New highly selective turn-on fluorescence receptor for the detection of copper (II)

Article abstract of DOI:10.1016/j.saa.2016.12.001

Three new receptors (1a–c) bearing a p-dimethylaminobenzamide fluorophore have been synthesized and evaluated in terms of their fluoroionophoric properties towards various metal ions. Notably, receptors 1a and 1c exhibited dramatic fluorescent enhancement towards Cu^2?+ in acetonitrile. Subsequent investigations revealed that the highly selective behavior of these receptors towards Cu^2?+ could be attributed to the Cu^2?+-mediated oxidative cyclization of these compounds to the corresponding 1,3,4-oxadiazoles. Solvent effects and quantum calculations indicated that 1a and 1c both possessed an intramolecular charge transfer channel, which could be obstructed by the oxidative cyclization of these receptors. Receptor 1a was successfully applied to the determination of the Cu^2?+ in drug sample with a low detection limit of 2.2?×?10^??8?mol?L^??1.

Full text of DOI:10.1016/j.saa.2016.12.001

Accepted Manuscript
New highly selective turn-on fluorescence receptor for the
detection of copper (II)
Qian Nan, Pu Rong, Yunbao Jiang, Rui Yang
PII:
DOI:
Reference:
S1386-1425(16)30717-X
doi: 10.1016/j.saa.2016.12.001
SAA 14814
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
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3 December 2016
3 December 2016
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ACCEPTED MANUSCRIPT
New highly selective turn-on fluorescence receptor for the
detection of copper (II)
Qian Nana, Pu Ronga, Yunbao Jiangb , and Rui Yanga*
a Key Laboratory on Luminescence and Real-Time Analysis, Ministry of Education, School of
Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, China
b Department of Chemistry, College of Chemistry and Chemical Engineering, and the MOE
Key Laboratory of Analytical Sciences, Xiamen University, Xiamen 361005, China
*Corresponding author. Tel: +86 023 68367060; Fax: +86 023 68254000
E-mail address:
(R. Yang)
 Corresponding author. Tel: +86 023 68367060 Fax: +86 023 68254000
E-mail address:
(R. Yang)
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Abstract: Three new receptors (1a–c) bearing a p-dimethylaminobenzamide fluorophore have
been synthesized and evaluated in terms of their fluoroionophoric properties towards various
metal ions. Notably, receptors 1a and 1c exhibited dramatic fluorescent enhancement towards
Cu2+ in acetonitrile. Subsequent investigations revealed that the highly selective behavior of these
receptors towards Cu2+ could be attributed to the Cu2+-mediated oxidative cyclization of these
compounds to the corresponding 1,3,4-oxadiazoles. Solvent effects and quantum calculations
indicated that 1a and 1c both possessed an intramolecular charge transfer channel, which could be
obstructed by the oxidative cyclization of these receptors. Receptor 1a was successfully applied to
the determination of the Cu2+ in drug sample with a low detection limit of 2.2×10−8 mol L−1.
Keywords: Fluorescence receptor; Copper (II); Oxidative cyclization; Intramolecular charge
transfer
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1. Introduction
The development of new methods for the detection and monitoring of trace elements continues
to attract considerable attention because of the critical roles played by these methods in biological
processes. In this regard, research towards the synthesis of fluorescence receptors capable of
exhibiting high levels of selectivity and sensitivity to specific metals remains an active area of
interest in chemistry [1-5]. Copper is an essential trace element that is vital for maintaining the
health of numerous organisms, including humans, and a large number of fluorescence receptors
have been developed for the detection of Cu2+ in recent years [6-13]. Among those reported,
receptors based on the binding [14-16] or reaction [17-19] of Cu2+ with a receptor, which results
in pronounced changes in the spectral properties of the receptor, represent one of the main
recognition mechanisms. However, Cu2+ is also an efficient fluorescence quenching agent because
of its paramagnetic characteristics and chelating properties, which has limited the development of
fluorescent receptors for the detection of Cu2+ [20-22]. For sensitivity reasons, receptors showing
fluorescence enhancement are generally favored over those exhibiting fluorescence quenching.
With this in mind, reaction-based receptors capable of overcoming the challenges posed by the
paramagnetic characteristics of Cu2+ fold great promise for the detection of this metal. In this
paper, we have developed a new strategy for the detection of Cu2+ based on an intramolecular
charge transfer (ICT) process, which is controlled by the presence of Cu2+. ICT is one of the most
important characteristics of any molecule, which can be controlled by the addition of various
organic donor and acceptor groups or organometallic moieties [23]. However, there have, to the
best of our knowledge, been no studies reported in the literature to date concerning ICT process
controlled by a metal ion-induced chemical reaction.
