基于MEMS技术的新型细胞传感器及其在细胞电生理中应用的研究
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摘要
随着生物医学工程和微机械加工技术(micro electronic mechanicalsystem，MEMS)的发展，生物医学传感器的微型化使其研究的范围已达到了细胞和分子水平。细胞拥有并表达着一系列分子识别的元件，如受体、离子通道、酶等，这些分子可以作为靶分析物，当有外界刺激时，将按照固有的细胞生理机制进行相应的生理功能活动。
基于MEMS技术的细胞传感器(cell-based biosensor)包括有微电极阵列(microelectrode array，MEA)、场效应管(field effect transistor，FET)阵列及光寻址电位传感器阵列(light-addressable potentiometric sensor，LAPS)等，它们可用于实现细胞胞外电位的记录。作为一种体外检测的新型细胞芯片技术，其实质就是在各阵列化的传感器芯片表面培养细胞，使细胞通过一层薄的电解液同芯片的电极或栅极相耦合，构成可以实现控制电路和神经系统双向通讯的生物芯片，从而对细胞的电生理特性进行传感测量。该技术以其可对多个细胞同时进行长期、无损检测的特点，已在药物筛选、环境检测等生物医学领域得到了初步的应用。同时，基于体加工技术的芯片技术同样适用于在体研究，从而在脑高级功能、神经修复、以及人工器官等研究领域展现出了诱人的前景。
目前胞外电生理芯片很多已实现了商品化，其存在的主要问题是测试参数的单一化、指标的不稳定、测试环境要求很高等。基于此，本论文首先从基于MEA的细胞传感器出发，介绍了自主设计的用于细胞胞外电生理测试的MEA，分析其基本原理和电学模型以及制作工艺，并研究了心肌细胞、嗅觉细胞在其表面的生长和电信号传递过程，此外，进行了8通道动作电位的并行检测试验。在此基础上，设计了一种基于细胞电生理检测的集成芯片，用于实现多种参数实时无损测量，包括胞外多种不同离子(H~+、K~+、Ca~(2+))的测试，胞外动作电位的检测，细胞贴壁的叉指电极(interdigital array，IDA)阻抗测量等，为集成化细胞传感器技术拓展了新的应用领域。
本论文的主要内容和贡献在于：
1．深入研究了细胞—MEA、细胞—IDA、细胞—LAPS器件耦合机理及测量模型。通过研究细胞膜的特点及膜上离子通道的通透性和导电性，分析了Au／Si／SiO_2界面特点对细胞接触的影响：在国外学者Kovacs、Fromherz等人建立的模型基础上，进一步提出了电活性细胞—金属、半导体界面的一种简化耦合模型；从而为本论文细胞传感器的设计提供了理论基础，也为实验结果的分析提供了重要的依据。
2．提出了一种用于细胞外电生理测试的微电极阵列的设计方法，分析了其工作原理、界面模型及基于MEMS技术的微加工制作工艺。研究了心肌、嗅觉等多种细胞等在其表面的生长和信号传递过程，进行了多通道动作电位的并行检测。实验结果表明，该细胞传感器可对多个细胞同时进行长期、无损的胞外测量，并具有制备简单、使用方便的特点，可应用于细胞电生理研究和药物检测等领域。
3．提出了一种细胞电阻抗和细胞胞外代谢离子与细胞动作电位同时检测的多功能集成芯片的设计方法，实现了细胞多种电生理参数的实时无损测量。通过电阻抗快速反映细胞在体外培养环境下的生长状况，采用MEA进行药物刺激下细胞动作电位的检测；采用LAPS进行细胞代谢产物中多种离子的浓度变化检测。芯片各部分同时工作，利用流动分析装置控制流向和速度，从而可以按一定顺序连续监测细胞从正常生理状态到药物刺激下的形态变化、动作电位以及代谢物质的改变，从多个方面揭示细胞生理活动的机理。该研究为集成化细胞传感器开拓了一个新的应用领域。
With the development of Biomedical Engineering and micro electronic mechanical system(MEMS), the research on micromation of cell-based biosensor (CBB) has reached on the cellular and molecular level. Cells provide and express a series of elements such as naturally evolved receptor, ion-channels, and enzymes that can be the targets of biological active analytes. When stimulated, the living cell responds and take actions: induce electronic activity, excrete something or absorb something. Cell-based biosensors that treat cells as biological sensing elements have the capacity to respond to analytes in a physiologically relevant manner.
