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.

Cytokine、Quantum dots、Environmental Hormone