先前研究已知綠色螢光蛋白發光團藉由其蛋白包覆，會使得發光團穩定而釋放強烈綠色螢光，相對的若是發光團脫離蛋白時，其螢光則會大幅下降。因 DNA 帶負電，所以本實驗將間位吡啶綠色螢光蛋白發光團類似物，進一步甲基化後使之帶正電，應用於 DNA 偵測上。利用可見光-紫外分光光譜儀以及螢光光譜儀檢測，並比較嵌合前後的光物理特性，發現於定量之 m4-Bn 逐漸滴定 DNA 後會使之吸收值及螢光強度皆上升，由 Benesi-Hildebrand plot 計算 m4-Bn 與 DNA 含氮鹼基之結合常數為 4.5x105。與已知之 DNA 螢光染劑如 EtBr、acridine orange 之結合常數相比，m4-Bn 與 DNA 的結合常數是較強的。因此我們認為 m4-Bn 可作為綠色螢光蛋白發光團類似物偵測 DNA 的前驅物。
過去的文獻顯示，螢光蛋白單分子之螢光光譜中發現具有閃爍現象。本實驗則利用綠色螢光蛋白發光團在不同溶液中，模擬其在蛋白中被牽制住之情況。利用 NMR、可見光-紫外分光光譜儀以及螢光光譜儀監測其光異構化轉換率、莫爾吸收係數、螢光量子產率並搭配 sigma-plot 軟體計算出熱異構化速率常數。將 p-HBDI 於溶劑 H2O 與 D2O 相比，發現溶於 H2O 中之中性分子與陰離子之熱異構化速率常數皆約為溶於 D2O 之值的兩倍。利用溶劑同位素效應及分餾係數計算，推測 p-HBDI 之 trans→cis 熱異構化反應中會與溶劑質子進行交換，其反應中間體會發生電荷轉移後回復為 cis conformation。將 p-HBDI 溶於不同介電常數之溶劑中，則可以由介電常數越高而其最大吸收波長與螢光放光波長紅位移現象越明顯之現象證明，當 p-HBDI 於溶劑中時發生電荷轉移之現象而之極性變高，因此於高極性之溶劑越能穩定它而使得紅位移現象發生。而若將 p-HBDI 放在高黏度溶劑如甘油中，其莫爾吸收係數及螢光量子產率較高，而熱異構化速率常數則遠小於其於低黏度溶劑水之值。將 p-HBDI 照射波長 350 nm 之光源後得其光異構化之 trans form ，並與其 cis form 比較後發現， p-HBDI 之 trans form 之螢光強度遠小於 cis form。並且將對位三級胺取代之綠色螢光蛋白發光團類似物(n3-Bn) 之 trans/cis form 比較也有相同結果。因此基於種種實驗結果，合理推測螢光蛋白之閃爍現象，因 p-HBDI trans form 之螢光極低，所造成閃爍效應中的無螢光態，於激發態衰退中有經過順反異構化，並且是透過 I-bond 之旋轉回 cis form 時則可以恢復至螢光態，因此使之產生一亮(螢光態)一暗(非螢光態)之閃爍現象。

We design a m‐pyridinium analogue of GFP chromophore(m4-Bn) to sense DNA. When it binds to DNA with a high binding constant of 4.5x105, both of the absorption and fluorescence emission intensity of m4-Bn are increased. Hence these results could be offered as evidence that m4-Bn can be a lead compound of GFP chromophore for fluorescence detection of DNA.
An interesting property of GFP shows that switching between fluorescent and nonfluorescent states are in the same timescale. However, the dynamic processes of this peculiar blinking behavior is still unclear. Hence we put p-HBDI in different solvents to simulate the states in GFP. The thermal-isomerzation rate constant of p-HBDI in H2O is twice as high as that in D2O. Calculating the ratio of kD2O/kH2O=2.041 by fractionation factors of solvent isotope effects, is the same as our hypothesis that p-HBDI, trans→cis thermal-isomerization will lead to proton transfer with solvent and also via charge transfer back to cis conformation. With a higher dielectric constant solvents p-HBDI, the absorption and fluorescence wavelength red shift phenomena would be greater. It proves that p-HBDI has a charged transfer in its transitional state that contributes to a higher dielectric constant solvents which stabilize it better. With p-HBDI in high viscosity solvents and excited by UV lamp with 350 nm wavelength, we can monitor the higher absorption and fluorescence emission values but with a much lower rate constant of thermal-isomerization. When we compare p-HBDI cis form with trans form, we find that the fluorescence quantum yield of trans form is much smaller. p-trimethylammonium analogues of GFP(n3-Bn) is the same as p-HBDI. Thus we can surmise blinking behavior of GFP is related to p-HBDI cis/trans-isomerzation, and its excited state decay of p-HBDI is via I-bond rotation to cis form.

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