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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 張煥宗(Huan-Tsung Chang) | |
| dc.contributor.author | Chuan-Kuo Chen | en |
| dc.contributor.author | 陳川國 | zh_TW |
| dc.date.accessioned | 2021-06-15T04:50:06Z | - |
| dc.date.available | 2012-08-03 | |
| dc.date.copyright | 2010-08-03 | |
| dc.date.issued | 2010 | |
| dc.date.submitted | 2010-08-02 | |
| dc.identifier.citation | 第一章:
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/45969 | - |
| dc.description.abstract | 此論文包含毛細管電泳技術開發與功能性金奈米粒子之合成與應用。毛細管電泳部分利用naphthalene-2,3-dicarboxaldehyde (NDA)染劑配合發光二極體誘導螢光偵測系統，同時對胺基酸與胺類分子進行分析，並且使用含有cetyltrimethylammonium bromide (CTAB)與acetonitrile (ACN)的poly(ethylene oxide) (PEO)聚合物溶液來提升分離效率。CTAB可有效降低PEO於毛細管壁之吸附並且提供了穩定又快速的反向電滲流。適當濃度的ACN (25%)使CTAB (20 mM)微胞對分析物有更好的選擇性，有效改善分離的效率。此方法可在4分鐘內完成14種胺基酸與生物胺分子的分析，並提供了良好的分離解析能力(理論板數0.9×105–6.4×105)與靈敏度(LODs~10 nM)，也成功地應用於啤酒樣品的分析。在功能性金奈米粒子部分，開發了一種新型且不需要標記分子的凝血酶檢測法。藉由纖維蛋白原修飾在金奈米粒子表面，當凝血酶與纖維蛋白原作用後，會產生不溶於水、纖維狀的纖維蛋白，使金奈米粒子聚集。透過金奈米粒子吸收變化，可進行高選擇性及高靈敏度的凝血酶偵測，其偵測線性範圍在0.1–10 pM，偵測極限為0.04 pM，並順利應用於血漿中凝血酶之測定，不受生物基質干擾。此外，凝血酶適合體(thrombin binding aptamer; TBA)可有效地與凝血酶接合(Kd~1–100 nM)，常見的凝血酶適合體為TBA15 (15個鹼基)與TBA27 (27個鹼基)。雖然TBA15的結合能力明顯較差(Kd~100 nM)，但TBA15可以直接影響凝血酶活性，而TBA27則不直接影響其活性。故可透過一段互補的DNA將TBA15與TBA27結合成具有較強抑制能力的DNA複合體(TBA15-TBA27)。利用此複合體的抑制能力與先前高靈敏度的凝血酶活性檢測方法，成功開發了新的DNA偵測比色法。此方法不需要繁複的平衡、清洗或溫度變化等處理步驟，其偵測線性範圍為0.050–2.0 nM，偵測極限為25 pM，並且能以肉眼進行single-nucleotide polymorphism (SNP)的觀測。 | zh_TW |
| dc.description.abstract | Three different analytical approaches are demonstrated in this thesis. First, naphthalene-2,3-dicarboxaldehyde (NDA)-amino acid and -amine derivatives were separated and detected by capillary electrophoresis in conjunction with light-emitting diode-induced fluorescence (LEDIF) detection using poly(ethylene oxide) (PEO) containing cetyltrimethylammonium bromide (CTAB). In the presence of CTAB and acetonitrile (ACN), adsorption of PEO on the capillary wall was suppressed, leading to generation of a fast and reproducible electroosmotic flow (EOF). In order to optimize separation resolution and speed, 100 mM Tris–borate solution (pH 7.0) containing 20 mM CTAB and 25% ACN was used to fill the capillary and to prepare 1.2% PEO that entered the capillary via EOF. The analysis of 14 NDA-amino acid and -amine derivatives by this approach was rapid (< 4 min), efficient [(0.9–6.4) × 105 theoretical plates], and sensitive [the LODs (S/N = 3) range from 9.5 to 50.5 nM]. The RSD values (n = 5) of the migration times and peak heights of the analytes for the intraday analysis were less than 1.5 and 1.2%, respectively. The