Oligonucleotides and nanomaterials for the analysis of biological and environmental samples

本論文主要目的為發展具有高靈敏度與選擇性汞離子之寡聚核苷酸分子(oligonucleotide)感測器；藉由電噴灑離子化質譜法(electrospray ionization mass spectrometry，ESI-MS)觀測寡聚核苷酸分子與配合基(ligand)於所形成之複合體(complex)中，兩者鍵結之比例；開發結合內標準品與表面輔助雷射脫附游離質譜法(surface-assisted laser desorption/ionization mass spectrometry，SALDI-MS)準確定量胺基酸硫醇(aminothiol)；利用汞-碲(mercury-tellurium，HgTe)奈米材料為SALDI-MS之無機基質，應用於胜肽(peptide)、蛋白質(protein)與蛋白質-藥物之複合體(protein-drug complexes)分析。本論文主要分成五個部份。第一章主要簡介核酸感測器(nucleic acid-based sensor)之架構與背景、質譜儀的基本原理以及現今兩種軟性游離法於質譜學之應用。在第二章中，主要描述藉由染料分子TOTO-3和由33個胸腺嘧啶組成之寡聚核苷酸分子T33所構成之螢光感測器，以測量水溶液中汞離子之含量。由於TOTO-3在無規捲曲(randomly coiled)之T33分子存在下，其螢光非常微弱；但在添加汞離子時，T33會因為序列中胸腺嘧啶(thymine)殘基與汞離子形成之胸腺嘧啶–汞離子–胸腺嘧啶配位對(thymine–Hg2+–thymine coordination pair)，T33則由原先無規捲曲之結構形成一固定構形(folded structure)，此時TOTO-3更能鑲嵌於固定構形之T33中而使TOTO-3螢光增強。在最佳化條件應用此種感測器時，相較其他金屬離子對於汞離子有極佳之選擇性與專一性，並可達到0.6 ppb之偵測極限。第三章節主要描寫利用ESI-MS、螢光與旋光分光儀(circular dichroism spectroscopy，CD)觀察溴化乙錠(ethidium bromide，EthBr)、聚胸腺嘧啶(poly-thymine，polyT)在汞離子存在下鍵結之情形。ESI-MS之結果顯露相較於其他金屬離子，汞離子可藉由thymine–Hg2+–thymine coordination與polyT於氣相中形成較強之親合力。此外，ESI-MS亦發現T33–EthBr–Hg2+ 複合體之形成，會因溶液中汞離子增加而更易存在；而此結果也與利用螢光與CD光譜法量測液相之情況相符。第四章主要介紹利用修飾N-2-mercaptopropionylglycine (MPG)之金奈米粒子，藉由此種奈米粒子作為SALDI-MS之內標準品後，應用於胺基酸硫醇之分析。此法主要先利用依未經修飾之金奈米粒子抓取分析物後，再混合吸附MPG之金奈米粒子直接進行SALDI-MS之分析。此分析方式對於三種胺基酸硫醇分子有良好之線性範圍(R2 = ca. 0.99)與再現性(相對標準誤差: <10%)。除此之外，此法也可以準確定量人類紅血球細胞中穀胱甘肽(glutathione，GSH)濃度，以及觀測添加抗發炎藥物sulfasalazine於MCF-7癌症細胞時GSH濃度之變化。最後一章介紹選擇利用HgTe奈米材料為SALDI-MS之無機基質，應用於胜肽、蛋白質與蛋白質-藥物之複合體分析。相較於其他奈米材料如金奈米粒子時，HgTe奈米材料有較低背景訊號干擾、較少分析物斷裂片段離子產生、alkali adducted analyte ions所形成之干擾較低以及可達150000 Da之質量分析上限等優點，此法對於分析血管收縮素I (angiotensin I)與牛血清蛋白(bovine serum albumin)可達200 pM與22 nM之偵測極限，並保有良好之實驗再現性(相對標準誤差 <25%)。此分析方法也已成功證明可以快速鑑定由大腸桿菌中轉化之重組蛋白質與觀測蛋白質-藥物之複合體之形成。這些實驗結果可以清楚顯現吾人可利用上述DNA分子與奈米材料，作為日後發展高專一性與高選擇性分析技術之研究素材。This thesis focuses on developments of highly sensitivity and selectivity oligonucleotides-based sensor for mercury detection, monitoring the binding stoichiometries of oligonucleotides/ligand noncovalent complexes by electrospray ionization mass spectrometry (ESI-MS), developing a practical method for the determination aminothiols through surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS) in conjunction with an internal standard, and using HgTe nanomaterials as matrices in the SALDI-MS analyses of peptides, protein-drug complexes, and proteins. The thesis is divided into five parts. Chapter one introduces the framework and background of nucleic acid-based sensors and basic principle and two most commonly used soft ionization techniques of mass spectrometry. In chapter two, fluorescence detection of Hg2+ in aqueous solution was demonstrated using a sensor composed of TOTO-3 and a polythymine oligonucleotide T33. The formation of folded structure of T33 induced by specific Hg2+-mediated thymine–Hg–thymine pairs between T-residues in the T33 sequence, allowing strong interaction of TOTO-3 with folded T33, leading to increased fluorescence. Under the optimum conditions, this TOTO-3/T33 sensor provided high sensitivity and specificity for Hg2+ over other metal ions in aqueous solutions with a limit of detection of 0.6 ppb. The third chapter describes the performance of ESI-MS, fluorescence and circular dichroism (CD) spectroscopies to explore the binding of ethidium bromide (EthBr) to non-self-complementary polyT strands in the absence and presence of Hg2+ ions. ESI-MS results revealed that Hg2+ ions have greater affinity, through T–Hg2+–T coordination, toward polyT strands than do other metal ions. These findings are consistent with fluorescence and CD results obtained in solution; they revealed that the T33–EthBr–Hg2+ complexes become more stable upon increasing the concentrations of Hg2+ ions. Chapter four describes an internal standard approach using d N-2-mercaptopropionylglycine (MPG) bound 4-nm-diameter gold nanoparticles (Au NPs) as an internal to improve the precision of SALDI-MS. This approach provided good quantitative linearity of the three aminothiols (R2 = ca. 0.99), with good reproducibility (relative standard deviations: <10%). The practicality of the SALDI-MS approach has been validated by the analyses of glutathione (GSH) in the lysates of human red blood cells and MCF-7 cancer breast cells in the presence and absence of the anti-inflammatory drug sulfasalazine. In the last chapter, mercury-tellurium (HgTe) nanomaterials were chosen as SALDI-MS matrices in the analyses of peptides, proteins, and protein-drug complexes. Relative to other commonly used nanomaterials like gold nanoparticles, HgTe nanostructures provide low background from metal clusters, fewer fragment ions, less interference from alkali adducted analyte ions, and higher mass range (150 000 Da). The present approach provides limits of detection (LODs) of 200 pM and 22 nM for angiotensin I and bovine serum albumin, respectively, with great reproducibility (RSD