1. Real-time Monitoring with a Portable Miniaturized Surface Plasmon Resonance System Clement E. Furlong, Research Professor, Departments of Medicine (Div. Medical Genetics) & Genome Sciences University of Washington, Seattle, WA Presented by: Brian Marquardt CPAC/UW

2. Spreeta sensing components Each Spreeta chip contains all of the optical components needed for sensitive SPR measurement of 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

3. 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 laboratory prototype

4. Touchscreen data display Selected SPR curve Detected levels (numeric) (bargraph) Sensor channel

5. Sensor surface chemistry Y Y Y Y Y  Glass substrate Gold layer (50 nM) Soluble protective coating Soluble 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 receptors Control receptors (usually antibodies) Designed NOT to respond to that agent Spreeta sensor chip Each Spreeta chip has 3 useable channels

6. System software Fundamentals of Surface Plasmon Resonance Fundamentals of Surface Plasmon Resonance Sensorgram

7. SPIRIT performs 24 simultaneous measurements of antibody binding Eight sensor chips Three active spots per sensor Analyte Detection event Flowcell

8. 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)

9. Detection of Larger Analytes Microbes Spores Viruses Proteins/Toxic Proteins Microbes Spores Viruses Proteins/Toxic Proteins

10. Signal Detection Analyte Detection and Signal Amplification

11. Signal Detection Analyte Detection and Signal Amplification

12. Signal Detection Analyte Detection and Signal Amplification

13. Signal Detection Analyte Detection and Signal Amplification

14. Detection Amplification/verification Detection of Microbes

15. Virus Detection Amplification

16. Detection of Staphylococcal Enterotoxin B

17. Detection of 5 ng/mL (5 ppb; 33pM) BotNT (denatured botulinum toxin)

18. Direct Detection of Ricin A Chain (64 ppb-320 ppb)

19. Detection of Cortisol by Competition Assay Lower arrows indicate return to no analyte

20. Standard Domoic Acid Concentration Curve in Clam Extracts

21. Other Useful Applications of SPR Sensing Nucleic Acid Analyses Many Other Molecular Interactions Nucleic Acid Analyses Many Other Molecular Interactions

22. Protein Nucleic Acids as Recognition Elements for DNA/RNA Very stable receptor on chip (Protein Nucleic Acid) Allows detection of target

23. Binding of a 79 bp DNA Probe to a Complementary PNA 16 mer on the Sensor Surface

24. Detection of Analytes in Complex Matrices (e.g., saliva, plasma, urine, stool extracts, sea water, fresh water, etc.)

25. Detection of 1 nM (28 ppb) SEB in seawater Staphylococcal enterotoxin B

26. Detection of 500 pM (14 ppb) SEB in urine 500 pM SEB Wash (urine) Amplification From: Naimushin et al., Biosensors and Bioelectronics 17:573

27. Detection of cortisol in saliva using the compound flow cell

28. Detection of Theophylline in Saliva Using the Compound flow Cell

29. Sequential Detection of 8 Analytes Y. pestis 10 6 CFU/ml Ovalbumin 10 ng/ml SEB 5 ng/ml F. tularensis 5 x 10 3 CFU/ml B. anthracis 5 x 10 6 CFU/ml Norwalk VLPs 5 x 10 9 particles/ml Ricin A chain 20 ng/ml BG Spores 9 x 10 4 CFU/ml

30. SPIRIT Team & Sponsors Medical Genetics Group: Dr. Clement Furlong Scott Soelberg Dr. Gary Geiss Dr. Rick Stevens Steve Near Matthew Probert Joshua Probert Nathaneal Swanson Dr. Paul Baker Electrical Engineering Group: Dr. Sinclair Yee Tim Chinowsky Peter Kauffman Jared Tritz Michael Grow Tony Mactutis Texas Instruments: Jerry Elkind Dwight Bartholomew John Quinn Sponsors: DOD Texas Instruments Center for Process Analytical Chemistry (CPAC), UW, Seattle Washington State Sea Grant, NIH/NIEHS