NSF Nanoscale Science and Engineering Grantees Conference, Dec 7-9, 2009
Grant # 0608978
NIRT: Actively Reconfigurable Nanostructured Surfaces for the Improved
Separation of Biological Macromolecules
NSF NIRT Grant 0608978
PIs: Ravi S. Kane1, Steve, Granick2, Sanat Kumar3
1
Rensselaer Polytechnic Insitute, 2University of Illinois at Urbana-Champaign, 3Columbia
University
Background: Controlling the recognition and transport of biomolecules is critical for the design
of numerous products in the pharmaceutical and biotechnology industries, among others; this
proposal is driven by this critical need. Our central idea is biologically inspired, specifically by
lipid rafts. We propose to combine theory and experiment to design actively reconfigurable
nanostructured surfaces that will significantly improve our ability to separate biomolecules in a
manner that is more facile and efficient than conventional separation strategies.
Results: We have investigated biomolecule adsorption and transport on reconfigurable surfaces.
We demonstrated that
the
clustering of peptides into “raftlike” membrane microdomains
significantly
enhances
the
efficiency of recognition of
proteins. Furthermore, the ability
to induce phase separation in
membranes could be used to
actively modulate the proteinbinding capabilities.
We also recently demonstrated
that the formation of raft-like
domains in supported lipid
bilayers provides control over the
adsorption and diffusion of DNA.
Specifically, we demonstrated the
ability to pattern the adsorption of
DNA on heterogeneous lipid
bilayers (Figure 1a and 1b). We
also found that the composition of
heterogeneous bilayers influences
the diffusivity of adsorbed DNA
Figure 1. Fluorescence micrographs of DNA adsorbed on bilayers
(Figure 1c) and that domains in
consisting of: (a) 10 mole% DSPC and (b)30 mole% DSPC. (c)
heterogeneous bilayers serve as
Diffusivity of DNA adsorbed on heterogeneous supported bilayers
as a function of mole% of the gel-phase lipid DSPC in the bilayer.
obstacles to the diffusion of
adsorbed DNA. In ongoing work,
we are characterizing DNA transport on supported bilayers in the presence of an applied external
electric field.
We have also been studying how larger DNA molecules diffuse on the lipid bilayer surface. A
curious result from these experiments has been the tendency of the DNA to collapse rather than
form the extended configurations that had been expected, as illustrated in the figure below
(Fig.2). In ongoing work, we are comparing the diffusivity of circular DNA on lipid bilayers
NSF Nanoscale Science and Engineering Grantees Conference, Dec 7-9, 2009
Grant # 0608978
supported on quartz, or unsupported in giant unilamellar vesicle (GUVs), to linear DNA of
similar number of base pairs.
In a first attempt to understand how
the transport of DNA on surfaces is
affected by the length of the DNA and
the external field, we have also
performed computer simulations of a
polymer chain of length N strongly
adsorbed at the solid-liquid interface in
the presence of explicit solvent to
delineate the factors affecting the chain
length N dependence of the polymer
lateral diffusion coefficient. We find that
surface roughness has a large influence,
and D scales as D ~ N-x, with x3/4 and
x1 for ideal smooth and corrugated
surfaces, respectively. The first result is
consistent with the hydrodynamics of a

Figure 2. Linear DNA, 48.5 kilo base pairs “particle” of radius of gyration Rg ~ N
long, stained with the fluorescent dye YOYO1, (=0.75) translating parallel to a planar
adsorbed at dilute surface coverage to supported interface, while the second implies that
lipid bilayers containing 10% cationic lipid the friction of the adsorbed chains
DOTAP and observed by epifluorescence dominates, as in unentangled polymer
imaging. We do not understand yet why some melts.
We have also met with encouraging
molecules are extended but not all of them.
success in using phospholipid bilayers to
explore the access of a biomolecule (streptavidin) to liposome-immobilized ligand (biotin) in
cases where the liposomes are stabilized against fusion by allowing nanoparticles to adsorb. It
was found that biomolecule binding persists over a range of nanoparticle surface coverages
where liposome fusion and large-scale aggregation is prevented. This indicated that liposome
outer surfaces, in the presence of stabilizers, remain biofunctionalizable, and may have bearing
on explaining the long circulation time of stabilized liposomes as drug delivery vehicles.
Nanoparticles may also be used as obstacles to control the diffusion of the adsorbed
biomolecules on surfaces.
