基于集成芯片的多功能细胞生理自动分析仪研究
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摘要
细胞传感器(Cell-Based Biosensor, CBB)采用活体细胞作为一级换能器,感受到刺激信号后将产生不同类型的特定变化,如细胞代谢、动作电位和电阻抗变化等。而与细胞相连的二级换能器可以检测到这些来自细胞的变化,并将这些测到的细胞信号转换为电学信号,从而送入电学系统。细胞传感器根据检测生理参数的不同,常用的二级换能器主要有微电极阵列(Microelectrodes Array, MEA)、光寻址电位传感器(Light Addressable Potentiometric Sensor, LAPS)、细胞电阻抗传感器(Electric Cell-substrate Impedance Sensor, ECIS)等。在进行细胞生理实验时,对单一参数的监测往往只能从某一方面反映出细胞的生理活动,而测试环境的复杂性(各种噪声源、长期测试后培养液对电极的腐蚀等)会干扰测试的结果,所以对细胞生理活动机理的揭示则需要同时对多种相关的生理参数进行综合分析才能完成。
为了实现胞外离子浓度(微环境检测)、胞外动作电位、以及电生理阻抗三种生理参数同时检测,采用电子技术与生物医学技术相结合的方法,研制了一种基于多功能集成芯片的全自动细胞生理参数分析仪。该仪器具有分析时间短、样品消耗少和效率高等优点。在测量过程中可自动调节测量腔中的温度、湿度及CO2浓度,以满足细胞的生长环境要求。同时通过自动进样系统可实现全自动分析功能。可广泛用于细胞生理、药物的筛选和环境检测等领域。此外,该仪器可实现对单细胞、细胞网络的传导以及药物作用后细胞生理状态的实时量化的检测。采用独特的芯片接口装置以及板卡式电路设计,该仪器适用于多种细胞以及多种不同集成芯片检测的需求。
本论文主要研究内容及创新点：
一、提出了多功能集成芯片的理论分析模型和仿真设计方法
本文提出了一种集成MEA、LAPS和ECIS为一体的新型细胞生理检测芯片的设计思想,解决了细胞胞外动作电位、胞外离子浓度以及细胞阻抗三种生理参数同时检测的一些关键问题。
首先研究了多功能集成芯片检测细胞生理参数的基本理论。通过对细胞作为一级换能器如何感受到外界刺激信号进行了分析,阐述了与细胞代谢、细胞动作电位、细胞膜阻抗特性、以及细胞黏附和迁移相关的细胞基本特性,并给出了细胞感受到外界刺激后不同类型输出信号与二级换能器的耦合模型,最终得出细胞传感器的理论模型。
其次对多功能集成芯片MEA传感单元、ECIS传感单元、以及LAPS传感单元部分的电学模型进行了研究。仿真结果得出集成芯片各传感单元的设计参数对检测结果的影响,优化了芯片的设计参数,为多功能集成芯片及仪器的设计提供了理论支持。
二、实现了多功能细胞生理分析仪的系统设计和加工制作
设计了集成芯片的ECIS、APS、MEA传感单元的相应检测电路,设计了环境控制系统对测量腔的温度、湿度、以及CO2浓度进行严格的控制,使得其维持在细胞最适合生长的环境,设计了自动进样系统控制进样泵和出样泵的通断及转速,从而完成培养液和药物的自动进样以及废液清洗,实现了自动化分析。
通过中央控制电路对整个生理采集信号的过程进行控制,并对获取的信号数据进行初步分析后,由USB接口传送到计算机上,计算机上设计了相应的软件实现数据的采集、存储、以及后期分析等功能。分析仪通过独特的芯片接口装置和电路按功能模块主板式设计,可适应不同的集成芯片设计。最终设计完成了一台多功能细胞生理分析仪样机,可灵活扩展以及向产业化方向发展,实现了细胞生理多参数分析的需求。
三、实现了细胞生理多功能参数的综合检测与分析
首先对分析仪样机进行了的测试评价,包括对LAPS、ECIS、MEA等集成芯片检测电路指标的测试,以及对环境控制系统以及自动进样系统的测试。结果表明,传感器芯片和仪器的性能指标可以满足实际分析的要求。
其次基于多功能集成芯片,进行了细胞生理参数检测研究,对本文研制的细胞生理分析仪的生物医学应用进行典型实验测试。利用集成芯片的LAPS传感单元对肾细胞在5-氟尿嘧啶作用下的酸化率变化进行了检测。利用集成芯片的MEA传感单元检测乳鼠心肌细胞在河豚毒素(TTX)作用下的胞外动作电位变化。利用集成芯片的ECIS传感单元检测细胞的迁移,生长增殖,以及死亡过程。实验结果表明分析仪可用于细胞多种生理参数的综合分析。
通过培养的心肌细胞在药物作用下的实验,表明该仪器可以在一次实验中同时检测到心肌细胞的阻抗、胞外动作电位以及胞外Ca2+浓度三种参数的变化过程。数据分析表明,将三种生理参数结合可以获到细胞在药物作用下的多种指标参数的评价,如药物对心脏毒性的评价等。初步的实验结果表明,本文研制的多功能细胞生理分析仪在未来的生物医药领域将有广泛的应用前景。
Cell-based Biosensors (CBB) exploit the naturally evolved sensitivity of cells in order to detect a broad range of biochemical agents as a cellular response. This cellular response is detected by a secondary transducer and converted into an electronic form suitable for analysis. Depending on the measured physiological parameters, commonly used secondary transducers are mainly Micro-electrodes Array (MEA), Light Addressable Potentiometric Sensor (LAPS), and Electric Cell-substrate Impedance Sensor (ECIS). During cellular physiology experiments, single parameter monitoring is often only from one aspect reflects the physiological activities of cells. And the complexity of test environment (various noise sources, long-term testing of the corrosion medium on the electrodes, etc.) may interfere with experimental results. So revealing the mechanism of cellular physiological activities needs the comprehensive analysis of a variety of related physiological parameters.
