Self-assembly in tailor-made polyfluorenes: Synergistic effect of porous spherical morphology and FRET for visual sensing of bilirubin

Article abstract of DOI:10.1021/ma4000946

Two new fluorene-based homo-(PDP-PF) and copolymers (PDPPF-co-Ph) were synthesized with a bulky 3-pentadecylphenoxy (PDP) group appended hexyl chains at the 9, 9′ position using Suzuki coupling polymerization. Investigation on the morphology of the polymers using microscopic techniques like TEM and AFM indicated formation of self-assembled nanostructures like vesicles by PDP-PF and porous spheres by PDPPF-co-Ph respectively. Dynamic as well as static light scattering studies (DLS, SLS) in THF also indicated the existence of self-assembled nanosized particles in solution with a shape factor (ρ) of 0.76 and 0.96 for PDP-PF and PDPPF-co-Ph, respectively, confirming the existence of vesicles in the case of the former and spherical particles in the case of the latter polymer. The favorable photophysical properties of the polyfluorenes were taken advantage of for the selective sensing of unbound bilirubin (BR) in THF. A high energy transfer efficiency of 86% upon addition of bilirubin with color change from blue (polyfluorene emission) to green (FRET-induced bilirubin emission) was observed with PDPPF-co-Ph. Steady state fluorescence measurements gave a minimum donor-acceptor distance of 36 A⁰ and time-resolved fluorescence decay measurements showed a reduction in average lifetime of PDPPF-co-Ph (from 450 to 240 ps) upon addition of bilirubin indicating efficient energy transfer. The open porous spherical assembly of PDPPF-co-Ph enabled better adsorption of the analyte, which along with the good spectral overlap resulted in greater efficiency for FRET-induced energy transfer. Sensing of unbound bilirubin was also attempted in THF/water solvent mixture in an effort to simulate the unbound (THF soluble) and bound (water-soluble) bilirubin equilibrium. Enhancement of bilirubin emission coupled with quenching of polyfluorene emission makes this approach adaptable for visual fluorimetric color change (blue to green) based sensor. Structural analogues such as biliverdin and porphyrin showed poor fluorescence quenching efficiency, thus highlighting the selectivity and sensitivity of the FRET-based sensing of bilirubin by the newly designed polyfluorene.

Full text of DOI:10.1021/ma4000946

Article
pubs.acs.org/Macromolecules
Self-Assembly in Tailor-Made Polyfluorenes: Synergistic Effect of
Porous Spherical Morphology and FRET for Visual Sensing of
Bilirubin
T. Senthilkumar and S. K. Asha*
Polymer & Advanced Material Laboratory, Polymer Science & Engineering Division, CSIR, NCL, Pune-411008, Maharashtra, India
S
* Supporting Information
ABSTRACT: Two new fluorene-based homo- (PDP−PF) and
copolymers (PDPPF-co-Ph) were synthesized with a bulky 3-
pentadecylphenoxy (PDP) group appended hexyl chains at the 9, 9′
position using Suzuki coupling polymerization. Investigation on the
morphology of the polymers using microscopic techniques like TEM
and AFM indicated formation of self-assembled nanostructures like
vesicles by PDP−PF and porous spheres by PDPPF-co-Ph
respectively. Dynamic as well as static light scattering studies (DLS,
SLS) in THF also indicated the existence of self-assembled nanosized
particles in solution with a shape factor (ρ) of 0.76 and 0.96 for
PDP−PF and PDPPF-co-Ph, respectively, confirming the existence of
vesicles in the case of the former and spherical particles in the case of
the latter polymer. The favorable photophysical properties of the
polyfluorenes were taken advantage of for the selective sensing of unbound bilirubin (BR) in THF. A high energy transfer
efficiency of 86% upon addition of bilirubin with color change from blue (polyfluorene emission) to green (FRET-induced
bilirubin emission) was observed with PDPPF-co-Ph. Steady state fluorescence measurements gave a minimum donor−acceptor
distance of 36 A0 and time-resolved fluorescence decay measurements showed a reduction in average lifetime of PDPPF-co-Ph
(from 450 to 240 ps) upon addition of bilirubin indicating efficient energy transfer. The open porous spherical assembly of
PDPPF-co-Ph enabled better adsorption of the analyte, which along with the good spectral overlap resulted in greater efficiency
for FRET-induced energy transfer. Sensing of unbound bilirubin was also attempted in THF/water solvent mixture in an effort to
simulate the unbound (THF soluble) and bound (water-soluble) bilirubin equilibrium. Enhancement of bilirubin emission
coupled with quenching of polyfluorene emission makes this approach adaptable for visual fluorimetric color change (blue to
green) based sensor. Structural analogues such as biliverdin and porphyrin showed poor fluorescence quenching efficiency, thus
highlighting the selectivity and sensitivity of the FRET-based sensing of bilirubin by the newly designed polyfluorene.
dye. Another example of energy transfer based acceptor
emission was demonstrated by Tapia et al., where energy
transfer occurred from conjugated polyelectrolyte to meso-
tetrakis-phenylporphyrinsulfonates resulting in decreased poly-
mer emission, followed by a corresponding increase in the
fluorescence of the porphyrin acceptors.12 In the scenario of
FRET-based enhanced acceptor emission, morphology of the
donor polymer plays a major role as desirable morphologies
like vesicles, porous assemblies, etc. enable close interaction of
the donor and acceptor units.13 Vesicular morphology is very
promising as drug delivery vehicles as they help encapsulate
hydrophilic/hyrophobic drug molecules in their interior cavity
depending on their structural constitution.14 In general,
researchers have sought the help of amphiphilic (hydrophilic/
hydrophobic combinations) structural modifications in poly-
mers to explore self-assembled architectures like vesicles.15
INTRODUCTION
■
Conjugated polymers provide a good platform for a wide
variety of applications such as light emitting diodes,1 polymer
solar cells,2,3 field effect transistors4 and sensors.5−8 The
delocalized π electrons in these semiconductors are sensitive
even to minor perturbations resulting in amplified signal
response due to which they find appications as chemical and
biosensors.9 The combination of amplified response and
sensitivity in conjugated polymer-based sensors makes them
superior compared to their small molecule counterparts.10
Generally, fluorescence-based sensors adopt three different
strategies: (a) fluorescence quenching (turn-off), (b) fluo-
rescence enchancement (turn-on), and (c) fluorescence
resonance energy transfer (FRET). Bazan et al., developed a
polyfluorene-based sensor that detected the hybridization of the
single stranded deoxyribinucleic acid (ssDNA) with specific
base sequence where the polyfluorene acted as the donor and
dye labeled ssDNA acted as the acceptor.11 FRET-induced
energy transfer from the polymer to dye enchanced the
emission of the dye compared to the direct excitation of the
Received: January 15, 2013
Revised: February 19, 2013
© XXXX American Chemical Society
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There have been reports where oligo/polyfluorenes modified
with hydrophilic units have been used for sensing application
based on the characteristic optical properties of the self-
assembled nanoparticles.16 Reports on the application of
conjugated homopolymers or conjugated polymers without
the required amphiphilic structure for self-assembly for
florescence-based sensing are very rare.
water solvated (bound) bilirubin and organic media soluble
(free) bilirubin. The selectivity and sensitivity of the bilirubin
sensing by the new polyfluorene was verified with structurally
analogous molecules like porphyrin which had very similar
absorption spectra with that of bilirubin. The tailor-made
polyfluorene structure presented here illustrates the importance
of self-assembling building blocks like the PDP unit which helps
induce self-organization even in the homopolymer, without
having to design amphiphilic block copolymers or polymers
incorporated with hydrophilic units like the oligooxyethylene to
induce phase separation.
