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  • 學位論文

毛細電泳質譜介面暨磁奈米粒子於毛細電泳質譜之應用

The development of capillary electrophoresis mass spectrometry interface and application of magnetic nanoparticles in capillary electrophoresis mass spectrometry

指導教授 : 何國榮

摘要


毛細電泳質譜具有高分離效率及高分析物鑑定能力等優點。然而毛細電泳銜接質譜時卻存在許多問題,例如:毛細電泳質譜介面的操作、不適合使用非揮發性的緩衝溶液及因使用毛細管而不易進樣大量體積而靈敏度較差等問題,皆減少毛細電泳質譜的應用性。本研究中提出數個方案來解決上述的問題。 使用三種常見的介面、鞘流溶液介面、低鞘流介面及無鞘流介面來探討其銜接晶片微胞電層析至質譜時,靈敏度之差異。使用十二烷基硫酸銨作為介面活性劑,在 10到40 mM的濃度,鞘流溶液介面的感度明顯差於無鞘流介面與低鞘流介面。比較無鞘流介面與低鞘流介面時,當介面活性劑濃度低於20 mM時,低鞘流介面的感度略低於無鞘流介面,反之當濃度超過20 mM時,低鞘流介面靈敏度則優於無鞘流介面。由於低鞘流介面具有少量的稀釋,因此當介面活性劑超過20 mM時,稀釋介面活性劑減少樣品訊號抑制造成訊號的提升超過稀釋造成樣品訊號的下降。此三種介面的感度差異主要來自於稀釋效應,然而當介面彼此間靈敏度差異不大時,流速效應才會變成一個顯著的因子。 為了使毛細電泳質譜更容易操作,利用PDMS來製作具有雙操作模式的毛細電泳質譜介面。由於PDMS為透明材質,透過顯微鏡操作時分離毛細管與電灑噴頭可有很好的連接。此介面包含一導電溶槽及銜接電灑噴頭與分離毛細管的插槽。銜接電灑噴頭的插槽位於導電溶槽的邊緣上,由插槽與電灑噴頭形成的空間中含有導電的電解質因此可進行導電。本介面具有兩種操作模式:無鞘流介面與低鞘流介面的操作。將電灑噴頭與介面接合處上的聚亞醯胺塗層保留時可操作為無鞘流介面,反之將此塗層移除時可操作成低鞘流介面。在此兩種操作模式下,其毛細電泳的分離解析度皆可維持。 為了增加非揮發性緩衝溶液於毛細電泳質譜的適用性,因此開發整體雙槽式介面以減少因使用非揮發性磷酸緩衝溶液產生的訊號抑制效應,此介面的設計中包含接合溶槽、導電溶槽與微通道。於接合溶槽中填充的磷酸緩衝溶液可使磷酸陰離子進入分離毛細管中以維持其分離效率。此介面包含導電溶槽的設計,因此可提供無鞘流及低鞘流等操作模式。介於接合溶槽與導電溶槽中的微通道則用於分析物的傳送。此介面已成功應用磷酸緩衝溶液系統於無鞘流及低鞘流的操作模式下分析抗組織胺與全氟羧酸。 此外,為了增加毛細電泳質譜的靈敏度,以硼酸修飾的磁奈米粒子來作為前濃縮的探針,此奈米粒子對於醣胜肽具有高度的選擇性,可以捕捉到所有來自於去唾液酸胎蛋白的醣胜肽。應用此磁奈米粒子將濃縮後的醣胜肽進行毛細電泳質譜時不僅可減少樣品的複雜度,且可作為前濃縮的探針。

並列摘要


Capillary electrophoresis mass spectrometry provides the advantages of high efficiency separation with high compound identification capability. However, several problems limit the utility of the capillary electrophoresis mass spectrometry. These problems include the operation and performance of a capillary electrophoresis mass spectrometry interface, incompatible with the nonvolatile buffer, and less sensitivity due to the use of a capillary-format column. To overcome these problems, several approaches were proposed in this work. Using ammonium dodecyl sulfate (ADS) as the surfactant, the response of three common interfaces, sheath liquid interface, sheathless interface and low sheath flow interface in the direct coupling of microchip micellar electrokinetic chromatography with electrospray ionization mass spectrometry was studied. In the range of 10 to 40 mM surfactant, a conventional sheath liquid interface provided poorer sensitivity than both sheathless interface and low sheath flow interface. At a surfactant concentration less than 20 mM, a low sheath flow interface exhibited less sensitivity than a sheathless interface, however it outperformed the sheathless interface above 20 mM concentration. At a surfactant concentration above 20 mM, signal reduction due to dilution of the analyte compensated by signal enhancement gained from a reduction in ion suppression effect. The difference in responses of the interfaces was mainly due to the dilution effect, whereas, the effect of flow rate became an important factor when the difference in responses between the interfaces was not significant. The utility of the polymethyl methacrylate (PMMA) microchip MEKC/MS using a low sheath flow interface was demonstrated by analysis of sulfonamides at 40 mM concentration. To simplify the CE/MS operation, a dual mode CE/MS interface based on a PDMS casting microdevice was proposed. Because of the transparency of the PDMS material, the separation column and the ESI emitter could be connected well by using a microscope. A hole fabricated on the edge of the conducting reservoir was designed for insertion of an ESI sprayer. Prior to the insertion of an ESI sprayer, the hole was filled with conducting electrolyte and thus the electric conduction could be established through the space formed between the ESI sprayer and hole. The interface could perform sheathless CE/MS operation because the formation of thin liquid film between the hole and ESI sprayer. The interface could perform the low flow CE/MS operation by removing the polyimide coating of the ESI sprayer because the formation of larger space (~15 μm). Because of the larger space, the make-up liquid could be delivered to the ESI sprayer. The CE separation integrity was preserved under both the sheathless and the low flow operation. To increase the utility of nonvolatile buffer in CE/MS, an integrated double junction interface based on a PDMS casting microdevice for the use of phosphate buffer without the ion suppression effect was developed. This interface consists of a junction reservoir, a conducting reservoir and a microchannel. Between junction reservoir and conducting reservoir is a microchannel for analyte transfer. The junction reservoir was designed to preserve the CE separation integrity because the phosphate anion flowed from the junction reservoir to the separation capillary. Whereas the conducting reservoir could be used to establish electric contact for sheathless CE/MS operation or deliver the make-up liquid for low flow CE/MS operation. The feasibility of this interface was demonstrated with the analysis of histamine antagonists under low flow CE/MS operation and perfluorocarboxylic acids under sheathless operation at low-pH buffer and high-pH buffer, respectively. The CE/MS sensitivity is rather limited due to the use of a capillary-format column. To improve the sensitivity, the functionalized magnetic nanoparticle (MNP) was used as concentrating probes. A simple strategy for the synthesis of boronic-acid functionalized MNP was proposed. The boronic-acid functionalized MNP shows a high selective enrichment of glycopeptides. For enrichment of the tryptic peptides derived from the asialofetuin (ASF), all the N-linked glycopeptides could be captured with this material. The use of this nanomaterial in the CE/MS not only reduces the sample complexity but also acts as concentrating probe.

參考文獻


[14] Chang, Y. Z., Chen, Y. R., Her, G. R., Anal. Chem. 2001, 73, 5083-5087.
[11] Chang, Y. Z., Chen, Y. R., Her, G. R., Anal. Chem. 2001, 73, 5083-5087.
1.6 References
[1] Jorgenson, J. W., Lukacs, K. D., Anal. Chem. 1981, 53, 1298-1302.
[2] Engelhardt, H., Beck, W., Schmitt, T., Capillary Electrophoresis: methods and potentials.

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