Fluorescence ratiometric assays of hydrogen peroxide and glucose in serum using conjugated polyelectrolytes

Article abstract of DOI:10.1039/b703856a

A new water-soluble cationic polyfluorene with boronate-protected fluorescein (peroxyfluor-1) covalently linking to the polymer backbone (PF-FB) was synthesized as a fluorescence probe to optically detect hydrogen peroxide (H₂O₂) and glucose in serum. The peroxyfluor-1 exists as a lactone form which is colorless and non-fluorescent. Without addition of H ₂O₂, the fluorescence resonance energy transfer (FRET) from fluorene units (donor) to peroxyfluor-1 (acceptor) is absent and only blue donor emission is observed upon excitation of the fluorene units. In the presence of H₂O₂, the peroxyfluor-1 can specifically react with H₂O₂ to deprotect the boronate protecting groups and to generate green fluorescent fluorescein. The absorption of fluorescein overlaps the emission of polyfluorene, which encourages efficient FRET from the fluorene units to the fluorescein. By triggering the shift in emission color and the ratio change of blue to green emission intensities, it is possible to assay H₂O₂ and its concentration change in buffer solution and in serum. The PF-FB probe can detect H₂O₂ in the range from 4.4 to 530 M. Since glucose oxidases (GOx) can specifically catalyze the oxidation of β-d-(+)-glucose to generate H₂O₂, glucose detection is also realized with the PF-FB probe as the signal transducer. Thus, an optical assay for hydrogen peroxide (H₂O ₂) and glucose in serum was created which combines a fluorescent ratiometric technique based on FRET with the light-harvesting properties of conjugated polymers. The Royal Society of Chemistry.

Full text of DOI:10.1039/b703856a

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PAPER
www.rsc.org/materials | Journal of Materials Chemistry
Fluorescence ratiometric assays of hydrogen peroxide and glucose in serum
using conjugated polyelectrolytes{
Fang He, Fude Feng, Shu Wang,* Yuliang Li and Daoben Zhu
Received 14th March 2007, Accepted 15th June 2007
First published as an Advance Article on the web 29th June 2007
DOI: 10.1039/b703856a
A new water-soluble cationic polyfluorene with boronate-protected fluorescein (peroxyfluor-1)
covalently linking to the polymer backbone (PF-FB) was synthesized as a fluorescence probe to
optically detect hydrogen peroxide (H2O2) and glucose in serum. The peroxyfluor-1 exists as a
lactone form which is colorless and non-fluorescent. Without addition of H2O2, the fluorescence
resonance energy transfer (FRET) from fluorene units (donor) to peroxyfluor-1 (acceptor) is
absent and only blue donor emission is observed upon excitation of the fluorene units. In the
presence of H2O2, the peroxyfluor-1 can specifically react with H2O2 to deprotect the boronate
protecting groups and to generate green fluorescent fluorescein. The absorption of fluorescein
overlaps the emission of polyfluorene, which encourages efficient FRET from the fluorene units
to the fluorescein. By triggering the shift in emission color and the ratio change of blue to green
emission intensities, it is possible to assay H2O2 and its concentration change in buffer solution
and in serum. The PF-FB probe can detect H2O2 in the range from 4.4 to 530 mM. Since glucose
oxidases (GOx) can specifically catalyze the oxidation of b-D-(+)-glucose to generate H2O2,
glucose detection is also realized with the PF-FB probe as the signal transducer. Thus, an optical
assay for hydrogen peroxide (H2O2) and glucose in serum was created which combines a
fluorescent ratiometric technique based on FRET with the light-harvesting properties of
conjugated polymers.
mechanism of fluoresceins with ROS-cleavable protecting
Introduction
groups (such as pentafluorobenzenesulfonyl fluoresceins and
peroxyfluor-1) have been developed,11,12 however these inten-
Hydrogen peroxide is a key reactive oxygen species (ROS) in
living systems. It plays an important role in physiological and
pathological processes.1 Emerging evidence proves the role of
tration changes of H2O2 in heterogeneous biological samples.
sity-based probes can not quantitatively measure the concen-
H2O2 as a second messenger in cellular signal transduction.2
In comparison to intensity-based methods, the fluorescence
Moreover, oxidative damage resulting from the cellular
resonance energy transfer (FRET) technique exhibits a large
imbalance of H2O2 is closely related to several human diseases
shift in emission profiles and provides a ratiometric fluores-
including Alzheimer’s disease,3 cardiovascular disorders4 and
cancers.5 H2O2 is generated by almost all oxidases, and so the
activity of oxidases, or the enzyme substrates, such as glucose,
can be quantitatively assayed by determining the H2O2
produced.6 Thus new diagnostic methods for detecting and
quantifying H2O2 are needed.
