即時的BIA感測晶片廣泛應用於生物醫學檢測，過去所發展的技術，SPR為最有發展之應用。過去表面電漿共振檢測方式，多探討角度量測及圖像式解析，以分辨生物分子進行生理反應後之對比。其中稜鏡系統量測方式，可檢測行專一特性的生物交互作用分析，精準量測非常微弱的光學性質變化，但由於檢測光點面積過大，並無法精細觀測管道內單一區域的特性及反應程度上做辨別。以光纖為基底之SPR感測器具有低成本優勢，但是卻很難與微流道系統整合。角度變化檢測有助於整合微流道系統做即時的檢測，但侷限於相對大的入射光點，與即時的觀測光學影像具有困難度。
本研究提出一個以TIRF物鏡的表面電漿共振系統，結合可替換狹縫晶片之自製光學元件，以取代表面電漿共振稜鏡系統，進行圖像捕捉和波長分辨SPR檢測微應用。面對不同折射率之樣本，可藉由更換光狹縫，調變光源入射離軸距離，取得最佳辨別的量測訊號。為驗證此一想法，以一簡單雷射光路的稜鏡系統，用以確認光學薄膜品質，以鎢絲燈做為燈源，結合全反射物鏡模組於倒立式顯微鏡組中，以激發SPR。研究中選擇MATLAB®做為光學模擬軟體，實驗中可以理論數據，校正系統穩定度與精確度。除了驗證系統可成功量測表面電漿共振訊號外，本研究利用離軸距為5.8 mm之光狹縫，精準量測樣本折射率於1.33到1.36之間之動態變化過程。光線通過所設計的1.00 mm孔徑之光遮片以獲得連續波長的光束。然後光束通過TM模態偏振片,並利用全反射式物鏡激發SPR。以此方法，入射光束可進一步凝結成小光點以提高空間解析度。此外，使用多模態光纖搜集反射光束且以高效能之光譜儀作為SPR分析。當檢測到SPR時，低數值孔徑物鏡使用於即時捕捉圖像。研究結果顯示5.8 mm距離的離軸距離具有較佳的SPR效能。
本研究成功於TIR物鏡上測量到SPR訊號。不論根據不同的入射角度改變off軸距離或是改變樣本的折射率，皆可以觀測SPR波長光強度之散失。此光學架構開發將可建立高空間解析度與高密度晶片系統的使用以整合與降低成本於製作SPR檢測平台。
光學微流體部分，研究中以鈉玻璃作為基板，採取濺鍍系統、表面改質以及蝕刻製程製作玻璃微流體晶片，再於高解析蓋玻片改質後濺鍍上45 nm黃金薄膜做為感測層。藉由填充高折射率耦合油於晶片與物鏡之間，降低光源在界面反射率。管道內注入自組裝薄膜分子進行黃金表面改質，同時以高解析度與高量子效應光譜儀於光路後端收光，切確及時地觀測表面改質過程。
實驗證明系統在50 μm週期結構上可清楚辨識，同時於晶片下方使用CCD拍攝或錄製掃描過程。此外更於單一定點量測不同折射率之樣本，以週期性交錯注入管道中，進行長時間動態量測，其中更利用黃金改質過後之表面官能基抓取酵母菌細胞，即時進行表面電漿共振量測。

