分子運算在生醫診斷上的應用─結合酵素反應與去氧核醣核酸之結構調控以研發鑑別流感病毒亞型之電化學偵測系統

Molecular Computation in Biomedical Diagnostics: A Structurally Controlled, Enzymatically Processed Bioelectrochemical Genosensor for Digital Subtyping of Specific Influenza Viruses

賴雨萱(Yu-Hsuan Lai)

指導教授 ： 何佳安

國立臺灣大學/生命科學院/生化科技學系/碩士(2013年)

https://doi.org/10.6342/NTU.2013.10048

摘要

有鑑於流感病毒傳播監控及臨床診斷工具的迫切需求，本研究發展了一流感病毒亞型鑑定平台，結合DNA三級結構的轉換調控與酵素連鎖反應，以達到專一而靈敏地同時偵測兩個重要抗原〔血液凝集素（hemagglutinin, HA）與神經氨酸苷酶（neuraminidase, NA）〕基因片段的目的。利用具莖環結構之去氧核醣核酸探針（stem-loop folded DNA, slfDNA）以及穩定的酵素反應處理，本研究所建構的生物電化學基因感測系統能快速地提供檢驗結論，避免潛在的人為誤判機會。為了更深入地了解由兩個目標核酸片段（gHA與gNA）共同引發組裝於一表面上的slfDNA的三級構型變化，本研究中使用不同的技術平台如螢光解離曲線、雷射掃描式共軛焦顯微鏡、計時安培分析法等，針對多個重要因子進行系統化地探討，包括序列組成及環境因子（反應系統中的鹽濃度、溫度）對slfDNA在水溶液中以及金膜表面上之構型摺疊穩定度的影響；不同的金電極表面修飾方法與分子組成抵抗核酸骨架及蛋白質非特異性吸附之效果；以及slfDNA與兩個目標基因所形成的雜合結構及其在感測表現上所產生的效應。實驗結果顯示，本研究所發展的感測平台能夠執行高度準確的流感病毒亞型鑑定 〔A/duck/Taiwan/DV30-2/2005 (H5N2)〕，在特定H5與N2基因共同存在時方呈現出一明確的陽性結果；而且，在此兩個目標基因同時存在的情況下所得到的電化學輸出訊號能夠顯著地與其他三種陰性組合（無H5與N2基因、只有H5基因、只有N2基因）區分出來（至少五倍以上的訊號差異）。此外，將光學與電化學實驗方法所得到的結果整合之後，我們發現gNA與slfDNA環狀部分的雜合行為可能引發slfDNA莖部結構的破壞，改變探針在平衡時的折疊狀態，進而大大影響亞型鑑定之準確性，且此效應與雜合的長度及位置有明顯的相關性。在此感測平台的定量分析表現中，只有H5或N2基因單獨存在時，即使在高濃度的情況下（0.5 μM）所得到的背景電流依然可以維持在一極低的範圍（< 5 nA），顯示此感測機制對於三種陰性組合不易產生偽陽性結果。本系統對於H5與N2基因共同存在時的偵測極限約為50 nM。此外，我們進一步將此感測平台應用於真實流感病毒基因體的檢測。結果顯示，在較為複雜的背景溶液中（未純化之非對稱聚合酶鏈鎖反應產物），本研究所發展的感測機制依然能準確且穩定的進行亞型鑑定，在H5與N2基因產物同時存在下得到一顯著訊號，並且在其他三種陰性組合中產生的電流依然維持在一極小之範圍（< 5 nA）。總結來說，據我們所知，本研究為目前第一個闡述將一slfDNA分子應用於雙重基因分析上的設計，透過調控生物分子構型轉換與生化反應的協同作用，產出一明確的分析結論。此篩檢技術具有高應用性與功能性，以及易擴展出多用途之靈活性，可望成為精密的尖端分子診斷平台，有效應用於多種而多變之流感病毒檢測。

關鍵字

流感病毒亞型鑑定 ； 電化學基因感測器 ； 莖環構型去氧核醣核酸 ； 雙基因檢測 ； 酵素訊號放大

並列摘要

To suffice the urgent need in surveillance and diagnostics of influenza virus, we herein report a sensor design coupling tailored combination of DNA structural switches and multi-enzymatic cascade reaction to specifically and sensitively identify dual target nucleic acid sequences which subsequently leads to subtyping of influenza viruses by hemagglutinin (HA) and neuraminidase (NA) simultaneously. Harnessed with a stem-loop folded DNA (slfDNA) and robust enzymatic processing, the bioelectrochemical genosensor is capable of deriving a rapid decision exclusive of potential personal errors made in data interpretation. To further the understanding of the dual-input induced structural disruption of slfDNA, we used Fluorescent Melting Curve Analysis, Laser Scanning Confocal Microscopic Analysis, and Chronoamperometric analysis to systematically investigate (i) the stability of slfDNA (either in solution or on interface) as functions of coded sequences toward various environmental parameters of reaction (i.e., temperature of reaction and salt concentration of buffer), (ii) optimum blocking procedure to reduce fouling and resist nonspecific adsorption of enzymes and DNA backbone, and (iii) the most advantageous architectures of slfDNA-target gene hybrid. The results reveal the feasibility of the proposed screening platform technology as a high-fidelity screening of specific subtype of influenza virus [(e.g., A/duck/Taiwan/DV30-2/2005 (H5N2)]. Digital interpretation reports “positive” upon the co-existence of both H5 gene (gH5) and N2 gene (gN2). The electrochemically outputted signal acquired in the simultaneous presence of both target segments is dynamically (5-fold higher) differentiated from the other three “negative” combinations (i.e., without gH5 and gN2, only with either gH5 or gN2). In particular, the integrated results obtained from the optical and electrochemical approaches exhibit that the interaction between NA gene and slfDNA plays a crucial role in activating the disruption of the structure of the stem region, which could substantially direct to a positive/negative output. In the quantitative regard of sensing performance, the background current remained at an ultralow level (< 5 nA) even with the existence of either H5 or N2 gene only at high concentrations (up to 0.5 μM), inferring a lowest possibility of false positive. The limit of detection (LOD) of this system was calculated as 50 nM (for both H5 and N2 segments). Moreover, the genosensor developed herein maintains accurate and reliable functionality toward analysis of unpurified amplicons generated from the genuine viral genome. In complex matrices, the amperometric signal acquired from positive H5N2 combination could be remarkably discriminated from negative testing scenarios. In summary, to the best of our knowledge, this is the first design and demonstration leveraging individual slfDNA across concurrent analysis of dual target genes, yielding a digital interpretation via the cooperated processing. Pursuant to the elegant characterizations of high applicability, extensible flexibility, and powerful utility, the screening platform technology exploits great promise to be a sophisticated molecular diagnostics against variable influenza viruses.

並列關鍵字

influenza virus subtyping ； electrochemical genosensor ； stem-loop folded DNA ； dual-gene detection ； enzymatic amplification

參考文獻

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