Here we report for the first time the sensing of bilirubin by
conjugated polyfluorenes with high selectivity and sensitivity.
Bilirubin is the breakdown product of hemoglobin and contains
a tetrapyrrole moiety with six intramolecular hydrogen bonds
existing in ridge tile conformation.17 Under normal physio-
logical conditions bilirubin is mostly present as the conjugated
form which is bound with human serum albumin (direct/
bound bilirubin), which makes it water-soluble. The uncon-
jugated or free fraction which is potentially toxic is usually
present in small amounts and is excreted after enzymatically
esterified with glucuronic acid.18 Abnormal levels of bilirubin −
either conjugated or unconjugated, detected in serum samples
is an indicator for disturbed bilirubin metabolism. For instance,
increased levels of conjugated bilirubin in serum could signal
disturbance of bile drainage as in obstructive jaundice or
increased levels of unconjugated bilirubin could be due to
abnormally high breakdown of hemoglobin (hemolysis).18,19
Therefore, monitoring the bilirubin concentration in body
fluids is a vital step in diagnosing liver disorders and jaundice.
Clinically, the levels of bilirubin (conjugated and unconjugated
bilirubin) is determined by the color change from yellow to red
produced by the diazotization reaction of bilirubin and
sulphanilic acid using spectrophotometer (total van den
Bergh reaction, 1914).18,20 The major disadvantage of the
assay is that it overestimates conjugated bilirubin and is time-
consuming (30 min to develop the maximum color).21 There
are only very few reports on fluorimetric bilirubin sensing; very
recently a spectrofluorometric method of determination of free
bilirubin by Kleinfeld et.al was described using mutated fatty
acid binding protein labeled with the fluorescent molecule
acrylodan or rhodamine-B, whose fluorescence changed upon
binding to bilirubin.22 Till date there are no reports on bilirubin
sensing using conjugated polymer as the fluorescent sensor. By
taking advantage of overlapping photophysical properties of
bilirubin and polyfluorenes we have developed a model
polyfluorene sensor to detect unconjugated bilirubin via
FRET process for the first time. Although simple dialkyl
polyfluorenes like 9,9′-dioctylpolyfluorenes also exhibited
complementary photophysical chracteristics to bilirubin, its
sensing efficiency was very low. In our molecular design we
incorporated bulky pentadecyl phenol (PDP) units (which are
well-known to have the ability to self-organize by way of alkyl
chain interdigitation as well as aromatic π stacking
interactions)23 at the 9,9′-position of fluorene with the
anticipation that the structurally modified polyfluorene would
be able to self-assemble into desired morphologies resulting in
close interaction with bilirubin. The self-assembled structures of
the PDP substituted polyfluorenes were studied in solution
state using static and dynamic light scattering (SLS and DLS),
and in solid state using microscopic techniques like TEM and
AFM. Steady states as well as time-resolved fluorescence
analysis were conducted to study the donor−acceptor
interaction parameters. An attempt was also made to sense
the unbound bilirubin in presence of bound bilirubin by
carrying out the fluorescence studies in THF/water (containing
NaOH) media, where an equilibrium was created between the
EXPERIMENTAL SECTION
■
Materials. 2,7-Dibromo-9H-fluorene, 3-pentadecylphenol, biliru-
bin, biliverdin, rhodamine, 5,10,15,20-tetrapyridyl-20H,25H-porphine,
Pd(PPh3)4, 1,4-benzenediboronic acid, sodium hydride, 1,6-dibromo-
hexane, 2-isopropoxy-4,4,5,5-tetramethyl-1,3,2-dioxaborolane, t-BuLi
and 1-bromooctane were purchased from Aldrich Company Ltd. and
were used as such. Potassium hydroxide, potassium carbonate and
ethanol were purchased from Merck Chemicals Ltd. Tetrahydrofuran
(THF), acetone and methanol were purchased locally and were
purified using standard procedures.
1
Measurements. H NMR was recorded on a Bruker-AVENS 200
MHz spectrometer. Chemical shifts are reported in ppm at 25 °C
using CDCl3 as solvent containing small amount of tetramethylsilane
(TMS) as internal standard. The purity of the compounds was
determined by elemental analysis as well as MALDI−TOF in
combination with size exclusion chromatography (SEC). Elemental
analysis was done by Thermofinnigan flash EA 1112 series CHNS
analyzer. The MALDI−TOF analysis was done on Voyager-De-STR
MALDI−TOF (Applied Biosystems, Framingham, MA) equipped
with 337-nm pulsed nitrogen laser used for desorption and ionization.
A 1 μM solution of the sample was premixed with DHB (2,5-
dihydroxybenzoic acid) matrix in CHCl3 and mixed well before
spotting on 96-well stainless steel MALDI plate by dried droplet
method for MALDI analysis. For small molecules SEC was performed
using polystyrene standards for the calibration in CHCl3 as eluent. The
flow rate of CHCl3 was maintained as 1 μL/min throughout the
experiments and the sample solutions at concentrations 3−4 mg/mL
were filtered and injected for recording the chromatograms at 30 °C.
The molecular weights of the polymers were determined by gel
permeation chromatography (GPC), which was performed using a
Viscotek VE 1122 pump, Viscotek VE 3580 RI detector, and Viscotek
VE 3210 UV/vis detector in tetrahydrofuran (THF) using polystyrene
as standards. UV−vis spectra were recorded using a Perkin-Elmer
Lambda-35 UV−vis spectrometer. The thermal stability of all the
model compounds and azo copolyesters were analyzed using a
PerkinElmer:STA 6000 thermogravimetric analyzer (TGA) under
nitrogen atmosphere from 40 to 900 °C at 50 °C/min. Differential
scanning calorimetry (DSC) was performed using a TA Q10 model.
2−3 mg of the sample was taken in aluminum pan, sealed and scanned
at 10 °C/min. The instrument was calibrated with indium standards
before measurements. The phase behaviors of the molecules were
analyzed using LIECA DM2500P polarized optical microscope
equipped with Linkam TMS 94 heating and cooling stage connected
to a Linkam TMS 600 temperature programmer. The transition from
isotropic to liquid crystalline phase was monitored by the evolution of
characteristic textures.
Photophysical Studies. Absorption spectra were recorded using
Perkin-Elmer Lambda 35 UV-spectrophotometer. Steady-state fluo-
rescence studies and time-resolved fluorescence lifetime measurements
were performed using Horiba Jobin Yvon Fluorolog 3 spectropho-
tometer having a 450 W xenon lamp for steady-state fluorescence.
Picosecond time-resolved-emission spectra (TRES) and fluorescence
lifetime decays were collected by a time-correlated single photon
counting (TCSPC) setup from IBH Horiba Jobin Yvon (U.S.) using a
375 nm diode laser (IBH, U.K., NanoLED-375 L, with a λmax =375
nm) having a fwhm of 89 ps as a sample excitation source. The
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Scheme 1. Synthesis of Monomers and the Polymerization Scheme
emission and excitation slit width was maintained at 1 nm throughout
the experiments, and the data was obtained in “S1c/R1” mode (to
account for the variations in lamp intensity).
