随着环境污染的日益严重，生物传感器成为环境监测的重要方法。在生 物传感器的制备过程中，生物活性物质的固定是其关键部分，常用的方法包 埋法、交联法等都会部分的降低其活性。Langmuir-Blodgett(LB)膜技术以其 生物相容性好，制备简单，操作方便等优点越来越引起科学家们的关注。 脂类分子由于是构成细胞生物膜的骨架成分，因此是制备 LB 膜首选的 材料，但是大量的研究表明，小分子的脂类分子 LB 膜存在耐热性和力学强 度差等缺点，从而在一定程度上限制了其应用。但是由高聚物类脂分子形成 的高分子薄膜的耐热性和力学强度等机械性能都可以得到很大的改善。 本研究选择一种带负电荷的新型两亲性高聚物(TPR)为成膜材料，通过 LB 膜技术将硝基苯降解菌固定，初步验证该法制备的生物传感器用于硝基 苯检测的可行性，并以硬脂酸进行对比研究。 研究分别以纯水、PAH 溶液、硝基苯降解菌液为亚相，高聚物和硬脂 酸为成膜物质的表面压-单分子面积曲线(即 π-A 曲线)，结果表明在气/液界 面均能形成稳定排列有序的单分子膜。 通过原子力显微镜、傅立叶红外光谱和接触角实验研究在不同表面压力 下转移到硅片上的 TPR LB 膜，其结果表明随着膜压的升高，得到的单分子 膜分布更加均匀，排列更加紧密，缺陷越少。 不同层数 TPR LB 膜修饰电极的循环伏安实验证明随着修饰电极的 LB 膜层数的增加，其峰电流值逐渐降低，且成线性关系，从而证实该超薄膜致 密均匀。 分别以高聚物和硬脂酸为成膜材料，通过 LB 膜法制备了固定硝基苯降 解菌的模拟微生物膜，并通过原子力显微镜观察了模拟微生物膜以及经过培 养后模拟生物膜的表面形貌。从原子力显微镜图片可以看出硝基苯降解菌稳 定存在，且分布均匀，以硬脂酸为成膜材料时，细菌以单个形式均匀附着， 以高聚物为成膜材料时，细菌以多个菌聚集的形式附着；被固定的硝基苯降 解菌能保持很好的活性，经过 24 h 的培养后菌体的数目明显增多。 研究制备的模拟微生物膜与硝基苯溶液作用后，硝基苯溶液浓度的变 化，以及采用循环伏安分析法对修饰电极及其在硝基苯溶液中的电化学特性 进行研究，并研究了不同 pH 值对其的影响。其结果表明硝基苯溶液的浓度哈尔滨工业大学工学硕士学位报告 在经过固定硝基苯降解菌的模拟微生物膜反应后明显降低，循环伏安图可以 看出修饰电极在硝基苯溶液中出现了一明显的还原峰，且峰电流值随着硝基 苯溶液浓度的增加而增大，有较好的线性关系，且体系 pH 的检测范围可在 4~11 之间。 以上实验数据完全说明用 LB 膜法制备的生物传感器用于硝基苯检测的 可行性。 关键词 微生物固定；LB 膜；高聚物；硬脂酸；硝基苯哈尔滨工业大学工学硕士学位报告 Abstract With the increasingly serious problems of environmental pollution, biosensor has become a significant part in environmental quality forecasting and warning and is widely employed in environmental monitoring. Immobiliazation of biomolecules play an important role in the preparation of biosensor, and the general methods including physical entrapment or covalent could reduce their activitis more or less. Langmuir-Blodgett(LB) technique have attracted much attention because of its good biocompatibility, simple preparation, covenient operation and other advantages. Lipid molecule is the preferred material of LB film preparation because it forms the skeleton of the cellular biomembrane. But various studies have shown that LB films of small molecular lipid compound are not good at mechanical and thermal stability, and its application is restricted. But polymer films of polymeric lipids have progressively gained importance because of their higher mechanical and thermal stability compared with lipid amphiphiles. A negative-charged terpolymer was chosen, and nitrobenzene degeneration bacterium were immobilized based on LB technique. Results proved that a microbial sensor based on LB technique for estimation of nitrobenzene can be used practicably. And all experiments were compared with stearic acid. The interfacial properties of terpolymer and stearic acid at pure water, positive polyelectrolyte PAH and liquid medium of nitrobenzene degeneration bacterium were investigated by π-A isotherm. Results showed that terpolymer and stearic acid formed stable monolayer at the air-water interface. AFM, FTIR and Contact Angle measurements were used to characterize the various physical properties of TPR LB films deposited on silicon substrates at different surface pressure. The results showed that the LB film packed tightly and orderly after the monolayers were deposited on silicon substrates at higher surface pressure by LB technique. The electrochemical properties were investigated with electrode coated with different number of TPR LB films. The results showed that cathodic peak哈尔滨工业大学工学硕士学位报告 intensity decreased with increasing of the number of TPR LB films on the electrode and there was a linear relationship between the intensity of the current and number of layers. We could conclude that TPR LB films were homogeneous and dense. The simulation of microbial membranes immobilized microbes were prepared by LB technique. Topographies of microbial membranes before and after microbial culture were observed via AFM. The AFM measurements proved the information that terpolymer and stearic acid were good for the immobilization of microbe. The microbe was found to distribute on the surface of terpolymer and stearic acid homongerously. It was interesting that only single microbe attached to stearic acid layer while 2~4 microbes were found to sticked to terpolymer layer. The number of microbes increased obviously and it showed that the microbes which were immobilized by terpolymer and stearic acid were active enough. The concentration of nitrobenzene was examined after the interaction between the simulation of microbial membranes and nitrobenzene solution. The electrochemical properties of modified electrode in nitrobenzene solution were studied by cyclic voltammetry. A notable cathodic peak was observed and cathodic peak intensity increased with increasing of the concentration of nitrobenzene. It was clearly that there was a linear relationship between the intensity of cathodic peak intensity and the oncentration of nitrobenzene. The modified electrode could be used in solution pH value in the range of 4~11. All results proved that a biosensor based on LB technique for estimation of nitrobenzene can be used practicably. Keywords Microbe immobility; Langmuir-Blodgett film; Terpolymer; Stearic- Acid; Nitrobenzene哈尔滨工业大学工学硕士学位报告 目 录 摘要...........................................................................................................................I Abstract ...................................................................................................................... III 第 1 章 绪 论..............................................................................................................1 1.1 生物传感器................................................................................................... 1 1.1.1 生物传感器概述.................................................................................... 1 1.1.2 生物传感器的分类................................................................................ 1 1.1.3 生物传感器的原理和特点.................................................................... 1 1.1.4 生物传感器的应用................................................................................ 2 1.2 生物活性单元的固定化技术....................................................................... 3 1.2.1 吸附法.................................................................................................... 3 1.2.2 共价结合法............................................................................................ 4 1.2.3 包埋法.................................................................................................... 5 1.2.4 交联法.................................................................................................... 6 1.2.5 超分子组装技术.................................................................................... 6 1.3 LB膜 .............................................................................................................. 8 1.3.1 LB膜概述................................................................................................ 8 1.3.2 LB膜成膜原理........................................................................................ 9 1.3.3 LB膜