer based on the cell-based biosensors was designed and achieved.
The overall structure of the sensor analyzer was designed as well as the detection circuits for ECIS, LAPS. The assembly and commissioning of the analyzer were accomplished. The cell experiments of drug screening, drug assessment and cytotoxicity assay were carried out and at last the effectiveness of the instruments were verified.
本论文的主要研究内容和创新性工作在于：
一、深入分析了叉指电极检测细胞生长的机理并应用于实际细胞阻抗传感器芯片的设计。
基于金属电极-电解液界面双电层模型,系统地研究了细胞与微电极耦合的电极阻抗模型。通过讨论不同尺寸电极设计用于检测细胞阻抗的结果,总结了细胞阻抗检测电极的优化准则。根据优化准则设计的细胞阻抗传感器芯片成功应用于了抗肝纤维化中药的初筛。
二、提出了一种光电复合传感器同时检测细胞生长和代谢的方法。
细胞阻抗和细胞代谢是细胞生理的两个十分重要的参数。前者反映细胞的粘附、增殖和凋亡过程中的细胞形态和数目变化,指示了细胞的生长阶段；后者反映细胞生理正常或异常时的能量代谢水平,指示了细胞的活性。本论文通过建立细胞与电解液-绝缘层-半导体结构(Electrolyte-Insulator-Semiconductor,EIS)的界面耦合模型,阐明了基于EIS检测细胞阻抗的原理。并根据EIS和LAPS分别检测细胞阻抗和细胞代谢的原理的不同,设计和搭建了专门的传感器测试系统。利用该测试系统成功测试了重金属镉对小鼠成纤维母细胞的细胞毒性。该方法弥补了ECIS和LAPS等方法只能检测单一细胞生理参数的不足；同时也简化了集成多参数检测的细胞传感器的设计方法。
三、提出了一种利用光寻址技术在芯片表面进行单细胞成像的方法。
通过模型分析揭示了该方法的可行性。同时,对EIS器件的光电稳定性进行了细致研究,给出了提高EIS器件稳定性的优化准则。该方法能够很好地弥补现有检测技术(如ECIS)的不足：只能分析群细胞行为,而缺少反映单细胞多样化分布信息的能力。该方法虽然还达不到光学显微镜的空间分辨率,但是它不需要构建精密的光路,成本低,操作简单,而且单细胞水平的分辨率也已经能够满足许多细胞实验的需求。
四、完成了基于细胞传感器的多功能和便携式自动分析仪器的硬件设计
设计了基于细胞传感器的多功能和便携式自动分析仪器的总体结构。完成了ECIS、LAPS两种细胞传感器检测电路的设计测试；完成了仪器的组装调试。开展了药物筛选、药物评价和药物毒性分析等细胞实验,验证了仪器的实效性。
Cell-based biosensors (CBBs) take live cells and transducers as primary and secondary sensing elements respectively. They can exhibit high-sensitivity, low-cost and high-throughput detection. Thus they have been gradually commercialized and widely applied in the fields of environmental toxicity, food safety and pharmacological screening. Electric cell-substrate impedance sensor (ECIS), a type of cell-based biosensors, is specially to detect the morphology and quantity of adhesive cells such as epithelial cells, fibroblasts, and visceral cells. With outstanding qualities such as non-invasiveness to cells, real-time monitoring, easy to fabricate and high-throughput, it has broad application prospects. Light-adressable potiential sensor (LAPS), also a type of cell-based biosensors, is specially to detect the ion concentration of the solution. It has the potential to detect the ion concentration of extracellular microenvironment of a single cell. Thus, it has been a research hotspot for years. In this dissertation, by combining the studies on ECIS and LAPS, novel cell-based biosensors based on optoelectronic detection principles were designed to meet the trends and needs of cell experiments:multi-parameter detection and single-cell analysis. Moreover, home-made sensor analyzer had been established for testing the cell-based biosensors designed in this paper.
The work mainly consisted of four parts as follows:
Firstly, detection mechanism of ECIS was systematically explored and the design of ECIS chip was achieved.
Based on the electrochemical attributes of metal-electrolyte interface, the electrode impedance model for cells and micro-electrode coupling was systematiclly studied.By discussing the results of detecting cellular impedance using electrodewith different sizes, the optimization criterion for cellular impedance sensing electrodes were summed up. ECIS chip according to the optimization criteria had been successfully applied in preliminary screening of antifibrotic Chinese medicine.
Secondly, an optoelectronic cell-based biosensor to simultaneously detect cell growth and metabolism was proposed.
The cell impedance and metabolism are two very important parameters of cell physiology. The former reflects the changes in cell morphology and quantity during cell adhesion, proliferation and apoptosis which indicating the growth status of cells; latter reflects the level of energy metabolism in the normal or abnormal cells which indicating the cell activity. By establishing the cell andelectrolyte-insulator-semiconductor (EIS) interface coupling model within this dissertation, the principle of detecting cellular impedance using EIS was explained.
Furthermore, according to the different detection principles of EIS and LAPS, special sensor testing system was designed and established to simultaneously detect cellular impedance and cellular metabolism. With the testing system, the toxicity of cadmium on mouse fibroblast cells was successfully tested. The method described here could make up for the shortage of ECIS and LAPS that could only detect one cell physiological parameter; it also simplifies the design of integrated multi-parameter biosensors.
Thirdly, a method for single-cell imaging on the chip surface by means of optical addressing was proposed.
The feasibility of the method was verified by model analysis. Then, a detailed study of the optoelectronic stability of EIS devices was carried out. Several optimization criterias for improving the stability of EIS device was revealed. The method can compensate the shortages of existing detection techniques (such as ECIS) which could only analyze behavior of cell population so that were lack of distribution information of single cells. Although this method hasn't yet reach the spatial resolution of an optical microscope, the merits including free of precision optical path, simple operation make it an patiential tool for various cell-based assays, in which single cellresolution is sufficient.
Fourthly, a multifunctional and portable automatic sensor analyzer based on the cell-based biosensors was designed and achieved.
The overall structure of the sensor analyzer was designed as well as the detection circuits for ECIS, LAPS. The assembly and commissioning of the analyzer were accomplished. The cell experiments of drug screening, drug assessment and cytotoxicity assay were carried out and at last the effectiveness of the instruments were verified.
引文
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