Synthesis of 1-(6-Bromohexyloxy)-3-pentadecylbenzene. 3-
Pentadecylphenol (10 g, 3.29 mmol) and KOH (9.21 g, 16.4 mmol)
were dissolved in ethanol (50 mL) and stirred for 30 min to obtain a
reddish black precipitate. 1,6-Dibromohexane (25 mL, 16.4 mmol) in
dry acetone (25 mL) was added very slowly to this reaction mixture
and heated to reflux for 24 h. After cooling to room temperature, the
solvent and excess 1,6-dibromohexane was distilled off and the product
extracted with dichloromethane. The organic layer was washed with
water, brine and dried with Na2SO4. The solvent was removed under
vacuum. The crude product was purified by silica gel column
chromatography using pet ether/ethyl acetate (v/v, 97/3) as eluent
to yield the product. Yield (18 g, 78%). 1H NMR (CDCl3, 200 MHz):
δ = 7.17 (t, 1H), 6.72 (t, 3H), 3.94 (t, 2H), 3.42 (t, 2H), 2.56 (t, 2H),
1.9−1.7 (m, 4H), 1.50 (m, 6H), 1.35−1.15 (m, 24H), 0.87 (t, 3H).
Synthesis of 2,7-Dibromo-9,9-bis(6-(3-pentadecylphenoxy)-
hexyl)-9H-fluorene. (1). 2, 7-dibromofluorene (3 g, 9.26 mmol) and
NaH (2.22 g, 92.6 mmol) were taken in a two-necked round-bottom
flask and purged with nitrogen. Dry THF (60 mL) was added to this
to obtain a dark red precipitate. (6-Bromohexyloxy)-3-pentadecylben-
zene (17.32 g, 37.04 mmol) was added very slowly to this reaction
mixture and heated to reflux for 12 h. The reaction mixture was
poured into water and the product was extracted with ethyl acetate.
The organic layer was washed with water, brine, and dried over
Na2SO4. The solvent was removed under vacuum. The crude product
was purified by silica gel column chromatography using pet ether/ethyl
acetate (v/v, 95/5). Yield: 6.4 g (64%). 1H NMR (CDCl3, 200 MHz):
δ = 7.55−7.4 (m, 6H), 7.15 (t, 3H), 6.8−6.6 (m, 6H), 3.83 (t, 3H),
2.55 (t, 3H), 1.98−1.90 (m, 4H), 0.89 (t, 6H), 0.62 (m, 4H). MALDI
(observed M+ Na. 1119.67, calculated 1120.21.) Anal. Found: C,
59.53%; H, 7.31. Calcd: C, 59.71; H, 7.23.
Transmission Electron Microscopy. For TEM measurements,
the samples in THF were deposited directly on a carbon coated
copper grid. No staining treatment was performed for the measure-
ment. A JEOL JEM-3010 electron microscope operating at 300 kV (Cs
= 0.6 mm, resolution 1.7 Å) was used for HR-TEM sample
observation. A Gatan digital camera (model 794, Gatan 1024 ×
1024 pixels, pixel size 24 × 24 μm) at 15 000−80 000× magnifications
was used to record micrographs.
Atomic Force Microscopy. AFM images were taken by using a
Multimode scanning probe microscope equipped with a Nanoscope IV
controller from Veeco Instruments, Inc. in the tapping mode using a
SiN probe, with a maximum scan size of 10 mm ×10 mm and with a
vertical range of 2.5 mm. For the AFM studies, samples were prepared
by drop-casting THF solution of the polymers onto the silicon wafer
and allowed to dry at ambient temperature before being subjected to
AFM analysis.
Dynamic and Static Light Scattering (DLS and SLS). DLS
measurements were carried out on a Zetasizer ZS 90 apparatus,
utilizing 633 nm red laser (at 90° angle) from Malvern Instruments.
The reproducibility of the data was checked at least three times using
independent polymer solutions.
The static light scattering experiment (SLS) was carried out using
3D-DLS spectrometer, from LS instruments, Switzerland. The
instrument consists of a He Ne laser having wavelength of 632.8 nm
attached to computer using the Lab view interface utilizing toluene as
reference. The measurement was performed in autocorrelation mode
from 20° to 134° by steps of 2°.
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Scheme 2. Structure of the Polyfluorenes and Bilirubin Used in the Study
1
Figure 1. H NMR spectra of PDPPF-co-Ph and PDP−PF recorded in CDCl3.
Synthesis of 2,2′-(9, 9-Bis(6-(3-pentadecylphenoxy)hexyl)-
9H-fluorene-2, 7-diyl)bis(4,4,5,5-tetramethyl-1,3,2-dioxaboro-
lane) (2). 2,7-Dibromo-9,9-bis(6-(3-pentadecylphenoxy)hexyl)-9H-
fluorene (1.35 g, 1.24 mmol) was dissolved in dry THF (20 mL)
and cooled to −78 °C. n-BuLi (1.5 mL, 2 M in hexane) was added
dropwise over a period of 45 min to the above THF solution, and the
reaction mixture was stirred for 1 h. At the same temperature, 2-
isopropoxy-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (0.7 mL, 4.93
mmol) was added over a period of 30 min and stirred for further 2
h. The reaction mixture was then slowly warmed to room temperature
and stirred for 12 h. For work-up, the reaction was quenched with
water (10 mL), and the reaction mixture was poured into water and
extracted with dichloromethane. The organic layer was washed with
water and brine and dried over Na2SO4. The solvent was removed
under reduced pressure. The crude product was purified by silica gel
column chromatography using pet ether/ethyl acetate (v/v, 90/10).
1
Yield: 0.75 g (51%). H NMR (CDCl3, 200 MHz): δ = 7.7−7.2 (m,
6H), 7.15 (t, 2H), 6.67 (m, 6H), 3.81 (t, 4H), 2.55 (t, 4H), 1.95 (m,
4H), 1.58 (m, 10H), 1.38 (s, 24H), 1.25 (m, 50H), 0.89 (t, 6H), 0.64
(m, 4H). MALDI: observed M + Na = 1214.22; calculated M + Na =
1214.41.
Polymerization. 2,7-Dibromo-9,9-bis(6-(3-pentadecylphenoxy)-
hexyl)-9H-fluorene (300 mg, 0.27 mmol), 2,2′-(9, 9-bis(6-(3-
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pentadecylphenoxy)hexyl)-9H-fluorene-2,7-diyl)bis(4,4,5,5-tetrameth-
yl-1,3,2-dioxaborolane) (diboronic ester) (325 mg, 0.27 mmol) and
tetrakis(triphenylphosphine) palladium (20 mg, 0.04 mmol) were
taken into a two necked round-bottom flask under nitrogen
atmosphere. Dry THF (8 mL) was added to the mixture. K2CO3
dissolved in water (2 mL) was added to the reaction medium. The
reaction mixture was heated to reflux for 36 h under nitrogen
atmosphere. The mixture was cooled down to room temperature and
added dropwise into a stirred solution of methanol (100 mL) in an
open vessel. The precipitate was isolated and dissolved in dichloro-
methane and filtered to remove the catalyst. The collected
dichloromethane solution was concentrated under reduced pressure
and purified by repeated precipitation from methanol (100 mL). The
precipitate was filtered, washed with methanol (50 mL), and dried
under high vacuum.
of the polymers are given in Supporting Information, Figure SI-
1e. The number and weight-average molecular weight (Mn,
Mw), polydispersity index, yield and 10% weight loss temper-
ature of the different polymers are given in Table 1. PDP−PF
Table 1. Sample Designation, Number and Weight Average
Molar Mass, Polydispersity Indices, Yield and 10 Wt % Loss
Temperature of the Polyfluorenes
number-
average
weight-
average
10% weight
loss
molecular molecular
weight
weight
polydispersity yield temperature
a
a
polymer
(Mn)
(Mw)
index (ĐM)
(%)
(°C)
PDP−PF
PDPPF-
co-Ph
19 600
18 600
53 000
46 000
3.5
2.4
75
78
370
408
Poly[2,7-(9,9-bis(6-(3-pentadecylphenoxy)hexyl)-9H-fluo-
rene)] (PDP−PF). 1H NMR (CDCl3, 200 MHz): δ (ppm) 7.66 (4H,
m), 7.48 (2H, m), 7.11 (2H, q), 6.65 (6H, m), 3.80 (4H, m), 2.51
(4H, m), 2.10 (4H, m), 1.58 (8H, m), 1.23 (60H, m), 0.87 (6H, m).
