A water soluble fluorescent polymer as a dual colour sensor for temperature and a specific protein

Article abstract of DOI:10.1039/c3tb21245a

We present two thermoresponsive water soluble copolymers prepared via free radical statistical copolymerization of N-isopropylacrylamide (NIPAm) and of oligo(ethylene glycol) methacrylates (OEGMAs), respectively, with a solvatochromic 7-(diethylamino)-3-carboxy-coumarin (DEAC)-functionalized monomer. In aqueous solutions, the NIPAm-based copolymer exhibits characteristic changes in its fluorescence profile in response to a change in solution temperature as well as to the presence of a specific protein, namely an anti-DEAC antibody. This polymer emits only weakly at low temperatures, but exhibits a marked fluorescence enhancement accompanied by a change in its emission colour when heated above its cloud point. Such drastic changes in the fluorescence and absorbance spectra are observed also upon injection of the anti-DEAC antibody, attributed to the specific binding of the antibody to DEAC moieties. Importantly, protein binding occurs exclusively when the polymer is in the well hydrated state below the cloud point, enabling a temperature control on the molecular recognition event. On the other hand, heating of the polymer-antibody complexes releases a fraction of the bound antibody. In the presence of the DEAC-functionalized monomer in this mixture, the released antibody competitively binds to the monomer and the antibody-free chains of the polymer undergo a more effective collapse and inter-aggregation. In contrast, the emission properties of the OEGMA-based analogous copolymer are rather insensitive to the thermally induced phase transition or to antibody binding. These opposite behaviours underline the need for a carefully tailored molecular design of responsive polymers aimed at specific applications, such as biosensing.

Full text of DOI:10.1039/c3tb21245a

Journal of
Materials Chemistry B
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PAPER
A water soluble fluorescent polymer as a dual colour
sensor for temperature and a specific protein†
Cite this: J. Mater. Chem. B, 2013, 1,
373
6
a
b
c
cd
e
Sahika Inal, Jonas D. K o¨ lsch, Frank Sellrie, J o¨ rg A. Schenk, Erik Wischerhoff,
b
a
Andr e´ Laschewsky* and Dieter Neher*
We present two thermoresponsive water soluble copolymers prepared via free radical statistical
copolymerization of N-isopropylacrylamide (NIPAm) and of oligo(ethylene glycol) methacrylates
(
OEGMAs), respectively, with
a
solvatochromic 7-(diethylamino)-3-carboxy-coumarin (DEAC)-
functionalized monomer. In aqueous solutions, the NIPAm-based copolymer exhibits characteristic
changes in its fluorescence profile in response to a change in solution temperature as well as to the
presence of a specific protein, namely an anti-DEAC antibody. This polymer emits only weakly at low
temperatures, but exhibits a marked fluorescence enhancement accompanied by a change in its
emission colour when heated above its cloud point. Such drastic changes in the fluorescence and
absorbance spectra are observed also upon injection of the anti-DEAC antibody, attributed to the
specific binding of the antibody to DEAC moieties. Importantly, protein binding occurs exclusively when
the polymer is in the well hydrated state below the cloud point, enabling a temperature control on the
molecular recognition event. On the other hand, heating of the polymer–antibody complexes releases a
fraction of the bound antibody. In the presence of the DEAC-functionalized monomer in this mixture,
the released antibody competitively binds to the monomer and the antibody-free chains of the polymer
undergo a more effective collapse and inter-aggregation. In contrast, the emission properties of the
OEGMA-based analogous copolymer are rather insensitive to the thermally induced phase transition or
to antibody binding. These opposite behaviours underline the need for a carefully tailored molecular
design of responsive polymers aimed at specific applications, such as biosensing.
Received 10th September 2013
Accepted 4th October 2013
DOI: 10.1039/c3tb21245a
www.rsc.org/MaterialsB
1,2
detection method. The output signal comes in a variety of
ways namely the lifetime, anisotropy or intensity of the emis-
Introduction
The need to continuously monitor and quantify an analyte or an sion of the uorescent probe. A well-exploited sensing strategy
environmental parameter in the fastest and cheapest way has via uorescence methods employs solvatochromic (environ-
been the driving force of sensor research for several decades. mentally sensitive) dyes which change their emission and/or
Given its facile operation, remote monitoring, easy read-out and
high sensitivity, uorescence is by far the most applied
absorbance properties with respect to physical or chemical
changes in their surroundings. Such environmental changes,
3
e.g., polarity or viscosity, can be induced by direct or indirect
interactions of the uorophore with target molecules whose
a
Institute of Physics and Astronomy, University of Potsdam, Karl-Liebknecht-Str. 24-25,
Potsdam, 14476, Germany. E-mail: neher@uni-potsdam.de
b
presence and concentration can be correlated with the extent of
Institute of Chemistry, University of Potsdam, Karl-Liebknecht-Str. 24-25, Potsdam, alterations of the photophysical spectra. However, many of
4–6
1
4476, Germany. E-mail: laschews@uni-potsdam.de
the conventional small molecule-based probes suffer from
c
UP Transfer GmbH, Am Neuen Palais 10, Potsdam, 14469, Germany
major limitations such as poor water solubility, low quantum
d
Hybrotec GmbH, Am M u¨ hlenberg 11, Potsdam, 14476, Germany
7
yield, and low structural stability. Moreover, due to the pres-
e
Fraunhofer Institute for Applied Polymer Research, Geiselberg Str. 69, Potsdam,
ence of different media and an uncountable variety of targets to
1
4476, Germany
detect, not only new sensing schemes but also new materials are
†
Electronic supplementary information (ESI) available: Photophysical data of the
1
mandatory.