CBB with MEMS technique, which include microelectrode array(MEA), field effect transistor array(FET), and the light-addressable potentiometric sensor(LAPS), can be applied as the secondary biosensor that coupled with living cells to realize the recording of extracellular electrophysiological signals. As a novel cell-based biochip, the principle of which is to culture varieties of cells on certain type of sensor array. When the cells are stimulated and electronically activated, the signals are transferred to the effective area of sensors, such as the metal electrodes of MEA and the gate of FET, and the two-way interface of cell-sensor can be constructed to make it feasible of transferring cellular signals to the processing amplifying units. Because of the advantage of extracellular CBB, e. g. long-term recording in non-invasive way, fast response, and easy fabrication, such biosensors have numerous applications including pharmaceutical screening, cellular physiological analysis, toxin detecting, peripheral nerve regeneration and environment monitoring, as well as in-vivo recordings, thus they are also promising in fields of neuronal prostheses and the reconstruction of damaged sense organs.
Nowadays, a diversity of CBBs are already commercially available, however, the main problems lie in the singleness of parameters, the instability of guide line and the critical environmental requirements. Thus this thesis first introduced MEA designed by ourselves for extracellular action potential monitoring, mainly focusing on the theories of cell-microelectrode interfacial model, the design and fabricating process, and the extracellular signal transferring process with different cell types, etc., cardiac myocytes and olfactory bulb neurons. Based on this, an integrated chip for detection of cell physiology was designed to realize the parallel monitoring of different parameters such as the metabolite(according to the H~+、K~+、Ca~(2+) level), the action potentials and the impedance change due to cell-IDA electrode attachment. The integrated chip deepens and widens the new fields of applications in CBBs.
The major contents and contributions of this thesis are given in the following aspects:
1. The model of the cell-silicon, cell-metal electrode interface and the detection model of MEA, IDA and LAPS have been demonstrated deeply. Firstly, the characteristic equations of transmembrane ionic current are given based on the conductance and permeability of cellular membrane. Secondly, we analyze the influence of Si/SiO_2 and Au interface on the cell-silicon interface model. Then, the cell-electrode model established by Kovacs group and the neuron-FET model established by Fromherz group in German have been deduced into the cell-silicon or cell-metal device model and simplified into the design of our experiment. Afterwards, the detection theory and model of certain sensor type has been discussed in detail, which is the theoretical foundations of cell-based biosensor design and provide the premise to explain the experiment results.
2. We designed and brought forward a novel MEA based on MEMS technique, including the theories, the design and fabricating process, and the system (hardware and software) implementation. Varieties of cell types, e.g. cardiac myocytes and olfactory bulb neurons, were cultured on the surface of MEA to validate the 8 parallel channels of CBBs. It offers the advantages of long-term recording in non-invasive way and easy fabrication, which facilitates the latter integration process, and can be applied in physiological analysis, peripheral nerve regeneration and environment monitoring.
3. The design and fabricating process of integrated cellular chip including MEA/IDA/LAPS unites are provided. Our efforts are directed to the parallel development, fabrication and integration of different sensors into miniaturized biochips for a multiparametric cellular monitoring with the multi parametric chip. Parallel and on-line acquisition of data related to different cellular targets will be required for advanced stages of drug screening, and the chip includes 3 main units: The IDA with the impedance measurement of cells for attachment evaluation is firstly detected for cellular impedance detection; The MEA with the voltage measurement of cells for extracellular action potential detection is secondly tested; The LAPS for the sensitive ions, e.g. H~+, K~+, Ca~(2+), from cellular metabolites in micro environment is thirdly measured. A set of automatic fluid flowing system is also introduced to control the process of inlet and outlet by software by changing the direction, the drugs and the velocity. Thus the continuous monitoring of cells from normal shapes to changes of cellular attachment, action potentials, metabolite according to stimulation is available. The primary cellular physiological experiments are done for further developments in cellular and molecular sensors.
