Photoinduced electron transfer (PET) based Zn2+ fluorescent probe: Transformation of turn-on sensors into ratiometric ones with dual emission in acetonitrile

Article abstract of DOI:10.1021/jp209061f

Ratiometric sensors for the detection of metal ions have gained increasing attention due to its self-calibration tendency for the environmental effects. In this context, we have synthesized and characterized a dual emitting ratiometric Zn²⁺ pro

Full text of DOI:10.1021/jp209061f

ARTICLE
pubs.acs.org/JPCA
Photoinduced Electron Transfer (PET) Based Zn2+ Fluorescent Probe:
Transformation of Turn-On Sensors into Ratiometric Ones with Dual
Emission in Acetonitrile
P. Ashokkumar, V. T. Ramakrishnan, and P. Ramamurthy*
National Centre for Ultrafast Processes, University of Madras, Taramani Campus, Chennai-600 113, India
S Supporting Information
b
ABSTRACT: Ratiometric sensors for the detection of metal ions
have gained increasing attention due to its self-calibration
tendency for the environmental effects. In this context, we have
synthesized and characterized a dual emitting ratiometric Zn2+
probe (1) having acridinedione as a fluorophore and N,N-bis(2-
pyridylmethyl)amine (BPA) as a receptor unit. Existence of two
different conformation of the molecule with photoinduced
electron transfer (PET) from amine moiety to the acridinedione
fluorophore leads to dual emission, namely locally excited
(425 nm) and anomalous charge transfer emission (560 nm) in aprotic solvents. In the presence of one equivalent of Zn2+, a
15-fold fluorescence enhancement in the locally excited state together with the quenching of charge transfer emission is observed.
The intensity changes at the two emission peaks allow a ratiometric detection of Zn2+ under PET signaling mechanism. The
utilization of PET process for the ratiometric fluorescence change will further signify the importance of PET mechanism in sensing
action. Addition of Zn2+ to 1 in acetonitrile/water mixtures shows a single emission peak with fluorescence enhancement.
the synthetic route. BPA can effectively quench the emission of
the fluorophore via PET; upon addition of Zn2+ ions, fluores-
cence enhancement is observed owing to the formation of BPA-
Zn2+ complex, which can efficiently block the PET process.
Because of the unchanged absorption and emission maxima,
ratiometric detection under PET signaling mechanism is not
achieved so far. Only a very few PET based sensors show
emission spectral shift due to the repulsive electrostatic interac-
tion between the excited-state dipole of the sensor molecule and
metal ion.13 Ratiometric detection by utilizing both PET and
ICT mechanisms is achieved.14 Similarly, a ratiometric fluores-
cence output by exploiting PET and FRET is also obtained;9a
however, a ratiometric change solely due to the operation of PET
process is not yet reported.
Unfortunately, most of the fluorescent probes reported for
Zn2+ are based on single emission intensity changes, which tend
to be affected by a variety of factors, such as instrumental
efficiency, probe molecule concentration, its stability under
photoillumination, and microenvironment around probe mole-
cule. These limitations can be circumvented by the use of
ratiometric probes, which are less prone for such problems.
Ratiometric fluorescent probes involve changes in the ratio of the
two emission peaks upon the addition of an analyte, which are
beneficial for increasing the dynamic range and providing built-in
I. INTRODUCTION
Among the trace elements involved in human metabolism,
Zn2+ is the most ubiquitous, and its physiological importance has
long been recognized.1 Generally, the total concentration of Zn2+
in different types of cells is quite varied, ranging from the nM
level to ∼0.3 mM.2 Thus, the highly selective, sensitive and
reversible detection of Zn2+ ions is of vital importance. In this
context, the development of fluorescence based sensors attracted
more attention due to its advantages such as high sensitivity of
the detection down to single molecule, local observation by
fluorescence imaging with subnanometer spatial resolution and
remote sensing by using optical fibers with a molecular sensor
immobilized at the tip.3 Until now, a variety of fluorescent Zn2+
sensors4ꢀ9 have been documented, based on photoinduced elec-
tron transfer (PET),5 internal charge transfer (ICT),6 excimer/
exciplex formation,7 chelation-enhanced fluorescence (CHEF),8
and fluorescence resonance energy transfer (FRET)9 mechan-
isms. To date, most of the available Zn2+ fluorescent sensors rely
on the PET mechanism, which exhibits an increase in the
emission intensity, but show little or no spectral shift in both
absorption and emission spectra.10 Lippard and co-workers
reported an elegant example of fluorescein-based Zn2+ chemo-
sensors (Zinpyr-1) containing the Zn2+ binding moiety, bis(2-
pyridylmethyl)amine (BPA).11 BPA is used as a chelator for Zn2+
due to its highly specific binding over other metal ions, and a
favorable kinetic and thermodynamic properties, which result in
quick formation of a stable Zn2+ complex.12 Furthermore, they
can be readily incorporated into a fluorophore, which simplifies
Received: September 20, 2011
Revised:
November 4, 2011
Published: November 09, 2011
r
2011 American Chemical Society
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Scheme 1. Structure of ADD Dyes
yield was determined by exciting the sample at 366 nm with the use of
quinine sulfate as the standard (ϕf = 0.546 in 0.1 N H2SO4).
