Impact of aggregation on fluorescence sensitivity of molecular probes towards nitroaromatic compounds

Article abstract of DOI:10.1039/c6tc00655h

We have designed and synthesized three tripods TIBP4, TIBP8 and TIBP12 possessing, respectively, OC₄H₉, OC₈H₁₇ and OC₁₂H₂₅ alkoxy chains on the biphenyl units and have investigated the effect of the chain length on their ease in aggregation and their efficiency in the detection of nitroaromatic compounds. Tripods TIBP8 and TIBP12 self-assemble to form densely populated nano-spheres (60-100 nm) in a water-DMSO (98:2) mixture, as shown by field-emission scanning electron microscopy, transmission electron microscopy and dynamic light scattering studies. TIBP4 which has shorter n-butyl alkyl chains does not undergo aggregation under these conditions. Tripods TIBP8 and TIBP12 which remain in a self-assembled state in water reveal amplified fluorescence quenching with PA, 2,4-DNP, TNT and Cl-DNB, and are associated with NAC induced disaggregation to well-dispersed particles. TIBP8 can detect as low as 10^-14 M PA and 2,4-DNP in solution and 2.29 × 10^-20 g cm^-2 (22.9 zg cm^-2) PA by contact mode and is nearly 6000-16000 times more selective towards PA and 2,4-DNP over TNT and Cl-DNB at 20% fluorescence quenching. However, the tripod TIBP12 can detect as low as 10^-14 M of each PA, 2,4-DNP, TNT and Cl-DNB and can find application as a general probe for these NACs. TIBP4 which remains in a molecularly dissolved state shows poor sensitivity (LOD 1 nM) towards NACs.

Full text of DOI:10.1039/c6tc00655h

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Impact of aggregation on fluorescence sensitivity of molecular
probes towards nitroaromatic compounds
Sana Sandhua, Rahul Kumara, Prabhpreet Singha*, Subodh Kumara*
Received 00th January 20xx,
Accepted 00th January 20xx
Abstract: We have designed and synthesized three tripods TIBP4, TIBP8 and TIBP12 possessing respectively OC4H9, OC8H17
and OC12H25 alkoxy chains on the biphenyl units and have investigated the effect of chain length on their ease in
aggregation and their efficiency in detection of nitroaromatic compounds. Tripods TIBP8 and TIBP12 self-assemble to form
densely populated nano-spheres (60-100 nm) in water - DMSO (98:2) mixture, as shown by field-emission scanning
electron microscopic, transmission electron microscopic and dynamic light scattering studies. TIBP4 which has shorter n-
butyl alkyl chains does not undergo aggregation under these conditions. Tripods TIBP8 and TIBP12 which remain in self-
assembled state in water, reveal amplified fluorescence quenching with PA, 2,4-DNP, TNT and Cl-DNB and are associated
with NAC induced dis-aggregation to well dispersed particles. TIBP8 can detect as low as 10-14 M PA and 2,4-DNP in
solution and 2.29 x 10-20 g/cm2 (22.9 zeptogm/cm2) PA by contact mode and is nearly 6000-16000 times more selective
towards PA and 2,4-DNP over TNT and Cl-DNB at 20% fluorescence quenching. However, tripod TIBP12 can detect as low
as 10-14 M each of PA, 2,4-DNP, TNT and Cl-DNB and can find application as general probe for these NACs. TIBP4 which
remains in moleculalry dissolved state shows poor sensitivity (LOD 1 nM) towards NACs.
DOI: 10.1039/x0xx00000x
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potential in amplifying4 the fluorescence signal on interaction
with NACs. The fluorescent organic aggregates also show
Introduction
Improving the sensitivity of a fluorescence probe1 for the
determination of a nitroaromatic compound is one of the major
challenge amongst chemists and other scientists working on
methods for detection of nitro aromatic explosives. In case of
molecularly dissolved probes2, the stoichiometric interaction of
the NAC with fluorescent probe results in modulation of only
its fluorescence but other molecules of the probe remain
unaffected. As a result, the change in fluorescence intensity is
directly proportional to the concentration of NAC and is
completed by equimolar or higher concentrations of the NAC.
In an alternative approach, if single molecule of NAC can
modulate the fluorescence of large number of fluorophores, the
sensitivity of the signal is drastically amplified. To achieve
such amplified fluorescence quenching or enhancement, the
conjugated polymers3 have shown tremendous potential but
difficulty in their synthesis and structural modifications and
poor stability have resulted in many demerits also.
exceptionally high sensitivity towards variety of analytes in
comparison to molecularly dissolved probes5. Such aggregates
are easily generated in situ by dissolving the concentrated
solution of an organic derivative into a nonꢀsolvent medium
that is a solvent in which the organic derivative has poor
solubility. This solvent is usually water and allows the
molecules of organic derivative to form well dispersed
aggregates in aqueous medium. Such amorphous aggregates
allow the single NAC species to remain in the vicinity of large
number of fluorophores and thus amplify the modulation of
fluorescence through electron, charge or resonance energy
transfer processes6.
