結合螢光修飾之DNA金奈米粒子及等溫放大法進行核酸偵測

Abstract

無論在臨床病源檢測或實驗室分析上，核酸的定量分析是相當常見且重要的技術，目前在核酸的偵測上最常使用到的訊號放大型式為聚合酶鏈鎖反應 (Polymerase Chain Reaction , PCR)。PCR放大技術的發展已相當地純熟，其靈敏度亦高；然而該方法需要反覆升降溫，其中造成的不便伴隨應用上的限制。因此，等溫放大法開始受到重視，過去的文獻報導過許多範例是利用等溫放大法結合不同的訊號偵測法以達到核酸偵檢的目的。其中，Exponential Amplification Reaction (EXPAR) 方法能在短時間內將短片段核酸分子放大至106~109倍，具有較其他等溫放大法更為省時、有效率的優點。奈米材料因具有諸多特殊性質使之備受矚目，金奈米粒子在生物醫學上的應用與發展在過去的十年間受到國際間相當的注目。金奈米粒子具有特殊的光學活性，表面電漿共振吸收則為奈米金所專屬的特性。與巨觀相的金所呈現亮黃色澤不同；當金的顆粒大小縮小至奈米尺度時 (大約小於可見光波長)，會產生強烈的吸收光特性，而金奈米粒子表面的自由電子雲會與520 nm波長的光產生電漿共振。在吸收這些光的能量後，金奈米粒子的自由電子雲會因而被極化，並隨著光波的頻率震盪。除此之外，金奈米粒子有能力透過能量或電子轉移導致螢光淬滅 (quenching fluorescence)。因此，本篇研究以具有螢光修飾之DNA金奈米粒子為材料，結合EXPAR及金奈米粒子的淬滅螢光特性，希望發展一較為簡單、快速的核酸偵測法。本研究成果證實了在金奈米粒子上的核酸模板能夠進行EXPAR的等溫放大反應，然而卻也發現到目標序列是影響靈敏度之重要因素，在經過實驗設計的改良後，此偵測方法可區分10 fM至100 nM的trigger濃度，其偵測極限約為1.5 pM。本研究發展出一通用的放大兼訊號偵測平台，未來期望可以透過一些目標物的轉換方法達到分析不同目標分析物的目的以拓展其應用性。

The detection and quantification of nucleic acid sequences for identification of pathogens or genetic markers is one of the fastest growing areas in clinical chemistry. In most cases, amplification is required before analysis of nucleic acid. Polymerase chain reaction (PCR) is known as the most commonly used and prominent amplification strategy. Despite the fact that PCR-based analytical methods offer advantages of high sensitivity, better precision, and rapidity, they often suffer from the limitation of high cost and the need for thermocycling. Isothermal nucleic-acid amplification strategy, on the other hand, permits less complex and cost-effective instrumentation set-up, leading to the fabrication of advantageous, low cost, promising point-of-care diagnostic that may further be employed in biomedical or clinical applications. In comparison with most of the isothermal amplification methods, which require long reaction time (from 30 min to several hours), exponential amplification reaction (EXPAR) is capable of rapidly amplifying short oligonucleotides up to 106 ~109-fold within minutes. In addition, EXPAR has been used in various fluorescence-based analytical methods. Gold nanoparticles (GNPs) possess unique optical and electrical properties, such as high absorption coefficient, scattering flux, luminescence, and conductivity. They also have ability to enhance electromagnetic fields, quench (or enhance) fluorescence, or catalyze reactions, providing opportunities to exploit these particles for sensing purposes. Compared to conventional fluorescence quenchers, GNPs is an excellent universal fluorescence quencher that have been used in various biomedical applications. In this study, a novel strategy for oligonucleotide detection using flourophore-modified DNA-functionalized AuNPs was designed and developed. It combined the fluorescence quenching property of gold nanoparticles and the rapid amplification of EXPAR. The proposed strategy can distinguish different concentrations of nucleic acid trigger, ranging from 10 fM to 100 nM. Moreover, in combination with other displacement methods, it can be applied in the detection of different analytes.