An automated multifunctional cellular physiological analyzer based on integrated chip, was developed using electronic technology and biomedical technology. This instrument achieved the simultaneous detection of three kinds of physiological parameters, such as extracellular ion concentrations (micro-environment detection), extracellular action potentials, and electrophysiological impedance. The instrument has several advantages such as shorter analysis time, low sample consumption and high efficiency. Temperature, humidity and CO2 concentration in the measuring cavity can be automatically adjusted during the measuring process, in order to meet environmental requirements in cell growth. At the same time, the fully automated analysis was achieved by the flow analysis system. The instrument can be widely used in cell physiology analysis, drug screening and environmental monitoring and other fields. In addition, the instrument enables real-time quantitative detection of the conduction of single cells and cell networks, and the cellular physiological state after the effects of drugs. Based on the unique design of chip-instrument interface and circuit board, the instrument is applicable to a variety of cells and different designs of multifunctional integrated chip.
The major contents and innovations of this thesis are given as the following aspects.
1.Numerical design and simulation of multifunctional integrated chip
A multifunctional integrated chip was designed, which integrates three kinds of sensors as MEA, LAPS, and ECIS. The chip can simultaneously detect the extracellular action potentials, the state of cell growth and adhesion and extracellular micro-environment.
Theoretical analysis of multifunctional integrated chip was used to understand the basic theory of cell-based biosensor detecting physiological parameters. We analyzed how cells receipt the stimulation signals as primary transducers. Here, we focused on the basis of cellular functions as they relate to cellular metabolism, electrical activity in cells, cellular membrane impedance characteristics, and cell adhesion and motion. The model of cells coupled to the secondary transducers was established by analyzing cellular responses to the outside stimulation. Based on these results, the model of cell-based biosensor was established.
We analyzed the different functional units of the integrated chip, and established the electrical model of MEA sensor unit, ECIS sensor unit and LAPS sensor unit. Numerical model and simulation help us design of multifunctional integrated chip and cellular physiological analyzer.
2.Design and processing of automated multifunctional cellular physiological analyzer
The corresponding detection circuit of MEA, LAPS, ECIS sensing unit on the integrated chip, was designed. Temperature, humidity and CO2 concentration in the measuring cavity can be automatically adjusted during the measuring process, in order to meet environmental requirements in cell growth. Flow control circuit controls on-off and speed of valve, injection pump and output pump. Thus automatic analysis is achieved, by auto sampling the culture medium and drugs as well as washing the waste liquid.
The central control circuit controls the process of physical signal acquisition, preliminary analysis of the signal data, and transfer to computer by USB interface. The designed software on PC enables data collection, storage, and post-analysis. Integrated chip cultured with cells was coupled to the instrument by a special interface. At last, a prototype of multifunctional cellular physiological analyzer was completed, which can achieve the needs of cellular multi-parameter analysis.
3. Detection and analysis of cellular physiological parameters for drugs
Evaluation of the instrument, mainly include calibration of sensing unit detection circuit calibration, as well as the testing of environment and drug control system. The results indicate that the instrument can be used for analysis of cell physiological parameters.
