Received Date: January 16, 2013; Accepted Date: February 11, 2013; Published Date: February 15, 2013
Citation: Chereddy NR, Thennarasu S, Mandal AB (2013) A Photo-Induced Electron Transfer Based Chemosensor for the Selective Detection of Zn2+ Ions. Biochem Anal Biochem 1:126. doi:10.4172/2161-1009.1000126
Copyright: © 2013 Chereddy NR, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Visit for more related articles at Biochemistry & Analytical Biochemistry
Aim: The chemosensors permitting naked eye detection of toxic metal ions are user-friendly, portable and obviate the requirement of sophisticated equipments. The objective of the present study was to develop a PET (photoinduced electron transfer) based chemosensor for Zn2+ that changes its color upon binding to Zn2+ allowing naked eye detection.
Methods: A new 4-piperazino-1,8-naphthalimide based fluorescent probe 1 was synthesized and its structure was determined using NMR and XRD techniques. The solvatochromic effects on the absorbance and fluorescence characteristics of 1 in aqueous and non-aqueous media were explored. Metal ion competition experiements were performed to monitor the interference of common ions like Li+, Na+, K+, Cu2+, Mg2+, Ca2+, Cr3+, Mn2+, Fe2+, Co2+, Ni2+, Zn2+, Cd2+, Hg2+ and Pb2+.
Results and conclusion: The significance of the solvatochromic effect in colorimetric and fluorometric detection of Zn2+, and turn-off sensing of Cu2+ via metal ion displacement have been emphasized. In non-aqueous environment, probe 1 acts as a turn-on chemosensor for Zn2+ and as a turn-off chemosensor towards Cu2+ and thereby, enables the detection of Zn2+ and Cu2+ ions in two different modes. In aqueous environment, the probe 1 acts only as a turn-off chemosensor for Cu2+ ion
Chemosensor; Naked eye detection; Solvatochromic effect; Photo-induced electron transfer
Zinc, following iron, is the second most abundant transition metal in humans. About 2-3 g of zinc is present in an adult human body . Zinc (II) ions play several roles in biological processes such as enzyme regulators, DNA binding or recognition motifs, catalytic centres and neural signal transmission . The most important role for zinc is as a structural cofactor in metalloproteins . The disruption of zinc pools, located primarily in muscle and bone  can lead to a number of diseases such as type I and type II diabetes [5,6], neural malfunction  particularly Alzheimer’s disease  and certain cancers . Zinc is now recognized as an important factor in the regulation of apoptosis . Hence, design and synthesis of new chemosensors for the selective determination of zinc in trace levels is attaining importance. Moreover, the d electronic configuration  of Zn (II) makes it inactive and forbids UV-Vis spectroscopic determination. Therefore, new fluorescent indicators that show “off-on” signal in response to the presence of zinc metal ions will be advantageous in the detection and imaging of zinc sources as well as zinc contamination.
4-amino-1,8-naphthalimide is one of the most attractive fluorochromes because of its desirable properties, such as an excellent photostability, high luminescence efficiency, large Stokes shift and easy modification of the molecular structure [11,12]. Several probes for Zn2+ and Cu2+ have been reported [13-31] with 4-amino-1,8-naphthalimide as the signalling moiety, but none of them are useful for detection of both the metal ions on duel “off-on” or “on-off” modes. Generally, the fluorescence of 4-piperazino-1,8-naphthalimide derivatives is quenched due to PET arising from the piperazine nitrogen [32-35]. In the present study, binding of a Zn2+ ion quenches the PET process, resulting in the fluorescence emission from the chemosensor. Since the stability of metal complexes are influenced by the solvent composition, and solvent medium plays an important role on the metal ion selectivity [36-37]. It is imperative to study the effect of solvents while addressing metal ion selectivity and designing chemo sensors.
