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Title 環境微生物基因晶片之技術建立及應用(2/4)- 基因晶片應用在化學毒性評估
Abstract 本「環境微生物基因晶片之技術建立及應用（2/4）-基因晶片應用在化學毒性評估」為建立創新檢測環境荷爾蒙之研究計畫，建立酵母菌檢測法與單股核酸適配體 (Aptamer) 專一性之環境荷爾蒙篩選平台。酵母菌雙質體系統以bisphenol-A、p’p’-DDE、Estrone、17β estradiol及genistein等五種環境荷爾蒙來測試，此酵母菌系統不論用螢光偵測或β-Galactosidase活性偵測之結果於偵測極限、EC、TEQ等方面都接近，以17β estradiol為例，螢光偵測極限為5x10-11（g/L），於10-9~10-11（g/L）之線性達R2=0.98，EC50為2.1x10-10（g/L）；β-Galactosidase活性的偵測極限為5x10-12（g/L），於10-6~10-12（g/L），線性達R2=0.97，EC50為1.4x10-9（g/L）。研究結果本系統於bisphenol-A、p’p’-DDE、Estrone、17β estradiol及genistein等五種環境荷爾蒙的偵測極限、EC、TEQ等均優於現行文獻之酵母菌系統達100倍以上。單股核酸適配體平台採用之微珠量子點 (quantum dots) 檢測技術（「量子點奈米基因檢測系統」（計畫編號：EPA-101-U1U4-04-001）），設計1種bisphenol-A 與2種17β estradiol等環境荷爾蒙之專一性核酸適配體，我們利用Discovery Studio 3D完成bisphenol-A與 17β estradiol的微珠適配體的條件預測，經以五種環境荷爾蒙作專一性測試，17β estradiol（E2）微珠適配體的專一性最好，在100nM的環境荷爾蒙以下均有專一性分析能力。另2組適配體只有在環境荷爾蒙非常低的濃度時有專一性，故專一性還未達最佳化。17β estradiol微珠適配體系統均未達最佳線性，但BPA微珠適配體系統線性達到R2=0.9411；BPA之偵測極限達250ρM，17β estradiol之偵測極限為500ρM，敏感性為100%無偽陰性發生。
EngTitle
EngAbstract The main purpose of this project “Quantum Dots-BioChip Detection System for Environmental Microbes (2/4) -Application of Gene Chip in Endocrine Disrupter Substance Assay” is to combine two technologies included yeast screening and aptamer screening. The double expression dual plasmid yeast system can be applied for primary screening and the aptamer test can detect the specific Endocrine Disrupter Substance. Five different endocrine disrupter substance including bisphenol-A、p’p’-DDE、Estrone、17β estradiol and genistein were tested in our yeast system. The results of detection limit, EC and TEQ are similar from fluorescence assay and β-Galactosidase activity assay of our yeast system. For example, detection limit of 17β estradiol in fluorescence test is 5x10-11 (g/L) with R2=0.98 among 10-9~10-11 (g/L) and EC50 is 2.1x10-10 (g/L). For β-Galactosidase activity of 17β estradiol in this system, the detection limit of our system is 5x10-12 (g/L) with R2=0.97 among 10-6~10-12 (g/L) and EC50 is 1.4x10-9 among 10-9~10-11 (g/L). Our system is 100 times of sensitivity than current yeast system. The aptamer will be applied with previous technology that was developed by previous EPA project (Gene-Quantum Dots Detection System for environmental pathogen, (EPA-101-U1U4-04-001)). This novel technology utilized microbead linked with specific oligonucleotide and detected by quantum dots. To develop the aptamer system, we used 3D softare to simulate aptamer structures that are specific to Bisphenol A and 17β estradiol. We have designed one BPA-aptamer and two 17β estradiol-aptamer structures and tested by bisphenol-A、p’p’-DDE、estrone、17β estradiol and genistein. 17β estradiol of aptamer-microbead system have 100% specificity below 100nM. Both aptamer- microbead systems have 100% sensitivity with 1nM of detection limit. However, the quality and quantity detection ability of both aptamer- microbead systems required future research to improve
ProjectYear 103
SponsorOrg 環檢所
ExecutingOrg 中原大學
PublicFullVersionURL http://epq.epa.gov.tw/project/filedownload.aspx?fid=78872