1 Development of Immunoassays for the Detection of Markers of Human and Environmental Exposure Department of Entomology University of California @ Davis.
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1
Development of Immunoassays for the Detection of Markers of Human and Environmental Exposure
Department of EntomologyUniversity of California @ Davis
bdhammock@ucdavis.edu
Bruce D. Hammock, Ph.D.
Shirley J. Gee
2
UC Davis/NIEHS Superfund Hazardous Substances Basic Research Program
“Biomarkers of Exposure to Hazardous
Substances”
University of California, Davis
3
1. Transport, Transformation and Remediation of Perchlorate and VOCs in the Vadose Zone
2. Aquatic Biomarkers in Site Characterization and Remediation
3. Development and Implementation of Immunoassays for Human and Environmental Monitoring
4. Biomarkers of Exposure to Pulmonary Toxicants
5. Development and Applications of Integrated Cell-Based Bioassays
6. Assessing Adverse Effects of Environmental Hazards on Reproductive Health in Human Populations
7. Thermal Remediation
8. Development of Rapid, Miniaturized Sensors for Use in the Detection of Environmental Toxins
9. Epidemiology Studies
PROJECTS
4
A. Analytical Chemistry Core
B. Statistical Analysis of Toxics Measurements
C. DNA Microarray Core
D. Training Core
F. Administrative Core
CORES
5
RESEARCH TOPICS ADDRESSED BY PROJECTS
• ANALYTICAL METH 1, 3, 5, 8, A, B
• BIOMEDICAL 2, 3, 4, 5, 6, 9
• BIOSTATISTICS B, C
• BIOINFORMATICS 1, 2, 3, 4, 5, B, C
• CBPI 6, 9, E
• ECOLOGY 1, 2
• ENGINEERING 1, 7, 8
• EPIDEMIOLOGY 6, 9
• HYDROGEOLOGY 1
• MECHANISM 1, 2, 4, 5, 6
• MICROBIOLOGY 1
• METABOLISM 1, 2, 3, 4, 5, A
• MOLECULAR BIOL 1, 2, 4, 5,
• REMEDIATION 1, 7
• ON SITE PROJECTS 1, 9
• TOXICOLOGY 3, 4, 5, 6, A
• TRANSPORT 1, 2
6
O
O
O
Cl
ClCl
Cl Cl
ClCl
Cl
Cl
Cl Cl
Cl
Cl
2,3,7,8-Tetrachlorodibenzo-p-dioxin 2,3,7,8-Tetrachlorodibenzofuran 3,4,3',4,'5-Pentachlorobiphenyl
Halogenated Aromatic Hydrocarbons
7
Chemical and Biological Techniques to Detect and QuantitateHalogenated Dioxins and Related Chemicals
Instrumental Immunoassay Bioassays
Biological/Toxic Potency Estimates of a Complex Mixture are Based on TCDD Equivalency Factors (TEFs) for the Specific
Chemicals Present in the Mixture.
