利用逆微乳化系統輔助合成幾丁聚醣微 / 奈米顆粒以進行酵素固定及其衍生應用

Abstract

本研究利用水、采酮 X-100、己醇以及甲基環己烷所製造出來的逆微乳化系統輔助合成出幾丁聚醣奈米顆粒 (chitosan nanoparticles, CNPs)、幾丁聚醣-過氧化酶複合奈米顆粒 (chitosan-HRP complex nanoparticles, C-HRP-cNPs) 以及幾丁聚醣微米膠囊 (chitosan microcapsules, CMCs)，並利用共軛焦顯微鏡、電子顯微鏡以及動態光散射儀觀察其材料的大小與形貌。而從實驗結果發現 CNPs 的大小可以藉由微乳化系統溫度變化來控制，而改變幾丁聚醣的濃度以及混合過氧化酶的比例可以直接影響 CNPs 與 C-HRP-cNPs 的形貌以及穩定度。 同時，為了測試幾丁聚醣奈米材料的酵素固定能力，以過氧化酶 (horse radish peroxidase, HRP) 作為測試酵素固定在三種材料上，並製作出表面修飾過氧化酶之幾丁聚醣奈米顆粒 (Surface modified HRP-CNPs, surHRP-CNPs)、幾丁聚醣-過氧化酶複合奈米顆粒以及包裹過氧化酶之幾丁聚醣微米膠囊 (Encapsulated-HRP CMCs, eHRP-CMCs)。隨後分析三種材料之過氧化酶的固定量以及其酵素活性，結果發現合成過程中各種幾丁聚醣奈米材料的合成參數會影響過氧化酶的固定量以及對於環境穩定度。 最後本研究將固定過氧化酶之幾丁聚醣材料應用在免疫分析上作為分析的訊號來源，觀察出不同合成條件以及不同修飾狀況之幾丁聚醣顆粒在免疫分析系統中表現的差異，並同時影響整體免疫分析產生訊號的能力。

In this study, the reverse microemulsion system, consisting of water, Triton X-100, hexanol and methylcyclohexane was applied for the synthesis of chitosan nanoparticles (CNPs), chitosan-HRP complex nanoparticles (C-HRP-cNPs) and chitosan microcapsules (CMCs). The physical properties such as the morphology and the diameter of these three kinds of chitosan materials were characterized by confocal laser scanning microscope (CLSM), scanning electron microscope (SEM) and dynamic light scattering (DLS). Our study indicated that the size control of CNPs could be achieved by temperature adjustment; while varying chitosan concentration and the ratio of incorporated HRP influenced significantly the morphology and the stability of CNPs and C-HRP-cNPs . To investigate the feasibility of three chitosan-based solid supports for enzyme immobilization, horse radish peroxidase (HRP) was employed as the model enzyme. Surface modified HRP-Chitosan nanoparticles (surHRP-CNPs), C-HRP-cNPs , and Encapsulated-HRP Chitosan nanocapsules (eHRP-CMCs) were prepared, and the total enzyme amounts and the corresponding activity of immobilized HRP were quantified to evaluate the immobilization processes. Different synthetic conditions contributed to varied quantity and stability of immobilized HRP in each type of chitosan nanomaterials. Finally, we employed the HRP-immobilized chitosan nanomaterials as signal generator for the immunoassay applications. The ligand recognition interface and the performance of signal generation in the assay were found to be greatly influenced by the different fabrication procedures of chitosan nanomaterials.