A Novel Thiosemicarbazide-Based Fluorescent Chemosensor for Hypochlorite in Near-Perfect Aqueous Solution and Zebraﬁsh

: A novel thiosemicarbazide-based ﬂuorescent sensor ( AFC ) was developed. It was successfully applied to detect hypochlorite (ClO − ) with ﬂuorescence quenching in bis-tris buffer. The limit of detection of AFC for ClO − was analyzed to be 58.7 µ M. Importantly, AFC could be employed as an efﬁcient and practical ﬂuorescent sensor for ClO − in water sample and zebraﬁsh. Moreover, AFC showed a marked selectivity to ClO − over varied competitive analytes with reactive oxygen species. The detection process of AFC to ClO − was illustrated by UV–visible and ﬂuorescent spectroscopy and electrospray ionization–mass spectrometry (ESI–MS).


Introduction
Concern for the recognition of reactive oxygen species (ROS) has increased because of the significant role of ROS in physiological and pathological processes [1][2][3]. ClO − , which is one of the significant ROS, is critically important in the human immune system, and has effective antibacterial and anti-inflammatory properties [4][5][6][7]. In addition, quantification of ClO − is so important in the environmental system because it is significantly used in industrial fields, for example, as disinfectant and bleaching agent [8][9][10]. Abnormal amounts of ClO − in organisms cause several diseases, such as inflammation and cardiovascular disease [11][12][13][14][15]. Hence, it is absolutely critical to develop selective and practical sensors for determining the amount of ClO − in life systems [16][17][18][19][20].
Various analytical methods for the detection of ClO − , such as colorimetric analysis, fluorescent detection, electrochemistry, and spectrophotometry, have been developed so far [21][22][23]. Fluorescence analysis, one of the analytical methods, has the merits of high sensitivity, specificity, fast response time, and manageability [24][25][26][27]. A number of fluorescent ClO − sensors have been developed in the past decade, with several functional groups like hydrazide, thioether, thione, thioester, and C=N bond [28][29][30][31][32][33]. Nevertheless, many of them have some problems, such as poor water solubility, complicated synthesis methods, and nonbiological application. Therefore, it is necessary to develop fluorescent chemosensors with good water solubility and biological application.
Acridine and its derivatives are good fluorophores for chemosensors with high fluorescence quantum yield [34,35]. Moreover, amino acridine could readily form conjugated Schiff bases with aldehyde or ketone through the imine formation [36][37][38]. On the other hand, thiourea moiety is hydrophilic and well known to interact with reactive oxygen species like ClO − [39][40][41][42][43]. Hence, we expected that a compound with thiourea moiety linked to amino acridine may be a water-soluble chemosensor capable of detecting ROS like hypochlorite.
Here, we present a distinctly hypochlorite-specific fluorescent chemosensor, AFC, based on acridine moiety. Sensor AFC showed obvious fluorescent quenching and spectral variation with ClO − . In particular, AFC could monitor ClO − in zebrafish and environmental samples. With ESI-MS (electrospray ionization-mass spectrometry) analysis and 1 H NMR titration, the sensing process of AFC for ClO − was proposed.

Materials and Equipment
All the reagents and solvents used for synthesis and spectroscopic measurements were purchased from Sigma-Aldrich. A Varian spectrometer (Mercury) was used to get 13 C NMR (100 MHz) and 1 H NMR (400 MHz) spectra. Elemental analysis for C, H, N, and S was carried out by using a Vario Macro/Micro-Cube elemental analyzer. PerkinElmer UV/Visible and fluorescence spectrometers were employed for UV-VIS and fluorescent measurements. A single-quadrupole ACQUITY QDa was employed to get ESI mass data.

Synthesis of AFC
An amount of 1 × 10 −3 mol of FHC was dissolved in EtOH (7 mL). Then, 1 × 10 −3 mol of 9-aminoacridine (AAD) was dissolved in the solution. The mixture was stirred overnight, until the yellow powder precipitated. The yellow powder filtered was scrubbed with ether. Yield, 48%. 1

General Procedures
A stock solution of AFC was prepared by dissolving AFC (0.05 mmol) in DMSO (5.0 mL). An aqueous NaClO solution (500 µmol, 11%) was diluted in distilled water to make a concentrated solution (100 mM). Stock solutions of varied anions and ROS were prepared in bis-tris buffer. Fluorescent and UV-visible data were recorded in a near-perfect aqueous media (10 mM, bis-tris, pH 7.0).

