A Ratiometric Fluorescent Probe for N₂H₄ Having a Large Detection Range Based upon Coumarin with Multiple Applications

Abstract

Although hydrazine (N₂H₄) is a versatile chemical used in many applications, it is toxic, and its leakage may pose a threat to both human health and environments. Consequently, the monitoring of N₂H₄ is significant. This study reports a one-step synthesis for coumarin-based ratiometric fluorescent probe (FP) CHAC, with acetyl as the recognition group. Selected deprotection of the acetyl group via N₂H₄ released the coumarin fluorophore, which recovered the intramolecular charge transfer process, which caused a prominent fluorescent, ratiometric response. CHAC demonstrated the advantages of high selectivity, a strong capacity for anti-interference, a low limit of detection (LOD) (0.16 μM), a large linear detection range (0–500 μM), and a wide effective pH interval (6–12) in N₂H₄ detection. Furthermore, the probe enabled quantitative N₂H₄ verifications in environmental water specimens in addition to qualitative detection of N₂H₄ in various soils and of gaseous N₂H₄. Finally, the probe ratiometrically monitored N₂H₄ in living cells having low cytotoxicity.

1. Introduction

Hydrazine (N₂H₄) is a significantly active alkali as well as a reducing agent that has been widely applied in medicine, pesticides, materials, and dyes [1,2,3]. Furthermore, N₂H₄ is a well-known, high-energy fuel broadly employed in propulsion systems such as missiles and rockets [4,5]. However, N₂H₄ is a highly toxic substance, whose leakage in manufacturing, usage, transferring, and disposal can cause serious environmental problems. Furthermore, N₂H₄ can enter the human body via breathing and skin contact, thus leading to serious damage of the kidneys, liver, central nervous system, and lungs, which can also induce gene mutation or cancers [6,7,8]. The U.S. Environmental Protection Agency (EPA) has classified N₂H₄ as a candidate carcinogen with a 10 ppb (0.31 μM) threshold limit value [9]. Consequently, the development of simple and reliable methods that enable the detection of N₂H₄ trace amounts in the environment and living organisms is of utmost importance.

Several analysis technologies have been proposed to detect N₂H₄, such as chromatography [10], spectrophotometry [11], electrochemistry [12], potentiometry [13], and surface-enhanced Raman spectroscopy [14]. However, the majority of these technologies are costly, laborious, and require complex analytical procedures, greatly restricting their application to living biological specimens. Fluorescence-based methods, meanwhile, have been broadly applied to verify different analytes, including metal ions, anions, and biomolecules, because of their simple operation, high sensitivity, good selectivity, non-invasiveness, and good biocompatibility [15,16]. Several selective N₂H₄ fluorescent probes (FPs) with various recognition mechanisms [17,18], including selective deprotection [19,20,21,22]; reaction with diketones to form rings [23,24]; reactions with aromatic aldehydes, dicyanovinyl, or monocyanovinyl to form hydrazones [25,26,27,28,29]; cleavage of carbon–carbon double bonds to form hydrazones [30,31]; and hydrazine-induced ring-opening reactions [32,33] have been reported. However, many of these probes still possess disadvantages, such as their complex synthesis, narrow pH range, low sensitivity, and small detection range, which have limited their application. Furthermore, most hydrazine-based FPs adopt a fluorescence turn-on or turn-off strategy, and these single-emitting FPs are easily interfered with by probe concentrations, instrumental factors, and the test environment. Meanwhile, ratiometric FPs—based on ratios of emission intensity with two different wavelengths—can eliminate the possible external interference mentioned above and are thus more sensitive and accurate [34,35]. Consequently, an easy and reliable method allowing development of an effective ratiometric FP for N₂H₄ captures is of the utmost importance.

