臺灣博碩士論文加值系統

　　光纖式粒子電漿共振感測器 (Fiber Optic Particle Plasmon Resonance，簡稱FOPPR) 為新穎的偵測技術，利用貴金屬 (如：金奈米粒子) 吸收漸逝波能量產生粒子電漿共振進行分析物感測，此技術具有免標記、即時偵測、高靈敏度、專一性等優點。
　　本實驗室在過去的研究中，已成功建立FOPPR結合液相層析串聯質譜儀 (Liquid Chromatography/Mass Spectrometry, LC/MS) 的連接介面，欲利用MS確認FOPPR所檢測的蛋白質種類。FOPPR對於蛋白質利用免疫分析原理的偵測有良好成果，但因蛋白質的電噴灑游離化效率不佳，導致實驗受到限制，常無法順利的結合。為改善此問題，利用蛋白質經過消化處理 (digestion) 產生的胜肽 (peptides)，提高離化效率以增進訊號強度，並經由Ziptip去鹽，即可在不經LC的情況下直接進MS，節省偵測時間。本研究目的為建立質譜儀測定蛋白質的方法，以牛血清白蛋白 (Bovine Serum Albumin) 做為樣品，利用trypsin進行消化，經過Ziptip的處理後，進入質譜儀偵測，成功將消化後的peptide質譜圖利用Mascot進行辨識，並且透過特定peptide達成20至200 µg/mL濃度的偵測，和FOPPR的檢測範圍 (0.01-100 μg/mL) 部分重疊，未來欲合併兩者為一可行的方法。

Fiber Optic Particle Plasmon Resonance (FOPPR) is a novel technology, which is based on coating functionalized gold nanoparticles-modified on unclad optical fiber for biomolecules detection. Particle plasmons are charge density collective oscillation confined to metallic nanoparticles. When the incident photon frequency is resonant with the collective oscillation of the conduction electron of the nanoparticle, a peak in the extinction spectrum of the nanoparticle is manifested. When the analytes bind to the nanoparticle surface, the local refractive index (RI) surrounding of these nanoparticles changes, resulting in increasing plasmon absorbance and a shift in the PPR wavelength. FOPPR technology has advantages of label-free, real-time detection, high sensitivity, and specificity for biochemical molecules detection.
In the past, the integrated system of FOPPR biosensor with liquid chromatography–mass spectrometry has already been developed in our group to facilitate analyte structure confirmation. FOPPR technology has the ability for protein quantitative detection, but the previous results indicate the difficulties of obtaining the protein mass spectrum because of the inefficient electrospray ionization of intact biomolecules of high molecular weight. In the study, the procedures of digesting protein as peptide fragments and then desalting using Ziptip devices were used to overcome this problem.
We have developed Bovine Serum Albumin (BSA) determination method at low concentration level (≤ 200 μg/mL) using mass spectrometry. The mass spectra of BSA peptide was confirmed with database and provided a good linearity of signal intensity versus BSA concentration in the range of 20-200 μg/mL. This range is compatible with that of typical FOPPR determinations (0.01-100 μg/mL).

摘要 I
Abstract III
目錄 VI
圖目錄 VIII
表目錄 X
第一章 緒論 1
1-1 前言 1
1-2 光纖式粒子電漿共振感測器 4
1-2-1 光纖基本構造 4
1-2-2 光纖內傳導原理與漸逝波 5
1-2-3 粒子電漿共振 8
1-2-4 自我組裝單層 9
1-2-5 免標記光纖式電漿子共振感測器 11
1-3 質譜儀 13
1-3-1 質譜儀 13
1-3-2 離子源 15
1-3-3 質量分析器 18
1-4 研究動機 20
第二章 實驗方法 26
2-1 實驗藥品 26
2-2 實驗儀器設備 29
2-3 光纖式電漿子共振感測器 30
2-3-1 光纖製備與清洗 30
2-3-2 金奈米粒子製備 31
2-3-3 修飾金奈米粒子與自我組裝單層 32
2-3-4 晶片封裝與儀器架構工作原理 35
2-4 蛋白質消化處理 37
2-5 質譜儀與液相層析串聯質譜儀參數設定 40
2-6 蛋白質辨識軟體(Mascot) 42
第三章 結果與討論 45
3-1 質譜儀與液相層析串聯質譜儀條件探討 45
3-2 Data dependent acquisition 47
3-3 控溫方法 48
3-4 去鹽方法 49
3-5 Ziptip和digestion效率 51
3-6 Digestion BSA於質譜儀檢測結果 55
3-7 BSA於PBS buffer中進行digestion之質譜偵測探討 58
3-8 BSA於FOPPR檢測結果 60
第四章 結論 64
第五章 參考文獻 65
附錄 69

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