Optical "blinking" Triggered by Collisions of Single Supramolecular Assemblies of Amphiphilic Molecules with Interfaces of Liquid Crystals

Article abstract of DOI:10.1021/jacs.9b13360

We report that incubation of aqueous dispersions of supramolecular assemblies formed by synthetic alkyl triazole-based amphiphiles against interfaces of thermotropic liquid crystals (LCs; 4-cyano-4′-pentylbiphenyl) triggers spatially localized (micrometer

Full text of DOI:10.1021/jacs.9b13360

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Article
Optical “Blinking” Triggered by Collisions of Single Supramolecular
Assemblies of Amphiphilic Molecules with Interfaces of Liquid Crystals
Michael Tsuei, Manisha Shivrayan, Young-Ki Kim, Sankaran Thayumanavan, and Nicholas L. Abbott
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.9b13360 • Publication Date (Web): 21 Feb 2020
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Optical “Blinking” Triggered by Collisions of Single Supramolecular
Assemblies of Amphiphilic Molecules with Interfaces of Liquid Crystals
Michael Tsuei1†, Manisha Shivrayan2†, Young-Ki Kim1, S. Thayumanavan2*, and Nicholas L. Abbott1*
1 Smith School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY 14853, USA
2 Department of Chemistry, University of Massachusetts Amherst, Amherst, MA 01003, USA
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ABSTRACT: We report that incubation of aqueous dispersions of supramolecular assemblies formed by synthetic alkyl
triazole-based amphiphiles against interfaces of thermotropic liquid crystals (LCs; 4-cyano-4’-pentylbiphenyl) triggers
spatially localized (micrometer-scale) and transient (sub-second) flashes of light to be transmitted through the LC. Analysis
of the spatio-temporal response of the LC supports our proposal that each optical “blinking” event results from collision of
a single supramolecular assembly with the LC interface. Particle tracking at the LC interface confirmed that collision and
subsequent spreading of amphiphiles at the interface generates a surface pressure-driven interfacial flow (Marangoni flow)
that causes transient reorientation of LC and generation of a bright optical flash between crossed polarizers. We also found
that dispersions of phospholipid vesicles cause “blinks”. When using vesicles formed from 1,2-dilauroyl-sn-glycero-3-
phosphocholine (DLPC), we measured the frequency of blinking to decrease proportionally with the number density of
vesicles in the aqueous phase, consistent with single vesicle events, with the lifetime of each blink dependent on vesicle
size (800 ± 80 nm to 150 ± 30 nm). For 100 μM of DLPC, we measured vesicles with a diameter of 940 ± 290 nm to generate
47 ± 9 blinks min-1 mm-2, revealing that the fraction of vesicle collisions resulting in fusion with the LC interface is ~10-3.
Overall, the results in this paper unmask new non-equilibrium behaviors of amphiphiles at LC interfaces, and provide fresh
approaches for exploring the dynamic interactions of supramolecular assemblies of amphiphiles with fluid interfaces at the
single-event level.
such as optical birefringence and elasticity, and enables
LCs to amplify and transduce molecular-level events at
INTRODUCTION
Dynamic interactions of amphiphilic assemblies,
including vesicles, at biological interfaces underlie
processes as diverse as transmission of synaptic signals,1
blebbing of microvesicles from cells for intercellular
communication2-3 and endocytosis.4 Key aspects of these
processes have been recapitulated using synthetic
amphiphiles, such as the fusion of vesicles into
monolayers5 or bilayers at surfaces of solids.6-8 In this
paper, we report the observation that interfaces of water-
immiscible liquid crystals (LCs), when incubated against
aqueous dispersions of synthetic and biological
amphiphiles and viewed using crossed-polarizers
(transmission mode), exhibit transient bright domains.
We establish that the “blinking” events arise from non-
equilibrium states of the LC caused by dynamic
interactions of the LC with single supramolecular
assemblies formed by the amphiphiles (including vesicles).
interfaces into easily visualized and quantifiable changes in
optical signals.11-14 These characteristics have made LCs
exemplary materials for reporting a range of molecular
stimuli and events, including photo isomerizations,15-20
enzymatic reactions,21 formation of DNA complexes,22
directed assembly of oligopeptides23-24 and interfacial
interactions of ions.25-26
Of relevance to the study reported herein, a number of
past studies of LCs have focused on understanding the
equilibrium orientations of LCs at aqueous interfaces
decorated by amphiphiles such as phospholipids (1,2-
dilauroyl-sn-glycerol-3-phosphocholine (DLPC), Scheme
24,
27-28
1b),12,
synthetic
surfactants
(e.g.
