可选择性捕获循环肿瘤细胞的三维聚合物软链的初步探索

发布时间：2018-08-03 17:01

【摘要】：恶性肿瘤是种严重的疾病,每年大约有820万人死于癌症。目前检测肿瘤的领域里的黄金标准是“活体组织检查”,而对循环肿瘤细胞(CTC)的检测,则可以作为“液体活检”部分替代传统方法,实现对癌症的无创、快速检测。然而CTC在血液里含量稀少,大约10亿个血细胞中只有1~100个,因此CTC的捕获和富集非常有挑战性。目前CTC的富集和检测领域已经涌现出很多方法,已有研究证明纳米结构有助于提高对CTC的检测效果,并且相比于无机纳米材料,柔性有机的纳米结构对CTC的捕获具有较好的效果。因此,基于前人经验,为进一步提高捕获效率,本课题制备了具有可捕获CTC潜力的三维聚合物软链平台。为了实现高效率捕获CTC的三维软链平台的制备,本课题首先利用电化学手段将表面引发原子转移自由基聚合(ATRP)的引发剂和具有抗非特异性黏附能力的分子聚合沉积到导电介质表面,然后通过表面引发ATRP的方法制备出具有生物功能化及抗非特异性黏附能力的三维分子软链。通过这种方法得到的捕获平台,其本身具有良好的抗非特异性黏附能力,又可以通过控制分子链密度,分子链长和生物素功能化单体所占组分来调控最终的捕获能力,理论上具有可控的高效特异性捕获ctc的潜力。捕获平台的具体实现方法如下。本课题通过溴代异丁酰溴与羟基化3,4-乙烯二氧噻吩(edot-oh)发生消去反应,合成具有表面引发atrp能力的含溴乙烯二氧噻吩(edot)衍生物(edot-br),并通过核磁共振波谱分析,确定了此表面atrp引发剂的化学结构。本课题通过两步法合成出了具有抗非特异性粘附能力的单体(含磷酸胆碱的edot衍生物)。第一步利用edot-oh与2-氯-2-氧-1,3,2-二氧磷杂环戊烷(cop)之间的消去反应生成edot-cop;第二步利用edot-cop与三甲胺的开环加成反应,最终获得edot-pc。通过核磁共振波谱对edot-pc进行分析,确定了此分子的化学结构,并证明了其具有极好的抗非特异性粘附能力。本课题通过在乙腈中加入高氯酸锂作为掺杂离子,加入琥珀酸二辛酯磺酸钠(dss)促进edot-pc的溶解性,最终将edot-br和edot-pc稳定的共聚合沉积到导电基片上。并通过x射线光电子能谱分析(xps)对共沉积膜的表征,证明本课题已经成功将edot-br和edot-pc共聚到导电基片上,并且可以通过调整溶液中edot-br的含量来控制导电基片上引发剂的密度。通过原子力显微镜的表征,通过循环伏安法电化学聚合可以获得表面粗糙度很低的聚合物膜。通过接触角表征,证明了通过调整聚合物中的edot-pc的含量可以对聚合物膜的亲水性进行良好控制。本课题合成了两种生物素功能化的单体M-Biotin和Hema-Biotin,并在此基础上探索由导电高分子表面引发M-Biotin/Hema-Biotin与MPC的ATRP共聚合。为了获得稳定的表面引发ATRP共聚合方法,本课题尝试了三种不同的催化剂体系:一种为CuBr/联吡啶(bpy);一种是CuBr2/bpy/抗坏血酸(VC);一种是CuBr2/三(2-吡啶甲基)胺(TPMA)/VC。最终通过QCM表征分子链与链霉亲和素及模型蛋白的耦合效果,确定了CuBr2/TPMA/VC在本体系的表面引发ATRP聚合中具有最优效果。另外,此体系下生成的分子链同样具有极好的抗非特异性粘附能力,这种能力是进一步优化分子链捕获能力的前提。为了最大化分子链上的抗体表达,本课题进一步优化了引发剂密度,生物素功能化共聚单体的组成和聚合时间(链长度)。通过QCM表征分子链与链霉亲和素的耦合能力,最终探索出了在引发剂密度为10%,生物素功能化共聚单体含量为5%,ATRP聚合时间6h的条件下,可以实现分子链对链霉亲和素和模型蛋白的最大化耦合,同时保持较低的链密度。通过接触角表征,发现随着聚合时间的增长,ATRP聚合生成的分子刷的亲水性会略有增强,超过6h以后会稳定在10°左右,证明了本体系具有良好的亲水性。
[Abstract]:Malignant tumor is a serious disease. About 8 million 200 thousand people die of cancer every year. The gold standard in the field of cancer detection is "biopsy", and the detection of circulating tumor cells (CTC) can be used as a "liquid biopsy" part to replace the traditional method to achieve non-invasive and rapid detection of cancer. However, CTC is in the blood. There are few 1~100 in about 1 billion blood cells, so the capture and enrichment of CTC is very challenging. Many methods have emerged in the field of CTC enrichment and detection. Research has shown that nanostructures help to improve the detection of CTC. And compared to inorganic nanomaterials, the flexible organic nanostructures are to CTC Therefore, based on the previous experience, in order to further improve the capture efficiency, we have prepared a three dimensional polymer soft chain platform with the potential to capture CTC. In order to realize the preparation of the three-dimensional soft chain platform with high efficiency to capture CTC, this topic first uses electrochemistry to induce the surface of the free radical polymerization of atomic transfer. The ATRP initiator and molecular polymerization with anti non specific adhesion ability are deposited on the surface of the conductive medium, and then the three-dimensional molecular soft chain with the ability of biofunctionalization and anti non specific adhesion is prepared by the method of surface initiation ATRP. The capture platform obtained by this method has good anti non specificity. Adhesion ability can control the ultimate capture ability by controlling the molecular chain density, molecular chain length and the component of biotin functionalized monomers. In theory, it has the potential of controllable high efficiency and specificity to capture CTC. The specific realization method of the capture platform is as follows. This subject uses bromo isobutylyl bromide and hydroxylated 3,4- ethylene two oxygen thiophene (e