We, in our earlier reports7 have shown that the
pꢀterphenyl
based molecular probes undergo selfꢀassembly to rod like
morphology and these aggregates reveal highly sensitive and
selective amplified fluorescence quenching with PA to detect
PA as low as 10ꢀ12 M in solution and 2.29 × 10ꢀ20 g/cm2 in
contact mode. The biphenyl based tripod8 remains in
molecularly dissolved state, encapsulates PA in its tripodal
cavity but could detect only 1 nM PA. This enhanced
sensitivity of probe7 in selfꢀassembled state could be ascribed to
increased probability of electron / charge transfer or RET from
electron–rich fluorophores to electronꢀdeficient NAC in the
aggregate particle in comparison to molecularly dissolved state.
Amongst other approaches, the aggregation of small organic
molecules into nanoꢀfibers has shown their tremendous
a.Department of Chemistry, Guru Nanak Dev University, Amritsar 143005, Punjab,
India
Electronic Supplementary Information (ESI) available: [Spectra of compounds and
some photophysical studies are available as supplementary information]. See
DOI: 10.1039/x0xx00000x
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In literature long alkyl chain bearing imidazolium TIBP8 and TIBP12 have been recorded as 5 mM solution in
derivatives are well known to undergo aggregation to form DMSOꢀd6
micelle, vesicle or even liquid crystalline state9. We envisaged
that alkoxyꢀbiphenyl based tripods, depending on the length of
alkyl chain, can undergo aggregation in water and such
aggregates would show enhanced sensitivity towards PA and
other NACs than that observed with probes existing in
molecularly dissolved state.
.
Herein, we have designed three tripods TIBP4, TIBP8
and TIBP12 which differ from each other only in the length of
the alkoxy chain (OC4H9, OC8H17 and OC12H25) present on the
biphenyl unit. We have found that tripods TIBP8 and TIBP12
in water (2% DMSO) selfꢀassemble to form densely populated
spherical aggregates as observed by FEꢀSEM, TEM and DLS
studies, whereas TIBP4 does not undergo aggregation and
remains in molecularly dissolved state. The aggregates of
TIBP8 and TIBP12 undergo amplified fluorescence quenching
with NACs and can detect as low as 10ꢀ14 M PA in solution and
2.29 x 10ꢀ20 g/cm2 PA by contact mode. The aggregates of
TIBP8 and TIBP12 are found to be more sensitive to NACs by
103 to 105 times in comparison to molecular solution of TIBP4
.
Figure 1: (a) Partial NOESY spectrum of TIBP4 showing cross peaks and signal
assignments; (b) 1H NMR signal of N-phenyl moiety in three tripods
UV-VIS and fluorescence studies of TIBP4, TIBP8 and TIBP12
in binary mixtures
To investigate the aggregation behavior of these tripods,
we performed their absorbance and fluorescence measurements
in DMSO and DMSOꢀH2O binary mixtures with different
fractions of water. UVꢀVis and fluorescence spectra were
recorded at 5 µM concentration. UVꢀVis spectrum of TIBP4 in
DMSO exhibits absorption maxima at 292 nm (ε = 77600). On
increasing the fraction of water in DMSO, the absorption
maxima of TIBP4 is gradually blueꢀshifted to 282 nm. The
extinction coefficient (ε0 value of the absorption band at 292
nm also gradually decreases and attains minimum value (ε =
53400) in 95% aqueous medium (Figure 2a, SIꢀ1). For all
fluorescence studies, 290 nm was used as excitation
wavelength. The DMSO solution of TIBP4 reveals emission
maxima at 434 nm, which on increasing the amount of water is
gradually redꢀshifted to 454 nm (Φ = 0.05)11 and is associated
with gradual decrease in fluorescence intensity up to 95% water
(Figure 2, SIꢀ1).
