新型纳米材料用于构建电化学生物传感器及癌症治疗

发布时间：2018-03-08 15:12

本文选题：Bcl-2　切入点：Bax　出处：《南京大学》2017年硕士论文　论文类型：学位论文

【摘要】：癌症已经成为人类健康的主要威胁之一。癌细胞与正常细胞不同,有无限增殖、可转化和易转移三大特点,能够无限增殖并破坏正常的细胞组织。癌症的早期诊断与治疗是提高癌症患者生存率的关键,一直是相关领域研究热点。但是传统的诊疗技术通常存在在诊断过程中出现假阳性或在癌症给药治疗过程中产生毒副作用等缺点。电化学传感技术是一种将电化学响应与生物分子识别技术结合的技术,具有装置简单、成本低廉、灵敏度高及稳定性好等优点,因此在检测癌症相关生物分子方面受到广泛关注。另一方面,纳米材料由于其尺寸小,生物相容性好,独特物理化学性质,易修饰等优点,被广泛用于临床诊疗,药物分析,能源和环境等领域。本论文正是基于以上研究背景,开发新型功能化的纳米材料,将其应用于电化学生物传感领域,并探索了其在癌症的早期诊断和治疗中的应用。主要内容如下:1.通过双标记的电化学免疫传感器同时检测癌细胞凋亡调节因子Bcl-2和BaxBcl-2家族在细胞凋亡调控中有重要的作用。其中,Bcl-2和Bax常被用于监控癌细胞凋亡以及评估抗癌药物药效。本工作设计了一种新型的双标记免疫夹心电化学传感器用于同时检测癌细胞凋亡调节因子Bcl-2和Bax。首先将还原氧化石墨烯(RGO)悬浮液滴涂在玻碳电极(GC)表面,形成RGO基底,用于固定两种蛋白的捕获抗体(anti-Bcl-2 I和anti-Bax I)。用BSA封闭电极上多余的活性位点后,电极可捕获Bcl-2和Bax抗原蛋白,并进一步捕获Bcl-2和Bax信号抗体。信号抗体可以提前制备。首先将介孔二氧化硅(SmS)的表面分别连接CdSeTe@CdS量子点(QDs)和Ag纳米簇(NCs),再分别连接Bcl-2信号抗体(anti-Bcl-2II)和Bax信号抗体(anti-Bax Ⅱ),得到两种蛋白对应的信号探针(anti-Bcl-2 Ⅱ-Cd-SmS和anti-Bax Ⅱ-Ag-SmS)。接下来将构建好的传感电极浸于酸性溶液中,得到含有Ag+和Cd2+的溶液,利用阳极溶出伏安法(ASV)检测该溶液中的离子浓度。Bax和Bcl-2的量与Ag和Cd的氧化峰强度呈正比。由于信号探针显著的信号放大作用,所构建的电化学免疫传感器显示了对Bax和Bcl-2的高灵敏检测,同时具有良好重复性和稳定性。该传感器被进一步用于检测尼洛替尼处理过的K562细胞中Bax和Bcl-2的含量。结果证明本工作构建的电化学平台通过于Bax/Bcl-2的比值可以用于药效和凋亡程度的评估,对癌细胞的诊断和治疗具有重要意义。2.DNA聚合酶诱导发卡DNA自组装,用于靶向药物递送及构建电化学生物传感器由于遵循严格的核酸碱基配对原则,DNA作为构建结构的材料基元受到广泛关注。在本工作中,我们通过一种简单的DNA聚合酶介导的发卡DNA自组装(PDHA)方法,得到一种多功能DNA纳米材料并证明其在多方面有应用前景,如生物医药和生物传感。反应体系中包含两种发卡DNA,一旦加入连有适配体的引发链,不活泼的发卡DNA会在DNA聚合酶作用下被激活,最终生长成适配体修饰的DNA纳米自主装结构(AptNAs)。通过AptNAs与荧光基团和药物(Dox)作用,我们可以将其用于靶向癌细胞,实现药物递送。结果证明该DNA纳米材料具有高载药量及好的生物相容性。我们进一步将PDHA用于构建无标记的生物传感平台来检测microRNA-21(miR-21)。通过微分伏安法(DPV)检测到的电流强度与miR-21呈正比。该生物传感器显示了对miR-21的高灵敏检测,检测限低至0.75 fM,检测范围为8个数量级。同时还表现了良好的重现性、特异性及稳定性。而且可以进一步用于检测MCF-7细胞中的miR-21实际含量。证明该DNA纳米自组装材料不仅在癌症治疗在癌症诊断方面也有潜在的应用前景。
[Abstract]:Cancer has become one of the major threats to human health. Different cancer cells and normal cells, unlimited proliferation, transformation and metastasis three characteristics, can proliferate and destroy the normal tissue. Early diagnosis and treatment of cancer is the key to improve the survival rate of patients with cancer, has been a hot research field. But the traditional treatment technology usually exists in the diagnosis of false positives or to produce toxic side effects in cancer treatment process. The disadvantages of electrochemical sensing technology is a technology combining electrochemical response and molecular biology identification technology, has the simple structure, low cost, high sensitivity and good stability, so it was widely attention in the detection of cancer related molecular biological aspects. On the other hand, nano materials because of its small size, good biocompatibility, unique physical and chemical properties, easy modification, etc. Which is widely used in clinical diagnosis, drug analysis, energy and environment field. This thesis is based on the above research background, the development of new functional nano materials, its application in the field of electrochemical biosensor, and explore its application in cancer diagnosis and treatment. The main contents are as follows: 1. by double mark the electrochemical immunosensor for simultaneous detection of apoptosis regulatory factors Bcl-2 and BaxBcl-2 family plays an important role in regulating the cell apoptosis. Among them, Bcl-2 and Bax are often used for monitoring apoptosis of cancer cells and evaluate the efficacy of anticancer drugs. We design a double labeled immuno sandwich novel