建構電流式生訊感測器對格蘭氏陰性菌之偵測

Abstract

本篇論文研究為發展快速且靈敏的電流式生訊感測器應用於格蘭氏陰性菌的偵測。此電流式生訊感測器 (GM (-)-bacterial biosensor) 可辨識格蘭氏陰性菌是藉由在碳膠電極(SPCE)表面塗覆幾丁質來固定格蘭氏陰性菌的醣類辨識蛋白，本研究當中我們選擇常見的大腸桿菌當作模式陰性菌株，藉此驗證此感測器的可行性。當大腸桿菌因醣類辨識蛋白而吸附在碳膠電極時，我們利用同時接有二茂鐵 (ferrocene) 和甘露糖官能基(mannosyl)的奈米金粒子(f/m-GNP)進行辨識然後偵測，實驗結果發現每一個奈米金粒子直徑約為31奈米，粒子表面平均接有910個二茂鐵分子。此外，利用伏安法與植物凝集素聚集實驗證明：奈米金具有鍵結E. coli的能力。最後我們利用計時電流法，在無須額外添加反應試劑的情況下，對大腸桿菌進行定量分析。結果顯示，二茂鐵分子中心的二價鐵離子氧化成三價鐵離子時產生的法拉第電流訊號與E.coli濃度，兩者在2.92×104 ~ 2.92×108 CFU/mL範圍內呈現線性關係(R2 = 0. 9737)，其偵測極限為2.92×104 CFU/mL。對於格蘭氏陰性菌，我們所建構之電流式生訊感測器具有高度專一性。

An amperometric biosensor was developed in this study for the rapid and sensitive detection of gram-negative bacteria This amperometric biosensor (GM(-)-bacterial biosensor) was fabricated by immobilizing a gram-negative bacteria-sensitive glycoprotein on the screen-printed carbon paste electrode (SPCE) with chitosan. Escherichia coli was used a model of gram-negative bacteria to investigate the availability of the fabricated biosensor. The attached E. coli cells on the SPCE were then detected by the ferrocene and mannose-conjugated gold nanoparticle (f/m-GNP), ferrocene and mannosyl groups. Studies show that the developed f/m-GNP is water-dispersible with a diameter of around 31 nm and contains an average of 910 ferrocenes per f/m-GNP. The capability of f/m-GNPs to bind E. coli was demonstrated by cyclic voltammetry and its ability to aggregate with ConA. The reagentless detection of E. coli was performed by chronoamperometry. The results show that the faradaic anodic current (Fe2+ to Fe3+) is linearly proportional to the E. coli concentration between 2.92×104 and 2.92×108 CFU/mL (R2 = 0.9737). The lowest detection limit of GM(-)-bacterial biosensor was approximately 2.92×104 CFU/ml. The developed GM(-)-bacterial biosensor exhibits a extreme high specificity to gram-negative bacteria.