Fluorescence decays were recorded by using an IBH time-correlated
single-photon counting spectrometer as reported elsewhere.19 NMR
spectra were recorded on JEOL 500 MHz instrument in deuterated
solvents as indicated; chemical shifts are reported in ppm and
0
coupling constants (JX‑X ) are reported in Hz. ESI-MS were per-
formed on an ECA LCQ Thermo system with ion-trap detection in
positive and negative mode. Elemental analyses (C, H, and N) were
taken on a Euro EA Elemental analyzer.
General Procedure for Metal Ion Binding Studies. Com-
pound 1 was dissolved in the respective solvents. Metal per-
chlorate stock solutions (9.11 ꢁ 10ꢀ3 M) were prepared in
acetonitrile and the titrations were carried out by adding small
volumes (1ꢀ5 μL) of the metal ion to dye (3.5 mL) in quartz
cuvette. After the addition of metal ion to the cuvette, using a
microliter syringe, the solution was shaken well and kept for
1 min before recording any measurements. Complex stoichiome-
try has been determined from the continuous variation technique
(Job’s plot),20 based on the difference in integral area of
fluorescence spectra ΔF (ΔF = F0 ꢀ F) of dye observed in the
presence of metal ions. Equimolar solutions of 1 and metal ions
were prepared and mixed to standard volumes and proportions in
order that the total concentration remained constant. ΔF values
were calculated by measuring the integral area of fluorescence
spectra of dye in the absence (F0) and presence (F) of the
corresponding concentration of metal ion. Subsequently, ΔF
were plotted for the metal ion against mole fraction (xi =
[dye]/([dye] + [metal ion])).
correction for environmental effects.15 Ratiometric probes for
metal ions have been the subject of a series of investigations in
recent years.16 So far, the reported ratiometric sensors utilized
ICT process, which exhibits a shift in both the absorption and
emission maxima.6a For example, cation complexation with an
electron donor group reduces the electron-donating character
and thus reducing the conjugation, which results in a blue shift of
the absorption and emission maxima together with a decrease of
the molar absorptivity. In contrast, the metal ion binding to
the acceptor group enhances its electron-withdrawing character,
which results in a red shift in both absorption and emission
maxima. However, most currently available ratiometric sensors
for Zn2+ are still not satisfactory due to the small emission
shifts.17 Here, we report our new strategy for the development of
the ratiometric fluorescent sensor based on the PET signaling
mechanism by using acridinedione (ADD) as a fluorophore and
BPA moiety as a Zn2+ chelator. These two moieties are linked
covalently through nonconjugated fashion, which minimizes the
electronic communication between the receptor unit and fluoro-
phore moiety in the ground state. On excitation, an efficient
PET occurs from BPA to ADD fluorophore, which results in a
lower fluorescence quantum yield with dual emission, one peak at
425 nm and another peak at 560 nm in aprotic solvents. The large
difference in the peak position will be an advantage for the
ratiometric detection of Zn2+.
