A NOVEL REPORTER GENE- BASED BIOASSAY SYSTEM FOR DIOXINS - - PowerPoint PPT Presentation
A NOVEL REPORTER GENE- BASED BIOASSAY SYSTEM FOR DIOXINS DETERMINATION IN SOLID WASTE Bin Zhao 06/28/2019 State Key Laboratory of Environmental Chemistry and Ecotoxicology Research Center for Eco-Environmental Sciences Chinese Academy of
State Key Laboratory of Environmental Chemistry and Ecotoxicology Research Center for Eco-Environmental Sciences Chinese Academy of Sciences
O O Cl Cl Cl Cl
USA and JAPAN EUROPE CHINA EU Dioxin Emission Inventory Stage I EU Dioxin Emission Inventory Stage II Emission standard Waste incineration 2000/76/EC Foodstuffs 2006/1881/EC Industrial emissions 2010/75/EU Emission standard 1994 MSIWs 59 FR 48198 2000 HWIs 65 FR 47937 EPA 2005 The Inventory
Goverment of Japan
Emission standard In Various fields Emission standard MSIWs GB 18485-2001 HIWs GB 18484-2001 Landfill of IWs GB 16889-2008 Steelmaking Plant GB 28664-2012 Waste water GB 3544-2008 GB 18918-2002
HRGC-HRMS
Concentration of congeners TEF----TEQ BIOASSAY EROD ELISA Reporter gene
Expensive Strict condition Low throughput
Luminis cence LUCIFERASE
+ Luciferin
AhR
ARNT AhR
ARNT
AhR AhR DRE
Luciferase gene
Dioxins Toxicological mechanism of dioxins: AhR signaling pathway
CYP1A1
Reporter Gene
CALUX (Chemically Activated LUciferase gene eXpression )
US EPA SW846- 4435,TOX 21 2013 Chile SAG CALUX in birds European Union 2005 Belgium NVRIP dioxins detection in feed and soil 2009 Revision EC No152/2009 DR-CALUX in feed 2011 EC No 252/2012 DR-CALUX in food Japan JIS K 0463-2009 Environmental samples 2018 China Draft comments (HJ) in solid waste AU Queensland government air pollution assessment
China Patent 201410099507.7
10 20 30 15 5 25 35
TCDD (M)
10-9 10-10 10-11 10-12 10-13 10-14 10-8
MDL: 0.1 pM
Control)
10 20 30 15 5 25 40
DMSO 1nM TCDD
35
Our Plasmid Reference
China Patent: 201410100654.1
DMSO
10-14 10-13 10-12
TCDD （ M ）
MDL: 0.1 pM
2000 4000 6000
Reference
10 30 20 50
DMSO 1nM TCDD Our Sensor
40
standard curve of TCDD Stability of standard curve Minimum response to TCDD
Stability Repeatability reproducibility
0.1pM
Fly ash of Municipal Solid Waste Fly ash of Hazardous Waste
Samples extraction and cleaning
n=45 n=6
Correlation between bioassay and HRGC-HRMS in Fly ash samples
Unpublished data
Listed as China National Monitoring Method for Solid Waste Management
n=45
Zhang et al. Environ. Sci. Technol. 2018
0,1 0,2 0,3 0,4 0,5 0,6 1,6 3,2 4,8 6,4 8 9,6 f(x) = R² = 0
HRGC-HRMS analysis (pg-TEQ/g ww)
CBG 2.8D Cell Bioassay (pg BEQ/g ww)
1 2 3 4 5 6 7 8 2 4 6 8 10 12
f(x) = 1,1x + 0,55 R² = 0,91 HRGC-HRMS analysis (pg-TEQ/g fat)
C B G 2 .8 D C e ll B io a s s a y (p g B E Q /g fa t)
Milk Seafood
n= 13 n= 18
New standard curve
Pollut ants
Natural Product Metabo lites
Toxicity Effect Assessment Zhang et al. Environ. Sci. Technol. 2018
Activity Screening
Ginsenoside
Health Risk Assessment
Tryptophan metabolites
Kyn
Indole compounds
Liu et al. Nat. Commun. 2017
EHP 2011 119, A112-113