傳統鑑定病原菌之方法大多需要一至兩天的培養時間甚至更久，使得過程繁複且耗時，因此非培養式的快速檢測法就成為目前時代潮流的趨勢。本研究利用磁性奈米粒子(magnetic nanoparticles，MNPs)所具備之高比表面積與超順磁之特性製備出針對革蘭氏陽性菌具有專一性之細菌探針，並在外加磁場(3000G)的輔助下達到快速基質分離與濃縮之目的。透過液相層析電灑游離質譜儀(LC-ESI/MS)的分析，並搭配網路資料庫之比對，達到鑑定菌種之目的。
本研究係利用化學共沉澱法進行MNPs之製備，並修飾萬古黴素(Vancomycin)於磁性奈米粒子之表面。續以穿透式電子顯微鏡(TEM)、傅利葉轉換紅外光譜儀(FTIR)與熱重分析儀(TGA)等進行特性分析來確認細菌探針之性質。將細菌探針(200 mg)應用於革蘭氏陽性菌(2×108 cfu/mL)如:金黃色葡萄球菌(Staphylococcus aureus)及仙人掌桿菌(Bacillus cereus)之分析試驗。本研究中以冷凍/解凍、超音波震盪以及微波輔助破菌等方法分別測試細菌破菌效果，最後選定最佳之冷凍/解凍法萃取細菌蛋白質，結合液相層析電灑游離質譜儀之蛋白質分析技術與網路資料庫之比對，可比對出S. aureus與B. cereus的特有蛋白質，利用這些菌種特有的蛋白質作為生化指標來加以鑑定菌種種類。
本研究所製備之細菌探針可專一的從基質中分離出革蘭氏陽性菌，輔以LC-ESI/MS分析之蛋白質層析滯留時間、分子量及資料庫比對，可成功鑑定出不同之菌種。比起傳統分析方法，可快速從食物基質中分離出細菌，並初步鑑定細菌種類。

Rapid identification of pathogens is of most importance considering the timeliness of therapy treatment. Traditional methods usually taking at least two days for pathogens incubation and detection fail to meet the aforementioned challenge. Recent progress in magnetic nanoparticles (MNPs) research has gained attention of their use for sample pretreatment and separation. These magnetic nano-materials could be modified with different kinds of functional group targeting to specific compounds and easily be isolated from matrix. Higher efficiencies and fast speed of analysis is also expected based on the property of high surface area per unit volume. This motivated us to couple these potential advantages to develop a technique of rapid identification for pathogens.
MNPs prepared by chemical co-precipitation and modified with vancomycin were used as bacterial probes (MNPs-Van) which are specific to Gram-positive bacteria. Vancomycin was successfully immobilized onto the surface of MNPs with an average diameter of 10 nm as demonstrated by TEM, TGA and FTIR analysis. An aliquot of 200 mg bacterial probes (MNPs-Van) were applied to separate and trap Gram-positive bacteria (2×108 cfu mL-1)such as Staphylococcus aureus and Bacillus cereus from matrix with the assistance of external magnetic field (3000 G). After testing ultrasonication, freeze/thaw and microwave assisted cell lysis, we choose freeze/thaw method to extract the bacteria proteins. Specific biomarkers of proteins extracted from trapped bacteria were successfully identified by LC-ESI-MS analysis followed by database search.
The detection of bacteria by bacterial probes (MNPs-Van) by LC-ESI-MS analysis and protein database search was successfully developed in this study. Bacteria could be identified based on individual protein molecular weight and retention time.

1. Hedberg, C., Food-related illness and death in the United States. Emerging Infectious Diseases 1999, 5, (6), 840-841.
2. 行政院衛生署. http://www.doh.gov.tw/
3. Mims, C. P., J.; Roitt,I.; Wakelin, D.; Williams, R. Medical microbiology. C.V. Mosby: 1998.
4. 中國國家標準檢索系統. http://www.cnsonline.com.tw/
5. Perry, J. D.; Freydiere, A. M., The application of chromogenic media in clinical microbiology. Journal of Applied Microbiology 2007, 103, (6), 2046-2055.
6. Sapsford, K. E.; Bradburne, C.; Detehanty, J. B.; Medintz, I. L., Sensors for detecting biological agents. Materials Today 2008, 11, (3), 38-49.
7. Cousins, D. L.; Marlatt, F., An evaluation of a conductance method for the enumeration of enterobacteriaceae in milk. Journal of Food Protection 1990, 53, (7), 568-&.
