【作者】 贾学恩；

【导师】 姚守拙； 谢青季；

【作者基本信息】 湖南师范大学 ， 分析化学， 2009， 博士

【摘要】 细胞传感分析新方法的建立、细胞兼容性界面的构建对促进生物医药、生物技术及组织材料工程发展具有重要作用。研究肿瘤相关抗原的高效电化学免疫分析新方法对临床癌症早期预警具有现实意义。纳米材料因其特殊的光、电、磁、热、力学等性能,在生化分析与生物传感领域显示出重大的应用潜力。本学位论文在简要综述了细胞传感分析以及电化学免疫分析的进展、压电传感技术以及纳米材料在生化分析领域的应用等的基础上,利用石英晶体微天平（QCM）和电化学技术在细胞行为监测以及蛋白质免疫传感等方面开展了一些研究工作,主要内容如下:1.以QCM对人乳腺癌细胞（MCF-7）在裸金电极上的粘附、生长行为进行了实时监测。结合显微镜对照实验讨论了QCM响应与细胞数目和表面覆盖度的关系。结果发现,在细胞粘附过程中,频率（f₀）和动态电阻（R₁）改变是电极表面质量、粘密度以及应力综合作用的结果,并且一定范围内与细胞的覆盖度成正比。2.以QCM实时、动态监测了MCF-7细胞在壳聚糖/多壁碳纳米管（CS/MWCNTs）和壳聚糖（CS）修饰电极上的粘附生长行为。讨论了同步记录的谐振频率和动态电阻响应,发现细胞在CS/MWCNTs修饰电极上粘附所引起的压电响应均大于CS修饰电极,表明前者更适于细胞的粘附和生长。采用MTT实验、荧光显微镜、循环伏安以及电化学阻抗谱（EIS）对细胞培养前后的电极进行了表征,支持了QCM法的结论。3.提出了一种悬浮细胞的磁固定和电化学检测新方法。制备了正电性磁性纳米粒子（CMNPs）,利用其与细胞的静电作用,在电极上磁固定悬浮细胞人白血病K562细胞,以QCM技术监测了磁固定过程。对所固定K562细胞进行循环伏安测试,所得阳极电流峰峰高与细胞活性成正比,籍此考察了抗肿瘤药5-氟尿嘧啶（5-FU）的细胞毒性,与常规MTT法吻合。4.提出了一种仿生复合纳米材料界面固定贴壁肿瘤细胞和电化学检测的新方法。通过溶液吸附法合成羟基磷灰石-多壁碳纳米管（HA-MWCNTs）并将其修饰在电极表面,以QCM技术研究了成骨肉瘤细胞MG-63在HA-MWCNTs修饰电极上的粘附生长过程。HA-MWCNTs具有良好的生物亲和性和导电性。对所固定的MG-63细胞进行循环伏安测试,所得阳极电流峰峰高与细胞活性成正比,优化了实验条件,考察了细胞培养过程中细胞活性以及抗肿瘤药5-氟尿嘧啶（5-FU）的细胞毒性,与常规MTT分析法结果一致。5.以QCM-EIS联用技术原位动态监测了抗人IgG在几种修饰电极上的吸附和与人IgG的免疫反应。分别制备了戊二醛、纳米金（nanoAu）以及多壁碳纳米管（MWCNTs）修饰金电极,记录和讨论了抗体吸附及免疫反应所引起压电石英晶体（PQC）的谐振频率（f₀）、动态电阻（R₁）以及电化学阻抗等参数。以循环伏安以及电化学阻抗谱法对抗体吸附和抗体-抗原反应前后的电极进行了表征。比较了抗人IgG在不同表面的吸附量以及免疫活性,结果表明,纳米金修饰电极的吸附量最大,其次是多壁碳纳米管修饰电极,而抗体在几种表面的活性基本一致。同时,利用所得频率和界面阻抗参数计算了免疫反应的结合常数。6.基于金微电极以及纳米金制备了一种超灵敏阻抗型甲胎蛋白（AFP）免疫传感器。通过金微电极（100μm直径）自组装纳米金,实现AFP抗体固定,循环伏安扫描以及原子力显微镜（AFM）对纳米金固定抗体前后的电极表面进行了表征。优化了实验条件,与常规金圆盘电极进行了比较,并对不同浓度AFP抗原进行了检测,线性范围为0.005-50 ng mL^-1,检测限为0.76 pg mL^-1。结果表明,该免疫传感器具有超高灵敏度、良好的重现性和抗非特异性吸附性能,有一定的潜在应用价值。7.基于载有电子媒介体的纳米复合物膜制备了一种新型无标记安培型癌胚抗原（CEA）免疫传感器。首先合成金纳米粒子和甲苯胺蓝掺杂二氧化硅纳米粒子,以壳聚糖为交联剂,将两者混合滴干成膜制备了免疫修饰电极。考察优化了实验条件,并对CEA进行了检测,线性范围为1-80 ngmL^-1,检测下限为0.65 ng mL^-1。实验结果表明,该免疫传感器具有良好的伏安响应,纳米粒子的协同作用使响应电流信号大大提高。这种传感器检测时的非特异性吸附小,灵敏度和稳定性较高。更多还原

