二氧化矽氣凝膠之修飾及其在生物感測上之應用

Modification of Silica Aerogel and Application on Biosensors.

李彥廣(Yen-Kuang Li)

指導教授 ： 陳玉惠

中原大學/理學院/化學研究所/博士(2008年)

https://doi.org/10.6840/cycu200800079

摘要

中 文 摘 要
本研究利用氣凝膠為基材與生物體結合，開發具生物感測能力之生物氣凝膠載體。依材料特性與應用需求，以包覆方式及化學表面修飾固定化方式分別進行。
在包覆方面，本研究選用肉眼即能快速判別其活性之紅/綠螢光蛋白為模型，開發出一種全新製程的生物氣凝膠 (bioaerogel) 材料。其主要係使用TEOS為矽源，並以離子熔液 (ionic liquid) 為溶劑兼模板藉溶膠-凝膠 (sol-gel) 聚合反應製備而成。所製成之包覆紅/綠螢光蛋白生物氣凝膠之性質，利用FTIR、SEM、TEM、TGA、29Si固態核磁共振及BET檢測；結果顯示材料有很高的表面積及孔隙，並且其結構相當穩定。包覆在生物氣凝膠中之螢光蛋白，其穩定性及活性，則由螢光光度計及共聚焦雷射掃描顯微鏡 (confocal laser scanning microscopy) 觀察探討之。此外，另選擇"纖維素水解酵素"做為實際檢測之酵素，檢測被包覆在生物氣凝膠中之酵素催化活性之變化，並回收重複反應，研究其重複使用之可能性。
在化學表面修飾固定化方面，首先將氣凝膠表面改質成胺基或環氧基後，在玻璃基材上形成氣凝膠點，開發出新型三維立體氣凝膠基因及蛋白質晶片。此三維立體結構氣凝膠層的表面經改質後，表面官能基可捕捉抗體及DNA等生物分子，得以分別進行三明治免疫反應 (sandwich immunoreaction) 或雜交(hybridization)反應。利用三維立體結構表面積大之特性，提高單位面積之鍵結量，達到提高晶片掃描影像訊號及偵測靈敏度之目的。

關鍵字

生物晶片 ； 生物氣凝膠 ； 化學表面修飾固定化 ； 包覆 ； 紅/綠螢光蛋白

並列摘要

Abstract
In this research, the silica aerogel was utilized as a matrix by the sol-gel polymerization and the proper chemical surface modification to encapsulate or immobilize the selected proteins and DNAs to prepare the bioaerogels and 3-D biochips.
In the encapsulation, recombinant red/green fluorescent proteins, DsRed and EGFP, were chosen as the model proteins to prepare the protein-encapsulated bioaerogels, DsRed-SAG and EGFP-SAG. The bioaerogels were prepared by the sol-gel polymerization of tetraethyl orthosilicate (TEOS) with an ionic liquid as the solvent and pore-forming agent. These DsRed-SAG and EGFP-SAG bioaerogels were characterized by Fourier Transformation Infrared (FTIR), Scanning Electron Microscopy (SEM), Transmission Electron Microscope (TEM), solid-state 29Si MAS NMR spectroscopy, Thermogravimetric Analysis (TGA) and Brunauer-Emmett-Teller (BET) measurements. The results showed that the as-prepared bioaerogels had high surface area and porosity, and the silica network exhibited only little shrinkage during the drying process. The stability of the bioaerogels were monitored by fluorescence spectroscopy and confirmed by confocal laser scanning microscopy. In addition, a cellulase-encapsulated bioaerogel was also prepared and characterized by the similar method. The cellulase’s enzymatic activity in the bioaerogel was further investigated by the hydrolysis of waste paper cellulose. Also studied was the recycle possibility of the bioaerogel.
In the immobilization, the surface of the as-prepared silica aerogel was grafted with an amine or epoxy group to form the functional aerogel. These functional aerogels were further developed to form the three-dimensional DNA and protein biochip by doting the aerogel on a glass microscopy slide followed by grafting with a DNA probe and a capturing antibody, respectively. The functionalities of the as-prepared DNA biochip and protein biochip were verified by performing hybridization and a sandwich immunoassay, respectively and investigating the intensities of the labeled species. It was found that compared with those on the traditional two-dimensional flat glass surfaces, the signal intensities of the as-prepared aerogel biochips detected were amplified for several orders of magnitudes mainly due to the enormous surface area of the aerogel.

並列關鍵字

biochip ； Bioaerogel ； immobilization ； encapsulation ； red/green fluorescent proteins

參考文獻

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