Fluorescent sensory microparticles that "light-up" consisting of a silica core and a molecularly imprinted polymer (MIP) shell

Article abstract of DOI:10.1002/anie.201300322

From darkness came light: Incorporation of urea-based fluorescent dyes in an anion-imprinted thin polymer shell coated onto silica microparticles leads to a unique and highly enantioselective fluorescent "light-up" response to analytes (see scheme, MIP molecularly imprinted polymer). Copyright

Full text of DOI:10.1002/anie.201300322

Angewandte
Chemie
Communications
Sensor Particles
W. Wan, M. Biyikal, R. Wagner,
B. Sellergren,*
K. Rurack*
&&&& — &&&&
Fluorescent Sensory Microparticles that
“Light-up” Consisting of a Silica Core and
a Molecularly Imprinted Polymer (MIP)
Shell
Form darkness came light: Incorporation
of urea-based fluorescent dyes in an
anion-imprinted thin polymer shell
a unique and highly enantioselective
fluorescent “light-up” response to ana-
lytes (see scheme, MIP molecularly
imprinted polymer).
coated onto silica microparticles leads to
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DOI: 10.1002/anie.201300322
Sensor Particles
Fluorescent Sensory Microparticles that “Light-up” Consisting of
a Silica Core and a Molecularly Imprinted Polymer (MIP) Shell**
Wei Wan, Mustafa Biyikal, Ricarda Wagner, Bçrje Sellergren,* and Knut Rurack*
Molecularly imprinted polymers (MIPs) are an established
and powerful medium for the selective enrichment and
separation of chemical species, in particular for small organic
molecules carrying functional groups. They are generally
assembled by polymerization of a mixture of functional
monomers and cross-linkers in the presence of a template that
is analogous to the target compound. After extraction of the
template, cavities which are complimentary in shape, size, and
electronic or hydrogen-bonding demand remain in the cross-
linked polymer matrix, ready to selectively recognize and
bind the target molecule.[1] Because their preparation, hab-
itus, and function show parallels to a certain molecular
recognition system from nature, MIPs are frequently referred
to as “artificial antibodies”.[2] Although MIPs can accomplish
rather advanced analytical tasks, such as chiral discrimina-
tion,[3,4] the key step for future success in a broader range of
applications is introducing additional functional features.
One important goal concerns the integration of a signaling
element so that the binding events can be directly assessed
with a sensitive technique, such as fluorescence.[5] Such
signaling MIPs would be a sensor material and would
expand the application of MIPs in fluorometric analysis
beyond the detection of fluorescent analytes,[6] the fluores-
cence tagging of analytes prior to detection in the MIP,[7] and
displacement assays.[8] In the latter two approaches, the MIP
is only used for separation. Detection has to be carried out in
a second, discontinuous step, which is not ideal for sensor
applications.
MIPs in which fluorescent moieties are directly incorpo-
rated in the polymer are scarce. Moreover, the quenching of
a covalently embedded dye, lacking designated receptor sites,
can only be employed for analytes which are potent quench-
ers.[9] The perhaps most obvious approach, the covalent
integration of a fluorescent probe monomer into a MIP,
however has only seldom been accomplished,[10–14] and
especially examples showing directional recognition at a des-
ignated binding site[12–14] or fluorescence enhancement upon
analyte binding are rare.[13] The most appealing type, fluoro-
genic MIPs that simply “light up” in an analytically useful
wavelength range upon binding of the analyte have, to our
knowledge, not been reported.[15,16]
To develop MIPs that show an enhancement of fluores-
cence upon analyte binding and perform well in molecular
recognition, we chose N-carbobenzyloxy-l-phenylalanine
(Cbz-l-Phe or Z-l-Phe, Scheme 1) as our template. It is an
established building block in peptide synthesis and a frequent
model target for the development of MIPs for enantiopurity
control of synthetic peptides.[4] We constructed the fluores-
cent monomer 1, from a nitrobenzoxadiazole (NBD) fluo-
rophore carrying a directly fused urea group as the carbox-
ylate recognition site, a short ethylene spacer, and methacry-
late polymerizable unit (Scheme 1). When equipped with an
electron-donating group in the 4-position, NBD dyes show
intense absorption and fluorescence bands at around 450 and
550 nm, respectively, arising from an intramolecular charge
transfer (ICT) process,[17] and have thus been used as
molecular probes for many years.[18] Accordingly, we reasoned
that introduction of the moderate electron-donating 4-urea
group and subsequent binding of an electron-rich carboxylate
guest at this Y-shaped hydrogen-bonding site should lead to
bathochromic shifts and an increase in NBD fluorescence.
