Chemosensory performance of molecularly imprinted fluorescent conjugated polymer materials

Article abstract of DOI:10.1021/ja0748027

Fluorescent conjugated polymers are an attractive basis for the design of low detection limit sensing devices owing to their intrinsic signal amplification capability. A simple and universal method to rationally control or fine-tune the chemodetection sel

Full text of DOI:10.1021/ja0748027

Published on Web 11/29/2007
Chemosensory Performance of Molecularly Imprinted
Fluorescent Conjugated Polymer Materials
Jiahui Li, Claire E. Kendig, and Evgueni E. Nesterov*
Department of Chemistry, Louisiana State UniVersity, Baton Rouge, Louisiana 70803
Received June 29, 2007; E-mail: een@lsu.edu
Abstract: Fluorescent conjugated polymers are an attractive basis for the design of low detection limit
sensing devices owing to their intrinsic signal amplification capability. A simple and universal method to
rationally control or fine-tune the chemodetection selectivity of conjugated polymer materials toward a desired
analytical target would further benefit their applications. In a quest of such a method we investigated a
general approach to cross-linked molecularly imprinted fluorescent conjugated polymer (MICP) materials
that possess an intrinsic capability for signal transduction and have potential to enhance selectivity and
sensitivity of sensor devices based on conjugated polymers. To study these capabilities, we prepared an
MICP material for the detection of 2,4,6-trinitrotoluene and related nitroaromatic compounds. We found
the imprinting effect in this material to be based on analyte shape/size recognition being substantial and
generally overcoming other competing thermodynamically determined trends. The described molecularly
imprinted fluorescent conjugated polymers show remarkable air stability and photostability, high fluorescence
quantum yield, and reversible analyte binding and therefore are advantageous for sensing applications
due to the ability to “preprogram” their detection selectivity through a choice of an imprinted template.
to 1/c for a three-dimensional network.5 This simple statistical
Introduction
reasoning alone can explain the greatly improved amplifying
ability of CPs in the aggregated or solid state. The highest
chemosensory efficiency of the CP based materials was found
for the detection of electron-deficient analytes such as nitroaro-
matic compounds, which cause fluorescence quenching via the
electron-transfer mechanism.6 Despite dramatic improvements
in sensitivity, it still remains a challenging task to obtain a
selective response toward a particular analyte, a condition that
is required in order to reduce the false alarm rates of the
detecting devices. It would be also conceivable to develop a
simple and general way to change or fine-tune the chemode-
tection selectivity. The molecular shape of a target analyte is a
natural feature that can be employed to achieve this goal through
enhanced molecular recognition. This approach has been long
and successfully used in molecular imprinting (MI), a versatile
technique to create affinity in solid matrices through controlled
selection of functional groups location and shape recognition.7
The idea of MI, as first put forward by Wulff8 and Mosbach,9
The development of robust and sensitive platforms for real-
time analytical detection is important in a broad range of
practical areas. Fluorescence-based chemosensing, when analyte
binding produces an attenuation in the light emission, has a
remarkable advantage over other detection schemes due to its
high sensitivity and detection simplicity.1 Conjugated polymers
(CPs) have found wide applications as a foundation for building
fluorescent sensors.2 The remarkable sensitivity of CP based
sensor devices as compared to small molecule sensors is
attributed to CPs’ intrinsic ability to provide an amplified
response to an analyte binding event.3 This amplification, first
demonstrated by Swager in 1995,4 stems from the efficient
energy transfer in CPs that allows excitation energy from large
areas to be effectively funneled into the sites where analyte
binding occurred. This effect is the most remarkable in
aggregated systems and solid films, where it is greatly enhanced
by intermolecular three-dimensional exciton migration via a
Fo¨rster-type mechanism. It was previously demonstrated that
the number of steps required for a randomly walking exciton
to find a randomly distributed trap (analyte-bound site) with
concentration c reduces from 1/c2 for a one-dimensional array
(5) Montroll, E. W. J. Phys. Soc. Jpn. 1969, 26 Suppl., 6-10.
(6) (a) Yang, J.-S.; Swager, T. M. J. Am. Chem. Soc. 1998, 120, 11864-11873.
(b) Yang, J.-S.; Swager, T. M. J. Am. Chem. Soc. 1998, 120, 5321-5322.
(c) Sohn, H.; Sailor, M. J.; Magde, D.; Trogler, W. C. J. Am. Chem. Soc.
2003, 125, 3821-3830. (d) Liu, Y.; Mills, R. C.; Boncella, J. M.; Schanze,
K. S. Langmuir 2001, 17, 7452-7455.
(7) Molecularly Imprinted Polymers: Man-Made Mimics of Antibodies and
Their Applications in Analytical Chemistry; Sellegren, B., Ed.; Elsevier:
Amsterdam, 2001; (b) Molecular and Ionic Recognition with Imprinted
Polymers; Bartsch, R. A., Maeda, M., Eds.; ACS Symposium Series 703;
American Chemical Society: Washington, DC, 1998. (c) Zimmerman, S.
C.; Lemcoff, N. G. Chem. Commun. 2004, 5-14. (d) Wei, S.; Jakusch,
M.; Mizaikoff, B. Anal. Chim. Acta 2006, 578, 50-58.
(8) Wulff, G.; Sarhan, A. Angew. Chem., Int. Ed. Engl. 1972, 11, 341.
