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Real-time Monitoring with a Portable Miniaturized
Surface Plasmon Resonance System
Clement E. Furlong, Research Professor, Departments
of Medicine (Div. Medical Genetics) & Genome SciencesUniversity of Washington, Seattle, WA
Presented by: Brian Marquardt CPAC/UW
Spreeta sensing components
Each Spreeta chip contains Each Spreeta chip contains all of the optical all of the optical components needed for components needed for sensitive SPR measurement sensitive SPR measurement of biomolecular interactionsof biomolecular interactions
• Spreeta SPR components developed in collaboration with UW with TI
• Miniaturized, robust, high performance devices.
• Inexpensive in large quantity
• Excellent manufacturing capabilities and quality control
The SPIRIT system(Surface Plasmon Instrumentation for the Rapid Identification of Toxins)
The SPIRIT system(Surface Plasmon Instrumentation for the Rapid Identification of Toxins)
• Compact, lightweight (lunchbox size, 6 lb.)
• High performance• 24 simultaneous
measurements • Low power (5W)
allows portable operation
• Automated
Current laboratoryprototype
Touchscreen data display
Selected SPR curveSelected SPR curve
Detected levelsDetected levels(numeric)(numeric)
Detected levelsDetected levels(bargraph)(bargraph)
Sensor channelSensor channel
Sensor surface chemistry
Y Y Y Y Y
Glass substrateGlass substrate
Gold layer (50 nM)Gold layer (50 nM)
Soluble protective coatingSoluble protective coating (dextran/trehalose) allows long-term dry storage at room temperature
Target receptors:Target receptors:
(usually antibodies)Designed to capture a specific agent or analyte e.g.:
•Toxins•Viruses•Spores•Bacteria
Control receptorsControl receptors (usually antibodies) Designed NOT to respond to that agent
Spreeta sensor chip
Each Spreeta chip has Each Spreeta chip has 3 useable channels3 useable channels
0
0.2
0.4
0.6
0.8
1
0 20 40 60 80
Ө Degrees
Re
fle
cti
vit
y
1.3367
1.3368
1.3369
1.337
1.3371
1.3372
1.3373
1.3374
1.3375
1.3376
1.3377
0 5 10
Time, MinR
efr
ac
tiv
e In
de
x
System software
Fundamentals of Fundamentals of
Surface Plasmon Surface Plasmon ResonanceResonance
Fundamentals of Fundamentals of
Surface Plasmon Surface Plasmon ResonanceResonance
Sensorgram
SPIRIT performs 24 simultaneous measurements of antibody
bindingEight sensor chips
Three active spots per sensor
Analyte
Detection event
Flowcell
Examples of Assays Possible with SPR
• Whole microbial cells -(F.tularensis, E. coli, Y. pestis)
• Spores -(e.g., anthrax)
• Viruses with or without amplification -(e.g. Norwalk, flu)
• Proteins by direct detection with or without amplification/verification-(protein toxins, industrial proteins, therapeutics)
• Small molecular weight analytes using displacement or competition assays-(e.g., domoic acid, cortisol, insecticides, toxic chemicals, TNT & other small organics)
Detection of Larger AnalytesDetection of Larger Analytes
• Microbes
• Spores
• Viruses
• Proteins/Toxic Proteins
• Microbes
• Spores
• Viruses
• Proteins/Toxic Proteins
Signal Detection
Analyte Detection and Signal Amplification
1.3368
1.3370
1.3372
1.3374
1.3376
1.3378
1.3380
1.3382
0 50 100 150
Time, min
Ref
ract
ive
ind
ex
Signal Detection
Analyte Detection and Signal Amplification
Analyte Detection and Signal Amplification
1.3368
1.3370
1.3372
1.3374
1.3376
1.3378
1.3380
1.3382
0 50 100 150
Time, min
Ref
ract
ive
ind
ex
Signal Detection
Analyte Detection and Signal Amplification
Analyte Detection and Signal Amplification
1.3368
1.3370
1.3372
1.3374
1.3376
1.3378
1.3380
1.3382
0 50 100 150
Time, min
Ref
ract
ive
ind
ex
Signal Detection
Analyte Detection and Signal Amplification
Analyte Detection and Signal Amplification
1.3368
1.3370
1.3372
1.3374
1.3376
1.3378
1.3380
1.3382
0 50 100 150
Time, min
Ref
ract
ive
ind
ex
Detection and Verification of F. Tularensis (105 cfu/ml)
1.3388
1.3390
1.3392
1.3394
1.3396
0 2 4 6 8 11 13 15 17 19 21 23
Time (min)
Rel
ativ
e R
efra
ctiv
e In
dex
anti-F.T #1
anti-F.T #2
anti-F.T. #3
anti-Bot A NT #1
anti-Bot A NT #2
anti-Bot A NT #3
Active channels
Reference channels
Detection
Amplification/verification
Detection of MicrobesDetection of Microbes
Virus DetectionVirus Detection
