新型生物传感器及其在相关生物物理研究中的应用

发布时间：2017-12-30 21:32

本文关键词：新型生物传感器及其在相关生物物理研究中的应用　出处：《中国科学技术大学》2016年博士论文　论文类型：学位论文

【摘要】：低温生物学和细胞力学是现代生物医学工程领域中的两个重要分支。本博士论文以冷冻保存血小板为主线,阐述了作者为解决血小板冷冻保存这一世界性难题,在上述两个领域中所做的研究工作。本文第一部分主要针对血小板冷冻保存环节中所面临的如何更快速精确的测量生物材料导热系数、如何更深入的认识低温冷冻期间水的跨膜传输特性和如何更准确的预测胞内冰形成概率这三个问题。笔者详细介绍了一种基于微纳米加工技术,可以对生物材料的微观热物性进行高精度自动化测量的微电子机械系统的研发工作;同时,还详细阐述了使用该套系统在探索生物材料微尺度传热以及细胞冷冻损伤过程中所进行的基础性研究。笔者通过微纳米加工技术将传统的热物性探针微型化,并将热探针的制造工艺工业化,将热探针的精度校准和测量过程自动化和标准化。笔者成功的在4英寸硅晶圆上将作为微探针能量发生单元和导热系数测量单元的尺寸进一步降低成线宽仅为3微米、线间距仅为5微米的蛇形高密度金属热丝。同时,微探针的分割切片技术也升级成为自由度及加工精度都更高的感应耦合等离子体深硅刻蚀技术,能够更快更容易的制造出尺寸和形状都更为多样的微型热物性探针(单针局部测量或阵列式多针多点测量)。通过对不同低温保护剂和不同生物材料在不同温度下进行大量且系统的导热系数测量,笔者发现使用微纳米加工工艺制造出的微型探针拥有更小的热容,更紧凑的探针阵列,能更直接地与被测样品接触,通过使用不同结构的热探针可以更准确、更灵敏、更可靠的对生物及其相关材料进行单点微创式热传导系数的测量,或者获得某个特定区域内的热物性分布数据。整套芯片的制备过程已经完成了从微纳米加工与传统精密制造工艺的混合制造到纯微纳米加工的转型,能够像生产集成电路一样,实现高良品率、高物理和电学相似性的大批量生产,完美解决了传统热传导系数测量设备与方法在临床应用和科学研究上的缺陷与不足(只能对材料进行离体检测,设备内部结构复杂,繁琐的测量前准备工作,灵敏度低,制造难度大等)。与此同时,我们的实验结果还揭示出一直被广泛采用的热探针精度标定过程存在严重缺陷,当测量温度和标定温度不一致时,通过常规标定过程所得出的热探针系统精度校准常数会导致最终测量结果出现较大误差。因此,我们提出了一套新的标定与测量流程,有效确保了热探针系统精度校准常数的准确性,使得最终测量结果的分析与处理过程标准化、统一化、精准化。并且根据所获得的实验数据,通过使用联合拟合、方程组合等数据分析方法,笔者还首次提出了一种基于Second-order Polynomial和Filippov equation的可用于预测不同浓度的二元低温保护剂溶液在不同温度下导热系数的双因素(温度和浓度)理论公式。通过使用研发出来的微纳米热探针和低温显微镜平台,笔者还对细胞的跨膜水传输特性和胞内冰形成概率进行了深入的学习。由于实验条件的限制,笔者使用了人子宫颈癌细胞作为概念证明实验里的实验对象。对人子宫颈癌细胞在不同降温速率下体积的变化情况进行了仔细深入的观察与分析,对人子宫颈癌细胞的冷冻响应特性(跨膜水传输性质和胞内冰形成概率)进行了系统的研究。使用Mazur提出的跨膜水传输模型,联合拟合得出人子宫颈癌细胞细胞膜的水传输参数,从而进一步预测在其他冷冻环境下细胞跨膜水传输的响应规律。笔者在实验中发现胞内冰的形成主要分为Darkening和Twitching两种类型。这两种不同类型的胞内冰形成机制很可能与最初所形成的胞内冰大小密切相关。通过使用Toner提出的胞内冰生成概率模型对实验结果进行拟合,笔者还发现该模型存在较为严重的弊端和局限性,因此笔者通过引入临界体积这一新参数,提出了一种新的能更准确预测胞内冰生成概率的改进型理论模型。利用微纳米加工技术大规模生产微尺度热探针是现代科学技术与传统生物热物理测量技术的完美结合。在传统宏观生物材料热传导系数测量技术的基础上,从微尺度、微创和集成的角度上,更加准确、可靠和全面地研究生物活体材料及其相关材料的微尺度传热及损伤特性,这不仅可以帮助认识和发现生物材料在微观传热过程中的本质规律,还可以在生物材料或器官的低温冷冻保存、低温或聚能外科手术及相关传热领域中,提供必要的研究测量技术和理论依据。本文的第二部分主要针对冷冻血小板复温后所面临的如何快速可靠的对血小板功能进行定量测量的问题。笔者详细阐述了一种基于BioMEMS技术对对血小板收缩力进行定量检测的细胞微力测量系统的研发和使用该系统对血小板综合性功能进行评估的探索过程。激活后的血小板,其中后期的功能表达主要表现在用产生的血小板收缩力来强化和巩固前期形成的松散的止血拴,让止血栓能更牢固地粘附在受损血管的裂口处,继续起到止血,维护血管壁完整性的作用。在本文中,我们提出用血小板收缩力来做为检验其中后期功能的新指标,并详细阐述了一种通过利用阵列微柱式微力感测系统对血小板收缩力直接进行微力测量的技术。笔者通过微纳米加工技术制造出具有不同直径(600纳米-3微米)、不同高度(5-15微米)和不同微柱间距(1-3微米)的微柱阵列,而且用该微力感测系统首次测量并研究了处于纳牛顿量级上半静态下的血小板收缩力与外界生物蛋白、刺激剂和刺激时间等参数之间的关系,更首次在高倍微显条件下动态观测并分析了血小板收缩力的整个产生、发展和变化过程。通过阵列微柱式微力感测系统,血小板收缩力首次被高精度的测量出来,由此,为解决血小板功能性检测这一世界性难题,提供了新的解决思路和办法。为早期发现血小板功能异常,指导血小板抗凝药物开发和阐明相关疾病的形成机理,提供了必要的技术设备和奠定了一定的理论基础。总的说来,本论文针对血小板在低温保存和复温后功能检测这两个方面进行了系统深入的研究。成功研发出的两种新型微纳米生物传感器,可以很好的用于检测生物材料的热物理和力学特性。在其他相关生物医学领域的研究中,我们提出的新的微纳米生物医学微全分析系统系统与检测方法也有着广泛的应用前景。
[Abstract]:Low temperature biology and cell mechanics are two important branches of modern biomedical engineering. This thesis takes the cryopreserved platelets as the main line, elaborated the author to solve the cryopreservation of platelets which is a worldwide difficult problem, the research work done in the above two areas. The first part of this paper mainly for the cryopreservation of platelets facing link in how to quickly and accurately measuring thermal conductivity of biomaterials, how the transmembrane transport characteristics of water during freezing and a deeper understanding of how to more accurately predict intracellular ice formation probability of these three questions. The author introduces a kind of micro nano processing technology based on micro electro mechanical system, can high precision automatic measurement the micro thermal properties of biological materials research and development work; at the same time, also elaborated the system used in the exploration of micro scale biological material Based on the heat and cell freezing injury process. Through the micro nano processing technology will be thermal probe micro traditional manufacturing technology, industrialization and heat probe, the precision of the calibration and measurement of process automation and thermal probe. The standard of success in 4 inch silicon wafers, as micro the