開發大腸桿菌之雙訊號多巴胺全細胞生物感測器

Abstract

多巴胺為一兒茶酚胺類之神經傳導物質且在人體中扮演著不可或缺的角色，而不正常的多巴胺濃度會導致一些疾病的產生，如：帕金森氏症以及亨丁頓舞蹈症。因此檢測多巴胺是一門很重要的課題。我們設計了一組感測多巴胺之大腸桿菌全細胞生物感測器，運用大腸桿菌中單胺類調控組作為感測機制，並以紅色螢光蛋白作為訊號來源，針對多巴胺濃度能夠有良好的相關係數以及偵測極限 (1.43M)，且將紅色螢光蛋白置換為紫茉莉之4,5-多巴雙加氧酶 (MjDOD)，4,5-多巴雙加氧酶能將左旋多巴轉換為甜菜醛胺酸 (具有432nm特徵吸收峰，為甜菜黃色素之前驅物)，推測多巴胺也能透過4,5-多巴雙加氧酶催化而形成同樣具有432nm特徵吸收峰的6-去羧基甜菜醛胺酸，置換4,5-多巴雙加氧酶後在偵測多巴胺以及左旋多巴時，同時具有相當不錯的相關係數以及偵測極限，也經由酵素的置換，我們成功地消除了苯乙胺以及苯乙醛的干擾。最後我們結合紅色螢光蛋白以及4,5-多巴雙加氧酶，設計出足以區分苯乙胺、多巴胺、左旋多巴以及腎上腺素等四種類似物，透過偵測紅色螢光以及432nm特徵吸收峰，這四種類似物的訊號消長不盡相同，因此可以製作其特徵訊號圖譜。此生物感測器有著相當不錯的偵測極限以及區分多巴胺類似物的特性，在未來偵測與多巴胺相關的疾病能夠更加精準且有效。Dopamine, a catecholamine neurotransmitter, plays an important role in mammalian central nervous system. Abnormal concentration of dopamine in biological fluids causes several diseases such as Parkinson’s and Huntington’s disease. Therefore, it is an important research topic to develop a quantitative method to accurately estimate the level of dopamine. In this study, we designed a whole-cell biosensor for dopamine detection using monoamine regulon in Escherichia coli and RFP (red fluorescence protein) as a signal output. The detection of limit was 1.43M. Afterward, we replaced RFP with MjDOD (4,5-DOPA extradiol dioxygenase from Mirabilis Jalapa) . L-DOPA can be converted into betalamic acid (a precursor of betaxanthin and absorb at 432nm) by MjDOD. We proposed that dopamine can be converted into 6-decarboxylated betalamic acid by MjDOD. This biosensor could detect dopamine and L-DOPA with relatively high selectivity and without the interferences of phenethylamine and phenylacetaldehyde. Finally, phenethylamine, dopamine, L-DOPA, and (-)-Epinephrine can be distinguished with as-developed dual-signal biosensor carrying RFP and MjDOD simultaneously and each analogue has a unique fingerprint profile. With feature, this biosensor could potentially improve the accuracy and specificity in the diagnosis of dopamine.