Fluorescence self-quenching of a mannosylated poly(p-phenyleneethynylene) induced by Concanavalin A

Article abstract of DOI:10.1021/ja802094s

We report that static quenching of a mannosylated conjugated polymer (sugar-PPE) by Concanavalin A is positively dependent upon sugar-PPE concentration, that is, the recorded Stern-Volmer constants increase with increasing sugar-PPE concentration. Comparison with data obtained from isothermal titration calorimetry (ITC) display the increased sensitivity of the quenching method when compared to ITC. The proposed mechanism suggests the interaction of two or more chains of PPE with one Con A molecule leading to a quenched sugar-PPE-Con A construct. Copyright

Full text of DOI:10.1021/ja802094s

Published on Web 05/13/2008
Fluorescence Self-Quenching of a Mannosylated Poly(p-phenyleneethynylene)
Induced by Concanavalin A
Ronnie L. Phillips, Ik-Bum Kim, Laren M. Tolbert,* and Uwe H. F. Bunz*
School of Chemistry and Biochemistry, 901 Atlantic DriVe, Georgia Institute of Technology,
Atlanta, Georgia 30332-0400
Received March 20, 2008; E-mail: uwe.bunz@chemistry.gatech.edu
Scheme 1. Synthesis of the PPE 5 via Pd-Catalyzed Coupling of 1
to 2 Followed by Dipolar Cycloaddition
Conjugated polymers (CP) have attained considerable status in
the detection and quantitation of biologically active molecules.1
CPs have either been used in the solid state as sensory coatings for
electrodes, or, transparently in solution as species that change their
absorption spectra and more sensitively, their emission spectra upon
exposure to a specific analyte. They are often superior to noncon-
jugated, nonfluorescent polymeric scaffolds,2 as they elegantly
combine recognition and transmission elements. We now probe a
protein/CP interaction using fluorescence quenching of the mannose-
substituted poly(p-phenyleneethynylene) (PPE) 5 by Concanavalin
A (Con A), the tetrameric lectin of the jack bean,3 and investigate
its unusual mechanism.
The optical/fluorescence-based detection of biologically important
species in solution profits from the endless synthetic variability of
CPs. Different tailored backbones are fitted to the intended purpose
by the attachment of suitable recognition elements. The sensing or
probing of biomolecular targets by CPs rests on either (a)
fluorescence resonance energy transfer, (b) ratiometric response or
(c) quenching of the fluorescence after binding to a specific analyte.
All three of these mechanisms are of significant interest and have
demonstrated use in the probing of biomolecules. The properties
that make CPs so much more powerful than small dyes are (a)
their facility for acting as molecular antennae, in which one exciton
can “patrol” up to one hundred repeat units, (b) their ability for
supporting multivalent interactions,4 and, (c) most complex and
least well understood, their aggregation phenomena, which can lead
to a phenomenally large signal amplification that allows detection
of zeptomolar concentrations of DNA by CPs.5
The quenching of the fluorescence of PPEs by paraquat deriva-
tives was first described by Swager and Zhou in a classic paper.6
The PPE-analyte interactions could be correctly described by the
Stern-Volmer formalism assuming static quenching, induced by
excited-state electron transfer from the PPE to paraquat. The
molecular antenna effect led to signal amplification by a factor of
up to 100 when compared to similar monomeric fluorophores. Wudl
and Whitten7 later demonstrated superquenching when examining
the interaction of paraquat with a sulfonated PPV and explained
the observed thousand-fold gain in sensitivity by a combination of
the antenna effect compounded by paraquat-induced aggregate
formation. While quenching with paraquat involves excited-state
electron transfer, Fo¨rster energy transfer can also be employed to
quench the fluorescence of a water soluble conjugated polymer by
the addition of the deeply colored cytochrome C as demonstrated
by Heeger et al.8 A combination of mechanisms is believed to be
responsible for the superquenching ability of gold nanoparticles
on the fluorescence of conjugated polymers.9 However, the quench-
ing of CP fluorescence can also occur if the analyte does not carry
any obvious chromogenic or electron transfer center. This is the
case in the interaction of PPEs with several proteins such as
lysozyme, etc.,10 or more importantly, Con A (tetrameric at pH
7.2 in phosphate buffer (PB)) as a model protein for biotoxins such
as Ricin or E. coli toxin.11,12
The classic tool for investigating quenching is the Stern-Volmer
equation:
Fo ⁄ F[Q] ) 1 + KSV[Q]
(1)
in which Fo/F[Q] is the ratio of the initial fluorescence intensity Fo
and the fluorescence intensity F[Q] in the presence of the quencher
Q at a concentration [Q]. KSV is the Stern-Volmer constant and
may refer to static quenching if the complex between fluorophore
and quencher is preformed and does not undergo diffusion or to
dynamic quenching if diffusion occurs during the excited-state
lifetime. In such a case, KSV represents the equilibrium constant.
In static quenching it is assumed that the quencher and the
fluorophore form a nonfluorescent ground-state complex and the
slope of Fo/F[Q] for different concentrations of Q provides the
binding constant of fluorophore to quencher. In cases of static
quenching the fluorescence lifetime of the fluorophore is indepen-
dent of the concentration of added quencher Q. This is generally
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2008 American Chemical Society

