An Aggregation-Induced Fluorescence Probe for Detection H2S and Its Application in Cell Imaging

Monitoring hydrogen sulfide (H2S) in living organisms is very important because H2S acts as a regulator in many physiological and pathological processes. Upregulation of endogenous H2S concentration has been shown to be closely related to the occurrence and development of tumors, atherosclerosis, neurodegenerative diseases and diabetes. Herin, a novel fluorescent probe HND with aggregation-induced emission was designed. Impressively, HND exhibited a high selectivity, fast response (1 min) and low detection limit (0.61 μM) for H2S in PBS buffer (10 mM, pH = 7.42). Moreover, the reaction mechanism between HND and H2S was conducted by Job’s plot, HR-MS, and DFT. In particular, HND was successfully employed to detect H2S in HeLa cells.


Introduction
H 2 S is considered to be an important endogenous gaseous transmitter and plays a crucial role in the regulation of intracellular redox status, angiogenesis and anti-inflammatory effects in living organisms [1][2][3].It is generated from L-cysteine or catalyzed by cysteine sulfide β-synthases and cysteine sulfide γ-lyase [4].Evidence showed that the upregulation of H 2 S concentration is closely related to the occurrence and development of tumors, atherosclerosis, neurodegenerative diseases, diabetes and so on [5][6][7][8][9].Therefore, it is necessary to construct a new H 2 S instantaneous detection method with high sensitivity and selectivity to evaluate H 2 S levels in biological systems.
Currently, many H 2 S-activatable small-molecule fluorescent probes have been developed based on different reaction mechanisms, such as the removal of copper ion via copper sulfide precipitation, nucleophilic addition or the reduction of azides/or nitros to amines mediated by the thiolysis of dinitrophenyl ether [7,[10][11][12][13][14][15].However, some of them are based on hydrophobic organic fluorescent groups, which have the disadvantage of poor water solubility and easy aggregation to form precipitates in aqueous solutions, thereby reducing the responsivity to the analyte and limiting the practical application of organic fluorescent molecular probes in biological imaging.Due to the advantages of excellent photostability and biocompatibility of aggregation-induced emission (AIE), a lot of fluorescent probes with AIE effect for H 2 S have been synthesized.However, most of the fluorescent probes with AIE effect are based on the molecular skeleton of tetrastyrene (TPE) [16,17] and have relatively complex molecular structures, which increases the difficulty of AIE molecular design and synthesis.Therefore, the design of efficient AIE bioluminescent materials with simple molecular structures and simple synthesis remains a challenge.
In this paper, a novel AIE activity fluorescent probe, named 2-hydroxy-1-naphthalaldehyde N-(dimethylamino)benzaldehyde (HND) was conducted.The mechanism was tested by Job's plot, HR-MS, and DFT, and revealed that the two C=N double bonds in the structure of HND were used to provide a receptor unit for H 2 S. Compared with early reports (Table S1), HND exhibits a comparable limit of detection and a fast response.Furthermore, HND realized the detection of H 2 S in HeLa cells.

AIE Effect of HND in Solution
To better understand the AIE effect of HND, the fluorescence properties of HND in a mixed solvent system of DMSO and H 2 O were first investigated.As an exception, HND showed no fluorescence signal when dissolved in the good solvent DMSO, while the fluorescence signal at 536 nm could be turned on and gradually increased with the increase in water volume fraction; an 8.23-fold increase was observed when the water volume fraction increased to 60% (as shown in Figure 1a).With the water volume fraction increased from 60% to 100%, the fluorescence signal reduced slightly.The photographs given in Figure 1d show the color change in HND with increasing water content.A clear yellow fluorescence was observed under a 365 nm lamp.These results indicated that the compound HND is a typical aggregation-induced emission (AIE) active compound in the aqueous medium [18].Herein, DMSO is a good solvent and water is a poor solvent for HND [19].The reason for the fluorescence enhancement of HND in DMSO is because the radiation channel was opened, leading to an increase in fluorescence intensity due to the restricted intramolecular rotations (RIRs) of the coumarin matrix and thiosemicarbazide [20].
reports (Table S1), HND exhibits a comparable limit of detection and a fast response.Furthermore, HND realized the detection of H2S in HeLa cells.

