核酸、蛋白质物化过程和聚合物性质压电传感研究

【作者】 毛友安；

【导师】 魏万之；

【作者基本信息】 湖南大学 ， 分析化学， 2002， 博士

【摘要】 压电石英晶体体声波传感器具有响应谱广、灵敏度高、易实现数字化等独特优点。本论文将压电传感技术用于生物分析化学和聚合物科学领域，在DNA、蛋白质在固-液界面上的物理化学过程研究和聚合物分析表征方面开展了一系列探索性工作，取得了一系列很有价值的研究成果。本论文的主要研究工作如下。 1．提出了用压电石英晶体阻抗分析技术(PQCI)实时监测小分子诱导DNA与蛋白质交联形成过程的新方法，并用这种新方法监测了甲醛诱导下DNA与溶菌酶的交联反应过程。基于对PQCI技术提供的多维信息的分析，定量研究了交联过程的动力学，首次报道了交联反应过程的动力学参数，如交联结合过程的结合速率常数和解离速率常数，以及交联反应的平衡常数。 2．提出了用PQCI技术实时监测以DNA为靶标的药物分子与DNA结合过程的新方法，并用这种新方法监测了抗癌药物米托蒽醌与固定在压电传感器表面上的双股小牛胸腺DNA的结合过程。DNA的固定采用逐层自组装方法。基于对PQCI多维信息的分析，定量研究了结合过程的动力学。得到的结合平衡常数与文献值合理地一致。首次报道了结合过程的结合速率常数和解离速率常数。 3．从理论上推导出了一个描述蛋白质在固一液界面上吸附过程的动力学的压电响应模型，用PQCI技术监测了人血清白蛋白(HSA)和牛血清白蛋白(BSA)从pH 7.4的缓冲溶液(PBS)中吸附到裸银电极和聚苯乙烯修饰电极表面的过程，以及溶菌酶吸附到裸银电极和巯基乙酸修饰电极表面的过程。利用压电响应模型拟合了吸附过程中频移响应的实验数据。结果表明，HSA和溶菌酶的吸附过程可以用包含两步反应的动力学方程来描述。对BSA吸附过程而言，在其浓度较低时，吸附只涉及第一步反应；浓度较高时，在聚苯乙烯修饰表面上的吸附过程包含两步反应。通过数学拟合，本文得到了这些吸附过程的反应速率常数。所得到的数据显示出这些蛋白质在不同条件下吸附过程动力学上的差别。 4．提出了用PQCI技术实时监测药物小分子与蛋白质结合过程的新方法，并用这种新方法监测了盐酸小檗碱与BSA的结合过程，定量研究了结合过程的动力学，首次报道了该结合反应过程的动力学参数，如结合速率常数、解离速率常数和结合反应平衡常数。 5．提出了用串联式压电石英晶体传感技术(SPQC)研究离子型表面活性剂与蛋白质之间相互作用过程的新方法，并用这种新方法研究了溴化十六烷基三甲基铵和十二烷基磺酸钠与溶菌酶在水溶液中的相互作用过程。与表面张力法、 湖 南 大 学 博 士 学 位 论 文FTJR法和圆二色性法相比，新方法优点在于实验装置和实验操作简单，分析成本低；与电导法相比，新方法具有较高的灵敏度。 6，提出了一种测定聚合物粘均分子量的新方法一流过式压电石英晶体粘度传感技术，构建了带有流动检测池的实验装置，并用这种新方法测定了未知聚乙烯醇（PVAL）试样的分子量。结果表明，新方法较之传统的毛细管粘度计法，具有明显的优点，如简便快速、仪器价廉便携、可用于连续测定等。 7．基于压电传感器对与其接触的聚合物稀溶液的粘度变化的动态电阻响应，提出了一种研究聚合物分子间热力学相互作用的新方法，并用这种新方法研究了聚乙二醇（PEG）与 PVAL之间的相互作用，得到了该聚合物共混体系的热力学系数之值。结果表明，这两种聚合物分子间存在吸引性相互作用。新方法的一个重要特征是可以用于连续测定。更多还原

【Abstract】 Piezoelectric quartz crystal bulk acoustic wave sensing techniques have many advantages, such as broad sensing spectrum, high sensitivity, simple construction and easy realization for digitization. In the present thesis, the piezoelectric sensing techniques were applied for the study in the fields of bio-analytical chemistry and polymer science, actually for the study of the physical and chemical processes of DNA and proteins at the solid-liquid interface and the analysis and characterization of polymers. A series of valuable and important results were obtained. The main work of the thesis could be summarized as follows.1. A novel method for monitoring, in real-time, the small molecule - induced DNA -protein cross-linking process with piezoelectric quartz crystal impedance (PQCI) technique was proposed. The method was used to monitor the formaldehyde -induced DNA - lysozyme cross-linking formation process. On the basis of the analysis of the multi-dimensional information provided by the PQCI technique, the kinetics of the cross-linking process was quantitatively studied. The kinetic parameters, such as the binding and dissociation rate constants and the equilibrium constant for the cross-linking, were first reported in the world.2. A novel method for the real-time monitoring of the binding process of the DNA-targeted drug molecule to DNA with the PQCI technique was proposed. The method was used to monitor the binding process of an anticancer drug, mitoxantrone, to the double stranded calf-thymus DNA immobilized on the surface of the piezoelectric sensor. The DNA immobilization was realized by layer-by-layer self-assembly. The kinetics of the binding process was quantitatively studied. The value of the binding equilibrium constant obtained here was in reasonable agreement with the reference value. The values of the binding and dissociation rate constants were first reported .3. A piezoelectric response model for describing the kinetics of the protein adsorption process at a solid-liquid interface was theoretically derived. The adsorption processes of human serum albumin (HSA) and bovine serum albumin (BSA) from phosphate buffered saline (PBS, pH 7.4) onto bare and polystyrene (PS) -modified silver electrode surfaces and of lysozyme onto bare and thioglycollic acid -modified silver electrode surfaces were in situ monitored with the PQCI technique. The response model was used to fit the experimental data of frequency shift for these adsorption processes. It was shown that the adsorption processes of HSA and lysozyme could be described by a kinetic equation involving two consecutive reactions. At lower concentration, BSA adsorption only involved the first reaction. At higher concentration, BSA adsorption onPS-modified surface involved two consecutive reactions. The regression values of the reaction rate constants for these adsorption processes were obtained. These data exhibited the difference in the protein adsorption kinetics under different conditions. ?4. A novel method for the real-time monitoring the drug binding process to protein with the PQCI technique was proposed. The method was used to monitor the binding process of berberine hydrochloride to BSA. The kinetics of the binding process was quantitatively studied and the kinetic parameters, such as the binding and dissociation rate constants and the binding equilibrium constant, were first reported in the world.5. A new method for probing the interaction process between ionic surfactant and protein with series piezoelectric quartz crystal sensing technique (SPQC) was developed. The method was used to examine the interaction process of cetyltrimethyl ammonium bromide and sodium dodecyl sulfate with lysozyme in aqueous solution. Compared with the surface tension method, FT-IR and CD method, the new method was of its advantages, such as simple更多还原