(9) Andersson, L.; Sellergren, B.; Mosbach, K. Tetrahedron Lett. 1984, 25,
5211-5214.
(1) (a) Callan, J. F.; de Silva, A. P.; Magri, D. C. Tetrahedron 2005, 61, 8551-
8588 and references therein. (b) Special issue on “Luminescent Sensors”
Coord. Chem. ReV. 2000, 205, 1-232.
(2) (a) McQuade, D. T.; Pullen, A. E.; Swager, T. M. Chem. ReV. 2000, 100,
2537-2574. (b) Thomas, S. W., III; Joly, G. D.; Swager, T. M. Chem.
ReV. 2007, 107, 1339-1386. (c) Yang, C. J.; Pinto, M.; Schanze, K.; Tan,
W. Angew. Chem., Int. Ed. 2005, 44, 2572-2576. (d) Liu, B.; Bazan, G.
C. J. Am. Chem. Soc. 2006, 128, 1188-1196.
(3) (a) Swager, T. M. Acc. Chem. Res. 1998, 31, 201-207. (b) 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) Zhou, Q.; Swager, T. M. J. Am. Chem. Soc. 1995, 117, 12593-12602.
9
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J. AM. CHEM. SOC. 2007, 129, 15911-15918
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A R T I C L E S
Li et al.
is very simple. A template with attached (covalently or
noncovalently) functional monomers is copolymerized in the
presence of cross-linking agents that results in formation of a
polymer network around the template. Subsequent removal of
the template leaves binding cavities that are complementary in
shape and functionality to the template molecule. Although it
has been long postulated that binding to the correctly preorga-
nized functionalities inside the imprinted cavity plays a decisive
role in selectivity of molecular imprinting, it has been recently
demonstrated that it is the molecular shape which is crucially
important in analyte recognition.10 This understanding opens
up the prospect for obtaining high selectivity based on the shape
recognition alone, therefore bringing about the possibility of
preparing high recognition sites even for the analytes that lack
specific binding groups.11
were no systematic studies on using chemically synthesized
fluorescent molecularly imprinted conjugated polymers, where
more sensitive optical detection of analyte binding could be
readily employed. We were naturally interested in filling this
gap and studying the possibility of preparing fluorescent MICPs
with the enhanced selectivity toward a particular analyte of
choice. Since a common land-mine explosive 2,4,6-trinitrotolu-
ene (TNT) 1 is a practical and desirable target for sensor design,
we investigated the possibility of preparing “TNT-imprinted”
fluorescent CPs and studied the properties of the resulting
materials.16
The vast majority of MI methods uses “conventional”
aliphatic, such as vinyl and acrylic, polymers. These traditional
materials for MI generally show good selectivity but do not
possess the ability for intrinsic signal transduction required for
sensing applications.12 To solve this dilemma, incorporating
fluorescent dye reporters within the imprinted cavity of tradi-
tional molecularly imprinted polymers has been proposed.13 Still
such materials suffer from low sensitivity. In contrast, the
concept of molecularly imprinted CPs (MICPs) described
herein utilizes CPs as both imprinting materials and analytical
signal transducers. We presumed that, like typical fluorescent
CPs, the MICP materials would possess high chemodetection
sensitivity, coupled with the possibility of adjusting the detection
selectivity by simply changing or modifying the template.
Therefore, they could be considered as an interesting and general
way to improve the selective response of CP based chemosensor
materials.
Results and Discussion
Preparation of MICP Materials. Three-dimensionally cross-
linked poly(p-phenylene vinylene)s (PPVs) were a natural choice
for the current study as they show some degree of conforma-
tional flexibility around the conjugated backbone that is required
for shaping the imprinted cavity. In addition, PPVs are highly
emissive in the solid state,17 and their fluorescent properties are
strongly affected by the environment and the presence of
quenchers.18 Three major components are necessary for prepar-
ing a molecularly imprinted material: a monomer, a cross-linker,
and a template. As cross-linked polymers are not soluble, we
paid special attention to the design of the synthetic strategy
which would allow synthesis of these materials in a form suitable
for further studies and applications.
At the time we initiated this research, there were only a few
previous reports concerning studies on molecularly imprinted
CPs. Cross-linked polypyrroles obtained by electropolymeriza-
tion were most often used due to the convenience of this
polymerization technique. In 1992, Sauvage et al. prepared a
cross-linked polypyrrole matrix imprinted with metal cations.14
They demonstrated that removal of the cationic template resulted
in a stable three-dimensional porous structure, which kept the
“memory” of the template and could be recomplexed again.
Since that milestone paper, there was some further development
toward building sensors based on molecularly imprinted elec-
tropolymerized polypyrrole, polyphenol, poly(phenylenedi-
amines), etc.15 In all the reports, electrochemical detection of
analyte binding was used. To the best of our knowledge, there
Emulsion polymerization at a water-organic interface is a
robust approach to prepare cross-linked micro- and nanoparticles
of conjugated polymers.19 Suzuki coupling, although not widely
used for PPV synthesis,20 was preferred as it requires mild
conditions. The synthesis of the monomers for polymerization,
diiodide 5 and bis-vinylboronate 7, is shown in Scheme 1. The
trans bis-vinylboronate monomer 7 was prepared in good yield
by using hydroboration of the precursor bis-acetylene 6. We
found that this reaction barely proceeds in the absence of catalyst
(15) Some recent references: (a) Namvar, A.; Warriner, K. Biosens. Bioelectron.
