Dynamic Imine Chemistry at Complex Double Emulsion Interfaces

Article abstract of DOI:10.1021/jacs.9b06852

Interfacial chemistry provides an opportunity to control dynamic materials. By harnessing the dynamic covalent nature of imine bonds, emulsions are generated in situ, predictably manipulated, and ultimately destroyed along liquid-liquid and emulsion-solid interfaces through simple perturbation of the imine equilibria. We report the rapid production of surfactants and double emulsions through spontaneous in situ imine formation at the liquid-liquid interface of oil/water. Complex double emulsions with imine surfactants are stable to neutral and basic conditions and display dynamic behavior with acid-catalyzed hydrolysis and imine exchange. We demonstrate the potential of in situ imine surfactant formation to generate complex surfactants with biomolecules (i.e., antibodies) for biosensing applications. Furthermore, imine formation at the emulsion-solid interface offers a triggered payload release mechanism. Our results illustrate how simple, dynamic interfacial imine formation can translate changes in bonding to macroscopic outputs.

Full text of DOI:10.1021/jacs.9b06852

Article
Cite This: J. Am. Chem. Soc. XXXX, XXX, XXX−XXX
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Dynamic Imine Chemistry at Complex Double Emulsion Interfaces
†
‡
,‡
,†
Cassandra A. Zentner, Francesca Anson, S. Thayumanavan,* and Timothy M. Swager*
†
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States
‡Department of Chemistry, University of Massachusetts − Amherst, Amherst, Massachusetts 01003, United States
*
S Supporting Information
ABSTRACT: Interfacial chemistry provides an opportunity to
control dynamic materials. By harnessing the dynamic covalent
nature of imine bonds, emulsions are generated in situ,
predictably manipulated, and ultimately destroyed along
liquid−liquid and emulsion−solid interfaces through simple
perturbation of the imine equilibria. We report the rapid
production of surfactants and double emulsions through
spontaneous in situ imine formation at the liquid−liquid
interface of oil/water. Complex double emulsions with imine
surfactants are stable to neutral and basic conditions and
display dynamic behavior with acid-catalyzed hydrolysis and
imine exchange. We demonstrate the potential of in situ imine
surfactant formation to generate complex surfactants with
biomolecules (i.e., antibodies) for biosensing applications.
Furthermore, imine formation at the emulsion−solid interface offers a triggered payload release mechanism. Our results
illustrate how simple, dynamic interfacial imine formation can translate changes in bonding to macroscopic outputs.
INTRODUCTION
through reactions with the primary amines of lysine residues.
We demonstrate herein that single or double emulsions can be
synthesized in one step via imine surfactants generated in situ
by the interfacial reaction between an amine and an aldehyde,
bypassing postemulsification functionalization (Figure 1a).
The reversibility of imine bond formation has proved useful
in creating materials with dynamic behaviors including in 2D
■
Double emulsions are of significant importance in food
1
2−6
7
applications, drug delivery,
and cell-encapsulation. The
fabrication of complex emulsions (e.g., oil-in-oil-in-water,
water-in-oil-in-water) has primarily focused on microfluidic
techniques and phase-separation emulsification, with particular
interest in developing responsive double emulsion systems.
For example, responsive double emulsions can provide a
detectable optical readout when responding to external stimuli,
8
−12
1
9,20
21
22
assemblies,
receptor design, micelles,
been limited examples of imine surfactant stabilized
biomimicry, systems chemistry, drug/
23
24−26
27,28
and vesicles.
There have
13−15
making them an effective sensing platform technology.
29−32
emulsions.
