Jun Zhang1, Qiang Wu2, Bangliang Yu3, Chunwei Yu4. 1. Laboratory of Environmental Monitoring, School of Tropical and Laboratory Medicine, Hainan Medical University, Haikou 571101, China. jun_zh1979@163.com. 2. Faculty of Laboratory Medicine, School of Tropical and Laboratory Medicine, Hainan Medical University, Haikou 571101, China. wuqiang001001@aliyun.com. 3. Department of Pharmacy, Hainan Medical University, Haikou 571101, China. yubangliang@126.com. 4. Laboratory of Environmental Monitoring, School of Tropical and Laboratory Medicine, Hainan Medical University, Haikou 571101, China. cwyu@yic.ac.cn.
Abstract
A new fluorescent probe P derived from naphthalimide bearing a pyridine group has been synthesized and characterized. The proposed probe P shows high selectivity and sensitivity to Cu2+ in aqueous media. Under optimized conditions, the linear response of P (2 μM) toward Cu2+ was 0.05-0.9 μM in ethanol-water solution (3:2, v:v, 50 mM HEPES, pH 7.4), and the detection limit was 0.03 μM.
A new fluorescent probe P derived from class="Chemical">naphthalimide beariclass="Chemical">ng a class="Chemical">n class="Chemical">pyridine group has been synthesized and characterized. The proposed probe P shows high selectivity and sensitivity to Cu2+ in aqueous media. Under optimized conditions, the linear response of P (2 μM) toward Cu2+ was 0.05-0.9 μM in ethanol-water solution (3:2, v:v, 50 mM HEPES, pH 7.4), and the detection limit was 0.03 μM.
Development of optical sensors for the detection of environmental targets has been an class="Chemical">actively research topic receclass="Chemical">ntly. Because of its simplicity aclass="Chemical">nd high seclass="Chemical">nsitivity, the fluoresceclass="Chemical">nce techclass="Chemical">nique has become a powerful tool amoclass="Chemical">ng the methods available for chemical seclass="Chemical">nsors [1]. Most class="Chemical">n class="Chemical">metal ions play important roles in living systems and have an extreme eco-toxicological impact on the environment and humans. Among these various metal ions, Cu2+ is both a significant environment pollutant and an essential trace element in biological systems [2], so detecting the presence of Cu2+ has received considerable attention. So far, many Cu2+-selective fluorescent probes have been successfully devised [2-24]. It is unfortunate that only a few examples of “off-on” type probes are available due to the fluorescence quenching nature of paramagnetic Cu2+ [2-20]. In most practical applications, fluorescence quenching changes in fluorescence intensity can be interrupted by many other poorly quantified or variable factors such as photobleaching, probe molecule concentration, the environment around the probe molecule (pH, polarity, temperature, and so on), and stability under illumination, etc. [21-23]. To increase the selectivity and sensitivity of a measurement for analytical purposes, probes in which the binding of Cu2+ leads to a fluorescence enhancement are desirable. Therefore, there is still room to develop highly sensitive and selective “off-on” probes for Cu2+ in neutral aqueous media.
For the construction of a highly efficient probe for a target, it is necessary to choose an efficient fluorophore and consider the geometry of the coordination sites of the target [2,3,23]. class="Chemical">Naphthalimide derivatives, which are widely used as fluoresceclass="Chemical">nt dyes, have excelleclass="Chemical">nt photophysical properties, such as high fluoresceclass="Chemical">nce quaclass="Chemical">ntum yields, large Stokes shifts, stroclass="Chemical">ng absorptioclass="Chemical">n baclass="Chemical">nd aclass="Chemical">nd stability. Furthermore, the recogclass="Chemical">nitioclass="Chemical">n moiety should be prelimiclass="Chemical">narily coclass="Chemical">nsidered iclass="Chemical">n desigclass="Chemical">niclass="Chemical">ng probes because they are respoclass="Chemical">nsible for the selectivity aclass="Chemical">nd biclass="Chemical">ndiclass="Chemical">ng efficieclass="Chemical">ncy of the whole probe. class="Chemical">n class="Chemical">According to Hard-Soft-Acid-Base theory, O and Ndonor atoms established the high affinity for Cu2+ [14]. With this intention, a Cu2+-specific “off-on” type fluorescent probe P derived from naphthalimide with N and O as coordination sites was designed and synthesized (Scheme 1).
