CBMSE Overview

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CBMSE Overview
Center for Bio/Molecular Science and
Engineering Study of complex bio/molecular
systems with the aim of understanding how biology
has solved difficult structural and sensing
problems and mimicking biological materials and
their self-assembly. Director Joel M.
Schnurhttp//cbmsews1.nrl.navy.mil/
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Bio/Molecular Science and Engineering

• We are learning important lessons from nature by
  understanding and when appropriate, mimicking,
  biological materials and their self-assembly.

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CENTER GENERAL PROGRAM AREAS

• Optical Biosensors
• Bio/Molecular Materials
• Stabilized Biomaterials
• Nano-scale manipulations
• Controlled Sustained Release
• Bio/molecular and Cellular Arrays
• Surface Modification and Patterning
• Environmental Monitoring and Cleanup
• Advanced Materials from Self-Assembly
• Liquid Crystal Based Electro-Optic Materials
• Supra-molecular and Bio-based Self-Assembly

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Where we were
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1979
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1984
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1986
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NRL BIO/MOLECULAR ENGINEERING PROGRAMS

• Premise Utilized The Ability Of Biological
  Molecules To Self Organize The Fabrication
  Molecular Systems Of Interest To The Navy
• Primary Emphasis Non-medical Applications
• Program Elements
• Novel Materials And Processing Methods
• Chemical Sensors
• Obscuration
• Chemical And Biological Diagnostics
• Blood Surrogates
• Microelectronics And Hybrid Devices

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LIPIDS

• PROPERTIES
• SELF ORGANIZATION
• MICROSTRUCTURES
• PROTEIN INCORPORATION
• FILM FABRICATION
• CHEMICAL MILIFICATIONS
• POLYMERIZABLE
• ATTACH MONOCLONALS

• POTENTIAL APPLICATIONS
• BLOOK SURROGATES
• ULTRA SENSITIVE DETECTORS
• DIAGNOSTICS
• SEPARATION TECHMIZUES
• PROTECTIVE COATINGS
• SENSORS

1986
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1988
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1987
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1987
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1988
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1990
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1990
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1991
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1992
Jeff Calvert ARI Presentation
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1992
ARI
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Jeff Calvert ARI Presentation
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1992
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Where we are
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Bio/Molecular Science and Engineering

• Important lessons are to be learned from
  understanding, and when appropriate mimicking,
  biological materials and their self-assembly.

1998
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CENTER GENERAL PROGRAM AREAS

• Optical Biosensors
• Bio/Molecular Materials
• Stabilized Biomaterials
• Nano-scale manipulations
• Controlled Sustained Release
• Bio/molecular and Cellular Arrays
• Surface Modification and Patterning
• Environmental Monitoring and Cleanup
• Advanced Materials from Self-Assembly
• Liquid Crystal Based Electro-Optic Materials
• Supra-molecular and Bio-based Self-Assembly

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CBMSE 1998
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Biocomposites and Bio\Molecular Materials

• Develop advanced biomaterials, devices,
  biocomposite materials and sensors by taking
  advantage of the knowledge of biological
  materials

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Electroclinic Liquid Crystals

• R. Shashidhar, B. Ratna, J. Naciri, and M. Spector

Develop ultra-fast electro-optic materials with
analog gray scale capabilities.
Siloxane-based liquid crystals exhibit a large
field-induced optical tilt, with very small layer
shrinkage.
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Conducting Polymer Based LCD

• R. Shashidhar

Development of new conducting polymer with
improved transparency and conductance for use in
a plastic, reflective liquid crystal display

• Sunlight readable
• Large viewing angle
• Light weight and rugged
• Low power consumption
• Bistable (no power needed to maintain image)
• Curved displays

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Pyroelectric Liquid Crystals

• R. Shashidhar

Development of ferroelectric liquid crystals with
high sensitivity to IR radiation for use in
uncooled IR sensors

• Developed new class of siloxane liquid crystals
• Focal plane array using thin films (lt 1 mm) in
  development
• Expected NETD lt 10 mK

