A Fluorescent Probe Based on the Hydrazone Schiff Base for the Detection of Zn2+ and its Application on Test Strips

A novel fluorescent probe SHK for Zn2+ detection was designed based on the hydrazone Schiff base, successfully synthesized by Suzuki coupling and condensation reactions. The probe SHK in DMSO/H2O showed extremely weak fluorescence. However, the solution exhibited an intensive yellow-green emission with the introduction of Zn2+. In contrast, negligible fluorescence change was observed when other metal ions were added, suggesting a high selectivity of SHK for Zn2+ detection. The Job’s Plot analysis revealed that a 1:1 stoichiometric adduct SHK-Zn2+ formed during the Zn2+ sensing. The binding constant of the complex was determined to be 184 M− 1, and the detection limit for Zn2+ was calculated to be 112 µM. Moreover, the probe SHK achieved selective fluorescence sensing for Zn2+ on test strips, which guaranteed its practical application prospect.


Introduction
The development of selective and sensitive chemosensors for detecting metal ions has received more and more attention due to their essential roles in medicine, living systems, the environment, and so on [1][2][3]. Among various detecting methods, the fluorescent chemosensor is considered one of the most effective tools due to its high sensitivity, rapid response rates, nondestructive detection, and relatively simple handling [4][5][6]. The luminescent intensity of the chemosensor increases linearly with the concentration of metal ions. Zinc, the second most abundant in the human body after iron, plays a vital role in the human brain and is an active component in the growth and reproduction of all organisms [7][8][9]. Appropriate intake of Zn 2+ ions has specific benefits for the human body. However, too high or too low levels of Zn 2+ can cause profound human disease states [10,11]. For example, physical growth retardation and neurological disorders like Alzheimer's can result from Zn 2+ deficiencies [12,13]. Furthermore, excess Zn 2+ in water can lead to severe environmental pollution and phytotoxic effects [14,15]. Therefore, developing excellent fluorescent probes for detecting Zn 2+ in a given environment is necessary and significant [16,17].
Schiff bases, an essential class of fluorescent sensors, feature multiple nitrogen-oxygen-rich coordination sites for chelation metal ions [18][19][20]. In common, the Schiff base derivatives display extremely weak fluorescence due to the photo-induced electron transfer (PET) process and -C = N-isomerization [21]. In contrast, the fluorescence intensity enhances drastically after coordinating with metal ions resulting from the inhibition of the PET process and the facilitation of the chelation-enhanced fluorescence (CHEF) [22]. Moreover, the fluorescence color has a distinct red shift in response to metal ions, achieving naked-eye recognition [23]. Schiff base fluorescent sensors could detect Zn 2+ in living cells and actual waters with a low limit detection order of 10 − 10 M [24][25][26]. In addition, Schiff bases could be made into a rewritable paper to detect Zn 2+ qualitatively, showing great potential in the anti-counterfeiting and on-site monitoring fields [27,28].
Based on the inherent nitrogen recognition sites, hydrazone Schiff bases attract considerable attention due to their specific coordination ability towards metal ions and excellent water solubility [29][30][31][32]. Therefore, in this work, we developed a 1 3 novel naked-eyes probe SHK for highly selective and sensitive Zn 2+ ions detection based on functional hydrazone, which was constructed from 2-hydrazinobenzothiazole and triphenylamine hydroxyl carbaldehyde. SHK could selectively and sensitively detect Zn 2+ ions by fluorescence. When added with Zn 2+ ions, SHK displayed a turn-on green fluorescence in DMSO/H 2 O (9:1, v/v) aqueous media, which was not inferred by other metal ions. And the complex mechanism of Zn 2+ ions and probe SHK was verified by NMR and HR-MS. Furthermore, the probe SHK can be made into a simple test paper for the qualitative detection of Zn 2+ .

