Bifunctional Janus beads made by “sandwich” microcontact printing using click
chemistry
Tobias Kaufmann,a M. Talha Gokmen,b‡ Stefan Rinnen,c Heinrich F. Arlinghaus,c Filip Du Prezb* and Bart Jan
Ravooa*
a
Organisch-Chemisches Institut, Westfälische Wilhelms-Universität Münster, Corrensstraße 40, 48149
Münster, Germany. E-mail: [email protected]; Fax: +49 251 83 36557; Tel: +49 251 83 33287
b
Department of Organic Chemistry, Ghent University, Krijgslaan 281 S4, 9000 Gent, Belgium. E-mail:
[email protected]; Fax: +3292644972; Tel: +329 2644503
c
Physikalisches Institut, Westfälische Wilhelms-Universität Münster, Wilhelm-Klemm-Straße 10, 48149
Münster, Germany
† Electronic supplementary information (ESI) available: Synthesis and surface analytical data. See DOI:
10.1039/c2jm16807c
‡ Currently staff scientist at Life Technologies, Svelleveien 29, 2004 Lillestrom, Norway.
Published in Journal of Materials Chemistry 2012 DOI: 10.1039/C2JM16807C
This article describes the preparation of spherical Janus particles by microcontact printing. A set of three
different polymer beads (diameter ca. 170 μm), each bearing different functional groups at their surface are
used to covalently attach distinct functional molecules exclusively on opposing poles of the beads. The
covalent modification of the beads involves three different prototypes of click chemistry as originally defined
by Sharpless,1 which are the epoxide ring opening (ERO), copper catalysed alkyne azide cycloaddition (CuAAC)
or thiol-yne addition (TYA) reactions. These reactions are compared with regard to their advantages and
disadvantages in the context of “sandwich” microcontact chemistry. The success of surface modification of the
beads is verified by fluorescence microscopy and 3D-time of flight secondary ion mass spectrometry
measurements and is further supported by reference experiments on planar surfaces bearing the same
surface functionality and analysed by X-ray photoelectron spectroscopy, secondary ion mass spectrometry,
atomic force microscopy and fluorescence microscopy. Furthermore we demonstrate that sandwich
microcontact printing can also be performed on smaller polymer beads with a diameter of ca. 5 μm. The broad
scope of surface chemistry in combination with the simple experimental setup makes this method attractive
to a wide range of material science applications, since it combines orthogonality of surface functionalization
with high pattern fidelity.
1. Introduction
Janus particles are objects that have two different
functionalities on opposing faces. Their name is
derived from the ancient Roman god Janus, who
was believed to have two faces on his head,
looking into opposing directions.2-4 In a materials
science perspective, Janus particles have different
properties on opposing poles. These distinct
properties can give rise to amphiphilic behavior,5
adsorption at liquid-liquid phase boundaries,6
catalytic activity,7,8 magnetic response,9 cell
targeting,10
colour,11
fluorescence12
and
combinations thereof. The anisotropic character of
Janus particles makes them potentially attractive
for various applications. One possible field of
application is the use of amphiphilic Janus particles
as “solid surfactants” in order to emulsify liquids.13
However, amphiphilic Janus particles have even
more far-reaching implications than just their
amphiphilic nature, since they possess the
feasibility to assemble in suprastructures.14-16
Granick et. al. recently showed that such particles
can be assembled into highly ordered kagomé
lattices by fine tuning of their electrostatic
interactions.14 This behavior has been investigated
in terms of assembly kinetics, which can help to
understand the interaction of amphiphilic
colloids.15 Other applications are based on site
specific biological recognition,10, 17 demonstrating
that site specific cell targeting is possible using
properly modified particles. Also bar-coded
detection has been shown to be feasible by Janus
particles.18, 19 These broad opportunities to use
Janus particles are the reason why they recently
have attracted a lot of attention.2-4, 20-23
The preparation of Janus particles usually relies on
one of two strategies: post modification, which can
usually also be described as a “top-down”
approach, or the exploitation of intrinsic features
of the particle synthesis, representing a “bottomup” approach. The concept of post modification is
probably more straightforward, as it can
essentially be applied to any object. So far, this
method comprised a first step of particle
immobilization on a surface8, 14, 15 or inside a
protecting matrix,24-28 followed by vapor or
solution based chemical functionalization of the
exposed particle surface. This approach benefits
from its applicability to a wide variety of substrates
(since almost every material can be covered with a
metal film by vapor deposition) and can be
versatile in functionality as metals can in turn be
modified with self-assembled monolayers.14, 15
Related techniques used etching29 or radiation30, 31
techniques and very recently a multistep matrix
assisted patterning procedure to produce
multipatch Janus particles exploiting contacts of
particles in a colloidal crystal has been presented.32
However, these methodologies have a number of
drawbacks, most strikingly the fact that usually
only one side of the particle is functionalized and
rather complex multistep strategies have to be
employed to selectively modify the opposing side,
Figure 1: Schematic illustration of the preparation of
polymer Janus particles by “sandwich” microcontact
chemistry: Polymer particles comprising reactive groups
are adsorbed between two stamps loaded with
different “inks”. The inks having the complement
reactive group react with the particles in the area of
contact upon initiation of the reaction. Functional Janus
particles are obtained after lift-off and extensive rinsing.
