Solution-based single molecule imaging of surface-immobilized conjugated polymers

Article abstract of DOI:10.1021/ja311874f

The photophysical behavior of conjugated polymers used in modern optoelectronic devices is strongly influenced by their structural dynamics and conformational heterogeneity, both of which are dependent on solvent properties. Single molecule studies of these polymer systems embedded in a host matrix have proven to be very powerful to investigate the fundamental fluorescent properties. However, such studies lack the possibility of examining the relationship between conformational dynamics and photophysical response in solution, which is the phase from which films for devices are deposited. By developing a synthetic strategy to incorporate a biotin moiety as a surface attachment point at one end of a polyalkylthiophene, we immobilize it, enabling us to make the first single molecule fluorescence measurements of conjugated polymers for long periods of time in solution. We identify fluctuation patterns in the fluorescence signal that can be rationalized in terms of photobleaching and stochastic transitions to reversible dark states. Moreover, by using the advantages of solution-based imaging, we demonstrate that the addition of oxygen scavengers improves optical stability by significantly decreasing the photobleaching rates.

Full text of DOI:10.1021/ja311874f

Article
pubs.acs.org/JACS
Solution-Based Single Molecule Imaging of Surface-Immobilized
Conjugated Polymers
Paul A. Dalgarno,†,∥ Christopher A. Traina,§ J. Carlos Penedo,†,‡ Guillermo C. Bazan,*,§
and Ifor D. W. Samuel*,†
‡
†Organic Semiconductor Centre, SUPA, School of Physics and Astronomy and Biomedical Science Research Complex (BSRC),
University of St. Andrews, North Haugh, St Andrews, Fife, KY16 9SS, United Kingdom
§Department of Materials and Chemistry and Biochemistry, Centre for Polymers and Organic Solids, University of California, Santa
Barbara, California 93106, United States
S
* Supporting Information
ABSTRACT: The photophysical behavior of conjugated
polymers used in modern optoelectronic devices is strongly
influenced by their structural dynamics and conformational
heterogeneity, both of which are dependent on solvent
properties. Single molecule studies of these polymer systems
embedded in a host matrix have proven to be very powerful to
investigate the fundamental fluorescent properties. However,
such studies lack the possibility of examining the relationship
between conformational dynamics and photophysical response
in solution, which is the phase from which films for devices are
deposited. By developing a synthetic strategy to incorporate a biotin moiety as a surface attachment point at one end of a
polyalkylthiophene, we immobilize it, enabling us to make the first single molecule fluorescence measurements of conjugated
polymers for long periods of time in solution. We identify fluctuation patterns in the fluorescence signal that can be rationalized
in terms of photobleaching and stochastic transitions to reversible dark states. Moreover, by using the advantages of solution-
based imaging, we demonstrate that the addition of oxygen scavengers improves optical stability by significantly decreasing the
photobleaching rates.
matrix, such as PMMA or polystyrene.12 There have been
several key discoveries, such as the observation of energy
INTRODUCTION
■
Conjugated polymers are the enabling materials for polymer
funneling within the polymer to the lowest available
LEDs,1 lasers,2 solar cells,3,4 and field-effect transistors,4 all of
chromophore.7 Additional work has shown detailed vibronic
which show significant potential for modern optoelectronic
technologies. Despite this commercial potential, much remains
unknown about the underlying photophysical behavior of the
structure,13 photon antibunching,14,15 narrow spectral line
width, and nonemissive dark states16 and revealed the potential
for single polymer organic spintronics17 along with device
polymers. Conjugated polymers are large, complex macro-
optimization through understanding film annealing at the
molecules in which the electronic and photophysical functions,
including emission quantum yield, charge carrier mobility,
molecular limit.18−20 This wealth of information, which is
unavailable with ensemble measurements, highlights the
spectral characteristics, and optical stability, are all intrinsically
significant impact of single molecule studies for understanding
linked to polymer conformation, aggregation, and, ultimately,
chromophore interaction. Moreover, given that the polymer
conjugated polymers.