TEFs are Derived from the Relative Toxic Potency of each HAHand is Directly is Related to their Relative Ability to Activate
the Ah Receptor (AhR) and AhR Signaling Pathway
8
ELISAProject 3
GLC-MSCore A
CALUXProject 5
TCDDor
UNKNOWN
ELISAProject 3
GLC-MSCore A
BIOASSAY DRIVENFRACTIONATION
Core A
ARBProject
1
USGSProject
2
SERUMProject
6
SOOTProject
7
9
ANALYTICAL PROBLEMS
• ANALYTICAL COSTS TOO HIGH
• ANALYSIS TIME TOO LONG
• ANALYSIS NOT FIELD PORTABLE
• ANALYSIS NOT IN ANALYSTS’ HANDS
• NOT ADAPTABLE TO MULTIPLE ANALYTES
10
TYPICAL ANALYTICAL COSTS
ATRAZINE
PARAQUAT
GLYPHOSATE
TCDD
$50 - $150/Sample
$200 - $500/Sample
$500 - $1000/Sample
$1,500 - $5,000/Sample
12
DISADVANTAGES
• Sensitivity
• Cross reactivity/Interferences
• Reagent availability
• New technology
• Large sample load required
• Difficult to apply to multianalyte analysis
13
A B C D1
2
3
4
Absorbance
Log Concentration
Polystyrene
Coating Antigen
E Enzyme coupled to Second Antibody
First antibody
Analyte
Substrate to Product
A
B
CD
14
Immunoassays Developed
• Triazines– Atrazine– Simazine– Hydroxytriazines– N-Dealkylated triazines– Atrazine mercapturate
• Thiocarbamates– Molinate– Thiobencarb
• Paraquat• Amitrole• Bentazon• Bromacil• Carbaryl• Diflubenzuron
– Other benzoylphenyl ureas• Fenoxycarb• Glyphosate• Alternaria toxins• Bacillus thuringiensis -endotoxin• Bacillus thuringiensis -exotoxin• 2,4,5-Trichlorophenoxyacetic acid
• Trichlopyr• Triton Series X and N detergents• Octyl and nonyl phenol• Urea Herbicides
– Monuron– Diuron– Linuron
• Naphthalene and metabolites• Nitrophenols (other nitroaromatics)• Pyrethroids
– Fenpropathrin– Esfenvalerate– Permethrin– Deltamethrin– 3-Phenoxybenzoic acid– Metabolites and conjugates
• Dioxins• General Mercapturates
– S-benzyl mercapturate• General Glucuronides
– Phenyl glucuronide– Thiophenyl glucuronide
15
Cl
ClO
O
CN
O
O
O
CN
O
ClO
O
CN
O
F
Cl
Cl
O
OO
Cl
Cl
Major Pyrethroids Used in California
Permethrin (57.5%) Cypermethrin (22.6%)
Cyfluthrin (7%) Esfenvalerate (6%)
*In 1998, a total of 650,000 lbs pyrethroids were applied in California
16
O O
O Cl
Cl
OO
O Cl
Cl
H2N
Hapten Design for Compound-specific Assay (I)
Target Compound Hapten
Permethrin
• Close mimic of analyte• Distal attachment site• Four carbon handle optimal
17
Hapten Design for Class-specific Assay
Target Compound Hapten
OO
OCOOH
OO
O
COOH
CN
O
O
HO
O
CHO
Type I Pyrethroids(without CN)
Type II Pyrethroids(with CN)
All Pyrethroids(with PB)
18
% C
ontr
ol A
bsor
banc
e
0.001 0.01 0.1 1 10 100 1000 100000
20
40
60
80
100
Concentration (ppb)
permethrinI50 = 5 ppbLDL = 0.001 ppb
Standard Curve for Permethrin
19
020406080
100120
Perm
ethrin
Phenoth
rin
Cyper
met
hrin
Esfen
vale
rate
Delta
met
hrin
Cyflu
thrin
Fenpro
pathrin
PBA
Cro
ss-r
eact
ivit
y %
Permethrin-specific Immunoassay (Selectivity)
OO
OC l
C l
Permethtrin
I50 = 2.5 ppbLOQ = 0.01 ppb (SPE)
ni ni ni ni
*ni = less than 10 % inhibition at 10 ppm
20
Relationship between Permethrin Measured by ELISA and GC-MS
Y = 1.211x - 0.068 R2 = 0.900, n = 27
Permethrin (GC-MS, ppb)
0.001
0.01
0.1
1
10
100
0.001 0.01 0.1 1 10 100
y = x
Per
met
hri
n (E
LIS
A, p
pb)
21
Role of Immunochemistry in Trace Analysis
• Direct immunochemical analysis• Prioritization of samples for other methods• Immunoaffinity cleanup for other methods• Post separation detection• HPLC/Microbore LC• Capillary electrophoresis• TLC
22
ELISA: A Complementary Technology
ELISA: A Complementary Technology
Analytical Chemist
MS GC CE HPLC
IR SFC UV/VISImmunoassay
23TCDD (pg/well)
0.001 0.01 0.1 1 10 100 1000 10000
Ab
so
rban
ce
0.0
0.1
0.2
0.3
0.4
0.5
0.6
O
O
Cl
Cl
Cl
Cl
IC50 = 4 pg/well
24
Strategies for detection in Strategies for detection in environmental samplesenvironmental samples
• Increased throughput• Increased sensitivity• Increased specificity
• Small volumes• Minimized pre-processing of samples• Minimized autofluorescence
Achieved by
Emission spectra of lanthanides
25
Department of Environmental ToxicologyUniversity of California @ Davis
msdenison@ucdavis.edu
Development, Validation and Application of Recombinant CellBioassay Systems for Rapid Detection of Dioxins and
Related Halogenated Aromatic Hydrocarbons
Michael S. Denison, Ph.D.