Coumarins have been broadly utilized to develop FPs for distinguished analytes because of their high quantum yield, large Stokes shift, low cytotoxicity, and easy modification [36,37]. The introduction of reactive masking groups on the 7-hydroxylgroup of coumarin is an indispensable step in the design of coumarin-based N₂H₄ probes, whereby the masking groups are deprotected in the presence of N₂H₄, which causes significant fluorescence changes, mainly because of inhibition and the recovery of the intramolecular charge transfer (ICT) procedure concerning 7-hydroxycoumarin derivatives. Among these reported N₂H₄ probes based on 7-hydroxycoumarin, most of them belong to the turn-on variety [19,38,39,40,41,42,43,44,45,46,47,48], while ratiometric probes are rarely reported [49]. The current investigation prepared an ICT-based ratiometric FP CHAC for N₂H₄ detection via a simple, one-step reaction (Scheme 1). Although CHAC has been previously reported [50], as far as we know, this is the first time it has been used as an FP. The probe CHAC utilized a 7-hydroxycoumarin derivative CHOH as the fluorophore and an acetate ester as the recognition site. We deem that the selective ester cleavage in the probe by N₂H₄ will restore the push–pull electronic structure of CHOH and enhance the ICT effect, which will lead to a significant red shift with regard to the fluorescence emission spectra, thereby achieving ratiometric detection of N₂H₄. The experiment data showcased that CHAC exhibited perfect selectivity, a low detection limit (0.16 μM), a large detection range (0–500 μM), and a wide, effective pH range (6–12). Also, we successfully utilized CHAC to monitor N₂H₄ in environmental specimens as well as in living cells.

2. Results and Discussion

2.1. Design and Synthesis of the Probe CHAC

According to the design strategy of coumarin-based FPs [37], we introduced an electron-donating group (hydroxyl) at the 7-position and an electron-withdrawing group (4-chlorophenyl) at the 3-position of coumarin to form a push–pull π-conjugated system that could enhance the ICT effect, thereby resulting in an increase in fluorescence intensity and the emission wavelength of the fluorophore CHOH. The probe CHAC was prepared by introducing the recognition group acetyl to the hydroxyl group of CHOH. As an electron-withdrawing group, the acetyl group reduced the push–pull electron conjugation effect in CHAC. After reaction with N₂H₄, CHAC removed the acetyl group to release CHOH with a strong push–pull character, resulting in a significant red shift in the emission spectrum. As shown in Scheme 1, we prepared CHAC through a one-step condensation reaction. To investigate the response mechanisms of CHAC towards N₂H₄, the fluorophore CHOH was prepared via ester hydrolysis from CHAC. The CHAC and CHOH structures were verified through ¹H NMR, ¹³C NMR and HRMS (Figures S1–S6).

2.2. Optical Properties

We tracked the UV-Vis and fluorescence spectra (FS) with regard to the probe CHAC for the absence and presence of N₂H₄ in DMSO:PBS buffer (10 mM, 4:6 v/v, pH = 7.4). The free probe (10 μM) showed obvious absorption at 327 nm (ε = 2.39 × 10⁴ M⁻¹·cm⁻¹) (Figure S7a). The addition of N₂H₄ (100 equiv) to the CHAC solution caused a decrement in the absorption peak at 327 nm accompanied by a novel band centered at 402 nm, which was consistent with that of CHOH (10 μM) (ε = 2.09 × 10⁴ M⁻¹·cm⁻¹). Upon excitation at 340 nm, the CHAC (5 μM) exhibited a single emission centered at 420 nm, which was red-shifted to 480 nm on its reaction with N₂H₄ (100 equiv), thus corresponding to a fluorescence color alternation from blue to cyan (Figure S7b). The resulting fluorescence spectrum was almost identical to that of CHOH (5 μM). We also measured the quantum yields (QYs) of CHAC and CHOH in the test solution, with quinine sulfate as the reference. The results show that both CHAC (Φ = 0.65) and CHOH (Φ = 0.46) produce high QYs. These data thus confirmed that CHAC could be utilized for fluorescent ratiometric detection of N₂H₄ via its transformation into CHOH mediated by N₂H₄.