For the present study, we synthesized a 4-piperazino-1,8- naphthalimide based fluorescent probe 1 for the selective detection of Zn2+ ions. The solvatochromic effects of water and the routinely used non-aqueous solvent CH3CN on the stability of 1-Zn2+ complex were encountered during the investigation. Interference from other competing metal ions was also investigated. The paramagnetic ion Cu2+ quenched the fluorescence of 1 as expected, even though Cu2+-induced enhancement in fluorescence emission were observed with rhodamine based chemosensors [37-39]. The effect of the routinely used solvents water and acetonitrile on the selective detection of Zn2+ and Cu2+ ions, and the mechanistic aspects involved in complex formation were explored.
Dry acetonitrile and double distilled water were used in all experiments. All the materials for synthesis were purchased from commercial suppliers and used without further purification. The solutions of metal ions were prepared from the corresponding chloride salts. Absorption spectra were recorded on a SPECORD 200 PLUS UV-Visible spectrophotometer. Fluorescence measurements were performed on a Cary Eclipse fluorescence spectrophotometer. NMR spectra were recorded using a JEOL-ECP500 MHz spectrometer operated at 500 MHz. ESI-MS spectra were obtained on a Thermo Finnigan LCQ Advantage MAX 6000 ESI spectrometer. Absorption and fluorescence measurements were made using a 3.0 mL cuvette.
Synthesis of bromoacetylamino quinoline (o)
The bromoacetylamino quinoline was synthesized in a single-step procedure. To a mixture of 8-aminoquinoline (0.72 g, 5.0 mmol) and triethylamine (0.60 g, 6.0 mmol) in dichloromethane (DCM) (20 mL) at 0ºC, bromoacetylbromide (1.21 g, 6.0 mmol) was added drop-wise, and the reaction mixture was stirred at room temperature for 3 h. The progress of the reaction was monitored using TLC. After completion, the reaction mixture was washed with water and subjected to column chromatography (silica gel 100-200 mesh) and eluted using hexaneethtyl acetate (98:2) mixture to get 1.06 g (80%) of bromoacetylamino quinoline (B) in pure form as pale yellow solid.
Synthesis of naphthalimide derivative 1
To a solution of 4-bromo-1,8-naphthanoic anhydride (1.0 g, 3.6 mmol) in EtOH maintained at 80ºC, propylamine (1.0 mL) was added slowly. The resulting mixture was stirred for 1h and cooled to room temperature. The precipitate formed was filtered and used in the next step. To this product, 4-bromo-N-propyl-1,8-naphthanamide, (0.7 g, 2.2 mmol) in DMSO, piperazine (1.00 g, 11.6 mmol) and K2CO3 (0.42 g, 3.0 mmol ) were added and stirred at 120ºC for 3h. After completion of the reaction, the mixture was extracted with DCM, concentrated and subjected to column chromatography (silica gel 100-200 mesh) and eluted using ethyl acetate to get 0.50 g (70%) of 4-piperazinyl-N-propyl- 1,8-naphthalimide (p). In order to prepare the desired compound 1, 4-piperazinyl-N-propyl-1,8-naphthalimide (p) (0.32 g, 1.0 mmol) and K2CO3 (0.14 g, 1.0 mmol) were dissolved in DMF. To the resultant mixture, bromoacetylamino quinoline (o) (0.30 g, 1.1 mmol) was added and stirred at room temperature overnight. After completion of the reaction, the mixture was extracted with DCM, concentrated and subjected to column chromatography (silica gel 100-200 mesh) and eluted using hexane-ethtyl acetate (80:20) mixture to get 0.40 g (80%) of probe 1 as yellow solid.
