A Practical Hydrazine-Carbothioamide-Based Fluorescent Probe for the Detection of Zn 2+ : Applications to Paper Strip, Zebraﬁsh and Water Samples

: A practical hydrazine-carbothioamide-based fluorescent chemosensor TCC (N-(4-chlorophenyl)-2-(thiophene-2-carbonyl)hydrazine-1-carbothioamide) was applied for Zn 2+ detection. TCC exhibited selective ﬂuorescence emission for Zn 2+ and did not show any interference with other metal ions. In particular, TCC was utilized for the detection of Zn 2+ in paper strips, zebraﬁsh and real water samples. TCC could detect Zn 2+ down to 0.39 µ M in the solution phase and 51.13 µ M in zebraﬁsh. The association ratio between TCC and Zn 2+ was determined to be 2:1 by ESI-mass and Job plot. The sensing mechanism of TCC for Zn 2+ was illustrated to be a chelation-enhanced ﬂuorescence process through spectroscopic experiments and theoretical calculations.


Introduction
Zinc is a crucial trace nutrient for organisms and the second-most plentiful transition metal in the body [1][2][3][4]. For decades, zinc has been noted for its pivotal roles involved in biological processes, such as the growth of living organisms, neural signal transmission and gene transcription [5][6][7][8]. Due to the various functions of zinc in biological processes, however, an unbalance of zinc has been associated with various pathological troubles [9][10][11].
Particularly, zinc deficiency in the human body results in a severe effect on impaired taste, depressed immunity, delayed sexual maturation and growth defects [12]. In contrast, too much zinc can lead to neurodegenerative damage, including infantile diarrhea, Alzheimer's disease, diabetes and Parkinson's disease [13,14]. Thus, there is an imperative need to develop tools that can prevent undue exposure to zinc in living organisms.
Hitherto, several studies have reported that fluorescent probes based on naphthalene, coumarin, phenanthrene, anthracene, rhodamine, antipyrine and triazole have been applied to the sensing of Zn 2+ [27][28][29][30][31][32][33]. However, there are still many disadvantages, such as complex synthesis processes and difficulty in bioimaging. Thus, it is necessary to develop an easily accessible fluorescent chemosensor for detecting zinc in biological systems.
Thiourea has attracted attention for its capability to bind to metals [34,35]. In particular, the sulfur atom in thiourea prefers to chelate with soft metal ions, such as Zn 2+ and Hg 2+ , through the hard-soft acid base theory [36][37][38]. In order to selectively detect only Zn 2+ with the thiourea moiety, we intended to endow a hard character to the thiourea by combination with hydrazine with hard base nitrogen atoms [39,40].
Moreover, hydrazine has a water-soluble character [41]. To keep these properties in mind, we designed and found the compound TCC, including the thiourea and hydrazine moieties, as reported in the literature [42,43]. We applied TCC as a sensor with the expectation that it could coordinate well to zinc ion and might be soluble in water for biological applications.
Herein, we address a practical hydrazine-carbothioamide-based fluorescent sensor TCC for detecting Zn 2+ . TCC exhibited selective fluorescence emission for only Zn 2+ and all the other cations did not interfere with the fluorescence emission of TCC to Zn 2+ . Significantly, TCC was a suitable chemosensor capable of detecting Zn 2+ with practical applications, such as real water samples, paper-strips and zebrafish. TCC could detect down to 0.39 µM of Zn 2+ in the solution phase and 51.13 µM of Zn 2+ in zebrafish. The sensing interaction of TCC for Zn 2+ was demonstrated by ESI-mass, 1 H NMR titration, calculations, fluorescent experiments and UV-vis titration.

Materials and Equipment
All the chemicals were supplied by Sigma-Aldrich (Burlington, MA, USA). A Varian spectrometer was employed to obtain 1 H NMR and 13 C NMR. Perkin Elmer model spectrometers were employed to obtain the absorption and fluorescent spectra. ESI-mass measurements were conducted using a Thermo MAX instrument (Molecular Devices, San Jose, CA, USA).

Job Plot
Two stock solutions, TCC (2.0 × 10 −2 M) and Zn 2+ (1.0 × 10 −2 M), were prepared as described in titration section. We diluted 100 µL of the TCC stock in 49.9 mL buffer to give 4 × 10 −5 M, and 200 µL of the Zn 2+ stock was diluted to 49.9 mL buffer to afford 4 × 10 −5 M. We delivered 0.3-2.7 mL of the diluted TCC to the UV-vis cell. The diluted Zn 2+ was delivered to the cells to provide 3 mL. After blending them for 5 s, fluorescent data were obtained.

Water Sample
To analyze the utilization of TCC for Zn 2+ in real water samples, tap and drinking water were prepared in our laboratory. A TCC stock (2.0 × 10 −2 M) was prepared as described in titration section. We added 6 µL of the TCC stock to a 2.99 mL water sample containing Zn 2+ (8.00 µM). After blending for 5 s, fluorescent data were obtained.

