本文選題：生物材料 + MicroBCA��； 參考：《西南交通大學(xué)》2012年碩士論文

【摘要】：植入物的生物相容性是生物醫(yī)用材料需滿足的最基本要求。從材料表面與生物內(nèi)環(huán)境相互作用調(diào)控的角度,對生物材料進(jìn)行表面修飾改性是提高材料生物相容性、滿足臨床應(yīng)用要求的關(guān)鍵。表面生物化是近年研究及應(yīng)用最為廣泛的方法之一,此方法不會(huì)影響材料的本體性能,可基本保持所固定的生物分子的活性。對與蛋白質(zhì)相互作用的表面進(jìn)行活性及量化表征,可更深入地了解蛋白質(zhì)和表面之間的相互作用,以及生物材料表面修飾等研究。 本文主要通過MicroBCA法對Fn蛋白修飾的石英玻璃、純鈦薄膜、氧化鈦薄膜表面進(jìn)行定量表征,并結(jié)合目前應(yīng)用廣泛的FTIR, XPS、QCM等手段對其進(jìn)行跟蹤分析。以期為探尋不同接枝形式對蛋白接枝量的變化情況,尋求最適接枝方式起一定的指導(dǎo)作用,服務(wù)于實(shí)際應(yīng)用領(lǐng)域。 (1)對于穩(wěn)定性較好的石英玻璃樣品,經(jīng)過化學(xué)清洗、多聚賴氨酸化、共價(jià)固定Fn多步處理后,采用MicroBCA檢測手段檢測,結(jié)果顯示石英玻璃表面涂覆多聚賴氨酸后接枝Fn的固定量為308±14ng/cm2。同時(shí)還使用XPS全譜及C/N/O高分辨、水接觸角、酸性橙Ⅱ等手段對每一階段處理樣品進(jìn)行了定性或定量表征,了解了各步處理樣品表面的特性變化情況。 (2)通過非平衡磁控濺射在石英玻璃表面沉積制得了純鈦薄膜。經(jīng)過堿活化、多聚賴氨酸化或硅烷化兩種固定形式,將Fn分子共價(jià)固定在純鈦表面。采用MicroBCA法對石英基底純鈦薄膜表面的Fn蛋白進(jìn)行定量,結(jié)果顯示以多聚賴氨酸形式接枝Fn的固定量為311±16ng/cm2,以硅烷化形式接枝的Fn蛋白分子在鈦薄膜表面的固定量約為767ng/cm2。此外還將目前應(yīng)用較為廣泛的QCM檢測與其作了比較,兩種手段得到的Fn的固定量極為接近,可以說明所測得樣品表面固定的Fn的量具有一定準(zhǔn)確性。實(shí)驗(yàn)中還通過水接觸角、傅立葉變換紅外光譜、XPS等分析證實(shí)了各步處理的樣品表面的特性變化情況。 (3)利用非平衡磁控濺射設(shè)備,在石英玻片表面沉積制得氧化鈦薄膜。MicroBCA法檢測到石英基底氧化鈦薄膜表面以多聚賴氨酸形式接枝Fn的固定量為386±6ng/cm2。同樣還結(jié)合傅立葉變換紅外光譜、X射線光電子能譜、水接觸角及酸性橙反應(yīng)等檢測表征了各步處理后表面性質(zhì)和化學(xué)成分的變化,證實(shí)了經(jīng)過本文的特殊處理后,可以將Fn分子更有效地共價(jià)固定到氧化鈦表面。 (4) MicroBCA檢測結(jié)果說明共價(jià)固定Fn的不同材料表面會(huì)對其固定的量產(chǎn)生一定的影響,同時(shí)本文研究表明不同形式的接枝蛋白手段也會(huì)影響蛋白的接枝量。 (5)本文在不改變材料表面的性質(zhì),而從改變基底的角度減小或消除干擾所造成的假陽性值,使得固定化蛋白定量結(jié)果穩(wěn)定且可靠。但此法還存在很多問題,后續(xù)有待科研工作者進(jìn)一步進(jìn)行研究及優(yōu)化。 綜上所述,本文針對石英玻璃、純鈦薄膜、氧化鈦薄膜材料表面修飾的蛋白分子進(jìn)行了量化研究,通過QCM和MicroBCA兩種方法對材料表面蛋白的含量進(jìn)行了研究,得到了有重要參考價(jià)值的實(shí)驗(yàn)結(jié)果,為生物材料表面生物化修飾與改性研究中生物大分子的定量表征提供了可借鑒的方法。
[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.

【學(xué)位授予單位】：西南交通大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R318.08

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