摘要
本论文以“聚合物／蛋白质核壳纳米粒子的合成及在葡萄糖传感器中的应用研究”为主题,利用铜离子引发体系在蛋白上接枝聚合物,制备了壳层为蛋白的核壳纳米粒子；通过激光光散射(LLS)、透射电镜(TEM)、Zeta电位和X射线光电子能谱(XPS)等分析技术表征了粒子的核壳结构；通过带耗散的石英晶体微天平(QCM-D)和LLS研究了酶与壳层为蛋白的纳米粒子之间的相互作用以及酶在粒子上的吸附性质；利用壳层为蛋白的纳米粒子固定酶制备了生物传感器,并结合循环伏安法和恒电位安培法详细表征了生物传感器的测试性能。主要结果如下：
首先,利用铜离子引发体系,我们制备出核层为聚甲基丙烯酸甲酯(PMMA)壳层为牛血清白蛋白(BSA)的PMMA-BSA核壳纳米粒子。通过TEM表征,直接观察到了PMMA-BSA纳米粒子的核壳结构。Zeta电位实验表明粒子和BSA的Zeta电位随溶液pH值变化基本一致,说明粒子的壳层为BSA。结合XPS测试,对比分析了PMMA-BSA纳米粒子和BSA的表面成分,为PMMA-BSA纳米粒子的壳层是BSA提供了直接的证据。在实验上,我们研究了温度、BSA与MMA质量比和铜离子浓度对聚合反应的影响,并利用索氏提取和酸水解法,得到聚合生成的均聚PMMA (homo-PMMA)和接枝到BSA上的共聚PMMA (graft-PMMA),通过分析它们的结构,来研究聚合反应的原理。结果表明铜离子介入的MMA在BSA上的接枝聚合属于自由基聚合反应,并表现出乳液聚合的特征。铜离子通过与BSA络合,在加热下发生氧化还原,产生自由基,引发MMA在BSA上的接枝聚合,产生的PMMA和BSA在水溶液中自组装形成PMMA-BSA核壳纳米粒子。通过控制聚合反应中BSA与MMA的质量比,我们可以制备出粒径分布较窄,半径范围在60-110 nm的PMMA-BSA核壳纳米粒子。由于壳层为蛋白,使得PMMA-BSA纳米粒子具有良好的生物相容性。
其次,研究了PMMA-BSA纳米粒子对葡萄糖氧化酶(GOx)的固定。在实验上,利用LLS和QCM-D表征方法,详细研究了GOx与PMMA-BSA纳米粒子之间的相互作用及GOx在PMMA-BSA纳米粒子上的吸附过程。结果表明,GOx是以静电吸引为驱动力吸附到PMMA-BSA纳米粒子上。当溶液pH值小于GOx等电点4.2或大于PMMA-BSA等电点5.1时,GOx和PMMA-BSA纳米粒子表面带同种电荷,静电排斥阻碍了GOx在粒子表面的吸附；当溶液pH值介于PMMA-BSA和GOx等电点之间时,GOx通过静电吸引固定到PMMA-BSA粒子表面。QCM-D实验结果表明GOx在PMMA-BSA纳米粒子上的吸附有两个动力学过程：第一个过程是由静电吸引作用导致的GOx在PMMA-BSA纳米粒子表面的快速吸附过程；第二个过程是由吸附在粒子上的GOx构象调整,产生了一些新的吸附位点,使GOx继续吸附的过程。利用紫外光谱法测试了吸附后GOx的活性,结果表明与自由GOx相比,吸附到PMMA-BSA纳米粒子上的GOx活性保留了80%以上。并且吸附后的GOx耐热性也获得很大提高。在50℃下恒温35小时之后,GOx自由酶的活性损失了64%,而吸附到粒子上的GOx仅损失了28%的活性。
在这些实验基础上,我们利用PMMA-BSA纳米粒子固定GOx,制备了电流型葡萄糖传感器。通过循环伏安法和恒电位安培法,表征了传感器的测试性能。在氧气存在下,传感器对葡萄糖具有良好的催化作用,循环伏安实验表明传感器具有典型的扩散控制的电化学特征。在0.4 V的工作电位下,传感器对葡萄糖的线性响应范围为0.2-9.1 mM,灵敏度高达44.1μA mM-1 cm-2,响应时间仅为6s,并且传感器在0.25 V的低电位下,对葡萄糖仍具有较高的电流响应密度(25.8μAmM-1 cm-2),表明传感器可以通过低电位工作降低干扰物的影响。在相同条件下制备的传感器,对1 mM葡萄糖的响应电流的相对标准偏差仅为4.9%；另外,传感器在含有1mM葡萄糖的磷酸盐缓冲溶液(0.05 M,pH 7.4)中,以50 mV／s的扫描速度在0-0.6 V的范围内扫描50圈,氧化电流未见明显减小,表明传感器具有良好的重现性和稳定性。我们还考察了传感器的储存稳定性,结果表明传感器在室温下储存23天响应电流仅下降4%,储存30天后,仍具有82%的响应电流。优异的操作稳定性和储存性能使得我们制备的传感器具有潜在的应用价值。
With a title of "Synthesis of Polymer/Protein Core-Shell Nanoparticles and Their Application in Glucose Biosensors", the dissertation focuses on the synthesis of core-shell nanoparticles with a protein shell by copper mediated graft copolymerization; the core-shell structure of these particles are characterized in detail by a combination of laser light scattering (LLS), transmission electronic microscopy (TEM), Zeta potential and X-ray photoelectron spectra (XPS) techniques; the interaction and adsorption of enzyme on these particles with a protein shell are studied by using a quartz crystal microbalance with dissipation (QCM-D) and LLS; the biosensor is constructed by immobilizing enzyme on these particles with a protein shell, and the analytical performance of the biosensor is investigated by cyclic voltammetry and chronoamperometry. The main results are as follows:
First, well-defined core-shell poly(methyl methacrylate)-bovine serum albumin (PMMA-BSA) particles have been prepared by direct graft copolymerization of methyl methacrylate (MMA) on bovine serum albumin (BSA) with a trace amount of copper ions. The core-shell structure of PMMA-BSA particles was confirmed by the TEM analysis. The pH dependence of the Zeta potential of PMMA-BSA particles and pure BSA is similar, indicating BSA is coated on the PMMA-BSA particles as a shell. The XPS analysis reveals that the XPS surveys of the PMMA-BSA particles and pure BSA are similar, further demonstrating that the surface of the PMMA-BSA particles is covered by BSA. Effects of the reaction temperature, the mass ratio of BSA to MMA, and the copper ions concentration on the copolymerization have been studied. The principle of copolymerization was investigated by