NIRT: Hierarchical Bionanomanufacturing

1
NIRT Hierarchical Bionanomanufacturing
PI Robert L. Clark1. Co-PIs Ashutosh
Chilkoti2, Eric Toone3, and Stefan Zauscher1
Mechanical Engineering and Materials Science1,
Biomedical Engineering2, Chemistry3, Duke
University
Abstract
II) Localized Actuation of Thermally Responsive
Polymers and Polypeptides
III) Fabrication of Bioconjugated and Hybrid
Polymeric Nanostructures by Field-Induced
Scanning Probe Lithography
Here we report on progress on our NIRT on
hierarchical nanomanufacturing. I) First we
report on a holographic technique we adopted that
provides a noninvasive laser-based approach for
adaptable, real-time nanofabrication and
nanomodification of (bio)-polymeric materials
substrates. II) We then discuss a technique we
are developing in which optically addressable
metal nanoparticles display large spectral shifts
as a function of subtle changes in distance from
conductive films, and hence, represent a
promising way to measure angstrom-scale distances
in potential biosensor applications. III) Finally
we report on a process we developed for the
chemical modification of protein resistant
polymer brushes for the fabrication of protein
arrays by field-induced scanning probe
lithography.
We are currently developing a novel and highly
sensitive method of sensing nanometer-scale
distances between single nanoparticles (NPs) and
conductive surfaces. Once characterized, this
method will be used to indicate thermally
responsive polymer actuation that is localized to
specific regions in-between metal nanoparticles
and a conductive film.  Here, the high-energy
focusing capabilities of nanoparticles in
proximity to metal films upon laser irradiation
will cause a temperature increase, and likely
trigger polymer actuation, in a highly localized
manner.
Highly controlled patterning of polymeric and
biomolecular nanostructures on surfaces is a
critical step in the fabrication of biomolecular
devices and sensors. We use field-induced
scanning probe lithography (FISPL) to chemically
modify polymer brushes to allow conjugation of
biomolecules. Surface-confined, non-fouling
(i.e., protein resistant) poly(oligo(ethylene
glycol) methyl methacrylate) (p(OEGMA)) brushes
were prepared on silicon substrates by
surface-initiated atom transfer radical
polymerization (SI-ATRP) in a grafting-from
approach. These p(OEGMA) brushes were then
patterned directly on the nanoscale by FISPL,
generating chemical functionalities that allowed
for subsequent protein conjugation (Schematic and
AFM images below). Although our approach works,
we do not yet have a complete physical-chemical
understanding of the process. We hypothesize that
-CH3 groups are oxidized in the presence of high
electric fields and converted to -COOH groups.
After chemical modification, protein conjugation
is achieved on the patterned areas via
biotin-streptavidin coupling. With this approach
we were able to create periodic BSA protein
arrays with a feature width of 130 nm.
Non-specific adsorption of protein was
dramatically reduced due to the non-fouling
nature of the polymer.
Au
Au-coated glass
I) Three-dimensional Dynamic Mask-Less
Holographic Lithography
Spectroscopy of Nanoparticles near Gold Films
A method is presented for dynamic,
computer-controlled, maskless beam-steering, by
spatial light modulators (SLMs), to address
specific locations on arrays with large spatial
and temporal selectivity. The dynamic maskless
holographic lithography (DMHL) approach is
ideally suited to trigger and direct
nanofabrication in the optical near-field through
easily controllable far-field, broad beam
illumination sources. The beam from the laser is
centered on the computer-controlled SLM,
reflected into the microscope and directed
upwards through the objective to illuminate the
specimen plane. The optical system ensures the
plane of the SLM is imaged to and completely
fills the rear aperture of the microscope
objective. Beam propagation from the rear
aperture through the objective results in
reconstruction of the original image on the
micro/nanoscale.
