【作者】 杨政鹏；

【导师】 司士辉；

【作者基本信息】 中南大学 ， 应用化学， 2008， 博士

【摘要】 纳米材料及相关技术的应用为当今生物医学、功能材料、能源科学等领域的研究提供了新的技术平台。纳米结构单元构筑的纳米材料在磁性、光电性质、化学活性、催化等方面均表现出常规材料所不具备的性能，目前纳米生物检测、仿生纳米材料合成、生物大分子（如酶，DNA）功能化纳米粒及纳微孔中生物分子受限反应等方面研究已成为研究的热点。本论文将纳米材料、膜制备技术、压电石英晶体传感及其它表面分析技术结合起来，研究了纳微多孔材料上胆红素及牛血清白蛋白吸附、尿素酶及葡萄糖氧化酶在纳微多孔材料上催化行为以及羟基磷灰石仿生钙化等过程，获得了纳微多孔材料上特殊的生化作用规律，对于发展纳米生化检测新技术、实现酶高效固载与催化、合成仿生矿化材料等具有重要意义。论文主要研究工作概括如下：一、人体中过量游离胆红素（BR）沉积到各种组织细胞膜上将引发BR代谢紊乱，导致多种疾病。本研究借助于石英晶体微天平（QCM）考察了模拟细胞膜（磷脂双层膜）上BR的沉积过程及影响因素，探讨其致病机理。着重探讨了BR在纳米TiO₂膜上的吸附与光催化降解行为，以期为BR引发疾病的治疗提供新思路。UV-vis和IR光谱研究证实了BR在纳米晶体TiO₂膜上的吸附，QCM测量结果表明溶液的pH、离子强度、浓度以及温度影响BR的吸附。BR的吸附量随着BR浓度的增加而增大；然而温度和离子强度的增加，BR吸附量却明显下降。pH的影响较为复杂，当pH在2-4范围内变动时，BR吸附量略有上升；然而当pH上升到4-8范围内时，吸附量随着pH增大快速增加；当pH大于8时，吸附量则出现下降趋势。UV光照下吸附BR的光催化降解研究表明TiO₂膜能再生并重复使用。目前，纳米TiO₂膜应用于去除BR仍处于实验阶段，实际应用有待进一步研究。二、基于溶胶凝胶分子印迹技术，以纳米TiO₂溶胶为基质印迹了牛血清白蛋白和尿素酶分子。石英晶体微天平研究表明纳米TiO₂印迹膜稳定性好，印迹分子在纳米TiO₂非印迹与印迹膜上的吸附分别符合Langmuir和Allosteric吸附模型；印迹分子在纳米TiO₂印迹膜上吸附量随溶液浓度和pH的增加而增大，然而随离子强度的增加而减小。钛丝基体表面修饰一层纳米TiO₂印迹膜，尿素酶固载后构建了一种廉价的、易于微型化的尿素生物传感器；电位响应测量表明该方法制备的尿素生物传感器稳定性好，对尿素检测响应速度快（25s）、线性范围宽（8μM-3 mM）。三、采用两步阳极氧化法制备了纳米多孔氧化铝膜，尿素酶固载于氧化铝纳米多孔膜中构筑了压电尿素生物传感器。借助ESPS／FIA传感体系监测酶反应，测量结果表明固载于多孔氧化铝中的酶具有高催化活性。纳米孔中尿素酶固载条件优化如下：2．0 mg／mL尿素溶液（pH 7．5，25℃），固载时间2．5 h，大尺寸氧化铝膜。研究发现戊二醛交联60 min后壳聚糖涂覆构建的压电尿素生物传感器用于尿素检测具有响应速度快（30 s）、低检测限（0．2μM）、宽线性范围（0．5μM-3mM）、高选择性（0．92-1．03）、良好重现性（S．D．=0．02，n=6）及长期贮存稳定性（贮存30天后，酶活性保留了76％）等优点。实际样品测量表明该传感器可用于尿样中尿素快速检测，在临床检验与环境监测等领域具有应用前景。四、单酶分子经由表面修饰和原位聚合制备了一种新的无机／有机聚合物网状结构包裹的磁性单酶纳米粒（SENs）。TEM、FTIR和XRD分析表明合成的SENs呈球形、多分散状，直径大约为50 nm，包裹酶的纳米壳由Fe₃O₄／聚（吡咯-N-丙烷基磺酸）复合物组成。电磁测量表明SENs的电导率为2．7×10^-3S．cm^-1，具有超顺磁性，饱和磁强度为14．5 emu．g^-1，矫顽力为60 Oe。与自由酶相比，包裹酶不仅活性显著增强，而且对溶液pH和温度变化、有机溶剂影响及长期贮存过程都具有良好的稳定性，在生物检测与传感、酶催化工程等领域有着潜在的应用前景。五、采用石英晶体微天平（QCM）技术现场研究了纳米TiO₂表面Cu（Ⅱ）、Hg（Ⅱ）的吸附与光化学还原过程。结果表明Cu（Ⅱ）的吸附过程符合准二级动力学反应，反应速率常数约为0．09 g·mmol^-1·min^-1；Hg（Ⅱ）的吸附动力学过程可用准一级方程描述，吸附平衡常数约为3．9×10⁵L．mol^-1。Cu（Ⅱ）、Hg（Ⅱ）的吸附量均受到溶液pH、浓度和共存阴离子的影响；在pH=4的溶液中，其饱和吸附量分别为1．5和0．85mmol．g^-1。UV光照下，Cu（Ⅱ）发生光还原反应，频率逐渐下降；然而光照初始阶段，TiO₂表面水光解产生的质子可使吸附的Hg（Ⅱ）发生脱附，且Hg（Ⅱ）浓度和pH越大，脱附现象越明显，随后Hg（Ⅱ）光还原沉积逐渐占主导地位，频率下降。此外，Cu（Ⅱ）、Hg（Ⅱ）的光还原沉积速率受溶液pH和有机物的影响，pH越高，光沉积速率越大，有机物的加入使光化学还原速率显著加快。六、基于Cu（Ⅱ）、Hg（Ⅱ）离子界面物理化学过程的研究，采用SEM、FTIR、XRD、EDX和QCM研究了模拟体液（SBF）中负电荷纳米TiO₂膜上羟基磷灰石矿化动态过程中成核、生长及结晶行为。结果发现羟基磷灰石的形成过程存在两个不同的阶段，在初始阶段，SBF溶液中的Ca²⁺离子首先结合到负电荷的TiO₂膜表面；随后，在界面上形成的钛酯钙与PO₄^3-离子发生作用，并生成了羟基磷灰石核。成核之后，TiO₂膜不再作为成核的中心，初始阶段形成的羟基磷灰石变成了成核与成长的新中心，过饱和SBF溶液中的Ca²⁺、PO₄^3-以及其它微量离子(CO₃^2-和Mg²⁺等)自发沉积到初始阶段形成的羟基磷灰石层上，并最终生成了羟基磷灰石沉淀。根据QCM随时间的频率变化，求得了羟基磷灰石成核与成长的速率常数（K₁和K₂），结果发现对同样浓度的SBF溶液，K₁值高于K₂，说明成核阶段的反应速率高于成长阶段反应速率。更多还原

【Abstract】 The application of nanomaterial and related technology provides a new platform for the development of current biomedicine, functional material, energy science, and so on. Compared with the bulk materials, nanostructured materials composed of nanostructure building blocks possess unique properties in many aspects, such as magnetism, photoelectricity, chemical activity, catalysis, and so on. At present, nanobiodetection, synthesis of biomineralized material, nanoparticles functionalized by biological macromolecules （i.e., enzyme and DNA） and restricted reaction of biomolecules in nanometer space are the hotspots of current research. In this thesis, piezoelectric quartz crystal sensing combined with nanomaterial, membrane preparation technology and other surface analysis techniques has been employed to study the adsorption of bilirubin and bovine serum albumin, catalysis of urease and glucose oxidase, and biomimetic calcification of apatite on nanoporous materials, and we aim to cognize the special laws of biochemical action on nanoporous materials. The present study is essential for the development of new biochemical