Yield: 75%.
POF
9000
22 000
36 100
2.4
1.7
80
75
420
411
POF-co-
Ph
20 300
Poly[(1,4-phenylene)-2,7-(9,9-bis(6-(3-pentadecylphenoxy)-
hexyl)-9H-fluorene)] (PDPPF-co-Ph). The copolymer PDPPpF-co-
Ph was prepared by following the same procedure but using 1,4-
benzene diboronic pinacolate ester as the comonomer. Yield: 78%. 1H
NMR (CDCl3, 200 MHz): δ (ppm) 7.77 (4H, m), 7.64 (4H, m), 7.48
(2H, m), 7.12 (2H, q), 6.65 (6H, m), 3.79 (4H, m), 2.52 (4H, m), 2.0
(4H, m), 1.58 (8H, m), 1.23 (60H, m), 0.87 (6H, m).
a
Measured by size exclusion chromatography (SEC) in tetrahydrofur-
an (THF), calibrated with linear, narrow molecular weight distribution
polystyrene standards.
had a Mw of 53, 000 with a polydispersity index (ĐM) of 3.5 and
PDPPF-co-Ph had a Mw of 46, 000 with a ĐM of 2.4. The
molecular weight and polydispersity values were comparable
with the polyfluorenes reported in the literature.25
POF and POF−Ph were synthesized following literature
procedures.24
Poly[2,7-(9,9-dioctylfluorene)] (POF). 1H NMR (CDCl3, 200
MHz): δ (ppm) 7.85 (2H, d), 7.68 (4H, m), 2.15 (4H, m), 1.2 (24H,
m), 0.8 (6H, t). Yield: 80%.
Thermal Analysis and Liquid Crystallinity. The 10%
weight loss temperature determined using TGA (plot given in
Supporting Information, SI-1f) indicated that all polymers were
thermally stable up to 350 °C. The family of dialkylpoly-
fluorenes is well-known for their ability to exhibit thermotropic
nematic liquid crystalline phases.26 The thermotropic liquid
crystalline tendency of the PDP-based homo and copolymers
PDP−PF and PDPPF-co-Ph were analyzed using both
differential scanning calorimetry (DSC) and polarized light
microscopy (PLM). Both polymers were sluggish to crystallize,
therefore, they were subjected to rapid cooling from isotropic
to − 70 °C and then a heating and cooling cycle were recorded
at a slow rate of 2 °C/min. The DSC thermograms and
polarized optical microscopic images of PDP−PF and PDPPF-
co-Ph in their second heating and cooling cycles respectively
are given in Supporting Information (SI-2). PDP−PF showed a
glass transition at −24 °C, followed by a sharp endothermic
transition at −1.8 °C with an enthalpy of 9.3 J/g and a melting
transition at 39 °C with an enthalpy of 0.95 J/g in the heating
cycle. While cooling from the isotropic melt, a sharp
crystallization was observed at −13 °C followed by a glass
transition at −30 °C. PDPPF-co-Ph exhibited only one
transition at −20 °C both in the heating and cooling cycle as
seen in the DSC thermogram. Clear nematic thread-like
patterns of PDP−PF and PDPPF-co-PF polymers could be
observed under the PLM after rapid quenching of the isotropic
sample using liquid nitrogen. Observation under the PLM
indicated that PDP−PF melted at around 40 °C whereas
PDPPF-co-Ph melted at a higher temperature of 116 °C.
Sluggishness of the polymers PDP−PF and PDPPF-co-Ph to
crystallize is ascribed to the increasing flexibility imparted by
the pentadecyl units.27
Poly[(1,4-phenylene)-2,7-(9,9-dioctylfluorene)] (POF−Ph).
1H NMR (CDCl3, 200 MHz): δ (ppm) 7.85 (4H, d), 7.65−7.5
(2H, m), 2.1 (4H, m), 1.15 (24H, m), 0.85 (6H, t). Yield: 75%.
RESULTS AND DISCUSSION
■
Synthesis and Characterization. The synthesis of PDP−
PF and PDPPF-co-Ph are shown in Scheme 1. The dibromo
monomer 2,7-dibromo-9,9-bis(6-(3-pentadecylphenoxy)hexyl)-
9H-fluorene (1) and the dioxaborolane monomer, 2,2′-(9,9-
bis(6-(3-pentadecylphenoxy)hexyl)-9H-fluorene-2,7-diyl)bis-
(4,4,5,5-tetramethyl-1,3,2-dioxaborolane) (2), were synthesized
in 50−64% yield and the details of synthesis are given in the
Experimental Section. The structure and purity of the
monomers were confirmed by 1H NMR, MALDI, and
elemental analysis (Supporting Information SI-1a−f). The
2,7-dibromo-9,9-dioctylfluorene and its boronic ester were
synthesized following reported procedure.24,25 The Suzuki
polymerization of the monomers 1 and 2 in the presence of
Pd(PPh3)4 catalyst afforded poly(2,7-dimethyl-9,9-bis (6-(3-
pentadecylphenoxy)hexyl)-9H-fluorene) (PDP−PF) in 75%
yield. The monomer 1 was coupled with phenyl boronic ester
to obtain the copolymer (PDPPF-co-Ph). Poly[2,7- diyl-(9,9-
dioctylfluorene)] (POF) and its copolymer with a phenyl
group, poly[(1,4-phenylene)-2,7-diyl-(9,9-dioctylfluorene)]
(POF-co-Ph) were also synthesized as benchmark polymers
to compare the photophysical and sensing properties. Scheme 2
shows the structure of all the polymers that were used in the
present study along with the structure of bilirubin. Figure 1
compares the labeled proton NMR spectra of the PDP−PF and
1
copolymer PDPPF-co-Ph. The broadening of the peaks in H
Self-Assembly. The PDP-substituted polyfluorene has a
rod−coil structure with the rigid polyfluorene backbone
forming the rod segment and the hexyloxy appended PDP
unit forming the coil segment. In general, rod -coil structures
modified with hydrophilic and hydrophobic groups have been
NMR indicated reasonably high molecular weights. The
number-average molecular weight (Mn) and polydispersity of
the polymers were determined by gel permeation chromatog-
raphy (GPC) using THF as solvent. The GPC chromatograms
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Figure 2. (a, c) TEM images of PDP−PF (top) and PDPPF-co-Ph (bottom) at 1 × 10−5 M concentration drop cast on carbon coated copper grids.