基于MEMS技术的细胞传感器(cell-based biosensor)包括有微电极阵列(microelectrode array，MEA)、场效应管(field effect transistor，FET)阵列及光寻址电位传感器阵列(light-addressable potentiometric sensor，LAPS)等，它们可用于实现细胞胞外电位的记录。作为一种体外检测的新型细胞芯片技术，其实质就是在各阵列化的传感器芯片表面培养细胞，使细胞通过一层薄的电解液同芯片的电极或栅极相耦合，构成可以实现控制电路和神经系统双向通讯的生物芯片，从而对细胞的电生理特性进行传感测量。该技术以其可对多个细胞同时进行长期、无损检测的特点，已在药物筛选、环境检测等生物医学领域得到了初步的应用。同时，基于体加工技术的芯片技术同样适用于在体研究，从而在脑高级功能、神经修复、以及人工器官等研究领域展现出了诱人的前景。
目前胞外电生理芯片很多已实现了商品化，其存在的主要问题是测试参数的单一化、指标的不稳定、测试环境要求很高等。基于此，本论文首先从基于MEA的细胞传感器出发，介绍了自主设计的用于细胞胞外电生理测试的MEA，分析其基本原理和电学模型以及制作工艺，并研究了心肌细胞、嗅觉细胞在其表面的生长和电信号传递过程，此外，进行了8通道动作电位的并行检测试验。在此基础上，设计了一种基于细胞电生理检测的集成芯片，用于实现多种参数实时无损测量，包括胞外多种不同离子(H~+、K~+、Ca~(2+))的测试，胞外动作电位的检测，细胞贴壁的叉指电极(interdigital array，IDA)阻抗测量等，为集成化细胞传感器技术拓展了新的应用领域。
本论文的主要内容和贡献在于：
1．深入研究了细胞—MEA、细胞—IDA、细胞—LAPS器件耦合机理及测量模型。通过研究细胞膜的特点及膜上离子通道的通透性和导电性，分析了Au／Si／SiO_2界面特点对细胞接触的影响：在国外学者Kovacs、Fromherz等人建立的模型基础上，进一步提出了电活性细胞—金属、半导体界面的一种简化耦合模型；从而为本论文细胞传感器的设计提供了理论基础，也为实验结果的分析提供了重要的依据。
2．提出了一种用于细胞外电生理测试的微电极阵列的设计方法，分析了其工作原理、界面模型及基于MEMS技术的微加工制作工艺。研究了心肌、嗅觉等多种细胞等在其表面的生长和信号传递过程，进行了多通道动作电位的并行检测。实验结果表明，该细胞传感器可对多个细胞同时进行长期、无损的胞外测量，并具有制备简单、使用方便的特点，可应用于细胞电生理研究和药物检测等领域。
3．提出了一种细胞电阻抗和细胞胞外代谢离子与细胞动作电位同时检测的多功能集成芯片的设计方法，实现了细胞多种电生理参数的实时无损测量。通过电阻抗快速反映细胞在体外培养环境下的生长状况，采用MEA进行药物刺激下细胞动作电位的检测；采用LAPS进行细胞代谢产物中多种离子的浓度变化检测。芯片各部分同时工作，利用流动分析装置控制流向和速度，从而可以按一定顺序连续监测细胞从正常生理状态到药物刺激下的形态变化、动作电位以及代谢物质的改变，从多个方面揭示细胞生理活动的机理。该研究为集成化细胞传感器开拓了一个新的应用领域。
With the development of Biomedical Engineering and micro electronic mechanical system(MEMS), the research on micromation of cell-based biosensor (CBB) has reached on the cellular and molecular level. Cells provide and express a series of elements such as naturally evolved receptor, ion-channels, and enzymes that can be the targets of biological active analytes. When stimulated, the living cell responds and take actions: induce electronic activity, excrete something or absorb something. Cell-based biosensors that treat cells as biological sensing elements have the capacity to respond to analytes in a physiologically relevant manner.
