H2S Sensors: Synthesis, Optical Properties, and Selected Biomedical Applications under Visible and NIR Light

Hydrogen sulfide (H2S) is an essential signaling gas within the cell, and its endogenous levels are correlated with various health diseases such as Alzheimer’s disease, diabetes, Down’s syndrome, and cardiovascular disease. Because it plays such diverse biological functions, being able to detect H2S quickly and accurately in vivo is an area of heightened scientific interest. Using probes that fluoresce in the near-infrared (NIR) region is an effective and convenient method of detecting H2S. This approach allows for compounds of high sensitivity and selectivity to be developed while minimizing cytotoxicity. Herein, we report a review on the synthesis, mechanisms, optical properties, and selected biomedical applications of H2S sensors.


Introduction
Hydrogen sulfide (H 2 S) is the third most important endogenous signal gas that cells use [1,2], after carbon monoxide and nitric oxide. It mediates multiple physiological and pathological processes [3,4]. While crucial to normal biological function, H 2 S can prove an extremely toxic gas at higher concentrations. Some basic physicochemical and thermodynamic properties of H 2 S are listed in Table 1 [5].
A simple molecule, H 2 S is a marker of some of society's most prevalent maladies. Cardiovascular disease, diabetes, and Down's syndrome have all been associated with an increased level of H 2 S [6]. It is known to regulate myriad biological functions in vivo; oxidative stress, insulin signaling, and cell growth and death are all mediated by this species [7,8]. Energy available to the cell can also be affected by levels of H 2 S because it is involved in the inhibition of adenosine triphosphate (ATP) [9][10][11][12][13].
Biosynthesis of H 2 S in mammals is carried out by the enzymes cystathionine βsynthase (CBS), 3-mercaptopyruvate sulfotransferase , and cystathionine γ-lyase (CSE) [14]. The endogenous pathways are laid out in Scheme 1 [15]. Homocysteine acts as the substrate for CBS, with H 2 S and cystathionine being liberated. The second mode involves a "desulfurization" reaction, whereby sulfur is cleaved from cysteine directly without its oxidation. Furthermore, it has been reported that the activities of these two enzymes in different cells and tissues are correlated to several health disorders [16][17][18]. For these reasons, H 2 S has become an important medium in the detection of disease [19][20][21].
Traditional methods of H 2 S detection, such as electrochemical analysis and gas chromatography, require a sample of living cells from target tissues [22,23]. However, since H 2 S can exist at levels as low as 30 µM in human blood, such methods are inadequate for detecting the analyte in the blood [24]. With these limitations in mind, the discovery of highly sensitive and selective detection methods for H 2 S is of peak interest [25][26][27][28][29][30]. Traditional methods also present the inherent drawbacks of trauma from conducting biopsies, inconsistent concentrations of the analyte within the target tissue, and an overall more Table 1. Basic physicochemical and thermodynamic properties of H 2 S. Reprinted with permission from Ref. [5]. Copyright ©2018 American Chemical Society.

Properties Data
Dipole  Table 1. Basic physicochemical and thermodynamic properties of H2S. Reprinted with permission from Ref. [5]. Copyright ©2018 American Chemical Society.

. Nucleophilic Addition to the Conjugated System for H2S Detection
Under the physiological condition of pH 7.6, H2S was deprotonated to HS − . This species was then able to be added to the conjugated system by way of nucleophilic attack onto the electrophilic center of the probe. In this way, cyanine dyes were able to detect H2S. Such a mechanism has four different modes of signal transduction. These include intramolecular charge transfer (ICT) [61][62][63], twisted intramolecular charge transfer [64][65][66] (TICT), fluorescence resonance energy transfer (FRET) [67][68][69], and photoinduced electron transfer (PET) [72][73][74]. Each of these processes involves the transfer of energy within the probe in the presence of the analyte. These mechanisms are discussed individually under the banner of H2S detection by way of nucleophilic addition to a conjugated system.

Intramolecular Charge Transfer (ICT)
This is the process of converting light energy into chemical energy. An example is photosynthesis, where plants use sunlight to carry out key charge separation processes in the production of food. The ICT process depends on the relationship between intensities of the donor and acceptor in the system. The donor resides at the HOMO energy level, and the acceptor resides at the LUMO. Moreover, the energy gap between the two can serve to insulate or conduct depending on the circumstances, which determines the electronic coupling degree between the two orbitals [61][62][63].