Additionally, imine bond equilibria are pH
However, responsive emulsions that rely on premade
surfactants limit the ability to use biomolecules as surfactants
at the interface for biosensing applications. Presynthesized
amino-acid-based surfactants are a step toward functional
biocompatible surfactants; however, they lack the specificity
sensitive, allowing for manipulation of the bond and therefore
the structures of double emulsions (Figure 1b). Although there
are reported examples of in situ molecular surfactant
generation for single emulsions, including deprotonation of
3
3−39
16
carboxylic acids and metallosurfactants
single and double emulsions,
and Pickering
necessary for sensing applications. Although postemulsifica-
30,31
we are unaware of any prior
tion functionalization of responsive biological components has
1
7,18
reports of double emulsions fabricated via in situ molecular
surfactant synthesis.
been reported,
these processes introduce complications
and additional preparation steps. An additional issue with
premade molecular surfactants is that they need to have
appreciable solubility in one of the emulsion phases. This
solubility requirement complicates the development of more
complex surfactants. Generating surfactants from precursors
via in situ interfacial synthesis during emulsion formation
prevents additional manufacturing steps and provides access to
surfactants that are ordinarily not used as a result of solubility
restrictions or synthetic complexity.
In this study, we demonstrate the fabrication and
manipulation of complex double emulsions as a consequence
of the in situ formation of imines at emulsion interfaces. Imine
formation greatly simplifies the fabrication of complex double
emulsions, reducing a multistep process to a single-step
method. The dynamic nature of this reaction allows the
manipulation of emulsion interfaces through pH changes and
associated imine exchange. In addition, dynamic imine
Dynamic covalent bonds provide an avenue to impart
materials with responsive behavior. Imine formation, specifi-
cally, provides a versatile interface with biological materials
Received: June 28, 2019
©
XXXX American Chemical Society
A
DOI: 10.1021/jacs.9b06852
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Journal of the American Chemical Society
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Figure 2. Dynamic double emulsions. (a) Morphological changes of
dynamic double emulsions of HC (red) and FC (white) with
adjustments in γ balance. Top and side view images of the droplets at
each morphology are shown. Typical FC solvents used are denser
than the HC solvent; thus phases align with gravity. (b) Example of
commercially available HC and FC-surfactants.
Figure 1. Interfacial imine formation scheme. (a) Non-amphiphilic
amine/protein and aldehyde reagents in separate immiscible phases to
form an imine surfactant through interfacial formation during
emulsification. (b) By lowering the pH of the system or through
imine exchange, the amphiphilic properties of the imine surfactant are
lost causing changes in morphology or emulsion stability. (c) Amine-
functionalized surface and aldehyde reagent in an emulsion to form an
imine-functionalized surface and triggered payload release.
Figure 3. In situ formation of imine surfactants and double emulsions.
The aldehyde, selectively soluble in HC or FC, is dissolved in the
dispersed phases. HC and FC are heated above miscibility and are
added to an amine-enriched continuous phase. Interfacial surfactant
formation via imine condensation promotes single-step emulsification.
formation at the emulsion−solid interface produces reactive
wetting and controlled payload release (Figure 1c).
RESULTS AND DISCUSSION
Phase Separation Emulsification and Dynamic Dou-
ble Emulsions. A facile fabrication of double emulsions is
enabled by thermally induced phase separation. In this
method, the dispersed phase, comprising two immiscible oils
■
water-soluble amine and oil-soluble aldehyde that undergo a
rapid in situ interfacial reaction to produce imine surfactants
(Figure 3). To achieve exclusive interfacial imine formation, we
chose the amine to be soluble only in the water phase, and the
aldehyde to be soluble exclusively in only the HC or the FC oil
phases. Additionally, the amine and aldehyde reagents were
chosen to be non-amphiphilic to prevent inherent surfactant
character from generating emulsions without imine formation.
As a result, stable double emulsions require imine formation
via the in situ method.