N-Acylhydrazones have been reported to be typical chemical reaction receptors for Cu2+
because they undergo an oxidative cyclization reaction in the presence of this metal ion [24].
Herein, we report the coupling of three different N-acyhydrazones with the ICT fluorophore
p-dimethylaminobenzamide, to afford the corresponding benzoylhydrazones 1a–c (Scheme 1).
These receptors were synthesized with the aim of developing a deeper understanding of the
influence of the ICT fluorophore on molecule characteristics of Cu2+ recognition. The
experimental results showed that receptors 1a and 1c displayed high levels of selectivity towards
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Cu2+, which led to large increases in the fluorescence intensities of these compounds in ACN and
ACN/H2O buffer solutions. These changes in the fluorescence properties were attributed to the
Cu2+-mediated oxidative cyclization of the weakly fluorescent receptor molecules to give the
corresponding 1,3,4-oxadiazoles, which are rigid and highly fluorescent. These new
1,3,4-oxadiazole systems also combined with Cu2+ to quench the long-wavelength emissions, as
well as introducing new short-wavelength emissions. We evaluated the properties of these
receptors and found that they both exhibited intramolecular charge transfer (CT) characteristics,
whereas no charge separation occurred in the corresponding 1,3,4-oxadiazoles. The recognition of
Cu2+ by these receptors therefore led to a change in the CT properties of these compounds from an
on state to an off state, representing a sophisticated switch for the recognition of Cu2+.
Receptor 1a was also successfully used to determine the presence of Cu2+ in the traditional
Chinese medicine azurite, further highlighting the potential practical value of this process. Azurite
contains natural copper sulfate, which can exist in a variety of different structural forms, including
granular, radial and massive structures. Azurite can be used to treat various ailments, including
fever, red eye pain, ear ache and lachrymatory effects and epilepsy, and is widely used as a powder
or solid tablet. Notably, no methods have been reported to date for the determination of azurite.
2. Experimental
2.1. Apparatus
Fluorescence correlation spectroscopy experiments were conducted on a Hitachi F-2500
spectrofluorophotometer (Hitachi Company, Tokyo, Japan) using excitation and emission slits of
5 nm. Fluorescence quantum yields were measured using quinine sulfate as the standard (0.55 in
0.50 mol L−1 H2SO4) [25]. Absorption spectra were recorded on a UV-8500 spectrophotometer
(Shimadzu Corporation, Kyoto, Japan) using a quartz cell with a path length of 1 cm. Verification
tests were conducted using a TAS-986FLAA (flame atomic absorption) spectrophotometer (Persee
Co.,Ltd, Beijing, China).
All of the pH measurements taken in this study were recorded on a pHS-3C pH meter
(Shanghai Scientific Instruments Company, Shanghai, China). 1HNMR and 13CNMR spectra were
recorded in DMSO-d6 on a Varian Unity+ 500 MHz, Bruker AV300 and Bruker AV600 MHz
spectrometer using TMS as an internal reference standard.
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The geometries of the ground and first singlet excited states were optimized under vacuum
using the hybrid density functional Becke-3-Lee-Yang-Parr (B3LYP) and the single-excitation
configuration interaction (CIS) method, respectively in combination with the 6-31G* basis set. All
of these calculations were completed using the GAUSSIAN 2003 software package.
2.2. Reagents
p-(N,N-Dimethylamino)benzaldehyde was purchased from China National Chemicals Group
(Shanghai, China). Azurite was purchased from Haocao flagship store (Bozhou, China). All of the
other reagents used in this study were purchased from Sinopharm Reagent Co., Ltd (Beijing,
China) and used without further purification. metal ion solutions were prepared from the
corresponding perchlorate salts. Solvents for spectroscopic investigation were purified prior to
being used and checked to ensure they contained no fluorescent impurities at the used excitation
wavelength being used in the study. Tris-HCl buffer solutions were prepared using 0.01 mol L−1
Tris and the appropriate amount of HCl using a pH meter. Buffer solutions were prepared using
phosphoric acid,boric acid and acetic acid to allow for the determination of the traditional Chinese
medicine azurite. With the exception of acetonitrile (ACN), which was purchased as the HPLC
grade, all of the chemicals used in this study were purchased as the analytical grade. Distilled
water was used to prepare the aqueous solutions.
2.3. Synthesis of receptors 1a−c
The target compounds were readily synthesized using a series of simple reactions. The initial
esterification of the corresponding benzoic acids with ethanol gave the ethyl benzoates, which
were reacted with hydrazine hydrate to give the corresponding benzoylhydrazines. These
compounds were then heated at reflux with p-(N,N-dimethylamino)benzaldehyde for 6 h to give
the desired targets 1a–c, as shown in Scheme 1.