electrochemical sensor for the simultaneous detection of apoptosis in cancer cells regulator of Bcl-2 and Bax. for the first reduction of graphene oxide (RGO) suspension was coated on glassy carbon electrode (GC) surface, the formation of RGO substrate for fixed two protein trap The obtained antibodies (anti-Bcl-2 I and anti-Bax I). BSA closed electrode active site redundant after the electrode can capture Bcl-2 and Bax antigen, and further to capture Bcl-2 and Bax signal. The signal can advance antibody antibody preparation. The mesoporous silica (SmS) surface are respectively connected with CdSeTe@CdS quantum dots (QDs) and Ag nanoclusters (NCs), then connect the Bcl-2 signal and Bax signal (anti-Bcl-2II) antibody antibody (anti-Bax II), get the signal corresponding to the two kinds of protein probe (anti-Bcl-2 II -Cd-SmS and anti-Bax II -Ag-SmS). The next will be to build a good sensing electrode immersed in acid solution, obtained solution containing Ag+ and Cd2+ and using the method of anodic stripping voltammetry (ASV) was proportional to the strength of the oxidation peak in the solution ion concentration.Bax and the amount of Bcl-2 and Ag and Cd detection. Due to amplification of the signal probe significantly, electrochemical immunosensor built Shows a high sensitive detection of Bax and Bcl-2, and also has good repeatability and stability. The content of Bax and Bcl-2 of the sensor was further used for the detection of nilotinib treated K562 cells. The results showed that the ratio of the construction work by electrochemical platform in Bax/Bcl-2 can be used to evaluate the efficacy and the degree of apoptosis, diagnosis and treatment of cancer cells has important significance for.2.DNA polymerase Induced Hairpin DNA self-assembly for targeted drug delivery and construction of biosensors due to follow strict nucleic acid base pairing, DNA as a building material base element structure has attracted extensive attention. In this work, we assembled by issuing a simple DNA DNA polymerase mediated (PDHA) method, a multi functional DNA nano materials and demonstrate its application prospect in many aspects, such as bio pharmaceutical and biological sensing response. The Department contains two hairpin DNA, once connected with aptamer triggered chain, hairpin DNA inactive will be activated in the DNA polymerase. The final growth of DNA nano aptamer modified self assembly structure (AptNAs). By AptNAs and fluorescent group and drug (Dox), we can be used for targeting cancer cells, to achieve drug delivery. The results showed that the DNA nano materials with high loading and good biocompatibility. We will further build PDHA for biosensing platform without marker to detect microRNA-21 (miR-21). Through differential voltammetry (DPV) to detect the current intensity was proportional to the miR-21. The biosensor shows high sensitive detection of miR-21, detection limit as low as 0.75 fM, the detection range is 8 orders of magnitude. At the same time also showed good reproducibility, specificity and stability. It can be used to detect MCF-7 cells The actual content of miR-21 shows that the DNA nano self assembled materials have potential applications not only in cancer diagnosis but also in cancer diagnosis.

【学位授予单位】：南京大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：TP212.3;R730.5;O657.1

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