Despite the remarkable insights delivered by such techniques,
host-matrix-based single molecule imaging remains limited.
Highly dilute films represent only one of four defining polymer
film in the device can inherit the structural state from the
processing solution,5,6 controlling the conformation in solution
is a crucial step to rationally improve device performance.
Consequently, there exists a major challenge to reveal the
fundamental relationship between polymer shape, conforma-
tion, and function at the single polymer level and in doing so
environments (bulk solution, film, isolated solution and isolated
film) each of which display very different photophysical
properties.21 In each case, the functionality is defined by
disorder and aggregation. Only intrachain interactions are
provide an understanding of polymer behavior that will directly
possible in isolated environments, whereas in bulk solutions
link to device performance.
both intrachain and interchain interactions need to be
Single molecule studies of conjugated polymers have been
established in recent years7−11 with the majority of work
considered. Solvent properties dominate conformation and
focusing on MEH-PPV7−9 in addition to polyfluorenes10 and
polythiophenes.11 Almost exclusively the established procedure
is to probe single polymer chains embedded in an inert host
Received: December 5, 2012
© XXXX American Chemical Society
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therefore behavior, in solution. This also means that they
strongly influence the properties of films that are deposited
from solution. Not only can this result in difficulty in isolating
single chains from small aggregates in single molecule studies21
but also leads to the well-established relationship between
solvent choice and device functionality.5,6,22,23 Of the four
defining environments, isolated chains in solution are, in
principle, the most fundamental with behavior defined primarily
by solvent properties and intrachain interactions. In addition it
provides a uniquely dynamic environment in which polymer
conformation can be directly manipulated and correlated to
photophysical properties.
Solution-based single molecule imaging is unfortunately
extremely challenging with no established strategy. In order to
observe dynamic fluctuations a single polymer must be studied
for long periods of time, requiring an effective immobilization
strategy such that the backbone still preserves its conforma-
tional dynamics. Recent work on slowly diffusing MEH-PPV
chains in a high viscosity, toluene solution with a low molecular
weight polystyrene base approaches a possible solution.24
However, high viscosity solvents are required, and nonspecific
adsorption offers limited control over polymer immobilization.
An alternative strategy has employed water-soluble MPS-PPV
encapsulated in lipid vesicles.25 However, vesicles are a closed
system and are limited to aqueous environments.
We respond in this paper to these challenges by establishing
a new strategy that demonstrates the proof of principle for the
effective immobilization and isolation of single polymers in a
fluid solution medium. We utilize end-group site-specific
immobilization of conjugated polymers, specifically biotin
functionalized poly(3-dodecylthiophene) (bi-P3DDT), to
immobilize at only one end, allowing for the chain to extend
freely into the solvent and adopt dynamic conformations
dictated only by solvent properties. We demonstrate the ability
to interrogate single polymer dynamics for extended time
periods, until photobleaching (>minutes). In addition we show
that by taking advantage of the flexibility of a solution-based
environment, it is possible to restrict the photobleaching rate
and increase the total fluorescence output from a single
polymer more than 4-fold by the addition of a water compatible
oxygen scavenger agent.
Figure 1. Single-molecule imaging of surface-immobilized conjugated
polymers. (a) Schematic of the immobilization of a conjugated
polymer chain to a substrate via biotin/neutravidin interactions. The
polymer chain was designed to contain a biotin unit at a single end,
bound to a single neutravidin protein in turn bound to a single BSA
protein adsorbed on the glass surface. (b) Representative fluorescence
image of immobilized molecules (70 × 70 μm).
strongly absorbing and sufficiently emissive with a well-defined
structure and low polydispersity (PDI). Furthermore, only one
chain end should be biotinylated, while the other chain end
must remain unattached to permit extension of the chain into
the solvent. We therefore selected an end-functionalized
poly(3-alkylthiophene) material as a target. Synthetic protocols
have been developed to prepare well-defined poly(3-
alkylthiophene)s in a controlled manner by chain-growth
Kumada catalyst-transfer polycondensation procedures.28,29
These nickel-catalyzed methods give rise to low-polydispersity,
regioregular materials with tunable average molecular weights
that can be modulated via the initial catalyst to monomer ratio.