monomer (2), temperature dependent absorbance and time-resolved PL spectra of
ꢀ
P1, steady-state PL spectra of the monomer (2) at 20 and 50 C, concentration
In this context, synthesis and characterization of a large
variety of thermoresponsive polymers bearing covalently
dependent PL spectra of P1, decay curves of P1 in the presence of AB, evolution
of absorbance, steady state and time resolved PL spectra of aqueous P1 solution
upon heating and AB injection, evolution of absorbance and emission of the
heated P1 solution upon monomer addition, decay curves of P2 below/above its
7–9
attached uorophores has been reported. Typically, induced
by an increase in solution temperature above their lower critical
solution temperature (LCST), these polymers undergo a solu-
bility transition which involves drastic, yet reversible changes in
ꢀ
LCST, ELISA tests on all polymers and the monomer at 4 C, and the inuence
of AB on the turbidity prole of P2. See DOI: 10.1039/c3tb21245a
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chain conformation and association behaviour. The chains, ethylmethacrylate (MEO
Paper
10
2
MA) and oligo(ethylene glycol) meth-
which are extended and exposed to water at lower temperatures, acrylate (OEGMA) monomers. The resulting NIPAm-based
collapse and aggregate leading to the formation of relatively less copolymer reports the LCST-type phase transition via a
mobile and less polar, polymer-rich domains at higher remarkable uorescence output. Such a change in uorescence
temperatures. The covalent incorporation of solvatochromic characteristics is also induced as a monoclonal anti-DEAC
dyes, which are sensitive to the hydration state of polymer antibody is injected into the solution. This biological sensing
chains, is the key methodology to obtain a uorescent output of can be (de)activated simply by tuning the conformation of
this adaptive behaviour, and thereby to develop schemes that polymer chains with temperature. We suggest that the correla-
9
exploit the LCST transition for optical sensing. The uorophore tion of uorescence enhancement with the solution tempera-
senses the changes in its micro-environment generated as a ture and antibody concentration should pave the way for new
result of the polymer phase transition, and transforms these strategies of the detection and quantication of multiple
1
1–21
into a marked uorescence read-out,
e.g., typically a hyp- stimuli using responsive polymers.
22
sochromic (blue) shi and enhanced uorescence intensity.
The NIPAm-based platform is furthermore intriguing as a
Taking advantage of the delicate solubility of amphiphilic biological assay. Above the LCST of the polymer, a fraction of
polymer chains in water, changes in the dye microenvironment the bound antibody is released from the polymer-attached
can as well be induced by stimuli other than temperature. For DEAC sites out to the solution. In the presence of the DEAC-
instance, thermoresponsive N-isopropylacrylamide (NIPAm) functionalized monomer in this mixture, the released antibody
based microgels bearing CdS quantum dots and phenylboronic binds to the monomer enabling rather effective inter-chain
acid groups swell upon binding of the glucose molecules to aggregation/collapse. This competitive binding mechanism can
23
boronic acid moieties. In this swollen state of the microgel, be directly monitored from the polymer's temperature–turbidity
the emission of the quantum dots is quenched. Similarly, prole. Remarkably, when the same dye is attached to the pol-
Ohwada et al. reported a uorogenic ion sensor based on ymethacrylate bearing OEG side chains, we nd that the uo-
NIPAm units, an ion-receptor, and a solvatochromic dye. Upon rescence properties are almost insensitive to the polymer phase
interaction of the receptor with target ions, the polymer coils transition as well as to the presence of the antibody. This con-
undergo a three-dimensional conformational change to a trasting behaviour stresses the importance of structural
globular form due to increased inter-segmental interactions, considerations for the design of such molecular sensors.
18
leading to an enhanced uorescence signal. Such systems
relying on the specic functionality, i.e., recognition unit of the
polymer, enable sensing of a particular stimulant via uores-
cence. In order to generate a dramatic change around the u-
orophore by the receptor–analyte interactions, the mandatory
condition is nano-sized control of the placement and distribu-
Materials and methods
Synthetic procedures
0
2
9
-(2 -Methoxyethoxy)ethyl methacrylate (MEO
2
MA, Aldrich,
5%, stabilized with 100 ppm 4-methoxyphenol (MEHQ)
24
tion of receptor and uorescent moieties, which generally
requires rather challenging synthetic procedures. On the other
hand, by utilizing merely the sensitivity of certain sol-
vatochromic probes, few studies reported the detection of metal
ions directly by changes in the uorescence. The ions change
the emission of the probes attached to thermoresponsive units
and 300 ppm 2,6-di-tert-butyl-4-methylphenol) and oligo-
(
ethylene glycol) monomethylether methacrylate (OEGMA,
ꢁ
1
Aldrich, M
2
n
¼ 475 g mol , stabilized with 100 ppm MEHQ and
00 ppm 2,6-di-tert-butyl-4-methylphenol) were passed through
0
a basic alumina column to remove inhibitors before use. 2,2 -
Azobisisobutyronitrile (AIBN, Acros, 98%) was crystallized from
methanol before use. Other chemicals were purchased and used
without further purication as received: N-isopropylacrylamide
25
in a water–ethanol environment, or incorporated into micro-or
14,24
nano-gels.