26
Chemical and Biological Techniques to Detect and QuantitateHalogenated Dioxins and Related Chemicals
Instrumental Immunoassay Bioassays
Biological/Toxic Potency Estimates of a Complex Mixture are Based on TCDD Equivalency Factors (TEFs) for the Specific
Chemicals Present in the Mixture.
TEFs are Derived from the Relative Toxic Potency of each HAHand is Directly is Related to their Relative Ability to Activate
the Ah Receptor (AhR) and AhR Signaling Pathway
27
Spectrum of Toxic and Biological Effects Producedby TCDD in Different Species and Tissues______________________________________________
• Immunotoxicity• Hepatotoxicity• Wasting Syndrome• Tumor Promotion• Dermal Toxicity (Chloracne)• Teratogenicity• Lethality• Uroporphyrin Accumulation (Porphyria)• Endocrine Disruption• Modulation of Cell Growth, Proliferation and
Differentiation• Induction of Gene Expression
Cytochrome P4501A1/2 and 1B1UDP-Glucuronosyl Transferase 1*06Glutathione S-Transferase YaQuinone Reductase-Aminolevulinic Acid SynthaseProstaglandin Endoperoxide H Synthase 2
The biological and toxicological effects of TCDD and related halogenated aromatic hydrocarbons (HAHs) are mediated by the Ah Receptor (AhR)
28
AhR Signal Transduction Pathway
TranslationNew Polypeptides
Increased Cytochrome P-4501A1
ARNT
Exogenous and Endogenous Ligands
Endogenous Ligands
Other Gene Products
mRNA
DREs CYP1A1
DREs Other Genes
Other Factors?Translocation
Proteosome
Degradation
AhR hsp90
XAP2 hsp90p23
Toxicity
29
AhR Signal Transduction Pathway
TranslationNew Polypeptides
Increased Cytochrome P-4501A1
ARNT
Exogenous and Endogenous Ligands
Endogenous Ligands
Other Gene Products
mRNA
DREs CYP1A1
DREs Other Genes
Other Factors?Translocation
Proteosome
Degradation
AhR hsp90
XAP2 hsp90p23
Toxicity
30
Development of a Ah Receptor-Based Bioassay Bioassay Systemfor Detection and Relative Quantitation of Dioxin and Related HAHs
TCDD + AhR
TCDD:AhR
TCDD:AhR*
TCDD:AhR:DRE
TranscriptionalActivation
Ligand Binding
Transformation
DNA Binding
Gene Expression*
31
CALUX (Chemically-Activated Luciferase Expression) Cell Bioassay
TranslationNew Polypeptides
Increased Cytochrome P-4501A1
ARNT
TCDD HAHs PAHs
Luciferase Activity
mRNA
DREs CYP1A1
DREs Luciferase
Other Factors?Translocation
AhR hsp90
XAP2 hsp90
32
CALUX Bioassay Procedure
H1L6.1c3 Mouse HepatomaCells Plated into 96-Well
Microplates
Chemicals Added to Each Well and
Incubated for 24 hours
Wells are Washed, CellsLysed, and Luciferase
Activity Measured in a Microplate Luminometer
33
10 -710 -810 -910 -1010 -1110 -1210 -1310 -140
1
2
3
4
5
6
7
TCDD Concentration (M)
Lu
cif
era
se A
cti
vit
y (
RLU
/ug
pro
tein
)
TCDD Dose Dependent Induction of Luciferase Activityin Stably Transfected Mouse Hepatoma (Hepa1c1c7) Cells
EC50 = 10 pMMDL = 1 pM
Current Microplate AssayMDL = 0.03pg/assay
34
10 -510 -610 -710 -810 -910 -1010 -1110 -1210 -1310 -140
2
4
6
8
2378-TCDD
2378-TCDF
23478-PCDF
12478-PCDD
3344-TCB
33445-PCB