or
dodecyltrimethylammonium bromide (DTAB))11-12,
29-31
peptide-polymer amphiphiles.21,
Adsorption of these
32
amphiphiles from aqueous dispersions results in
reorientation of the LCs in a manner that is dependent on
the structure and the concentration of the amphiphiles.13,
29, 33-35 Various factors have been identified to influence the
orientations of LCs at amphiphilic-decorated aqueous
interfaces, including interdigitation of the aliphatic tails of
the amphiphiles into the LCs,11-15, 27-28, 32-33 disorder induced
The LCs used in our study are formed from 4-cyano-4’-
pentylbiphenyl (5CB) (Scheme 1a). As a single component
at room temperature, 5CB forms a liquid within which the
5CB molecules exhibit long-range orientational order (a
so-called nematic LC phase).9-10 The long-range ordering of
molecules within LCs gives rise to anisotropic properties
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in the LCs due to frustrated packing of mesogens at the
LC Ordering Transitions Induced by O1. In our initial
interface32, 36 and electrical double layer interactions.26, 37
experiments, films of LC were hosted within 20 μm-thick
metal grids supported on dimethyloctadecyl[3-
(trimethoxysilyl)propyl]ammonium chloride (DMOAP)-
treated glass microscope slides (which orient nematic 5CB
along the surface normal (Fig. 1a)) and then submerged
into aqueous 10 mM phosphate-buffered saline (PBS)
containing 25 µM O1 (Fig. 1a-f). When viewed through
crossed polarizers in transmission mode, the LC films
initially exhibited a bright optical appearance with dark
brushes (Schlieren texture), indicative of tangential LC
alignment at the aqueous interface (planar anchoring; Fig.
1a,b). Subsequently, the LC displayed a time-dependent
progression of interference colors with retardance values
of 1400 ± 70 nm (0 min, Fig. 1b), 650 ± 210 nm (20 min, Fig.
1c), 300 ± 140 nm (40 min, Fig. 1d) and 0 nm (60 min, Fig.
1e). The progression of retardance values is consistent with
a continuous tilting of the LC at the aqueous interface,
from planar to perpendicular (homeotropic) anchoring
(Fig. 1b-e) with increasing time. The end point of the
continuous transition is a dark optical state, corresponding
to homeotropic anchoring of the LC (Fig. 1e,f). Overall,
these observations are consistent with spontaneous
adsorption of O1 onto the LC interface, triggering the
reorientation of the LC. We also used LC films supported
on polyimide-coated glass substrates that caused planar
anchoring of LC. When these LC films were immersed in
25 μM O1 in PBS, the LCs also underwent orientational
transitions, indicating that adsorption of O1 causes a
change in the lowest free energy orientation (so-called easy
axis) of the LC at the aqueous-LC interface (see Section
S1).12-13, 15, 32, 44
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In this paper, we move beyond prior studies of the
equilibrium properties of amphiphile-decorated LC
interfaces to explore non-equilibrium states induced by
dynamic interactions of amphiphilic assemblies. As noted
above, this investigation was motivated by the observation
of transient flashes of light transmitted through LC
interfaces contacting aqueous solutions of alkyl-triazole
oligomers (O1, Scheme 1c) that we were studying to
develop structure-property relationships for branched
amphiphilic oligomers at LC interfaces. Scheme 1c shows
the structure of the oligomer, which includes three design
motifs; alkyl chains, oligoethylene glycol and a triazole
ring. The alkyl chains facilitate anchoring of O1 on the LC
interface and the oligoethylene glycol moieties were used
as hydrophilic arms to promote solubility of O1 in aqueous
solutions. The triazole ring, which was incorporated as part
of the synthetic strategy, also increases the rigidity of O1
and introduces asymmetry that impacts the organization
of the amphiphiles at LC interfaces. Ongoing studies are
addressing the effects of oligomer rigidity on LC ordering
and will be reported elsewhere.
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Subsequent to our initial observations with O1, we
found that optical blinking occurred with other
amphiphilic systems (e.g. phospholipids). Our results
reveal that the non-equilibrium states of the LC are
generated by collisions of single amphiphilic assemblies
with the LC interface, leading to transient interfacial flows
that reorient the LCs.38-39 The results presented in this
paper hint at broad utility of this phenomenon for probing
the presence and properties of amphiphilic assemblies.
Specifically, the approach enables characterization of
interfacial events triggered by individual assemblies rather
than large populations of assemblies,40-41 does not require
use of fluorescent labels (or other molecular probes)40 and
exploits unique attributes of LC interfaces, such as the high
mobility necessary to observe Marangoni phenomena (in
contrast to solid-supported amphiphile assemblies).42-43
Figure 1: a,f) Schematic illustrations (side view) and b,e)
corresponding micrographs (top view, crossed polarizers) of
aqueous-LC interface exhibiting a,b) planar and e,f)
homeotropic anchoring of LCs with respect to the aqueous-LC
interface upon adsorption of O1. b-e) Sequential micrographs
(top view, crossed polarizers) of the LC interface after b) 0, c)
20, d) 40 and e) 60 min of contact with the aqueous O1
solution (25 μM). Scale bar is 200 μm.
