利用螢光鑽石奈米粒子為親和性探針結合質譜分析技術於細菌的鑑定分析

目前鑑定細菌的傳統方法 PCR 及 ELISA ，由於需要耗費較多的分析時間及成本，並且需要專業技術人員操作，因此我們開發出一種新穎的材料，聚精氨酸-羧基螢光奈米鑽石（PA-FND），不僅可以應用在螢光標記細菌表面，也可以結合 MALDI-TOF MS 細菌分析技術，達到有效快速鑑定細菌種類。過去文獻報導，精氨酸被應用在抗菌胜肽上，由於精氨酸之胍基團帶正電，革蘭氏陽性菌與革蘭氏陰性菌表面皆帶負電，因此可藉由精氨酸提高抗菌胜肽與細菌間的靜電吸引力，增加抗菌胜肽的抗菌能力。因此我們將螢光奈米鑽石表面修飾上聚精氨酸，使聚精氨酸結合螢光奈米鑽石（PA-FND）形成親和性探針，可與細菌形成多價鍵結，利用聚精氨酸-羧基螢光奈米鑽石可吸附細菌之能力，結合螢光奈米鑽石的螢光標記與 MALDI-TOF MS 的細菌蛋白質分析，達到快速偵測及鑑定細菌的效果。由實驗結果顯示，聚精氨酸-羧基螢光奈米鑽石與細菌，經過 30 分鐘的混合後，可清楚看見離心管底部有聚精氨酸-羧基螢光奈米鑽石與細菌的聚集物，利用共聚焦顯微鏡下可以觀察到， E. coli 與 B. subtilis 與聚精氨酸-羧基螢光奈米鑽石之螢光共位影像，將影像放大時，可觀察到聚精氨酸-羧基螢光奈米鑽石吸附在細菌表面，因此可證明，聚精氨酸-羧基螢光奈米鑽石的確可以吸附細菌，並可利用其螢光特性標記細菌，作為親和性螢光探針。而 MALDI-TOF MS 的實驗結果顯示，藉由聚精氨酸-羧基螢光奈米鑽石吸附細菌後，結合 MALDI-TOF MS 的分析，可以得到細菌蛋白質的質譜圖，經分析發現加入聚精氨酸-羧基螢光奈米鑽石，並不會干擾質譜分析結果。而且經由 MALDI-TOF MS 分析可發現， E. coli 與 B. subtilis 所產生的蛋白質訊號位置並不相同，因此可藉此區分不同的菌種。希望未來可以將聚精氨酸-羧基螢光奈米鑽石作為親和性螢光探針，結合 MALDI-TOF MS 分析細菌技術，及市售鑑定微生物之軟體 Biotyper ，應用在臨床抗抗生素細菌之測定。

關鍵字

螢光鑽石奈米粒子；聚精氨酸；細菌；基質輔助鐳射脫附離子化飛行時間質譜儀

並列摘要

Efficient methods for rapid and accurate identification of bacteria are critical for adequate antibiotic therapy. This study presents a new approach for rapid detection and identification of bacteria using fluorescent nanodiamond labeling and MALDI- TOF MS. 100 nm fluorescent nanodiamonds (FNDs) were surface-functionalized with polyarginine (PA) to manipulate bacterial adhesion through multivalent binding and to act as affinity labeling agents. After a 30-min incubation of PA-coated FNDs with bacteria followed by a short low-speed centrifugation, visible FND-bacterial aggregates were formed at the bottom of the sample vial. Confocal microscopy studies and bacterial adhesion kinetics revealed that both gram-negative bacteria (Escherichia coli) and gram-positive bacteria (Bacillus subtilis) show highly efficient adhesion to the surface of PA-coated FNDs. The FND-bacterial aggregates were further subjected to MALDI-TOF MS analysis for rapid and accurate identification of bacterial species. Bacteria fewer than 105 cells and different species in bacterial mixture (E.coli and B. subtilis) were clearly identified by comparing specific bacterial protein mass patterns. In this study, we demonstrated PA-coated FND is an outstanding affinity probes which is a facile, rapid, culture-free approach for bacteria identification.

並列關鍵字

Fluorescent nanodiamond ； Polyarginine ； Bacteria ； MALDI-TOF MS

參考文獻

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利用螢光鑽石奈米粒子為親和性探針結合質譜分析技術於細菌的鑑定分析

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