Synthesis and Mechanism of H2S Probes Based on ICT
An example of a H2S probe performing on the basis of ICT is the morpholinostyryl probe 3, as illustrated in Scheme 2. It was synthesized by Li et al. following the reaction of indolium salt 1 with aldehyde 2. Under reflux in ethanol, a yield of 70% was achieved. The probe behaves as a switch, with fluorescence turning on and off in the absence and presence of H2S, respectively. This occurs because H2S, behaving as the nucleophile, attacks the indolium moiety to break conjugation. The result was a loss of fluorescence in the presence of the H2S at levels as low as 30 μM [61]. Scheme 2. Synthetic route and detection mechanism of H2S probe 3.
In the previous example of a H2S probe, the detection is indicated by a loss of fluorescence. A different probe was synthesized by Yong Liu et al. to have a hypsochromic shift upon binding with the H2S analyte. Scheme 3 shows the production of probe 6 following a two-step reaction. First, the bromine of compound 4 is substituted with 4-vinylpyridine under basic conditions using a Pd(Ac)2 catalyst to yield compound 5. Next, the conjugated system is extended following a Vilsmeier-Haack coupling with an indolium salt. The resulting probe 6 contains two separate sites where the fluorescence Scheme 2. Synthetic route and detection mechanism of H 2 S probe 3.
In the previous example of a H 2 S probe, the detection is indicated by a loss of fluorescence. A different probe was synthesized by Yong Liu et al. to have a hypsochromic shift upon binding with the H 2 S analyte. Scheme 3 shows the production of probe 6 following a two-step reaction. First, the bromine of compound 4 is substituted with 4-vinylpyridine under basic conditions using a Pd(Ac) 2 catalyst to yield compound 5. Next, the conjugated system is extended following a Vilsmeier-Haack coupling with an indolium salt. The resulting probe 6 contains two separate sites where the fluorescence wavelength can be altered. Protonation can occur at the pyridine moiety to result in a bathochromic shift, Molecules 2023, 28, 1295 5 of 32 and the indolium moiety can undergo nucleophilic attack in the presence of H 2 S to give a hypsochromic shift. As it is able to distinguish between H 2 S present in acidic versus nonacidic conditions, probe 6 can be useful in detecting the H 2 S analyte in different parts of the body. This is possible because the fluorescence wavelength can easily be measured by spectroscopy, and whichever shift is observed can indicate the environment in which the analyte was present. For instance, tumors are known to be more acidic than the surrounding healthy tissue while blood is normally slightly alkaline [62]. wavelength can be altered. Protonation can occur at the pyridine moiety to result in a bathochromic shift, and the indolium moiety can undergo nucleophilic attack in the presence of H2S to give a hypsochromic shift. As it is able to distinguish between H2S present in acidic versus nonacidic conditions, probe 6 can be useful in detecting the H2S analyte in different parts of the body. This is possible because the fluorescence wavelength can easily be measured by spectroscopy, and whichever shift is observed can indicate the environment in which the analyte was present. For instance, tumors are known to be more acidic than the surrounding healthy tissue while blood is normally slightly alkaline [62].

Scheme 3.
Synthetic route and detection mechanism of probe 6.
A novel on/off NIR fluorescent probe was developed by Fengzao Chen et al. which is unique in that it can detect H2S simultaneously by cyclization to form the six-member ring. Its synthesis and ICT mechanism are laid out in Scheme 4, involving three steps before being able to bind to H2S. The aldehyde derivative 7 and the 1,3-dicarbonyl compound 8 are refluxed under basic conditions to yield coumarin derivative 9. The resulting ketone group of 9 is formylated via a Vilsmeier-Haack coupling reaction with DMF in the presence of POCl3 to yield compound 10. Subsequent condensation with malononitrile produced compound 11 at a yield of 60%. This step is carried out at room temperature using EtOH and piperidine as solvents, and it results in the conjugated system being lengthened, as well as the addition of the strongly electron-donating cyano groups. This final product is then able to detect H2S under physiological conditions. The deprotonated species HS − substitutes the chloride to give a thiol which then undergoes a proton transfer with one of the cyano groups. A six-membered ring is formed, and this can be detected by the shift in fluorescence wavelength [63]. A novel on/off NIR fluorescent probe was developed by Fengzao Chen et al. which is unique in that it can detect H 2 S simultaneously by cyclization to form the six-member ring. Its synthesis and ICT mechanism are laid out in Scheme 4, involving three steps before being able to bind to H 2 S. The aldehyde derivative 7 and the 1,3-dicarbonyl compound 8 are refluxed under basic conditions to yield coumarin derivative 9. The resulting ketone group of 9 is formylated via a Vilsmeier-Haack coupling reaction with DMF in the presence of POCl 3 to yield compound 10. Subsequent condensation with malononitrile produced compound 11 at a yield of 60%. This step is carried out at room temperature using EtOH and piperidine as solvents, and it results in the conjugated system being lengthened, as well as the addition of the strongly electron-donating cyano groups. This final product is then able to detect H 2 S under physiological conditions. The deprotonated species HS − substitutes the chloride to give a thiol which then undergoes a proton transfer with one of the cyano groups. A six-membered ring is formed, and this can be detected by the shift in fluorescence wavelength [63].

