微型电化学生物传感器的设计及其在细胞生化参数检测中的应用研究
发布时间：2018-08-12 18:24
【摘要】：活体细胞对外界刺激如药物、环境因素变化和受体作用等产生响应,会引起细胞代谢等功能性变化,外在表现为生化参数的改变。利用微型化电化学生物传感器,可以将细胞培养在传感器表而,细胞内的生理变化引起胞外环境的变化,胞外的电解质溶液与传感器之间形成电化学池,从而将这些参数的变化通过二级换能器转换为电信号输出,最终实现定量化监测和细胞水平的分析。将传感器微型化和建立良好的细胞-芯片耦合是关键问题所在。鉴于此,采用微机械加工技术开展的基于细胞传感器的微电极阵列、叉指型阻抗传感器、场效应传感器等具有良好的生物相容性,得到了广泛的研究,并已在药物筛选、环境监测等领域得到了初步应用。 本文从微型电化学生物传感器的设计和制备等关键技术入手,对基于电解质/金属-绝缘层-半导体(Electrolyte/metal-insulator-silicon, EIS/MIS)结构和和光伏检测技术的传感器、金微电极、碳微电极等传感器进行了研究,实现了离体原代肾细胞在传感器表而的良好耦合以及胞外酸化率、胞外氧化还原电位检测、葡萄糖溶液浓度检测,为EIS/MIS结构半导体器件拓展了新的生物学应用领域。此外,本文将碳微加工(Carbon micro-electromechanical systems, C-MEMS)工艺用于碳微电极的制备,实现了碳电极的微型化,成功应用于胰岛素溶液的检测,为细胞分泌的研究提供了一种新的可行性方法。本论文的主要内容和贡献如下 1.提出了一种新的有效的细胞胞外氧化还原电位检测的方法 细胞胞外氧化还原电位(Extracellular redox potential, ERP)反应了细胞外微环境的氧化还原状态,与细胞的增殖、分化、凋亡及癌细胞侵蚀都有着密切的关系。本文采用基于MIS结构的传感器和流动注射微腔体联用的方法,为细胞水平的氧化还原电位测试提供了一个可行的解决方案。测试无需添加其他氧化还原介质。研究表明,线粒体电子传递链对ERP有较大的影响。 2.提出了一种从细胞释放H+和自由电子活性两种角度研究细胞生命代谢活动的集成传感器的设计和可行性 本文提出了同时检测胞外酸化率和胞外氧化还原电位的一种集成传感器的设计,该集成传感器的性能可与单独的EIS、MIS传感器相媲美。此外,本文对正常生理状态下的细胞酸化率和ERP两种参数进行了初步的研究,获得了与单独测试类似的结果,验证了该设计的可行性。该设计有望应用于揭示细胞代谢机理的研究和药物分析测试中。 3.研制了一种新的电位型葡萄糖传感器 本文将PPy-GOD (Polypyrrole-glucose oxidase)超薄膜修饰在MIS结构传感器上获得了比使用同种修饰薄膜的其他类型传感器更高的灵敏度。MIS传感器所采用的光伏检测技术与流动注射微腔体联用,有利于提高传感器本身的灵敏度和响应时间。 4.提出了将C-MEMS工艺制备碳微电极用于胰岛素的检测 采用C-MEMS工艺将光刻胶热解制备的碳微电极具有较好的电化学性能,在其表面电沉积纳米RuOx颗粒之后实现了胰岛素的电化学检测,这是继玻碳电极、碳糊电极、碳纤维微电极之后,首次将C-MEMS应用于胰岛素测试的研究。采用C-MEMS工艺的优势在于,可根据实际检测需要灵活设计电极的结构和尺(?),有利于胰岛素传感器的微型化和集成化。
[Abstract]:In vivo cells respond to external stimuli such as drugs, changes in environmental factors and receptors, which can cause functional changes in cell metabolism and external changes in biochemical parameters. An electrochemical cell is formed between the electrolyte solution and the sensor, and the changes of these parameters are converted into electrical signals by a two-stage transducer for quantitative monitoring and cell level analysis. Microelectrode arrays, interdigital impedance sensors and field effect sensors based on cell sensors have been extensively studied for their good biocompatibility, and have been preliminarily applied in drug screening and environmental monitoring.
Starting with the design and fabrication of micro-electrochemical biosensors, the electrolyte/metal-insulator-silicon (EIS/MIS) structure and photovoltaic sensors, gold microelectrode, carbon microelectrode and other sensors are studied in this paper. The in vitro primary kidney cells can be found in vivo. The good coupling of the sensor surface and the detection of extracellular acidification rate, extracellular redox potential and glucose solution concentration have opened up new biological applications for EIS/MIS semiconductor devices. In addition, the Carbon micro-electromechanical systems (C-MEMS) process has been applied to the preparation of carbon microelectrodes, which has been realized. The miniaturization of carbon electrode, which has been successfully applied to the detection of insulin solution, provides a new feasible method for the study of cell secretion.