although this is possible.26, 32 Furthermore, the
variety of materials that can be vapor deposited is
limited and the harsh conditions and potential use
of aggressive reagents and solvents is often
incompatible with delicate compounds like
bioactive materials. The second strategy of using
intrinsic features of the particle synthesis such as
phase separation, immiscibility of two phases or
immediate solidification of polymers after mixing is
often used in combination with microfluidics. In
this approach a two phase mixture of which at
least one includes the monomer is injected into an
aqueous carrier fluid as small droplets, which are
photopolymerized immediately after formation.11,
12, 18, 33-36
Here the delicate interplay of interfacial
energies determines the outcome. Other, related
techniques exist, where phase separated copolymers are crosslinked in films37-39 or micelles40
and the Janus particle formation is driven by selfassembly of covalently connected polymer blocks
that do not mix. These techniques are attractive as
a result of the virtually unlimited range of chemical
functionality that can be incorporated into the
polymer architecture, the impressive control of
particle shape and the low cost equipment.
Drawbacks of these methods include that also here
that delicate components may be incompatible
with solvents and chemicals used for the synthesis
or the other way round, that many chemicals may
disturb the polymerization or bead forming
process. Also, the common approach of
microfluidic copolymerization is limited to the
higher micrometer scale. A further disadvantage of
this approach is the need of larger amounts of
functional material as it is directly added to the
monomer mixture (when polymer particles are
considered). At the same time, most of the
additive will be embodied in the interior of the
Janus beads and will therefore be useless for
surface functionality, unless the particles are
porous.
We have recently developed a method to prepare
Janus polymer particles based on microcontact
printing as a post modification technique, which
only uses minimal amounts of functional material
that is selectively deposited at the surface of the
polymer bead, anchors the functional molecules
covalently on the particle under mild conditions
and – most importantly – modifies both faces
within one easy step. We refer to this technique as
“sandwich” microcontact printing. 41 A related
approach was reported by Granick et al, who
showed the site selective attachment of reactive
fluorescent silanes on opposing poles of silica
particles by microcontact printing, yielding
bicolored Janus particles.42 The developed method
relies on the principles of microcontact chemistry,
which describes the covalent attachment of
functional molecules (“inks”) on complementary
functionalized surfaces (“substrate”), using an
elastomeric stamp made from PDMS as the
pattern feature.43 Polymers offer a grateful
playground for such kind of surface chemistry,
since it is possible to include a wide range of
functional groups in a polymer by selecting the
respective monomer. Examples of microcontact
chemistry at polymer surfaces are still very rare,
however.44 We use reactive polymer beads as
substrates to link ink molecules at the surface by
microcontact chemistry. These ink molecules are
attached strictly on opposing poles of the beads, as
the beads are sandwiched between two flat
stamps that have been inked with two different
inks (Figure 1).
To this end, each flat stamp is loaded with a
bifunctional molecule, which has to meet two
criteria: first, it should bear a functionality that one
wants to transfer to one pole of the bead, and
second the ink molecule should comprise a
functional group that can potentially react with
functional groups at the surface of the bead. Of
course, the second criteria is not a strict
requirement, as this technique could equally well
work for ink deposition by physisorption or any
non-covalent interaction of the ink molecules with
the substrate surface. However, covalent
immobilization of the ink on the polymer substrate
is most robust and very versatile.
In this article we significantly broaden the scope of
sandwich microcontact printing on polymer beads,
starting from the previously described epoxide ring
opening (ERO) sandwich microcontact chemistry
with amine inks on large epoxy polymer beads and
subsequently expanding the range of surface
reactions to copper catalyzed azide alkyne
cycloaddition (CuAAC) and thiol-yne addition (TYA)
“click” reactions. The success of surface
modification is demonstrated by 3D-time of flight
secondary ion mass spectrometry (ToF-SIMS)
measurements on polymer beads (which shows
the fidelity of chemical texture at the surface),
confocal laser scanning microscopy (CLSM, which
gives an insight into the 3D functionalization of the
beads) as well as fluorescence microscopy. Results
obtained on spherical polymer particles are
supported by control experiments on planar
surfaces that bear the same surface functionality
but can additionally be analyzed by XPS and AFM.
Furthermore we show that this method is able to
establish unprecedented patterned patches, such
as stripe patterned patches exclusively on one pole
of a particle. Finally, we also demonstrate that
sandwich microcontact printing can be applied on
polymer beads of only ca 5 µm diameter. It should
be noted that the printing procedure needs to be
adapted to the size of the particles: when printing
on large beads (170 μm), the particles can simply
be deposited onto one of the inked stamps and the
second stamp is brought into contact on top of the
beads. A printing press avoids sliding of the upper
stamp on top of the beads. On the other hand,
small particles (5 μm) need to be deposited in a
monolayer that is subsequently picked up with the
ink loaded side of a stamp. Additionally, reaction
conditions including light sources, photoinitiators,
catalysts and temperature have to meet the
requirements of the chemical reaction under
investigation.
Figure 2: Light microscope image of nonporous epoxide
bead (e) and fluorescence microscopic image of printed
stripe pattern of rhodamine amine thereon (f).
