Responsive inverse opal hydrogels for the sensing of macromolecules

Article abstract of DOI:10.1002/anie.201500674

Dual responsive inverse opal hydrogels were designed as autonomous sensor systems for (bio)macromolecules, exploiting the analyte-induced modulation of the opal's structural color. The systems that are based on oligo(ethylene glycol) macromonomers additionally incorporate comonomers with various recognition units. They combine a coil-to-globule collapse transition of the LCST type with sensitivity of the transition temperature toward molecular recognition processes. This enables the specific detection of macromolecular analytes, such as glycopolymers and proteins, by simple optical methods. While the inverse opal structure assists the effective diffusion even of large analytes into the photonic crystal, the stimulus responsiveness gives rise to strong shifts of the optical Bragg peak of more than 100nm upon analyte binding at a given temperature. The systems' design provides a versatile platform for the development of easy-to-use, fast, and low-cost sensors for pathogens. A successful marriage: The combination of smart hydrogels and inverse opal structures unites simplicity with efficacy for sensing macromolecules. While the inverse opal structure provides structural color and a large accessible interface for binding, the induced phase transition of the analyte-responsive hydrogel produces strong optical effects. The resulting spectral shifts can surpass 100nm and are easily detected.

Full text of DOI:10.1002/anie.201500674

Angewandte
Chemie
International Edition: DOI: 10.1002/anie.201500674
German Edition: DOI: 10.1002/ange.201500674
Responsive Materials Very Important Paper
Responsive Inverse Opal Hydrogels for the Sensing of
Macromolecules**
Jean-Philippe Couturier, Martin Sꢀtterlin, Andrꢁ Laschewsky,* Cornelia Hettrich, and
Erik Wischerhoff*
Abstract: Dual responsive inverse opal hydrogels were
designed as autonomous sensor systems for (bio)macromole-
cules, exploiting the analyte-induced modulation of the opalꢂs
structural color. The systems that are based on oligo(ethylene
glycol) macromonomers additionally incorporate comono-
mers with various recognition units. They combine a coil-to-
globule collapse transition of the LCST type with sensitivity of
the transition temperature toward molecular recognition
processes. This enables the specific detection of macromolec-
ular analytes, such as glycopolymers and proteins, by simple
optical methods. While the inverse opal structure assists the
effective diffusion even of large analytes into the photonic
crystal, the stimulus responsiveness gives rise to strong shifts of
the optical Bragg peak of more than 100 nm upon analyte
binding at a given temperature. The systemsꢂ design provides
a versatile platform for the development of easy-to-use, fast,
and low-cost sensors for pathogens.
Scheme 1. Analyte binding induced volume changes of three-dimen-
sional photonic crystal sensors, exemplified by inverse opal hydrogels
(IOHs).
response to changes of the overall hydrophilicity of the system
because of the binding of the analyte (Scheme 1). Such sensor
devices are of striking simplicity, enabling a priori even visual
detection without the need for complicated equipment or
especially trained operators. Accordingly, hydrogel-based
photonic crystal sensors have been explored successfully for
several low-molar-mass analytes.[1b–3]
However, systems for the sensing of large analytes, such as
macromolecules, viruses, or even bacteria, are virtually
missing.[1b,d,e] In the few reported cases, the maximal shifts
of the interference maximum were small, hardly exceeding
20 nm at the outmost.[4]
This is presumably due to a limited change of the
hydrophilicity of the IOHs upon the binding of the analyte,
as well as to the problem of fast and effective diffusion of
macromolecules into the photonic crystal, as needed to induce
a sufficiently big change of the grating, and thus of the optical
signal. To overcome these problems, we speculated on the use
of dual responsive inverse opal hydrogels (IOH), which can
undergo a volume phase transition. While the IOH structure
will facilitate the infiltration of the sensor hydrogel by the
analyte through large channels, the stimulated phase tran-
sition will induce a strong signal.
