利用MicroBCA法对三种材料表面固定的Fn分子进行定量表征研究

发布时间：2018-04-22 11:44

本文选题：生物材料 + MicroBCA　；参考：《西南交通大学》2012年硕士论文

【摘要】：植入物的生物相容性是生物医用材料需满足的最基本要求。从材料表面与生物内环境相互作用调控的角度,对生物材料进行表面修饰改性是提高材料生物相容性、满足临床应用要求的关键。表面生物化是近年研究及应用最为广泛的方法之一,此方法不会影响材料的本体性能,可基本保持所固定的生物分子的活性。对与蛋白质相互作用的表面进行活性及量化表征,可更深入地了解蛋白质和表面之间的相互作用,以及生物材料表面修饰等研究。本文主要通过MicroBCA法对Fn蛋白修饰的石英玻璃、纯钛薄膜、氧化钛薄膜表面进行定量表征,并结合目前应用广泛的FTIR, XPS、QCM等手段对其进行跟踪分析。以期为探寻不同接枝形式对蛋白接枝量的变化情况,寻求最适接枝方式起一定的指导作用,服务于实际应用领域。 (1)对于稳定性较好的石英玻璃样品,经过化学清洗、多聚赖氨酸化、共价固定Fn多步处理后,采用MicroBCA检测手段检测,结果显示石英玻璃表面涂覆多聚赖氨酸后接枝Fn的固定量为308±14ng/cm2。同时还使用XPS全谱及C/N/O高分辨、水接触角、酸性橙Ⅱ等手段对每一阶段处理样品进行了定性或定量表征,了解了各步处理样品表面的特性变化情况。 (2)通过非平衡磁控溅射在石英玻璃表面沉积制得了纯钛薄膜。经过碱活化、多聚赖氨酸化或硅烷化两种固定形式,将Fn分子共价固定在纯钛表面。采用MicroBCA法对石英基底纯钛薄膜表面的Fn蛋白进行定量,结果显示以多聚赖氨酸形式接枝Fn的固定量为311±16ng/cm2,以硅烷化形式接枝的Fn蛋白分子在钛薄膜表面的固定量约为767ng/cm2。此外还将目前应用较为广泛的QCM检测与其作了比较,两种手段得到的Fn的固定量极为接近,可以说明所测得样品表面固定的Fn的量具有一定准确性。实验中还通过水接触角、傅立叶变换红外光谱、XPS等分析证实了各步处理的样品表面的特性变化情况。 (3)利用非平衡磁控溅射设备,在石英玻片表面沉积制得氧化钛薄膜。MicroBCA法检测到石英基底氧化钛薄膜表面以多聚赖氨酸形式接枝Fn的固定量为386±6ng/cm2。同样还结合傅立叶变换红外光谱、X射线光电子能谱、水接触角及酸性橙反应等检测表征了各步处理后表面性质和化学成分的变化,证实了经过本文的特殊处理后,可以将Fn分子更有效地共价固定到氧化钛表面。 (4) MicroBCA检测结果说明共价固定Fn的不同材料表面会对其固定的量产生一定的影响,同时本文研究表明不同形式的接枝蛋白手段也会影响蛋白的接枝量。 (5)本文在不改变材料表面的性质,而从改变基底的角度减小或消除干扰所造成的假阳性值,使得固定化蛋白定量结果稳定且可靠。但此法还存在很多问题,后续有待科研工作者进一步进行研究及优化。综上所述,本文针对石英玻璃、纯钛薄膜、氧化钛薄膜材料表面修饰的蛋白分子进行了量化研究,通过QCM和MicroBCA两种方法对材料表面蛋白的含量进行了研究,得到了有重要参考价值的实验结果,为生物材料表面生物化修饰与改性研究中生物大分子的定量表征提供了可借鉴的方法。
[Abstract]:Biocompatibility of implants is the most basic requirement for biomedical materials. The surface modification of biomaterials is the key to improve the biocompatibility of biomaterials and meet the requirements of clinical application from the angle of the interaction between the surface of the material and the biological environment. The surface physicochemical is the most widely used research and application in recent years. One of the methods, this method does not affect the properties of the material, and can basically maintain the activity of the fixed biomolecules. The surface activity and quantitative characterization of the proteins interacting with the protein can be used to understand the interaction between the protein and the surface, and the surface modification of the biomaterials.
In this paper, the surface of Fn modified quartz glass, pure titanium film and titanium oxide film was quantitatively characterized by MicroBCA method, and it was tracked and analyzed by the methods of FTIR, XPS and QCM, which were widely used at present. It serves in the field of practical application.
(1) for the quartz glass samples with good stability, after chemical cleaning, polylysine and covalent fixed Fn multistep treatment, MicroBCA detection was used. The results showed that the fixed amount of Fn was 308 + 14ng/cm2. after the coating of polylysine on the surface of quartz glass, and the XPS full spectrum and C/N/O high resolution, water contact angle and acidity were also used. Each phase of the sample was qualitatively or quantitatively characterized by orange II, and the characteristics of the surface of the samples were analyzed.
(2) a pure titanium film was deposited on the surface of quartz glass by non-equilibrium magnetron sputtering. After alkali activation, polylysine or silanized two fixed forms, Fn molecules were fixed on the pure titanium surface. The MicroBCA method was used to quantify the Fn protein on the surface of the pure titanium film on the quartz substrate. The results showed that the graft was grafted with polylysine. The fixed amount of Fn is 311 + 16ng/cm2. The fixed amount of Fn protein grafted on the surface of the titanium film is about 767ng/cm2.. In addition, it is also compared with the widely used QCM detection. The fixed amount of Fn obtained by the two means is very close, which shows that the amount of Fn fixed on the surface of the sample has a certain accuracy. In addition, the characteristics of the surface of the samples were confirmed by the analysis of water contact angle, Fu Liye transform infrared spectroscopy and XPS.
(3) using the non-equilibrium magnetron sputtering equipment, the.MicroBCA method for the deposition of titanium oxide film on the surface of quartz glass has been made to detect the immobilization of Fn on the surface of the silica based titanium oxide film on the surface of polylysine with a fixed amount of 386 + 6ng/cm2., which is also combined with Fu Liye transform infrared spectroscopy, X line photoelectron spectroscopy, water contact angle and acid orange reaction, etc. The change of surface properties and chemical composition after each step was characterized, and it was proved that after the special treatment of this paper, the Fn molecules could be more effectively covalently fixed to the surface of titanium oxide.
(4) the results of MicroBCA test show that the surface of different materials with a covalent fixed Fn will have a certain influence on the fixed quantity. At the same time, this paper shows that different forms of graft protein can also affect the graft quantity of the protein.
(5) this paper does not change the properties of the surface of the material, but reduces or eliminates the false positive value caused by the change of the substrate, which makes the quantitative results of the immobilized protein stable and reliable. However, there are many problems in this method, and further research and optimization are needed by the researchers.
To sum up, this paper quantifies the surface modified protein molecules of quartz glass, pure titanium film and titanium oxide film. The content of the surface protein of the material is studied by two methods of QCM and MicroBCA, and the experimental results with important reference value are obtained, which is the study of the biological modification and modification of the biomaterial surface. The quantitative characterization of biological macromolecules provides a reference method.

【学位授予单位】：西南交通大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R318.08

【参考文献】