整合液相色層分析及表面增強型拉曼散射於單一微流道檢測平台

Abstract

細菌的鑑定及分類已廣泛應用於許多領域，例如: 食物、水質、敗血症等檢測。表面增強型拉曼散射 (surface-enhanced Raman scattering, SERS) 光學檢測技術因具有高專一性及非侵入性的優點，目前已被應用於細菌偵測的研究領域，其偵測的細菌指紋光譜是由細菌代謝的嘌呤衍生物所貢獻，此代謝混合物產生的光譜非常複雜，為了解決這個問題，我們發展了一個結合液相色層分析 (liquid chromatography, LC) 及表面增強型拉曼散射進行混合物分離及原位檢測的微流道平台 (LC-SERS microfluidic platform) 。首先，本論文使用具有螢光的FITC及R6G混合物進行此微流道系統功能表現的測試，藉由螢光訊號的記錄和表面增強型拉曼散射光譜偵測，此系統能夠成功地將兩種分子進行分離及原位檢測。接著，兩種具有相似表面增強型拉曼散射光譜的大腸桿菌代謝物―次黃嘌呤 (hypoxanthine) 及腺嘌呤 (adenine) 混合物亦驗證了此系統於生物分子的檢測表現。最後，尿嘧啶 (uracil) 及腺嘌呤 (adenine) 的實驗除了評量此系統的功能表現外也用來測試我們進一步建置的異地偵測平台。總結本論文之LC-SERS微流道平台有兩個重要的特色，其一，此平台之空間體積較傳統機台小一個數量級以上，其二，此平台能夠進行自動化並即時的流體控制，有效的減少樣本及試劑之使用量和交叉污染的問題。綜合以上所述，此高度整合之微型化LC-SERS流道系統平台能夠幫助細菌的偵測及監控並降低檢測成本，以實現更廣泛的臨床應用。

Bacteria identification and characterization reveal much important information in various fields, such as: food safety, water quality, sepsis and so on. Surface-enhanced Raman scattering (SERS) has been applied for bacteria identification due to its highly specific and non-invasive features. However, the SERS spectra of bacteria are usually very complicated due to their complex metabolite composition of purine derivatives. To address the above problem, we developed a microfluidic platform which enables on-chip liquid chromatography (LC) separation of multiple molecules followed by in situ SERS detection. A mixture of fluorescent FITC and R6G dyes was tested in the system to confirm its functionality. Their retention times were revealed by time-lapsed fluorescence imaging and SERS detection. Next, the system separated a mixture of hypoxanthine and adenine— the main purine metabolites of E. coli—and identified them with acquired SERS signatures. Then, a uracil and adenine mixture was tested to evaluate the performance of the platform. In the end, an ex situ SERS detection was introduced. The LC-SERS microfluidic platform holds two important features. First, the developed microfluidic LC column is just few centimeters long that is one order of magnitude shorter than commercial HPLC columns. Second, the microfluidic flow control system connected to the LC-SERS microfluidic device allows for automatic and real-time fluidic control, greatly reducing sample consumption and preventing any potential cross contamination. Accordingly, this highly integrated and miniaturized LC-SERS microfluidic system could greatly facilitate identification and monitoring of bacteria, relocating the entire process from modern microbiology laboratories with expensive facilities to actual clinical settings, which in turn enables punctually available diagnosis data.