10
(
e.g., hydrocarbon (HC) and fluorocarbon (FC) oils), is
heated above its upper critical temperature. The homogeneous
single phase) mixture of HC and FC oils is then emulsified in
(
an aqueous continuous phase containing surfactants. Upon
cooling, the two oils separate to yield double emulsions with
uniform compositions and a structure determined by the
surfactants in the system. In the present studies, HC-
surfactants and FC-surfactants in the continuous phase
stabilize the HC/water (HC/W) and FC/water (FC/W)
interfaces, respectively. Tuning the balance of the interfacial
tension (γ) at the water interfaces of HC and FC (γHC/W and
γFC/W, respectively) using HC and FC-surfactants generates
morphologies that range from encapsulated to Janus droplets
To validate our interfacial reactions, we investigated a proof-
of-concept scheme with monofunctional amine polymer
monomethoxypolyethylene glycol amine (amine 5, MW
1000) that reacts with FC-soluble compounds 1−2 or HC-
soluble carbonyl compounds 3−4 at the FC/W and HC/W
interfaces (Figure 4). The reactions were initially analyzed by
1
H-NMR to elucidate the extent of reaction of 1−4 with 2-(2-
(Figure 2). The perfect Janus morphology (equal hemi-
(2-methoxyethoxy)ethoxy)ethanamine (mPEG NH ) to eval-
3
2
spheres) is achieved when γHC/W = γFC/W. When γHC/W
≫
uate potential substrates (Supporting Information 3.2).
Substrates 1−3 showed quantitative conversion to the imine
within 30 min, while benzaldehyde 4 showed only 5%
γFC/W, a higher surface area at FC/W interface over HC/W is
preferred leading to HC-in-FC-in-water (HC/FC/W) encap-
sulated droplets, and the inverse is necessary to attain FC-in-
HC-in-water morphology (FC/HC/W). Adjusting the mass
ratio and/or the effectiveness of the two surfactants would lead
to changes in the balance between γHC/W and γFC/W and thus
changes in the droplet morphology, thereby creating a dynamic
system.
conversion in THF-d at room temperature. Although these
8
conditions vary from those at the target interfaces, they do
reflect some of the intrinsic reactivity of the components.
We confirmed that the precursor substrates are incapable of
stabilizing double emulsions individually by evaluating if the
reagents 1−5 display inherent surfactant capabilities (Support-
ing Information 3.3 and 3.4). Substrates 1−4 (at or below 200
mM) and amine 5 (at or below 1 mM) did not exhibit
In Situ Surfactant and Phase Separation Emulsifica-
tion. We report a new emulsification method that uses a
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Figure 4. Interfacial imine formation scope and results. (a) Overall interfacial imine formation reaction. (b) FC-surfactant 6 derived from FC-
soluble reagents 1 or 2. Top view images of HC/FC/W morphology emulsions stabilized by FC surfactant 6. (c) Proposed imine formation at HC/
W interface, with HC-soluble reagent 3, but no stable emulsion droplets were obtained. (d) HC-surfactant 8 derived from HC-soluble reagent 4.
Top view images of FC/HC/W morphology emulsions stabilized by HC surfactant 8, with top and side view morphology depictions. Black, FC-
soluble reagent; red, HC-soluble reagents; blue, water-soluble reagents. Scale bar = 100 μm.
surfactant behavior, and double emulsions formed with these
individual components coalesced over the experimental time
frame. We subsequently demonstrated the in situ generation of
surfactant to stabilize emulsion droplets for each reaction in
Figure 4. On the basis of the model studies, 1−4 are expected
to form imines by condensation with 5 at either the FC/W or
the HC/W interfaces. For each emulsification, we reacted 1
mM of amine 5 with 200 mM of HC/FC soluble reagents 1−4
in a neutral pH continuous phase (Supporting Information 3.4
for optimization). As expected with the generation of FC-
surfactant 6 from 1 or 2 with amine 5, the diethylbenzene/
HFE-7500 droplets adopt a HC/FC/W morphology (FC
encapsulating HC in water) and are stable with minimal
coalescence for more than 10 months (Figure 4b). Although
complete conversion was not observed in NMR model studies,
HC-surfactant generated from benzaldehyde 4 and amine 5
Figure 5. Pendant drop analysis of imine formation. 200 mM
produces stable FC/HC/W morphology double emulsions
solutions of substrates 1−4 in HFE-7500 (1, 2), diethylbenzene (3),
(
toluene/HFE-7500 and FC43 (9:1)) as a result of the in situ
and toluene (4) were dispersed in water or 1 mM amine 5. In situ
emulsification was successful only for substrates that display some
surface activity (1, 2, and 4), reducing interfacial tension in water.