(E)-N'-(4-(dimethylamino)benzylidene)benzohydrazide (1a): This compound was purified by
repeated recrystallization from ethanol/water and characterized by 1H and 13C NMR spectroscopy.
1H NMR (500MHz, DMSO-d6) δ (ppm): 11.56 (s, 1H), 8.30 (s, 1H), 7.89 (d, J = 8.4 Hz, 2H), 7.51
(t, J = 7.4 Hz, 5H), 6.76 (d, J = 8.8 Hz, 2H), 2.97 (s, 6H). 13C NMR (126 MHz, DMSO-d6) δ
(ppm): 162.91, 151.67, 148.89, 133.83, 131.56, 128.58, 128.51, 127.54, 121.65, 111.90, 39.83.
(E)-N'-(4-(dimethylamino)benzylidene)-2-hydroxybenzohydrazide (1b): This compound was
purified by repeated recrystallization from N,N-dimethylformamide/water and characterized by 1H
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and 13C NMR. 1H NMR (500 MHz, DMSO-d6) δ (ppm):11.64 (s, 1H), 8.30 (s, 1H), 7.88 (d, J =
9.3 Hz, 1H), 7.57 (d, J = 8.9 Hz, 2H), 7.41 (s, 1H), 7.02 6.87 (m, 2H), 6.77 (d, J = 8.9 Hz, 2H),
2.98 (s, 6H). 13C NMR (126MHz, DMSO-d6) δ (ppm): 164.59, 159.39, 151.82, 149.91, 133.73,
128.79, 128.24, 121.29, 118.93, 117.38, 115.73, 111.87, 39.81.
(E)-N'-(4-(dimethylamino)benzylidene)-2-methoxybenzohydrazide (1c): This compound was
1
purified by repeated recrystallization from ethanol/water and characterized by H and 13C NMR
spectroscopy. 1H NMR (600 MHz, DMSO-d6) δ (ppm):11.16 (s, 1H), 8.18 (s, 1H), 7.62（d, J= 7.2
Hz, 1H）, 7.52 (d, J= 8.4 Hz, 2H), 7.49(t, J= 7.8 Hz, 1H), 7.16 (d, J= 8.4 Hz, 1H), 7.05 (t, J= 7.2
Hz, 1H), 6.76 (d, J= 9.0 Hz, 2H), 3.88 (s, 3H), 2.98 (s, 6H). 13C NMR (75 MHz, DMSO-d6) δ
(ppm): 162.08, 157.05, 151.89, 148.37, 132.38, 130.08, 128.72, 128.10, 123.99, 121.99, 120.78,
112.21, 56.00, 39.83.
Scheme 1: Synthesis of compounds 1a−c.
3. Results and discussion
3.1. Absorption spectrum behaviors of receptor 1a in the presence of Cu2+ in ACN
The absorption spectrum of receptor 1a in ACN contained two bands at 352 and 231 nm with
molar absorption coefficients of 4.28×104 and 1.86×104 M−1 cm−1, respectively, which are
indicative of the (π, π*) transition character of this compound (Fig. 1). The addition of Cu2+ to a
solution of 1a in ACN led to a reduction in the intensity of the band at 352 nm, as well as the
appearance of two new bands (from 250 to 325 nm and 390 to 450 nm). Several other transition
metal ions were also tested, including Cd2+, Ni2+, Zn2+, Al3+, Fe3+, Hg2+ and Pb2+, which all had
some influence on the absorption spectrum of 1a. In contrast, the effects of Ca2+, Mg2+, K+, Co2+,
Fe2+, Mn2+, Cu+ and Ba2+ were negligible (Fig. S1). Based on the absorption spectra of receptor 1a
in the presence of different amounts of Cu2+, we selected an isosbestic wavelength of 310 nm as
the optimum excitation wavelength for all of the subsequent fluorescence measurements.
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Fig. 1 Absorption spectra of 1a (1.5×10−5 mol L−1) in ACN in the presence of increasing amounts
of Cu2+ (0.0–6.0×10−5 mol L−1).