2. Results and discussion
2.1. Printing on large epoxide beads (170
μm diameter)
In previous work we could demonstrate a novel,
straightforward technique for realizing Janus
structures on polymer microbeads (170 µm
diameter) by sandwiching microcontact printing.41
As illustrated in Figure 1, this procedure relies on
contacting beads with two inked stamps from
opposing sides. We have shown that using this
technique it was possible to covalently fix distinct
fluorophores on opposing poles of epoxide
functional
beads
using
ERO
chemistry.
Furthermore we prepared Janus spheres of two
carbohydrate ligands on one bead, which was able
to selectively bind one of two proteins from a
mixed solution. Also, we have printed a magnetic
ink in combination with a fluorescent ink. The
magnetic ink was comprised of amine-modified
magnetite nanoparticles covalently linked to the
polymer beads, which allowed control over
alignment of the Janus particles.
Figure 2 demonstrates an unprecedented
dimension of “patterned patterning” of polymer
microbeads. This stripe pattern was realized by
printing onto the beads with a stripe patterned
stamp instead of a flat one. Pattering a spherical
particle with stripes strikingly depicts one major
advantage of sandwich printing compared to all
other techniques to prepare Janus beads, which is
the precise control of ink attachment on the bead.
Detailed insight into the site specifity of chemical
modification of the beads was obtained by CLSM.
Stacked slices of fluorescence images through a
bead bearing a printed stripe pattern of
fluoresceine amine convincingly demonstrate that
microscale stripe patterns can be obtained on
beads as well as on flat surfaces (Figure 3).
Approximately one third of the bead surface is
modified with the stripe pattern, depending on the
applied pressure. However, the pressure during
printing must not be chosen too high, as at some
point the pressure destroys the pattern fidelity,
probably due to compression of the PDMS stamp.
Figures 4 c and f were optimized to demonstrate
the integrity of the stripe pattern also in the center
of the image. To this end, the brightness and
Figure 3: CLSM images of stripe patterns of Fluorescein
amine printed on solid epoxide beads; 3D-Volume plots
(a, d), 3D-surface plots (b, e) with partly adjusted
contrast/brightness (c, f).
Figure 4: ToF-SIMS images of printed patterns of
fluorine containing ink 1 (top) printed onto epoxide
polymer beads. Images are calculated from different
anionic fragments given in the image caption.
contrast of single images of the stack had to be
adjusted, since otherwise slices recorded from the
top of the bead are overexposed (see Figure 3 b,
e).
Precise information about the chemical texture of
printed beads could be obtained by ToF-SIMS
measurements. To this end, a fluorine containing
ink (4-(2,2,2-trifluoroethylthio)butan-1-amine, 1)
was synthesized as a sensitive probe. In this case,
flat stamps were used to print the ink on the poles
of porous epoxide beads. ToF-SIMS measurements
of the printed beads show the local composition of
the bead surface. Fragments originating from ink 1
clearly derive from opposing poles of the bead in
the same resolution known from fluorescence
experiments (Figure 4).
ToF-SIMS images are constructed by mapping the
local origin of fragments on a specimen surface.
The most characteristic signals of ink
decomposition are fluorine (Figure 4 a) and the
sulfur (Figure 4 b) containing fragments. The
chemical texture of these signals perfectly
resembles the structure of fluorophores obtained
in previous experiments41 and therefore gives
additional proof for successful site selective
epoxide surface. Although the nucleophilicity of
silanol groups is significantly lower than that of
primary amines, this undesired side reaction
cannot be excluded at elevated temperatures. The
lower reaction temperature can be balanced to
some extent by increasing reaction time.
Figure 5: Surface analysis of (patterned) epoxide SAMs
on silicon wafer; ToF-SIMS images of ink 1 printed in
stripe pattern (a-d), AFM image of ink 1 printed with a
stripe patterned stamp (e), overlay of fluorescence
images of a fluoresceine amine dot pattern and a
rhodamine amine hole pattern (using a flat stamp, f)
and XPS spectra of unmodified epoxide SAM (g) and
after printing ink 1 with a flat stamp (h-i).
surface functionalization. Furthermore, the total
ion count images show that the contrast of the
chemical pattern is not an artifact due to the
topology of the bead. Comparing the different
images, one can also see that fragments derived
from polymer decomposition (e.g. carbon
fragments, Figure 4 c) show no structures (see also
Figure S 1e, f), whereas fragments that are
exclusive for printed regions (Figure 4 a,b, see also
Figure S 1 a d) show a clear contrast and are not
limited to fluorine (see Figure S 1).
Further support for successful surface modification
of epoxide terminated substrates was obtained by
different analysis on epoxide SAMs build on
ultraflat surfaces of silicon wafer. Figure 5 shows a
collection of these data. Interestingly, reactions by
microcontact chemistry on epoxide SAMs needed
different reaction conditions than epoxide beads:
the temperature for printing on epoxide SAMs had
to be lowered to not more than 45°C since
otherwise the stamp would be glued to the
substrate. This phenomenon was never observed
on epoxide beads, which in turn need higher
reaction temperatures of 120°C, since otherwise
the resulting pattern was significantly worse. We
attribute this result to the much higher density of
functional groups on an epoxide SAM. The oxidized
PDMS stamp bears silanol groups, i.e. nucleophilic
moieties,44 which potentially can react with the
Ink 1 was also printed on epoxide terminated
SAMs using a patterned stamp, which could
equally well be imaged using ToF-SIMS, yielding
images of very good pattern resolution
(Figure 5 a-d) that confirm site selective ink
transfer. For example fragments from the epoxy
silane coating (Figure 5 a and Figure 5 b) show the
inverse stamp pattern, whereas fragments that can
clearly be associated with fluorine ink
decomposition (F- (Figure 5 c) or OH- (Figure 5 d)
from the opened epoxide) show the expected
stripe patterns. The full set of ToF-SIMS images
constructed from different fragment signals are
shown in the supporting information (Figure S 2).