AIE Effect of HND in Solution
To better understand the AIE effect of HND, the fluorescence properties of HND in a mixed solvent system of DMSO and H2O were first investigated.As an exception, HND showed no fluorescence signal when dissolved in the good solvent DMSO, while the fluorescence signal at 536 nm could be turned on and gradually increased with the increase in water volume fraction; an 8.23-fold increase was observed when the water volume fraction increased to 60% (as shown in Figure 1a).With the water volume fraction increased from 60% to 100%, the fluorescence signal reduced slightly.The photographs given in Figure 1d show the color change in HND with increasing water content.A clear yellow fluorescence was observed under a 365 nm lamp.These results indicated that the compound HND is a typical aggregation-induced emission (AIE) active compound in the aqueous medium [18].Herein, DMSO is a good solvent and water is a poor solvent for HND [19].The reason for the fluorescence enhancement of HND in DMSO is because the radiation channel was opened, leading to an increase in fluorescence intensity due to the restricted intramolecular rotations (RIRs) of the coumarin matrix and thiosemicarbazide [20].Particle size analysis was a useful method to further verify the formation of aggregates of HND.As shown in Figure 1b, the average particle size of HND (5.0 µM) in DMSO solution was 45.69 nm, while the average diameter in PBS (10 mM, pH = 7.40) was approximately 128.52 nm, indicating that HND does indeed have aggregates in aqueous solutions.Furthermore, the diameter of HND was supplemented by transmission electron microscopy (TEM).The TEM results showed that the particle size of HND is approximately 100 nm, which is consistent with the results of particle size analysis (128.52 nm).In addition, TEM showed that the morphology of HND was almost elliptical and it had a structural feature with a darker color in the center and a thin film coating at the periphery (Figure S4).
To better understand the properties of AIE, we conducted analyses of the fluorescence spectra of HND (5.0 µM) in different CH 3 OH/glycerin mixtures.As shown in Figure 1c, the fluorescence intensity at 536 nm increased with the increase in glycerol fractions.The results indicated that the sticky glycerin could block the nonradiative intramolecular rotation [18].This means that HND is a typical AIE compound [13,19].

Selectivity and Competition
Many H 2 S probes based on nucleophilic addition reaction have been reported in recent years [15,[21][22][23][24].To identify the interaction between HND and H 2 S (Na 2 S was employed as H 2 S donor), as shown in Figure 2a, an extremely strong yellow fluorescence with λ max = 536 nm was observed in PBS buffer solution (10 mM, pH = 7.40).However, the fluorescence intensity of HND (5.0 µM) was dramatically reduced after the addition of 100 eq.H 2 S in PBS buffer solution (10 mM, pH = 7.40).The fluorescence intensity of HND was decreased to almost no fluorescence.There are other biologically relevant reactive sulfurs (RSS), such as Na 2 SO 4 , Na 2 SO 3 , NaHSO 4 , NaHSO 3 , Na 2 S 2 O 3 , L-Cys and GSH, which could react with HND; here, it is necessary to verify the specificity of HND.In addition, many anions and cations, such as PO 4 3− , HCO 3 2− , I − , etc., were also investigated.The results indicated that no detectable responses were observed when 100 eq. of other RSS or many anions were added to HND, respectively.The response of HND to H 2 O 2 was also conducted; it was found that H 2 O 2 could not respond to HND.
Molecules 2024, 29, x FOR PEER REVIEW 3 of 10 Particle size analysis was a useful method to further verify the formation of aggregates of HND.As shown in Figure 1b, the average particle size of HND (5.0 µM) in DMSO solution was 45.69 nm, while the average diameter in PBS (10 mM, pH = 7.40) was approximately 128.52 nm, indicating that HND does indeed have aggregates in aqueous solutions.Furthermore, the diameter of HND was supplemented by transmission electron microscopy (TEM).The TEM results showed that the particle size of HND is approximately 100 nm, which is consistent with the results of particle size analysis (128.52 nm).In addition, TEM showed that the morphology of HND was almost elliptical and it had a structural feature with a darker color in the center and a thin film coating at the periphery (Figure S4).
To better understand the properties of AIE, we conducted analyses of the fluorescence spectra of HND (5.0 µM) in different CH3OH/glycerin mixtures.As shown in Figure 1c, the fluorescence intensity at 536 nm increased with the increase in glycerol fractions.The results indicated that the sticky glycerin could block the nonradiative intramolecular rotation [18].This means that HND is a typical AIE compound [13,19].