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Liquid Crystal Elastomers

• B. R. Ratna

Development of a biomimetic actuator material
that approximates muscle in its dexterity,
flexibility, and efficiency.
Properties Skeletal Muscle Current status
Stress 350kPa 310kPa
Strain 25 40
Strain rate 3 10 s-1
Contraction Anisotropic Anisotropic
Dynamic mechanical measurements show that 10
S-1 is achievable
Anisotropic contraction/elongation
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Liquid Crystal Elastomer An Abiotic Muscle
Technology challenge Development of a
biomimetic actuator material that approximates
muscle in its dexterity, flexibility, and
efficiency.
Approach Develop an excitable liquid crystal
elastomer that mimics a skeletal muscle in its
properties.
Current Status
Advantages Stimulated by by external fields
Temperature, electric and optical field.
Possibility of introducing multifunctionality
Adaptive feedback control.
Properties Skeletal Muscle Current status
Stress 350kPa 310kPa
Strain 25 40
Strain rate 3 10 s-1
Contraction Anisotropic Anisotropic
Contact Dr. B. R. Ratna Ratna_at_ccs.nrl.navy.mil
Acknowledgement DARPA
Dynamic mechanical measurements show that 10
S-1 is achievable
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Hierarchical Structures in Biology

• Use biology as a role model to think small and to
  learn to rationally control molecular
  interactions leading to property control at
  various size scales.

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Chemical Modification of Surfaces Utilizing
Non-Covalent Interactions

• S. L. Brandow, W. J. Dressick, M.-S. S Chen, and
  T. L. Schull

Investigation of the fundamental properties
governing hydrophobic interactions between
solution phase species and molecular groups
localized on a surface.
e-
Ni
Ni
Cl
Cl
Cl
Cl
Cl
Cl
Metallization
50 kV e-beam
Substrate

• Electron induced desorption of adsorbate upon
  exposure to 50 kV electrons
• Fabrication of reactive template with feature
  resolution to 40 nm

Tour
SEM Image of Metallized sample following
RIE/prior to Ni strip (Exposure conditions 500
mC/cm2)
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Surface Modification and Patterning
PROBLEM Develop new approaches for fabricating
two- and three-dimensional nanostructured
materials with tailored chemical and biochemical
functionalities.
APPROACH Surface patterning using AFM, STM,
e-beam, proximity x-ray probe and Deep UV
lithography
STM Patterned Siloxane Film Following
Metallization and Pattern Transfer by Reactive
Ion Etching (Trench Width 15 nm)

• IMPACT
• Electronics
• Si IC Lithography
• Integrated Optics/MEMS
• Selective CVD/Interconnects
• Displays/Optical Materials
• Non-contact LC Alignment
• Conducting Polymers for Flexible Displays
• Biological Surface Recognition
• 3-D Biosurfaces
• Sensors

Selective Reaction of Fluorescent Cy3.5 Dye on
-NH2 Groups Grafted to Irradiated PVBC
Collaborations with NRL ESTD (6860,6870,6804)
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Template-Directed Molecular Imprinting

• B. P. Gaber and M. A. Markowitz

Develop highly selective ceramic catalysts and
adsorbents for synthetic, detection, and sensor
applications in harsh environments.
Micelle-forming surfactant
Imprint surfactant
SiO2 precursor
Functionalized silanes

• Development of process to surface-imprint metal
  oxide surfaces
• Formation of homogeneous molecular recognition
  sites demonstrated
• Formation of enantioselective catalysts
• Formation of highly selective adsorbents for
  chemical agent detection/sensing

Tour
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Molecular ImprintingCatalysts and Sorbents

• B. P. Gaber and M. A. Markowitz

Selective Organophosphonate AdsorbentsIncrease
in Adsorption of Soman Hydrolysis Product PMP by
Silica Particles Surface-imprinted with PMP
Enantioselective AmideHydrolysis Catalysts
Particles imprinted with L-substrate analog
Silica Functional Groups
Increase in Binding ()
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Self-Assembled Virus Nanoblocks