2.3. Reaction Time and pH Effects

The kinetics of the ratio responses of the probe CHAC to N₂H₄ and the stability of the probe itself were then investigated in DMSO:PBS buffer (10 mM, 4:6 v/v, pH = 7.4). In the absence of N₂H₄, the fluorescence intensity ratio (I₄₈₀/I₄₂₀) did not change during the testing duration, thus producing the inference that the probe was highly stable in the test solution under neutral conditions (Figure 1a). When 100 equiv of N₂H₄ was added, I₄₈₀/I₄₂₀ gradually increased as the reaction time increased, and a plateau was achieved within 30 min. Consequently, the reaction time was set to 30 min.

The pH effects upon N₂H₄ recognition by CHAC were investigated. In the absence of N₂H₄, the I₄₈₀/I₄₂₀ value did not change in pH ranges from 3 to 12, thus indicating that the free probe was very stable under acidic and alkaline conditions (Figure 1b). Upon incubating the probe with N₂H₄, I₄₈₀/I₄₂₀ gradually increased in the pH range of 3–12, and this increase was particularly significant in the pH range 6–12. This may be because alkaline conditions can prevent N₂H₄ from binding to hydrogen cations and also promote the hydrazinolysis of CHAC. Therefore, the probe CHAC was more suitable for the detection of N₂H₄ under neutral and alkaline conditions. A pH value of 7.4 was thus chosen to explore N₂H₄ in biological samples.

2.4. Fluorescence Spectra (FS) of Probes Titrated with N₂H₄

The detection limit and linear range were determined via fluorescence titration of the probe CHAC with N₂H₄ (from 0 to 1000 μM). As the concentration of N₂H₄ increased, CHAC fluorescence emission at 480 nm gradually increased, while the fluorescence emission at 420 nm decreased markedly (Figure 2a). When the N₂H₄ concentration reached 500 μM, I₄₈₀/I₄₂₀ reached a plateau (Figure 2b). The plot of the I₄₈₀/I₄₂₀ ratio of CHAC versus N₂H₄ concentrations showcased a perfectly linear relationship (R² = 0.9944) in the 0–500 μM ranges (inset of Figure 2b). The limit of detection (LOD) of CHAC was computed as 0.16 μM following 3σ/K (σ: the standard deviation of blank measurements; K: the linear regression line slope), which is less than the U.S. EPA standard (0.31 μM). Consequently, the CHAC probe demonstrates excellent sensitivity and can be used for the quantitative detection of N₂H₄. Compared to previously reported N₂H₄-based FPs (Table S1), CHAC features a large linear detection range, a low LOD, broad effective pH ranges, and versatile application in regard to N₂H₄, which makes CHAC more effective in detecting N₂H₄.

2.5. CHAC Selectivity and Anti-Interference Performance

To test CHAC selectivity for N₂H₄, we treated CHAC with various competitive analytes in a DMSO:PBS buffer (10 mM, 4:6 v/v, pH = 7.4). As illustrated in Figure 3a, when 100 equiv of related species—such as Cl⁻, Br⁻, F⁻, ${\mathrm{SO}}_4^{2-}$, I⁻, CH₃COO⁻, ${\mathrm{H}}_2{\mathrm{PO}}_4^{-}$, ${\mathrm{HSO}}_3^{-}$, ${\mathrm{N}}_3^{-}$, K⁺, Na⁺, Ca²⁺, Mg²⁺, Al³⁺, Cd²⁺, Fe³⁺, Fe²⁺, Zn²⁺, ammonia, aniline, urea, thiourea, hydroxylamine, EDA, Cys, Hcy, or GSH—was incubated with CHAC (5 μM), no obvious variation in CHAC fluorescence characteristics was captured. However, adding 100 equiv N₂H₄ caused a significant shift in fluorescence emission from 420 to 480 nm. Furthermore, anti-interference experiments showed that the coexistence of all the relevant competing species mentioned above had few effects upon N₂H₄ detections by CHAC (Figure 3b). It was thus concluded that CHAC responds to N₂H₄ with perfect selectivity, and that the possibly coexisting interferential species did not interfere with this detection.