Additionally, selection of an appropriate catalyst/initiator
allows for controlled introduction of a functional group onto
a single chain end. For example, it has been demonstrated that a
catalyst/initiator of the type L2Ni(Br)Ar, where L is a
phosphine ligand and Ar is an aryl group, can initiate the
polymerization by transfer of the aryl group to the active
thiophene monomer.30−34 A substituted aryl ring, such as a 4-
(triisopropylsilylethynyl)phenyl group, would offer a reactive
handle for postpolymerization functionalization by click
chemistry after deprotection to the active acetylene function-
ality.35,36 Thus, as shown in eq 1, where TIPS corresponds to
RESULTS AND DISCUSSION
■
Polymer Molecular Design and Synthesis. Our strategy
for solution-based imaging of conjugated polymers is based on
the specific interaction between the small molecule biotin and
neutravidin, which is one of the strongest noncovalent binding
events known in nature, with association constants in the
picomolar range. Such an interaction is commonly used in a
variety of purification and analysis methods in molecular
biology and when implemented at the single molecule level has
provided a platform that is revolutionizing our understanding of
biomolecular structure and function.26,27 As shown in Figure
1a, biotin binds to the multivalent neutravidin protein, which in
turn binds to biotinylated bovine serum albumin (BSA)
adsorbed to glass or quartz surfaces, providing a single anchor
point that isolates the molecule from the substrate surface.
Importantly the single point of attachment allows for
conformational freedom such that the polymer structure can
respond to solvent-mediated environmental effects.
triisopropylsilyl, the catalyst/initiator 2 was synthesized from
the mixed phosphine nickel complex 1 by oxidative addition of
1-bromo-4-(triisopropylsilylethynyl)benzene.37 In this reaction,
the aryl bromide was treated with a THF solution of 1 and
allowed to stir for 44 h, after which time the solvent was
removed. The residue was then taken up in toluene and
precipitated with pentane to give TIPS-ethynyl containing 2.
1
The H NMR spectrum of 2 is consistent with the proposed
structure, showing methyl and methine signals arising from the
TIPS protecting group at 1.22 and 1.15 ppm, respectively. Two
resonances in the 31P NMR spectrum at 58 and 39 ppm
indicate the presence of two distinct phosphorus environments,
reflecting the asymmetric nature of the complex.
Complex 2 was thus used to prepare the target biotin-
terminated poly(3-dodecylthiophene), as depicted in Scheme 1.
First, a THF solution of the dibromothiophene 3 was treated
The experimental platform for single molecule imaging in
Figure 1 imposes specific requirements for the conjugated
polymeric material to be studied. The polymer should be
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Scheme 1. Synthetic Entry into the End-Group Biotinylated Polythiophene Bi−P3DTT
with isopropylmagnesium chloride−lithium chloride complex
to give the monobromo, monomagnesio reactive monomer,
and this solution was quickly added to the polymerization
initiator 2, at a monomer:initiator ratio of 100:1. After 5 min of
reaction time, the polymerization was terminated with
ethylmagnesium bromide to give TIPS-ethynyl-phenyl end-
functionalized poly(3-dodecylthiophene) (4). The resulting
polymer was characterized by gel permeation chromatography
(GPC) using a mobile phase of CHCl3 + 0.25% triethylamine,
which gave a number average molecular weight (Mn) of 19 kDa,
which would correspond at face value to ∼75 repeat units and a
PDI of 1.4, as estimated relative to poly(styrene) standards. It is
worth noting here that the Mn values of conjugated polymers
obtained by GPC are often overestimated when calculated
against poly(styrene) due to the large differences in hydro-
the proton source, to yield polymer 5. The course of the
deprotection reaction was followed by monitoring the
disappearance of the TIPS protons at 1.25 ppm, resulting in
an end-functionalized polymer which was active for copper-
catalyzed azide−alkyne cycloaddition reactions. The biotin
functionality was thus introduced by treatment of 5 with biotin-
dPEG3+4-azide (azide-containing biotin molecule) under click
reaction conditions of CuI/triethylamine in THF to give bi-
1
P3DDT. H NMR spectroscopy is unable to confirm biotin
attachment, as the spectrum for bi-P3DDT is very similar to
that of precursor 5, with signals due to the biotin group likely
overlapping with the more intense peaks of the thiophene
repeat units. Therefore, FTIR spectroscopy is used to confirm
the presence of the biotin functional group; bands at 1700 and
1650 cm−1 in the IR spectrum of bi-P3DDT, which correspond
to carbonyl groups in the biotin molecule and linker, are not
present in the spectra of precursor 5.
