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CPAC Summer InstituteCPAC Summer InstituteJune 15-17, 2008
Real Time Biosensor Systems & Protein ProductionReal Time Biosensor Systems & Protein Production
••Clement E. Furlong, Scott D. Soelberg, Richard Stevens and Peter Kaufmann Clement E. Furlong, Scott D. Soelberg, Richard Stevens and Peter Kaufmann Departments of Medicine Departments of Medicine
(Div. Medical Genetics) & Genome Sciences(Div. Medical Genetics) & Genome Sciences
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Fundamentals of Fundamentals of Fundamentals of Fundamentals of
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Surface Plasmon ResonanceSurface Plasmon ResonanceSurface Plasmon ResonanceSurface Plasmon Resonance
0
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Ө DegreesӨ Degrees
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System software
Sensorgram
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Our goal is to reduce the size and cost of SPR Our goal is to reduce the size and cost of SPR technology from a ~$300 000 desk top device to a technology from a ~$300 000 desk top device to a technology from a ~$300,000 desk top device to a technology from a ~$300,000 desk top device to a portable system that costs under $30,000portable system that costs under $30,000
This system is designed around gthe miniature TI-SPR chip, designed by José Melendez and teamand team.
Spreeta sensing componentsSpreeta sensing componentsSpreeta sensing componentsSpreeta sensing componentsS SPRS SPR•• Spreeta SPR components Spreeta SPR components developed in collaboration developed in collaboration with UW with TIwith UW with TI
Each Spreeta chip contains Each Spreeta chip contains all of the optical all of the optical components needed for components needed for
•• Miniaturized, robust, high Miniaturized, robust, high performance devices. performance devices.
I i i l titI i i l tit components needed for components needed for sensitive SPR measurement sensitive SPR measurement of biomolecular interactionsof biomolecular interactions
•• Inexpensive in large quantityInexpensive in large quantity
•• Excellent manufacturing Excellent manufacturing capabilities and qualitycapabilities and qualitycapabilities and quality capabilities and quality controlcontrol
The SPIRIT systemThe SPIRIT systemThe SPIRIT systemThe SPIRIT systemyy(Surface Plasmon Instrumentation for the Rapid Identification of Toxins)(Surface Plasmon Instrumentation for the Rapid Identification of Toxins)
yy(Surface Plasmon Instrumentation for the Rapid Identification of Toxins)(Surface Plasmon Instrumentation for the Rapid Identification of Toxins)
•• Compact, Compact, lightweight (lunchbox lightweight (lunchbox size, 6 lb.)size, 6 lb.)
Current laboratoryprototype
•• High performanceHigh performance•• 24 simultaneous 24 simultaneous
measurementsmeasurementsmeasurements measurements •• Low power (5W) Low power (5W)
allows portable allows portable titioperationoperation
•• SemiSemi--AutomatedAutomated
Touchscreen data displayTouchscreen data displayTouchscreen data displayTouchscreen data display
Selected SPR curveSelected SPR curve Detected levelsDetected levels(bargraph)(bargraph)
Sensor channelSensor channel
Detected levelsDetected levels(numeric)(numeric)
Sensor surface chemistrySensor surface chemistrySensor surface chemistrySensor surface chemistryEach Spreeta chip has 3 Each Spreeta chip has 3 useable channelsuseable channels
Soluble protective coatingSoluble protective coating(dextran/trehalose)allows long-term dry storage at
useable channelsuseable channels
room temperatureControl receptorsControl receptors(usually antibodies)Designed NOT to respond to that agent
Y Y Y Y Y Ψ Ψ Ψ Ψ ΨGold layer (50 nM)Gold layer (50 nM)
Target receptors:Target receptors:(usually antibodies)Designed to capture a specific agent or
l t Y Y Y Y Y Ψ Ψ Ψ Ψ Ψ
Glass substrateGlass substrate
analyte e.g.:
•Toxins•Viruses•Spores•Bacteria Spreeta Spreeta
sensor chip
Storage of Gold SlidesStorage of Gold Slidesgg120140
wet Dextran 1
6080
100of
1 d
ay w
02040
erce
nt o
Control
Dextran 2
00 50 100 150 200 250 300 350
Time (days)
Pe
Anti-Alkaline-phosphatase(AP)-antibody-coated slides were dried with a thin layer of 10 mM Tris, pH 8.0, 2.5% trehalose
d 2 5% d t Aft t d d t lid tt dand 2.5% dextran. After extended storage, slides were wetted, exposed to AP and analyzed for AP activity.