2.6. The Proposed Detection Mechanism

Based on the reported literature [38,40] and the results of UV-Vis and emission spectra (Figure S7), the probe CHAC response mechanism with N₂H₄ was proposed (Scheme 2). When a sufficient amount of N₂H₄ was added, it acted as a nucleophile to attack the acetoxy group in CHAC, and a hydrazinolysis reaction occurred that released the fluorophore CHOH. The proposed sensing process was further verified via HPLC and HRMS. From the HPLC chromatograms (Figure 4), the probe CHAC exhibited a main peak with a 7.22 min retention time. Upon post-incubation with N₂H₄, the CHAC peak gradually disappeared as the N₂H₄ concentration increased. A new peak also appeared at 6.31 min and gradually increased in intensity, which was attributed to CHOH. Furthermore, a main peak at m/z 271.0168 was discovered in the MS spectrum of the CHAC solution after the reaction with N₂H₄ (Figure S8), which was concordant with the deprotonated molecular weight of CHOH ([M–H]⁻ 271.0167). All these results indicated the conversion of CHAC into CHOH by N₂H₄.

To help us understand the photophysical properties of probe CHAC before and after reaction with N₂H₄, we used the Gaussian 16 program [51] to perform time-dependent density functional theory (TDDFT) calculations at a B3LYP/6-311G(d,p) level. A polarizable continuum model (PCM) [52] with a dielectric constant of 78.54 was used to simulate a solvent environment for the calculation. As demonstrated in Figure 5, in the process of excitation from the ground state (S₀) to the lowest excited state (S₁), the probe CHAC and the fluorophore CHOH showed significantly different π-electron distributions. In the HOMO of CHAC and CHOH, the electron density was distributed evenly throughout the whole molecule, while that in the LUMO of these two molecules displayed a tendency to transfer to coumarin. Compared with the electron density in the LUMO of CHOH, which was found more locally on the pyranone ring of coumarin, that in the LUMO of CHAC was spread mainly over the entire coumarin, indicating that the acetyl group weakened the ICT process in the probe CHAC. In addition, the HOMO-LUMO gap of CHOH (2.783 eV) was slightly weaker compared with the probe CHAC (2.946 eV), which was concordant with the red shift of FS post-hydrazinolysis.

2.7. N₂H₄ Determinations for Water Samples

To verify the potential application of the probe we designed, we employed CHAC to capture N₂H₄ in water samples using the addition method. Tap and lake water were selected as representatives of actual water samples. The results revealed that the N₂H₄ concentrations detected by this probe were consistent with the added concentrations (

Table 1. N₂H₄ detection in water samples.

Sample	Added (μM)	Found (μM)	Recovery (%)	RSD (%)
Tap water	0	Not detected	—	—
	5.00	5.36	107.2	1.1
	25.00	25.51	102.0	0.8
	50.00	52.07	104.1	3.6
Lake water	0	Not detected	—	—
	5.00	5.13	102.2	2.8
	25.00	24.91	99.5	0.8
	50.00	51.02	102.0	3.3

). Recoveries for the determination of N₂H₄ were between 99.5% and 107.2%, thus indicating that CHAC can be utilized as a practical means for the detection of N₂H₄ in environmental specimens.

2.8. Gaseous N₂H₄ Detections

Another practical application of CHAC in N₂H₄ vapor capture was also investigated by loading CHAC onto filter paper. We pre-treated filter papers via the probe by immersing them in a methanol solution (1 mM) and then drying them. The filter papers were then placed in airtight jars that included distinguished N₂H₄ concentrations (0, 0.1%, 0.5%, 1%, 5%, 10%, and 20%), and were then exposed to N₂H₄ vapor for 0.5 h at room temperature. The results revealed that under a 365 nm light, N₂H₄ concentration increased and the fluorescent color of the filter paper gradually turned from deep blue to cyan (Figure 6). N₂H₄ vapor concentrations of 0–20% resulted in a noticeable difference in the CHAC color on the filter paper, which indicated that the probe CHAC could also be conveniently utilized for the qualitative detection of gaseous N₂H₄.