The terminal biotin of bi-P3DDT was then probed for
surface immobilization reactivity. Streptavidin-coated agarose
beads were treated with a THF solution of bi-P3DDT, and
after 2 h incubation time, the beads were collected and washed
to remove nonbound polymer. The bi-P3DDT treated beads
showed strong orange luminescence under UV (365 nm)
illumination, indicating biotin-mediated surface binding of bi-
P3DDT to the steptavidin present on the beads. A control
experiment conducted in a similar way but with a poly(3-
dodecylthiophene), which did not contain a terminal biotin,
yielded beads with negligible luminescence. Thus, the presence
of a terminal biotin moiety was confirmed, and moreover, this
functional group proved effective for immobilizing the polymer,
as required for single molecule experiments.
Spectral Characteristics of bi-P3DDT. To demonstrate
the principle of our solution-based immobilization method-
ology, we chose to immobilize bi-P3DDT from a 90:10 (v/v)
solution of aqueous buffer (pH 7.5) and tetrahydrofuran
(THF) and to image in a 100% buffer solution. Bi-P3DDT is
soluble in THF which is fully water miscible. A 90:10 (v/v)
mixture of aqueous buffer:THF was chosen for several reasons:
First, the poor solvent properties of this mixture will be
expected to predominantly induce a well-known collapsed coil
conformation.38 Second, as proof-of-principle for solution-
based single molecule studies, the change in refractive index
between the quartz surface and the aqueous medium is
adequate to use wide-field total-internal reflection (TIR) as the
single molecule imaging method. This improves the signal-to-
1
dynamic volume between the two types of polymers. The H
NMR spectrum of 4 is dominated by the methylene protons of
the dodecyl side chain, which appear between 2.90 and 1.35
ppm, and the terminal methyl signal at 0.95 ppm. Resonances
due to the aromatic thiophene protons are visible at 7.19 ppm.
1
The H NMR spectrum also displays a small doublet at 1.25
ppm, which has been assigned to the methyl groups of the
triisopropylsilyl-ethynyl group, indicating successful end group
incorporation. The signal at 1.75 ppm represents protons from
one methylene group of the thiophene dodecyl side chain and
for each unit represents 2 protons. A comparison of the
integrated intensity from the methylene resonance at 1.75 ppm
and the TIPS signal at 1.25 ppm gives a ratio of 2H:0.35H,
which corresponds to approximately one TIPS-ethynyl end
group per 51 thiophene repeat units. This ratio of one reactive
end group per 51 repeat units is lower than the initial monomer
to initiator ratio used during polymerization. However, upon
further investigation, this difference is not surprising. It has
been shown that activation of the 2,5-dibromo-3-alkyl
thiophene premonomer with organomagnesium compounds
results in a 4:1 mixture of reactive vs nonreactive monomers,
depending on magnesium insertion at the 5- or 2-position of
the thiophene. Furthermore, the polymerization was terminated
with ethylmagnesium bromide after 5 min polymerization time
so as to ensure polymer end-capping with an inert alkyl group.
