Amplifying fluorescent conjugated polymer sensor for singlet oxygen detection

Article abstract of DOI:10.1039/c9cc04123k

A "higher energy gap" concept was applied towards designing an efficient turn-on amplifying sensor for singlet oxygen-an important biomedical and environmental monitoring analytical target. The concept is based on modulation of intramolecular energy trans

Full text of DOI:10.1039/c9cc04123k

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Amplifying Fluorescent Conjugated Polymer Sensor for Singlet
Oxygen Detection
Chun‐Han Wangb and Evgueni E. Nesterov*a,b
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
A “higher energy gap” concept was applied towards designing an reaction with a target analyte, this group would be converted to
efficient turn‐on amplifying sensor for singlet oxygen – an a lower‐energy fluorophore where photoexcitation energy
important biomedical and environmental monitoring analytical could be funnelled to via intramolecular exciton migration,
target. The concept is based on modulation of intramolecular generating an amplified turn‐on response.4
Although
energy transfer in fluorescent conjugated polymers through the promising, this scheme is severely limited by the need to use a
formation of a higher energy gap “roadblock” upon reaction with a reactive group which forms a lower‐energy fluorescent
target analyte. The polymer sensor incorporates 1,4‐disubstituted chromophore upon reaction with analyte. Indeed, such
tetracene units which act as reactive sites for singlet oxygen. The analytical reactions are not very common, and they are simply
resulting polymer sensor demonstrates significant fluorescent not available for many practically important analytical targets.
signal amplification and a broader analyte detection range relative
to a corresponding small‐molecule sensor.
Conjugated polymers (CPs) make a popular choice for designing
fluorescent sensors because of the intrinsic signal amplification
associated with intra‐ and interchain photoexcitation energy
(exciton) migration.1,2 In particular, CP‐based sensors deliver
better detection sensitivity relative to the corresponding small‐
molecule sensors. A typical CP amplifying fluorescent sensor
utilizes exciton migration to a lower energy gap site created by
analyte binding. Such a scheme is common for the detection of
fluorescence‐quenching electron‐deficient analytes (e.g.
nitroaromatic explosives) and is used in the design of turn‐off
sensors (i.e. where interaction with analyte results in amplified
fluorescence quenching through a photoinduced electron
transfer mechanism).3 However, from a practical standpoint,
turn‐on sensors which increase fluorescent intensity upon
analyte binding could be more convenient than turn‐off
sensors, but it still remains a challenge to develop and validate
a general design scheme towards turn‐on sensing which could
utilize signal amplification ability of CPs. One previous scheme
Fig. 1. General mechanism of the “higher energy gap” control of CP fluorescence:
was based on incorporating an analyte‐specific reactive group
as part of the CP’s ‐electron conjugated backbone. Upon
reaction of an analyte (red circle) with the CP receptor generates a higher energy
gap site in the ‐conjugated backbone which decreases the exciton diffusion
length in the CP, therefore causing an increase of the intensity of fluorescent
emission.
a.Department of Chemistry and Biochemistry, Northern Illinois University, DeKalb,
Illinois 60115, USA
As an alternative to this approach, we recently proposed a
“higher energy gap” design concept towards fluorescent turn‐
on chemosensors which is free of the limitations of the previous
scheme (Fig. 1).5 Like in the previous scheme, an analyte‐
b Department of Chemistry, Louisiana State University, Baton Rouge, Louisiana,
70803, USA
E‐mail: een@niu.edu
Electronic Supplementary Information (ESI) available: Detailed synthetic and
experimental procedures, compounds characterization, and additional data and
figures. See DOI: 10.1039/x0xx00000x
reactive group has to be incorporated as part of the CP ‐
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electron backbone.
The critical (and essentially only) through a [4+2]‐cycloaddition (Diels–Alder reaction), yet these
requirement to such a group is that, upon reaction with a target sensors normally operate through a turn‐off pathway, and none
analyte, it should generate a species with a higher energy (i.e. of them demonstrate signal amplification.
HOMO‐LUMO) gap. The generated higher energy gap site in the
In this report, we describe an amplifying turn‐on fluorescent
CP backbone would act as a “roadblock” diminishing the sensor P1 which was designed using the “higher energy gap”
efficiency of exciton migration (photoexcitation energy concept (Scheme 1a). The sensor is a poly(arylene vinylene)
transfer) occurring via
a
through‐bond (Dexter‐type) conjugated polymer which incorporates 1,4‐tetracene unit as
1
mechanism,6 thus decreasing the overall exciton migration part of the
‐electron conjugated backbone. Reaction of O2
length. The shorter exciton migration length would result in a with the tetracene unit yields an endo‐peroxide moiety which
fluorescent emission enhancement in a turn‐on sensor. Since features a higher HOMO‐LUMO gap compared to the initial
this phenomenon is mechanistically related to intramolecular tetracene. A key design approach was to use the 1,4‐
exciton migration in the CP
from the same amplification phenomenon as in the case of preferentially react with 1O2 at a central benzene ring and not
fluorescent‐quenching turn‐off chemosensing. Importantly, in at the ring which is part of the CP ‐conjugated backbone. In
contrast to the previous approach, there is no need to generate the former case, the reaction with 1O2 would result in increasing
a fluorescent chromophore unit upon the analyte reaction, HOMO‐LUMO gap at the reaction site without disrupting the
‐electron system, it will benefit bisubsituted tetracene unit for the sensor as this unit would

