【作者】 刘敏；

【导师】 钱国栋； 刘爱萍；

【作者基本信息】 浙江大学 ， 材料科学与工程， 2015， 硕士

【摘要】 选择合适的基底材料构建细胞传感器(Cell Sensor),实现对体外细胞行为的有效调控和实时检测在细胞生理及病理研究、疾病诊断及药物筛选等领域具有重要意义。类金刚石(DLC)薄膜凭借低成膜温度、良好的耐摩擦磨损能力、耐酸碱腐蚀及长期的稳定性、生物相容性好等特性成为制备细胞传感器的优选材料。但DLC薄膜的高内应力、低电催化活性及生物化学惰性限制其作为生物电极材料的使用。本文将元素掺杂及化学分子修饰应用于DLC薄膜的改性,并探讨了改性对薄膜微观结构、细胞相容性及电化学性能的影响。采用磁过滤阴极真空电弧(FCVA)技术,以氮气(N2)、磷烷(PHa)为气体掺杂源,或在石墨阴极靶中掺入适量的硼粉或铂粉实现掺杂DLC薄膜的制备。采用原子力显微镜(ArM)、拉曼(Raman)光谱及X-射线光电子能谱(XPS)表征薄膜的表面形貌和微观结构。结果显示,掺杂没有破坏薄膜的光滑平整表面,且没有显著改变薄膜的非晶结构,但增加了sp2杂化碳含量。PCI2神经细胞的粘附、增殖及凋亡实验表明,B、N、P元素的掺杂提高了薄膜的细胞相容性,但Pt的掺入起了相反的作用。培养5天后,相比于未掺杂DLC薄膜,掺P薄膜上细胞密度增加约60%,凋亡率降低约40%,而N、Pt共掺的薄膜上细胞密度降低约40%,凋亡率提高约30%。电化学测试显示,不同元素掺杂的薄膜对双氧水(H2O2)的电催化能力不同,其中N、P的掺杂能更有效地提升薄膜的催化活性。通过共价键合方式在掺氮类金刚石(DLC：N)薄膜表面分别连接化学活性分子多巴胺(DA)、三氨基苯硼酸(APBA)及三磷酸腺苷(ATP),实现对掺杂薄膜的进一步改性。修饰后的薄膜微观结构没有明显变化,粗糙度略有增加(约0.3 nm)。经APBA及ATP修饰后的薄膜具有更好的细胞相容性,DA修饰获得了相反的效果。培养3天后,APBA及ATP修饰的薄膜上PC12细胞的活性约为修饰前的两倍,而DA修饰薄膜的上细胞活性仅约为修饰前的40%。化学分子修饰提高了薄膜对H2O2的催化能力,APBA修饰的薄膜检测1H202浓度的灵敏度约增加为原来的4倍,因而体外检测细胞受激释放H2O2的能力也随之提高。光刻法制备的DLC：N阵列电极检测H2O2浓度的灵敏度约为原薄膜电极的10～30倍。总之,元素掺杂及化学分子修饰能同时实现对DLC薄膜电化学性能及细胞相容性的调控,通过选择合适的掺杂元素及化学分子可以获得适宜的细胞传感器基底材料。更多还原

【Abstract】 To select the appropriate substrate material for cell sensor and realize the effective control and real-time detection of cell behaviors in vitro is of great significance in the study of cell physiology and pathology, medical diagnosis, drug screening and other areas. With low deposition temperature, good resistance to friction and wear, perfect acid and alkali corrosion-resistant ability and long-term stability, good biocompatibility and other characteristics, diamond-like carbon (DLC) film is regarded as a preferred material for cell sensor substrate. However, the intrinsic compressive stress, low electro-catalytic activity and biochemical inertness inhibit its use as biological electrode material. In this paper, element doping and chemical molecular modification was applied to modify the DLC film, and the effects of modification on the microstructure, cell compatibility and electrochemical properties of the film were discussed.Differently doped DLC films were prepared by using nitrogen (N2), phosphine (PH3) as gas doping source, or by adding boron powder or platinum powder to the graphite cathode target with the filtered cathodic vacuum arc (FCVA) technique. Atomic force microscope (AFM), Raman spectrum and X-ray photoelectron spectroscopy (XPS) were carried out to character the performances of the DLC films. Our results showed that element doping did no harm to the smooth surface and only slightly changed the amorphous structure of the film by a little increase of the amount of the sp2 hybridized carbon. The adhesion, proliferation and apoptosis experiments of PC 12 cells showed that B, N, P doping improved the cell compatibility of DLC film, but the incorporation of Pt had the opposite effect. After 5-day culture, compared to the undoped DLC film, the cell density on the phosphorus doped film increased by about 60% and the apoptotic rate reduced by about 40%, while the cell density on the film with nitrogen and platinum codoping decreased by about 40% and the apoptosis rate increased by 30%. The electrochemical test showed that the DLC films doped with different elements had distinct catalytic abilities on hydrogen peroxide, especially, N and P doping obviously enhanced the catalytic ability.Through covalent bonding, the surface of nitrogen doped DLC (DLC:N) films were connected with chemical active molecules such as dopamine (DA), amino phenylboronic acid (APBA) and adenosine triphosphate (ATP), respectively, to achieve further modification of the doped DLC films. The microstructure of the modified films did not change obviously, and the surface roughness only slightly increased by about 0.3 nm. The APBA and ATP modified films had better cell compatibility, while DA modification had the opposite effect. After 3-day culture, the cell activity on APBA and ATP modified DLC:N films were about two times of that on the unmodified film. For the DA modified film, the cell activity is reduced. Chemical molecular modification enhanced the catalytic ability to H2O2 oxidation and the detcetion sensitivity of the APBA modified film is increased up to about 4 times when compared to the original film, so the film can be better used to dectect H2O2 released by the stimulated cells in vitro. The electrocatalytic activity of the DLC:N array electrode prepared by photolithography was also significantly enhanced by 10～30 times.In summary, the regulation of electrochemical properties and cell compatibility of the DLC film can be realized at the same time by element-doping or chemical molecular modification. Via choosing appropriate doping elements and modification molecules, satisfactory cell sensor substrate originated from DLC films can be obtained.更多还原