Highly efficient quenching of nanoparticles for the detection of electron-deficient nitroaromatics

Article abstract of DOI:10.1002/pola.26824

Reported herein is the highly efficient quenching of fluorescent organic nanoparticles by 2,4-dinitrotoluene and 2,4,6-trinitrotoluene. These fluorescent nanoparticles are formed from the hydrophobic collapse of fluorescent polymer chains and display quen

Full text of DOI:10.1002/pola.26824

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Highly Efficient Quenching of Nanoparticles for the Detection of
Electron-Deficient Nitroaromatics
Patrick Marks,1 Sage Cohen,2 Mindy Levine1
1Department of Chemistry, University of Rhode Island, 51 Lower College Road, Kingston, Rhode Island 02881
2South Kingstown High School, South Kingstown, Rhode Island
Correspondence to: M. Levine (E-mail: mlevine@chm.uri.edu)
Received 29 April 2013; accepted 9 June 2013; published online 26 June 2013
DOI: 10.1002/pola.26824
ABSTRACT: Reported herein is the highly efficient quenching of
fluorescent organic nanoparticles by 2,4-dinitrotoluene and
2,4,6-trinitrotoluene. These fluorescent nanoparticles are
formed from the hydrophobic collapse of fluorescent polymer
chains and display quenching efficiencies that are in line with
the highest reported literature values. Moreover, the fluores-
cent quenching occurs only for the fluorescent nanoparticles,
and not for the precursor polymer solutions, which display
marked insensitivity to the presence of nitroaromatics. This
aggregation-dependent fluorescent quenching has numerous
applications for the detection of small-molecule electron-defi-
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KEYWORDS: conducting polymers; fluorescence; nanoparticles
“re-precipitation,”14,15 and (b) synthesis in mini-emulsions
stabilized by surfactants.16 Despite the ubiquity of such
nanoparticles in biological detection schemes,17 they have
been used only rarely for explosive detection. In one exam-
ple, polythiophene nanoparticles were used for DNT detec-
INTRODUCTION Researchers have had remarkable success in
developing detection methods for electron-deficient nitroaro-
matic explosives such as 2,4,6-trinitrotoluene (TNT), and for
the nonexplosive but structurally related 2,4-dinitrotoluene
(DNT) (Chart 1).1–3 These detection methods have significant
practical applications for national security and have been
used in airport screening systems4 and in the detection of
land mines in Iraq and Afghanistan.5 Such methods include
the use of amplified fluorescent quenching of conjugated pol-
y(phenyleneethynylenes)6,7 and other fluorescent polymers,8
quenching of fluorescent silica nanoparticles,9 and quenching
of metallic nanoparticles.10 Using such methodology, detection
limits as low as 1 fg (femtogram) have been obtained.11
tion, with a reported quenching constant of 2706 M21 18
.
Oligo(tetraphenyl)silole nanoparticles have also been used,
with a quenching constant of up to 5000 M21 in the pres-
ence of DNT.19 The quenching of other fluorescent nanopar-
ticles by nitroaromatics has not been reported.
Reported herein is the use of fluorescent nanoparticles fabri-
cated by re-precipitation methods from 2,1,3-benzooxadiazole-
alt-polyfluorene (PFBO, Compound 5) and 2,1,3-benzothiadia-
zole-alt-polyfluorene (PFBT, Compound 6) for the detection of
DNT and TNT (Chart 2). Quenching constants up to 6.9 3 103
M21 were observed for PFBO nanoparticles exposed to DNT
(0.17 mM DNT), and 1.2 3 104 M21 for PFBT nanoparticles
in the presence of TNT (0.14 mM TNT). These quenching effi-
ciencies clearly establish the general use of fluorescent nano-
particles in nitroaromatic-induced quenching schemes.
Many of the previously reported detection methods rely on
the quenching of fluorescent polymers in the solid state, for
example, in spin-cast thin films or in capillary tubes. The flu-
orescence quenching in such aggregated states is generally
higher than quenching observed in solution, due to the abil-
ity of the fluorescent polymers to have inter-chain exciton
migration in addition to intra-chain migration.12
Another way to ensure inter-chain communication between
polymer chains is to confine them in noncovalently linked
fluorescent nanoparticles, often referred to as “polymer
dots.”13 Such particles can be fabricated in a number of
ways, including: (a) the hydrophobic collapse of the polymer
chain when introduced into an aqueous solvent, also called
EXPERIMENTAL
Materials and Methods
All chemicals were purchased from Sigma-Aldrich Chemical
Company, with the exception of DNT (purchased from MP
Biomedicals, Inc.) and TNT (ampules of 1 mg/mL solution in
Additional Supporting Information may be found in the online version of this article.