generation of surfactant 8 (Figure 4d). It is possible only a
small amount of surfactant 8 is necessary to stabilize the
emulsion; thus, the low conversion exhibited in model system
is not problematic. Unexpectedly, we did not observe double
emulsions using the aliphatic aldehyde 3 and amine 5. We
attribute this result to the instability of imine 7. Synthetic
attempts to premake and isolate imine 7 were unsuccessful as a
result of its susceptibility to hydrolysis (Supporting Informa-
tion 3.5). Imine 8, derived from benzaldehyde 4, however, is
hydrolytically more stable than 7 (Supporting Information 3.6
and 4.3).
tension reduction is insufficient to stabilize double emulsions.
Addition of amine 5 to the continuous phase further reduces γ
to 3.84 and 4.35 mN/m by reaction with aldehyde 1 and
hydrate 2, respectively. Similarly, benzaldehyde 4 assembles at
the toluene/water interface to reduce γ from 38.3 to 20.4 mN/
m, and with the addition of amine 5, γ is lowered to 7.61 mN/
m. Aldehyde 3 showed no surfactant activity, and the addition
of amine 5 did not reduce the overall interfacial tension. We
concluded that the intrinsic interfacial activity of 1, 2, and 4 is
an important component to the success of emulsion formation.
Reagents that lower γ assemble at the oil/W interface to allow
reaction with amine 5, which is constrained to the continuous
phase. Interfacial organization may in fact promote imine
Pendant drop analysis reveals the impact of imine formation
on interfacial tension (Figure 5). The fluorous soluble reagents
1
and 2 are surface active, reducing γ from 43.3 mN/m to 11.1
and 10.40 mN/m, respectively, when 200 mM of the reagents
in HFE-7500 were dispersed in water; however, this interfacial
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Figure 6. Imine exchange studies in emulsion droplets. (a) Toluene/HFE-7500 and FC-43 (9:1) droplets stabilized by imine surfactant 8 and
Zonyl. Morphology changes from FC/HC/W to HC/FC/W with imine exchange and the transformation of imine surfactant 8 to non-amphiphilic
imine 10. (b) Diethylbenzene/HFE-7500 droplets stabilized by imine surfactant 6 and Tween 20. With imine exchange, coalescence and the
appearance of microdroplets was observed. Scale bar = 100 μm.
formation and could reconcile the low conversion observed in
model NMR studies for benzaldehyde 4 with 5. The lower
propensity to assemble at the interface, along with hydrolytic
instability, explains the lack of emulsification with aldehyde 3.
We observed an interesting fluoride elimination in the
reaction of 1 and 2 with mPEG NH , resulting in an α,β-
of the double emulsions when no cosurfactant is present.
Comparative hydrolysis of imine surfactant 6 and 8 was
explored through model imine compounds in THF-d8
(Supporting Information 4.3 and Table S2). On the basis of
these studies, we expect a higher hydrolysis rate of imine
surfactant 8 as compared to 6, with the model compounds
initially hydrolyzing in 5 min and >1 h, respectively.
Hydrolysis-induced morphology changes were studied in a
Janus emulsion using imine surfactants 6 and 8 with
cosurfactants Tween 20 and Zonyl, respectively. The Janus
emulsion was generated in situ at neutral pH, and the pH was
then lowered to below 3 by addition of 1 M HCl. As expected,
double emulsions stabilized by surfactant 8 and Zonyl
displayed instant changes in morphology upon acidification
from near perfect Janus to HC/FC/W morphology, thereby
signifying the destruction of surfactant 8 (Figure S16).