3.2. Fluorescence spectrum behaviors of 1a in the presence of Cu2+ in ACN
As a solution in ACN, compound 1a emitted weak fluorescence at 409 nm with a total
fluorescence quantum yield (Ф) of 0.0079. The addition of Cu2+ to a solution of 1a in ACN led to
a dramatic increase in the fluorescence of 1a (i.e., up to 160-fold). The addition of less than 1
equiv. of Cu2+ to a solution of 1a in ACN led to a red-shift in the long-wavelength emission from
409 to 458 nm, as well as a sharp increase in in the fluorescence intensity (Fig. 2a). The addition
of more than 1 equiv. of Cu2+ to a solution of 1a in ACN led to a considerable decrease in the in
the intensity of the long-wavelength emission at 458 nm. This process also led to a pronounced
increase in the intensity of the emission band centered at 360 nm, with an isoemissive point at 408
nm (Fig. 2b). The paramagnetic character of this metal meant that the addition of more Cu2+ to
this solution after the fluorescence intensity at 360 nm had reached its maximum resulted in the
quenching of the fluorescence emission (Fig. 2c). The unpredictable fluorescence behavior of this
system in the long-wavelength emission region inspired us to investigate the recognition
mechanism of this receptor in greater detail.
Fig. 2 Fluorescence spectra of 1a (1.5×10−5 mol L−1) in ACN in the presence of increasing
amounts of Cu2+ (a: 0–1.5×10−5 mol L−1; b: 1.5–6.0×10−5 mol L−1; c: 6.0×10−5–4.0×10−5 mol L−1).
3.3. Solvent effects and quantum calculations
p-Dimethylaminobenzamide is a typical fluorophore with intramolecular charge transfer (ICT)
characteristics. We synthesized two derivatives of p-dimethylaminobenzamide, compounds 1b
and 1c, to develop a deeper understanding of the crucial role played by this moiety in receptor 1a.
The absorption and fluorescence spectra of 1a–c were measured in several different solvents,
including cyclohexane (CHX), diethyl ether (DEE), ethyl acetate (EAC) and acetonitrile (ACN),
which have been arranged in order of increasing polarity. The maximum absorption peaks for
compounds 1a–c showed a continuous red-shift (Table. 1) as the polarity of the solvents increased
from CHX to ACN, indicating that a ground-state charge transfer (CT) could occur [26].
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Consideration of the fluorescence spectra of receptors 1a–c revealed that they all contained a dual
fluorescence emission (Table
1
and Fig. S2), which was attributed to their
p-dimethylaminobenzamide moiety. The short-wavelength emissions in 1a and 1c were found to
be independent of the solvent polarity, whereas the long-wavelength emissions of these
compounds showed a considerable blue-shift with increasing solvent polarity. This result was
characteristic of the charge transfer properties of the excited state, and the dual fluorescence was
therefore attributed to the LE and CT states [27]. The long wavelength emissions of 1b were very
weak in aprotic solvents, where the intensity as dependent on the solvent polarity. In contrast, the
long wavelength emissions of this receptor showed a large blue-shift in the protic solvent
methanol, most likely because this solvent prevented the formation of intramolecular hydrogen
bonds between the oxygen atom of the carbonyl group and hydrogen atom of the hydroxyl group
in 1b. The lack of an intramolecular hydrogen bond would therefore inhibit the excited-state
intramolecular proton transfer (ESIPT) process [28].
Table 1
The maximal absorption and emission wavelengths of 1a−c in CHX, DEE, EAC and ACN.
To develop a deeper understanding of the emission characteristics of these receptors, we studied
the geometries of the first singlet excited states of 1a and 1c. All calculations in this work are
performed with the Gaussian 09 programs[29]. All the geometric structures of first excited stateare
fully optimized using B3LYP/6-31G* method in the framework of density-functional theory
(DFT)[30]. In the process of optimization, the solvent effect of CH3CN is taken into consideration
through a self-consistent reaction field (SCRF) method[31] with the polarized continuum model
(PCM)[32]. The HOMO, LUMO and LUMO+1molecular orbitals of Reactants and Products are
obtained in Figure 1 and 2, respectively, at the B3LYP/6-31G* calculation level (isovalue=0.02).
The results shown in Fig. 3 revealed that the HOMO orbitals of 1a and 1c were mainly
localized on the dimethylaminobenzoyl moiety, whereas the π electrons in the LUMO orbitals
were delocalized over the whole molecule. These results therefore indicated that the transition
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from HOMO to LUMO was accompanied by
a
charge transfer (CT) from the
dimethylaminobenzoyl moiety to the whole molecule [33]. Furthermore, the quantum calculations
revealed important CT channels in 1a and 1c.
Fig. 3 HOMO, LUMO and LUMO+1 molecular orbitals of 1a and 1c at the B3LYP/6-31G*
calculation level (Isovalue=0.02).
