Development of Immunoassays for the Detection of
Markers of Human and Environmental Exposure
Bruce D. Hammock, Ph.D.
Shirley J. Gee
Department of Entomology
University of California @ Davis
bdhammock@ucdavis.edu
1
UC Davis/NIEHS Superfund Hazardous
Substances Basic Research Program
“Biomarkers of Exposure to Hazardous
Substances”
University of California, Davis
2
PROJECTS
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
3
CORES
A.
Analytical Chemistry Core
B.
Statistical Analysis of Toxics Measurements
C.
DNA Microarray Core
D.
Training Core
F.
Administrative Core
4
RESEARCH TOPICS
ADDRESSED BY PROJECTS
•
•
•
•
•
•
•
•
•
•
ANALYTICAL METH
BIOMEDICAL
BIOSTATISTICS
BIOINFORMATICS
CBPI
ECOLOGY
ENGINEERING
EPIDEMIOLOGY
HYDROGEOLOGY
MECHANISM
1, 3, 5, 8, A, B
2, 3, 4, 5, 6, 9
B, C
1, 2, 3, 4, 5, B, C
6, 9, E
1, 2
1, 7, 8
6, 9
1
1, 2, 4, 5, 6
•
•
•
•
•
•
•
MICROBIOLOGY
METABOLISM
MOLECULAR BIOL
REMEDIATION
ON SITE PROJECTS
TOXICOLOGY
TRANSPORT
1
1, 2, 3, 4, 5, A
1, 2, 4, 5,
1, 7
1, 9
3, 4, 5, 6, A
1, 2
5
Halogenated Aromatic Hydrocarbons
Cl
O
Cl
Cl
Cl
Cl
Cl
Cl
Cl
O
Cl
2,3,7,8-Tetrachlorodibenzo-p-dioxin
Cl
O
Cl
Cl
Cl
2,3,7,8-Tetrachlorodibenzofuran 3,4,3',4,'5-Pentachlorobiphenyl
6
Chemical and Biological Techniques to Detect and Quantitate
Halogenated 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 HAH
and is Directly is Related to their Relative Ability to Activate
the Ah Receptor (AhR) and AhR Signaling Pathway
7
ARB
Project
1
TCDD
or
UNKNOWN
USGS
Project
2
CALUX
Project 5
BIOASSAY DRIVEN
FRACTIONATION
Core A
SERUM
Project
6
SOOT
Project
7
ELISA
Project 3
ELISA
Project 3
GLC-MS
Core A
GLC-MS
Core A
8
ANALYTICAL PROBLEMS
• ANALYTICAL COSTS TOO HIGH
• ANALYSIS TIME TOO LONG
• ANALYSIS NOT FIELD PORTABLE
• ANALYSIS NOT IN ANALYSTS’ HANDS
• NOT ADAPTABLE TO MULTIPLE ANALYTES
9
TYPICAL ANALYTICAL COSTS
ATRAZINE
$50 - $150/Sample
PARAQUAT
$200 - $500/Sample
GLYPHOSATE
$500 - $1000/Sample
TCDD
$1,500 - $5,000/Sample
10
ADVANTAGES
•
•
•
•
•
•
Sensitive
Selective
Rapid
Cost Effective
Applicable
Adaptable
11
DISADVANTAGES
•
•
•
•
•
•
Sensitivity
Cross reactivity/Interferences
Reagent availability
New technology
Large sample load required
Difficult to apply to multianalyte analysis
12
A
B
A
B
C
D
C
1
D
Log Concentration
2
Polystyrene
Coating Antigen
First antibody
3
Analyte
Substrate to Product
4
E
Enzyme coupled to
Second Antibody
13
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
14
– Thiophenyl glucuronide
Major Pyrethroids Used in California
Cl
Cl
Cl
O
O
Cl
O
O
O
F
Cl
O
O
O
CN
Cypermethrin (22.6%)
Permethrin (57.5%)
Cl
O
O
Cl
O
O
CN
CN
Cyfluthrin (7%)
Esfenvalerate (6%)
*In 1998, a total of 650,000 lbs pyrethroids were applied in California
15
Hapten Design for Compound-specific Assay (I)
Target Compound
Hapten
O