Compound 1 was prepared from 4-amino-NBD[19] and 2-
isocyanatoethyl methacrylate by using 4-(dimethylamino)-
pyridine as a catalyst and butylhydroxytoluene as a stabilizer.
Before attempting molecular imprinting, it is important to
choose a solvent that is suitable for complex formation
between probe monomer and the template as well as for
polymerization, in this case for RAFT (reversible addition–
fragmentation chain-transfer) polymerization. This technique
promised to be the way to obtain the MIP matrix as a thin
polymer film on silica microparticles (MPs). RAFT polymer-
ization is not only quasi-living, but also leads to more-
homogeneous networks, more-accessible sites, and hence
higher binding capacities.[20] MPs were the format of our
choice because they can be used as individual sensor units yet
can also be integrated in sensor membranes. Investigation of
[*] W. Wan, Dr. M. Biyikal, Dr. K. Rurack
Fachbereich 1.9 Sensormaterialien
BAM Bundesanstalt fꢀr Materialforschung und -prꢀfung
Richard-Willstꢁtter-Strasse 11, 12489 Berlin (Germany)
E-mail: knut.rurack@bam.de
Dr. R. Wagner, Prof. Dr. B. Sellergren
INFU Institut fꢀr Umweltforschung, Fakultꢁt fꢀr Chemie
Technische Universitꢁt Dortmund
Otto-Hahn-Strasse 6, 44221 Dortmund (Germany)
E-mail: B.Sellergren@infu.uni-dortmund.de
Prof. Dr. B. Sellergren
Department of Biomedical Sciences, Malmç University
SE-205 06 Malmç (Sweden)
E-mail: borje.sellergren@mah.se
[**] This work was supported by the DFG (RU 1622/1-1, SE 777/15-1)
and the Innovationsfonds of BAM/BMWi. We thank S. Selve
(Technische Universitꢁt Berlin) for TEM images, A. Zehl and U.
Kꢁtel (Humboldt Universitꢁt Berlin) for elemental analysis support
and C. Mꢀgge (HUB), D. Pfeifer, and W. Altenburg (BAM Div. 1.3)
for NMR support.
Supporting information for this article (details of syntheses and
characterization of compounds and materials; instrumental details;
additional NMR, TEM, TGA, FTIR, and optical spectroscopic data)
is available on the WWW under http://dx.doi.org/10.1002/anie.
201300322.
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Scheme 1. A) Preparation of a highly fluorescent MIP (1). Extraction and rebinding of the template switch between high and low fluorescence (2).
Preparation of the non-imprinted polymer (NIP, 3) and analytically relevant nonspecific binding (4) and cross-sensitivity (5) reactions
complementing the performance parameters. B) Co-monomers and C) other analytes investigated.
1 with the tetrabutylammonium (TBA) salt of Z-l-Phe in
various solvents suitable for RAFT polymerization (acetoni-
trile, chloroform, tetrahydrofuran) revealed that chloroform
is the best choice, because both the spectral shift and
fluorescence enhancement induced by complex formation
are most pronounced in this solvent (Figure 1 and Figure S1
and S2 in the Supporting Information).
One of the crucial points in using urea units for
carboxylate recognition through the formation of two direc-
tional hydrogen bonds is to adjust the acidity of the urea
protons to such an extent that strongest possible complex
formation is achieved yet undesired deprotonation is
avoided.[21] Deprotonation would give rise to nondirectional
electrostatic interactions at the expense of directional H-
bonding and would hamper the formation of cavities with
desired binding sites. Deprotonation is usually detected in the
titration spectra of ureas and carboxylates by a strongly red-
shifted (often by about 100 nm) absorption band.[22] In
addition, anionic ICT dyes are usually nonfluorescent which
is detrimental for fluorescence sensing,[23] and an anionic
probe is much less selective in recognizing a hydrogen-
bonding guest. Figure 1A shows that in CHCl3, only after
addition of a 7-fold excess of Z-l-Phe, an absorption band at
about 520 nm develops, which is tentatively ascribed to
deprotonation (for further NMR spectroscopic support, see
Section 7 of the Supporting Information).
