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研究生:王哲偉
研究生(外文):Che-Wei Wang
論文名稱:毛細管組裝式聚二甲基矽氧烷微流體裝置於毛細電泳質譜介面之開發與應用
論文名稱(外文):Interface Development for CE/MS Using Capillary-Assembled Poly(dimethylsiloxane) Microfluidic Devices
指導教授:何國榮何國榮引用關係
口試委員:韓肇中傅明仁陳逸然賴建成
口試日期:2013-07-23
學位類別:博士
校院名稱:國立臺灣大學
系所名稱:化學研究所
學門:自然科學學門
學類:化學學類
論文種類:學術論文
論文出版年:2013
畢業學年度:101
語文別:中文
論文頁數:152
中文關鍵詞:毛細電泳質譜聚二甲基矽氧烷反向流無鞘流介面鞘流介面環糊精胜肽動態pH接合面前濃縮電灑法溶劑輔助進樣口游離法
外文關鍵詞:capillary electrophoresis mass spectrometrypoly(dimethylsiloxane)counterflowsheathless interfacesheath-flow interfacecyclodextrinspeptidedynamic pH junctionpreconcentrationelectrospray ionizationsolvent-assisted inlet ionization
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毛細電泳電灑法質譜由於整合毛細電泳的分離效率及質譜的分析物鑑定能力而成為極具發展潛力的一項分析技術。然而現行毛細電泳質譜卻因靈敏度不如液相層析質譜而降低其實務的應用性。造成靈敏度不佳的三個主要原因包含傳統鞘流介面的稀釋效應、使用非揮發性緩衝溶液造成訊號抑制以及線上濃縮策略的不足等。雖然有許多介面已開發用來解決上述對於靈敏度的限制,但提升靈敏度的同時卻也伴隨耐用性較差及操作較複雜的缺點而無法普遍地被使用。因此本研究開發以毛細管組裝式聚二甲基矽氧烷(PDMS)微流體裝置為主體的新式介面,並針對上述限制提出不同的介面設計,藉由此類裝置的製作簡單且組裝方便之特點,達到兼顧分析靈敏度及介面操作性之訴求。除了開發以電灑法為離子化方式的介面外,本研究也針對一種於2011年發展的新游離技術:溶劑輔助進樣口游離法,進行毛細電泳質譜介面之開發及效能評估。
使用奈灑介面如低流速鞘流介面或是無鞘流介面雖可減緩傳統介面的稀釋問題,但由於電灑噴頭需拉尖以操作在奈灑模式,使得噴頭堵塞及破損的問題經常發生。本研究開發PDMS薄膜噴頭式無鞘流介面,此介面整合了內徑未拉尖的薄膜噴頭及液體薄膜導電之無鞘流設計於微流體裝置中。此內徑50 μm、厚度130 μm的三角噴頭可於流速範圍在30至350 nL/min內提供穩定的電噴灑。本研究成功將介面操作在低電滲流(60 nL/min)及高電滲流(210 nL/min)的毛細電泳質譜分析,由於內徑未拉尖而改善噴頭阻塞的問題,並證實能與使用10 μm拉尖噴頭的無鞘流介面有相同分析靈敏度。
利用中性環糊精分離帶正電荷的分析物時,環糊精會隨著電滲流進入質譜端造成樣品訊號抑制。本研究提出反向流輔助-整體雙槽式介面以提升非揮發性環糊精緩衝溶液系統在進行對掌異構物分析時的靈敏度,此介面包含液體接合槽、導電容槽及位於兩槽間的一組上下層的十字型微通道,分離毛細管與電灑噴頭分別組裝至液體接合槽端及導電容槽端,透過介面設計使得當一壓力流從垂直分離途徑的微通道引入時,可直接流往液體接合槽。因此可藉由直接調控流往液體接合槽的反向流速介於環糊精與正電荷分析物的正向流速之間,將環糊精阻擋在液體接合槽而分析物仍能順利往質譜偵測端移動,降低環糊精在質譜偵測時的背景干擾及訊號抑制。本研究成功應用此介面於2-羥丙基-β-環糊精緩衝系統並得到分析物約7.4倍的訊號提升。
利用反向流輔助電動進樣於毛細電泳線上濃縮技術可提供相當優異的濃縮效果,原因在於反向流的施加可減緩樣品堆積面往前移動的速度,故可以得到更長時間的進樣。然而無論是無鞘流或鞘流介面均受限於介面的設計而難以從電噴灑端引入反向流至毛細管內。本研究在毛細電泳質譜之胜肽分析上建立反向流輔助電動進樣-動態pH接合面濃縮策略,首先開發反向流相容之導電液體薄膜式無鞘流介面,利用PDMS微流體裝置的設計,使一壓力流引入裝置後,其中有部份可分流至分離毛細管。隨後將此介面應用於反向流輔助電動進樣之動態pH接合面濃縮策略中,在電動進樣的同時引入適當反向流以減緩pH堆積面的前進速度,藉此延長進樣時間,成功對胜肽標準品及蛋白質水解胜肽樣品產生高濃縮倍率並維持原有的毛細電泳分離效果。
