基于微流控芯片电泳的单细胞及线粒体内多组份活性物质的同时检测
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摘要
地球上的动、植物种类繁多，形态结构变化多样，但它们都是由细胞组成的。细胞是生命体的基本形态结构和功能单位，生命的运动都在细胞内得以实现，对细胞的研究是生命科学的出发点。细胞同外界进行物质、能量、信息交换的过程里，始终处于动态平衡之中，正常生理条件和特殊的病理状态下，机体在细胞水平的表达上是不同的。因此，了解动、植物细胞内各种组分的种类、水平、变化，将有助于对动、植物细胞基本生理功能的理解。
动、植物细胞受内源或外源性因素作用而产生大量活性氧、活性氮自由基等，这些自由基的水平与生物的生理、病理密切相关。一方面，体内适当水平的自由基为机体维持正常功能所必需，它们参与信号转导、基因表达及细胞生理功能调控；另一方面，当自由基在体内生成过多，因其高反应活性，则易引起许多病理性变化。此外，自由基在体内处于不断产生、瞬间转化的动态平衡之中,一定生理条件下自由基的互相转化会导致自由基失衡，进而引起机体损伤。伴随着自由基的生成与存在，生物体内也形成了保护细胞免受氧化损伤的自由基清除、抗氧化防御系统，谷胱甘肽体系就是维持细胞内微环境氧化还原状态的重要体系。谷胱甘肽通过清除生命体内的自由基维持细胞的氧化还原平衡，使细胞免遭氧化伤害。
由于对细胞内自由基、清除剂的生成、转化机制、生理作用的研究还远未达到今天生命科学发展所要求的精细和精确，很多机制的探讨仍缺乏细胞、亚细胞水平上自由基产生、变化与自由基的清除防御、损伤之间的基本关系探索。造成对细胞氧化胁迫深层次研究难以开展的原因是缺少对细胞内自由基及谷胱甘肽等调控氧化还原态的活性小分子物种的有效分析方法。基于此，本论文工作以微流控芯片电泳技术作为平台，选择肝癌（HepG2）细胞、大鼠肾上腺嗜铬细胞瘤（PC12）细胞作为研究对象，旨在建立高灵敏度、高选择性,能同时识别和检测细胞内多组份活性物质（自由基、谷胱甘肽）的新方法。本研究为在细胞内、分子水平上研究动、植物氧化胁迫提供了新策略与新方法，对自由基细胞生物学研究具有重要的科学意义。论文研究内容如下：
建立了微流控芯片电泳-激光诱导荧光（MCE-LIF）方法用于凋亡细胞线粒体提取液中谷胱甘肽（GSH）和过氧化氢（H_2O_2）的同时测定。自行合成的有机硒荧光探针Rh-Se-2和二（对甲基苯磺酸酯基）二氯荧光素（FS）分别用作检测GSH和H_2O_2的荧光探针；自身不发荧光的Rh-Se-2与GSH反应生成量子产率较高的荧光产物罗丹明110（Rh-110）；无荧光的FS与H_2O_2反应生成强荧光物质二氯荧光素（DCF）；两种荧光探针对于他们的目标分子都表现出很好的选择性。得到了MCE-LIF同时检测GSH和H_2O_2的优化条件，实现了GSH和H_2O_2的同时、快速检测。我们利用该方法检测了HepG2细胞线粒体提取液中GSH和H_2O_2的含量，还进一步同时检测了经抗癌药物阿霉素和光动力学疗法诱导的HepG2细胞凋亡过程中线粒体提取液内两种活性物质的含量变化。
应用微流控芯片电泳-激光诱导荧光（MCE- LIF）方法同时检测了细胞线粒体提取液中的活性氧/活性氮自由基—即超氧阴离子（O_2~(·-)）和一氧化氮（NO）。荧光探针2-氯-1，3-二苯并噻唑啉环己烯（DBZTC）和3-氨基，4-氨甲基-2’，7’-二氯荧光素（DAF-FM）分别被用于检测线粒体提取液中的O_2~(·-)和NO。得到了MCE-LIF同时检测O_2~(·-)和NO的优化条件，脱氢抗坏血酸（DHA）和抗坏血酸（AA）对DAF-FM检测NO的潜在干扰，可以通过加入抗坏血酸氧化酶（AO）对AA催化氧化后结合微流控芯片电泳分离而得以消除。利用该方法检测了HepG2细胞和PC12细胞线粒体提取液中的O_2~(·-)和NO，证实了这种方法具有简单，快速，重现性好和高效的优点；该方法还被进一步用于检测白藜芦醇诱导的HepG2细胞和β-淀粉样蛋白诱导的PC12细胞凋亡过程中线粒体提取液O_2~(·-)和NO的水平变化。
建立了微流控系统同时检测单个PC12细胞内O_2~(·-)和NO的分析方法。基于自行搭建的微流控分析系统，在具有辅助通道的“十字”芯片上，采用电动门式进样和激光诱导荧光检测，实现了细胞采样、单细胞捕获、电场溶膜、电泳分离和活性氧（O_2~(·-)）/活性氮（NO）自由基同时检测的多步集成。利用荧光染料和荧光微球的流形实验证实了进样方法的有效性，并进行了单细胞连续进样的实验探索。利用该方法检测了单个PC12细胞中的O_2~(·-)和NO。