Figure 1 also reveals that favorable sensing features can
be expected for the MIP. As well as bathochromic shifts of
about 30 and 10 nm in absorption and fluorescence, respec-
tively, complex formation between 1 and the template leads to
pronounced fluorescence enhancement (Figure 1B). In con-
trast, in MeCN and THF, both polar solvents with electron
lone pairs that can facilitate deprotonation, the bathochromic
shifts and fluorescence enhancement upon H-bonding are less
pronounced and deprotonation occurs already at equimolar
concentrations (Figure S1, S2). Analysis of the titration data
of 1 and Z-l-Phe in CHCl3 according to a 1:1 binding model
yielded a complex formation constant logK = 5.11 (Fig-
ure S3), which is suitable for imprinting.[24]
Being aware of the influence of species concentration on
hydrogen bonding and deprotonation equilibria from studies
by other groups,[25] a crucial step was to assess the spectro-
scopic features of the actual pre-polymerization mixture.
Figure 1C shows that at equimolar concentrations (1 mm) of
1 and Z-l-Phe, the amount of 1À formed in CHCl3 is higher
than at 5 mm (Figure 1A). However, in accordance with the
Figure 1. Absorption (A) and fluorescence (B) spectra of 1 (c=5 mm) in CHCl3 in the absence (black) and presence of Z-l-Phe/TBA (red: 0.4, 1,
3 equiv Z-l-Phe; green: 5 equiv; orange: 7, 10, 15 equiv; blue: 20 equiv). Absorption spectra (C) of the pre-polymerization mixtures of
1 (c=1 mm) for the NIP in CHCl3 (black) and the MIP for stoichiometric imprinting of Z-l-Phe/TBA in CHCl3 (green), THF (blue) and
MeCN (red).
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low-concentration data, stoichiometric imprinting should
suffer less from this unwanted side reaction in CHCl3 than
in MeCN and THF (Figure 1C). In addition, studies of the
temperature-dependent dissociation revealed that the com-
plex is sufficient stable at elevated temperatures (up to 808C),
which are used in the curing step of the polymerization (here
actually 70 8C, Figure S4).
Having established the signaling response, we prepared
the sensing material. For this purpose, we chose core–shell
MPs because MPs are a very versatile platform and can be
employed in many different formats. In addition, thin
polymer shells on robust core particles can be reproducibly
prepared by surface-initiated polymerization[26] and MIP
matrices a few nanometers thick promise to yield fast
response times and permit the quantitative extraction of the
template in a comparatively short time.[27]
Silica particles of about 300 nm were used as the support.
This strategy has the advantages of offering a higher density
of the silica core, which facilitates the handling of the beads in
various applications, and that silica MPs are well-suited for
optical detection techniques. As well as 1 and Z-l-Phe/TBA,
EDMA (ethylene glycol dimethacrylate) was used as a mod-
erately polar cross-linker and various (aromatic and aliphatic)
monomers of different polarity were employed as additional
co-monomers (Scheme 1, Table S1). To achieve a homogene-
ous polymer layer around the SiO2 core, a RAFT agent (2-(2-
cyanopropyl)dithiobenzoate, CPDB) was coupled onto the
aminosilane-activated SiO2 surface prior to MIP formation.
Representative TEM images of the core–shell MIP particles
revealing a shell thickness of approximately 10 nm are given
in the Supporting Information.
The performance of the sensor particles was assessed in
chloroform by titration with the designated analyte Z-l-Phe,
the potential competitors Z-d-Phe as the enantiomeric twin,
and Z-l-Glu as a conformationally closely related amino acid
having two possible H-bonding sites instead of one. As
detailed in Table S1, particles CS8 prepared with benzylme-
thacrylate (BMA) as the co-monomer yielded the highest
fluorescence enhancement upon addition of Z-l-Phe and the
best discrimination of MIP versus non-imprinted polymer
(NIP) as well as the best discrimination against Z-d-Phe and
Z-l-Glu. After comparing polymers prepared using the other
co-monomers, we ascribe this beneficial effect to the presence
of the benzyl groups, which offer additional aromatic moieties
for further p–p interactions with the phenyl groups of Phe and
Cbz (see Table S1 and description in the Supporting Infor-
mation).
A typical fluorescence response of CS8 toward Z-l-Phe is
shown in Figure 2A,B. The advantage of the thin MIP shell is
also clear as full equilibration is obtained in about 15 s
(Figure S5). Moreover, Figure 2 also illustrates the different
responses of CS8-MIP and -NIP and the discrimination
against the two other protected amino acids investigated.[28]
Again, a fluorescence enhancement is obtained for CS8 and
Z-l-Phe, although not as strong as that of neat 1 in CHCl3
(Figure 1). This difference is because the environment around
immobilized 1 in the particle shell is more polar as shown by
the red-shifted absorption maximum at 412 nm of 1 in CS8
suspended in CHCl3. The red shift resembles the solvato-
chromic shifts of 1 in MeCN or MeOH (both 411 nm) and
contrasts with 1 in CHCl3 (403 nm) or toluene (404 nm). If we
further consider that the fluorescence quantum yield Ff of
NBD dyes increases with increasing solvent polarity[17] and
Ff(1) was determined to be 0.002 in CHCl3, 0.01 in THF, and
0.024 in MeCN, a Ff = 0.015 for CS8 supports the conclusion
drawn from the absorption measurements.[29] For single-
particle assays such an intrinsically higher fluorescence of
particles containing 1 is advantageous because sufficient
signal intensity can be expected across the entire concen-
tration range. Analytical studies of CS8 finally revealed
a quantification limit in a cuvette-based experiment of about
3 mm and a detection limit (3s) of 60 nm for Z-l-Phe/TBA in
CHCl3.
