三股螺旋奈米開關於生物分析平台的開發與應用

Abstract

即時定量聚合酶連鎖反應（Real-time quantitative polymerase chain reaction, qPCR） 為分子生物學核酸檢驗的主流方法，然而此法需搭配高成本的溫度循環設備及多款螢光探針，以實現多重檢測的能力。本論文研究致力開發一新穎的多重表面電漿共振（Surface Plasmon Resonance, SPR）生醫感測平台，藉由使用不同pH感應性之三股螺旋DNA奈米開關（Triplex DNA nanoswitches, TDS）作為訊號輸出的調控鈕。三股螺旋DNA透過Hoogsteen作用力，使得第三股序列插入到特定的雙股核酸之間。此過程透過調節序列中T−A•T和C−G•C+比例，以達成對結構的pH感應性控制。我們透過調整反應環境的pH值，以掌控三股和雙股之間構型的轉換，期望進一步調整SPR的訊號。我們將這項平台技術稱為TDS-SPR感測平台。本論文研究的第一部分致力於運用TDS技術結合金奈米粒子（Gold nanoparticle, AuNPs）形成AuNPs@TDS探針，進行目標核酸片段的捕獲並增強SPR訊號，以開發無需進行擴增（Nucleic Acid Amplification-Free）的核酸檢測平台，並應用於定量具有膀胱癌特異性之微小核糖核酸。藉由調整反應環境pH，我們得以調控TDS的構型，使AuNPs脫離SPR感測金片。在相對應的pH值下，AuNPs釋放所引起的SPR訊號變化量與miRNA濃度呈正相關，成功實現在單一SPR平台上檢測兩個目標分析物。此平台的偵測極限（Limit of detection, LOD），對應miR-183及miR-155，分別為4.35 pM和9.41 pM。此外透過AuNPs釋放之SPR訊號變化量反推濃度的大小，能夠忽略AuNPs在感測金片上的非特異性吸附訊號，進一步提升訊噪比。同時，此平台能夠有效區分膀胱癌患者和健康受試者的尿液樣本。第二部分的研究主題延續TDS-SPR訊號輸出平台的應用，首先設計三維結構（Three-way junction, 3WJ）探針並用來捕捉目標核酸片段，接著結合恆溫核酸指數擴增法（Exoponational amplification reaction, EXPAR）快速放大核酸片段。最後透過使用不同pH感應性之TDS，成功開發出可以同時偵測兩種登革熱病毒核酸之SPR感測平台。藉由探針序列的優化，配合上最佳條件的酵素濃度、反應溫度及反應時間，我們可以實現一鍋化（one-pot）EXPAR的放大反應，簡化實驗步驟並縮短實驗時間。此外，透過TDS-SPR平台，我們成功實現兩種目標核酸的區分，凸顯了該技術在生物醫學檢測領域的應用潛力。

Although real-time quantitative PCR (RT q-PCR) remains the gold standard for nucleic acid detection, it requires repeated temperature cycling and multiple fluorophores for multiplexing. Herein, we introduced triplex DNA nanoswitches (TDS) with distinct pH responsiveness to establish a multiplexed surface plasmon resonance (SPR) biosensing platform (TDS-SPR assay). Triplex DNA, formed by Hoogsteen base pairing, involves the insertion of a third strand into specific double-strand DNA. The pH responsiveness, controlled by regulating the T-A•T and C-G•C+ ratios in the sequence, enables the construction of programmable DNA nanoswitches. In the first project, we integrated TDS with gold nanoparticles (AuNPs) to form AuNPs@TDS probes. These probes efficiently captured bladder cancer-specific microRNAs (miR-183 and miR-155) and enhanced SPR signals. By manipulating the TDS conformational changes with different pH buffers, the controlled release of AuNPs from the sensing surface resulted in proportional SPR signal changes, correlating with the concentration of miRNA. This platform achieved the simultaneous detection of two target analytes on a single SPR platform. The limit of detection (LOD) was calculated to be 4.35 pM and 9.41 pM for miR-183 and miR-155, respectively. Minimizing the non-specific adsorption of AuNPs was achieved by using the responses induced by the release of AuNPs as the signal output. The platform successfully differentiated between bladder cancer patients and healthy controls using urine samples. In the second project, a three-way junction (3WJ) probe was employed for target nucleic acid capture, coupled with an Exponential Amplification Reaction (EXPAR) for rapid nucleic acid amplification at a constant temperature. We optimized the primer sequence, enzyme concentration, reaction temperature, and reaction time to achieve the one-pot amplification reaction. Subsequently, pH-responsive TDS were incorporated, enabling the detection of two different Dengue virus RNA sequences based on SPR signal changes at corresponding pH values. This one-pot reaction streamlined experimental steps and time, demonstrating the potential for efficient and dual-target nucleic acid detection. This platform technology, using TDS-assisted SPR systems, presents a user-friendly and promising approach for diverse biomedical applications.