UVꢀVis spectrum of TIBP8 in DMSO exhibits absorption
maxima at 292 nm (ε = 73400). The solutions of TIBP8 with
increasing fraction of water result in gradual blueꢀshift of the
maxima to 280 nm (ε = 38200) up to 60% water fraction. On
further increasing water fraction up to 98%, the absorption
maxima and absorption intensity do not show any measurable
change. On excitation at 290 nm, DMSO solution of TIBP8
exhibits emission maximum at 430 nm. On increasing water
fraction up to 80%, the emission maxima redꢀshifts to 448 nm
along with gradual decrease in its fluorescence intensity. On
Scheme 1: Synthesis of tripods TIBP4, TIBP8 and TIBP12
Synthesis of tripods TIBP4, TIBP8 and TIBP12
The compounds 2a-2c were synthesized by the procedure
reported in literature9b (for details see SI) The reaction of 2a
with 1,3,5ꢀtris(bromomethyl)ꢀ2,4,6ꢀtriethylbenzene
(3) in
acetonitrile at reflux temperature gave white solid in 89 %
o
yield, m.p. 240 C. The presence of Cꢀ2H at δ 9.72, H4ꢀIm
at δ 8.43, H5ꢀIm at δ 8.04 (confirmed by NOESY spectrum
of TIBP4, Figure 1a) along with NꢀCH2 singlet at δ 5.64
confirms the formation of TIBP4. Similarly, 2b on refluxing
with tribromide (
yield, m.p. 248 C and the reaction of 2c with tribromide (
3
) in acetonitrile gave TIBP8 in 86.7 %
o
3
)
gave TIBP12 in 73.6 % yield, m.p. 251ꢀ254 oC. The 1H
NMR spectra of these tripods show that Hc and Hd protons in
TIBP12 appear as AB quartet with coupling constant J1 = 8.5
Hz and ∆ = 31 Hz (Figure 1). On moving to TIBP8 and
TIBP4, the ∆ value is decreased to 21.5 and 12 Hz,
respectively. Presumably, the increase in chain length results in
aggregation of molecules and increases the compactness10 of
the arms in the tripod TIBP12
. ,
1H NMR spectra of TIBP4
2 | J. Name., 2012, 00, 1-3
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further increasing the fraction of water to 90ꢀ98%, the emission PA, show the presence of well dispersed particles with sizes
maxima is blue shifted to 410 nm (Φ = 0.06)11 with increase in between 20ꢀ60 nm (Figure 3e) which is in agreement with DLS
its intensity (Figure 2b, Figure SIꢀ2). This blue shift of the studies (Figure 3d). In the presence of 0.1 equiv. of PA the
emission maxima from 448 to 410 nm is ascribed to the aggregates are not observed in DLS and FEꢀSEM studies. The
aggregation of TIBP8 molecules in DMSOꢀH2O mixtures with formation of densely populated aggregates of TIBP8 and their
> 90% water fraction.
disaggregation to well dispersed and smaller aggregates is also
confirmed by TEM studies (Figure 3c, 3f). There are only
couple of reports7,12 where the aggregates of molecular probes
undergo disꢀaggregation in the presence of nitro aromatic
explosives.
Figure 2: The comparison of change in intensity in UV-Vis and Fluorescence spectra of
TIBP4, TIBP8, and TIBP12 in binary mixtures with different fractions of water in DMSO
Tripod TIBP12 in its UVꢀVis spectrum elicits the blue shift
of the absorption maxima by 12 nm from 290 to 278 nm in
solution with 40% water fraction and does not undergo further
change in its maxima at higher fractions of water. The
recording of fluorescence spectra of TIBP12 in binary mixtures
reveals blueꢀshift of 16 nm from 428 to 412 nm (Φ = 0.07)
associated with significant decrease in the fluorescence
intensity in 40% water fraction and points to its aggregation in
solutions with > 40 % water fraction (Figure 2, SIꢀ3).
These results clearly show that TIBP4 undergoes linear
decrease in its absorbance for all fractions of water (Figure 2),
whereas this linear decrease in absorbance is stopped at 80%
and 40% water fractions in case of TIBP8 and TIBP12,
respectively. Similarly, in case of fluorescence studies, the
fluorescence intensity of TIBP4 decreases linearly up to 90%
water fraction, but TIBP8 and TIBP12 undergo decrease in
fluorescence up to 80% and 40% water fractions, respectively.
Therefore, TIBP4 remains in molecularly dissolved state in
solutions with all water fractions, TIBP8 achieves aggregate
state in solutions with > 80% water fraction and TIBP12 starts
aggregating in solutions with > 40% water fraction.
Figure 3: DLS, SEM and TEM images of (a-c) TIBP8 (10-6 M); (d-f) TIBP8 + PA (10-7 M);
(water + 2 % DMSO)
Similarly, DLS studies on solution of TIBP12 (H2O:
DMSO; 98:2)show the formation of aggregates with average
size 60 nm, which in the presence of PA undergo
disaggregation to average 30 nm size particles (Figure 4a, 4d).
FEꢀSEM and TEM images of the film obtained from the
solution of TIBP12 show the formation of densely populated
aggregates (Figure 4b, 4c). However, thin film of the solution
of TIBP12 containing 0.1 equivalent of PA reveals the
formation of well dispersed aggregates (Figure 4e, 4f).
Therefore, TIBP8 and TIBP12 exhibit similar morphological
changes in H2OꢀDMSO (98:2) solution to form spherical
aggregates which in the presence of 0.1 µM PA (0.1 equivalent)
undergo disaggregation to form well dispersed particles.
Aggregation – disaggregation studies by DLS, FE-SEM and
TEM techniques
We further investigated the difference in aggregation
behaviour of TIBP8 and TIBP12 under these conditions using
dynamic light scattering (DLS) studies of the solutions and FEꢀ
SEM and TEM studies of the thinꢀfilms of TIBP8 and TIBP12
solutions with and without PA.