cence measurement,13 which is not affected by external non-
specific events. To date few studies have been demonstrated to
detect H2O2 using ratiometric probes.14
In comparison to small molecule counterparts, the electronic
structure of conjugated polymers coordinates the action of a
large number of absorbing units. The excitation energy along
the backbone of the conjugated polymer is transferred to an
energy/electron acceptor, resulting in an amplified detection
signal.15 Therefore, water-soluble conjugated polymers have
attracted more attention as the optical platforms of sensitive
chemical and biological sensors.16 Recently, we have designed
a new ratiometric system based on a conjugated polymer to
detect H2O2 and glucose with high sensitivity and broad pH
working range.17 However, this sensing system is unworkable
with the increase of ion strength of the assay buffer solution
because the main driving force is electrostatic interaction,
which restricts its application in blood samples. In this
contribution, we design a new conjugated polymer (PF-FB)
with boronate-protected fluorescein covalently linked to the
polymer backbone to eliminate the electrostatic interaction. As
expected, this new polymer probe shows good potential to
detect H2O2 and glucose both in buffer solution and in serum.
Since fluorescence spectroscopy is one of the most useful
analytical tools in bioanalysis,7 many fluorescent probes have
been developed for the detection of H2O2. Fluorescent probes,
such as dihydro analogues of fluorescent dyes,8 phosphine-
based fluorophores9 and lanthanide coordination complexes,10
have been designed, however, the lack of water compatibility,
requirement of external activating enzymes, low selectivity
toward H2O2 over other ROS or narrow pH working range
(6.6–7.2) limit their practical application. Recently, probes
with high selectivity toward H2O2 based on the deprotection
Beijing National Laboratory for Molecular Sciences, Key Laboratory of
Organic Solids, Institute of Chemistry, Chinese Academy of Sciences,
Beijing, 100080, P. R. China. E-mail: wangshu@iccas.ac.cn;
Fax: +86-10-62636680; Tel: +86-10-62636680
{ Electronic supplementary information (ESI) available: synthesis
details. See DOI: 10.1039/b703856a
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Experimental
Materials and measurements
2,7-Dibromofluorene, bis(pinacolato)diboron and the catalyst
Pd(dppf)Cl2 were purchased from the Pacific Chemsource Inc
(China). 3-Iodophenol (4) and benzene-1,2,4-tricarboxylic
acid (5) were from Alfa. Di-tert-butyl dicarbonate was
from Acros. N-Hydroxysuccinimide was from Aldrich.
1-(69-Bromohexyloxy)-2,5-dibromo-4-methoxybenzene (1),18
2,7-dibromo-9,9-bis(69-bromohexyl)-fluorene (8)19 and 1,4-
bis(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzene (9)20
were synthesized according to the procedures in the literature.
The synthetic details of the monomers and polymers are
described in the ESI.{ 1H NMR and 13C NMR were collected
on a 400 MHz AC Bruker spectrometer. Mass spectra were
recorded on an AEI-M850-MS or an APEXII-electrospray
ionization (ESI) instrument. Elemental analyses were
carried out with a Flash EA1112 instrument. GPC was
performed on a Water Styragel system using polystyrene
as a calibration standard with THF as the eluent. UV-vis
absorption spectra were taken on a JASCO V-550 spectro-
photometer and the fluorescence spectra were measured
using a Hitachi F-4500 fluorometer with a Xe lamp as
excitation source.
Scheme 1 Schematic representation of the H2O2 assays.
Synthesis and optical properties of the polymer PF-FB
The procedures for the synthesis of the monomers and
polymers are shown in Scheme 2. Reaction of 1 with excess
sodium azide in anhydrous DMF gives 2 in 95% yield.
Reduction of 2 with PPh3 in THF–H2O (v/v 44 : 6) and
subsequent protection with di-tert-butyldicarbonate yields 3
in 86% yield. Compound 6 is obtained by reaction of
3-iodophenol (4) with benzene-1,2,4-tricarboxylic acid (5)
in methanesulfonic acid in 36% yield. Reaction of 6 with
bis(pinacolato)diboron in the presence of potassium acetate
and Pd(dppf)Cl2 in DMSO yields boronic ester 7 in 27% yield.