Selectivity and Competition
Many H2S probes based on nucleophilic addition reaction have been reported in recent years [15,[21][22][23][24].To identify the interaction between HND and H2S (Na2S was employed as H2S donor), as shown in Figure 2a, an extremely strong yellow fluorescence with λmax = 536 nm was observed in PBS buffer solution (10 mM, pH = 7.40).However, the fluorescence intensity of HND (5.0 µM) was dramatically reduced after the addition of 100 eq.H2S in PBS buffer solution (10 mM, pH = 7.40).The fluorescence intensity of HND was decreased to almost no fluorescence.There are other biologically relevant reactive sulfurs (RSS), such as Na2SO4, Na2SO3, NaHSO4, NaHSO3, Na2S2O3, L-Cys and GSH, which could react with HND; here, it is necessary to verify the specificity of HND.In addition, many anions and cations, such as PO4 3-, HCO3 -, NO2 -, Br -, NO3 -, F -, CO3 2-, Cl -, C2O4 2-, I -, etc., were also investigated.The results indicated that no detectable responses were observed when 100 eq. of other RSS or many anions were added to HND, respectively.The response of HND to H2O2 was also conducted; it was found that H2O2 could not respond to HND.In addition, a lot of metal ions, such as Mn 2+ , Cu 2+ , Ag + , Co 3+ , Fe 3+ , Al 3+ , Ca 2+ , K + , Ni 2+ , Cd 2+ , Cr 3+ and so on, which coexist in the human body, were also investigated; except for Cu 2+ , all other tested metal ions have no interference with HND (Figures S5 and S6).All of the results showed that HND has high selectivity for H2S.In addition, a lot of metal ions, such as Mn 2+ , Cu 2+ , Ag + , Co 3+ , Fe 3+ , Al 3+ , Ca 2+ , K + , Ni 2+ , Cd 2+ , Cr 3+ and so on, which coexist in the human body, were also investigated; except for Cu 2+ , all other tested metal ions have no interference with HND (Figures S5 and S6).All of the results showed that HND has high selectivity for H 2 S.
To further demonstrate the ability of HND to respond to H 2 S in the presence of other biologically relevant reactive RSS, anions and H 2 O 2 , the competition of HND was also examined (Figure 2b).It was found that HND could also respond to H 2 S in the presence of other biologically relevant reactive RSS, anions and H 2 O 2 , and the fluorescence intensity was similar to that of HND directly responding to H 2 S.This informed us that the RSS, anions and H 2 O 2 tested had a negligible interfering effect on the detection of H 2 S by HND.Similarly, when metal ions and H 2 S coexisted, the decrease in fluorescence intensity of HND was also observed (Figure S6).

H 2 S Response by Probe HND
Figure 3a shows the relationship between HND and the concentrations of H 2 S. The fluorescence intensity of HND in PBS buffer solution (10 mM, pH = 7.40) at 536 nm decreased with the gradual addition of H 2 S at different concentrations.When 435 µM H 2 S was added, a 5.71-fold quenching of the fluorescence was observed.The fluorescence color changed from light yellow to colorless (shown in Figure 3a inset).A good linear relationship was observed between the fluorescence intensities at 536 nm, as well as the H 2 S concentration from 25 to 210 µM; moreover, the value of the linear correlation R 2 was 0.9862 (Figure 3a inset).Followed by the equation LOD = K × Sb1/S [25], the detection limit for H 2 S was estimated to be 0.61 µM.Compared with the early results (as shown in Table S1), the LOD value is consistent with the reports by Lu and Jose [26][27][28] and good compared with the reports by Zhang [29,30].Additionally, the pH-dependent responses of probe HND and HND to H 2 S were carried out.From Figure S7, it can be seen that probe HND could only respond to H 2 S at pH 7.42.At other pH values, such as 4.00, 6.86, 9.18 and 10.01, HND could not respond to H 2 S. Therefore, PBS buffer (10 mM, pH 7.4) was chosen as the reaction system.CIE chromaticity was used to better understand the chromaticity changes in the fluorescence spectra of HND upon the addition of H 2 S [31].CIE chromaticity coordinates were also calculated from the emission spectrum [32].A two-dimensional space (XY plane) could be found in the CIE where each point of the chromaticity map stands for a particular color.From Figure 3b, the CIE chromaticity of HND was found to be x = 0.3818, y = 0.5219, while after the addition of H 2 S, the CIE chromaticity was converted to be x = 0.3618, y = 0.4174.This is an indication of a gradual shift in the color (Figure S8).
Response time is one of the most important parameters for a sensor.Therefore, kinetic studies were conducted to investigate the response time.A total of 10 eqiv. of H 2 S was added to HND (5.0 µM) and the fluorescence intensity at 536 nm was monitored over time until the spectrum became constant (Figure 3c).The results showed that it takes almost 1 min for HND to react completely with H 2 S.This is a fast reaction rate compared to the first report [13,28,33,34].
Furthermore, the absorption spectra of HND (15 µM) treated with different concentrations of H 2 S in PBS buffer solution (10 mM, pH = 7.40) were recorded.As shown in Figure 3d, four peaks at 457 nm, 427 nm, 404 nm and 355 nm were observed; with the addition of H 2 S, the peaks at 457 nm, 427 nm, 404 nm and 355 nm were decreased and two new peaks at 438 nm and 326 nm appeared and gradually increased.An isoabsorption point at 329 nm was found.The isoabsorptive point indicated that a new compound was formed between HND and H 2 S [31,35].mated to be 0.61 µM.Compared with the early results (as shown in Table S1), the LOD value is consistent with the reports by Lu and Jose [26][27][28] and good compared with the reports by Zhang [29,30].Additionally, the pH-dependent responses of probe HND and HND to H2S were carried out.From Figure S7, it can be seen that probe HND could only respond to H2S at pH 7.42.At other pH values, such as 4.00, 6.86, 9.18 and 10.01, HND could not respond to H2S.Therefore, PBS buffer (10 mM, pH 7.4) was chosen as the reaction system.

