以螢光共振能量轉移原理發展對銀離子具有選擇性的生物感測器

Abstract

銀離子獨特的抗菌能力被廣泛的被應用於醫療與生技產業，然而環境中過多的銀離子卻也會造成人類與生物體的危害。生物體為了保持體內金屬離子的平衡，發展一系列金屬調節機制，避免金屬離子可能引起細胞毒性。在實驗中，我們利用CupR建構以螢光共振能量轉移之生物感測器。CupR為MerR家族轉錄調控蛋白，來自耐金屬貪銅菌 (Cupriavidus metallidurans CH34) 。此生物感測器對銀離子具有良好專一性與選擇性，利用%FRET對銀離子濃度作圖，其結果在濃度0 - 10 µM具有良好的線性關係，最低偵測極限為0.45 µM，低於WHO所規範銀離子濃度之數值。我們嘗試比較感測器在不同溫度以及pH值時的蛋白質穩定性，並且測試不同的溶劑介質中，都有良好的檢測結果。此外，將蛋白質感測器製作於瓊脂糖凝膠中，增加感測的攜帶性及穩定性。Silver ions are widely used for biomedical applications and antimicrobial agents. However, silver ions are highly poisonous and could be harmful and accumulate in human and living organisms. In order to maintain the balance of metal ions, organisms evolve series of metal regulation mechanisms to prevent the accumulation of metal ions to reduce cytotoxicity. In this work, we used CupR to construct a fluorescence resonance energy transfer (FRET) -based protein biosensor. CupR is a MerR family transcriptional regulator protein (from Cupriavidus metallidurans CH34). This biosensor exhibited high specificity and selectivity for silver ions. There was a linear relationship between %FRET and silver concentration response ranging from 0 to 10 µM. The limit of detection was 0.45 µM which was less than the maximal levels accepted by WHO. We examined the performance of biosensor at different temperature and pH to compare the stability of proteins. The samples in different aqueous media were successfully detected by our biosensor. Furthermore, agarose protein gel was used to increases the feasibility of portable and stable biosensors.