無機材料應用於質譜分析與致病菌之檢測

Abstract

基質輔助雷射脫附游離質譜法為目前常被使用於分析生化樣品的質譜法，然而在此質譜法中基質分子的使用造成在質譜低質量範圍的背景干擾，因而限制了於小分子量的分析物的應用。另外，以基質輔助雷射脫附游離質譜法進行含微生物之檢體的分析時，樣品中干擾物的存在影響了菌種在質譜中的可被辨識性。針對這些問題，本論文利用具不同特性的無機材料發展分析方法改善基質輔助雷射脫附游離質譜法於分析時所遭遇的問題。

本論文使用溶膠凝膠材料鍵結傳統2,5-二羥基苯甲酸分子做為基質輔助雷射脫附游離質譜法的基質，利用此一基材具有吸收雷射光的特性，且因為2,5-二羥基苯甲酸被固定於溶膠凝膠中再鐳射光照射下不易被游離，因而可以避免使用傳統基質所產生的背景干擾，可針對不同的小分子樣品進行基質輔助雷射脫附游離質譜法分析。同時因為2,5-二羥基苯甲酸分子可均勻的分佈於溶膠凝膠材料中，因此也改善了傳統基質與樣品混合共結晶時所產生的樣品集中點的問題。而結合晶片式的偵測方法可以縮短分析時間，並應用於蛋白質消化反應的快速分析。

此外，本論文中亦成功製作表面修飾上萬古黴素的氧化鐵磁性奈米粒子做為親和辨識致病菌的探針，可於溶液中成功萃取目標菌種，降低檢體中之基質干擾，經基質輔助雷射脫附游離質譜法分析後可經由線上微生物蛋白質資料庫的比對以確定菌種的身分。此探針可成功應用於尿液檢體的前處理，有效親和辨識微量的致病菌如金黃色葡萄球菌及腐生葡萄球菌，可降低尿液檢體中含有複雜基質的干擾問題。利用此探針前處理樣品後經質譜分析針對金黃色葡萄球菌及腐生葡萄球菌在尿液中之偵測極限約為106 cfu/mL。

本論文的最後一部份中進行螢光性的磁性奈米粒子之研究，將此粒子表面修飾上萬古黴素以當作可親和辨識致病菌親的探針，此探針由於具有螢光性質，因此可在親和辨識目標菌種後，利用基質輔助雷射脫附游離質譜法分析致病菌的身分並同時使用螢光光譜進行菌種濃度分析，對金黃色葡萄球菌之偵測極限約為105 cfu/mL。

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has been widely used to analyze diverse samples. However, difficulties may arise as employing MALDI-MS to the analysis of small molecules owing to unavoidable matrix interference appearing in the low-mass range of mass spectra. Additionally, the presence of complex matrices in real samples containing pathogenic bacteria generally suppress the ion signals generated from the microorganism cells when MALDI-MS is employed for characterization of microorganism strains. Inorganic materials such as sol-gels and magnetic particles with different features were employed to the development of appropriate analytical methods to solve the problems that generally take place during MALDI-MS analysis.

A novel matrix, which could dramatically reduce matrix interference in the low mass region, generated from sol-gel/2,5-dihydroxybenzoic acid (DHB) hybrid material was demonstrated. 2,5-DHB was covalently bound to the network structure of sol-gels, so it was hard to be directly desorbed during laser irradiation. Therefore, the backgrounds ions contributed from 2,5-DHB could be reduced. This sol-ge/2,5-DHB derived material is a suitable matrix for the analysis of small molecules. Furthermore, 2,5-DHB molecules were homogeneously distributed in the sol-gel structure, so the problem of sweet spots arising from poor co-crystallization in conventional MALDI matrix could be avoided. Additionally, chip-based approach by directly spin-coating the sol-gel/2,5-DHB material on glass slides could simplify the steps for MALDI sample preparation. Furthermore, on-chip tryptic digest of proteins was directly carried out on the chip. This approach does not only reduce the digestion time, but also the digest on the chip can be directly subjected into a mass spectrometer for analysis without requiring extra steps.

A method for fabricating vancomycin onto the surface of magnetic nanoparticles was demonstrated. The generated probes have the capacity of recognizing target bacteria from sample solutions. The bacteria enriched by the probes were characterized by MALDI MS. The ions and mass spectral pattern generated from the target bacteria appearing in the MALDI mass spectra can be subjected into protein databases for protein matching. The results can be employed for rapid identification of bacterial strains. This approach has been demonstrated to effectively reduce the interference from complex samples and successfully probing trace of bacteria such as Staphylococcus saprophyticus and Staphylococcus aureus from a urine sample. The detectable cell concentration for both S. saprophyticus and S. aureus in a urine sample was ~106 cfu/mL.

In the final part of this thesis, a method for fabricating fluorescent dye-embedded magnetic nanoparticles immobilized with vancomycin for pathogenic bacteria was demonstrated. Owing to the fluorescent feature of the probes, the target bacteria tapped by the fluorescent probes could be directly detected using a spectrofluorometer for quantitative analysis. MALDI-MS was used to characterize bacterial strains trapped by the affinity probes. The lowest detectable cell concentration of S. aureus by MALDI-MS after enrichment by the affinity probes was ~105 cfu/mL.