核酸適合體金奈米粒子於生物感測及酵素抑制之應用

Abstract

本篇論文主要利用核酸適合體金奈米粒子(Apt-Au NPs)開發出高選擇性、高靈敏度的生化光學感測器，並探討其於生物分子之感測及酵素活性之抑制等生物分析範疇之應用。首先，結合標的物誘導核酸適合體產生構型改變及金奈米粒子在鹽類環境造成聚集現象的特性，本研究使用Apt-Au NPs為基礎的比色裝置來進行腺苷與其磷酸化衍生物(三磷酸腺苷)之感測，此裝置的靈敏度與選擇性，可藉由調控鹽類濃度、變化金奈米表面結合核酸適合體之密度及添加其他物質等方式來改善。此外，本實驗亦成功地結合腺苷接合核酸適合體(adenosine-binding aptamer, AptAdo)、血小板衍生性生長因子接合核酸適合體(platelet-derived growth factor (PDGF)-binding aptamer, AptPDGF)、金奈米粒子及DNA鍵結染料Oligreen (OG)開發出一種免標記(label-free)的螢光分析感測器，並將其應用於尿液腺苷之偵測。在感測混合液中，AptAdo及AptPDGF分別用於辨識分析物及放大偵測訊號，而由實驗結果得知，使用AptAdo.來增強標的物之濃度，可以得到更佳的偵測極限。最後，為測量Apt-Au NPs對酵素活性的抑制效果，本研究選用了兩種核酸適合體－RT1t49 (Aptpol) 及 ODN 93 (AptRH)－它們分別與人類免疫缺乏病毒反轉錄酶(Human immunodeficiency virus type 1 reverse transcriptase, HIV-1 RT)上的聚合酶端(polymerase site, pol) 及核糖核酸酶端(RNase H site, RH) 作用。實驗結果顯示，聚合酶核酸適合體金奈米粒子(Aptpol-Au NPs)及核糖核酸酶端核酸適合體金奈米粒子(AptRH-Au NPs)對於人類免疫缺乏病毒反轉錄酶之聚合現象有很好的抑制效果，且40Aptpol-Au NPs(表示一個金奈米粒籽表面修飾40 條Aptpol)在濃蛋白基質(100 μM BSA)中仍有很好的專一性，且具備不受核酸酶作用之穩定度。此外，本研究亦發現同時添加兩種不同核酸適合體，因其彼此間產生增強作用(Synergy)，可觀察到抑制效果的提升，相關研究具有抑制HIV病毒藥物開發之潛力。綜合上述研究結果，Apt-Au NPs確實為在偵側、診斷及疾病治療等方面極具應用價值之生物分析工具。

In this thesis, a highly selective and sensitive optical biosensor using aptamer-modified gold nanoparticles (Apt-Au NPs) was developed for bio-analytical applications, including the determination of biomolecules and the inhibition of enzyme activity. Combinating with the properties of target-induced conformational changes of aptamers and the salt-induced aggregation of Au NPs, a colorimetric approach using Apt-Au NPs had been designed for the determination of adenosine and its phosphorylated derivatives. The sensitivity of this sensing assay could be improved by evaluating the effects of several parameters, such as the ionic strength, the aptamer density on the Au NPs, and the presence of additives. In addition, I also developed a label-free fluorescence analytical sensor—comprising an adenosine-binding aptamer (AptAdo), platelet-derived growth factor (PDGF)-binding aptamer (AptPDGF), Au NPs, and the DNA-binding dye Oligreen (OG)—for the determination of adenosine. In the sensing mixture, AptAdo and AptPDGF are for the recognition of adenosine and for the amplification of fluorescence signal, respectively. The LOD for adenosine could been further lowered when using AptAdo-Au NPs for the enrichment of adenosine. To demonstrate the inhibition of enzymatic activity by Apt-Au NPs, I selected two aptamers, RT1t49 (Aptpol) and ODN 93 (AptRH), that binds to polymerase site (pol) and RNase H site (RH) of Human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT), respectively. With the advantages of specific, increased inhibition, AptRH-Au NPs and Aptpol-Au NPs were considered as the highly effective inhibitors for HIV-1 RT polymerization. The stability and specificity of the 40Aptpol-Au NPs remain almost the same in the presence of DNase I and BSA, respectively. Moreover, I found that the combination of 40AptRH-Au NPs with Aptpol led to the synergistic inhibition, which hold great potential to be an effective and alternative drug for HIV. Apt-Au NPs indeed could be applied to the bio-analytical tools for the detection, diagnosis and therapeutic of diseases.