Imaging in Zebrafish
Under the previous conditions were cultured zebrafish embryos [45]. An amount of 66 µL of a stock AFC solution (15.2 mM) was diluted to 20 mL bis-tris buffer. The zebrafish embryos (6 days old) were treated with the diluted AFC (50 µM) for 20 min and then smoothly washed with E2 media to get rid of the excess of AFC. Afterward, the zebrafish were divided into two groups. One was control group and the other group was experimental group. In the experimental group, the zebrafish were further dealt with 50 µM of ClO − for 15 min and scrubbed with E2 media. The zebrafish were narcotized by adding ethyl-3-aminobenzoate methanesulfonate. The fluorescence images of the zebrafish were obtained by a fluorescent microscope.

Results and Discussion
Chemosensor AFC was obtained by the imine formation reaction of 9-aminoacridine and FHC (Scheme 1). It was verified by 1 H NMR, 13 C NMR, and ESI-MS. The detecting process of AFC to ClO − was studied by UV-VIS spectroscopy, fluorescent spectroscopy, and 1 H NMR titration. osensors 2021, 9, x FOR PEER REVIEW 3 of 11 tized by adding ethyl-3-aminobenzoate methanesulfonate. The fluorescence images of the zebrafish were obtained by a fluorescent microscope.

Results and Discussion
Chemosensor AFC was obtained by the imine formation reaction of 9-aminoacridine and FHC (Scheme 1). It was verified by 1 H NMR, 13 C NMR, and ESI-MS. The detecting process of AFC to ClO − was studied by UV-VIS spectroscopy, fluorescent spectroscopy, and 1 H NMR titration. Scheme 1. Synthesis of AFC.

We examined the fluorescent responses of AFC to varied anions (Br
and Cl − ) and ROS species (H2O2, AcOOH, and tBuOOH) in buffer ( Figure 1). Sensor AFC exhibited an intense fluorescence emission at 455 nm with excitation at 350 nm (Ф = 0.8438). When 290 equivalents of varied anions were added, respectively, to the AFC solution, only ClO − induced a distinct decrease in fluorescence emission (Ф = 0.0197). By contrast, the other anions did not make substantial changes in fluorescent intensity, and AcOOH showed some increase in intensity at 455 nm. This result verified that chemosensor AFC could be served as an efficient fluorescent sensor for selectively detecting ClO − .   tized by adding ethyl-3-aminobenzoate methanesulfonate. The fluorescence images of the zebrafish were obtained by a fluorescent microscope.