2007, 22, 2018-2024. (b) Ramanavicˇius, A.; Ramanavicˇiene, A.; Ma-
linauskas, A. Electrochim. Acta 2006, 51, 6025-6037. (c) Shiigi, H.;
Kijima, D.; Ikenaga, Y.; Hori, K.; Fukazawa, S.; Nagaoka, T. J. Electro-
chem. Soc. 2005, 152, H129-H134. (d) Ho, K.-C.; Yeh, W.-M.; Tung,
T.-S.; Liao, J.-Y. Anal. Chim. Acta 2005, 542, 90-96. (e) Panasyuk, T.
L.; Mirsky, V. M.; Piletsky, S. A.; Wolfbeis, O. S. Anal. Chem. 1999, 71,
4609-4613.
(16) Other approaches to TNT detection using molecularly imprinted materi-
als: (a) Gao, D.; Zhang, Z.; Wu, M.; Xie, C.; Guan, G.; Wang, D. J. Am.
Chem. Soc. 2007, 129, 7859-7866. (b) Bunte, G.; Hu¨rttlen, J.; Pontius,
H.; Hartlieb, K.; Krause, H. Anal. Chim. Acta 2007, 591, 49-56. (c) Walker,
N. R.; Linman, M. J.; Timmers, M. M.; Dean, S. L.; Burkett, C. M.; Lloyd,
J. A.; Keelor, J. D.; Baughman, B. M.; Edmiston, P. L. Anal. Chim. Acta
2007, 593, 82-91.
(17) Moratti, S. C. In Handbook of Conducting Polymers, 2nd ed.; Skotheim,
T. A., Elsenbaumer, R. L., Reynolds, J. R., Eds.; Marcel Dekker: New
York, 1998; pp 343-361.
(18) (a) Rose, A.; Zhu, Z.; Madigan, C. F.; Swager, T. M.; Bulovic´, V. Nature
2005, 434, 876-879. (b) Kim, Y.; Zhu, Z.; Swager, T. M. J. Am. Chem.
Soc. 2004, 126, 452-453. (c) Chang, C.-P.; Chao, C.-Y.; Huang, J. H.; Li,
A.-K.; Hsu, C.-S.; Lin, M.-S.; Hsieh, B. R.; Su, A.-C. Synth. Met. 2004,
144, 297-301.
(19) (a) Hittinger, E.; Kokil, A.; Weder, C. Angew. Chem., Int. Ed. 2004, 43,
1808-1811. (b) Weder, C. Chem. Commun. 2005, 5378-5389.
(20) Terao, J.; Tang, A.; Michels, J. J.; Krivokapic, A.; Anderson, H. L. Chem.
Commun. 2004, 56-57.
(10) (a) Sibrian-Vazquez, M.; Spivak, D. A. J. Am. Chem. Soc. 2004, 126, 7827-
7833. (b) Spivak, D. A.; Campbell, J. Analyst 2001, 126, 793-797.
(11) As an example of highly selective molecular imprinting of a nonfunction-
alized template, imprinting of hexachlorobenzene was recently reported:
Das, K.; Penelle, J.; Rotello, V. M. Langmuir 2003, 19, 3921-3925.
(12) (a) Dickert, F. L.; Forth, P.; Lieberzeit, P.; Tortschanoff, M. Fresenius’ J.
Anal. Chem. 1998, 360, 759-762. (b) Hillberg, A. L.; Brain, K. R.;
Allender, C. J. AdV. Drug DeliVery ReV. 2005, 57, 1875-1889. (c) Haupt,
K. Chem. Commun. 2003, 171-178.
(13) Some selected examples: (a) Turkewitsch, P.; Wandelt, B.; Darling, G.
D.; Powell, W. S. Anal. Chem. 1998, 70, 2025-2030. (b) Manesiotis, P.;
Hall, A. J.; Emgenbroich, M.; Quaglia, M.; De Lorenzi, E.; Sellergren, B.
Chem. Commun. 2004, 2278-2279. (c) Takeuchi, T.; Mukawa, T.; Matsui,
J.; Higashi, M.; Shimizu, K. D. Anal. Chem. 2001, 73, 3869-3874. (d)
Rathbone, D. L.; Bains, A. Biosens. Bioelectron. 2005, 20, 1438-1442.
(e) Henry, O. Y. F.; Cullen, D. C.; Piletsky, S. A. Anal. Bioanal. Chem.
2005, 382, 947-956. (f) Matsui, J.; Higashi, M.; Takeuchi, T. J. Am. Chem.
Soc. 2000, 122, 5218-5219.
(14) Bidan, G.; Divisia-Blohorn, B.; Lapkowski, M.; Kern, J.-M.; Sauvage, J.-
P. J. Am. Chem. Soc. 1992, 114, 5986-5994.
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Molecularly Imprinted Fluorescent Conjugated Polymers
A R T I C L E S
Scheme 1. Synthesis of Monomers for Polymerization
Figure 1. (A) Schematic representation of a template imprinted into a CP
network. Red dots correspond to cross-linking units. (B) Fluorescent optical
micrograph of polymer P2 (suspension in dioxane, illuminated through a
blue filter in the excitation channel). (C) Scanning Electron Microscopy
(SEM) image of polymer P1. (D) SEM image of polymer P2. Note the
highly porous, spongy morphology of P2.
