使用全細胞生物感測器在微流道設備中同時測定L-苯丙胺酸、苯乙胺和苯乙酸

Abstract

苯乙胺 (PEA) 是一種重要的神經傳遞物質，容易被氧化成苯乙酸 (PA)。PEA 和 PA 都是人體中苯丙胺酸 (Phe) 的代謝產物，並且是診斷苯丙酮尿症(PKU) 的重要指標。在這項研究中，透過全細胞生物感測器的多重輸出信號可同時並特異性地檢測三種分析物 (PEA、PA 和 Phe)。 tyrP 啟動子用於反應 Phe水平控制綠色螢光蛋白信號的產生， FeaR 調節劑用於指示 PEA 的存在，而 Paa 調節劑則用於檢測 PA，三種感測器的組合可以根據各別顏色輸出對三種分析物進行半定量而不會產生交叉干擾。透過優化各種模組件 (核醣體結合位點和螢光蛋白)，以確保快速生成螢光信號，並將生物感測器運用在微流道設備中，以減少即時檢驗中的樣品消耗。此外，本研究進一步針對苯丙酮尿症患者所開發的雙信號輸出生物感測器，能特異性偵測 Phe 及 PA，以減少混合菌液的步驟，更方便患者的操作及檢測。The neurotransmitter phenylethylamine (PEA) is highly susceptible to oxidization to produce phenylacetic acid (PA). The fact that PEA and PA are both metabolites of phenylalanine (Phe) in humans makes them important indicators in the diagnosis of phenylketonuria (PKU). In this study, we developed a whole-cell biosensor for the simultaneous detection of these three analytes (Phe, PEA, and PA). The tyrP promoter is used to control the production of green fluorescent protein signals in response to Phe levels. The FeaR regulon is used to indicate the presence of PEA, whereas the Paa regulon is used for the detection of PA. The combination of three sensor strains together makes it possible to semi-quantify three analytes according to unique color outputs without cross interference. We sought to optimize various modular components (ribosomal binding sites and fluorescent proteins) to ensure the rapid generation of fluorescent signals. Lastly, the biosensors were implemented within a microchannel device to reduce sample consumption in point-of-care assays. Furthermore, the dual-signal output biosensor developed for PKU patients can specifically detect Phe and PA to reduce the steps of mixing and make it easier for patients to operate.