Introduction

Zinc plays crucial roles in a variety of biological processes including apoptosis, regulators of gene expression, and neural signal transmitters or modulators [1, 2]. Deficiency of Zn2+ can lead to unbalanced metabolism, which is responsible to some problems such as retarded growth in children, the decrease of the immunological defense, eye lesion and some skin diseases [3]. Overloading Zn2+ level in human body is also associated with some serious neurological disorders such as Alzheimer’s and Parkinson’s diseases [4–6]. As a consequence, considerable attention has been devoted to the development of fluorescent chemosensors for Zn2+ detection [7–13], because fluorescence techniques can offer distinct advantages such as low cost, simplicity, good sensitivity, and capability of real-time detection [14].

Although a large number of Zn2+ selective fluorescent sensors have been well established, many reported Zn2+ sensors still encountered a difficulty in distinguishing Zn2+ from Cd2+ [15–18], because Cd2+ is in the same group in the periodic table with Zn2+ and usually induces a comparable fluorescent response to that of Zn2+. Therefore, development of small molecular fluorescent sensors that can clearly distinguish Zn2+ from Cd2+ is still challenging and imperative. Fluorescence Zn2+ sensors generally contain two parts, a Zn2+ receptor unit that can selectively binds Zn2+ ion and a signaling unit that can responses the recognition event by fluorescence changes. Besides the well-known dipicolylamine (DPA) Zn2+ ion chelator, 2-picolylamine also has been widely used as the Zn2+ binding unit [19–21]. Although a number of fluorophores such as anthracene [22–25], coumarin [10, 26, 27], boron dipyrromethene [28, 29], fluorescein [30], rhodamine [31, 32], cyanine [13, 33], benzimidazole [34–36], and benzoxazole [19, 21, 37, 38] have been employed to construct Zn2+ selective fluorescent sensors, benzothiazole derivatized fluorescent sensors for Zn2+ detection are still rare [39–41]. Therefore, we are encouraged to design and synthesize a novel fluorescent sensor based on benzothiazole fluorophore.

Owing to the potential ESIPT property of 2-(2′-aminophenyl)benzothiazole (APBT) derivatives, design and synthesis of new Zn2+ selective fluorescent sensors based on APBT fluorophore aroused our great interest. In this work, a simple and effective APBT-based fluorescence sensor (1) has been designed and prepared (Scheme 1). Sensor 1 displays highly selective response to Zn2+ with the appearance of a strong longer-wavelength emission band, which makes 1 has an excellent ability to discriminate Zn2+ from Cd2+ and other metal ions. Sensing mechanism studies reveal that sensor 1 bind with Zn2+ through the amide form and the newly developed longer-wavelength emission band is attributed to the ESIPT fluorescence emission.

Scheme 1
scheme 1

Synthesis of fluorescent sensor 1

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Experimental Section

General Methods and Materials

Unless otherwise stated, solvents and reagents were of analytical grade from commercial suppliers and were used as received. Compound 2 was prepared following the method previously described [42]. 1H NMR and 13C NMR spectra were recorded on Agilent 400-MR spectrometer, chemical shifts (δ) were expressed in ppm and coupling constants (J) in Hertz. High-resolution mass spectroscopy (HRMS) was measured on a Bruker micrOTOF-Q mass spectrometer (Bruker Daltonik, Bremen, Germany). Low-resolution mass spectroscopy (LRMS) was measured on an Agilent 1100 series LC/MSD mass spectrometer. Fluorescence measurements were performed on a Sanco 970-CRT spectrofluorometer (Shanghai, China). The pH measurements were made with a Model PHS-25B meter (Shanghai, China).

Synthesis of Compound 3

A solution of 2-chloroacetyl chloride (0.34 g, 3 mmol) in dry CH2Cl2 (5 mL) was added dropwise to a solution of compound 2 (0.44 g, 2 mmol) and 4-dimethylaminopyridine (DMAP, 0.5 g, 4.1 mmol) in dry CH2Cl2 (5 mL) in an ice bath. The resultant was stirred for 2 h at room temperature and then the solvent was removed by rotary evaporation. The desired product 3 was recrystallized from acetone. Yield: 70 %. m.p. 144.1–144.5 °C. 1H NMR (400 MHz, CDCl3) δ 13.25 (s, 1H), 8.80 (d, J = 8.4 Hz, 1H), 8.04 (d, J = 8.1 Hz, 1H), 7.90 (dd, J = 18.5, 7.9 Hz, 2H), 7.57–7.46 (m, 2H), 7.43 (t, J = 7.6 Hz, 1H), 7.22 (t, J = 7.6 Hz, 1H), 4.31 (s, 2H). 13C NMR (100 MHz, CDCl3) 172.6, 170.9, 157.5, 141.7, 138.4, 137.2, 135.4, 132.2, 131.3, 129.8, 127.7, 126.0, 125.3, 48.9. LRMS (API-ES+) calcd. for C15H12ClN2OS [M+H]+, 303.0, found 303.0.