Here, rather than being only a scaffold, the
polymer properties, i.e., the intrinsic response, can be exploited
to endow a second functionality or to adjust the detection effi-
ciency in terms of selectivity and sensitivity.
(
4
NIPAm) (Acros, 99%, stabilized with 500 ppm MEHQ),
-(diethylamino)-2-hydroxybenzaldehyde (Synthon, 99%),
7
diethylmalonate (Merck, >98%), piperidine (Acros, 99% extra
pure), sodium hydroxide (Chemsoulte, 99%), pyridine (Merck,
Motivated by these previously reported designs, we envis-
aged to construct a proof of concept, multifunctional water
soluble material which can report changes in solution temper-
ature as well as in analyte concentration through its emission
prole. For this purpose, we combined the fundamental ability
of the responsive polymer to react on temperature changes with
the sensitivity of the probe's uorescence to a specic, targeted
biological macromolecule. Instead of utilizing an additional
>
99%), 2-aminoethylmethacrylate hydrochloride (Acros, 90%
stabilized with 500 ppm phenothiazine), thionyl chloride
Acros, 99.7%), and phosphate buffered saline (PBS, Fluka).
(
Synthesis of 7-(diethyl amino)-2-oxo-2H-chromene-3-
carboxylic acid (7-(diethylamino) 3-carboxy coumarin) (1)
moiety for molecular recognition, here, the uorophore itself 4-Diethylaminosalicylaldehyde (3.86 g, 0.02 mmol), dieth-
ꢁ
1
acts as the recognition unit. We incorporated a solvatochromic ylmalonate (6.4 g, r ¼ 1.06 g mL , 0.04 mmol), and piperidine
ꢀ

uorophore,
7-(diethylamino)-coumarin-3-carboxylic
acid (82 mL) were stirred in 60 mL of ethanol at 120 C for 10 h. The
(
DEAC) via statistical copolymerisation into two well-estab- mixture was then cooled to room temperature, and 60 mL of
ꢀ
lished types of thermoresponsive polymers based on NaOH (2 M) was added. The mixture was stirred at 120 C for
either NIPAm or on
0
a
mixture of 2-(2 -methoxyethoxy)- 30 min. The pH of the reaction mixture was adjusted to ca. 2 by
6
374 | J. Mater. Chem. B, 2013, 1, 6373–6381
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titration of hydrochloric acid while cooling the mixture in an ice
OEGMA-based copolymer (P2). DEAC-functionalized mono-
ꢁ1
bath. The orange precipitate formed was ltered off and crys- mer (2) and a mixture of OEGMA (475 g mol ) and MEO MA
2
ꢀ
tallized in ethanol. Yield: 2.89 g (11.06 mmol, 55%); mp. 230 C. with ca. 1 mol% of AIBN were dissolved in DMF (10 wt%). The
+
Mass spectrum (ESI) signal at 262.1079 [M + H] , 284.0899 [M + ask was sealed with a rubber septum; the mixture was purged
+
+
+
ꢀ
Na] found: 262.1092 [M + H] , 284.0924 [M + Na] . Elemental with dry nitrogen for 30 min, and heated in an oil bath to 65 C
analysis (C14
): calcd: C ¼ 64.36%, H ¼ 5.79%, N ¼ for 17 h. The mixture was then diluted with de-ionized water,
.36%, found: C ¼ 64.03%, H ¼ 5.60%, N ¼ 5.79%; FT-IR (cm
selected bands: 2982 n(CH ), 2935 n(CH ), 1737 n(C]O), 1669 membrane, molecular weight cut off 4000–6000). The puried
H
15NO
4
ꢁ
1
5
)
and dialyzed against de-ionised water (Roth, ZelluTrans
2
2
1
n(C]O), 1611 n(C]C), 1582 n(C]C); H NMR (300 MHz, polymer was isolated by freeze drying, to yield a yellow gluey
CDCl ): d ¼ 12.35 (s, 1H, OH), 8.65 (s, 1H, C]CH), 7.45 (d, J ¼ mass (labelled p(MEO MA-co-OEGMA): 875 mg MEO MA,
3
2
2
1
2
9
.0 Hz, 1H, CH
), 6.71 (dd, J ¼ 9.0 Hz, J ¼ 2.4 Hz, 1H, 555 mg OEGMA, 13 mg AIBN, 24 mg (2), 10 mL ethanol, yield:
aromatic
CHaromatic), 6.53 (d, J ¼ 2.2 Hz, 1H, CH
), 3.50 (q, J ¼ 1.33 g (90%) of P2).
aromatic
13
7
(
1
.1 Hz, 4H, CH
75 MHz, CDCl
): d ¼ 165.6, 164.5, 158.2, 153.9, 150.4, 132.0,
11.0, 108.7, 105.8, 97.0, 45.4, 12.5.