Silicone moldingSilicone moldinggg
•• Versatile technique for production of Versatile technique for production of q pq pprecision flowcells & other fluidic precision flowcells & other fluidic componentscomponents
Antibody 1
Flow channelsformed by fishing line
Antibody 2
Antibody 3
Flowcell cast from mold
Fixture for derivatizing individual channelsFixture for derivatizing individual channelsFixture for derivatizing individual channelsFixture for derivatizing individual channels
SPIRIT performs 24 simultaneous SPIRIT performs 24 simultaneous ppmeasurements of antibody bindingmeasurements of antibody binding
Eight sensor chipsDetection event
Eight sensor chips
Toxin
Silicon flow cell Silicon flow cell i f i hi f i hinterfaces with interfaces with TE controller TE controller ((±±0.010.01oo C)C)
Three active spots per sensor
Flowcell
Snap-in block of 8 sensor chips
Examples of Assays Possible with SPRExamples of Assays Possible with SPRExamples of Assays Possible with SPRExamples of Assays Possible with SPR
•• Whole microbial cells Whole microbial cells --((F.tularensis, E. coli, Y. pestisF.tularensis, E. coli, Y. pestis))
•• Spores Spores (e g anth a )(e g anth a )--(e.g., anthrax)(e.g., anthrax)
•• Viruses with or without amplification Viruses with or without amplification --(e.g. Norwalk, flu)(e.g. Norwalk, flu)
P t i b di t d t ti ith ith tP t i b di t d t ti ith ith t•• Proteins by direct detection with or without Proteins by direct detection with or without amplification/verificationamplification/verification--(protein toxins, industrial proteins, therapeutics)(protein toxins, industrial proteins, therapeutics)
•• Small molecular weight analytes using displacement orSmall molecular weight analytes using displacement orSmall molecular weight analytes using displacement or Small molecular weight analytes using displacement or competition assayscompetition assays--(e.g., domoic acid, cortisol, insecticides, toxic chemicals, TNT & other small (e.g., domoic acid, cortisol, insecticides, toxic chemicals, TNT & other small organics)organics)
Detection of Larger AnalytesDetection of Larger AnalytesDetection of Larger AnalytesDetection of Larger AnalytesDetection of Larger AnalytesDetection of Larger AnalytesDetection of Larger AnalytesDetection of Larger Analytes
• Microbes• Microbes• Microbes
• Spores
• Microbes
• Spores
• Viruses
•Proteins/Toxic Proteins
• Viruses
•Proteins/Toxic Proteins•Proteins/Toxic Proteins
•Small molecule toxins
•Proteins/Toxic Proteins
•Small molecule toxins
Analyte Detection and Signal AmplificationAnalyte Detection and Signal Amplification
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Signal Detection
Analyte Detection and Signal AmplificationAnalyte Detection and Signal AmplificationAnalyte Detection and Signal AmplificationAnalyte Detection and Signal Amplificationy py p
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Signal Detection
Analyte Detection and Signal AmplificationAnalyte Detection and Signal AmplificationAnalyte Detection and Signal AmplificationAnalyte Detection and Signal Amplificationy py p
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Signal Detection
Analyte Detection and Signal AmplificationAnalyte Detection and Signal AmplificationAnalyte Detection and Signal AmplificationAnalyte Detection and Signal Amplificationy py p
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Signal Detection