The Boc-protected copolymer Boc-Br-PF is prepared by
palladium-catalyzed Suzuki cross-coupling reaction21 between
one equivalent of monomers 3 and 8 with 1,4-bis(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)benzene (9) in the pre-
sence of 2.0 M aqueous K2CO3 and Pd(dppf)Cl2 in THF (43%
yield). The gel permeation chromatography (GPC) analyses
show that the weight-average molecular weight (Mw) and
number-average molecular weight (Mn) of Boc-Br-PF are
60210 and 24660, respectively, with the polydispersity index
(PDI) of 2.44. The polymer Boc-Br-PF is treated with 30%
trimethylamine–ethanol solution to obtain water-soluble
Boc-NMe3-PF followed by Boc-deprotecting with trifluoro-
acetic acid (TFA) to give NMe3-NH2-PF containing free
primary amino groups. Compound 7 is linked covalently to the
side chain of NMe3-NH2-PF in the presence of N-hydroxy-
succinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide hydrochloride (EDCI) to afford target copoly-
mer PF-FB.
Assays for H2O2 and glucose
To a solution of PF-FB in phosphate buffer (50 mM, pH =
7.2) or in serum was added H2O2 at room temperature. After
incubating for 20 minutes, the fluorescence spectra were
measured with an excitation wavelength of 360 nm.
For the detection of glucose in serum, the serum was
saturated with oxygen gas for 30 minutes. To a solution of
PF-FB in serum were added glucose and glucose oxidase
(GOx), respectively. After incubating for 1 hour, the fluores-
cence spectra were measured with an excitation wavelength of
360 nm.
Results and discussion
Design of the fluorescent probe PF-FB for H2O2
Our new H2O2 assay is illustrated in Scheme 1. The boronate-
protected fluorescein (peroxyfluor-1) is covalently linked to
the side chain of water-soluble polyfluorene (PF-FB). The
peroxyfluor-1 exists as a lactone form which is colorless and
non-fluorescent.12,14 FRET from the fluorene units (donor) to
the peroxyfluor-1 (acceptor) is absent and only blue donor
emission is observed upon excitation of the fluorene units. In
the presence of H2O2, the peroxyfluor-1 can specifically react
with H2O2 to deprotect the boronate protecting groups and to
generate fluorescent fluorescein (Fl)12,14 which acts as the
FRET acceptor.16l In this case, efficient intramolecular FRET
from fluorene units to fluorescein is observed. In comparison
to our previous cationic PF/peroxyfluor-1 system,17 the new
PF-FB probe eliminates the effect of electrostatic interactions
on the FRET efficiency. By triggering the shift in emission
color and the ratio change of blue to green emission intensities,
it is possible to assay H2O2 and its concentration change in
buffer solution and in blood samples.
The photophysical properties of PF-FB were investigated in
water. As shown in Fig. 1, the UV-vis absorption spectrum of
PF-FB exhibits a maximum peak at 375 nm, which is
attributed to the p–p* transition of the polymer backbone.
Upon excitation at 360 nm, the emission spectrum shows a
maximum peak at 420 nm with a shoulder peak at 445 nm,
which is characteristic of polyfluorenes.22 As expected, the
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Scheme 2 Synthetic route to the monomers and copolymers.
Assays for H2O2
The emission maximum of PF-FB itself ([PF-FB] = 1.5 6
1026 M in repeat units (RU)) in phosphate buffer solution
(50 mM, pH 7.2) appeared at around 420 nm and no emission
of the peroxyfluor-1 unit was observed at the excitation
wavelength of 360 nm (Fig. 2a). Adding H2O2 ([H2O2] =
1.76 mM) into the solution of PF-FB led to a significant
quenching of fluorene unit emission at 420 nm and the
appearance of the peroxyfluor-1 unit emission at 527 nm. The
efficient FRET confirmed that a fluorescent fluorescein as
energy acceptor was generated from the reaction of peroxy-
fluor-1 and H2O2. Noted that the FRET ratio of fluorescein
to fluorene emission intensities (I527nm/I420nm) increased from
0.17 in the absence of H2O2 to 1.01 after H2O2 treatment,
where the 6-fold FRET ratio was enhanced due to the FRET.
For meeting the detection requirement in blood samples (for
example, serum), the sensing probe needs to be efficient for
large ion strength and a wide pH range. To examine the effect
of ionic strength on the FRET from the fluorene units to the
fluorescein, the fluorescence spectra of PF-FB ([PF-FB] =
1.5 6 1026 M) were studied in the presence of H2O2
Fig. 1 UV-vis absorption and emission spectra of PF-FB in water.
