Joshua Probert, Scott D. Soelberg, Peter Kaufmann

and Clement E. Furlong

Departments of Medicine (Div. Medical Genetics) & Genome Sciences, University of Washington

CPAC Summer Institute,

July 21, 2011

Portable Surface Plasmon Resonance

Instrumentation for Rapid, Versatile

Biodetection

PROBLEM: CHEMICAL AND BIOLOGICAL HAZARDS A Growing Challenge to Public Safety

2

Food supply

Terrorism

Environment

The Challenge – Detection of Molecular Hazards

Anthrax

Melamine

Shiga Toxin

E. coli

4

The Challenge – Fast Detection in the Field

Salmonella

Ricin

E. coli

Paralytic & Amnesiac Toxin

Anthrax

0

0.2

0.4

0.6

0.8

1

0 20 40 60 80

Angle (pixel number)

Lig

ht

Inte

ns

ity

θ

-50

150

350

550

750

0 5 10 15Time (min)

RIU

x 1

0E

-6

System Software

Fundamentals of

Surface Plasmon Resonance

• Reduced Fluidics Size

• Compact, lightweight (lunchbox size, 6 lb.)

• Up to 24 simultaneous measurements for military, food industry, medical and other applications

• Low power (5W) allows portable operation with rechargeable battery

• Semi-Automated, Self Contained Storage and Readout

• Touchscreen Interface For Control and Simple Data Display

• Flow cell interfaces with TEC (±0.01 C)

Current laboratory platform

Portable Multi-channel SPR System

MBARI AUV

Old and New Fluidics

Reduced Fluidics Footprint

Reduced Electronics Footprint

Current Interface

New Interface (Mock-Up)

GO

Magnetic Nanoparticles

• Magnetic 40 nm

nanoparticles improve sensitivity by concentrating, purifying, and amplifying the signal.

• Effective for detection of low levels of Proteins, Small Molecules, Bacteria/viruses and larger (competition) in complex media

400 nm

probing

distance*

Gold (SPR) Surface

Flow Area

Magnetic Nanoparticles Amplify the SPR signal

Stepwise Detection of Staphylococcus enterotoxin B

(SEB)

1 Sensor

Ready 2 3

1

10

100

1000

10000

0 10 20 30 40 50 60

Time (min)

Rela

tive R

IU

10 ng/ml SEB (13 RIU)

50 ug/ml biotinylated

anti-SEB monoclonal

(24 RIU)

Streptavidin

nanobeads (1075 RIU)

1

3

2

C NO C

NC

B

NO

C NC

A NO C

NC

Buffer Waste

Sensors

Sample +

Nanobead

Mixture

Magnetic Separation

Column

Pump

1) Load Sample

2) Buffer Wash

(Magnet On)

3) Flow to Sensors

(Magnet Off)

Fluidic Diagram for Automated IMS

Separation and Analyte Detection

U

Servo Motor Controlled Magnet

Magnet OFF Magnet ON

ANALYTE ATTACHED TO THE SURFACE

•Small Analytes:

•Estriol, Cortisol, Domoate…

•Same analyte on the surface

Detection of small molecules by SPR

requires an indirect Competition Assay

NO ANALYTE PRESENT

Time

Re

sponse

Competition Assay

ANALYTE PRESENT

Time

Re

sponse

Competition Assay

ELISA vs. SPR

0.00%

20.00%

40.00%

60.00%

80.00%

100.00%

120.00%

0.25 2.5 25 250

SPR ELISA

Domoate Assay

-20

0

20

40

60

80

100

120

140

0 2 4 6 8 10 12 14

0ng

1ng

10ng

100ng

1000ng

Bulk

Shift

Binding

Area of

Analysis

Examples of Assays Performed 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 or with 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)

SPR will detect any ligand in sufficient concentration to which a binding partner (usually antibody) has been obtained, i.e.:

-50

0

50

100

150

200

0 20 40 60 80 100 120 140 160 180Time (min)

RIU

Y. pestis

106 CFU/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

Ovalbumin

10 ng/ml

Multiplex Measurement Capability:

Sequential Detection of 8 Analytes

Ongoing Collaborations

• Dr. Laurie Connell, University of Maine-PNA (peptide nucleic acid) probes for detection of Alexandrium (red tide) mRNA for species identification.

• Dr. Jian Payandeh in Professor William Catterall lab, UW – defining the molecular interactions between sodium channels and their toxin inhibitors.

MBARI

http://www.mbari.org/AUV

The UW-Furlong Group

• Professor

Dr. Clement Furlong

• Sensor Team

Scott Soelberg

Peter Kaufmann

Joshua Probert

• Biomarker/PON Team Rebecca Richter Dr. Toby Cole Stephanie Suzuki Dr. Rick Stevens

• Sponsors:

Center for Process Analytical Chemistry (CPAC), UW, Seattle DOD Texas Instruments Washington State Sea Grant NSF/NIEHS NW Center for Human Health and Ocean Studies