applying protein plastics in chemical defense
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Applying Protein Plastics in Chemical Defense
October 2003
3636 Boulevard of the AlliesPittsburgh, PA 15213
Keith LeJeune, PhDCEO and Co-founder
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Background
• Founded Dec 1998
– Keith LeJeune, CEO, Agentase LLC
– Alan Russell, Director of McGowan Institute for Regenerative Medicine,University of Pittsburgh and Pittsburgh Tissue Engineering Initiative
• Proprietary technology permitting the direct chemical incorporation ofenzymes within polymers during polymer synthesis
• Advantages over conventional techniques for enzyme immobilization
– No leaching of activity from support material– Desensitization of enzyme to environment– Durability and reusability– Substantial loading capacity for protein– High degree of activity retention– Opportunity to incorporate indicating compounds within polymers (sensor)
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Technology Applications
• Chemically active surfaces
• Decontamination of hazardous chemicals– Pesticides
– Chemical weapons (DFPase, OPAA, OpdA, OPH)
• Sensors– Co-polymerize enzymes and indicating compounds
– Color change illustrates enzyme activity
– Used to detect
• Substrates (Reactants)
• Inhibitors
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Decontamination• Optimization of enzyme-polyurethane for agent detoxification
– Improve physical properties for removing agent from surfaces• Moisten polymers in tap water• Achieve > 99.5% removal of 40mg GD on a 80 cm2 surface in 3rd party experiments
– Steel plates – Plastic surfaces – Ceramic tile
– Improve catalytic activity of enzyme polymers
Time (min)
0 50 100 150 200 250 300
Mas
s of
ana
lyte
in
sol
utio
n (µ
g)
0
10000
20000
30000
40000
5000060000
GDPinacolyl methylphosphonate
55 mg GD in 100 ml buffer with ~1.0g polymer containing less than 3mg of enzyme
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Issues limiting practical utility of employingpolymers for CW decontamination
• Enzyme specificity• Need for buffer capacity
– Hydrolysis of moderate concentrations of agent reduces pH– Initial pH outside acceptable range for enzyme catalysis– Not practical to be required to “buffer” a surface or solution prior to
decontamination
• Incorporation of buffer capacity within polymers during synthesis
– Buffered polymers ~1g moistened in tap water decontaminate (>99.5%) 40mg GD in < 2 hrs.
0%
25%
50%
75%
100%
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Initial methyl parathion Concentration (mM)
Det
oxi
fica
tio
n
Enzyme polymer
Buffered enzyme polymer
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First Product: NerveNerve agent Sensor
Agentase’s sensor provides a clear, intuitive, and easy toread response for nerve agent contamination
Clean Clean ContaminationContamination
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How does the TL Sensor work?
• Two polyurethane-based fabric layers– Enzyme Polymer Layer– Substrate Polymer Layer
• Dynamic pH equilibrium– Cholinesterase
• Produces butyric acid• pH optimum at ~8.0
–– UreaseUrease• Produces NH4
+ and OH-
• pH optimum below 70
0.5
1
1.5
2
2.5
5 6 7 8 9 10
pH
En
zym
e a
cti
vit
y [
rela
tiv
e] pH equilibrium
UreaseUrease ChE
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Benefits of the described technology
• Versatility in sampling– Surfaces – Water – Air
• Response– Positive response in seconds
– Response intensity proportional to contamination level
• Surface compatibility– Steel Plates, flat and grooved – Plastic – Aluminum– Finished Wood – Ceramic floor tile – Glass – Leaves – Skin Tissue – Soil
• Great resistance to interference because of dynamic equilibriumapproach
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Performance of Nerve Agent sensor
• Storage stability and shelf-life– > 31 days at 140oF
– > 6 months at 105oF
• Temperature effects– Sensor operational at subzero and high temperatures (tested at 158oF)
• High resistance to chemical interference– Environment tested include:
• Diesel, auto & aviation fuel, antifreeze, ethanol, vinegar, toluene, fire-fightingfoam, sea water and several pesticides & household cleaners
• Tests conducted in 1% or greater concentrations in solution and and 1%interferent saturated air
• Training version available– Classroom and operational exercises
Correlates to more than 18 months atroom temperature
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Live agent validationAgent Total Mass Mass / cm2 Color
(2 min)Color
(25 min)Green (Y/N) Result
GD 100 ug 1.25 µg/cm2 Red Red N20 ug 0.25 µg/cm2 Red Red N
8.0 ug 0.10 µg/cm2 Red Red N4.0 ug 0.05 µg/cm2 Some Red Red N1.0 ug 0.0125 µg/cm2 Some Red Red N Limit0.2 ug 0.0025 µg/cm2 Yellow Yellow N
VX 100 ug 1.25 µg/cm2 Red Red N20 ug 0.25 µg/cm2 Red Red N
8.0 ug 0.10 µg/cm2 Some Red Red N4.0 ug 0.05 µg/cm2 Slight Red Red N Limit1.0 ug 0.0125 µg/cm2 Yellow Yellow N0.2 ug 0.0025 µg/cm2 Yellow Yellow N
Water Blank Plate a 0.00 µg/cm2 Yellow Lime green YBlank Plate b 0.00 µg/cm2 Yellow Lime green Y
Blank Activated Only 0.00 µg/cm2 Yellow Lime green Y
Summary of TL Sensor data from ECBC Report 0033-080702: 7 August 2002
• Surface detection limits verified in UK• GA - between 99 and 22 ng/cm2 • GB - between 99 and 22 ng/cm2
