Design and synthesis of monofunctionalized, water-soluble conjugated polymers for biosensing and imaging applications

Article abstract of DOI:10.1021/ja202877q

Water-soluble conjugated polymers with controlled molecular weight characteristics, absence of ionic groups, high emission quantum yields, and end groups capable of selective reactions of wide scope are desirable for improving their performance in various

Full text of DOI:10.1021/ja202877q

ARTICLE
pubs.acs.org/JACS
Design and Synthesis of Monofunctionalized, Water-Soluble
Conjugated Polymers for Biosensing and Imaging Applications
Christopher A. Traina, Ronald C. Bakus II, and Guillermo C. Bazan*
Department of Materials and Chemistry and Biochemistry, Center for Polymers and Organic Solids, University of California,
Santa Barbara, California 93106, United States
S Supporting Information
b
ABSTRACT: Water-soluble conjugated polymers with controlled molecular
weight characteristics, absence of ionic groups, high emission quantum yields,
and end groups capable of selective reactions of wide scope are desirable for
improving their performance in various applications and, in particular, fluor-
escent biosensor schemes. The synthesis of such a structure is described herein.
2-Bromo-7-iodofluorene with octakis(ethylene glycol) monomethyl ether
chains at the 9,90-positions, i.e., compound 4, was prepared as the reactive
premonomer. A high-yielding synthesis of the organometallic initiator
(dppe)Ni(Ph)Br (dppe = 1,2-bis(diphenylphosphino)ethane) was designed and implemented, and the resulting product was
characterized by single-crystal X-ray diffraction techniques. Polymerization of 4 by (dppe)Ni(Ph)Br can be carried out in less than
30 s, affording excellent control over the average molecular weight and polydispersity of the product. Quenching of the
polymerization with [2-(trimethylsilyl)ethynyl]magnesium bromide yields silylacetylene-terminated water-soluble poly(fluorene)
with a photoluminescence quantum efficiency of 80%. Desilylation, followed by copper-catalyzed azideꢀalkyne cycloaddition
reaction, yields a straightforward route to introduce a wide range of specific end group functionalities. Biotin was used as an example.
The resulting biotinylated conjugated polymer binds to streptavidin and acts as a light-harvesting chromophore to optically amplify
the emission of Alexa Fluor-488 chromophores bound onto the streptavidin. Furthermore, the biotin end group makes it possible to
bind the polymer onto streptavidin-functionalized cross-linked agarose beads and thereby incorporate a large number of optically
active segments.
’ INTRODUCTION
for the hydrophobic nature of the backbone. Biosensors con-
structed from conjugated polyelectrolytes often rely on Coulombic
interactions for binding to analytes or probe molecules.31,32
While effective for binding to the desired substrate, these charges
may also cause nonspecific binding to other species, potentially lead-
ing to increased background noise or false-positive readings.33,34
Additionally, the sensitivity of these sensors can be compromised
in certain environments, such as those containing high salt con-
centrations, as these ions can screen charges and disrupt sensorꢀ
target interactions.35
There are conjugated polymer-based biosensors that utilize
covalently bound recognition probes for binding to analytes, such
as the biotin-conjugated poly(phenyleneethynylene)s reported by
Swager and co-workers.36 The syntheses of such polymers
often utilize step-growth procedures, whereby monomers with
appended biotargeting molecules are copolymerized with those
containing no biofunctionalization. Thus, the probes in these
materials are distributed randomly along the backbone. Varia-
bility in sensing is reasonable under these circumstances, parti-
cularly when the statistical characteristics of the polymer chains
are also taken into account.
