endotoxin-induced ordering transitions in liquid crystalline
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Endotoxin-Induced Ordering Transitions in Liquid Crystalline Droplets
Nicholas L. AbbottDepartment of Chemical and Biological Engineering
University of Wisconsin-Madison
Email: abbott@engr.wisc.edu
1
H2O (+ ions)oil
oil
Oil-in-Water Emulsion
2
C N
H2O (+ ions)
nematic LC
LC
LC
Oil-in-Water Emulsions
5CB
and 3
Surface Anchoring
n
o+Wasin2(s-e)/2
Wa~10-3-10-2mJ/m2
ns
ne
e
s
interface
(surface director)
(easy axis)
z
x
y
Elasticity of Liquid Crystals
2
3
2
2
2
1 21
21
21 nnKnnKnKF
d
Splay Twist Bend
Ki~10-11N
m = - ½ m = + ½m = +1
Topological Defects
4
C N
H2O (+ ions)
nematic LC
LC
LC
Bipolar Configuration
Radial Configuration
Common Configurations of LCs in EmulsionDroplets
5
8μm: Increasing Surfactant concentration
Surfactant-Induced Anchoring Transition
0.0mM 0.2mM 0.4mM 0.6mM 0.8mM 1.0mM
Water
5CB
Surfactant
O S OO
O
Na
SDS
(Bright-field micrographs) (Equilibrium states)6
Endotoxin (Lipopolysacharide, LPS)
Endotoxin
OO
OO
O
HO
OP
O
O-
-O
NH
O
OO
O
OHO
OO
HO
NHO
HO
O P
O
O-
O-
Lipid A
7
Current assays for endotoxinare based on blood of horseshoe crabs
Endotoxin inducescoalgulation of horseshoe crab blood 8
C Bipolar
F RadialED
BA
Scale bar is 5um.
Ordering Transitions in Micrometer-Sized Droplets of Nematic 5CBInduced by Endotoxin
endotoxin-free
1 g/ml of endotoxin
OO
OO
O
HO
OP
O
O-
-O
NH
O
OO
O
OHO
OO
HO
NHO
HO
O P
O
O-
O-
Lipid A
9
0 2 4 6 8 100
10
20
30
40
50
60
70
80
90Pe
rcen
tage
of L
C D
ropl
ets
with
R
adia
l Con
figur
atio
n [%
]
LPS Concentration [pg/mL]
N=5; n=818
N=4; n=589
N=5; n=1142
N=5; n=1125
N=4; n=877
N>15; n>10000 20 40 60 80 100
0
10
20
30
40
50
60
70
80
90
Perc
enta
ge o
f LC
Dro
plet
s w
ith
Rad
ial C
onfig
urat
ion
[%]
LPS Concentration [pg/mL]
N=5; n=632
N=5; n=589
N=4; n=469
N=4; n=386
N>15; n>1000
Influence of Endotoxin Concentration on Bipolar-to-Radial Ordering Transition
pM concentrations 10
0 2 4 6 8 100
10
20
30
40
50
60
70
80
90P
erce
ntag
e of
LC
Dro
plet
s w
ith
Rad
ial C
onfig
urat
ion
[%]
LPS Concentration [pg/mL]
N=5; n=818
N=4; n=589
N=5; n=1142
N=5; n=1125
N=4; n=877
N>15; n>10000 20 40 60 80 100
0
10
20
30
40
50
60
70
80
90
Perc
enta
ge o
f LC
Dro
plet
s w
ith
Rad
ial C
onfig
urat
ion
[%]
LPS Concentration [pg/mL]
N=5; n=632
N=5; n=589
N=4; n=469
N=4; n=386
N>15; n>1000
Influence of Endotoxin Concentration of Bipolar-to-Radial Ordering Transition
~300 Lipid A molecules per LC dropletScience, 332, p1297-1300, 2011.
11
How Many LPS Molecules are Causing the Ordering Transition?
5 μm1pg/mL LPS
40uL LPS solution+
0.16 nL 5CB emulsion dropletsCalculation:
1) 0.16 nL 5CB correspond to 1415 LC droplets.2) 40μL, 1pg/ mL LPS solution corresponds to 481,600 LPS molecules.
