發展可程式編輯之自動化樣品稀釋系統及螢火蟲螢光素酶反應自發放光顏色變化之觀測

Abstract

分析化學仰賴基本的化學知識、新儀器的發展以及解釋實驗數據的能力。在碩士班的研究當中，我著重在發展自動化儀器系統以及與生物放光相關之生化實驗。第一個專題中(第二章)我發展了一個可程式編輯之自動化樣品稀釋系統，此系統主要用來簡化傳統的化學稀釋程序，可用於製備不同濃度的標準品，以利實驗校正；稀釋亦可用來降低複雜樣品的基質效應。在此稀釋系統中，小段的樣品溶液和溶劑交替注入流動的管線中，並在管線中混合均勻。稀釋係數由所注入樣品和溶劑的體積比例定義。小段的樣品及溶劑可在管內藉著平流、擴散、紊流等方式混合均勻。第二個專題(第三章)為螢火蟲螢光素酶催化反應之生物/化學放光的基礎研究，主要觀測結果為酵素催化反應中自發放光之顏色由綠色轉為紅色。三磷酸腺苷是螢光素酶催化反應之受質之一，當三磷酸腺苷溶液以人為方式週期性地添加到反應之中，或是三磷酸腺苷在一個自催化反應的幫助下被自動合成，在以上兩種情況下，螢火蟲螢光素酶催化反應的自發放光顏色發生變化。在自催化反應中，顏色變化時酸鹼值並無改變。透過分析酵素反應放光影像的紅色和綠色通道的數值，指出紅色放光和綠色放光可能為兩種不同的發光動力學，也就代表同時存在兩種或兩種以上的放光途徑，然而放光途徑已在螢光素酶化學領域中被討論。基於得到的實驗結果，研究放光反應的特性必須同時收集光譜圖和即時的影像數據。以上的觀測結果在使用螢光素酶的生物影像具有潛在的應用，因為在雙色發光系統中未預期之顏色改變將會影響對於結果的判讀。螢火蟲螢光素酶所催化的自發放光顏色變化可用在生物醫學光電系統，生物醫學光電系統中需要放射光的即時顏色變化。總結，我的研究包含了稀釋系統的儀器設計以及螢火蟲螢光素酶反應自發放光顏色的變化觀測。

Analytical science relies on the knowledge of basic chemistry as well as the ability to develop new instrumentation and to interpret experimental data. In my graduate research work, I focused on the development of automated instrumentation and biochemical studies involving bioluminescence. My first project (Chapter 2) encompassed development of an automated programmable on-line sample dilution system. The developed dilution system simplifies a common chemical procedure—dilution—which is carried out in the chemistry laboratories to prepare standard solutions with different concentrations for assay calibration, and to reduce matrix effects while handling complex samples. Here, plugs of sample solution and solvent are injected interchangeably to a flow line. The ratio of plug volumes determines the dilution factor. The mixing of the sample and solvent plugs is achieved due to dispersion (advection, diffusion, turbulent mixing). My second project (Chapter 3) encompassed a fundamental study of bio/chemi-luminescence in the reaction catalyzed by unmodified firefly luciferase enzyme. The major observation is the spontaneous color change from green to red. Adenosine triphosphate (ATP) is the substrate of the luciferase reaction. When ATP solution was added periodically, or when ATP was synthesized in the same test tube with the aid of an autocatalytic reaction occurring simultaneously, a temporal color change occurred. In the latter case, the color change was not accompanied by pH change. Numerical analysis of the red and green channels points to dissimilar kinetics of the green and red luminescence, suggesting the existence of two or more luminescence pathways, what is discussed in the context of the current understanding of the luciferase chemistry. Based on these results, characterization of luminescence reactions must always include collection of both spectral and temporal data. The above finding has practical implications on the implementation of dual-color biosensing/imaging protocols involving luciferase enzymes, in which case an unexpected color change could lead to artefactual results. The spontaneous luminescence color switching of firefly luciferase can be utilized in the engineered biophotonic systems where there is a need to produce temporal changes of the color of emitted light. In summary, my graduate research work contributed to the development of instrumentation for chemistry laboratory (dilution system), and led to an unexpected observation of temporal color changes of luciferase luminescence.