Atomic force microscopy (AFM) on stripe patterns
of ink 1 printed onto epoxide SAMs (Figure 5 e)
documents
the
patterned
surface
functionalization. Also fluorescence microscopy on
flat surfaces (epoxide SAMs on glass object
carriers) provided important information, since it
was possible to print one nucleophilic fluorophor
(fluoresceine amine) in a dot pattern and printing a
second fluorophore (rhodamine amine) selectively
into the interspaces of the first pattern by using a
flat (not patterned) stamp (Figure 5 f). Printing was
performed at room temperature for 7 h. The
exclusiveness of both resulting patterns (dots from
the first printing and holes from the second
printing step) revealed two facts: first, not
contacted and hence unreacted epoxide moieties
within the interspaces of the first pattern remain
reactive towards other inks and second, the
density of the first pattern is rather high, as no
fluorescence of the second ink (rhodamine, one of
the strongest fluorophores) is visible within the
dots of the first pattern. The overlay of both
fluorescence images is shown in Figure 5 f, the
whole set of images is provided in the supporting
information (Figure S 5). Additional proof of
covalent attachment of ink 1 on epoxide SAMs was
obtained from X-ray photoelectron spectroscopy
(XPS) (Figure 5 g-i), which cannot be recorded on
polymer beads due to electrostatic charging of the
sample. XPS spectra give valuable information
about the elemental composition at the surface of
a substrate and confirm the covalent nature of
attachment between ink and substrate as can be
seen by the high resolution carbon XPS signal
(Figure 5 g, h). Comparison of the C1s signal within
the recorded XPS spectra before (Figure 5 g) and
after printing ink 1 with a flat stamp (Figure 5 h)
shows that the amount of carbon atoms that are
exclusively bound to other carbon, hydrogen and
sulfur atoms rises at the expense of carbon atoms
bound to other heteroatoms (i.e. the 1s electrons
having a higher binding energy). Furthermore, the
elements of nitrogen and fluorine can only be
detected after printing.
Figure 6: SEM images of 5 µm epoxide functional beads;
500-times (a), 2000-times (b), 15.000-times (c) and
100.000-times (d) magnification, respectively.
2.2. Printing on small epoxide beads (5 μm
diameter)
Having established a reliable protocol for sandwich
printing of amine inks onto “large” epoxide beads,
a major objective was to investigate whether the
sandwich microcontact printing technique is also
applicable to much smaller polymer beads. To this
end, beads of smaller diameter were prepared by
precipitation
polymerization.
The
optimal
procedure was a slightly modified literature
protocol using divinyl benzene (DVB) as crosslinker
and glycidyl methacrylate (GMA) as the functional
co-monomer.45 Using one percent of total
monomer mixture together with 2 mol% of AIBN
(with respect to polymerizable double bonds) in a
solvent mixture of acetonitrile and toluene (80:20)
and polymerizing this mixture for 24 h at 60°C
yielded epoxide functionalized polymer beads of
an approximate diameter of 5 µm. Scanning
electron microscopy (SEM) images of these beads
show an acceptable monodispersity (some smaller
beads are also visible) as well as the rough surface
of each single bead (Figure 6). Evaluation of the
SEM images revealed a distribution of the bead
diameter between 4-8 µm with a mean diameter
of 4.7 µm and a standard deviation of 4.2 (Figure S
8). Elemental analysis confirmed the expected
composition within acceptable tolerances: C (calc):
83.50 %, H (calc): 8.05 %; C (found) 82.50 %, H
(found) 7.77 %.
Furthermore the synthesized beads were analyzed
by FT-IR spectroscopy (Figure S 7), revealing that
the beads contain functional epoxide moieties that
are required for further derivatization. Epoxide
groups could be identified by the absorption bands
for the symmetric (837 cm-1) and antisymmetric
(908 cm-1) epoxide ring deformation as well as the
band for ring breathing (1258 cm-1).46
These beads were subjected to sandwich
microcontact printing in order to obtain Janus
particles. The printing procedure as depicted in
Figure 1 had to be modified, since single µm sized
beads behave completely different compared to
beads with a diameter of hundreds of µm. This
becomes clear immediately after preparation,
since one cannot recognize single particles but only
aggregates; the beads macroscopically behave like
dust. After some optimization it was found, that
the small microbeads could be arranged in almost
perfect monolayers when aggregates of these
particles were ground between an object carrier
and a coverslip. Optical microscopy (differential
interference contrast, Figure S 7) and SEM (Figure
6) illustrate the efficacy of this simple procedure. It
is obvious from the images that this gentle grinding
does not affect the integrity of the spheres. The
second challenge is to pick up these particles,
which can easily be realized by adsorbing particles
on a sticky, ink loaded PDMS stamp by multiple
contacting of the “ground monolayer”. This
procedure also explains why the printed beads
Figure 7: Fluorescence (a-c, e-g) and light microscopy
images (d, h) of small epoxide polymer beads.