practicality of this approach was validated by quantitative determination of 10 amino acids and amines in a beer samples within 4 min. Secondly, a novel, label-free, colorimetric assay – using fibrinogen (Fib) and gold nanoparticles (Au NPs) –was developed for the highly selective and sensitive detection of thrombin. Addition of fibrinogen to a solution of Au NPs (average diameter: 56 nm) led to ready conjugation, forming Fib–Au NPs through electrostatic and hydrophobic interactions. Introduction of thrombin (a serine protease) into the Fib–Au NPs solutions in the presence of excess fibrinogen induced the formation of insoluble fibrillar fibrin–Au NPs agglutinates through the polymerization of the unconjugated and conjugated fibrinogen. After centrifugation, the absorbance at 532nm of the supernatants decreased upon increasing the concentration of thrombin. This Fib–Au NP probe provided high sensitivity [limit of detection (LOD): 0.04 pM] for thrombin, with remarkable selectivity over other proteins and proteases. The range of linearity for the absorbance against the thrombin concentration was 0.1–10 pM (R2 = 0.96). This approach provided an LOD for thrombin that is lower than those obtainable using other nanomaterial- and aptamer-based detection methods. The utility of this Fib–Au NP probe was validated through separate analyses of thrombin and Factor Xa at picomolar levels in plasma samples—without the need for sample pretreatment. This technique appears to have practical potential in the diagnosis of diseases associated with coagulation abnormalities and cancers (e.g., pulmonary metastasis). Last, detection of DNA hybridization was demonstrated using a Fib-Au NPs-based assay. Two thrombin binding aptamers (TBAs)－TBA15 (15 bases long) and TBA27 (27 bases long)－that are specific towards thrombin were used to form a TBA15-TBA27 assembly in the presence of a complementary DNA (cDNA). The TBA15-TBA27 assembly relative to TBA15 and TBA27 provided a greater inhibition activity for thrombin, showing bivalent binding capacity. The activity of thrombin decreased upon increasing the concentration of cDNA. This new sensing strategy provides high sensitivity [limit of detection (LOD): 25 pM] and remarkable specificity for cDNA. To test the practicality, another probes [TBA15’ (P-TBA15’) and TBA27’ (P-TBA27’)] were used for the detection of the single-nucleotide polymorphism (SNP) responsible for hepatocellular carcinoma. Unlike conventional approaches, this method requires neither postsynthetic modification of the probe oligonucleotides nor precise temperature control for SNP typing. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-15T04:50:06Z (GMT). No. of bitstreams: 1 ntu-99-R97223207-1.pdf: 2855917 bytes, checksum: e51c7afbb9463e94c400ec16238aed30 (MD5) Previous issue date: 2010 | en |