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dissolved in THF was quickly added to 8 mL of deionized
water, while sonicating the water. After addition of the poly-
mer solution, nitrogen was bubbled through the solution for
1 h to remove the THF.
Fluorescence Quenching Experiments
In quenching experiments, two solutions were made: one
containing dilute particles in water with acetonitrile (Solu-
tion A), and one with dilute particles in water and DNT in
acetonitrile (Solution B).
CHART 1 Electron-deficient analytes under investigation.
acetonitrile were purchased from Ultra Scientific). 1H NMR
spectra were obtained using a Bruker 300 MHz spectrome-
ter. Ultraviolet-visible (UV-Vis) spectra were obtained using
an Agilent 8453 spectrometer equipped with a photodiode
array detector. Fluorescence spectra were obtained using a
Shimadzu RF-5301PC spectrophotofluorimeter and inte-
grated versus wavenumber. Dynamic light scattering (DLS)
data were acquired using a Malvern Zetasizer Instrument.
Gel permeation chromatography (GPC) data were acquired
using a Waters GPC liquid chromatography system with an
internal refractive index detector and two Waters Styragel
HR-5E columns. Retention times were calibrated against
polystyrene standards (Polymer Laboratories, Amherst, MA)
to produce number average molecular weight (Mn) and
weight average molecular weight (Mw) values. Thin films
were spun using a Laurell Technologies Spin Processor.
The water-acetonitrile solutions were allowed to equilibrate
for 1 h before analysis. Absorbance spectra of all particle sol-
utions were recorded, and the absorbance at the excitation
wavelength was checked to be below 0.1 absorbance units.
A total of 2.5 mL of the particle solution in water/acetoni-
trile (Solution A) was added to the cuvette, and the fluores-
cence was recorded. Three 500 mL aliquots of the DNT/
particle solution (Solution B) were added to the cuvette, and
the fluorescence was re-recorded after each addition. The
cuvette was then filled with 1.25 mL Solution A and 1.25 mL
Solution B. Three 500 mL aliquots of B were added, and fluo-
rescence recorded after each addition. Finally, the fluores-
cence of Solution B by itself was recorded.
All PFBO solutions were excited at 400 nm, and all PFBT sol-
utions were excited at 420 nm.
Polymer Synthesis
The synthesis of polymers PFBO and PFBT followed literature-
reported procedures.20,21 The synthetic schemes for each poly-
mer are shown below (Schemes 1 and 2).
The fluorescence emission spectra were integrated versus
wavenumber on the X-axis. Plotting the concentration of ana-
lyte (DNT or TNT) in molarity on the X-axis versus Io/I (ini-
tial integrated emission in the absence of the analyte divided
by the integrated emission in the presence of the analyte) on
the Y-axis yielded Stern-Volmer plots. The data were fit to
linear relationships to determine the Stern-Volmer quenching
constants. The R2 values for all linear fits were above 0.93.
GPC
The molecular weight and size distribution of the newly syn-
thesized polymers were determined by GPC, and the results
are shown below:
PFBO 5:
Thin Film Quenching
Mn 5 6.1976 3 103 g mol21
Mw 5 8.8652 3 103 g mol21
PDI 5 1.43
Thin films were spun-cast on a square glass cover slip
(dimensions: 1 cm 3 1 cm), by adding 0.2 mL of a 0.1 mg
mL21 solution of THF to the slip, then spinning it at 1000
rpm for 20 s. Thin film quenching was assessed by adding
10 mg of DNT (Compound 2) to a cuvette, and covering the
cuvette for 2 h. After 2 h, the polymer thin film was dropped
in, the cuvette was covered, and fluorescence intensity was
immediately recorded. The intensity was recorded at a single
wavelength in 1 s intervals for 20 min. (Polymer 5: Excita-
tion at 430 nm; emission monitoring at 500 nm; polymer 6:
excitation at 450 nm; emission monitoring at 533 nm).