Results and Discussion
Chemosensor AFC was obtained by the imine formation reaction of 9-aminoacridine and FHC (Scheme 1). It was verified by 1 H NMR, 13 C NMR, and ESI-MS. The detecting process of AFC to ClO − was studied by UV-VIS spectroscopy, fluorescent spectroscopy, and 1 H NMR titration. Scheme 1. Synthesis of AFC.   Spectroscopic titrations were implemented to investigate the physical responses of AFC to ClO − (Figure 2). In addition to ClO − , the intensity of the fluorescence emission of AFC at 455 nm gradually decreased, and the detection limit (C DL = 3σ/k) for ClO − turned out to be 58.7 µM ( Figure S1). In the same way, UV-VIS titration was carried out (Figure 3). The result showed a consistent increase of absorbance at 320 and 490 nm and a decrease of absorbance at 400 nm with an apparent isosbestic point at 420 nm. In addition, the time-dependent UV-VIS change of AFC showed that AFC was stable enough for 1 h ( Figure S2). Spectroscopic titrations were implemented to investigate the physical responses of AFC to ClO − (Figure 2). In addition to ClO − , the intensity of the fluorescence emission of AFC at 455 nm gradually decreased, and the detection limit (CDL = 3σ/k) for ClO − turned out to be 58.7 μM ( Figure S1). In the same way, UV-VIS titration was carried out ( Figure  3). The result showed a consistent increase of absorbance at 320 and 490 nm and a decrease of absorbance at 400 nm with an apparent isosbestic point at 420 nm. In addition, the time-dependent UV-VIS change of AFC showed that AFC was stable enough for 1 h ( Figure S2).   Spectroscopic titrations were implemented to investigate the physical responses of AFC to ClO − (Figure 2). In addition to ClO − , the intensity of the fluorescence emission of AFC at 455 nm gradually decreased, and the detection limit (CDL = 3σ/k) for ClO − turned out to be 58.7 μM ( Figure S1). In the same way, UV-VIS titration was carried out ( Figure  3). The result showed a consistent increase of absorbance at 320 and 490 nm and a decrease of absorbance at 400 nm with an apparent isosbestic point at 420 nm. In addition, the time-dependent UV-VIS change of AFC showed that AFC was stable enough for 1 h ( Figure S2).   The binding process of AFC to ClO − could be demonstrated with the result of the ESI-mass experiment ( Figure S3). The peak at m/z = 211.294 can be assigned as [AAD-O + H + ] + (calcd, m/z = 211.090). In addition, we can assign the peak at m/z = 232.287 as [FHC + MeOH + H + ] + (calcd, m/z = 232.080). The outcome suggests that the C=N bond of AFC would be cleaved by ClO − to produce FHC and AAD. Then, AAD was further oxidized to AAD-O by another ClO − . To get more information on the cleavage of AFC, 1 H NMR titration was conducted ( Figure 4). Consequently, the imine proton (H6) of AFC disappeared due to the cleavage of the imine bond. The amine protons (H5 and H5′) of AAD-O and the aldehyde proton (H6′) of FHC appeared.  To further understand the sensing mechanism, we investigated the fluorescent and UV-VIS changes of AAD and FHC upon the addition of ClO − (290 equivalents). The fluorescent intensity of AAD was substantially decreased by adding ClO − , suggesting the oxidation of AAD into AAD-O ( Figure S4). The UV-VIS spectra of AAD showed an increase of absorbance at around 490 nm ( Figure S5). On the other hand, FHC with/without ClO − showed no fluorescence intensity and an increase in UV-VIS absorbance at 280 nm ( Figures S6 and S7). Therefore, these observations and the results of the ESI-MS and 1 H NMR titration drove us to propose that the C=N bond of AFC was cleaved by ClO − , and then the resultant AAD was further oxidized to AAD-O by another ClO − (Scheme 2).

Spectroscopic Investigations of Chemosensor AFC to ClO
Chemosensors 2021, 9, x FOR PEER REVIEW 6 of 11 To further understand the sensing mechanism, we investigated the fluorescent and UV-VIS changes of AAD and FHC upon the addition of ClO − (290 equivalents). The fluorescent intensity of AAD was substantially decreased by adding ClO − , suggesting the oxidation of AAD into AAD-O ( Figure S4). The UV-VIS spectra of AAD showed an increase of absorbance at around 490 nm ( Figure S5). On the other hand, FHC with/without ClO − showed no fluorescence intensity and an increase in UV-VIS absorbance at 280 nm ( Figures S6 and S7). Therefore, these observations and the results of the ESI-MS and 1 H NMR titration drove us to propose that the C=N bond of AFC was cleaved by ClO − , and then the resultant AAD was further oxidized to AAD-O by another ClO − (Scheme 2).

Scheme 2. Sensing process of AFC by ClO
To inspect the capability of AFC as a ClO − sensor, we conducted a competitive test in the presence of ClO − mixed with other anions of the same equivalents ( Figure 5). The result demonstrated that all other analytes did not disturb the detection of ClO − by AFC. Therefore, sensor AFC could be applied as an efficient chemosensor for ClO − without the interference of other analytes. Moreover, the pH condition is critical for cellular behaviors and physiological processes. To evaluate the pH dependence of AFC, we measured fluorescent intensity in the range of pH 6-9 ( Figure 6). AFC displayed intense fluorescence at pH 6-9, and the addition of ClO − to AFC induced fluorescence quenching at pH 7-9. These outcomes imply that AFC could successfully detect ClO − at pH 7-9. In addition, fluorescent analysis in the real samples including tap and drinking water was implemented for the practicality of probe AFC. The trustworthy values of recoveries and relative standard deviation (RSD) gave proof of the potential application of AFC to detect ClO − in real samples (Table 1).