One of the most challenging problems in the conventional
procedures for molecularly imprinted materials preparation is
postpolymerization removal of a template.22 This commonly
demands harsh conditions and cannot be accomplished com-
pletely. Consequently, if an actual analyte capable of fluores-
cence quenching was used as a template, even a small residual
amount would result in strong “self-quenching” thus signifi-
cantly diminishing or even eliminating the analytical response
of the system. A strategy to overcome this difficulty is to choose
a nonquenching surrogate template which possesses a shape
similar to that of the actual analyte. We chose 2,4,6-triisopro-
pyltoluene 2a as a nonquenching template for preparing a “TNT-
imprinted” polymer due to the similarity between 2a’s and
TNT’s molecular shapes.23 The template-bearing monomer 11a
was prepared following the route in Scheme 1.
a
Reagents and conditions: (a) I2, HIO3, aq. H2SO4, reflux, 12 h; (b)
BBr3, CH2Cl2, 25 °C, 48 h, 42% over two steps; (c) R-OTs, KI (cat.), K2CO3,
2-propanone, 100 °C, 48 h, 81%; (d) Me3SiCtCH, Pd(PPh3)4 (cat.), CuI
(cat.), i-Pr2NH-toluene, 70 °C, 48 h; (e) KOH, MeOH, 25 °C, 40 min,
41% over two steps; (f) pinacolborane, ZrCp2HCl (cat.), ClCH2CH2Cl, 65
°C, 72 h, 54%; (g) t-BuLi, THF, -75 °C, 1 h; (h) (CH2O)n, -75 °C f 25
°C, 12 h, 72%; (i) DMAP, CH2Cl2, 25 °C, 24 h, 11a (80%), 11b (70%).
but becomes highly efficient in the presence of a catalytic
amount of ZrCp2HCl21 and can serve as a convenient and
general method to access highly valuable trans bis-vinylbor-
onates.
The polymerization of a mixture of comonomers 5 and 7,
the template-bearing monomer 11a (10 mol %), and the cross-
linker 1,3,5-triiodobenzene 12 (30 mol %) was carried out in a
water-ethanol-toluene medium at high-speed stirring (∼1000
rpm) in the presence of sodium dodecyl sulfate as a surfactant
(Scheme 2).24 This resulted in the cross-linked material P1 as
nonsoluble fluorescent spherically shaped micrometer-size
particles. For comparison, we also carried out the polymerization
under the same conditions but in the absence of the cross-linker
12 to yield the linear polymer P3 that was soluble in common
organic solvents. We then proceeded to find a suitable method
to cleave the ester bond between the template and the polymer,
in order to remove the template. After trying different ap-
proaches, we determined that lithium n-propyl mercaptide in
HMPA worked the most efficiently.25 Thus, the treatment of
P1 microparticles with an excess of this reagent at room
In designing an imprinted system, the judicious selection of
the side groups on a conjugated backbone is highly important.
In our case, an imprinted cavity is likely to be formed between
separate PPV strands that are rigidly held together by cross-
linking units rather than a single chain “wrapping” around the
template (Figure 1A). Therefore, the flexible long-chain sub-
stituents could improve the fitting of the imprinted cavity to a
template by getting entangled in the three-dimensional (3D)
network during the polymerization. These “trapped” chains can
possibly fill the small voids between the template and the cavity
which cannot be filled by the growing rigid 3D conjugated
framework, and they shall stay in place after template removal.
Based on this consideration, we chose the diethylene glycol side
groups for the present study. One extra advantage these side
groups can offer is the possibility of stabilizing interactions
between their ether oxygen atoms and electron-deficient core
of the nitroaromatic analytes that could result in stronger binding
between the analyte and the imprinted cavity.
(22) (a) Ki, C. D.; Oh, C.; Oh, S.-G.; Chang, J. Y. J. Am. Chem. Soc. 2002,
124, 14838-14839. (b) Dirion, B.; Lanza, F.; Sellergren, B.; Chassaing,
C.; Venn, R.; Berggren, C. Chromatographia 2002, 56, 237-241.
(23) Molecules 1 and 2a show the opposite electrostatic potential distribution.
Although this may result in differences in the template/analyte electrostatic
interactions with the imprinted cavity, our experimental results demonstrated
that the matching shape/size is a dominant factor.
(24) See Supporting Information for experimental details, procedures, and
characterizations.
(25) Bartlett, P. A.; Johnson, W. S. Tetrahedron Lett. 1970, 4459-4462.
(21) Pereira, S.; Srebnik, M. Organometallics 1995, 14, 3127-3128.
9
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A R T I C L E S
Li et al.
Scheme 2. Synthesis and Structures of Polymers P1-P6
a
Reagents and conditions: (a) Pd(PPh3)4 (cat.), K2CO3, sodium dodecyl sulfate, H2O-EtOH-toluene, 75 °C, 72 h; (b) n-PrSLi, HMPA, 25 °C,
ultrasonication, 1.5 h, then HCl, MeOH.