2
), 1.27 (t, J ¼ 7.1 Hz, 6H, CH
3
), C NMR
3
Preparation of the monoclonal anti-DEAC antibody
The monoclonal antibody G100-HB9 was generated by standard
26
hybridoma technology using electrofusion. In brief, spleen
Synthesis of 2-(7-(diethylamino)-2-oxo-2H-chromene-3-
carboxlic acid amido) ethyl-methacrylate (2)
cells of Balb/c mice immunized with a DEAC–KLH-conjugate
keyhole limpet hemocyanin, emp biotech, Berlin, Germany)
(
ꢁ
1
Thionyl chloride (0.33 mL, r ¼ 1.64 g mL , 4.59 mmol) was
carefully added to the mixture of 7-(diethylamino)-2-oxo-2H-
chromene-3-carboxylic acid (1, 1.00 g, 3.83 mmol) in pyridine.
The colour of the mixture changed from green to red and then
were fused with the myeloma cell line P3 ꢂ 63Ag8.653 (ATCC
CRL-1580). Positive clones were screened for binding DEAC–
ovalbumin (emp biotech) in an indirect ELISA and veried in a
competitive ELISA using free DEAC. Clone G100-HB9 was mass-
produced in serum free medium and the antibody was puried

nally to black. Upon stirring this mixture for 1 h at room
temperature, 2-aminoethylmethacrylate hydrochloride (755 mg,
.67 mmol) was added to the mixture. Aer stirring overnight at
27
on protein A columns to avoid contamination with SLPI.
4
room temperature, the solvent was evaporated, the residue was
dissolved in a small amount of acetone, and this solution was
Photophysical characterization
poured into water. The yellow precipitate was collected via Absorption spectra of solutions were recorded with a Varian
ꢀ

ltration. Yield: 1.28 g (3.31 mmol, 86%); mp. 103 C. Mass Cary 5000 spectrometer. Turbidity measurements were per-
+
spectrum (ESI) signal at 373.1763 [M + H] , found: 373.1735 [M + formed on a Varian Cary 50 UV-VIS photometer. The trans-
+
H] . Elemental analysis: calcd: C ¼ 64.5%, H ¼ 6.5%, N ¼ mittance of polymer solutions at 700 nm was monitored as a
ꢁ
1
7
.52%, found: C ¼ 62.9%, H ¼ 6.25%, N ¼ 7.55%; FT-IR (cm
)
function of temperature (with a heating–cooling cycle at rates of
ꢀ
ꢁ1
selected bands: 3334 n(NH), 2975 n(CH ), 2926 n(CH ), 1698 0.1 C min , cell path length of 12 mm). Steady-state uores-
2
2
1
n(C]O), 1650 d(NH), 1616 n(C]C), 1583 n(C]C); H NMR (300 cence spectra were recorded with a HORIBA Jobin Yvon Fluo-
MHz, CDCl
): d ¼ 9.08 (s, 1H, CONH), 8,70 (s, 1H, C]CH), 7.43 rolog-3. The samples in PMMA cells of 1 cm path length were
3
(
d, J ¼ 9.0 Hz, 1H, CHaromatic), 6.65 (d, J ¼ 9.0 Hz, 1H, excited at the wavelength of their maximum absorption and the
CHaromatic), 6.50 (s, 1H, CHaromatic), 6.20 (s, 1H, emission was detected at the front face. PL quantum efficiencies
C]CH2,methacrylate), 5.60 (s, 1H, C]CH2,methacrylate), 4.32 (t, J ¼ were determined with a Hamamatsu C9920 set-up, including an
5
4
.5 Hz, 2H, CH ), 3.76 (q, J ¼ 5.6 Hz, 2H, CH ), 3.46 (q, J ¼ 7.1, integrating sphere combined with a photonic multi-channel
2
2
H, CH ), 1.98 (s, 3H, CH
), 1.24 (t, J ¼ 7.1, 6H, CH ) analyzer. The time-resolved uorescence measurements were
2
3,methacrylate
3
1
3
C NMR (75 MHz, CDCl ) d ¼ 167.4, 163.5, 162.9, 157.9, 152.8, performed by using
a
single photon counting setup
3
1
48.4, 131.3, 126.2, 110.2, 108.6, 96.8, 63.6, 45.3, 38.6, 18.5, 12.6. (TCSPC) with a Becker&Hickl PML-spectrometer (modied Oriel
MS-125). As the excitation source, a picosecond diode laser at
4
05 nm was operated at a repetition rate of 20 MHz. The
Synthesis of the copolymers
detector comprises a Becker&Hickl PML-16-C-1 (modied
Hamamatsu) multi-alkaline photomultiplier. Considering that
NIPAm-based copolymer (P1). DEAC-functionalized mono-
mer (2) and NIPAm with ca. 1 mol% of AIBN were dissolved in
ethanol. The ask was sealed with a rubber septum. The
i¼n
X
ꢁt=si
I
PLðtÞ ¼
a
i
e
;
(1)
mixture was purged with dry nitrogen for 30 min, and heated to
i¼1
ꢀ
6
5 C for 17 h. The solution was cooled, the solvent evaporated,
th
i i
where s is the lifetime and a is the amplitude of the i
component, the intensity-averaged uorescence lifetime was
calculated as
and the residue was dissolved in a small amount of acetone. The
solution was precipitated into diethyl ether. The precipitate was
redissolved in acetone, and precipitated into diethyl ether, at
least thrice, until the supernatant liquid became colourless. The
polymer was dried to give a yellowish powder (labelled pNIPAm:
,
i¼n
i¼n
X
X
2
hsi ¼
a
i
s
i
a
i
s
i
(2)
i¼1
i¼1
1
8
.0 g NIPAm, 14 mg AIBN, 34 mg (2), 10 mL ethanol, yield:
60 mg (81%) of P1).