Conjugated polymers offer a set of optoelectronic and light-
harvesting properties that are unique for macromolecular mate-
rials and that are relevant for various emerging technologies.1ꢀ8
For instance, thin films of these materials find applications in
solution-processed field effect transistors,9 polymer light-emitting
diodes,10 and plastic solar cells.11,12 There is ample evidence that
bulk behavior is influenced by the molecular characteristics of
single chains, interchain relationships, and the processing history
of the material.13ꢀ17 Solvent interactions are important for deter-
mining the latter, and there is a significant body of work asso-
ciated with the need to understand self-assembly in solution prior
to film formation.18
Various conjugated polymer materials have also been studied
as useful platforms for facilitating optically amplified chemical
and biological fluorescent sensors.19ꢀ22 The π-electron deloca-
lization increases the electronic coupling between segments along
the chain, thereby allowing excited states to interact with analytes
within a larger sample volume, relative to small-molecule coun-
terparts.23 Environmental perturbations are therefore more
easily detected. Solubility in aqueous media is a typical require-
ment for sensing biological targets. Conjugated polymers used
for this purpose include pendant groups with ionic (i.e., con-
jugated polyelectrolytes)24ꢀ26 or highly polar functionalities, for
example, oligo(ethylene glycol) segments,27ꢀ30 that compensate
Received: March 30, 2011
Published: July 13, 2011
r
2011 American Chemical Society
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Scheme 1. Synthesis of Premonomer 4a
Figure 1. Structural and physical attributes of an ideal biosensing
poly(fluorene) material.
On the basis of the discussion above, one can envision several
desirable properties of an ideal conjugated polymer for biosensor
applications, namely, high photoluminescence quantum yield,
water solubility, uniformity in chain length, resistance to nonspecific
interactions, and a single site for biorecognition (Figure 1). To
the best of our knowledge, such materials have not been reported
and their preparation would require taking full advantage of
modern polymer synthesis techniques. Current conjugated poly-
mer syntheses generally involve Suzuki37 or Stille38 reaction schemes
to couple a bis(halo)aryl species to an arylbisboronate or -bisstannane,
respectively, and under these conditions, high molecular weight
polymers have been generated.39,40 These step-growth polymer-
izations may suffer from lack of control, necessitating purification
of the crude product; furthermore, significant variations in physical
properties can occur between different batches of the same poly-
mer.41,42 As such, the wide scope of conjugated polymer biosensor
applications can benefit from improved synthetic procedures.
In this paper we detail a general synthetic approach for
accessing water-soluble conjugated polymers with excellent mole-
cular weight uniformity and with chain end functionalization
comprising reactive groups capable of selective postsynthesis
modification reactions of wide scope. The work utilizes a well-
defined, highly active transition-metal initiator for mediating
controlled polymerization and demonstrates “click” chemistry
protocols amenable for introduction of specific probes. As a final
point, we show that the overall process allows biotin incorpora-
tion and that this terminal functionality has been shown to be
reactive through solution and surface binding experiments to
commercially available soluble streptavidin protein and strepta-
vidin-conjugated microspheres, respectively.
a Synthetic conditions: (i) tosyl chloride, triethylamine, ether, 0 °C to
room temperature, 18 h; (ii) (a) NaH, THF, 2 h, (b) 1, THF, 19 h;
(iii) NaI, acetone, reflux, 15 h; (iv) (a) KH, THF, 1.5 h, (b) 3, THF, 14 h.
characteristics over thiophene-based materials, especially in aqueous
media, but metalꢀhalogen exchange is slower for 2,7-dibromo-
9,90-dialkylfluorene systems, often requiring more than 8 h to
form the active metalated species. Furthermore, this treatment
can result in unreacted alkyl Grignard in solution or the genera-
tion of bismetalated fluorene species, both of which hinder chain
growth characteristics.47 In contrast, it has been shown that
2-bromo-7-iodofluorene premonomers subjected to similar con-
ditions undergo metalꢀhalogen exchange exclusively with iodide,
generating themonometalatedactivespecieswithin1h.48 Therefore,
a 2-bromo-7-iodofluorene core was selected for our study.