temperature upon ultrasonication followed by extended washing
with various solvents in a Soxhlet extractor yielded the highly
fluorescent material P2 (Figure 1B). Examination of P2 with
Scanning Electron Microscopy (SEM) revealed a highly porous,
spongy morphology which greatly contrasts the smooth surface
morphology of the precursor P1 (Figure 1C and 1D). It is likely
that the porous morphology of the material P2 resulted from
washing out the less cross-linked fraction of the polymer by
HMPA, therefore effectively increasing the surface area and
providing access to a larger number of imprinted cavities (Vide
infra). The extent of the template removal was determined by
comparing the FT-IR spectra of polymers P1 and P2 (Figure
2). The spectra are identical, except for the band corresponding
to the stretching carbonyl frequency. In P1 this band appears
at 1710 cm-1, while in P2 the presence of an additional band
at 1722 cm-1 can be attributed to the conversion from ester to
free carboxylic acid upon the cleavage of the template-polymer
Figure 2. FT-IR spectra of polymers P1 and P2. The carbonyl stretching
linkage. Based on the relative intensities of the two bands in
P2, we could estimate that approximately 50% of the template
was removed in this polymer. Similar postpolymerization
treatment of the linear polymer P3 with n-PrSLi in HMPA
resulted in polymer P4 showing virtually no residual template
frequency bands used to determine the extent of template removal are
marked with a blue arrow, and the area around these bands is expanded in
the inset. The frequency shift of the CdO band is ∼12 cm-1
.
the “TNT-like” template 2a. The corresponding MICP P6 was
obtained after removal of the template from P5 using n-PrSLi
in HMPA (Scheme 2). The FT-IR analysis similar to that
described above revealed practically quantitative removal of the
template in P6 (Figure S1 in Supporting Information).
Photophysical Properties of the Polymers. Analysis of
spectral properties of the obtained materials revealed a noticeable
difference between the cross-linked (P1, P2, P5, and P6) and
linear (P3 and P4) polymers. The cross-linked materials display
small hypsochromic shifts in both absorption and fluorescence
1
by H NMR analysis.
For comparison purposes, and to further investigate the
validity of our imprinting hypothesis, we also prepared a cross-
linked polymer P5. This polymer was prepared based on the
same monomers and reaction conditions as P1 with the
exception of using, instead of 11a, the template-bearing
monomer 11b (Scheme 2). This allowed incorporating toluene
2b as a “wrong”, smaller size template structurally similar to
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Molecularly Imprinted Fluorescent Conjugated Polymers
A R T I C L E S
Figure 3. Absorption (left) and fluorescence (right) spectra of P1-P6. Dashed lines correspond to spectra in CH2Cl2 solution (for P3, P4); solid lines
correspond to spectra acquired in spin-cast films. The absorption spectra for P1, P2, P5, and P6 were acquired from suspensions in CHCl3. Extinction
coefficients in solution (per repeating unit): P3 ꢀ(481 nm) 21 000, P4 ꢀ(439 nm) 13 000. Fluorescence quantum yields: P1 ∼0.03, P2 ∼1.00, P3 0.23
(solution), <0.001 (solid), P4 0.33 (solution), 0.007 (solid).
spectra relative to the linear analogues (Figure 3). This can be
attributed to shorter π-electron conjugation in the former as the
conjugation is diminished due to bending of the CP backbones
to accommodate the template.26
noticeable fluorescence quenching for all six polymers. In order
to obtain quantitative characteristics of the quenching process
to allow comparison of detection efficiency for the different
polymers, vapor-phase Stern-Volmer experiments were carried
out. In a typical Stern-Volmer experiment, a substrate is
exposed to various concentrations of a quencher, and the drop
in fluorescence intensity is described by eq 1:
There is a noticeable spectral difference between the template-
containing precursor (e.g., P1) and the MICP material (P2).
Removal of the template resulted in substantial hypsochromic
shifts both for the absorption (∼40 nm for P1-P2 pair) and
for the fluorescence bands (∼20 nm for P1-P2 pair). This trend
is quite consistent, does not seem to depend on the template (it
also clearly showed up for the P5-P6 pair), and therefore can
be considered as a characteristic spectral signature of successful
template removal. It is likely that this behavior reflects some
reorganization of the polymer fragments adjacent to the
imprinted cavity after the template is removed (possibly, even
some collapsing of the cavity-forming CP inside the emptied
imprinted cavity), resulting in decreasing conjugation length and
attenuation of the exciton migration efficiency. With this
assumption, one would expect seeing more drastic changes in
the case of formation of a larger imprinted cavity as compared
with formation of a smaller cavity. Indeed, this is the case for
the related polymer pairs P1-P2 and P5-P6. Removal of the
substantially less bulky template 2b in the latter case resulted
in smaller spectral changes. While this interesting behavior
definitely requires further investigation, it does serve as ad-
ditional evidence in favor of the molecularly imprinted nature
of polymers P2 and P6.
I
0 ) 1 + KSV[Q] ≈ 1 + K′ t
(1)
SV
I
In eq 1, I0 is the initial fluorescence intensity without analyte,
I is the fluorescence intensity with added analyte of concentra-
tion [Q], and KSV is the Stern-Volmer constant.27 For gas-phase
measurements, when a sample is exposed to an extremely low
and steady concentration of an analyte vapor, and assuming that
the quenching process is diffusion-controlled, the concentration
of the analyte permeating into the sample and causing fluores-
cence quenching (equilibrium solubility) will be approximately
proportional to the analyte’s partial pressure (Henry’s law).28
With the steady pressure, the total amount of analyte absorbed
by the sample should be proportional to the exposure time t,
and therefore the time t can be used in quantitative experiments
as a variable instead of the concentration [Q] in the eq 1.29
Although this assumption is only approximately valid, it allows
running Stern-Volmer experiments for extra-low concentrations
of gas-phase analytes using a relatively simple experimental
setup. For our experiments, we used borosilicate glass slides
with a layer of attached polymer microparticles prepared by
spin-casting from their dilute suspensions in chloroform. The
glass slides were placed inside a sealed quartz cuvette containing
small amounts of various solid analytes deposited onto cotton
gauze to ensure constant vapor pressure, and the cuvette was
immediately placed into a fluorimeter to record the sample’s
fluorescence spectra at particular time intervals. A representative
Interestingly, molecularly imprinted polymer P2 is highly
emissive, with the fluorescence quantum yield approaching
100%. This remarkably increased emission efficiency can be
attributed to a more rigid 3D conjugated framework which
diminishes vibrationally/rotationally coupled electronic deactiva-
tion processes therefore reducing the rate of nonradiative decay.