2.9. N₂H₄ Determinations in Soils

Subsequently, the probe CHAC applications in soil samples were also explored [53]. Sandy, field, and humus soils were collected from the surrounding area as experimental samples. First, 1 g sandy soil/field soil/ humus soil was added to 3 mL of probe CHAC solution (5 μM). We discovered strong blue fluorescence under 365 nm UV light (Figure 7a), indicating that the soil itself did not influence the fluorescence properties of CHAC. Another 1 g of the abovementioned soil samples was first treated with N₂H₄ solution (2 mM), and then transferred to a solution of 3 mL CHAC (5 μM). After 30 min, the solution showed strong cyan fluorescence under 365 nm UV light. The supernatant of soil solution was then analyzed via fluorescence spectrometry. The data highlighted that the fluorescence intensity ratio I₄₈₀/I₄₂₀ of the soil solution treated with N₂H₄ was significantly higher than that without N₂H₄ (Figure 7b), which demonstrated that CHAC could be utilized for the ratiometric detection of N₂H₄ in soils.

2.10. Toxicity of Probe CHAC and Imaging of Cells

The cytotoxicity of the probe CHAC (10–50 μM) on MC3T3-E1 cells was evaluated using the MTT assay. These data revealed that, even after treatment with the 50 μM probe CHAC for 1 day, the cells maintained a viability of more than 90%, thus indicating the very low cytotoxicity of the probe CHAC (Figure S9).

The imaging results of CHAC in MC3T3-E1 cells are shown in Figure 8. Under excitation at 405 nm, the cells showed fluorescence in neither blue nor green channels (Figure 8b,c). If we treated cells with CHAC alone for 0.5 h, strong fluorescence appeared in the blue channel, and almost no green fluorescence was found (Figure 8f,g). For cells pre-incubated with CHAC for 0.5 h and with N₂H₄ for another 0.5 h, the fluorescence in the blue channel disappeared, and strong fluorescence appeared in the green channel (Figure 8j,k), which indicated that the probe CHAC possessed effective cell membrane permeability to enable the fluorescent ratiometric detection of exogenous N₂H₄ in living cells.

3. Materials and Methods

3.1. Materials and Apparatus

2,4-Dihydroxybenzaldehyde, 4-chlorophenylacetic acid, triethylamine, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), glutathione (GSH), cysteine (Cys), homocysteine (Hcy), and quinine sulfate were purchased from Shanghai Macklin Biochemical Co., Ltd (Shanghai, China). Acetic anhydride, ethylenediamine (EDA), aniline, urea, thiourea, hydroxylamine, N₂H₄, and various inorganic salts were purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Mouse embryonic osteoblasts (MC3T3-E1) were purchased from the National Experimental Cell Resource Sharing Platform (Beijing, China). All solvents used in the experiments were of commercially analytical or spectroscopic grade. Ultrapure, de-ionized water was utilized in the experiments.

NMR spectra were recorded on a Bruker Avance 400 MHz spectrometer (Bruker, Bremen, Germany), employing TMS as an internal reference. HRMS spectra were recorded using an Agilent 6540 Q-TOF spectrometer (Agilent, Santa Clara, CA, USA). UV-Vis spectra were measured using a Shimadzu UV-2450 spectrophotometer (Shimadzu, Kyoto, Japan). FS were recorded using a Hitachi F-4600 fluorescence spectrometer. pH values were determined utilizing a Leici PHSJ-4A acidometer (Leici, Shanghai, China). Cell imaging was performed using Leica Stellaris 5 confocal microscopy (Leica, Wetzler, Germany). Cytotoxicity tests were carried out on a DNM-9602G enzyme-labeled instrument (Perlong, Beijing, China). HPLC analysis was performed using a Shimadzu Prominence LC-20A liquid chromatograph (Shimadzu, Kyoto, Japan).

