基于有机纳米功能膜信号增强的生物传感器检测c-Myc蛋白

发布时间：2018-11-06 20:33

【摘要】：c-Myc蛋白是一种使细胞无限增值的原癌基因产物，在调节DNA合成、细胞分化、凋亡以及细胞周期的进程中起着极其重要的作用，其过度表达易使细胞转化为恶性表型。目前，许多生物学方法如酶联免疫吸附分析法（ELISA）和聚合酶链反应法（PCR）等，费时费力、成本高，因此快速检测c-Myc含量的变化，对于癌症的预防、诊治有着非常重要的意义。本文针对如何增强生物传感器响应信号的关键问题，结合电化学分析技术简便、快速、可实时输出的特点，利用有机纳米功能材料的独特性能，制备了三种信号放大的新型电化学免疫生物传感器，用于实际样品中c-Myc肿瘤蛋白的检测，优于传统生物学方法，在癌症预防、治疗等生物医学、分子生物学领域具有十分重要的研究价值和应用前景。主要内容如下： 1.采用层层自组装方法，制备了一种基于双抗体夹心层修饰金电极的信号增强电化学生物传感器，即先通过组装L-半胱氨酸、戊二醛，固定c-Myc单克隆抗体(C-Ab1)，形成C-Ab1单抗修饰电极，可识别致癌基因c-Myc蛋白；再结合上第二抗体Anti-MouseIgG(H+L)Antibody(C-Ab2)，形成C-Ab1/c-Myc/C-Ab2双抗夹心修饰电极，响应信号大幅度增强，传感性能优于C-Ab1单抗修饰电极。通过电化学阻抗和循环伏安行为探讨了双抗夹心法信号增强的机理，其阻抗值与c-myc浓度对数在0.043nM～430nM范围内成良好的线性关系，最低检测限也降低至25.76pM。该传感器制备简单，选择性、重现性、稳定性和再生性好，在鼠血清样品中测得c-Myc的回收率在97.4%～103.7%之间，表明该方法可用于实际肿瘤样品中c-Myc的检测，在生物医学领域具有潜在的应用价值。 2.以圆盘金电极为基底，制作了一种信号放大的新型生物传感器，GNPs(Goldnanoparticles)标记C-Ab2/c-Myc/C-Ab1修饰电极，并运用交流阻抗法和循环伏安法探讨了该传感器的性能。研究了抗癌基因c-Myc蛋白在该修饰电极上的直接电化学反应。实验结果表明，采用GNPs标记的C-Ab2,即GNPsC-Ab2修饰电极比无GNPs标记修饰电极的电化学响应大，信号更强。该传感器（C-Ab1/c-Myc/C-Ab2-GNPs）的阻抗值与c-Myc浓度对数在4.3pM～43nM范围内成良好的线性关系，最低检测限也降低至2.18pM。与方法1所制备的传感器相比该传感器具有较高的灵敏度，较好的重现性，良好的选择性等优点。在1%鼠血清中测得的c-Myc蛋白的回收率在96.3%～108.9%之间，表明该方法可用于实际样品中c-Myc的检测。 3.在方法1和方法2的基础上，以金圆盘电极为基底，依次组装1,6-己二硫醇、GNPs、L-cys、戊二醛、固定c-Myc单克隆抗体（C-Ab1），形成C-Ab1单抗修饰电极，识别c-Myc蛋白，结合GNPs标记C-Ab2实现信号的放大。该传感器（GNPs/C-Ab1/c-Myc/C-Ab2-GNPs）的阻抗值与c-Myc浓度对数在0.86pM～8.6nM范围内成良好的线性关系，最低检测限也降低至0.86pM。在鼠血清中测得的c-Myc蛋白的回收率在95.2%～104.7%之间，在致癌基因c-Myc蛋白检测方面具有应用价值。
[Abstract]:C-Myc protein is a proto-oncogene product which can increase the value of cells. It plays an important role in regulating the synthesis of DNA, cell differentiation, apoptosis and cell cycle. Overexpression of c-Myc protein can easily transform cells into malignant phenotypes. At present, many biological methods, such as enzyme linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR), are time-consuming and costly. Diagnosis and treatment are of great significance. This paper aims at the key problem of how to enhance the response signal of biosensor, and combines the characteristics of electrochemical analysis technology, such as simple, fast and real-time output, and makes use of the unique properties of organic nano-functional materials. Three novel electrochemical immunosensors with amplified signal were prepared for the detection of c-Myc tumor proteins in practical samples. These biosensors were superior to traditional biological methods in the biomedicine of cancer prevention and treatment. Molecular biology has very important research value and application prospect. The main contents are as follows: 1. A kind of signal enhanced electrochemical biosensor based on double antibody sandwich layer modified gold electrode was prepared by layer self-assembly method. Firstly, the c-Myc monoclonal antibody (C-Ab1) was fixed by assembling L- cysteine and glutaraldehyde. The modified electrode of C-Ab1 monoclonal antibody can recognize