Chemical Concentration (M)
Lu
cif
era
se A
cti
vit
y
(R
LU
/ug
pro
tein
)
Activation of the CALUX Cell Bioassay by PCDDs, PCDFs and PCBs
35
WHO Toxic Equivalency Factors (TEFs) and CALUXRelative Potency (REP) Factors for Chlorinated Dibenzo-
p-Dioxins, Dibenzofurans and Biphenyls._______________________________________________
Compound WHO-TEF CALUX REP
_______________________________________________
2378-TCDD 1 1.00 ± 0.0112378-PeCDD 1 0.73 ± 0.1123478-HxCDD 0.1 0.075 ± 0.014123678-HxCDD 0.1 0.098 ± 0.017123789-HxCDD 0.1 0.061 ± 0.0121234678-HpCDD 0.01 0.031 ± 0.008OCDD 0.0001 0.00034 ±0.00008
2378-TCDF 0.1 0.67 ± 0.0112378-PeCDF 0.05 0.14 ± 0.0423478-PeCDF 0.5 0.58 ± 0.08123478-HxCDF 0.1 0.13 ± 0.02123678-HxCDF 0.1 0.14 ± 0.03123789-HxCDF 0.1 0.11 ± 0.02234678-HxCDF 0.1 0.31 ± 0.061234678-HpCDF 0.01 0.024 ± 0.0071234789-HpCDF 0.01 0.044 ± 0.010OCDF 0.0001 0.0016 ± 0.0005
PCB 77 0.0005 0.0014 ± 0.0004PCB 81 0.0001 0.0045 ± 0.0012PCB 114 0.0005 0.00014 ± 0.00002PCB 126 0.1 0.038 ± 0.007PCB 156 0.0005 0.00014 ± 0.00002PCB169 0.01 0.0011 ± 0.0003_______________________________________________
36
Bioassay Systems - Considerations
Structural Diversity of AhR Agonists and/or Antagonists:Potential for False Positives in HAH Detection
O
O
O
O
O
Cl
ClCl
Cl Cl
ClCl
Cl
Cl
Cl Cl
Cl
Cl
2,3,7,8-Tetrachlorodibenzo-p-dioxin 2,3,7,8-Tetrachlorodibenzofuran 3,4,3',4,'5-Pentachlorobiphenyl
CH3
3-Methylcholanthrene -Naphthoflavone Benzo(a)pyrene
Halogenated Aromatic Hydrocarbons and Polycyclic Aromatic Hydrocarbons
O
O
Cl
Cl
N
N
N
H
2
O
H
Guanabenz
N
H
2
S
C
H
3
2(Methylmercapto)aniline
N
H
2
N
H
2
1,5-Diaminonaphthalene
N
N
S
N
O
CH2
OCH3
CH3CH3
Omeprazole
CH3O
N
C
H
3
H
5-Methyl-2-phenylindole
N
H
2
O
H
(1S,2R)-(-)-cis-1-Amino-2-indanol
N
H
N
H
N
H
2
N
O
2
O
2
N
O
C
H
3
SRN-P2:109,NH2
N
N
H
2
C
H
3
1-Methyl-1-phenylhydrazine
N
N
N
H
CN
CF3
SKF71739
S
N
C
H
3
N
H
2
2-(4-Amino-3-methylphenyl) benzothiazole
N
S
S
O
N
O
O
S
Cl
ClCl
Cl
2,3,7,8-Tetrachlorodibenzo-p-dioxin
CH3YH439
CH3
CH3
H
• Promiscuous ligand binding by the AhR requires appropriate extraction and chemical clean-up methods to remove unwanted compopunds
37
Flow Diagram for Analysis of Samples by CALUX and GC/MS
Sample
Extraction and Chemical Clean-up
CALUX Bioassay Analysis
Positive
Negative
No Compounds that Activate the Ah Receptor and/or
Contains Compounds that Block Activation (i.e. Antagonists)
Analysis by HRGC/MS
Estimate of relative
activity
Total HAHsPCDD/PCDF
&PCB
38
Bioassay Systems - Considerations • Calculation of relative biological potency of an unknown sample extract
10 -710 -810 -910 -1010 -1110 -1210 -1310 -140
1
2
3
4
5
6
7
TCDD Concentration (M)
Resp
on
se
10 110 210 310 410 5106
10 710 80
1
2
3
4
5
6
7
Dilution Factor
EC50 = 20pM
ED50 = 1000-fold dilution = 20pM Thus, the original sample contains the equivalent (TEQ) of 20nM TCDD
EC50
EC50 = 1000-fold dilution = 20pMThus the original extract contains the
equivalent (bioassay-TEQ) of 20 nM TCDD
39.