Mechanism Investigation
HR-MS was used to further investigate the mechanism involved in the fluorescence quenching between HND and Na 2 S. The HR-MS of HND shows a strong peak at m/z = 318.1606[HND + H] + (Figure S2).After the addition of an excess of Na 2 S, a new peak appears at m/z = 418.1619[HND -HS + CH 3 OH + H] + , which indicates clearly that the C=N of HND were formed through nucleophilic addition by H 2 S in aqueous solution (Figure 4).The recognition mechanism is similar to the other reports [36][37][38][39].
DFT with B3LYP/6-31G(d) (the Gaussian 09) was carried out to further investigate the electron distributions and orbital energies of HND and H 2 S [40,41].From Figure 5, we find that the highest occupied molecular orbitals (HOMOs) and the lowest occupied molecular orbitals (LOMOs) of HND were mainly distributed over the whole molecule, so HND showed a strong fluorescence due to the absence of electron transfer.The HOMOs and LUMOs of HND -HS were mainly distributed on the 2-hydroxy-1-naphthylaldehyde group.The HOMO-LUMO energy gaps of HND and HND -HS were 9.1417 ev and 10.3809 ev, respectively.The HOMO-LUMO band gap of HND -HS was higher than that of HND, which could be clearly explained by the blue shift in the emission maxima of HND -HS [32,42].
HR-MS was used to further investigate the mechanism involved in the fluorescence quenching between HND and Na2S.The HR-MS of HND shows a strong peak at m/z = 318.1606[HND + H] + (Figure S2).After the addition of an excess of Na2S, a new peak appears at m/z = 418.1619[HND -HS + CH3OH + H] + , which indicates clearly that the C=N of HND were formed through nucleophilic addition by H2S in aqueous solution (Figure 4).The recognition mechanism is similar to the other reports [36][37][38][39].DFT with B3LYP/6-31G(d) (the Gaussian 09) was carried out to further investigate the electron distributions and orbital energies of HND and H2S [40,41].From Figure 5, we find that the highest occupied molecular orbitals (HOMOs) and the lowest occupied molecular orbitals (LOMOs) of HND were mainly distributed over the whole molecule, so HND showed a strong fluorescence due to the absence of electron transfer.The HOMOs and LUMOs of HND -HS were mainly distributed on the 2-hydroxy-1-naphthylaldehyde group.The HOMO-LUMO energy gaps of HND and HND -HS were 9.1417 ev and 10.3809 ev, respectively.The HOMO-LUMO band gap of HND -HS was higher than that of HND, which could be clearly explained by the blue shift in the emission maxima of HND -HS [32,42].

Fluorescence Imaging
The applicability of HND in biological systems was investigated by analyzing H2S imaging in living cells.Bright yellow fluorescence was obtained when HeLa cells were incubated with 20 µM of HND for 20 min.However, the bright yellow fluorescence of HeLa cells was gradually quenched when the cells were treated with HND for 20 min and then treated with H2S (the concentrations of H2S were 20 µM and 40 µM, respectively) for another 20 min (Figure 6b,c).The results indicated that HND has good biocompatibility and is able to image different concentrations of H2S in living cells.

Fluorescence Imaging
The applicability of HND in biological systems was investigated by analyzing H 2 S imaging in living cells.Bright yellow fluorescence was obtained when HeLa cells were incubated with 20 µM of HND for 20 min.However, the bright yellow fluorescence of HeLa cells was gradually quenched when the cells were treated with HND for 20 min and then treated with H 2 S (the concentrations of H 2 S were 20 µM and 40 µM, respectively) for another 20 min (Figure 6b,c).The results indicated that HND has good biocompatibility and is able to image different concentrations of H 2 S in living cells.