8. Blackstone, G. M.; Nordstrom, J. L.; Vickery, M. C. L.; Bowen, M. D.; Meyer, R. F.; DePaola, A., Detection of pathogenic Vibrio parahaemolyticus in oyster enrichments by real time PCR. Journal of Microbiological Methods 2003, 53, (2), 149-155.
9. Fricker, M.; Messelhausser, U.; Busch, U.; Scherer, S.; Ehling-Schulz, M., Diagnostic real-time PCR assays for the detection of emetic Bacillus cereus strains in foods and recent food-borne outbreaks. Applied and Environmental Microbiology 2007, 73, (6), 1892-1898.
10. Huletsky, A.; Giroux, R.; Rossbach, V.; Gagnon, M.; Vaillancourt, M.; Bernier, M.; Gagnon, F.; Truchon, K.; Bastien, M.; Picard, F. J.; van Belkum, A.; Ouellette, M.; Roy, P. H.; Bergeron, M. G., New real-time PCR assay for rapid detection of methicillin-resistant Staphylococcus aureus directly from specimens containing a mixture of staphylococci. Journal of Clinical Microbiology 2004, 42, (5), 1875-1884.
11. Loy, A.; Bodrossy, L., Highly parallel microbial diagnostics using oligonucleotide microarrays. Clinica Chimica Acta 2006, 363, (1-2), 106-119.
12. Liu, R. H.; Yang, J. N.; Lenigk, R.; Bonanno, J.; Grodzinski, P., Self-contained, fully integrated biochip for sample preparation, polymerase chain reaction amplification, and DNA microarray detection. Analytical Chemistry 2004, 76, (7), 1824-1831.
13. Anhalt, J. P. F., C., Identification of bacteria using mass spectrometry. analytical chemistry 1975, 47, 219-225.
14. Fang, J. S.; Barcelona, M. J.; Alvarez, P. J. J., A direct comparison between fatty acid analysis and intact phospholipid profiling for microbial identification. Organic Geochemistry 2000, 31, (9), 881-887.
15. Heller, D. N.; Murphy, C. M.; Cotter, R. J.; Fenselau, C.; Uy, O. M., Constant neutral loss scanning for the characterization of bacterial phospholipids desorbrf by fast atom bombardment. Analytical Chemistry 1988, 60, (24), 2787-2791.
16. Karas, M.; Hillenkamp, F., Laser desorption ionization of proteins with molecular masses exceeding 10000 daltons. Analytical Chemistry 1988, 60, (20), 2299-2301.
17. Bakhtiar, R.; Nelson, R. W., Mass spectrometry of the proteome. Molecular Pharmacology 2001, 60, (3), 405-415.
18. Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M., Electrospray ionization for mass-spectrometry of large biomolecules. Science 1989, 246, (4926), 64-71.
19. Lo, A. A. L.; Hu, A.; Ho, Y. P., Identification of microbial mixtures by LC-selective proteotypic-peptide analysis(SPA). Journal of Mass Spectrometry 2006, 41, (8), 1049-1060.
20. Dworzanski, J. P.; Deshpande, S. V.; Chen, R.; Jabbour, R. E.; Snyder, A. P.; Wick, C. H.; Li, L., Mass spectrometry-based proteomics combined with bioinformatic tools for bacterial classification. Journal of Proteome Research 2006, 5, (1), 76-87.
21. Salzano, A. M.; Arena, S.; Renzone, G.; Dambrosio, C.; Rullo, R.; Bruschi, M.; Ledda, L.; Maglione, G.; Candiano, G.; Ferrara, L.; Scaloni, A., A widespread picture of the Streptococcus thermophilus proteome by cell lysate fractionation and gel-based/gel-free approaches. Proteomics 2007, 7, (9), 1420-1433.
22. Ho, Y. P.; Hsu, P. H., Investigating the effects of protein patterns on microorganism identification by high-performance liquid chromatography-mass spectrometry and protein database searches. Journal of Chromatography A 2002, 976, (1-2), 103-111.
23. http://www.sustainpack.com/nanotechnology.html
24. Ferrari, M., Cancer nanotechnology: Opportunities and challenges. Nature Reviews Cancer 2005, 5, (3), 161-171.
25. Johnson, C. J.; Zhukovsky, N.; Cass, A. E. G.; Nagy, J. M., Proteomics, nanotechnology and molecular diagnostics. Proteomics 2008, 8, (4), 715-730.
