利用可重複使用之矽奈米線場效應電晶體偵測小分子核醣核酸

Detection of MicroRNA with a Reusable Silicon Nanowire Field-Effect Transistor

曾令瑋(Ling-Wei Tseng)

指導教授 ： 陳逸聰

國立臺灣大學/理學院/化學研究所/碩士(2012年)

https://doi.org/10.6342/NTU.2012.02332

摘要

小分子核糖核酸（microRNA）為非蛋白質編碼且含有22個核苷酸的核糖核酸分子，其對於目標RNA可進行分解或轉譯抑制，因此在動、植物中扮演重要的調控角色。研究指出，當植物受病毒入侵後，已存在於植物體中的miRNA能作為提前對抗感染的防禦機制，成熟miRNA所形成的RNA誘導沉默複合體（RNA-induced silencing complex, RISC）會將互補的目標病毒mRNA切斷，使病毒無法轉譯成蛋白質，進而達成防禦的目的。由於病毒感染植物後，特定miRNA的量會急遽增加，因此本論文中將藉由靈敏之矽奈米線場效電晶體（silicon nanowire field-effect transistor, SiNW-FET）來偵測特定miRNA，以決定植物感染與否。此外在哺乳動物方面，近期研究發現miRNA的突變或過量表現與人類癌症有關，若抑制腫瘤之miRNA的生物合成步驟中有缺陷，或是會致癌的miRNA於不當時間或組織中過度增殖，皆會造成腫瘤的產生。因此，特定miRNA在癌症細胞及正常細胞中的數量會有所差異，且能作為生物標誌物。在本論文中，我們將使用SiNW-FET偵測miRNA生物標誌物，以作為一種早期診斷的方法。SiNW-FET由於擁有極佳的靈敏度、高度選擇性、不需標記、以及即時偵測等優點，可廣泛的應用在生物及化學物種的偵測，包含蛋白質、核酸、小分子、和病毒等。首先，我們利用化學鍵結方法將DNA探針以雙硫鍵修飾於SiNW-FET表面，而後可藉由DNA- RNA雜交來偵測特定的目標miRNA。當檢測結束後，以二硫蘇糖醇（dithiothreitol, DTT）將雙硫鍵切斷，便可再次進行偵測，而達成重複使用SiNW-FET元件的目的。檢測將分為三部分，首先測單一片段的合成RNA，結果顯示SiNW-FET可以成功得藉由DNA作為檢測RNA的探針。接著，則自植物之總RNA中，偵測與植物葉片生長有關的miR159之特定RNA ，並以人類的總RNA作為控制組，實驗結果說明了SiNW-FET有良好的選擇性，能由不同片段的RNA中偵測到目標序列，並且證明SiNW-FET擁有重複使用的能力，再現性佳。最後，我們選擇乳癌生物標誌物之一的miR21，作為目標來比較癌細胞和正常細胞。在高濃度下，由於目標物在SiNW-FET表面達到飽和，因而難以區別癌細胞與正常細胞，但當濃度逐漸降低後，即可觀察出miR21在癌細胞中的量，明顯比正常細胞來的多。此說明SiNW-FET的高靈敏度，在低濃度下偵測能力不減，且能顯著分辨致病與健康的樣品。由以上實驗得知SiNW-FET不但提供了很好的平台來偵測miRNA的生物標記，並可作為診斷植物致病與癌症樣品的有效工具。

關鍵字

矽奈米線場效電晶體 ； 生物感測器 ； 小分子核糖核酸 ； 癌症生物標誌物

並列摘要

MicroRNAs (miRNA) of around 22 nucleotides and long non-protein-coding RNAs can play an important regulatory role in animals and plants by targeting mRNA for cleavage or translational repression. Recent research finding shows that miRNAs, which had already existed in a cell before viruses invaded, help to serve as an advanced preparation to counteract infection. Viral mRNA might be regulated by the cleavage directed by miRNA-programmed RNA-induced silencing complex (RISC). Since plant virus infections resulted in a dramatic increase in miRNA, we detected specific miRNA with a sensitive silicon nanowire field-effect transistor (SiNW-FET) biosensor to confirm if the plant was infected. In addition, recent studies have evidenced that miRNA mutation or mis-expression is responsible for various human cancers. The reduction or deletion of some significant miRNAs, acting as tumor suppressors, might lead to the tumor formation during miRNA biogenesis. The amplification or overexpression of the specific miRNA at inappropriate time or in wrong tissues would also result in tumor formation. Therefore, different amounts of specific miRNA from cancer cells and normal cells could make miRNA act as a biomarker for cancer diagnosis. In this study, SiNW-FETs were utilized to detect a specific miRNA biomarker for early cancer diagnosis. Based on its high sensitivity and selectivity, real-time response, and label-free detection capabilities, SiNW-FET has been widely used in a variety of detections, including protein, nucleic acid, small molecules, and virus. Here, we immobilized DNA probes on the SiNW-FET surface via a disulfide linker, which was then applied to detect target RNAs through DNA-RNA hybridization. After detection, dithiothreitol (DTT) was employed to cleave the disulfide linker, concurrently removing the DNA-RNA complex and allowing for the SiNW-FET device reusable. Our experiments show that a DNA-modified SiNW-FET is able to probe target miRNAs from a cocktail solution, such as the detection of miR159 in total RNA extracted from leaves. Moreover, the reusable SiNW-FET has made it possible to fast screen various target RNAs by easily replacing different DNA probes. Finally, miR21, a biomarker of breast cancer, was selected as the target sequence to diagnose cancer and normal cells. However, it was difficult to distinguish cancer cells from normal ones at high sample concentrations, because of the binding saturation on the SiNW-FET sensing device. Fortunately, distinction became apparent as the concentration decreased, where the amount of miR21 from cancer cells was measured to be more than that from normal cells. As a conclusion, a SiNW-FET modified with specific DNA probes not only provides an excellent platform for miRNA biomarker detections, but also exhibits the potential in biomedical applications for early cancer diagnosis.

並列關鍵字

silicon nanowire field-effect transistor ； biosensor ； microRNA ； cancer biomarker

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