probe energy generating unit and the thermal conductivity measurement unit to further reduce the size of Cheng Xiankuan is only 3 microns, line spacing is only a serpentine high density metal wire 5 microns. At the same time, the segmentation technology of micro probe also upgraded inductively coupled plasma deep silicon etching technology has become a degree of freedom and accuracy are higher, faster and easier to create the size and shape of more micro thermal probe diversity (local measurement or single needle array of multi needle multipoint measurement). According to the different cryoprotectants and different students By measuring the thermal conductivity of a material and system at different temperatures, the author found that the heat capacity with smaller use of micro probe micro nano processing technology to produce the probe array is more compact, more direct contact with the sample, through the thermal probe using different structure can be more accurate, more sensitive measurement single point of minimally invasive thermal conductivity of biological and related materials are more reliable, or obtain the thermal distribution data of a specific area of the whole chip. The preparation process has been completed from the mixture of micro nano processing and traditional precision manufacturing technology to manufacture pure micro nano processing transformation, as production the integrated circuit, to achieve high yields, high physical and electrical properties similar to mass production, the perfect solution to the traditional heat conduction coefficient measuring device and method in the clinical application and scientific research. Defects and shortcomings (only the materials were measured in vitro, the internal structure of complex equipment, preparation work, tedious before the measurement sensitivity is low, difficult to manufacture etc.). At the same time, our results reveal that the thermal probe has been widely used in precision calibration process in serious defects, when the measured temperature and temperature calibration consistent, through the conventional calibration precision calibration of thermal probe system constant the process will lead to the final measurement result error. Therefore, we put forward a new set of calibration and measurement process, effectively ensure the accuracy of thermal probe system precision calibration constant, standard analysis and process of making the final measurement results, unified and accurate. And according to the experimental data, by using the combined fitting equation, combined data analysis methods, the author also proposed a method based on Second-or Der Polynomial and Filippov equation can be used in two factor two yuan of cryoprotectant solutions at different temperatures and thermal conductivity prediction of different concentration (temperature and concentration). By using the theoretical formula developed by the micro nano scaled heat and low temperature probe microscope platform, the author on the cell membrane water transport properties and intracellular ice formation the probability of in-depth study. Due to the limitation of experimental conditions, the author used the human cervical cancer cells as a proof of concept in the experimental subjects. Observation and in-depth analysis carefully carried on a human cervical cancer cell in the volume changes at different cooling rates, on human cervical cancer cell freezing response (characteristics of transmembrane water transport and intracellular ice formation probability) was studied. Mazur proposed transmembrane water transport model, combined with the fitted cells of human cervical carcinoma cells Water transmission parameters of membrane, thus predicting response of cells in other frozen environment of transmembrane water transport. The further experiments found that the intracellular ice formation is mainly divided into two types of Darkening and