溶劑輔助進樣口游離法為新式的液相游離技術,在不加任何高電壓、氣體及雷射之下,樣品可藉由真空吸力被拉進離子源加熱傳輸管內並產生游離,並產生與電灑法相似的質譜圖及分析靈敏度。本研究針對此技術開發PDMS組裝式控速介面,此裝置將分離毛細管、200 μm的拉尖導電塗佈噴頭以及鞘流輸送管組裝至PDMS基座,毛細電泳的電迴路僅需將噴頭接地即完成而不需施加額外的電壓,可簡化操作及避免電灑法常見之放電現象。在鞘流溶液流速2 μL/min的補充下,經毛細電泳分離的胜肽標準品可順利被拉近質譜端游離偵測並得到不錯的靈敏度,初步證實其方法可行性。


Capillary electrophoresis/electrospray ionization-mass spectrometry (CE/ESI-MS), hyphenating the high separation efficiency by CE and the powerful ability of analyte identification by MS, has been a promising analytical tool. However, poorer sensitivity of CE/MS than that of LC/MS makes the routine services to CE/MS platforms less applicable. The major reasons for losing sensitivity in CE/MS include dilution effects of conventional sheath-liquid interfaces, ion suppressions by non-volatile electrolytes, and insufficiency of preconcentration methods. Interfaces had been reported to solve the above-mentioned problems, however, were often not user-friendly and robust. In this study, three CE/MS interfaces with properties of the simple fabrication and facile assembly were developed using capillary-assembled poly(dimethylsiloxane) (PDMS) microfluidic devices to improve the sensitivity. In addition to ESI, a CE/MS interface for solvent-assisted inlet ionization (SAII), an ionization approach first introduced in 2011, was developed. The performance of CE/SAII-MS was also invastigated.
The use of nanospray-based interfaces, such as low sheath-flow interfaces and sheathless interface, could alleviate the problem of sample dilution. However, tapered tips acting as the ESI sprayers are likely to clog and break. In this work, a simple sheathless CE/MS interface was developed by integrating PDMS membrane emitters and liquid-film electric conduction. Using a 125-μm-thick triangular emitter with a 50-μm-diameter micro-channel, a stable electrospray was obtained from 30 to 350 nL/min. CE/MS analysis in low-EOF (60 nL/min) and high-EOF (210 nL/min) conditions demonstrated the utility of the interface. Because the i.d. of the emitter was not tapered, the problems of clogging of tip damaging were alleviated. The performance of this PDMS-based interface was comparable to that of a sheathless interface with a 10-μm tapered emitter.