O
O
O
Cl
Cl
O
O
Cl
Cl
H2N
Permethrin
• Close mimic of analyte
• Distal attachment site
• Four carbon handle optimal
16
Hapten Design for Class-specific Assay
Hapten
Target Compound
O
Type I Pyrethroids
(without CN)
O
O
CN
Type II Pyrethroids
(with CN)
COOH
O
O
COOH
O
O
All Pyrethroids
(with PB)
HO
O
CHO
O
17
Standard Curve for Permethrin
% Control Absorbance
100
80
60
40
20
permethrin
I50 = 5 ppb
LDL = 0.001 ppb
0
0.001 0.01
0.1
1
10
100
1000 10000
Concentration (ppb)
18
Cross-reactivity %
Permethrin-specific Immunoassay (Selectivity)
120
100
80
60
40
20
0
O
Cl
Cl
O
O
Permethtrin
I50 = 2.5 ppb
LOQ = 0.01 ppb (SPE)
ni
ni
ni
ni
*ni = less than 10 % inhibition at 10 ppm
19
Relationship between Permethrin Measured by ELISA and GC-MS
Permethrin (ELISA, ppb)
100
y=x
10
1
0.1
Y = 1.211x - 0.068
R2 = 0.900, n = 27
0.01
0.001
0.001
0.01
0.1
1
10
100
Permethrin (GC-MS, ppb)
20
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
21
ELISA:
A Complementary Technology
MS
GC
CE
HPLC
Analytical Chemist
IR
Immunoassay
SFC
UV/VIS
22
Absorbance
0.6
0.5
Cl
O
Cl
0.4
Cl
O
Cl
0.3
0.2
0.1
IC50 = 4 pg/well
0.0
0.001
0.01
0.1
1
10
100
1000
10000
TCDD (pg/well)
23
Strategies for detection in
environmental samples
• Increased throughput
• Increased sensitivity
• Increased specificity
Achieved by
Emission spectra of lanthanides
• Small volumes
• Minimized pre-processing of samples
• Minimized autofluorescence
24
Development, Validation and Application of Recombinant Cell
Bioassay Systems for Rapid Detection of Dioxins and
Related Halogenated Aromatic Hydrocarbons
Michael S. Denison, Ph.D.
Department of Environmental Toxicology
University of California @ Davis
msdenison@ucdavis.edu
25
Chemical and Biological Techniques to Detect and Quantitate
Halogenated 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 HAH
and is Directly is Related to their Relative Ability to Activate
the Ah Receptor (AhR) and AhR Signaling Pathway 26
Spectrum of Toxic and Biological Effects Produced
by 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 1B1
UDP-Glucuronosyl Transferase 1*06
Glutathione S-Transferase Ya
Quinone Reductase
g-Aminolevulinic Acid Synthase
Prostaglandin Endoperoxide H Synthase 2
The biological and toxicological effects of TCDD and related halogenated
aromatic hydrocarbons (HAHs) are mediated by the Ah Receptor (AhR)
27
AhR Signal Transduction Pathway
Exogenous and
Endogenous
Ligands
Endogenous
Ligands
hsp90 p2 3
AhR
XAP2 hsp90
DREs
CYP1A1
ARNT
Other
Factors?
Translocation
mRNA
DREs Other Genes
Proteosome
Degradation
New Polypeptides
Increased Cytochrome
P-4501A1
Translation
Other Gene
Products
Toxicity
28
AhR Signal Transduction Pathway
Exogenous and
Endogenous
Ligands
Endogenous
Ligands
hsp90 p2 3
AhR
XAP2 hsp90
DREs
CYP1A1
ARNT
Other
Factors?
