AFOSR

Natural Materials & Systems

March 2011

Dr. Hugh C. De Long

Interim Director
AFOSR/RSL
Air Force Office of Scientific Research
Distribution A: Approved for public release; distribution is unlimited. 88ABW-2011-0778
 2011 AFOSR SPRING REVIEW
2306 DX PORTFOLIO OVERVIEW
Name: Hugh C. De Long
Portfolio Description:
The goals of this program are to: 1) study, use, mimic, or alter how
biological systems accomplish a desired (from our point of view) task, and
2) enable them to task-specifically produce natural materials and systems.
Both goals are to advance or create future USAF technologies.

List Portfolio Sub-Areas*:

2306 DX Biomimetics
2306 DX Natural Materials
2306 DX Natural/Synthetic Interfaces
2306 EX Extremophiles

*All four sub-areas include

international programs
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 Scientific Challenges
• Bio-camouflage – To achieve rapid adaptive coloration.
– Payoff: Would make current camouflage ideas obsolete.
• Peptide mediated materials synthesis – Looking to learn how materials
either bind to or grow on peptides.
– Payoff: Get to a predictive state for sequence or material.
• Structural Coloration – Identifying the role of hierarchical self-assembly
– Payoff: Create new empirically supported paradigm of development
of highly ordered biophotonic nanostructures.
• Cell-Directed Assembly – Using cells to build nanostructured
architectures with engineered bio/nano interfaces
– Payoff: Understand cellular behavior and allow understanding of
biology at individual cell level
• Chromophores – To discover or engineer new chromophores in desired
wavelength regions
– Payoff: New materials in desired wavelength regions 3
 Transformational Opportunities
• Bio-Camouflage – (cephalopods sole animal group with controllable skin
texture) important to learn how and when they are deployed in changing
backgrounds
– will transform approaches to mission coats and enable an efficient
(passive) mechanism to regulate brightness & contrast
• Rational design of peptides for material synthesis and assembly – engineered
material and material systems
– leads to new method to fabricate sensors; other multifunctional materials
• Soft Lithography - a unique approach to synthesize and position single sub-
10-nm nanoparticles
– create nanostructures small enough to behave as single particle devices
(e.g., sensors) integrated with prefabricated circuitry.
• Structural Coloration – will look to understand the biological strategies
involved in the manipulation of light, and structural color
– it will produce new processing technologies to fabricate bio-inspired
tunable optical architectures with optimal structure and properties at
4
multiple length scales.
 Other Organizations That Fund
Related Work
• Chromophores – I currently have two grants plus work in AFRL. The work of
other organizations is almost exclusively on reporter technology. The interest
of the AFOSR program is on wavelength, intensity, and lifetime as it pertains
to marking items.
• Silk – DARPA has contributed to my existing program. ARO has a single
grantee. NSF has several single PI grants.
• Structural Coloration/Bio-Camouflage – MURI with ONR focused on vision
aspect. ARO has a single grant with ICB PI. NSF has just single PI grants.
• Biomolecular assembly – A number of funding organizations are interested in
this area, so the AFOSR program is focused on soft lithography, peptide
binding, and self or directed assembly for materials. AFRL program works
closely with this group for both relevance and guidance.
• Extremophiles – NASA has funded this area and focused on the origins of life.
The focus of the AFOSR program is on radiation protection mechanisms, bio-
templating, and biopolymers that can exist in extreme environments. ARO is
focused on spore formers.

