摘要
细胞是组成生物机体的基本结构和功能的单位。初期的细胞学是以研究细胞的形态和结构为主要内容。最近几十年,随着现代科学技术,特别是由于分子生物学的发展,使细胞学的研究内容焕然一新,对于细胞的研究已逐渐发展成为一门研究细胞显微结构和超微结构化学组成的科学;生命活动的一些基本过程如物质代谢、运动、发育、繁殖以及遗传等重要机制,正在细胞水平上进行日益深入地研究。同时,现代细胞化学技术又吸引了多学科的先进技术,更深入地阐明了细胞的增殖与分化、遗传与变异,为疾病的病理和病因提供科学依据。目前,细胞学已逐渐发展成为从显微水平、亚显微水平和分子水平三个层次上深入探讨细胞生命活动的学科。
细胞的分析研究已经成为分析化学前沿学科领域中的热门话题,研究细胞化学不仅有利于确定细胞功能和代谢及细胞内的化学成分,而且还有利于确定细胞特征,进行疾病的病理和有关药理的机制研究。目前,细胞生物学、分子生物学等生命科学前沿学科向分析化学提出的课题集中在多肽、蛋白质、核酸等生物大分子分析以及生物药物分析和超痕量、超微量生物活性物质,如单个细胞内生物物质的分析。在生物无机分析领域中,痕量元素分析已集中到元素在生物组织层、单个细胞甚至细胞膜中的微分布及其结合形式等方面。使用超微量样品的微痕量分析,检测限达到fg级水平。
生物现象最基本的过程是电荷运动,人体运动、大脑信息传递及细胞膜的结构与功能机制都涉及到电化学过程,可以说,电化学是生命科学的基础学科。热点研究领域之一——生物电分析化学便是建立在电分析化学、生物物理学、生物化学、电生物物理学、电生理以及电化学等数门学科基础上的独立学科。在电化
微型电化学传感器的研究及其在细胞分析中的应用一论文摘要
学的基本原理和实验方法基础之上,生物电分析化学已经迅速发展成为研究和探
讨生命现象本质的最合适、最有力的工具之一。
本论文创新性地以多种灵敏度高、选择性好的微型电化学修饰传感器,对细
胞损伤和修复过程中胞内外的多种生命活性物质的水平变化进行了研究。以心肌
细胞、内皮细胞、平滑肌细胞等心血管细胞为研究对象,以酶、纳米材料等为修
饰剂,研制了多种用于监测细胞内外NO、次黄嘿吟、细胞色素C及超氧阴离子
自由基的微型传感器,对细胞损伤及给药修复后的信息物质的水平变化进行了深
入研究,建立了细胞实时在线监测的新方法。因此,本论文不仅拓展了电分析化
学的研究领域,对促进细胞生物学和临床科学的发展具有比较重要的理论意义和
科学价值,同时在开发治疗心脏病等心血管疾病药物方面也具有广泛的应用前
景。
第一章:序言
本章对研究细胞的传统生物技术方法、分析化学在细胞研究中的应用以及
生物体中信息物质及检测技术研究进展等方面进行了比较详尽的综述,着重综述
了毛细管电泳(CE)分析法、微柱分离法、微电极电化学法在细胞分析中的应用,
同时对细胞中重要的信息分子,包括:一氧化氮、超氧阴离子、嗓吟碱以及细胞
色素C的性质、生物合成及生理作用、生理功能进行了阐述。讨论了开展本论
文研究的目的和意义并归纳了本论文研究的创新之处。
第二章:一氧化氮微型传感器的研制及其应用于细胞分析的研究
NO是神经、心血管系统细胞间信息传递的重要调节因子,作为第二信使和
神经传递物质而起着不同的功能,同时还是宿主免疫反应中的一种细胞毒性因
子:作为内皮舒张因子(ED孙),NO可以释放到相邻的平滑肌,使血管舒张;
NO也可以扩散到血流中抑制血小板的凝聚。由于NO在神经生理、免疫、病理
华东师范大学申请博士学位论文
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微型电化学传感器的研究及其在细胞分析中的应用一论文摘要
及药理等方面的研究都具有极为重要的作用,实现NO的直接实时测定,成为进
一步探索NO功能的必要手段。但由于生物体内NO易逸、不稳定,且浓度极低,
实时测定尤为困难。微电极电化学法具有电极探头微小、响应快速、灵敏、选择
性好等特点,非常适用于NO的实时动态检测。本论文制备了一种新型的
Nafi。可Au溶胶修饰微铂传感器,对NO有较高的灵敏度和良好的选择性,检测
限为5.ox 10一“m。比。本论文还探讨了该修饰微传感器对No的催化氧化机理,
并以该微传感器为工作电极组成三电极系统,研究了在L一精氨酸(L一Aig)和乙
酞胆碱(Ach)及黄酮类药物刺激下平滑肌细胞内的NO释放,以及在L一抢g、
Ach刺激下心肌细胞内的NO释放。拓展了NO的检测方法,具有广阔的应用前
景,并对生理、病理、临床医学研究都具有重要的价值。
第三章:超氧阴离子自由基微传感器的研制及在内皮细胞研究中的应用
近年来,超氧阴离子在心肌缺血和再灌注损伤研究中受到广泛关注。在缺血
一再灌注损伤部位,自由基形成增多,其强烈的氧化作用引起血管内皮细胞和组
织细胞的损伤,导致器官功能障碍。由于02一具有强氧化性、半衰期短、且生物
体内的浓度较低(约为10-5.、10气1℃比)等特点,实现实时动态分析必须发展高灵敏度、
高选择性、可靠性好及响应快速的检测技术。
Living beings were constructed with some single cells, and substances inside are most important to cell's surviving, reproduce, damage, and death. Therefore, studies upon functions of organisms from single cells level and molecules level, are spotlight and significant in present days. Cultured cells kept up some characteristics of tissue. The model of cells in hypoglycemia and hypoxia can mimic the morbid state of intracorporal tissue or organs in the absence of other species, such as neutrophils and nerve corpuscles. In order to fight for myocardial ischemic necrosis and protect myocardium, lots of model studies were performed for mechanism research. In this paper, three parts of work have been performed in the thesis following an overall review:
Chapter One: Critical Reviews