Single NP, TIR Illumination (SPP LSP)
SPP and LSP Deconvolution
We also found that both (a) raised features and
(b) trenches can be created on the p(OEGMA)
brushes by controlling the contact force between
the tip and the substrate. While the feature size
of the patterns could be controlled by adjusting
the patterning parameters such as applied
voltage, relative humidity and tip velocity. We
note that if a negative tip bias or no bias is
applied with respect to the substrate, no
appreciable changes are observed. This highlights
the directionality of the electrochemical process
and shows that physical interactions between the
tip and the sample alone cannot be responsible
for the formation of the patterns.
Layer-by-layer (LBL) assembly of oppositely
charged polyelectrolytes (PELs) was used to
control the distance between NPs and gold films.
The surface plasmon polariton (SPP) resonance
propagating along the gold film red shifts as the
number of PEL layers increases (left plot, green
trend). Single NP spectra can be acquired using
two different methods of illumination i) dark
field (DF) or ii) total internal reflectance
(TIR) illumination. Dark field illumination
primarily excites the localized surface plasmon
(LSP) resonance of the NP. Single NP spectra,
obtained using DF illumination, are increasingly
blue-shifted as the NPs are spaced further away
from the high-dielectric gold film (left plot,
blue trend). While TIR illumination primarily
excites the SPP resonance of the gold film, LSP
resonances of NPs in close proximity to the film
are also excited via an evanescent field. For
this reason, single NP spectra, obtained using
TIR illumination, show a convoluted dependence on
layer height that is dominated by the
blue-shifting LSP when NPs are less than 14 nm
away from the gold film, and by red-shifting
(SPP-dominated) when NPs are further than 14 nm
from the gold film (right plot). These data
suggest that the SPR of NPs is shifted by 12 nm
for every 1-nm change in distance from 0  6 nm
and 7 nm for every 1-nm change in distance from
6  14 nm. With a high resolution grating this
experimental design is potentially capable of
sensing distance changes on the order of
Ångstroms!
a)
b)
Localized Optothermal ELP Actuation
We have made use of the chemical changes on the
brush surface that occur due to the oxidative
nature of the electrochemical patterning process
and demonstrate that they can be used to create
bioconjugated nanostructures. Our unique
patterning approach can potentially form the
basis for the fabrication of a large range of
novel polymeric and biomolecular nanostructures
that may find application as biosensors or
substrates for the precise presentation of
biomolecular queues to cells. We are currently
developing the tools and procedures to expand the
lithographic approach into a massively parallel,
anodization stamping process.
Micro/Nano Patterning with Photopolymer
Initial system testing was performed using an
optically responsive epoxy (Norland Photopolymer
63). (a) CAD-generated pattern (b) SEM image
taken normal to the substrate surface showing 880
nm to 2.5 ?m feature widths. Note the letter L
has fallen over due to post-processing after
patterning. (c) SEM images taken at a 40 and
(d) 60 from the surface show the height of the
features ranging between 3-5 microns. With an
optimized system and improved feedback control,
resolution could be improved to at least the
diffraction limit of 250 nm.
a)
b)
Acknowledgments
We acknowledge support through NSF-NIRT 0609265.
We also acknowledge the substantial contributions
to the optothermal NP interrogation by Prof.
David Smith and Jack Mock at Duke University.
c)
d)
Preliminary experiments are aimed at localized
actuation of elastin-like polypeptides (ELP, is a
thermally responsive polypeptide) sandwiched
between single NPs and a gold film, using laser
heating. The schematic experimental setup is
shown above the plot (above right) shows a shift
of 30 nm in the SPR response of a single
nanoparticle after 1 min of laser exposure.
Although a spectral shift of this magnitude would
indicate a change in distance between the NP and
the gold film, it cannot yet be concluded that
this shift is due to thermally induced changes in
ELP conformation. Current studies are focused on
characterizing spectral properties of NPs in
close proximity to gold films with water as the
surrounding medium to provide a distance
calibration for localized ELP actuation
experiments.