Selective sensing of metalloproteins from nonselective binding using a fluorogenic amphiphilic polymer

Article abstract of DOI:10.1021/ja063544v

Nonconjugated fluorogenic amphiphilic polymers containing an anthracene chromophore exhibit fluorescence quenching in the presence of metalloproteins, although the binding of the polymer to proteins is not selective. The reason for this difference is that

Full text of DOI:10.1021/ja063544v

Published on Web 08/01/2006
Selective Sensing of Metalloproteins from Nonselective Binding Using a
Fluorogenic Amphiphilic Polymer
Britto S. Sandanaraj, Robert Demont, Sivakumar V. Aathimanikandan, Elamprakash N. Savariar, and
S. Thayumanavan*
Department of Chemistry, UniVersity of Massachusetts, Amherst, Massachusetts 01003
Received May 21, 2006; E-mail: thai@chem.umass.edu
Development of new biosensors for the detection of proteins is
an active area of research due to the implications in proteomics,
medical diagnostics, and pathogen detection.1 Designing artificial
scaffolds for this purpose has been of interest, because of their
inherently robust characteristics that make them suitable for device
fabrication. Conjugated polyelectrolytes have received particular
attention for sensing proteins, due to their water solubility and fluor-
escence properties.2,3 More specifically, the possibility of energy
or electron transfer from the polymer to the biological analyte, re-
sulting in a fluorescence change, renders conjugated polymers useful
Figure 1. (A) Fluorescence spectra of polymer 4 with various concentra-
tions of metalloprotein (Cc) in 5 mM sodium phosphate buffer (pH 7.4).7
Inset: Stern-Volmer plot of the fluorescence spectra shown in panel A.
(B) Fluorescence spectra of polymer 4 with various concentrations of non-
metalloprotein (lysozyme).7
for sensing metalloproteins. However, it has been shown through
a set of careful experiments that such a change in fluorescence be-
havior could also be achieved through binding of non-metallopro-
teins.4 The planarization/deplanarization of the conjugated poly-
electrolyte backbone upon interaction with the target analyte results
in a chromic change of the polymer.5 We conceived that covalent
incorporation of a fluorophore in a nonconjugated amphiphilic ho-
mopolymer nanoassembly, introduced by us in the literature recent-
ly,6 provides a unique solution to circumvent this issue: (i) The
polymers form micelle-type assemblies with sizes of about 40 nm
and afford optically clear assemblies in water. (ii) The proximity
of the hydrophobic fluorophore to the solvent-exposed carboxylate
units to which the proteins bind should aid efficient energy- or elec-
tron-transfer events. (iii) Since any possible conformational change
associated with the binding event is unlikely to change the inherent
fluorescence characteristics of a pendent chromophore, the response
will be specific to metalloproteins. With these characteristics, we
hypothesized that, even if the polymers bind nonspecifically to the
positively charged surface of the proteins, response in terms of a
fluorescence change will occur only with proteins that contain the
appropriate energy or electron-donating/accepting functionalities,
i.e., metalloprotein cofactor. We disclose here that our polymer was
indeed selective in its fluorescence response to metalloproteins.
The structure of our target polymer is represented by 4 in Scheme
1.7 Polymer 4 is designed so that the hydrophobic fluorescent
anthracene present at the core of the nanoparticle6 will act as a
transducer for sensing, whereas the carboxylate groups presented
at the solvent-exposed periphery of the assembly act as ligands for
protein binding. The absorption and emission spectrum of polymer
4 reveals that the anthracene functionality is efficiently incorporated
onto the polymer backbone and that incorporation into the polymer
or aggregation in water does not alter the inherent electronic
properties of the anthracene moiety.7 The ability of polymer 4 to
sense proteins was studied by analyzing the change in the anthracene
emission with increasing concentration of the protein.
Scheme 1. Synthesis of Polymer
centrations of lysozyme to the solution had no effect on anthracene
fluorescence. The pI’s of Cc and lysozyme are 10.2 and 11.0,
respectively. Therefore, we assumed that the nature of the interac-
tions of our amphiphilic polymer assembly with these two proteins
would be similar. A fluorescence decrease occurs with Cc, since
the porphyrin functionality in this protein is capable of quenching
the excited state of anthracene by energy or electron transfer.
However, lysozyme does not have a photoactive or electroactive
functionality to access anthracene’s excited-state energy.
Concentration-dependent quenching was used to generate a
Stern-Volmer plot (Figure 1A, inset). The slope obtained from
the plot of the concentration of the protein Vs the ratio of fluor-
escence peak intensities (I0/I) is the Stern-Volmer quenching
constant (KSV). For Cc, this value was 2.0 × 105. The higher value
of KSV and the nonlinear behavior indicate a binding-induced
fluorescence quenching, i.e., a static quenching.
To test the generality of this sensing mode, we tested the
fluorescence response of polymer 4 in water to 12 different proteins
(Figure 2). Four of these proteins are metalloproteins, while the
other eight proteins are non-metalloproteins. Independent of the pI
of the protein, none of the non-metalloproteins affect fluorescence
changes in the polymer, and thus the KSV value is close to 0 (Figure
2). On the other hand, all metalloproteins exhibited KSV values
ranging from 104 to 106, illustrating the selective response of our
nonconjugated, fluorescent polymer to metalloproteins. We have
shown that polymers of type 4 can bind to proteins such as
chymotrypsin.8 Therefore, it is clear that the selectivity is in the
response and not in binding. It is important to recognize that the
To test our hypothesis regarding the selective response to
metalloproteins, we compared the fluorescence response of polymer
4 to that of cytochrome c (Cc, a metalloprotein) and lysozyme (a
non-metalloprotein). The results are shown in Figure 1. It is clear
that the fluorescence from the anthracene moiety in 4 reduced with
the increasing concentration of Cc, while addition of similar con-
9
10686
J. AM. CHEM. SOC. 2006, 128, 10686-10687
10.1021/ja063544v CCC: $33.50 © 2006 American Chemical Society

