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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 張煥宗 | |
dc.contributor.author | Li-Yi Chen | en |
dc.contributor.author | 陳莉沂 | zh_TW |
dc.date.accessioned | 2021-06-16T03:51:24Z | - |
dc.date.available | 2017-03-13 | |
dc.date.copyright | 2015-03-13 | |
dc.date.issued | 2014 | |
dc.date.submitted | 2015-01-17 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/55209 | - |
dc.description.abstract | 本論文著重於結合發光金奈米點與微膠複合材料以建立高靈敏度與選擇性的光學探針,並將其應用於生物樣品中常見的蛋白質與重金屬離子之檢測。另一方面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),將此功能性微膠奈米材料(胰蛋白酶/金奈米粒子/微膠) 進一步應用在活體細胞樣品中進行蛋白質檢測。本論文共分成四個章節。第一章節簡述螢光金奈米材料與微膠發展及應用現況與軟性游離技術於質譜學之應用。第二章節,主要利用表面修飾11-巰基十一酸(11-mercaptoundecanoic acid, 11-MUA)的金奈米點(gold nanodots, Au NDs),簡稱11-巰基十一酸‒金奈米點(11-MUA–Au NDs),透過光激發放光(photoluminescence)產生消光(quenching)現象進行血紅蛋白偵測。此機制來自於11-巰基十一酸‒金奈米點與高鐵血紅素(hemin)中的鐵二價離子產生氧化還原反應,同時利用高自旋態鐵三價離子之訊號變化作為輔助證明。結果顯示,實驗組含有牛血清蛋白的11-巰基十一酸‒金奈米點,可提高在生理緩衝溶液中對於血紅蛋白的偵測靈敏度達0.5 nM並展現良好的選擇性。在第三章節,製備聚N-異丙基丙烯醯[poly (N-isopropylacrylamide), PNIPAM]微膠結合發光金奈米點,並應用於汞離子偵測。汞離子所產生的光激發放光消光現象可來自於汞合金(amalgam)之形成與金奈米點–微膠粒子之聚集。同時,利用此技術可在含有500 mM氯化鈉(NaCl)的溶液中偵測汞離子,其偵測極限(limits of detection)為1.9 nM。此一具有選擇性與靈敏度之探針也可應用至代表性魚類樣品中的汞離子偵測。在第四章節,利用溫度敏感性微膠包覆胰蛋白酶與金奈米粒子針對人類子宮頸癌(HeLa)細胞裂解液中細胞質之細胞色素c的含量進行探討,該組合可在微波能量照射下,加速對細胞色素c的消化作用以達到精確定量的目的,則利用金奈米粒子作為基質的SALDI-MS並結合GR-10胜肽作為內標準品,初步實驗結果,可在樣品與內標準品的質譜訊號比值中得到良好的線性關係(R2 = 0.98),且對細胞色素c的偵測極限可達10 nM。 | zh_TW |
dc.description.abstract | This dissertation focuses on developments of high sensitivity and selectivity optical probes for the detection of proteins and heavy metal ions in biological sample, including photoluminescent gold nanodots and hybrid nanomaterials with microgels. The functional microgel nanomaterials (trypsin/gold nanoparticle/microgels) is further applied to detect proteins through surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS) in conjugation with an internal standard. The dissertation was structured with four chapters. In Chapter 1, the detailed background of photoluminecent gold nanomaterials and microgels, and basic principle of soft ionization techniques of mass spectrometry were widely described. In Chapter 2, the developed approach, using photoluminescent 11-mercaptoundecanoic acid–gold nanodots (11-MUA–Au NDs), was applied for the detection of hemoglobin through photoluminescence (PL) quenching. The mechanism of quenching, which occurred through redox reactions between the 11-MUA–Au NDs and the Fe(II) atoms of hemin units, was supported by an increase in the signals (G 2.0 and 5.9) of high-spin state Fe(III) ions. This approach using bovine serum albumin blocked 11-MUA–Au NDs provided good sensitivity for hemoglobin of 0.5 nM in biological buffer and with great selectivity over other non-heme–containing proteins. Furthermore, we also validated the practicality of this approach through the determination of the concentrations of hemoglobin in diluted human blood samples. In Chapter 3, poly(N-isopropylacrylamide) microgels (PNIPAM MGs) incorporated with photoluminescent Au NDs had been prepared and used for the detection of mercury ions (Hg2+). Based on Hg2+-induced photoluminescence quenching due to the formation of Au–Hg amalgam and formation of Au NDs–PNIPAM MGs aggregates, this approach provided limits of detection for Hg2+ of 1.9 nM in biological buffer solutions containing 500 mM NaCl. This selective and sensitive Au NDs–PNIPAM MG probe has been applied to the determination of the concentration of Hg2+ in a representative fish sample. In Chapter 4, temperature sensitive MGs containing trypsin and gold nanoparticles have been employed for the digestion of cytochrome c (Cyt c) under microwave irradiation. For further quantitation of Cyt c in cell lysates, SALDI-MS was applied for detection by using gold nanoparticles as the matrix and GR-10 peptide as an internal standard. The internal standard SALDI-MS approach provided linearity (R2 = 0.98) of MS signal ratio (I1168.6/I1067.6) of the tryptic digested peptide (m/z 1168.6) to GR-10 peptide (m/z 1067.6), with a limit of detection of 10 nM. This approach has been validated by the analysis of the concentration of cytoplasmic Cyt c the lysates of HeLa cells. | en |
