開發Fenton-like催化螢光連線分析系統應用於活體動物大腦葡萄糖動態變化偵測

Development of Online Fenton-like Catalytic Fluorescence Analytical Systems for In-vivo Monitoring Extracellular Brain Glucose

陳震宇(Chen-Yu Chen)

指導教授 ： 孫毓璋

國立清華大學/原子科學院/生醫工程與環境科學系/碩士(2013年)

摘要

由於人類對於宇宙真理永無止盡的好奇，促使科學家們積極投入分析技術的研究與開發，而日益精進的各項分析技術則使人類終於得以稍稍窺探生物體中各種機制的箇中精妙。做為絕大多數生命與中樞大腦的主要能量來源，葡萄糖代謝行為牽涉到的能量流轉一直以來都是生物醫學領域亟欲掌握的關鍵資訊；尤其以阿茲海默症與帕金森氏症等各種大腦機能性異常的疾病，幾乎無可避免地連帶影響特定腦部區域產生明顯的能量代謝變化。因此，如何提供大腦特定區域的葡萄糖動態資訊便成為相關研究中，一個相當受到重視的議題。
綜觀目前在探討葡萄糖偵測的文獻中，主要技術分別為以測繪能力見長的正子斷層掃描、靈敏度掛帥的電化學探針與新興的奈米螢光技術。然而，縱然葡萄糖檢測技術發展歷史悠久，實質檢測上仍遇到相當多的困難與瓶頸，如提供特定區域分析資訊的空間解析能力、長時間分析抵抗環境干擾的能力以及於生物體中使用奈米材料的諸多疑慮。為克服上述問題並提供方便、便宜且可進行長時間精準監測的解決方案，本研究成功開發兩套分析效能卓越的檢測技術，分別為以盤式儀進行高通量分析的檢測技術與自動化連線即時監測系統。根據實驗結果可知，本研究所建立的分析技術兼具微透析探針取樣的空間解析度與螢光探針的高靈敏度二項優點。
本研究也是目前文獻中，首度將螢光連線分析方法與銅離子催化Fenton-like反應成功結合的分析技術。與未經催化增強的螢光方法相比，本研究將整體分析時間縮減4倍以上，且靈敏度提升130倍以上，並具有偵測極限100 μM、量測線性範圍100 μM至10 mM的優異表現。
根據本研究於活體動物實驗所獲得的結果，當大腦細胞間液的離子平衡遭到高量鉀離子入侵而破壞時，將立即誘發周圍腦細胞及神經細胞極化及去極化的反應，而能量攝取也會因而大幅增加，於短時間內即造成胞外葡萄糖濃度急速下降，而當刺激因素消除後，其生理平衡機制隨即啟動，迅速使胞外葡萄糖濃度回復正常水準。藉本研究中獲得的各項實驗成果可知，以本研究發展的高通量的連線分析系統，確實可對活體動物大腦細胞間液中的葡萄糖濃度進行定量，並監測其長時間動態變化。

關鍵字

葡萄糖 ； Fenton催化反應 ； 螢光 ； 線上分析

並列摘要

Development of methods for direct and rapid measurement of glucose concentration in the intact organ of animals permits the study of the kinetics of the energy balance in the brain. To monitor the dynamic variation in the concentration of extracellular glucose in rat brain, in this study, we designed two novel glucose monitoring systems, which are comprised of microdialysis sampling and Cu2+-enhanced fluorescence turn-on detection devises. To measure the content of glucose in microdialysate samples, we used glucose oxidase to transform glucose into H2O2, and used 2',7'-dichlorfluorescin-diacetate (DCFH-DA) as fluorescent probe. However, a long incubation time was needed (60 min at room temperature) prior to the fluorescence measurement if only DCFH-DA was treated to react with H2O2.
To conquer this embarrassments, we have successfully used a Fenton-like reaction involving the conversion of H2O2 to hydroxyl radical by aqueous Cu2+ ions, which acted as ionic catalyst, to not only shortened the incubation time, but also reached a higher sensitivity. Based on the analytical results obtained, the detection limit of our developed systems was as low as 100 μM, which was low enough for the determination of basal concentration of extracellular glucose in the brain (2 mM). Furthermore, the linear range was ranging from 100μM to 10 mM, which was also wide enough to cover the brain extracellular glucose range of rats. It also indicated that our developed systems can not only be used for the continuous monitoring of brain glucose, but provided necessary evidence for the study of brain energy metabolism.
According to the results obtained from the animal experiments, the extracellular glucose concentration would drop simultaneously due to the raised energy consumption rate when the original ionic balance of extracellular environment had been perturbed by treating high doses of K+ ion. However, based on the natural mechanism, the balance of glucose would be rebuilt when the extracellular potassium concentration returned to normal value. Based on the superiority and uniqueness of our developed methods, it is expected that sufficient solid evidences would clarify the mechanism of brain glucose metabolism.

並列關鍵字

Glucose ； Fenton Reaction ； Fluorescence ； on-line analysis

參考文獻

13 行政院衛生署國民健康局. 遠離糖尿病完全學習手冊. (2006).

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