Introduction

Mercury is a highly toxic and hazardous pollutant with recognized accumulative and persistent characteristics in the environment and biota [1]. Excessive exposure of the vital organs and tissues to mercury will lead to the dysfunction of the brain, kidney, and stomach, and to central nervous system defects [2, 3]. Given these environmental and toxicological concerns, there is a considerable interest in the development of new detection methods for Hg2+ in the environment and in biological samples [4]. Current techniques for Hg2+ screening usually require expensive and sophisticated instrumentation. However, fluorescent chemosensors detect Hg2+, at relatively low cost, high selectivity, sensitivity, and simplicity. The design of fluorescent chemosensors is mainly based on intramolecular charge transfer [5, 6], through bond energy transfer [7], fluorescence resonance energy transfer [8–10], and photoinduced electron transfer (PET). Currently, PET is an active field in supramolecular chemistry because of the ‘fluorophore-spacer-receptor’ format [11]. A number of Hg2+-selective based PET fluorescent chemosensors have therefore been reported in recent years [12–16].

Although many chemosensors for Hg2+ are successful, there are some factors, such as water insolubility, fluorescence quenching by many heavy- and transition-metal ions, interference by protons, cross-sensitivity toward other metal ions, and nonreversible responses, which limit their application in biological and environmental systems [14]. Thus, to overcome these disadvantages and further broaden their application, there is a great need for the development of improved simple chemosensors capable of detecting Hg2+.

Among many fluorophores, naphthalimide has been widely used as a signaling handle for the design of functional supramolecules [17] because of its advantageous optical characteristics, such as a large stokes shift, high fluorescence quantum yield, modest excitation and emission wavelengths, high photostability, and a high absorption coefficient [18–22]. We previously reported an Hg2+-selective fluorescent chemosensor, which was composed of two aminonaphthalimide fluorophores and a 2,6-bis(aminomethyl)- pyridine receptor [13] and could be used for the real-time detection of Hg2+ inside a single living cell [23]. However, other metal ions, such as Zn2+, Cd2+, Pb2+, and Ag+, also caused slight fluorescence enhancement.

Based on our previous works, in this study, we report a novel PET fluorescent chemosensor, H1, which is composed of a naphthalimide fluorophore and a 6-[(quinolin-8-yloxy)methyl]pyridine-2-ylmethanamine receptor. This fluorescence chemosensor for Hg2+ has effectively solved the problem of interferences from other transition-metal ions. In addition, it shows reversible and fast responses toward Hg2+ in a neutral buffered aqueous solution.

Experimental Section

Apparatus and Reagents

Fluorescence steady state spectra and fluorescence lifetime were obtained on a Hitachi F-4500 (Tokyo, Japan) and Edinburgh instruments FLS920 (Livingston, UK), respectively. UV–vis absorption spectra were measured on a Puxi TU-1901 (Beijing, China) spectrophotometer. All pH measurements were made with a Sartorius basic pH-meter PB-10 (Göttingen, Germany). 1H NMR and 13C NMR spectra were recorded on a Bruker AV-400 spectrometer with chemical shifts recorded as ppm (in CDCl3, TMS as internal standard). Infra-red spectral data were measured with Nicolet Avatar-370. Mass spectral analyses were carried out on an Agilent 6310 ESI-Ion Trop Mass spectrometer (Santa Clara, CA, USA).

A stock solution of 50 mM Hg2+ was prepared in double-distilled water. A 0.1M HEPES buffer solution (pH 7.2) was used. Double-distilled water was used throughout the experiments. All chemicals were purchased from commercial suppliers and used without further purification. N-butyl-4-butylamino- 1,8-naphthalimide (Φ s = 0.81) in absolute ethanol was used as a quantum yield standard [21]. The fluorescence quantum yield (Φ fl) was calculated using the relative method according to the equation:

$$ {\Phi_{\text{fl}}} = {\Phi_{\text{s}}} \times \left( {F/{F_{\text{s}}}} \right) \times \left( {{A_{\text{s}}}/A} \right) \times {\left( {n/{n_{\text{s}}}} \right)^{{2}}} $$

Where Φ s is the reported quantum yield of the standard, F and F s are the area of sample and standard solutions under the emission spectra, A and A s are the absorbance of sample and standard solutions at the excitation wavelength (390 nm), respectively; n s and n are refractive indexes of ethanol and methanol–water (1:9, v/v) solution, respectively.