the oncogene c-Myc protein. Combined with the second antibody Anti-MouseIgG (H L) Antibody (C-Ab2), the C-Ab1/c-Myc/C-Ab2 double antibody sandwich modified electrode was formed, and the response signal was greatly enhanced. The sensing performance of the modified electrode was better than that of the C-Ab1 monoclonal antibody modified electrode. Through electrochemical impedance and cyclic voltammetry, the mechanism of signal enhancement by double antibody sandwich method was discussed. The linear relationship between the impedance value and the logarithm of c-myc concentration was found in the range of 0.043nM~430nM, and the minimum detection limit was reduced to 25.76 pm. The biosensor was simple, selective, reproducible, stable and reproducible. The recovery rate of c-Myc in mouse serum was between 97.4% and 103.7%, which indicated that the method could be used for the detection of c-Myc in actual tumor samples. It has potential application value in biomedical field. 2. A novel biosensor, GNPs (Goldnanoparticles) labeled C-Ab2/c-Myc/C-Ab1 modified electrode based on disk gold electrode was fabricated. The performance of the sensor was investigated by means of AC impedance method and cyclic voltammetry. The direct electrochemical reaction of anticancer gene c-Myc protein on the modified electrode was studied. The experimental results show that the electrochemical response and signal of C-Ab2 modified by GNPs, that is, GNPsC-Ab2 modified electrode, is larger and stronger than that of non-GNPs labeled modified electrode. The impedance of the sensor (C-Ab1/c-Myc/C-Ab2-GNPs) has a good linear relationship with the logarithm of c-Myc concentration in the range of 4.3pM~43nM, and the minimum detection limit is reduced to 2.18 pM. Compared with the sensor prepared by method 1, the sensor has the advantages of high sensitivity, good reproducibility and good selectivity. The recovery rate of c-Myc protein in 1% rat serum was between 96.3% and 108.9%, which indicated that the method could be used for the detection of c-Myc in real samples. 3. On the basis of methods 1 and 2, the gold disk electrode was used as the substrate to assemble 1 hexamethanediol, GNPs,L-cys, glutaraldehyde, and fixed c-Myc monoclonal antibody (C-Ab1) to form a C-Ab1 monoclonal antibody modified electrode. The c-Myc protein was recognized and the signal was amplified by GNPs labeled C-Ab2. The impedance of the sensor (GNPs/C-Ab1/c-Myc/C-Ab2-GNPs) has a good linear relationship with the logarithm of c-Myc concentration in the range of 0.86pM~8.6nM, and the minimum detection limit is reduced to 0.86 pm. The recovery rate of c-Myc protein in mouse serum was between 95.2% and 104.7%, which was valuable in the detection of oncogene c-Myc protein.
【学位授予单位】：长沙理工大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R318.0

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