Real-time biomolecule interaction analysis (BIA) sensor chips are widely used in biomedical testing. Over the developed techniques, surface plasmon resonance shows the most promising for these applications. Optic fiber based SPR sensors exhibit the advantage of low-cost but it is difficult to integrate with microfluidic systems. The angle variation detection is promising to integrate with microfluidic system for real-time detection. However, conventional angle-variation SPR detection schemes are usually limited by the relatively big size of the incident light spot and the difficulty for real-time optical image observation. In addition, an expensive goniometer is required to obtain precise angle measurement. Therefore, it is essential to develop a system for simultaneously optical image observation and SPR detection without using the moving part.
This work presents a TIRF objective-based surface plasmon resonance (SPR) system for simultaneously image capturing and wavelength- resolved SPR detecting for microfluidic applications. The light from a low-cost tungsten bulb passes through a designed light stop and polarizer is used as the light source for SPR detecting. The light spot is reduced to around 15 m after passing through the 60X objective lens, resulting in a higher spatial resolution for SPR detection in microfluidic channels. Moreover, the designed confocal scheme makes the system capable of simultaneously optical observation and SPR detection without scanning the wavelength or switching the optical system. Results show that the developed system is capable of detecting samples of refractive index in the range from 1.0 to 1.38 with a sensitivity of around 3417 nm/RIU. The developed system provides a simple yet high performance method for on-site detecting the SPR spectra anywhere inside a microfluidic device.
The system was constructed under a home-built microscope system. A tungsten bulb was used for the light source for SPR excitation. The light passed through a designed light-stop with a 1.0 mm hole to obtain light beam with continuous wavelength. The light beam then passed through the polarizer (TM mode) and deflect using a TIRF objective lens for SPR excitation. With this approach, the incident light beam can be further condensed into a small light spot such that the spatial resolution can be enhanced.
The system setup for simultaneously image capturing and high spatial SPR detection. The reflected light beam was collected using a multimode optic fiber and analyzed with a high performance spectrometer for SPR analysis. A low N.A. objective lens was used for simultaneously capturing the image while SPR detection. Therefore, microscopic image and SPR analysis can be achieved in a simple system without moving or scanning parts. Results show that the off-axis distance of 5.8 mm has the greater SPR performance.
This study has measured the signal of SPR upon TIR Objective successfully. Whether we changed the d off-axis distance corresponding to a different angle of incidence, or changed in the refractive index of the sample, we all find the obvious SPR wavelength of the light intensity lost. This optical architecture development will create a high spatial resolution and the utilization of high-density chip system which will integrate and reduce the cost of making the SPR measurement stage.

中文摘要 I
Abstract III
目錄 V
圖目錄 VIII
表目錄 XII
簡寫表 XIII
符號表 XIV
第一章 緒論 1
1.1 研究動機 2
1.2 研究目標 3
1.3 章節概要 4
第二章 基礎原理與文獻回顧 5
2.1 表面電漿波原理 5
2.1.1 漸逝場(Evanescent Field) 5
2.1.2 表面電漿與金屬色散原理與電漿子(Plasmon)理論 6
2.1.3 偏振光學原理馬克斯威方程 9
2.1.4 表面電漿激發原理 13
2.1.4.1 稜鏡耦合方式 13
2.1.4.2 光柵式耦合 16
2.1.4.3 波導耦合(Waveguide coupling) 17
2.2 表面電漿共振激發與量測方式 19
2.2.1 調變角度量測 19
2.2.2 調變波長量測 20
2.2.3 光強度量測 20
2.3 表面電漿於生醫免疫分析之運用 20
2.3.1 薄膜自組裝表面改質與免疫分析 21
2.3.2 表面電漿共振用於生醫檢測之方法 24
2.3.3 表面電漿共振顯微術 26
2.3.4 全反射式物鏡螢光生醫檢測系統 28
第三章光學系統架設與晶片製程 30
3.1 光學系統架設 30
3.1.1 倒立式螢光顯微鏡 30
3.1.2 TIRF表面電漿共振偵測系統原理 32
3.1.3 光遮擋片設計與物鏡基本原理 33
3.1.4 基本實驗架構 34
3.1.5 即時掃描與影像捕捉 35
3.2 晶片製程 36
3.2.1 標準玻璃晶片製程 36
3.2.2 鈉玻璃蝕刻速率探討 37
3.2.3 表面電漿共振金薄膜製程及驗證 39
3.2.4 表面電漿共振生醫晶片製程與設計 41
3.3 光譜儀訊號分析 44
3.3.1 光訊號擷取系統 44
3.3.2 數位訊號處理與模擬方法 46
第四章 實驗結果與討論 51
4.1 金薄膜特性量測 51
4.2 稜鏡式表面電漿共振 52
4.3 物鏡式波長解析表面電漿共振系統量測結果 52
4.3.1 光離軸距離對表面電漿共振之影響 52
4.3.2 折射率改變與表面電漿共振波長 55
4.4 管道內SPR量測 59
4.4.1 表面改質製程與黃金黏著層之貼附能力探討 59
4.4.2 自組裝單分子層之表面改質與生醫量測 60
4.5 動態偵測單點之SPR解析與掃描方式探討 62
第五章 結論與未來展望 65
5.1 結論 65
5.2 未來展望 66
參考文獻 67
附錄A 74
附錄B 76
附錄C 77
附錄D 80

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