自動化洗菌之微流道與微流井晶片整合於表面增強拉曼散射進行細菌抗藥性檢測

An Automatic Medium Exchanging Microwell Microfluidic Device for SERS-based Antibiotics Susceptibility Test

廖證傑(Cheng-Chieh Liao)

指導教授 ： 黃念祖

國立臺灣大學/電機資訊學院/生醫電子與資訊學研究所/碩士(2020年)

https://doi.org/10.6342/NTU202003307

摘要

血流感染(Blood stream infection, BSI)是重要的全球公衛議題，也是急重症領域中的重大挑戰之一。即時且正確地使用抗生素是其治療關鍵。然而現行微生物檢測技術耗時且無法及時給予臨床醫師藥物選擇的參考資訊。為了提升抗生素用藥的精準度及降低近年來快速發展的抗藥性菌株，建立新的快速微生物檢測技術有其必要性。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性試驗。過去幾年，本研究團隊已使用表面增強拉曼光譜技術進行了一系列的微生物檢測實驗，發展出一套可使用在臨床檢體的快速抗生素敏感性檢測方法 (SERS-AST)。相較於傳統的細菌培養方式，此技術所需時間可縮短至16 ~ 18小時，且結果正確率高。為了繼續朝臨床應用端前進，我們希望將具有自動化與微環境控制優點的微流道系統整合至現行的SERS-AST流程中，以精簡化其中重複性高或複雜、耗時的多道步驟，如樣本的離心清洗、稀釋、濃縮等。預期不僅能節省人力、時間成本，亦降低了潛在的操作錯誤風險，同時，經由將細菌均勻地捕捉於尺寸最佳化的微流井中，將可提供小面積中多量測點的效益。目前的結果顯示我們的微流道自動化洗菌方式可達到和手動操作幾乎相同的效果，並且在2~3小時內完成大腸桿菌與金黃葡萄球菌的藥敏性試驗。

關鍵字

微流井 ； 自動化洗菌 ； 表面增強拉曼散射 ； 細菌抗藥性檢測

並列摘要

In the intensive care unit area, bloodstream infection has always been one of the most important public health issues in the world. Timely and effective antibiotics used to control infection is crucial for survival. However, the current antimicrobial testing technology is time-consuming and cannot provide clinicians timely drug selection. To improve the accuracy of antibiotic treatment and reduce the rapid evolution of drug-resistant strains, the establishment of new rapid microbial detection technology is necessary. Surface-enhanced Raman scattering (SERS) technology has been used in bacterial detection and antibiotics susceptibility tests (AST) due to its high specificity and label-free feature. In the past few years, our research team has used SERS technology to conduct a series of microbial testing experiments, and developed a rapid AST. Compared to the traditional bacterial culture method, the required time can be shortened to 16-18 hours with highly accurate results. To integrate with clinical workflow, we integrate the microfluidics system to automate sample processes, such as centrifugal-based washing, dilution, and concentration of samples in SERS-AST process. The system can not only saves the man power and time but also reduces the potential operating human errors. By capturing bacteria in microwells with optimized size uniformly, it could also provide the benefits of multiple-point measurement in a small sensing area. Overall, our automated microfluidic system enables a great bacteria washing followed by the drug sensitivity test of E. coli and S. aureus within 2-3 hours.

並列關鍵字

Microwell ； Automatic medium exchange ； Surface-enhanced Raman scattering ； Antibiotics susceptibility tests

參考文獻

1. Rudd, K.E., et al., Global, regional, and national sepsis incidence and mortality, 1990–2017: analysis for the Global Burden of Disease Study. The Lancet, 2020. 395(10219): p. 200-211.

2. Puskarich, M.A., et al., Association between timing of antibiotic administration and mortality from septic shock in patients treated with a quantitative resuscitation protocol. Critical care medicine, 2011. 39(9): p. 2066.

3. Puttaswamy, S., et al., A Comprehensive Review of the Present and Future Antibiotic Susceptibility Testing (AST) Systems. Archives of Clinical Microbiology, 2018. 09(03).

4. Khan, Z.A., M.F. Siddiqui, and S. Park, Current and Emerging Methods of Antibiotic Susceptibility Testing. Diagnostics (Basel), 2019. 9(2).

5. Jorgensen, J.H. and M.J. Ferraro, Antimicrobial susceptibility testing: a review of general principles and contemporary practices. Clin Infect Dis, 2009. 49(11): p. 1749-55.