本论文以微流控芯片电泳技术为研究平台，完成了凋亡细胞线粒体提取液中GSH和H_2O_2的同时测定；凋亡细胞线粒体提取液中O_2~(·-)和NO的同时测定；并完成了单细胞内O_2~(·-)和NO的同时检测。这些活性物质的同时检测有助于了解其各自的生物学功能以及在复杂生命活动过程中它们之间的作用机制。本论文内容从研究对象上，实现了从细胞群体匀浆样品到体现细胞异质性的单细胞样品中活性物质同时检测的跨越；从研究层面上，完成了影响细胞氧化还原态及活性氧/活性氮物质基本关系的探索。研究工作未见报道，为生命科学领域的自由基生物学研究提供了新途径、新方法。
Earth is a planet with a magnificent variety of plants and animals, they are diverse inmorphology and structure. But they are all consist of cells. Cell is the structural andfunctional unit of an organism, i.e., it is the basic unit of structure. A cell in itself is thesmallest part of an organism, which is capable of functioning independently and can carryout the fundamental duties of life like reproduction and metabolism. Under normalphysiological conditions and specific pathological conditions, the expression of organismat the cellular level is different. Awareness of the various types of reactive species, theirlevels and changes within the plant and animal cells will contribute to the understanding ofthe basic physiological functions of plant and animal cells.
Affection of cells by endogenous or exogenous factors will result in the production ofreactive oxygen species and reactive nitrogen species, whose levels are closely related tophysiology and pathology. On one hand, appropriate levels of free radicals in the organismare required to maintain normal functions. They are involved in signal transduction,regulation of gene expression and cellular physiological function. On the other hand, whenfree radicals are overproduced in the organism, they will result in many pathologicalchanges because of their high reactivity. What’s more, production of free radicals is in aconstant, instantaneous transformation state in the organism. Accompanied by theformation of free radicals, there exists a scavenging, antioxidant defense systems, whichwill protect cells from oxidative injury. Among them, glutathione (GSH) system is animportant part in maintaining cellular microenvironmental redox state. GSH protects cellsfrom oxidative damage by depleting free radicals and maintaining plant and animalintracellular redox balance.