Synthesis of Sensor 1

Compounds 3 (0.30 g, 1.5 mmol) and 2-picolylamine (0.195 g, 1.8 mmol) were dissolved in DMF and were stirred overnight at room temperature. After removing the solvent under reduced pressure, the residue was extracted with ethyl acetate. The organic layer was dried over Na2SO4, filtered and evaporated. The crude product was purified by silica gel column chromatography to afford sensor 1 as yellow solids (0.12 g, 65 %). 1H NMR (400 MHz, DMSO-d 6 ) δ 12.96 (s, 1H), 8.82 (d, J = 8.4 Hz, 1H), 8.53 (d, J = 4.8 Hz, 1H), 8.16 (d, J = 8.0 Hz, 1H), 7.96 (d, J = 8.0 Hz, 1H), 7.70 (d, J = 8.0 Hz, 1H), 7.65 (t, J = 8.0 Hz, 1H), 7.56 (t, J = 8.0 Hz, 1H), 7.45–7.39 (m, 3H), 7.29–7.24 (m, 2H), 3.95 (s, 2H), 3.45 (s, 2H); 13C NMR (100 MHz, DMSO-d 6 ):171.7, 167.5, 153.0, 149.3, 137.3, 137.0, 133.6, 132.2, 131.9, 130.6, 127.0, 126.3, 124.2, 123.0, 122.7, 122.6, 122.4, 121.0, 54.3, 53.0. HRMS (ESI+) calcd. for C21H19N4OS [M+H]+ 375.1280, found 375.1270.

Procedures of ion Sensing

Doubly distilled water was used for all experiments. Sensor 1 was dissolved in aqueous solution (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) was employed as buffer agent, HEPES/CH3CN=4/6, v/v, HEPES 20 mM, pH = 7.4) to afford the test solution (10 μM). Titration experiments were carried out in 10-mm quartz cuvettes at 25 °C. Metal ions (as chloride or nitrate salts, 10 mM) were added to the host solution and used for the titration experiment.

Result and Discussion

Synthesis

The synthetic route to sensor 1 is depicted in Scheme 1, which involves the synthesis of APBT (2), amidation of 2 with chloroacetic chloride, and nucleophilic substitution of 3 with 2-picolylamine. The obtained compound 1 is fully characterized by 1H NMR, 13C NMR, and HRMS spectroscopy, and the results are in full agreement with the presented structure.

Fluorescence Recognition of Zn2+

The metal ion binding behavior of 1 was examined by fluorescence spectroscopic studies. As shown in Fig. 1, sensor 1 showed a weak fluorescence in HEPES/CH3CN=4/6 (v/v, HEPES 20 mM, pH = 7.4) solution at 348 nm, which is assigned to the normal excited state emission of 1. Upon addition of Zn2+, a new strong emission band centered at 485 nm was observed. Concomitantly, the original emission at 348 nm enhanced slightly. In addition, the Zn2+ induced fluorescence change is naked eye detectable (Fig. 1, inset). However, other tested cations promoted no distinct emission shift and enhancement. These results indicate that sensor 1 has an excellent selectivity to Zn2+ and can clearly discriminate Zn2+ from Cd2+ and other metal ions. The influences of other potential competitive metal ions on Zn2+ recognition were then evaluated. In the presence of some metal ions such as Ag+, Pb2+, Sr2+, Ba2+, Cd2+, Mn2+, Cr3+, and Al3+, further addition of Zn2+ ion still generated the similar fluorescence changes (Fig. 2). Co-existence of some metal ions including Co2+, Ni+, Fe2+, and Fe3+ could quench the fluorescence intensity of 1-Zn2+ solution at 485 nm to different extent. Notably, Cu2+ can completely quench the fluorescence emission of 1-Zn2+ solution at 485 nm. This phenomenon is often encountered in many fluorescent sensors due to the paramagnetic nature of Cu2+ [43, 44]. In addition, the biologically abundant metal ions of Na+, K+, Ca2+, and Mg2+ displayed no hinder effects on Zn2+ recognition even they were used in high concentration as 1 mM. Thus, sensor 1 has a good anti-jamming ability to other metal ions except Cu2+.