Scheme 2. Sensing process of AFC by ClO
To inspect the capability of AFC as a ClO − sensor, we conducted a competitive test in the presence of ClO − mixed with other anions of the same equivalents ( Figure 5). The result demonstrated that all other analytes did not disturb the detection of ClO − by AFC. Therefore, sensor AFC could be applied as an efficient chemosensor for ClO − without the interference of other analytes. Moreover, the pH condition is critical for cellular behaviors and physiological processes. To evaluate the pH dependence of AFC, we measured fluorescent intensity in the range of pH 6-9 ( Figure 6). AFC displayed intense fluorescence at pH 6-9, and the addition of ClO − to AFC induced fluorescence quenching at pH 7-9. These outcomes imply that AFC could successfully detect ClO − at pH 7-9. In addition, fluorescent analysis in the real samples including tap and drinking water was implemented for the practicality of probe AFC. The trustworthy values of recoveries and relative standard deviation (RSD) gave proof of the potential application of AFC to detect ClO − in real samples (Table 1).

In Vivo Imaging in Zebrafish
In order to test the sensing feasibility of the biological application of AFC to ClO − fluorescent bioimaging, experiments were conducted with zebrafish ( Figure 7). We first incubated zebrafish with AFC (50 µM), followed by treatment with ClO − (50 µM). While the zebrafish treated with only probe AFC exhibited a green fluorescence in the swim bladder and eyes, the zebrafish with additional treatment of ClO − showed no fluorescence signal. The bioimaging experiments demonstrated the detecting ability of AFC to trace ClO − in living organisms. Importantly, AFC is the second fluorescent turnoff sensor for ClO − applicable to both real water samples and zebrafish [46][47][48][49][50][51].
In order to test the sensing feasibility of the biological application of AFC to ClO − fluorescent bioimaging, experiments were conducted with zebrafish ( Figure 7). We first incubated zebrafish with AFC (50 μM), followed by treatment with ClO − (50 μM). While the zebrafish treated with only probe AFC exhibited a green fluorescence in the swim bladder and eyes, the zebrafish with additional treatment of ClO − showed no fluorescence signal. The bioimaging experiments demonstrated the detecting ability of AFC to trace ClO − in living organisms. Importantly, AFC is the second fluorescent turnoff sensor for ClO − applicable to both real water samples and zebrafish [46][47][48][49][50][51][52][53].

Conclusions
A novel thiosemicarbazide-based chemosensor AFC for detecting ClO − was synthesized from the reaction of aminoacridine and a new aldehyde group synthesized from formic hydrazide. Probe AFC selectively recognized ClO − over other anions including ROS in aqueous solution. With ClO − , probe AFC showed remarkable fluorescence

Conclusions
A novel thiosemicarbazide-based chemosensor AFC for detecting ClO − was synthesized from the reaction of aminoacridine and a new aldehyde group synthesized from formic hydrazide. Probe AFC selectively recognized ClO − over other anions including ROS in aqueous solution. With ClO − , probe AFC showed remarkable fluorescence quenching. The limit of detection of AFC for ClO − was calculated to be 58.7 µM. Additionally, probe AFC could be applicable for quantitative analysis in real water samples and zebrafish. Importantly, AFC is the second fluorescent turnoff sensor for ClO − applicable to both real water samples and zebrafish. The dependable results in this study shows that AFC could be used as an efficient chemosensor for detecting ClO − in aqueous solution and small organisms by a fluorescent quenching method.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/chemosensors9040065/s1. Table S1: Fluorescent turnoff chemosensors for recognizing hypochlorite in aqueous solutions. Figure S1: Determination of the detection limit of AFC for ClO − based on the change of intensity at 455 nm. Figure S2: The time-dependent UV-VIS change (400 nm) of AFC with/without ClO − . Figure S3: Positive-ion ESI mass spectrum of AFC upon the addition of NaClO. Figure S4: Fluorescent change of AAD with/without ClO − . Figure S5: UV-VIS change of AAD with/without ClO − Figure S6: Fluorescent change of FHC with/without ClO − . Figure S7

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. Informed Consent Statement: Not applicable.

Conflicts of Interest:
The authors declare no conflict of interest.