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Paper
A cuvette holder equipped with a Peltier element was used to
The analytical data of these copolymers are summarised in
perform temperature-dependent measurements. Each spec- Table 1. The polymerization was performed such that it yielded
trum is acquired aer reaching equilibrium, i.e., assuring that roughly the same degree of polymerisation and dye concentra-
both the intensity and the shape of the spectrum are stable tion for both polymers. Because of the higher average molar
15 minutes aer reaching the desired temperature.
w n
mass of the repeat unit, the M and M of P2 are larger than
those of P1. The incorporation of the dye into the copolymer
structure was quantied via UV-VIS spectroscopy. As expected,
the incorporation of the hydrophobic dye shied the LCST of
original polymers to lower temperatures. The fact that the
average number of uorophores per polymer chain is greater
than one might result in photophysical characteristics different
than those of the isolated monomer.
Competitive enzyme-linked immunosorbent assay (ELISA)
tests
To evaluate the binding of the monoclonal antibody (G100-HB9)
to the DEAC-containing polymers, a competitive ELISA was
performed. G100-HB9 was added together with different dilu-
tions of polymers or DEAC-labelled monomer 2, to the DEAC–
ovalbumin coated and blocked (5% NCS in PBS) microtiter plate
for 1 h. Aer washing with tap water, a goat anti-mouse-IgG
antibody conjugated to horseradish peroxidase (HRP) was
added and the ELISA stained with a tetramethylbenzidine
As their photophysical properties depend strongly on
polarity, viscosity, pH, and hydrogen bonding ability of the
microenvironment, 7-aminocoumarins have received consider-
30,31
able interest for chemo-sensing applications.
Due to the
28
push–pull character of the conjugated system, upon electronic
(TMB) substrate according to standard protocols.
excitation, the excited state forms an intramolecular charge
2
transfer (ICT) state. For some derivatives of 7-aminocoumarins,
Results and discussion
depending on the strength of the electron donating and
32
21,29
accepting capabilities of the related moieties, the amino group
undergoes an internal rotation upon photoexcitation, resulting
in full charge separation through a perpendicularly orientated
In analogy with a recently reported procedure,
we prepared
the uorescent thermoresponsive polymers by free radical
copolymerisation of the DEAC-functionalized methacrylate
monomer with an appropriate amount of NIPAm or with the
mixture of MEO MA and OEGMA. Fig. 1 shows the synthesis of
2
the uorescent monomer (2) and the chemical structure of the
22
donor orbital with respect to that of the acceptor. This is called
a twisted ICT (TICT) state, the formation of which is facilitated
in polar solvents, and which decays non-radiatively leading to a
33–35
decreased yield of emission from the ICT state.
A complete
resulting copolymers.
list of photophysical ndings of the DEAC-functionalized
monomer dissolved in a variety of solvents can be found in
Table S1.† Due to its ICT character, when the monomer is dis-
solved in solvents of high polarity, the emission maximum
shis bathochromically accompanied with a decrease in
2
quantum yield and uorescence lifetime. These effects are
more pronounced in protic solvents. Also, the quantum yield is
considerably enhanced in solvents of high viscosity, ascribed to
inhibition of the TICT state.
As shown in Fig. 2, we observe marked changes in the
emission properties of the aqueous P1 solution upon increasing
its temperature. In the photoluminescence (PL) spectra, the
ꢀ
formerly two, weakly emitting bands at 20 C evolve into a single
emission component at higher temperatures. While the red-
emitting band disappears, the intensity of the higher energy
emission increases by about 6 fold, with the maximum shiing
to shorter wavelengths. The quantum yield of the dye attains a
value even higher than that measured in a structurally similar
solvent, i.e., N-methylformamide (4: 9%). These ndings point
to a highly rigid/viscous environment which limits encounters
of the dye with water and enhances encounters between poly-
mer segments. Accordingly, absorbance and time-resolved PL
measurements exhibit marked changes upon increasing the
solution temperature, e.g., an abrupt increase in scattering
intensity and a longer lifetime extracted for emission at 475 nm
(Fig. S1 and S2†).
Note that the emission spectrum of the aqueous monomer
solution exhibits no such response to temperature in the
examined range (Fig. S3†). Therefore, the temperature-triggered
changes in the emission prole of P1 are attributed to its phase
Fig. 1 Synthesis of the DEAC-functionalized monomer (2) and of the thermor-
esponsive copolymers: labelled pNIPAm (P1), and labelled p(MEO
OEGMA) (P2).
2
MA-co-
6
376 | J. Mater. Chem. B, 2013, 1, 6373–6381
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Journal of Materials Chemistry B
Table 1 Molecular characterisation and thermoresponsive properties of the copolymers
appa
n
ꢁ1
app
w
appa
n
b
c
d
e
ꢀ
f
ꢀ
Polymer
M
[kg mol
]
M
/M
DP
Dye content [% mol]
NC
Cloud point [ C]
T (50%) [ C]
P1
P2
23
44
1.9
2.5
196
177
1.1
0.9
2.2
1.6
20
41
23
44
a
b
Determined by size exclusion chromatography in DMF, using polystyrene standards. Nominal number average of monomer repeat units per
c
polymer chain. Calculated from the UV-VIS spectra of the monomer and the copolymer solutions in CHCl according to Beer–Lambert's law
3
ꢁ1
ꢁ1
d
e
(molar extinction coefficient (3): 35.000 L mol cm ). Average number of uorophores per polymer chain. Onset of the decrease of
ꢁ1 f
ꢁ1
transmission in PBS solution (2 g L ). Temperature at which the transmission is reduced by 50% in PBS solution (2 g L ).