【Abstract】 Researches on novel cytosensors and biocompatible interfaces are of great significance for advances of biomedical engineering,biotechnology,and tissue engineering.The development of new and effective electrochemical biosensors for tumor relevant antigens is important for tumor’s early diagnosis in practice. Nanomaterials of special nanostructure possess interesting physical and chemical properties,making them highly promising in bioanalysis and biosensing applications.In this dissertation,the recent progress of cell-based biosensing and electrochemical immunoassay,application of nanomaterials and quartz crystal microbalance（QCM）in bio/chemo fields are reviewed,and a series of studies on cell adhesion,detection of cell viability,adsorption of proteins and immunosensor fabrication were carried out with QCM and electrochemical techniques.The studies are summarized as follows.1.The gold-electrode QCM was used to monitor the one-day incubation of human breast cancer cells（MCF-7）.In combination with an optical microscope simulation experiment that shows the cell-population pictures at various stages,the QCM responses to the cells’ adhesion,spreading and proliferation on the electrode surface are discussed.The resonant frequency（△f₀）and the motional resistance（△R₁）responses were found mainly from mixed effects of mass,viscodensity and surface stress,and in proportion to the cell coverage.2.The QCM was used to monitor the adhesion of mammalian cells on a chitosan（CS）/multiwalled carbon nanotubes（MWCNTs）composite modified gold electrode.The human breast cancer cells（MCF-7）were adhered to and grown on the CS/MWCNTs film modified gold surface or a net CS film modified gold surface,and the process of which was continuously monitored and displayed by changes of the△f₀ and△R₁ of the QCM.The attachment rate and viability of the cells when proliferating on the two surfaces were detected by the MTT assay.The presence and state of cells on the electrode surface were confirmed by the fluorescent microscopy.Cyclic voltammetry and electrochemical impedance spectroscopy of the ferricyanide/ferrocyanide couple were examined before and after the cell adhesion.3.A simple protocol was proposed for rapid magnetic isolation,immobilization and electrochemical detection of leukemia K562 cells via their decoration with cationic magnetic nanoparticles（CMNPs）.The CMNPs modified living cells could be rapidly isolated from the medium and immobilized firmly on the electrode surface via a magnetic field.The cyclic voltammetric experiments of the cells immobilized at glassy carbon electrode exhibited an irreversible anodic current peak whose height is proportional to cell viability, and the 5-fluorouracil cytotoxicity results obtained from the proposed magnetic-electrochemical method were supported by conventional MTT assays. 4.A new biocompatible interface made up of hydroxyapitite-multiwalled carbon nanotubes（HA-MWCNTs）nanocomposite was developed for adhesion and electrochemical detection of human osteoblast-like cells （MG-63）.The HA-MWCNTs nanocomposite was synthesized by self-assembling nano-hydroxyapatite onto multiwalled carbon nanotubes in solution.The QCM was used to monitor the adhesion of MG-63 cells on HA-MWCNTs modified gold electrode.The nanocomposites interface showed improved immobilization capacity for cells and good biocompatibility for preserving the activity of immobilized living cells.The living cells immobilized on an HA-MWCNTs modified glassy carbon electrode exhibited an irreversible anodic peak response being positively associated with the density and viability of cells,which was used to describe the cells growth and evaluate the effectiveness of antitumor drug 5-fluorouracil（5-FU）on the cells.The obtained 5-FU cytotoxicity results agreed well with those from conventional MTT assays.5.The QCM,in combination with electrochemical impedance spectroscopy （EIS）,has been utilized to monitor in situ anti-human IgG adsorption on several Au-based surfaces,bare Au,nanogold/4-aminothiophenol（4AT）/Au, and multi-walled carbon nanotubes（MWCNT）/Au,and succeeding human IgG reactions.The resonant frequency（f₀）and the motional resistance（R₁）of the piezoelectric quartz crystal（PQC）as well as electrochemical impedance parameters were measured and discussed.Cyclic voltammetry（CV）and electrochemical impedance spectroscopy（EIS）of the ferricyanide/ferrocyanide couple were examined before and after electrode modification,antibody adsorption and antibody-antigen reactions.We found that the amount for antibody adsorption was the greatest on the colloid Au modified surface,and that at MWCNT ranked the second,while the specific bioactivity was almost identical on the four kinds of surfaces.Two parameters simultaneously obtained at the colloid Au modified surface,△f₀ and△C_s（interfacial capacitance）,have been used to estimate the association constant of the immunoreaction.6.An ultrasensitive impedance immunosensor for the detection of human alpha fetoprotein（AFP）has been fabricated based on Au microelectrode and gold nanoparticle（nanoAu）.A nanoAu monolayer was assembled on the Au microelectrode（d=100μm）to immobilize AFP antibody（anti-AFP）,the electrode before and after anti-AFP modification was characterized by cyclic voltammetry（CV）and atom force microscopy（AFM）.The factors influencing the performance of the immunosensor were studied in detail. Different concentrations of AFP antigen were determined and the results were compared with the normal Au disk electrode.The Rot response was proportional to logarithm of AFP concentration ranging from 0.005-50 ng mL^-1and the detection limit was 0.76 pg mL^-1.The presented immunosensor is ultrasensitive,selective,and promising for clinical immunoassays.7.A novel lable-free amperometric immunosensor for the determination of carcinoembryonic antigen（CEA）based on nanocomposite containing electron mediator has been developed.Firstly gold nanoparticle（nanoAu） and toluidine blue doped-silica nanoparticle（TB-silica）were synthesized, after mixing with chitosan（CS）,the nanoAu/CS/TB-silica nanocomposite was cast on the electrode surface to prepare immuno electrode.The factors influencing the performance of the amperometric immunosensor were studied in detail.Different concentrations of CEA antigen were determined and the current response was proportional to CEA concentration ranging from 1 to 80 ng mL^-1and the detection limit was 0.65 pg mL^-1.The nanocomposite modified electrode exhibited excellent redox reversibility and sensitivity due to synergistic effect of nanoparticles.Tests performed with this immunosensor showed high selectivity and stability.更多还原