1. a new effective method for extracellular redox potential detection is proposed.
Extracellular redox potential (ERP) reflects the redox state of the extracellular microenvironment and is closely related to cell proliferation, differentiation, apoptosis and cancer cell erosion. Potential testing provides a viable solution without adding any other redox medium. Studies have shown that mitochondrial electron transfer chains have a greater impact on ERP.
2. The design and feasibility of an integrated biosensor for the study of cell life metabolism from the point of view of H+ release and free electron activity are presented.
An integrated sensor for simultaneous detection of extracellular acidification rate and extracellular redox potential is proposed in this paper. The performance of the integrated sensor is comparable to that of the EIS and MIS sensors. In addition, the acidification rate and ERP parameters under normal physiological conditions are preliminarily studied, and the results are similar to those obtained by the single sensor. The results show that the design is feasible and is expected to be applied to reveal the mechanism of cell metabolism and drug analysis.
3. a new potential glucose sensor has been developed.
In this paper, PPy-GOD (Polypyrrole-glucose oxidase) ultrathin film was modified to obtain higher sensitivity than other sensors using the same modified film on MIS structure sensor.
4. it is proposed to prepare carbon microelectrodes for the detection of insulin by C-MEMS process.
Carbon microelectrode prepared by pyrolysis of photoresist with C-MEMS process has good electrochemical performance. After electrodepositing nano-RuOx particles on its surface, the electrochemical detection of insulin was realized. This is the first time that C-MEMS was applied to insulin measurement after glassy carbon electrode, carbon paste electrode and carbon fiber microelectrode. The advantage is that the electrode structure and ruler can be flexibly designed according to the actual detection needs, which is conducive to the miniaturization and integration of insulin sensors.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R318.6
本文编号：2179912
[Abstract]:In vivo cells respond to external stimuli such as drugs, changes in environmental factors and receptors, which can cause functional changes in cell metabolism and external changes in biochemical parameters. An electrochemical cell is formed between the electrolyte solution and the sensor, and the changes of these parameters are converted into electrical signals by a two-stage transducer for quantitative monitoring and cell level analysis. Microelectrode arrays, interdigital impedance sensors and field effect sensors based on cell sensors have been extensively studied for their good biocompatibility, and have been preliminarily applied in drug screening and environmental monitoring.
Starting with the design and fabrication of micro-electrochemical biosensors, the electrolyte/metal-insulator-silicon (EIS/MIS) structure and photovoltaic sensors, gold microelectrode, carbon microelectrode and other sensors are studied in this paper. The in vitro primary kidney cells can be found in vivo. The good coupling of the sensor surface and the detection of extracellular acidification rate, extracellular redox potential and glucose solution concentration have opened up new biological applications for EIS/MIS semiconductor devices. In addition, the Carbon micro-electromechanical systems (C-MEMS) process has been applied to the preparation of carbon microelectrodes, which has been realized. The miniaturization of carbon electrode, which has been successfully applied to the detection of insulin solution, provides a new feasible method for the study of cell secretion.
1. a new effective method for extracellular redox potential detection is proposed.
Extracellular redox potential (ERP) reflects the redox state of the extracellular microenvironment and is closely related to cell proliferation, differentiation, apoptosis and cancer cell erosion. Potential testing provides a viable solution without adding any other redox medium. Studies have shown that mitochondrial electron transfer chains have a greater impact on ERP.
2. The design and feasibility of an integrated biosensor for the study of cell life metabolism from the point of view of H+ release and free electron activity are presented.
An integrated sensor for simultaneous detection of extracellular acidification rate and extracellular redox potential is proposed in this paper. The performance of the integrated sensor is comparable to that of the EIS and MIS sensors. In addition, the acidification rate and ERP parameters under normal physiological conditions are preliminarily studied, and the results are similar to those obtained by the single sensor. The results show that the design is feasible and is expected to be applied to reveal the mechanism of cell metabolism and drug analysis.
3. a new potential glucose sensor has been developed.
In this paper, PPy-GOD (Polypyrrole-glucose oxidase) ultrathin film was modified to obtain higher sensitivity than other sensors using the same modified film on MIS structure sensor.
4. it is proposed to prepare carbon microelectrodes for the detection of insulin by C-MEMS process.
Carbon microelectrode prepared by pyrolysis of photoresist with C-MEMS process has good electrochemical performance. After electrodepositing nano-RuOx particles on its surface, the electrochemical detection of insulin was realized. This is the first time that C-MEMS was applied to insulin measurement after glassy carbon electrode, carbon paste electrode and carbon fiber microelectrode. The advantage is that the electrode structure and ruler can be flexibly designed according to the actual detection needs, which is conducive to the miniaturization and integration of insulin sensors.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R318.6
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