T
hree-dimensional photonic crystals, namely synthetic opals
and inverse opals, are attractive platforms for autonomous
chemical sensing and colorimetric bioassays. Characteristi-
cally, their structural color is modified by the binding of an
analyte to appropriate functional groups incorporated into
the photonic crystal.[1] This changes the conditions for the
constructive interference of reflected light. While the color
changes may be induced either by modulating the refractive
index or the spacing of the grating, the latter is more
effective.[2] In the case of bioassays, the colloidal crystal is
favorably made of a hydrogel, which swells or shrinks in
[*] Ing. J.-P. Couturier, Dr. M. Sꢀtterlin, Prof. Dr. A. Laschewsky
Institut fꢀr Chemie, Universitꢁt Potsdam
Karl-Liebknecht-Str. 24-25, 14476 Potsdam-Golm (Germany)
E-mail: laschews@uni-potsdam.de
Homepage: http://www.chem.uni-potsdam.de/groups/apc/
Dr. C. Hettrich
Fraunhofer-Institut fꢀr Zelltherapie und Immunologie
Institutsteil Bioanalytik und Bioprozesse IZI-BB
Am Mꢀhlenberg 13, 14476 Potsdam-Golm (Germany)
Dr. E. Wischerhoff
Accordingly, we explored new IOH systems based on
copolymers of oligo(ethylene glycol) monomers 1 and 2
(Figure 1) for sensing macromolecules. Such copolymers are
known to be biocompatible and to show low-fouling behav-
ior,[5] a prerequisite for every bioassay. Importantly, they
exhibit also a lower critical solution temperature (LCST) type
phase transition in aqueous media, which can be tuned by the
copolymer composition.[5b,c,6] Moreover, they can easily
incorporate additional functional comonomers, which bear
recognition units for binding specific analytes. If the analyte
Fraunhofer Institut fꢀr Angewandte Polymerforschung IAP
Geiselbergstr. 69, 14476 Potsdam-Golm (Germany)
E-mail: erik.wischerhoff@iap.fraunhofer.de
Homepage: http://www.iap.fraunhofer.de/en.html
[**] Financial support from the German Federal Ministry of Education
and Research (BMBF), initiative “Spitzenforschung & Innovation in
den neuen Lꢁndern”, cooperative project “Das Taschentuchlabor—
Impulszentrum fꢀr Integrierte Bioanalyse”, FKZ 03IS2201 B/C.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201500674.
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Table 1: Composition of selected responsive copolymers and IOHs
based on 1 and 2 (fixed molar ratio 85/15).
Sample functional
comonomer
cross-linker
cloud
comonomer content [mol%] content [mol%] point [8C][a]
5a
4a
4a
4b
4b
15
7
0
19
0
0
19
0
27.0
IOH-a
5b*
5b
IOH-b 4b
5c
10
10
14
10
13
–
39.2[b]
42.4[c]
4c
4c
none
34.5
30.9
IOH-c
5d
19
0
Figure 1. Monomers used for producing the hydrogel matrix (1 and 2),
its cross-linking (3), and functionalization (4a–c). The mannose
moiety of 4b was protected (R=acetyl) during polymerization, and
deprotected (R=H) in the final copolymer and IOH systems.
IOH-d none
–
19
[a] Cloud points measured by turbidimetry in 0.3 wt% PBS solution
(pH 7.4). [b] Before deprotection. [c] After deprotection.
binding modifies the overall hydrophilicity, the LCST of such
systems is shifted.[7]
content (Table 1). Moreover, in order to obtain IOH films of
sufficient mechanical stability for handling, the thin mono-
mer-filled opal template films were covered by a compara-
tively thick comonomer film of about 100 mm thickness,
resulting in about 5 mm thin IOH layer supported by the
corresponding bulk hydrogel film after curing and template
removal.
