用于希瓦氏菌操控及检测的微流体平台研制与应用研究

发布时间：2018-05-14 05:35

本文选题：微流体芯片 + 介电泳　；参考：《中国科学技术大学》2017年博士论文

【摘要】：人类社会即将面临能源危机和环境污染等困境,希瓦氏菌污染治理和绿色能源方面表现出巨大的潜力,因而获得到了广泛关注并掀起了研究热潮。然而,过去的研究主要集中于群体水平至生物膜水平,不易探究精确的机理。直至2010年,基于单细菌或若干细菌(下文简称为细菌层次)的多项精彩工作发表,希瓦氏菌的两种电子传递机制获得证明,这些工作的成功基于微纳技术的引入。因此,希瓦氏菌细菌层次上的深入探究及定量检测也需依赖于微纳技术和微流控技术。微流体技术因其众多优势已经被广泛应用于分析化学、生命科学、药学及环境科学中,然而在微流体平台上进行的原核细胞研究还处于起步阶段,因原核细胞形状差异大、尺寸较小(亚微米级别)、无规则自由运动等特点使得相关研究进展困难。本文将研究对象聚焦于原核生物中具有特色的电化学活性菌的代表希瓦氏菌,目标是实现希瓦氏菌在细菌层次的快速有效且无标记的定量检测,以支持后续进一步地探究。实现希瓦氏菌在细菌层次的检测前提是制备细菌层次的样品,而这种样品制备也只能借助于微纳技术及微流体相关技术。考虑到微流体芯片具有微型化与集成化两大特征,本文的思路是先开发微型化的单元模块,主要为细菌层次样品制备的操控模块及相关的检测模块,然后借助于开发这些单元模块的经验,制作集成化的微流体芯片,实现希瓦氏菌细菌层次的操控及检测。希瓦氏菌操控模块的章节中,先后研制了三种操控基底,分别为被动操控的微结构阵列、侧壁微电极的介电泳主动操控及非接触式微电极的主动操控。这些操控模块均能实现快速高通量无标记的希瓦氏菌操控,其中被动微结构阵列无法提供稳定的固定。本章也同时提出了希瓦氏菌的介电泳操控模型,建立了完整的操控体系。三种操控模块的研制为后续微流体中操控模块的开发与集成提供了基础及经验。希瓦氏菌检测模块的章节中,针对研究对象希瓦氏菌,开发了微流体中的光及电信号相关的检测模块。光学模块以聚二甲基硅氧烷(Polydimethylsiloxane,PDMS)膜为核心,以内锁结构的接口为配件,开发了一体成型工艺制作PDMS微流体,支持在线监测微流体通道并提升了通道中获取希瓦氏菌精确信息的可能性。电学模块中为下游的希瓦氏菌检测制作了大面积的双层电极基底,采用了测量电极-聚甲基丙烯酸甲酷(Polymethylmethacrylate,PMMA)绝缘层-介电泳操控电极三层结构的设计。同时也提出了微流体漏液模型和解决方案。最后,以单元模块开发积累的经验,设计制造了集成了介电泳捕获、荧光检测、拉曼检测的微流体平台,用于希瓦氏菌在细菌层次上样品制备和检测,实现了快速有效无标记可计数的希瓦氏菌的定量捕获及检测。借助于模拟分析,我们对介电泳操控电极进行优化,设计了有微阱结构的钝化层,能够与微流体平台的其它部分协同,实现三点功能,即重排电场分布、确定捕获区及测量区。微阱中被捕获固定的希瓦氏菌可进行精确的数目统计,结果表明捕获的希瓦氏菌数目及尺寸与微阱尺寸相关。最后对捕获的希瓦氏菌进行了初步的拉曼光谱检测,验证了微流体芯片同时具有样品检测的功能。
[Abstract]:The human society is facing the difficulties of energy crisis and environmental pollution. The pollution control and green energy show great potential. Therefore, we have received extensive attention and raised the upsurge of research. However, the past research focused on the level of the group to the biofilm level and was not easy to explore the precise mechanism until 2010. The two electron transfer mechanisms of habelli have been proved to be based on the introduction of micronanotechnology. Therefore, the deep exploration and quantitative detection on the level of the bacteria at the hagabelli bacteria need to be dependent on micro nanotechnology and microfluidic technology. Because of its many advantages, fluid technology has been widely used in analytical chemistry, life science, pharmacy and environmental science. However, the study of prokaryotic cells on the microfluidic platform is still in its infancy. Due to the large difference in the shape of the prokaryotic cells, the small size (sub micron grade), and the free movement of the cells, the related research progress is difficult. The aim is to realize the rapid, effective and unmarked quantitative detection of the bacteria at the bacterial level, in order to support further exploration. The sample preparation can only be prepared by micro nano technology and microfluidic related technology. Considering the micromation and integration of micro fluid chips, two characteristics are considered. The idea of this paper is to develop miniaturized unit modules, mainly for the control module and related detection modules prepared by bacteria level samples, and then to develop these sheets. With the experience of the meta module, integrated microfluidic chips are fabricated to realize the manipulation and detection of the bacteria level of the H. valsalis. In the chapter of the Bush control module, three kinds of manipulated substrates are developed, respectively, the passive manipulated microstructures, the main dynamic manipulation of the side wall microelectrodes and the active manipulation of the non-contact microelectrodes. The control module can achieve fast and high flux and unmarked hirwelli control, in which the passive microstructural array can not provide stable fixation. This chapter also proposes a hirvellic electrophoresis manipulation model and a complete control system. The development and integration of the three control modules is the development and integration of the control module in the continuous microfluidic. For the foundation and experience. In the chapter of the detection module of the H. bush, the detection module related to light and electrical signals in the microfluidic is developed for the study object of the study object. The optical module uses the Polydimethylsiloxane (Polydimethylsiloxane, PDMS) membrane as the core and the interface of the inner lock structure as the accessory, and develops the integrated forming process to make PDMS The microfluidic supports the on-line monitoring of the microfluidic channel and increases the possibility of obtaining the exact information of the hevale bacteria in the channel. In the electrical module, a large area of the double layer electrode substrate was fabricated for the detection of the lower hevale bacteria in the downstream, and the measuring electrode - Polymethylmethacrylate (PMMA) insulating layer - dielectrophoretic manipulation electrode The three layer structure is designed. At the same time, the micro fluid leakage model and solution are also proposed. Finally, with the experience of the unit module development, the microfluidic platform integrated with diphoretic capture, fluorescence detection and Raman detection is designed and manufactured, which is used for the preparation and detection of the samples of the bacteria at the level of bacteria. The quantitative capture and detection of the number of hewelli bacteria. With the aid of simulation analysis, we optimized the electrophoretic manipulated electrode and designed a passivation layer with micro well structure. It can cooperate with the other parts of the microfluidic platform to realize the three point function, that is to rearrange the electric field distribution, determine the capture area and the measurement area. The results showed that the number and size of the collected hewsv were related to the size of the microwell. Finally, a preliminary Raman spectrum detection was carried out for the collected hewsv bacteria, which proved that the microfluidic chip had the function of sample detection at the same time.

【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】：2017
【分类号】：TH79

【参考文献】