摘要：分子信標（molecular beacons）泛指一種具備分子開關（molecular switches）特性之人工合成寡去氧核糖核苷酸（oligodeoxyribonucleotides），這類寡核苷酸會因為和標的物（如核酸或蛋白質）進行錯合配位作用後產生立體結構變化。由於分子信標的一端標記著施體分子（donor molecule, e.g., fluorescent dye）、另一端標記著受體分子（acceptor molecule, e.g., quencher），所以當分子信標和生物體內或生物體外之無標記（label-free）標的物進行專一辨識作用而導致立體結構改變的同時會產生（螢光）共振能量轉移之訊號變化供光學感測器讀取判別，因此分子信標為相當重要的DNA分子檢測工具。最近的研究進展顯示，分子信標的端點標記也能使用氧化還原媒介分子（redox mediators）並作為定點照護電化學生物感測器（point-of-care electrochemical biosensors）之辨識元素。另一方面，分子信標的設計亦具備多元的面貌。藉由適當的核酸序列設計與分子構裝，分子信標不僅可以扮演著高性能DNA分子探針的角色，也可以變成人工抗體或人工酵素。受到上述DNA感測技術進程之啟發，本計畫擬利用三年（2009.08-2012.07）的時間利用分子信標並針對核酸、蛋白質與小分子標的物開發一系列無標記、超靈敏之定點照護生物感測器。計畫執行過程中有三種電化學分子信標會被廣泛地研究與探討 – DNA信標、適體（aptamer）信標及催化式信標（或稱DNA觸媒, DNAzyme）。這三種電化學分子信標分別會被應用於核酸感測器發展（針對病毒與致病菌檢測，第一年）、免疫感測器發展（針對蛋白質生物指標分子檢測，第二年）及生化感測器發展（針對代謝物感測，第三年）。為了增進三種信標分子感測器將來在醫學檢測上的實用性，具有高度生醫指標意義之標的物分子將被選擇為待測物。計畫第一年所發展之核酸感測器將用來偵測高病原性禽流感病毒（H5N1）及肺結核桿菌（M. tuberculosis）之特異性PCR放大片段；計畫第二年所發展之免疫感測器將用來檢測人類凝血蛋白、免疫球蛋白E（IgE）及血小板衍生生長因子；計畫第三年所發展之生化感測器將用來感測血液中之葡萄糖與膽固醇。除上述內容，在分子信標生物感測器的發展過程中，計畫團隊亦會導入材料科學與奈米生技方面之跨領域創新元素。計畫之終極目標在於促進電化學DNA感測器在定點照護生醫檢測之全方位應用同時提供如目前市售血糖儀一樣使用方便、迅速且準確之電子式DNA感測平台技術。
Abstract: Molecular beacons are tailor-made strands of oligodeoxyribonucleotides labeled with a donor molecule at one terminus and an acceptor molecule at the other. They act like molecular switches and feature binding-induced conformational changes. Accordingly, an optical signal change resulted from resonance energy transfer can be detected when a molecular beacon specifically bind to its target. Recently, it has been demonstrated that molecular beacons can also be labeled with redox reporters to serve as promising recognition elements for point-of-care electrochemical biosensors. Moreover, molecular beacons have shown versatile facets. With an appropriate design on the oligonucleotide sequence and introducing some molecular construction, they can not only be a high-performance DNA probe but also play a role of artificial antibody or artificial enzyme. Inspired by the above advances of DNA sensing technology, the present project aims to develop label-free, ultrasensitive point-of-care biosensors for nucleic acids, proteins and small molecules based on molecular beacons in three years (from Aug. 2009 to Jul. 2012). Three types of electrochemical molecular beacons – DNA beacons, aptamer beacons and catalytic beacons (or DNAzymes) will be studied and engineered to develop nucleic acid sensors (for virus and pathogen detection), immunosensors (for protein biomarker detection) and biochemical sensors (for metabolite detection) in the first, second and third years, respectively. To enhance the feasibility of the three types of molecular beacon biosensors in clinical diagnostics, the target molecules of great biomedical meaning are chosen in this project. In the first year, the nucleic acid sensors that detect polymerase chain reaction (PCR) amplicons from avian influenza (H5N1) and Mycobacterium tuberculosis (MTB) will developed. In the second year, the immunosensors that detect human -thrombin, human immunoglobulin E (IgE) and platelet-derived growth factor (PDGF) will be developed. In the third year, the biochemical sensors that detect glucose and cholesterol will be developed. In addition, interdisciplinary innovations from the aspects of material sciences and nanobiotechnology will also be incorporated during the development of molecular beacon biosensors. The ultimate goal of the present project is to facilitate the application of the electrochemical DNA sensing technology to a wide variety of point-of-care diagnostics and to provide a diversity of quick, accurate electronic bioassay platforms as convenient as a glucose meter.
