用於偵測生物標記物之電容式生物感測器開發

Abstract

生物感測器一直以來透過靈敏度和高特異性快速地針對待測分子進行的檢測，在各個領域具有高度的應用價值。近年來，醫療檢驗體系中為了落實照護點的概念，儘管有許多高精度的技術和設備結合了半導體製程或是微奈米加工技術將檢測機構微小化帶來了便利性，卻帶來許多挑戰難以成為市售的產品，也因此提高了相對簡易且成本較低的檢測機構開發需求。本文將以低成本且可攜帶的電容生物感測器的開發為主旨，以研究電化學感測器為基礎探討常見的界面分子結合策略以及利用阻抗頻譜分析和電阻、電容、電感零件分析測試儀（LCR 錶）分析自組裝單層（SAM）絕緣度和蛋白修飾後的電容尺度，再以類比轉數位模組結合網印碳電極組成電子式的生物感測器透過電容變化實現分子感測之目的。本文以自製金電極取代市售柱狀金電極，提高分析效率，在製備好自組裝單層後將蛋白透過交聯劑固定在金電極，由阻抗量測確效，研究過程中電化學量測工具有不同分析的適性及絕緣性，阻抗過大會造成分析上的不準確性。最後選用實驗室開發的類比轉數位模組結合拋棄式網印碳膠電極，以聚丙烯酸樹脂作為感測界面絕緣層，再以硝化纖維素薄膜作為吸附層，將10 mg/ml為蛋白（抗原）固定化在感測界面進行抗體結合。目前在結果部分可以在電容值變化上確認生物辨識元件之步驟，透過雷射顯微鏡分析感測介面上的絕緣層與吸附層物理性狀，了解生辨識元件之製程效果，以電容值變化分析抗原固定以及抗體結合之成果，同時，利用雙辛可寧酸測定法（Bicinchonininc acid assay, BCA）和增強型化學發光試劑（Enhanced chemiluminescence, ECL）光呈色分別對抗原以及抗體進行吸附與結合效果的確效與電容變化結果比對，在不同濃度抗原吸附在感測界面後整體電容訊號變化倍率提升了約5倍（𝚫Capacitance/fold 值從0.01%提升至0.05%），最後在抗原抗體結合後可以看到電容下降的現象，在抗體濃度為0-25 𝜇g/ml具有分辨性，達到檢測之目的，日後若能調整訊大小或優化電極與或是感測界面的材料，進而將感測的敏感度提高、訊號放大，相信必能在分子檢測上提供另種選擇。

Biosensors have always been valuable in various fields due to their ability to quickly and accurately detect target molecules. Recently, there has been a movement to implement the Point-of-Care Testing concept in medical testing systems by combining high-precision technologies and equipment with semiconductor processes or micro-nano processing technologies to miniaturize detection mechanisms and improve convenience. However, this has created challenges in commercializing these technologies and thus increased the demand for simpler, more cost-effective detection mechanisms. This thesis focuses on the development of a low-cost and portable capacitive biosensor. It explores common interface molecule binding strategies based on electrochemical sensors and analyzes the insulation and protein-modified capacitance degrees using impedance spectroscopy and LCR meter analysis of self-assembled monolayers (SAMs). A capacitive biosensor is then constructed using an analog-to-digital converter module combined with screen-printed carbon electrodes to achieve molecular sensing through changes in capacitance. Homemade gold electrodes are used instead of commercial cylindrical gold electrodes to improve analysis efficiency. After the SAM is prepared, the protein is fixed to the gold electrode via a crosslinker, and impedance measurements confirm the effectiveness of the process. During the study, it is found that the electrochemical measurement tool has different suitability and limitation for analysis, and excessive impedance can cause inaccuracies in analysis. To overcome this problem, a disposable screen-printed carbon electrode is combined with a laboratory-developed analog-to-digital converter module. A polyacrylic acid resin is used as the insulation layer, and a nitrocellulose film is used as the adsorption layer to immobilize the protein (antigen) at the sensing interface for antibody binding at a concentration of 10 mg/ml. This study has confirmed that changes in capacitance value can be used to confirm the steps of the biometric recognition component. By analyzing the physical properties of the insulation layer and adsorption layer on the sensing interface through laser microscopy, the processing effect of the biometric recognition component can be understood, and capacitance value changes can be analyzed for the results of antigen fixation and antibody binding. Additionally, the Bicinchoninic Acid (BCA) Assay and Enhanced chemiluminescence (ECL) cold light colorimetry were used to verify the adsorption and binding effects of antigens and antibodies, and were compared to the capacitance change results. Different concentrations of antigens were applied to the sensing interface resulting in an overall increase in the capacitance signal ratio of approximately 5 times (the ΔCapacitance/fold value increased from 0.01% to 0.05%). The antigen-antibody binding led to a decrease in capacitance, providing a resolution for antibody concentrations of 0-25 𝜇g/ml and meeting the detection objectives. In the future, amplifying the signal and increasing the sensor's sensitivity can be achieved through signal size adjustment or optimization of the electrode and sensing interface materials, providing an alternative choice for molecular detection.