Fluorescent ratiometric sensing of pyrophosphate via induced aggregation of a conjugated polyelectrolyte

Article abstract of DOI:10.1039/c0cc01332c

Direct detection of pyrophosphate (PPi) in aqueous solution is demonstrated using a cationic poly(phenylene-ethynylene) with polyamine side chains. Pyrophosphate-induced polymer aggregation causes a significant spectroscopic change, which in turn allows quantification of dissolved PPi using ratiometric signals.

Full text of DOI:10.1039/c0cc01332c

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Fluorescent ratiometric sensing of pyrophosphate via induced aggregation
of a conjugated polyelectrolytew
Xiaoyong Zhao*z and Kirk S. Schanze*
Received 10th May 2010, Accepted 16th June 2010
DOI: 10.1039/c0cc01332c
Direct detection of pyrophosphate (PPi) in aqueous solution is
demonstrated using a cationic poly(phenylene-ethynylene) with
polyamine side chains. Pyrophosphate-induced polymer aggre-
gation causes a significant spectroscopic change, which in turn
allows quantification of dissolved PPi using ratiometric signals.
concentration, solution conditions and instrument settings.
Herein, we report a ratiometric sensor design for pyro-
phosphate (PPi) which utilizes two distinct fluorescence signals
corresponding to the ‘‘free chain state’’ and ‘‘aggregate state’’,
respectively. The current sensor shows high selectivity to PPi
over phosphate (Pi) in aqueous solutions. Since discrimination
between PPi and Pi is of crucial importance to assays for
detecting many enzymes,11 we believe the current system
using a ratiometric response will provide a facile platform
for CPE-based enzyme assays with built-in corrections.
In a series of studies, we have shown that poly(phenylenꢀe-
Conjugated polyelectrolytes (CPEs) are a class of water-soluble
polymers characterized by a p-conjugated backbone with ionic
solubilizing side groups.1,2 Chen et al. reported the first example
ꢀ
of using PPV-SO3 as an ultrasensitive probe for the biotin–
avidin binding event.3 Ever since, this family of materials
has been intensively studied for chemical and biological
sensor applications owing to their superior signal amplifica-
tion compared to small molecule dyes.4 Regardless of the
nature of various analytes, e.g. DNA, proteins and ions, the
signal transduction has been mainly based on three different
mechanisms: photoinduced electron transfer quenching, fluores-
cence resonance energy transfer, and analyte-induced pertur-
bation of polymer chain conformation.5 Many sensory systems
based on these mechanisms rely on fluorescence intensity
change at a single wavelength or require labeling of the probe
and/or the analyte with special dyes. In contrast, signal
transduction via analyte-induced polymer aggregation has
received less attention.6 This mechanism takes advantage of
the multivalent interaction between the polymer and the
analyte to induce absorption and/or fluorescence modulation.
Thus it does not require any extra labeling steps and allows the
analytes to be extended to molecular species without optical or
quenching properties.
ethynylene)s (PPEs) with linear ionic groups (SO3ꢀ, CO2
,
and NR3+) undergo spontaneous aggregation in aqueous
solution as signaled by a broad emission band with low
quantum yields (B5%).12–14 In order to retain the optical
properties of these materials in aqueous solution, we designed
and synthesized a diiodo-monomer (1) with branched poly-
amine side chains, as shown in Scheme 1. This monomer was
prepared through multi-step synthesis (see ESIw) and then
co-polymerized with 1,4-diethynylbenzene (2) under standard
Sonogashira coupling conditions.13 The polymerization is a
typical step-growth condensation yielding an organic-soluble
polymer (PPE-NHBoc) with a number-averaged molecular
weight of 24 kD and a polydispersity of 4.3. The neutral
polymer was then hydrolyzed using 4 M HCl in dioxane
affording the water-soluble polymer, PPE-NH3Cl with a
quantitative yield. The complete removal of the tert-butyloxy-
carbonyl (tButyl) groups was confirmed by the absence of the
singlet at d 1.40 ppm assigned to the protecting group (tBoc) in
1
the H NMR spectrum and the disappearance of carbamate
(NHCOOtButyl) absorption at 1692 cmꢀ1 in the infrared spectrum
Pyrophosphate is an anion that plays a significant role in many
biological processes, such as cellular ATP hydrolysis, DNA
and RNA polymerizations and many enzymatic reactions.7,8 It
is also known that abnormal PPi levels can cause vascular
calcification leading to severe medical conditions.9 In an
earlier contribution, we demonstrated the first example of a
PPi sensor based on an anionic CPE with carboxylate side
chains.10 The sensor design combines the efficient intrachain/
interchain exciton migration of CPEs with association–
dissociation of the fluorescence-quenching cupric ion, affording
PPi detection in the nanomolar range. The system shows high
sensitivity and selectivity to PPi in buffered aqueous solution;
however, like many intensity-based sensors, the sensory response
is subject to interferences such as variation of polymer
(Fig. S1 and S2, ESIw).