Scheme 1: Molecular structures of a) 4-cyano-4’-
pentylbiphenyl
(5CB),
b)
1,2-dilauroyl-sn-glycerol-3-
phosphocholine (DLPC), c) amphiphilic branched oligomer
(O1) containing alkyl-triazole (n = 13) and pentaethylene
glycol domains and d) PEGylated lipid (DOPE-PEG).
We compared the duration of the dynamic anchoring
transition (60 ± 30 min at 25 uM of O1) to the time required
for diffusion of O1 assemblies from the bulk aqueous phase
RESULTS AND DISCUSSION
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to the LC interface (50 min; using an experimentally Fourth, subsequent to our observations of blinking in
measured diffusivity of 3.7 x 10-13 m2/s; see Section S2) and
determined that the dynamics of the anchoring transition
are controlled by diffusion of O1 to the LC interface.
Surprisingly, however, after the LC had transitioned to the
homeotropic orientation, we observed localized
(micrometer-scale) and transient (sub-second) flashes of
light at the LC interface when viewed between crossed
polarizers (transmission mode) (Fig. 1e, red circles, Video
S1). Below we refer to the transient flashes of light as
“blinking”. For an aqueous phase containing 25 μM of O1,
directly following the observation of homeotropic
anchoring, we measured 45 ± 4 blinks min-1 mm-2 of LC
interface.
homeotropically oriented samples, closer examination of
LC samples that had not yet transitioned to the
homeotropic orientation revealed evidence of blinks with
footprints and durations similar to those observed in
homeotropically aligned samples (see Section S5 for
images and additional description).
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Characterization of Blinking Events At LC
Interfaces. Additional optical microscopy performed at
high magnification led to four key observations regarding
the blinking events. First, when viewed between crossed
polarizers, blinking events generated transient Maltese
crosses (Fig. 2a-d). This observation suggested that the
blink was generated by a radially symmetric rotation of the
LC orientation away from the surface normal (Fig. 2e
inset). This interpretation was supported by measurement
of the optical retardance of the LC during the blink and
calculation of the angular rotation of the orientation of the
LC away from the surface normal (tilt angle of the LC).10, 45
By assuming the LC tilt angle within the film to be
dominated by the elastic energy of the LC, we estimate the
average tilt angle of the LC (see Section S3) during the
blinking event in Fig. 2b to be θavg ~ 23 ± 4° (see below and
Section S9 for a more detailed model of the LC tilt).
Figure 2: a-d) Sequential micrographs (crossed polarizers, top
view) of a single blinking event at the LC interface contacting
an aqueous O1 solution (CO1= 25 μM). e) Corresponding
transmittance profiles (red, green, blue) representing the
intensity of light transmitted through the grid squares during
three individual blinking events. Lines are drawn to guide the
eye. Inset illustrates the director profile (side view) across
the LC film during a blinking event. f) histogram of blinking
lifetimes (n=100, bin size: 0.1 s). g) histogram of blinking
footprint (n=100, bin size: 10 μm). h-k) Sequential
micrographs (bright field) of a single blinking event at the LC
interface in contact with aqueous O1 (CO1= 25 μM).
Second, we quantified the temporal and spatial
characteristics of individual blinks by analyzing the
intensity of light transmitted through a single 285 μm x 285
μm area of the LC interface. We found that the lifetime of
a blink was typically subsecond but that it varied from one
event to the next (Fig. 2f). The mean size of the footprint
of the blink was ~100 μm, but it varied substantially
between events (from 20 μm to 250 μm, Fig. 2g). While
individual blinking events were transient, we observed the
blinking phenomena to persist at LC interfaces in contact
with dispersions of O1 for hours.
Correlation
Between
LC
Blinking
and
Concentration of O1 in the Aqueous Phase. We
determined that blinking ceased when the aqueous
dispersion of O1 in contact with the LC was replaced by PBS
free of O1 (see Section S6). This result indicates that
blinking requires the presence of O1 in the bulk aqueous
phase. Furthermore, after 48 hrs of contact with PBS free
of O1, the LC remained in the homeotropic orientation,
indicating slow desorption of O1 from the LC interface at
surface concentrations of O1 that sustain homeotropic
anchoring (see below for evidence of desorption of O1).
Third, when using bright field microscopy (no
polarizers), we observed that a small fraction of blinks were
preceded by the presence of micrometer-sized particles
near the site of the blink. The particles were observed to
disappear from the field of view during the blink (see
Section S4). The majority of blinks, however, occurred in
the absence of visible particles (Fig. 2h-k). These
observations revealed that micrometer-sized particles can
lead to, but were not required for, the generation of the
blinks. The particles were not observed between crossed
polarizers, indicating that they are not LC droplets. When
combined, these observations led us to the hypothesis that
the particles were O1 aggregates and that blinking was a
consequence of the interaction of O1 aggregates (the
majority of which are too small to be imaged) with the LC
interface. Below, we explore this proposal.