NMR and mass data of napthalimide derivative 1
1]H NMR (CDCl3, 500 MHz), δ (ppm): 1.02 (t, J=7.6 Hz, 3H, NCH2CH2CH3), 1.78 (m, 2H, NCH2CH2CH3), 3.05 (s, 4H, Piperazine- CH2), 3.51 (d, 2H, N-CH2-C=O), 3.49 (m, 4H, Piperazine-CH2), 4.15 (t, 2H, NCH2CH2CH3), 7.31(d, J = 8.0 Hz, 1H, Ar-H), 7.46 (m, 1H, Ar-H), 7.55 (m, 2H, Ar-H), 7.71 (t, J=8.0 Hz, 1H, Ar-H), 8.17 (dd, J=1.5 Hz, 1H, Ar-H), 8.44 (d, J=8.0 Hz, 1H, Ar-H) 8.58 (m, 2H, Ar-H), 8.79 (dd, J=1.5 Hz, 1H, Ar-H) 8.85 (dd, J=1.0 Hz, 1H, Ar-H), 11.49 (s, 1H, Amide-NH). 13C NMR (CDCl3, 100 MHz), δ (ppm): 11.66, 21.53, 41.90, 53.43,53.59, 62.49, 115.07, 116.73, 117.10, 121.73, 121.99, 123.42, 125.91, 126.28, 129.97, 130.25, 131.25, 132.57, 134.26, 136.37, 139.08, 148.70, 155.86, 164.15, 164.57,168.75. ESI-MS: calcd for C30H29N5O3 m/z (M+) 507.2, found (M+H)+ 508.3.
Synthesis and structure determination of probe 1
The 4-piperazino-1,8-naphthalimide based fluorescent probe 1 was synthesized in four convenient steps as outlined in figure 1 and characterized using NMR (Figure S1-S2, ESI), ESI-Mass (Figure S3, ESI), and single crystal X-ray crystallographic techniques (Figure 2, CCDC 888111). The proton NMR spectrum of 1 in CDCl3 displayed a triplet at 7.6 ppm, a triplet 4.15 ppm and a multiplet at 1.78 ppm arising from the propyl group attached to the naphthalimide moiety. The two broad peaks at 3.48 and 3.05 ppm were assigned to the two types of methylene protons on the piperazine moiety. The presence of a doublet at 3.51 ppm corresponding to the linker moiety (N-CH2-C=O) confirms the attachment of 8-aminoquinoline to the 4-piperazino-1,8- naphthalimide derivative. The signals seen in the 13C NMR spectrum are in good agreement with the proposed structure. ESI-Mass analysis of the probe 1 also supports the proposed structure. Finally, X-ray diffraction analysis of single crystals of the probe 1 confirmed the three dimensional structure (Figure 2).
Metal ion selectivity and sensitivity of probe 1
The fluorescence of 4-piperazino-1,8-naphthalimide derivatives are usually quenched either completely or partially due to PET [32-35]. During our screening experiments involving different solvents and metal ions, probe 1 showed significant quantum of fluorescence in aqueous and non-aqueous media indicating partial quenching due to PET process. The fluorescence emission intensity of probe 1 in neat CH3CN was decreased upon addition of incremental quantities of water, indicating the ability of water to interfere with the life time of the excited state of probe 1, presumably, facilitating the non-radioactive decay. Hence, we investigated the probe’s ability for the detection of different metal ions in aqueous and non-aqueous media.
A 10 μM solution of probe 1 in CH3CN emits green fluorescence, as shown in figure 3. Addition of various metal ions (50 μM) like Li+, Na+, K+, Mg2+, Ca2+, Cr3+, Mn2+, Fe3+, Co2+, Ni2+, Cd2+, Hg2+ or Pb2+ does not change either the colour or the fluorescence intensity of the solution. Interestingly, addition of 50 μM Zn2+ produces a distinct colour change due to fluorescence enhancement, while 50 μM Cu2+ turns off the fluorescence signal. This dual performance of probe 1 enables the naked-eye detection of both Zn2+ and Cu2+ ions in ‘On’ and ‘Off’ modes, respectively (Figure 3a). However, when the solvent system is changed from CH3CN to CH3CN/H2O (1:1) mixture, the probe 1 fails to discriminate Zn2+ from other metal ions while retaining the ‘Off’ signal for Cu2+ (Figure 3b). It appears, therefore, that the high polarity of aqueous CH3CN (1:1 CH3CN/H2O) medium effectively nullifies the Zn2+-induced enhancement in the fluorescence of 1, and thereby, permits only Cu2+ selective ‘turn-off’ sensing.