Twitching. The formation mechanism of these two different types of intracellular ice is likely to be closely related to the initial form intracellular ice size. By using the Toner of intracellular ice formation probability model to fit the experimental results, the author also found that the model has disadvantages and limitations of the more serious, so the author introduces a new parameter of the critical volume, proposes a new improved theoretical model can more accurately predict intracellular ice generation probability. Using the micro nano processing technology for large-scale production of micro scale thermal probe is a perfect combination of modern science and technology and traditional bio physical measurement technology. In the traditional macro biological material thermal conductivity Based on the measurement technique, from micro scale, minimally invasive and integrated perspective, more accurate, reliable and comprehensive study of biological materials and related materials in micro scale heat transfer and damage characteristics, which can not only help understand and find the essence of the law of biological materials in micro heat transfer process, can also be frozen in biology material or organ preservation, field surgery and related energy at low temperature or heat transfer, provide the necessary measurement technology research and theoretical basis. The second part mainly for frozen platelets after rewarming faced by how fast and reliable quantitative measurement of platelet function. The author elaborates a kind of BioMEMS technology research and development quantitative detection of platelet contractile force on cells based on micro force measuring system and use the system to evaluate the comprehensive functions of the explored platelet Cheng. After activation of platelet function, the late expression mainly in loose platelet contractile force generated to strengthen and consolidate the early formation of the hemostatic plug to let the more firmly in the gap of damaged blood vessels, continue to maintain hemostasis, integrity of vessel wall in this role., we propose to use platelet contractile force to test a new index which later function, and elaborated a micro column array by using the micro force sensing system of platelet contractile force directly to micro force measurement technology. The micro nano processing technology to produce with different diameter (600 nm -3 micron). Different height (5-15 microns) and different micro column spacing (1-3 microns) micro column array, and the micro force sensing system for the first time measurement and study in nano platelet contractile force and semi static under Newton on Bound protein, the relationship between stimuli and stimulus parameters such as time, for the first time in high times slightly under the condition of dynamic observation and analysis of platelet contractile force of the whole generation, development and change process. The micro column array micro force sensing system for the first time, platelet contractile force was measured with high precision, thus, in order to solve the detection of platelet function which is a worldwide difficult problem, provides new ideas and solutions. For the early detection of abnormal platelet function, the formation mechanism of guidance of anticoagulant drug development and clarify the related diseases, provides the necessary technical equipment and laid a theoretical foundation. In general, this paper aresystematically platelet function during cold storage and rewarming after these two aspects. Successfully developed two new micro nano bio sensor, can be very good for the detection of students The thermophysical and mechanical properties of materials and materials have been widely applied in other related biomedical fields.

【学位授予单位】：中国科学技术大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：TP212.3;R33

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