Fluorescent Paper-Strips
The TCC-paper strips were provided by soaking the filter papers in TCC (2 × 10 −2 M, DMF) and drying them. TCC-paper strips were added to 1 mM of metal ions in buffer. After drying, their photographs were taken.

Zebrafish Imaging
The 6-day-old zebrafish were reared under our former conditions [44]. Before proceeding with the imaging experiment, we prepared a TCC stock (2.0 × 10 −2 M) and a Zn 2+ stock (1.0 × 10 −2 M). We added 50 µL of the TCC stock to 19.95 mL E2 media. The zebrafish were incubated with TCC (50 µM) in E2 media with 0.3% DMSO for 15 min and then washed with E2 media.
The zebrafish were separated into four groups. One was a control group, and the other groups were further treated with 150, 250 or 500 µM of Zn 2+ for 15 min. The zebrafish were anesthetized by ethyl-3-aminobenzoate methanesulfonate. A few seconds later, we conducted all the imaging experiments using a fluorescence microscope. With Icy software, the mean fluorescence intensity of the images was analyzed.

Theoretical Studies
Theoretical calculations for TCC and TCC-Zn 2+ were studied using the Gaussian 16 program [45]. The DFT method was employed for geometry optimizations [46,47]. The B3LYP and 6-31G(d,p) basis set was employed for all atoms except Zn 2+ [48,49]. In the case of TCC-Zn 2+ , the LANL2DZ basis set was applied to Zn 2+ [50][51][52] . None of the imaginary frequency appeared in the optimized-patterns and local minima of TCC and TCC-Zn 2+ were verified. The solvent effect of water was dealt with IEFPCM [53]. The thirty probable UV-vis transition states were calculated with the TD-DFT method based on the energy-optimized patterns of TCC and TCC-Zn 2+ .
Fluorescence and UV-vis titrations were conducted to examine the sensing property of TCC for Zn 2+ (Figures 2 and 3). As different concentrations of Zn 2+ (0-1.7 equiv) were added to TCC, the fluorescence emission at 450 nm constantly increased until 1.6 equiv of Zn 2+ was added. UV-vis titration was also performed under the same condition. Upon addition of Zn 2+ into TCC, the absorbance of 340 nm consistently increased and that of 270 nm decreased until Zn 2+ reached at 1.6 equiv. There was an evident isosbestic point at 288 nm, which signifies that the interaction of TCC and Zn 2+ provided a product.  A Job plot was employed to apprehend the association ratio of TCC for Zn 2+ (Figure 4). The greatest fluorescence emission at 450 nm appeared at a molar fraction of 0.7, which means that TCC and Zn 2+ formed a complex with a 2:1 association ratio. The ratio was also proven by ESI-MS ( Figure S5  From the definition of IUPAC (C DL = 3σ/k) [54], the detection limit for Zn 2+ was calculated to be 0.39 µM ( Figure 5). This was much lower than the drinking water standard (76 µM) stipulated by the World Health Organization (WHO) [55]. More importantly, the value is the lowest among those formerly addressed for hydrazine-carbothioamide-based fluorescent Zn 2+ chemosensors (Table S1) [34,39,[56][57][58]. The association constant (K) of TCC-Zn 2+ was given as 2 × 10 8 M −2 from Li's equation ( Figure S6). To determine an appropriate sensing mechanism between TCC and Zn 2+ , 1 H NMR titrations were conducted ( Figure S7). When 0.5 equiv of Zn 2+ was added to TCC, the peak of thiourea protons (H 4 , H 5 and H 6 ) shifted downfield. Upon the addition of Zn 2+ up to 2.0 equiv, the integral value of H 4 decreased to half, indicating that the proton H 4 of one of two TCC molecules was deprotonated by binding with Zn 2+ . Thus, we predicted that both the nitrogen of amide and the sulfur of thiourea would bind to Zn 2+ . Based on the results of the ESI-mass, Job plot and 1 H NMR titration, a proper structure of Zn 2+ -2·TCC was suggested (Scheme 2).

Scheme 2.
The proposed response mechanism of TCC for Zn 2+ .
A competition test was performed to understand a probing ability of TCC toward Zn 2+ . The fluorescent spectra of TCC were recorded in the presence of Zn 2+ along with other cations (Figure 6). There was no interference in the fluorescent spectra of TCC for detecting Zn 2+ , indicating that TCC was an excellent sensor to detect Zn 2+ without interference from other cations. The pH test of TCC and Zn 2+ -2·TCC was conducted in different pH conditions (pH 6-9) ( Figure S8). For TCC, there was no fluorescence emission from pH 6 to 9. Meanwhile, the fluorescence intensity of Zn 2+ -2·TCC was prominently increased between pH 7 and 9. This outcome signified that TCC may be utilized for sensing Zn 2+ at pH 7-9. To ensure the practical availability of TCC, a fluorescent paper-strip application was performed under fluorescence lamp (λ ex = 365 nm) (Figure 7). Among the various metals, TCC could detect only Zn 2+ with definite fluorescent emission. The results suggested that TCC was able to detect Zn 2+ in the paper-applied phase. The application of TCC in real samples was conducted to inspect the practical utility of TCC (Table 1). Reliable recoveries and R.S.D. values were observed in both drinking and tap water samples, meaning that TCC has a great potential to be employed as a reliable tool for monitoring Zn 2+ in real samples.