ꢁ1
Fig. 2 The evolution of the PL spectra of P1 in PBS (0.15 g L ) with temperature,
recorded at an excitation wavelength of 400 nm.
transition, which sets in as the solution temperature is raised
ꢀ
above 20 C (cf. Table 1). Notably, the emission spectra of the
monomer and of the polymer solution in PBS do not resemble
each other, i.e. the monomer spectrum involves only the peak
centred at about 475 nm. For the polymer, the species respon-
sible for the emission at longer wavelengths also exhibit a
ground-state absorption (Fig. S1†). With its relatively long
emission lifetime (>10 ns), this feature is only encountered in
aqueous solutions of P1 below its LCST and disappears when
ꢁ4
ꢁ1
the P1 concentration is lower than 10 g L (Fig. S4†). These
ndings evidence the presence of DEAC aggregates, promoted
presumably by interchain associations. Although it is generally
favoured that the photophysical properties of the label are

ꢁ
1
ꢀ
Fig. 3 (a) Fluorescence spectra of P1 solution in PBS (0.005 g L ) at 15 C
containing varying amounts of the antibody [from 6.7 nM to 660 nM]. The
direction of the arrows shows the increase in antibody concentration. The exci-
preserved upon incorporation into the polymer structure, the tation wavelength is 400 nm. (b) The relative change of the maximum emission
intensity as a function of antibody concentration. Data are taken from Fig. 3a.
possibility to modulate the relative emission intensity of the two
emitting species, i.e., the single dye and its aggregates, by a
stimulus, such as the temperature here, makes this polymer very
promising and advantageous to construct a dual-colour sensor.
ꢁ
1
polymer concentration of 0.005 g L , the presence of the
Apart from the distinguished uorescence response to vari- antibody in the solution at any concentration between about 6
ations in solution temperature, P1 alerts the presence of a and 300 nM invokes correspondingly different emission inten-
monoclonal antibody through its optical spectra. When the sity values, evident from the linear region shown in Fig. 3b.
anti-DEAC antibody is introduced into the aqueous P1 solution, Also, as scattering does not interfere, the antibody-induced
remarkable changes in the PL characteristics are induced changes in the absorbance spectrum are clearly visible, and
(Fig. 3). Despite the similarity to those triggered by increasing qualitatively correlated with its concentration (Fig. 4).
temperature, these changes are more drastic: while the aggre- In Fig. 3 and 4, almost complete disappearance of the
gate band is suppressed, the emission from the isolated dye spectroscopic features attributed to DEAC aggregates evidences
moieties at shorter wavelengths builds up. The slight blue shi their disruption which might indicate changes in chain
PL
max
in l
points to a more hydrophobic environment around the conformation/association. In parallel to these, the presence of
dye. As more protein is present in the solution, we measure a antibody in the solution shis the LCST of P1 to somewhat
longer lifetime of the emission at 475 nm (Fig. S5†). For a higher temperatures accompanied by lowered turbidity values
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37
specic binding of Avidin to the biotin moieties. The direction
of the LCST shi depends thus on the type of the analyte, on the
recognition unit, and on the nature of interactions between
these groups and water. It must be emphasised that pNIPAm
synthesised analogously to P1 but devoid of DEAC moieties does
not exhibit changes in its turbidity prole in the presence of the
antibody (Fig. 5b). This reveals that the LCST shi of P1 is
caused by the specic binding of the antibody to DEAC, rather
than a complexation with NIPAm units. Apparently, when the
antibody captures a dye, the co-aggregated chains are separated
and further aggregation is inhibited. Within the binding pocket
of the antibody, the dye has a rigid, water-free, and relatively
ꢁ
1
ꢀ
Fig. 4 Absorbance spectra of P1 solution in PBS (0.005 g L ) at 15 C containing nonpolar environment. This situation may be comparable to
varying amounts of the antibody [from 6.7 nM to 660 nM]. The direction of the
the enhanced uorescence of DEAC dyes upon their inclusion
arrow shows the increase in antibody concentration.
34
in the hydrophobic nanocavities of cyclodextrins. The increase
in emission intensity was attributed to the inhibition of the
TICT state in such an environment.
(
Fig. 5a). Here, in addition to the increased hydrophilicity that
the polymer chains attain upon complexation with the anti-
body, steric hindrance might play a role in retarding and/or
hindering the interchain aggregation.
Nevertheless, the effect of the interaction of a thermores-
ponsive polymer with another macromolecule is a priori not
obvious. For instance, Rimmer et al. reported that binding of
highly branched pNIPAm to a Gram negative bacterium
decreased the LCST of the polymer by more than 20 C. On the
other hand, the phase transition temperature of a biotinylated
OEGMA-based copolymer shied to higher temperatures upon
ꢀ
Injection of the antibody into the solution of P1 at 40 C, i.e.
well above its LCST, does affect neither the shape nor the
intensity of the PL spectrum. However, when this mixture is
ꢀ
cooled down to 15 C, we once again detect marked photo-
physical changes indicative of the binding event (for the
evolution of PL and absorbance spectra with the relevant heat-
ing and injection steps, see Fig. S6†). Regulating the access of a
recognition unit linked to a thermoresponsive polymer by the
ꢀ
36
particular temperature sensitivity of the chains has been
38–40
reported before.