General Procedure for H1 Synthesis

The chemosensor (H1) was synthesized from 2-chloromethyl-6-[(quinolin-8-yloxy)methyl]pyridine (V1) and 4-(piperazin-1-yl)-N-[(2-(2-hydroxyl)ethoxy)ethyl]-1,8-naphthalimide (V2) [24], as follows (Scheme 1).

Scheme 1
scheme 1

Synthesis of H1

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V1 (0.15 g, 0.52 mmol) and V2 (0.22 g, 0.60 mmol), in the presence of K2CO3 (0.11 g, 0.80 mmol) were dissolved in 15.0 mL of dry acetonitrile. The reaction mixture was stirred under a nitrogen atmosphere and heated under reflux for 5 h. After removal of the solvent, the residue was purified by silica column chromatography with CHCl3/methanol to afford H1 as 0.28 g (yield: 87 %) of a yellow oil. The structure of H1 was characterized by infra-red, 1H NMR, 13C NMR and electrospray ionization mass spectrometry (ESI-MS).

IR (KBr) ν

3353.4, 2926.0, 2854.8, 1687.7, 1649.3, 1572.6, 1539.7, 1506.8, 1457.5, 1375.3, 1315.1, 1232.9, 1189.0, 1117.8, 1084.9, 1052.1, 997.3, 821.9, 783.6, 750.9, 668.5 cm−1.

1H NMR (CDCl3, 400 MHz) δ (*10−6)

2.58 (br, 1-H, 1H), 2.89 (br, 12-H, 4H), 3.35 (br, 11-H, 4H), 3.67-3.69 (m, 2-H, 3-H, 4H), 3.84 (t, 4-H, J = 5.4 Hz, 2H), 3.87 (s, 13-H, 2H), 4.44 (t, 5-H, J = 5.4 Hz, 2H), 5.58 (s, 17-H, 2H), 7.08 (dd, 18-H, J 1 = 2.0 Hz, J 2 = 6.8 Hz, 1H), 7.22 (d, 9-H, J = 8.0 Hz, 1H), 7.36-7.43 (m, 14-H, 19-H, 20-H, 3H), 7.46 (dd, 22-H, J 1 = 4.0 Hz, J 2 = 8.4 Hz, 1H), 7.58 (d, 16-H, J = 7.6 Hz, 1H), 7.69 (t, 7-H, 15-H, J = 7.4 Hz, 2H), 8.15 (dd, 21-H, J 1 = 1.6 Hz, J 2 = 8.4 Hz, 1H), 8.43 (d, 8-H, J = 8.4 Hz, 1H), 8.52 (d, 6-H, J = 8.4 Hz, 1H), 8.59 (d, 10-H, J = 7.2 Hz, 1H), 9.00 (dd, 23-H, J 1 = 1.6 Hz, J 2 = 4.4 Hz, 1H).

13C NMR (CDCl3, 100 MHz) δ (*10−6)

39.59, 53.29, 53.68, 62.12, 64.67, 68.79, 71.81, 72.45, 110.00, 115.18, 116.64, 120.25, 120.38, 121.99, 122.46, 123.29, 125.89, 126.38, 126.82, 129.79, 130.24, 130.84, 131.60, 133.12, 136.20, 137.69, 140.64, 149.74, 154.34, 156.48, 157.08, 157.91, 164.60, 165.09.

ESI MS

m/z [M+H]+ Calcd. 618.27, Found 618.0.

