製備抗凝血?核酸適合體-鉑錯合物與其應用在凝血?及抗凝血?素之比色法檢測; Preparation of a DNA aptamer-Pt Complex and its Use in the Colorimetric Sensing of Thrombin and Anti thrombin Antibodies

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Title:	製備抗凝血?核酸適合體-鉑錯合物與其應用在凝血?及抗凝血?素之比色法檢測;Preparation of a DNA aptamer-Pt Complex and its Use in the Colorimetric Sensing of Thrombin and Anti thrombin Antibodies
Authors:	蕭伊迪;Yi-di Siao
Contributors:	化學工程與材料工程研究所
Keywords:	凝血酶;酵素;核酸;生物檢測;適合體;enzyme;thrombin;DNA;aptamer;bioassay
Date:	2008-07-08
Issue Date:	2009-09-21 12:32:15 (UTC+8)
Publisher:	國立中央大學圖書館
Abstract:	本研究主要利用核酸適合體(DNA aptamer)及鉑錯合物(K2PtCl4)製備核酸適合體-鉑錯合物(aptamer-Pt complexes)，其具有核酸適合體(aptamer)的專一鍵結能力及催化過氧化反應的催化活性。利用改變不同合成環境尋找催化活性之最佳化合成條件，與蛋白質酵素比較。並選擇凝血酶為分析物及對應的核酸適合體(thrombin binding aptamer)，發展發展核酸酵素連結適合體分析法及競爭型核酸酵素連結合適體分析法，對核酸適合體-鉑錯合物之功能加以驗證。同時對核酸適合體-鉑錯合物做熱穩定性、保存時間、穩定性之特性分析。進一步篩選分子量，提升核酸適合體-鉑錯合物之檢測效率。催化活性最佳化的結果顯示，反應條件在90℃、pH9和8小時有最佳的催化活性，鉑錯合物的水解與核酸適合體的活化程度扮演重要的角色。核酸酵素連結適合體分析法的的結果顯示，抗凝血酶核酸適合體-鉑錯合物可以專一性鍵結凝血酶，並無需蛋白質酵素的幫助下催化過氧化呈色反應，成功檢測凝血酶，檢測極限達0.4?M，所使用檢體100?l，依粒徑篩選核酸適合體-鉑錯合物的結果，檢測效率提高了13倍。競爭型核酸酵素連結合適體分析法同樣成功檢測抗凝血酶素(anti-thrombin)，與市售的檢測試劑相比，在低濃度有較佳的檢測效率。比較蛋白質酵素辣根過氧化酶，催化活性Kcat低200倍，Km值則高150倍，但在熱穩定性及隨時間活性衰退的實驗中，核酸適合體-鉑錯合物則表現出優異的特性。本研究製備核酸適合體-鉑錯合物具有專一性鍵結能力、催化活性、熱穩定性、活性不易隨時間衰退等優點，在取代臨床檢測及基礎研究被廣泛使用之抗體與酵素上，極具發展潛力。 DNA aptamers carrying Pt nanoparticles were prepared by the reaction of DNA aptamers (without functionalization with biotin, thiol or other reactive groups) with K2(PtCl4) in solution at 60-90 °C. The DNA-Pt complexes possessed peroxidase enzymatic activity while retaining the specific binding ability of the aptamers. The enzymatic reaction of these complexes obeyed Michaelis-Menten kinetics. Km for the DNA-Pt complex was found to be on the same order as Km for hemin and hemin-DNA complex, but two orders of magnitude higher than that of horseradish peroxidase. The rate of the reaction catalyzed by the DNA-Pt complex, kcat, was found to be on the same order as that of hemin and hemin-DNA complex, but two orders of magnitude lower than that of horseradish peroxidase. Two types of DNAzyme-linked aptamer assays (DLAAs) were developed using these complexes, which successfully detected target proteins, with the sandwich type of DLAA targeting thrombin and the competitive type of DLAA targeting anti-thrombin IgA/G/M in serum. The DNA-Pt complexes retained their peroxidase enzymatic activity even after heat treatment. DLAAs having high thermal stability were developed using these complexes, which were free of animal and plant matter because neither antibodies nor horseradish peroxidase were used in their synthesis.
Appears in Collections:	[National Central University Department of Chemical & Materials Engineering] Electronic Thesis & Dissertation

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