Experimental Section
Reagents and Instruments
All of the materials were analytical reagent grade and used without further purification. The class="Chemical">metal ioclass="Chemical">ns aclass="Chemical">nd aclass="Chemical">nioclass="Chemical">ns salts employed are class="Chemical">n class="Chemical">NaCl, MgCl2·6H2O, CdCl2, HgCl2, CaCl2·2H2O, FeCl3·6H2O, CrCl3·6H2O, Zn(NO3)2·6H2O, AgNO3, CoCl2·6H2O, MnCl2·4H2O, CuCl2·2H2O, NiCl2·6H2O, PbCl2, NaClO, NaNO3, Na2CO3, NaCl, NaAc, NaClO4, KBr and Na2HPO4, respectively. NMR spectra were recorded in DMSO-d6 at 25 °C on a Bruker WM-300 spectrometer (Fällanden, Switzerland). Electrospray ionization (ESI) analyses were performed on a Thermo TSQ Quantum Mass Spectrometer (Waltham, MA, USA). UV-Vis spectra were obtained on a Beckman DU-800 spectrophotometer (Bremen, Germany) with 1 cm quartz cell at 25 °C. Fluorescence measurements were carried out on a HORIBA Fluoromax-4 luminescence spectrometer (Paris, France). Fluorescence imaging was performed by confocal fluorescence microscopy on an Olympus FluoView Fv1000 laser scanning microscope (Osaka, Japan). pH values were measured with a PBS-3C pH-meter (Shanghai, China).
A stock solution of P (1 mM) was prepared in class="Chemical">DMSO. To 5 mL glass tubes, P (10 μL, 1 mmol) aclass="Chemical">nd a proper amouclass="Chemical">nt of class="Chemical">n class="Chemical">Cu2+ stock solution (1.0 mmol) were added succesively and then diluted with ethanol-water solution (3:2, v:v, 50 mM HEPES, pH 7.4). The resulting solution was thoroughly mixed. For all measurements, excitation and emission slit widths were 2 nm, excitation wavelength was 360 nm.
Results and Discussion
pH Effects on P and P with Cu2+
The influence of pH on the fluorescence response of probe P was determined first (Figure 1). At pH below 5.7, the fluorescence response of P was affected by pH to some extent. With the increase of pH from 5.7 and 10.0, “off-on” fluorescence signals at 432 nm were mainly caused by the addition of class="Chemical">Cu2+. This iclass="Chemical">ndicated that the receptor gradually captured class="Chemical">n class="Chemical">Cu2+ and formed the P-Cu2+ complex. In this work, pH 7.4 was chosen as an optimum experimental condition in that P could work with very low background fluorescence.
Figure 1.
Influence of pH on the fluorescence spetra of P (2 μM, ■) and P (2 μM, ▼) plus Cu2+ (50 μM) in ethanol-water solution (3:2, v:v). The pH was modulated by adding 1 M HCl or 1 M NaOH in HEPES buffers.
Fluorescence Spectral Response of P
An important feature of P was its selectivity toward class="Chemical">Cu2+ over other competitive species, aclass="Chemical">nd the selectivity experimeclass="Chemical">nts for probe P were coclass="Chemical">nducted as showclass="Chemical">n iclass="Chemical">n Figure 2. Fluoresceclass="Chemical">nce spectral chaclass="Chemical">nges of P were examiclass="Chemical">ned with additioclass="Chemical">n of class="Chemical">n class="Chemical">metal ions and anions including Na+, K+, Ag+, Mg2+, Ca2+, Zn2+, Pb2+, Cd2+, Co2+, Ni2+, Mn2+, Hg2+, Cu2+, Cr3+, Fe3+, Al3+, S2−, SO42−, SCN−, NO3−, CO32−, Cl−, Ac−, ClO4−, Br− and HPO42−. An obvious enhancement of fluorescence intensity at 432 nm was observed only upon addition of Cu2+, which was attributable to the complexation between P and Cu2+. In contrast, no obvious changes were observed in the case of other metal ions and anions. Moreover, to check the interferences from other metal ions and anions on the fluorescence signal of Cu2+, competition experiments were performed between Cu2+ and selected metal ions and anions (Figure 3). When selected metal ions and anions (50 μM) were added into ethanol-water solution (3:2, v:v, 50 mM HEPES, pH 7.4) of P (2 μM) containing Cu2+ (10 μM), the emission spectra displayed a similar pattern to that with Cu2+ alone. This experiment clearly demonstrated that selected metal ions and anions even in higher concentrations did not interfere the Cu2+ detection, which made it applicable for Cu2+ sensing in the real sample.
Figure 2.
(a) Fluorescence spectra of P (2 μM) with different metal ions or (b) anions (50 μM) in ethanol-water solution (3:2, v:v, 50 mM HEPES, pH 7.4).
Figure 3.
(a) Fluorescence response of P (2 μM) to 10 μM of Cu2+ or to the mixture of 50 μM individual metal ions with 10 μM of Cu2+ in ethanol-water solution (3:2, v:v, 50 mM HEPES, pH 7.4); (b) Fluorescence response of P (2 μM) to 10 μM of Cu2+ or to the mixture of 50 μM individual anions with 10 μM of Cu2+.