Conversely, when imine surfactant 6 and Tween 20 stabilized
Janus emulsions were subjected to the same acidification, small
changes in morphology were observed over 5 min (Figure
S17). In the absence of cosurfactants, acidified double
emulsions stabilized with imine surfactant 8 burst, while
imine surfactant 6 stabilized emulsion showed only partial
destruction with some coalescence (Figures S18 and S19).
These studies indicate emulsions stabilized by imine surfactant
6 are less sensitive to acidic pH.
The dynamic nature of the imine surfactants was further
probed through imine exchange. We hypothesized that after in
situ interfacial formation of surfactants 6 and 8, an addition of
excess propylamine to the continuous phase would lead to
imine exchange. Upon successful exchange, the amphiphilic
imines 6 and 8 would generate non-amphiphilic imines 11 and
10 (Figure 6). Such a transformation would then lead to
changes in morphology. We validated imine exchange through
model solution NMR studies (Supporting Information 4.8 and
4.9). The addition of 10 equiv of propylamine (relative to
amine 5) to Janus emulsions having surfactant 8 with Zonyl
cosurfactant transformed the morphology to the anticipated
HC/FC/W form (Figure 6a). No morphology changes were
observed in Janus emulsions stabilized by Tween 20 and Zonyl
in the presence of benzaldehyde 4 only, indicating that the
morphology changes are a result of imine exchange of imine 8
to form 10 and not simply the formation of imine 10 (Figure
S22). Alternatively, surfactant 6 and Tween 20 stabilized
3
2
unsaturated imine (Supporting Information 3.2). This
4
0−42
behavior finds numerous precedents in literature.
Accordingly, we propose that an amine compound acts both
as a base and as a nucleophile to yield the α,β-unsaturated
imine (Supporting Information 3.7 and Figure S9). An
important characteristic of the proposed mechanism is that
formation of the α,β-unsaturated imine is under equilibrium
control, retaining the desired reversible behavior. Hydrolysis of
the system was confirmed by using a model imine, which was
formed with mPEG NH and hydrate 2 in situ in THF-d
8
3
2
(
Supporting Information 3.8). Hydrolysis to the α,β-
unsaturated aldehyde occurs with the addition of water or 1
M HCl, demonstrating the reversibility of the imine bond
(Figure S11). The mechanism of formation, hydrolysis, and
interfacial generation of the α,β-unsaturated imine are further
detailed in Supporting Information 3.7−3.11.
Dynamic Nature at Liquid−Liquid Interface. As
discussed, the imine bond is dynamic, and changes in pH
will impact the formation rates and resulting equilibria.
Accordingly, we studied the dependence of in situ
emulsification on pH. Emulsification with hydrate 2 or
benzaldehyde 4 with amine 5 was successful at neutral or
basic pH (7−11), and we did not observe stable emulsions at
pH < 3. Pendant drop analysis of the reaction showed that at
higher pH values, γ was indeed lower (Supporting Information
4
.1 and 4.2). At pH 5, double emulsions formed initially, but
significant coalescence was observed within 1 h. Although low
γ was observed in pendant drop analysis for hydrate 2 and
amine 5 at pH 5, we concluded that at lower pH, hydrolysis
leads to coalescence. The pH sensitivity of formation provides
further evidence that in situ imine formation is crucial to
stabilize emulsions.
Changes in the pH also affect the imine−aldehyde equilibria.