Rhodamine amine (a) and dansylcadaverine (b) Janus
beads, overlayed images (c) and fluoresceine amine (e)
and dansylcadaverine (f) Janus beads, overlay (g).
show a much better monodispersity than the
originally synthesized batch (see SEM images,
Figure 6): during adsorption of the bead monolayer
from the object carrier, only the larger beads are
contacted by the stamp, while the smaller beads
remain at the substrate. In other words, smaller
beads are simply excluded from the printing
procedure. This fact might be regarded as a
disadvantage of the sandwich microcontact
printing technique, since not every single bead will
be modified by this procedure. On the other hand,
this feature can equally be considered as an
advantage exclusive to this technique, since this
method works well with less monodisperse
samples and contains an intrinsic size selection.
Also the workup had to be adjusted to the new
dimensions of the beads, since a simple filtration
of the beads is no longer possible due to the pore
size of the frit. Therefore these small beads were
cleaned by a multiple sonication and centrifugation
procedure in different solvents.
As a first proof of principle experiment again
different amine-conjugated fluorophores were
printed on the 5 µm epoxy beads (Figure 7).
Printing either red and blue or green and blue
fluorophores onto the opposing poles of small
epoxide functional polymer beads works equally
well. The pattern fidelity is the same as on the
previously used larger beads and the obtained
functionalization by microcontact printing is strictly
orthogonal. From this, we conclude that the size of
substrates applicable to our sandwich printing
approach could be reduced by almost two orders
of magnitude.
Figure 8: Fluorescence images of rhodamine alkyne (a)
and dansyl alkyne (b) printed on azide beads; overlay of
both images (c), inset: light microscope image of
sample.
2.3.
Printing alkynes on azide beads
The second major goal of our investigation was the
demonstration of the chemical versatility of
sandwich printing. The results described in the
next sections collect examples of two additional
chemical reactions which were successfully
employed to yield bifunctional Janus beads. In
these experiments, exclusively “large” beads (170
Figure 9: Fluorescence microscopy images of rhodamine
alkyne (a) and a fluorogen (b) printed on azide beads;
overlay of fluorescence images (c) and light microscope
image (inset).
µm) have been used. The first type of reaction is
the copper catalyzed alkyne azide cycloaddition
(CuAAC), which is a well-established “click”
reaction for surface functionalization, also in the
field of microcontact chemistry.47-49 To this end,
azide terminated polymer beads were prepared as
already described50 by solution reaction of epoxide
beads with sodium azide. These beads were then
printed with alkyne terminated inks (Figure S9).
As a first experiment again two different
fluorophores were printed on opposing poles of
the beads, yielding nicely resolved Janus particles
(Figure 8).
These results clearly demonstrate that the
technique of sandwich microcontact printing can
be applied to a wider range of spherical particles
using suitable surface chemistry. Another
convincing result is the preparation of Janus beads
using one fluorophore and one fluorogen, which is
only fluorescent after the CuAAC reaction due to
the formation of a conjugated triazole moiety.
Figure 9 shows the obtained bifunctional Janus
beads.
It should be noted that during recording of the
fluorescent images in Figure 9, the camera settings
were adjusted in such a way that no green color
was detected, since these azide beads show a
green-blue background fluorescence themselves.
Therefore the apparent color of the fluorogen,
when compared to the same experiment on planar
azide SAMs (Figure S 11), is slightly different.
However, these obtained results on azide beads, in
combination with reference data on azide
terminated SAMs, clearly prove that it is not only
possible to attach distinct inks on opposing poles
of the spheres, but also to perform this in a
covalent way, since otherwise the fluorogen pole
would not be fluorescent at all.51 Further evidence
for successful microcontact chemistry using CuAAC
could be obtained by reference experiments on
azide SAMs, which are provided in the supporting
information (Figure S 11 and Figure S 12).
Figure 10: Fluorescence microscopy of thiol Janus
beads, showing rhodamine alkyne (a, d), dansyl
alkyne (b), fluoresceine alkyne (e) fluorescence,
overlay of images (c, f), respectively.
2.4.
Printing alkynes on thiol beads
The third reaction that was employed to prepare
Janus particles is the photochemical thiol-yne
addition (TYA) reaction of alkynes with thiol
terminated polymer beads. This type of reaction
has recently received a lot of attention – also in the
field of surface and polymer science52, 53 including
microcontact printing54 – since it is very fast, highly
efficient and triggered by UV irradiation under mild
conditions. Because of these admirable properties,
it has also been accounted to the quickly growing
family of “click” reactions.55 However, it can be
argued whether the thiol-yne reaction in general
does actually meet all the criteria installed by
Shparpless.56 Anyway this reaction was found to
work very well in the context of microcontact
chemistry, which will be shown in the following
section.
The synthesis of thiol functional polymer beads has
already been described elsewhere.52 The alkyne
terminated inks used for CuAAC reactions could be
used for TYA reactions as well (Figure S 10). The
printing procedure was adapted from previous
work:54 in brief, the stamps where inked with
alkyne
solutions
containing
DMPA
as
photoinitiator, freshly reduced thiol beads were
sandwiched between two inked stamps, and a high
intensity UV-LED (365 nm) was put on top of the
upper stamp to trigger the reaction. The images
obtained after printing blue, red or green alkyne
fluorophores on the poles of thiol beads (Figure
10) clearly show that also this reaction is suitable
for sandwich microcontact printing.