It is likely that some unreacted monomer remained in solution
at the time the polymerization was quenched.
Incorporation of the biotin functionality begins with
deprotection of the ethynyl group by using tetrabutylammo-
nium fluoride (TBAF) in THF with several drops of water as
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noise ratio compared to conventional illumination methods
and, at the same time, allows us to obtain a much higher
statistical characterization of polymer behavior compared to, for
example, confocal methods. Lastly, an aqueous-based medium
offers the possibility of exploring the effect of oxygen scavenger
systems on single polymer photostability. Although, oxygen
scavengers based on in situ glucose oxidation are highly
effective and widely used in aqueous solution to inhibit
photobleaching in organic chromophores, to date, their effect
on single conjugated polymers has never been reported.
To verify the optical behavior of bi-P3DDT in our
immobilization medium, Figure 2 shows the absorption (Figure
SM TIRF microscopy was performed at varying incident
powers, defined as the estimated power density at the interface
between the quartz slide and the solution medium, from 0.015
to 0.74 W/cm2. Images were collected in custom built relay
optics with a 550 nm long pass filter and an emCCD camera
(Andor, Belfast). Figure 1b shows that our immobilization
protocol produced ∼200−400 isolated, bright spots spatially
distinct on the emCCD field of view. We report correlated
concentration-dependent changes in the number of spots and
distinct single step bleaching and blinking events, see below,
providing confidence we are observing, at least in part,
individual polymer chains or small aggregates. To confirm
that the observed spots are due to specific immobilization of
the biotinilated polymer onto the BSA-neutravidin-coated
quartz surface and not caused by nonspecific adsorption, we
performed control experiments using both uncoated and BSA-
only functionalized surfaces, Figure S1. Under both of these
conditions, for the same incubation period and with sample
concentrations up to 10 times those described above, we report
no immobilized molecules. We are therefore confident we are
observing single immobilized P3DDT molecules imaged in
solution.
Oxygen-Dependent Photostabilization. We first inves-
tigated polymer photostability by taking advantage of the
significant degree of flexibility that solution-based imaging
allows in modifying the environment of the molecules. One of
the most significant impacts for biomolecular imaging has been
the use of oxygen scavengers to significantly reduce photo-
bleaching.26 We use the common oxygen scavenger imaging
buffer, glucose oxidase, 165 U/ml and catalase, 2170 U/ml in a
6% glucose/buffer solution. Figure 3 shows the normalized
Figure 2. Absorption (a) and fluorescence emission spectra (b)
obtained for bi-P3DDT in THF (black) and 10:90 THF:aqueous
buffer (red) solutions. Fluorescence spectra were collected at 532 nm
excitation wavelength.
2a) and fluorescence (Figure 2b) of a bulk solution of bi-
P3DDT in pure THF and 90:10 buffer:THF solution. Bulk
solutions were prepared with a saturated bi-P3DDT THF
solution (<0.33 mg/mL), stirred for 4 h, and filtered through a
1.45 μm filter. For bulk spectroscopy measurements, this stock
was further diluted by a factor of 10 either in pure THF or
aqueous buffer. In the aqueous buffer there is a significant
red-shift ( ∼100 nm) for both absorption and emission
accompanied by a reduction in fluorescence intensity and the
appearance of two distinct peaks at 650 and 725 nm. These
spectral signatures suggest collapsed and aggregated chains, a
phenomenon that was also observed for poly(3-dodecylth-
iophene) in poor solvent mixtures.38 Single molecule
fluorescence was excited at 532 nm and collected over the
complete bandwidth of the bulk fluorescence emission by
collecting all fluorescence above 550 nm.