5
 Program Trends
• Chromophores/Bioluminescence – Bio-X STT phase 1 focus. One of
its discoveries are now used by AFRL TDs, Navy & several Univ PI’s
• Bio-camouflage –New PBD 709 program for FY09 looking at
iridiphores, leucophores, chromatophores, papillae, & control system.
This program is linked to a new program in AFRL/RX in FY2011.
• Structural Coloration – new area, several PIs & new MURI (Harvard)
• Biopolymers – Mainly silk but looking at other biopolymers. The silk
work is well integrated with AFRL; many exchanges of personnel &
material.
• Biomolecular assembly – New MURI at Georgia Tech, rest has
remained constant.
• Peptide Mediated Materials Synthesis – The efforts are focused on
discovering the nature of the mechanism behind this.
• Extremophile survival –Focus shifted to materials within cells that
work under these conditions. Looking at mechanisms of protein
activity under extreme conditions with the goal to transfer good ideas
into weaker systems. Fewer PIs left that perform this type of work. 6
 Recent Transitions
• Endogenous Green Fluorescent Protein (GFP) from Amphioxus (UCSD) transitioned to
AFRL/RX & RH as well as Navy and other University PIs
• Thin film flexible device with unique optical properties for military applications-multi-
laminated structure to mimic cephalopod skin (AFRL/RX) transitioned (AFRL & Army 6.2)
• Encapsulation of enzymes in silk films and fibers for decon applications (AFRL/RX)
(transition to DTRA program)
• Peptide Functionalized Nanoparticles for Environmental Sensing (AFRL/RX) – UES, Inc.
& Woolpert, Inc
• Intracellular detection and tagging demonstrated with Aptamer Coated Nanoparticles
that release reporter flares (NWU) – single base-pair mismatch selectivity – licensed to
Aurasense, Inc
• Pretreatment using IL on Corn Stover (NCSU) transitioned to Novozyme as only system
that will allow them to commercially process corn as a cellulosic biofuel.
• Tetratopic Phenyl Compounds, Related Metal-Organic Framework Materials, (NWU).
Product is currently commercialized by Sigma-Aldrich Corp.
• Silk-electronic interfaces for disposable optical transmitters (Tufts U) – transitioned to
the DARPA chemical communications program
• Use of low light digital spectrograph analysis on biomaterials (UCSD) - Ken Browne
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(GenProbe)
 Bio-inspired Supramolecular Enzymatic Systems
PI: C. A. Mirkin; Co-PIs: J. T. Hupp, S. T. Nguyen, J. F.
Stoddart, M. A. Ratner
• No versatile assembly methods
Status Quo
for radicals and highly reactive
species are available.
• Detection schemes for non-
biological self-amplification are
non-existent .
• Allosterism in supramolecular
systems is not well-established.
• Radically-enhanced molecular
New Insights & Approaches
recognition properties enable
access to stable radical dimers in
the mechanically interlocked
assemblies.
• Rational design of supramolecular
building blocks and the associated
assembly methods can provide a
platform to switchable catalysts.
• Elucidation of catalytic mechanism
for enhanced rates in MOFs via
advanced Grand Canonical Monte
Carlo simulations.
• Supramolecular assembly
technique can be implemented for
synthesis of enzyme mimics,
which delineate the principles for
recognition and catalysis.
Main Achievements • First proof-of-principle
demonstration of artificial PCR.
• Significantly advanced chemical
• A series of structurally
Quantitative Impact
toolkit and theoretical knowledge for
the construction of abiotic
unprecedented porphyrin-based
MOF catalysts was developed.
supramolecular enzyme mimics.
200 µm • Learned how to control pore
• Created abiotic systems which can activation and catenation in
allosterically catalyze MOFs.
polymerization, self-amplify small • First allosteric single-site catalyst
molecule substrate, selectively was developed.
recognize Cu(II) ions, control the
orientation of multiple catalytic
porphyrin sites in MOFs and radical
pairs in mechanostereochemical
assemblies, and selectively bind
small molecule guests in highly
porous materials.
• Established theoretical foundation
for studying catalytic porphyrin
MOFs in solution.
End Goals
• Materials for small molecule
separations and storage.
• Selective sensors for chemical
and biological targets.
• High density information storage.
• Compact energy generation,
conversion and storage materials.
8
Allosteric Switch – Synthesis and
Concept

9
Artificial PCR via WLA

10
 Summary

 Created and studied the first artificial mimic of PCR.

 Designed and synthesized a triple-decker allosteric supramolecular
catalyst which can catalyze an allosterically controlled polymerization
reaction.
 Developing general routes towards controlled allosteric regulation with
complex substrates via relevant coordination chemistry.
 Currently investigating multiple heteroligated structures which can act
as allosteric enzyme mimics in the context of substrate recognition,
amplified detection and catalytic bond activation.
 Colorimetric detection of Cu(II) ions using DNA modified gold
nanoparticles has been accomplished, new detection scheme using
infinite coordination polymer nanoparticles is currently pursued. 11
 Structural Coloration
• Structural coloration is common in nature (insect wings, bird feathers,
fish scales, cephalopods, cat eyes)
• Accomplished through diffraction, interference, and differences in
refractive indices (most common form is the multilayer reflector)
• Nature’s design can be used to inspire a new generation of (dynamic)
optical materials and devices

Multilayer Reflectors
light

(Vukusic) high r.i.

low r.i.

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 Chromatophores
(Oregon State U)
4 µm
Iridophores
(AFRL)