Translocation
mRNA
DREs Other Genes
Proteosome
Degradation
New Polypeptides
Increased Cytochrome
P-4501A1
Translation
Other Gene
Products
Toxicity
29
Development of a Ah Receptor-Based Bioassay Bioassay System
for Detection and Relative Quantitation of Dioxin and Related HAHs
TCDD + AhR
Ligand Binding
TCDD:AhR
Transformation
TCDD:AhR*
DNA Binding
TCDD:AhR:DRE
Gene Expression*
Transcriptional
Activation
30
CALUX (Chemically-Activated Luciferase Expression) Cell Bioassay
TCDD
HAHs
PAHs
DREs
AhR hsp90
XAP2 hsp90
CYP1A1
ARNT
Other
Factors?
Translocation
mRNA
DREs Luciferase
Translation
New Polypeptides
Increased Cytochrome
P-4501A1
Luciferase
Activity
31
CALUX Bioassay Procedure
H1L6.1c3 Mouse Hepatoma
Cells Plated into 96-Well
Microplates
Chemicals Added to
Each Well and
Incubated for 24 hours
Wells are Washed, Cells
Lysed, and Luciferase
Activity Measured in a
Microplate Luminometer
32
TCDD Dose Dependent Induction of Luciferase Activity
in Stably Transfected Mouse Hepatoma (Hepa1c1c7) Cells
7
Luciferase Activity (RLU/ug protein)
6
5
EC50 = 10 pM
MDL = 1 pM
Current Microplate Assay
MDL = 0.03pg/assay
4
3
2
1
0
10 -14
10 -13
10 -12
10 -11
10 -10
10 -9
10 -8
10 -7
TCDD Concentration (M)
33
Activation of the CALUX Cell Bioassay by PCDDs, PCDFs and PCBs
8
2378-TCDD
12478-PCDD
2378-TCDF
23478-PCDF
6
Luciferase Activity
(RLU/ug protein)
3344-TCB
33445-PCB
4
2
0
10 -14
10 -13
10 -12
10 -11
10 -10
10 -9
10 -8
10 -7
10 -6
10 -5
Chemical Concentration (M)
34
WHO Toxic Equivalency Factors (TEFs) and CALUX
Relative Potency (REP) Factors for Chlorin ated Dibenzop-Dioxin s, Dibenzofurans and Biphenyls .
_______________________________________________
Compound
WHO-TEF
CALUX REP
_______________________________________________
2378- TCDD
12378- PeCDD
123478- HxCDD
123678- HxCDD
123789- HxCDD
1234678- HpCDD
OCDD
1
1
0.1
0.1
0.1
0.01
0.0001
1.00 ± 0.01
0.73 ± 0.1
0.075 ± 0.014
0.098 ± 0.017
0.061 ± 0.012
0.031 ± 0.008
0.00034 ±0.00008
2378- TCDF
12378- PeCDF
23478- PeCDF
123478- HxCDF
123678- HxCDF
123789- HxCDF
234678- HxCDF
1234678- HpCDF
1234789- HpCDF
OCDF
0.1
0.05
0.5
0.1
0.1
0.1
0.1
0.01
0.01
0.0001
0.67 ± 0.01
0.14 ± 0.04
0.58 ± 0.08
0.13 ± 0.02
0.14 ± 0.03
0.11 ± 0.02
0.31 ± 0.06
0.024 ± 0.007
0.044 ± 0.010
0.0016 ± 0.0005
PCB 77
0.0005
0.0014 ± 0.0004
PCB 81
0.0001
0.0045 ± 0.0012
PCB 114
0.0005
0.00014 ± 0.00002
PCB 126
0.1
0.038 ± 0.007
PCB 156
0.0005
0.00014 ± 0.00002
PCB169
0.01
0.0011 ± 0.0003
_______________________________________________
35
Bioassay Systems - Considerations
Structural Diversity of AhR Agonists and/or Antagonists:
Potential for False Positives in HAH Detection
CH3
Cl
N
NNH2
Cl
O
O
S