26. Kumar, R. V.; Koltypin, Y.; Cohen, Y. S.; Cohen, Y.; Aurbach, D.; Palchik, O.; Felner, I.; Gedanken, A., Preparation of amorphous magnetite nanoparticles embedded in polyvinyl alcohol using ultrasound radiation. Journal of Materials Chemistry 2000, 10, (5), 1125-1129.
27. Corr, S. A.; Rakovich, Y. P.; Gun'ko, Y. K., Multifunctional magnetic-fluorescent nanocomposites for biomedical applications. Nanoscale Research Letters 2008, 3, (3), 87-104.
28. Mornet, S.; Vasseur, S.; Grasset, F.; Duguet, E., Magnetic nanoparticle design for medical diagnosis and therapy. Journal of Materials Chemistry 2004, 14, (14), 2161-2175.
29. Neuberger, T.; Schopf, B.; Hofmann, H.; Hofmann, M.; von Rechenberg, B., Superparamagnetic nanoparticles for biomedical applications: Possibilities and limitations of a new drug delivery system. Journal of Magnetism and Magnetic Materials 2005, 293, (1), 483-496.
30. Pautler, R. G.; Fraser, S. E., The year(s) of the contrast agent - micro-MRI in the new millennium. Current Opinion in Immunology 2003, 15, (4), 385-392.
31. Yu, L. S. L.; Uknalis, J.; Tu, S. I., Immunomagnetic separation methods for the isolation of Campylobacter jejuni from ground poultry meats. Journal of Immunological Methods 2001, 256, (1-2), 11-18.
32. Lin, Y. S.; Tsai, P. J.; Weng, M. F.; Chen, Y. C., Affinity capture using vancomycin-bound magnetic nanoparticles for the MALDI-MS analysis of bacteria. Analytical Chemistry 2005, 77, (6), 1753-1760.
33. Chang, S. Y.; Zheng, N. Y.; Chen, C. S.; Chen, C. D.; Chen, Y. Y.; Wang, C. R. C., Analysis of peptides and proteins affinity-bound to iron oxide nanoparticles by MALDI MS. Journal of the American Society for Mass Spectrometry 2007, 18, (5), 910-918.
34. Cottingham, K., Bio on the terror front lines. Analytical Chemistry 2006, 78, (1), 18-23.
35. Gu, H. W.; Ho, P. L.; Tsang, K. W. T.; Wang, L.; Xu, B., Using biofunctional magnetic nanoparticles to capture vancomycin-resistant enterococci and other gram-positive bacteria at ultralow concentration. Journal of the American Chemical Society 2003, 125, (51), 15702-15703.
36. Benveniste, H.; Drejer, J.; Schousboe, A.; Diemer, N. H., Elevation of the extracellular concentrations of glutamate and aspartate in rat hippocampus during transient cerebral-ischemia monitored by intracerebral microdialysis. Journal of Neurochemistry 1984, 43, (5), 1369-1374.
37. Johnson, B. H.; Hecht, M. H., Recombinant proteins can be isolated from escherichia-coli-cells by repeated cycles of freezing and thawing. Bio-Technology 1994, 12, (13), 1357-1360.
38. Lill, J. R.; Ingle, E. S.; Liu, P. S.; Pham, V.; Sandoval, W. N., Microwave-assisted proteomics. Mass Spectrometry Reviews 2007, 26, (5), 657-671.
39. 紀柏享，楊末雄，孫毓璋, 微波消化之方法與應用. chemistry 1998, 56, 269-284.
40. Choi, I.; Choi, S. J.; Chun, J. K.; Moon, T. W., Extraction yield of soluble protein and microstructure of soybean affected by microwave heating. Journal of Food Processing and Preservation 2006, 30, (4), 407-419.
41. Smith, P. K.; Krohn, R. I.; Hermanson, G. T.; Mallia, A. K.; Gartner, F. H.; Provenzano, M. D.; Fujimoto, E. K.; Goeke, N. M.; Olson, B. J.; Klenk, D. C., Measurement of protein using bicinchoninic acid. Analytical Biochemistry 1985, 150, (1), 76-85.
42. Keith, L.H.; Gron, L.U.; Young, J. L., Green analytical methodologies, Chemical Reviews 2007, 107, (6), 2695-2708