- 學位論文
酶固定化與生物分子貼附測試於抗沾黏智能型水凝膠
Enzyme Immobilization and Biomolecules Attachment test on anti-foulingIntelligent Hydrogels
摘要
本研究使用帶有羧酸基團的2-carboxyethyl acrylate(CA)單體及三級胺基團的(2-dimethyl amino)ethyl methacrylate(DMAEMA)單體，依6:4莫爾比例混合後(C6D4)，並藉由自由基聚合反應製備出類雙離子型結構水凝膠，此水凝膠經XPS分析量測出，其CA與DMAEMA的實際反應莫爾比約為1:1。[1] 使用微量光譜量測儀與雷射共軛焦顯微鏡觀察蛋白質與螢光大腸桿菌於類雙離子水凝膠表面上之貼附情形，研究發現此水凝膠具備抗蛋白質沾黏與細菌貼附之特性[1]。接下來實驗主要關注於水凝膠的雙功能性測試，指水凝膠經過酶固定化後能具備生物催化活性並同時保有抗生物分子的沾黏與抗細菌之貼附，因此之後實驗進一步將C6D4水凝膠以偶聯劑莫爾比EDC:NHS=5:2或單純EDC進行改質，再利用pH=4.5、6、7的緩衝溶液環境將脂肪酶或溶菌酶固定至水凝膠。待固定完後，依據實驗架構會分別進行活性測試、表面化學鑑定(XPS)、貼附測試(BSA、LYZ、Fibrinogen、螢光大腸桿菌)。 實驗結果利用表面元素鑑定(XPS)證明酶有共價鍵結至C6D4水凝膠中，並可進行特定生物催化反應(脂肪酶:水解酯類；溶菌酶:分解大腸桿菌)。水凝膠固定酶之後，便再次進行蛋白質吸附與菌貼附之實驗，經結果證明水凝膠仍然保有抗蛋白質吸附與抗菌貼附之特性。
並列摘要
Hydrogel is a versatile biomedical material seen wide range of applications. In this study, we mixed 6:4 molar ratio of the monomers of 2-carboxyethyl acrylate (CA) with carboxylic acid and (2-dimethyl amino) ethyl methacrylate (DMAEMA) with tertiary amine groups, followed by radical polymerization, to prepare pseudo-zwitterionic hydrogel. This hydrogel was confirmed by XPS analysis to possess near-equal molar ratio of CA and DMAEMA. Using microplate spectrophotometer and laser confocal microscopy to observe attachment situation of proteins and fluorescent E. coli on the surface of pseudo-zwitterionic hydrogel, it was found out that the hydrogel have the properties of anti-protein adsorption and anti-bacterial attachment. The next experiment focused on bifunctional test of hydrogel, which is the ability of biocatalysis after enzyme immobilization, whilst still antifouling at the same time. It was proposed that such pseudo-zwitterionic smart hydrogel could be switched from the anti-fouling to protein adsorption favored status by configuring environmental pH values. As a result, the C6D4 hydrogel was modified by molar ratio of coupling agent EDC:NHS = 5:2 or simply EDC at pH range from 4.5-7 to optimize protein immobilization based on activity test followed by chemical surface identification (XPS), and attachment test (BSA, LYZ, Fibrinogen, fluorescent E. coli). XPS analysis provided evidences of immobilized enzyme, and the enzyme could carry out specific biocatalytic reactions(lipase: hydrolysis of esters; lysozyme: decomposition of E. coli). After enzyme immobilized on the hydrogel, the results from attachment experiments demonstrated that the hydrogel still retained, even improved, the anti-fouling properties. In summary, we have successfully combined bio-functionalization and anti-fouling characteristics within this Intelligent hydrogel with a systematical approach.