Detection of 1 nM (28 ppb) SEB in seawaterDetection of 1 nM (28 ppb) SEB in seawaterDetection of 1 nM (28 ppb) SEB in seawaterDetection of 1 nM (28 ppb) SEB in seawaterDetection of 1 nM (28 ppb) SEB in seawaterDetection of 1 nM (28 ppb) SEB in seawaterDetection of 1 nM (28 ppb) SEB in seawaterDetection of 1 nM (28 ppb) SEB in seawater
1.33973
1.33974
n
1.3397
1.33971
1.33972
ve in
dex,
n
1 33967
1.33968
1.33969
Ref
ract
iv
1.33966
1.33967
0 20 40 60 80Time minTime, min
Detection of 500 pM (14 ppb) SEB in urineDetection of 500 pM (14 ppb) SEB in urineDetection of 500 pM (14 ppb) SEB in urineDetection of 500 pM (14 ppb) SEB in urineDetection of 500 pM (14 ppb) SEB in urineDetection of 500 pM (14 ppb) SEB in urineDetection of 500 pM (14 ppb) SEB in urineDetection of 500 pM (14 ppb) SEB in urine
Amplification
500 pM SEBWash(urine)
From: Naimushin et al., Biosensors and Bioelectronics 17:573
Detection of MicrobesDetection of MicrobesC t D t ti Li it 10C t D t ti Li it 1033 f / lf / l
Detection and Verification of F. Tularensis (105 cfu/ml)
Current Detection Limit: 10Current Detection Limit: 103 3 cfu/mlcfu/ml
1.3396
dex
Detection
Amplification/verification
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Ind
anti-F.T #1anti-F.T #2anti-F.T. #3anti Bot A NT #1Active channels
e ec o
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tive
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r anti-Bot A NT #1anti-Bot A NT #2anti-Bot A NT #3
Active channels
1.3388
1.3390
Rel
a Reference channels
0 2 4 6 8 11 13 15 17 19 21 23Time (min)
Virus DetectionVirus DetectionNorwalk VLP Detection
Reference Subtracted, 0.75 mL Sample, p
180
10^6 PFU/ml Norwalk VLPs Anti-Norwalk Amp
120140160180
Norwalk
Amplification
406080
100
RIU
Amplification
-40-20
020
0 2.5 5 7.5 10 12.5 15 17.5 20 22.5Time (min)
-6040 Time (min)
Quantitative Detection of Quantitative Detection of QQStaphylococcal Enterotoxin BStaphylococcal Enterotoxin B
9 0E-05
1.33515
1.33520
1.33525
, RIU 6.0E-05
7.0E-05
8.0E-05
9.0E-05
RIU/
min
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enso
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3.0E-05
4.0E-05
5.0E-05
B bi
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2 0E-06
4.0E-06
6.0E-06
8.0E-06
1.0E-05
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0 20 40 60 80Ti i
Se
0.0E+00
1.0E-05
2.0E-05
0 20 40 60 80 100
SEB t ti MSE
B
0.0E+00
2.0E-06
0 1 2 3 4
Time, min SEB concentration, nM
Detection of 5 ng/mL (5 ppb; 33pM) Detection of 5 ng/mL (5 ppb; 33pM) g pp pg pp pBotNT BotNT (denatured botulinum toxin)(denatured botulinum toxin)
1.332261.33228
1.3323Anti-Bot-toxin
Reference
1.33221.332221.33224
RI AmplifyDetect
1.332141.332161.33218
1.33211.33212
0 10 20 30 40Time (min)
Direct Detection of Ricin A Direct Detection of Ricin A Direct Detection of Ricin A Direct Detection of Ricin A Direct Detection of Ricin A Direct Detection of Ricin A Chain (64 ppbChain (64 ppb--320 ppb)320 ppb)
Direct Detection of Ricin A Direct Detection of Ricin A Chain (64 ppbChain (64 ppb--320 ppb)320 ppb)( pp( pp pp )pp )( pp( pp pp )pp )
14)
8
10
12
14
x10-
6RIU
/min
)
0.00005
0.00007
0.00009
ubtr
acte
d R
IU 100 nM Ricin A Chain50 nM Ricin A Chain20 nM Ricin A ChainNo Ricin A Chain
0
2
4
6
Bin
ding
Rat
e (x
0 00001
0.00001
0.00003
Bac
kgro
und-
s
00 100 200 300 400
Ricin A Chain Concentration (nM)
B-0.000010 200 400 600 800 1000
Time (seconds)
B