Vapor-Phase Fluorescence Quenching. Exposure of P1-
P6 to saturated TNT vapors at room temperature resulted in
(26) Alternatively, the decrease in conjugation length may be due to cross-
conjugation in meta-substituted cross-linker units, which disrupts the
π-electron conjugation in the ground state (but enhances the conjugation
in the excited state; see: Thomson, A. L.; Gaab, K. M.; Xu, J.; Bardeen,
C. J.; Martinez, T. J. J. Phys. Chem. A 2004, 108, 671-682). This effect
requires further studies which will be reported elsewhere.
(27) Turro N. J. Modern Molecular Photochemistry; University Science
Books: Mill Valley, CA, 1991.
(28) Masoumi, Z.; Stoeva, V.; Yekta, A.; Pang, Z.; Manners, I.; Winnik, M. A.
Chem. Phys. Lett. 1996, 261, 551-557.
(29) Note that the Stern-Volmer constants KSV and K′SV in eq 1 are different
and are expressed in different units.
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A R T I C L E S
Li et al.
types of cavities are present, such a behavior can be analyzed
using the modified Stern-Volmer equation:30
I0
1
1
)
+
(2)
fa KaSV[Q]
∆I fa
In eq 2, I0 is the initial fluorescence intensity without analyte,
∆I ) I0 - I is the fluorescence intensity drop with added analyte
of concentration [Q], fa is the fraction of easily accessible
cavities, and KaSV is the Stern-Volmer constant for quenching
inside these cavities. A plot in the coordinates from eq 2 is
shown as an inset to Figure 5. Its linearity does agree with the
proposed model of having two types of imprinted cavities. From
this plot, the fraction of easily accessible cavities fa was
estimated to be 0.9. Although this value is very approximate
and should be considered cautiously, it is in good agreement
with the highly porous nature of polymer P2, when not only
cavities close to the surface but also cavities well inside the
polymer microparticles are accessible through the network of
interconnecting channels. From a practical standpoint, this
property is of ultimate significance for chemical sensing
materials.
Figure 4. Quenching of fluorescence emission of polymer P2 upon
exposure to TNT vapors at room temperature at various time intervals.
Analysis of TNT quenching data for polymers P1-P6 (Figure
5) allows us to differentiate between fluorescence quenching
due to selective recognition of the analyte by molecularly
imprinted cavities and quenching by the analyte molecules
nonselectively bound at the surface of the sensor material. In
principle, the effect of such nonspecific surface associations with
electron-deficient TNT can be significantly amplified by the
efficient 3D exciton migration resulting in a noticeable nonse-
lective fluorescence quenching. Indeed, it is this quenching mode
which produces highly sensitive but not selective detection of
electron-deficient analytes by the traditional non-imprinted CPs.6
In the case of template-containing precursor polymers P1, P3,
and P5, as well as the spin-cast film of linear polymer P4, such
nonspecific fluorescence quenching by surface-bound TNT is
the primary quenching pathway; since the surface area is
relatively small, the magnitude of the analytical response is
noticeable but not significant.31 The template removal procedure
produces a material with a highly porous morphology (Figures
1C and 1D). Due to the greater surface area, the increased
probability of the analyte nonspecific binding is a possible
reason for the overall enhanced quenching response of polymer
P6, since selective recognition of TNT by P6’s much smaller
imprinted cavity seems unlikely. It is natural to assume that
the “TNT-imprinted” polymer P2 which was prepared under
conditions similar to those for P6 would have exhibited a
somewhat similar quenching efficiency had there been no
specific recognition by imprinted cavities involved. Therefore,
P2’s almost 2-fold higher quenching response upon TNT
exposure (Figure 5) stems from the selective recognition of TNT
due to molecular imprinting.
Figure 5. Stern-Volmer plots for polymers P1-P6 fluorescence quenching
with saturated vapors of TNT at room temperature. Inset: modified Stern-
Volmer plot for polymer P2.
set of fluorescence spectra obtained in this way for the exposure
of polymer P2 to TNT vapors is shown in Figure 4.
The vapor-phase Stern-Volmer plots obtained for polymers
P1-P6 are shown in Figure 5. As expected, the “TNT-
imprinted” polymer P2 showed better sensitivity to TNT than
its non-imprinted linear analogue P4 or template-containing non-
imprinted precursors P1, P3, and P5. An extremely important
finding was that polymer P6 imprinted with the “wrong”
template also showed lower sensitivity to TNT. From these
observations, we could conclude that the enhanced sensitivity
of P2 is consistent with its “TNT-imprinted” nature, where the
complementary imprinted cavities provide better recognition for
TNT as compared to the other polymers with no imprinted
cavities (P1, P3-P5) or with substantially smaller size, “wrong”
imprinted cavities (P6). The Stern-Volmer plot for polymer
P2 initially shows a linear behavior consistent with the eq 1;
however, upon longer exposure it displays downward curvature
(Figure 5). The downward curvature is likely to reflect a
saturation phenomenon, when the analyte first permeates into
easily accessible imprinted cavities (linear part of the plot),
which is followed by much slower absorption into less accessible
cavities. With a very approximate assumption that only two
In order to further understand the distinguished behavior of
P2 and eliminate the possibility that this special behavior was
simply due to the highly porous morphology of this material,
(30) Lakowicz, J. R. Principles of Fluorescence Spectroscopy, 3rd ed.;
Springer: New York, 2006.
