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dc.contributor.author黃馨儀en_US
dc.contributor.authorHuang, shin-yien_US
dc.contributor.author陳月枝en_US
dc.contributor.author帕偉鄂本en_US
dc.contributor.authorChen,Yu-Chieen_US
dc.contributor.authorUrban, Pawel L.en_US
dc.date.accessioned2014-12-12T01:57:58Z-
dc.date.available2014-12-12T01:57:58Z-
dc.date.issued2012en_US
dc.identifier.urihttp://140.113.39.130/cdrfb3/record/nctu/#GT079925579en_US
dc.identifier.urihttp://hdl.handle.net/11536/49911-
dc.description.abstract奈米技術的快速發展,也提升科學領域在多方面的進展。舉例來說,奈米材料常被應用於發展簡單且快速的分析方法,但是在人工合成的奈米材料可被進一步應用之前先行瞭解其物理及化學特性如尺寸大小等等是相當重要的課題。但現有的技術中卻受限於現有技術的限制,如小於1奈米的材料樣品其尺寸便很難由現有技術而被進行量測,而且大多現有的分析儀器多半複雜且昂貴,因此發展簡單的方法有其必要性。所以本論文利用流體層析法、泰勒分散法以及質譜法發展可以針對不同尺寸大小的奈米材料進行量測、分離及分析,及利用奈米材料為探針結合質譜法進行生物樣品分析方法之發展,本論文的研究主要分成三個部分,第一個部分為發展偵測樣品粒徑及分離奈米尺寸樣品的方法,此裝置包含自製的氣體壓力系統,毛細管以及紫外光吸收光譜偵測器,結合流體層析與泰勒分析可以達到同時進行奈米材料的水合半徑之量測以及進行不同尺寸大小的奈米材料之分離效果。我們也發現使用此系統分析蛋白質的水合半徑,和文獻報導的實驗值僅有10%以下的誤差率,而且此系統也進一步被用來研究微生物細胞間的作用力,可藉以分辨因細胞間有作用力產生聚集而造成在流體層析中顯現的滯留時間及層析峰分佈產生的差異。。 在本論文第二個部分,我們發展了結合流體層析與線上結合質譜法界面的發展,可應用在觀測不同流體壓力下蛋白質的構形變化,研究中使用了胺基酸、胜肽與蛋白質當作樣品。我們發現當外加流體壓力大於1.5 psi時,蛋白質可以處於它們的原始構形,如小於此值則反之。我們懷疑在毛細管中之流體提供的剪應力會對蛋白質結構造成影響,當我們施加較低的壓力時,流速較低,蛋白質停留在毛細管中較久進而延長剪應力對於蛋白質構形的影響,進而使蛋白展產生變性的結果。但是在進行胺基酸,奈米材料以及小分子的分析中,則沒有觀察到粒徑改變的情形發生,可能是因為這些小尺寸樣品的結構較堅固且沒有太多構形可改變。在本部分的研究中我們成功地證實使用拉尖的毛細管為界面,以結合流體層析與質譜法可以應用於尺寸大小不同的樣品之粒徑測量與分子量之分析。 本論文的最後一部分是開發了以磁性奈米粒子為分析富含組胺酸樣品的分析平台,可以在含複雜基質樣品中快速分析具有組胺酸標記的樣品。由於磁性奈米粒子具有藉由施加外加磁場便可以快速濃縮與分離的優點,我們利用表面修飾有氧化鋁層的氧化鐵磁性奈米粒子做為組胺酸標記分子的親和探針。此氧化鋁磁性奈米粒子對於組胺酸標記分子的解離常數值約為10-5 M左右,因此在複雜樣品中對於組胺酸標記分子有很好的選擇能力。為了加速分析的過程,我們將整個實驗移到雷射輔助脫附游離質譜儀的樣品盤上進行操作,結果可使樣品需要量下降至1-2 μL左右且可將濃縮萃取時間減少至30秒內。同時,我們也證明了可以在10分鐘內快速完成樣品盤上即時萃取濃縮及進行目標物的酵素消化反應,並進行雷射輔助脫附游離質譜分析。最後,實驗中也證明此方法可以應用於快速分析經基因工程改造的大腸桿菌所表達富含有組胺酸標記的類志賀毒素之蛋白質片段。 總體而言,在本論文中,我們成功開發了三種可以快速定性樣品粒徑大小、進行流體層析分離、結合質譜法以及分析生物樣品的新穎方法我們提出了第一個利用流體層析進行分析細胞間相互作用的方法;此外,我們也建立了一個非常簡易的流體層析線上結合質譜系統的界面而可用於生物分子的分析;最後,我們也成功地發展了以磁性奈米粒子為基材之平台,可應用於快速且靈敏地偵測在複雜樣品中富含組胺酸的生物分子的方法。這三種新開發的方法應該有潛力於被進一步擴展應用於其他的奈米材料以及生物分子的分析之研究。zh_TW
dc.description.abstractThe rapid growth of nanotechnology has benefited science in many aspects. For example, nanoparticles are commonly used in simple and fast analytical assays. Nevertheless, a thorough characterization of newly synthesized nanomaterials always needs to be conducted. The existing techniques are not always readily applicable to determination of size of extremely small nanoparticles (i.e. < 1 nm). Furthermore, most of existing tools are quite expensive. In this thesis, analytical methods for characterizing nanomaterials, as well as nanoparticle-based analytical methods for the analysis of biological samples, were developed. All of them are based on the implementation of hydrodynamic flow (e.g. hydrodynamic chromatography (HDC), Taylor dispersion analysis (TDA)) and mass spectrometry (MS). This thesis is composed of three parts. In the first part, the research goal was to determine hydrodynamic radii for nanoparticles such as nanodiamonds and platinum clusters using HDC combined with TDA. The home-built experimental system used in this study was composed of a gas pressurization system, a fused silica capillary, and an ultraviolet imaging absorption detector. Later on, the size determination and the separation of different nanoparticle-containing samples – in one run – were demonstrated. The deviation of hydrodynamic radii of bovine serum albumin and myoglobin, measured using this approach, from those reported in literature, was below 10%. In another application of the same experimental system, interactions between yeast and Escherichia coli J96 cells could be assayed. The