Leucophores
(Woods Hole)
5 µm

J. B. Messenger (2001) Biol. Rev. 76 473-

528.
Cephalopods: Multi-
Layered Structure
uses both structural
and pigment 5 µm
13
SEM Micrograph by Hanlon &
coloration Mathger
 Determination of the Minimal Functional Units of
Wendy Crookes-Goodson and Rajesh Naik, AFRL/RXBN
• Mimicry of cephalopod
structural color is impossible
STATUS QUO
without basic understanding of
components involved.
• Structure-function relationship
of reflectins is unknown
• Limited characterization of the
protein has been performed.
• Little systematic analysis of
the protein family has been
performed since 2004.
• Reflectin sequence
database is deeper than
NEW INSIGHTS
anticipated, and can provide
information to influence
functional studies.
• Reflectins can be
processed and tested
systematically to determine
structure-function
relationships.
Reflectins
MAIN ACHIEVEMENTS IMPACT ACHIEVED
• Using a subset of sequences available, have already
begun to identify residues that are highly conserved • Identification of > 50
amongst reflectins. reflectin sequences that can
QUANTITATIVE IMPACT
be used for bioinformatics
PER(W/Y)(M/F)DMSx(W/Y)(Q/S)MDMxGR(W/Y)MDxxGR(H/Y)xxP(F/Y) analysis
• Alignment of repeats from
• Gathered evidence that reflectins are present outside 10 different reflectins already
of cephalopods, in Tridacna, the giant clam. show high conservation in
certain residues of the
repeats.
• Formation of thin films using
reflectin variants and
development of spectral
analysis techniques
• Cloned and began expression of reflectin constructs
to examine impact of repeat number on reflectin
• Cloning and
activity. Assays showing that only 2 repeats may be
characterization of non-
END OF PHASE GOAL
necessary for function.
cephalopod reflectin
• Generation of consensus
• Established method for casting reflectin sequence
thin films of reflectin for • Understanding of
characterization (with Kaplan relationship between primary
Group). Determining optical sequence and function in
conditions for reproducibility of vitro, that can be used to
films and methods to quantify develop model for in vivo
spectral characteristics of films. function.
14
 Reflectins in Iridophores
Bacterial Expression of Reflectin Reflectin Solubility Issues
native
Ref1a reflectin Amino acid composition of Reflectin 1a • Ionic detergents
amino % amino %
• Ionic liquids
acid of total acid of total • DMSO
Tyr 20.8 Phe 3.2 • HFIP
Met 14.8 Glu 1.4 • GuHCl
74% Arg 11.7 His 1.4
Asn 9.9 Cys 1.1
Gly 8.5 Thr 0.4
Asp 8.1 Val 0.4
Ser 5.3 Ala 0
Pro 4.6 Ile 0
Gln 4.6 Leu 0
Trp 3.9 Lys 0
reflectin 1b
• No known homologs Subdomain 5
Thin Films of Reflectin Characterize Optical Properties

specular light

15

15% (w/w) 10% (w/w) scattered light

 Optical malleability: light diffusers & dynamic 3-D texture in
cephalopod skin - Roger Hanlon, MBL, Woods Hole, MA
Bio-inspired approach to development of diffuse & textured camouflage materials
Efficient light diffusers.
Commercially available diffusers
rely on high-index contrast (e.g.,
Status Quo
using titania nanoparticles), and
are physically abrasive
Adaptable 3-D skin texture.
3.D texture has to date not been
incorporated in modern
camouflage materials. Texture is
added in the form of additional
structures such as plant
materials etc.
Cephalopod skin –
New Insights & Approaches
Inspiration for camouflage
materials.
Leucophores appear to provide
both high reflectivity and near-
Lambertian scattering with a
low-index contrast system.
They are flexible and work
under wet and dry conditions.
Papillae produce dynamic 3-D
skin texture along a broad
continuum from smooth to
highly rugose. The visual
sensori-motor system of rapid
adaptive camouflage provides
novel bio-inspired approaches
to materials science
Main Achievements
Leucophore ultrastructure and gross anatomy
have revealed first visualization in 3-D of
leucophores. Cell culture of leucophores has
been initiated. A new microscopy technique
has been developed 200to
µmsimultaneously
measure the full spectral reflectance of
scattering thin films such as leucophores or
iridophores.
Leucophore Iridophore
Papillae gross anatomy and kinematics have
been obtained in 3 species. Initial studies on
neural control of papillae were conducted.
The ultrastructure of leucophores enables
nearly perfect diffusion of incident light.
Papillae are under neurological control and the
animal is able to achieve a range of textural
states.
Current impact
1 – Quantified spectral reflectance
Quantitative Impact
and ultrastructural properties of
leucophores of one cephalopod
species. The protein appears not to
be a known reflectin. Initial 3-D
structure of leucophore paves way
for accurate modeling of light
interactions between spheres &
platelets. Cell culture will enable
focused measurements
2 – Morphology and neural control of
individual papillae appear complex;
morphology and neuroanatomy
require priority study at this juncture.
Planned Experiments
Characterize, measure, & model
different cephalopod & fish
leucophores & papillae
Research goals
End Goals
Structural coloration is rarely
achieved with proteins. With AFRL.
We are characterizing this yet-
unidentified protein for potential
future applications. This study also
aims to elucidate the biomechanics
and neural control of 3-D skin
papillae that provide dynamic textual 16
camouflage.
 Leucophore whiteness:
(white + reflectivity)