(b, d) Tapping mode AFM images of PDP−PF (top) and PDPPF-co-Ph (bottom) at 1 × 10−5 M concentration coated on silicon wafers. The
corresponding height section analysis are also shown. (e, f) Schematic diagram showing the self-assembly of PDP−PF and PDPPF-co-Ph into
vesicles and porous spherical aggregates.
shown to exhibit nanophase separation into various morphol-
ogies. Dialkyl-substituted polyfluorenes, in particular 9,9-
dioctylfluorene, have been shown to self-assemble into hairy
rod structures,28 whereas polyfluorenes with poly(ethylene
oxide) side chains have been shown to form fluorescent
micellar structures in aqueous solution.29 The self-assembly
behavior of the two polymers PDP−PF and PDPPF-co-Ph in
tetrahydrofuran (THF) as solvent was studied. Both polymers
had good solubility in THF and a 1 × 10−5 M solution in THF
was drop cast onto TEM grids and the solvent was allowed to
evaporate at room temperature. PDP−PF formed vesicles with
an average diameter of 300−500 nm as shown in Figure 2a top
where a thin wall could be clearly differentiated by the dark
contrast. AFM was also recorded by casting the sample in THF
onto silicon wafers. Figure 2b shows the tapping-mode AFM
height profile clearly showing spheres with a small crater-like
depression characteristic of vesicles. The average diameter
observed from AFM was around 400 nm. In sharp contrast, the
PDPPF-co-Ph formed soap bubble-like porous spherical
structures with average diameter of 300−700 nm (Figure 2c).
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Figure 3. (a) Volume−average size distribution of the two polymers PDP−PF and PDPPF-co-Ph in THF (5 × 10−6 M) obtained by dynamic light
scattering (DLS) analysis. (b) Static light scattering (Guinier plot) for PDP−PF and PDPPF-co-Ph (5 × 10−6 M) in THF.
More TEM images of the porous structures of PDPPF-co-Ph
and vesicles of PDP−PF are given in Supporting Information
(SI-3). The AFM height profile (Figure 2d) also clearly
distinguished them as spheres and not vesicles.
3a. This was in good correlation with the size obtained from
TEM and AFM measurements. The slight increase in size of the
self-assembled structures in film is probably due to aggregation
upon solvent evaporation. The hydrodynamic radius (RH) of 96
and 99 nm was extracted from the dynamic light scattering
experiments for PDPPF-co-Ph and PDP−PF respectively. In
the static light scattering experiment, the intensity of the
scattered light measured at different angles is plotted against q2,
where q is the magnitude of the scattering vector. This plot
known as the Guinier plot is shown in Figure 3b for PDPPF-
co-Ph and PDP−PF. Rg values of 73 and 95 nm were obtained
for PDPPF-co-Ph and PDP−PF respectively. The ratio
between Rg/RH obtained from the static and dynamic light
scattering experiments respectively, called shape factor (ρ) is a
well-known indicator for the morphology of the assemblies in
solution (ρ ∼ 0.77 for hard sphere, ρ ∼ 1 for vesicles, and ρ ∼
1.7 for the coil conformation).36 The ratio Rg/RH was obtained
as 0.76 for PDPPF-co-Ph and 0.96 for PDP−PF confirming the
existence of spherical particles for the former and vesicles for
the latter in solution. Thus, the observations from the light
scattering experiments strongly supported the morphology
observed using the various microscopic techniques.
Photophysical Properties. Figure 4 depicts the absorption
and emission characteristics of all the polymers used in the
present study in THF solvent along with that of the absorption
spectrum of bilirubin. The characteristic broad featureless
absorption of the polymers with absorption maxima at 350 and
366 nm for PDP−PF and PDPPF-co-Ph respectively is
assigned to the π−π* transition of the polyfluorene backbone.
Compared to the absorption of dialkyl-substituted polyfluor-
enes like 9,9′-dioctylpolyfluorene which has absorption wave-
length maxima at 383 nm, the absorption maximum of PDP−
PF was blue-shifted by 33 nm. The introduction of bulky
hexyloxy pentadecyl phenol unit twisted the polyfluorene
backbone resulting in reduced conjugation length as indicated
by the DFT energy minimized structure discussed earlier. The
degree of backbone twisting was lessened in the case of
copolymer PDPPF-co-Ph because of the presence of the rigid
phenyl unit. Figure 4 also compares the normalized emission
spectra (dotted lines) of the polymers in THF upon excitation
at their absorption wavelength maxima. The polymers exhibited
emission spectra with clear vibronic transitions at 409, 432, and
Energy minimization was carried out for one repeat unit of
PDP−PF and PDPPF-co-Ph using DFT,30,31 which showed
that the PDP unit in PDP−PF with the C15 alkyl chain was
extended outward resulting in a torsion angle of ∼62° with the
plane of the fluorene ring. The presence of two hexyloxy
pentadecyl phenyl units on each repeating unit could be
expected to put a strain on the polyfluorene backbone resulting
in reduced conjugation of the backbone. This was proved by
the blue shift in the absorption spectra of PDP−PF compared
to POF, as discussed later on. Figure 2e shows a schematic
diagram representing the self-assembly of PDP−PF into
vesicles. Bilayer structures are formed by the interdigitation of
the C15 alkyl chains of the PDP units, which would further
transform into vesicles due to the curvature formed by twisting
of the strained polyfluorene backbone. The extra phenyl ring on
each repeating unit of the polyfluorene backbone in the case of
PDPPF-co-Ph could be expected to lessen the strain felt by the
backbone leading to a more open porous structure with
accessible interior volumes. Figure 2f shows the schematic
representation of self-assembly in PDPPF-co-Ph resulting in
formation of open and interconnected porous spheres. Small
structural changes in fluorene oligomers have been shown to
result in differences in torsion angles between neighboring units
and different molecular shapes eventually resulting in strong
differences in aggregation behavior.32,33 The differences in the
extent of twisting/torsion angle of the polyfluorene backbone is
expected to be the driving force causing strong differences in
the mode of self-assembly in the two polymers PDP−PF and
PDPPF-co-Ph.
Rod−coil polymers are known to exist in self-assembled
vesicles and micelles in solution and to retain these structures
in film form upon evaporation of solvent.34 Information
regarding the size and morphology of the particles in solution
can be obtained from dynamic and static light scattering
experiments (DLS and SLS).35 Dynamic light scattering (DLS)
analysis in THF showed a good autocorrelation and indicated
presence of particles with average diameter ∼200 and 260 nm
for PDP−PF and PDPPF-co-Ph polymers as shown in Figure
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PDP−PF and PDPPF-co-Ph showed good overlap with the
absorption spectra of bilirubin (shown in Figure 4) indicating
the possibility of fluorescence resonance energy transfer
(FRET) from polymer to bilirubin. Figure 5a shows the effect
of addition of varying concentrations of bilirubin (0.1 × 10−6 to
10 × 10−6 M) on the absorption spectra of PDPPF-co-Ph (5 ×
10−6 M). No change was observed in the absorption spectra of
polyfluorene upon addition of bilirubin, but a new peak at 453
nm corresponding to bilirubin absorption became more
prominent at higher concentrations of bilirubin. Figure 5b
shows the corresponding emission spectra, upon excitation at
367 nm. On addition of bilirubin, there was a drastic reduction
in the characteristic emission of polymer at 410 nm. The inset
in Figure 5b shows the same figure with the unquenched
emission of PDPPF-co-Ph removed for better clarity. Figure 5c
shows the same spectra after normalizing at polyfluorene
emission maxima highlighting the FRET-induced bilirubin
emission at 502 nm. Bilirubin is known to emit at 502 nm with
low quantum efficiency (<0.00002 at 25 °C in (CHCl3).40 The
bilirubin emission at 502 nm increased in the intensity with
obvious color change in solution from blue to green. The inset
in Figure 5c shows photographs taken under a hand-held UV
lamp highlighting the visual color change at various intervals of
addition of bilirubin. The gradual evolution of color change
upon addition of bilirubin to PDPPF-co-Ph is also explained
using the CIE diagram where the emission at various
concentrations was plotted in the CIE graphical representation
in Supporting Information (SI-4). This visual color change
makes quick and easy monitoring of bilirubin levels a reality.
