【作者】 周峰；

【导师】 陈红； 袁琳；

【作者基本信息】 武汉理工大学 ， 生物材料学， 2012， 博士

【摘要】 微纳米拓扑结构化表面近年来在生物医学领域的应用正受到越来越广泛的关注,如在传感检测和组织工程材料领域,这类材料有着独特的优势。但长期以来制备这种表面的方法存在多种不足。因而,通过一种简便易行的方法在多种材料表面制备这类表面,将有助于加快其在生物医学领域的理论研究和应用拓展。本论文的主要工作是通过简便易行的葡萄糖还原法在常见材料表面构建金纳米粒子聚集体(GNPL)构成的微纳米级拓扑结构。一方面考察了这种表面在应用于传统的酶联免疫吸附测试(ELISA)检测中的效果；另一方面,我们对其进行了化学改性修饰使之具备抗蛋白质吸附能力并接枝上能与细胞特异性结合的多肽甘氨酸-精氨酸-甘氨酸-天冬氨酸-酪氨酸(GRGDY),考察了这种微纳米结构化粗糙表面对L929成纤维细胞与表面之间的特异性结合作用的影响。进一步地,我们考察了修饰有能与特定癌细胞选择性结合的核酸适体的表面对混合癌细胞中特定细胞的选择性捕获能力。具体研究内容如下：1.利用葡萄糖还原法在多种材料表面制备了微纳米级三维结构的GNPL修饰的表面,考察了这种方法所制备的GNPL修饰酶标板在应用于ELISA中的检测效果。用金相显微镜和场发射扫描电子显微镜(FESEM)对这种GNPL的表面形貌和结构进行了表征。通过放射性同位素标记法测试了不同形貌的GNPL修饰表面对模型蛋白人纤维蛋白原(Fg),人血清白蛋白(HSA)和溶菌酶(LYZ)吸附量的影响,并进一步考察了所吸附LYZ的活性。考察了这种GNPL表面特殊的浸润性现象,以及捕获抗体固定方式对ELISA检测效果的影响。考察了这种GNPL修饰酶标板在对癌症标志蛋白人癌胚抗原(CEA)以及人抗凝血酶原(AT),兔免疫球蛋白(IgG)和人纤连蛋白(Fn)的检测效果。研究发现：这种GNPL形貌和对蛋白的吸附量可以很方便地受反应液用量来控制。新制备出的GNPL经不同方法处理后,分别出现出超亲水和超疏水两种完全相反的浸润状态,并且超亲水态的GNPL在应用于ELISA时效果最好。这种GNPL修饰板能显著提高ELISA对所测试蛋白的检测能力,且具有优异的选择性和稳定性。这种改进方法简便易行、低成本、易于推广、可以作为具有更高检测能力的酶联免疫测试方法的一个重要补充。2.通过表面化学改性的方法在GNPL表面制备了具有抗蛋白非特异性吸附能力并同时能与细胞发生特异性结合的表面。研究了GNPL表面粗糙度对L929成纤维细胞与该表面的特异性结合作用的影响。采用表面引发原子转移自由基聚合(SI-ATRP)在GNPL表面修饰上聚甲基丙烯酸寡聚乙二醇酯(POEGMA),并以其为间隔臂链接上能与细胞发生特异性作用的GRGDY短肽。通过场发射扫描电子显微镜(FESEM)、水接触角仪、表面粗糙度仪、椭圆偏振光谱仪和X-射线光电子能谱仪(XPS)对GNPL表面理化性质进行了表征。利用放射性同位素标记方法考察了修饰前后表面对蛋白Fg吸附量的影响,并利用ELISA测试了表面对血浆中Fn的吸附量。用L929成纤维细胞考察了细胞与表面之间的特异性结合作用,以及表面粗糙度因素对这种作用的影响。结果表明：这类未经修饰的GNPL表面自身显著抑制了L929细胞的黏附生长,而在修饰POEGMA并链接GRGDY分子后,表面微纳米拓扑结构不仅赋予各粗糙表面更加优异的抗蛋白吸附性能,并且能极大地增强细胞与表面特异性结合分子的作用,显著增强了表面的细胞相容性。该研究为基于细胞与材料相互作用的各种研究和应用提供了一种很有前景的表面修饰策略。3.利用同样的方法在金片表面制备了表面粗糙度递增的GNPL结构,并通过自组装的方法在其表面修饰上能与特定的癌细胞特异性结合的核酸适体(Aptamer, APT)TD05,考察了这种功能化表面对癌细胞混合液中特定的Ramos细胞的选择性捕获效果。采用水接触角仪、椭圆偏振光谱仪对修饰前后的表面进行了表征。分别在无血清和含血清两种不同条件下,考察了人B细胞白血病细胞Ramos和人T细胞白血病细胞CCRF-CEM(简称为CEM)在修饰前后的各GNPL表面的竞争性黏附行为。研究表明：无论是在含血清还是无血清培养条件下,两种细胞对GNPL表面都有着更高的亲和力。更重要的是,在修饰有APT后,GNPL表面粗糙度的增大对于该表面对Ramos细胞的选择性捕获作用有显著的增强作用,特别是在无血清条件下,其对Ramos细胞的选择性捕获能力是对照细胞CEM的18倍。该研究表明,这种GNPL修饰材料在癌症早期诊断,干细胞分离装置的制备等领域有着潜在应用前景。总之,本论文以葡萄糖还原法制备的GNPL微纳米结构化表面为研究基材,考察了这种方法对多种材料的适用性,以及反应液用量对这种微纳米结构化材料表面形貌和粗糙度的影响。并重点以这种微纳米结构化材料修饰的酶标板和金片为基材,研究了这类表面对蛋白吸附量及活性的影响,考察了将其用于ELISA中的效果。进一步地,我们研究了这种材料表面的粗糙度因素对表面与细胞之间特异性结合的影响,以及在修饰特异性配体后对癌细胞选择性捕获作用的影响。本文的工作为一类简便易行的微纳米拓扑结构修饰表面的制备及其在生物医学领域的研究提供了一定的理论指导并初步展示了其潜在应用价值。更多还原