PFBT 6:
Mn 5 6.3503 3 103 g mol21
Mw 5 1.7003 3 104 g mol21
PDI 5 2.68
Nanoparticle Fabrication
Nanoparticles were formed following literature-reported pro-
cedures.22 Briefly, 2 mL of a polymer solution (0.01 g L21
)
RESULTS AND DISCUSSION
The newly synthesized polymers were characterized by 1H
NMR, GPC, UV-Vis absorption spectroscopy, and fluores-
cence spectroscopy, and the results are summarized in
Table 1. The fluorescent nanoparticles were characterized
by absorption and fluorescence spectroscopy (Table 2),
and the formation of the particles was confirmed by DLS
measurements.
CHART 2 Structures of the fluorescent organic polymers.
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SCHEME 1 Synthesis of PFBO 5.
SCHEME 2 Synthesis of PFBT 6.
Remarkably, the fluorescent nanoparticles demonstrated
highly efficient quenching in the presence of DNT and TNT,
whereas the fluorescent polymers (from which the particles
were synthesized) displayed no fluorescence quenching. All
the quenching efficiencies were fitted to the Stern-Volmer
equation and displayed excellent linear fits. A summary of
the Stern-Volmer constants for each particle-analyte combi-
nation is shown in Table 3, and examples of the quenching
are shown in Figure 1.
a. Low limits of detection: The concentration of analytes
required to achieve full quenching are shown in Table 3
(0.17 and 0.14 mM for DNT and TNT, respectively). How-
ever, in all cases, noticeable quenching was observed at
substantially lower analyte concentrations. The addition of
as little as 29 mM of TNT to the nanoparticle solutions
resulted in quenching, as did the addition of 23 mM of
DNT. Although these detection limits are substantially
greater than those reported by Trogler and coworkers23
and Rochat and Swager24 for analogous thin-film systems,
the use of nanoparticles to achieve quenching in this case
provides operational advantages.
This quenching displayed the following key features:
TABLE 1 Properties of the PFBO and PFBT Polymers
TABLE 2 Properties of the PFBO and PFBT-Derived
Nanoparticles
PFBO 5
PFBT 6
kmax abs
kmax em.
Mw
413 nm
452 nm
PFBO particles (5)
PFBT particles (6)
507 nm
8.8652 3 103 g mol21
532 nm
1.7003 3 104 g mol21
kmax abs
411 nm
534 nm
24.4 nm
458 nm
536 nm
18.2 nm
kmax em.
PDI
1.43
2.68
Average size (DLS)
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TABLE 3 Stern-Volmer Constants Obtained for Each Analyte-
Fluorophore Combination (Maximum Analyte Concentrations
in Parentheses)
DNT
TNT
PFBT particles
PFBO particles
4169 M21
11974 M21
(0.17 mM DNT)
6867 M21
(0.14 mM TNT)
6737 M21
(0.17 mM DNT)
(0.14 mM TNT)
PFBT polymer
PFBO polymer
No quench
No quench
No quench
No quench
3.13% acetonitrile in water; 2.5 3 1024 mg mL21 nanoparticles; 420 nm
excitation wavelength for PFBT and 400 nm excitation wavelength for
PFBO.
b. Requirement of aggregation: Although polymer-derived
nanoparticles demonstrated efficient quenching, the poly-
mers dissolved in tetrahydrofuran solution displayed little
to no quenching under otherwise identical conditions.
Moreover, polymer-derived thin films, like polymer-derived
nanoparticles, were efficiently quenched by DNT, which
confirms that noncovalent aggregation of the polymer
chains is necessary for efficient energy transfer. These thin
films were fabricated from Polymers 5 and 6 (see experi-
mental section and ESI for details), and their fluorescence
emission was monitored in the presence of DNT vapor
FIGURE 2 Quenching of fluorescent thin films in the presence
of DNT vapor. Polymer 5 emission monitored at 500 nm; Poly-
mer 6 emission at 533 nm.
(Fig. 2). After 20 min, the fluorescence emission of Poly-
mer 5 in the film decreased to 10% of its maximum value,
and the emission of Polymer 6 decreased to 48% of its
initial value.
c. Significant solvent dependence: The quenching of Polymer
6-derived nanoparticles also displayed
a
substantial
FIGURE 1 Examples of fluorescence quenching of (a) PFBT (6) particles with DNT (b) PFBO (5) particles with DNT (c) PFBT (6) par-
ticles with TNT and (d) PFBO (5) particles with TNT. Stern-Volmer plots are included as insets for each example.
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caused the particle diameter to increase from 18 to 58 to 78
nm. This solvent-induced swelling caused the individual
polymer chains in the nanoparticles to separate, which lim-
ited inter-chain aggregation and thus the nitroaromatic-
induced quenching efficiencies.