Halogenated Aromatic Hydrocarbons and Polycyclic Aromatic Hydrocarbons
Cl
O
Cl
O
Cl
2,3,7,8-Tetrachlorodibenzo-p-dioxin
CH3O
Cl
1-Methyl-1-phenylhydrazine
O
Cl
Cl
Cl
N N
Cl
2,3,7,8-Tetrachlorodibenzofuran 3,4,3',4,'5-Pentachlorobiphenyl
CH3
NH2
N
H
OH
SKF71739
(1S,2R)-(-)-cis-1-Amino-2-indanol
S
SCH3
O
N
H
NH2
CH3
S
NH2
Benzo(a)pyrene
5-Methyl-2-phenylindole
CH3
O
S
O
-Naphthoflavone
N
H
CN
O
3-Methylcholanthrene
CH3
OCH 3
CF3
O
CH3
CH3
Omeprazole
Cl
Cl
Cl
O
2,3,7,8-Tetrachlorodibenzo-p-dioxin
Cl
Cl
Cl
Cl
N
CH2
N
N
NH2
2(Methylmercapto)aniline
Cl
N
Cl
YH439
H3 C
H
N
NH2
O
Guanabenz
1,5-Diaminonaphthalene
CH3
O
CH3
N
H
H
N
O2N
SRN-P2:109,NH2
NH2
N
NH2
S
NO2
2-(4-Amino-3-methylphenyl)
benzothiazole
• Promiscuous ligand binding by the AhR requires appropriate extraction and
chemical clean-up methods to remove unwanted compopunds
36
Flow Diagram for Analysis of Samples by CALUX and GC/MS
Sample
Extraction and
Chemical Clean-up
PCDD/PCDF
&
PCB
Estimate
of relative
activity
Total HAHs
CALUX Bioassay
Analysis
Negative
Positive
Analysis by
HRGC/MS
No Compounds that Activate
the Ah Receptor and/or
Contains Compounds that Block
Activation (i.e. Antagonists)
37
Bioassay Systems - Considerations
• Calculation of relative biological potency of an unknown sample extract
EC50 = 1000-fold dilution = 20pM
ED50the
= 1000-fold
dilution
= 20pM
Thus
original
extract
contains the
Thus,
the
original
sample
contains
6
equivalent
(bioassay-TEQ) of 20 nM TCDD
7
7
6
Response
the equivalent (TEQ) of 20nM TCDD
5
5
4
4
3
3
EC50 = 20pM
2
2
1
1
0
EC50
0
10 -14
10 -13
10 -12
10 -11 10 -10
10 -9
T CDD Concentration (M)
10 -8
10 -7
10 8
10 7
10
6
10 5
10 4
10 3
10 2
10 1
Dilution Factor
38
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
.
39
Comparativ e GCMS/CALUX Result s from
the Umea Round Robin Analysis of
Environmental Samples for Chlorinated
Dibenzo-p-Dioxin s,
Dibenzofurans
and
Biphenyls .
______________________________________
TEQ (ng/g sample)
Sample
GC/MS
CALUX
______________________________________
Soil A
0.3
0.16
Soil B
0.17
1.2
Soil C
0.19
0.6
Soil D
0.30
1.06
Ash A
Ash B
Ash C
0.40
0.04
0.42
0.13
0.04
0.50
Solution F
222
448
Solution H
3.42
8.39
______________________________________
40
Double-Blind Validation Results
Comparison of CALUX and HR GC/MS
Analysis of Ash and Exhaust Gas Samples
Ash
Exhaust Gas
41
Double-Blind Validation Results
Comparison of CALUX and HR GC/MS
Analysis of Soil and Human Fat Samples
HRGCMS, pg TEQ/gram
1000
Soil
R2 = 0.854
100
10
1
0.1
0.01
0.1
1
10
100
1000
CALUX, pg TEQ/gram
HRGCMS(pgTEQ/g fat)
100
Human Fat
80
R = 0.8051
60
40
20
0
0
20
40
60
CALUX Assay(pgTEQ/g fat)
80
100
42
Environmental Samples Contain Additional
Ah Receptor Active HAHs?