Results and Discussion

Hg2+-Titration and Spectral Responses

The changes in UV–vis spectra of H1 with the gradual addition of Hg2+ in a buffered aqueous solution (pH 7.2) were investigated (Fig. 1). The absorption band appeared at 405 nm (ε = 1.1 × 104 M−1·cm−1) was observed in the absence of Hg2+. When Hg2+ (0–2 equiv) was added to a solution of H1 (10 μM) in methanol–water (1:9, v/v), the band centered at 405 nm progressively shifted to 390 nm (ε = 1.3 × 104 M−1·cm−1), indicating that the electron-donating nitrogen atoms of the receptor, in the ground state, participated in H1/Hg2+ complexation. The presence of two isosbestic points, at 303 nm and 413 nm, indicated the coexistence of free H1 and the H1/Hg2+complex.

Fig. 1
figure 1

The absorption responses of H1 (10 μM) upon addition of Hg2+ in HEPES buffered aqueous solution (methanol/water = 1:9, v/v, pH 7.2)

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The changes in the emission spectrum of H1 (10 μM) and the intensity, monitored at 550 nm, with stepwise addition of Hg2+ is showed in Fig. 2. At first, H1 has a weak fluorescent intensity (Ф 0 = 0.13). The fluorescence intensity of H1 increased significantly upon gradual addition of Hg2+. In contrast, this emission (Ф = 0.42) was saturated by ≥1 equiv of Hg2+. Significantly, the intensity at 550 nm increased linearly with the Hg2+ concentration, which indicated that H1 could potentially be used for the quantitative determination of Hg2+. The linear equation was found to be y = 4.3076 x + 1.082 (linearly dependent coefficient: R 2 = 0.9914). The detection limit was calculated to be 63 nM (3σ/slope). This behavior is also diagnostic for a H1/Hg2+formation of a complex with 1:1 stoichiometry. According to reported methods [25, 26], the associated constant was determined to be 1.11 × 105 M−1.

Fig. 2
figure 2

The fluorescence responses of H1 (10 μM) upon addition of Hg2+ in HEPES buffered aqueous solution (methanol/water = 1:9, v/v, pH 7.2). Excitation wavelength was set at 390 nm with excitation and emission slit at 1.0/5.0 nm

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Effect of pH on Chemosensor Performance

The pH of the environment around the fluorescent chemosensor usually affects its performance because of protonation or deprotonation of the fluorophore [27]. The influence of pH on H1 was determined in methanol–water (1:9, v/v) solution. Figure 3 shows the UV–vis spectroscopic changes; there are three isosbestic points at 260 nm, 320 nm and 405 nm. The band centered at 385 nm progressively red shifted to 415 nm with increasing pH.

Fig. 3
figure 3

UV–vis absorption spectra of H1 (10 μM) upon influence of pH in aqueous solution (methanol/water = 1:9, v/v)

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The fluorescence responses of H1 (10 μM) in methanol/water solution (1:9, v/v) were adjusted with varying amounts of perchloric acid and NaOH (Fig. 4). The fluorescence intensity increased about 13-fold and the maximum emission wavelength blue-shifted from 560 nm to 545 nm with a pH decrease from 9.0 to 5.3. From pH 2.5 to 4.9, the fluorescence intensity of the H1 steadily increased (Fig. 5). H1 contains two main proton receptors, namely a piperazinyl group and a quinolinyl group. The receptors have sufficiently different pK a values (7.78 for N-benzoylpiperazine versus 4.60 for 8-methylquinoline as models) [28, 29] for them to be stepwise protonated. Gan et al. [30] have studied the luminescent properties and PET of naphthalimides with piperazine substituents; their studies suggested that the fluorescence intensity of the 4-amino-1,8-naphthalimide fluorophore did not decrease under acid conditions. The quinoline moiety contains a pyridyl nitrogen group, which is able to act as a weak base under acidic conditions [31]. A PET quenching process from the fluorescent signaling unit to the quinolinium cation receptor is therefore responsible for the fluorescent properties of H1.

