優化全細胞生物感測器對苯丙胺酸與食品檢測之應用

Abstract

苯丙胺酸是人體中的必需胺基酸，醫學研究發現患者若患有苯丙酮尿症，他們的血液和尿液中苯丙胺酸濃度會升高。我們實驗室以往使用全細胞生物感測器偵測苯丙酮尿症時，因結構類似物酪胺酸干擾，故我們建構了雙質體全細胞生物感測器來解決這個問題，一個質體含有苯丙胺酸轉運蛋白(PheP)，可增加苯丙胺酸利用效率；另一個質體含有突變PtyrP啟動子(mTCG)開啟紅色螢光蛋白，以降低酪胺酸影響。使用 19 種胺基酸和苯丙酮尿症的常見代謝物進行選擇性試驗，與單質體全細胞生物感測器相比，所有化合物的干擾均顯著降低。苯丙酮尿症患者透過藥物與飲食控制苯丙胺酸體內濃度，故我們期望開發檢測食品的全細胞生物感測器，使用碳酸飲料為真實樣品，雙質體系統在複雜的環境中，亦能不受基質干擾準確地檢測我們的分析物。Phenylalanine (Phe) is an essential amino acid in the human body. According to medical researchs, patients with phenylketonuria have increased levels of phenylalanine in their blood and urine. Using a whole-cell biosensor to detect phenylalanine is challenging due to the interference of structural analogs (e.g. Tyr). Herein, we constructed the dual-plasmid whole-cell biosensor to solve the problem. One plasmid contains a phenylalanine transporter, which increases the intracellular availability of phenylalanine. The other plasmid contains a red fluorescence protein driven by mutated Tyrp promoter, which makes the promoter region less likely to be affected by tyrosine. Nineteen amino acids and the common metabolites of phenylketonuria were used to perform the selectivity test. The results showed that all compounds have a significant reduction in interference compared with the single plasmid whole-cell biosensors. Finally, we chose soft drinks as the real sample. The higher selectivity is the major advantages of this dual-plasmid whole-cell biosensor.