DLS measurements of TIBP8 (1 µM, H2OꢀDMSO; 98;2)
solution show the formation of aggregates with sizes ranging
between 45ꢀ105 nm (average size 60 nm) with PDI 0.364
(Figure 3a). FEꢀSEM and TEM images of thin film obtained
from solution of TIBP8 (1 µM) show the formation of
spherical aggregates with ~ 60 nm size (Figure 3b, 3c). Thin
films obtained from the solution of TIBP8 containing 0.1 µM
Figure 4: DLS, SEM and TEM images of (a-c) TIBP12 (10-6 M); (d-f) TIBP12 + PA (10-7 M);
(water + 2 % DMSO)
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UV-Vis and fluorescence studies of TIBP4, TIBP8 and DNP, TNT and ClꢀDNB, we performed the fluorescence
TIBP12 with NACs titrations. The FI of TIBP4 (5 µM, H2OꢀDMSO, 98:2) at 454
In the present study, we have chosen H2O – DMSO (98:2) as nm (λex 290 nm) gradually decreases on addition of aliquots of
solvent to investigate the interactions of tripods with NACs in NACs and achieves plateau on addition of 30 µM PA, 50 µM
the solution. In this solvent medium, TIBP4 remains in 2,4ꢀDNP, 70 µM TNT and 200 µM ClꢀDNB (Figures 6, SIꢀ4).
molecularly dissolved state, where as TIBP8 and TIBP12 At lower concentrations of NACs, the fluorescence quenching
achieve aggregate state. The aromatic compounds investigated follows SternꢀVolmer equation, Io/I = 1 + KSV [Q]. The Sternꢀ
are phenol, 2ꢀ nitrophenol (2ꢀNP), 4ꢀnitrophenol (4ꢀNP), 2,4ꢀ Volmer constant for PA (6.25 x 105 Mꢀ1) is larger in
dinitrophenol (2,4ꢀDNP), picric acid (PA), 4ꢀhydroxybiphenyl comparison to KSV values for 2,4ꢀDNP (5.03 x 104 Mꢀ1), TNT
(4ꢀOHBP); 2ꢀnitrotoluene (2ꢀNT), 2,4ꢀdinitrotoluene (2,4ꢀ (1.84 x 104 Mꢀ1) and ClꢀDNB (1.35 x 104 Mꢀ1). The lowest
DNT), dinitrobenzene (DNB), 2ꢀchloronitrobenzene (2ꢀClNB), detection limits are found to be 2.5 x 10ꢀ8 M for PA, 5 x 10ꢀ7
M
trinitrotoluene (TNT); 3ꢀchloronitrobenzene (3ꢀClNB), 4ꢀ for 2,4ꢀDNP, 1 x 10ꢀ6 M for TNT and ClꢀDNB. TIBP4 shows
chloroꢀnitrobenzene (4ꢀClNB), chlorodinitrobenzene (Clꢀ fluorescence quenching efficiency in the order PA > 2,4ꢀDNP >
DNB), 2ꢀnitroaniline (2ꢀNA) and 2,4ꢀdinitroaniline (2,4ꢀDNA). TNT > ClꢀDNB.
The UVꢀVis absorption spectra of all three tripods show
negligible change on addition of 1 equivalent of these aromatic
compounds. Therefore, these tripods have only weak
interactions with aromatic compounds, in the ground state.
Figure 5: The fluorescence quenching efficiency of the solutions of TIBP4, TIBP8 and
TIBP12 (5 µM) in water-DMSO (98:2) on addition of NACs 0.5 µM
The solutions of TIBP8 (5 µM) undergo fluorescence
quenching by 92, 89, 73 and 51 percent, respectively with 0.5
µM (0.1 equiv.) each of PA, 2,4ꢀDNP, TNT and ClꢀDNB
(Figure 5). Similarly, the addition of 0.1 equivalent of PA, 2,4ꢀ
Figure 6: Fluorescence spectra of TIBP4 , on gradual addition of (a) picric acid; (b) TNT
DNP, TNT and ClꢀDNB, to the solution of TIBP12 elicits
efficient fluorescence quenching of 94, 93, 73 and 66 %,
respectively. The other NACs have insignificant effect on the
(c) Plot of Io/I vs [NAC] of fluorescence titration of TIBP4 with PA, 2,4-DNP, TNT and Cl-
DNB; (d) Plot of FI (I/Io) vs [PA] / [TNT] at concentrations > 1 µM showing fluorescence
enhancement at 335 nm
FI of TIBP8 and TIBP12 solutions. Tripod TIBP4 exhibits
only 1ꢀ10% fluorescence quenching on addition of 0.5 µM of
Characteristically, TIBP4 forms fluorescent complexes with
these NACs. However, TIBP4 reveals 62, 45, 25 and 14.5 %
maxima centered at 340 nm and 332 nm at > 1 equivalent
fluorescence quenching, respectively with 10 µM (2 equiv.) of
concentrations of PA and TNT. These complexes exhibit linear
PA, 2,4ꢀDNP, TNT and ClꢀDNB. The other NAC’s have
change in its fluorescence intensity at ~ 335 nm between 2ꢀ30
insignificant effect on the FI of TIBP4 solution.