(31) Diffusion-controlled analyte penetration from the surface into the bulk of
the solid material will depend on the solubility of the analyte in the polymer
and may also affect the quenching efficiency, especially at longer exposure
times. Still, considering the relative similarity between the polymers under
study, it is expected to provide only a minor contribution.
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15916 J. AM. CHEM. SOC. VOL. 129, NO. 51, 2007

Molecularly Imprinted Fluorescent Conjugated Polymers
A R T I C L E S
Table 1. Structures and Properties of the Analytes Used in the Quenching Selectivity Studiesa
TNT
DNT
BDNB
NA
AQ
DNN
CA
DDQ
PO
VPb
E
1
18
2c
-0.99
-0.69
0.56
0.07
-1.21
-0.47
0.28
0.02
-0.97
-0.71
0.56
0.05
-0.90
-0.78
0.33
950
8
0.03
-1.16
-0.52
0.28
Red, Vd
-0.77
-0.91
1.0
-1.01
-0.67
3.50
-0.05
-1.63
0.83
0.61
-2.29
0.39
0.11
∆G0, eV
K
K
SV(P2)e
SV(P1)e
0.11
0.39
0.11
0.11
0.11
0.06
0.44
0.22
a VP, equilibrium vapor pressure (relative to TNT); ERed, electrochemical reduction potential (relative to SCE); ∆G°, Gibbs free energy for the electron
transfer from the polymer’s excited state to a given quencher; KSV(P2) and KSV(P1), experimental Stern-Volmer constant for fluorescence quenching of
b
polymers P2 and P1, respectively (relative to TNT). The data from: Handbook of Physical Properties of Organic Chemicals; Howard, P. H., Meylan, W.
c
d
M., Eds.; CRC Press: Boca Raton, FL, 1997. Estimated by extrapolation. Experimentally measured in CH2Cl2 solutions (0.1 M Bu4NPF6 as supporting
e
electrolyte) with Pt working electrode. Determined from the slope of the initial straight sections of corresponding Stern-Volmer graphs.
we carried out a comparative study using a group of analytes
with different molecular shapes and sizes which can interfere
with TNT detection. These analytes are listed in Table 1 along
with their corresponding equilibrium vapor pressure, reduction
potential, and Gibbs free energy change (∆G°) for the electron
transfer from the excited polymer to a particular quencher. Since
fluorescence quenching involves an electron transfer from the
excited polymer to the LUMO of a quencher, the magnitude of
∆G° determines the thermodynamic “driving force” of the
quenching process, and it was estimated using the Rehm-Weller
equation:32
∆G° ) EOx(P/P+•) - ERed(Q/Q-•) - E00
(3)
In this equation, EOx(P/P+•), ERed(Q/Q-•), and E00 are the
oxidation potential of polymer P2, the reduction potential of a
quencher, and the energy difference between ground and first
excited singlet states of the polymer, respectively. As polymer
P2 was not soluble, we used the values determined for the
related polymer P4. The value of EOx(P/P+•) (0.82 V vs SCE)
was determined electrochemically, and E00 (2.5 eV) was
obtained from the intersecting wavelength of the normalized
absorption and fluorescence spectra. As can be seen from Table
1, the electron transfer is a highly exergonic process for each
analyte, with the exergonicity being either comparable to that
of TNT or significantly exceeding it. Therefore, if the quenching
process was simply thermodynamically controlled (as in the case
of nonspecific surface associations), each of these compounds
would demonstrate similar or better quenching of P2’s fluo-
rescence than TNT does.
Figure 6. Stern-Volmer plots for polymer P2 fluorescence quenching with
saturated vapors of the analytes listed in Table 1 (at room temperature).
6) and can be used to quantify the selectivity of P2’s
chemosensing response. The outcome of the quenching experi-
ments was consistent with the preliminary expectations, with
the majority of the analytes (including likely field-use interfer-
ents such as a common agricultural insecticide paraoxon (PO))
not interfering significantly with the polymer’s fluorescence,
and non-imprinted precursor polymer P1 exhibiting much
smaller quenching response (mostly due to the nonspecific
analyte binding). It was not affected even by exposure to 2,3-
dichloro-5,6-dicyano-1,4-benzoquinone (DDQ), despite the re-
markably high exergonicity of the electron-transfer quenching
for this analyte. This means that even when the electron transfer
is favored from the thermodynamic standpoint (which is
especially significant for DDQ), P2’s analytical response is
strongly determined by the imprinting effect. The analytes with
the size/shape not matching the imprinted template are not
capable of entering the imprinted cavity and causing fluorescent
quenching.33
The last two lines in Table 1 give the relative values of Stern-
Volmer constant KSV for the imprinted polymer P2 and the non-
imprinted precursor P1 fluorescence quenching with different
analytes. They correspond to the slopes of the initial straight
sections of the gas-phase Stern-Volmer plots (shown in Figure
(32) Rehm, D.; Weller, A. Isr. J. Chem. 1970, 8, 259-271.
