固定生醫單體之生物活性與結構穩定性之探討

Study of Bioactivity and Structural Stabilization of Immobilized Biomolecules

田育彰(Yu-Chang Tyan)

指導教授 ： 廖峻德

中原大學/工學院/醫學工程研究所/博士(2003年)

https://doi.org/10.6840/cycu200200618

摘要

利用低溫高密度氧氣微波電漿活化聚丙烯不織布，經電漿處理數秒可使其表面產生高度的氧化基團。隨之接枝稀釋的丙烯酸單體，使表面具官能基與親水性的結構，丙烯酸接枝量為187±0.54 mg/cm2。再以EDC為煤合劑固定生物單體：膠原蛋白、明膠蛋白、肝素及幾丁聚醣，使其表面具有生物相容特性，擴充醫療用途。固定生物單體試片經血液及細菌測試，以瞭解其表面在血液相容與滅菌等特性。其中，以固定蛋白質試片進行滅菌程序，得到所需要的適度劑量，文中並探討蛋白質結構受到輻射或能量破壞而影響其生物活性。本研究使用減弱全反射-傅立葉轉換紅外光譜儀及化學分析電子能譜儀對改質基材進行化學結構分析；以染色或重量比較法定量生醫單體之固定量。實驗結果顯示：接枝丙烯酸的羧基（O=C-OH）與生物單體的胺基（-NH2）形成共價鍵結的醯胺鍵結（O=C-NH）。而鍵結的生醫單體之官能基均可被辨識。四種固定的生物分子經血液凝固測試顯示均仍保有原來之生物特性。其中，肝素會因酸鹼環境或離子強度之不同而影響固定量與生物活性。將具有生物活性的固定膠原蛋白試片以紫外線或加馬射線照射，會造成膠原蛋白結構之a-螺旋、醯胺I及II鍵結的破壞。再對不同劑量照射的試片測試部分活化凝血脢時間、凝血時間的改變、纖維蛋白濃度變化，並計數紅血球及血小板在材料試片上的吸附率及以電子顯微鏡觀察吸附材料表面之血小板外型，均受到紫外線照射時數或加馬射線劑量增加而明顯變化。從”保有生物活性”的觀點，適當的照射強度分別為：紫外線20小時、加馬射線10 KGy。滅菌測試的結果：紫外線照射20小時或加馬射線照射7.5 KGy以上可以達到滅菌之效果。因此，在考慮生物活性且達到滅菌效果下，可將加馬射線照射降至7.5 KGy，然而，紫外線照射20小時雖可達到滅菌效果，但其生物活性明顯下降。

關鍵字

聚丙烯不織布 ； 微波電漿 ； 滅菌測試 ； 生物單體 ； 生物活性

並列摘要

Low-temperature high-density O2 microwave plasma was utilized for the activation of non-woven polypropylene (PP) fabric. With several seconds of plasma treatment, highly oxidative groups were created at surface. The diluted acrylic acid (AAc) was subsequently graft-copolymerized to obtain a surface with functional and hydrophilic structure. The grafting amount was 187±0.54 mg/cm2. To originate biocompatible function for biomedical uses, bio-molecules: type III collagen, gelatin, heparin and chitosan, were then immobilized on the fabric using EDC as coupling agents. The biomolecule-immobilized substrates were carried out blood compatible and sterilization tests to characterize their surface properties. The collagen-bonded PP non-woven fabric was employed for sterilization test and for the index to find out an appropriate dose. The structural denaturation of immobilized collagen induced by irradiation or energy accumulation was also discussed. Using Fourier-Transformed Infrared with Attenuated Total Reflection and Electron Spectroscopy for Chemical Analysis, we examined the chemical structures of samples with different treatments. To quantify the immobilized amount of biomolecules, coloring or weighting method was applied. Experimental result demonstrated that The O=C-OH group of the grafted pAAc was covalently bonded with biomolecule and formed the amide bond (O=C-NH). The functional groups of the immobilized biomolecules were well identified. Using blood clot tests, these four kinds of the immobilized biomolecules still retained their original bioactivities. Nevertheless, the immobilized quantity and the bioactivity of the immobilized heparin were influenced by the processing pH environment and ionic strength. The bioactive collagen-immobilized samples were then sterilized using UV-254nm or gamma ray irradiation, which provoked a-helix, amide I and II damages. Subsequently, the sterilized samples in different degrees were assessed by activated partial thromboplastin time, thrombin time, and fibrinogen concentration tests. By means of cell counter and Scanning Electron Microscopy, we counted red blood cells and platelets adhesion in the modified porous matrix, as well as the morphologies of platelets. The above-mentioned observations were significantly changed with the increase of UV exposure time or gamma ray irradiation dose. From “upholding bioactivity” point of view, the optimized UV exposure time should be less than 20 hrs or gamma ray irradiation dose less than 10 KGy. Still, from our sterilization test, the gamma ray irradiation dose could be reduced to 7.5 KGy, whereas the UV exposure time should be higher than 20 hrs. In the latter case, UV exposure would significantly diminish the bioactivity of the immobilized collagen.

並列關鍵字

Bioactivity ； Microwave plasma ； PP non-woven Fabric ； Biomolecules ； Sterilization.

參考文獻

72. 許濤, ”紫外線對植物生長的影響-DNA修補作用” 1998紫外線研討會論文集, 海洋大學水產生物技術研究所.

37. R. K. Murray, Harper’s Biochemistry, 665-687. Appleton & Lange, Prentice-Hall International Inc., 1994.

8. Y. C. Tyan, J. D. Liao, K. Y. Hsu and Y. D. Wu, ”Comparative study of immobilized collagen onto varied porous fabric surfaces activated by O2 microwave plasma” BME’99 Conference, 203~204, 1999.

56. B. D. Ranter, A. B. Johnston and T. J. Lenk, “Biomaterial surfaces” Journal of Biomedical Materials Research, 21: 59-89, 1987.

3. A. S. Hoffman, “Adsorption and immobilization of proteins on gas discharge-treated surfaces” Journal of Applied Polymer Science-Applied Polymer Symposium, 46: 341-359, 1990.