We varied the concentration (0-25 µM) of O1 incubated
against the LC and documented the blinking frequencies
after 1 hour of incubation (Fig. 3a). Below a threshold
concentration of 1 μM O1, no blinking was observed. Above
the threshold, we measured the blinking frequency to
increase in an approximately sigmoidal manner as a
function of O1 concentration (Fig. 3a). We hypothesized
that the concentration-dependent frequency of blinking
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shown in Fig. 3a was related to the concentration of O1 properties of O1 assemblies and/or LC interfaces are not
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assemblies in the aqueous dispersions and thus frequency
of collisions of O1 assemblies with the LC interface. By
using dynamic light scattering, however, we found that the
average sizes of the assemblies formed by O1 increased
from ~100 nm to ~1000 nm (Fig. 3b, see Section S7) with
concentration of O1 in the bulk aqueous solution. This
result led us to conclude that the concentration-
dependence of the blinking frequency reported in Fig. 3a
likely reflects changes in both the number and size of O1
assemblies in the PBS.
changed by the presence of such a high concentration of
urea. We note also that additional measurements reported
below reveal that amphiphilic assemblies smaller than 250
nm can generate blinks.
Motion of the LC Interface During a Blink. To
further characterize the dynamic state of the LC interface
during a blink, we tracked the time-dependent locations of
tracer particles deposited onto the LC interface. In this
experiment, glass microspheres (3 µm in diameter) were
dispersed onto the surface of a film of LC in contact with
25 μM O1 in PBS. Fig. 4a-c shows a series of micrographs
(polarizers crossed at 30° to enable imaging of the tracer
particles) obtained during a blinking event. By tracking the
displacements of the glass microspheres, we established
that blinking was accompanied by motion of the LC at the
interface49 (Fig. 4d). The radially directed interfacial flow
originated from the center of the blinking event and
displaced microspheres up to 100 μm away from the center
of the blink. This observation is consistent with a flow
driven by a surface pressure gradient that decreases
radially from the center of the blinking event.
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Figure 3: a) Blinking frequency at different concentrations of
O1 in the aqueous solution. Inset shows a magnified plot at
low O1 concentrations. b) O1 aggregate number average
diameters with respect to O1 concentration in the aqueous
solution. Inset shows autocorrelation functions using light
scattering angles of 75°, 90°, 110° and 140° for aqueous
solutions containing 25 μM O1 incubated for 4 hours at 25 °C.
a,b) Data points show mean values and the error bars
represent 1 standard deviation (n=3).
Figure 4: Micrographs of LC films between polarizers crossed
at 30° illustrating a) a micrometer-sized O1 aggregate (red
circle) above a LC thin film surrounded by glass microspheres
(3 μm in diameter), followed by b) a single blinking event and
the c) disappearance of the O1 aggregate. The aqueous
solution contains 25 µM O1. d) trajectories and e) velocity
profile of microspheres as a function of distances stemming
radially from the center of a blinking event (n=130, bin size: 10
μm). f-h) Schematic illustrations depicting the mechanism of
blinking, showing f) collision of O1 aggregate with LC interface
that leads to g) Marangoni flow at the aqueous-LC interface
followed by h) rapid spreading of O1 at the LC interface that
results in cessation of flow.
As an additional test of the connection between
blinking and O1 aggregate size, we added urea to change
the size and properties of O1 aggregates46-48 (see Section
S8). Dynamic light scattering measurements conducted
using 25 µM O1 in 8 M urea revealed that urea caused the
size of O1 assemblies to decrease sixfold to 250 nm ± 180
nm. Upon submerging a LC film into an aqueous solution
containing 8 M urea and 25 µM O1, blinking ceased, but the
anchoring of the LC changed from planar to homeotropic,
consistent with adsorption of O1 onto the LC interface. A
control experiment performed with 8 M urea in the
absence of O1 did not trigger an anchoring transition or
blinking of the LC. These results support our hypothesis
that the size of O1 assemblies influence the blinking
phenomenon, although we cannot rule out that dynamic
We mapped the interfacial flow by analyzing the motion
of 130 microspheres (see Materials & Methods). The
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microsphere velocities were then plotted against distance Blinking Caused by Vesicles of DLPC. Next we
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from the center of each blinking event (Fig. 4e). As shown
in Fig. 4e, the microsphere velocity decreased with
increasing distance from the center of the blink.
Additionally, we measured the maximum particle
velocities to coincide with the peak optical signal from the
blink, with particle velocities decreasing with subsequent
diminishing optical signal. After the optical signal
associated with the blink ceased along with particle
motion, a local depletion of tracer particle density was
observed in the region of the blink.
investigated whether LC blinking is observed with
amphiphiles other than O1. We focused on vesicles formed
from the phospholipid DLPC because, unlike O1, for which
size and concentration of O1 assemblies are coupled (Fig.