In addition to the functional inverse opal series IOH-a to
IOH-c, we prepared analogously IOHs devoid of recognition
units as references (series IOH-d, Table 1). Also for com-
parative model studies in aqueous solution, we synthesized
the analogous soluble copolymers without cross-linker (sam-
ples 5a–d, Table 1).
Cloud points of the soluble copolymer analogues of the
IOHs in aqueous buffer solution (phosphate buffered saline,
PBS) are listed in Table 1. They indicate an LCST-type phase
transition of the basic comonomer composition at 30.98C.
Functionalization by monomers 4a–c inevitably modulated
the overall hydrophilicity of the copolymers and thus changed
the cloud points to a small extent. Importantly, the addition of
the corresponding ligands (see Figure 2 for a selection)
induced a notable shift of the cloud points. For instance, the
addition of fructose or of the xylose-derived copolymer PXM
(see below) increased the cloud point of 0.3 wt%
This dual responsiveness allows an induced phase tran-
sition under isothermal conditions, if the binding of the
analyte shifts the phase transition temperature from below to
above the sensing temperature, or vice versa.[7b] Within an
appropriately chosen temperature window, this should pro-
duce much larger volume changes than simple swelling/
deswelling effects because of the primary modulation of the
hydrophilicity of the system by the binding of an analyte.
For a proof of concept, we focused on established model
recognition systems, such as the binding of vicinal diols as in
certain glycopolymers by benzoboroxol,[8] of lectins by
specific sugars,[9] and of avidin by biotin.[7b,10] Hence, we
synthesized the recognition group bearing comonomers 4a–c,
functionalized by a boronic acid half-ester,[8a] an azidoman-
nose derivative, and
a
biotin moiety,[11] respectively
(Figure 1). The functional IOHs were produced in three
steps. First, monodisperse silica nanoparticles were prepared
through the Stçber process[12] and assembled into a colloidal
crystal. Then, the voids were infiltrated by appropriate
monomer mixtures and polymerized. Finally, the template
nanoparticles were removed (Scheme 2).
solutions in PBS of copolymer 5a by 8.48C or by
5.98C, respectively, while the weakly or non-
binding related molecules ribose, maltopentaose
(PMA), or poly(vinylalcohol) (PVA) produced
only marginal shifts (< 0.58C). Similarly, the
addition of the protein avidin increased the
Scheme 2. Synthesis of inverse opal hydrogels (IOHs): a) Monodisperse silica par-
ticles (diameter 420 nm) are assembled into a synthetic opal of about 5 mm
thickness. b) The template opal is infiltrated by the precursor monomer mixture,
which is subsequently photopolymerized to give a filled hydrogel. c) Removal of the
template particles by dilute HF produces the dual responsive IOH.
cloud points of 5c by 6.78C, while the addition
of other, non-binding proteins, such as bovine
serum albumin (BSA), does not affect the cloud
point. These results demonstrate the basic double
responsiveness of the functionalized copolymers
toward the temperature as well as molecular
The nature and content of the cross-linker in the sensor
IOH must be carefully adapted, to allow effective contact/
penetration of the macromolecular analytes onto/into the
hydrogel walls, while ensuring a still sufficient refractive index
difference between the hydrogel matrix and the water-filled
voids. Good results were obtained with the oligomeric cross-
linker 3 in quantities of 19 mol% of the total comonomer
recognition processes.
When appropriately cross-linked, the thermoresponsive-
ness of these copolymer systems is preserved in inverse opal
hydrogels. This results in their pronounced shrinking upon
heating, with the concomitant marked shift of the Bragg peak
position and of the corresponding color. This is exemplified
for IOH-a in Figure 3. Characteristic for copolymers of 1 and
2
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Figure 2. Selection of potential low-molar-mass and polymeric ligands
tested for binding to boroxol-containing copolymer 5a and IOH-
a (sugars shown in their preferential b-pyranose form).