3.4. Fluorescence spectrum behaviors of 1b and 1c in the presence of Cu2+ in ACN
The fluorescence spectra of receptors 1b and 1c were measured in the presence of different
concentrations of Cu2+ to develop a deeper understanding of the effects of Cu2+ on the
fluorescence behavior of 1a. Receptor 1b bearing a phenolic -OH group exhibited no fluorescent
response to any of the metals examined in the current study, including Cu2+ in ACN. The lack of a
fluorescent response in this case was attributed to the existence of an ESIPT channel in 1b,
leading to a decrease in the fluorescence quantum yield [34]. In contrast to receptor 1b, receptor
1c bearing a -OCH3 group instead of a phenolic -OH group was found to be much more
responsive to Cu2+. As a solution in ACN, receptor 1c emitted faint fluorescence with a total
fluorescence quantum yield (Ф) of 0.022. This value increased up to 35-fold in the presence of
Cu2+, with the fluorescence spectrum of 1c becoming increasingly similar to that of 1a. The
addition of a small amount of Cu2+ led to a blue-shift from 477 to 453 nm as well as a
considerable increase in the emission intensity (Fig. 4a). The addition of more than 1 equiv. of
Cu2+ resulted in a slight decrease in the intensity of the band at 453 nm, which was accompanied
by a significant increase in the fluorescence intensity centered at 380 nm (Fig. 4b). After reaching
its maximum value, the addition of more Cu2+ simply quenched the fluorescence (Fig. 4c). These
results therefore indicated that receptor 1c is highly selective for Cu2+ and that it most likely
functions in a similar manner to 1a.
Fig. 4 Fluorescence spectra of 1c (1.0×10−5 mol L−1) in ACN in the presence of increasing
amounts of Cu2+ (a: 0–1.0×10−5mol L−1; b: 1.0–4.0×10−5mol L−1; c: 4.0×10−5–3.0×10−4mol L−1).
The excitation wavelength was 315 nm.
3.5. Recognition mechanism
Frontier molecular orbital analysis of receptors 1a and 1c showed that the potential binding
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sites and electron donor atoms were located on the hydrazide-hydrazine moiety. If the recognition
of Cu2+ by these receptors was driven exclusively by an intramolecular charge transfer process
induced by Cu2+ then the fluorescence bands in the spectra of 1a and 1c would both be expected to
show a blue-shift in same solvent [35]. However, the fluorescence spectra of 1a showed a red-shift
after the addition of Cu2+ to a solution of 1a. It is thus not possible to explain the complex changes
observed in the spectra of this receptor in the presence of Cu2+ based on an intramolecular charge
transfer mechanism alone concentrations of KMnO4 (0–4.5×10−5 mol L−1).
Fig. 5 Fluorescence spectra of 1a (1.5×10−5 mol L−1) in ACN in the presence of increasing
amounts of KMnO4 (0–4.5×10−5 mol L−1) (red lines) and fluorescence spectra of 1a+KMnO4
solution in the presence of increasing amounts of Cu2+ (0–1.5×10−5 mol L−1) (black lines).
The fluorescence emission of a receptor can not only be affected by metal ion coordination but
can also be effected by metal ion-promoted reactions [36]. It has been reported that
N-acylhydrazones can undergo an oxidative cyclization to afford 1,3,4-oxadiazoles in the presence
of several oxidants, including Cu(ClO4)2 [37-39]. With this in mind, we hypothesized that Cu2+
would act as an oxidant prior to forming a stoichiometric ratio with 1a (i.e., 1:1 ratio), and that this
oxidant activity would result in the oxidative cyclization of 1a to give the corresponding
1,3,4-oxadiazole. KMnO4 has been widely used as an oxidant for the preparation of
1,3,4-oxadiazoles from hydrazide-hydrazones [40]. Thus, we recorded the fluorescence spectra of
1a in ACN in the presence of several different concentrations of KMnO4. A comparison of these
data with those obtained for a solution of 1a in ACN in the presence of Cu2+ revealed several
similar spectral variations with a red-shift from 409 to 457 nm. This comparison also revealed a
30-fold increase in the fluorescence intensity following the addition of 3 equiv. of KMnO4 to a
solution of 1a in ACN (Fig. 5). Compared with the Cu2+, KMnO4 has a low reduction speed with
1a in ACN, thence the 1a-KMnO4 solution was placed 12 hours to obtain complete reaction.
Afterwards, Cu2+ was added to the 1a-KMnO4 solution. As shown in Fig. 5, it was found that
fluorescence at long wavelength decreased and fluorescence at short wavelength increased
simultaneously, which was consistent with that of 1a and Cu2+(fig.2a and 2b). Taken together,
these data demonstrate that the changes observed in the emission properties in the
long-wavelength region of these spectra could be attributed to the Cu2+-mediated oxidative
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cyclization of receptor 1a to give the corresponding 1,3,4-oxadiazole.. Based on this result, we
have proposed a mechanism for this process, which is shown in Scheme 2.