The gradual color change with varying concentration of
bilirubin signals the corresponding change from normal to
abnormal levels of bilirubin.
Similar fluorescence quenching experiments were conducted
for PDP−PF and also for the dioctyl substituted benchmark
polymers POF and POF-co-PH and are given in the
Supporting Information, SI-5. Figure 5d compares the change
in fluorescence intensity for the four polymers as a function of
varying bilirubin to polymer concentration. There was a 10-fold
reduction in the emission of PDPPF-co-Ph for 0.1 mol %
addition of bilirubin. The homopolymer PDPD−PF also
showed a reduction in emission intensity upon addition of
bilirubin although the reduction was gradual and not drastic as
in the case of the phenyl copolymer PDPPF-co-Ph. Compared
to the PDP substituted polyfluorenes, the performance of the
poly 9, 9-dioctylfluorene homopolymer (POF) and copolymer
(POF-co-Ph) were very poor with marginal decrease in
emission intensity. The quenching of polymer fluorescence
upon addition of bilirubin was not accompanied by changes in
the polymer absorption spectra (or excitation spectra; see
Supporting Information, SI-6) which ruled out charge transfer
between polymer and bilirubin. The good overlap in the
emission spectra of polymer and absorption spectra of bilirubin
suggested energy transfer as the most plausible reason for the
quenching of polymer fluorescence. The FRET ratio calculated
as the ratio of integrated area of bilirubin emission from 482 to
700 nm to that of polyfluorene emission in the range 360 to
482 nm for the four polymers as a function of increasing
bilirubin concentration was compared in Figure 5e. At all
bilirubin concentrations, PDPPF-co-Ph exhibited the highest
FRET efficiency compared to the other polymers. The FRET
efficiency at higher bilirubin concentrations was ∼7 times
higher for PDPPF-co-Ph compared to the PDP−PF.
Figure 4. Stack plot of normalized absorption and emission spectra of
all polymers along with the absorption spectrum of bilirubin in THF.
Polymers were excited at the respective absorption wavelength
maxima.
462 nm for PDPPF-co-Ph and at 404, 422, and 456 nm for
PDP−PF corresponding to 0−0, 0−1, and 0−2 transitions
respectively from the lowest singlet excited state. The
fluorescence quantum yield of the two polymers PDP-Ph and
PDPPF-co-Ph along with that of POF and POF-co-Ph
polymers were determined using 0.1 OD solutions in THF
relative to quinine sulfate (Φfl = 0.546) as the reference and the
data is given in Table 2. The PDPPF-co-Ph exhibited the
Table 2. Fluorescence Quantum Yields ΦPF, Spectral
Overlap Integral J(λ), Forster Radii R , and Energy Transfer
̈
0
Efficiency of the Donor Polyfluorenes with Bilirubin as
Acceptor together with the Calculated Donor−Acceptor
Distance (r)
energy
transfer
efficiency
(%)
donor−
acceptor
distance r
(Å)
J(λ) 1015
(M‑1 cm‑1
(nm)4)
Forster
̈
donor
polymer
distance
a
ΦPF
R0 (Å)
PDP−PF
PDPPF-
co-PF
0.61
0.78
1.04
1.40
46.16
48.99
52
86
45.55
36.2
POF
0.72
0.66
1.50
1.38
50.42
50.50
33
43
56.7
POF-co-
Ph
51.39
a
The fluorescence quantum yields were obtained using 0.1 OD
quinine sulfate (Φ = 0.546) solution at 360 nm in 0.1 M H2SO4
solution as a standard.
highest quantum yield of 78%, whereas PDP−PF had a
quantum yield of ∼61% only. Although bulky groups are
known to enhance the quantum yield significantly by retarding
detrimental polymeric aggregation,37 if the bulky groups result
in considerable twisting of the conjugated polymer backbone
the result would be a reduction in emission efficiency and
quantum yield.38
Fluorescence Sensing of Bilirubin. The targeted analyte
molecule bilirubin (structure shown in Scheme 2) undergoes
structural isomerization into another isomer (lumirubin) upon
prolonged exposure to light.39 Hence all our photophysical
studies with bilirubin in THF were carried out in dim light and
bilirubin stock solutions were kept away from light and stored
below room temperature (∼10 °C). The emission spectra of
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Figure 5. (a) Absorption spectra of PDPPF-co-Ph (5 × 10−6 M) upon addition of various concentrations of bilirubin (0.1 × 10−6 M to1 × 10−5 M)
in THF. (b) Fluorescence spectra of PDPPF-co-Ph (5 × 10−6 M) upon addition of various concentrations of bilirubin (0.1 × 10−6 to 1 × 10−5 M) in
THF. Inset shows the same figure without the initial unquenched emission of PDPPF-co-Ph. Excitation wavelength was 367 nm. (c) Normalized
(∼410 nm) emission spectra highlighting the FRET-induced bilirubin emission at 502 nm. Inset shows the photograph of the emission observed
under a hand-held UV lamp. (d) Fluorescence intensity of polymers at 410 nm as a function of [bilirubin]/[polymer] ratio. (e) FRET ratio
(integrated area of bilirubin emission to that of polyfluorene emission; ABR(482−700)/APF(360−482)) of the polymers at various concentration of bilirubin.
Since FRET is a distance-dependent radiationless transfer of
energy from a donor to acceptor molecule it can be used to
investigate molecular interactions between the donor and
acceptor. The FRET efficiency is good for donor−acceptor
distances in the range of ∼10−100 Å and it depends on the
spectral overlap of the donor emission and the acceptor
absorption (spectral overlap integral “J”) and the relative
orientation of the donor and acceptor transition dipole
moments (orientation factor “k”) must be approximately
parallel. The various parameters for calculating donor−acceptor
for the PDP-co-Ph polymer with a maximum value of 86%
followed by the PDP−PF homo polymer (52%). POF and
POF-co-Ph gave efficiency values of 43% and 33%, respectively.
The energy transfer efficiency is related to the Forster distance
̈
by eq 2 in the Supporting Information. The Forster distances
̈
for the four polymers were similar and in the range of 46−50 Å,
which was within the maximum separation distance (20−60 Å)
between donor and acceptor over which resonance energy
transfer can occur.41 From the known values of E and R0, the
actual distance between the donor and acceptor“r” was
determined using eq 1 in the Supporting Information and is
shown in Table 2. Comparing the values of the donor−acceptor
distances of the four polymers it can be seen that PDPPF-co-
Ph had the shortest distance of ∼36 Å, whereas the other
polymers had larger D−A distances (>45 Å).
distance (r) like the spectral overlap integral J(λ), Forster
̈
distance (R0), and energy transfer efficiency (E) were calculated
for 0.1 OD solutions of the polyfluorenes in the absence and
presence of bilirubin (1 × 10−5 M) in THF (Supporting
Information, SI-7) and the calculated values are given in Table
2. Details of the calculations and the equations used are given in
the Supporting Information. The energy transfer was highest
On the basis of these observations a plausible mechanism of
sensing is depicted schematically in Figure 6. The spectral
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Figure 6. Schematic representation of the mechanism of FRET-induced bilirubin sensing in PDPPF-co-Ph and PDP−PF.