C O M M U N I C A T I O N S
Figure 3. (A) Fluorescence spectra of polymer 4 in the presence of various
concentrations of methyl viologen.7 Inset: Stern-Volmer plot of the
fluorescence spectra shown in panel A. (B) Fluorescence spectra of polymer
4 in the absence (red line) and in the presence (blue line) of proflavin.7
Figure 2. KSV values of different proteins: ferritin (KSV ) 1.0 × 106), Cc
(KSV ) 2.0 × 105), myoglobin (KSV ) 5.7 × 104), CcP (KSV ) 6.6 × 104),
and all other non-metalloproteins (KSV = 0).
proflavin at 373 nm, by itself, at the same concentration does not
afford a significant fluorescence. The KSV value for this process
was found to be 4.6 × 104 M-1. These results suggest that both
energy- and electron-transfer mechanisms could operate in this
sensing event. We could not resolve which mechanism is dominant
in the metalloprotein binding studies due to the inherent lack of
fluorescence from protein cofactors; this will, however, be a focus
of our studies using time-resolved spectroscopy.
In summary, we have shown that a nonconjugated, fluorescent,
amphiphilic polymer can recognize proteins nonspecifically but
responds only to metalloproteins. The reasons for this selective
response are that (i) the cofactors in metalloproteins can quench
the excited state of the fluorescent polymer by an energy- or
electron-transfer process and (ii) there are no complications that
arise from fluorescence response to binding-induced conformational
changes. While these polymers are selective to metalloproteins,
these results are not specific to a particular metalloprotein at this
time. Designing amphiphilic polymer surfaces that exhibit such
features is a part of the ongoing efforts in our laboratories.
KSV value itself represents not only the binding affinity of the
metalloproteins with the polymer but also the relative ability of
the protein to quench the excited state of anthracene. For example,
ferritin exhibits the highest KSV; this is likely to be due to the fact
that a single protein binding event brings hundreds of bound Fe2+
,
the key electron-accepting functionality, close to the otherwise
fluorescent anthracene moiety. On the other hand, the greater KSV
of Cc, compared to those of cytochrome c peroxidase (CcP) and
myoglobin, could arise from the difference in binding affinities due
to the differences in pI. Note, however, that pI alone is not a good
indicator of the ability of a protein to bind to a charged polymer
surface.9 Moreover, this simple pI-dependent binding affinity expla-
nation, and therefore its impact on KSV, is complicated by the fact
that shifts in electronic spectra of porphyrins are possible (and there-
fore changes in redox potentials and energy-accepting capabilities)
upon binding to a species with a complementarily charged surface.10
We were interested in identifying the possible modes of
fluorescence quenching in these polymers. There are mainly two
limiting mechanisms of fluorescence quenching: energy transfer
and electron transfer. Among the metalloproteins studied, the
mechanism of quenching for ferritin is likely to be based on electron
transfer, since there is no chromophore that could accept the excited-
state energy from anthracene. The other three proteins have por-
phyrin cofactors, the absorbance spectra of which overlap well with
the emission spectra of anthracene. Therefore, a Fo¨rster-type energy
transfer is possible. But, the redox potentials of these proteins are
such that an electron-transfer-based quenching is also viable.
Usually, the fluorescence emission from the energy acceptor upon