Experimental
All chemicals and reagents were used as received from chemical companies without further purification. Column chromatography was performed using silica gel (200 ~ 300 mesh) as a stationary phase. The synthetic route of probe SHK was shown in Scheme 1. The target compound SHK was synthesized in the moderate yield from 2-hydrazinobenzothiazole via Suzuki coupling and condensation reactions (Scheme 1). FT-IR spectra were recorded by Thermo Scientific Nicolet 6700 Fourier transform infrared spectrometer in KBr pellets. 1 H and 13 C-NMR measurements were performed by a DRX-600 spectrometer (Bruker Biospin) using tetramethylsilane as an internal standard. High-resolution ESI (HR-ESI) mass spectrometry spectra were acquired on a Thermo Scientific Q Exactive Mass Spectrometer. Elemental analyses were performed on a PE 2400 II Elemental analyzer (PerkinElmer). UV-vis spectra were measured on a UV-3600 spectrophotometer. Fluorescence spectra were recorded on FL-6500 spectrophotometer (PerkinElmer). After being cooled to room temperature, 30 mL of water was added and extracted with CH 2 Cl 2 (50 mL × 2). The combined organic layers were washed with water (50 mL × 2) and brine (50 mL) and dried over anhydrous magnesium sulfate. Evaporation of the solvent under reduced pressure to get the crude product, which was purified by chromatography on a silica gel column using PE/EA (5:1,v/v) as an eluent to afford triphenylamine hydroxyl carbaldehyde as a yellow powder (yield: 70%). 1 (Fig. S3). 13