In addition, the titration of P with various amounts of class="Chemical">Cu2+ iclass="Chemical">n class="Chemical">n class="Chemical">ethanol-water solution (3:2, v:v, 50 mM HEPES, pH 7.4) were studied (Figure 4).
Figure 4.
Fluorescence spectra of P (2 μM) in ethanol-water solution (3:2, v:v, 50 mM HEPES, pH 7.4) in the presence of different amounts of Cu2+. Inset: Fluorescence intensity at 432 nm as a function of Cu2+ concentration.
As the class="Chemical">Cu2+ coclass="Chemical">nceclass="Chemical">ntratioclass="Chemical">n iclass="Chemical">ncreased, the fluoresceclass="Chemical">nce emissioclass="Chemical">n iclass="Chemical">nteclass="Chemical">nsity at 432 class="Chemical">nm gradually iclass="Chemical">ncreased class="Chemical">n class="Chemical">accordingly. The linear fluorescence enhancement of P (2 μM) to Cu2+ was obtained in the range of 0.05–0.9 μM (R = 0.999; inset of Figure 4). The limit of detection (LOD) was 0.03 μM, based on 3 × δblank/k (where δblank is the standard deviation of the blank solution and k is the slope of the calibration plot).
The Binding Mechanism
To quantify the complexation ration between P and class="Chemical">Cu2+, a Job plot experimeclass="Chemical">nt was carried out by keepiclass="Chemical">ng the total coclass="Chemical">nceclass="Chemical">ntratioclass="Chemical">n of P aclass="Chemical">nd class="Chemical">n class="Chemical">Cu2+ at 10 μM (Figure 5). The results suggested that a 1:1 complex of P with Cu2+ was formed, which was supported by the presence of a peak at m/z 659.2 corresponding to P-Cu2+ in the ESI-MS spectrum of the components of the mixture of P and 1 equivalent Cu2+ in ethanol (Supplementary Material, Figure S4). The 1H-NMR spectra also indicated the binding of P with Cu2+ (Supplementary Material, Figures S5 and S6). The association constant K was determined from the slope to be 6.2 × 105 M−1, by plotting the fluorescence intensity 1/(F − F0) against 1/[Cu2+].
Figure 5.
Job's plot for P-Cu2+ complex, keeping the total concentration of P and Cu2+ as 10 μM.
From Figure 2, no significant changes in fluorescence spectra were observed when probe P was exposed to other class="Chemical">metal ioclass="Chemical">ns aclass="Chemical">nd aclass="Chemical">nioclass="Chemical">ns. We believe that this is due to a rapid isomerizatioclass="Chemical">n of the C=class="Chemical">n class="Chemical">N double bond in the excited state [26,27], though other mechanisms such as photoinduced electron transfer (PET) may also contribute to it. Notably, by adding Cu2+, the fluorescence character of P was different from free P and other metal ions and anions, its fluorescence at λem 432 nm was turned from “off” to “on”. The enhancement of fluorescence was likely due to restriction of acyclic C=N isomerization in the Schiff base upon addition of Cu2+ [25-27]. Accordingly, the proposed binding mode of P with Cu2+ can be illustrated as in Scheme 2. It is believed that this process is reversible, which has been proved by a test using EDTA-Cu2+ (Figure 6). As seen, in absence of Cu2+, probe P had a weak fluorescence. Addition of Cu2+ led to a reversible coordination with the ligand, resulting in an appearance of fluorescence enhancement at λem 432 nm. Thus, an “off-on” based fluorescent probe for Cu2+ was implemented.
Figure 6.
Fluorescence spectra in ethanol-water solution (3:2, v:v, 50 mM HEPES, pH 7.4). a: P (2 μM); b: P (2 μM) + Cu2+ (50 μM); c: P (2 μM) + Cu2+ (50 μM) + EDTA (100 μM); d: P (2 μM) + Cu2+ (50 μM) + EDTA (100 μM) + Cu2+ (100 μM); e: P (2 μM) + Cu2+ (50 μM) + EDTA (200 μM) + Cu2+ (100 μM).
Conclusions
In summary, an efficient “off-on” probe P for class="Chemical">Cu2+ was proposed. Our studies showed that P was a highly selective aclass="Chemical">nd seclass="Chemical">nsitive probe for class="Chemical">n class="Chemical">Cu2+, which could work in neutral aqueous solution media and has great potential use in environmental sensing applications. These results open up new possibilities for the construction of “off-on” probes for other metal ions.
Figure 1.
Figure 2.
Figure 3.
Figure 4.
Figure 5.
Figure 6.