Real-time Monitoring with a Portable Miniaturized Surface Plasmon Resonance System Clement E. Furlong, Research Professor, Departments of Medicine (Div.

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



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



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



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


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



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


1.34225

1.3423

1.34235

1.3424

1.34245

1.3425

2000 2500 3000 3500 4000Time (seconds)

RIU

Reference

Theophylline

Saliva only



-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

Real-time Monitoring with a Portable Miniaturized Surface Plasmon Resonance System Clement E. Furlong, Research Professor, Departments of Medicine (Div.

Documents

analyte slide

ppb slide

signal amplification

detection of cortisol

quality control slide

direct detection of

brian marquardt cpacuw

agent spreeta sensor