Research on the formation of free radicals within cells, their scavenging andtransformation mechanism are far from being reached. But developments in the lifesciences today still require precise and accurate information on them. The reason thatdeeper research on cells’oxidative stress is difficult to carry out is mainly because oflacking of effective analytical methods for determination of free radicals and glutathione.Based on this, we choose microchip electrophoresis technology as our research platform, and selected HepG2 cells and PC12 cells as our research cell targets, aiming at establishinga simultaneous determination method of cellular reactive species (free radicals, GSH). Thestudy is of great scientific importance, not only for the research on free radical cell biology,particularly for cellular oxidative stress, but also provides new strategies and methods forstudy on oxidative stress in plants and animals at the cellular level. Three parts areincluded as follows:
The application of microchip electrophoresis with laser-induced fluorescence(MCE-LIF) detection to simultaneously determine GSH and H_2O_2in mitochondria extractwas described. Organoselenium probe Rh-Se-2 and bis(p-methylbenzenesulfonate)dichloro- fluorescein (FS) synthesized in our laboratory were utilized as fluorescent probesfor GSH and H_2O_2, respectively. Rh-Se-2, which is non-fluorescent, reacts with GSH toproduce rhodamine 110 (Rh110) with high quantum yield. Similarly, non-fluorescent FSreacts with H_2O_2and produces dichlorofluorescein (DCF) accompanied by drasticfluorescence enhancement. Both probes exhibit good sensitivity toward their respectivetarget molecule determination. Fast, simple and sensitive determination of GSH and H_2O_2was realized within 37 s using a running buffer of 50 mM mannitol, 40 mM HEPES (pH7.4) and an electric field of 360V/cm for separation. The MCE-LIF assay was utilized toinvestigate the levels of GSH and H_2O_2in mitochondria extract isolated from HepG2 cells.The method was further extended to observe situations of the two species in mitochondriaextract of HepG2 cells experiencing cell apoptosis that were induced by doxorubicin andphotodynamic therapy.
Next we applied MCE-LIF method for concurrent determination of reactive oxygenspecies (ROS) and reactive nitrogen species (RNS), i. e. superoxide (O2-·) and nitric oxide(NO) in mitochondria, using fluorescent probes 2-chloro-1,3- dibenzothiazoline-cyclohexene (DBZTC) and 3-amino,4-aminomethyl-2',7'-difluorescein (DAF-FM),respectively. Potential interference of intracellular dehydroascorbic acid (DHA) andascorbic acid (AA) for NO detection with DAF-FM were eliminated through oxidation ofAA on addition of ascorbate oxidase, followed by subsequent MCE separation. Fluorescentproducts of O_2~(·-)and NO, that is, DBZTC oxide (DBO) and DAF-FM triazole (DAF-FMT)realized excellent baseline separation within 1 min with a running buffer of 40 mM Tris solution (pH 7.4) and a separating electric-field of 500 V/cm. Using the method, weinvestigated the levels of DBO and DAF-FMT in mitochondria extract isolated fromnormal HepG2 cells and PC12 cells, and further extended the method to detect DBO andDAF-FMT level change in mitochondria extract isolated from apoptotic HepG2 cellsinduced by reseveratrol, and PC12 cells induced by amyloidβpeptide, which was provedto be simple, fast, reproducible and efficient.
Based on the electrokinetic gated injection, we established the automation andintegration of multiple single-cell operations, including cell sampling, single cell loading,single cell cytolysis, electrophoresis separation and ROS/RNS simultaneous detection, on asimple cross microchip with assistant channel using a a microfluidic system. Fluid shapesof fluorescent dyes and microsphere confirms the validity of the method, and we nextinvestigated the possibility of single-cell continuous injection. With the method, wedetermined O_2~(·-)and NO in single cells.
Using microchip electrophoresis technology as our research platform, we realizedsimultaneous determination of GSH and H_2O_2, O_2~(·-)and NO in mitochondria extractisolated from apoptotic cells, then we attempted to determine O_2~(·-)and NO in single cellsconcurrently. Determination of intracellular bioactive species will afford beneficialinformation related to cell metabolism, signal transduction, cell function and diseasetreatment. Simultaneous measurement of those species with the method will helpunderstand their distinctive functions in addition to the interaction between them, and willprovide a new insight into the role that those species play in biological systems. Ourresearch realized determination of biological samples from cellular extracts to single cell,and investigated the factors that influence cellular redox state and the basic relationshipbetween ROS and RNS. The research work has not been reported previously and hasprovided new strategy and new method for free radical biology in life science field.