Scheme 2. Proposed mechanism for the response of these receptors to Cu2+.
Several electrospray ionization-mass spectrometry (ESI-MS) experiments were conducted to
provide experimental evidence to support this conclusion. The analysis of a solution of 1a (104
mol L−1) in ACN in the absence of Cu2+ by ESI-MS revealed the presence of a single mass ion
with an m/z value of 268.1 (Fig.S3a), which was attributed to [1a+H]+. However, the analysis of
this mixture by ESI-MS following the addition of 1 equiv. of Cu2+ revealed the presence of three
different peaks with m/z values of 145.0, 147.0 and 266.1 (Fig.S3b). The peaks with m/z values of
145.0 and 147.0 were attributed to the isotopic peaks of [Cu(I)(CH3CN)2]+, which suggested that
1a was being oxidized by Cu2+ to give Cu+ as a reduction product [41]. In contrast, the peak with
an m/z value of 266.1 was attributed to the 1,3,4-oxadiazole, which would have been formed by
the Cu2+-induced oxidative cyclization of 1a according to the proposed kinetics process shown in
Fig.S4.
The first singlet excited states of the 1,3,4-oxadiazoles resulting from the oxidative cyclization
of receptors 1a and 1c were also studied. The results obtained following the analysis of their
frontier molecular orbitals revealed that the π electrons were located across the entire molecule in
both cases, and that the CT channels closed after the oxidative cyclization of receptors 1a and
1c(Fig. 6). The nitrogen and oxygen atoms in the 1,3,4-oxadiazoles could combine with Cu2+ to
form complexes. This would explain why the addition of more Cu2+ to 1a in ACN led to a gradual
decrease in the fluorescence intensity of the band at 458 nm after it had reached its maximum.
This would also explain the increase observed in the fluorescence emission at 360 nm (Scheme 2).
The subsequent addition of a small amount of protic solvent such as methanol led to a decrease in
the intensity of the short-wavelength emission at 360 nm, which was accompanied by an increase
in intensity of the long-wavelength emission (Fig. S5). This change was attributed to coordination
between the binding sites in the 1,3,4-oxadiazoles and Cu2+, which induced the formation of a
complex with the 1,3,4-oxadiazoles and Cu2+. This result further illustrated that the changes
observed in the long-wavelength region of the spectra resulted from the formation of the
corresponding 1,3,4-oxadiazoles rather than the formation of a simple complex between Cu2+ and
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the receptor 1a or 1c.
(a)
(b)
Fig. 6 HOMO, LUMO and LUMO+1 molecular orbitals of two different 1,3,4-oxadiazoles at the
B3LYP/6-31G* calculation level (Isovalue=0.02).
(a) and (b) were the oxidation product of receptors 1a and 1c, respectively.
3.6. Selectivity
The selectivities of receptors 1a and 1c for Cu2+ were evaluated in the presence of a variety of
other metal ions to investigate the effects of these ions on the fluorescence intensities of receptor
1a and 1c. Several metals were tested, including Zn2+, Hg2+, Pb2+, Cd2+, Ni2+, Ca2+, Mg2+, K+, Ba2+,
Al3+, Fe2+, Co2+, Fe3+, Cu+ and Mn2+, which were added to ACN solutions of receptors 1a and 1c.
The results revealed that Cu2+ was the only metal to have a significant effect on the fluorescence
intensities of 1a and 1c (Fig. S6). These results also showed that the fluorescence response of 1a
to Cu2+ was unaffected by the presence of any other metal ion, even when the contaminating ion
was present at a much higher concentration than that of Cu2+ (Fig. 7), indicating a good selectivity
for Cu2+. Notably, the response of receptor 1a to Cu2+ could be readily distinguished by the naked
eye under a UV lamp, as shown in Fig. S7. The high selective recognition of 1a and 1c towards
Cu2+ could be attributed not only to coordination of N and O atoms, a typical example is the
selective binding of amino acids to Cu2+, but also to Cu2+ involved oxidative cyclization reaction.
This is also a reason for the high sensitivity of Cu2+ recognition.
Fig. 7 Plot of the fluorescence enhancement (F) at 360 nm versus the concentrations of Cu2+ and
several other metal ions for 1a in ACN. Inset: the fluorescence spectra of 1a (1.5×10−5 mol L−1) in
the presence of 1 equiv. of Cu2+ and 1 equiv. of Cu2+ plus 20 equiv. of several other metal ions.
3.7. Fluorescence spectrum behaviors of 1a in the presence of Cu2+ in ACN/H2O buffer
solutions
To investigate the practical application of these receptors for the detection of Cu2+, we
evaluated the responses of compounds 1a–c to ACN/H2O solutions containing Cu2+ ions.