Leucophores: passive cells that

manipulate light (i.e. no muscles or
nerves to power the cells)
Leucosomes: light diffusers composed of
protein, which may be different from
iridophores
Spherical – 250-1250nm dia - scattering

Purpose:
1) shadow mitigation
2) maximum tonal contrast for disruptive coloration & pictorial
relief act as a light base layer upon which patterning is
applied in artificial architectures
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 LEUCOPHORES
Near-perfect diffusers of white light in all directions;
no nerves or muscles associated with them (i.e. passive)

BASIC QUESTIONS:
-what combinations of leucosomes and iridophores produce different grades of
“whiteness?”
-how do leucophores diffuse light equally in all directions?
-what are the basic capabilities of leucophores from cuttlefish or octopus? 18
 2. PAPILLAE

How do the papillae morph in shape (biomechanics,

etc.)?
What are the neural control mechanisms?
How does an octopus/cuttlefish view the
background monocularly to assess and reproduce
3D texture in the skin?
19

Can materials scientists engineer such capabilities?

 Status of achievement of
Program Goals
LEUCOPHORES
-Aim 1: Spectrometry - New microscope and objectives acquired; isolation
and culture of leucophores for high-mag spectroscopy.
-Aim 2: Ultrastructural analysis - 3D structure of leucophores produced;
determined temporal and spatial relationships between leucophores and
iridophores.
-Aim 3: Mathematical modeling - Initial model construction.
-Aim 4: Protein chemistry - Leucosomes are not same reflectin protein as
in iridophores; tissue samples sent to AFRL.

PAPILLAE
-Aim 1: Ultrastructural analysis - Extensive skin samples obtained and
prepared (4 Octopus; 1 cuttlefish)
-Aim 2: Neurophysiological control - Experiments on local and peripheral
stimulation of papillae (3 species)
-Aim 3: Videography - Excellent field acquisition of octopus; lab
kinematics from neurophysiology experiments
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 NEURAL REGULATION OF
CHROMATOPHORE FUNCTION IN CEPHALOPODS
N. Tublitz, U. Oregon

CURRENT STATUS MAIN ACHIEVEMENTS NEXT STEPS

 Chromatophore  Novel FaRP gene coding for  Use digital
addition aim several unique FMRFamide acquisition system
completed neuropeptides has been to ID
 Good progress on cloned and sequenced chromatophore
FaRP role & transmitters
transmitter ID  Spatial expression pattern of  Chromatophore
aims FaRP gene in cuttlefish brain motoneuron
has been determined electrophysiology
NEW INSIGHTS
 Process of  Principles governing the
chromatophore insertion of new LONG TERM GOAL
maturation chromatophores in the skin To understand the
have been identified neural mechanisms
& principles
 Chromatophore maturation underlying body
and development have been
patterning in
elucidated for all three 21
chromatophore types cephalopods
Aim 1: Neurotransmitter and neuro peptide
effects on Cuttlefish chromatophores

BEFORE AFTER

22
 Aim 1: Glutamate & FMRFa trigger different
responses in reddish-brown & yellow chromatophores

T’MITTER CHR.EFFECT BEHAV RESP

Glutamate Fast expansion Transient
FMRFa Slow expansion Sustained

CONCLUSIONS:
1) Transient patterns Glutamate
2) Sustained patterns FMRFamide
23
 Aim 2: PTHPFRFamide effects
on cuttlefish chromatophores

PTHPFRFamide:
1) Specificity: only black chromatophores
2) Selectivity: not all black ones
3) Causes chromatophores to pulsate 24
 Aim 4: Regeneration study

DAY 7 DAY 12

DAY 22 DAY 30 25
 SUMMARY: Neural Regulation of
Chromatophore Function in Cephalopods
1. All chromatophores have fast (glutamate) and slow
(FaRPs) excitatory inputs
2. Cuttlefish have at least 2 different FaRP genes that
encode 9 different FaRPs
3. FaRPs have differential effects on chromatophores
4. 2nd FaRP gene expression is limited to brain regions
involved in body patterning
5. There are specific principles governing the
insertion of new chromatophores
6. Newly added chromatophores are the same color at
first, then differentiate into separate color types
26
 SUMMARY

• This represents basic research support for a broad range of

biological science initiatives to provide the background and
innovation necessary for the future development of biomaterials,
devices and biosensors and their incorporation into future Air
Force weapons systems.
• To accomplish this, an increasing emphasis will be placed on
research that supports the following areas: biooptics,
composites, predator prey avoidance, directed self-assembly,
and biotemplating.
• Program Impacts:
– Understanding of allosteric enzyme operation and mimic
– Further understanding of Cephalopod camouflage system
– Discovery of New Chromophores and their Mechanism (2010)
– Importance of structural modalities in biopolymers to their
optical and strength components (2010) 27