Fluorene repeat units with oligo(ethylene glycol) side chains
were targeted so to obtain water soluble polymers. The length
of solubilizing oligo(ethylene glycol) group required some con-
sideration: a chain which was too short would not provide
the necessary solubility, whereas one that was too long would
dilute the emissive component of the material and could
potentially interfere with substrate binding. Fluorene-co-phenylene
polymers containing alternating tris(ethylene glycol) mono-
methyl ether and tris(ethylene glycol) tert-butyl ester side chains
show good organic solubility, but are not completely water-
soluble.49 2-Bromo-7-iodofluorene with octakis(ethylene glycol)
monomethyl ether chains at the 9,90-positions was therefore
selected. The octakis(ethylene glycol) chain was synthesized
from two tetrakis(ethylene glycol) subunits,50 as illustrated in
Scheme 1. First, tosyl groups were installed on both ends of
tetrakis(ethylene glycol) by treatment with tosyl chloride to give
the bis(tosylate) 1.51 Tetrakis(ethylene glycol) monomethyl
ether was then deprotonated with sodium hydride and slowly
added to a THF solution of 1 to yield octakis(ethylene glycol)
monomethyl ether tosylate, 2.52 Conversion of the terminal tosyl
group to iodide gave 3.53 Treatment of 2-bromo-7-iodofluorene
with potassium hydride in THF, followed by addition of 3,
afforded the premonomer 4 in good yield.
’ RESULTS AND DISCUSSION
General Considerations for Monomer Design. Chain
growth methods have been developed that generate well-defined
conjugated polymers for a limited number of systems.43 For
example, McCullough and co-workers44 and Rieke and co-workers45
reported polymerization of asymmetric monobromomonome-
tallothiophene monomers by (L)NiCl2 catalysts, where L is a
bidentate bisphosphine ligand. One can thereby generate regiore-
gular poly(3-alkylthiophenes) in good yield, with high degrees of
polymerization (DP) and low polydispersity indices (PDIs < 1.4).
The monohalomonometallo active monomer necessary for such
polymerizations is generated in situ and can be achieved through
metalꢀhalogen exchange with a Grignard reagent.46 This pro-
cess is effective for 2,5-dibromo-3-alkylthiophene premonomers,
requiring less than 1 h for full conversion to the active species.
Fluorene-based polymers usually provide superior emission
Initiator Selection. Grignard metathesis polymerizations
of 3-alkylthiophenes often rely on complexes such as [1,3-bis-
(diphenylphosphino)propane]nickel dichloride [(dppp)NiCl2]54,55
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Scheme 2. Synthesis of the (dppe)Ni(Ph)Br Catalyst/Initiatora
the nickel center and an out-of-plane twist of the ethyl backbone
for the dppe ligand.
Polymerization and End Functionalization. Initial screens
of the polymerization activity by using (dppe)Ni(Ph)Br were
conducted with 2,5-dibromo-3-dodecylthiophene. A high degree
of polymerization and low polydispersity were achieved (DP ≈
100ꢀ200, PDI ≈ 1.1) with short reaction times (∼5 min; see the
Supporting Information), and a linear dependence of molecular
weight could be demonstrated through adjustment of the initiator
to monomer ratio. Complex (dppe)Ni(Ph)Br also proved simi-
larly successful for polymerization of bis(alkoxy)phenyl and 9,90-
dialkylfluorene monomers (Supporting Information).
a Synthetic conditions: (i) 2 equiv of ethylmagnesium bromide, 2 equiv
of PPh3, diethyl ether, 2 h; (ii) bromobenzene, THF, 40 h.
Subsequent studies focused on the polymerization of 4, as
shown in Scheme 3. Treatment of 4 with isopropylmagnesium
chlorideꢀlithium chloride complex at ꢀ35 °C for 40 min
resulted in formation of the reactive species, which, in the
presence of (dppe)Ni(Ph)Br, quickly polymerized (<30 s) to
give the product as a yellow-green, waxy solid. Purification was
achieved through selective precipitation and extraction proce-
dures that took advantage of the high solubility of the product in
solvents such as CH2Cl2 and toluene, and its insolubility in
hexane. With a monomer to catalyst ratio of 40:1, GPC analysis
(DMF, 0.01% LiBr, calibrated against polystyrene standards)
provided an Mn in the 26000ꢀ30000 range with PDI values of
1.3ꢀ1.4. It is well-known that discrepancies can exist between the
true molecular weight and that measured by GPC.65 The polymer
molecular weight was therefore measured by dynamic light
scattering (DLS) techniques in DMF to obtain a value of 32 000,
which is similar to that obtained by GPC. The number of repeat
units can therefore be estimated using Mn and the mass of each
repeat unit (897 g/mol) to give a DP of between 29 and 33.