Therefore, there are
340 LPS molecules per LC dropletor
300,000 nm2 of LC interface (on average) per LPS
3m
340 LPS molecules
0.3μm2/ LPS
[i.e., ~10-5 of saturation monolayer coverage] 12
Density of Lipid A Needed to Trigger Ordering Transition via Surface Anchoring Effects:Langmuir Schaeffer Transfer
At a planar interface, surface anchoring changes ordering of LC at ~1.3 nm2/lipid A molecule
O
OH O
O
OHHN
OOH O
PHO OHO
OOHO
O
P OHHOO
O
O O
O
NH
O
O
O
Lipid A structure
80 100 120 140 160 180 200 220 240
0
10
20
30
40
50
Sur
face
Pre
ssur
e [m
N/m
]
Area/Molecule [Å/Lipid A]
115Å2 148Å2
Planar Interface
13
Response of LC Droplets to Endotoxin is Highly Selective
DOPC
DLPC
SDS
LPS
OO
OO
O
HO
OP
O
O-
-O
NH
O
OO
O
OHO
OO
HO
NHO
HO
O P
O
O-
O-
1mg/ml
1µg/ml
1ng/ml
Con
cent
ratio
n of
Ads
orba
te R
equi
red
to
Indu
ce R
adia
l Con
figur
atio
n
Lipid A DLPC DOPC SDS
1pg/ml
Science, 332, p1297-1300, 2011. 14
Microfluidic channels
Polymer dispersed LC Films (PDLC)(Drzaic, P. S., Liquid Crystal Dispersions, 1995)
(Weitz D. A. et al., Langmuir, 2000)
Polydisperse/polymer embedded
Serial/difficult below ~3m
LC Emulsions: Effects of Confinement
Order??
15
Bulk elastic free energy ~K*R
Orientation-dependent surface energy ~W *R2
R ~ K/W ~ 1m
Size-Dependent Ordering in LC Droplets
Lavrentrovich O. D., Liquid Crystals, 1998
A B
Bipolar Configuration (R > 1m)Surface Effects > Elastic Effects
Homogeneous Configuration (R < 1m)Elastic Effects > Surface Energetics
Decreasing Radius
(K ~ 1 pN and W ~ 10-3 mJ/m2)
Prototypical Example
Bulk elastic constant
Surface anchoring energy
16
SiO2 SiO2
SequentialDeposition of
polymers
1Polymeric
capsuleLC-filled Polymeric
capsule3
5CB loading
2
Silica etching(HF treatment)
10m 8m 5m 3m 1m 700nm
Bright-field images of LC droplets of different sizes
Synthesis of LC Droplets with Independent Controlof Surface Chemistry and Size
Collaborator: Frank Caruso University of Melbourne
Gupta J. K.,Sivakumar S., Caruso F., Abbott N. L., Angewandte Chemie, 2009
4
Remove polymer(H-bonded systems)
17
C F H K
D G I L
E J M
d 3m d 1md 700nm
A B
Size-Dependent Order
Polarized
Brightfield
Bipolar Pre-radial Radial
Gupta J. K.,Sivakumar S., Caruso F., Abbott N. L., Angewandte Chemie, 2009
18
ionconfigurat bipolar
favorsionconfigurat radial
favors
22411 223 WRRKRKF rbp
Observed size-dependent ordering in LC droplets
d 3md 700nm
Bipolard 1mPre-radial Radial
Miller and AbbottSoft Matter, 2013.
RK2RK5WR2RK4RK8FFF 24112
2411bprrbp
(Lavrentovich et al., Phys. Rev. Lett. 1994) (Zumer et al., Phys. Rev. E. 1994) (Dubois-Violette et al., J. Phys. (Paris) Colloq. 1969)
W
K23K
R1124
c
if
19
E J M
d 3m d 1md 700nm
Size-Dependent Configuration
Bipolar Pre-radial Radial
Configuration driven byK24?
Configuration dominatedby surface anchoring
Is the influence of endotoxin dependent on the LC droplet size?20
Influence of LC droplet size on endotoxin-triggered ordering transitions
21
• Percentage of droplets exhibiting a radial configurationdecreases with increasing droplet diameter
• Solid bar = 100 pg/mL endotoxin
• Open bar = no endotoxin
• 86,000 droplets in solution
Threshold radius ~ 5 µm
A CB
Polarized micrographs of nematic 5CB in contact with aqueous phases containingendotoxin. (A) with endotoxin-free solution. (B) after 24 hr incubation in 1μg/mL endotoxin solution. (C) < 2 minute incubation in 1mg/mL endotoxin solution. Scale bars are 300μm.