For imine surfactants 6 and 8, hydrolysis at low pH cleaves the
imine and destroys the amphiphilic properties. Disassembly of
the surfactants leads to morphological changes when a
competing cosurfactant is present, or coalescence/destruction
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Figure 7. In situ generation of FC-surfactants with proteins. (a) FC-surfactant generated with anti-β-actin-FITC and hydrate 2, expected emulsion
morphology, confocal image (left), and optical image (right) of nonspherical behavior. Red arrow highlights the string of droplets created by
interactions between the antibodies. (b) FC-surfactants with hydrate 2 and either anti-β-actin-FITC or amine 5, which stabilize the emulsion
interface, expected morphology, confocal image (left), and optical image (right). (c) Agglutination of salmonella antibody-functionalized emulsions
8
in response to 10 HKST cells/mL. Macroscopically, the droplets changed from transparent to opaque, rendering a QR code unreadable upon
agglutination. (d) FC-surfactant generated with Protein A and hydrate 2, the resulting morphology, and optical image. (e) Confocal image of
Protein A conjugated emulsion tagged with IgG-FITC antibody. (f) 3D side view image of selective Protein A attachment in a Janus emulsion
stabilized by Protein A surfactant and Tween 20. Scale bar = 100 μm.
4
3
droplets exhibited some coalescence and formation of
microdroplets within the double emulsions upon addition of
exploited in indirect ELISA techniques, may be responsible
for the agglomeration that prevents well-behaved phase
separation for precision droplets. In addition, antibodies with
multiple lysine residues can serve as cross-linkers between
individual droplets. To circumvent this behavior, amine 5 was
added as a cofunctionalization agent to disrupt these undesired
processes (Figure 7b and Supporting Information 6.1).
Similarly, when Tween-20 was instead employed as a
cosurfactant during emulsification with in situ antibody-imine
surfactants, perfect Janus emulsions were obtained (Figure
S30). We confirmed that the antibodies are bound at the
surface using confocal microscopy and anti-β-actin-FITC stain,
with and without added amine 5 or Tween 20. In the absence
of amine 5 or Tween 20, a higher density of the antibodies is
observed at the defects, indicating that a local overabundance
of antibodies is responsible for the nonspherical droplets and
phase-separation issues. Although a small decrease in
fluorescence was observed in the presence of amine 5 or
Tween 20, the multiple primary amine units on each antibody
ensure successful conjugation (Figure S30). Importantly, anti-
β-actin-FITC was observed only at the FC/W interface,
showing the successful conjugation of antibody to hydrate 2.
Avidin, salmonella, and listeria antibodies exhibited the same
behavior as anti-β-actin (Figure S30).
1
0 equiv of propylamine (Figure 6b). The microdroplets
formed by the production of imine 11, which contains both a
HC and a FC component, indicating that 11 behaves as a HC/
FC surfactant (Supporting Information 4.10−4.13).
Bioconjugation at Liquid−Liquid Interface. We were
motivated to extend the in situ method of double emulsion
fabrication to more complex protein−surfactants, as conjugat-
ing proteins to double emulsions can be used to generate
15,18
biosensors for pathogens and viruses.
In previous methods,
proteins were conjugated to the oil/W interface through
standard bioconjugation reactions with reactive surfactants on
preformed droplets. Attaching proteins to oil/W interfaces
through this additional fabrication step can pose problems
when scaling-up emulsification for commercial applications.
We hypothesized that lysine units on the surface of proteins
would allow for one step in situ fabrication via imine formation
at the oil/W interface.
We functionalized droplets with four antibodies, anti-β-
actin-FITC, avidin antibody, salmonella antibody, and listeria
antibody, to determine the fidelity of the in situ method to
generate antibody-decorated emulsions. Droplets of dieth-
ylbenzene/HFE-7500 with hydrate 2 were reacted with each
antibody in 10 mM HEPES buffer, with the expectation that a
FC-surfactant would form, thereby giving a HC/FC/W
morphology (Figure 7a and Supporting Information 6.1). All
four antibody systems gave nonspherical droplets upon cooling
with incomplete phase separation of the HC and FC phases
and strings of bound droplets that are unusable for biosensors.
