偵測生物及環境樣品之功能性奈米材料及分子適合體

本論文主題為利用蛋白質修飾之量子點(quantum dots, QDs)及DNA修飾之金奈米粒子(gold nanoparticles, Au NPs)等功能性奈米材料和分子適合體(aptamer)來開發高選擇性及靈敏度的酵素、汞(Hg2+)、鉛(Pb2+)及單一核苷酸多型態(single nucleotide polymorphisms, SNPs)之光學感測器。本論文共分成六章。在第一章中，先概述感測器的架構及其發展背景。在第二章中，我們利用蛋白質修飾的QDs藉由螢光共振能量轉移(fluorescence resonance energy transfer, FRET)的原理來開發胰蛋白酶(trypsin, Try)及其抑制劑(trypsin inhibitor, TI)之感測器。將鍵結有rhodamine isothiocyanate (RITC)的牛血清蛋白(bovine serum albumin, BSA)修飾於綠色CdTe QDs表面而產生FRET，當溶液中有Try存在時，由於其對BSA有消化作用，使兩者之轉移效率下降而導致螢光比例(IF574/IF520)改變，藉由此變化，便可成功的偵測Try及TI，且TI的偵測極限可至250 pM。第三章描述控制DNA修飾於Au NPs表面的密度來開發Hg2+之感測器。藉由金-硫鍵結可修飾具有胸腺嘧啶核苷酸(thymidine, T)序列的DNA於Au NPs表面，當表面密度較大時(> 60 DNA/Au NP)，由於T-Hg2+的結合使得DNA由直線型轉變成髮夾狀結構，進而造成立體障礙使得部分DNA脫離Au NPs表面並和溶液中的染劑(OliGreen)結合而放出螢光，此螢光強度隨Hg2+濃度上升而增加，並可得到一線性範圍從0.05-2.5 μM (R2= 0.98)。在第四章中，我們藉由Au NPs聚集會使表面電漿共振(surface plasmon resonance, SPR)位移的特性結合由T序列組成之DNA來開發一可用肉眼觀測的Hg2+感測器。由於T-Hg2+-T的形成造成DNA的構型及電荷密度產生改變，以致於其吸附且保護Au NPs的能力有所不同，因此藉由提高系統中鹽類濃度即可由肉眼區分出Hg2+的存在與否。第五章介紹使用T序列特性開發之螢光SNPs感測器。設計一具有待測DNA辨識端的環狀(loop)及T序列組成的柄狀(stem)結構之molecular beacons (MBs)，當互補DNA與MBs結合後再和嵌合式染劑ethidium bromide (EthBr)結合來偵測。由於完全互補之DNA (perfectly matched, DNApm)和MBs的結合力會較單點突變序列(single-base mismatched, DNAmm)的要強，在某特定Hg2+濃度下，DNApm會存在雙股的型態(較Hg2+之結合力強)，反之DNAmm則呈現無雙股存在(較Hg2+之結合力弱)的髮夾狀立體結構，因此DNApm會得到較強之螢光訊號。於最後一章中，我們使用thrombin-binding aptamer (TBA)結合遮蔽試劑開發雙金屬Hg2+及Pb2+之感測器。由於直線型的TBA和Pb2+及Hg2+結合會分別形成G-quartet及髮夾狀之立體結構，因此修飾於TBA兩末端之染劑會因距離拉近而有FRET產生，再搭配遮蔽試劑phytic acid及random DNA/NaCN的使用即可分別偵測Pb2+及Hg2+至300 pM及5.0 nM。

關鍵字

量子點；金奈米粒子；分子適合體；酵素；汞；鉛；單一核苷酸多型態

並列摘要

This thesis describes highly selective and sensitive optical sensors for enzyme, mercury(II), lead(II), and single nucleotide polymorphisms (SNPs) using functional nanomaterials, including proteins modified quantum dots (QDs) and DNA modified gold nanoparticles (Au NPs), and aptamers. The thesis is divided in six chapters. In the first chapter, the framework and background of sensors were discussed. In chapter two, protein-conjugated QDs were used for detecting trypsin (Try) and trypsin inhibitor (TI) through fluorescence resonance energy transfer (FRET). Green-fluorescent CdTe QDs served as the energy donors and rhodamine isothiocyanate (RITC) conjugated to bovine serum albumin (BSA-RITC) was the acceptor. By simply mixing the two fluorophores, FRET occurred when BSA-RITC bound to the CdTe QDs. When Try was used to digest BSA, the FRET efficiency decreased and the fluorescence intensity ratio (IF574/IF520) decreased, allowing the detection of Try and TI. The LOD for TI was down to 250 pM. The third chapter describes control of the surface DNA density on Au NPs for selective and sensitive detection of mercury(II). When Hg2+ ions interacted with the thymine (T) units of the DNA molecules bound to the Au NPs through Au-S bonds, the conformations of these DNA derivatives changed from linear to hairpin structures, causing the release of some of the DNA molecules from the surface of the Au NPs into the bulk solution to react with OliGreen (high density, > 60 DNA/Au NP). The fluorescence of OliGreen-DNA complexes increased with increasing concentration of Hg2+, and Hg2+ could be detected at concentrations over the range 0.05-2.5 μM (R2= 0.98). In chapter four, the detection of mercury(II) was created by Hg2+-DNA complexes inducing the aggregation of Au NPs, and the sensor was detectable by naked eye as a result of the shift of surface plasmon resonance (SPR) for the aggregated Au NPs. The probe for sensing Hg2+ using the formation of DNA-Hg2+ complexes through T-Hg2+-T coordination to control the negative charge density of the DNA strands—thereby varying their structures—adsorbed onto Au NPs. Therefore, the ability of protection of them in the presence and absence of Hg2+ was different and we can easily distinguish between them in the high concentration of salt. Chapter five describes the fluorescence detection of SNPs using a thymine-based molecule beacon (MB). A T7-MB-T7, which contains a 19-mer loop and a stem comprising a pair of seven T bases, forms double-stranded structures with target DNA molecules, leading to increases in the fluorescence of ethidium bromide (EthBr) as a result of intercalation. The interactions of the beacon with perfectly matched (DNApm) and single-base mismatched (DNAmm) DNA strands were stronger and weaker, respectively, than those with Hg2+ ions. As a result, the fluorescence of a solution containing DNApm was higher than that of a corresponding solution containing DNAmm, because the former had a greater number of intercalation sites for EthBr. In the last chapter, the sensor of two metal ions was created using the thrombin-binding aptamer (TBA), a random coil structure that changed into a G-quartet structure and a hairpin-like structure upon binding Pb2+ and Hg2+ ions, respectively. As a result, the fluorescence decreased through FRET between the fluorophore and quencher labeled on the termini of TBA. These changes in fluorescence intensity allowed the selective detection of Pb2+ and Hg2+ ions at concentrations as low as 300 pM and 5.0 nM using this TBA probe in the presence of phytic acid and a random DNA/NaCN mixture, respectively.

並列關鍵字

quantum dots (QDs) ； gold nanoparticles (Au NPs) ； aptamer ； enzyme ； mercury ； lead ； single nucleotide polymorphisms (SNPs)

參考文獻

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偵測生物及環境樣品之功能性奈米材料及分子適合體

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