Detection of small molecule toxins by SPRDetection of small molecule toxins by SPRDetection of small molecule toxins by SPRDetection of small molecule toxins by SPRis a more difficult taskis a more difficult taskis a more difficult taskis a more difficult task
Displacement AssayDisplacement AssayR
espo
nse
Time
R
Displacement AssayDisplacement Assay
Antibody Loading
Sensor Ready Condition
Antibody Loading
Res
pons
e
Time
R
Displacement AssayDisplacement Assay
Introduction of Target
Sample Detection Mode
Introduction of Target
Res
pons
e
Time
R
Displacement AssayDisplacement Assay
Introduction of Target
Sample Detection Mode
Introduction of Target
Res
pons
e
Time
R
Displacement AssayDisplacement Assay
Antibody Displacement
Displacement of Target Analog
Antibody Displacement
Res
pons
e
Rate proportional to analyte (target) concentration
Time
R
The main advantage of the displacement The main advantage of the displacement i th t th i t i th t th i t assay is that the expensive components assay is that the expensive components
of the assay (antibodies or receptors) of the assay (antibodies or receptors) b t i d d b d b t i d d b d can be retained under a membrane and can be retained under a membrane and
reused many times.reused many times.
Competition AssayCompetition Assay
ANALYTE ATTACHED TO THE SURFACEANALYTE ATTACHED TO THE SURFACE
••Small Analytes:Small Analytes:••Estriol, Cortisol, Domoate…Estriol, Cortisol, Domoate…••Same analyte on the surfaceSame analyte on the surfaceSame analyte on the surfaceSame analyte on the surface
Competition AssayCompetition AssayNO ANALYTE PRESENTR
espo
nse
Time
R
Competition AssayCompetition AssayANALYTE PRESENT
Res
pons
e
Time
R
Competition AssayCompetition Assayp yp y
No target presentNo target present
nse
Ti
Res
po
Samples 1, 2, and 3
Time
Detection of Cortisol by Competition AssayDetection of Cortisol by Competition Assay
Cortisol Competition 2-24-04c 10 nM
1.3388
1.3389 BSABSA Cortisol
1 nM 750 pM
1.3387 RIU
BSA-CortisolHSA-GD
2 nM 1000 nM Estriol
13385
1.3386 5 nM Lower arrows indicate returnto no analyte
1.3384
1.3385
0 1000 2000 3000 4000 50000 1000 2000 3000 4000 5000Time (seconds)
External Compound Flow CellExternal Compound Flow CellExternal Compound Flow CellExternal Compound Flow Cell
Vacuum
Detection of cortisol in saliva Detection of cortisol in saliva Detection of cortisol in saliva Detection of cortisol in saliva using the compound flow cellusing the compound flow cell
0 0073
0.0074
RIU
Saliva plus 28 nM Cortisol
0 0073
0.0074
RIU
Saliva plus 28 nM Cortisol
0.0072
0.0073
ubtr
acte
d
Saliva plus 14 nM 0.0072
0.0073
ubtr
acte
d
Saliva plus 14 nM
0.0071
eren
ce-s
u
Saliva only
pCortisol0.0071
eren
ce-s
u
Saliva only
pCortisol
0.0069
0.007
Ref
e
0.0069
0.007
Ref
e
17 27 37 47 57 67 77Time (minutes)
17 27 37 47 57 67 77Time (minutes)
Quantification of CortisolQuantification of CortisolQuantification of CortisolQuantification of Cortisol100
ol s
ampl
e)
100
of n
o co
rtiso
10
ion
slop
e (%
1
1 10 100
Det
ect
0.1
Cortisol concentration (ng/ml)
1 10 100
Detection of Domoic Acid by Detection of Domoic Acid by yyCompetition AssayCompetition Assay
Collaboration with Dr. Vera Trainer’s team at NOAATrainer s team at NOAA
Standard Domoic Acid ConcentrationStandard Domoic Acid ConcentrationC i Cl E t tC i Cl E t tCurve in Clam ExtractsCurve in Clam Extracts
Stevens et al. Harmful Algae
Comparison of HPLC/SPR AssaysComparison of HPLC/SPR AssaysComparison of HPLC/SPR AssaysComparison of HPLC/SPR Assays