动、植物细胞受内源或外源性因素作用而产生大量活性氧、活性氮自由基等，这些自由基的水平与生物的生理、病理密切相关。一方面，体内适当水平的自由基为机体维持正常功能所必需，它们参与信号转导、基因表达及细胞生理功能调控；另一方面，当自由基在体内生成过多，因其高反应活性，则易引起许多病理性变化。此外，自由基在体内处于不断产生、瞬间转化的动态平衡之中,一定生理条件下自由基的互相转化会导致自由基失衡，进而引起机体损伤。伴随着自由基的生成与存在，生物体内也形成了保护细胞免受氧化损伤的自由基清除、抗氧化防御系统，谷胱甘肽体系就是维持细胞内微环境氧化还原状态的重要体系。谷胱甘肽通过清除生命体内的自由基维持细胞的氧化还原平衡，使细胞免遭氧化伤害。
由于对细胞内自由基、清除剂的生成、转化机制、生理作用的研究还远未达到今天生命科学发展所要求的精细和精确，很多机制的探讨仍缺乏细胞、亚细胞水平上自由基产生、变化与自由基的清除防御、损伤之间的基本关系探索。造成对细胞氧化胁迫深层次研究难以开展的原因是缺少对细胞内自由基及谷胱甘肽等调控氧化还原态的活性小分子物种的有效分析方法。基于此，本论文工作以微流控芯片电泳技术作为平台，选择肝癌（HepG2）细胞、大鼠肾上腺嗜铬细胞瘤（PC12）细胞作为研究对象，旨在建立高灵敏度、高选择性,能同时识别和检测细胞内多组份活性物质（自由基、谷胱甘肽）的新方法。本研究为在细胞内、分子水平上研究动、植物氧化胁迫提供了新策略与新方法，对自由基细胞生物学研究具有重要的科学意义。论文研究内容如下：
建立了微流控芯片电泳-激光诱导荧光（MCE-LIF）方法用于凋亡细胞线粒体提取液中谷胱甘肽（GSH）和过氧化氢（H_2O_2）的同时测定。自行合成的有机硒荧光探针Rh-Se-2和二（对甲基苯磺酸酯基）二氯荧光素（FS）分别用作检测GSH和H_2O_2的荧光探针；自身不发荧光的Rh-Se-2与GSH反应生成量子产率较高的荧光产物罗丹明110（Rh-110）；无荧光的FS与H_2O_2反应生成强荧光物质二氯荧光素（DCF）；两种荧光探针对于他们的目标分子都表现出很好的选择性。得到了MCE-LIF同时检测GSH和H_2O_2的优化条件，实现了GSH和H_2O_2的同时、快速检测。我们利用该方法检测了HepG2细胞线粒体提取液中GSH和H_2O_2的含量，还进一步同时检测了经抗癌药物阿霉素和光动力学疗法诱导的HepG2细胞凋亡过程中线粒体提取液内两种活性物质的含量变化。
应用微流控芯片电泳-激光诱导荧光（MCE- LIF）方法同时检测了细胞线粒体提取液中的活性氧/活性氮自由基—即超氧阴离子（O_2~(·-)）和一氧化氮（NO）。荧光探针2-氯-1，3-二苯并噻唑啉环己烯（DBZTC）和3-氨基，4-氨甲基-2’，7’-二氯荧光素（DAF-FM）分别被用于检测线粒体提取液中的O_2~(·-)和NO。得到了MCE-LIF同时检测O_2~(·-)和NO的优化条件，脱氢抗坏血酸（DHA）和抗坏血酸（AA）对DAF-FM检测NO的潜在干扰，可以通过加入抗坏血酸氧化酶（AO）对AA催化氧化后结合微流控芯片电泳分离而得以消除。利用该方法检测了HepG2细胞和PC12细胞线粒体提取液中的O_2~(·-)和NO，证实了这种方法具有简单，快速，重现性好和高效的优点；该方法还被进一步用于检测白藜芦醇诱导的HepG2细胞和β-淀粉样蛋白诱导的PC12细胞凋亡过程中线粒体提取液O_2~(·-)和NO的水平变化。
建立了微流控系统同时检测单个PC12细胞内O_2~(·-)和NO的分析方法。基于自行搭建的微流控分析系统，在具有辅助通道的“十字”芯片上，采用电动门式进样和激光诱导荧光检测，实现了细胞采样、单细胞捕获、电场溶膜、电泳分离和活性氧（O_2~(·-)）/活性氮（NO）自由基同时检测的多步集成。利用荧光染料和荧光微球的流形实验证实了进样方法的有效性，并进行了单细胞连续进样的实验探索。利用该方法检测了单个PC12细胞中的O_2~(·-)和NO。
本论文以微流控芯片电泳技术为研究平台，完成了凋亡细胞线粒体提取液中GSH和H_2O_2的同时测定；凋亡细胞线粒体提取液中O_2~(·-)和NO的同时测定；并完成了单细胞内O_2~(·-)和NO的同时检测。这些活性物质的同时检测有助于了解其各自的生物学功能以及在复杂生命活动过程中它们之间的作用机制。本论文内容从研究对象上，实现了从细胞群体匀浆样品到体现细胞异质性的单细胞样品中活性物质同时检测的跨越；从研究层面上，完成了影响细胞氧化还原态及活性氧/活性氮物质基本关系的探索。研究工作未见报道，为生命科学领域的自由基生物学研究提供了新途径、新方法。