Fig. 1
figure 1

The fluorescence spectra of 1 (10 μM) upon addition of various metal ions, including Zn2+, Cd2+, Cr3+, Mn2+, Hg2+, Ag+, Pb2+, Fe2+, Fe3+, Ni2+, Co2+, Cu2+, Ba2+, Al3+, Sr+ (3 equiv. of each) and Na+, K+, Ca2+, Mg2+ (100 equiv. of each) in aqueous solution (HEPES/CH3CN=4/6, v/v, HEPES 20 mM, pH = 7.4). Inset: Fluorescence color changes of 1 solution before and after addition of Zn2+ under irradiation at 365 nm

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Fig. 2
figure 2

Fluorescence intensity changes of 1 (10 μM) to various metal ions in aqueous solution (HEPES/CH3CN=4/6, v/v, HEPES 20 mM, pH = 7.4) at 485 nm. The black bars represent the fluorescence intensity of 1 solution in the presence of 3 equiv. of miscellaneous metal ions; the red bars represent the fluorescence intensity of the above solution upon further addition of 3 equiv. of Zn2+

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Fluorescence Titrations and Detection Limit

To obtain a better insight into the Zn2+ sensing property of 1, fluorescence titration experiments with addition of increasing amounts of Zn2+ were carried out (Fig. 3). Upon stepwise increasing in Zn2+ concentration, the fluorescence intensity of 1 solution at 348 nm increased slowly, but the intensity at 485 nm increased sharply. The fluorescence spectra changes stopped when 3 equiv. of Zn2+ was added. Moreover, time course examination reveals that the response of 1 to Zn2+ can finish within 1 min (Fig. 3, inset), indicating its rapid response to Zn2+.

Fig. 3
figure 3

Fluorescence spectra of 1 (10 μM) in aqueous solution (HEPES/CH3CN=4/6, v/v, HEPES 20 mM, pH = 7.4) in the presence of different amounts of Zn2+. Inset: Time-dependence fluorescence intensity of 1 solution in the presence of 3 equiv. of Zn2+

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To check its practical utility, the fluorescence detection limit of 1 for Zn2+ was evaluated. Based on the fluorescence titration data at 485 nm, plotting of the normalized fluorescence intensity of (I min−I)/(I min−I max) versus log[Zn2+] afforded a nice linear relationship (R = 0.99861), the point at which this line crossed the ordinate axis was regarded as the detection limit [45, 46], which was calculated to be 5.37 × 10−6 M (Fig. 4). This result demonstrates that sensor 1 is highly sensitive for Zn2+ recognition with micromolar level detection limit.

Fig. 4
figure 4

Normalized intensity of 1 solution (10 μM) against Log[Zn2+] in the low Zn2+ concentration range

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Determination of Binding Stoichiometry

To determine the binding stoichiometry of 1 and Zn2+, Job’s plot analysis was conducted. As depicted in Fig. 5, a maximum fluorescence intensity was observed when the mole fraction of Zn2+ is 0.5, indicating the 1:1 binding stoichiometry of 1 and Zn2+. Meanwhile, nonlinear least-squares fitting of the titration profiles (Fig. 6) employing a 1:1 binding mode equation led to a nice nonlinear curve (R 2 = 0.9969) [47], which also strongly supports the 1:1 interaction between 1 and Zn2+, and the association constant K a was calculated to be 2.97 × 106 M−1.

Fig. 5
figure 5

Job’s plot for 1 and Zn2+ solution with a total concentration as 10 μM in aqueous solution (HEPES/CH3CN=4/6, v/v, HEPES 20 mM, pH = 7.4). The fluorescence intensity was monitored at 485 nm

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Fig. 6
figure 6

Nonlinear fitting of fluorescence intensity of 1 solution (10 μM) against Zn2+ concentration (emission at 485 nm)

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pH Effect and Reversibility

To apply 1 in more complicated systems, the influence of pH on the fluorescence of 1 and 1+Zn2+ was examined (Fig. 7). As for sensor 1, the fluorescence intensity was barely affected when the pH ranged from 1 to 14. Upon addition of Zn2+, 1 solution exhibited strong fluorescence emission between pH 7 and 8. This result demonstrates that sensor 1 is especially suitable to detect Zn2+ at near neutral pH conditions. The reversible Zn2+ binding behavior was further evaluated by alternative addition of Zn2+ and EDTA to 1 solution. The fluorescence intensity of the solution exhibited alternative enhancing and quenching processes (Fig. 8), indicating that the Zn2+ recognition event is reversible.