‐
which opens up a breadth of efficient potentially applicable conjugation across the polymer backbone – a main condition for
chemical reactions available for almost any analyte of interest. the “higher energy gap” principle to be operative. This
Earlier, we applied the “higher energy gap” concept towards preferential reactivity is driven mostly by increasing aromatic
developing turn‐on fluorescent sensors for organophosphate stabilization of the corresponding endo‐peroxide cycloaddition
agents as well as for hydrogen sulfide.5 In the present work, we products. Indeed, DFT computations (at B3LYP/6‐31G* level of
demonstrated how this concept can be used in the design of an theory) on a truncated 1,4‐bisubstituted tetracene unit
amplifying fluorescent sensor for singlet oxygen (1O2).
indicated significantly higher (12.4‐13.0 kcal/mol) stabilization
of the endo‐peroxides formed at the one of the central benzene
rings relative to the endo‐peroxide formed at the 1,4‐positions
(Fig. S1 in the Supporting Information).
Scheme 1. a) Structures of polymer sensor P1 and corresponding small‐molecule
sensor M1, as well as the reaction with singlet oxygen 1O2 to generate a higher
energy gap site in the CP backbone. b) Synthesis of polymer P1
.
Scheme 2. Synthesis of 1,4‐dibromotetracene 1.
The CP P1 with number‐average molecular weight Mn 11.8
kDa (as determined by GPC vs. polystyrene standards, Fig. S2 in
the SI) was prepared using Suzuki polymerization of 1,4‐
dibromotetracene 1 and bis‐vinylboronate monomer 2 (Scheme
1b). Preparation of 1,4‐dibromotetracene has not been
previously described in literature due to the difficulty of
selective functionalization at the 1 and 4 positions in tetracene
(since direct functionalization of tetracene through electrophilic
aromatic substitution occurs at its central benzene rings).
Therefore, we developed a convenient indirect method towards
Singlet oxygen is a highly reactive excited state of molecular
oxygen which is responsible for photochemical oxidative
damage of biological molecules and organic optoelectronic
materials, and its high cytotoxicity makes 1O2 an important
species in photodynamic therapy treatment.7 A number of
small molecules and conjugated polymers that sense 1O2
specifically through changes in intensity of fluorescence have
been reported.8,9 These systems contain either a small‐
1,4‐dibromotetracene (Scheme 2).
commercially available 2,3‐dimethylaniline with NBS occurred
selectively and yielded 4‐bromo‐2,3‐dimethylaniline
Diazotization followed by Sandmeyer reaction led to 1,4‐
dibromo‐2,3‐dimethylbenzene which was selectively
brominated at both benzylic positions to yield 1,4‐dibromo‐2,3‐
bis(dibromomethyl)benzene An in situ formation of a
Bromination of
3
.
4
5
.
reactive 1,4‐dibromo‐5,6‐bis(methylene)‐1,3‐cyclohexadiene
intermediate and its subsequent Diels‐Alder reaction with 1,4‐
1
molecule or CP chromophore connected to a O2‐responsive
1
unit such as anthracene which can react with dienophile O2
naphthoquinone gave 1,4‐dibromo‐6,11‐tetracenequinone 6 10
.
2 | J. Name., 2012, 00, 1‐3
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Reduction of
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6
and subsequent dehydroxylation produced
1
in spanning until 630 nm (Fig. 2a). The intense peak at 290 nm is
characteristic of tetracene structure,9 and was also prominent
a total 11% yield.
In addition to CP sensor P1
corresponding small‐molecule sensor M1 to be used as a as 1,4‐dibromotetracene
,
we also prepared
a
in the absorption spectra of small‐molecule sensor M1 as well
A characteristic vibronically
benchmark for measuring fluorescent detection amplification structured tetracene absorption band between 400 and 520 nm
by P1 in became a featureless shoulder at around 500 nm in M1, and
1.
.
1
was not easily identifiable in P1. The small‐molecule compound
M1 also displayed a strong absorption band with a maximum at
385 nm which could be attributed to the
‐electron conjugation
across the (arylene vinylene) system (Fig. 2a). The longer
‐
conjugation length in CP P1 resulted in a bathochromic shift of
this band in the polymer’s spectrum, where it also overlapped
with the tetracene absorption band, producing an overall broad
featureless absorption in the spectrum of P1
.
The fluorescence spectrum of M1 showed a broad band with
a maximum at 475 nm and a shoulder at 575 nm. The polymer
P1 spectrum displayed a significantly bathochromically shifted
emission band with an overall very low intensity making P1
essentially non‐fluorescent. The low fluorescent intensity of P1
was not surprising considering that other, previously described
structurally related CPs also showed low fluorescence.5
However, without additional photophysical studies, we cannot
offer a reliable general explanation for this observation.
To test chemosensing response to 1O2, we used
photoirradiation of the sensor solution in the presence of Rose
Bengal sensitizer. In order to avoid interference of the
sensitizer with UV/vis absorption and fluorescence
spectroscopy experiments, we prepared Rose Bengal
functionalized polystyrene beads.13 The photoirradiation was
carried out in a standard 1 cm rectangular cuvette upon stirring.
When the stirring was stopped, the Rose Bengal functionalized
beads would rapidly precipitate to the bottom of the cuvette,
allowing performing spectroscopic measurements in situ,
without interference from the sensitizer. Thus, solutions of P1
or M1 in DMSO were stirred with Rose Bengal functionalized
beads under oxygen and irradiated using a 500 W tungsten‐
halogen lamp for specified time periods.
In excellent
agreement with the “higher energy gap” concept, the CP sensor
P1 exhibited a strong and near‐linear increase in fluorescent
intensity, with the total increase (as an integrated intensity ratio
F/F0) more than 20 times (Fig. 2c, d). The significant
enhancement in fluorescent intensity was especially
remarkable since it occurred over the essentially dark initial
fluorescent background of P1 (Fig. 2e), thus resulting in a high
signal‐to‐background detection ratio. The control experiments
(irradiation with no Rose Bengal sensitizer or exposure to
oxygen with no photoirradiation) did not produce any
significant increase in fluorescent intensity (Fig. S3 and S4 in the
SI). In comparison to the polymer sensor P1, fluorescence
intensity enhancement of the small‐molecule sensor M1 upon
1O2 exposure in the same conditions was practically negligible
(Fig. 2b, d). This illustrated the significant analyte detection
amplification in the turn‐on fluorescent sensor designed using
the “higher energy gap” concept.
Fig. 2. a) Normalized UV/vis absorption and fluorescence spectra of polymer P1
small‐molecule sensor M1 and 1,4‐dibromotetracene in DMSO (conc. 0.02
mg/ml). b) Change in absorbance and fluorescence spectra of small‐molecule
sensor M1 (10
M solution in DMSO) upon exposure to 1O2. c) Change in
upon exposure to 1O2. d) Change of fluorescence intensity of polymer P1 and
small‐molecule sensor M1 upon exposure to 1O2. The intensity is expressed as a
ratio of integrated area of a fluorescent band at each point divided by the area of
the fluorescent band before exposure to 1O2. e) Photograph of a P1 solution
before and after exposure to 1O2 upon irradiation with a hand‐held UV lamp.
,
1

