臺灣博碩士論文加值系統

表面電漿共振(Surface Plasmon Resonance, SPR)是一種對物質折射率的微小改變，以及低濃度生物分子結合反應十分靈敏的偵測方式。利用在感測晶片表面固化生物分子作為探針(probe)，可使表面電漿共振感測儀具有不需對待測分子進行螢光物質標定、對生物分子反應的動態測量、專一性反應測量和大量平行檢測等優點，因此對抗體抗原結合、DNA雜合反應或酵素與受質等反應來說是十分方便的工具。
有關表面電漿共振感測儀的偵測方式，依其調變原理可分為入射角調變、入射光波長調變、反射光強度量測和反射光相位量測等方式，其中以相位方式最為靈敏。然而自Nelson等人(Nelson et al., 1996)提出相位量測方式以來，相關的研究似乎便僅止於偵測極限的探討，完全忽略了動態測量的優勢。因此本研究的目標便是希望以一套自組的表面電漿共振感測儀，藉由測量反射光相位變化的方式，對綿羊免疫球蛋白(sheep IgG)與其相應抗體間的結合反應進行動態測量，達到計算其反應速率常數的目的。
實驗結果顯示：再現性測試方面感應晶片在六次相同濃度的量測，訊號變化的CV値僅6.76%；而從改變流速的實驗結果發現，流速在150~600μl/min之間變化時，訊號的變化率沒有明顯的改變，顯示在這個範圍內的流速變化對反應速率的影響可以忽略；計算綿羊免疫球蛋白與其抗體的速率常數結果顯示，結合速率常數為(1.02±0.17)×104M-1s-1，解離速率常數為(3.01±1.43)×10-3s-1。相較於商用儀器Biacore X的測量結果，本系統所測得之結合速率常數較低，可能原因是樣品溶液在管路中流動距離太長，導致樣品濃度被稀釋，以及結合速率常數被低估。
由於表面電漿共振所造成的反射光相位變化具有對折射率變化的高靈敏性與極窄的線性區間之特性，因此適合在極小範圍的濃度變化進行測量，與商用儀器大範圍濃度變化測量的訴求不同。因此，以相位偵測原有的靈敏性和本研究展示的動力學分析結果，相信在低濃度生物分子的反應測量上，還是能展現其應用價值。

Surface plasmon resonance (SPR) is highly sensitive to biomolecular interactions and refractive index (RI) changes. SPR biosensor has advantages of label-free with analyte, real-time and specific interaction detections, and high throughput measurement. Through immobilization of biomolecules on the sensor chip as a probe, SPR is widely used in antibody-antigen binding, DNA hybridization, enzyme-substrate interaction, and etc.
There are four kinds of SPR detection: angular interrogation, wave- length interrogation, intensity measurement, and phase measurement. Among these methods, phase measurement is the most sensitive one. Since Nelson et al. (Nelson et al., 1996) proposed the phase measurement method, to our knowledge, the latter published work have focused mainly on improving detection limit and other bio-applications and none of them has applied to dynamic measurement in biomolecular interactions. Here, I present the study by using a homemade phase-detection based SPR system of measuring the kinetic parameters from antibody-antigen binding.
First, I tested the repeatability of kinetics by six samples in the same concentration; the results show a good repeatability of 6.76% in CV value. Second, I tested the velocity effect to antibody binding ability by varying the flow rate from 150 to 600 μl/min; the results indicate that the difference of binding rate is small (within 8.84%), implying that we can fix at any velocity in the tested range to conduct our experiment. Finally, I conducted the dynamically binding test of antigen-antibody using both the homemade SPR system and a commercial Biacore X SPR system. The association (ka) and dissociation (kd) rate constants obtained from the homemade system were (1.02±0.17) × 104M-1s-1 and (3.01±1.43) × 10-3s-1, respectively. Comparing to the Biacore X, I found that the ka value obtained from the home-made system was smaller than that obtained from the Biacore X. This discrepancy was considered mainly from the dilution effect, in which the adjacent running buffer diluted the sample solution adjacent to the running buffer; mass transport effect was considered to be the second reason to affect the association rate constant.
Phase-based SPR system has characteristics of high sensitivity and short linear range in measurement whereas commercial instrument, based on the angular interrogation, has relative poor sensitivity and longer measurement range. Therefore, the phase-based SPR system would reveal more powerful in the low concentration-end of dynamic biomolecular interaction measurement.

中文摘要 i
英文摘要 iii
誌謝 v
目錄 vi
圖目錄 viii
表目錄 x
第一章 緒論 1
1.1 前言 1
1.2 文獻回顧 3
1.2.1 表面電漿共振感測儀 3
1.2.2 親合性與速率常數的測定 8
1.3 研究動機 9
1.4 論文大綱 10
第二章 原理 11
2.1 表面電漿共振的相位特性 11
2.2 相位訊號的偵測原理 16
2.3 動力學參數分析 18
2.3.1 反應速率方程式 18
vii
2.3.2 協同性與空間障礙 22
2.3.3 質量傳輸 24
2.3.4 總結 26
第三章 材料與方法 27
3.1 實驗架構 27
3.1.1 外差光源 27
3.1.2 表面電漿共振感測裝置 29
3.1.3 流體傳輸系統 30
3.2 實驗材料 33
3.3 實驗步驟 35
第四章 實驗結果與討論 37
4.1 固化條件的選擇與結果 37
4.2 訊號再現性測試 41
4.3 流速的選擇 42
4.4 速率常數的計算 43
4.5 Biacore X 的測量結果 47
4.6 結果比較與討論 49
4.7 解離現象的討論 51
第五章 53
viii
參考文獻 55

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