Although the particles would be suitable for the direct
assessment of enantiomeric purity during the synthesis of
stereochemically pure drugs in the pharmaceutical industry
provided organic solvents are employed, binding studies in
neat water did not yield a measureable response. Apparently,
the thin polymer shell, though prepared from rather nonpolar
building blocks, is not hydrophobic enough to shield the
hydrogen-bonded complexes efficiently from competing
water molecules. To enable the system to detect analytes in
Figure 2. A) Fluorescence spectra of CS8 in CHCl3 in the absence (black) and presence of Z-l-Phe/TBA (red: 0.001–0.085 mm; blue: 0.09 mm).
B) Normalized fluorescence changes DF/F0 (with DF=FÀF0) versus analyte concentration for CS8 and Z-l-Phe (red squares), CS8 and Z-d-
Phe (blue triangles), CS8 and Z-l-Glu (circles), and CSN8 and Z-l-Phe (crosses; CSN8 is the NIP analogue of CS8). Note that the slight decrease
at over 0.1 mm is due to the onset of deprotonation. C) Fluorescence spectra of CS8 in the CHCl3 phase in the absence (black) and upon addition
of various amounts of Z-l-Phe/TBA to the water phase (red: 0.1–0.7 mmol; blue: 1.4 mmol; after stirring and phase separation). Inset:
Corresponding fluorescence titration curves of CS8 (squares) and CSN8 (crosses) with Z-l-Phe (symbols as in B).
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nandez-Sanchez, A. Fernandez-Gutierrez, Biosens. Bioelectron.
2011, 26, 3331 – 3338.
aqueous media, we developed a phase-transfer method. In
a 10 mm quartz cell, CS8 (10 mg) was suspended in CHCl3
(2 mL) before water (1 mL) was added. 0.1–1.4 mmol of the
target Z-l-Phe (as TBA salt) were then added under stirring,
and the system was left to settle for 5 min before the
fluorescence spectra were measured. Subsequently, the
amount of Z-l-Phe that remained in the aqueous phase was
measured by HPLC (control experiments of identical regime
yet in the absence of sensor particles were conducted to
account for the partioning of Z-l-Phe in the neat biphasic
system). Figure 2C shows representative titration spectra and
curves, verified by the indirect determination with HPLC. It is
clear that not only the fluorescence “light-up” of CS8 with Z-
l-Phe is preserved in the extraction-based approach, but that
nonspecific binding is even reduced (cf. CSN8 data in
Figure 2C, inset); the selectivity and response time are also
preserved.
In conclusion, we have demonstrated for the first time the
integration of a fluorogenic monomer into a MIP matrix that
absorbs and fluoresces in an analytically important wave-
length range and that “lights up” upon binding to the analyte.
The amount of nonspecific binding and the enantiomeric
discrimination observed are promising when compared with
previous values for MIP sensing systems utilizing fluores-
cence[11,30] or other detection techniques, such as quartz
crystal microbalance (enantiomeric discrimination less than
2).[31] Integration of the MIP as a thin shell on silica
microparticles makes handling of the sensor particles straight-
forward and assay response times fast. We could also
demonstrate that such a system cannot only be directly
employed in pharmaceutical process-control in organic sol-
vents, but also in a simple phase-transfer assay, which allows
the assaying of analytes in aqueous solution. Very recent work
by others[16] and the work presented by us herein suggest
a bright future for fluorescent MIP sensor particles.
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Received: January 14, 2013
Revised: April 18, 2013
Published online: && &&, &&&&
Keywords: core–shell particles · enantioselectivity ·
.
fluorescence · molecularly imprinted polymers · sensors
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[28] Control experiments using polymers imprinted with Z-d-Phe as
the template resulted in comparable imprinting factors and
enantioselectivities as those obtained for the Z-l-Phe imprinted
particles In addition, the amino acids employed are virtually
nonfluorescent under the spectroscopic conditions used (see
Figure S6).
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