3), the number of lipid vesicles in a dispersion can be
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controlled by dilution without change of size.53,
Additionally, the size of DLPC vesicles can be controlled by
extrusion through filters containing pores.53,55 We also
viewed DLPC as a primitive model of biological vesicles,
and thus an observation of blinking with DLPC would hint
at potential relevance of our observations to studies of
vesicle-based events in biological systems.
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We used the above-described measurements of the
interfacial velocities of the LC to provide an additional
estimate of the tilt angle of the LC induced by the flow
accompanying a blink. From Fig. 4e, we calculated the LC
surface velocity at a distance of 10 µm from the center of
the blinking event (x=10 µm) to be ~100 µm/s. In Section
S9, we present a detailed model that evaluates the LC tilt
across the film during a blink from a balance of viscous and
elastic stresses. This model predicts the average tilt of the
LC to be θavg = 38 ± 4°. A similar estimate can be obtained
by equating the viscous stress to the elastic torque at the
LC-aqueous interface, yielding
In our initial experiments with DLPC, films of LC were
contacted with aqueous 10 mM PBS containing 100 µM
DLPC (as vesicles with diameters of 940 ± 290 nm; see
below for additional characterization). The LCs
±
transitioned to homeotropic orientations after 7
2 min.
These samples, when viewed through crossed polarizers in
transmission mode, revealed the presence of blinks with
characteristics qualitatively similar to O1. Blinking with the
DLPC system, however, ceased several minutes after the
samples had assumed a homeotropic orientation whereas
O1-induced blinking continued for hours. Similar to O1
samples, blinking was observed in DLPC systems prior to
(see Section S10) and immediately following the LCs
assuming a homeotropic orientation (see Section S11).
Quantitative differences in blinking time scales are
discussed below.
2
2
∂
∂
∂
(
∂
αLC ( vx/ y) = KLC
θ/ y )
(1)
훼
LC
where
is an effective LC viscosity that is a function of
퐾
~
LC tilt (see Section S9 for a more complete analysis),
LC
10 ―11
N
푣푥
is the elastic constant of the LC,
is the LC
surface velocity, y ~ 20 μm is the LC layer thickness, and θ
is the tilt angle of the LC at the aqueous interface.9-10, 44, 49
This interfacial stress balance leads to θavg ~ 35°. We note
that incorporation of internal viscous stresses into our
estimate of the LC orientation leads to a larger surface tilt
angle than that estimated assuming the LC orientation is
dominated by elastic stresses only (see above).
The observations described above, when combined,
lead us to propose that each blinking event involves three
essential steps. First, an O1 aggregate in the aqueous phase
collides with the aqueous-LC interface (Fig. 4a,f). Second,
fusion and adsorption of O1 molecules from the aggregate
with the LC interface generates a lateral O1 surface
concentration gradient. This O1 surface concentration
gradient generates a surface pressure gradient that induces
a Marangoni flow away from the collision site (Fig. 4b,g).
The shear stress generated by the Marangoni flow is
sufficient in magnitude to overcome the elastic torque of
the LC, leading the LC to reorient away from the
homeotropic orientation.38,50-52 The change in LC
orientation (and thus optical axis) causes the optical
appearance of the LC to change from dark to bright49 (Fig.
4b). Third, due to rapid spreading of O1 across the LC
interface, the surface tension gradient dissipates with time
and the Marangoni flow ceases. In the absence of flow, the
elastic torque of the LC causes the LC to return to the
initial perpendicular orientation. The return of the
homeotropic LC orientation causes a bright to dark
transition in optical appearance (Fig. 4c,h).
Figure 5: a) Blinking frequency as a function of DLPC
concentration in the aqueous solution showing linear
relationship. Inset shows magnified plot at low concentrations
of DLPC. (b,c) histograms representing (b) number average
vesicle diameters before and after extrusion through a 220 nm
filter (n=100, bin size: 50 nm) and (c) blinking footprints
(n=100, bin size: 5 μm). Green inset shows large blinking
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footprint before vesicle extrusion. Red inset shows small
confidence interval, see Section S13). Overall, this result
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blinking footprint after vesicle extrusion. (c) Scale bar is 100
μm. (a) Data points show mean values and the error bars
represent 1 standard deviation (n=3).
and others presented below establish that a decrease in
vesicle size correlates with a decrease in blinking footprint
size. This result hints at the utility of blinking as a means
of probing heterogeneity in a population of vesicles (or
other amphiphilic assemblies) at the single vesicle level.
To understand the relationship between the number of
phospholipid vesicles in an aqueous dispersion and
blinking frequency, we diluted aqueous dispersions of
vesicles (100 μM DLPC stock solution with PBS) to
concentrations as low as 0.05 μM DLPC (Fig. 5a). The
blinking frequencies were found to be first order in the
concentration of DLPC in the dispersion (Fig. 5a). The
proportional relationship between blinking frequency and
the number of vesicles in the aqueous phase supports our
hypothesis that blinking arises from single vesicle collision
events. Second, we found that LC blinking occurs at
concentrations above but not below 100 nM. Because the
critical aggregation concentration (CAC) of DLPC is 100
nM (Fig 5a inset), this result provides further support for
our conclusion that blinking is caused by collisions of
DLPC vesicles with the LC interface.