When using neutral cyclodextrins (CDs) with analysis of positive-charged compounds in chiral CE/MS, nonvolatile CDs could cause severe ion suppressions due to its introduction to MS by EOF. To improve the utility of nonvolatile CDs in chiral CE/MS, a counterflow-assisted double-junction interface was developed. A liquid junction reservoir, a conducting reservoir, and a two-leveled cross-type microchannel between two reservoirs were integrated into a single PDMS device. The separation capillary and ESI sprayer were connected to the liquid junction reservoir and conducting reservoir, respectively. By means of a specific design for the interface, a pressure flow induced from the microchannel perpendicular to CE pathway could be introduced to the liquid junction reservoir. In this way, neutral CDs migrating out of the separation capillary were hold in the liquid junction reservoir by applying a proper counterflow to compensate the electrophoretic velocity of CDs. Because positive-charged analytes migrated faster than CDs, they were able to migrate toward MS. By using this approach, a 7.4-fold signal enhancement in 2-hydroxypropyl β-cyclodextrin electrolyte systems was achieved.
Coupling counterflow-assisted electrokinetic injection with on-line preconcentration strategies in CE had shown a superior preconcentration performance, because the sample injection time was prolonged by retarding the stacking boundary based on a counterflow. However, the intrinsic configurations of either sheathless or sheath-flow interfaces resulting in unavailability of counterflow make this approach yet to conduct on CE/MS. In this study, a preconcentration approach based on dynamic pH junction and counterflow-assisted electrokinetic injection was developed for CE/MS platforms. The proposed preconcentration method was conducted on CE/MS using a sheathless interface consisting of a capillary-assembled PDMS microdevice allowing liquid-film electric conduction and a counterflow inside the separation capillary. A hydrodynamic counterflow was introduced during electrokinetic injection to retard the pH boundary having an electrophoretic velocity in the direction of CE outlet. Accordingly, longer injection times were achieved without a loss of CE separation. The utility of the proposed system was demonstrated with analysis of peptide standards and tryptic peptides.
