利用鋸齒電極以優化微型免標定電化學生物感測器之靈敏度

Abstract

聯合國人口基金會 (United Nations Population Fund, UNFPA) 指出，21世紀最重要的趨勢之一為人口老化，而慢性病為老年人主要面臨的健康問題，且慢性病每年在醫療保健上占了絕大部分的支出，因此「及早發現、及早治療」的觀念漸漸深植人心，預防醫學也逐漸成為醫療的主要趨勢，因此以定點照護為訴求的生物感測器逐漸興起。此外心血管疾病是全球慢性病中的頭號死因，能夠以快速且低成本的方式及早檢測心血管疾病並進行治療是必要的。因此本論文提出一個能夠檢測 S100 beta protein 與 C-reactive protein (CRP) 兩種心血管疾病的生物標記物，希冀能夠開發出免標定高靈敏的定點照護生物感測器。 為了符合定點照護的應用，體積小、成本低、操作簡易及具有足夠的靈敏度等特性是優先的考慮要素。雖然光學式生物感測器具有高靈敏度的優點，但相反的，亦具有體積大、成本高與光路校正不易等問題，因此不適合開發成定點照護的儀器。而電化學阻抗式生物感測器具有成本低、體積小、免標定與校正方便等優點，因此，本論文採用電化學阻抗量測的方法作為檢測的基礎，將三極式電化學量測的三種電極 (工作電極、輔助電極及參考電極) 縮小到一個生物晶片上，並採用微型化之微流體生物晶片量測機構來架設系統。本論文採用電腦模擬電化學電解液電流密度以及原子力顯微鏡量測表面電位來研究能提升生物晶片之檢測靈敏度的方法，並發現於工作電極與輔助電極相鄰處因為電極邊緣效應而有較高的電解液電流密度及表面電位，若於電極邊緣設計上鋸齒結構，其電極尖端效應可更進一步提高電解液電流密度。因此，本論文將工作電極與輔助電極相鄰的電極邊緣加長並於其上加上鋸齒結構，藉以提昇電化學量測效率。此外增加之電極邊緣亦可提高生物分子鍵結於電極邊緣的機率，並進一步提高生物分子被量測到的機率。 本研究利用4-aminothiophenol (4-ATP) 以及cysteamine作為連結分子，分別進行心血管疾病生物標記物 S100 beta protein 與 CRP 的抗體－抗原交互反應，並利用電化學阻抗分析法，驗證微流體生物晶片之可行性。實驗結果顯示，隨著抗原濃度增大，電子傳遞電阻的變化量 (ΔRet) 與其呈現相當線性之關係，可見生物晶片檢測之穩定性。整體的線性量測區間為27 pM (10 ng/ml) 至270 nM (10 μg/ml)，檢測極限可達27 pM (10 ng/ml)。本實驗結果也顯示了本論文所開發的鋸齒電極生物晶片的設計可有效提昇電化學阻抗量測的靈敏度，並說明了本生物感測器具有發展成定點照護或手持式生物感測器的潛力。

According to United Nations Population Fund (UNFPA), population aging is one of the most important trends in the 21st century. Chronic diseases have become the major health problem of the elderly and cost a large expense in health care every year. Therefore, the concept of “early detection, early treatment” is growing in popularity in typical citizen’s mind. The preventive medicine thus gradually becomes the main trend. All of which indicate that point-of-care (PoC) biosensors are becoming ever more important. Since cardiovascular disease (CVD) is the leading cause of death among chronic diseases worldwide, it is necessary to detect CVD biomarkers by rapid and low cost methods so as to treat the disease early. This thesis thus focuses on developing a label-free electrochemical biosensor with high sensitivity for PoC application to detect CVD biomarkers, i.e., S100 beta protein and C-reactive protein (CRP). To fit the PoC application, a biosensor with advantages such as small size, low cost, ease of operation, label-free, and possessing enough sensitivity are primary design factors. Although typical optical biosensors possess high sensitivity, they face limitation such as miniaturization, high cost, and complexity in light beam alignment, etc. All of which limit typical optical biosensors for PoC application. In contrast, electrochemical impedance biosensors have properties needed for PoC biosensors. Therefore, electrochemical methods were used as the basis of the detection methods adopted in this thesis. A more stable three-electrode configuration, including working electrode (WE), counter electrode (CE), and reference electrode (RE), was miniaturized and fit onto a biochip. A miniaturized microfluidic biochip measurement device was used as the platform to develop the system. To improve the detection sensitivity associated with the reduced size in the biochip, computer simulation of electrolyte current density and atomic force microscopy (AFM) for measuring surface potential were used to investigate several potential effective possibilities. It was found that the electrolyte current density and surface potential on the edge near the WE and CE was higher. The zigzag structure designed on the edge could further increase the electrolyte current density. Therefore, in this thesis, the edge of the WE and CE was lengthened and added zigzag structure without changing the area of the WE and CE and maintained the gap between the two electrodes, which raised measurement efficiency. Moreover, the increased edge/area ratio could increase the probability of the bio-molecules binding to the edge of the electrode, which certainly further raised the probability to detect the bio-molecules. The 4-aminothiophenol (4-ATP) and cysteamine were used as linkers to perform antibody-antigen interaction tests for S100 beta protein and CRP biomarkers with an attempt to verify the microfluidic biochip’s feasibility. Results showed that it had a good logarithmic linear relationship between the electron transfer resistance (ΔRet) and a logarithm of the concentrations of bio-molecules. The dynamic range was from 27 pM (10 ng/ml) to 270 nM (10 μg/ml), and the detection limit was 27 pM (10 ng/ml). With the designed zigzag electrode, an electrochemical impedance biosensor developed in this dissertation shows a great potential to be used for point-of-care applications.