Guided by the result above that blinking footprint size
correlates with vesicle size, we explored changes in
blinking that accompany vesicle destruction in mixed
lipid/surfactant systems. Here, the aqueous solution
comprised mixtures of DTAB and DLPC, resulting in the
formation of mixed DTAB/DLPC assemblies. Dynamic
light scattering measurements of DLPC and DTAB
mixtures confirmed a decrease in assembly size with
increasing DTAB concentrations (see Section S14).
Concurrent with the change in vesicle size, the blinking
footprint also decreased. However, we observed a complex
relationship between blinking frequency and DTAB
concentration, which we do not yet fully understand but
suspect reflects competitive dynamics of DTAB and DLPC
adsorption and spreading at the LC interface.
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We considered it likely that only a small fraction of
collisions of vesicles of DLPC with the LC interface
generated fusion events and thus blinks. To test this
prediction, we performed dynamic light scattering
measurements of dispersions of vesicles in aqueous 100 μM
DLPC. From the measured average diameters of the
vesicles (940 ± 290 nm) and corresponding diffusivities, we
estimated that ~106 vesicles collide with a 100 mm2 area of
the LC interface every minute. By using the measured
blinking frequency of 47 ± 9 blinks min-1 mm-2, we
calculated the probability that a collision between a DLPC
vesicle and the LC interface results in fusion and
subsequently blinking to be ~10-3 (see Section S12).
Comparison of Blinking with O1 and DLPC. The
results above establish that blinking is observed with both
O1 assemblies and vesicles of DLPC. We note, however,
that key differences were observed between these two
systems. In particular, as shown in Fig. 6, O1-induced
blinking at LC interfaces is sustained for hours, whereas
DLPC-triggered blinking persists for ~10-15 mins after
contact of the LC with the dispersion of amphiphiles. The
duration over which LC blinking with DLPC is observed is
consistent with the time scale required for the LC interface
to saturate with the phospholipid (~15 mins).60 The long
duration over which O1 is observed to generate blinks,
however, suggests that an additional mechanism must be
active to prevent saturation of the LC interface with O1
after a few minutes. Additionally, in contrast to DLPC,
inspection of Fig. 6 reveals that O1 exhibits a local
minimum in blinking frequency with incubation time.
To further explore our proposal that only a small
fraction of vesicles colliding with the LC interface
subsequently fuse and spread along the interface to cause
a blink, we performed measurements using vesicles
prepared from lipids conjugated to polyethylene glycol56-58
(DOPE-PEG, Scheme 1d). Consistent with past
observations that repulsive steric interactions of PEGylated
lipids inhibit spontaneous fusion of liposomes58, we did not
observe blinking of LC interfaces when using the
PEGylated lipid.
Next, we investigated the influence of DLPC vesicle size
on LC blinking. We extruded vesicles through
a
polyvinylidene fluoride (PVDF) filter with a pore diameter
of 220 nm and compared the blinking generated by the
vesicles before and after extrusion (1 minute after contact
between the aqueous DLPC and LC film). To minimize
lipid loss during extrusion, we pretreated the filter with 1
mL of aqueous 25 μM DLPC59 and the dispersion of DLPC
vesicles was passed once through the PVDF filter. We
measured the distributions of vesicle sizes (Fig. 5b) and LC
optical footprint sizes, both before and after extrusion of
the vesicles (Fig. 5c). Prior to vesicle extrusion, the vesicle
and blinking footprint diameters were 800 ± 80 nm and 70
± 5 μm, respectively, whereas following extrusion, they
decreased to 150 ± 30 nm and 20 ± 1 μm, respectively (95%
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Figure 6: Blinking frequency as a function of time for LC thin
The observations reported in this paper generate a
1
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4
5
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7
8
films in contact with an aqueous solution containing 100 µM
DLPC (black squares) and 25 µM O1 (red circles). Data points
show mean values and the error bars represent 1 standard
deviation (n=3).
number of interesting questions. For example, we infer
that the fraction of vesicles of DLPC colliding with the LC
interface that generate a blink to be approximately 10-3.
Additional studies are needed to fully understand the
factors that control this sticking probability, although we
predict that it will depend strongly on the properties of the
amphiphiles within their assemblies61-62 (e.g., LC or gel-like
state of phospholipids within bilayers). We also do not yet
understand how the properties of the LC impact the
collision, interactions and spreading of amphiphilic
assemblies (e.g., LC dynamic rheological properties and
interfacial properties such as charge). We envisage that
ionic strength,63-65 pH66-69 and viscosity70-74 of the aqueous
phase will play a key role in regulating the interactions of
vesicles with the LC interface.32 Additionally, Marangoni
stresses at the aqueous-LC interface during a blinking
event will generate localized flow in the aqueous phase,
which in turn may facilitate the transfer of amphiphilic
assemblies from the aqueous bulk onto the LC interface.