Apparently, DEAC is accessible to the
antibody only when P1 chains adopt a well-extended confor-
mation, whereas their collapse blocks the binding sites.
Therefore, by exploiting the intrinsic response of pNIPAm to
temperature, i.e., changing the conformational state of the
chains, we can switch the protein sensing on and off.
The control of biological sensing with temperature led us to
the question whether the antibody bound to P1 is available for
competitive binding. Fig. 6 shows that aer treatment with a 20
fold excess of DEAC molecules (the functional monomer 2), the
-
1
mixture of P1 (0.01 g L ) with 0.17 mM of antibody exhibits a “re-
clouding”. This ternary mixture was prepared by injecting the
DEAC labelled monomer into the solution, comprising of P1
ꢀ
ꢀ
and the antibody, either at 15 C followed by heating to 40 C or
ꢀ
directly at 40 C (lines c and d, respectively). Scattering is clearly
identied by the appearance of an absorption tail which extends
towards the NIR region. Note that the addition of the uores-
cent monomer to pure P1 solution does not introduce such a
scattering contribution (Fig. S7†). In all experiments, the heat-
ing times and rates were identical, excluding the effect of
annealing on the size/density of globules formed, therefore on
the scattering intensity.
The increase in turbidity (compared to the binary mixture of
P1 with the antibody) in the presence of the DEAC-labelled
monomer indicates that a fraction of the antibody is released
ꢀ
from the polymer-attached DEAC sites at 40 C. Apparently, the
detached antibody forms new complexes with the monomer
available in the solution so that P1 chains can more efficiently
collapse and inter-aggregate.
ꢁ
1
Fig. 5 Temperature–transmission profiles of (a) P1 solution in PBS (0.1 g L
)
containing varying amounts of the antibody (AB) (0, 0.03, 0.17, and 0.33 mM) and
ꢁ1
The question then remains whether this competitive binding
(
b) a solution of unlabelled pNIPAm in PBS (0.01 g L ) in the presence (red
ꢀ
triangles) and absence of the antibody (0.17 mM) (black squares).
occurs already at 15 C or whether the process necessitates
6
378 | J. Mater. Chem. B, 2013, 1, 6373–6381
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ꢁ
1
Fig. 6 Absorbance spectra of aqueous solutions of P1 (0.01 g L ) recorded at
Fig.
7
Temperature–transmission profile of the aqueous solution of P1
ꢀ
ꢁ1
4
0
C: (a) pure P1 solution, (b) the mixture of P1 with antibody (0.17 mM), (c) the
(0.01 g L , black squares), containing 0.17 mM of antibody (red triangles), and
containing both the antibody (0.17 mM) and the DEAC-labelled monomer (4 mM)
(green stars). The profiles were recorded for the same solutions labelled a, b, and
d of Fig. 6.
ternary mixture of P1 with antibody (0.17 mM) and with the DEAC-labelled
ꢀ
monomer (4 mM) added at 15 C before heating, and (d) the ternary mixture of P1
with antibody (0.17 mM) and with DEAC-labelled monomer (4 mM) added at
ꢀ
ꢀ
4
0 C. The heating time after reaching the designated temperature, i.e., 40 C was
identical and lasted for 60 min.
sensor assays utilizing thermoresponsive polymers. Scheme 1
depicts the proposed mechanism of sensing with P1.
elevated temperatures. One way to address this is to examine the
effect of monomer addition at 15 and at 40 C on the uorescence
In comparison, we also examined the effect of temperature
on photophysical properties of the OEGMA-based copolymer
P2. To our surprise, no change in emission properties could be
associated with its phase transition. Fig. 8 demonstrates the
temperature independent shape of the PL spectrum. As the
ꢀ
spectrum of the binary mixture at the respective temperature. As
pointed out above (cf. Fig. 2), the emission spectrum of the P1
solution below its LCST includes an aggregate-assigned band at
ca. 575 nm. While this feature is still visible in the spectrum of

uorescence intensity decreases gradually with the increase in
temperature, the average uorescence lifetime also shortens
Fig. S9†). These observations are attributed to the heat-facili-
ꢀ
the P1 solution at 40 C, it becomes fully suppressed upon
ꢀ
addition of a sufficient amount of the antibody at 15 C, below
(
the LCST (cf. Fig. 3a). Here, we detect that the aggregate emission
22,42
tated non-radiative deactivation processes.
ꢀ
reappears as the monomer is injected into the mixture at 40 C
Although copolymers like P2 exhibit an LCST-transi-
(
Fig. S8a†), while no such effect is present for the low tempera-
29,37,43
PL
tion,
the constant lmax below and above the cloud point
ꢀ
ture spectrum of the ternary mixture prepared at 15
C
suggests that the dye's environment is not affected by phase
transition. This is quite impressive considering the distinct
response of DEAC to pNIPAm phase transition. This depen-
dence of the dye's uorescence response to the chemical
structure of the polymer was observed for another sol-
vatochromic uorophore, a naphthalimide derivative, incorpo-
rated into NIPAm- or OEGMA-based copolymers analogous to
(Fig. S8b†). This shows that below the LCST, the antibody is not
released from the polymer. The driving force of the observed
ꢀ
antibody detachment at 40 C is yet not clear, but it might be
related to a temperature-activated process, e.g., increased
mobility/diffusion of the sensing components or the dehydration
of the chains. There have been examples in which the latter was
assigned as the trigger of similar kinds of dissociation mecha-
nisms. For example, Stayton and co-workers reported that the
biotin bound-streptavidin functionalized-NIPAm chains released
a signicant amount of the biotin upon heating the mixture
21
P1 and P2, respectively. Certain peculiarities about the
40,41
above its LCST.