Recently, ADD, a class of laser dye has been exploited as a
signaling unit in sensor molecule due to the occurrence of both
PET and ICT in a single molecule.18 Here, we have synthesized
BPA linked ADD (1), which exhibits a dual emission in aprotic
solvents due to the locally excited (LE) and PET promoted
charge transfer (CT) state. The origin of the dual emission has
been confirmed by comparing with two other ADD derivatives,
which have p-dimethylaminobenzene and methyl groups at the
ninth position of the ADD moiety (Scheme 1). In the presence of
Zn2+, compound 1 shows a fluorescence enhancement in the LE
state with the disappearance of PET promoted CT state that
allows a selective ratiometric detection of Zn2+ by utilizing PET
signaling mechanism.
Synthesis. The synthetic routes to sensor 1 is outlined in
Scheme 2. Compounds 1aꢀc and the other compounds (2 and
3) employed in this investigation as illustrated in Scheme 1 were
synthesized following the procedure reported in the literature.18a,21
9-(4-(Bis(pyridine-2-ylmethyl)amino)phenyl)-3,3,6,6-tetra-
methyl-3,4,6,7,9,10-hexahydro-1,8(2H,5H)acridinedione (1). A
mixture of amino-acridinedione 1c (0.75 g, 2.0 mmol), 2--
(chloromethyl)pyridine hydrochloride (0.67 g, 4.1 mmol), and
sodium carbonate (0.47 g, 7.4 mmol) in 20 mL ethanol was
refluxed under a nitrogen atmosphere for 12 h. The reaction
mixture was concentrated by evaporating the solvent; the residue
was dissolved in 20 mL of aqueous solution of sodium hydroxide
and extracted three times with dichloromethane. The combined
organic layer was dried with magnesium sulfate, filtered and
evaporated in vacuo. The crude product was purified by silica gel
chromatography using CHCl3/MeOH (97:3) as an eluent to
isolate pure compound 1 (0.62 g, 57%) as a yellow powder. Mp
209ꢀ211 °C; IR (KBr): 3273 [br. (NH)], 1636 [vs conj. CO)],
1367 [s. (conj. ꢀCdCꢀ)] cmꢀ1; 1H NMR (500 MHz, DMSO-
d6) δ (ppm): 0.81 and 0.94 (2s, 12H, gem-dimethyl); 1.94 and
2.08 (2d, 4H, J = 16.1 Hz, C2 and C7 ꢀCH2); 2.26 and 2.33 (2d,
4H, J = 17.0 Hz, C4 and C5 ꢀCH2); 4.61 (s, 1H, C9ꢀH); 4.69 (s,
4H, ꢀCH2); 6.39 (d, 2H, J = 8.5 Hz, ADD ArꢀH); 6.80 (d, 2H,
J = 8.6 Hz, ADD ArꢀH); 7.20 (m, 4H, ArꢀH); 7.65 (t, 2H,
II. EXPERIMENTAL SECTION
ArꢀH); 8.49 (d, 2H, J = 4.5 Hz, ArꢀH); 9.12 (s, 1H, NH); 13
C
General Information. Dimedone was purchased from Lancaster
(India) Ltd. 4-Nitrobenzaldehyde and 2-(chloromethyl)pyridine
hydrochloride and all metal ions as their perchlorates were purchased
from Sigma Aldrich Chemicals Pvt. Ltd. The solvents used for the
spectral studies were of HPLC grade. Absorption spectra were
recorded using Agilent 8453 diode array spectrophotometer. Emis-
sion; excitation and 3D contour spectra were recorded on HORIBA
JOBIN YVON Fluoromax-4P spectrometer. Fluorescence quantum
NMR (125 MHz, DMSO-d6) δ (ppm): 27.6 (CH3), 29.4 (CH3),
30.9 (CH), 32.7 (C), 40.5 (CH2), 50.8 (CH2), 57.4 (CH2),
111.9 (CH), 112.3 (C), 121.6 (CH), 122.6 (CH), 128.6 (CH),
136.0 (C), 137.2 (CH), 146.4 (C), 149.4 (C), 149.8 (CH), 159.8
(CH), 195.0 (C); MS (ESI): m/z = 547.81 [M + 1]+; elemental
analysis (%) calcd for C35H38N4O2 (546.70): C 76.89, H 7.01, N
10.25. Found: C 76.78, H 7.04, N 10.29.
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Scheme 2. Synthetic Routes to Compound 1
Figure 2. Fluorescence excitation spectra of 1 in acetonitrile: (a) λem at
560 nm, (b) λem at 425 nm.
absorption maximum. Compound 1 shows a single emission in
protic solvents, whereas in aprotic solvents, it exhibits a dual emission.