Applications of the CALUX Dioxin Cell Bioassay
1. Biological Samples• Blood (whole serum and extracts)• Breast Milk• Tissue Extracts
2. Environmental Samples• Soil and Sediment• Ash• Emission (PUF)• Pulp and Paper
3. Food Samples• Animal Fats (oil and fats)• Milk and Butter• Animal Feeds
40
Comparative GCMS/CALUX Results fromthe Umea Round Robin Analysis of
Environmental Samples for ChlorinatedDibenzo-p-Dioxins, Dibenzofurans and
Biphenyls.
______________________________________ TEQ (ng/g sample)
Sample GC/MS CALUX______________________________________
Soil A 0.3 0.16
Soil B 0.17 1.2Soil C 0.19 0.6
Soil D 0.30 1.06
Ash A 0.40 0.13
Ash B 0.04 0.04Ash C 0.42 0.50
Solution F 222 448Solution H 3.42 8.39
______________________________________
41
Double-Blind Validation ResultsComparison of CALUX and HR GC/MS Analysis of Ash and Exhaust Gas Samples
R2 = 0.947
0.01
0.1
1
10
100
1000
10000
100000
1 10 100 1000 10000 100000
CALUX, pg TEQ/gram
R2 = 0.918
0.0001
0.001
0.01
0.1
1
10
100
1000
0.01 0.1 1 10 100 1000
CALUX, pg TEQ/m3
Ash
Exhaust Gas
42
Double-Blind Validation Results Comparison of CALUX and HR GC/MSAnalysis of Soil and Human Fat Samples
R2 = 0.854
0.01
0.1
1
10
100
1000
0.1 1 10 100 1000
CALUX, pg TEQ/gram
HR
GC
MS
, pg
TE
Q/g
ram
R = 0. 8051
0
20
40
60
80
100
0 20 40 60 80 100CALUX Assay(pgTEQ/ g fat)
HRGC
MS(p
gTEQ
/g f
at)
Soil
Human Fat
43
Environmental Samples Contain Additional Ah Receptor Active HAHs?
R2 = 0.947
0.01
0.1
1
10
100
1000
10000
100000
1 10 100 1000 10000 100000
CALUX, pg TEQ/gram
R2 = 0.918
0.0001
0.001
0.01
0.1
1
10
100
1000
0.01 0.1 1 10 100 1000
CALUX, pg TEQ/m3
Ash
Exhaust Gas
R2 = 0.854
0.01
0.1
1
10
100
1000
0.1 1 10 100 1000
CALUX, pg TEQ/gram
HR
GC
MS
, pg
TE
Q/g
ram
Soil
R = 0. 8051
0
20
40
60
80
100
0 20 40 60 80 100CALUX Assay(pgTEQ/ g fat)
HRGC
MS(p
gTEQ
/g f
at) Human Fat
44
Bioassay Systems - Further Considerations • Calculation of relative biological potency of an unknown sample extract• Promiscuous ligand binding by the AhR requires appropriate extraction and chemical clean-up methods for each bioassay
• Discrepancy between bioassay and instrumental analysis needs to be resolved a. Use of bioassay-specific REPs for TEQ estimates from instrumental analysis b. False positives or other AhR-active HAHs
R2 = 0.918
0.0001
0.001
0.01
0.1
1
10
100
1000
0.01 0.1 1 10 100 1000
CALUX, pg TEQ/m3
45
101.1.01.001.0001.0001
.001
.01
.1
1
10
WHO TEF Values for Dioxin-Like HAHs
CA
LU
X R
EP
Valu
es f
or
Dio
xin
-Lik
e H
AH
s
Comparison of GC/MS TEF and CALUX REP Values
46
Tiered Flow Diagram for Sample Screening
POSITIVERun immunoassay
NEGATIVENo compounds that bind
productively to Ah receptor
POSITIVERun GC/MS for
confirmation
NEGATIVENo TCDD-like
compounds
BOTH NEGATIVEUse CALUX to drivechemical purification
PCBELISA
TCDDELISA
CALUX
POSITIVERun GC/MS for
confirmation
NEGATIVENo TCDD-like
compounds
ELISA Also suggeststhat there are more 2,3,7-PCDD/Fs thanGC/MS indicates.