C O M M U N I C A T I O N S
Figure 1. Stern-Volmer graph for different PPE concentrations: dark blue,
[PPE] ) 5 × 10-7 M; turquoise, [PPE] ) 2.5 × 10-7 M; green, [PPE] )
1.25 × 10-7 M; gold, [PPE] ) 6.25 × 10-8 M.
Figure 2. Master plot of all quenching data, including fit to eq 3 and eq
4.
the case for PPEs, which have a lifetime of 0.3-0.4 ns.13 The other
necessary prerequisite for a straightforward application of the
Stern-Volmer formalism is that a one-to-one complex forms
between quencher and fluorophore. It is often argued that at low
quencher/ fluorophore concentrations this assumption is valid. One
should also note that the concentration of the fluorophore does not
show up in this analytical expression of the Stern-Volmer
equation.14 However, for numerous examples of fluorescence
quenching of CPs by small molecule quenchers published in the
literature the polymer concentration does matter.15 The observed
Stern-Volmer “constants” are inversely dependent upon the
concentration of the CP. As a consequence KSV decreases with
increasing concentration of CPs.
Con A displays sensitive quenching of the fluorescence of
mannosylated PPEs.11 As Con A does not contain an easily
identifiable electron or charge transfer center we decided to
investigate the mechanism of this quenching. We chose the PPE 5
as a suitable object for our investigation, as it (a) carries a negative
charge and (b) has the mannose residue attached to the PPE chain
by a 20-atom tether, suggesting easy interaction of 5 with Con A.
The synthesis of 5 is displayed in Scheme 1; in the last step the
pre-PPE 3 is desilylated and a copper catalyzed 1,3-dipolar
cycloaddition to 4 is performed to give 5 in high yield.16
Figure 1 displays the dependence of the fluorescence quenching
of 5 on the concentration of Con A, the classic Stern-Volmer
formalism, for different concentrations of the polymer 5. It is
immediately noticeable that, at low concentrations of Con A, the
S-V plot is linear. At higher concentrations of the quencher Q,
the plot is also linear but with a different slope. Surprisingly, the
extracted initial KSV is proportional to the PPE concentration as
well, which is contrary to the existence of a 1:1 complex, but rather
commensurate with a 1:2 complex. If the concentration of PPE 5
is below 3 × 10-8 M, no quenching whatsoever is observed.
The lifetime of the PPE excited-state was measured as 450 ps
and did not depend on [Con A], in agreement with a purely static
quenching mechanism. The presence of a “two-phase” S-V plot,
as opposed to a second order plot, suggests the intervention of two
such quenching mechanisms. The general treatment of such kinetics
is provided by eq 3, where f1 and f2 represent the relative fractions
of quenching mechanisms (f1 + f2 ) 1.0) and KSV1 and KSV2 are
the respective Stern-Volmer constants.17 Using the data in Figure
1 and eq 2, we determine by least-squares analysis the equilibrium
constants shown in Table S2 and Figure 2. Figure 3 illustrates that
KSV1 is linearly dependent on [PPE] and KSV2 is partially dependent
on [PPE]. We also notice that the two rate constants differ by a
factor of 104, but that, within experimental error, the contribution
Figure 3. Plot of Ksv1 and Ksv2 and of f1 [PPE]: dark blue ) Ksv1; gold )
f1 × 109; green ) Ksv2 × 104.
of each process to quenching remains nearly constant, that is, ca.
0.3 for KSV1 and 0.7 for KSV2
.
F[Q] ⁄ Fo ) f1 ⁄ (1 + KSV1[Q]) + f2 ⁄ (1 + KSV2[Q])
(2)
F[Q] ⁄ Fo ) f1 ⁄ (1 + K′SV1[Q][PPE]) + f2 ⁄ (1 + K′SV2[Q][PPE]) (3)
The near linear dependence of the KSV values suggests that the
quenching involves a complex of two PPE molecules. Indeed, if
we assume that KSV(obs) ) K′SV[PPE], we can fit all the data in
Figure 1 to a master equation, eq 3, as shown in Figure 2. The rms
error for this least-squares treatment is a factor of 2 over that for
the individual fits. Using this treatment,18 we obtain a K′SV1 ) 1.1
× 1015 L2 mol-2 (31%) and K′SV2 ) 2.5 × 1011 L2 mol-2 (69%).
An alternate fit when static quenching is involved is to use the
concept of quenching volume, based upon the Perrin model, in
which fluorophores within the quenching volume Vq quench with
100% efficiency and those without quench with 0% efficiency.19
Again, with the two quenching complexes, this model reduces to17
F[Q] ⁄ Fo ) f1 ⁄ (exp(Vq1[Q][PPE])) + f2 ⁄ (exp(Vq2[Q][PPE]))
(4)
Use of a nonlinear least-squares fit to this equation provided the
fit shown in Figure 2 by the dashed line. We note the total sum of
squares for this fit is 4 times that for equation 3. However, since to
a first approximation exp(x) ) 1 + x , we note that Vq and KSV are
almost indistinguishable. The fit to equation 4 yields Vq1 ) 7.6 ×
1014 and Vq2 ) 2.1 × 1011, nearly identical to the KSV values.
To get a better understanding for the association processes that
occur, we performed isothermal titration calorimetry (ITC) by
adding the polymer solution to a solution of Con A in 0.01 M
phosphate buffer at a pH of 7.2. We obtained a binding ratio of N
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C O M M U N I C A T I O N S
Acknowledgment. We thank the Department of Energy (Grant
DE-FG02-04ER46141 to U.H.F.B. and Grant DE-FG05-85ER45194
to L.M.T.) for generous financial support and Prof. Dr. Cornelia
Bohne for helpful discussions.
Supporting Information Available: Synthetic details and NMR
spectra for 5. This material is available free of charge via the Internet
at http://pubs.acs.org.
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The exquisite sensitivity, the ease of data collection and data
evaluation make the quenching of polymeric fluorophores alluring
and-despite the large discrepancies with the ITC derived associa-
tion constants-uniquely useful and promising for further sensory
and probe-type applications.
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