The absorption and emission spectra of PPE-NH3Cl exhibit
a muted solvent dependence in comparison to its analogue
Department of Chemistry, University of Florida, Gainesville, FL,
USA. E-mail: kschanze@chem.ufl.edu; Fax: +1-352-392-2395
w Electronic supplementary information (ESI) available: Synthetic
details and characterization; absorption and fluorescence spectra at
varied pH. See DOI: 10.1039/c0cc01332c
Scheme 1 Synthesis of cationic poly(phenylene-ethynylene) with
z Current address: Department of Chemical Engineering, MIT,
Cambridge, MA, USA. Fax: + 1-617-258-2395; E-mail: xyzhao@mit.edu
branched polyamine side groups.
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polymers with linear ionic groups. Specifically, many CPEs
with linear side chains exhibit optical properties that are
strongly dependent on the solvent.2 Typically, addition of
water (‘‘poor solvent’’) to methanol (‘‘good solvent’’) results
in the emergence of a new absorption band at longer wave-
length and the replacement of a strong emission band at
shorter wavelength with a red-shifted, weak and broad band.
In clear contrast, PPE-NH3Cl shows a negligible change of
the absorption band with a maximum at 405 nm in water
(pH B 6.0) the same as that in methanol solution, as shown in
Fig. 1. For both solutions, a strong and sharp fluorescence
assigned to the 0–0 band with lmax = 432 nm is observed for
the polymer. The polymer retains approximately 50% of its
quantum efficiency in water (F = 0.23) compared to that in
methanol (F = 0.45) and maintains a similar vibrational
structure. The optical properties were found to be strongly
dependent on the solution pH (Fig. S3, ESIw). For pH r 6.5,
the polymer shows similar spectra as in deionized water, and
varying pH in this range resulted in minimal spectral changes.
An abrupt and significant change in both absorption and
emission spectra is observed between pH 7.0 and 8.0. Further
increasing of pH only leads to slight spectral changes. The
potentiometric titration curve (Fig. S4, ESIw) of the polymer
indicates a three-step deprotonation process around pH 4, 8
and 10, which corresponds well with the pKa values of the
polyamine ligand.15 Combined with the spectral changes of
the polymer solution with pH, it is clear that deprotonation of
the side chains leads to a significant reduction of the electronic
repulsion and induces aggregation of polymer chains through
hydrophobic and p–p stacking interactions.16
Fig.
2
Absorption (left) and fluorescence (right) spectra of
PPE-NH3Cl in buffered solutions (pH = 6.5) with increasing PPi
concentration ([PPE-NH3Cl] = 10 mM). Inset: fluorescence images of
the solutions before and after the addition of PPi (1 equiv.).
with resolved vibrational structure (433–455 nm) decreases in
intensity with increasing PPi concentration accompanied by
enhancement of the green emission band at lmax = 520 nm. This
large red-shift (B90 nm) of the emission spectra is due to the
efficient intermolecular exciton coupling among polymer chains
with close proximity, leading to a lower energy ‘‘aggregate state’’.
The blue-to-green transition is visually readily observable under
UV illumination (Fig. 2, inset).