Results and Discussion
Firstly, we investigated the fluorescence change of SHK towards different metal ions. As shown in Fig. 1, the fluorescence of SHK without any metal ions was extremely weak in DMSO/H 2 O (9:1, v/v) with the emission maximum at 430 nm. However, a noticeable fluorescence enhancement was observed when Zn 2+ or Al 3+ ions were added into the DMSO/H 2 O (9:1, v/v) solution of SHK within 10 s, respectively. And the emission maximum was red-shifted to 530 nm with the introduction of Zn 2+ or Al 3+ ions. The interference experiments of probe SHK to Zn 2+ have been performed by the addition of other metal ions except for Al 3+ (Fig. S5). Most competitive cations (Cr 3+ , Cd 2+ , Co 2+ , Na + , Ca 2+ , Ni 2+ , Pb 2+ , Mn 2+ , Pt 2+ , Mg 2+ , Hg 2+ , Ba 2+ , Ag + , K + and Li + ) induced a little effect on the fluorescence intensity, giving a negligible interference for the detection of Zn 2+ . However, Cu 2+ and Fe 3+ ions quenched about 30% of fluorescence intensity, which was ascribed to the relatively strong complexation between SHK and the two ions, even in the presence of Zn 2+ . Therefore, SHK showed good selectivity and sensitivity for Zn 2+ over most competing metal ions. The weak fluorescence in free SHK solution was due to the photoinduced electron transfer (PET) process involving lone pairs of electron N atoms, which blocked the radiation of the fluorescent group [21-24, 33, 34]. The turn-on fluorescence responses of SHK towards Zn 2+ and Al 3+ ions were ascribed to the inhibition of the PET process and the promotion of chelation-enhanced fluorescence (CHEF) after binding to metal ions [21-24, 33, 34].
We found that adding tetrabutylammonium fluoride (TBAF) can cover the influence of Al 3+ ions to probe SHK in DMSO/H 2 O. When 10 Equiv. TBAF was added into the solutions of SHK containing Zn 2+ and Al 3+ ions; the fluorescence of the former was strengthened distinctly while the latter one was suppressed significantly, with almost no fluorescence in SHK + Al 3+ +TBAF system (Fig. 2) [35]. Therefore, the introduction of TBAF can be used to distinguish Zn 2+ from Al 3+ ions. The selectivity of SHK towards different metal ions in DMSO/H 2 O (9:1, v/v) was exhibited in Fig. 3 (TBAF was added into the solution of Zn 2+ and Al 3+ ions), and the probe SHK could be used to identify and detect Zn 2+ .
To explore the chemosensing behavior of SHK towards Zn 2+ , we performed the fluorescent titration of SHK with Zn 2+ ions in DMSO/H 2 O (9:1, v/v). As shown in Fig. 4a, the probe SHK exhibited a weak emission at 430 nm in DMSO/ a) H 2 O (9:1, v/v). When Zn 2+ ions were added into the solution of SHK, the peak at 430 nm decreased. A new peak at 530 nm was observed and intensified with the increasing amount of Zn 2+ ions. For the fluorescent detection of Zn 2+ , a linear relationship between SHK and Zn 2+ concentration was displayed in DMSO/H 2 O (9:1, v/v) with a correlation coefficient of 0.991. The regression equation was F/ F 0 = 0.0165 x + 1.2715 (10 − 5 M), and the limit of detection was calculated to be 112 µM (Fig. 4b).
To determine the stoichiometric ratio of the probe SHK with Zn 2+ , Job-plots of SHK with Zn 2+ were calculated from the continuous variation of the fluorescent emission intensity during the titration process [21][22][23][24]. A plot of fluorescent intensity attained to a maximum at a molar ratio of ca. 0.5, indicating a 1:1 stoichiometry of Zn 2+ to SHK in the complexing process (Fig. 5). In addition, the association constant (184 M − 1 ) between SHK and Zn 2+ ions was obtained using the Benesi-Hildebrand equation (Fig. S6).
We also studied the absorbance spectrum of SHK with Zn 2+ . With the gradual addition of Zn 2+ into the solution of SHK in DMSO/H 2 O (9:1, v/v), the absorption intensity at 335 nm maintained strong, and a new band at 425 nm became intense (Fig. S7). When adding about 120 Equiv. of Zn 2+ , the absorption intensity of the solution in the visible region reached the maximum, which suggested that the absorbance reached the saturated value. At the same time, the color of the solution changed from colorless to light green. The corresponding association constant between SHK and Zn 2+ ions was 144 M − 1 (Fig. S8). Furthermore, the limit of detection was calculated to be 69 µM (Fig. S9). The probe SHK can hold good stability in DMSO/H 2 O (9:1, v/v) (Fig. S10). 1 H NMR, HR-ESI and FT-IR spectra were conducted to understand the detection mechanism of SHK to Zn 2+ . Before the addition of Zn 2+ , the protons (-OH, -NH, and -HC = N) of SHK in DMSO-d 6 were at 8.45 ppm, 7.83 ppm, and 7.68 ppm. Upon the addition of Zn 2+ ions (2 Equiv.), the protons were shifted to 8.53 ppm, 7.89 ppm, and 7.75 ppm (Fig. 6). According to the reported binding modes of hydrazone Schiff base to metal ions [29,31,33,34], we proposed that probe SHK chelated to Zn 2+ through N atoms of benzothiazole and hydrazine, and O atom of phenol (Fig. 6), which was further confirmed by HR-ESI and FT-IR spectra. The positive ion mass spectrum of SHK upon the addition of 2.0 equivalent Zn 2+ displayed the m/z peak at 575.0862, which was assigned as [SHK + Zn − H] + (Calcd. 575.0884) (Fig. 7), suggesting the formation of 1:1 complexes and the deprotonation of the phenolic hydroxyl (-OH) in the binding process. And the m/z peak at 513.179 (Calcd. for [SHK + H] + = 513.1749) contributing from SHK was also observed. As revealed in Fig. S11, the FT-IR spectra of probe SHK exhibited clear stretching peaks at 3190 cm − 1 (O-H), 3063 cm − 1 (N-H), 1631 cm − 1 (C = O), and 1618 cm − 1 (C = N). After SHK chelating to Zn 2+ , the peak at 3190 cm − 1 disappeared, while C = O and C = N stretching moved to a lower frequency region 1622 cm − 1 and 1613 cm − 1 , respectively. In contrast, the N-H stretching peak at 3063 cm − 1 remained unchanged [24,27,28].
To check the practical applicability of SHK, we prepared dipsticks by coating paper strips with DMSO/H 2 O (9:1, v/v) solution of SHK and TBAF (molar ratio = 1:10). After drying, various metal ions solution (1 mmol/L) were dripped onto the strips and then dried with a blow-drier. As we can see from Fig. 8, the color of test strips containing Zn 2+ ions changed from non-emission to strong yellow-green fluorescence within 5 s. In contrast, the others had no apparent color change under the UV lamp's illumination. Hence, this exciting result showed that such test papers had potential applications in fast detecting the existence of Zn 2+ ions in a specific system [18,22,24,27,28]. Compared to other reported Zn 2+ ion probes (Table S1), probe SHK exhibited a comparable Stokes shift in recognition of Zn 2+ and negligible interference from other metal ions. Furthermore, a high contrast fluorescence change was observed in Zn 2+ test strips.

Conclusion
This work describes the synthesis and characterization of a novel probe SHK for detecting Zn 2+ ions in DMSO/H 2 O (9:1, v/v). UV-visible and fluorescence studies were performed to determine the selectivity of various metal ions. The probe SHK was weakly fluorescent and enhanced its fluorescence property significantly after adding Zn 2+ ions into the solution of SHK. The binding of probe SHK to Zn 2+ ions could be attributed to the formation of a 1:1 complex, as confirmed by Job's plot. The limit of determination was about 112 µM by fluorescence (69 µM by absorbance). We also found that test papers had potential applications in fast detecting Zn 2+ ions in a specific system.