Langmuir, 2009, DOI:10.1021/la90222gDNA diffusivity decreases with increase in the mole fraction of the gel-phase lipid.

Epifluorescence tracking of naked GUVs (top) and microsphere adsorbed GUVs (bottom) reveal binding induced charge separation and slaved motion of lipids.

5µm

a

b c

DMPC

DMTAP+

Before particle binding

After binding

Lipids diffusion slaved by particle

-

0 30 60 90 120 150

-20

-15

-10

-5

0

Bin

din

g e

nth

alp

y (

10

6 cal/m

ol)

cnanoparticle

/cliposome

DMPC 20C DMPC/DMTAP 20C DMPC/DMTAP 40C

Enhanced cooperative binding

Particle binding induces DMTAP segregation at

liquid phaseCDMTAP 15%→50%

Isothermal titration calorimetry measures the binding energy quantitatively and shows that binding induces charge separation and facilitates further binding

Advance made here: particle binding induces segregation of lipid in membrane and gathers their reins

In progress: coupled diffusion of lipids and particles

Random walkers on a fluctuating

lipid tube

For Corrugated Surfaces D~N-1

Model Chain Diffusion on Surfaces

For Smooth Surfaces D~N-3/4

Experiments Show D~N-3/2

Include Surface Defects to Explain Data

Transition to experimental data when defect spacing is less than chain size

DNA mobility on homogeneous bilayers and vesicles

Adsorption of ss-DNA on supported cationic lipid bilayer composed of DMTAP or DOTAP

0.001

0.01

0.1

1

10

0.001 0.01 0.1 1 10DLipid (µm2/s)

DD

NA (

µm

2 /s)

Diffusivity of the adsorbed DNA () plotted as a function of the lipid (DMTAP) diffusivity. Also shown is the diffusivity of bacteriorhodopsin in liposomes plotted as a function of lipid mobility in the presence of protein at different Lipid(L)/Protein(P) ratios. L/P = 140 () and L/P = 30 () (adapted from Peters et al ,PNAS, 79,4317(1982))

Mobility of ss DNA tracks lipid mobility

Diffusion of large DNA on giant unilamellar vesicles

Diffusion of linear DNA

Diffusion of circular DNA

0.23m2/s0.2m2/s

No D

D=0.44m2/s

48.5kbp linear DNA (3.2*107g/mol) trajectory (black) on DOPC DOTAP (10%) GUV. The three smaller red circles show trajectories from same DNA molecule. Scale of this image is 40 m x 40 m. It is exciting to see that the DNA diffusivity differs from spot to spot, and from time to time, even in the absence of external electric field for electrophoresis.

2D projection of 2.6*107 g/mol circular DNA trajectory on giant lipid vesicle surface. From a plot of mean-square displacement against time, the straight line implies a translational diffusion coefficient of D = 0.66 m2/sec.

X2 (m

2 )

ConclusionsLipid mobility controls the diffusivity of short biopolymer adsorbates.Formation of raft-inspired lipid bilayers enhances the effectiveness of polyvalent recognition as well as adsorbed enzyme stability.Domain formation in lipid bilayers and biomolecule recognition can be actively controlled by adding divalent cations.Domains in lipid bilayers provide control over the adsorption and transport of DNA.Nanoparticles stabilize phospholipid vesicles by preventing vesiclefusion even at high vesicle concentrations.Nanoparticles do not interfere with receptor binding or functionalization of bilayer lipids.Nanoparticle binding can locally induce phase transitions in lipid membranes.

Education and Outreach: The project has already contributed to the

training and development of four graduate students (Krishna Athmakuri, Jeffrey Litt, Chakradhar Padala, and Yan Yu), one undergraduate student (Andrew Devine) and a high school student (Kevin Crimmins). Students are introduced to an interdisciplinary research environment and gain expertise in topics ranging from soft materials to nanotechnology, biophysics, transport phenomena, and biomaterials. Further training is provided through outreach efforts, such as presentations to high school students in the New Visions High School Program and to a high school teacher, Ms. Tammy Borland.

Confocal Images of GUVs containingi)5% and ii-iv)20% cholesterol.

Characterization by FRET of peptide clustering due to cholesterol dependentphase separation

Controlling Biomolecule Stability and Recognition Using Raft-Mimetic Lipid Bilayers

Actively Induced Phase Separation

Enhanced efficiency of recognitionusing raft-mimetic liposomes

Phase separation leads to a 2 order of magnitude increase in polyvalent recognition

Phase separation provides a general mechanism to increase efficiency of polyvalent recognition

Confocal image of Ca2+

induced phase separation of a GUV

Peptide-functionalizedlipid

Gel-Phase lipids

Fluid-Phase lipids

Angew. Chem. 119, 2257 (2007)

No cholesterol

5% cholesterol20% cholesterol

0

0.1

0.2

0.3

0.4

21 42 54 63 84

Diff

usiv

ity(

m2 /s

ec)

# of bases of ssDNA (N)

Novelty:Actively reconfigurable, nanostructures to control biomolecule adsorption and transport. This biologically-inspired approach seeks to implement, in the bioseparations context, the concept of lipid rafts. 