(GOLD STANDARD)(GOLD STANDARD)
Rapid Sample Clean UpRapid Sample Clean Up--p p pp p pConcentrationConcentration
Analyte
Analyte-immuno b dmagnetic bead
complex
Rapid Immuno Magnetic Bead Separation (IMS)Rapid Immuno Magnetic Bead Separation (IMS)Rapid Immuno Magnetic Bead Separation (IMS)Rapid Immuno Magnetic Bead Separation (IMS)
Rapid Immuno Magnetic Bead Separation (IMS)Rapid Immuno Magnetic Bead Separation (IMS)p g p ( )p g p ( )
Design for Ciguatoxin Detection ProtocolDesign for Ciguatoxin Detection Protocol
50 nm bead
50 nm bead
50 nm bead
Concentration, Verification, Amplification ProtocolConcentration, Verification, Amplification Protocol
1400 StreptavidinStreptavidin Microbeads (1075 RIU) 3
50 nM900
RIU 10 ng/ml
SEB
anti-SEB-biotin
(24 RIU)
50 nMParamagnetic
Bead
400
SEB (13 RIU)
(24 RIU)
1 21Sensor 2 3-100
0 20 40 60Time (min)
11 Ready 2 3
Concentration, Amplification and Verification Concentration, Amplification and Verification
of SEB from a Stool Sampleof SEB from a Stool Sample500
of SEB from a Stool Sampleof SEB from a Stool Sample
300
400 1 ng/ml SEB from a spiked stool sample
200
300
RIU
100
-100
0
0 2 4 6 8 100 2 4 6 8 10Time (min)
1/03 1/03 –– 6/03: Airborne SPR Sensing 6/03: Airborne SPR Sensing ggTest FlightsTest Flights
Variviggen instrumented with SPR sensors
Protein Nucleic Acids as Recognition Protein Nucleic Acids as Recognition El t f DNA/RNAEl t f DNA/RNA
Protein Nucleic Acids as Recognition Protein Nucleic Acids as Recognition El t f DNA/RNAEl t f DNA/RNAElements for DNA/RNAElements for DNA/RNAElements for DNA/RNAElements for DNA/RNA
Very stable receptor on chip
(Protein Nucleic Acid)
Allows detection of target
Binding of a 79 bp DNA Probe to a Complementary PNA Binding of a 79 bp DNA Probe to a Complementary PNA 16 mer on the Sensor Surface16 mer on the Sensor Surface
60
70
1000 ng/ml probe
40
50
U
1000 ng/ml probe
100 ng/ml probeApprox 60 RIU bound
10
20
30RIU 10 ng/ml probe
Reference (Avidinw ithout PNA)
Approx 30 RIU bound
-10
0
10
0 2 4 610
Time (min)Approx 2 RIU bound
Sequential Detection of 8 AnalytesSequential Detection of 8 Analytes
200
100
150Ovalbumin 10 ng/ml
SEB 5 ng/ml
F. tularensis 5 x 103 CFU/ml
Norwalk VLPs 5 x 109 particles/ml BG Spores
0
50RIU
Y. pestis
106 CFU/ml B. anthracis5 x 106 CFU/ml
5 x 109 particles/ml
Ricin A chain 20 ng/ml
9 x 104CFU/ml
-50
0
-1000 20 40 60 80 100 120 140 160 180
Time (min)
Inexpensive Production of AntibodiesInexpensive Production of AntibodiesInexpensive Production of AntibodiesInexpensive Production of Antibodies
Advantages of Shark IgNARsAdvantages of Shark IgNARsAdvantages of Shark IgNARsAdvantages of Shark IgNARs
Single poly peptide chain
12-15kD
Single poly peptide chain
Stanfield et al Science 305-1770Stanfield et al. Science 305 1770
Large scale production in E. coli – based bioreactors
Staby Expression SystemStaby Expression SystemStaby Expression SystemStaby Expression System
Poison = CcdB
Antidote = CcdA
Target = DNA
From Eurogentec Staby manual
Expression of IgNARExpression of IgNAR--cytb cytb fusionfusionfusionfusion
IgNAR-cytb
uced
uced
uced
pSCodon-IgNAR-cytB
BlaT7 pro
lac op
ccdA Not
indu
cIn
duce
d
Not i
nduc
Indu
ced
Not i
nduc
Indu
ced
6465 bp
pBR Rep
lac I
66
45
66
45
66
45
ileX glyT
proLleuW
argU
45
36
29
24
20
45
36
29
24
20
45
36
29
24
2020
14
6
20
14
6
20
14
6
The heme-binding domain of mouse cytochrome b5 has a red color allowing has a red color allowing visualization of expression.