Imaging in Zebrafish
To identify the biological applications of TCC for Zn 2+ , imaging experiments were achieved with zebrafish ( Figure 8). When the zebrafish were treated with TCC (50 µM) for 15 min, there was no fluorescence in the swim bladder (Figure 8(a 2 )). However, as the amounts of Zn 2+ increased to 150, 250 and 500 µM (Figure 8(b 2 -d 2 )), the fluorescence in the swim bladder gradually increased. In the swim bladder, the detection limit for Zn 2+ was analyzed to be 51.13 µM with the Icy software ( Figure S9). These results illustrate that TCC may be applied to trace Zn 2+ in live organisms.

Calculations
Optimized patterns of TCC and Zn 2+ -2·TCC were investigated according to the analyses of the ESI-mass and Job plot. As shown in Figure 9, TCC had a twist structure with a dihedral angle of −101.27 • for 1C, 2N, 3N and 4C, whereas the coordination of Zn 2+ to two TCC molecules displayed a more rigid tetrahedral structure (dihedral angle = 175.18 • ). The bond distances related to coordination of Zn 2+ to TCC were calculated to be 1.992 Å for 2N-Zn 2+ and 2.341 Å for 5S-Zn 2+ , which are in the range of the general bond distances for binding with Zn 2+ [59,60].
Both TCC and its complex state showed similar transition characters, and the rigidity in the complex state of TCC increased. Thus, fluorescent 'turn-on' sensing would be caused by chelation-enhanced fluorescence process [61]. When TCC was converted into the complex state with Zn 2+ , the reduction of nonradiative transitions, such as rotations and vibrations, would lead to the enhancement of radiative transitions, like fluorescence.
Referring to various spectroscopic experiments and theoretical calculations, we present a plausible sensing model of Zn 2+ by TCC (Scheme 2).

Conclusions
We presented a practical hydrazine-carbothioamide-based fluorescent chemosensor TCC that could effectively detect Zn 2+ in aqueous media. Probe TCC could detect Zn 2+ among the other metal ions through selective fluorescence emission. In addition, TCC could clearly recognize Zn 2+ with competition from metal ions. Particularly, TCC could be used as a practical probe capable of detecting Zn 2+ in paper-strip, zebrafish and real water samples.
The detection limit of TCC for Zn 2+ was calculated to be 0.39 µM in the solution phase and 51.13 µM in zebrafish. Importantly, the value in the solution phase is the lowest among those formerly addressed for hydrazine-carbothioamide-based fluorescent Zn 2+ chemosensors. The binding mode of TCC for Zn 2+ was revealed to be a 2:1 by the Job plot and ESI-mass. The detecting mechanism of TCC toward Zn 2+ was described as the chelation-enhanced fluorescence process based on the results of spectroscopic studies and theoretical calculations.
Future study will focus on the development of hydrazine-carbothioamide-based chemosensors, which may operate at long excitation wavelengths for fluorescence bioimaging. In addition, we will consider the development of an integrated system with portable fluorescent recognition or smartphone-based sensors [62,63].

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/chemosensors10010032/s1, Table S1: Examples of hydrazinecarbothioamide-based fluorescence chemosensors for detecting Zn 2+ . Figure S1: 1 H NMR spectrum of TCC. Figure S2: 13 C NMR spectrum of TCC. Figure S3: Positive-ion ESI-mass spectrum of TCC (100 µM). Figure S4: Solubility of TCC in distilled water based on the absorbance at 320 nm. Solubility was calculated to the TCC-saturated solution with linear fitting curve of TCC (0, 40, 80, 120, 160 and 200 µM). Figure S5: Positive-ion ESI-mass spectrum of TCC (100 µM) upon the addition of Zn 2+ (1 equiv). Figure S6: Li's equation plot (at 450 nm) of TCC (40 µM) based on fluorescence titration, assuming 2:1 stoichiometry for association between TCC and Zn 2+ . Figure S7: 1 H NMR titration of TCC (10 mM) upon the addition of different amounts of Zn 2+ (0-2.0 equiv). Figure S8: Fluorescence intensity of TCC and TCC-Zn 2+ at a pH range of 6 to 9. Figure S9: Quantification of the mean fluorescence intensity in Figure 8a 2 -d 2 . Figure S10: (a) The theoretical excitation energies and the experimental UV-vis spectrum of TCC. (b) The major electronic transition energies and molecular orbital contributions of TCC. Figure S11: The major molecular orbital transitions and excitation energies of TCC and the Zn 2+ -2·TCC complex. Figure S12 Institutional Review Board Statement: The maintenance of zebrafish was approved by the Institutional Animal Care and Use Committees at the Seoul National University of Science and Technology. Ethical review and approval were waived for this study because early-life stages of zebrafish (<120 hpf) are not protected according to the European Union (EU) Directive 2010/63/EU.