Ash
HRGCMS, pg TEQ/gram
1000
Soil
R2 = 0.854
100
10
1
0.1
0.01
0.1
1
10
100
1000
CALUX, pg TEQ/gram
100
HRGCMS(pgTEQ/g fat)
Exhaust Gas
Human Fat
80
R = 0.8051
60
40
20
0
0
20
40
60
CALUX Assay(pgTEQ/g fat)
80
100
43
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
44
Comparison of GC/MS TEF and CALUX REP Values
CALUX REP Values for Dioxin-Like HAHs
10
1
.1
.01
.001
.0001
.0001
.001
.01
.1
1
10
WHO TEF Values for Dioxin-Like HAHs
45
Tiered Flow Diagram for Sample Screening
CALUX
NEGATIVE
No compounds that bind
productively to Ah receptor
POSITIVE
Run immunoassay
POSITIVE
Run GC/MS for
confirmation
PCB
ELISA
NEGATIVE
No TCDD-like
compounds
BOTH NEGATIVE
Use CALUX to drive
chemical purification
TCDD
ELISA
NEGATIVE
No TCDD-like
compounds
POSITIVE
Run GC/MS for
confirmation
ELISA Also suggests
that there are more
2,3,7-PCDD/Fs than
GC/MS indicates.
What could it be?
46
One Possibility for the Higher Equivalent Estimates: PB/CDDs/Fs
Halogenated Aromatic Hydrocarbons
Br
Cl
O
Cl
Cl
Cl
O
Cl
Cl
Cl
O
OH
O
Br
Cl
Br
Br
Br
2,3,7,8-Tetrachlorodibenzo-p-dioxin
2,3,7,8-Tetrachlorodibenzofuran Polybrominated Diphenyl Ether
Sources:
Formed during PDBE Synthesis
Burning of PBDE Containing Materials
Photochemical Reactions of PBDEs
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
Br
Br
O
Br
Br
Br
Br
Br
Br
O
Br
O
Br
Polybrominated Dibenzofurans (PBDFs)
Polybrominated Dibenzo-p-dioxins (PBDDs)
47
Brominated Dioxins and Related HAHs are Relatively Potent Activators
12000
10000
2378-TCDD, n=11
PCB 169, n=6
PBB 169, n=5
2378-TBDD, n=4
RLU
8000
6000
4000
2000
0
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
48
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
2,3,7,8-TeBDD
0.05
0.068
0.09
0.034
0.004
0.0076
1,2,3,7,8-PeCDD
1,2,3,7,8-Pe BDD
0.62
0.87
0.17
0.39
0.031
0.025
2,3-DiB-7,8-DiCDD
2-B-3,7,8-TriCDD
0.012
0.12
0.0073*
0.035
0.00049*
0.0025
1,2,4,7,8-PeCDD
1,2,4,7,8-PeBDD
34.0
12.9
11.2
6.17
2.82
0.195
Immature 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).
49
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)
50
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
51
Cell Bioassay Systems - Development
Microplates containing cells can be sealed and stored at room
temperature for up to 14 days with little loss of TCDD-inducible
luciferase activity.
12000
Day 1
Day 14
Seal
Luciferase Activity (RLU/well)
10000
8000
6000
4000
2000
0
.001
Viewplate microplate
.01
.1
1
TCDD (ng/ml)
with well sealer
This development now allows plates to be prepared off-site and
52
mailed to the site for extract treatment and analysis.
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.