Amplification
Detection of Staphylococcal Enterotoxin B
1.33495
1.33500
1.33505
1.33510
1.33515
1.33520
1.33525
0 20 40 60 80Time, min
Sen
sor
resp
on
se, R
IU
0.0E+00
1.0E-05
2.0E-05
3.0E-05
4.0E-05
5.0E-05
6.0E-05
7.0E-05
8.0E-05
9.0E-05
0 20 40 60 80 100
SEB concentration, nM
SE
B b
ind
ing
rat
es, R
IU/m
in
0.0E+00
2.0E-06
4.0E-06
6.0E-06
8.0E-06
1.0E-05
0 1 2 3 4
Detection of 5 ng/mL (5 ppb; 33pM) BotNT (denatured botulinum
toxin)
1.33211.332121.332141.332161.33218
1.33221.332221.332241.332261.33228
1.3323
0 10 20 30 40Time (min)
RI
Anti-Bot-toxin
Reference
AmplifyDetect
Direct Detection of Ricin A Chain (64 ppb-320 ppb)
Direct Detection of Ricin A Chain (64 ppb-320 ppb)
0
2
4
6
8
10
12
14
0 100 200 300 400
Ricin A Chain Concentration (nM)
Bin
din
g R
ate
(x10
-6R
IU/m
in)
-0.00001
0.00001
0.00003
0.00005
0.00007
0.00009
0 200 400 600 800 1000
Time (seconds)
Bac
kgro
un
d-s
ub
trac
ted
RIU 100 nM Ricin A Chain
50 nM Ricin A Chain
20 nM Ricin A Chain
No Ricin A Chain
Detection of Cortisol by Competition Assay
Cortisol Competition 2-24-04c
1.3384
1.3385
1.3386
1.3387
1.3388
1.3389
0 1000 2000 3000 4000 5000 Time (seconds)
RIU
BSA BSA-Cortisol HSA-GD
5 nM
2 nM
1 nM 750 pM
10 nM
1000 nM Estriol
Lower arrows indicate returnto no analyte
Standard Domoic Acid Concentration
Curve in Clam Extracts
Other Useful Applications of SPR SensingOther Useful Applications of SPR Sensing
• Nucleic Acid Analyses
• Many Other Molecular Interactions
• Nucleic Acid Analyses
• Many Other Molecular Interactions
Protein Nucleic Acids as Recognition Elements for DNA/RNA
Protein Nucleic Acids as Recognition Elements 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 16 mer on the Sensor Surface
Binding of a 79 bp DNA Probe to a Complementary PNA 16 mer on the Sensor Surface
Detection of Analytes in Complex Matrices
(e.g., saliva, plasma, urine, stool extracts, sea water, fresh water, etc.)
Detection of Analytes in Complex Matrices
(e.g., saliva, plasma, urine, stool extracts, sea water, fresh water, etc.)
Detection of 1 nM (28 ppb) SEB in seawater
Staphylococcal enterotoxin B
Detection of 1 nM (28 ppb) SEB in seawater
Staphylococcal enterotoxin B
1.33966
1.33967
1.33968
1.33969
1.3397
1.33971
1.33972
1.33973
1.33974
0 20 40 60 80
Time, min
Re
fra
cti
ve
in
de
x,n
Detection of 500 pM (14 ppb) SEB in urine
Detection of 500 pM (14 ppb) SEB in urine
500 pM SEBWash(urine)
Amplification
From: Naimushin et al., Biosensors and Bioelectronics 17:573
Detection of cortisol in saliva using the compound
flow cell
0.0069
0.007
0.0071
0.0072
0.0073
0.0074
17 27 37 47 57 67 77Time (minutes)
Ref
eren
ce-s
ub
trac
ted
RIU
Saliva only
Saliva plus 28 nM Cortisol
Saliva plus 14 nM Cortisol
0.0069
0.007
0.0071
0.0072
0.0073
0.0074
17 27 37 47 57 67 77Time (minutes)
Ref
eren
ce-s
ub
trac
ted
RIU
Saliva only
Saliva plus 28 nM Cortisol
Saliva plus 14 nM Cortisol
Detection of Theophylline in Saliva
Using the Compound flow Cell
Detection of Theophylline in Saliva
Using the Compound flow Cell
1.34225
1.3423
1.34235
1.3424
1.34245
1.3425
2000 2500 3000 3500 4000Time (seconds)
RIU
Reference
Theophylline
Saliva only
Saliva plus 2 MTheophylline
Saliva plus 1 MTheophylline
1.34225
1.3423
1.34235
1.3424
1.34245
1.3425
2000 2500 3000 3500 4000Time (seconds)
RIU
Reference
Theophylline
Saliva only
Saliva plus 2 MTheophylline
Saliva plus 1 MTheophylline
-100
-50
0
50
100
150
200
0 20 40 60 80 100 120 140 160 180
Time (min)
RIU
Sequential Detection of 8 Analytes
Y. pestis
106 CFU/ml
Ovalbumin 10 ng/ml
SEB 5 ng/ml
F. tularensis 5 x 103 CFU/ml
B. anthracis 5 x 106 CFU/ml
Norwalk VLPs 5 x 109 particles/ml
Ricin A chain 20 ng/ml
BG Spores 9 x 104 CFU/ml
SPIRIT Team & Sponsors• Medical Genetics
Group:Dr. Clement FurlongScott Soelberg Dr. Gary GeissDr. Rick Stevens Steve NearMatthew Probert Joshua ProbertNathaneal SwansonDr. Paul Baker
• Electrical Engineering Group:Dr. Sinclair YeeTim ChinowskyPeter KauffmanJared TritzMichael GrowTony Mactutis
• Texas Instruments: Jerry Elkind Dwight Bartholomew John Quinn
• SponsorsSponsors:: DOD Texas Instruments Center for Process Analytical Chemistry (CPAC), UW, Seattle Washington State Sea Grant, NIH/NIEHS
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