Extension of the polymerization time beyond 30 s yielded only
minimal gains in molecular weight at the expense of broader PDI
values.
Figure 2. POV-Ray depiction of the molecular structure of (dppe)Ni-
(Ph)Br using 50% ellipsoids. Solvent and hydrogen atoms were omitted
for clarity (C, gray; Br, red; Ni, green; P, orange).
or [1,2-bis(diphenylphosphino)ethane]nickel dichloride [(dppe)-
NiCl2],56,57 and it has been shown that each chain end of the
polymer may be active for monomer addition.58 End functiona-
lization has been achieved by quenching of the polymerization
with a suitable Grignard reagent (RMgX), generating MgX2 and
R-terminated polymer.59,60 For systems catalyzed by (dppp)-
NiCl2, double end functionalization is a possibility due to reaction
at each terminus. Controlled 3-alkylthiophene polymerization
and selective end functionalization have also been demonstrated
using complexes of the type LNi(Ar)X, where L is a bidentate
bisphosphine ligand, Ar is an aryl group, and X is a halogen.61ꢀ63
For these reactions one chain end of the polymer corresponds to
the Ar group. Initial screens in our laboratory demonstrated a
faster activity with (dppe)NiCl2, relative to (dppp)NiCl2, for the
polymerization of 2,5-dibromo-3-dodecylthiophene. For example,
120 s after monomer addition, (dppp)NiCl2 yielded a polymer
with a number average molecular weight (Mn) of 14 000, while
(dppe)NiCl2 provided a product with Mn = 21 000, as deter-
mined by gel permeation chromatography (GPC) relative to
polystyrene standards.
Quenching of polymerizations of 4 with [2-(trimethylsilyl)-
ethynyl]magnesium bromide was carried out to examine the
efficiency of introducing a silylacetylene end group, as shown in
1
Scheme 3. H NMR analysis of the purified product revealed
three resonances between 8.0 and 7.8 ppm, which correspond to
the three unique aromatic proton environments.66 Signals due to
the methylene protons from the pendant oligo(ethylene glycol)
chains appear between 3.6 and 2.6 ppm,67 and the singlet attrib-
uted to the trimethylsilyl-protected ethynyl group appears at
0.28 ppm.68 These data are consistent with the structure of the
product 5 in Scheme 3. An estimate of end group incorporation
was calculated to gauge the efficiency of this procedure. An
Mn = 29 000 polymer (as measured by GPC) should contain
1
On the basis of the preceding discussion, the complex
cis-(bromo)(phenyl)[1,2-bis(diphenylphosphino)ethane]nickel,
(dppe)Ni(Ph)Br in Scheme 2, was chosen as the initiator64
and was synthesized in two steps from commercially available
starting materials. First, a suspension of (dppe)NiCl2 in the
presence of PPh3 was treated with 2 equiv of ethylmagnesium
bromide to afford (dppe)Ni(PPh3)2. Oxidative addition of bromo-
benzene by (dppe)Ni(PPh3)2 resulted in formation of the target,
(dppe)Ni(Ph)Br. The structure of (dppe)Ni(Ph)Br was con-
approximately 32 units. Each aromatic H NMR resonance
accounts for two protons on the repeat unit, and therefore, a
polymer with 32 repeat units should give rise to an integrated
value of 64 protons for each aromatic resonance. For 100% end
group incorporation, the trimethylsilyl group should integrate for
9 H, and a ratio of (trimethylsilyl)ethynyl to each aromatic
resonance of 9:64 or 0.14 should result. The 1H NMR spectrum
of 5 gives an integral ratio of 8:64 or 0.125 from resonances at
0.28 ppm ((trimethylsilyl)ethynyl end group) and 8.0 ppm (one
set of backbone aromatic protons). Division of this experimental
value by the theoretical maximum, as calculated above, repro-
ducibly demonstrated 80ꢀ90% acetylene end group incorpora-
tion into the polymer. End group incorporation was further
confirmed using MALDI-TOF mass spectrometric analysis of a
low molecular weight oligomer, prepared similarly to the polymer
1
firmed using H, 13C, and 31P NMR spectroscopy, elemental
analysis, and X-ray crystallography. Two doublets at 55 and 36
ppm are observed in the 31P NMR spectrum, indicating two
inequivalent phosphorus environments.62 As shown in Figure 2,
the solid-state structure as determined by single-crystal X-ray
diffraction studies shows a pseudosquare planar geometry about
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Scheme 3. Polymerization and End Functionalization of 4a
a Synthetic conditions: (i) iPrMgCl LiCl, THF, ꢀ35 °C, 40 min; (ii) (a) (dppe)Ni(Ph)Br, room temperature, 20 s, (b) [2-(trimethylsilyl)-
3
ethynyl]magnesium bromide, THF, 1.5 h.
