微透析取樣技術結合高效能液相層析法進行細菌生長過程中氧化壓力標記之動態分析研究

Combining microdialysis sampling and high-performance liquid chromatography for dynamic monitoring of oxidative stress marker during bacterial growth periods

林昕潔(Hsin-Chieh Lin)

指導教授 ： 黃友利

高雄醫學大學/醫學院/醫學研究所碩士班/碩士(2016年)

摘要

有氧環境下生存之微生物，在生長週期內受環境中活性氧化物質所導致的氧化性傷害是無可避免的，而其相關性早已被研究證實。為達到探討微生物之氧化性傷害之目的，現行研究方法、取樣以及樣品前處理步驟通常較為繁瑣與耗時。因此，開發一個能有效簡化取樣步驟且能連續取樣的樣品前處理技術有其必要性。本研究中，以開發「脂質過氧化產物於微生物生長過程之動態變化」的技術為目的。基於微透析取樣技術即時、連續取樣、樣品純化以及取樣過程對微生物生長影響低等優點，從而被使用於微生物培養液中脂質過氧化代謝終產物－丙二醛之連續取樣。在實驗中主要針對(1)建立微透析取樣技術於微生物培養基中最適化取樣參數進行探討以及(2)毒性微量元素對微生物生長週期中氧化性傷害之影響。依研究結果顯示，對於培養基中丙二醛的平均探針回收率為78.6 ± 0.9%；方法精密度之相對標準偏差為2.7 - 7.6%。為評估方法之實際應用性，以所建立之分析方法進行24小時連續檢測標準化菌株生長週期間丙二醛濃度之動態變化。此分析系統之建立於微生物抗性機轉或生物孵育等研究議題有其研究發展潛力。

關鍵字

微透析 ； HPLC-FL ； 肉湯培養基 ； 動態監測 ； 脂質過氧化

並列摘要

Organisms that grow aerobically are routinely exposed to oxidative stress in the form of reactive oxygen species. Monitoring the dynamic variations of oxidative stress allows us to understand its role in basic cellular function and determine mechanisms of antioxidation. In this study, microdialysis (MD) sampling was employed for continuous monitoring of the formation of malondialdehyde (MDA) in a bacterium-inoculated culture broth. To test the practicality of this approach, oxidative stress was induced by cadmium and then a 60-min interval was selected to collect sufficient amounts of dialysate for high performance liquid chromatography with fluorescence (HPLC-FL) detection. After optimization of this simple to operate, simultaneous, and continuous method for dynamic monitoring of MDA during periods of bacterial growth, a retrodialysis technique and a no-net-flux method were used to assess the probe recovery and analytical performance of the proposed system. The mean probe recovery of MDA was 78.6 ± 0.9%, with intra- and inter-day precisions of 2.7–6.1 and 3.5–7.6%, respectively. To evaluate the practicality of this method, the dynamic variations in the concentrations of MDA in standardized bacterial species (Staphylococcus aureus, ATCC® 29213TM) were monitored continuously for 24h. The analytical results confirmed that this MD sampling technique combined with HPLC-FL detection can be used to accurately and continuously monitor the levels of MDA in microbially inoculated culture broths.

並列關鍵字

Microdialysis ； High performance liquid chromatography ； Culture broth ； Dynamic monitoring ； Malondialdehyde

參考文獻

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