環境微生物基因晶片之技術建立及應用(4/4)—建立流式細胞儀之多重細胞激素微珠檢測平台
| 中文摘要 | 本「環境微生物基因晶片之技術建立及應用(4/4)- 建立流式細胞儀之多重細胞激素微珠檢測平台」計畫為一融合既有的酵母菌檢測法與新的細胞激素檢測之計畫。因此本計畫是建立酵母菌之雙表現載體系統做為環境荷爾蒙的初篩工具與細胞激素偵測平台做為專一性偵測工具。細胞激素偵測平台採用之前開發的微珠量子點檢測技術，此技術是利用量子粒的特性配合微珠嵌連之專一性單股核酸探針，來同時解決定性、定量與標定問題。於酵母菌之雙表現載體系統中，我們已完成建構雙表現質體，可表達human estrogen receptor (hER-)， estrogen responsive elements (EREs) 及 reporter gene –BGFP與β-galactosidase。以螢光偵測時，五種環境荷爾蒙於酵母菌系統中最佳線性範圍為10-2~10-9 g/L，線性為R2=0.88~0.98。以β-galactosidase來偵測，酵母菌系統中最佳線性範圍為10-2~10-7 g/L，線性為R2=0.81~0.99。於細胞激素偵測平台，我們針對五種細胞激素完成control RNA的線性評估。對IL-1、IL-6、LT-、TNF-與SPP1的偵測極限為6.25pg，線性可達R2=0.99。五種細胞激素之微珠-量子點系統之專一性達96%以上。以藥物刺激A549細胞來產生細胞激素，並以微珠-量子點系統評估，線性達R2=0.92~0.99。靈敏度高，可達至少10-10g，無偽陰性。不同濃度之藥物刺激A549細胞後以QPCR及微珠-量子點系統的檢測的相關性(除SPP1)達R=0.71~0.99。 | ||
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| 中文關鍵字 | 細胞激素、量子點、環境荷爾蒙 | ||
基本資訊
| 專案計畫編號 | EPA-105-E3S5-02-03 | 經費年度 | 105 | 計畫經費 | 1900 千元 |
|---|---|---|---|---|---|
| 專案開始日期 | 2016/02/22 | 專案結束日期 | 2016/12/31 | 專案主持人 | 陳中庸 |
| 主辦單位 | 環檢所 | 承辦人 | 許令宜 | 執行單位 | 中原大學 |
成果下載
| 類型 | 檔名 | 檔案大小 | 說明 |
|---|---|---|---|
| 期末報告 | 環境賀爾蒙-cytokine-期末報告 (定稿本).pdf | 4MB |
Quantum Dots-BioChip Detection System for Environmental Microbes (4/4)-The Flow Assay System of cytokines by Quantum Dots Detection SyStem
| 英文摘要 | The main purpose of this project “Quantum Dots-BioChip Detection System for Environmental Microbes (4/4)- The Flow Assay System of cytokines by Quantum Dots Detection SyStem” is to combine two technologies included yeast screening and cytokine detection system. The double expression plasmid in yeast system can be applied for primary screening for environmental hormone and the cytokine detection system for the specific cytokine. The cytokine detection system will be applied by previous technology that was developed by previous EPA projects. This novel technology utilized microbead linked with specific oligonucleotide and detected by qutatum dots. The advantage of this is high accuracy for quantity and quality to monitor target samples. In double expression plasmid yeast system, we have finished the expression plasmid. This plasmid will express human estrogen receptor (hER-), estrogen responsive elements (EREs) and reporter gene including BGFP and β-galactosidase. For BGFP detection, those environmental hormones can be detected in our yeast expression system for linear detection between 10-2~10-9 g/L, and R2=0.88~0.98. For β-galactosidase detectin, those environmental hormone can be detected in our yeast expression system for linear detection between 10-2~10-7 g/L, and R2=0.81~0.99. To evaluate 5 cytokines incluing L1-, IL-6, LT-, TNF- and SPP1 in our microbead-QD system, the control RNAs were prepared by in vitro transcription assay. The detection limit of 5 cytokines is around 6.25 pg with R2=0.99。The specificity of those 5 cytokines is up to 96%. In addition, the A549 cells were treated with various drugs to induce the cytokines and tested in microbead-QD system, the data demonstrated our system has high sensitivity up to 10-10g with R2=0.92~0.99 without false negative. The comparison between Q-PCR and microbead-QD system showed the high relationship with R=0.71~0.99 except SPP1. | ||
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| 英文關鍵字 | Cytokine、Quantum dots、Environmental Hormone | ||