Turning the heat on conjugated polyelectrolytes: An off-on ratiometric nanothermometer

Article abstract of DOI:10.1039/c5cc08541a

We report a self-referenced ratiometric nanothermometer based on short conjugated polyelectrolytes. An amphiphilic macromolecule destabilizes the polymer π-π stacking and makes it possible to shift the equilibrium between the less emissive aggregated state (520 nm) and the brighter individual chain (450 nm) within 20.0°C and 70.0°C.

Full text of DOI:10.1039/c5cc08541a

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Turning the heat on conjugated polyelectrolytes:
an off–on ratiometric nanothermometer†
Cite this:DOI: 10.1039/c5cc08541a
Ghinwa H. Darwish, Ali Koubeissi, Tharallah Shoker, Samir Abou Shaheen and
Pierre Karam*
Received 14th October 2015,
Accepted 9th November 2015
DOI: 10.1039/c5cc08541a
www.rsc.org/chemcomm
We report a self-referenced ratiometric nanothermometer based probe and (ii) it must possess a two distinct temperature
on short conjugated polyelectrolytes. An amphiphilic macromolecule dependent emission states.18
destabilizes the polymer p–p stacking and makes it possible to shift
Conjugated polyelectrolytes (CPEs) are a class of polymers
the equilibrium between the less emissive aggregated state (520 nm) with high extinction coefficients and exceptional brightness.19–22
and the brighter individual chain (450 nm) within 20.0 8C and 70.0 8C. Their photophysical and photochemical properties are highly
dependent on their microenvironment. Whether dissolved in
Temperature is a fundamental variable that controls and drives hydrophobic or hydrophilic media, their fluorescence emission
many chemical, biological and physical processes.1–4 Nowadays, dramatically changes.23–25 In particular phenylene based polymers
there is a tremendous drive to explore and understand processes exhibit a pronounced spectral emission shift when going from the
at the nanoscale level in many fields of science and technology.5–7 aggregated to the free chain.26 As such, they have been employed
For instance, a nanotemperature probe will provide a powerful as piezochromic probes27 and membrane reporters.28
tool to unravel the biological functions induced or correlated
Herein, we report on the use of short phenylene based
with local cellular temperature fluctuations. Mapping localized conjugated polyelectrolytes to develop a single component
hot spots in engineered nanothermoelectric devices is also of reversible nanothermometer with a ratiometric readout, an
fundamental importance.8
excellent sensitivity and a wide linear range. For practical
Many promising approaches have been recently developed.5,6,9,10 purposes, we will report the ratiometric signal readout as the
Non-invasive methods, such as fluorescent or lifetime-based probes, ratio of the integrated intensity between 435 nm and 509.9 nm
are advantageous because they allow accurate signal readout (IBlue) and between 510 nm and 650 nm (IRed).
with minimal interference with the system of interest.11–15 An
In water, short poly(phenylene ethynylene) carboxylate (PPE-CO2),
ideal probe must remain stable over time to avoid frequent driven by the p–p stacking and the hydrophobic-backbone
calibration and false readings. A fluorescence signal is, however, interactions, aggregates. Subsequently, its fluorescence emission
prone to many external factors which might compromise is mostly from excimer species leading to a broad, red-shifted and
the thermometric response. Probes with ratiometric signals structureless spectra with a maximun emission at 520 nm and a
minimize errors produced by the light source fluctuations, the quantum yield of 0.14 Æ 0.04 (Fig. 1B).26 In ethanol, PPE-CO2 is
detector instability, and the optical setup, as well as being well dissolved and mostly emits from disaggregated individual
unaffected by changes in fluorophore concentration.16,17 They polymer chains with a well-structured emission band at 450 nm
often consist of two subunits each with a different thermal and a quantum yield of 0.62 Æ 0.03 (Fig. 1B).26 This distinct
response property. Any fluctuation in the excitation source is difference in the spectral signatures of the aggregated polymers,
then eliminated by the signal ratio. However, fluorophores versus individual chains, satisfied our aforementioned criteria and
photobleach at different rates and are quenched by different made PPE-CO2 an appealing material for us to test its thermal
species. We therefore searched for a material that satisfies two response. Poly(phenylene ethynylene) carboxylate with an average of
important criteria; (i) it must be a single component fluorescent 7 monomer repeating units was therefore prepared using Sonaga-
shira coupling following previously reported procedures.26,29