The excitation wavelength is 360 nm.
typical fluorescein absorption band centered at 480 nm and
emission band centered at 530 nm were not observed, which
proves that no FRET occurs from the fluorene units to
boronate-protected fluorescein. The fluorescence quantum
yield (QY) of PF-FB is calculated to be 0.165 with 9,10-
diphenylanthracene in cyclohexane as the standard.13
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Fig. 2 (a) Emission spectra of PF-FB in phosphate buffer (50 mM, pH 7.2) in the absence and presence of H2O2. (b) Emission spectra of PF-FB in
the presence of H2O2 in phosphate buffer (pH 7.2) with varying ion strength from 50 to 1500 mM. (c) Emission spectra of PF-FB in the presence of
H2O2 in phosphate buffer (50 mM) with varying pH value from 4.6 to 10.1. [PF-FB] = 1.5 6 1026 M, [H2O2] = 1.76 mM. The excitation
wavelength was 360 nm.
([H2O2] = 1.76 mM) at a pH value of 7.2 with varying ion
strengths. Fig. 2b shows that there was only a slight decrease in
the FRET efficiency upon varying the buffer ionic strength
from 50 to 1500 mM, which confirmed that the fluoran
pendant was covalently linked to the polymer side chain and
the emission of fluorescein originated from the intramolecular
FRET. To check for the working pH range of the PF-FB
probe, the effect of medium pH values on the fluorescence
spectra of PF-FB ([PF-FB] = 1.5 6 1026 M) was investigated
in the presence of H2O2 ([H2O2] = 1.76 mM) in phosphate
buffer (50 mM). Since the absorption and fluorescence of
fluorescein are both pH dependent,23 a pH dependent FRET
ratio was also observed (Fig. 2c). At pH 4.6, inefficient
FRET from fluorene units to fluorescein was observed. The
FRET ratio (I527nm/I420nm) was 0.46 at pH 6.2 and 0.68 at
pH 10.1, that is, only an approximate 30% difference for the
FRET ratio was exhibited over a pH range from 6.2 to 10.1.
The above results exhibit that PF-FB can detect H2O2 for
large ion strength and a wide pH range. Thus, PF-FB affords
the chance to detect H2O2 in biological samples, for example,
in serum.
To examine the ability of PF-FB to detect H2O2 in bio-
logical samples, assay experiments were performed in serum
Fig. 3 (a) Emission spectra of PF-FB and (b) FRET ratio
solution. Fig. 3a shows the fluorescence spectral changes of
(I527nm/I420nm) as a function of the H2O2 incubating time. [PF-FB] =
PF-FB as a function of H2O2 incubating time. In these
1.5 6 1026 M, [H2O2] = 4.4 mM. The experiments were performed
experiments, H2O2 ([H2O2] = 4.4 mM) was added to a solution
in phosphate buffer (50 mM, pH 7.2) containing 20% serum. The
of PF-FB ([PF-FB] = 1.5 6 1026 M) in phosphate buffer
(50 mM, pH 7.2) containing 20% serum, and the emission
excitation wavelength was 360 nm.
spectra were measured at 1 min intervals over 13 minutes by
concentration was observed over the H2O2 concentration
the excitation at 360 nm. The initial solution of PF-FB showed
range from 4.4 to 530 mM, where the emission intensity was
less FRET from fluorene units to fluorescein. After adding
obtained at the H2O2 incubating time of 20 minutes with a
H2O2, the intensity of emission band centered at 420 nm was
standard commercial fluorometer (Hitachi F-4500, Japan)
gradually decreased and that centered at 527 nm was gradually
equipped with a xenon lamp excitation source and a photo-
increased with the incubating time from 0 to 10 minutes and the
multiplier tube (Fig. 4b). This indicates that the PF-FB can
FRET ratio (I527nm/I420nm) reached a plateau after 10 minutes
also be utilized as a probe to assay the H2O2 concentration
(Fig. 3b). These observations indicated that PF-FB can be
quantitatively.
utilized as a probe to detect H2O2 in real time.