moieties.74 Several biologically relevant organoazides are com-
mercially available or can be readily synthesized; therefore, this
reaction was a logical choice for attaching relevant functional
probes. Typical reactions involve a terminal alkyne,75 and thus, it
was necessary to deprotect the terminal group of polymer 5.
Desilylation of this alkyne can be accomplished by treatment
of 5 with potassium carbonate in methanol,76 as illustrated in
Scheme 4, to yield the deprotected alkyne-bearing polymer 6.
Figure 4 depicts a comparison of the upfield region (0.5ꢀ0 ppm)
for the 1H NMR spectra of 5 and 6. It is evident that product 6
lacks the signal from the terminal trimethylsilyl protons, as was
seen in the spectrum of 5; no differences in polymer solubility
before and after desilylation were observed.
After deprotection, the terminal alkyne of 6 is active for click
chemistry. To determine an approximate yield of this final step,
polymer 6 was treated with a test reagent, (3-azidopropyl)tri-
Figure 3. Absorption (left) and photoluminescence (right) spectra of
polymer 5 in water.
methylsilane, in the presence of Cu(II)/sodium ascorbate in a
THF/H2O/tert-butyl alcohol solution77 to give triazole-coupled
product 7, as shown in Scheme 4. This test reagent was selected
to provide a unique signal in the 1H NMR spectrum that can be
but with a higher initiator:monomer ratio (Supporting In-
formation). The resulting mass distribution exhibits peaks at
1968.0, 2864.4, and 3761.9 amu, corresponding to n = 2, 3, and 4
fluorene repeat units (MW = 896.51), respectively, with phenyl
and (trimethylsilyl)ethynyl end groups. These data confirm the
presence of the desired end groups on the majority of product.
Optimization studies showed that success of end group incor-
poration depends on the polymerization time and decreases if the
terminating Grignard reagent is added more than 30 s after
initiation. Therefore, care was taken to determine the optimal
polymerization time (20 s) that achieved high molecular weight
polymer while still retaining satisfactory end group incorporation.
Polymer 5 is soluble in polar organic solvents, including
CH2Cl2, CHCl3, methanol, ethanol, acetonitrile, and toluene.
Furthermore, it can be dissolved in water and aqueous buffer
solutions. Figure 3 shows the UVꢀvis absorption and emission
properties in water, which are similar to those of other poly-
(fluorene)s.69 Solutions of 5 display a broad absorption peak with
a maximum (λmax) at 387 nm in CHCl3, while the λmax for
aqueous solutions occurs at 405 nm, with a shoulder at 387 nm;
the extinction coefficient is on the order of 4 ꢁ 104 Mꢀ1 cmꢀ1, as
calculated per repeat unit. Solution-state photoluminescence
measurements reveal a quantum yield of 0.8 in aqueous solution,
which is comparable to those of other highly efficientwater-soluble
fluorene-based polymers.70,71 Emission maxima are located at
420 nm in CHCl3 and 425 nm in water.
used to determine reaction efficiency. Furthermore, GPC mea-
surements of 5, 6, and 7 showed no major differences in Mn or
PDI, indicating no change in the hydrodynamic volume of
polymer due to potential undesirable side reactions. Therefore,
1
a ratio was calculated between H NMR integrated intensities
from the appended trimethylsilyl-containing test reagent and
backbone aromatic proton resonances in 7. Polymers 5 and 7
each contain a terminal trimethylsilyl moiety, the former from
initialin situ endfunctionalizationand the latter from thefinal click
1
reaction. The ratio of the integrated H NMR peak intensities
between (trimethylsilyl)ethynyl and one set of backbone aromatic
protons in 5 was compared to the ratio obtained for 7, and the
values were found to be virtually identical (0.125 vs 0.120),
indicating nearly quantitative conversion and successful postpoly-
merization incorporation of azide-containing organic reagents.
