新型雙酵素系統生化檢測器：偵測水楊酸酶新型隱藏式螢光探針合成

A novel bienzyme-platform biosensor：the synthesis of new latent fluorophore for salicylate hydroxylase.

鄧竣任(Chun-Jen Teng)

指導教授 ： 黃聲東

國立臺北科技大學/工程學院/化學工程研究所/碩士(2009年)

摘要

隱藏式螢光探針(Latent fluorophore)主要運用化學反應的方式將螢光分子作改質，使螢光分子的螢光消失或螢光的強度下降，並能藉由化學、催化反應將螢光分子釋放；設計三種不同程序方式合成對於羥化酵素水楊酸酶( Salicylate hydroxylase)具有專一作用之新型隱藏型式螢光探針SF。新型隱藏式螢光探針螢光顯色主要運用水楊酸酶( Salicylate hydroxylase)及NADH在有氧的情況下，使得水楊酸(Salicylate acid)進行去碳酸基反應(Decarboxylation)和羥化反應(Hydroxylation reaction)得到Catechol，再經由quinone-methide-rearrangement reaction釋放螢光分子。新隱藏式螢光探針對於偵測水楊酸酶(Salicylate hydroxylase)有很高的靈敏性，而螢光分子釋放在生理溫度的緩衝溶液中是自發且不可逆之過程。螢光分子所釋放出來的訊號(597nm)具有獨特的識別性質。本實驗結合雙酵素系統搭配脫氫酵素(dehydrogenase)氧化分析物，造成NAD+還原成NADH應用於偵測3-hydroxybutyrate(0.2~2 μM)，具有線性關係，其未來應用於生化檢測系統開發深具價值。

關鍵字

水楊酸酶隱藏型式螢光探針 ； 雙酵素系統

並列摘要

We synthesized a new latent fluorogenic probe SF to detect salicylate hydroxylase. Salicylate hydroxylase catalyzes a stoichiometric conversion of salicylate acid to catechol in the presence of NADH and oxygen. The fluorogenic chemical transformation of SF triggered by SHL is through a tandem reaction, decarboxylation, hydroxylation, and quinine-methide-rearrangement reaction, which are spontaneous and irreversible at physiological temperature in aqueous media. The fluorescence signal revealed by this process is specific and exhibited in the spectrum region with emission maxima at 597nm. Furthermore, we established a bienzyme system composed a dehydrogenase (3-hydroxybutyrate dehydrogenase) and Salicylate hydroxylase (SHL) to detect 3-hydroxybutyrate. The novel fluorimetric indicator SF demonstrated a good linear relationship in detecting 3 –hydroxybutyrate in 0.2 to 2.0 µM range, which presents to the applicability for the construction of biosensors in the future.

並列關鍵字

Salicylate hydroxylase ； Latent fluorophore ； Bienzyme system

參考文獻

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