本研究之目的在於建構可應用於活體內 (in vivo) 進行局部 (in situ) 特定蛋白質濃度監控之免疫探針感測系統。此系統結合感測與再生機制，將干涉式光纖、紫外光光纖與微型毛細管整合至一針狀光阻結構中，並於外緣包覆微透析膜完成可重複使用之免疫探針。檢測時，待測之蛋白質分子可經由擴散方式穿過透析膜進入探針內，藉由觀察其與光纖表面接合所造成的干涉圖譜位移達到偵測目地。
實驗中利用奈米金球增加干涉光程之差異，以提升蛋白質接合訊號靈敏度。經由在光纖表面進行共振腔製作與半反射金薄膜蒸鍍，並透過自組裝分子、氨基酸分子修飾，可偵測出接合在光纖表面之奈米金球 (30nm粒徑)，並達到1×1010 particles/ml之偵測極限。此外，根據理論推導與實驗驗證結果，可得知光譜位移量與表面金球接合之顆粒數、大小成正比。應用於免疫檢測部分則先以修飾抗體 (anti-rabbit IgG) 之奈米金球溶液注入探針內，當待測蛋白質-細胞色素抗體 (anti-cytochrome C antibody) 由外部溶液擴散進入時可與其結合，並透過光纖表面修飾之細胞色素C (cytochrome C) 進行免疫辨識。運用此方式可測量之抗體濃度偵測極限可達10 ng/ml。
為了達到重覆使用以期於定點進行連續偵測，本研究結合新型局部酸洗技術，利用聚乙二醇 (PEG) 包覆光誘發鄰-硝基苯甲醛 (o-nitrobenzaldehyde; o-NBA) 分子，以多模態光纖導入紫外光並以聚焦方式控制其解離氫離子範圍，可使感測器表面免疫鍵結解構而能再度進行感測，並且局限氫離子之分布使其不至於對探針外部環境造成影響。實驗結果並顯示鄰-硝基苯甲醛溶液經紫外光照射可於20秒內由酸鹼值7下降至3.5以下，足夠用以酸洗免疫接合分子表面。此外，此探針感測系統最快之檢測週期可控制在8分鐘以內，未來可針對活體內特定蛋白質分子之微量濃度變化進行量測，例如神經傳導或疾病發生時相關酵素或蛋白質之釋放，以解決目前研究上無法於特定位置快速得知其分子濃度的缺點。

This study reports a novel perfusion-based micro opto-fluidic system (PMOFS) as a reusable immunosensor for in-situ and continuous protein detection. The PMOFS includes a fiber optic interferometry (FOI) sensor housed in a micro-opto-fluidic chip covered with a microdialysis membrane. It features immune sensing and surface regeneration mechanism for continuous detection.
A method using gold-nanoparticles (GNPs) to enhance immune-sensing signal is proposed. It is suggested that an enlarged index mismatch and an elongated optical path by GNPs conjugated on recognition proteins will contribute most to signal enhancement in the interference fringe shift. Theoretical and experimental results show that the interference fringe shift is linearly related to both the amount and size of the GNPs binding on the sensor surface. The detected signal for 30 nm GNPs can reach a lowest detection limit of 18 pM (1010 particles/ml). Besides, surface regeneration of the FOI sensor was achieved through local pH level manipulation by means of a photoactive molecule, o-Nitrobenzaldehyde (o-NBA), which triggered the dissociation of immune complexes. Experimental results showed that the pH level of the o-NBA solution can be reduced from 7.0 to 3.5 within 20 seconds under UV irradiation, sufficient for an effective elution process. The o-NBA molecules, contained within poly(ethylene glycol) diacrylate (PEG) complexes, were trapped within the sensing compartment by the microdialysis membrane and would not leak into the outside environment. The pH variation was also limited in the neighborhood of the sensor surface, resulting in a self-contained sensing system.
In-situ immune detection and surface regeneration of the sensing probe has been successfully carried out for two identical cycles by the same sensing probe, and the cycle time can be less than 8 minutes, which is so far the fastest method for continuous monitoring on protein/peptide molecules. In addition, the interference fringe shift of the sensor shows a linear relationship to the concentration of anti-cytochrome C antibody solution and the detection limit approaches 10 ng/ml. This sensory mechanism has the potential to be applied for in-situ, in-vivo monitoring immune recognition signals in the future.

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