exciting the energy donor is considered to be a clear evidence for
energy transfer. However, in our case, such a distinction is difficult,
since Fe-porphyrins are inherently poor fluorescence emitters.
To investigate whether both energy- and electron-transfer
quenching is possible with this polymer, we have carried out the
following model studies. We have shown before that our homo-
polymer-based polyelectrolyte assemblies are capable of binding
small molecules with complementary charges.11 Methyl viologen
(MV2+) is a cationic molecule that can accept an electron from the
excited state of anthracene but does not have the possibility of
accepting its energy. We found that MV2+ was able to quench the
fluorescence of anthracene with a KSV of 9.3 × 103 M-1 (Figure
3A). On the other hand, proflavin is a cationic dye which has an
absorption spectrum that overlaps perfectly with the emission
spectrum of anthracene. Therefore, the latter molecule can accept
energy from anthracene. If this molecule does accept the energy, it
should be observable through an emission from proflavin upon
excitation of anthracene. Indeed, when anthracene was excited at
373 nm, a significant amount of fluorescence arose from proflavin
with a concomitant decrease in fluorescence from anthracene
(Figure 3B). Control experiments showed that directly exciting
Acknowledgment. The authors thank the National Institutes of
Health (GM-65255), Office of Naval Research, and the NSF-
supported Center Hierarchical Manufacturing for financial support.
Supporting Information Available: Detailed procedure for the
synthesis of monomers and polymer and fluorescence spectra of
polymer 4 with different proteins. This material is available free of
charge via the Internet at http://pubs.acs.org.
References
(1) (a) Kodadek, T. Chem. Biol. 2001, 8, 105-115. (b) Wang, D.; Gong, X.;
Heeger, P. S.; Rininsland, F.; Bazan, G. C.; Heeger, A. J. Proc. Natl.
Acad. Sci. U.S.A. 2002, 99, 49-53. (c) Pinto, M. R.; Schanze, K. S. Proc.
Natl. Acad. Sci. U.S.A. 2004, 101, 7505-7510. (d) Wiskur, S. L.; Ait-
Haddou, H.; Lavigne, J. J.; Anslyn, E. V. Acc. Chem. Res. 2001, 34, 963-
972. (e) McQuade, D. T.; Pullen, A. E.; Swager, T. M. Chem. ReV. 2000,
100, 2537-2574. (f) Wright, A. T.; Anslyn, E. V. Chem. Soc. ReV. 2006,
35, 14-28. (g) Baldini, L.; Wilson, A. J.; Hong, J.; Hamilton, A. D. J.
Am. Chem. Soc. 2004, 126, 5656-5657.
(2) (a) Fan, C.; Plaxco, K. W.; Heeger, A. J. J. Am. Chem. Soc. 2002, 124,
5642-5643. (b) Wilson, J. N.; Wang, Y.; Lavigne, J. J.; Bunz, U. H. F.
Chem. Commun. 2003, 1626-1627.
(3) Chen, L.; McBranch, D. W.; Wang, H.-L.; Helgeson, R.; Wudl, F.;
Whitten, D. G. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 12287-12292.
(4) Kim, I.-B.; Dunkhorst, A.; Bunz, U. H. F. Langmuir 2005, 21, 7985-
7989.
(5) Nelson, T. L.; O’ Sullivan, C.; Greene, N. T.; Maynor, M. S.; Lavigne, J.
J. J. Am. Chem. Soc. 2006, 128, 5640-5641.
(6) Basu, S.; Vutukuri, D. R.; Shyamroy, S.; Sandanaraj, B. S.; Thayumanavan,
S. J. Am. Chem. Soc. 2004, 126, 9890-9891.
(7) See Supporting Information for details.
(8) Sandanaraj, B. S.; Vutukuri, D. R.; Simard, J. M.; Klaikherd, A.; Hong,
R.; Rotello, V. M.; Thayumanavan, S. J. Am. Chem. Soc. 2005, 127,
10693-10698.
(9) Renner, C.; Piehler, J.; Schrader, T. J. Am. Chem. Soc. 2006, 128, 620-
628.
(10) Sanghera, N.; Pinheiro, T. J. Protein Sci. 2000, 9, 1194-1202.
(11) Basu, S.; Vutukuri, D. R.; Thayumanavan, S. J. Am. Chem. Soc. 2005,
127, 16794-16795.
JA063544V
9
J. AM. CHEM. SOC. VOL. 128, NO. 33, 2006 10687