dc.description.provenance | Made available in DSpace on 2021-06-16T03:51:24Z (GMT). No. of bitstreams: 1 ntu-103-D99223204-1.pdf: 8072671 bytes, checksum: 1a0c745505afdc783d6f30f0845f94e8 (MD5) Previous issue date: 2014 | en |
dc.description.tableofcontents | 誌謝 i
中文摘要 ii Abstract iv Contents vi Figure Contents x Table Contents xv Chapter 1 Introduction 1 1.1 Gold Nanoparticles (Au NPs) 1 1.2 Photoluminescent Gold Nanoclusters (Au NCs) 2 1.2.1 Synthesis and Optical Properties of Au NCs 3 1.2.2 Applications of Luminescent Au NCs 6 1.3 Microgels 14 1.3.1 Synthesis and Characterization of Microgel 15 1.3.2 Application of Microgels 16 1.4 Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry (MALDI-MS) 17 1.5 Surface-Assisted Laser Desorption/Ionization Mass Spectrometry (SALDI-MS) 18 1.6 Motive of Research 19 1.7 References 21 Chapter 2 Using Photoluminescent Gold Nanodots to Detect Hemoglobin in a Single Drop of Blood 41 2.1 Introduction 42 2.2 Experimental Section 44 2.2.1 Chemicals 44 2.2.2 Preparation of photoluminescent 11-MUA–Au NDs 44 2.2.3 Characterization of 11-MUA–Au NDs 45 2.2.4 PL Quenching by Hemin and Heme-Containing Proteins 46 2.2.5 Detection of Hb in biological buffer 46 2.2.6 Detection of Hb in diluted blood samples 47 2.3 Result and discussion 48 2.3.1 Hemin-induced PL quenching of 11-MUA–Au NDs 48 2.3.2 Stern–Volmer plots 51 2.3.3 Detection of Hb in diluted blood samples 52 2.4 Conclusions 54 2.5 References 55 Chapter 3 Synthesis of Photoluminescent Au NDs–PNIPAM Hybrid Microgel for the Detection of Hg2+ 69 3.1 Introduction 70 3.2 Experimental Section 72 3.2.1 Chemicals 72 3.2.2 Synthesis of PNIPAM MGs 72 3.2.3 Preparation of Au NDs–PNIPAM MGs 73 3.2.4 Characterization 73 3.2.5 Detection of Hg2+ 74 3.2.6 Analysis of Fish Samples 75 3.3 Result and discussion 75 3.3.1 Characterization of the Au NDs–PNIPAM MGs 75 3.3.2 Optical Properties and Stability of Au NDs–PNIPAM MGs 76 3.3.3 Detection of Hg2+ Ions Using Au NDs–PNIPAM MGs 79 3.3.4 Detection of Hg2+ in Fish Samples 80 3.4 Conclusions 81 3.5 References 82 Chapter 4 Functional Microgels Assisted Tryptic Digestion and Quantification of Cytochrome c through Internal Standard Mass Spectrometry 98 4.1 Introduction 99 4.2 Experimental Section 101 4.2.1 Chemicals 101 4.2.2 Preparation of PNIPAM–co–MAA MGs 101 4.2.3 Preparation of 13-nm Au NPs 102 4.2.4 Preparation of Au NPs/MGs 102 4.2.5 Characterization of Au NPs/MGs 103 4.2.6 Preparation of Trypsin-Immobilized Au NPs/MGs (TR/Au NPs/MGs) 103 4.2.7 Microwave-Accelerated Protein Digestion Using TR/Au NPs/MGs 104 4.2.8 Capture of Cyt c in Standard Solution 105 4.2.9 Capture of Cytoplasmic Cyt c from Cultured Cells 105 4.2.10 SALDI-MS Analysis 106 4.3 Result and discussion 107 4.3.1 Preparation and Characterization of Au NPs/MGs 107 4.3.2 Immobilization of Trypsin in Au NPs/MGs 108 4.3.3 SALDI-MS for the Analysis of Tryptic Digest of Cyt c 109 4.3.4 Optimization of Digestion Conditions 110 4.3.5 Detection of Cyt c by SALDI-MS 112 4.4 Conclusions 114 4.5 References 116 Conclusions and Prospects 134 Publications 136 Conferences 138 | |
dc.language.iso | en | |
dc.title | 開發金奈米材料與微膠之複合材料及其生物分析之應用 | zh_TW |
dc.title | Development of Gold Nanomaterials and Microgels Based Hybrid Materials for Bioanalysis Application | en |
dc.type | Thesis | |
dc.date.schoolyear | 103-1 | |
dc.description.degree | 博士 | |
dc.contributor.oralexamcommittee | 戴桓青,許邦弘,吳秀梅,黃志清,胡焯淳 | |
dc.subject.keyword | 發光金奈米點,金奈米粒子,聚N-異丙基丙烯醯,微膠,血紅蛋白,細胞色素c,汞離子,表面輔助雷射脫附游離質譜法, | zh_TW |
dc.subject.keyword | photoluminescent gold nanodots (Au NDs),gold nanoparticles (Au NPs),poly(N-isopropylacrylamide),microgels,hemoglobin,cytochrome c,mercury ion,surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS), | en |
dc.relation.page | 138 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2015-01-17 | |
dc.contributor.author-college | 理學院 | zh_TW |
dc.contributor.author-dept | 化學研究所 | zh_TW |
顯示於系所單位: | 化學系 |
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