measurement technology, realization of high efficiency enzyme immobilization and catalysis, synthesis of biomineralized material, and so on. The main work could be summarized as follows:1. Deposition and accumulation of extra free bilirubin（BR） in body tissues will initiate disorders in the metabolism of BR and cause various diseases. The deposition process and affecting factors of BR on mimic cell membranes （phospholipid bilayer） were first investigated using QCM to know its mechanism of causing illness, and then mainly studied the adsorpton and photochemical decomposition of BR at the nanometer TiO₂, the purpose of the work would explore new way to treat these dicreases caused by BR. The adsorption of BR at nanometer TiO₂ was verified with UV/vis and IR spectra. QCM measurements indicated that the amount of adsorbed BR increased with increasing BR concentration and decreased with increasing temperature and ionic strength. The effect of pH was complicated, the amount of BR adsorbed increased slightly in the pH range of 2-4, and then increased rapidly in the pH range of 4-8, finally decreased at pH > 8. The photodegradation of adsorbed BR at nanometer TiO₂ during UV illumination showed that the TiO₂ films could be regenerated and used repeatedly. At present, the removal of BR by naometer TiO₂ films is only referring to laboratory use, and not for practical use.2. The bovine serum albumin （BSA） and urease imprinted TiO₂ films were prepared via surface sol-gel process using nano-sized TiO₂ sol as imprinted matrix. QCM study indicated that the imprinted TiO₂ film possessed good stability, the adsorption behavior of imprinted molecules onto non-imprinted and imprinted TiO₂ films fitted into Langmuir and Allosteric model respectively. The adsorption amount of imprinted molecules onto imprinted film increased with the increasing concentration and pH, while decreased with the increase of ionic strength. By immobilizing urease to imprinted TiO₂ film modified at the surface of titanium silk, a cheap and miniaturized urea biosensor was developed. Potential measurements indicated that the obtained urea biosensor had a good stability, and exhibited shorter response time （25 s） and wider linear range （8μM-3 mM）.3. A novel piezoelectric urea biosensor has been developed for urea determination, based on the immobilization of urease to nanoporous alumina membranes prepared by two-step anodization. The ESPS/FIA monitoring indicated that the enzymes immobilized into porous alumina possessed high activity. Factors affecting urease immobilization were discussed and the optimized immobilization conditions obtained were pH of 7.5, urease concentration of 2.0 mg/mL, temperature of 25℃, immobilization time of 2.5 hours and relatively big pore dimension. In addition, it was observed that the urea biosensor prepared by glutaraldehyde reticulation for 60 min and followed by chitosan coating exhibited shorter response time （30 s）, lower detection limit （0.2μM）, wider linear range （0.5μM-3 mM）, high selectivity （0.92-1.03）, better reproducibility （S.D. = 0.02, n = 6） and good long-term storage stability （with about 76% of the enzymatic activity retained after 30 days）. The practical application of the urea biosensor not only demonstrated the feasibility of urea detection in urine sample, but also meant that a urea biosensor with low cost and anti-jamming was obtained in our study. Such sensors might be widely applied to medical and environmental fields in the future.4. Magnetic single-enzyme nanoparticles （SENs） encapsulated within a composite inorganic/organic polymer network were fabricated via the surface