CBB with MEMS technique, which include microelectrode array(MEA), field effect transistor array(FET), and the light-addressable potentiometric sensor(LAPS), can be applied as the secondary biosensor that coupled with living cells to realize the recording of extracellular electrophysiological signals. As a novel cell-based biochip, the principle of which is to culture varieties of cells on certain type of sensor array. When the cells are stimulated and electronically activated, the signals are transferred to the effective area of sensors, such as the metal electrodes of MEA and the gate of FET, and the two-way interface of cell-sensor can be constructed to make it feasible of transferring cellular signals to the processing amplifying units. Because of the advantage of extracellular CBB, e. g. long-term recording in non-invasive way, fast response, and easy fabrication, such biosensors have numerous applications including pharmaceutical screening, cellular physiological analysis, toxin detecting, peripheral nerve regeneration and environment monitoring, as well as in-vivo recordings, thus they are also promising in fields of neuronal prostheses and the reconstruction of damaged sense organs.
Nowadays, a diversity of CBBs are already commercially available, however, the main problems lie in the singleness of parameters, the instability of guide line and the critical environmental requirements. Thus this thesis first introduced MEA designed by ourselves for extracellular action potential monitoring, mainly focusing on the theories of cell-microelectrode interfacial model, the design and fabricating process, and the extracellular signal transferring process with different cell types, etc., cardiac myocytes and olfactory bulb neurons. Based on this, an integrated chip for detection of cell physiology was designed to realize the parallel monitoring of different parameters such as the metabolite(according to the H~+、K~+、Ca~(2+) level), the action potentials and the impedance change due to cell-IDA electrode attachment. The integrated chip deepens and widens the new fields of applications in CBBs.
The major contents and contributions of this thesis are given in the following aspects:
1. The model of the cell-silicon, cell-metal electrode interface and the detection model of MEA, IDA and LAPS have been demonstrated deeply. Firstly, the characteristic equations of transmembrane ionic current are given based on the conductance and permeability of cellular membrane. Secondly, we analyze the influence of Si/SiO_2 and Au interface on the cell-silicon interface model. Then, the cell-electrode model established by Kovacs group and the neuron-FET model established by Fromherz group in German have been deduced into the cell-silicon or cell-metal device model and simplified into the design of our experiment. Afterwards, the detection theory and model of certain sensor type has been discussed in detail, which is the theoretical foundations of cell-based biosensor design and provide the premise to explain the experiment results.
2. We designed and brought forward a novel MEA based on MEMS technique, including the theories, the design and fabricating process, and the system (hardware and software) implementation. Varieties of cell types, e.g. cardiac myocytes and olfactory bulb neurons, were cultured on the surface of MEA to validate the 8 parallel channels of CBBs. It offers the advantages of long-term recording in non-invasive way and easy fabrication, which facilitates the latter integration process, and can be applied in physiological analysis, peripheral nerve regeneration and environment monitoring.
3. The design and fabricating process of integrated cellular chip including MEA/IDA/LAPS unites are provided. Our efforts are directed to the parallel development, fabrication and integration of different sensors into miniaturized biochips for a multiparametric cellular monitoring with the multi parametric chip. Parallel and on-line acquisition of data related to different cellular targets will be required for advanced stages of drug screening, and the chip includes 3 main units: The IDA with the impedance measurement of cells for attachment evaluation is firstly detected for cellular impedance detection; The MEA with the voltage measurement of cells for extracellular action potential detection is secondly tested; The LAPS for the sensitive ions, e.g. H~+, K~+, Ca~(2+), from cellular metabolites in micro environment is thirdly measured. A set of automatic fluid flowing system is also introduced to control the process of inlet and outlet by software by changing the direction, the drugs and the velocity. Thus the continuous monitoring of cells from normal shapes to changes of cellular attachment, action potentials, metabolite according to stimulation is available. The primary cellular physiological experiments are done for further developments in cellular and molecular sensors.
引文
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