µM of PA and 10 µM
̶ 100 µM of TNT with maximum
Therefore, TIBP8 and TIBP12 derivatives which exist in
aggregate state undergo amplified fluorescence quenching in
the presence of < 0.1 equiv. of the above discussed four NACs,
but TIBP4 which remains in molecularly dissolved state
exhibits smaller quenching of fluorescence even at 2 equiv. of
these NACs. This provides the proof of the concept that the
probes which attain aggregate state exhibit remarkably
enhanced sensitivity towards NACs than those probes which
remain in molecularly dissolved state.
fluorescence intensity enhancement by 170 and 280%,
respectively (Figure 6d). The formation of fluorescent
complexes by NACs is relatively unꢀknown phenomenon.
Tripods TIBP8 and TIBP12, which exists in aggregate state
in H2O (2% DMSO) reveal more efficient fluorescence
quenching with PA, 2,4ꢀDNP, TNT and ClꢀDNB at 410 nm
(Figure 7, SIꢀ5, SIꢀ6). The plots of I/Io vs [NAC] show nonꢀ
linear change and do not follow SternꢀVolmer equation Io/I = 1
+ KSV [Q]. Therefore, KSV values of TIBP8 and TIBP12 for
the detection of PA, 2,4ꢀDNP, TNT and ClꢀDNB have been
calculated by exponential equation I/Io = AeKsv[Q] + B using
Further to find out the limits of detection and SternꢀVolmer
constant values of these tripod derivatives towards PA, 2,4ꢀ
4 | J. Name., 2012, 00, 1-3
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origin software (Figure 7). KSV values for TIBP8 and TIBP12 However, this upꢀfield shift of PA protons is significantly lower
have been found in the order 1011 to 1010 for PA, 2,4ꢀDNP and than ~ 0.8 ppm upꢀfield shift in case of earlier reported
TNT which are higher by the order of 3ꢀ5 in comparison to biphenyl8 based tripod. 1H NMR spectra of TIBP8 and TIBP12
those determined for TIBP4 (Table SIꢀ1). Such high Ksv values in DMSOꢀd6-water (1:1) show broad peaks due to the
are well known for the nonꢀconjugated polymers or aggregation of these molecules at high concentrations. The
aggregates13. For determining the selectivity of TIBP8 towards addition of PA to these solutions resulted in precipitation.
these NACs, the concentrations of these NACs at 20%
fluorescence quenching has been determined (Table 1), which spectrum of fluorophore points to the presence of RET process
is found to be 5x10ꢀ12 M (PA), 5x10ꢀ11 M (2,4ꢀDNP), 3x10ꢀ8
in their interaction. Fluorescence spectra of TIBP8 and
The overlap of UVꢀVis spectra of NAC with fluorescence
M
(TNT) and 8x10ꢀ8 M (ClꢀDNB). Under these conditions, TIBP8 TIBP12 show nearly 50% overlap with absorption spectra of
is selective towards PA by 10, 6000, 16000 times in PA and 2,4ꢀDNP but poor spectral overlap with TNT and Clꢀ
comparison to 2,4ꢀDNP, TNT and ClꢀDNB. The detection DNB molecules (Figure SIꢀ8). Therefore, TIBP8 and TIBP12
limits (3σ/m)14 for PA, 2,4ꢀDNP, TNT and ClꢀDNB are might follow, at least partly, RET process for efficient
calculated to be 1 x 10ꢀ14 M, 1 x 10ꢀ14 M, 5 x10ꢀ14 M and 5 x10ꢀ fluorescence quenching with these NACs. The proximity of
12 M, respectively.
fluorophores in the aggregate state with NAC molecules and
fast electron transfer from ground state of fluorophore to the
NAC further increases the efficiency of fluorescence
quenching. The fluorescence spectrum of TIBP4 shows poor
overlap with the absorption spectrum of PA and efficiency of
RET process is decreased. The lesser proximity of the
fluorophores with an NAC molecule due to its molecularly
dissolved state also attributes to its lower sensitivity towards
NACs.
Contact mode method for detection of PA
Paper strips coated with TIBP8 reveal observable
fluorescence change on addition of 10 µl solutions of 10ꢀ17 M ꢀ
10ꢀ9 M PA and at higher concentrations only black area is
observed (Figure 8). 10 µl of 10ꢀ17 M solution of PA is
equivalent to 2.29 x 10ꢀ20 g PA. The steadyꢀstate fluorescence
spectra of these paper strips were recorded. The plot of Io/I vs
log [NAC] shows the linear increase in fluorescence quenching
efficiency on moving from 10ꢀ17ꢀ10ꢀ9 M PA (Figure 9).