Materials
All reagents were purchased through a commercial method and used without further purification. 1H-NMR was obtained using a Bruker DRX 400 spectrometer with TMS as the internal standard.High-resolution mass spectrometry (HR-MS) was performed on a Triple TOF TM 5600 + system.Fluorescence spectra were obtained at room temperature using a CRT-970 fluorescence spectrophotometer.The absorption spectra were measured by Shimadzu UV1800.Imaging of probe HND and HND with Na2S was conducted on a confocal laser scanning microscope L700 with HeLa cells.

The Synthesis Process
As shown in Scheme 1, first, the mixture of ethanol (30 mL), 2-hydroxynaphthalene-1-carbaldehyde (0.344 g, 2.0 mmol) and hydrazine hydrate (400 µL) were first refluxed in a flask.After 10 h, the reaction was stopped by TLC.Yellow solids were obtained, named 2-hydroxynaphthalene-1-carbaldehyde hydrazone (1).Next, 1 (0.279 g, 1.5 mmol) and 4-(dimethylamino)benzaldehyde (0.1491 g, 1.0 mmol) were mixed in 30 mL ethanol, a few drops of DMF was added as a catalyst, and then the above mixture was heated and refluxed for 24 h.The yellow powder HND was obtained after the mixture was cooled and washed by ethanol three times; then, it was recrystallized by CH3CH2OH.Yield, 0.17 g

Materials
All reagents were purchased through a commercial method and used without further purification. 1H-NMR was obtained using a Bruker DRX 400 spectrometer with TMS as the internal standard.High-resolution mass spectrometry (HR-MS) was performed on a Triple TOF TM 5600 + system.Fluorescence spectra were obtained at room temperature using a CRT-970 fluorescence spectrophotometer.The absorption spectra were measured by Shimadzu UV1800.Imaging of probe HND and HND with Na 2 S was conducted on a confocal laser scanning microscope L700 with HeLa cells.

The Synthesis Process
As shown in Scheme 1, first, the mixture of ethanol (30 mL), 2-hydroxynaphthalene-1carbaldehyde (0.344 g, 2.0 mmol) and hydrazine hydrate (400 µL) were first refluxed in a flask.After 10 h, the reaction was stopped by TLC.Yellow solids were obtained, named 2-hydroxynaphthalene-1-carbaldehyde hydrazone (1).Next, 1 (0.279 g, 1.5 mmol) and 4-(dimethylamino)benzaldehyde (0.1491 g, 1.0 mmol) were mixed in 30 mL ethanol, a few drops of DMF was added as a catalyst, and then the above mixture was heated and refluxed for 24 h.The yellow powder HND was obtained after the mixture was cooled and washed by ethanol three times; then, it was recrystallized by CH 3 CH 2 OH.Yield, 0.17 g (54%).

Fluorescence Spectral Property
Na2S was dissolved in water and used as the donor of H2S [36].Stock solution of probe HND (10 −3 M) was prepared in DMSO.The fluorescence spectra of HND with H2S were obtained by the addition of various concentrations of Na2S to HND (5.0 µM) in PBS buffer (10 mM, pH 7.4).The fluorescence spectra were excited by 420 nm.

Conclusions
In conclusion, a novel fluorescent probe HND with AIE activity was synthesized.The results showed that HND could be used for the detection of H2S in aqueous solution and HeLa cells.HND exhibited obvious fluorescence quenching and excellent selectivity/competitiveness toward H2S with a limit of detection of 0.64 µM and a response time of 1 min.We assumed that H2S reacted with HND through nucleophilic addition reaction and the mechanism was proven by HR-MS and DFT calculations.In addition, HND was successfully applied to image the H2S in HeLa cells.

Figure 3 .
Figure 3. (a) The fluorescence titration curve of 5.0 µM HND in the presence of different concentrations of H2S.Inset: (i) The corresponding fluorescence images of HND and HND mixed with H2S Figure 3. (a) The fluorescence titration curve of 5.0 µM HND in the presence of different concentrations of H 2 S. Inset: (i) The corresponding fluorescence images of HND and HND mixed with H 2 S under 365 nm UV lamp illumination.(ii) Changes in emission intensity at 536 nm with incremental additions of H 2 S. (b) CIE diagram of the HND in PBS buffer solution (10 mM, pH = 7.40).(c) Changes in emission intensity of HND (5.0 µM) at 536 nm in the presence of 10 equiv. of H 2 S as the function of time.(d) Absorption spectra of HND (15 µM) treated with different concentrations of H 2 S.

Figure 4 .
Figure 4. ESI mass spectrum of HND upon addition of excess Na2S.

Figure 4 .
Figure 4. ESI mass spectrum of HND upon addition of excess Na 2 S.