Single Molecule Imaging. To prepare individual polymers
for single molecule immobilization, a solution of bi-P3DDT in
THF was prepared as described in the previous section. This
stock solution was then diluted 100 times in THF and further
stirred for 4−5 h. This final solution was diluted 10 times in the
buffer medium before being incubated on a BSA-neutravidin
precoated microchannel. After 15 min the channel was
thoroughly rinsed with fresh buffer and the sample was ready
for imaging. In aqueous medium the channel is robust against
leakage and evaporation for several hours, and molecular
imaging from the same channel is reported for periods of time
in excess of 5 h. Additionally the channel is robust against any
form of contamination from channel breakdown, which
dominates when imaging is attempted in pure organic solvents.
The configuration is such that replacement of the imaging
medium is possible at any time during the experiment, allowing
for directly comparative experiments under different environ-
mental conditions.
Figure 3. Effect of oxygen scavenger on polymer photostability. The
normalized sum of intensity traces from 1200 individual molecules as a
function of excitation power is shown (a) without and (b) with
scavenging buffer. (c) Fitted photobleaching lifetimes as a function of
power from data shown in (a) and (b).
time-dependent variation in the fluorescence signal as a
function of excitation power in the absence (Figure 3a) and
presence (Figure 3b) of oxygen scavenger imaging buffer. The
observed fluorescence trajectory at each excitation power is the
result of adding together the individual trajectories of more
than 1000 molecules. Over two decades of excitation power
there is between a 4- and 10-fold stabilization of the
fluorescence in the presence of oxygen scavenger, quantified
in Figure 3c by plotting the longer lifetime component of a
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biexponential fit to equivalent data from Figure 3a,b at 10
different excitation powers. Although each decay is biexponen-
tial (Figure S2), they are all dominated by the longer lifetime
component which we attribute to the photobleaching
dynamics.39 Full analysis of the photobleaching lifetimes, the
short and long lifetime contributions and stabilization are
shown in Figure S2. We therefore demonstrate that oxygen
scavenging is a viable avenue for photostabilization of
conjugated polymers. Importantly this stabilization allows us
to reduce photobleaching and therefore make clear measure-
ments of the distinct molecular behavior from each molecule.
No further stabilization of the fluorescence emission, including
removal of transiently populated dark states (blinking), was
observed when adding triplet-state quenchers, such as Trolox
(derivative of vitamin E) or β-mercaptoethanol.
Single Molecule Behavior. We now categorize molecular
behavior into distinct patterns by inspection of the individual
fluorescence trajectories. In all data sets, at all excitation powers
and for both with and without oxygen scavengers, we
consistently identify four main trace types. Examples of three
of these fluorescence types are shown in Figure 4. The
background of MEH-PPV in high viscosity solvent.24 Non-
reversible photobleaching is prominent for all trace types, but
when oxygen scavenger is present, emission can be stabilized
for many minutes, even at high excitation power.
Figure 4d shows the statistical distribution of single molecule
fluorescence behavior in the presence and absence of oxygen
scavenger classified following the criteria described previously.
In the presence of oxygen scavengers, a total of 1200 molecules,
taken evenly from data at 0.015, 0.3, and 0.59 W/cm2, were
analyzed. The relative ratio of trace types is, to within 5%,
consistent between excitation powers, Figure S3. Molecular
behavior is dominated by trace type A, 45%, with bright rapidly
unresolved fluctuations with little or no characteristic single
molecule blinking. There is an even distribution (20%) of trace
types B and C. For data without oxygen scavengers, where 550
molecules are analyzed, (Figure 4d, blue) molecular behavior is
dominated (50%) by exponential decays, with roughly 20% of
types A and B.
The molecular classifications reveal a number of interesting
points: First, there is a notable contribution, 20%, of traces that
show distinct, single step blinking or bleaching events, trace
type B. This behavior is typical for collapsed, single chains cast
from bad solvents7,40,41 and can be attributed to energy
funneling to the lowest-energy chromophore or Forster transfer
̈
induced photogenerated quenching.42 Second, there is a lack of
clear multilevel blinking from any molecules, often seen in
MEH-PPV. This could be attributed to very few active
chromophores, to chain quenching as discussed above42 or to
nonemissive dark states.16 Third, there is a range of distinct
molecular behaviors for identical environmental conditions of
which the statistics remain unchanged over two decades of
excitation power, Figure S3. Fourth, oxygen scavenging shifts
the dominated trace type from exponential decays without
scavenger (type C) to rich transient dynamics with scavenger
(type A), without affecting the proportion of single step
blinking/bleaching traces.