second part of this thesis was focused on the determination of the conformational changes of biomolecules using hydrodynamic flow (HDF) combined with MS. Amino acids, peptides, and proteins were used as the test samples. The protein remained folded when the applied pressure was set at ≧ 1.5 psi (≧ 10 kPa). We suspect that such a low pressure provided a low flow rate in the HDF capillary, and the transported proteins could reach unfolded state as they were subjected to the shear stress induced by the laminar flow. However, hydrodynamic radii of nanoparticles and small molecules, such as amino acids, were not affected by the hydrodynamic pressures used because these analytes have more rigid structures. The combination of HDF and MS on-line was also demonstrated by using a facile interface. The capillary outlet was tapered and placed close (~ 1 mm) to the orifice of an quadrupole time-of-flight mass spectrometer. Using this configuration, molecular weights of the test analytes could be obtained in the same HDF-MS runs. The aim of the final part of this thesis was to develop a magnetic nanoparticle (MNP)-based platform for rapid enrichment and analysis of poly-histidine (His)-tagged species present complex samples. Taking advantage of the magnetic properties of MNPs, isolation and separation could be easily achieved following the enrichment. In this study, iron oxide/alumina core/shell (Fe3O4@Al2O3) MNPs were generated and used as the affinity probes for histidine tagged peptides/proteins. The dissociation constant of the complex of Fe3O4@Al2O3 and His6 was estimated to be: 10-5 M. Thus, the selectivity of the Fe3O4@Al2O3 toward His-tagged peptides/proteins was good. To make this method sensitive and fast, the Fe3O4@Al2O3 MNP-based enrichment was further conducted on the sample target for matrix-assisted laser desorption/ionization (MALDI)-MS. An aliquot of ~ 2 μL of the sample solution was sufficient to perform the analysis. The enrichment could be completed within ~ 30 s. When conducting enzymatic digestion of target proteins, the entire analysis – including enrichment, enzymatic digestion, and MALDI-MS analysis could be completed within 10 min. Eventually, to demonstrate the suitability of this method for analyzing for real samples, His-tagged Shiga-like toxins present in complex cell lysates were used as model samples. Overall, in this project, three analytical methods for characterizing nanoparticles and biological samples have been successfully developed. The results are novel. For example, to the best of our knowledge, it is the first report on the combination of the application of HDC to assay cell interactions. Furthermore, a very straightforward coupling of the HDF platform and MS was demonstrated, and thoroughly tested. Finally, an MNP-based method for rapid and sensitive detection of His-rich species was demonstrated. These methods have the potential to enhance future studies involving nanoparticles and biological samples.en_US
dc.language.isoen_USen_US
dc.subject流體zh_TW
dc.subject層析zh_TW
dc.subject泰勒分析zh_TW
dc.subject質譜分析zh_TW
dc.subject多組胺酸物種zh_TW
dc.subject奈米材料zh_TW
dc.subjecthydrodynamic flowen_US
dc.subjectchromatographyen_US
dc.subjectTaylor dispersion analysisen_US
dc.subjectmass spectrometryen_US
dc.subjectpoly-histidine (His)-tagged speciesen_US
dc.subjectnanomaterialsen_US
dc.title發展流體與質譜法於奈米粒子與生物樣品之分析zh_TW
dc.titleDevelopment of methods based on hydrodynamic flow and mass spectrometry for the analysis of biomolecules, cells, and nanoparticlesen_US
dc.typeThesisen_US
dc.contributor.department應用化學系碩博士班zh_TW
Appears in Collections:Thesis