Endotoxin-free 1μg/mL 1mg/mL
Ordering Transitions in Thin Films of Nematic 5CB Induced by Endotoxin
22
2 to 4 4 to 6 6 to 8 8 to 10 > 100
10
20
30
40
50
60
70
80
90
100
Rad
ial C
onfig
urat
ion
[%]
Diameter Range [m]
Influence of Confinement on Response to 10 pg/ml of Endotoxin
LC
LC
K24 predisposes droplets toendotoxin-inducedordering transitions
23
Influence of Endotoxin on Free Energy of the LC Droplets
24
22411
rbp WR2RK2RK3F
Threshold radius ~ 5 µm
J10F 17rbpm5R
Standard Free Energy of Self-Association of Endotoxin
ln CACnaggregatio xkTG
moleculesendotoxin 700for 10 17 JG naggregatio
Steady-state fluorescence measurements indicate that the critical aggregation concentration (CAC) of endotoxin is ~10 µg/mL (CACs reported for lipid A are similar).
Aurell et al., Biochemical and Biphysical Research Communications. 1998.
OO
OO
O
HO
OP
O
O-
-O
NH
O
OO
O
OHO
OO
HO
NHO
HO
O P
O
O-
O-
25
Influence of Endotoxin on Free Energy of the LC Droplets
26
22411
rbp WR2RK2RK3F
JG 1710
Similar in magnitude to the standard free energy
of association of ~103
endotoxin molecules
Threshold radius ~ 5 µm
J10F 17rbpm5R
Change in free energy associated with a bipolar-to-radial ordering transitionin a LC emulsion droplet
endotoxinexbp
exr
rbp FFFF
rbpF
endotoxinrbp FRKRKWRRKRKF
24112
2411 25248
endotoxinrbp FWRRKRKF 2
2411 223
Surface anchoring energy of LCat the aqueous interface
Easy axis of LCis tangentialto droplet interface
27
thisinterface
C N
H2O (+ ions)
nematic LC
LC
LC
28
LC Ordering Transition Probes Formation of Electrical Double Layer on the “Oily Side” of the Interface
Salt solution
LC phase
Ψ
zZeta potential
‐1
water~ 80
para= 19.7perp= 6.7
High local E‐field
Carlton and Abbott, Langmuir 2012 and 2013
29
Zeta-Potentials of Nematic 5CB-Aqueous Interface
5CB droplets, 100 mM NaCl
LC (oil)
LC (oil)
C N
5CB
30
Influence of ionic strength and pH on bipolar-to-radial transition in LC droplets induced by 10 pg/ml
of endotoxin
5.0 5.5 6.0 6.5 7.0 7.5 8.0
0
10
20
30
40
50
60
70
80
90
100
Rad
ial C
onfig
urat
ion
[%]
pH0 10 20 30 40 50 60 70 80 90 100
0
10
20
30
40
50
60
70
80
90
100
Rad
ial C
onfig
urat
ion
[%]
Ionic Strength [mM]
endotoxin2
2411rbp FWR2RK2RK3F
W(pH, ionic strength)Miller and Abbott, Soft Matter 2013
31
O
OHO
OOH
NHO
HO
OPHOOH
O
OO
HOO
POH
HOO
OOO
OHN
OO
O
endotoxin2
2411rbp FWR2RK2RK3F
How does Lipid A (or Endotoxin) Trigger the Ordering Transition in the LC Droplets?
How does the endotoxinstabilize the radial configuration relative to thebipolar configuratoin
32
Self-Assembly of Endotoxin
Seydel et al., Chemical Immunology. 2000.Brandenburg et al., Journal of Structural Biology. 1992.
MolecularConformation
Supramolecular Aggregate Structures
Phase Diagram for Endotoxinfrom S. Minnesota
Increasing chain fluidity promotes formation of inverted micelles
Phase behavior measured by SAXS
Endotoxin has a negative spontaneous curvature (co)33
Response of LC Droplets to Endotoxin is Highly Selective
DOPC
DLPC
SDS
LPS
OO
OO
O
HO
OP
O
O-
-O
NH
O
OO
O
OHO
OO
HO
NHO
HO
O P
O
O-
O-
1mg/ml
1µg/ml
1ng/ml
Con
cent
ratio
n of
Ads
orba
te R
equi
red
to
Indu
ce R
adia
l Con
figur
atio
n
Lipid A DLPC DOPC SDS
1pg/ml
Science, 332, p1297-1300, 2011. 34
Response of LC Droplets Correlates with Spontaneous Curvature of Lipids
DLPC DPPC Lipid ASDS
co > 0forms micelles
co > 0form vesicles
co < 0forms inverted micelle structure
Seydel et al., Chemical Immunology. 2000.Strandberg et al., Biochimica et Biophysica Acta. 2012.Zimmerberg et al., Nature Reviews Molecular Cell Biology. 2005.
