Applications of Inorganic Materials to Mass Spectrometric Analysis and Detection of Pathogenic Bacteria

• Main Authors: Ya-Shiuan Lin, 林亞玄
• Other Authors: Yu-Chie Chen
• Format: Others
• Language: zh-TW
• Published: 2006
• Online Access: http://ndltd.ncl.edu.tw/handle/23392479432064010635

博士 === 國立交通大學 === 應用化學系所 === 95 === 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.