Fig. 2 summarizes the fluorescence intensity changes
obtained for PPE-CO2-7 upon controlled heating between
20.0 1C and 70.0 1C with a step increment of 5.0 1C. The
The Department of Chemistry, American University of Beirut, P.O. Box 11-0236,
Beirut, Lebanon. E-mail: pierre.karam@mail.aub.edu.lb
† Electronic supplementary information (ESI) available: Detailed information on
fluorescence intensity increase was accompanied by a gradual
blue-shift (from 520 nm to 485 nm) and a slight decrease
the PPE-CO2-7 synthesis, NMR spectra of the synthesized molecules and Fig. S1–
S9. See DOI: 10.1039/c5cc08541a
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Fig. 3 (A) Fluorescence emission spectrum of PPE-CO2-7 in complexation
with PVP acquired at different temperatures between 20.0 1C and 70.0 1C with
a 5.0 1C increment. (B) Integrated intensity ratio before (dark area in A; IBlue
)
Fig. 1 (A) Schematic and chemical structure of the short anionic conjugated
polyelectrolyte poly(phenylene ethynylene) carboxylate with an average of
7 monomer repeating units (PPE-CO2-7) and the amphiphilic polymer
polyvinylpyrrolidone (PVP). (B) Normalized absorption (solid) and emission
(dashed) spectra of PPE-CO2-7 in 10 mM HEPES and 150 mM NaCl (pH 7.0)
buffer (blue) and ethanol (red) upon excitation at 420 nm.
and after (light area in A; IRed) the isoemissive point at 510 nm versus
temperature. The thermal response was fitted into a second degree
polynomial function which gave a maximum relative sensitivity of 2.78%
at 20.0 1C. The error bars represent the standard deviation of ten
independent measurements. The maximum and minimum represent the
lowest and highest recorded values at each measured temperature. (C)
Fluorescence emission spectrum of PPE-CO2-7 in complexation with PVP
during heating and cooling cycles at 20.0, 30.0, 40.0, 50.0 and 60.0 1C. (D)
Cycling a solution of PPE-CO2-7: PVP between 20.0 1C and 55.0 1C. The
lines connecting the experimental points are for visual aid. All experiments
were performed in 10 mM HEPES and 150 mM NaCl (pH 7.0) buffer.
to break down the strong hydrophobic backbone interactions in
an anionic conjugated polyelectrolyte.30 To this end, PPE-CO2-7
was mixed with PVP (Mw = 55 000) at an optimized concentration
of 1 : 10 (CPE : PVP) polymer ratio. At room temperature, the CPE
fluorescence emission increased by 1.5 fold in the presence of
PVP (Fig. S2, ESI†). In addition to the previously reported peak
at 520 nm, a new blue-shifted maximum emission centered at
485 nm was observed coinciding with the previously observed
emission peak reported when PPE-CO2-7 solution was heated to
70.0 1C and attributed to a quasi-destabilized state.
Fig. 2 (A) Fluorescence emission spectra of PPE-CO2-7 (5 mg mlÀ1) in
10 mM HEPES and 150 mM NaCl (pH 7.0) buffer acquired between 20.0 1C
and 70.0 1C at 5.0 1C intervals upon excitation at 420 nm. (B) Ratio of the
integrated signal before (dark area in A; IBlue) and after (light area in A; IRed
)
510 nm versus temperature. The error bars represent the standard deviation
of ten independent measurements. The maximum and minimum represent
the lowest and highest recorded value at each measured temperature. Fitting
(B) into a second degree polynomial function gave a relative sensitivity of
0.99% at 20.0 1C and maximum relative sensitivity of 2.06% at 55.0 1C.
The prepared hybrid complex was subjected to a gradual
increase in temperature between 20.0 1C and 70.0 1C with a step
followed by an increase in the emission intensity. Plotting the increment of 5.0 1C (Fig. 3A). As a result, the emission peak at
ratio of the integrated signals versus the applied temperature 520 nm decreased concomitantly with an increase in the
gave an exponentially changing thermal response with a relative individual chain polymer emission peak at 450 nm. We ascribe
calculated sensitivity of 0.99% at 20.0 1C and a maximum the observed temperature dependence on fluorescence emission
relative sensitivity of 2.06% at 55.0 1C. Even though promising, intensity to the interconversion between the aggregated and the
the fluorescence shift was not significant hence limiting its individual polymer chains. We speculate that upon addition of
practical applications. The experiment also revealed that the PVP, PPE-CO2-7 aggregates are initially destabilized by the
CPE, even at temperatures as high as 70.0 1C, did not achieve a amphiphilic polymer and are further disaggregated due to the
disaggregated state as we have originally aimed for. We therefore increased vibrational and rotational freedom of the macromolecules
attributed the observed peak at 485 nm to a quasi-destabilized at higher temperatures. Subsequently, fluorescence from
state of the PPE-CO2-7 aggregates (Fig. S1, ESI†).