Influence of solvent on Zn2+ and Cu2+ complex formation
The absorbance characteristics of probe 1 (10 μM) in CH3CN and CH3CN/H2O (1:1) mixture containing equimolar quantities of different metal ions are shown in Figure 4. In neat CH3CN, probe 1 exhibits two absorbance maxima, one corresponding to quinoline moiety (~242 nm) and the other arising from naphthalimide moiety (~408 nm). Addition of various metal ions (10 μM), excepting Zn2+ and Cu2+, does not induce any changes in the absorption pattern of 1 (Figure 4a). The addition of Zn2+ (10 μM) causes a red shift in the absorbance maximum of quinoline moiety (from ~242 to ~255 nm). However, no significant changes are observed in the absorbance maximum of naphthalimide moiety (Figure 4a). This marked red shift in the absorbance maximum of quinoline moiety and the absence of any significant changes in the absorbance maximum of naphthalimide moiety clearly indicate that Zn2+ is bound to the probe 1, but not to the piperazine ‘N’ attached to the naphthalimide moiety. A similar titration in CH3CN/H2O (1:1) mixture (Figure 4b) shows the absence of any red shift in the absorbance maximum of quinoline moiety, and indicates the destabilizing effect of water in the formation and stability of 1-Zn2+ complex.
A similar study with Cu2+ provides a different picture of the destabilizing effect of water on the stability of 1-Cu2+ complex (Figures 4a and 4b). The addition of Cu2+ (10 μM) shifts the absorbance maximum of quinoline moiety (red shift: from ~242 to ~255 nm) and that of naphthalimide moiety (blue shift: from ~408 to ~399 nm) in neat CH3CN. The blue shift is more pronounced in CH3CN/H2O (1:1) mixture (from ~399 to ~389 nm). These shifts in absorbance clearly indicate that Cu2+ is bound to the piperazine ‘N’ attached to the naphthalimide moiety, and thereby, decreases the electron donating capacity of the latter in both the solvents.
The variations in the absorbance pattern of probe 1 (10 μM) in neat CH3CN, in response to the addition of serial concentrations of Zn2+ and Cu2+ are shown in figure 5. In both the cases, the absorbance at ~255 nm reached the maximum at 20 μM concentrations of metal ions. However, the incremental increase in the peak intensity at ~255 nm is more pronounced on the addition of Cu2+ ions (Figure 5b) than on the addition of Zn2+ ions (Figure 5a). This observation sheds light on the relative stabilities of the respective complexes formed.
In a similar experiment carried out in 1:1 CH3CN/H2O medium, significant variations in the absorbance of probe 1 (10 μM) are observed only upon addition of serial concentrations of Cu2+ ions. Upon addition of one equivalent of Cu2+, the absorbance maximum at ~255 nm reaches the maximum level (Figure 6b) suggesting the formation of a 1:1 complex with probe 1. Such a complex formation with Zn2+ is not observed even after the addition of 30 μM Zn2+ ions as indicated by the absence of the red shifted peaks at ~255 nm arising from the absorbance maximum of quinoline moiety (Figure 6a).
Solvent assisted dual mode sensing of Zn2+ and Cu2+
Since the fluorescence of probe 1 is quenched by water, all fluorescence measurements were carried out in 100% CH3CN for dual detection of Zn2+ and Cu2+ ions. The effect of different metal ions on the fluorescence emission intensity of probe 1 (10 μM) is shown in figure 7a. Whereas the addition of Zn2+ (10 μM) enhances the fluorescence to a significant level, the addition of Cu2+ (10 μM) quenches the fluorescence emission intensity almost completely. Confirmation of the opposing contributions of Zn2+ and Cu2+ is seen in the fluorescence spectra of 1 in the presence of serial concentrations of Zn2+ and Cu2+ ions (Figures 7b and 7c). Other common metal ions do not affect the fluorescence emission spectrum of probe 1. These results reveal that probe 1 could be used to detect Zn2+ and Cu2+ from normally co-existing metal ions, albeit, at two different modes.