Earth is a planet with a magnificent variety of plants and animals, they are diverse inmorphology and structure. But they are all consist of cells. Cell is the structural andfunctional unit of an organism, i.e., it is the basic unit of structure. A cell in itself is thesmallest part of an organism, which is capable of functioning independently and can carryout the fundamental duties of life like reproduction and metabolism. Under normalphysiological conditions and specific pathological conditions, the expression of organismat the cellular level is different. Awareness of the various types of reactive species, theirlevels and changes within the plant and animal cells will contribute to the understanding ofthe basic physiological functions of plant and animal cells.
Affection of cells by endogenous or exogenous factors will result in the production ofreactive oxygen species and reactive nitrogen species, whose levels are closely related tophysiology and pathology. On one hand, appropriate levels of free radicals in the organismare required to maintain normal functions. They are involved in signal transduction,regulation of gene expression and cellular physiological function. On the other hand, whenfree radicals are overproduced in the organism, they will result in many pathologicalchanges because of their high reactivity. What’s more, production of free radicals is in aconstant, instantaneous transformation state in the organism. Accompanied by theformation of free radicals, there exists a scavenging, antioxidant defense systems, whichwill protect cells from oxidative injury. Among them, glutathione (GSH) system is animportant part in maintaining cellular microenvironmental redox state. GSH protects cellsfrom oxidative damage by depleting free radicals and maintaining plant and animalintracellular redox balance.