Fig. 4
figure 4

Fluorescence spectra of H1 (10 μM) upon influence of pH in aqueous solution (methanol/water = 1:9, v/v). Excitation wavelength was set at 390 nm with excitation and emission slit at 1.0/5.0 nm

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Fig. 5
figure 5

pH titration profiles of the H1 and H1/Hg2+ complex in aqueous solution (methanol/water = 1:9, v/v). Excitation wavelength was set at 390 nm with excitation and emission slit at 1.0/5.0 nm

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To study the applicability of H1, the effects of pH on the fluorescence response of H1/Hg2+ were investigated. The experiments were carried out in the pH range 2.0–12.0, with the concentration of H1 fixed at 10 μM and that of Hg2+ at 50 μM (Fig. 5). At pH values higher than 9.0, the fluorescence intensity of H1/Hg2+ and that of H1 become closer to each other, probably because the formation of a hydroxo-complex of Hg2+ is favored under these conditions [32]. From pH 9.0 to 4.0, the fluorescence intensity steadily increased, which indicated that H1 and Hg2+ effectively formed a complex. However, when the pH decreased from 3.6 to 3.0, the fluorescence intensity decreased slightly; this might be attributable to the large number of protons transforming the quinolinyl group into a quinolinium cation [33, 34]. This behavior may not favor for the formation of H1/Hg2+complexes. At pH < 3.0, the intensity returned to the original H1 value, indicating complete dissociation, and thus Hg2+ no longer affected the fluorescence intensity of H1.

Selectivity Studies

The selectivity of H1 for various metal ions was investigated. The selectivity of H1 for Hg2+ over various other detected metal ions was high. Even chemically closely related metal ions (e.g., Cd2+ and Pb2+) did not quench or generate fluorescence (Fig. 6). H1 shows a very weak emission as a result of the efficient PET quenching of the excited state of the 4-amino-1,8-naphthalimide moiety by the lone pair of electrons on the nitrogen atom in piperazine. On adding Hg2+, the fluorescence intensity of H1 increases 4-fold. The recognition process was shown to respond to Hg2+ via inhibition of PET. This showed that H1 could discriminate Hg2+ from other metal ions fluoroscopically.

Fig. 6
figure 6

Fluorescence responses of H1 (10 μM) at 550 nm in HEPES buffered aqueous solution (methanol/water = 1:9, v/v, pH 7.2) after the addition of 50 μM of various metal ions. Excitation wavelength was set at 390 nm with excitation and emission slit at 1.0/5.0 nm

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Figure 7 shows the results of an experiment to explore further the use of H1 as an ion-selective fluorescent chemosensor for Hg2+. The fluorescence changes in the chemosensor were highly specific for Hg2+ in the presence of other abundant cellular metal ions (e.g., Na+, K+, Mg2+, and Ca2+), essential transition-metal ions in cells (e.g., Zn2+, Fe2+, Co2+, and Ni2+), and environmentally relevant heavy metal ions (e.g., Ag+, Pb2+, Cr3+, and Cd2+). Excess amounts of these metal ions were added to 50 μM Hg2+ in a buffered methanol/water (1:9, v/v) solution and the fluorescence responses of the chemosensor were detected and then compared with that of a buffer aqueous solution containing only 50 μM Hg2+. H1 showed almost unchanged responses to Hg2+ before and after addition of other interfering metal ions. Cu2+ had a low negative interference effect on this fluorescent assay for Hg2+. These results indicate that H1 is highly selective and has great potential for biomedical and environmental applications.

Fig. 7
figure 7

Fluorescence responses of H1 (10 μM) to Hg2+ (50 μM) in the presence of other metal ions (50 μM) at 550 nm in HEPES buffered aqueous solution (methanol/water = 1:9, v/v, pH 7.2). Excitation wavelength was set at 390 nm with excitation and emission slit at 1.0/5.0 nm

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Response Time and Hg2+ Binding Reversibility for H1