3.2. Synthesis of the Probe CHAC

2,4-Dihydroxybenzaldehyde (0.54 g, 3.61 mmol) and 4-chlorophenylacetic acid (0.93 g, 5.45 mmol) were dissolved in 8 mL acetic anhydride, and then 2 mL triethylamine was added. The mixture was heated to 120 °C and stirred for 7 h. The end of the reaction was monitored using TLC (EtOAc:petroleum ether = 1:3). After cooling, the white precipitate was collected, washed using water, dried, and then recrystallized with ethanol to obtain CHAC (0.81 g, yield 71%). ¹H NMR (400 MHz, DMSO-d₆): δ 8.31 (s, 1H), 7.82 (d, 1H, J = 5.4 Hz), 7.76 (d, 2H, J = 5.1 Hz), 7.54 (d, 2H, J = 5.1 Hz), 7.32 (s, 1H), 7.20 (d, 1H, J = 5.4 Hz), 2.32 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆): δ 168.8, 159.4, 153.5, 152.8, 140.4, 133.4, 133.3, 130.3, 129.6, 128.3, 124.9, 118.9, 117.3, 109.7, 20.9. HRMS m/z calcd. for C₁₇H₁₂ClO₄ [M+H]⁺: 315.0419, found: 315.0419.

3.3. Synthesis of the Fluorophore CHOH

CHAC (0.60 g, 1.91 mmol) was added to a mixture with 4 mL acetone and 8 mL concentrated hydrochloric acid, and the mixture was heated to 80 °C and stirred for 4 h. TLC (EtOAc:petroleum ether = 1:2) showed that CHAC was consumed. After cooling to room temperature, the yellow precipitate was collected, cleaned using water, and dried to collect CHOH (0.44 g, 85% yield). ¹H NMR (400 MHz, DMSO-d₆): δ 10.65 (s, 1H), 8.19 (s, 1H), 7.73 (d, 2H, J = 5.6 Hz), 7.68 (d, 1H, J = 5.7 Hz), 7.49 (d, 2H, J = 5.6 Hz), 6.83 (dd, 1H, J = 5.6 Hz, J = 1.0 Hz), 6.76 (s, 1H). ¹³C NMR (100 MHz, DMSO-d₆): δ 161.5, 159.9, 155.0, 141.4, 133.9, 132.6, 130.1, 130.0, 128.2, 120.81, 113.5, 111.9, 101.7. HRMS m/z Calcd for C₁₅H₉ClNaO₃ [M+Na]⁺: 295.0132, found: 295.0137.

3.4. Preparations for Spectral Measurement

The stock solution (5 mM) of CHAC was prepared in DMSO. Stock solutions (10 mM) of sodium salts (I⁻, F⁻, ${\mathrm{SO}}_4^{2-}$, ${\mathrm{HSO}}_3^{-}$, ${\mathrm{H}}_2{\mathrm{PO}}_4^{-}$, Cl⁻, Br⁻, CH₃COO, and ${\mathrm{N}}_3^{-}$), cations (K⁺, Na⁺, Ca²⁺, Mg²⁺, Al³⁺, Cd²⁺, Fe³⁺, Fe²⁺, and Zn²⁺), and amino-containing analytes (ammonia, aniline, urea, thiourea, hydroxylamine, Cys, EDA, Hcy, and GSH) were obtained by dissolving them in deionized water. The test solutions were prepared as follows: CHAC (3 μL, 5 mM) and the analyte solution were added to a tube and diluted to 3 mL using DMSO: PBS buffer (10 mM, 4:6 v/v, pH = 7.4). We incubated the resulting solution for 0.5 h at room temperature and the fluorescence spectrum was recorded at an excitation wavelength of 340 nm. Emission and excitation slit widths were 5 nm and 2.5 nm, respectively, and the voltage was 700 V.

