摘要：（一） 計畫中文摘要。（五百字以內）
即時定量聚合酶鏈鎖反應(qPCR)技術已被證明是一種有效的病原標的診斷工具，可針對流感或人畜共通疾病例如非典型肺炎(SARS)，禽流感(H5N1)和新流感(H1N1)等病毒進行檢測與確認。為了確實預防與控制新興流行性疾病，可針對病原核酸進行快速且精確檢測的可攜式qPCR裝置需求日益增加，最近已有相關基於螢光SYBR Green或TaqMan 探針的光學qPCR微小化裝置相繼問世。然而將微小化的光學設備、加熱/冷卻循環系統與供應電源和PID控制元件組裝成微小、可攜式的感測設備是一項極具挑戰性的任務，並且會降低qPCR的偵測靈敏度。相形之下，電化學技術非常適合於發展定點照護qPCR感測器，並且具同時擁有小尺寸與高靈敏度的可能性。自從可攜式血糖儀投入醫療市場後，電化學研究工作者在近十年來已經針對DNA發展感測平台，同時就在近幾年電化學real-time PCR技術也被陸續報導。然而，目前電化學qPCR生物感測器仍屬於萌芽階段，需要有更多的科學家與技術投入以達成此一目標。有鑑於此，本團隊提出為期一年的「電化學即時監控生物體外核酸擴增反應：邁向電子式qPCR感測器」計畫，其具體目標如下：(1) 開發一種高敏度，高準確性且兼容PCR與其它恆溫DNA擴增方法的無標記電化學DNA感測器；(2) 使用一種三維網狀導電奈米碳管結構和氧化還原聚合物的混成電極修飾層以增強電化學DNA感測之靈敏性；(3) 最佳化探針及間隔分子設計，以求大幅度提升雜交效率，進而達到專一性的電化學DNA感測；(4) 評估以電化學方式進行即時定量感測體外擴增流感病原核酸標的之可行性。為了達到上述目標，相對應的研究方法概述於下：(1) 將寡核苷酸(oligo)固定在金線電極進行電化學DNA感測，而DNA鑲嵌氧化還原媒介分子(如: 亞甲基藍)和氧化還原DMA分子信標皆會被用於此感測系統的研究；雜交程序所誘導的訊號變化則採用安培法和交流循環伏安法進行比較，並且重複確認PCR 加熱循/冷卻環偵測訊號的再現性和可靠性。(2) 一種能提高電子傳輸和收集效率的混成薄膜將被用於修飾金電極表面。此混成薄膜是由三維網狀導電奈米碳管結構與矩陣式聚(3,4-二氧乙基塞吩)和間隔連結的氧化還原媒介分子(如:二茂鐵，奎寧和甲苯胺藍)進行鑲嵌，以此提供一種物理和化學渠道使雜交所誘導的電子快速傳輸至電極表面。(3) 以三明治檢測模型進行探針設計，使用兩種寡核苷酸探針(類似上游和下游引子)和標的物進行雜交來增強檢測效率。並比較oligo(dT)、oligo(dA)和碳分子間隔對間隔條件進行優化。且用微陣列技術對嵌入式DNA檢測的探針與間隔進行設計，並以圓二色光譜進行DNA信標設計。(4)本檢測系統使用專一性的H1N1 病毒DNA序列，其目標序列將藉由基因重組方式轉殖至大腸桿菌內，以架構具有非目標DNA背景干擾之模擬病原，並進行分子生物層次之電化學qPCR感測；除了PCR外，恆溫DNA鏈置換擴增技術也將被應用於電化學DNA感測評估。綜言之，為實現電化學qPCR生物感測器，本計畫將發展許多新技術，因此我們預期能藉由此計畫的研究成果發表極具影響力的期刊論文、並創造有價值的發明專利和技術轉讓，為台灣大學前瞻創性研究注入新活力。
Abstract: I. Abstract (maximum 500 words)
Quantitative real-time polymerase chain reaction technique (qPCR) has been proven as an effective laboratory tool for identification of the pathogen targets that cause emerging flu pandemics or zoonoses, such as SARS, H5N1, and H1N1 viruses. For better pandemic control and prevention, the need of a portable qPCR device for on-site rapid and accurate surveillance of pathogenic nucleic acid targets is growing and drives the recent miniaturization of an optical qPCR instrument based on fluorescent SYBR Green or TaqMan probes. But miniaturization of optics, a thermocycler device, and related power and PID control elements into a compact hand-held device is highly challenging and tends to reduce qPCR’s fluorescence sensitivity. In contrast, electrochemical technique is well-suited for developing a point-of-care qPCR device that features both small size and high detection sensitivity. After bringing glucose meters into the health care market, electrochemists have developed various DNA detection platforms in the past decade, and real-time PCR with concurrent electrochemical detection has been initiated very recently. Yet, an electrochemical qPCR biosensor is still a frontier to explore and requires many scientific and technological efforts to achieve the goal. Accordingly, a one-year project entitled Electrochemical Real-time Monitoring of In Vitro Amplification of Nucleic Acids: Toward Electronic qPCR Biosensors is proposed here. The specific aims of this project are
1. Developing a label-free, electrochemical DNA sensing approach that is ultrasensitive, accurate and compatible with PCR and other isothermal DNA amplification method.
2. Using a hybrid electrode modified layer composed of a 3D conductive nano-network and a redox polymer matrix to enhance the sensitivity of the electrochemical DNA sensing approach.
3. Optimizing the probe design and spacer condition to maximize the hybridization efficiency and thus the specificity of the electrochemical DNA sensing approach.
4. Assessing the electrochemical DNA sensing approach for real-time, quantitative monitoring of in vitro amplification of an influenza nucleic acid target.
To achieve the specific aims, the corresponding methods are summarized below.
1. A fine gold wire will be used as the electrode to anchor the oligonucleotide probe to carry out the electrochemical DNA sensing. Both the DNA-intercalating redox mediator (e.g., methylene blue) and redox DNA beacon will be investigated for the reporter system. Amperometry and alternative cyclic voltammetry will be used and compared for collecting the hybridization-induced signal. The signal reproducibility and reliability with successive PCR thermal cycles will be checked.
2. A hybrid thin film that improves the electron-transport and electron-collecting efficiency will be used to modify the gold wire surface. The hybrid thin film will be composed of a 3D conductive MWCNT network and a poly(3,4-ethylenedioxythiophene) matrix grafted with spacer-linked redox mediators (such as ferrocene, quinine and TBO), so it provides both physical and chemical channels for “ fast transporting” the hybridization-induced electrons to the electrode surface.
3. For probe design, a sandwich detection format using two oligo probes (resembling upstream and downstream primers) to hybridize one target will be adopted to increase the detection specificity. For spacer optimization, oligo(dT), oligo(dA) and carbon spacers will be compared. Microarrays will be used to examine the probe and spacer design for intercalating-based DNA detection. Circular dichroism will be used to examine the design for beacon-based DNA detection.
4. H1N1-specific sequence in DNA format will be used to assess the applicability of this work, and the target sequence will be cloned to an E. coli cell for simulating the electrochemical qPCR detection of a nucleic acid sequence embedded inside the virus shell and mixed with non-target genomic DNA. In addition to Taq PCR, the strand displacement amplification – an isothermal DNA amplification method will be tested with the electrochemical DNA sensing as well.
To sum up, the project will develop several new technologies that are indispensible for realizing an electronic qPCR biosensor. The anticipated outcomes will be able to turn into high-impact journal papers, valuable patents and technology transfers and will bring in creative ideas for further frontier and innovative research for NTU. The details of the project will be disclosed in the formal proposal.