It appears that antibody−antibody interactions, such as those
We endeavored to ascertain if binding fidelity is retained
after random attachment via in situ imine formation, which is
vital to employing antibody-decorated double emulsions as
biosensors. Accordingly, we studied the response of salmonella
antibody surfactant stabilized Janus emulsions to heat killed
Salmonella typhimurium (HKST). In our previous work, Janus
emulsions decorated with salmonella antibodies displayed
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Figure 8. Imine-formation induced selective wetting. (a) Selective wetting on amine-functionalized surface through imine formation in single
emulsions. (b) Amine-functionalized substrates: poly lysinse (left) and APTMS surfaces (right). (c) APTMS surface after reactive wetting with
hydrate 2. (d) (Tridecafluoro-1,1,2,2-tetrahydrooctyl)trichlorosilane-functionalized glass surface. (e) Scheme of double emulsion payload release.
(f) Hydrate 2 enriched diethylbezene/HFE-7500 double emulsion bursting on an APTMS surface.
morphology changes in response to HKST via a surfactant
precious reagents. Finally, the retention of enzymatic activity of
human carbonic anhydrase II (hCAII) was probed to
determine if in situ imine formation has an impact on activity.
To this end, the catalytic hydrolysis of 4-nitrophenyl acetate by
hCAII was studied before and after emulsification, with no
decrease in activity observed (Figure S33). In situ imine
formation with biomolecules proves to be a quick, robust route
to biosensors.
15
cleavage scheme; here we instead demonstrated agglutina-
tion in response to HKST. Through the binding of antibodies
on different individual droplets to the same HKST cells,
emulsions tilt and agglutinate (Figure 7c). As a result of
selective antibody functionalization, the FC/W interfaces
orient toward each other with pathogen binding. Upon
agglutination, the originally optically clear emulsions become
opaque, allowing for the detection of HKST via a simple “on/
off” analysis with a QR code and smartphone reader for the
Emulsion−Solid Interfacial Imine Formation. The
control of interfacial tension through imine bond equilibria
provides control for the formation, manipulation, and
destruction of double emulsions at the oil−water interfaces.
However, no spatial control is achieved as a result of diffusion
of the amine through the continuous phase. The ability to
manipulate emulsions through dynamic imine bond formation
can be further extended to the emulsion−solid interfaces,
where spatial control is introduced. Imine formation at solid
14
presence of HKST (Figures 7c and S31). Binding affinity was
retained with the in situ surfactant formation, paving the way
toward biosensor fabrication with this new technique.
We have examined other proteins to study how size and
lysine content impacts bioconjugation. While keeping protein
concentration constant, emulsions were successfully generated
with hydrate 2 and proteins comprising a broad range of sizes
and lysine content (Supporting Information 6.6 and Table S3).
While size did not impact success, proteins with higher surface
lysine density (>0.3 lysine units per kDa) enabled successful in
situ emulsification. To demonstrate selective protein con-
jugation at the FC/W interface, Protein A decorated droplets
were successfully fabricated and then tagged with comple-
mentary binding antibody IgG-FITC, and exhibited interfacial
attachment, as observed by confocal microscopy (Figure 7d,e
and Supporting Information 6.8). The morphology was then
adjusted with Tween 20 to obtain a near perfect Janus
morphology to demonstrate selective protein conjugation at
the FC/W interface (Figure 7f). Protein A double emulsions
were stable to the exchange of the continuous water phase to
HEPES buffer in the absence of additional surfactants or
proteins, thereby signifying the strong association of the new
imine surfactant to the interface. Furthermore, the continuous
phase could be reused in up to three successive trials without
degradation of performance, allowing for the exhaustive use of
4
4,45
interfaces has been explored for biomolecule adhesion,
46
quantification of amine density, adsorption of gaseous
47
30,31
aldehydes, and in Pickering emulsions.
