微流道抗生素濃度梯度產生器整合於表面增強拉曼散射平行進行細菌抗藥性檢測

A Microfluidic Antibiotic Concentration Gradient Generator Integrating Surface enhanced Raman Spectroscopy for Multiparallel Antimicrobial Susceptibility Testing

林上竣(Shang-Jyun Lin)

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(2021年)

https://doi.org/10.6342/NTU202104148

摘要

為了提供適當的抗生素治療，臨床應用上的標準做法是將從病人檢體分離出來的菌株進行抗藥性檢測（antimicrobial susceptibility test，AST），來選擇合適的抗生素種類和劑量，同時也可以避免抗生素的濫用。然而，目前的AST方法仍有耗時、耗力、低準確性等缺點。為了提高準確性，具有高專一性和免標定等優點的表面增強拉曼散射（surface-enhanced Raman spectroscopy，SERS）開始被應用在細菌檢測和抗藥性檢測上。在本論文中，我們設計了一個微流道裝置，具有可用來建立抗生素濃度梯度的側流道（side channel），及在液體流動時，可將細菌保留在其中的微流井（microwell）。透過使用針筒幫浦同時注入細菌培養液與抗生素溶液，此裝置可以自動建立2、4、8、16、32、64 μg/mL等六個抗生素濃度。在螢光珠模擬細菌的實驗中，得知微流井可保留80%以上的螢光珠避免被沖走。接著，當細菌在建立好的各個抗生素濃度下培養三個小時後，即可整合SERS檢測技術進行AST（SERS-AST）。本論文研究共針對兩株細菌進行AST，分別為氨苄青黴素（ampicillin）非抗藥性與抗藥性的大腸桿菌（Escherichia coli）。所有微流井的SERS訊號皆會被量測，經分析即可測得最小抑菌濃度（minimum inhibitory concentration，MIC），且該結果與標準AST作法的結果一致。此SERS-AST方法不僅只需要少量（20 μL）細菌溶液與單一微流道裝置與即可測得MIC，更是將整個AST過程從一天大幅縮短至5小時，如此一來可以幫助醫生及早修正施用的抗生素種類和劑量，進一步提高病患存活率並減少抗生素的濫用。

關鍵字

微流道 ； 濃度梯度 ； 細菌 ； 抗藥性檢測 ； 表面增強拉曼散射

並列摘要

To ensure appropriate antibiotic treatment, antimicrobial susceptibility test (AST) is a standard method in clinical therapies for selecting proper antibiotic treatment and preventing antibiotic misuse or overuse. However, current AST methods still suffer from time-consuming, label-intensive, and low accuracy issues. To address above issues, surface-enhanced Raman spectroscopy (SERS) technology has been used in bacterial detection and AST based on its high specificity and label-free features. In this thesis, we designed a microfluidic device with branch channels for antibiotic concentration gradient generation and microwells for trapping bacteria during the fluidic introduction, including reagent gradient generation and culture medium removal steps. Operated by the syringe pump, the bacteria culture medium and antibiotics are injected into the microfluidic device to automatically generate a wide range of antibiotic concentrations, from 2 to 64 μg/mL, which increased by the power of two. In the fluorescent beads experiments, over 80% of beads were trapped inside the microwells, which prevents the beads be washed away during gradient generation. Then, after 3-hour-incubation, the device is integrated with SERS substrate for SERS-AST. Finally, two Escherichia coli strains, susceptible and resistant to ampicillin, are applied in AST. The SERS signal of all microwells in each side channel was analyzed to obtain the minimum inhibitory concentration (MIC). The result is consistent with the gold standard method. With this microfluidic device in SERS-AST, only 20 μL of bacteria solution and a single chip are required to obtain MIC. Moreover, this SERS-AST process only requires 5 hours, much faster than the current gold standard methods. Therefore, the antibiotic treatment can be modified earlier, which can increase the survival rate and prevent antibiotic misuse or overuse.

並列關鍵字

microfluidic ； concentration gradient ； bacteria ； antimicrobial susceptibility test ； surface-enhanced Raman spectroscopy

參考文獻

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