以丙型干擾素檢測來驅動橢圓偏振干涉儀與電化學阻抗分析儀整合之研究

Abstract

全世界有1/3的人口是肺結核帶原者，由於目前的檢測方法費時且人力密集，很難篩檢龐大的潛在病患，故本團隊以肺結核的指標蛋白質丙型干擾素（IFN-γ）為檢測目標，期望找到免標定，免多道沖洗步驟，可即時反應、檢驗快速的方法。 為驗證本團隊開發之橢偏儀，將橢偏儀與商用電化學工作站CHI進行同步量測，以達到相互驗證之目的。研究過程中，以丙型干擾素為生物樣本、用本團隊研發之CS20S當連結分子，將IFN-γ抗體接在生物晶片上。為使兩種儀器能順利整合，設計一流道機構與生物晶片可同時量測橢偏移相位訊號與電化學阻抗頻譜(EIS)的電阻抗訊號，並用LabVIEW程式控制馬達移動，便於產生不同角度入射光來尋找表面電漿共振角，其相位訊號的校正及量測也用程式來輔助簡化。 本研究的實驗結果顯示電化學與橢偏儀在高濃度(0.5~1 μM )時有一致性，接隨著濃度增加其相位、阻抗也增加，較低的濃度(0.125~0.25 μM )時，電化學可能要考慮等電點(isoelectric point，pI)的影響，因為IFN-γ的pI值是9.61，在pH7.4的緩衝溶液中帶正電，會吸引氧化還原對，而空間障礙會阻止氧化還原對靠近電極，兩種因素互相競爭，造成IFN-γ的濃度在0.125~0.25 μM 時，靜電作用力的效應比空間障礙明顯，使得阻抗隨濃度下降，當濃度提升到0.5~1 μM 時，空間障礙比靜電作用力明顯，阻抗隨濃度上升。而橢偏儀的檢測則不受pI值影響的影響，相位皆隨濃度增加。

About one third of the world’s population is infected with tuberculosis (TB) bacteria. As the screening technique is still time-consuming and labor-intensive; it is hard to pursue large scale TB screening. With an attempt to alleviate this problem, our team chooses IFN-γ as our target protein so as to pursue a label-free, no rinse step requirement, real-time, and rapid detection technique. To verify the ellipsometry and interferometry developed by our group before, we executed the measurements of ellipsometry and commercial electrochemical work station CHI simultaneously to detect Interferon gamma (IFN-γ). We also use the linker CS20S developed within the team to bind IFN-γ-Ab onto the biochip. Besides, we designed a flow channel and corresponding chip to integrate ellipsometer and electrochemical detection within the same platform. This platform provides us with an opportunity to measure the phase change of ellipsometry and the electronic signal of Electrochemical Impedance Spectroscopy (EIS) simultaneously. To change the angle of the incidence light beam, we used LabVIEW to control the motor so as to identify the proper surface resonance angle. Signal calibration and measurements are also carried out by the LabVIEW program. The results of this research show the trend of EIS and ellipsometry agree well in most ranges. In higher concentration samples (0.5~1 μM ) measurement cases, the phase and resistance increase when the concentration increases. On the other hand, in measuring lower concentration samples (0.125~0.25 μM ), the EIS signal may be affected by the isoelectric point(pI) value. More specifically, the pI value of IFN-γ is 9.61 and is positive charged in pH 7.2 running buffer. More specifically, IFN-γ is attractive to redox couple in the above-mentioned condition and the steric hindrance may prevent charge carrier from getting closer. The two factors compete with each other, which result electrostatic attraction domination in low concentration so as to make resistance decrease. On the other hand, steric hindrance dominates in high concentration, which makes resistance increase. On the other hand, ellipsometry does not get affected by the pI value so the phase increases with concentrations in both high and low concentrations.