absorbance and fluorescence spectra of polymer P1 (17 M solution in DMSO)
Despite possessing tri(ethylene glycol) (TEG) solubilizing
groups, both P1 and M1 showed insufficient solubility in water.
Alternatively, we decided to use DMSO solvent in these studies.
DMSO is a popular solubility and penetration enhancer in
biomedical research,11 and it has been occasionally used as a
medium for 1O2 related previous studies.12
Further analysis of the spectroscopic results and
corresponding 1H NMR data allowed to gain deeper insight into
the 1O2 sensing response. Reaction of the small‐molecule
The UV‐vis absorption spectrum of polymer P1 showed an
intense peak at 290 nm, and a broad lower‐intensity shoulder
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sensor M1 with O2 resulted in the gradual decrease of the the importance of the through‐bond (Dexter‐type) mechanism
tetracene absorbance at 500 nm, whereas the absorption band of intramolecular energy migration in fluorescent CPs. On
at 385 nm (corresponding to the
the (arylene vinylene) system) persisted (and actually increased turn‐on sensors for many other important analytes of interest.
its absorbance) (Fig. 2b). This indicated that the reaction with We gratefully acknowledge support of this research by the
1O2 occurred at one of the central benzene rings of 1,4‐ National Science Foundation (grant number CHE‐1362686).
disubstituted tetracene system, and the extended ‐conjugated
‐electron conjugation across practical side, this concept can be used for designing amplifying