What could it be?
47
O
O
O
O
Br
O
H
Br
Br
Br
Br
Halogenated Aromatic Hydrocarbons
Cl
ClCl
Cl Cl
ClCl
Cl
2,3,7,8-Tetrachlorodibenzo-p-dioxin 2,3,7,8-Tetrachlorodibenzofuran Polybrominated Diphenyl Ether
O
Br
Br
Br
Br
Br
O
Br
Br
Br
Br
Br
O
Polybrominated Dibenzofurans (PBDFs)Polybrominated Dibenzo-p-dioxins (PBDDs)
Sources:Formed during PDBE SynthesisBurning of PBDE Containing MaterialsPhotochemical Reactions of PBDEs
One Possibility for the Higher Equivalent Estimates: PB/CDDs/Fs
2,3,7,8-PBDD/F found in PBDE workers@ n.d - 500pg/g blood lipid.
Mo-OctBDF produced from combustion of DBDE-polypropylene matrices
48
0
2000
4000
6000
8000
10000
12000
1,E-13 1,E-12 1,E-11 1,E-10 1,E-09 1,E-08 1,E-07 1,E-06 1,E-05
Concentration, M
RL
U2378-TCDD, n=11PCB 169, n=6PBB 169, n=52378-TBDD, n=4
Brominated Dioxins and Related HAHs are Relatively Potent Activators
49
In Vivo ED50 Values for Toxic and Induction Effects of Select PHDDs in the Rat
Congener In Vivo ED50 (µmol/kg body weight)Body Weight Loss Thymic Atrophy Gene Induction
2,3,7,8-TeCDD 0.05 0.09 0.0042,3,7,8-TeBDD 0.068 0.034 0.0076
1,2,3,7,8-PeCDD 0.62 0.17 0.0311,2,3,7,8-PeBDD 0.87 0.39 0.025
2,3-DiB-7,8-DiCDD 0.012 0.0073* 0.00049*2-B-3,7,8-TriCDD 0.12 0.035 0.0025
1,2,4,7,8-PeCDD 34.0 11.2 2.821,2,4,7,8-PeBDD 12.9 6.17 0.195Immature male Wistar rats (n=4), 14 days after a single intraperitoneal dose.Adapted from IPCS Env. Health Criteria #205 - data from Mason et al. (1987); Safe et al. (1989).
50
AhR Gene Expression Cell Bioassay Systems
Advantages
• Sensitive, relatively rapid and easy to carry out• Amenable to high throughput analysis• Relatively inexpensive compared to instrumental analysis• A significant amount of validation data is available for the CALUX bioassay • Overestimate TCDD equivalents in environmental samples (new dioxin-like HAHs?)
