利用相位式表面電漿共振系統檢測免疫球蛋白鍵結之應用分析;Immunoglobulin Binding Analysis by Utilizing a Surface Plasmon Resonance Phase-Measurement System

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题名:	利用相位式表面電漿共振系統檢測免疫球蛋白鍵結之應用分析;Immunoglobulin Binding Analysis by Utilizing a Surface Plasmon Resonance Phase-Measurement System
作者:	林萱;Lin,Shiuan
贡献者:	照明與顯示科技研究所
关键词:	表面電漿共振;生物分子;Surface Plasmon Resonance;Immunoglobulin
日期:	2012-07-31
上传时间:	2012-09-11 18:43:19 (UTC+8)
出版者:	國立中央大學
摘要:	本研究之動態式表面電漿共振感測器是以相位移干涉為基礎，以偵測器擷取光強訊號，代入五步相移法進行解相。由實驗結果可得知系統於3600秒下相位穩定度為1.2214度、系統靈敏度為1.1465✕10^4 (Degree/RIU)、系統折射率解析度可達1.0653✕10^-4 (RIU)。利用鹽水實驗可得折射率與相位差關係，進而去推算未知鹽水溶液折射率，經折射率計量測與實驗後內插所得之折射率，兩者誤差約為0.023 %。於免疫球蛋白量測部分，利用濃度分別為200 μg/ml、100 μg/ml、50 μg/ml、25 μg/ml之Anti-IgG與固定於金膜上之IgG進行鍵結，所對應相位變化分別為：78.7476°、58.8533°、41.0506°、33.1399°，由相位與時間關係圖去計算結合速率常數k_a與解離速率常數k_d，其值分別為4.1139✕10^4M^-1s^-1與2.8768✕10^-4s^-1。經由鹽水實驗與生物分子鍵結量測可得知SPR相位訊號隨待測液濃度改變之關係式，故可利用此關係曲線來進行未知濃度檢測。A dynamic surface-plasmon-resonance (SPR) sensor was developed based on interferometric phase measurements. Temporal intensity signals were acquired by photodetectors and processed by the Schwider-Hariharan five-step algorithm to obtain the phases. The achieved phase-detection stability was 1.2214 degrees in a 3600-second period and the system sensitivity was 1.1465×10^4 degree/RIU (refractive index unit). The corresponding system resolution was 1.0653×10^-4 RIU. Salt-water mixture measurements were performed to characterize the relation between the measured phase difference and the refractive-index variation of the specimen. Several known concentrations of salt-water mixtures were flowed into the SPR system to measure the phases. A refractometer was utilized to measure the refractive indices. The results showed a linear correspondence between the phase difference and the refractive index. The measurement of an unknown salt-water mixture showed a relative refractive-index error of about 0.023 % between the refractometer measurement and the linear interpolation result from the SPR system.For the antibody-antigen binding experiments, the phase differences caused by the injection of anti-IgG with concentrations of 25 μg/ml, 50 μg/ml, 100 μg/ml and 200 μg/ml were measured to be approximately 33.1399°, 41.0506°, 58.8533°, and 78.7476°, respectively. From the relation between the time and phase difference, the association rate constant ka and the dissociation rate constant kd were calculated to be 4.1139×10^4 M^-1s^-1 and 2.8768×10^-4 s^-1, respectively. By utilizing the relation between the SPR signals and the concentrations of the analyte from salt-water mixture measurements and antibody-antigen binding experiments, the concentration of unknown solutions can be estimated by this SPR system.
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