• B. R. Ratna

Self-organization of cowpea mosaic virus
particles on surfaces leads to extended
orthogonal pattern.
Reconstruction of a cowpea mosaic virus showing
icosahedral symmetry.
Diameter 28.4 nm
Finger height 250nm (10 viruses) Average Finger
width 600nm 40nm
In collaboration with J. Johnson (Scripps
Research Institute)
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Evolution of NRL Materials Research on Lipid
Tubules
Tubule-based dielectric composites (ONT)
1983-1990
Tubules discovered at NRL DARPA Program Metal
Clad Tubules
Tubule controlled release demonstrated
1991
1994
Field Emitting Cathodes Demonstrated
1996 lipid wall thickness control demonstrated
Chiral Models developed CD Experiments Start
Tubule antifouling patent issued
6.2 NRL funding for Tubule Antifouling paint
Successful tubule Antifouling in Australia 1997
RTP, Biocompatibles CRADAs for controlled
release 1996
lipid nanotubules fabricated-2000
Nano particle patterning demonstrated on lipid
tubules 1999
Polypeptide nano tubules fabricated 2001
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Self-Assembled Lipid Tubules

• P.E Schoen, A. Singh, R. Price, D. Zabetakis, M.
  S. Spector , J. Selinger, J. M. Schnur

Development of materials from chiral
self-assembly of synthetic phospholipids.
diameter 0.5 mm length 10 - 100 mm
Low temperature nanotubule phase in 11 mixture
of DC8,9PC and DNPC, which melts into a helical
ribbon phase at room temperature.1
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Imaging Tubules using Anionic Particles
Electron micrograph of nanoparticle caps at the
end of DC8,11PC tubules after sequential
absorption of oppositely charged polymers
(PSS-/PEI) and silica spheres.
Nanoparticle helices inside DC8,11PC/DC8,9PEOH
(2) tubules after sequential absorption of
charged polymers PEI/PSS-/PEI and 45 nm silica
spheres.
Y. M. Lvov et al., Langmuir 16, 5932 (2000)
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Metallized Lipid Tubules

• P. E. Schoen, D. Zabetakis, and J. M. Schnur

Electroless plating techniques have been used to
clad tubules with copper, nickel, gold, iron,
paladium, silica, and chitosan.

• Encapsulation of antifoulingagents for
  controlled release
• Electroactive composites forhigh dielectric
  material (RAM)

0.5 mm
SEM of copper-coated tubule
Tour
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Release from Lipid Based Tubules

• Larger diameter (0.5u)
• Aspect Ratio 10-200
• Lipid or Metal Coated
• Some control of structure possible
• Rational control of release demonstrated
• may be utilized in multi-purpose coatings
• May be used to entrap
• nanoparticles
• higher molecular weight polymers
• Demonstrated effectiveness over 1 year in field
  conditions
• Subject of 2 CRADA agreements

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Biology for Energy Transduction and Sensing

• Harness natures mechanisms for molecular
  recognition to make new materials and highly
  sensitive detection and transduction systems

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Electroactive Biomimetic Systems

• A. Singh and I. Stanish

Develop Stable Biomaterials for Charge Storage
Systems and Electron Coupling Applications
Polymerized Vesicles Chemisorbed on Au
Electron Transport
Bare Au
Bare Au
Morphology by ESEM
Tour
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Ocean Floor Bio-Fuel Cell

• L. Tender

Harvest continuous power from ocean floor from
oxidation of sediment organic matter with
seawater oxygen.
Ocean Sediment Carbon Anaerobic Reactor
Net reaction C6H12O6 O2 ? CO2 H2O
DGo -3190 kJ
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Ocean Floor Bio-Fuel Cell69-6049-01, Leonard M.
Tender

• Objective Harvest energy at Seafloor to Operate
  Autonomous Marine Deployed Instrumentation
• Approach Design, build, and demonstrate
  2-electrode devices that sit on ocean floor and
  generates electrical power by oxidizing marine
  sediment organic matter with seawater oxygen