Based on multifunctional integrated chip, several experiments were conducted to detect cellular parameters for typical experimental test. LAPS sensor unit on the integrated chip was used to detect acidification rate changes of the kidney cells under the action of 5-fluorouracil. MEA sensor unit on the integrated chip was used to detect extracellular action potential changes of SD rat myocardial cells under the action of TTX. ECIS sensor unit on the integrated chip was used to detect cell extension, growth, proliferation, and death process. At last, the instrument is used to detect the extracellular action potential, cell metabolism, and cell impedance simultaneously. It is used to study the acute toxicity effect of adriamycin acting on the HL-1 cardiac cell line on the electrophysiological point. The results indicated that the multifunctional cellular physiological analyzer can be widely used in future based on the capability of simultaneous detection of three kinds of parameters.
为了实现胞外离子浓度(微环境检测)、胞外动作电位、以及电生理阻抗三种生理参数同时检测,采用电子技术与生物医学技术相结合的方法,研制了一种基于多功能集成芯片的全自动细胞生理参数分析仪。该仪器具有分析时间短、样品消耗少和效率高等优点。在测量过程中可自动调节测量腔中的温度、湿度及CO2浓度,以满足细胞的生长环境要求。同时通过自动进样系统可实现全自动分析功能。可广泛用于细胞生理、药物的筛选和环境检测等领域。此外,该仪器可实现对单细胞、细胞网络的传导以及药物作用后细胞生理状态的实时量化的检测。采用独特的芯片接口装置以及板卡式电路设计,该仪器适用于多种细胞以及多种不同集成芯片检测的需求。
本论文主要研究内容及创新点：
一、提出了多功能集成芯片的理论分析模型和仿真设计方法
本文提出了一种集成MEA、LAPS和ECIS为一体的新型细胞生理检测芯片的设计思想,解决了细胞胞外动作电位、胞外离子浓度以及细胞阻抗三种生理参数同时检测的一些关键问题。
首先研究了多功能集成芯片检测细胞生理参数的基本理论。通过对细胞作为一级换能器如何感受到外界刺激信号进行了分析,阐述了与细胞代谢、细胞动作电位、细胞膜阻抗特性、以及细胞黏附和迁移相关的细胞基本特性,并给出了细胞感受到外界刺激后不同类型输出信号与二级换能器的耦合模型,最终得出细胞传感器的理论模型。
其次对多功能集成芯片MEA传感单元、ECIS传感单元、以及LAPS传感单元部分的电学模型进行了研究。仿真结果得出集成芯片各传感单元的设计参数对检测结果的影响,优化了芯片的设计参数,为多功能集成芯片及仪器的设计提供了理论支持。
二、实现了多功能细胞生理分析仪的系统设计和加工制作
设计了集成芯片的ECIS、APS、MEA传感单元的相应检测电路,设计了环境控制系统对测量腔的温度、湿度、以及CO2浓度进行严格的控制,使得其维持在细胞最适合生长的环境,设计了自动进样系统控制进样泵和出样泵的通断及转速,从而完成培养液和药物的自动进样以及废液清洗,实现了自动化分析。
通过中央控制电路对整个生理采集信号的过程进行控制,并对获取的信号数据进行初步分析后,由USB接口传送到计算机上,计算机上设计了相应的软件实现数据的采集、存储、以及后期分析等功能。分析仪通过独特的芯片接口装置和电路按功能模块主板式设计,可适应不同的集成芯片设计。最终设计完成了一台多功能细胞生理分析仪样机,可灵活扩展以及向产业化方向发展,实现了细胞生理多参数分析的需求。
三、实现了细胞生理多功能参数的综合检测与分析
首先对分析仪样机进行了的测试评价,包括对LAPS、ECIS、MEA等集成芯片检测电路指标的测试,以及对环境控制系统以及自动进样系统的测试。结果表明,传感器芯片和仪器的性能指标可以满足实际分析的要求。
其次基于多功能集成芯片,进行了细胞生理参数检测研究,对本文研制的细胞生理分析仪的生物医学应用进行典型实验测试。利用集成芯片的LAPS传感单元对肾细胞在5-氟尿嘧啶作用下的酸化率变化进行了检测。利用集成芯片的MEA传感单元检测乳鼠心肌细胞在河豚毒素(TTX)作用下的胞外动作电位变化。利用集成芯片的ECIS传感单元检测细胞的迁移,生长增殖,以及死亡过程。实验结果表明分析仪可用于细胞多种生理参数的综合分析。
通过培养的心肌细胞在药物作用下的实验,表明该仪器可以在一次实验中同时检测到心肌细胞的阻抗、胞外动作电位以及胞外Ca2+浓度三种参数的变化过程。数据分析表明,将三种生理参数结合可以获到细胞在药物作用下的多种指标参数的评价,如药物对心脏毒性的评价等。初步的实验结果表明,本文研制的多功能细胞生理分析仪在未来的生物医药领域将有广泛的应用前景。
Cell-based Biosensors (CBB) exploit the naturally evolved sensitivity of cells in order to detect a broad range of biochemical agents as a cellular response. This cellular response is detected by a secondary transducer and converted into an electronic form suitable for analysis. Depending on the measured physiological parameters, commonly used secondary transducers are mainly Micro-electrodes Array (MEA), Light Addressable Potentiometric Sensor (LAPS), and Electric Cell-substrate Impedance Sensor (ECIS). During cellular physiology experiments, single parameter monitoring is often only from one aspect reflects the physiological activities of cells. And the complexity of test environment (various noise sources, long-term testing of the corrosion medium on the electrodes, etc.) may interfere with experimental results. So revealing the mechanism of cellular physiological activities needs the comprehensive analysis of a variety of related physiological parameters.
