開發以4,5-多巴雙加氧酶為基礎之全細胞生物感測器用於檢測二價銅離子及多巴胺

Abstract

左旋多巴經由全細胞生成之4,5-多巴雙加氧酶能將其催化成甜菜醛胺酸，透過加入2-胺基對苯二甲酸 (2-AIPA) ，可進一步生成具有紅螢光的甜菜色素 (2-AIPA-BX)。本研究開發了一種簡單且經濟的方法，可大量合成甜菜色素用於二價銅離子檢測，在銅離子濃度為25~500 μM的範圍內，有隨著濃度提升而紅螢光下降的趨勢，呈現出良好的線性 (R = 0.99) 且最低偵測極限為21.92 μM，也成功將2-AIPA-BX應用於人工尿液樣品及紙基材上的檢測，以簡單、低樣品耗量、低成本且便攜式的設置進行二價銅離子的檢測。此外，本研究也經由4,5-多巴雙加氧酶上特定的胺基酸位置突變以改變其反應的選擇性，成功使其反應選擇性從左旋多巴變成多巴胺，證實此方法的可行性及未來發展性。

In the human body, copper ions participate in the catalysis of many enzymes. In this study, we developed a simple and economical method for the green synthesis of Cu2+ sensors based on 2-aminoterephthalic acid–betaxanthin (2-AIPA–BX). 2-AIPA–BX was synthesized by coupling 2-aminoterephthalic acid and betalamic acid produced from DOPA-extradiol-4,5-dioxygenase in situ. It has a high degree of selectivity for Cu2+ over interfering metal ions. In the range of Cu2+ concentration of 25~500 μM, 2-AIPA-BX had a tendency to lessen the fluorescence as the concentration increases, the result showed a good linearity and the limit of detection was 21.92 μM. Additionally, we successfully incorporated 2-AIPA–BX into paper-based analytical devices to facilitate simple, inexpensive, and portable setup with lower sample consumption for onsite monitoring of environmental and biological samples. Dopamine is a neurotransmitter that plays an important role in the function of the central nervous system and cardiovascular system. In this study, the specific amino acid mutation on DOPA 4,5-dioxygenase was used to change the selectivity of its reaction, and its reaction activity was successfully changed from L-DOPA to dopamine, confirming the feasibility and future development of this method.