As part of our studies of the interaction of charged biomolecules with reconfigurable surfaces
– lipid bilayers – we have also been studying the influence of adsorbate-induced reconfiguration
on transport through the bilayer. Specifically, we have been studying the ability of polycationic
biomolecules to form pores in planar lipid bilayers. In this study we used a peptide composed of
nine arginine residues as a model peptide. The lipid bilayer composition used was a 3:1 molar
mixture of the lipids DOPC and DOPG, which are zwitterionic and negatively charged
respectively. This composition yields a good model of the charge density of a typical cell
membrane. Our results (Fig. 3) demonstrate the ability of a model polycationic biomolecule to
induce transient openings in a reconfigurable bilayer.
Scientific Uniqueness This work is unique in that it demonstrates the ability to control
biomolecule adsorption and transport on and through reconfigurable surfaces (Figures 1-3).
NSF Nanoscale Science and Engineering Grantees Conference, Dec 7-9, 2009
Grant # 0608978
Figure 3. Plot of current across a DOPC/DOPG (3:1) bilayer versus time. (a) Current trace prior to
peptide addition, (b) current trace 0-10 min after peptide addition shows transient current fluctuations, (c)
current trace 10-20 minutes after peptide addition shows an increase in baseline current, (d) current after
calcium addition decreases over 50 minutes back to zero baseline current.
Impact on Industry The design, purification, sensing, and controlled delivery of biomolecules
is a major window of opportunity in the development of new high-value industrial markets –
particularly in the biotechnology and pharmaceutical sectors. This work will combine theory and
experiments to design actively reconfigurable nanostructured surfaces to control the adsorption
and transport of biological molecules. The understanding of biomolecule recognition and
transport provided by this work will impact the design of novel technologies for biosensing,
bioseparation, drug delivery, as well as the design of novel therapeutics.
An exceptionally notable aspect of this work is that for the first time a synergistic,
multidisciplinary combination of theory and experiment is being used to design biologically
inspired actively reconfigurable nanostructured surfaces to control biomolecule adsorption and
transport. The fundamental underpinnings resulting from this work will significantly improve
our ability to separate biomolecules in a manner that is more facile and efficient than
conventional separation strategies. The ability to control biomolecule recognition and transport
is critical for the design and product of a wide range of industrial products.
References
[1] For further information about this project email Ravi Kane (kaner@rpi.edu), Steve Granick
(granick@mrl.uiuc.edu), or Sanat Kumar (sk2794@columbia.edu).
[2] Padala, C.; Cole, R.; Kumar, S.; Kane, R. S. “Lipid Mobility Controls the Diffusion of Small Biopolymer
Adsorbates”, Langmuir, 2006, 22, 6750.
[3] Rai, P. R.; Saraph, A.; Ashton, R. ; Poon, V. ; Mogridge, J. ; Kane, R. S. “Raft-inspired Polyvalent Inhibitors of
Anthrax Toxin: Modulating Inhibitory Potency by Formation of Lipid Microdomains”, Angewandte Chemie
International Edition, 2007, 46, 2007.
[4] Kane, R. S. “Fabricating complex polymeric micro- and nanostructures: Lithography in microfluidic devices”,
Angewandte Chemie International Edition, in press.
[5] Desai, T. G.; Keblinski, P.; Kumar, S. K.; Granick, S. “Modeling Diffusion of Adsorbed Polymer with Explicit
Solvent”, Physical Review Letters, 2007, 98, 218301.
[6] Zhang, L.; Dammann, K.; Bae, S. C.; Granick, S. “Ligand-Receptor Binding on Nanoparticle-Stabilized
Liposome Surfaces, ” Soft Matter 2007, 3, 551.
[7] Herce, H.D.; Garcia, A. E.; Litt, J.; Kane, R.S.; Martin, P.; Enrique, N.; Rebolledo, A.; Milesi, V. “ArginineRich Peptides Destabilize the Plasma Membrane, Consistent with a Pore Formation Translocation Mechanism of
Cell Penetrating Peptides”, Biophysical Journal, 2009, in press.
[8] Litt, J.; Padala, C.; Asuri, P.; Vutukuru, S.; Athmakuri, K.; Kumar, S.; Dordick, J.; Kane, R. S “Enhancing
Protein Stability by Adsorption onto Raft-Like Lipid Domains“, Journal of the American Chemical Society,
2009, in press.
[9] Athmakuri, K.; Padala, C.; Litt, J.; Cole, R.; Kumar, S.; Kane, R. S. “Controlling DNA Adsorption and Diffusion
on Lipid Bilayers by the Formation of Lipid Domains“, Langmuir, 2009, in press.