individual polymer chains gives rise to the distinctive and
We next searched for a material that would destabilize brighter peak at 450 nm.
the strong p–p interactions but not completely dissolve the
We then compared the fluorescence intensities coming from
conjugated polyelectrolyte backbone at room temperature. individual and aggregated polymers by integrating the intensity
In fact, our laboratory has previously exploited the extra- before and after the isoemissive point. The ratiometric variation
ordinary amphiphilic properties of polyvinylpyrrolidone (PVP) was plotted against the change in temperature. A maximum relative
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sensitivity of 2.78% at 20.0 1C was computed (Fig. 3B). This
value is substantially larger than previously reported fluorescent
ratiometric probes.17,31 The ratiometric response was reproducible
with small standard deviation calculated from ten runs. The
isoemissive point observed at 510 nm clearly reflects the two state
models that govern this system. In addition, the substantial
spectral shift in emission from 520 nm to 450 nm makes the
material ideal for implementation in microscopy imaging setups.
Light could be hence split into two imaging channels to accurately
map temperatures within diffraction limited spots. PPE-CO2 with a
longer polymer chain (108 repeating units) was also prepared
and tested, in complexation with PVP, over the same temperature
range. Upon plotting the ratiometric thermal response versus
temperature, a maximum relative sensitivity of 3.97% was obtained
at 20.0 1C (Fig. S3, ESI†). The results show that irrespective of
the polymer chain length, the PPE-CO2/PVP complex has an
excellent thermal sensitivity.
Fig. 4 Photographic images acquired using a DSLR Nikon 5100 camera
(shutter speed
= 40 ms) for a CPE solution prepared using PVP
(Mw = 55 000) at a 1 : 10 (PPE-CO2-7 : PVP) polymer ratio illuminated with
a UV lamp at 20 1C and 70 1C.
relative sensitivity of 2.78% at 20.0 1C. The shift in the fluorescence
emission was significant to allow practical microscopy imaging at
the nanoscale level. The probe signal was highly reproducible with
no hysteresis and most importantly an off–on signal response. We
believe that this strategy can be easily extended to other conjugated
polyelectrolyte systems. We are currently exploiting the effect of
different PVP molecular weights as well as the effect of the
conjugated polyelectrolyte length.
This work was supported by Lebanese National Council for
Scientific Research (LCNRS #102901) and the University
Research Board (URB #102848) at the American University of
Beirut (AUB). The authors are also thankful for the Kamal A.
Shair Central Research Science Lab (KAS CRSL) of the Faculty of
Arts and Sciences at AUB for providing access to their facilities.
A
common limitation in polymer-based thermometers
is hysteresis which adversely affects the probe performance;
different output signals could be recorded for the same
temperature value during a cycle of heating and cooling.32 In
our case, the ratiometric response was independent of the
cycling direction with a signal repeatability between 98.5%
and 99.8%, at 20.0 1C and 70.0 1C, respectively (Fig. S6, ESI†).
As shown in Fig. 3C, the solid lines are those obtained during
the heating run, and the dashed lines denote the spectra
obtained during the cooling run. A careful look reveals that
the emission intensity slightly increases for the 20.0, 30.0, 40.0
and 50.0 1C during the cooling run possibly due to polymer–CPE
restructuring, but the ratiometric nature of the probe minimizes
the adversity of this effect (Fig. S6, ESI†). At 60.0 1C, the cooling
and heating runs overlap.
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We next investigated the probe stability by performing
multiple testing runs. We recorded spectra at 20.0 1C and
55.0 1C (Fig. 3D). Over the 14 cycles, the probe ratiometric
response showed high reversibility with only 2.5% deviation
from the average at 20.0 1C and 2.1% at 55.0 1C revealing the
lack of hysteresis and a great stability over the two hours and
half of the experiment.
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´
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photostability when compared to pristine PPE-CO2-7 (Fig. S7, ESI†)
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