Finally, although we establish that the sizes of DLPC
vesicles impact the characteristic features of the blinks
(footprint), the lower limit to vesicle size that generates a
measurable blink is unknown.
We hypothesized that blinking by O1 was sustained for
hours by desorption of O1 from the LC interface into the
LC bulk, thus enabling further collisions and spreading of
O1 aggregates from the aqueous solution onto the
interface. Several observations support this hypothesis.
First, we measured the temperature at which the 5CB
transitioned from a nematic to an isotropic phase (TNI) to
change following incubation with O1 but not DLPC,
consistent with desorption of O1 but not DLPC into the LC
bulk (see Section S15). Second, we determined that pre-
loading O1 into 5CB suppressed blinking upon incubation
of the 5CB against an aqueous dispersion of O1. This result
is consistent with the presence of O1 in the LC bulk
decreasing the rate of desorption of O1 from the interface
into the LC bulk, thus slowing the rate of blinking (see
Section S15).
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Additional support for the hypothesized role of O1
desorption into the LC bulk was obtained by incubating a
LC film against a 25 μM O1 aqueous solution for 24 hours.
Between crossed polarizers, we observed a ~80% decrease
in blinking frequency after 24 hrs. We note, however, that
while we conclude that sustained blinking is enabled by O1
desorption (see Section S15), we do not yet understand why
O1 exhibits a local minimum in blinking frequency after 5
mins (Fig. 6). Additional studies are needed to elucidate
the complex kinetics associated with O1 blinking. Our
results, however, clearly establish that blinking is a
consequence of a delicate interplay of O1 adsorption from
the aqueous bulk onto the LC interface, and desorption of
O1 from the interface into the LC bulk.
The results in this paper also suggest a range of future
directions of inquiry, such as determining if biologically-
derived vesicles (e.g., exosomes) induce blinking of LCs.
This direction of investigation has the potential to yield
important results as variations in composition of vesicles
shed from eukaryotic cells in biological systems can
75
provide key information regarding cell health.3,
Such
studies may offer the basis of novel analytical methods for
detecting and characterizing microvesicles. It is also
possible that the system reported in this paper might be
used to screen for peptides, other additives (e.g., ions) or
molecular recognition groups that facilitate fusion of
vesicles at interfaces. These and other ideas building from
the study reported in this paper will be described in future
publications.
CONCLUSIONS
In conclusion, this paper reports that dynamic, non-
equilibrium interfacial states of LCs can be triggered by
interactions with individual supramolecular assemblies of
amphiphilic molecules with the LC interface. We have
discovered reversible, spatially localized, sub-second
transitions in LC orientation that arise from collisions of
single amphiphilic assemblies dispersed in aqueous
solution with the LC interface. Collision and subsequent
rapid spreading of amphiphiles along the LC interface give
rise to a surface tension gradient that triggers transient and
localized interfacial (Marangoni) flows. The Marangoni
flows realign the LC, inducing a localized change in the
optical appearance of the LC (a “blink”).38, 51-52 Due to the
rapid spreading of amphiphiles at the LC interface, the
interfacial tension gradient dissipates within seconds,
causing flow to cease and thereby allowing the LC to relax
to its equilibrium orientation. We conclude that some
amphiphiles (O1) in our study desorb from the LC interface
into the LC, thus enabling further adsorption of
amphiphilic assemblies, leading to blinking that is
sustained over hours.
MATERIALS AND METHODS
Materials. The nematic LC 4-cyano-4’-pentylbiphenyl
(5CB) was purchased from HCCH (Jiangsu Hecheng
Display Technology Co., Ltd). Fisher Finest Premium
Grade glass slides were purchased from Fisher Scientific
(Pittsburgh,
aminopropyltrimethoxysilylchloride
PA).
N,N-dimethyl-N-octadecyl-3-
(DMOAP) was
purchased from Acros Organics. Purification of water (18.2
MΩ cm resistivity at 25 °C) was performed using a Milli-Q
water system (Millipore, Bedford, MA, USA). DLPC was
purchased from Avanti (Alabaster, AL, USA).
Synthesis of O1.
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surface area defined by the nine TEM grids (0.731 mm2) and
1
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3
4
plotted as a function of concentration. Unless otherwise
specified, blinking frequencies generated by DLPC vesicles
were determined 1 minute after contact of the LC with
aqueous DLPC dispersions.
5
6
7
8
Dynamic Light Scattering.