Importantly, at this temperature, the released
antibody cannot re-bind to the DEAC sites of P1 as chain collapse
and aggregation partly block the accessibility of the polymer-
bound dye. Instead, it remains stably bound to the low molar
mass dye present excessively in the solution. The relatively rm
nature of the new antibody–DEAC complexes is evidenced by the
temperature–transmission prole of the ternary mixture that was
cooled down to room temperature and reheated, shown in Fig. 7.
The ternary mixture displays a situation that is intermediate
Scheme 1 Dual responsiveness of P1 to the presence of antibody and to
between the presence of only pure P1 (no interference to inter- increasing the solution temperature above the cloud point. In the absence of
antibody and/or at low temperatures, the emission from the DEAC label is weak.
chain aggregation) and the case where P1 solution is saturated
with the antibody (full suppression of inter-aggregation).
Upon formation of globules at elevated temperatures and/or binding of the
antibody at low temperatures, the emission becomes intense. When heated, the
We, therefore, suggest that the competitive binding mecha-
antibody attached to P1 chains is released out to the solution where it forms new
nism is activated only above the cloud point of the polymer and
complexes with the DEAC-labelled monomer. This promotes inter-aggregation of
that this proof of concept experiment might pave the way for P1 chains.
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TICT state, this new environment might be less hydrophobic
than that provided by the polymer chains, leading to the slight
red-shi in the emission spectrum. On the other hand, the
antibody complexion with a certain fraction of the dyes might
enhance the water exposure of some others as a result of the
expansion of the chains. A detailed analysis on the processes
responsible for such spectral changes is on the way.
Conclusions
We demonstrate the synthesis and photophysical character-
isation of two thermoresponsive polymers bearing a covalently
Fig. 8 The effect of temperature on the fluorescence characteristics of P2 in PBS attached TICT-type DEAC dye, promising for optical sensing
ꢁ
1
(
0.15 g L ). The excitation wavelength is 400 nm. The direction of the arrow
applications. In aqueous solutions of the OEGMA-based poly-
mer, the quantum yield of DEAC is relatively high. The dye is
mostly insensitive to the polymer phase transition, and to the
anti-DEAC antibody. In contrast, aqueous solutions of the
NIPAm-based polymer exhibit a very weak emission at low
temperatures or in the absence of antibody, which increases
dramatically above the LCST or upon binding of the antibody.
Heating of the solution of antibody-bound polymer above its
cloud point releases a fraction of the immobilized antibody. In
the presence of the DEAC-labelled monomer in this mixture, the
detached antibody binds competitively to the low molar mass
monomer allowing the free polymer chains to aggregate.
Consequently, on one hand, this work presents a water soluble
ꢀ
shows the increase in the solution temperature from 20 to 65 C.
aqueous solubility and phase transition behaviour of the P2-like
copolymer were found out, presumably associated with the lack
of intermolecular hydrogen bonding and the limited dehydra-
tion of long OEG side chains. When bound to a poly-
methacrylate backbone, it seems that the rather hydrophobic
microenvironment of the uorophore is preserved due to a
particular arrangement of neighbouring molecules. Therefore,
although P2 is relatively emissive in aqueous solutions (4: 25%),
it does not feature the temperature sensing ability of P1.
We, lastly, addressed the effect of antibody binding on the
optical spectra of P2. It was proven by ELISA tests that the
antibody also binds to the DEAC moieties of P2, upon which the
LCST shis to slightly higher temperatures (Fig. S11 and S12†).
However as opposed to P1, the emission peak displays a bath-
ochromic shi of ca. 8 nm with a slight decrease in its intensity
while there is no signicant change in the absorbance spectrum
polymer with
a distinct uorescence response towards
temperature and a biological macromolecule. On the other
hand, by comparing two analogous DEAC-labelled polymers,
the need to design a specic polymer structure is stressed, e.g.,
composed of alternative thermo-sensitive blocks, one of which
only produces a marked uorescence signal of the thermal
transition, or of the binding event.
(
Fig. 9). With respect to PL spectra, similar observations were
reported for a chromophore-modied cyclodextrin upon its
44
interaction with guest molecules. Bound to the guest mole-
cule, the chromophore inherently located in the interior of the
Acknowledgements
hydrophobic cavity of dextrin was excluded into the polar The work was funded by the BMBF-Initiative “Spitzenforschung
aqueous solution. Although the dyes which are bound to the und Innovation in den neuen L ¨a ndern” within the cooperative
antibody have now less segmental mobility, i.e. an inhibited project “Taschentuchlabor – Impulszentrum f u¨ r Integrierte
Bioanalyse” (FKZ 03IS2201B). S.I. thanks Sina Reiter (University
of Potsdam) for her assistance in the lab and Sandor Dippel
(University of Potsdam) for turbidity measurements.
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