Optical Properties of ICT H 2 S Probes
Referring to the on/off H 2 S probe synthesized by Li et al. in Scheme 2, probe 3 was shown to absorb light at a wavelength of 520 nm and fluoresce at 596 nm, as shown in Figure 1A. Its fluorescence intensity was then measured at 600 nm in the presence of H 2 S at varying concentrations. Figure 1B reveals that the signal of probe 3 was strongest with the analyte absent, but its signal was unobservable at a concentration of 100 µM of H 2 S. These results prove that probe 3 is a useful H 2 S sensor [61].

Optical Properties of ICT H2S Probes
Referring to the on/off H2S probe synthesized by Li et al. in Scheme 2, probe 3 was shown to absorb light at a wavelength of 520 nm and fluoresce at 596 nm, as shown in Figure 1A. Its fluorescence intensity was then measured at 600 nm in the presence of H2S at varying concentrations. Figure 1B reveals that the signal of probe 3 was strongest with the analyte absent, but its signal was unobservable at a concentration of 100 μM of H2S. These results prove that probe 3 is a useful H2S sensor [61].  , which displayed a bathochromic shift upon detection of the H2S analyte, was also found to exhibit spectral changes at varying pH of H2S. The absorption and fluorescence spectra of probe 6 at different pH values are shown in Figure 2A, B, respectively. Furthermore, probe 6 is highly sensitive to H2S under acidic conditions ( Figure 2A). As presented in Figure 2B, probe 6 has good selectivity of H2S over various other sulfur-containing molecules such as SO4 2− , S2O3 2− , and cysteine [62].