9
J. AM. CHEM. SOC. VOL. 129, NO. 51, 2007 15917

A R T I C L E S
Li et al.
One remarkable and clearly unexpected observation was that
exposure of P2 to vapors of 2,4-dinitrotoluene (DNT) caused
much stronger fluorescence quenching than even TNT (Figure
6). Moreover, a Stern-Volmer plot for the DNT quenching is
practically linear over the entire time span of the experiment
and does not show saturation behavior (downward curvature)
typical for other analytes. Although DNT’s vapor pressure at
room temperature is higher than that of TNT, its ability to
quench by an electron-transfer mechanism is substantially
diminished as reflected in the corresponding ∆G° values.
Furthermore, the non-imprinted precursor P1 was only slightly
affected by DNT exposure. Therefore, the magnitude of P2’s
response to DNT clearly indicates that the imprinted cavity
provides a better fit to this analyte. Two factors are probably
responsible for this. First, the surrogate template 2a used for
“TNT imprinting” in P2 does not perfectly match the TNT
molecular shape. The second factor is likely to be related to
partial collapsing of the imprinted cavity upon removing the
template leading to decreasing the “free volume” of the cavity.
The experimental reflection of such reorganization is also seen
in the characteristic hypsochromic shifts of absorption and
fluorescence maxima observed upon template removal (Vide
supra). As a result, DNT rather than target TNT seems to be
the best match to the P2’s imprinted cavities. Contrarily, P2’s
fluorescence was practically not affected by exposure to 1-tert-
butyl-2,4-dinitrobenzene (BDNB) that was specially prepared
to compare with DNT. Both DNT and BDNB possess similar
∆G° values for electron transfer (Table 1); therefore the
influence of electronic factors on quenching selectivity is almost
negligible. Thus, the dramatic difference in the magnitude of
fluorescence quenching by these two analytes can only be
understood in terms of the molecularly imprinted nature of
polymer P2, when the bulkier tert-butyl group in BDNB makes
entering the imprinted cavity practically impossible.
It is important to mention that the molecularly imprinted
polymer P2 is remarkably air-stable and photostable. The
polymer microparticles covered glass slides prepared for the
experiments described herein were stored in air for 3 months
without significant diminishing of their fluorescence intensity.
This high stability greatly contrasts with the low stability of
the linear analogue P4; thin films of P4 degrade and become
completely nonemissive after only a few hours of exposure to
air. Absorption of DNT and TNT by P2 is also completely
reversible (Figure 7). Exposure of this polymer to DNT or TNT
vapors for 10 min causes a substantial decrease in fluorescence
intensity (almost complete for DNT); however, the fluorescence
of the samples markedly recovers after keeping them for 10
min in air; the fluorescence recovers completely in 1 h. Such
exposure to the quenchers with subsequent recovery of fluo-
rescence can be done repeatedly, without any noticeable
irreversible quenching. In contrast, thin films of the linear
polymer P4 become irreversibly quenched upon exposure to
Figure 7. Quenching and recovery of fluorescence of P2 with TNT and
DNT. Solid line: initial fluorescence. Dash line: fluorescence immediately
after 10 min of exposure to TNT (DNT) vapors. Dash-and-dot line: recovery
of fluorescence after 10 min of exposure to air. Fluorescence of both samples
was completely recovered after 1 h of exposure to air.
DNT or TNT (this irreversible quenching also coincides with
irreversible oxidative degradation by air). This stability and
analytical response reversibility increase the value of cross-
linked molecularly imprinted conjugated polymers for potential
chemosensing applications.
Conclusions
We demonstrated the principle possibility of molecular
imprinting in three dimensionally cross-linked fluorescent
conjugated polymers. The imprinting effect that is based on
analyte shape/size recognition is substantial, although it is not
capable of completely overriding the effect of nonspecific
surface binding of electron-deficient analytes. Nevertheless, the
new molecularly imprinted fluorescent conjugated polymer
materials, as intrinsically amplifying signal transducers, are
advantageous for sensing applications due to the ability to
“program” the required detection selectivity through a choice
of imprinted templates. While in the current study the “TNT-
imprinted” material turned out to be a much better sensor for
DNT, it may be possible to improve the fitness of the imprinted
cavity to TNT by fine-tuning the surrogate imprinting template.
Further experimental studies on various factors capable of
influencing the imprinting effect, as well as gaining a deeper
understanding of the nature and origin of this phenomenon, are
required to optimize the performance of these materials and are
currently underway.
Acknowledgment. This research was supported by the
National Science Foundation (CAREER Award CHE-0547895),
Louisiana Board of Regents (LEQSF Pfund-45), and LSU
through start-up funding. Kind appreciation is due to Dr. W.
Ong for helping with electrochemical measurements.
Supporting Information Available: Detailed synthetic and
experimental procedures and additional data. This material is
available free of charge via the Internet at http://pubs.acs.org.
(33) The anomalously high sensitivity of P2 to chloranil (CA) can be explained
by a combination of very high exergonicity for the electron transfer
quenching and significantly (103 compared to TNT) higher vapor pressure.
In addition, CA’s shape and size are somewhat close to those of TNT which
allows this compound to enter some of the more easily accessible imprinted
cavities.
JA0748027
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15918 J. AM. CHEM. SOC. VOL. 129, NO. 51, 2007