End Group Conjugation for Biospecificity. To demonstrate
the applicability of this system to biosensing and imaging
applications, it was necessary to attach an appropriate model
reagent to the chain end, so as to provide a handle for studying
probeꢀtarget specificity. For this purpose, we chose the biotinꢀ
streptavidin interaction, which is one of the strongest noncova-
lent interactions78 and is often utilized as a model for biosensor
applications79,80 and as an affinity pair in standard bioassays.81,82
Furthermore, derivatized streptavidin proteins and azide-containing
biotinreagents arecommercially available. As showninScheme4,
using a procedure similar to that for the preparation of 7, biotin-
dPEG3+4-azide83 (an azide-containing biotin with an oligo-
(ethylene glycol) spacer; see the Supporting Information for
the full structure) was reacted with polymer 6, and the product, 8,
End Group Functionalization via Click Chemistry. The
copper-catalyzed azideꢀalkyne cycloaddition reaction72 is one
type of click chemistry73 that is useful for installing a wide variety
of functionalities to organic compounds bearing alkyne or azide
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Scheme 4. Postpolymerization Click Conjugation of 5a
a Synthetic conditions: (i) K2CO3, methanol, room temperature, 5 h; (ii) RN3, CuSO4 5H2O/sodium ascorbate, THF/tert-butyl alchol/H2O, room
3
temperature, 24 h. See the Supporting Information for the molecular structure of 8.
Scheme 5. Schematic for FRET Detection of Binding between
8 and StreptavidinꢀAlexa Fluor-488
Figure 4. 1H NMR spectra (0.5ꢀ0 ppm) of 5 (top), 6 (middle), and 7
(bottom), highlighting deprotection of the (trimethylsilyl)ethynyl
end group of 5 (0.28 ppm) and subsequent click reaction of 6 with
(3-azidopropyl)trimethylsilane to generate 7 (0.02 ppm).
are assigned to carbonyl signals from biotin84 and amide groups
in the linker. Additionally, no azide stretch was evident at
2100 cmꢀ1, verifying removal of all unbound biotin reagent.
The ability of the biotin-functionalized polymer terminus of 8
to bind to a streptavidin receptor was used as a model assay to
determine specific probeꢀanalyte interactions. Streptavidin con-
jugates with appended chromophores are commercially available
and were used to establish binding through fluorescence reso-
nance energy transfer (FRET) experiments. FRET is a nonra-
diative transfer of energy from a donor absorbing moiety to an
acceptor fluorophore.85 A typical experiment involves excitation
of a donor, followed by energy transfer to and emission by the
acceptor, resulting in donor emission quenching and sensitiza-
tion of acceptor luminescence. The efficiency of energy transfer is
related to the spectral overlap between the donor and acceptor
but, more importantly for the purpose of our experiments, is
dependent on the spatial distance between the two and, thus, is
useful for verification of binding interactions.86,87 Typical effec-
tive FRET distances range between 1 and 10 nm, as energy
transfer efficiency decays as 1/r6, with r being the distance
between donor and acceptor.88 A streptavidin reagent with
appended Alexa Fluor-488 organic dye molecules (5 mol of
dye/mol of streptavidin) was selected as the binding partner due
Figure 5. Comparison of FT-IR spectra of 6 (blue) and 8 (green),
highlighting new carbonyl bands (1708 and 1658 cmꢀ1) in 8 from
successful attachment of biotin reagent. The absence of the azide stretch
(2100 cmꢀ1) indicates efficient removal of the starting material.
was purified by dialysis against methanol. GPC analysis of 8
revealed no major differences in Mn or PDI, relative to those of 5
or 6. No resonances specific to the appended biotin moiety were
1
observed in the H NMR spectrum of 8, likely due to overlap
with polymer resonances, and therefore, NMR spectroscopy was
unsuitable for determination of conjugation success. However,
Fourier transform infrared (FT-IR) spectroscopy bands specific
to the biotin fragment were useful in demonstrating attachment.