All fluorophores show good patch resolution, even
though the thiol beads are transparent and difficult
to image properly since light diffraction and
reflection at the bead surface often produce
artifacts. Nevertheless it was also possible to print
and visualize a rhodamine stripe pattern on one
pole of the beads (Figure 11), whichrepresents one
of the outstanding features of sandwich
microcontact printing: polymer beads can be
surface modified with a tailor-made micropattern
on one pole exclusively, due to the contact
between stamp and substrate.
One interesting aspect when comparing the TYA
reaction with ERO and CuAAC is the speed of
reaction. Time dependent printing experiments
were done in order to establish the minimum
printing time that is necessary to create Janus
beads. For this study rhodamine and dansyl alkyne
inks were used. The results displayed in Figure 12
indicate that even after one min of printing the
alkyne inks onto thiol beads, the reaction has
proceeded to a significant extent. The pattern
quality visibly improves after printing for 5 min,
but not anymore when printing for even longer
times. This demonstrates the main advantage of
TYA reaction in microcontact chemistry, namely
that it is extremely fast. For the experiments
shown in Figure 12 a, 2 mM of alkyne ink and 1
mM of DMPA were used; an increase of both
concentrations might speed up the reaction even
further.54
2.5.
Figure 11: Fluorescence images of rhodamine alkyne
stripe pattern on thiol terminated polymer beads.
Comparison of surface chemistries
After demonstrating the versatility of three
chemical reactions to locally modify polymer beads
using sandwich microcontact printing, it is of
Figure 12: Fluorescence microscopic images of time
dependent printing of dansyl (first column) and
rhodamine alkyne (second column) on thiol beads.
Images are recorded through a UV filter (first column)
and a green filter (second column) and were
superimposed (after adjusting brightness and contrast)
(third column). The inset of the last image in each row
shows the bright field image of the respective sample.
interest to compare the three examined surface
reactions (Table 1).
Solution based organic syntheses have different
demands on reaction conditions than surface
chemistry and microcontact chemistry in
particular. First, a reagent can have different
reactivity when it diffuses freely in solution or
when its degree of freedom is drastically reduced
by surface attachment. Therefore it is reasonable
that reactions at surfaces tend to need longer
reaction times and/or more drastic reaction
conditions. Second, there are practical limitations
to microcontact chemistry. One of the most
important ones concerns restrictions to the choice
of solvent, since only alcohols, water and
acetonitrile are compatible with the PDMS stamps,
and most other solvents either swell or dissolve
the polymer. Furthermore, printing under inert
atmosphere is challenging (unless printing is
carried out in a glove box). And third, also the
chemical properties of the PDMS stamps itself
have to be considered. Oxidized PDMS stamps
have a certain amount of silanol groups at their
surface, which in principle can also react with
electrophilic surface bound molecules (e.g.
epoxides).
The epoxide substrates used for ERO are
surprisingly stable under ambient conditions,
which make elevated temperatures necessary to
react them with nucleophilic inks in an acceptable
period of time. CuAAC is much faster (1 h reaction
time instead of 4 h for ERO) and does not need
that much activation energy (60°C instead of 120°C
for ERO). However, major drawbacks of this
reaction are the instability of the Cu(I) catalyst and
its residues, which might be disadvantageous for
certain applications. The instability of the catalyst
makes it necessary to form its reactive species in
situ, which means practically that four different
solutions have to be mixed on the stamp. Also it
has to be considered that small azide compounds
might be unstable and even explosive. Concerning
the TYA reaction, this is by far the fastest reaction
to modify surfaces covalently. By choosing the
proper initiator one can choose if either heat or UV
light irradiation is used to start the reaction.
However, also this reaction has disadvantages,
most of all the instability of free thiols, which tend
to oxidize easily. This makes it necessary to
synthesize/deprotect the thiol functionality prior
to printing or to work under inert atmosphere,
which is virtually impossible without using a glove
box. Also this reaction is known to be less tolerant
to the presence of certain functional groups such
as amines.
In summary, the ERO reaction seems to be the
most versatile candidate for our sandwich
microcontact printing technique, since it is
amenable to a wide scope of reactive inks.