3.5. Determination of Quantum Yield (QY)

The QYs of the probes CHAC and CHOH were determined using quinine sulfate (Φ = 0.54 in 0.1 M H₂SO₄) as a reference. To obtain reliable data, the absorbance of all solutions should not be higher than 0.05. The QY was calculated using the following equation [54]:

$${\Phi }_r={\Phi }_s\times \left(\frac{F_S}{F_r}\right)\times \left(\frac{A_r}{A_s}\right)\times {\left(\frac{n_s}{n_r}\right)}^2$$

(1)

where Φ_r and Φ_s represent the QY of the sample and the reference, respectively. Fs and F_r denote the integral areas of the Fs of the sample and the reference under the same test conditions, respectively. A_s and A_r denote the absorbance of the sample and the reference, respectively. n_s and n_r indicate the refractive index of the sample and the reference in the corresponding solvent, respectively.

3.6. HPLC Detection

The test concentrations of both CHAC and CHOH were 5 μM. The injection volume was 10 μL. The mobile phase was CH₃OH/H₂O (7:3 v/v) and the flow rate was 1 mL/min. A Diamonsil C18(2) column (150 mm × 4.6 mm, 5 μm) was employed as the chromatographic column.

3.7. Detection of N₂H₄ in Water Samples

A standard addition approach was utilized to determine N₂H₄ content in water samples [55]. Our team obtained tap and lake water aliquots from our laboratory and Jian Lake, respectively. The water samples were filtered through a microporous membrane to remove impurities, and then replaced with a PBS buffer for fluorescence spectrometry. Various N₂H₄ concentrations were spiked into the water samples and subsequently analyzed using CHAC. Every group of experiments was performed simultaneously 3 times.

3.8. Cytotoxicity Test

We culturedMC3T3-E1cells in a DMEM containing 10% FBS at 37 °C under CO₂ atmosphere (5%). Initially, we cultured MC3T3-E1 cells in 96-well plates for 1 day and allowed them to adhere. Different concentrations of CHAC (10, 20, 30, 40, and 50 μM) were then added and incubated for another 1 day. The culture media in the wells were removed and an MTT solution (5 mg/mL, 20 μL) was added to every well. Medium was then supplemented and incubated for an additional 4 h under the same conditions. After removing solution from the well, we added DMSO (150 μL/well) to dissolve the formed formazan precipitate. To calculate the cytotoxicity, we captured solution absorbance at 490 nm via a microplate reader.

3.9. Living Cell Fluorescence Imaging (FI)

MC3T3-E1 cells cultured under the aforementioned conditions were used for cell FI experiments. We incubated CHAC (10 μM) and MC3T3-E1 cells at 37 °C for 0.5 h, washed them 3 times with PBS buffer, and divided them into 2 groups. One group was directly imaged utilizing a fluorescence confocal microscope, while the other group of cells was treated with N₂H₄ (500 μM), incubated for an additional 0.5 h, and cleaned 3 times with PBS prior to imaging experiments. Blank MC3T3-E1 cells were used as the controls. We performed FI utilizing a confocal laser scanning microscope at 420–460 nm and 480–520 nm upon excitation at 405 nm.

4. Conclusions

We developed a new ratiometric N₂H₄ FP, CHAC, through a simple one-step reaction. The N₂H₄ presence produced a significant ratiometric alteration in the fluorescence emission curve by cleaving the acetate and releasing the coumarin fluorophore. The response mechanism of CHAC for N₂H₄ was validated through HRMS, HPLC, and TDDFT computations. CHAC exhibited high selectivity, strong anti-interference, a low LOD, a large detection range, and a broad pH application range. In addition, the CHAC-loaded filter paper strips could detect N₂H₄ vapor effectively via fluorescence color change. More importantly, we successfully employed CHAC for N₂H₄ detection in various water and soil specimens, as well as in ratiometric imaging regarding N₂H₄ in live cells with low toxicity. These data indicate that CHAC could be employed as a useful tool to capture N₂H₄ in environmental and biological systems.