本實驗為設計分子間之級聯螢光共振能量轉移。此系統使用芘，3-乙醯基-7-N,N-二乙基香豆素(香豆素衍生物)和吖啶橙之乙醇溶液。芘和吖啶橙之混和溶液顯示有限的螢光共振能量轉移，因此藉由香豆素衍生物連結由芘到吖啶橙之螢光共振能量轉移。在此三種染料系統下，香豆素衍生物扮演雙重角色，其接受來自芘的能量(接受者)以及提供吖啶橙能量(捐贈者)。此現象可藉由三成份間的光譜重疊解釋，即: 芘的螢光帶重疊於香豆素衍生物的紫外光可見光吸收帶以及香豆素衍生物的螢光帶重疊於吖啶橙的紫外光可見光吸收帶。另外，藉由染料溶液的螢光強度與紫外光可見光吸收度來計算螢光共振能量轉移之相關參數，參數如下: 芘的量子效率(Ф)和能量轉移效率(ФT)、芘與香豆素衍生物之分子間距離(r1)、芘與吖啶橙之分子間距離(r3)以及芘與香豆素衍生物間的能量轉移速率常數(kT)。在此，會影響分析及計算之其它相關參數亦被仔細探討。例如:pH與螢光之相關性以及利用芘的I3/I1 和I5/I1之比值來分析染料混和溶液之極性變化。由上述之參數與條件，進而確認此三種染料之級聯螢光共振能量轉移之最大能量轉移效率。此外，香豆素衍生物加入芘溶液後，芘的猝滅行為可藉由Stern-Volmer定律分析得知。本研究之結果亦可應用於生物化學以及光電裝置之研究。

In this study, an intermolecular cascade fluorescence resonance energy transfer (cascade FRET) was designed. For this system, ethanol solutions of pyrene (Py), 3-acetyl-7-N,N-diethylcoumarin (AC) and acridine orange (AO) were used. While the mixture of Py and AO solutions showed very limited FRET, it was observed that AC bridged the FRET from Py to AO. In the three dyes system, AC played a dual role, an acceptor for Py and a donor for AO. This phenomenon can be explained by the spectral overlaps between three components; between the emission band of Py and the UV-visible absorption band of AC, and between the emission band of AC and the UV-visible absorption band of AO. The parameters related to the FRET were calculated from the emission intensity and UV-visible absorbance of the solutions: quantum yield (Ф) of Py, energy transfer efficiency (ФT) of Py, distance (r1 or r3) between Py and AC or AO, respectively, rate constant (kT) for energy transfer between Py and AC. For these analyses, other parameters which could affect the calculations were carefully examined, such as the pH dependency on fluorescence and the polar variations in the mixed solutions, by the I3/I1 and I5/I1 of Py. Then, the condition to provide the maximum energy transfer efficiency was confirmed for the cascade FRET consisting of three components. Collaterally, the quenching behavior on Py was analyzed by Stern-Volmer law for AC added into the Py solution. These results are applicable to develop biochemical probes and photoelectric devices.

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