基于石墨烯电极的细胞内嘌呤代谢传感研究

发布时间：2018-01-12 09:21

本文关键词：基于石墨烯电极的细胞内嘌呤代谢传感研究　出处：《佳木斯大学》2016年硕士论文　论文类型：学位论文

【摘要】：目的构建具有高选择性、灵敏性和稳定性的电化学生物传感器,以实现对细胞质中鸟嘌呤(G)、黄嘌呤(X)、腺嘌呤(A)和次黄嘌呤(HX)的原位检测。通过研究正常细胞与癌变细胞电化学信号之间的关系,建立可用于评估细胞活性、监控细胞癌变的电化学方法。方法本文以离子液体与化学还原石墨烯的杂化材料修饰玻碳电极(RGI-GCE)作为工作电极,以人乳腺癌(MCF-7)细胞和小鼠胚胎成纤维(BALB/3T3)细胞作为细胞模型,采用线性扫描伏安法(LSV)同时对细胞质中的腺嘌呤和次黄嘌呤进行检测。并且以多金属氧酸盐与还原石墨烯泡沫复合修饰电极(POM@3DGF-GCE)作为工作电极,采用循环伏安法(CV)对人乳腺癌细胞细胞质中鸟嘌呤和黄嘌呤进行同时检测。利用扫描电子显微镜、透射电子显微镜、拉曼光谱仪、X射线粉末衍射等手段对传感器的表面形貌和内部组成进行表征。采用高效液相色谱法(HPLC)对细胞电化学监测细胞内嘌呤代谢进行可靠性验证。结果实验结果表明RGI-GCE和POM@3DGF-GCE电极材料中离子液体及多金属氧酸盐被吸附在碳材料表面。RGI-GCE电极在低嘌呤表达的MCF-7和BALB/3T3细胞中产生三个电化学信号,分别归属于鸟嘌呤/黄嘌呤氧化峰(0.7 V),腺嘌呤氧化峰(0.94 V)以及次黄嘌呤氧化峰(1.0 V)。这种电极材料首次实现了腺嘌呤和次黄嘌呤信号的同时检测。利用RGI-GCE电极可检测到细胞癌变后细胞裂解液中腺嘌呤和次黄嘌呤信号的增长率分别为300%和317%,成功利用电化学方法对细胞癌变过程实现监控。对于POM@3DGF-GCE电极,附着于石墨烯泡沫表面的多金属氧酸盐具有良好的氧化还原活性。利用POM@3DGF-GCE对MCF-7细胞原位裂解液进行循环伏安法扫描时,产生两个电化学信号,分别归属于鸟嘌呤(1.048 V)和黄嘌呤(1.128 V)的氧化峰信号;在电化学传感器领域,首次实现了鸟嘌呤和黄嘌呤的同时检测。利用高效液相色谱法证实了上述细胞电化学方法的可靠性。结论本课题采用纳米材料与不同种类的碳材料进行杂化,成功制备出可以检测MCF-7和BALB/3T3细胞中四种嘌呤的信号的生物传感材料。所制备出的电化学传感器具有高选择性、灵敏性和稳定性,可监测BALB/3T3细胞发生转化时其细胞质中嘌呤碱基含量的变化,为细胞内嘌呤代谢的原位检测提供了新的电化学方法。
[Abstract]:Objective to construct an electrochemical biosensor with high selectivity, sensitivity and stability for the determination of guanine guanine in cytoplasm. In situ detection of adenine (A) and Hypoxanthine (HX). By studying the relationship between the electrochemical signals of normal cells and cancerous cells, we established a method for evaluating cell activity. Methods the glassy carbon electrode modified with ionic liquid and chemically reduced graphene was used as the working electrode. Human breast cancer MCF-7 cells and mouse embryonic fibroblast BALB / 3T3) cells were used as cell models. Adenine and Hypoxanthine in cytoplasm were detected simultaneously by linear scanning voltammetry. The composite electrode was modified with polyoxometalate and reduced graphene foam. POM @ 3DGF-GCEs are used as working electrodes. The contents of guanine and xanthine in the cytoplasm of human breast cancer cells were detected simultaneously by cyclic voltammetry. Scanning electron microscopy, transmission electron microscope and Raman spectrometer were used to detect guanine and xanthine in the cytoplasm of human breast cancer cells. The surface morphology and internal composition of the sensor were characterized by X-ray powder diffraction. High performance liquid chromatography (HPLC) was used to characterize the surface morphology and internal composition of the sensor. The reliability of cell electrochemical monitoring of intracellular purine metabolism was verified. Results the results showed that ionic liquids and polyoxometalates were adsorbed in RGI-GCE and POM@3DGF-GCE electrode materials. The RGI-GCE electrode on the surface of carbon materials produced three electrochemical signals in MCF-7 and BALB/3T3 cells with low purine expression. It belongs to the oxidation peak of guanine / xanthine (0.7 V). Adenine oxidation peak 0.94 V) and Hypoxanthine oxidation peak 1.0 V). By using RGI-GCE electrode, we can detect the growth rate of adenine and Hypoxanthine signal in cell lysate after canceration, respectively. 300% and 317%. The electrochemical method was successfully used to monitor the process of cell carcinogenesis. For the POM@3DGF-GCE electrode. Polyoxometalates attached to the surface of graphene foam have good redox activity. POM@3DGF-GCE was used to scan MCF-7 cell in situ by cyclic voltammetry. Two electrochemical signals, assigned to the oxidation peak of guanine (1.048V) and xanthine (1.128V), were produced. In the field of electrochemical sensors. For the first time, the simultaneous detection of guanine and xanthine was realized. The reliability of the electrochemical method was confirmed by HPLC. Conclusion the nanocomposites and different kinds of carbon materials are used for hybrid. The biosensors which can detect the four purine signals in MCF-7 and BALB/3T3 cells were successfully prepared. The electrochemical sensors were highly selective, sensitive and stable. It can be used to monitor the changes of purine base content in the cytoplasm of BALB/3T3 cells during transformation, which provides a new electrochemical method for in situ detection of purine metabolism in cells.
【学位授予单位】：佳木斯大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q2-33;TP212

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