An automated multifunctional cellular physiological analyzer based on integrated chip, was developed using electronic technology and biomedical technology. This instrument achieved the simultaneous detection of three kinds of physiological parameters, such as extracellular ion concentrations (micro-environment detection), extracellular action potentials, and electrophysiological impedance. The instrument has several advantages such as shorter analysis time, low sample consumption and high efficiency. Temperature, humidity and CO2 concentration in the measuring cavity can be automatically adjusted during the measuring process, in order to meet environmental requirements in cell growth. At the same time, the fully automated analysis was achieved by the flow analysis system. The instrument can be widely used in cell physiology analysis, drug screening and environmental monitoring and other fields. In addition, the instrument enables real-time quantitative detection of the conduction of single cells and cell networks, and the cellular physiological state after the effects of drugs. Based on the unique design of chip-instrument interface and circuit board, the instrument is applicable to a variety of cells and different designs of multifunctional integrated chip.
The major contents and innovations of this thesis are given as the following aspects.
1.Numerical design and simulation of multifunctional integrated chip
A multifunctional integrated chip was designed, which integrates three kinds of sensors as MEA, LAPS, and ECIS. The chip can simultaneously detect the extracellular action potentials, the state of cell growth and adhesion and extracellular micro-environment.
Theoretical analysis of multifunctional integrated chip was used to understand the basic theory of cell-based biosensor detecting physiological parameters. We analyzed how cells receipt the stimulation signals as primary transducers. Here, we focused on the basis of cellular functions as they relate to cellular metabolism, electrical activity in cells, cellular membrane impedance characteristics, and cell adhesion and motion. The model of cells coupled to the secondary transducers was established by analyzing cellular responses to the outside stimulation. Based on these results, the model of cell-based biosensor was established.
We analyzed the different functional units of the integrated chip, and established the electrical model of MEA sensor unit, ECIS sensor unit and LAPS sensor unit. Numerical model and simulation help us design of multifunctional integrated chip and cellular physiological analyzer.
2.Design and processing of automated multifunctional cellular physiological analyzer
The corresponding detection circuit of MEA, LAPS, ECIS sensing unit on the integrated chip, was designed. Temperature, humidity and CO2 concentration in the measuring cavity can be automatically adjusted during the measuring process, in order to meet environmental requirements in cell growth. Flow control circuit controls on-off and speed of valve, injection pump and output pump. Thus automatic analysis is achieved, by auto sampling the culture medium and drugs as well as washing the waste liquid.
The central control circuit controls the process of physical signal acquisition, preliminary analysis of the signal data, and transfer to computer by USB interface. The designed software on PC enables data collection, storage, and post-analysis. Integrated chip cultured with cells was coupled to the instrument by a special interface. At last, a prototype of multifunctional cellular physiological analyzer was completed, which can achieve the needs of cellular multi-parameter analysis.
3. Detection and analysis of cellular physiological parameters for drugs
Evaluation of the instrument, mainly include calibration of sensing unit detection circuit calibration, as well as the testing of environment and drug control system. The results indicate that the instrument can be used for analysis of cell physiological parameters.
Based on multifunctional integrated chip, several experiments were conducted to detect cellular parameters for typical experimental test. LAPS sensor unit on the integrated chip was used to detect acidification rate changes of the kidney cells under the action of 5-fluorouracil. MEA sensor unit on the integrated chip was used to detect extracellular action potential changes of SD rat myocardial cells under the action of TTX. ECIS sensor unit on the integrated chip was used to detect cell extension, growth, proliferation, and death process. At last, the instrument is used to detect the extracellular action potential, cell metabolism, and cell impedance simultaneously. It is used to study the acute toxicity effect of adriamycin acting on the HL-1 cardiac cell line on the electrophysiological point. The results indicated that the multifunctional cellular physiological analyzer can be widely used in future based on the capability of simultaneous detection of three kinds of parameters.
引文
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