Results Prototype Deployed Device (not
benefited from ongoing electrode optimizations)
Generating gt100-mwatt/meter2 continuos power for
over 8 months. Real Time Monitoring at
http//209.158.75.87/shorestation.htm Science,
Vol 295, p483 (2002)
ImpactLong-term (Perpetual) Uninterrupted
Instrument Operation
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Bio-detection Challenge
National Museum of Wales Canary and coal miner
Circa 1890
Time magazine Japanese police raid on Aum
Shinrikyo compound (1995)
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BW Detection Spanning Multiple Ranges
Point Detection
Long Range
Diagnostic
Currently In use IBAD particle sizer/counter,
particle wet cyclone sampler, membrane
colorimetric ticket
Lidar for stand-off particle detection
Assay methodologies/technologies Fiberoptic/flow
immunosensor Nucleic acid assays
UAV with fiber optic biosensor payload
Z-chip
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Activity Detection Technologies Concept and
Perspectives

• is it physically present?
• Require previous known information on gene
  sequence, mass, or antibody
• what are the physiological consequences of
  acute or chronic exposure?
• Responds to CBW, live vs. dead, TICs, TIMs and
  gives functional response information for
  diagnosis and treatment

• Molecular Strategies
• Spectroscopic
• Mass Spec
• Antibodies
• Nucleic Acids
• Living Systems Strategies
• Cells
• Tissues
• Organisms

Structure
Activity
Sensitivity depends on physiological Function
Effective Dose (ED), Lethal Dose (LD) PD likely
high, False positives higher than negatives
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Cells Amplify Responses

• Established technologies

Immunoassays
Polymerase Chain Reaction (PCR)
Chemical multiplication of DNA
Labeled Antibody
Pathogen
Antibody
Substrate
Sensitivity limit 5 - 20 particles Slow
(7 - 60 minutes for pristine sample) Best
specificity if test properly designed
Sensitive to contamination
Insensitive (1000s of particles reqd.)
Slow (minutes to 10s of minutes) False
positives and negatives

• CANARY sensor concept
• Extremely fast
• Highly sensitive
• Low nonspecific binding

Pathogens
B Cell
Photons in lt 1 second after binding
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Approach Comparison for Molecular Biosensor
Technologies

• Biosensors utilize a biological component as a
  sensing element compatible with electrical,
  chemical or optical transduction.
• Molecular biosensors include

Functional Information
Generic Threat Sensitivity
Identification Assay
X
Nucleic Acids
X
Antibodies
X
Enzymes
X
Ion channel/receptors
X
X
Cell-based sensors
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Optical Biosensors Practical Criteria for BWD

• Necessary
• Fully automated, simple to use
• Minimal logistical burden
• Very low false positives
• Sensitivity appropriate for use
• Size/weight appropriate for use
• Rugged reliable
• Works for bacteria, viruses and toxins

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Sandwich Fluoroimmunoassay









Optical Waveguide
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Fiber Optic Biosensor

• G. P. Anderson F Ligler

RAPTORTM Portable device that can simultaneously
monitor for up to four environmental toxins in a
1 ml sample

• Completely self-contained, programmable
  instrument integrates optics, fluidics,
  electronics, and software into one compact system
• 2 RAPTORs with assays for 6 environmental
  agents are currently in use
• 2nd generation RAPTOR-Plus in production by
  Research International
• Field Tested with 8 validated assays

Tour
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Flow Immunosensor

• A. W. Kusterbeck

Rapid, on-site detection of explosives and other
small molecular weight chemicals using a
displacement assay.

• Adaptable to field analysis of environmental
  contaminants
• Measures TNT RDX in environmental samples at
  low levels (ppt)
• Determines the concentration of analyte in
  samples containing high concentrations of other
  explosives
• Demonstrated successfully in field trials at U.S.
  military bases

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The flow sensor now being evaluated by EPA
Problem Closure of U.S. military bases and
concern for environmental security have led to a
need for rapid, on-site measurements of
explosives and other small molecular weight
chemicals

• Critical Issues
• Matrix effects from complex environmental samples
• Biosensor stability
• Instrument calibration procedures for accurate
  measurements