As shown in Figure 5, the FT-IR spectrum of precursor polymer
6 contains bands near 1650 and 1600 cmꢀ1; after the click
reaction, new bands at 1710 and 1660 cmꢀ1 are observed, which
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to suitable overlap between the emission of 8 and the absorption
of the dye (λmax,abs = 495 nm, λmax,em = 520 nm). Binding
between streptavidin and the terminal biotin of 8 should bring
the optical partners into close proximity, resulting in efficient
FRET. The overall process is illustrated in Scheme 5. In contrast,
no interaction should exist between a control polymer (5 or 7)
and streptavidin.
Thus, a solution of 8 (172 nM, calculated in terms of polymer
chains on the basis of the Mn from GPC) in Tris buffer was
treated with aliquots of the streptavidinꢀAlexa Fluor-488 dye
conjugate (8.6 nM, calculated per protein molecule). As shown
in Figure 6, before streptavidinꢀdye addition, excitation at
405 nm gave a typical polymer emission spectrum with a
maximum near 425 nm. After introduction of one aliquot of
streptavidinꢀdye, reduced polymer emission was observed with
concomitant appearance of dye emission at 520 nm. The solution
was treated with another aliquot of streptavidinꢀdye, and further
attenuated polymer emission and dye luminescence enhance-
ment can be observed. This process, shown in Figure 6, was
repeated until no further decrease in polymer luminescence or
increase in dye emission was detected, and the solution concen-
tration of streptavidinꢀdye was noted (34.4 nM). It is known
that streptavidin is a tetrameric protein capable of binding
up to four biotin molecules,89 and thus, the concentration of
biotin-binding sites should be equal to 4 times that of streptavidin
protein molecules. The saturation concentration of streptavidin
for this experiment, after which no further FRET was observed,
therefore corresponds to an equivalent biotin-binding site con-
centration of 138 nM (i.e., 34.4 nM ꢁ 4).90 A comparison of this
biotin-binding site concentration relative to the concentration of
polymer chains in solution (172 nM) gives a ratio of ∼0.8. As
noted earlier, GPC data and 1H NMR spectra of 5 are consistent
with approximately 80ꢀ90% (trimethylsilyl)ethynyl end group
incorporation. Comparison between the NMR spectra of 5 and 7
indicates nearly quantitative click reactivity of this terminal
alkyne, and therefore, approximately 80ꢀ90% of the polymer
chains of 8 should contain a biotin end group. Thus, the ratio of
streptavidin-bound polymer calculated from the FRET experi-
ment (0.8) is approximately equal to the ratio of biotin-termi-
nated polymer estimated from 1H NMR spectroscopy and GPC
data (0.8ꢀ0.9), indicating excellent end group binding activity.
An appropriate control experiment was performed using a poly-
mer which contained no biotin terminus, 5 or 7 (Scheme 5). As
anticipated, addition of streptavidinꢀdye to a solution of 5 or 7
gave rise to no significant polymer emission quenching or dye
luminescence upon excitation at 405 nm (Supporting Information).
The absence of energy transfer between 5or 7and the streptavidinꢀ
dye conjugate highlights the resistance of these polymers to non-
specific interactions.
It should also be noted that significant enhancement of the
Alexa Fluor dye emission was observed upon streptavidinꢀ8
binding due to FRET sensitization, as compared to direct
excitation at 495 nm. As shown in Figure 6, at the concentrations
used for this experiment, excitation at the polymer absorption of
405 nm gives rise to a greater than 5-fold increase in dye emission
intensity as compared to direct dye excitation. This type of signal
enhancement has been observed in other conjugated polymerꢀ
organic dye systems due to the increased absorption cross-
section provided by the polymer, coupled with efficient energy
transfer.19,25,26,91,92 As expected, no signal enhancement is
evident for the control system, streptavidinꢀAlexa Fluor-488
and 5.