modification and in situ aqueous polymerization of separate enzyme molecule. The analyses of TEM, FTIR and XRD indicated that the synthesized SENs with about 50 nm in diameter were spherical in shape, quite polydisperse and the nanoshell entrapping enzyme was composed of Fe₃O₄/poly（pyrrole-N-propylsulfonic acid） composites. Electrical and magnetic measurements revealed that the magnetic SENs had a conductivity of 2.7×10^-3S.cm^-1, and were superparamagnetic with a saturation magnetization of 14.5 emu.g^-1 and a coercive force of 60 Oe. Compared with free enzyme, encapsulated enzyme exhibited a strong tolerance to the variation of solution pH and temperature, organic solvent and long-term storage, thus showing significantly enhanced enzyme performance and stability. The magnetic SENs with high activity and stability would find potential applications in many fields, such as biological detection and sensing, enzymatic catalysis and so on.5. The adsorption and photochemical reduction process of Cu（Ⅱ） and Hg（Ⅱ） at the surface of nanometer TiO₂ were investigated using in situ quartz crystal microbalance （QCM）. It was found that the adsorption of Cu（Ⅱ） onto active sites of nanocrystalline fit the pseudo-second-order reaction reaction, and that the rate constant of the reaction was estimated about 0.09 g·mmol^-1·min^-1; whereas the adsorption equilibrium constant of Hg（Ⅱ） was about 3.9×10⁵ L.mol^-1 based on the pseudo-first-order kinetic model. The adsorption amount of Cu（Ⅱ） and Hg（Ⅱ） depended on pH、concentration and coexisting anions, and the saturated amounts of adsorbed Cu（Ⅱ） and Hg（Ⅱ） were approximately 1.5 and 0.85 mmol·g^-1 at pH 4, respectively. During UV illumination, the frequency of QCM decreased gradually, which meaned the photoreduction deposition of Cu（Ⅱ） from the solution; whereas as for Hg（Ⅱ）, at the initial stage of UV illumination, the protons produced in photodegradative reactions of water could cause the desorption of adsorbed Hg（Ⅱ） from the surface of TiO₂, and the degree of desorption increased with the increase of both the concentration of Hg（Ⅱ） and pH value of solution, then the frequency decreased due to the strength of photochemical deposit reaction of Hg（Ⅱ）. In addition, the photodeposition rates of Cu（Ⅱ） and Hg（Ⅱ） increased with increasing pH of solution, and the rate of photoreduction enhanced significantly in the presence of the organisms.6. Based on the study of physical and chemical process of Cu（Ⅱ）, Hg（Ⅱ） ions at the interface, the nucleation, growth and crystal of apatite induced by negatively charged nanometer TiO₂ coatings soaked in simulated body fluid （SBF） were investigated using SEM, FTIR, XRD, EDX and QCM. Two different stages were clearly observed in the process of apatite formation, indicating two different kinetic processes. At the first stage, the calcium ions in SBF were initially attracted to the negatively charged TiO₂ surface, and then the calcium titanate formed at the interface combined with phosphate ions, consequently forming apatite nuclei. After the nucleation, the TiO₂ surface did not act as the center of nucleation, and the apatite formed at the first stage became the new center of nucleation and growth; the calcium ions, phosphate ions and other minor ions (i.e., CO₃^2- and Mg²⁺) in supersaturated SBF deposited spontaneously on the original apatite coatings to form apatite precipitates. In terms of the in situ frequency shifts, the growth rate constants of apatite （K₁ and K₂） were estimated respectively at two different stages. It was found that the reaction rate at the first stage was obviously higher than that at the second stage.更多还原