However, paper strips coated with TIBP4 show measurable
change with PA in concentration range 10ꢀ13 to 10ꢀ7 M both
under UV light and steady state fluorescence measurements. In
case of TIBP12 coated paper strips, on addition of 10 µl
Figure 7: Plot of I/Io vs log [NAC] M of fluorescence titration against PA, 2,4-DNP, TNT
and Cl-DNB (a) TIBP8 (b) TIBP12
Table 1: [NAC] at which 20 % of fluorescence quenching of probe
Probe
PA
1x10ꢀ6
5x10ꢀ12
10ꢀ12
2,4ꢀDNP
TNT
ClꢀDNB
18 x10ꢀ6
M
8x10ꢀ8
M
TIBP4
TIBP8
TIBP12
M
3 x10ꢀ6
5x10ꢀ11
3x10ꢀ12
M
8 x10ꢀ6
3x10ꢀ8
5x10ꢀ13
M
M
M
M
M
M
M
2x10ꢀ12
M
Tripod TIBP12 undergoes > 50% fluorescence quenching
with 0.1 equivalent of PA, 2,4ꢀDNP, TNT and ClꢀDNB which
implies 50% fluorescence of TIBP12 molecules is quenched by
10% PA molecules. TIBP12 elicits poor selectivity towards
these NACs. In fact, TIBP12 can determine PA, 2,4ꢀDNP, TNT
and ClꢀDNB with equal ease. In comparison with TIBP8, the
sensitivity of TIBP12 towards TNT and ClꢀDNB has
remarkably improved.
Mechanism of interaction of TIBP4, TIBP8 and TIBP12 with
NACs
1
In order to rationalize the interaction of TIBP4 with PA, H
NMR spectrum of TIBP4 before and after addition of PA was
recorded in DMSOꢀd6-water (7:3) at 5 mM concentration. In 1H
NMR spectrum of the solution of 1:1 mixture of TIBP4 and
PA, the upꢀfield shift of 2H singlet of PA from δ 8.62 (in H
NMR spectrum of PA) to 8.39 (ꢁ δ = 0.23) points to the
Figure 8: Photographs of fluorescence quenching (under 365 nm UV light) of tripod
coated paper strips and 10 µl of different concentrations of PA.(A-E) TIBP4 (A) Paper
strip with a drop of water; (B) 10-13 M PA; (C) 10-11 M PA; (D) 10-9 M PA; (E) 10-7 M PA;
1
(F-J) TIBP8 (F) Paper strip with a drop of water; (G) 10-17 M PA; (H) 10-15 M PA; (I) 10-13
M
PA; (J) 10-11 M PA. (K-O) TIBP12 (K) Paper strip with a drop of water; (L) 10-17 M PA; (M)
10-15 M PA; (N) 10-13M PA; (O) 10-11 M PA
encapsulation of PA in the cavity of TIBP4 (Figure SIꢀ7).
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solution of water of PA solution, it took > 4 minutes for the drop to
be absorbed by the paper strip and exhibits some significant change
in fluorescence intensity only on addition of 10ꢀ13 M or higher
concentrations of PA. Probably, TIBP12 coated paper strips are too
hydrophobic to allow the aqueous droplet to absorb on it.
Synthesis of tripods TIBP4/ TIBP8/ TIBP12: The solution of
1,3,5ꢀtris(bromomethyl)ꢀ2,4,6ꢀtriethylbenzene (0.18 mmol,
79.38 mg) and 2a (0.54 mmol, 218 mg) in acetonitrile was
refluxed for 40 h. The solid separated during the course of the
reaction was filtered to get TIBP4 as white solid. The solid was
found to be pure. Similar reactions of 2b and 2c gave TIBP8
and TIBP12, respectively.
TIBP4: Yield 89 %; m.p. 240oC. 1H NMR (500 MHz, DMSOꢀ
d
6) : δ 0.96 (t, 9H,
1.43ꢀ1.50 (m, 6H, 3 x CH2), 1.73 (quintet, 6H,
CH2), 2.82 (d, 6H, = 6.5 Hz, 3 x CH2), 4.01 (t, 6H,
3 x CH2), 5.64 (bs, 6H, 3 x CH2), 6.98 (d, 6H, = 9 Hz, ArH),
7.62 (d, 6H, = 8.5 Hz, ArH), 7.86 (q, 12H, ArH), 8.04 (s, 3H,
ImꢀH), 8.43 (s, 3H, ImꢀH), 9.72 (s, 3H, ImꢀC2H); 13C NMR
(500 MHz, DMSOꢀ 6): δ 14.2, 15.8, 19.2, 31.2, 67.8, 115.5,
121.9, 123.2, 123.9, 127.7, 128.4, 128.8, 130.8, 133.6, 135.3,
J
= 7.5 Hz, 3 x CH3), 0.99 (bs, 9H, 3 x CH3),
= 6.5 Hz, 3 x
= 6.5 Hz,
J
J
J
J
J
d
Figure 9: (a) Front surface steady-state fluorescence quenching of TIBP8 with PA. 10 μl
of 10-17-10-9 M concentrations of PA added. (b) Plot of fluorescence intensity of TIBP8
vs log [PA] M
141.6, 148.4, 159.4. HRMS-ESI
:
calculated for
C72H81Br3N6O3, m/z 578.2777 [Mꢀ2Brꢀ]2+, 354.5404 [Mꢀ3Brꢀ
]3+; found 578.2758 [Mꢀ2Brꢀ]2+, 354.2116 [Mꢀ3Brꢀ]3+.
