液相層析電灑游離質譜儀搭配官能化磁性奈米粒子以濃縮及偵測食品中毒病原菌

Bacteria capture using functionalized magnetic nanoparticles to facilitate the LC-ESI-MS analysis of foodborn pathogens

劉怡靜(Yi-Ching Liu)

指導教授 ： 凌永健

國立清華大學/理學院/化學系/碩士(2008年)

https://doi.org/10.6843/NTHU.2008.00342

摘要

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。

關鍵字

磁性奈米粒子 ； 食品病原菌 ； 液相層析電灑游離質譜儀

並列摘要

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.

並列關鍵字

HASH(0x1b1de5c0) ； HASH(0x1b1de660) ； HASH(0x1b1de700)

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