The metal ion competition experiments exemplifies that Zn2+ (10 μM) induced fluorescence enhancement of probe 1 is unaffected by even five-fold excess amounts of other metal ions like Li+, Na+, K+, Mg2+, Ca2+, Cr3+, Mn2+, Fe3+, Co2+, Ni2+, Cd2+, Hg2+ and Pb2+. However, complete quenching of fluorescence is observed by the addition of an equimolar quantity of Cu2+, indicating the higher binding affinity for Cu2+ as compared to Zn2+ (Figure S4, ESI). The higher binding affinity of probe 1 for Cu2+ over Zn2+ is confirmed by the binding constants calculated  for 1-Cu2+ (5.8×105 M-1) and 1-Zn2+ (3×105 M-1) complexes. Further, addition of Cu2+ (10 μM) to probe 1 equilibrated with the other metal ions (50 μM) also results in complete quenching of fluorescence (Figure S5, ESI).
It appears, therefore, that upon addition of Cu2+ to the fluorescent 1-Zn2+ complex, Cu2+ replaces Zn2+ in the complex leading to the formation of a non-fluorescent 1-Cu2+ complex (Figure 8). Moreover, addition of EDTA to 1-Cu2+ complex restores the fluorescence characteristics of probe 1. This indicates that the formation of 1-Zn2+ and 1-Cu2+ complexes is reversible and that the changes in the fluorescence characteristics of probe 1 in response to the addition of Zn2+ and Cu2+ ions are due to complex formation (Figure S6, ESI) and not due to any catalytic action.
Thus, the UV-Visible and fluorescence experiments clearly establish Zn2+ induced red shift of the absorbance of quinoline moiety in probe 1. This red shift in the absorbance could be attributed to the change in the electronic delocalization of the quinoline moiety upon Zn2+ binding. Further insight into the reason as to why these changes occur in the absorption and fluorescence emission upon addition of Zn2+, could be gained using NMR and ESI-MS techniques.
Binding sites for Zn2+ and Cu2+ on probe 1 are different
Analysis of the 1H-NMR spectra of the probe 1 alone, and in the presence of serial concentrations of Zn2+ (Figure S7, ESI) clearly show that the piperazine protons labelled as ‘a’ in figure 9, shift to down field, and that the intensity of the amide proton decreases while the intensity of the other protons remain unchanged. The deshielding effect experienced by the piperazine protons labelled as ‘a’ is not observed with the piperazine protons labelled as ‘b’ in figure 9.
The 13C-NMR spectra recorded under identical conditions (Figure S8, ESI) show the disappearance of the amide carbonyl resonance at ~170 ppm, indicating its involvement in complex formation. Taken together, the NMR data confirm the involvement of the piperazine ‘N’ and carbonyl ‘O’, in 1-Zn2+ complex formation and the induction of extended conjugation with the quinoline moiety. This also explains the red shift in the absorbance maximum of quinoline moiety upon 1-Zn2+ complex formation. Since the piperazine ‘N’ has the ability to quench the fluorescence of the naphalimide moiety by PET process, the 1-Zn2+ complex formation would decrease the electron density at piperazine ‘N’, reduce the effect of PET process on napthalimide moiety, and thereby, enhance the fluorescence intensity. This proposition is further supported by ESI-MS spectrum (Figure S6, ESI). The ESI-MS spectrum of 1-Zn2+ complex also confirms the 2:1 ligand:metal geometry of the fluorescent complex with subsequent deprotonation of amide ‘NH’ as shown in figure 9. Further, addition of Cu2+ to the fluorescent 1-Zn2+ complex, leads to the formation of a non-fluorescent 1-Cu2+ complex via metal ion displacement (Figure 8).
The synthesis, structure determination and physicochemical properties of a chemosensor that emits fluorescence only upon complex formation with Zn2+, are described. Probe 1 selectively detects Cu2+ in aqueous environment by fluorescence “on-off” mechanism. In non-aqueous medium, it acts as a dual sensor for Zn2+ and Cu2+, albeit, in two different modes. The mechanistic aspects involved in metal ion detection are demonstrated. Water plays a significant role in colorimetric and fluorometric detection as well as metal-ligand complex formation. The observations of this study emphasize the importance of solvent screening during the development of new fluorescent probes for the selective detection of specific metal ions.