TIBP8: Yield 86.7 %, m.p. 248oC; 1H NMR (500 MHz,
For further exploring the applicability of TIBP8 and TIBP12 for
detection of PA vapour, thin films of TIBP8 and TIBP12 (20 µL, 20
µM) were fabricated through drop cast method on preꢀcleaned glass
plate surface and were allowed to dry in incubator at 25oC. The
fluorescence intensity of the thinꢀfilms decreased gradually on
exposure to PA vapour at regular intervals of time. In case of film of
TIBP8, 18 % fluorescence quenching was observed on exposure of
DMSOꢀd6
CH3), 1.27ꢀ1.32 (m, 30H, CH2), 1.40ꢀ1.46 (m, 6H, 3 x CH2),
1.74 (quintet, 6H, = 6.5 Hz, 3x CH2), 2.81 (bs, 6H, 3 x CH2),
3.99 (t, 6H, = 6.5 Hz, 3 x CH2), 5.64 (bs, 6H, 3 x CH2), 6.96
(d, 6H, = 8.5 Hz, ArH), 7.60 (d, 6H, = 9 Hz, ArH), 7.83 (d,
6H, = 9 Hz, ArH), 7.87 (d, 6H, = 8.5 Hz, ArH), 8.07 (s, 3H,
) : δ 0.87 (t, 9H, J = 7 Hz, 3 x CH3), 1.00 (bs, 9H, 3 x
J
J
J
J
J
J
30 seconds to saturated vapour pressure of PA, whereas TIBP12 ImꢀH), 8.43 (s, 3H, ImꢀH), 9.68 (s, 3H, ImꢀC2H); 13C NMR
underwent only 4.7 fluorescence quenching under similar (500 MHz, DMSOꢀ
6): δ 14.8, 16.0, 23.0, 26.4, 29.6, 29.6,
%
d
conditions. The overall fluorescence quenching efficiency was found 29.7, 32.1, 48.3, 68.5, 115.9, 122.3, 123.7, 124.5, 128.1, 128.8,
to be 55 % in case of TIBP8 and 40 % in case of TIBP12. In both 129.2, 131.2, 134.2, 135.8, 142.0, 159.8. HRMS-ESI :
calculated for C83H103Br3N6O3, m/z 655.3637 [Mꢀ2Brꢀ]2+,
the cases, plateau was achieved after 300 secs of exposure (Figure
410.6030 [Mꢀ3Brꢀ]3+; found 654.8922 [Mꢀ2Brꢀ]2+, 410.6436
SIꢀ9). These results are in consonance with higher sensitivity of
TBP12 over TIBP8 as observed above.
[Mꢀ3Brꢀ]3+.
TIBP12: Yield 73.6 %, m.p. 251ꢀ254oC; 1H NMR (500 MHz,
CONCLUSIONS
DMSOꢀd6
60H, CH2), 1.40ꢀ1.44 (m, 6H, 3 x CH2), 1.73 (t, 9H,
3 x CH3), 3.97 (t, 6H, = 6.5 Hz, CH2), 5.65 (s, 6H, 3 x CH2),
)
:
δ 0.84 (t, 9H,
J
= 7 Hz, 3 x CH3), 1.24ꢀ1.33 (m,
J
= 7.5 Hz,
In summary, tripods TIBP8 and TIBP12 selfꢀassemble in
aqueous medium and exhibit amplified fluorescence quenching
with PA, 2,4ꢀDNP, TNT and ClꢀDNB. TIBP4 under these
conditions remains in molecularly dissolved state and shows
poor sensitivity towards PA and other NACs. These results
unambiguously highlight the significance of selfꢀassembled
materials in developing highly sensitive probes for NACs.
TIBP8 exhibits 20% fluorescence quenching with PA at 6000ꢀ
16000 times lower concentration than that observed with TNT
and ClꢀDNB. TIBP8 can detect as low as 10ꢀ14 M PA in
solution and 2.29 x 10ꢀ20 g/cm2 PA by contact mode. However,
TIBP12 is equally sensitive towards all four NACs and can
detect as low as 10ꢀ14 M each of PA, 2,4ꢀDNP, TNT and Clꢀ
DNB and can find application as general sensor for NACs. The
increased hydrophobic character of TIBP12 restricts its
applications in contact mode.