Research on the formation of free radicals within cells, their scavenging andtransformation mechanism are far from being reached. But developments in the lifesciences today still require precise and accurate information on them. The reason thatdeeper research on cells’oxidative stress is difficult to carry out is mainly because oflacking of effective analytical methods for determination of free radicals and glutathione.Based on this, we choose microchip electrophoresis technology as our research platform, and selected HepG2 cells and PC12 cells as our research cell targets, aiming at establishinga simultaneous determination method of cellular reactive species (free radicals, GSH). Thestudy is of great scientific importance, not only for the research on free radical cell biology,particularly for cellular oxidative stress, but also provides new strategies and methods forstudy on oxidative stress in plants and animals at the cellular level. Three parts areincluded as follows:
The application of microchip electrophoresis with laser-induced fluorescence(MCE-LIF) detection to simultaneously determine GSH and H_2O_2in mitochondria extractwas described. Organoselenium probe Rh-Se-2 and bis(p-methylbenzenesulfonate)dichloro- fluorescein (FS) synthesized in our laboratory were utilized as fluorescent probesfor GSH and H_2O_2, respectively. Rh-Se-2, which is non-fluorescent, reacts with GSH toproduce rhodamine 110 (Rh110) with high quantum yield. Similarly, non-fluorescent FSreacts with H_2O_2and produces dichlorofluorescein (DCF) accompanied by drasticfluorescence enhancement. Both probes exhibit good sensitivity toward their respectivetarget molecule determination. Fast, simple and sensitive determination of GSH and H_2O_2was realized within 37 s using a running buffer of 50 mM mannitol, 40 mM HEPES (pH7.4) and an electric field of 360V/cm for separation. The MCE-LIF assay was utilized toinvestigate the levels of GSH and H_2O_2in mitochondria extract isolated from HepG2 cells.The method was further extended to observe situations of the two species in mitochondriaextract of HepG2 cells experiencing cell apoptosis that were induced by doxorubicin andphotodynamic therapy.
Next we applied MCE-LIF method for concurrent determination of reactive oxygenspecies (ROS) and reactive nitrogen species (RNS), i. e. superoxide (O2-·) and nitric oxide(NO) in mitochondria, using fluorescent probes 2-chloro-1,3- dibenzothiazoline-cyclohexene (DBZTC) and 3-amino,4-aminomethyl-2',7'-difluorescein (DAF-FM),respectively. Potential interference of intracellular dehydroascorbic acid (DHA) andascorbic acid (AA) for NO detection with DAF-FM were eliminated through oxidation ofAA on addition of ascorbate oxidase, followed by subsequent MCE separation. Fluorescentproducts of O_2~(·-)and NO, that is, DBZTC oxide (DBO) and DAF-FM triazole (DAF-FMT)realized excellent baseline separation within 1 min with a running buffer of 40 mM Tris solution (pH 7.4) and a separating electric-field of 500 V/cm. Using the method, weinvestigated the levels of DBO and DAF-FMT in mitochondria extract isolated fromnormal HepG2 cells and PC12 cells, and further extended the method to detect DBO andDAF-FMT level change in mitochondria extract isolated from apoptotic HepG2 cellsinduced by reseveratrol, and PC12 cells induced by amyloidβpeptide, which was provedto be simple, fast, reproducible and efficient.
Based on the electrokinetic gated injection, we established the automation andintegration of multiple single-cell operations, including cell sampling, single cell loading,single cell cytolysis, electrophoresis separation and ROS/RNS simultaneous detection, on asimple cross microchip with assistant channel using a a microfluidic system. Fluid shapesof fluorescent dyes and microsphere confirms the validity of the method, and we nextinvestigated the possibility of single-cell continuous injection. With the method, wedetermined O_2~(·-)and NO in single cells.
Using microchip electrophoresis technology as our research platform, we realizedsimultaneous determination of GSH and H_2O_2, O_2~(·-)and NO in mitochondria extractisolated from apoptotic cells, then we attempted to determine O_2~(·-)and NO in single cellsconcurrently. Determination of intracellular bioactive species will afford beneficialinformation related to cell metabolism, signal transduction, cell function and diseasetreatment. Simultaneous measurement of those species with the method will helpunderstand their distinctive functions in addition to the interaction between them, and willprovide a new insight into the role that those species play in biological systems. Ourresearch realized determination of biological samples from cellular extracts to single cell,and investigated the factors that influence cellular redox state and the basic relationshipbetween ROS and RNS. The research work has not been reported previously and hasprovided new strategy and new method for free radical biology in life science field.
引文
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