The final two points suggest a complex conformational
landscape of the molecules under study. The bad solvent
environment is expected to induce collapsed chains, Figure 2,
which in principle should give rise to single step blinking and
bleaching events remnant of trace type B.7,39,40 However only
20% of traces demonstrate such behavior. The majority show
behavior expected from a multichromophoric system. In the
absence of oxygen scavenger this manifests itself as that of an
exponentially decreasing intensity produced by the gradual
photobleaching of multiple chromophores. With scavenger,
bleaching is removed, and the dynamics in trace type A suggest
continuous emission from multiple emitters each with differing
blinking and intensity dynamics. However, this is clearly an
oversimplification. Our proof of principle experiment limits
further investigation, but we tentatively conclude that we are
observing behavior from two classes of molecular states. One
class is stable, tightly collapsed single chains with clear, single
step blinking dynamics dominated by efficient energy funneling
or quenching behavior (type B). The other class is either small
aggregates of multiple emitters or dynamic, collapsed chains
undergoing constant conformational restructuring which
prevents funneling/quenching behavior (mostly types A and
C depending on the environment).
Figure 4. (a−c) Representative trajectories of the single molecule
fluorescence patterns observed for bi-P3DDT in aqueous medium.
Only trajectories in the presence of oxygen scavenger buffer are shown.
Trajectories were obtained with 0.59 W/cm2 incident excitation power
and with 50 ms integration rate. (d) Statistical distribution of single
molecule fluorescence patterns in the presence (red) and absence of
oxygen scavenging additives (blue).
classification of the observed fluorescence traces was carried out
following a number of criteria based on intensity, level of
dynamic fluctuation, presence of discrete fluorescence states
and photobleaching. The specific patterns can be described as
follows: (A) rapid fluctuations (millisecond time scale) between
two or more highly emissive states occasionally interlaced with
transitions to dark states lasting several seconds; (B)
trajectories dominated by fluctuations between a low emitting
state and a dark state both lasting for several seconds,
reminiscent of blinking transitions to triplet states commonly
observed in single chromophore organic dyes; (C) trajectories
showing a highly emissive state exponentially decaying to
background level, similar to that observed in highly aggregated
systems; and (D) the fourth classification “other”, which is not
shown, corresponds to a wide variety of trace types that show
no easily classified behavior.
Autocorrelation Analysis. To investigate in further detail
the dynamics, we calculated the autocorrelation function of
representative fluorescence trajectories corresponding to the
dynamic groups A and B. This analysis is commonly used to
For the three main classes we do not observe the complex,
multileveled photoblinking dynamics reported from collapsed
MEH-PPV chains cast from poor solvent.9 Nor do we observe
the two state blinking behavior over a constant bleaching
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extract the underlying hidden dynamics in fast fluctuating
systems providing a global macroscopic rate constant that
groups the sum of the forward and backward interconversion
rates.43 We observe similar autocorrelation dynamics between
group types A and B (see Figure S4) and therefore group the
autocorrelation results as a single set, Figure 5. The
conformations, each of them having different quantum yields.
Indeed, NMR and photophysical studies of MEH-PPV
morphology in low-quality solvents indicate that microscopic
variations in the degree of polymer packing may account for up
to two-thirds of variation in quantum efficiency.23
CONCLUSION
■
To summarize, we have demonstrated single molecule
spectroscopy of immobilized single polymer chains in solution.
A novel type of conjugated polymer, namely bi-P3DDT, has
been designed that contains the biotin structural element for
selective end-group binding to neutravidin-modified substrates.