A critical review with regard to the cells assaying was presented, including the study of cell with traditional methods, application of analytical chemistry towards cell analysis, the development of detection technology for messengers of biological systerm, especially, the CE, micro-column separation, microelectrodes. In addition, the characteristic, biosynthesis and biological functions of some important messenger molecular, such as, nitric oxide, superoxide, purine and Cyt C was also reviewed.
Chapter Two: Preparation of Nitric Oxide Microsensor and Its Application towards Cells Analysis
The aim of this work was to fabricate microelectrodes as a current approach for the in situ monitoring of nitric oxide release from living cells stimulated by some exogenic agonists. For the purpose, a novel sensitive, selective and stable nitric oxide (NO) microsensor is constructed, which is modified by nano Au colloid and Nafion. Determining by Atomic Forced Microscope, the diameter of Au colloid particles is from 7 to 14 nanometers. The detection of NO is based on the nano Au particles catalysis of
NO oxidation at an anodic potential of +0.74V (vs. SCE). The microsensor showed a low detection limit, high selectivity and sensitivity for NO determination. The oxidation current (measured by differential pulse amperometric technique) was linear with NO concentration ranging from 1.0x10-7 to 4.0x10-5 mol/L with a calculated detection limit of 5.0x10-8 mol/L (S/N=3). Using the microsensor, the direct, real time production of NO in the smooth muscle cells and myocardial cells was continuously measured.
Chapter Three: Fabrication of Superoxide Microsensors and its Application toward Endothelial Cells
A novel superoxide microsensors based on platinum/palladium microparticles and electropolymerized pyrrole was fabricated for the measurement of extracellular endothelial cells superoxide. The Pt/Pd-PPy modified microsensors were evaluated as superoxide sensor. The amperometric responses to superoxide were monitored at the potential of O.OOV(vs SCE) in HBSS. The sensor was proved to have a high sensitivity, selectivity and short response time. The detection limit is 12 nmol/L (S/N=3). The life period (at least 1 month) of sensors is longer than that of enzyme electrodes. The potential interference from some endogenous electroactive substances in biological system, such as H2O2, UA, neurotransmitters and their metabolites, at the concentrations higher than those in biological system, could be eliminated by further coating the Pt/Pd modified electrode with a poly pyrrole fihn. The method was applied to the measurement of superoxide production in endothelial cells.
Chapter Four: Study on the Change of Purin during the Process of Injury and Respairation of Myocardial Cells
(1): A novel hypoxanthine (Hx) microsensor was constructed. In this work, Nafion Xanthine oxidase and Au colloid were immobilized on the surface of Pt microelectrode, respectively. The enzyme biosensor displayed a quick and sensitive response to Hx. In physiological condition, the biosensor showed a low detection limit, high selectivity and sensitivity for Hx determination. The oxidation current
(measured by i-t) was linear with Hx concentration ranging from 2.0x10
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