Figure 7. (a) Fluorescence lifetime measurement for the polymer [PDPPF-co-Ph] = 5 × 10−6 M at various Bilirubin concentration (1 × 10−6 M to 1
× 10−5 M) collected at 410 nm by using nanoLED of 375 nm. (b) Plot of the average lifetime of PDP−PF and PDPPF-co-Ph as a function of
bilirubin concentration.
overlap of the polyfluorene emission with bilirubin absorption
was in the similar range for all the polymers used in the present
study. The physical proximity of the analyte to the polymer is a
major deciding factor in these energy transfer processes. Figure
6 depicts the open porous spherical self-assembled aggregates
of PDPPD-co-Ph which allows easy access to the bilirubin
molecules whose diameter ranges <5 nm,42 into the interior of
the spherical assembly (rPF‑BR ∼36 Å). This results in a more
efficient transfer of energy from PF to BR upon excitation of
PF. On the other hand, the closed vesicular assembly of PDP−
PF allows interaction of BR only on the periphery. BR cannot
penetrate the clear demarcating walls of the polymer vesicles
limiting their proximity to rPF‑BR >45 Å. The synergistic effect of
morphology and efficient energy transfer thus acts in favor of
PDPPD-co-Ph enabling naked eye detection of bilirubin using
hand-held UV lamp.
Fluorescence Lifetime Studies. Fluorescence lifetime of
the polyfluorenes was obtained by picosecond time-correlated
single photon counting (TCSPC) technique. Time resolved
fluorescence decays were collected at 410 nm (polyfluorene
emission) with excitation at 375 nm in THF as solvent at 25
°C. The lifetime decay analysis of bilirubin could not be carried
out due to the very weak nature of its fluorescence.
Polyfluorenes are known to exhibit complex fluorescence
decay behavior in solution which is both solvent and
temperature dependent.43 The fluorescence decay of PDPPF-
co-Ph could be fitted to a biexponential fit with lifetimes of 211
ps (0.35%) and 495 ps (0.65%) (χ2 = 1.005). The lifetime
reduced sharply with increasing bilirubin concentration reach-
ing biexponential fit values of 38 ps (0.30%) and 253 ps
(0.70%) (χ2 = 1.06) for the highest bilirubin content of 1 ×
10−5 M. Figure 7a shows the time-resolved fluorescence decay
of PDPPF-co-Ph upon addition of varying concentrations of
bilirubin. The decay of the polymer alone in the absence of
bilirubin is also given for comparison. A sharp change in the
decay pattern identifiable as two distinct regions − one where
the slope was similar to that of the polymer alone (>0.2 ps) and
the other where the slope changed with the different bilirubin
concentration (<0.2 ps) was clearly discernible in the PDPPF-
co-Ph bilirubin system. Similar time-resolved fluorescence
decay was collected for the all other polymers also upon
addition of varying amounts of bilirubin (given in Supporting
Information, Table S1). The average lifetime ⟨τ⟩ calculated as
⟨τ⟩ = Σaiτi2/ΣaiτI,44 (where “a” is the amplitude (normalized
contribution)), was plotted as a function of bilirubin
concentration for the two PDP substituted polyfluorenes and
given in Figure 7b. It clearly showed that the average
fluorescence lifetime of the homopolymer PDP−PF did not
decrease much upon bilirubin addition; in fact, after the first
addition of bilirubin the lifetime remained constant indicating
very low energy transfer. On the other hand, the drastic
reduction in the average fluorescence lifetime in the case of
PDPPF-co-Ph upon addition of bilirubin clearly gave evidence
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Figure 8. The normalized (at the wavelength of maximum fluorescence intensity) TRES with a gated time from 0.084 to 4.2 ns: (a) PDPPF-co-Ph
and (b) PDP−PF. (c, d) Contour plots representing the three-dimensional time−intensity−wavelength data collected in the wavelength range 390
to 570 nm using nano LED of 375 nm.
Figure 9. (a) Nanomolar sensing of all polymers. [polymer] = 1 × 10−5 M and [bilirubin] = 1x 10−9 M. (b) Bar graph denoting fluorescence
emission response profiles of PDPPF-co-Ph on addition of various analytes. [PDPPF-co-Ph] = 0.1OD M, [analyte] = 1 × 10−5 M.
for efficient fluorescence energy transfer from polymer to
bilirubin.
times (0.084 to 4.2 ns) during the fluorescence decay. Figure 8
a, b compares the reconstructed normalized (at the wavelength
of maximum emission) TRES for PDP-co-Ph and PDP−PF
respectively during the time 0.084 to 4.2 ns, which was very
similar to the normalized emission spectra in presence of
bilirubin. The steady increase in the evolution of the bilirubin
emission beyond 500 nm as a function of time could be clearly
observed. Beyond 4.0 ns the polymer fluorescence was almost
completely quenched and the bilirubin emission had increased
4-fold in the case of PDPPF-co-Ph. In sharp contrast, the same
time gated spectra for PDP−PF showed that the polymer
Additional insight into the different energy transfer efficiency
of the two polymers PDP-co-Ph and PDP−PF toward bilirubin
was obtained using time-resolved emission spectra (TRES)
based experiments. In the picosecond TRES experiment the
time-resolved decays at a number of wavelength (390−570 nm)
across the emission spectrum were collected after the addition
of 1 × 10−6 M bilirubin to a 5 × 10−6 M solution of the
polymer in THF. A two-dimensional fluorescence emission
spectra was constructed from the intensities sampled at discrete
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emission was quite high compared to bilirubin emission, which
clearly demonstrated the poor energy transfer efficiency of
PDP−PF toward bilirubin compared to the copolymer
PDPPF-co-Ph. Parts c and d of Figure 8 also show the
contour plots representing the three-dimensional time−
intensity−wavelength data showing the evolution of the
FRET-induced bilirubin emission.
Sensitivity and Selectivity. The sensitivity of the bilirubin
sensing was determined by adding nanomolar (1 × 10−9 M)
solution of bilirubin to the polymers PDP−PF, PDPPF-co-Ph,
POF, and POF-co-Ph (concentration of the polymers: 1 × 10−5
M), followed by excitation at the respective absorption
wavelength maximum. Figure 9a compares the fluorescence
spectra of the four polymers before and after addition of
nanomolar concentration of bilirubin. Among all the polymers
PDPPF-co-Ph showed the highest fluorescence quenching of
11% for nanomolar concentrations of bilirubin (1 × 10−9 M)
where as no fluorescence quenching was observed at this low
level of bilirubin in the case of the other polymers.