Discrimination between PPi and Pi is an important issue for
PPi sensing as the two anions coexist under many circumstances.8
To demonstrate the feasibility of our current system to selectively
detect PPi, we tested the response of PPE-NH3Cl to Pi under
the same conditions. As expected, neither absorption nor
emission spectra show distinct changes to Pi even at higher
concentrations (Fig. S5, ESIw). We ascribed the different
response to the ability of PPi to ‘‘crosslink’’ the polymer
chains carrying polyamine ligands, thus inducing polymer
aggregation, as illustrated in Fig. 3. Although Pi can also
neutralize the polymer charges, clearly, it is unable to induce
the interpolymer interactions. We have also examined the
sensing selectivity of the current CPE sensor to other inorganic
anions including halide ions, carbonate, sulfate and biologically
important anions, such as adenosine monophosphate (AMP),
adenosine diphosphate (ADP) and adenosine triphosphate
(ATP). It is found that the current sensor is highly selective
to PPi compared to a number of tested inorganic ions. However,
the polymer’s fluorescence is affected somewhat by ADP
and ATP (Fig. S6, ESIw), therefore interference by these
species must be considered in developing assays based on the
PPE-NH3Cl/PPi sensor.
To investigate the analyte-induced aggregation for PPi
sensing, we next examined the response of this polymer to
PPi in 10 mM 2-(N-morpholino)ethanesulfonic acid (MES)
buffered solution at pH = 6.5. Fig. 2 shows the absorption
and fluorescence spectra of PPE-NH3Cl upon the addition of
PPi. As the PPi concentration increases, the absorbance at
lmax = 400 nm decreases and an absorption band at lmax
=
430 nm emerges to become the dominating peak. Consistent
with our earlier results and others,12,17 this red-shift corres-
ponds to aggregation-induced planarization of phenylene-
ethynylene backbone. In addition, a well-defined isosbestic
point at l = 410 nm is observed suggesting a conversion
between the ‘‘free chain state’’ and the ‘‘aggregate state’’ of the
polymer chains. The fluorescence spectra also reveal the clear
transition between the two states. The strong blue emission
Fig. 4 shows the ratiometric response of both the absorption
(A430/A400) and fluorescence (I510/I455) signals to the PPi
Fig. 1 Absorption and emission spectra of PPE-NH3Cl in methanol
and water ([PPE-NH3Cl] = 4 mM).
Fig. 3 Schematic illustration of PPi-induced polymer aggregation.
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design principles can be applied to other non-quenching
anionic species of interest.
We acknowledge the National Science Foundation
(Grant. No. CHE-0515066) for support of this work.
Notes and references
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Fig. 4 The ratiometric signals based on the absorption and fluores-
cence spectra of PPE-NH3Cl in buffered solutions with the addition of
PPi or Pi.
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concentration. A linear dependence of both signals was observed
in the PPi concentration range (0–10 mM). Importantly, the
use of a ratiometric signal allows quantification of PPi con-
centration without the need to utilize a Stern–Volmer based
calibration curve which is generated ex situ where varying
experimental conditions and artifacts such as sample bleaching
can affect the signal calibration.11a Also, the dual response
from both absorption and emission is of additional benefit for
accurate analyte detection. In contrast to the effects seen with
PPi, addition of Pi to the polymer solution leads to negligible
changes. This high selectivity for PPi over Pi enables the
current system to be used in biological assays in which these
two anions are involved. We estimate that the detection limit
of the current sensor to PPi is 340 nM (Fig. S7, ESIw).
Although the detection limit is slightly higher compared to
the sensor based on Stern–Volmer quenching,10 the ratiometric
signaling enables a simple and continuous method with in situ
calibration for measuring PPi concentration.
In summary, we have synthesized a cationic poly(phenylene-
ethynylene) with branched polyamine side chains and investi-
gated its application as a solution-based PPi sensor. This
polymer features a strong blue emission band with well-resolved
vibronic structure in water in contrast to many PPEs with
linear solubilizing groups. In buffered aqueous solution, the
spectroscopic properties of the polymer are sensitive to the
concentration of PPi with high selectivity over Pi and other
inorganic anions due to the analyte-induced aggregation
mechanism. The blue-to-green fluorescence change is readily
visible by the unaided eye. In addition, ratiometric signals
obtained from absorption and/or fluorescence spectra can be
easily and directly calibrated to provide the PPi concentration.
We are currently developing biological assays for enzymes
with PPi as substrates using this system and we believe similar
17 J. Kim and T. M. Swager, Nature, 2001, 411, 1030.
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