The increased susceptibility of Polymer 6 compared with
Polymer 5 is likely a result of the long alkyl chains on Poly-
mer
5
protecting the particles from swelling and
acetonitrile-induced collapse. The lower density of alkyl
chains on Polymer 6 increases the particle susceptibility.
d. Fluorescence lifetime decrease: Preliminary lifetime data
indicate that the fluorescence lifetime of the nanoparticles
is significantly decreased in the presence of nitroaromatic
quenchers (lifetime of Polymer 5 nanoparticles decreased
from 1.8 to 1.7 ns; Polymer 6 nanoparticles decreased
from 0.73 (major species) to 0.47 ns (major species).
These decreases indicate that dynamic quenching is likely
occurring in this system.25
FIGURE 3 Dependence of Ksv values on the acetonitrile/water
ratio. Measurements were obtained for PFBT nanoparticles (2.5
3 1024 mg mL21) in the presence of DNT.
The generality of this fluorescent quenching was assessed
by measuring the response of the nanoparticles to nitro-
benzene (Compound 3) and cyclohexanone (Compound 4)
(Chart 1). No significant quenching was observed for either
nitrobenzene or cyclohexanone, for either nanoparticles or
polymers. This selectivity is in line with similar literature
reports,26,27 which indicate improved selectivity for DNT
and TNT over nitrobenzene and other small-molecule
analytes.
dependence on the percentage of acetonitrile in the solution,
with higher amounts of acetonitrile leading to less efficient
fluorescent quenching. An example of such dependence is
shown in Figure 3. With 6.25% acetonitrile in water, the
quenching proceeded with KSV 5 804 M21, whereas a 50%
water-acetonitrile mixture yielded a KSV of 27 M21
.
To explain this solvent dependence, DLS measurements of
the particles in mixed solvents were collected, and the
results are shown in Figure 4. For Polymer 5 nanoparticles,
the addition of acetonitrile increased in the average particle
diameter from 24 to 38 nm, but further addition of acetoni-
trile led to no noticeable change in the particle size (par-
ticles in 3.1% and 50% acetonitrile displayed the same
average diameter).
Finally, the complete insensitivity of the polymers to the
presence of such nitroaromatics was determined by exposing
THF solutions of PFBO and PFBT polymers to DNT concen-
trations 100 times higher than those used for significant
quenching of nanoparticles. Even at such high concentra-
tions, NO detectable quenching of the polymer fluorescence
was observed (Fig. 5), which again indicates the significant
necessity of nanoparticle formation for the detection of
electron-deficient analytes.
Polymer 6 nanoparticles, by contrast, showed a marked
increase in average diameter with increasing amounts of
acetonitrile. Switching from 0% to 3.1% to 50% acetonitrile
FIGURE 4 DLS measurements of (a) polymer 5 nanoparticles and (b) polymer 6 nanoparticles in the presence of increasing
amounts of acetonitrile.
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FIGURE 5 Insensitivity of (a) PFBO and (b) PFBT polymers to 17.2 mM concentrations of DNT.
10 R. Tu, B. Liu, Z. Wang, D. Gao, F. Wang, Q. Fang, Z. Zhang,
Anal. Chem. 2008, 80, 3458–3465.
CONCLUSIONS
In summary, highly efficient quenching of fluorescent nano-
particles has been demonstrated with both TNT and DNT as
analytes. The high sensitivity (as determined by Ksv values)
and selectivity (no response to cyclohexanone or nitroben-
zene), means that such quenching has potentially significant
applications in turn-off detection schemes. Efforts toward
developing such systems are currently in progress and
results will be reported in due course.
11 A. Rose, C. G. Lugmair, Y.-J. Miao, J. Kim, I. A. Levitsky,
V. E. Williams, T. M. Swager, Proc. SPIE Int. Soc. Opt. Eng.
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12 T. L. Andrew, T. M. Swager, J. Polym. Sci., Part B: Polym.
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13 D. Tuncel, H. V. Demir, Nanoscale 2010, 2, 484–494.
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ACKNOWLEDGMENTS
S. Cohen thanks the American Chemical Society Project SEED
for support of this work, and P. Marks thanks the University of
Rhode Island Foundation for support of this work.
17 C. Wu, T. Schneider, M. Zeigler, J. Yu, P. G. Schiro, D. R.
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