Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/91372
標題: Aptamer-Based Impedimetric Biosensor for Allergen Der p2 detection
以配適體檢測塵蟎過敏原之電化學生物感測器
作者: 沈銘哲
Ming-Che Shen
關鍵字: aptamers;Der p2 dust mite allergen detection;nanostructured biosensors;electrochemical impedance spectroscopy;配適體;Der p2塵蟎過敏原檢測;奈米結構生醫感測器;電化學阻抗譜分析
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摘要: 
Dust mite in the Der p2 (DP2) is the main allergen to induce y rhinitis, eczema, and atopic dermatitis. When an allergic patient comes into contact with dust mite allergens, the TLR4 (Toll-Like Receptor 4) in the upper respiratory tracts induces allergy related IgE producing, hence eliciting an immune response. Currently, most of the protein based sensors for DP2 detection base on the immune response between specific antibody and antigen. This type of sensing principle requires many surface modification steps to immobilize the probe onto an electrode. The aptamer-based sensing mechanism uses the geometric structure of a specific designed aptamer to bind the target. Due to its advantages such as ease of synthesis, high purity, and high controllability, aptamer can effectively replaces the antibody or antigen probe in a immune response biosensor.
In this study, a novel aptamer-based impedimetric biosensor for allergen Der p2 detection was developed. An anodic aluminum oxide (AAO) membrane was prepared first. We then used the modified AAO barrier-layer surface as the template for nano electroforming of a nickel mold. After etching the AAO template off, a 3D nickel nano-mold of the concave nano structure array was created. The formed 3D nickel nano-mold was further used for replica molding of a nano-structure polycarbonate (PC) substrate by hot embossing. A gold thin film was then sputtered on the PC substrate to form the electrode. Finally, an orderly and uniform gold nanoparticles (GNPs) layer was electrochemically deposited on the 3D gold electrode. After immobilizing the specifically designed aptamer on the fabricated electrode, electrochemical impedance spectroscopy (EIS) analysis was used to detect the concentration of DP2. The sensitivity of the proposed scheme on the detection of the dust mite antigen DP2 was 2.088 /(ng/mLcm2) with dynamic detection range of 27.5 ng/mL~400 ng/mL, and detection limit of 16.47 ng/mL.
The aptamer-based impedimetric biosensor proposed in this study possesses advantages such as high sensitivity, low cost, high consistency. It can be useful for the rapid detection of rare molecules in an analyte.

塵蟎中之 Der p 2 (DP2) 是造成鼻炎、濕疹及異位性皮膚炎等過敏症狀的主要過敏原,當患者接觸到塵蟎過敏原時,透過TLR4 (Toll-Like Receptor 4) 類鐸受體會誘發過敏者體內特異性IgE的產生因而誘發免疫反應。目前大部分的蛋白質聚合物感測器多是利用抗體抗原之專一性為生物辨識元件(探針),而探針接附過程往往需要多道表面改質步驟將抗體固定於基板才能進行檢測,不僅耗時且費力。以配適體為生物辨識元件之感測器乃是與傳統探針辨識元件及接附機制截然不同之架構,主要是利用配適體與待測物之幾何結構熬合方式鍵結,配適體與蛋白質結合具有高特異性與高親合力。配適體具有合成方式簡單、純度高與可精準控制等優點,能有效取代免疫分析中之抗體或抗原,做為生醫檢測晶片探針之優勢。
本研究以AAO背阻障層之奈米半球陣列結構為基底,用以電鑄成形具凹型奈米半球陣列結構之鎳奈米模具,再利用熱轉印技術製做出與原AAO背阻障層之奈米半球陣列結構相似之PC基板,再於其上濺鍍均勻分佈之奈米金顆粒,形成複合奈米結構電極,接著將Der p2配適體自組裝於複合奈米結構金電極上,以電化學阻抗分析法檢測Der p2塵蟎過敏原。
藉由阻抗分析可確認aptamer探針可成功抓取DP2,且晶片不致產生非特異性接附。以不同濃度之DP2所建立之檢測標準曲線其檢測線性範圍為27.5 ng/mL~400 ng/mL,線性度可達0.988,檢測靈敏度與檢測極限分別為2.088 /(ng/mLcm2)與16.47 ng/mL。
本研究所提出之生醫檢測晶片架構除具高電極表面積,可大幅提升檢測靈敏度與檢測極限外,利用熱壓翻模技術可有效降低製作成本並確保基板之一致性,再以配適體辨識元件取代傳統之單株抗體,並結合電化學阻抗分析法之高靈敏性及響應速度快等特點,符合接附簡單、低成本、檢測範圍廣、即時檢測、可拋棄式之生醫感測器發展趨勢。
URI: http://hdl.handle.net/11455/91372
Rights: 同意授權瀏覽/列印電子全文服務,2018-08-28起公開。
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