利用生物方法檢測環境中的重金屬砷、鎘、汞

Abstract

近年來，高度的工業發展導致許多重金屬排放到環境中，由於重金屬不可被分解及易累積於生物體內之特性，目前已影響世界眾多人口的健康。化學檢測法雖然具有高靈敏度及準確度，然而其價格高以及檢測耗時，難以應用於大規模的樣品篩測，因此本研究欲利用生物方法檢測環境中的重金屬(砷、鎘、汞)，期能和化學方法互補。本研究分為2個目標，建構專一型及廣效型生物感測器用以檢測重金屬(砷、鎘、汞)。目標1為使用Escherichia. coli DH5α為宿主細胞，以arsR、cadC、merR與luxCDABE之基因組合，建構以生物冷光為訊號，分別可檢測砷、鎘、汞之專一型生物感測器，並應用於多重金屬之地下水檢測。結果顯示，本研究建構之專一型生物感測器，最低檢測極限分別為: 砷7.5 μg/l、鎘1 μg/l、汞5 μg/l，檢測時間皆為2-3小時之間即有偵測，於含多重金屬之地下水樣品的檢測上，砷以及汞之專一型生物感測器於地下水樣品中皆可準確的辨識砷(500 μg/l)與汞(20 μg/l)的含量。目標2為建構廣效型生物感測器，從陽明山小油坑土壤篩選現地嗜酸性鐵氧化菌，並利用重金屬干擾鐵氧化菌之鐵氧化原理，建構以顏色差異為訊號，可目測或是使用光度計法檢測水中重金屬之廣效型生物感測器，本研究篩選出之現地細菌Y10，經由16S rDNA片段定序及比對後，與Gram-positive iron-oxidizing acidophile Y0010具有100%相似，Y10之最佳生長環境為同時添加四硫磺酸根與葡萄糖之Thiobacillus caldus medium 中生長，最佳生長條件為溫度45℃、pH值2.5。菌株Y10能檢測出超過10倍環保署放流水濃度之混合重金屬，檢測時間為45分鐘，顯示其可做於廣效型生物感測器之應用。本研究所建構之專一型與廣效型生物感測器，具有成本低且檢測時間快速之優勢，可與化學分析方法進行互補，提升重金屬檢測效率。

In recent years, growing industry causes heavy metals releasing into the environment. Because of its non-degradable and high accumulation in living organisms, heavy metals have threaten human health in many countries. Despite chemical analysis has high sensitive and accuracy, it has limitation for large scale screening due to the high cost and time consuming. The goal of this study is to develop biological methods to detect heavy metals (arsenic, cadmium, mercury) in the environment in order to comprise current chemical methods. There are two specific aims in this study, including development of (i) specific and (ii) non-specific biosensors for the detection of heavy metals (arsenic, cadmium, mercury). In specific aim 1, three luminescent-based specific biosensors were constructed by transforming the arsR-luxCDABE, cadC-luxCDABE, merR-luxCDABE recombinant gene cassette into E. coli DH5 for detection of arsenic, cadmium, and mercury, respectively. These specific biosensors were also used for detecting multiple heavy metals in ground water. The results showed that the specific biosensor can detect arsenic 7.5 μg/l, cadmium 1 μg/l, mercury 5 μg/l in 2-3 hours. In addition, the arsenic and mercury biosensor can distinguish As3+ and Hg2+ accurately in ground water spiked with multiple heavy metals. In specific aim 2, one acidic bacterium Y10 was isolated and characterized from the soil collected from Yangmingshan. The non-specific biosensor was also examined based on the inhibition of the heavy metals to iron-oxidizing ability of this bacterium. Based on the 16S rDNA sequence analysis, the bacterium Y10 has 100% sequence similarity to Gram-positive iron-oxidizing acidophile Y0010. The results showed that Thiobacillus caldus medium supplymented with both S4O62- and glucose is the best growing medium for Y10 and the optimum temperature and pH is 45℃ and pH 2.5, respectively. In addition, Y10 can detect 10 x EPA effluent standard concentration of multiple heavy metals in 45 minute, suggesting that this bacterium can be used as non-specific biosensors to detect heavy metals. In conclusion, the low cost and rapid screening of specific and non-specific heavy metal biosensor (As, Cd, Hg) can not only compromise the current chemical analysis but also increase the efficiency of large scale screening.