Receptors 1a and 1c showed enhanced fluorescence emission spectra following the addition of
Cu2+ in 9:1 (v/v) mixture of ACN and H2O, with 1a showing a slightly higher level of
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enhancement towards Cu2+ than 1c (Fig. S8). In contrast to these two receptors, 1b had very little
impact on the fluorescence emission. Based on this result, receptor 1a was used in the subsequent
experiments. We initially examined the response of receptor 1a to a solution of Cu2+ in ACN/H2O
buffer solution over a range of different pH values, and pH 7.2 was found to be optimum for the
quantification of Cu2+ using 1a as a receptor (Fig. S9). A solution of 1a in a 9:1 (v/v) mixture of
ACN and H2O containing Tris-HCl buffer (pH = 7.2) showed very little fluorescence emission,
whereas the addition of Cu2+ led to a 200-fold increase in the fluorescence emission at 483 nm
(Fig. 8). However, no emission bands was observed at shorter wavelengths, even after the addition
of a large excess of Cu2+ (10 equiv.), presumably because the water molecules could prevent the
complexation of the Cu2+ ions to the 1,3,4-oxadiazole. Several other metal ions were tested,
including Zn2+, Hg2+, Pb2+, Cd2+, Ni2+, Ca2+, Mg2+, K+, Ba2+, Al3+, Fe2+, Co2+, Fe3+, Cu+ and Mn2+,
but had no discernible impact on the fluorescence spectra of 1a. This result therefore suggested
that receptor 1a could be used as a selective fluorescent receptor for the detection of Cu2+ in
ACN/H2O buffer solutions (Fig. S10).
The relationship between ΔF/F0 (ΔF = F–F0, where F0 and F are the fluorescence emission
intensities before and after the addition of Cu2+, respectively) and the concentration of Cu2+ in
10−6 mol L−1 (c) was measured under the conditions described above and found to be ΔF/F0 =
2.12×106c–16.56. The linear range of this method covered Cu2+ concentrations in range of
2.5×10−6 to 3.0×10−4 mol L−1 with a correlation coefficient of R2 = 0.9973. The detection limit of
this method was calculated using a previously reported procedure [38]. Briefly, the fluorescence
emission spectrum of 1a was measured 11 times and the standard deviation for the blank sample
was measurement (σ). The detection limit was then calculated using the following equation:
detection limit = 3σ/k, where k is the gradient of the fluorescence intensity versus the
concentration of Cu2+. In this case the detection limit was found to be 2.2×10−8mol L−1, which
indicated that receptor 1a was highly sensitive to the Cu2+ in the ACN/H2O buffer solutions.
Fig. 8 Fluorescence spectra of 1a (1.5×10−5 mol L−1) in ACN/H2O (9:1, v/v, pH=7.2)
3.8. Application of receptor 1a in azurite deterimination
Receptor 1a was used to determine the Cu2+ in azurite to evaluate the practicality and
efficiency of this new method. A sample solution of azurite (11.05 mg mL−1) was prepared by
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dissolving powdered azurite (0.5525g) in 50mL hydrochloric acid (0.2 mol L−1).
3.8.1. Procedure for evaluating the application of receptor 1a
The addition of a suitable amount of Cu2+ led to an increase in the intensity of the enhanced
fluorescence signal. The amount of Cu2+ contained in a single sample of azurite can be determined
theoretically using standard curves generated using a solution of 1a as shown in Fig.9. The linear
equation was ΔF= -97.72+25.53c (10−5 mol Lˉ1) where c is the concentration of Cu2+ and the
correlation coefficient was 0.995.
An aliquot of the sample solution of azurite (11.05 mg mLˉ1) prepared above was diluted 100
times with a 9:1 (v/v) mixture of ACN and water. The pH of the diluted sample solution was 3.00,
so buffer solution (pH = 3.12) was selected to replace the water in the mixture. The buffer solution
was prepared with phosphoric acid, boric acid and acetic acid. Notably, the H+ ions present in the
mixture could cause fluorescence quenching and the presence of other ions could interfere with
the results, which may explain the poor linearity of this plot.
Fig. 9 Linear plot of F versus the concentration of Cu2+ for the reaction of Cu2+ with 1a
3.8.2. Recovery and accuracy properties
The accuracy and precision characteristics of our newly developed method were determined
by the repeated analysis of samples containing five different concentrations of Cu2+. The recovery
values were determined to be in the range of 97.9–102.4  1.02–2.16% (Table 2), thereby
highlighting the accuracy of our new method for the determination of Cu2+. Consideration of the
results obtained for the different concentrations revealed that the average recovery was
99.781.71%, demonstrating the good accuracy and reproducibility of this method.