We hypothesized
that interfacial imine formation between an amine-function-
alized surface and an emulsion containing reactive aldehydes
would generate a locally hydrophobic surface that would cause
a triggered wetting of the emulsions (Figure 8a). Such
controlled wetting via interfacial reactions has potential
applications in programmed payload release from emulsions.
We initially evaluated imine formation at the emulsion−solid
interface using single emulsions containing hydrate 2 on
various amine-coated surfaces. Polylysine and (3-amino-
propyl)trimethoxysilane (APTMS)-functionalized glass were
used as the amine sources (Figure 8b). The polylysine surface
was chosen as a direct connection to our protein studies
performed at the oil/W interface. If the imine interfacial
reaction were to occur, the amine surface would transform into
a fluorous-functionalized surface, which should then wet the
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FC emulsion (Figure 8c). The amine surfaces were submerged
in Zonyl-containing aqueous solution, and single emulsions of
hydrate 2 in HFE-7500 (200 mM) were placed on top of the
substrates. For both amine substrates, instant wetting of the
FC emulsion occurred, signifying imine formation (Supporting
Information 7.3). There was no evidence of wetting in control
studies with pure HFE-7500 on an amine surface, and
additionally hydrate 2 enriched emulsions do not wet
unfunctionalized glass (Figure S34). The increase in hydro-
phobicity with imine formation was confirmed by contact angle
measurements before and after emulsion destruction (Table
S4). Interestingly, reactive wetting induced by in situ imine
formation displays instant, complete bursting of the emulsions
within a couple minutes, whereas slower, nonreactive wetting is
observed on a more hydrophobic fluoro-functionalized glass
surface over 10 min (Figure 8d). As a result, reactive wetting
through imine formation disrupts the emulsion interface to a
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/jacs.9b06852.
■
*
S
Instrumentation and materials; synthesis and character-
ization of 4-dodecoxy-benzaldehyde; emulsification
techniques; characterization of in situ imine formation;
model NMR studies of imine formation, hydrolysis, and
exchange; multivalent amine substrate for in situ imine
formation; biological amine emulsification techniques
and analysis; sensing and enzymatic activity studies;
surface functionalization; and surface wetting technique
and characterization (PDF)
AUTHOR INFORMATION
Corresponding Authors
tswager@mit.edu
thai@chem.umass.edu
■
*
*
greater extent than premade R -functionalized glass.
f
The selective spatial destruction and improved triggered
wetting on amine surfaces has utility in payload release. In a
double emulsion system, a payload can be protected within the
inner phase of the double emulsion and upon contact with an
amine surface emulsion bursting will release the payload
ORCID
S. Thayumanavan: 0000-0002-6475-6726
Timothy M. Swager: 0000-0002-3577-0510
(
Figure 8e). To demonstrate the potential of payload release,
Notes
double emulsions were fabricated with hydrate 2 in
diethylbenzene/HFE-7500 (stabilized by Zonyl), where
diethylbenzene is treated as the payload. Upon contact with
APTMS surface, the double emulsions burst. The FC (dyed
with a fluorous-perylene dye ) wets along the now fluorous-
functionalized surface, releasing diethylbenzene (Figure 8f and
Supporting Information 7.5). We envision this method could
be expanded to selective wetting between droplets decorated
with antibody along the FC/W interface and surfaces with
pathogens. Through the binding interaction, the surface
hydrophilicity could change, causing wetting and the payload
release of therapeutics. Further, through imine triggered
wetting and patterned amine surfaces, interesting opportunities
in spatial control of soft material placement are possible.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the National Science Foundation
Center for Chemical Innovation Grant #1740597, Center for
Autonomous Chemistry (CAC). We thank our colleagues in
the CAC for helpful discussions. Specifically, we thank Sanjana
Gopalakrishnan of the Rotello group (UMass Amherst) for
discussions on our emulsion-surfaces studies. We also thank
Nathan A. Romero and Suchol Savagatrup for insightful
comments and Kosuke Yoshinaga for the fluorous-perylene
dye.
48
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