Solvent-assisted inlet ionization (SAII) is a new liquid-phase ionization technique. The analyte/solvent solution could be introduced into the inlet tube due to a pressure drop from atmospheric pressure to vacuum and ionized without the use of voltage, nebulizing gas, or laser. The mass spectrum and sensitivity obtained by SAII had been reported to be similar to that by ESI. In this study, a sheath liquid interface for CE/SAII-MS was developed. This interface consisted of a PDMS substrate, a separation capillary, a sheath liquid-delivered capillary, a 200-μm tapered sprayer with conductive coatings. Because CE circuits were established by simply grounding the sprayer, the problems of tip clogging or ESI discharging were alleviated. Using a sheath-flow rate of 2 μL/min, peptides separated by CE could be drawn directly into the inlet tube and ionized with good sensitivity.


摘要 i
Abstract iv
第一章 序論 1
1-1前言 1
1-2毛細管電泳 3
1-2-1電泳淌度 3
1-2-2電滲透流 4
1-2-3毛細管區帶電泳 (CZE) 5
1-3電灑游離法 6
1-4毛細電泳電灑法質譜介面 11
1-5線性離子阱質譜儀 13
1-6參考文獻 15
第二章 聚二甲基矽氧烷薄膜式電灑噴頭於無鞘流毛細電泳電灑法質譜介面之開發 22
2-1前言 22
2-2材料與實驗方法 27
2-2-1藥品及材料 27
2-2-2聚二甲基矽氧烷薄膜噴頭式介面之製作 27
2-2-3儀器設備與操作 29
2-2-4直接注入電灑法質譜分析 29
2-2-5毛細電泳電灑法質譜 29
2-3結果與討論 31
2-3-1聚二甲基矽氧烷薄膜噴頭式無鞘流介面 31
2-3-1-1介面設計概念與結構 31
2-3-1-2三維模板灌鑄法:薄膜式噴頭模板之設計 32
2-3-1-3聚二甲基矽氧烷噴頭之化學雜訊干擾 33
2-3-2聚二甲基矽氧烷薄膜噴頭之電噴灑效能評估 35
2-3-3聚二甲基矽氧烷薄膜噴頭式無鞘流介面於毛細電泳質譜分析胜肽標準品 37
2-3-3-1高流速及低流速訊號強度之比較 37
2-3-3-2與低流速鞘流介面效能之比較 38
2-3-3-3薄膜噴頭式無鞘流介面於低濃度胜肽混合物之分析 39
2-4結論 40
2-5參考文獻 41
第三章 反向流輔助-整體雙槽式介面於非揮發性環糊精緩衝溶液毛細電泳電灑法質譜之應用 56
3-1前言 56
3-2材料與實驗方法 59
3-2-1藥品及材料 59
3-2-2反向流輔助-整體雙槽式介面之製作 59
3-2-3儀器設備與操作 61
3-2-4毛細電泳電灑法質譜 61
3-3結果與討論 63
3-3-1反向流輔助-整體雙槽式介面設計概念 63
3-3-2毛細電泳質譜分析胜肽標準品:施加反向流控制分析物泳動速度之效能評估 65
3-3-2-1單一胜肽標準品分析 65
3-3-2-2胜肽標準品混合物分析 66
3-3-3以2-羥丙基-β-環糊精毛細電泳質譜分析正電荷對掌異構物 68
3-3-4反向流輔助之2-羥丙基-β-環糊精毛細電泳質譜分析:移除背景訊號干擾及改善訊號抑制 70
3-3-4-1施加反向流以提升分析物靈敏度之策略 70
3-3-4-2以反向流阻止非揮發性環糊精進入質譜 70
3-3-4-3加入有機相於反向流以降低環糊精與分析物之間的作用力 71
3-4結論 72
3-5參考文獻 73
第四章 反向流輔助–動態pH接合面濃縮技術銜接毛細電泳電灑法質譜之介面開發與應用 84
4-1前言 84
4-2材料與實驗方法 87
4-2-1藥品及材料 87
4-2-2 PDMS微流體裝置之製作 87
4-2-3儀器設備與操作 88
4-2-4毛細電泳電灑法質譜 89
4-3結果與討論 91
4-3-1反向流相容–無鞘流毛細電泳質譜介面之製作及效能評估 91
4-3-1-1 PDMS微流體裝置設計 91
4-3-1-2評估反向流之分流比例 92
4-3-2反向流輔助電動進樣-動態pH接合面濃縮技術於胜肽標準品分析 93
4-3-2-1使用反向流輔助電動進樣對胜肽分析物進行動態pH接合面濃縮之概念 93
4-3-2-2反向流輔助電動進樣-動態pH接合面濃縮胜肽之可行性評估 95
4-3-2-3添加有機相於樣品溶液對濃縮效率之影響 96
4-3-2-4反向流對濃縮及分離效率之影響 97
4-3-2-5延長電動進樣時間以提升濃縮效率 98
4-3-3反向流輔助電動進樣-動態pH接合面濃縮技術於蛋白質水解胜肽之應用 99
4-4結論 101
4-5參考文獻 102
第五章 毛細電泳溶劑輔助進樣口游離法質譜介面之開發 123
5-1前言 123
5-2材料與實驗方法 125
5-2-1藥品及材料 125
5-2-2 PDMS微流體裝置之製作 125
4-2-3儀器設備與操作 126
5-2-5毛細電泳質譜分析 127
5-3結果與討論 128
5-3-1直接注入法評估SAII游離效能 128
5-3-1-1改變加熱傳輸毛細管溫度的效應 128
5-3-1-2溶劑組成及注入流速對分析物訊號強度之影響 128
5-3-1-3 SAII與ESI訊雜比差異探討 129
5-3-2使用SAII於毛細電泳質譜可行性評估 130
5-3-2-1 PMMA平躺式鞘流介面 130
5-3-2-2 PDMS組裝式控速鞘流介面 131
5-4結論 134
5-5參考文獻 135
總結 151


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第三章
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第五章
1.Hommerson, P.; Khan, A. M.; de Jong, G. J.; Somsen, G. W., Mass Spectrom Rev 2011, 30, 1096-1120.
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