以導電連結分子為基礎之創新電化學阻抗式生物感測器研發

Abstract

由於生活品質的提升，人們對於健康照護的需求也愈來愈重視，因此，標榜能夠快速檢測出生理狀態之生物感測器也愈來愈蓬勃發展。生物感測器依傳感器的不同可分為光、電、力、磁、熱、流、聲等多種檢測方法，其中，光學式生物感測器(如表面電將共振儀)雖然具有高靈敏度的優點，但是因為價格高昂以及光路校正不易等缺點，以致於到目前為止還無法實際應用在臨床的疾病檢測上。而目前最成功且已經被大量地應用在日常生活中的生物感測器就屬電化學式的血糖儀。雖然電化學生物感測器具有低成本、小體積等優點，但是因為蛋白質或是遺傳因子(DNA)本身等不具備氧化還原的特性，因此目前大多只能應用在以酵素為基礎的生物感測器上。 為了能夠擁有電化學感測器的優點又同時可以量測蛋白質的交互作用，本論文提出以導電連結分子為基礎之電化學阻抗式生物感測器，並利用該創新感測器來量測蛋白質間的交互作用。在本論文中，我們利用電化學循環伏安法以及阻抗分析法來量測數種導電連結分子以及傳統長鏈硫醇連結分子的導電特性。從實驗結果可以發現，當碳鏈愈短時，導電的效果愈好；此外，連結分子的官能基也會對阻抗造成很大的影響，然而，當生物分子與連結分子產生鍵結後，由於官能基已經被生物分子所取代，因此，其導電特性只與碳鏈的長度有關。我們也進而比較導電連結分子跟傳統長鏈硫醇分子鍵結生物分子後的阻抗值，實驗結果顯示，導電連結分子的阻抗值比傳統長鏈硫醇分子低2個等級。由於阻抗的降低(電流增加)，致使得訊雜比也相對提升2個等級，因此有機會改善偵測極限。為了確認該創新導電連結分子跟傳統的長鏈硫醇分子具有相同的生物分子鍵結能力，我們利用螢光顯微術來量測生物分子鍵結的量。此外，我們也利用酵素連結免疫分析的方法來驗證抗體-抗原間的專一性。最後，我們利用此創新阻抗式生物感測器來量測antiS100與S100間的交互作用，實驗結果顯示本感測器的線性量測區間為10 ng/ml到10 μg/ml，偵測極限約10 ng/ml。本實驗結果也顯示了本生物感測器極具有發展成定點照護或是手持式生物感測器的潛力。

With the rapid improvement of life quality, the demand of health care increases day by day. Thus, the development of biosensors which have the advantages of fast detection is growing year by year. Among all different biosensors, optical detection based system such as SPR is thought to have the best sensitivity. However, the high cost and complex alignment procedures make it hard to be a point-of-care device. Since electrochemical biosensors possess the advantages of low cost, small size and easy calibration, a glucosemeter which based on electrochemical measurement becomes one of the most successful biosensor nowadays. However, the fact that proteins and DNA themselves does not have reduction/oxidation properties prevents us from using electrochemical biosensor to detect proteins and DNA, etc. To have the advantages of electrochemical biosensor and also possess the ability of detecting protein interactions, we here proposed a label-free impedance immunosensor based on an innovative conductive linker. As the often used conventional long chain alkanethiol is a poor conductor, it is not a suitable material for use in a faradaic biosensor. In this thesis, we adopted a thiophene-based conductive bio-linker to form a self-assembled monolayer (SAM) and to immobilize the bio-molecules. We used cyclic voltammetry and impedance spectroscopy to measure the conductive characteristics of four kinds of conductive linkers and one conventional alkanethiol. From the experimental results, it is found that as the number of methylene chain decreased, the conductivity increased. Besides, the functional group has a great impact on the impedance. However, when bio-molecules immobilized on the SAM, the functional group was replaced by the bio-molecules. Thus, the conductivity is the function of methylene chain number only. We also compared the impedance baseline after bio-molecules immobilization of a conductive linker and a conventional alkanethiol. Results showed that the electron transfer resistance of this new conductive linker was 2 orders of a magnitude lower than the case using a conventional long chain alkanethiol linker. With the decreased impedance (i.e. increased faradaic current), we can obtain a higher signal/noise ratio such that the detection limit is improved. Using fluorescence microscopy, we verified that our new conductive linker has a protein immobilization capability similar to a conventional alkanethiol linker. Also, using S100 proteins, we verified the protein interaction detection capability of our system. Our obtained results showed a linear dynamic range from 10 ng/ml to 10 μg/ml and a detection limit of 10 ng/ml. With our new conductive linker, an electrochemical impedance biosensor shows great potential to be used for point-of-care applications.