characterizing the structure of PMMA homopolymer isolated by the Soxhlet extraction and PMMA grafted on BSA obtained by hydrolyzed with acid. The results reveal that copper mediated graft copolymerization of MMA on BSA is free radical polymerization, similar to an emulsion polymerization. The PMMA-BSA core-shell particles are formed by the self-assembly of those amphiphilic PMMA-BSA grafted copolymer chains, initiated by radical produced from copper complex with BSA under heating. The average particle size can be well controlled in the range 60-110 nm with a variation of the initial mass ratio of MMA to BSA. Such formed core-shell particles have a biocompatible BSA shell.
Further, we studied the immobilization of glucose oxidase (GOx) on such prepared PMMA-BSA particles. The interaction and adsorption of GOx on PMMA-BSA particles were investigated by a combination of QCM-D and LLS measurements. The results show that the adsorption of GOx on PMMA-BSA particles is driving by electrostatic interaction. Both PMMA-BSA and GOx are positively or negatively charged when pH<4.2 (the pI of GOx) or>5.1 (the pI of PMMA-BSA) so that the electrostatic repulsion leads to no adsorption of GOx on PMMA-BSA in these two ranges; the adsorption occurs at pH between these two pI values, at which they are oppositely charged. The QCM-D studies reveal that the adsorption kinetics contains two processes:the quick adsorption is due to electrostatic interaction; the second process is attributed to the continued adsorption of GOx on the new points produced by the conformation change of adsorbed GOx. The enzymatic activity was determined by UV/vis measurements. The result shows that the adsorbed GOx can retain more than 80% activity in comparison with free enzyme. Thermal stability studies reveal that the adsorbed GOx only losses 28% of its activity in comparison with a 64% activity loss of free GOx when they are incubated at 50℃for 35 h.
On the basis of these studies, we have constructed an amperometric glucose biosensor by immobilizing GOx on PMMA-BSA particles. The performance of prepared biosensors was tested by cyclic voltammetry and chronoamperometry. The testing results demonstrate the biosensor has a good performance in the oxidation of glucose. The cyclic voltammetry measurement reveals that the biosensor involves a typical diffusion-controlled electrochemical reaction and has a linear response in the glucose concentration range 0.2-9.1 mM with a sensitivity of 44.1μA mM-1 cm-2 and a short response time of 6 s under an working potential of 0.4 V. It is worth noting that the biosensor keeps a relatively high response current (25.8μA mM-1 cm-2) when a working potential 0.25 V is used; namely, it can be used to eliminate the interference by working at low potential. The reproducibility was tested by using six electrodes prepared in the same way and an identical glucose concentration (1 mM). The relative standard deviation is 4.9%, showing a good reproducibility of the biosensor preparation. The durability test demonstrated that there is nearly no change of the response current even after 50 successive scans in a glucose solution of 1 mM. More important, such prepared biosensors are thermally stable at the room temperature; namely, the response current of the biosensor only decreases～4% after they were stored for 23 days. After 30 days, the biosensor still retains～82% of its original response. The excellent operational and storage stability make these biosensors potentially useful in biomedical application.
引文
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