Optical Properties of ICT H2S Probes
Referring to the on/off H2S probe synthesized by Li et al. in Scheme 2, probe 3 was shown to absorb light at a wavelength of 520 nm and fluoresce at 596 nm, as shown in Figure 1A. Its fluorescence intensity was then measured at 600 nm in the presence of H2S at varying concentrations. Figure 1B reveals that the signal of probe 3 was strongest with the analyte absent, but its signal was unobservable at a concentration of 100 μM of H2S. These results prove that probe 3 is a useful H2S sensor [61].  , which displayed a bathochromic shift upon detection of the H2S analyte, was also found to exhibit spectral changes at varying pH of H2S. The absorption and fluorescence spectra of probe 6 at different pH values are shown in Figure 2A, B, respectively. Furthermore, probe 6 is highly sensitive to H2S under acidic conditions ( Figure 2A). As presented in Figure 2B, probe 6 has good selectivity of H2S over various other sulfur-containing molecules such as SO4 2− , S2O3 2− , and cysteine [62]. , which displayed a bathochromic shift upon detection of the H 2 S analyte, was also found to exhibit spectral changes at varying pH of H 2 S. The absorption and fluorescence spectra of probe 6 at different pH values are shown in Figure 2A,B, respectively. Furthermore, probe 6 is highly sensitive to H 2 S under acidic conditions ( Figure 2A). As presented in Figure 2B, probe 6 has good selectivity of H 2 S over various other sulfur-containing molecules such as SO 4 2− , S 2 O 3 2− , and cysteine [62].
The third H 2 S probe operating on the ICT principle, probe 11 (Scheme 4), was also studied to undergo changes to its fluorescence spectra at 490 nm by Fengzao Chen et al., as shown in Figure 3. Intensity of fluorescence took 30 min to reach a plateau in the presence of H 2 S, at which point there was only marginal increase in signal strength with time [63]. The third H2S probe operating on the ICT principle, probe 11 (Scheme 4), was also studied to undergo changes to its fluorescence spectra at 490 nm by Fengzao Chen et al., as shown in Figure 3. Intensity of fluorescence took 30 min to reach a plateau in the presence of H2S, at which point there was only marginal increase in signal strength with time [63]. The immortalized cells of Henrietta Lacks, who died of advanced cancer in 1951, were used to further bolster the ability of probe 3 (Scheme 2) to detect H2S in living cells. This sensor penetrated the cell membrane as shown by the contrast between bright-field and fluorescence images of cells incubated with probe 3 ( Figure 4A, B). Overlaying the brightfield and fluorescence images of cells treated with probe 3 verified this claim. Conversely, there was no visible fluorescence upon addition of H2S to the culture ( Figure 4C, D). These images indicate that probe 3 could effectively penetrate the membrane and was selective toward H2S in living cells [61].  The third H2S probe operating on the ICT principle, probe 11 (Scheme 4), was also studied to undergo changes to its fluorescence spectra at 490 nm by Fengzao Chen et al., as shown in Figure 3. Intensity of fluorescence took 30 min to reach a plateau in the presence of H2S, at which point there was only marginal increase in signal strength with time [63]. The immortalized cells of Henrietta Lacks, who died of advanced cancer in 1951, were used to further bolster the ability of probe 3 (Scheme 2) to detect H2S in living cells. This sensor penetrated the cell membrane as shown by the contrast between bright-field and fluorescence images of cells incubated with probe 3 ( Figure 4A, B). Overlaying the brightfield and fluorescence images of cells treated with probe 3 verified this claim. Conversely, there was no visible fluorescence upon addition of H2S to the culture ( Figure 4C, D). These images indicate that probe 3 could effectively penetrate the membrane and was selective toward H2S in living cells [61].