However, CuAAC and TYA reactions can be readily
employed as well, which demonstrates the wide
applicability of orthogonal surface modification by
sandwich printing. Certainly this method is not
limited to these three reactions. We emphasize
that it has already been shown by some of us that
thiol beads can react with various reagents via
different (“click”) reactions.52
3. Conclusions
In conclusion, the results presented in this paper
clearly show that it is possible to create well
resolved Janus structures on polymer beads (170
µm in diameter) employing three different types of
chemical reactions. Comparison of all the
parameters involved in successful surface
chemistry indicates that all of the three employed
reaction types are generally well suited for
Table 1. Comparison of surface reactions for sandwich printing
rctn. type
Advantages
ERO
Reagents
stable
at
ambient Low reactivity (high T, long reaction times);
conditions;
Ink has to be tolerant to high T
Applicable to any nucleophile;
Easy access to epoxide substrates
CuAAC
Faster than ERO;
Educts rather stable at ambient
conditions;
Selective reactivity;
Easy access to N3 or alkyne
substrates
Instability of in situ formed Cu(I) catalyst;
Toxicity of Cu(I/II) residues;
More restrictive concerning ink functionality;
Potential instability of N3 compounds
(explosive);
More complex procedure (mixing 4 solutions
ON STAMP)
TYA
Very fast;
Triggered by either light or heat;
Easy access to alkyne substrates/inks
Instability of thiol ink/substrate;
Need for suitable equipment (intense UV lamp
of proper λ);
UV can affect inks and substrates;
Incompatible with certain functionalities
(amines);
Thiols smell;
Difficult to apply to non-(UV)-transparent
substrates;
Side reactions of initiator
sandwich microcontact chemistry. ERO has the
major advantage of modesty in reaction conditions
(apart from temperature) and tolerance to ink
functionality. CuAAC on the other hand is
significantly faster and yields very stable products
whereas photochemical TYA reaction is
extraordinary fast. A second important result of
our work is the unique feature to our sandwich
printing method of hierarchical patterning of
spherical beads (creating patterned patches). To
the best of our knowledge, such particles are
impossible to realize with any other existing
approach apart from time consuming serial
lithographic methods.30 Furthermore the substrate
dimensions for sandwich printing could be reduced
by two orders of magnitude. We envisage that
even submicron spheres may be site selectively
modified using this method. Ultimately we expect
that our method will be particularly useful for the
preparation of Janus particles with recognition
patches that guide the self-assembly of Janus
particles into anisotropic nanomaterials.
Disadvantages
4. Experimental
Preparation of porous particles: Typically, particles
were prepared by using a tubing-needle based
microfluidic setup50. 2 m in length, 0.8 mm inner
diameter Tygon tubing served as the channel and
30G or 32G bent blunt needles were used for the
discrete monomer phase. Pumping rates were 1.2
mL/min and 0.60 mL/h for continuous and discrete
phases, respectively. The continuous carrier phase
was 3 wt% sodium lauryl sulfate (SDS) solution and
the discrete phase was composed of 20 vol%
ethylene
glycol
dimethacrylate
(EGDMA,
crosslinker), 30 vol% glycidyl methacrylate (GMA,
epoxy monomer), 50 vol% n-octanol (porogen) and
4 wt% (compared to EGDMA+GMA) 2,2dimethoxy-2-phenylacetophenone
(DMPA,
photoinitiator).
Preparation of small epoxide particles: Small
epoxide particles were synthesized by a modified
literature
procedure
via
precipitation
45
polymerization . In short, α,α` Azoisobutyronitrile
(AIBN) as initiator (10 mg, 6.1*10-5 mol, 2 mol%
w.r.t. polymerizable double bonds) was dissolved
in a monomer mixture of GMA (0.156 g, 1.1 mmol,
25% of monomers) and DVB (55%, 0.411 g, 3.2
mmol, 75% of monomers) before this solution was
diluted with a acetonitrile/toluene mixture (80:20).
The total monomer content was kept at 2 mol% or
less. The resulting clear solution was degassed by
bubbling argon for 15 min, then sealed under inert
atmosphere and polymerized at 60°C for 24 h at a
rotary evaporator at a speed of 20-30 rpm. After
completion of the reaction, the dispersion was
filtered over a Por 4 frit and washed extensively
with
acetone,
dichloromethane,
dimethylformamide, dichloromethane, methanol
and finally diethylether. Then the polymer particles
were collected in a centrifuge tube, sonicated for 1
min and subsequently centrifuged for 20 min with
a speed of 5000 rpm. The supernatant solution was
removed and the polymer particles resuspended in
methanol, briefly sonicated again for 1 min and
centrifuged again. This procedure was repeated
three times before the polymer particles were
transferred to a round bottom flask and the
solvent was removed. Afterwards the polymer
beads were collected and kept at 4°C until use.
Microcontact printing on large particles: Stamps
were made from poly(dimethyl siloxane) (PDMS,
Sylgard 184, Dow Corning). Oxidized stamps were
loaded with a few drops of ink solution (1 10 mM
in ethanol) for one min. Excess ink solution was
removed under a stream of argon for
approximately one min. A monolayer of polymer
particles was applied to one stamp before the
other stamp was put on top with the help of a
homemade, hand-operated press and then the
sandwich of stamp-particles-stamp was heated or
irradiated to initiate the reaction. When the
reaction was complete, the polymer particles were
collected in a frit and washed with water, ethanol,
acetone and diethylether. Samples were extracted
in hot ethanol for 10-12 h. The printing procedures
differed for the different reaction types in some
points:
a) Epoxide polymer beads: to the ink solutions of
amine-terminated
compounds
triethylamine
(~three drops per 5 mL) was added as a base. After
sandwiching the beads, the system was heated to
120°C for 4 h.
b) Azide polymer beads: on the oxidized stamp,
four different solutions were mixed. First, three
drops of a 10 mM alkyne ink solution in ethanol
was applied, before one drop of Cu(OAc)2 solution
(5 mM, EtOH), one drop of Tris[(1-benzyl-1H-1,2,3triazol-4-yl)methyl]amine (TBTA, 1 mM, EtOH) and
one drop of ascorbic acid (5 mM, EtOH) was added
after blow drying. The sandwiched beads were
then heated to 60°C for 1 h.
c) Thiol polymer beads: Prior to use, thiol beads
were stirred in a 1 M solution of DL Dithiothreitol
(DTT, Sigma) in DMF over night at room
temperature using a rotary evaporator. The
reduction of dithiols was then quenched by adding
excess of acetic acid, the beads were collected in a
frit and washed with DMF, acetone, ethanol and
diethylether (each acidified with acetic acid). Thiol
beads were stored under inert atmosphere until
use.