For example, in acetonitrile a shorter wavelength LE fluorescence at
425 nm and a new anomalous longer wavelength CT fluorescence
around 560 nm is observed. However, in methanol, a single emission
peak is observed at 440 nm. The observed dual emission of 1 in
aprotic solvents is similar to the earlier reported DMAADD (2)
molecule, which has a p-dimethylamino group as the electron donor
moiety.22 The existence of two different conformations of
DMAADD in the excited state leads to the dual emission in aprotic
solvents.
To confirm whether the similar reason is responsible for the
dual fluorescence in 1, we have carried out excitation (Figure 2)
and 3D contour spectral studies (Figure S1). The fluorescence
excitation spectrum of 1 show two different excitation maximum
at 370 and 362 nm, when monitored at emission maximum 425
and 560 nm, respectively. The excitation spectrum recorded at
the longer wavelength emission maximum shows a shoulder
around 315 nm, whereas such shoulder is not observed in the
excitation spectrum recorded at the shorter wavelength emission
maximum. So, the emission at 425 nm arises within the ADD
fluorophore (LE state); whereas the emission at 560 nm involves
BPA group as a donor and ADD moiety as an acceptor. Presence
of two different emission species was further confirmed from 3D
contour spectral studies. Compound 1 shows two contours at
excitation, emission wavelength 370, 427 and 362, 560 nm that
corresponds to LE and CT state, respectively. This clearly
confirms the presence of two different emission states of the
molecule with different origin of excitation. The existence of two
Figure 1. (a) Absorption and (b) emission spectra of 1 in methanol and
acetonitrile.
III. RESULTS AND DISCUSSION
(a). Photophysical Studies. Absorption and emission spectral
studies of 1 have been carried out in a series of protic and aprotic
solvents, which show two sets of results. The representative
spectra are presented in Figure 1. In aprotic solvents, 1 exhibits a
strong absorption around 360 nm due to the ICT from the ring
nitrogen to ring carbonyl oxygen center within the ADD
fluorophore. A shoulder around 315 nm is also observed, which
is assigned to the electronic transition of BPA group. However in
protic solvents, the shoulder appeared in the blue-shifted region
due to the protonation of amine moiety. The corresponding emis-
sion spectrum was recorded by exciting at its longest wavelength
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different conformation of the same molecule in the excited state
leads to the formation of a dual emission in aprotic solvents,
namely LE and PET promoted CT state as shown in Scheme 3.
In one of the conformations, an efficient PET from the electron-
rich amino moiety to the relatively electron-deficient excited
state of ADD fluorophore occurs that leads to the formation of a
new PET promoted CT state as observed in DMAADD 2.22 The
observed lower fluorescence quantum yield and shorter fluores-
cence lifetime (Table 1) of 1 also corroborate the operation of
PET process. In acetonitrile, compound 1 shows a fluorescence
quantum yield of 0.087 ((5%), which is very low when
compared to 3 (0.83 ( 2%), which has a methyl group at the
ninth position of the ADD.
absorption around 315 nm (Figure 3), which indicates that there
is an interaction between Zn2+ and the receptor unit in the
ground state. On the other hand, no change in the ADD
absorption maximum is observed, which confirms the absence
of any interaction between receptor unit and ADD fluorophore
in the ground state. Figure 4 presents the emission spectra of 1
with the addition of Zn2+ in acetonitrile. Addition of Zn2+ leads
Fluorescence decay of 3 obeys single exponential fit with the
lifetime of 5.65 ns, whereas 1 shows a biexponential decay (0.51
and 3.28 ns) at all emission wavelengths (Figure S2). The
shorter lifetime component is due to the PET promoted CT
state and the longer lifetime component is due to the PET
quenched LE state. In a similar manner, 2 also shows a
biexponential decay (0.66 and 1.98 ns) at emission wavelengths
below 530 nm due to the spectral overlap of both the LE and CT
states, whereas above 530 nm, only the CT state lifetime of 0.66
ns is observed. This clearly proves that the PET is more efficient
in compound 2 compared to 1 that exhibits a strong CT state
fluorescence above 530 nm. The observed lower CT/LE state
emission intensity ratio of 1 (0.52) compared to 2 (3.85; when
both excited at 366 nm) also supports our argument. The
presence of strong electron donor group in 2 leads to the
effective PET process, but the lack of selective metal ion
receptor group limits its usage as a sensor molecule. On the
other hand, the presence of selective Zn2+ chelator, BPA
enables compound 1 as a good candidate for the ratiometric
fluorescence sensor.