The Application of H 2 S Probes Based on ICT
The immortalized cells of Henrietta Lacks, who died of advanced cancer in 1951, were used to further bolster the ability of probe 3 (Scheme 2) to detect H 2 S in living cells. This sensor penetrated the cell membrane as shown by the contrast between bright-field and fluorescence images of cells incubated with probe 3 ( Figure 4A,B). Overlaying the brightfield and fluorescence images of cells treated with probe 3 verified this claim. Conversely, there was no visible fluorescence upon addition of H 2 S to the culture ( Figure 4C,D). These images indicate that probe 3 could effectively penetrate the membrane and was selective toward H 2 S in living cells [61].
Probe 6 (Scheme 3) was used to detect endogenous H 2 S in the lysosomes of A549 cells. To highlight the probe's ability to detect H 2 S, an assay was performed both with and without the presence of PMS (12-O-tetradecanoylphorbol 13-acetate, 4β,9α,12β,13α,20pentahydroxytiglia-1,6-dien-3-one 12-tetradecanoate 13-acetate), which was used to reduce endogenous H 2 S levels. Figure 5 illustrates that probe 6 did indeed detect the analyte within the lysosomes of the A549 cells (A-C), but the signal was reduced upon the addition of the H 2 S inhibitor, PMA (D-F). The addition of the inhibitor serves to prove the probe's selectivity toward H 2 S and not simply any sulfur-containing analyte. The study confirms the ability of probe 6 to detect endogenous H 2 S in the cell lysosome under the green channel [62]. 28  Probe 6 (Scheme 3) was used to detect endogenous H2S in the lysosomes of A549 cells. To highlight the probe's ability to detect H2S, an assay was performed both with and without the presence of PMS (12-O-tetradecanoylphorbol 13-acetate, 4β,9α,12β,13α,20-pentahydroxytiglia-1,6-dien-3-one 12-tetradecanoate 13-acetate), which was used to reduce endogenous H2S levels. Figure 5 illustrates that probe 6 did indeed detect the analyte within the lysosomes of the A549 cells (A-C), but the signal was reduced upon the addition of the H2S inhibitor, PMA (D-F). The addition of the inhibitor serves to prove the probe's selectivity toward H2S and not simply any sulfur-containing analyte. The study confirms the ability of probe 6 to detect endogenous H2S in the cell lysosome under the green channel [62].    , C), respectively. Reprinted with permission from Ref. [61]. Copyright ©2017 Royal Society of Chemistry.
Probe 6 (Scheme 3) was used to detect endogenous H2S in the lysosomes of A549 cells.
To highlight the probe's ability to detect H2S, an assay was performed both with and without the presence of PMS (12-O-tetradecanoylphorbol 13-acetate, 4β,9α,12β,13α,20-pentahydroxytiglia-1,6-dien-3-one 12-tetradecanoate 13-acetate), which was used to reduce endogenous H2S levels. Figure 5 illustrates that probe 6 did indeed detect the analyte within the lysosomes of the A549 cells (A-C), but the signal was reduced upon the addition of the H2S inhibitor, PMA (D-F). The addition of the inhibitor serves to prove the probe's selectivity toward H2S and not simply any sulfur-containing analyte. The study confirms the ability of probe 6 to detect endogenous H2S in the cell lysosome under the green channel [62].  Probe 11 (Scheme 4) was used to detect H 2 S levels in MCF-7 cells. As shown in Figure 6, a strong fluorescence signal was observed in the green channel from 470-530 nm with H 2 S present ( Figure 6(D2)), but hardly any was seen in the red channel from 620-680 nm ( Figure 6(D3)). Upon addition of probe 11 following treatment with the H 2 S inhibitor NEM, only red-channel signals were seen ( Figure 6(B4)). This means that, without H 2 S, probe 11 would show red channel signals. In the groups with supplemental H 2 S in addition to the inhibitor, the phenomenon was quite different; all others were only detectable in the green channel ( Figure 6C,D). H 2 S causes a blue shift, and the signal is shown in the green channel. These results proved that probe 11 has use as an effective tool in detecting H 2 S in MCF-7 cells [63].
NEM, only red-channel signals were seen ( Figure 6 (B4)). This means that, without H2S, probe 11 would show red channel signals. In the groups with supplemental H2S in addition to the inhibitor, the phenomenon was quite different; all others were only detectable in the green channel ( Figure 6C, D). H2S causes a blue shift, and the signal is shown in the green channel. These results proved that probe 11 has use as an effective tool in detecting H2S in MCF-7 cells [63]. (B) cells were preincubated with NEM and subsequently treated with probe 11; (C) cells pretreated with NEM followed by treatment with probe 11 and GSH; (D) cells pretreated with 0.6 mM NEM followed by probe 11 and then with H2S. Column 1, bright field; column 2, green channel; column 3, red channel; column 4, merged images of columns 1, 2, and 3. Reprinted with permission from Ref. [63]. Copyright ©2018 Royal Society of Chemistry.

Twisted Intramolecular Charge Transfer (TICT)
This is a process of electron transduction which is always linked by a single bond between donor and acceptor units. The twist of a single bond is an essential component of this process, unlike the ICT, which sees a lateral translation of electrons across a rigid structure. The TICT process occurs upon absorption of an electron. The excited molecular structure becomes twisted and then relaxes to the ground state, emitting light at a longer wavelength. Many TICT-based fluorescent probes are potential detectors for some small biological molecules [64][65][66].

Synthesis and Mechanism of H2S Probes Based on TICT
Mingguang Ren et al. synthesized the first H2S TICT probe 13 (Scheme 5) based on the BODIPY scaffold. Probe 13 was obtained by the reaction between compound 12 and the indolium salt under reflux in EtOH. The salt was condensed with the aldehyde group in compound 12 to form probe 13 at 60% yield. For this probe, the combination of the conjugated structure of indolium with the BODIPY group turned on the TICT process, which showed a strong fluorescence. In the presence of H2S, the indolium moiety underwent a nucleophilic attack, thereby quenching the TICT process. Probe 13 could detect the H2S levels in living cells [64]. (C) cells pretreated with NEM followed by treatment with probe 11 and GSH; (D) cells pretreated with 0.6 mM NEM followed by probe 11 and then with H 2 S. Column 1, bright field; column 2, green channel; column 3, red channel; column 4, merged images of columns 1, 2, and 3. Reprinted with permission from Ref. [63]. Copyright ©2018 Royal Society of Chemistry.