Fig. 7
figure 7

Fluorescence intensity (at 485 nm) of 1 and 1+Zn2+ at various pH values in aqueous solution (HEPES/CH3CN=4/6, v/v)

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Fig. 8
figure 8

Fluorescence intensity changes of 1 solution (10 μM) at 485 nm upon alternative addition of Zn2+ and EDTA

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Recognition Mechanism

The weak fluorescence emission of 1 at 385 nm can be attributed to the emission of its normal excited state, and the photoinduced electron transfer (PET) from aliphatic amine nitrogen to the excited state of phenylbenzothiazole moiety decreased its emission intensity. Binding with Zn2+ could suppress the PET process and allows fluorescence enhancement. The remarkable enhancement of longer-wavelength emission with a large Stokes shift (170 nm) of the 1-Zn2+ complex can be inferred as Zn2+ coordination promoted turn-on ESIPT emission [48].

To elucidate the proposed sensing mechanism and clarify the binding mode between 1 and Zn2+, 1H NMR spectra of 1 and 1- Zn2+ were compared (Fig. 9). In free 1, the NH protons signal for amide (Ha) and 2-picolylamine (Ha) appeared at 12.96 ppm (Fig. 9a). In the presence of 3.0 equiv. of Zn2+, this signal up-field shifted to 12.29 ppm (Fig. 9b), suggesting that 1 coordinates with Zn2+ through an amide form [26, 49]. The signal appeared at 8.82 ppm (Fig. 9a) can be tentatively assigned to He due to the possible existence of hydrogen bonding between He with amide O atom [35]. On addition of Zn2+, this signal up-field shifted to about 8.35 ppm (Fig. 9b), indicating the coordination of amide O atom with Zn2+, which weakened the hydrogen bonding. The signal of protons neighboring pyridine N atom (Hd) at 8.53 ppm up-field shifted to 8.50 ppm on addition of Zn2+. The methylene protons (Hb and Hc) signaling at 3.45 and 3.95 ppm, respectively. These signals down-field shifted to 3.70 and 4.09 ppm, respectively, on addition of Zn2+ (Fig. 9b), indicating that the alkyl amine nitrogen atom also coordinated with Zn2+. The proposed binding mode of 1 and Zn2+ was depicted in Scheme 2. Coordination of amide O atom with Zn2+ greatly increased the acidity of amide NH, which thus promotes H-transferred excited state efficiently.

Fig. 9
figure 9

1H NMR spectra of 1 in DMSO-d 6 in the absence and presence of 1.0 equiv. of Zn2+

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Scheme 2
scheme 2

The proposed sensing mechanism of 1 for Zn2+

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Application of Sensor 1 to Test Strips

To demonstrate the practical applicability of sensor 1 for the detection of Zn2+, we carried out a preliminary paper test strip experiment, as shown in Fig. 10. After immersing neutral filter papers into the acetonitrile solution of 1 (1 mM) and dried, Zn2+ solutions of with concentrations of 5 × 10−6 M and 5 × 10−5 M were prepared in buffered water (HEPES 20 mM, pH 7.4). When the dried test strips were dipped in different concentration solutions of Zn2+ for 1 min and dried in air respectively, the observed color change of test strips from non-fluorescent to be blue under UV light at 365 nm clearly. Therefore, the test strips experiment demonstrates the potential utility of 1 to detect Zn2+ ion.

Fig. 10
figure 10

Photographs of 1 on test strips after addition of HEPES buffered (pH 7.4) ZnCl2 solutions (left to right: no Zn2+, 5 μM and 50 μM Zn2+) under irradiation at 365 nm

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Conclusions

A new off-on fluorescent phenylbenzothiazole derivative (1) has been synthesized and proved to be a highly selective, sensitive and rapid recognition Zn2+ sensor. Sensor 1 binds Zn2+ via a 1:1 stoichiometry with an association constant of 2.97 × 106 M−1, and the detection limit is evaluated to be 5.37 × 10−6 M. Binding of Zn2+ at the receptor moiety leads to the quenching of PET state emission at 348 nm and the enhancing of ESIPT state emission at 485 nm, which is responsible for the fluorescence enhancement at the shorter-wavelength and longer-wavelength. We expect that this example will serve as practical tool for environmental samples analysis and biological studies.