Fig. 4a also shows the dependence of emission spectra
Assays for glucose
of PF-FB on the concentration of H2O2 with a fixed
PF-FB concentration in 20% serum solution ([PF-FB] =
1.5 6 1026 M in RU). It shows that the FRET efficiency
increased gradually with successive addition of H2O2. A linear
relationship between the FRET ratio (I527nm/I420nm) and H2O2
Many oxidases can catalyze the oxidation of their respective
substrates in the presence of oxygen to generate H2O2,6 which
suggests that PF-FB could be employed as the signal
transducer for the fluorescent sensing of the respective
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glucose to generate H2O2, leading to the efficient FRET
response of PF-FB. Fig. 5b shows the FRET ratio
(I527nm/I420nm) changes of PF-FB as a function of glucose
concentration in the concentration range from 0 to 15.4 mM.
With an increase of the glucose concentration from 0 to
7.7 mM, the value of I527nm/I420nm increases gradually and
reaches a plateau for glucose concentrations higher than
9.0 mM. It is well known that the normal clinical range for
glucose in blood is between 3.5 and 6.1 mM, and abnormal
glucose levels can reach as high as 20 mM.25 Thus, PF-FB
shows good potential to assay glucose in blood samples. Fig. 5c
shows the time curves of glucose detection. It is found that the
FRET ratio (I527nm/I420nm) values gradually increased with the
incubating time from 0 to 20 minutes and did not change much
after 20 minutes. It is still possible to detect glucose even within
five minutes, which demonstrates the rapid response of the
PF-FB to glucose in the presence of glucose oxidase.
Conclusions
In summary, a new conjugated polyfluorene containing a
boronate-protected fluoran pendent (PF-FB) is designed and
synthesized by the palladium-catalyzed Suzuki coupling reac-
tion. Optical assays for hydrogen peroxide and glucose in
serum are created which combine the fluorescent ratiometric
technique based on FRET with the light-harvesting properties
of conjugated polymers. In comparison to our previous
cationic PF/peroxyfluor-1 system,17 the new PF-FB probe
eliminates the effect of electrostatic interactions on the
intramolecular FRET. It shows a wide working pH range
and the intramolecular FRET was only slightly influenced by
the ion intensity of the assay buffer. This new polymer probe
shows good potential to detect H2O2 and glucose both in
buffer solution and in serum.
Fig. 4 (a) Emission spectra of PF-FB as a function of the H2O2
concentration. (b) FRET ratio (I527nm/I420nm) as a function of the
H2O2 concentration. [PF-FB] = 1.5 6 1026 M, [H2O2] = 0–4.4 mM.
The experiments were performed in phosphate buffer (50 mM, pH 7.2)
containing 20% serum. The excitation wavelength was 360 nm.
substrates. The oxidase and substrate chosen as a proof of
concept are glucose oxidase (GOx) and its specific b-D-(+)-
glucose. Fig. 5a compares the spectra before and after the
addition of glucose to the solution of PF-FB ([PF-FB] =
1.54 6 1026 M in RUs) and GOx (3.3 mg mL21) in oxygen
saturated phosphate buffer (50 mM) containing 20% of serum.
Upon adding glucose ([glucose] = 15.4 mM) and allowing
incubating at 37 uC for 1 hour to allow for complete oxidation
by GOx, efficient FRET from fluorene units to fluorescein was
observed. The control experiments showed that glucose itself
did not change the emission spectra of PF-FB. GOx itself
slightly increased the FRET efficiency, which resulted from the
cofactor of GOx, flavin adenine dinucleotide (FAD).24 These
results indicated that the GOx catalyzed the oxidation of
Acknowledgements
The authors are grateful for the financial support from the
‘‘100 Talents’’ program of Chinese Academy of Sciences, the
National Natural Science Foundation of China (20601027,
20574073 and 20421101), the National Basic Research
Program of China (No.2006CB806200) and the Major
Research Plan of China (No. 2006CB932100).
Fig. 5 (a) Emission spectra of PF-FB/GOx in the absence and presence of glucose. [PF-FB] = 1.54 6 1026 M, [GOx] = 3.3 mg mL21, [glucose] =
15.4 mM. (b) FRET ratio (I527nm/I420nm) as a function of the glucose concentration. [PF-FB] = 1.54 6 1026 M, [GOx] = 3.3 mg mL21, [glucose] =
0–15.4 mM. (c) FRET ratio (I527nm/I420nm) as a function of incubating time. [PF-FB] = 1.54 6 1026 M, [GOx] = 3.3 mg mL21, [glucose] = 7.7 mM.
The measurements were performed in oxygen saturated phosphate buffer (50 mM) containing 20% of serum and the excitation wavelength
was 360 nm.
3706 | J. Mater. Chem., 2007, 17, 3702–3707
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