表面電漿子共振(Surface Plasmon Resonance, SPR)的基本原理是當光從介電質材料入射至金屬或半導體介面時,在共振角的情形下,會產生一表面電漿波的漸逝波分佈,此一共振角度的產生與介質之介電常數有直接關係。利用此原理設計之表面電漿共振儀量的光學偵測方式有四種:角度調制法、光波長調制法、光強調制法、以及相位調制法。本研究選擇角度調制的方式量測共振角度的改變量或偏移量,以獲得待測物介質的光學特性。若將生化反應產生之分子層視為光學薄膜,則可以利用表面電漿共振儀量測反應前後的光學特性改變,計算出生化反應的曲線,可用以觀察生化反應的靜置反應與動態反應。此外,為增加系統的量測精度,提出相位偵測技術之光機設計並嘗試將此一創新架構加入至本系統中,以期角度偵測技術與相位偵測技術可以相輔相成。 在光學機構設計中,對於角度控制機構之設計最為困難,因為必須利用長距離的半徑及微小的移動量才能達成此精準的角度控制。本論文中利用拋物面的數學特性,延續研究團隊之研究經驗,建立以拋物鏡為基礎之表面電漿共振儀,此一特殊的光學設計,可將角度入射之控制,轉換為利用伺服馬達進行位置之精密控制,可輕易達到0.001度,將原本由機械控制之入射角度精度以光學元件取代,克服常見之旋轉平台造成之中心對位問題,或者免除精密機械手臂旋轉設計需求。利用此一特性建構完成表面電漿子共振儀之表面角度偵測,對系統進行初步測試成功,進一步進行生化反應之ELISA (Enzyme-Linked Immunosorbent Assays) 靜置反應,以及一系列不同濃度Human IgG和Anti-IgG的BIA (Biomolecular Interaction Analysis)動態反應量測。成功建立出生化反應的角度訊號改變對時間之變化圖,並從共振角的公式計算推出光學之折射率變化,之後利用反應濃度對折射率改變作圖,計算出兩種生物分子的親和力大約為4.9380×10-5。 由於部分獲得之分子親和力與文獻資料所得之生物分子親和力大小範圍有些許落差,故本研究除了提出系統可改進之部分外,並且著手設計量測精度更高之表面電漿共振儀相位偵測系統,提出創新Quadrature的干涉架構以及事先校正之演算法。
The instrument design for surface plasmon resonance (SPR) coupled heavily with the principle of surface plasmon waves. When a light beam impinges onto the interface between the dielectric media and metal media at a critical angle, a surface plasmon evanescent wave is produced. The value of the induced critical angle correlates with the permittivity of the media. The optical properties of the media can be obtained by calculating the amount of the angle shift. There are four optical configurations typically adopted for constructing the SPR, which are angular modulation, wavelength modulation, intensity modulation, and phase interrogation. In this study, angular modulation and phase information retrieval were chosen to pursue the further advancement of this technique. If the biomecular layers are taken as optical thin layers, the biomolecular interactions can be detected by SPR. Then the bio interaction curves were observed to analyze both the kinetic and static equilibrium reactions. In a typical optical mechanism, design to arrive at precise incident angle control is most difficult since a tiny displacement associated with a large radius is needed. By extending the many years of optical system design experience within our research team and by introducing some innovative ideas, a design based on a paraboloidal lens-based surface plasmon resonance instrument has been created. In this new design, the angle control can be transformed to a stage-type movement control. The 0.001 degrees incident angle control can be easily obtained by today’s precision servomotor stage management. The SPR instrument was shown to be feasible. Furthermore, using such a system to measure the SPR critical angle shift during a bio-molecular reaction on a biochip; the bio-chemical reaction is analyzed by using the measurement results obtained. Not only the ELISA (Enzyme-Linked Immunosorbent Assays) interaction was done, but also BIA (Biomolecular Interaction Analysis) experiments with human IgG and anti-IgG were successfully measured. The SPR angle shift to the time plots were obtained. The reflection index variations were also achieved. The RIeq/C versus RIeq showed the kinetic affinity between human IgG and anti-IgG is 4.9380×10-5. Since some of the kinetic affinity data measured is slightly different from the values obtained from previous studies, some ways to improve the measurement resolution were demonstrated. Besides, the design for phase interrogation SPR instrument developed by extending quadrature interferometer technique was also proposed and pursued. Suggestions on further improving its function were proposed as well.