Generation of Leaker StrainsGeneration of Leaker StrainsGeneration of Leaker StrainsGeneration of Leaker Strains
Phosphate binding protein productionPhosphate binding protein productionPhosphate binding protein productionPhosphate binding protein production
Non-leaker LeakerNon-leaker LeakerPB
P
PBP
Shoc
k
Shoc
k
bioreactor output(increasing time)
bioreactor output(increasing time)
Furlong and Sundstrom, J. Indus. Microbio. 30: 141-148
Protein purification systemProtein purification system
Pump 1
n 1
n 2
Fil 1
Pump 2 Col
umn
Col
umn
r 1
Filter 1
Fermentor
Sens
or
enso
r 2
Pump 3Fermentor S
Pump 4
Filter 2
Product OutWastePump 4 Product OutWaste
ConclusionConclusionThe portable SPR sensing system can The portable SPR sensing system can provide near realprovide near real--time monitoring for time monitoring for many different applications in many different applications in environmental monitoring, the food environmental monitoring, the food industry, pharmaceutical industry, medical industry, pharmaceutical industry, medical diagnostics and general research needs. diagnostics and general research needs.
SPR Team & SponsorsSPR Team & Sponsors•• Medical Genetics/Genome SciencesMedical Genetics/Genome Sciences
Dr. Clement FurlongDr. Clement FurlongScott Soelberg Scott Soelberg Dr. Gary GeissDr. Gary GeissDr Rick StevensDr Rick Stevens
•• Electrical Engineering Group:Electrical Engineering Group:Dr. Sinclair YeeDr. Sinclair YeeTim ChinowskyTim ChinowskyPeter KauffmanPeter KauffmanJared TritzJared TritzMichael GrowMichael GrowDr. Rick Stevens Dr. Rick Stevens
Steve NearSteve NearMatthew Probert Matthew Probert Joshua ProbertJoshua ProbertPeter KaufmannPeter Kaufmann
Michael GrowMichael GrowTony MactutisTony Mactutis
•• Texas Instruments: Texas Instruments: Jose MelendezJose MelendezJerry Elkind Jerry Elkind
•• NOAA TeamNOAA TeamDr. Vera TrainerDr. Vera TrainerDr. Jack Weckel Dr. Jack Weckel BB--TL EberhartTL EberhartS SpencerS Spencer
yyDwight Bartholomew Dwight Bartholomew John QuinnJohn Quinn
•• Seattle Sensor SystemsSeattle Sensor SystemsNathaneal SwansonNathaneal SwansonDr. Paul BakerDr. Paul BakerS SpencerS Spencer Dr. Paul BakerDr. Paul Baker
Financial Interests:Financial Interests:Sponsors:Sponsors: CEF SDS PK & PB holdSponsors:Sponsors: CEF, SDS, PK & PB hold
DOD stock in Seattle Sensor SystemsTexas Instruments Center for Process Analytical Chemistry (CPAC), UW, SeattleCenter for Process Analytical Chemistry (CPAC), UW, SeattleWashington State Sea Grant NSF/NIEHS NW Center for Human Health and Ocean Studies