Figure 3. Absorption spectra of 1 (12 μM) upon addition of Zn2+ in
acetonitrile.
(b). Metal Ion Binding Studies. Addition of Zn2+ to the
acetonitrile solution of 1 shows the disappearance of BPA moiety
Scheme 3. Dual Fluorescence of Dye 1 and the Zn2+ Induced
Disappearance of CT Emission
Figure 4. Emission spectra of 1 (12 μM) upon addition of Zn2+ in
acetonitrile; λex = 388 nm. Inset shows the emission spectra in the region
of 530ꢀ700 nm.
Table 1. Absorption, Emission Maxima, Molar Absorption Coefficient, Fluorescence Quantum Yield, and Fluorescence Lifetime
of 1, 2, and 3 in CH3CN
τf (ns)b
compound
λabs (nm)
molar extinction coeff. (log ε) Mꢀ1 cmꢀ1
λem (nm)
Φfa
λem at 425 nm
λem at 560 nm
1
361
3.85
425, 560
0.087 ((5%)
0.51 (28.7)
3.28 (71.3)
0.66 (36.4)
1.98 (63.6)
5.65 (100)
0.51 (87.4)
3.28 (12.6)
0.66 (100)
2
362
365
3.84
3.94
424, 550
426
0.006 ((5%)
0.83 ((2%)
327
5.65 (100)
a Fluorescence quantum yields were determined by exciting the sample at 366 nm using quinine sulfate as the standard (Φf = 0.546 in 0.1 N H2SO4).
b λex = 370 nm; error inshorter lifetime is (0.02 ns and inlonger lifetime is (0.03 ns; relative amplitude corresponding to the lifetime isgiven inthe bracket.
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to a 15-fold enhancement in the LE state fluorescence intensity
with the disappearance of CT state fluorescence. Fluorescence
intensity saturates when the concentration of Zn2+ reaches one
equivalent, which shows the 1:1 complexation between receptor
and Zn2+, and the stoichiometry was further confirmed from the
Job’s plot.20 The Job’s plot (Figure 5) showed the maximum
changes when the mole fraction of 1 was around 0.5, which is
characteristic of a 1:1 stoichiometric hostꢀguest binding. Bind-
ing of Zn2+ at the BPA moiety alters the conformation of the
molecule in to a single one, where PET process does not exist,
which results in the disappearance of CT state emission together
with the enhancement in the LE state intensity. The emission
intensity ratio, I425/I560, increases from 3.3 to 103, when the
concentration of Zn2+ is varied from 0 to 14 μM (Figure 6). A
linear increase in the ratio is observed in the concentration range
of 1.0 to 11.8 μM, which enables the quantitative detection under
ratiometric response. The large difference (135 nm) in the peak
position and the higher enhancement in the fluorescence in-
tensity will be an advantage for ratiometric detection. But, the
enhancement of the intensity at 425 nm is more compared to the
decrease in the intensity at 560 nm peak, which makes the
ratiometric detection is difficult, when compared to other
reported derivatives. However, the utilization of PET promoted
CT state quenching will be an additional example for the
ratiometric detection and it also signifies the usage of PET
signaling mechanism for the ratiometric detection. The detection
limit (DL) can be calculated with the equation,23 DL = 3S0/m,
where “m” is the calibration sensitivity of the fluorescence
intensity change (ΔF = F0 ꢀ F) versus [Zn2+], and “S0” is the
standard deviation of the blank signal (F0) obtained without
Zn2+. From this, the lower detection limit was found to be 98 nM
in acetonitrile.
Changes in the fluorescence intensity of 1 caused by other
metal ions, including Na+, K+, Mg2+, Ca2+, Mn2+, Fe2+, Co2+,
Ni2+, Cu2+, and Cd2+ were also measured as shown in Figure 7a.