1
mL of desired
concentrations of O1 (0-25 µM) in PBS were prepared in
glass tubes from a 100 µM O1 stock solution and allowed to
incubate for 4 hours at 25 °C. Following incubation,
dynamic light scattering measurements (Brookhaven
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Instrument) at 25 C were recorded at four different light
scattering angles (75°, 90°, 110° and 140°) and the
corresponding autocorrelation functions were normalized
using the Siegert relationship.76-77 The normalized
autocorrelation functions for each concentration of O1
were then rescaled76 to determine whether the measured
sizes of O1 aggregates in the aqueous solution were true
hydrodynamic diameters. A similar methodology was used
to determine the sizes of the dispersions of DLPC vesicles.
Scheme 2: Synthetic pathway of O1.
O1, 11 (Scheme 2), was synthesized via a 13-step
synthetic protocol from 3, 5-dihydroxy benzoic acid, 1. We
have previously reported the synthesis of 5 and 8 from 132.
Using
Dimethylaminopropyl)-N′-ethylcarbodiimide
hydrochloride (EDC-HCl) coupling between the alkyne 8
and di-tert-butyl (azanediylbis(ethane-2,1-diyl))
8,
9
was
synthesized
via
N-(3-
Particle Tracking at LC Interfaces. The positions of
130 glass microspheres (3 μm diameter) dispersed at the
aqueous-LC interface were recorded by video (30
ms/frame) during blinking events. The velocities were
calculated from the videos and plotted as a function of
distance of each microsphere from the center of each blink.
dicarbamate with a yield of 93%. Following this reaction,
10 was synthesized at 80% yield via a 2-step protocol by
first deprotecting tert-butyloxycarbonyl and its
subsequent substitution reaction with 5. The final step
involved
a copper-click reaction between 10 and
Safety Statement: No unexpected or unusually high
safety hazards were encountered.
tetradecane azide to synthesize O1 at 48% yield. The
characteristic peak of the propargyl group at 3.08 ppm
disappears and a peak corresponding to triazole proton at
1
ASSOCIATED CONTENT
7.61 ppm arises in H NMR, indicating the successful
alkyne-azide coupling. All compounds in this scheme were
Supporting Information
1
13
characterized via H, C NMR and ESI-MS spectroscopy.
Details of the synthesis of O1 are provided in SI.
Mechanism of LC reorientation (Section S1); Calculation of O1
aggregate diffusion time (Section S2); LC tilt estimate during
a blinking event (Section S3); Blinking using bright field
microscopy (Section S4); O1-induced blinking at planar/tilted
LC interfaces (Section S5); Role of O1 aggregates in blinking
(Section S6); Dynamic light scattering from aq O1 solutions
(Section S7); Disruption of O1 aggregates with urea (Section
S8); Theoretical estimate of LC tilt from a stress balance
(Section S9); DLPC-induced blinking at LC interfaces with
planar/tilted alignment (Section S10); DLPC-induced blinking
at LC interfaces with homeotropic alignment (Section S11);
Estimation of fusion probability as reported by blinking
(Section S12); Vesicle extrusion (Section S13); Disruption of
lipid vesicles by DTAB (Section S14); Desorption of
amphiphiles into the bulk LC (Section S15); Methods for
preparation of O1 aq dispersions, DMOAP-functionalization
of glass slides, and preloading LC with O1; Methods for
synthetic pathways, procedures and characterization of O1;
Movie of blinking (Video S1)
Preparation of Thin LC Films. Thin films of 5CB were
prepared by pipetting 0.5 µL of 5CB into the pores of 75
mesh (thickness 20 m; lateral pore size 285 μm)
transmission electron microscopy (TEM) grids supported
on a DMOAP-functionalized glass substrate (Details of
DMOAP functionalization are provided in SI). Excess 5CB
was then removed to produce a LC film with thickness of
20 µm.
Microscopy Observations. An Olympus BX41
microscope with 4x, 20x and 50x objectives, two rotating
polarizers and a Moticam 10.0 MP camera was used for
optical microscopy.
Determination of Blinking Frequency. Blinking
frequencies were measured by observing the optical
response of nine TEM grid squares (285 μm x 285 μm) filled
with LC. For measurements with O1, we recorded the
numbers of blinks over a 1 minute interval approximately
55 min, 60 min or 65 min after contact of the LC film with
the aqueous O1 dispersion. Individual blinking events were
identified by examining frames of videos. The blinking
events recorded during each of the 1 minute intervals were
averaged and used as one independent measurement.
Three independent measurements were averaged to obtain
each average blinking rate reported in this paper. The
average blinking frequencies were then divided by the LC
AUTHOR INFORMATION
Corresponding Author
*E-mail: thai@chem.umass.edu
*E-mail: nabbott@cornell.edu
Author Contributions
†These authors contributed equally to this work.
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Notes
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3
The authors declare no competing financial interest
4
5
ACKNOWLEDGEMENTS
We acknowledge support of this research from the Army
Research Office through W911NF-15-1-0568 and W911NF-
17-1-0575. The authors also thank Hector Fuster and
Karthik Nayani for their helpful comments on this work.
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