1mg/ml
1µg/ml
1ng/ml
Con
cent
ratio
n of
Ads
orba
te R
equi
red
to
Indu
ce R
adia
l Con
figur
atio
n
Lipid A DLPC DOPC SDS
1pg/ml35
Self-Assembly of Endotoxin with LC Droplets is Driven by Negative Spontaneous
Curvature?
36
Hypothesis: Self-Assembly of Lipid A at Sites of LC Defects
0 250 500 750 1000 1250 1500 1750 20000
20
40
60
80
100
Lase
r Lig
ht P
ower
Per
cent
age
Time [sec]
0 250 500 750 1000 1250 1500 1750 200020
25
30
35
40
Time [sec]
Fluo
resc
ent I
nten
sity
[a.u
.] Time Break 117 sec
Time Break 143 sec
Time Break 423 sec
Time Break 47 sec
A
5µm
Fluorescence microscopy shows evidence of endotoxin localization near the sites of defects
Measurements were performed with 20ug/mL BODIPY FL‐endotoxin concentration. 37
8μm: Increasing Surfactant Concentration
Surfactant-Induced Anchoring Transition
0.0mM 0.2mM 0.4mM 0.6mM 0.8mM 1.0mM
Water
5CB
Polymer coating
Surfactant
O S OO
O
Na
SDS
(Bright-field micrographs) (Equilibrium states)
38
Kinetic Pathway with 50ug/ml of Endotoxin (“Surface-Driven”)
Similar kinetic path for a surface-driven orderingtransition proposed by G. E. Volovik, O. D. Lavrentovich, Soviet Physics JETP 58, 1159-1165 (1983). 39
Kinetic Pathway with 10pg/ml of Endotoxin
O. O. Prischepa, A. V. Shabanov, V. Y. Zyryanov, JETP Letters 79, 257-261 (2004).
No transition state with a disclination loop
40
Flow Cytometry of LC Droplets
Top View
Sample Inlet
Flow Channel
Q = 1 mL/min
Zoomed Side View
D = 200 µm
Light source
Side Scatter (SSC)
Detector
Forward
Scatter (FSC) Detector
LC Droplet
Hydrodynamic Flow FocusingDetection of Forward- and Side-Scattered Light
Flow Cytometry Read-Out
41
Conclusions
LC droplets offer basis of a promising system for optical sensing of biologicalmolecules via ordering transitions in LC
- ordering transitions induced by 1-100pg/ml concentrations of endotoxinhave been observed
- selectively of response to endotoxin is high
- ordering transition is seen for a droplet size-range and surface anchoringenergies where elastic and surface contributions to free energy are in balance
- endotoxin-triggered ordering transition is not mediated by surface anchoring energy but rather is consistent with associationof endotoxin with defects
- specificity to lipid A over other lipids hints at formation of organizedassemblies of lipids in defects (center of LC droplet)
42
Some Unresolved Issues
What is the role of K24 in the size-dependent configuration of the droplets?
Are ionic effects size-dependent (Debye screening length is approaching radius of LC droplet)?
Do amphiphiles self-assemble in defects of LCs? If so, what dictatesthe dependence on molecular structure of the amphiphile?
endotoxin2
2411rbp FWR2RK2RK3F
43
Acknowledgements
Jugal GuptaI-Hsin LinVicki MeliJeff BrakeSarah TerenGaurav PranamiSelin Aytar
Frank Caruso (University of Melbourne)Christopher Murphy (Vet School, UW-Madison)Paul Bertics (Biomolecular Chemistry)Juan de Pablo (Chemical and Biological Engineering)Franz Himpsel (Physics)David Lynn (Chemical and Biological Engineering)Hongrui Jiang (Electrical Engineering)
NSF (MRSEC; Materials World Network)Army Research Office (Chemistry x2)NIH (National Cancer Institute x2)DoE (BES)
Jacob HunterMike KinsingerGary KoenigZhongqiang Yang Elise HuangJohn MullerRishabh Jain
Aaron LoweYiqun BaiBecky CarltonSantanu PalAnkit AgarwalDaniel MillerMatthew Wang
$$$
44
Lipid A DLPC DPPC SDS
45
46
DOPCco = -1/20
H
NH3+
O
O-
H
Na+P O-
OO
O
OOO
O
DOPSco = 1/14.4
DOPEco = -1/3
O‐Lyso PCco = 1/3.8
OHH
P O-
OO
N+
O
OO
L‐Lyso PCco = 1/5.8
O‐Lyso PEco = <1/40
47
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