Addition of paramagnetic transition metal ions such as Co2+ and
Cu2+ shows fluorescence quenching to a different extent (Figures
S3 and S4) at both the states and at all concentrations, whereas
the addition of Ni2+, Fe2+, Mn2+, and Cd2+ shows a relatively
lower fluorescence enhancement in the LE state at the lower
concentration and fluorescence quenching at the higher concen-
tration, as shown in Figure S5. On the other hand, the CT state
fluorescence is quenched at all concentrations. Binding of metal
ion at the receptor site suppresses the PET process and results in
the LE state fluorescence enhancement, whereas, the direct
interaction of transition metal ion with the fluorophore leads
to fluorescence quenching.24 From the above results, it is clear
that Cu2+ and Co2+ did not involve in the binding at the receptor
moiety, whereas Ni2+, Fe2+, Mn2+, and Cd2+ shows a weak
binding at lower concentration. On the other hand, the impor-
tant biorelated metal ions such as Na+, K+, Mg2+, and Ca2+, which
often exist at high concentrations in most of the living cells,
resulted in a negligible fluorescence change, as compared with
that of Zn2+ ions. This can be ascribed to the poor coordination
capability of these abundant metal ions with BPA receptor. These
results also agree quite well with those reported for Zn2+ probes
based on other fluorophores.6b,17e In addition, fluorescence
Figure 5. Job’s plot for the binding of 1 with Zn2+ in acetonitrile; λex
388 nm.
=
Figure 6. Fluorescence intensity ratio of 1 (12 μM) upon addition of
Zn2+ in acetonitrile; λex = 388 nm.
Figure 7. (a) Fluorescence intensity ratiometric response of 1 (12 μM) upon addition of various metal ions (3 equiv to that of Zn2+); (b) Fluorescence
intensity ratiometric response of 1 (12 μM) upon addition of Zn2+ in the presence of interfered metal ions in acetonitrile; λex = 388 nm.
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Table 2. Stability Constant of 1 with Various Metal Ionsa in Acetonitrile and Acetonitrile/Water (80:20, v/v) Mixture
solvent
Mn2+
Fe2+
Ni2+
Zn2+
Cd2+
Hg2+
acetonitrile
acetonitrile/water (80:20, v/v)
3.41 ꢁ 103
1.16 ꢁ 103
2.67 ꢁ 103
1.03 ꢁ 103
3.25 ꢁ 103
1.30 ꢁ 103
2.34 ꢁ 105
5.67 ꢁ 104
3.67 ꢁ 103
1.40 ꢁ 103
2.57 ꢁ 103
9.83 ꢁ 102
a Cu2+ and Co2+ do not involve binding with BPA moiety; Na+, K+, Mg2+, and Ca2+ show no appreciable change in the fluorescence intensity to measure
the stability constant.
Figure 9. Fluorescence intensity changes (at 437 nm) of 1 (12 μM) as a
function of water content in aqueous acetonitrile solution in the absence
Figure 8. Fluorescence lifetime decay of 1 (12 μM) upon addition of
and presence of Zn2+ (18 μM): λex = 388 nm.
Zn2+ in acetonitrile: λex = 388 nm, λem at 560 nm; (a) laser profile.
spectra of 1 recorded in the presence of Zn2+ ions (1.0 equiv) and
3.0 equiv of all the above competing metal ions further revealed
that other metal ions, except for Cu2+ and Co2+ ions, do not
interfere with Zn2+-induced fluorescence enhancement. The
observed quenching of the fluorescence by Co2+ and Cu2+
assures that no “false positive” turn-on response would be
induced by such metal ions in the analysis of samples. Fluores-
cence intensity ratio (Figure 7b) of 1 with Zn2+ in the presence of
above metal ions (except Cu2+ and Co2+) is observed to be
similar to that of 1 with Zn2+ alone (i.e., I425/I560 ∼ 100). The
well-known fluorescence quencher, Cu2+ ion can directly interact
with the ADD fluorophore, thus it can seriously interfere with the
fluorescence enhancement of 1 by Zn2+ ions. The stability
constant of the metal ion complex of 1 is evaluated from the
BenesiꢀHildebrand plot25 (Figure S6) of 1/I0 ꢀ I against [metal
ion]. Among the metal ions investigated, Zn2+ shows the binding
constant of 2.5 ꢁ 105 Mꢀ1 in acetonitrile, whereas other
transition metal ions, except Cu2+ and Co2+ show a weak binding
constant in the range of 2.3 ꢀ 3.7 ꢁ 103 Mꢀ1 as presented in
Table 2.
