使用微流道系統整合多孔性濾膜與表面增強拉曼散射應用於快速細菌檢測與抗生素藥敏試驗

Abstract

細菌的鑑定及分類在各個領域都扮演重要的角色，例如水質與食物的檢驗以及細菌感染的臨床診斷。尤其對於較嚴重的細菌感染症狀，如敗血症等，更仰賴即時且可靠的細菌檢驗技術，才能降低其致死率。然而，目前臨床所使用細菌檢驗方法須耗時一至三天，等待血液中的病原菌在血瓶機中培養到一定的濃度，以利後續的分析；若要進行細菌的抗生素藥敏試驗 (Antibiotic Susceptibility Test, AST)，則要再花費一至兩天的時間。因此，許多研究團隊致力於開發出新穎且更加快速的細菌檢測技術。表面增強拉曼散射 (surface-enhanced Raman scattering, SERS) 光學檢測技術，因具有高度專一性以及免分子標定的優點，已被應用於細菌檢測與藥敏試驗的研究領域。藉由分析細菌代謝物的分子光譜，此技術可減少抗生素藥敏試驗所花費的時間至數個小時；然而因病人血液中的病原菌濃度十分低，最耗時的血液培養流程仍是必須的步驟。為了解決這個問題，我們使用多孔性濾膜來捕捉並濃縮細菌，以增加細菌樣本的濃度進而縮短血液培養的流程。在此篇論文中，我們利用微流道系統整合多孔性濾膜與具有表面增強拉曼散射的底板，以達成細菌濃縮和其代謝物訊號的量測，藉此來檢測細菌並完成藥敏試驗。透過這個整合型系統，我們可以減少人工的操作步驟，在封閉的微流道中也能夠有較好的訊號均勻度，而最低可檢測到的細菌濃度則達到103/mL左右，因此能夠達成快速、免標定且即時的細菌檢測和抗生素藥敏試驗，並能大大減少檢驗中的人力使用與操作誤差。

Bacteria identification and characterization are significant issues in various research fields including microbial monitoring of water or food and clinical diagnosis of bacterial infection. A sensitive and reliable bacteria detection method is needed, especially for serious bacterial infection such as sepsis. Without timely and proper treatments, patients will suffer a high fatality rate. However, the conventional clinical bacteria detection process takes 1-3 days waiting for bacteria incubation in blood culture bottles in an automated culture system. Another 1-2 days are required for antibiotic susceptibility test (AST). Therefore, many researchers aim to develop rapid bacteria detection and AST techniques. Surface-enhanced Raman scattering (SERS), as a highly sensitive and label-free biomolecule detection method, can identify various bacteria species and understand antibiotic susceptibility by analyzing bacteria secreted metabolites. Although the time taken for AST can be reduced to several hours using SERS technique, the most time-consuming blood culturing process is still necessary since the bacteria concentration in patients’ blood is too low. To solve this problem, a bacteria enrichment technique, membrane filtration, is introduced in order to shorten the blood culturing time. In this thesis, a microfluidic system integrating membrane filtration and the SERS-active substrate was developed for performing on-chip bacterial enrichment and in-situ SERS measurements for bacteria detection and AST. With this integrated system, manual operating procedures are minimized and uniform SERS detection is ensured in the closed microfluidic chamber. Meanwhile, a meaningful SERS signal could be detected from a low concentration of bacteria (~103/mL). A rapid, label-free and real-time bacteria detection and AST technique can hence be achieved without intensive human labor and manual errors.