53
Acknowledgements and Support
University of California @ Davis
Michael Ziccardi, Joe Rogers, Patricia Garrison,
Bruce Hammock, Shirley Gee, Guomin Shan
Xenobiotic Detection Systems
George Clark, David Brown, Mick Chu
Hiyoshi Corporation (Japan)
Hiroshi Murata
Scientific Institute of Public Health, Brussels, Belgium
Leo Goeyens, Ilse van Overmeire
National Institutes of Environmental Health Sciences (NIEHS)
Superfund Basic Research Program - ESO4699, ES04911
54
Nanotechnology and
Biosensors
Ian Kennedy
Department of Mechanical and Aeronautical Engineering
and
Biomedical and Electrical Computer Engineering Graduate Groups
University of California Davis
Supported by the NIEHS Superfund Basic Research Program and
NSF NNI
55
Strategies for detection in
environmental samples
Increased throughput
Increased sensitivity
Increased specificity
Achieved by
Small volumes
Minimized pre-processing of samples
Minimized autofluorescence
New fluorescent labels
56
Novel technologies
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
materials
Quantum dots
Encapsulated lanthanide oxides
Absorbing labels such as C60
57
Quenching fluoroimmunoassay
in microdroplets
Competitive Quenching Fluoro ImmunoAssay (QFIA)
Antibody quenching
- TCP
analyte
- TCP- F
Labeled analyte
58
Assay for TCP
Calibration 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
mg/L
ELISA
110
QFIA
QFIA
200 mL
microdrop
0 100
0
90
I
1
80
*
/
) F 70
I
(
PBS
urine 1/50
A
60
50
IC50
2.74
4.2
0.45
LOD
0.2
0.36
0.04
mg/L
urine
IC50
22.5
LOD
2
40
10 -12 10 -11 10 -10 10 -9 10 -8 10 -7 10 -6 10 -5 10 -4
TCP, M
59
Micro cavity resonances
Intense optical field
created by internally
reflected fluorescence
from Rhodamine
60
Images of Rh emission
quenched by C60
Intensities within
droplets greatly
reduced when C60
is added to droplet
61
Detecting bacteria
Single E. coli detected
using optical resonances
without labeling
Couple to immunoassay
for specificity or use mode
structure for information
62
Microdroplets on a chip
Microdroplets of
aqueous sample
Biosensor chip
MIcrochannel filled with low
refractive index silicon oil
63
Trapped microdroplet with
long interrogation time
64
Lanthanide oxide
nanoparticles
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
65
Lanthanide oxides as
reporters for bioassays
57
58
La Ce
59
60
61
62
68
69
70
Pr Nd
P
Sm Eu Gd Tb Dy Ho Er
T
Yb Lu
m
63
64
65
66
67
71
m
66
Europium oxide spectra
Europium
80x10
3
Excitation spectrum
observed at 610 nm
Emission spectrum
excited at 466 nm
Intensity (arbitrary units)
70
60
50
40
30
20
10
350
400
450
500
550
600
650
Intensity (arbitrary units)
Wavelength (nm)
4x10
Emission spectrum
excited at 524 nm
6
Rhodamine
3
2
Excitation spectrum
observed at 548 nm
1
500
520
540
560
Wavelength (nm)
580
600
67
Gas phase synthesis
and functionalization
Antigen
Antibody
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.
68
Pure Eu2O3 particles
Photoluminescence Spectrum:
Pure Eu2O3; Monoclinic Dominant Peaks at 615 and 623 nm;
Phase; Gas-phase
Short Lifetime Due to Concentration
Synthesis.
Quenching Combined with Small Size.
69
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
70
Europium oxide lifetime
Lifetimes of 1 ms
or more
Eu:Y2O3
Biological
background
typically less than
10 ns
71
Functionalized
nanoparticles
Microwave
chemistry and gas
phase chemistry
used to add
protective SiO
layer and NH2
functional group
72
Optical properties of functionalized
particles
Optical properties of Eu2O3
unchanged by surface
treatment
Additional phosphors
obtained by doping
lanthanides into hosts
Capped with silane
Used in immunoassay for
atrazine
73
Nanophosphors separated
with microchip
electrophoresis
80 mm channel electrophoresis system
separated two size classes of nanoparticles
can be used for assays
74
Europium oxide label in
a competitive
immunoassay
Competition
Labeled
analyte
Ab
75
Europium oxide label in
a competitive
immunoassay
Separation
Magnetic bead
and 2 Ab
76
Europium oxide in a
pyrethroid metabolite
assay
Competitive assay for 3phenoxybenzoic acid (PBA)
with magnetic separation
IC50 =
2 x 10 –4 ng mL-1
Europium oxide particle
conjugated to the hapten
77
New nanoparticle assay
formats
Multi wavelength
labels can be used
to provide an
internal standard
for sandwich
immunoassays
78
Time gated detection
79
Eu phosphors in micro
channels
80
Fluorescence lifetimes
Eu lifetime can be
greater than 1ms
Fluorescein lifetime
of the order of 10 ns
81
MEMS sensors
Quartz transmits UV excitation
Glass blocks excitation light, transmits visible signal
82
Summary
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?
83
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