Time-resolved fluorescence studies will provide invaluable
information about the system when more than one emitting
species contribute to steady-state fluorescence intensities. Such
fluorescence lifetime based sensors for Zn2+ detection is already
reported.26 Here, we have carried out fluorescence lifetime
studies to confirm the involvement of PET process in the fluores-
cence signaling action. Compound 1 shows 0.51 (87.4%) and
3.28 ns (12.6%), when monitored at emission wavelength of
560 nm. Figure 8 shows the fluorescence decay of 1 with the
addition of various concentration of Zn2+ in acetonitrile. Addi-
tion of Zn2+ results in the disappearance of the shorter lifetime
component with the formation of a new lifetime component
(1.75 ns) along with an increase in the amplitude. Disappearance
of the shorter lifetime component confirms the suppression of
Figure 10. Emission spectra of 1 (12 mM) upon addition of Zn2+ in
water/acetonitrile (80:20, v/v): λex = 388 nm.
PET process and hence the disappearance of CT state emission
in the Zn2+ bound complex of 1 as presented in Scheme 3.
To optimize the conditions for practical applications in
environmental and biological samples, the effect of water on
the fluorescence emission of 1 in the absence and presence of
Zn2+ were investigated in acetonitrile solutions. Figure 9 shows
the effect of water content on the fluorescence behavior of 1 in
acetonitrile solution. As the percentage of water is increased, an
enhancement in the LE state emission intensity with the dis-
appearance of PET promoted CT state is observed. At 20% water
content, a single emission peak around 437 nm is observed.
The disappearance of PET promoted CT state indicates the
lesser extent of PET in these solvent mixtures. Addition of Zn2+
to the acetonitrile/water mixture solutions shows a fluorescence
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enhancement without any spectral shift due to the suppression of
PET process. The extent of enhancement was found to decrease
with an increase in the water content (Figure 9). The optimum
percentage of water for a better Zn2+ sensing was found to be 80:20
(acetonitrile/water, v/v) solvent mixture. Addition of Zn2+ to 1 in
this solvent mixture shows a 4.5-fold fluorescence enhancement as
shown in Figure 10. The observed lower extent of fluorescence
enhancement compared to that in acetonitrile also further supports
the decreased ease of PET in the acetonitrile/water mixtures. The
detection limit was found to be 167 nM in acetonitrile:water (80:20,
v/v) mixture, which is low when compared to the detection limit
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arrived in acetonitrile. The stability constant (Table 2) of 1 Zn2+
3
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IV. CONCLUSION
Anewdualfluorescent ADD derivative (1) bearing BPA receptor
has been synthesized and proved to be a highly selective and
sensitive Zn2+ sensor. Binding of Zn2+ at the receptor moiety leads
to the quenching of PET promoted CT state emission with an
enhancement in the LE state intensity under a ratiometric manner.
This ratiometric change is attributed to the suppression of PET
process during the chelation of Zn2+ in a 1:1 complexation ratio.
Fluorescence lifetime studies clearly prove the involvement PET
process in the fluorescence signaling action. The observation of
ratiometric fluorescence response under PET signaling mechanism
will expand the scope on PET based sensors for the ratiometric
detection. Sensing of Zn2+ is also achieved in a mixed acetonitrile/
water solutions, which shows a single emission with an enhance-
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’ ASSOCIATED CONTENT
S
Supporting Information. NMR spectra, 3D-contour
b
spectra of 1; fluorescence lifetime decay of ADD dyes, emission
spectra of 1 in the presence of various metal ions and Benesiꢀ
Hildebrand plot for 1 with Zn2+. This material is available free of
charge via the Internet at http://pubs.acs.org.
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’ AUTHOR INFORMATION
Corresponding Author
*Tel.: 091-44-24540962. Fax: 091-44-24546709. E-mail: prm60@
hotmail.com.
’ ACKNOWLEDGMENT
We thank the Department of Science and Technology (DST),
Government of India, for financial support through SERC
scheme Project No. DST/SR/S1/PC-31/2005. Financial sup-
port by DST-IRHPA is also gratefully acknowledged.
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