• Results
• Adaptable to field analysis of environmental
  contaminants
• Measures TNT RDX in environmental samples at
  low levels (ppt)
• Determines the concentration of analyte in
  samples containing high concentrations of other
  explosives
• Demonstrated successfully in field trials at U.S.
  military bases

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Array Biosensor

• F. S. Ligler, J. Golden, and C. R. Taitt

Development of ultrasensitive detection system
fluorescence imaging of antibody/antigen binding

• Identify simultaneously bacteria, viruses, and
  toxins at physiologically relevant levels
• Use remotely with air sampler or samples
  introduced by first responders
• Sensitivity equivalent to ELISA method
• Prototype has been field tested
• Current version provides 36 simultaneous assays
  (expansion planned to 100)
• Fully automated system advanced prototype under
  development

Tour
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Activity Detection Concept and Perspectives

• Changes in the cellular and tissue responses
  provide signature of threat and provide
• State change detection
• Classification of threat
• Probablistic identification based on library of
  responses

Single/multiple pathways within cells or tissue
are monitored following exposure to biological
and chemical threat agents

• Measured Responses Processing and Amplification
• gene expression
• DNA remodeling
• enzyme activities
• p38 activation/inhibition
• protein localization activation
• NF-?B activation/inhibition
• electrical activity
• Cell cycle events (apoptosis)
• Cytoskeletal rearrangements
• Space and time relationships

Blood Urine Mucous
Water Aerosol Food Material

• Activity detection can be used as a diagnostic
  or environmental sensor

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Genomic Based Detection
Objective To develop a near-real time broad
agent biological sensor that utilizes genetic
information to rapidly identify thousands of
biological organisms in parallel.
Current method
Sample
1-4 results
Project objective
Sample
1000s of results
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Impact of Microarray-based Sensor

• Ability to monitor a large number and variety of
  pathogens in an efficient and cost-effective
  manner

• High efficiency screening of food, water, or
  clinical samples
• 100 to 10,000-fold improvement over current
  screening capacity
• Simultaneous tracking of multiple biological
  markers for reliable target
  identification
• Universal sensor
• Highly sensitive and stable detection system
  useful for routine screening and/or
  epidemiological testing
• Easy-to-use sensor system that encourages testing
• Minimal handling system compatible with
  automation
• Minimal technical training
• Cost-and time-efficient tests

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Neuronal Network Detection

• Develop an Activity Detection System that
• detects ANY compound capable of interfering with
  normal mammalian central nervous systems function
• detects compounds at pertinent physiological
  levels, and
• provides data on general mechanims involved.

analog integration
period
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Cell-Based Biosensor

• D. Stenger and J. Pancrazio

Develop portable instrument capable of
characterizing neuropharmacologic responses to
unknown/unanticipated compounds.
Based on spike and burst properties, compounds
may be categorized into functional categories.
Spikes
Tour
Integrated Activity (Bursts)
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NRL Prototype System
Filter-amplifier boards.
Neuronal recording shipping chamber
Pumps valves
Pre-amplifiers
Li-ion batteries

• Fluidics and temperature control are integral to
  the overall design.
• System dimensions of 30cm x 40cm x 35cm, weighs
  10 kg, and has a battery lifetime of 8-10 hrs.
• Capable of high signal-to-noise recordings of
  extracellular potentials

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Where do we want to be and how do we get there?
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The Role of Modern Biology in the Development of
the Non Medical High Technologies of the 21st
Century
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What is our Future?

• Rugged Biomimitic Materials
• Interfacial Reactions of Cells at Surfaces
• Self-Assembling Colloidal Systems
• Biological Systems at the nanometer scale for
  Design and Fabrication
• Multifunctional Arrays

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What is in our Future? (Continued)

• Bio/Organic Electro-optic Sensors and Displays
• Real Time 3-D Holography
• Acoustic IR Materials for Advanced Imaging
• Ultra-Smart Materials for Controlled Release and
  Dynamic, Self Cleaning Filters
• Multianalyte Sensor and Integrated Sensor Suites
• Bio-based energy transduction for self powered
  sensors and devices

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What is in our Future? To be Continued!
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