Disadvantages/Limitations
• Experience in cell culture techniques necessary• Proper sample cleanup methods needed • Instrumentation - Luminescent microplate readers, cell culture facilities• Overestimate TCDD equivalents in environmental samples (false positives?)• Extracts containing chemicals that are directly toxic to cells can not be analyzed• Synergistic/antagonistic effects (over/underestimate potency)
51
Bioassay Systems - Further Considerations • Calculation of relative biological potency of an unknown sample extract
• Promiscuous ligand binding by the AhR requires appropriate extraction and chemical clean-up methods for each bioassay (in vitro assays require more extensive clean-up methodologies)
• Discrepancy between bioassay and instrumental analysis needs to be resolved a. false positives or other AhR-active HAHs b. Use of bioassay-specific REPs for TEQ estimates from instrumental analysis
• Toxic Equivalency Factors (TEFs) versus Relative Potency (REPs) Values TEQs (instrumental) versus Bioassay-TEQs
• Need method(s) to correct for extraction and clean-up efficiency and recovery since samples can’t simply be spike with AhR-active HAHs like 2,3,7,8-TCDD
• Establish quality control criteria for bioassay methodologies
• Require full validation studies (versus instrumental analysis) for different matrices
52
1.1.01.0010
2000
4000
6000
8000
10000
12000
Day 1Day 14
TCDD (ng/ml)
Luci
fera
se A
ctiv
ity (
RLU
/well)
Microplates containing cells can be sealed and stored at room temperature for up to 14 days with little loss of TCDD-inducible luciferase activity.
Viewplate microplate with well sealer
Seal
Cell Bioassay Systems - Development
This development now allows plates to be prepared off-site and mailed to the site for extract treatment and analysis.
53
Application and Utilization of Bioanalytical Methods for Dioxin Analysis
• Detection, quantitation and chemical identification of dioxin-like chemicals in a variety of matrices including:
• Environmental samples (soil, water, air)• Biological (blood, milk, tissues)• Food and feed• Commercial and consumer products
• Determination of the effectiveness of bioremediation, biodegradation and contamination clean-up procedures for dioxin-like chemicals.
• Identification and characterization of other classes of dioxin-like chemicals.
54
Acknowledgements and Support
University of California @ DavisMichael Ziccardi, Joe Rogers, Patricia Garrison,
Bruce Hammock, Shirley Gee, Guomin Shan
Xenobiotic Detection SystemsGeorge Clark, David Brown, Mick Chu
Hiyoshi Corporation (Japan)Hiroshi Murata
Scientific Institute of Public Health, Brussels, BelgiumLeo Goeyens, Ilse van Overmeire
National Institutes of Environmental Health Sciences (NIEHS) Superfund Basic Research Program - ESO4699, ES04911
55
Ian Kennedy
Department of Mechanical and Aeronautical Engineeringand
Biomedical and Electrical Computer Engineering Graduate Groups
University of California Davis
Supported by the NIEHS Superfund Basic Research Program andNSF NNI
Ian Kennedy
Department of Mechanical and Aeronautical Engineeringand
Biomedical and Electrical Computer Engineering Graduate Groups
University of California Davis
Supported by the NIEHS Superfund Basic Research Program andNSF NNI
Nanotechnology and Biosensors
Nanotechnology and Biosensors
56
Strategies for detection in environmental samplesIncreased throughputIncreased sensitivityIncreased specificity
Achieved by Small volumesMinimized pre-processing of samplesMinimized autofluorescenceNew fluorescent labels
57
Competitive immunoassays in microdroplets using fluorescence quenching
Microdroplet cavity resonances
Application of the microdroplet approach to analysis on a chip
Development of new labels using novel materialsQuantum dotsEncapsulated lanthanide oxidesAbsorbing labels such as C60
Novel technologies
58
Quenching fluoroimmunoassay in microdroplets
Antibody quenching
Competitive Quenching Fluoro ImmunoAssay (QFIA)
- TCP
- TCP- F
analyte
Labeled analyte
59
