基于有機(jī)納米功能膜信號增強(qiáng)的生物傳感器檢測c-Myc蛋白
發(fā)布時間：2018-11-06 20:33
【摘要】：c-Myc蛋白是一種使細(xì)胞無限增值的原癌基因產(chǎn)物，在調(diào)節(jié)DNA合成、細(xì)胞分化、凋亡以及細(xì)胞周期的進(jìn)程中起著極其重要的作用，其過度表達(dá)易使細(xì)胞轉(zhuǎn)化為惡性表型。目前，許多生物學(xué)方法如酶聯(lián)免疫吸附分析法（ELISA）和聚合酶鏈反應(yīng)法（PCR）等，費時費力、成本高，因此快速檢測c-Myc含量的變化，對于癌癥的預(yù)防、診治有著非常重要的意義。本文針對如何增強(qiáng)生物傳感器響應(yīng)信號的關(guān)鍵問題，結(jié)合電化學(xué)分析技術(shù)簡便、快速、可實時輸出的特點，利用有機(jī)納米功能材料的獨特性能，制備了三種信號放大的新型電化學(xué)免疫生物傳感器，用于實際樣品中c-Myc腫瘤蛋白的檢測，優(yōu)于傳統(tǒng)生物學(xué)方法，在癌癥預(yù)防、治療等生物醫(yī)學(xué)、分子生物學(xué)領(lǐng)域具有十分重要的研究價值和應(yīng)用前景。主要內(nèi)容如下： 1.采用層層自組裝方法，制備了一種基于雙抗體夾心層修飾金電極的信號增強(qiáng)電化學(xué)生物傳感器，即先通過組裝L-半胱氨酸、戊二醛，固定c-Myc單克隆抗體(C-Ab1)，形成C-Ab1單抗修飾電極，可識別致癌基因c-Myc蛋白；再結(jié)合上第二抗體Anti-MouseIgG(H+L)Antibody(C-Ab2)，形成C-Ab1/c-Myc/C-Ab2雙抗夾心修飾電極，響應(yīng)信號大幅度增強(qiáng)，傳感性能優(yōu)于C-Ab1單抗修飾電極。通過電化學(xué)阻抗和循環(huán)伏安行為探討了雙抗夾心法信號增強(qiáng)的機(jī)理，其阻抗值與c-myc濃度對數(shù)在0.043nM～430nM范圍內(nèi)成良好的線性關(guān)系，最低檢測限也降低至25.76pM。該傳感器制備簡單，選擇性、重現(xiàn)性、穩(wěn)定性和再生性好，在鼠血清樣品中測得c-Myc的回收率在97.4%～103.7%之間，表明該方法可用于實際腫瘤樣品中c-Myc的檢測，在生物醫(yī)學(xué)領(lǐng)域具有潛在的應(yīng)用價值。 2.以圓盤金電極為基底，制作了一種信號放大的新型生物傳感器，GNPs(Goldnanoparticles)標(biāo)記C-Ab2/c-Myc/C-Ab1修飾電極，并運用交流阻抗法和循環(huán)伏安法探討了該傳感器的性能。研究了抗癌基因c-Myc蛋白在該修飾電極上的直接電化學(xué)反應(yīng)。實驗結(jié)果表明，采用GNPs標(biāo)記的C-Ab2,即GNPsC-Ab2修飾電極比無GNPs標(biāo)記修飾電極的電化學(xué)響應(yīng)大，信號更強(qiáng)。該傳感器（C-Ab1/c-Myc/C-Ab2-GNPs）的阻抗值與c-Myc濃度對數(shù)在4.3pM～43nM范圍內(nèi)成良好的線性關(guān)系，最低檢測限也降低至2.18pM。與方法1所制備的傳感器相比該傳感器具有較高的靈敏度，較好的重現(xiàn)性，良好的選擇性等優(yōu)點。在1%鼠血清中測得的c-Myc蛋白的回收率在96.3%～108.9%之間，表明該方法可用于實際樣品中c-Myc的檢測。 3.在方法1和方法2的基礎(chǔ)上，以金圓盤電極為基底，依次組裝1,6-己二硫醇、GNPs、L-cys、戊二醛、固定c-Myc單克隆抗體（C-Ab1），形成C-Ab1單抗修飾電極，識別c-Myc蛋白，結(jié)合GNPs標(biāo)記C-Ab2實現(xiàn)信號的放大。該傳感器（GNPs/C-Ab1/c-Myc/C-Ab2-GNPs）的阻抗值與c-Myc濃度對數(shù)在0.86pM～8.6nM范圍內(nèi)成良好的線性關(guān)系，最低檢測限也降低至0.86pM。在鼠血清中測得的c-Myc蛋白的回收率在95.2%～104.7%之間，在致癌基因c-Myc蛋白檢測方面具有應(yīng)用價值。
[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.
【學(xué)位授予單位】：長沙理工大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R318.0
本文編號：2315396
[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.
【學(xué)位授予單位】：長沙理工大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R318.0
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