For printing, a 2 mM solution of the respective
alkyne terminated ink in ethanol, containing 1 mM
of 2,2-Dimethoxy-2-phenylacetophenone (DMPA)
was added on the oxidized PDMS stamp and after
drying and sandwiching the beads, they were
irradiated with an intense UV LED (365 nm) for the
given period of time.
Microcontact printing on small epoxide beads: A
small amount of polymer particles was put on an
object carrier and ground gently with a coverslip in
order to form a monolayer (see Figure S 7).
Subsequently beads were picked up with an ink
loaded stamp, turned upside down and been
sandwiched with a second inked stamp. The
sandwiched beads were then heated to 120°C for 4
h. After completion of the reaction, beads were
collected in centrifuge tubes, sonicated for 30 min
and then centrifuged at 5000 rpm for 20 min. This
procedure was repeated 4 times, twice using
methanol and twice using acetone as solvent. After
removing the supernatant solution after the last
centrifugation step, beads were transferred to
round a bottom flask, the solvent was removed
under reduced pressure and the beads were
dispersed in (fluorescence free) immersion oil or
PEG 600. This suspension was applied on a
coverslip, heated to 75°C for 2 h in order to collect
the beads at the bottom of the oil drop (one focal
plane) and was then examined by fluorescence
microscopy.
Instrumentation:
Light
microscopy
and
fluorescence microscopy images were recorded
using a Reflected Fluorescence System CKX41
(Olympus, Shinjuku, Tokyo/Japan) in combination
with a Kappa DX 20 L-FW camera (Kappa optronics
GmbH, Gleichen/Germany) and the operating
software Kappa ImageBase Control (version 2.7.2).
ToF-SIMS measurements were performed using a
type IV compatible ToF-SIMS instrument equipped
with a liquid metal ion gun (IONTOF GmbH,
Münster/Germany) utilizing Bi3+ clusters (25 keV)
as primary ions with a primary ion dose density of
up to 5*1013 ions/cm2. Confocal laser scanning
microscopy images on epoxide beads were
recorded using a Leica TCS SL (and DMRE)
microscope (Leica Camera AG, Solms/Germany)
with a continuous wave Ar Laser photon source
(λmax = 458, 476, 488 & 514 nm) of max. 200 mW
power and max. 50 mW power within the focal
plane in combination with Leica Confocal Software
as recording software (Version 2.5, build 1347).
Stacking and processing of the image slices was
done using ImageJ software (version 1.44p,
National Institutes of Health, Maryland/USA). SEM
images of small epoxide beads were recorded after
coating the sample with a thin film of gold using a
Zeiss AURIGA workstation (Carl Zeiss AG,
Oberkochen/Germany). A field emission gun with 5
kV acceleration voltage was employed in
combination with a Everhart-Thornley secondary
electron detector. Measuring conditions were 10-6
mbar at room temperature. SmartSEM (Vers.
5.04.05.00) was used as recording software. Size
distribution was determined employing ImageJ
software (version 1.44p, National Institutes of
Health, Maryland/USA). XPS measurements were
done on a Kratos Axis Ultra (Kratos Analytical Ltd,
Manchester/UK), using monochromated Al Kα
irradiation (1486.6 eV) and a pass energy of 20
meV for narrow scans. The obtained data was
processed utilizing CasaXPS software (version
2.3.15, Casa software Ltd, Teignmouth/UK). All
spectra were calibrated to a binding energy of 285
eV for the C 1s orbital. AFM images were obtained
using a Nano Wizzard 3 system (JPK Instruments
AG, Berlin/Germany) in combination with
processing
software
Gwyddion
(www.gwyddion.net, version 2.25). ATR-IR spectra
were recorded using a Varian 3100 FT-IR Excalibur
Series (Varian Inc. (now Agilent Technologies), Palo
Alto, California/USA) in combination with Varian
Resolutions Pro software (version 4.0.5.009).
Acknowledgements
The authors wish to thank Dr. Falko Schappacher
and Prof. Dr. Winter (Institut für Physikalische
Chemie,
Westfälische
Wilhelms-Universität
Münster) for SEM measurements and Ralf Rempe
and Prof. Dr. H.-J. Galla (Institut für Biochemie,
Westfälische Wilhelms-Universität Münster) for
CSLM measurements. Christian Schulz (OrganischChemisches Institut, Westfälische WilhelmsUniversität Münster) is gratefully acknowledged
for XPS measurements in the laboratory of Prof.
Dr. Winter (Institut für Physikalische Chemie,
Westfälische Wilhelms-Universität Münster). The
Belgian Program on Interuniversity Attraction
Poles initiated by the Belgian State, Prime
Minister’s office (Program P6/27) and the P2M
ESF-program are acknowledged for financial
support.
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