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