J
6.92 (d, 6H,
7.78 (d, 6H,
J
J
= 8.5 Hz, ArH), 7.56 (d, 6H,
= 8.5 Hz, ArH), 7.89 (d, 6H,
J
J
= 8.5 Hz, ArH),
= 8.5 Hz, ArH),
8.13 (s, 3H, ImꢀH), 8.42 (s, 3H, ImꢀH), 9.69 (s, 3H, ImꢀC2H);
13C NMR (500 MHz, DMSOꢀ
6) : δ 14.4, 15.9, 22.6, 26.0,
d
29.2, 29.2, 29.4, 29.5, 29.5, 31.8, 47.9, 68.1, 115.4, 121.8,
123.3, 124.2, 127.5, 128.3, 128.8, 130.8, 133.6, 135.2, 141.4,
148.5, 159.4; HRMS-ESI : calculated for C96H131Br3N6O3, m/z
747.9750 [Mꢀ2Brꢀ]2+, 472.3438 [Mꢀ3Brꢀ]3+; found 747.9715
[Mꢀ2Brꢀ]2+, 472.3411 [Mꢀ3Brꢀ]3+.
UV-Vis and Fluorescence Titrations
Stock solutions of TIBP4, TIBP8 and TIBP12 (1 mM) were
prepared in DMSO. For experiments with TIBP4, TIBP8 and
TIBP12, we have taken 3 ml of the solution that contains 15 µL
stock solution in DMSO, 45 µL of DMSO and 2.94 ml of water
in cuvette. Typically, aliquots of freshly prepared standard
solutions (10ꢀ1 M) of NACs in DMSO were used to record UVꢀ
Vis and fluorescence spectra.
Experimental Section
For general experimental information and instrumentation see
ref 7 and SI.
6 | J. Name., 2012, 00, 1-3
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Journal of Materials Chemistry C
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DOI: 10.1039/C6TC00655H
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D. Ding, S. Zhang, Y. Liu, B. Liu, J. Z. Sun and B. Z. Tang, J.
Mater. Chem., 2012, 22, 15128-15135; (c) J. Wang, X. Xu, L.
Shi, and L. Li, ACS Appl. Mater. Interfaces., 2013, 5, 3392-
3400; (d) A. Singh, T. Raj, T. Aree and N. Singh, Inorg. Chem.,
2013, 52, 13830–13832; (e) K. Tayade, A. Kaurb, S. Tetgurea,
G. K. Chaitanya, N. Singh and A. Kuwar, Analytica Chimica
Acta., 2014, 852, 196-202.
X. Sun, Y. Wang and Y. Lei, Chem. Soc. Rev., 2015, 44, 8019-
8061.
S. Sandhu, R. Kumar, P. Singh, A. Mahajan, M. Kaur and S.
Detection limit14
The detection limit was calculated based on the fluorescence
titration. To determine the S/N ratio, the emission intensity of
tripod (5 ꢂM) was measured 5 times and the standard deviation
of blank measurements was determined. The detection limit
was then calculated with the equation:
6
7
8
9
Detection limit = 3σbi/m
Where, σbi is the standard deviation of blank measurements; m
is the slope between intensity versus sample concentration. The
detection limit was measured to be 1 nM at S/N = 3.
Kumar, ACS Appl. Mater. Interfaces, 2015,
R. Kumar, S. Sandhu, P. Singh, G. Hundal, M. S. Hundal and S.
Kumar, Asian J. Org. Chem., 2014, , 805–813.
(a) A. A. Fernandez, L. T. Haan and Paul H. J. Kouwer, J.
Mater. Chem. A, 2013, , 354-357; (b) P. H. J. Kouwer and T.
7, 10491–10500.
3
1
M. Swager, J. Am. Chem. Soc., 2007, 129, 14042–14052; (c) J.
Cai and J. L. Sessler, Chem. Soc. Rev., 2014, 43, 6198-6213;
(d) B. Xin and J. Hao, Chem. Soc. Rev., 2014, 43, 7171-7187;
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Acknowledgements
This work was supported by Department of Science and
Technology, New Delhi (SR/S1/OCꢀ75/2012). We thank UGC
for UPE programme to the university and CAS status to the
department and DST for FIST programme. SS and RK
acknowledge DST and UGC for fellowships.
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Journal of Materials Chemistry C
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DOI: 10.1039/C6TC00655H
Impact of aggregation on fluorescence sensitivity of molecular probes
towards nitroaromatic compounds
Sana Sandhu, Rahul Kumar, Prabhpreet Singh, Subodh Kumar
Aggregation of the molecular probe increases its fluorescence sensitivity towards
nitroaromatic explosives by the order of 103 to 105 times.