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標題: | 以紙基微陣列與分子型二次離子質譜術進行高通量之
平行檢測與定量 High-Throughput Parallel Detection and Quantification of Peptides with Paper-based Microarray and Molecular Time-of-flight Secondary Ion Mass Spectroscopy (ToF-SIMS) |
作者: | Kuo-Jui Chu 朱國瑞 |
指導教授: | 薛景中 |
關鍵字: | 二次離子質譜儀,微陣列,高通量分析, Secondary ion mass spectrometry,microarray,high-throughput analysis, |
出版年 : | 2015 |
學位: | 碩士 |
摘要: | 二次離子質譜儀(SIMS)在表面分析上具有極高的靈敏度。加上近年來簇離子團(cluster ion)濺射技術的發展,二次離子質譜儀能夠產生高質量的離子破片,並分析未經同位素標記的混合樣品。而飛行式二次離子質譜儀(ToF-SIMS)屬於靜態二次離子質譜儀,在分析樣品後不對其造成改變,且能獲得全質譜以利分析。此外,利用高度聚焦的一次離子掃描樣品表面能獲得足夠的空間解析度,而且只需要少量的樣品消耗就能取得高解析度的分子分佈影像。結合微陣列(microarray)能善用上述優點進行高通量的樣品分析。近來由於成本低廉與製備容易,紙質的微陣列是與SIMS分析整合的絕佳選擇。在此研究中則採用玻璃纖維濾紙來製作微陣列,因為無機的玻璃纖維相較於一般的紙質濾紙在高質量區的質譜中有較低的背景質,較適合用來分析。玻璃纖維的濾紙會經octadecyltrichlorosilane (OTS)的自組裝單層膜(self-assemble monolayer)修飾成疏水性的表面後,再使用雷射雕刻機選擇性的製作出親水性的微陣列。接著不同濃度的胜肽(peptide)水溶液以1 μL分別滴在這些親水的圓形區域中,而尚未移除的疏水區域用來區隔每個粒滴。用來獲得二次離子影像的脈衝20 kV C60+聚焦至∼3 μm,離子通量為3.8×109 ion/cm2。大面積馬賽克影像是由700 μm×700 μm每幀256×256像素的影像拼成。經由實驗發現,胜肽分子的二次離子訊號相對強度正比於在水溶液中的濃度,透過不同胜肽分子濃度對二次離子相對強度的數據能繪製校準曲線用來定量混合的胜肽分子。藉由結合玻璃纖維基材的微陣列與影像二次離子質譜儀能高通量且低樣品消耗的分析混合胜肽樣品。 Secondary ion mass spectrometry (SIMS) is known as a powerful technique to perform surface analysis with high sensitivity. In recent development of cluster ion sputtering, current SIMS is capable to generate molecular ions of high mass that allow the parallel analysis of molecular mixtures without the need of labeling. With time-of-flight SIMS that operated within the static regime, the specimen is practically unaltered after analysis and the whole m/z pattern (up to a few thousand) can be acquired at the same time. Furthermore, with a focused primary ion that scans over the specimen, high spatial resolution images of molecular distribution on a surface could be obtained with minimal sample consumption. In order to utilize these advantages, a microarray of specimens could be prepared for automatic high throughput analysis of trace molecules. Recently, owing to its low-cost fabrication and ease of modification, paper-based microarray is an excellent candidate to be integrated with the SIMS analysis. In this work, glass microfiber filter paper was used to prepare the microarray because the inorganic glass microfiber is found to yields less background in the high mass (molecular ion) regime than cellulose-based paper. The filter paper was modified with octadecyltrichlorosilane (OTS) self-assemble monolayer (SAM) to form a hydrophobic surface. Hydrophilic microarray was then prepared by using a laser scriber that selectively removed the OTS layer with a designed pattern. 1 μL aqueous drops of various peptides at concentrations between 1.0 and 0.1 mM were then dispensed inside these circular patterns where the OTS SAM was removed while the remaining OTS layer serves as a barrier and separated each drop. Secondary ion images were acquired with a focused (~3 μm beam size) C60+ pulse operated at 20 kV and the primary ion dose was 3.8×109 ion/cm2. Large area mosaic image was obtained by stitching 700×700 μm2 frames of 256×256 pixel. The secondary ion intensities of peptides (e.g. M+Na+) normalized with respect to glass microfiber (SiOH+) were found to be proportional to their concentration in the solution. In other words, calibration curve for each peptide was generated for quantification and the detection limit is in the order of sub-pmol. By combining the paper-based micropad and imaging SIMS, this work demonstrated that the composition of mixed peptides can be determined with high throughput and low sample consumption. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/50761 |
DOI: | 10.6342/NTU201600246 |
全文授權: | 有償授權 |
顯示於系所單位: | 材料科學與工程學系 |
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