Transformative potential: The field of bio-separations is limited by use of passive surfaces. Our goal here is to remove this limitation. To accomplish this task, fundamental underpinnings are under development, those needed to design reconfigurable nanostructured surfaces that enable the separation of biomolecules in a manner that is far more facile and efficient than conventional strategies.

Potential Impact on Industry and Society: 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. The project will also contribute to the training of graduate, undergraduate, and high school students and expose them to a stimulating interdisciplinary research environment.

Novel materialsTransportRaft-mimetic bilayers

A + + +

DNA mobility on heterogeneous bilayersDNA adsorption and diffusion on heterogeneous bilayers

Fluorescence micrographs of DNA adsorbed on bilayers consisting of: (a) 10 mole% DSPC and (b) 30 mole% DSPC. (c) Diffusivity of DNA adsorbed on heterogeneous supported bilayers as a function of mole% of the gel-phase lipid DSPC in the bilayer.

DNA electrophoresis on heterogeneous bilayers

Fluorescence micrographs of 84mer ssDNA labeled with Texas Red dye in the presence of an electric field. (a) t = 0 min, (b) t =8 min and (c) t =16 min

DNA diffusivity (D) decreased with size (N) whereas drift velocity is nearly independent of N.

0

0.1

0.2

0.3

0.4

0.5

21 42 54 63 84

Dri

ft ve

loci

ty (

m/s

ec)

# of bases of ssDNA (N)

Isothermal titration calorimetry shows the affinity of nanoparticles to lipid membrane depends on the surface charge of particles. The binding can be divided into two categories: enthalpy driven, and entropy driven, accordingly. The binding strength is >>kT.

Positively charged particles

0 100 200 300 400 500 600 7000

200

400

600

800

1000

1200

H (

kcal/m

ol)

cnanoparticle

/cliposome

0 40 80 120

0.0

0.5

1.0

Heat

flo

w (

cal/sec)

Injection sequence (min)

0 100 200 300 400 500 600

-5000

-4000

-3000

-2000

-1000

0

1000

2000

H (

kcal/m

ol)

cnanoparticle

/cliposome

0 50 100 150 200 250 300-1.5

-1.0

-0.5

0.0

0.5

Heat

flo

w (

ccal/sec)

Injection sequence (min)

C

A

Negatively charged particles

Enthalpy driven

Entropy driven

0 300 600

0.48

0.52

0.56

0.60

GP

cnanoparticle

/cliposome

0 100 200 300

-0.3

0.0

0.3

0.6

cnanoparticle

/cliposome

Gen

era

l p

ola

rizati

on

400 450 500 550 6000.0

0.5

1.0

Em

issio

n (

a.u

.)

Wavelength (nm)

0100200600

B

A

Dielectric environment sensitive fluorescence demonstrates the ability of nanoparticles to locally induce liquid-gel phase transition in lipid membranes, which is the driven force for particle binding.

Negatively charged particles, Liquid-gel

Positively charged particles,Gel->liquid

Advance made here: particle binding modulate the membrane structure significantly In progress: particle packing and induced local curvature

gel

θ-+

liquid

+- θ- +

electrostatic

Particle binding induces local phase transition in lipid membranes

PNAS ,105, 18171-18175 (2008)

B

D

Advance made here: cationic nanoparticles stabilize phospholipid vesicles up to dense volume fractions without fusion, allowing ligand-receptor binding.

In progress: interactions with DNA, especially plasmid DNA.

Soft Matter 3, 551 (2007) J. Phys. Chem. C 111, 8233 (2007)

Fluorescence autocorrelation functions showing that streptavidin binds effectively to vesicle-attached biotin even when nanoparticles stabilize the liposomes to which biotin is attached.

0.1 1 101E-3

0.01

0.1

1

MS

D/ t

(m

2 / sec

)

Time (sec)

500 nm

Δπ=300 mM

Δπ=400 mM

t=0 min

t=20 min

Δπ=200 mM

Δπ=100 mM

0 100 200 300 400 500

0.0

0.2

0.4

0.6

0.8

1.0

Nor

mal

ized

Inte

nsity

Osmotic Pressure (mM)

Naked GUV Stabilized GUV

Epifluorescence images of naked GUVs (top)

and nanoparticle-stabilized GUVs (bottom) reveal enhanced osmotic stress tolerance for the latter.

Nanoparticle-stiffened phospholipid vesicles

DiI FITC-SBP Merged

Heterogeneous (rapid quench)Heterogeneous (slow quench)Homogeneous

Enzyme Adsorption onto Raft-Like Domains Increases Stability

JACS 131, 7107 (2009)

Adsorption of SBP onto small domains (5 nm) imparts greater stability than larger domains (13 nm)

NIRT: Actively Reconfigurable Nanostructured Surfaces for the Improved Separation of Biological Macromolecules

Ravi Kane, Steve Granick, and Sanat Kumar, CBET - 0608978

Particle binding induces charge separation and slaved motion