Surface Reactivity of Biotin-Functionalized Polymers. An-
other type of test was devised to examine the surface binding
reactivity93 of the biotin terminus of polymer 8. Streptavidin-
functionalized cross-linked agarose beads were incubated with a
solution of 8 (or 5, as a control). Upon removal of nonbound
polymer, beads treated with 8 were highly luminescent under UV
excitation, whereas no luminescence was observed for samples
treated with 5. Confocal microscopy images of individual parti-
cles confirmed this observation. Beads treated with 8 or 5 were
imaged under bright-field illumination and in luminescence
Figure 6. FRET from 8 (172 nM, excitation at 405 nm) to streptavidinꢀ
Alexa Fluor-488 (8.6 nM aliquots) showing decreased polymer emission
(425 nm) and increased dye emission (520 nm) with increasing strepta-
vidinꢀdye concentration. Saturation of dye emission was observed after
addition of four aliquots (34.4 nM streptavidinꢀdye). The orange
spectrum at the bottom corresponds to emission from direct dye excitation
(495 nm) for the highest streptavidinꢀAlexa Fluor-488 concentration,
highlighting signal enhancement due to FRET from 8.
Figure 7. Confocal microscope images of streptavidin-coated beads treated with 8 (A) or 5 (C) captured in luminescence mode (left panel) or with
bright-field illumination (right panel). The middle image (B) is an overlay of luminescence and bright-field images for a mixture of both sets of beads,
showing emission from beads treated with 8, but no emission is visible from beads treated with 5. The scale bar corresponds to 100 μm.
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ARTICLE
mode using 405 nm laser excitation. Figure 7A shows that beads
treated with 8 exhibit blue emission. However, no luminescence
was observed for those control particles treated with 5 (Figure 7C).
Figure 7B displays the emission from a batch where emissive
(treated with 8) and nonemissive (treated with 5) beads were
mixed together. Thus, polymers such as 8 provide well-defined
systems that are reactive for binding in solution and on surfaces
and hold promise as luminescent reporters for a number of
biological recognition applications.
’ AUTHOR INFORMATION
Corresponding Author
bazan@chem.ucsb.edu
’ ACKNOWLEDGMENT
Financial support from the Institute for Collaborative Bio-
technologies, the National Science Foundation (Grant DMR
1035480), and the Department of Energy is gratefully acknowl-
edged. We thank Alex Thomas for assistance with the confocal
microscopy measurements.
’ CONCLUSIONS
The work herein shows that it is possible to take advantage of
monomer and catalyst design, together with appropriate termi-
nation reactions, for the synthesis of water-soluble monofunctio-
nalized poly(fluorene)s with controlled molecular weight char-
acteristics and excellent incorporation of protected ethynyl end
groups. The organometallic initiator (dppe)Ni(Ph)Br allows poly-
merization reactions to be carried out in less than 30 s, yielding
poly(fluorene)s with high Mn and low PDI values. Similar success
in the preparation of other conjugated polymer structures is
anticipated. After facile deprotection, the monofunctionalized
poly(fluorene) reacts readily with azide-containing organic com-
pounds in nearly quantitative yield, and this chemistry has been used
to couple biorelevant targeting molecules to one of the polymer
chain ends. This synthesis strategy results in highly luminescent
materials, capable of engaging in biologically specific binding
interactions. In one demonstration, a biorecognition event is
monitored by the extent of energy transfer between a biotin-
terminated poly(fluorene) and an organic dye-conjugated streptavidin.
FRET between the polymer and dye due to biotinꢀstreptavidin
binding gives rise to decreased polymer luminescence and enhanced
dye emission and scales with increased concentration of streptavidinꢀ
dye. Substantial signal enhancement upon polymer excitation was
observed when compared to direct dye excitation. These materials
are also active for surface binding; streptavidin-coated beads showed
blue luminescence when treated with biotin-functionalized polymer,
whereas no emission was evident in a control experiment. These
solution and surface recognition capabilities make this material
attractive as a component of luminescent bioassays or for in vitro
tagging or imaging applications. More generally, a polymer, mono-
functionalized with an alkyne moiety as demonstrated herein, is a
versatile luminescent reagent platform which can be customized
for a particular application by reaction with an appropriate azide
starting material. Furthermore, this general scheme is also suitable
for fluorene monomers with different substituents at the 9,90-
positions, for applications where a highly luminescent, well-
defined, monofunctionalized material is important, but solubility
in organic solvents may be more relevant.
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b
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