電化學(EC) 生物感測器因其具有小型化、便攜性和降低成本等優勢，是最常被研究用於居家即時 (POC) 的生物感測器類型。本研究旨在以EC法為基礎開發符合POC要求的感測器，且得以能快速、靈敏地檢測感染中的生物標的物。為此，先對於雙電層(EDL)有較深入的概念釐清，其在檢測較低濃度之生物標的物時尤為重要。其次，利用電化學阻抗譜 (EIS) 技術以及對電路元件的分析和對電子轉移速率常數(k_et^0)的監測，可以提供受測試樣在添加前後在EDL特性上的變化。我們也對表面修飾進行探討，期望理出面對臨床檢體複雜環境於EIS量測時仍可適用之數值模擬參數解析。繼之，與直流偏壓交流電動(DC-ACEK)系統整合後，發現可在 60秒內收集標的物，且EIS量測是在具有高電導率的10 mM 磷酸鹽緩衝(PBS)液中進行。透過類似傳統親和性反應的「兩步驟式方案」操作，來分析腫瘤壞死因子-α (TNF-α)、介白素-6 (IL-6) 和 microRNA-155 (miR-155)，量測乃先以DC-ACEK促進標的物之雜交，經水洗瀝乾除去電極尚未結合物質後，再滴入EIS量測所需之含Fe(CN)64-/3-的氧化還原對(redox pair)之標準液等兩步驟來實施，全程可在5分鐘內完成，且被用於監測敗血症患者之血清試樣中如上所述三種生物標的物濃度之呈現走勢。此技術可檢測較低之標的物濃度(達0.1 pg mL-1)，且具有良好的線性，可作為醫師評估其預後的指標。此結果與傳統酵素連結免疫吸附測定法(ELISA) 相比較，也發現兩者間在屬較高的濃度(>10 pg mL-1)具有良好的正相關性，尤其DC-ACEK 在較低濃度下明顯具有3個指數以上的優異靈敏度。最後，我們更為解決兩步驟式方案中間須經水洗等較不便的問題，也開發了“一步驟式方案”，乃利用血清樣本先和含電化學氧化還原對的10 mM PBS之測試液，惟須先藉一小試管，以1:1比例混合後再引導至試片側向入口，即可透過微管道之親水性完成一種更簡單、快速的檢測方案。此種結合電動力學反應促進與電化學檢測，提供了能在兩分鐘內完成全程檢測的方法，更可滿足POC組件用於感染症早期診斷的要求。

Electrochemical (EC) biosensors are the most commonly studied type of biosensors for point-of-care (POC) due to their advantages, such as their potential for miniaturization, portability, and reduction of cost. This study aims to utilize EC biosensors in the fabrication of a POC device for the rapid and sensitive detection of biomarkers in infection. Prior to the biosensor fabrication, the concept of the electrical double layer (EDL) should be initially investigated, for its effects are more apparent in the detection of lower target biomarker concentrations, which can provide a great advantage in the early diagnosis of diseases. The utilization of the electrochemical impedance spectroscopy (EIS) technique along with the analysis of the electric circuit elements and monitoring of the electron transfer rate constant (k_et^0) can provide a view of the changes in the EDL before and after the addition of the modification layers. The optimized surface modification conditions were further utilized to fabricate a POC device to address the current limitations in the clinical setting. Integrating with DC-biased AC electrokinetics (DC-ACEK) allowed the target collection within 60 s, suspended in 10 mM phosphate buffer saline (PBS) solution with high conductivity. Tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and microRNA-155 (miR-155) were analyzed through a conventional “two-step protocol”, wherein the target hybridization was initially done followed by sample washing and detection stages utilizing the Fe(CN)64-/3- electrochemical reagent. This technique allowed the detection of lower target concentrations, reaching 0.1 pg mL-1 with good linearity and can be finished within 5 min. However, the separate detection still has the limitation of not being easy to operate. Therefore, a “one-step protocol” was further introduced, utilizing a 1:1 mixture of the serum sample and electrochemical reagent to facilitate easier and faster detection scheme. The mixture resulted in a high viscosity, causing an alteration in the electrochemical parameter from charge-transfer resistance (RCT) to double layer capacitance (QDL). A good correlation within 3 orders of magnitude was obtained with the traditional enzyme-linked immunosorbent assay (ELISA), specifically at higher concentrations (> 10 pg mL-1), with the DC-ACEK having superior sensitivity at lower concentrations. Combination of electrokinetic concentration and electrochemical detection provides a simple detection method that can be finished within 2 min, addressing the requirements for a POC device for the early diagnosis of infection.

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