以功能性蠶絲海綿作為捕捉病原菌的平台

Abstract

近年來，蠶絲蛋白（silk fibroin）常被用於生醫材料應用，其特點包含良好的機械強度、生物相容性、延展性，並具有良好的材料可修飾性進而可增加生物功能性。本研究以蠶絲蛋白作為生物材料支架，並經由生物製造之酵素修飾方式交聯可吸附多種病原菌之功能性分子，進而建立一個病原菌快速捕捉平台，可望作為新型的病原菌分離與篩選的裝置。具體來說，在蠶絲支架的製備上，我們首先將蠶絲經由冷凍乾燥法製成具多孔狀的蠶絲海綿，使得可比一般平面基質具有較大的孔隙度與作用面積。接著我們進一步調整蠶絲海綿的結構穩定性，經由乙醇處理後，能提升結構的結晶度，增強蠶絲海綿的的穩定性。在功能性修飾部分，我們進一步藉由酵素horseradish peroxidase (HRP)將基因改造之功能性蛋白質apolipoprotein H (ApoH)以二聚化形成雙酪氨酸共價鍵的方式鑲嵌在絲蛋白上。ApoH 為一種人體在免疫急性期會分泌出的載脂蛋白，具有體內先天免疫系功能，能以電荷吸引的特性吸附許多表面帶有負電荷的病原菌，像是病毒、革蘭氏陰性菌，甚至是寄生蟲等等。本研究所建構的病原菌捕捉功能性海綿已經證實可以捕捉大腸桿菌與諾羅病毒，將來將進一步改良此技術平台，預期在未來能夠運用於許多環境或生醫檢測，以及血液透析的病原菌分離醫材等開發。

Silk fibroin produced from silkworms has been widely used as biomaterials owing to its remarkable mechanical strength, extensibility, biocompatibility, and ease of biofunctionalization. In this research, we engineered the silk fibroin as a novel platform capable of capturing various species of pathogens by functionalizing pathogen targeting moiety. Specifically, we first generated the silk material into a porous silk sponge through the lyophilization procedure, yielding enhanced porosity and accessible surface area. The structural stability of our engineered sponge was further improved with ethanol treatment. Second, the sponge was functionalized with engineered apolipoprotein H (ApoH) by forming di-tyrosine crosslinking through the catalyzation of horseradish peroxidase (HRP). ApoH, an immune acute phase human plasma protein, serves as an innate immune defense system against infection and exhibits broad spectrum of pathogen binding ability, including viruses, Gram-negative bacteria and parasites. Finally, the platform was demonstrated to be effective in E. coli and norovirus binding. We envision the device we fabricated, yet to be tuned up, can potentially as a device for rapid isolation and high throughput screening of pathogens. Additionally, the technique could be further harnessed in hemodialysis for reducing the risk of infection in the future.