• GD - 22 ng/cm2 • VX - between 180 and 99 ng/cm2
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Practical limitations
• Temperature sensitivity– High temperature stability issue
• Shelf life is function of temperature• Co-package with Temp indicator
– Low temperature operation• Water in reservoir cap must be liquid• Thawed sensor can be used on frozen surface
• Incompatible environments– Excessively dirty surfaces
• Block ability to see polymer color
– Extremely acidic or basic environments• Immediate color change (purple/pink)
• Nerve agent point detection Only
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Additional applications of sensor technology• Expansion of detection capabilities to additional CW and TIC
– Polymerization chemistry compatible with most enzymes
– Many enzymes identified with sensitivity to target chemical hazards
Product Concept
• Pencil box for detection of CW agents and high-risk TICs
• Real-time continuous monitoring– Equilibrium reaction scheme used to trigger response
– Covalent attachment of sensing components within polymer
Product Concepts
• Water Monitor • Air monitor • Wearable badge
• Combination of parallel efforts• Array of wearable badges for operationally identifying chemical hazards
• Public venue air monitor for chemical hazards
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Additional applications of sensor technology
12 h
6 h
0 h
BlisterBlood
Nerve Clean
Inlet
Renewable substrate cartridge
Effluentcollector
Indicator lights
TL SensorTL Sensorfor nervefor nerveagent CWagent CW
Single-useSingle-usesensorssensors
for all CWfor all CWagentsagents
Water monitorWater monitorfor nerve agentsfor nerve agents
Wearable badge forWearable badge fornerve gasesnerve gases
Public venue gasPublic venue gasmonitor for CW agentsmonitor for CW agents
Real-time continuous waterReal-time continuous watermonitor for all CW agentsmonitor for all CW agents
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Point detection capabilities - Blood Agent sensor
• Prototype cyanide sensor– Identical in form and use to nerve agent sensor– Slightly improved shelf-life– Detection limits well below IDLH (µg)
• Excellent operational utilityfor example ...– Car– Brick– Wood– Steel– Concrete– Fire fighting foam
Clean surface Cyanide surface
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Performance
• Sensitivity– OSHA and IDLH levels (cyanogen bromide)
• Live agent tests scheduled at 3rd party facility– Hydrogen cyanide– Cyanogen chloride
5mg/m3 (OSHA)
Control25mg/m3 (IDLH)
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Demonstration of Blood Agent sensor
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Continuing expansion of agent detection capabilities
• Identified additional CW agents / TIC compatible with enzyme-based detection• Blister • Acetaldehyde • Hydrazine• Acrolein • Ammonia • Formaldehyde
• Development of Prototype kit for detection of CW / TIC
– Sensors for Nerve agents (B) and Blood agents (E) are fully functional
– Present activity• Devising kit and sensor construct to best fit end-user needs
• Adding additional hazardous agent detection capability
A B C D E F
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Continuous monitoring capabilities - Water monitor
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Response to ChE inhibitor
Signal Response - Effluent pH
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0 1 2 3
Time (hours)
pH
Addition of DFP to aqueous inlet
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Envisioned prototype - (Under construction)
Alarm and communication device
Power supplyand controller
EffluentReservoir
pH monitors
View cell with polymer
Color reader
Micro-pumpsWater Inlet
Substrate Cartridge
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Proof-of-concept demonstration: Wearable badge
• Model badge capable of detecting DFP vapor at low ppb levels• Product concept
– Single lightweight sensor (<50g) housing with multi-agent compatibility– Maintenance free operation (>12 hrs)– No external power requirement– Resistant to chemical interference– Integrated communications capability
Waste Reservoir
Gas-tight syringe withvalve to insert hazard
Polymer Sensor 1 Litercontainer
Lid withinjection portSubstrate Feed
0 0.5 2.5 5.0 10.0 min
Polymer response - post DFP vapor injection
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Contributors
• Supporting Agencies– Army Research Office (ARO) - Lee– Defense Threat Reduction Agency (DTRA) - Pollack, Hoefler– USMC - Becker– Defense Advanced Research Projects Agency (DARPA) - Rudolph
• Live agent testing facilities– UK Defense Evaluation and Research Agency (now dstl)– U.S. Army Soldier and Biological Chemical Command; Edgewood
Chemical Biological Forensic Analytical Center– Centre d'Etudes du Bouchet - Cazaux France
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Summary
• Enzyme polymerization has utility in decontamination anddetection
• Agentase nerve agent sensor is a proven product– Product validated by third parties
• Live agent (V and G-series) • Operational exercises
– Fielded product in both military and civilian environments
• Encouraging proof-of-concept data on 2nd generation products– Continuous monitoring devices
• Water• Air
– Detection kit with expanded CW/TIC capabilities• Blood• Blister• TICs