【Abstract】 The application of surfaces with micro-/nano structures in biomedical fields has drawn increasing attention in recent years, especially their notable advantages in innovative sensors and tissue engineering materials. However, it has long been difficult and costly in the preparation of such materials. Hence, developing a convenient method to form micro/nano structures on many materials of different nature is highly desirable, which will enable the transformation process of those surfaces in biomedical researches and applications.The main focus of this thesis is to build micro/nano topographical structures comprised of gold nanoparticle layer (GNPL) on material surfaces via a convenient glucose reduction process. And we first investigated the performances of GNPL modified ELISA plate in the applications of both indirect and sandwich format ELISA. Moreover, we modified the GNPL with protein-repellent polymer brush and conjugated it with GRGDY peptide, and studied the influence of GNPL roughness on the specific binding of L929fibroblasts. Finally, we conjugate tumor cell-specific aptamers (APT) TD05to GNPLs through surface self-assembly, and investigated its performances in selective capturing of specific tumor cells.First, we prepared micro/nano three dimensional GNPL on various materials by using glucose reduction method, and studied the performances of the GNPL modified ELISA plate in ELISA applications. Metallographic microscope and field emission scanning electron microscope (FESEM) were used to characterize the morphology of GNPL. And isotope labeling was used to monitor fibrinogen (Fg), human serum albumin (HSA) and lysozyme (LYZ) adsorption on GNPLs, and also, the activity of adsorbed LYZ was measured. Then, we studied the performances of the GNPL modified ELISA plates in ELISA by using cancer marker carcinoembryonic antigen (CEA), human antithrombin (AT), rabbit immonoglobulin (IgG), and human fibroncetin (Fn) as model analytes. We found that the morphologies of GNPL and the amount of adsorbed bioactive protein could be tuned simply by controlling the amount of reaction solution used. It is also shown that two opposite wetability states (superhydrophilic and superhydrophobic) of GNPLs can be formed, and the superhydrophilic state of GNPL was optimal in ELISA. The performances of GNPL in both indirect and sandwich ELISA showed the improved method can significantly enhanced the sensitivity and lowered the limit of detection. This improved method is convenient, universal and effective, and could be an ideal supplement to the conventional ELISA.In the second part of the thesis, we prepared low-fouling GNPL with cell-specific binding abilities. And investigated the effect of surface roughness on the specific binding between L929fibroblasts and the modified GNPLs. We modified GNPLs with poly(oligo(ethylene glycol) methyl ether methacrylate)(POEGMA) brush as spacers via surface-initiated atom transfer radical polymerization (SI-ATRP), and conjugate them with cell-specific peptide glycine-arginine-glycine-aspartic acid-tyrosine (GRGDY). FESEM, water contact angle, surface roughmeter, ellipsometry and X-ray photoelectron spectroscopy (XPS) were used to characterize the GNPLs before and after modifications. Isotope labeling was used to monitor Fg adsorption onto GNPLs before and after modification, and ELISA was used to study the Fn adsorption from human plasma. L929cells were used to investigate the specific binding between cells and GNPLs-POEGMA-GRGDY surface, and the effects of GNPLs roughness on this interaction. The results showed the pristine micro/nano rough structures significantly inhibited L929cell growth; however, after modification with POEGMA-GRGDY, the micro/nano structures greatly enhanced L929cell-specific interactions and improved the cell compatibility of surfaces while maintaining superior low-fouling ability compared with planar gold. Our findings demonstrated a promising and effective surface modification strategy for investigations and applications based on cell-surface interactions.Finally, we modified GNPLs of different roughness with TD05aptamers, a kind of tumor cell specific aptamer via self-assembly. And the selective binding effects of the functionalized GNPLs on the target tumor cell under serum-free and serum-containing culture conditions were investigated. Water contact angle and ellipsometry were used to characterize surface wetability and the thickness of polymer brush coatings on GNPLs before and after modification. The results demonstrated that the two kinds of tumor cells prefer to adhere on pristine GNPL surfaces compared to planar gold regardless of the presence of serum, and Ramos cells outnumbered slightly. More importantly, after the modification with TD05aptamer, the selective binding ability of GNPLs increased as surface roughness increased, especially, the number of adhered Ramos cells was18times higher than the CEM cells under serum-free conditions. Our results indicate that the properly functional ized GNPL holds great promise in future applications in cancer diagnosis, rare cell separation etc.In conclusion, this thesis mainly focused on the conveniently prepared GNPL modified surfaces, and investigated its universality on different materials, and the influence of the volume of reaction solution on the GNPL morphologies. The influence of GNPL on protein adsorption and activity were also investigated. Moreover, the performances of GNPL modified ELISA plate were studied detailedly, and satisfactory results were obtained. Furthermore, the influence of GNPL roughness on the specific binding between L929fibroblasts and GNPL surfaces, and the performances of APT functional ized GNPL in selective tumor cell binding were also investigated. Our work indicate that GNPL modified surfaces could find wide applications in biomedical research and applications.更多还原