(arylene vinylene) system remained mostly unaffected. To
determine what specific benzene ring in the 1,4‐disubstituted
tetracene unit reacted with 1O2, we carried out 1H NMR
monitoring of the reaction of M1 with 1O2. This reaction
produced a main product showing a distinct singlet peak at 6.61
ppm (corresponding to the bridgehead protons at the dioxygen
bridge in the endo‐peroxide). To assign this and other 1H NMR
signals to a specific endo‐peroxide, we carried out GIAO
computational studies on a truncated analogue of M1 and the
corresponding endo‐peroxides (Fig. S5 in the SI). The
computations confirmed that the dioxygen bridge was more
likely to form at the 5,12 positions of the tetracene unit, as the
calculated NMR shift for the corresponding bridgehead proton
was 6.72 ppm which was close to the experimentally observed
value. Other experimental chemical shifts also matched the
values calculated for this specific structure. This reactivity
Conflicts of interest
There are no conflicts to declare.
Notes and references
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pattern with O2 also agreed well with the UV/vis absorption
spectroscopy observations.
With this information, we could analyse the spectroscopic
data obtained for the polymer sensor P1. The UV/vis absorption
spectrum showed gradual decrease between 480 and 630 nm
due to the consumption of tetracene unit through its reaction
with O2 (Fig. 2c). At the same time, at the earlier stages of
photoirradiation, there was no decrease in the absorbance (or
even a slight increase) within the range of 360 – 480 nm where
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1
6
the ‐electron conjugation in the CP backbone was the main
contributor. These changes in the absorption spectra were
accompanied by a dramatic intensity increase in the fluorescent
emission band, but with essentially no emission wavelength
shift. Thus, formation of the endo‐peroxide at 5,12 positions of
the tetracene unit was responsible for a HOMO‐LUMO gap
increase which created “roadblocks” for the excitons migrating
7
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,
in the CP
‐conjugated backbone, and produced significant
turn‐on fluorescent response. We should also notice that a
longer exposure of the P1 sensor to 1O2 (at the later stages of
photoirradiation) led to
hypsochromic shift in the maximum of the emission band and a
smaller intensity increase as well as absorbance decrease of the
360 – 480 nm absorption band (traces corresponding to 40 and
60 min in Fig. 2c, d). Apparently, this was due to a subsequent
reaction of 1O2 at the 1,4 positions of tetracene ring resulting in
3
a noticeable (approx. 35 nm)
8
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breakdown of the ‐electron conjugation in the CP backbone.
In conclusion, we demonstrated how the simple “higher
energy gap” concept can be used in the design of an efficient
amplifying turn‐on CP fluorescent sensor. This concept is based
on hindering intramolecular exciton migration occurring via a
through‐bond (Dexter) mechanism when a higher energy
(HOMO‐LUMO) gap site is created in the polymer ‐electron
conjugated backbone upon reacting with a target analyte. From
a fundamental standpoint, the success of this design illustrates
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TOC graphic
A “higher energy gap” concept was used to design an
efficient conjugated polymer turn‐on amplifying fluorescent
sensor for singlet oxygen.
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