Table 2
Recovery studies for the determination of azuritum based on its reaction with FLP according to
our new method.
3.8.3. Trial experiment
The experiment results were examined using the standard addition method by atomic adsorption
spectrometry (AAS), and the results are shown in Tables 3 and 4. Figures. S11 and S12 show the
linear graphs obtained from the AAS experiments , the correlation coefficient were 0.99966 and
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0.99983. The actual Cu2+ contents of the test samples of azurite were determined to be 24.48 and
22.51%, respectively, representing an average Cu2+ content of 23.50%. The sample solution (11.05
mg mLˉ1) was then diluted 100 times with a 9:1 (v/v) mixture of ACN and water. A 500 µL aliquot
of this diluted material was then transferred to a cuvette containing 1800 mL of ACN and 200 mL
of buffer solution, and the final percentage mass content was 23.60%. This result was close to the
value of 23.50% obtained by AAS. Taken together, these data show that our newly developed
fluorometric method is highly sensitive with a low detection limit and good accuracy.
Table 3
Results for the detection of Cu2+ in azurite using the standard addition method by AAS
Table 4
Results for detection of Cu2+ in azurite using the standard addition method by AAS
4. Conclusion
We have designed and synthesized two simple, neutral receptors (1a and 1c), which showed
high levels of selectivity towards Cu2+ in ACN and ACN/H2O buffer solutions. Notably, receptor
1a was successfully applied to the detection of Cu2+ in practical samples with satisfied recovery
and accuracy characteristics. Although receptors 1a and 1c exhibited intramolecular charge
transfer (ICT) characteristics, they also showed dramatic fluorescence enhancement in response to
Cu2+. This enhancement effect was attributed to the Cu2+-mediated oxidative cyclization of the
non-fluorescent receptors to give the corresponding 1,3,4-oxadiazoles, which are rigid and highly
fluorescent. Furthermore, the ICT channels of these receptors were closed after they underwent
the oxidative cyclization process. We have therefore developed a new strategy for the construction
of fluorescent receptors.
Acknowledgments
This work was supported by the National Natural Science Foundation of China (Grant no.
20835005) and the Doctoral Foundation of Southwest University (SWUB2008044).
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Fig. 1 Absorption spectra of 1a (1.5×10−5 mol L−1) in ACN in the presence of increasing amount
of Cu2+ (0.0–6.0×10−5 mol L−1).
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Fig. 2 Fluorescence spectra of 1a (1.5×10−5 mol L−1) in ACN in the presence of increasing
amounts of Cu2+ (a: 0–1.5×10−5 mol L−1; b: 1.5–6.0×10−5 mol L−1; c: 6.0×10−5–4.0×10−5 mol L−1).
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Figure. 3 HOMO, LUMO and LUMO+1 molecular orbitals of 1a and 1c at the B3LYP/6-31G*
calculation level (Isovalue=0.02).
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Fig. 4 Fluorescence spectra of 1c (1.0×10−5 mol L−1) in ACN in the presence of increasing
amounts of Cu2+ (a: 0–1.0×10−5mol L−1; b: 1.0–4.0×10−5mol L−1; c: 4.0×10−5–3.0×10−4mol L−1).
The excitation wavelength was 315 nm.
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Fig. 5 Fluorescence spectra of 1a (1.5×10−5 mol L−1) in ACN in the presence of increasing
amounts of KMnO4 (0–4.5×10−5 mol L−1) (red lines) and fluorescence spectra of 1a+KMnO4
solution in the presence of increasing amounts of Cu2+ (0–1.5×10−5 mol L−1) (black lines).
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(a)
(b)
Fig. 6 HOMO, LUMO and LUMO+1 molecular orbitals of two different1,3,4-oxadiazoles at the
B3LYP/6-31G* calculation level (Isovalue=0.02).
(a) and (b) were the oxidation product of receptors 1a and 1c, receptively.
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Fig. 7 Plot of the fluorescence enhancement (F) at 360 nm versus the concentrations of Cu2+ and
several other metal ions for 1a in ACN. Inset: the fluorescence spectra of 1a (1.5×10−5 mol L−1) in
the presence of 1 equiv. of Cu2+ and 1 equiv. of Cu2+ plus 20 equiv. of several other metal ions.
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Fig. 8 Fluorescence spectra of 1a (1.5×10−5 mol L−1) in ACN/H2O (9:1, v/v, pH=7.2)
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Fig. 9 Linear plot of F versus the concentration of Cu2+ for the reaction of Cu2+ with 1a
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Scheme 1: Synthesis of compounds 1a−c.
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Scheme 2. Proposed mechanism for the response of these receptors to Cu2+.
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