| dc.description.tableofcontents | 謝誌……………………………………………………………………i
中文摘要…………………………………………………………iv 英文摘要………………………….........................vi 目錄……………………………………………............viii 圖目錄…………………….............................xiii 表目錄…………………………………………………………….xvi 著作發表………………………………………………..91 第一章、緒論 1 1.1 毛細管電泳部份 1 1.1.1 毛細管電泳基本介紹 1 1.1.1.1 電泳 1 1.1.1.2 電滲流 1 1.1.1.3 分離效率 2 1.1.1.4 影響分離效率的因素 3 1.1.2 毛細管電泳的分離模式 5 1.1.2.1 微胞電動力層析法 5 1.1.2.2 毛細管凝膠電泳法 6 1.1.3 聚合物溶液線上濃縮與分離生物樣品 6 1.1.4 發光二極體誘導螢光偵測系統 7 1.1.5 研究動機 7 1.2 功能性金奈米粒子部份 9 1.2.1 金奈米粒子之簡介 9 1.2.1.1 金奈米粒子的歷史與發展 9 1.2.1.2 金奈米粒子的合成方法 10 1.2.1.3 金奈米粒子的表面電漿共振 10 1.2.2 金奈米粒子的功能化修飾 11 1.2.2.1 金奈米粒子的修飾方式 11 1.2.2.2 DNA與金奈米粒子 12 1.2.2.3 蛋白質與金奈米粒子 13 1.2.2.4 功能性金奈米粒子的生醫應用 14 1.2.3 凝血酶與纖維蛋白原的簡介 14 1.2.3.1 凝血酶的簡介 14 1.2.3.2 纖維蛋白原與其聚合機制的簡介 16 1.2.4 研究動機 17 1.3 參考文獻 19 第二章、毛細管電泳之胺基酸與生物胺之分析 29 2.1導論 29 2.2 材料與方法 30 2.2.1 儀器裝置 30 2.2.2 藥品試劑與樣品 31 2.2.3 聚合物溶液( PEO )配製 32 2.2.4 樣品衍生化 32 2.2.5毛細管電泳分析步驟 33 2.3結果討論 33 2.3.1 反向電滲流的分離機制 33 2.3.2 添加ACN的影響 35 2.3.3 使用PEO的影響 36 2.3.4 不同CTAB濃度的影響 37 2.3.5 14種胺基酸與生物胺之標準品分離 38 2.3.6 真實樣品啤酒之分析 39 2.4結論 40 2.5參考文獻 41 第三章、利用功能性金奈米粒子開發凝血酶之免標記比色法 51 3.1 導論 51 3.2 材料與方法 52 3.2.1 實驗藥品 52 3.2.2 Fib-Au NPs 的製備 53 3.2.3 凝血酶標準品之偵測 54 3.2.4 血漿中凝血酶標準品之偵測 54 3.2.5 血漿中Factor Xa的偵測 54 3.3 結果與討論 54 3.3.1 Fib-Au NPs偵測凝血酶之機制與驗證 54 3.3.2 金奈米粒子大小的影響 56 3.3.3 Fib-Au NPs的選擇性 57 3.3.4 血漿中凝血酶的分析 58 3.3.5. 血漿中in situ產生凝血酶 59 3.4 結論 59 3.5 參考文獻 60 第四章、藉由酵素活性之調控發展DNA免標記比色法 74 4.1 導論 74 4.2 材料與方法 75 4.2.1 實驗藥品 75 4.2.2 Fib-Au NPs的製備 75 4.2.3纖維蛋白原濃度最佳化 75 4.2.4 利用調控凝血酶活性進行DNA的偵測 76 4.3 結果與討論 76 4.3.1 凝血酶活性調控原理與DNA偵測機制 76 4.3.2 Fib-Au NPs偵測凝血酶方法之改良 77 4.3.3 TBA15-TBA27複合體之凝血酶抑制 78 4.3.4 DNA偵測之靈敏度與選擇性 79 4.4 結論 80 4.5 參考文獻 81 第五章、總結論與展望 90 | |
| dc.language.iso | zh-TW | |
| dc.title | 毛細管電泳與功能性金奈米粒子之技術開發與應用 | zh_TW |
| dc.title | Development and Applications of Capillary Electrophoresis and Functionalized Gold Nanoparticles | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 98-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 劉春櫻,黃志清,孫毓璋,蔡素珍 | |
| dc.subject.keyword | 毛細管電泳,金奈米粒子,生物感測器,凝血酶,核酸適合體,單一核苷,酸多型態, | zh_TW |
| dc.subject.keyword | capillary electrophoresis (CE),gold nanoparticles (Au NPs),biosensor,thrombin,aptamer,single nucleotide polymorphisms (SNPs), | en |
| dc.relation.page | 91 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2010-08-03 | |
| 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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