Assay for TCPAssay for TCPCalibration curve for 2,4,6-TCP in PBS and in urine 1/50; 10 nM Af; 2.5 mg/ml Ab43; incubation time 45 min at room temperature
TCP, M
(I / I
AF)
*100
10 -12 10 -11 10 -10 10 -9 10 -8 10 -7 10 -6 10 -5 10 -440
50
60
70
80
90
100
110PBSurine 1/50
g/L urine
IC50 22.5
LOD 2
g/L ELISA QFIA
200 L
QFIA
microdrop
IC50 2.74 4.2 0.45
LOD 0.2 0.36 0.04
60
Intense optical field created by internally reflected fluorescence from Rhodamine
Micro cavity resonances
61
Intensities within droplets greatly reduced when C60 is
added to droplet
Images of Rh emission quenched by C60
62
Single E. coli detected using optical resonances without labeling
Couple to immunoassay for specificity or use mode structure for information
Detecting bacteria
63
Microdroplets on a chip
Biosensor chip
MIcrochannel filled with lowrefractive index silicon oil
Microdroplets ofaqueous sample
65
Sharp emission spectrum increases spectral sensitivity
Long lifetime emission permits gated detection
Surface treatment prevents quenching
No need for chelation
Magnetic moments useful for separation and for imaging contrast agents
No photobleaching
Lanthanide oxide nanoparticles
66
Lanthanide oxides as reporters for bioassays
57
La
58
Ce
59
Pr
60
Nd
61
P
m
62
Sm
63
Eu
64
Gd
65
Tb
66
Dy
67
Ho
68
Er
69
T
m
70
Yb
71
Lu
67
Europium oxide spectra
80x103
70
60
50
40
30
20
10
Inte
nsi
ty (
arb
itra
ry u
nit
s)
650600550500450400350
Wavelength (nm)
Excitation spectrumobserved at 610 nm Emission spectrum
excited at 466 nm
4x106
3
2
1
Inte
nsi
ty (
arb
itra
ry u
nit
s)
600580560540520500
Wavelength (nm)
Excitation spectrum observed at 548 nm
Emission spectrum excited at 524 nm
Europium
Rhodamine
68
Gas phase synthesis and functionalization
AntibodyAntigen
A functionalized nanoparticle containing lanthanide oxide can be used in an immunoassay that takes advantage of the specific binding between an antigen and a homologous antibody.
69
Pure Eu2O3 particles
Photoluminescence Spectrum: Dominant Peaks at 615 and 623 nm; Short Lifetime Due to Concentration
Quenching Combined with Small Size.
Pure Eu2O3; Monoclinic
Phase; Gas-phase Synthesis.
70
Composite Eu2O3/SiO2
particles
Eu2O3/SiO2 Composite
Nanoparticles; Higher-density Core with Lower-density Shell;
Gas-phase Synthesis.Photoluminescence Spectrum: Dominant Peak at ~ 615 nm;
Lifetime ~ 1 ms
71
Lifetimes of 1 ms or more
Biological background typically less than 10 ns
Europium oxide lifetime
Eu:Y2O
3
72
Microwave chemistry and gas phase chemistry used to add protective SiO layer and NH2 functional group
Functionalized nanoparticles
73
Optical properties of Eu2O3 unchanged by surface treatment
Additional phosphors obtained by doping lanthanides into hosts
Used in immunoassay for atrazine
Optical properties of functionalized particles
0
1000000
2000000
3000000
4000000
5000000
6000000
7000000
8000000
9000000
10000000
550 600 650 700 750
Wavelength / nmIn
ten
sity
0
10000
20000
30000
40000
350 400 450 500
Wavelength / nm
Capped with silane
74
80 m channel electrophoresis system
separated two size classes of nanoparticles
can be used for assays
Nanophosphors separated with microchip electrophoresis
77
Europium oxide in a pyrethroid metabolite assay
Competitive assay for 3-phenoxybenzoic acid (PBA) with magnetic separation
Europium oxide particle conjugated to the hapten
IC50
=
2 x 10 –4 ng mL-1
78
New nanoparticle assay formats
Multi wavelength labels can be used to provide an internal standard for sandwich immunoassays
81
Eu lifetime can be greater than 1ms
Fluorescein lifetime of the order of 10 ns
Fluorescence lifetimes
83
Nanoscale materials, engineered from the “bottom up”, can be functionalized for use in biology
Optical properties promise more sensitive detection in complex matrix, possibly reagentless
Eu labels demonstrated in immunoassays with up to 10 4 improvement in detection limits
Other properties of nanoscale materials can be useful (magnetic, thermophoretic, electrophoretic) for separation
Brilliant dust?
Summary
84
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