Figure 4. Temperature(T)-dependent swelling of functionalized IOHs,
in the presence of potential ligands, followed by the shift of the Bragg
peak (wavelength of maximum absorbance lmax). a) IOH-a: ꢀ =in PBS
~
*
buffer, =in the presence of fructose, =in the presence of PVA,
!
*
=in the presence of PMA, =in the presence of PXM (IOH-specific
analyte). b) Reference IOH-d (no recognition units): ꢀ =in PBS buffer,
*
=in the presence of PXM (all analyte concentrations 50 mm repeat
unit).
weakly binding polymers PVA and PMA on the deswelling
transition are negligible (Figure 4a). Remarkably, even
fructose hardly affects the transition of IOH-a, although this
moderately binding low-molar-mass ligand induces a notable
shift of the cloud point for the soluble copolymer analogues
(see above). In contrast, even the addition of the strongly
binding polymer PXM does not modulate the reference
inverse opal hydrogel IOH-d, which lacks recognition units
(Figure 4b). All these results indicate the successful and
specific sensing of macromolecules by the IOHs.
If the IOHs are kept at a constant temperature within the
transition window, the phase-transition-enhanced sensing can
be conducted under isothermal conditions. Importantly,
biologically relevant water-soluble polymers tend to add to
the hydrophilicity of the overall system, thus increasing its
phase-transition temperature. The resulting swelling transi-
tion of the hydrogels is much faster and easier to detect than
an induced collapse transition. For instance, the binding of the
specific ligand PXM to IOH-a at 378C induces a shift of the
Bragg peak by 130 nm. In fact, in the optimal temperature
range of 50–558C for the particular IOH, the shift amounts to
up to 200 nm. Such strong shifts are easy to detect by a simple
photometer, or even by the naked eye.
Figure 3. Temperature(T)-dependent optical properties of IOH-a swol-
len in PBS buffer: a) absorbance spectra, b) wavelength of the position
of the Bragg peak maximum (lmax).
2, the deswelling transition of the hydrogels stretches over
a rather broad temperature range,[13] in contrast to the sharp
cloud points that indicate the onset of the phase transition of
analogous soluble copolymers.
When adding specific polymeric ligands to the functional
IOHs, the temperature-induced shift of the Bragg peaks is
strongly modulated (Figure 4,5). This behavior is remarkably
selective. For instance, the strongly binding polymeric ligand
PXM induces a strong and sensitive shift of the deswelling
transition of IOH-a, which bears a boroxol moiety as
recognition group,[14] while the effects of the non-binding or
The IOH system based on comonomers 1–3 seems to be
a rather general platform for the sensing of macromolecules.
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*
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*
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If comonomer 4a with the boroxol recognition unit is
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the spectral shift is still larger than 20 nm (Figure 5b).
In summary, tailored dual responsive inverse opal hydro-
gels, which combine thermosensitivity with sensitivity toward
molecular recognition processes, enable the versatile sensing
of macromolecular analytes by simple optical means. Appro-
priately designed systems afford effective diffusion even of
large analytes into the photonic crystal, and undergo a big
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Keywords: hydrogels · photonic crystals · polymers ·
responsive materials · sensors
Received: January 23, 2015
Published online: && &&, &&&&
4
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Communications
Responsive Materials
A successful marriage: The combination
of smart hydrogels and inverse opal
structures unites simplicity with efficacy
for sensing macromolecules. While the
inverse opal structure provides structural
color and a large accessible interface for
binding, the induced phase transition of
the analyte-responsive hydrogel produces
strong optical effects. The resulting
spectral shifts can surpass 100 nm and
are easily detected.
J.-P. Couturier, M. Sꢁtterlin,
A. Laschewsky,* C. Hettrich,
E. Wischerhoff*
&&&& — &&&&
Responsive Inverse Opal Hydrogels for
the Sensing of Macromolecules
Angew. Chem. Int. Ed. 2015, 54, 1 – 5
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
5
These are not the final page numbers!