The synthesis involves a controlled polymerization reaction
with an initiator that imparts a masked acetylene functional
group. The latter can serve for straightforward incorporation of
various functionalities by reaction with substituted azides and
provides a flexible platform for further derivatization. Bi-
P3DDT can form the foundation for solution-based single
molecule imaging of site-specific immobilized polymers while,
in principle, retaining conformational freedom to be dictated by
solvent. As an example we have demonstrated this technique
with bi-P3DDT in a 90:10 aqueous:THF solution. In addition
to demonstrating photostabilization through the use of
standard oxygen scavenging, we observe largely heterogenic
behaviors separated into only two distinct classes: one showing
distinct single step blinking and bleaching and the other
displaying rapid intensity fluctuations resembling multiple
emitters. We demonstrated the principle in an intentionally
bad solvent, limiting molecular confirmation to tightly collapsed
chains. However, the methodology is compatible with confocal
imaging in organic solvents and can be combined with click
chemistry to covalently link the polymer to the surface thus
removing the need for immobilization protein.
Figure 5. Autocorrelation analysis of trace classification. (a−c)
Histogram of the fitted lifetime of the correlation function G(t)
from data at 0.015, 0.3, and 0.59 W/cm2 with oxygen scavenger. Insets
show a single example G(t) with biexponential fit. (d) The mean slow
component correlation lifetime from between 100 and 300 molecules
at each power, with std dev, as a function of excitation power.
autocorrelation function of each trace fitted well to a
biexponential decay with a minor component lifetime of
∼200 ms and a dominant slow component with a lifetime >5s.
Our primary result is that the mean of the slow component
lifetime, averaged over 100−300 molecules at each excitation
power, was dependent on the excitation intensity (Figure 5d)
and showed a 2−3 fold decrease with increasing excitation
power. On the other hand, the mean of the fast component
reflected dynamic events taking place close to our frame rate at
all excitation powers, Figure S7. These findings provide
evidence for the polymer exhibiting molecular processes at
two very different dynamic regimes. Although this is an
oversimplification and the nature of these processes will need
further investigation, we can still extract some conclusions.
First, our collection bandpass above 550 nm collects virtually
all of the polymer emission range, Figure 2, thus, the observed
intensity fluctuations are unlikely to be due to spectral diffusion.
Second, the discrete jumps between different emissive states
(type A) or between emissive and dark states (type B) suggest a
certain degree of cooperativity between conjugated regions of
the polymer as suggested for other organic polymers including
MEH-PPV and dPPV-PPyV.43 Third, the lack of multilevel
blinking and bleaching events suggests a more homogeneous
distribution of dynamic states for bi-P3DDT in solution over
host matrix environments. Fourth, the power dependence of
the slow component suggests an underlying photoblinking
mechanism responsible for the rapid transient dynamics.
Overall, the observation of a major population of fluorescence
trajectories in the presence of oxygen scavenger showing fast
dynamic exchange between highly emissive states in bi-P3DDT
and taking place in ∼200 ms time scale, as reflected by the fast
component of the autocorrelation function, points toward the
polymer structure dynamically sampling different packing
ASSOCIATED CONTENT
■
S
* Supporting Information
Confirmation of immobilization strategy; analysis of photo-
bleaching lifetimes in the presence and absence of oxygen
scavengers; classification of single molecule intensity trajecto-
ries as a function of excitation power; trace dependent
autocorrelation analysis; statistics of autocorrelation lifetimes;
fast correlation component and frame rate; materials; and
methods. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
idws@st-andrews.ac.uk; bazan@chem.ucsb.edu
Present Address
∥Institute of Biological Chemistry, Biophysics and Bioengineer-
ing, School of Engineering and Physical Sciences, Heriot-Watt
University, Edinburgh, EH14 4AS, United Kingdom.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
St Andrews acknowledges support from the Scottish University
Physics Alliance (SUPA) and funding from EPSRC (grant no.
EP/G061688/1). UCSB acknowledges funding from NSF
(grant DMR 103548).
F
dx.doi.org/10.1021/ja311874f | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

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