Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/16866
標題: 奈米材料輔助電噴灑質譜法於尿液中紫外光吸收劑與蛋白質電荷分佈之研究
Nanomaterial-Based Mass Spectrometry for Analysis of Ultraviolet Absorbers in Urine and the Charge-State Distribution of Protein
作者: 楊宗杰
Yang, Tzung-Jie
關鍵字: Nanomateril
奈米材料
Electrospray Mass spectrometry
電噴灑質譜法
出版社: 化學系所
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摘要: In this thesis, nanomaterial-based electrospray mass spectrometric approach was utilized for small molecular and macromolecular analysis. The thesis is divided in five chapters. The functionalized magnetic nanoparticles, Fe3O4/SiO2 was synthesized and then utilized for analysis of eleven ultraviolet absorbers in urine. The feasibility of the presented method was also evaluated. The linear range of proposed method were 10-1000 ng mL-1 for HBP, BP-3, BP-2, BP-6 and 4-MBC; 0.5-1000 ng mL-1, 0.5-500 ng L-1, 0.75-1000 ng mL-1 and 0.75-500 ng mL-1 for DHB, BP-12, DHMB and OD-PABA, respectively. LODs were ranged from 0.1 ng mL-1 to 8.8 ng mL-1 with R.S.D. below 10.8%. This approach was also applied for real samples analysis. The trace amount of DHMB in urine was detected and the concentration is 277.1 ng mL-1. The ambient mass spectrometry for one-droplet analysis was developed in this thesis. A 3.5 μL solution was dipped on a laboratory-made screen-printed plate coated with silver or graphite gel. The voltage of +5 kV was applied on the surface of the droplet and electrospray was performed when applying the voltage. The ultraviolet absorbers studied are including 3-(4-methylbenzylidene)-camphor (4-MBC), 2-ethyl-4-(dimethylamino)benzoate (OD-PABA) and 2-hydroxy-4-(octyloxy)-benzophenone (BP-12). To evaluate the effect on the ionization efficiency, the solution containing analytes and nanomaterials was dipped on the screen-printed substrate. From the results, the MWCNTs can enhance the intensity of the signal detected and short the analytical time. This technique demonstrates that the nanoparticles assisted electrospray ionization offers a high specific and high throughput screening for trace analysis. The analysis of protein conformation by ESI-MS was devoted in this study. This work outlines the changes to the charge-state distribution (CSD) and to improvement of the resolution of isotopic peak when the nanomaterial solution is infused to the ion source and co-electrosprayed with the protein-contained solution. The various conformation gold nanoparticles (AuNPs) and titanium dioxide nanoparticles (TiO2) were used in this study. Generally the ESI-MS of protein solution often show a monomodal distribution of charge-state peaks with a maximum charge-state and a most-intense charge-state. When AuNPs mixed with protein such as cytochrome c and ubiquitin, the mass resolution of isotopic peak in the mass spectrum were improved. The ESI-MS of protein solution was shown the average distribution of isotopic peaks with the most-abundant charge-state. For the assessment of myoglobin with AuNPs and TiO2, the charge-stated peaks of protein were shifted. The present study was demonstrated the effect of nanomaterials on the charge-states distribution of protein and the characteristics of isotopic peaks of protein in ESI-MS.
本論文研究主要是利用各式奈米材料結合電噴灑質譜法於小分子與生化分子分析之研究。利用二氧化矽修飾磁性奈米粒子應用於尿液中微量紫外光吸收劑之偵測,研究中首先針對合成之材料進行各項鑑定,包含IR、TEM、XRD、ESCA等光譜鑑定,之後進行樣品前處理之最佳化條件探討,並於實驗最佳化條件下,評估方法的可行性,根據實驗結果顯示,本實驗方法之線性範圍分別為於HBP、BP-7、BP-3、BP-2、BP-6與4-MBC等分析物介於10-1000 ng mL-1;對於DHB與BP-12分析物分別為0.5-1000 ng mL-1與0.5-500 ng L-1;對於DHMB與OD-PABA分析物分別為0.75-1000 ng mL-1與0.75-500 ng mL-1,線性相關係數為0.991以上,偵測極限介於0.1 ng mL-1 (OD-PABA)與 8.8 ng mL-1 (BP-2)之間,定量極限介於0.2 ng mL-1 (OD-PABA)與29.3 ng mL-1 (BP-2)之間,精密度介於1.8 %與10.8 %之間。應用於16個尿液樣品分析,於S5樣品中,測得尿液樣品中含有微量DHMB之成份,濃度為283.4 ng mL-1。 本實驗另發展一滴溶液分析大氣壓質譜法,於透明膠片上塗覆一層疏水性導電材料,根據實驗結果顯示,當在3.5 μL液滴表面施予不同電位時,液面與塗覆材料間的接觸角會隨著電位大小而改變,出現類似電潤濕的現象,且由離子訊號強度與CCD攝影機觀察其噴灑狀態,其訊號穩定度佳且持續時間較長,將其應用於奈米材料混合溶液分析,於尿液樣品中添加Fe3O4/SiO2與Fe3O4奈米材料對於分析物的訊號並無明顯提升,但在兩種不同磁性材料於分析物離子強度比較結果,使用Fe3O4/SiO2對於分析物的訊號可提升1.3(4-MBC)-2.1(BP-12)倍;添加MWCNTs則是分析物離子訊號相較於直接尿液樣品分析可提升2(4-MBC)-7(OD-PABA)倍,根據此實驗條件進行方法評估,方法線性範圍介於0.25-5 μg mL-1,且相對標準偏差低於16%。 由於蛋白質的電荷分佈與構型有關,因此利用電噴灑質譜法探討在不同奈米材料環境下,對於蛋白質電荷分佈型態之影響,實驗中分別探討不同尺寸金奈米粒子與二氧化鈦奈米粒子對於細胞色素C、肌紅蛋白與泛素電荷分佈之影響。於金奈米粒子研究部分,金奈米粒子對於細胞色素C與泛素於電噴灑質譜法分析下,其電荷分佈範圍並無顯著的改變,顯示這些蛋白質於金奈米材料環境下,其構型並不會因此受到改變,但是觀測細胞色素C與泛素之ESI-MS質譜圖則顯示其差異性。於細胞色素C之分析結果中,其每一帶電荷離子訊號峰由原先單一同位素訊號(monoisotopic peak),隨著金奈米粒子的比例增加,每一電荷離子訊號會呈現一高斯分佈型態,且同位素峰皆明顯表現;在泛素的分析結果則顯示於電噴灑游離法下會與金奈米粒子形成水合離子訊號,如[Ubq+Au+6H2O+6H]6+與[Ubq+Au+6H2O]6+。 在肌紅蛋白中分析結果則顯示隨著金奈米粒子比例的增加,其帶電荷數會隨著減少且落於高質量區,但在二氧化鈦溶液環境下,肌紅蛋白其帶電荷價數介於+11價與+20價之間,相較於在金奈米環境下,最強訊號之電荷價數由+21價趨於+17價分佈不同,推測二氧化鈦於ESI-MS分析過程中對肌紅蛋白帶電荷數的提升有所助益,且在此兩種奈米材料環境下,由於都未觀察到heme group (m/z 616.4)訊號峰的出現,因此推測肌紅蛋白結構並無任何改變。
URI: http://hdl.handle.net/11455/16866
其他識別: U0005-1407201120502300
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-1407201120502300
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