以功能性金奈米粒子結合漸逝波腔體震盪吸收光譜法觀察DNA間之作用力

Abstract

本研究是以金奈米粒子作為標籤，結合漸逝波腔體震盪吸收光譜法(evanescent-wave cavity ring-down absorption spectroscopy, EW-CRDS)來探測脫氧核糖核酸(deoxyribonucleic acid, DNA)的之間之作用，藉此計算出平衡常數(Ka)及自由能(ΔG)，並將此系統應用在偵測特定的核酸序列。 漸逝波腔體震盪吸收光譜法是利用兩片高反射率的鏡子形成一腔體(cavity)，使脈衝雷射在腔體中來回震盪，採集每次震盪露出的少部分訊號即得光強度衰減曲線。置入吸收物種於腔體內將縮短震盪時間，因此我們在腔體中置入一經特殊角度設計的稜鏡，使光在其內部表面產生全反射，並在反射點產生漸逝波(evanescent wave)。漸逝波腔體震盪光譜法即利用樣品吸收此漸逝波進而觀測樣品接觸表面的吸附等變化情形。由於測得的訊號基於光強度的遞減速度，並不受到雷射光脈衝的不穩定性所影響，而雷射光在腔體中來回震盪使其有效吸收路徑增加，因此相較於傳統吸收光譜法，EW-CRDS有較高的靈敏度，亦能做精確的測量。 實驗方法為將產生漸逝波的玻璃表面修飾上特定的核酸序列，另一互補序列則修飾上可吸收使用之雷射波長的金奈米粒子，利用核酸之間的氫鍵結合能力觀測。最後，本研究利用上述方式設計一個偵測目標DNA分子的三明治法(sandwich binding assay)，同樣利用金奈米粒子為標籤，藉由腔體震盪光譜法的高靈敏度作微量的偵測。

Evanescent wave cavity ring-down absorption spectroscopy (EW-CRDS) is employed to study the interaction between deoxyribonucleic acids (DNA) by functionalized gold nanoparticles (Au NPs). EW-CRDS is a surface sensitive technique based on the measurement of the decay rate of a pulsed laser light trapped in an optical cavity. The light undergoes total internal reflection (TIR) at an interface of a prism within the cavity and creates an evanescent field at the surface that is sensitive to small absorption changes and is particularly useful for investigating interfacial processes. EW-CRDS offers a significantly higher sensitivity than conventional absorption spectroscopy with a rather simple and straightforward experimental set-up. The high sensitivity results mainly from its independence of fluctuations of the light source and the extremely long effective path length realized in optical cavities. By applying this ultra-sensitive EW-CRDS to the observation of DNA, we were able to study the binding kinetics of DNA and obtain the association equilibrium constants (Ka) and the free energies (ΔG). Binding conditions such as changes in the salt concentration, buffer pH and temperature are systematically examined. This basic study gives further insight in the design of DNA detection for DNA mutation diseases.