Besides high sensitivity and selectivity, achieving a short response time and a reversible response for the analyte of interest in a complex matrix is critical in chemosensor development [35]. We studied the response times and chemical reversibility of the binding of H1 with Hg2+ in a buffered methanol/water (1:9, v/v) solution. The response time of the chemosensor to Hg2+ was quick, and a stable reading could be obtained within approximately 2 min (see Fig. 8). In addition, because of the high stability of the EDTA-Hg2+ complex (stability constant log K EDTA-Hg = 21.5) [36], it was expected that the addition of EDTA would liberate Hg2+ from the metal-ligand complex, releasing free H1. As shown in Fig. 9, on addition of 2 equiv of EDTA (20 μM) to the Hg2+ (10 μM) complex of H1 (10 μM) in buffered methanol/water (1:9, v/v) solution, a significant decrease in the fluorescence signal at 550 nm was observed. These results demonstrated that the Hg2+ binding of H1 in buffered aqueous solution is chemically reversible. The chemosensor could therefore be used for real-time tracking of Hg2+ in biological samples.

Fig. 8
figure 8

Time responses of H1 (10 μM) in the presence of Hg2+ (50 μM) in HEPES buffered aqueous solution (methanol/water = 1:9, v/v, pH 7.2). Excitation wavelength was set at 390 nm with excitation and emission slits at1.0/5.0 nm

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Fig. 9
figure 9

Fluorescence responses of H1/Hg2+ upon addition of EDTA in HEPES buffered aqueous solution (methanol/water = 1:9, v/v, pH 7.2). Excitation wavelength was set at 390 nm with excitation and emission slits at 1.0/5.0 nm

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Time-Resolved Fluorescence Studies

The exponential decay profiles of H1 and H1/Hg2+ were investigated. Figure 10 and 11 show fitting of the decay traces by deconvolution, taking into account the instrument impulse [37]. The fluorescence lifetime data of H1 and H1/Hg2+ in buffered methanol/water (1:9, v/v) solution are listed in Table 1. The goodness of fit was characterized by χ2 values of 1.08 and 1.03. In this study, direct excitation into the 4-amino-1,8-naphthalimide band (λ ex = 390 nm) and selective observation at 550 nm led, as expected, to very similar average lifetimes for the 1,8-naphthalimide fluorophore in H1 and in H1/Hg2+.

Fig. 10
figure 10

Fluorescence decay profile of H1 in HEPES buffered aqueous solution (methanol/water = 1:9, v/v, pH 7.2). Excitation and emission wavelength were set at 390/550 nm with excitation and emission slit at 13.0/13.0 nm

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Fig. 11
figure 11

Fluorescence decay profile of H1/Hg2+ in HEPES buffered aqueous solution (methanol/water = 1:9, v/v, pH 7.2). Excitation and emission wavelength were set at 390/550 nm with excitation and emission slit at 13.0/13.0 nm

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Table 1 Fluorescence lifetimes of the chemosensor and its metal complex
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For simplicity, the average lifetimes τ av, obtained using Eq. (1) (α i is the weighted pre-exponential factor), were calculated [38].

$$ {\tau_{{av}}} = \sum\limits_{{i = 1}}^{\text{n}} {{\alpha_i}{\tau_i}} \,{\text{with}}\,\sum\limits_{{i = 1}}^n {{\alpha_i} = 1} $$
(1)

For H1, τ av is 3.93 ns [τ 1 3.41 ns (86 %), τ 2 7.12 ns (14 %)]; after formation of H1/Hg2+ complex, the fluorescence lifetime (τ) is 3.83 ns (100 %). To better understand the fluorescence lifetime, further work aimed at investigating the intramolecular interactions between 1,8-naphthalimide and quinoline is under way, as well as the development of materials for Hg-contamination treatment

Conclusions

In summary, we designed and synthesized a novel fluorescent chemosensor based on the PET mechanism. The chemosensor exhibits reversible and fast responses toward Hg2+ in a buffered aqueous solution. In addition, this fluorescence chemosensor for Hg2+ has effectively solved the problem of interferences from other transition-metal ions. Furthermore, H1 is capable of quantitative detection of Hg2+ via a turn-on fluorescent response, with a linear range 0–10 μM; the detection limit was calculated to be 63 nM. The fluorescence quantum yield and lifetime of H1/Hg2+ were 0.42 and 3.83 ns, respectively. These selective and sensitive results may lead to the potential applications in managing environmental pollution and detecting biomedical samples.