The selectivity of PDPPF-co-Ph toward bilirubin was
investigated by analyzing sensing activity with structural
analogues of bilirubin like biliverdin and dyes like 5,10,15,20-
tetrapyridyl-23(H),25(H)-porphine (TPY) and rhodamine-B
(Supporting Information, SI-8). Biliverdin, which is another
hemee residue co exsists with bilirubin in plasma and bile and is
the structural analogue of bilirubin with a double bond at the
C10 position.45 The electronic absorption spectrum of
biliverdin has two peak maxima at 376 and 666 nm and it is
a nonfluorescent molecule. The spectral overlap for the
polyfluorene emission and the biliverdin absorption is almost
negligible. 5,10,15,20-tetra(4-pyridyl)-23H-25H-porphine
(TPY) is another structurally similar molecule, which can be
considered as the closed ring analogue of bilirubin. Their
absorption spectrum matches with the emission of the PDPPF-
co-Ph given in Supporting Information. Figure 9b compares the
percentage reduction of polymer (0.1 OD) fluorescence upon
addition of 1 × 10−5 M of various analytes such as bilirubin,
biliveridin, TPY and rhodamine B dye. Pronounced selectivity
was observed only for bilirubin as analyte compared to the
other structural analogues. This confirmed that the polymer
PDPPF-co-Ph had pronounced selectivity for bilirubin.
Biliverdin, the important structural analogue of bilirubin was
unable to quench the polymer fluorescence even at higher
concentrations.
Sensing in Water Medium. Under normal physiological
conditions bilirubin is present in the conjugated form as di- and
monoglucoronides (water-soluble) along with small amounts of
unconjugated bilirubin (water insoluble).39 The ability to sense
the unconjugated or free fraction of bilirubin in presence of the
water-soluble conjugated bilirubin, is a more challenging task.
The unconjugated bilirubin is insoluble in water because of its
ridge tile conformation.17 Literature reports the preparation of
Bilirubin stock solution in water by dissolving known amount
of bilirubin in presence of 10 mmol of NaOH.23 An experiment
was designed to simulate the bilirubin equilibrium in its water-
soluble conjugated form and water insoluble unconjugated
form by carrying out the sensing experiments in THF/water
(containing NaOH) media. Varying amounts (1 × 10−6 M to 1
× 10−5 M) of sodium salt of bilirubin were prepared in water.
The polyfluorene probe PDPPF-co-Ph being insoluble in water
was taken in THF (8 × 10−6 M). Separate experiments were
carried out where the polymer concentration was kept fixed and
the water solution of bilirubin (of different concentrations) was
added to it. Care was taken to see that the polymer aggregation
did not occur under the studied experimental conditions. For
instance, it was observed that the photophysical properties of
the polymer did not vary until a THF/water ratio of 6:4.
Beyond this volume ratio, polymer aggregate emission at 550
nm appeared which would be detrimental in sensing experi-
ments. Therefore, the THF/water ratio was fixed at (7.5: 2.5)
to be on the safe side to avoid polymer aggregation. The
sodium salt of the bilirubin in water was added in increasing
amounts (1 × 10−6 M to 1 × 10−5 M) to the polymer solution
in THF. Quenching of polymer fluorescence was observed
(Supporting Information, SI-9) upon addition of bilirubin,
which was due to the partitioning of bilirubin between water
and THF. It was obvious that the probe was sensitive only to
the free bilirubin present in the organic medium due to the
polymers insolubility in the aqueous medium. Although, this
partitioning of bilirubin between organic and aqueous medium
was not a true reflection of the actual concentrations present in
body fluids, the experiment was conducted to demonstrate the
probable ability of the polymer to sense free bilirubin in the
presence of conjugated form of bilirubin also. Further
experiments are ongoing to develop a fully water-soluble
polyfluorene probe which could be used for sensing albumin
conjugated bilirubin.
CONCLUSIONS
■
In summary, pentadecylphenol-appended polyfluorene homo-
polymer (PDP−PF) and phenyl copolymer (PDPPD-co-Ph)
were synthesized and characterized. The sensing efficiency of
both polymers toward the biologically important analyte
bilirubin was demonstrated by the quenching of polymer
fluorescence and FRET-based bilirubin emission. Although,
both polymers showed good overlap of their respective
emission spectra with the absorption spectra of bilirubin,
higher extent of quenching of polyfluorene emission at 410 nm
(86%) together with increased bilirubin emission at 502 nm (5
times) was observed only in the case of PDPPD-co-Ph. The
change in emission color from blue to green upon addition of
bilirubin could be easily detected using a hand-held UV lamp.
The differential self-assembling tendency of the polymers
open porous interconnected spherical assembly of PDPPD-co-
Ph versus vesicles for PDP−PFproved to play a crucial role
in their energy transfer efficiencies. A short donor (poly-
fluorene) to acceptor (bilirubin) distance of 36 A0 was obtained
for PDPPD-co-Ph in contrast to 46 A0 for PDP−PF using
steady state fluorescence-based calculations. Fluorescence
lifetime decay measurements also showed a significant
reduction in the average lifetimes of PDPPD-co-Ph upon
addition of bilirubin. The sensitivity of bilirubin detection by
PDPPD-co-Ph polymer was tested using nanomolar concen-
trations of bilirubin. PDPPD-co-Ph exhibited a 11% quenching
of its fluorescence upon addition of a 1 × 10−9 M solution of
bilirubin, whereas PDP−PF and dioctyl substituted bench mark
polyfluorene polymers did not exhibit any sensitivity to the
presence of bilirubin at such low concentrations. The selectivity
of detection was also investigated using structural analogues of
bilirubin like biliverdin and dyes like 5,10,15,20-tetrapyridyl-
23(H),25(H)-porphine (TPY) and rhodamine-B. Pronounced
selectivity was observed only for bilirubin as analyte compared
to the other structural analogues. We have thus highlighted the
successful application of tailor-made highly fluorescent
conjugated polymer based on polyfluorene for the sensitive
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ASSOCIATED CONTENT
* Supporting Information
■
S
Details of structural characterization of the monomers and
polymers and FRET-based calculations. This material is
available free of charge via the Internet at http://pubs.acs.
org/.
(26) Grell, M.; Bradley, D. D. C.; Inbasekaran, M.; Woo, E. P. Adv.
Mater. 1997, 9, 798−802.
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AUTHOR INFORMATION
Corresponding Author
*E-mail: sk.asha@ncl.res.in. Fax: 0091-20-25902615.
■
(29) Yao, J. H.; Mya, K. Y.; Shen, L.; He, B. P.; Li, L.; Li, Z. H.; Chen,
Z.-K.; Li, X.; Loh, K. P. Macromolecules 2008, 41, 1438−1443.
(30) Ahlrichs, R.; Bar, M.; Baron, H. P.; Bauernschmitt, R.; Bocker,
̈
̈
Notes
S.; Ehrig, M.; Eichkorn, K.; Elliott, S.; Furche, F.; Haase, F.; Haser, M.;
̈
Horn, H.; Huber, C.; Huniar, U.; Kattannek, M.; Kolmel, C.; Kollwitz,
M.; May, K.; Ochsenfeld, C.; Ohm, H.; Schafer, A.; Schneider, U.;
Treutler, O.; von Arnim, M.; Weigend, F.; Weis, P.; Weiss, H.
̈
The authors declare no competing financial interest.
̈
̈
ACKNOWLEDGMENTS
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TURBOMOLE (Version 6.2); Universitat Karlsruhe: Karlruhe,
̈
This work has been financially supported by the network
project NWP0054. The authors would like to thank Dr. Partha
Hazra, IISER-Pune for TRES and Fluorescence Lifetime facility
and Dr. Suresh Bhat, NCL-Pune for the Static light scattering
measurements. The authors also thank Mr. Anuj, Mrs. Pooja,
and Mr. Pankaj at NCL-Pune for the TEM and AFM analyses.
K.T.S. thanks CSIR-NCL for research fellowship.
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dx.doi.org/10.1021/ma4000946 | Macromolecules XXXX, XXX, XXX−XXX