Dot-oh) occurrence of elimination reaction, synthesis of two oxygen thiophene (EDOT) derivative (edot-br) containing the ability of surface induced ATRP, and the chemical structure of the ATRP initiator on this surface was determined by NMR spectroscopy. In this study, a monomer with the ability to resist nonspecific adhesion (EDOT derivatives containing phosphoric acid choline) was synthesized by the two step method. Biological). The first step is to produce edot-cop by the elimination reaction between edot-oh and 2- chloro -2- oxygen -1,3,2- two oxycyclopentane (COP); the second step using the open ring addition reaction of edot-cop and trimethylamine, and finally obtaining edot-pc. through nuclear magnetic resonance spectroscopy to analyze edot-pc, determine the chemical structure of the molecule, and prove it is excellent. By adding lithium perchlorate as doping ion in acetonitrile, adding sodium succinate two octyl sulfonate (DSS) to promote the solubility of edot-pc, the stable copolymerization of edot-br and edot-pc was finally deposited on the conductive substrate, and the characterization of the co deposition film was characterized by X ray photoelectron spectroscopy (XPS). The subject has successfully copolymerized the edot-br and edot-pc onto the conductive substrate and can control the density of the initiator on the conductive substrate by adjusting the content of edot-br in the solution. Through the atomic force microscope, the polymer film with low surface roughness can be obtained by cyclic voltammetry. Through the contact angle table, the surface of the polymer film can be obtained by the electrochemical polymerization of the cyclic voltammetry. It is proved that the hydrophilicity of the polymer membrane can be well controlled by adjusting the content of edot-pc in the polymer. Two kinds of biotin functionalized monomers M-Biotin and Hema-Biotin are synthesized, and on this basis, the ATRP copolymerization of M-Biotin/ Hema-Biotin and MPC is explored by the surface of the conductive polymer. On the surface of ATRP copolymerization, three different catalyst systems are tried: one is CuBr/ bipyridine (bpy), one is CuBr2/bpy/ ascorbic acid (VC), and the other is CuBr2/ three (2- pyridyl) amine (TPMA) /VC. (TPMA) /VC. finally through QCM to characterize the coupling effect of molecular chain with streptomycin and model protein, and determines the CuBr2/TPMA/VC On the surface of this system, it has the best effect in ATRP polymerization. In addition, the molecular chain generated under this system also has excellent anti non specific adhesion ability. This ability is the prerequisite to further optimize the ability of molecular chain capture. In order to maximize the expression of antibody on the molecular chain, the subject further optimizes the density of the initiator. The composition and polymerization time (chain length) of a functionalized copolymerized monomer. The coupling ability of molecular chains to streptavidin was characterized by QCM, and the maximum of the molecular chain to the chain of streptomycin and model protein could be found under the condition that the density of the initiator was 10%, the content of the biotin functionalized monomers was 5%, and the polymerization time of ATRP was 6h. With the increase of the polymerization time, it is found that the hydrophilicity of the ATRP polymerized molecular brush increases slightly with the increase of the polymerization time. It will stabilize at about 10 degrees after the 6h, which proves that the system has a good hydrophilic property.
【学位授予单位】：东华大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R730.4;O631

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