微流體整合石墨烯-適體生物感測器應用於心臟Troponin-I檢測之開發

Abstract

本論文以開發心臟病酵素Troponin-I (cTnI)生物感測器晶片為研究主題。臨床上對於急性冠心症(acute coronary syndrome, ACS)的診斷為症狀評估、心電圖判斷以及心臟酵素濃度的檢測。然而罹患癡呆症以及中風的病人，將難以獲得症狀的資訊，且心電圖有時候未有明顯的特徵顯示時，依據酵素濃度作為評判標準就有其必要性。近期的研究指出，cTnI釋放於血液中的濃度對於心肌組織壞損的程度具有相當優異的指標性且可以做為長期監控的生物標記，因此本論文以cTnI作為檢測的對象進行生物晶片之開發。應用於定點照護系統(point of care system)的生物晶片期望能具有使用方便、低濃度檢出以及高靈敏度的特點。在使用上若採用毛細力自行驅動檢測液體的方式，可省去外力驅動設備的需求。因此針對毛細力流體於矩形流道中的動態理論進行推導，由公式顯示檢測液體的表面張力、黏滯係數以及與流道材質之間的接觸角為所需參數，而其中對於流道深寬比與流速的關係可作為流道尺寸設計的依據。將不同比例的甘油與去離子水混和而成不同黏滯係數的溶液在經過聚對二甲苯表面改質的聚二甲基矽氧烷微流道內進行毛細力驅動實驗。從流體前緣位置與時間的關係，透過公式可獲得前進接觸角的變化，進而求得流速，再由流速估算流道表面產生的黏滯剪應力以及黏滯剪力，則可評估生物分子間的鍵結是否會被破壞而降低檢測的性能以及作為生物檢測器於微流道內位置設定的依據。在檢測元件方面，以石墨烯作為背閘極式場效電晶體的通道即為檢測區域，並用cTnI適體對石墨烯表面進行功能化改質以增進低濃度的檢測能力。經由元件製程與生物分子功能化改質後所獲得的電晶體特性皆為p型，由電晶體操作上可以發現在檢測區域滴下檢測液體後，轉移電導以及傳輸載子遷移率會隨著檢測濃度不同而改變，使得電晶體特型呈現n型摻雜。因此，轉移電導的變化可作為濃度量測的指標。本研究中的最低檢測濃度可達到0.01 ng/mL。在論文最後將聚二甲基矽氧烷與奈米氧化鐵粒子均勻混和，在微流道內製作具有內建磁場的微角椎，其具有層流流體混合與微磁珠捕捉的功能。

The topic of this dissertation is to develop a biosensor for detecting cardiac troponin-I (cTnI). In clinical, it is pathognostic to have acute coronary syndrome (ACS) in two abnormalities of three items that are clinical symptoms assessment, electrocardiography (ECG) diagnosis, and detection of the concentration of cardiac enzymes. However, it is sometimes not easy to determine clinical symptoms, such as the dementia and apoplexy suffered patients, or the ECG without obviously specific characteristic. Therefore, it is taken more attention to diagnose ACS with cardiac enzymes. Cardiac troponin I (cTnI) is one kind of cardiac enzymes. cTnI has been proved as the gold-standard and an attractive biomarker for presymptomatic diagnosis and suitable for long-term monitoring. The biochip with application in point of care system is expected convenient usage, low concentration detection and high sensitivity. Testing solution flows in biochip via capillary force for self-driving is performed without external equipment requirement. The dynamic equation of capillary flow in rectangular channel is derived. The surface tension, coefficient of viscosity and contact angle are parameters. The equation is available for design of channel dimension and location of biosensor in channel. Different mixing ratios of glycerol and deionized water to be testing solutions with different coefficients of viscosities are served into the parylene coated polydimethylsiloxane (PDMS) microchannel in the capillary force driving experiment. According to the relation of meniscus position and time, the variation of advancing contact angle can be obtained, and then the velocity can be calculated as well. The viscous shear stress and shear force near the surface of microchannel can be estimated. The information is provided to appraise if the binding of molecules would be broken and to design the location of biosensor in microchannel. The back-gate filed effect transistor (FET) is fabricated with graphene as transport channel (detecting area). The cTnI aptamer is immobilized on the surface of graphene for functionalization to enhance performance of low limit detection (LOD). All the characteristics of FET biosensors are p-type after fabrication process and bio-functionalization. Under the detecting test, the transconductance and carrier mobility in electrical characteristic of FET changes as the concentration of testing solution due to n-doping effect. When the concentration is greater, the variation of transconductance becomes larger. As a result, transconductance can be used to determine the concentration level of biomarkers in testing solution. The LOD in this research achieves at 0.01 ng/mL. The final part of this dissertation is to fabricate a microcone with build-in magnetic field in microchannel. It has functions of mixing microfluid and capturing microbeads.