Twisted Intramolecular Charge Transfer (TICT)
This is a process of electron transduction which is always linked by a single bond between donor and acceptor units. The twist of a single bond is an essential component of this process, unlike the ICT, which sees a lateral translation of electrons across a rigid structure. The TICT process occurs upon absorption of an electron. The excited molecular structure becomes twisted and then relaxes to the ground state, emitting light at a longer wavelength. Many TICT-based fluorescent probes are potential detectors for some small biological molecules [64][65][66].

Synthesis and Mechanism of H 2 S Probes Based on TICT
Mingguang Ren et al. synthesized the first H 2 S TICT probe 13 (Scheme 5) based on the BODIPY scaffold. Probe 13 was obtained by the reaction between compound 12 and the indolium salt under reflux in EtOH. The salt was condensed with the aldehyde group in compound 12 to form probe 13 at 60% yield. For this probe, the combination of the conjugated structure of indolium with the BODIPY group turned on the TICT process, which showed a strong fluorescence. In the presence of H 2 S, the indolium moiety underwent a nucleophilic attack, thereby quenching the TICT process. Probe 13 could detect the H 2 S levels in living cells [64].

Songjiao Li et al. designed H 2 S probe 16 to monitor levels of H 2 S in cells (Scheme 6).
This compound performs according to the TICT mechanism in the NIR region of the EM spectrum. Under reflux conditions in toluene, the pyridine ring of compound 15 is alkylated by the azide derivative 14 to produce probe 16 at a yield of 90% In the absence of the H 2 S analyte, probe 16 does not show a fluorescence signal due to the free rotation in the TICT state. Upon the addition of H 2 S, the pyridine undergoes nucleophilic attack, and the azide moiety is liberated. At this point, fluorescence was induced. In this way, probe 16 could target the mitochondrion and visualize H 2 S at different concentrations in HeLa cells [65]. Scheme 5. Synthetic route and detection mechanism of probe 13.

Songjiao Li et al. designed H2S probe 16 to monitor levels of H2S in cells (Scheme 6).
This compound performs according to the TICT mechanism in the NIR region of the EM spectrum. Under reflux conditions in toluene, the pyridine ring of compound 15 is alkylated by the azide derivative 14 to produce probe 16 at a yield of 90% In the absence of the H2S analyte, probe 16 does not show a fluorescence signal due to the free rotation in the TICT state. Upon the addition of H2S, the pyridine undergoes nucleophilic attack, and the azide moiety is liberated. At this point, fluorescence was induced. In this way, probe 16 could target the mitochondrion and visualize H2S at different concentrations in HeLa cells [65]. Scheme 6. Synthetic route and detection mechanism of probe 16.
Yongru Zhang et al. designed the dinitrophenyl H2S fluorescent probe 18. Basic conditions facilitated the condensation of the benzothiazole derivative with the aldehyde of compound 17 to produce probe 18 in 12% yield. The mechanism via which H2S was sensed occurs via the analyte undergoing a nucleophilic aromatic substitution on the dinitrobenzene ring. In the process, 2,3-dinitrobenzothiol was liberated, and the cyano group of the intermediate was reduced to an imine (Scheme 7). The formation of a six-membered ring brought conjugation to the molecule, and this could be detected by spectroscopy. In this way, probe 18 could detect endogenous levels of H2S within living cells [66]. Scheme 6. Synthetic route and detection mechanism of probe 16.
Yongru Zhang et al. designed the dinitrophenyl H 2 S fluorescent probe 18. Basic conditions facilitated the condensation of the benzothiazole derivative with the aldehyde of compound 17 to produce probe 18 in 12% yield. The mechanism via which H 2 S was sensed occurs via the analyte undergoing a nucleophilic aromatic substitution on the dinitrobenzene ring. In the process, 2,3-dinitrobenzothiol was liberated, and the cyano group of the intermediate was reduced to an imine (Scheme 7). The formation of a six-membered ring brought conjugation to the molecule, and this could be detected by spectroscopy. In this way, probe 18 could detect endogenous levels of H 2 S within living cells [66].  Figure 7A). The fluorescence intensity of probe 13 was shown to be stable across a wide pH range ( Figure 7B). As shown in Figure 7C, it took up to 3 min for a maximum detection of fluorescence to be detected. Probe 13 was reported to have high specificity to H2S, as supported by Figure  7D; signals were negligible for a dozen other analytes, such as cysteine, SO3 2− , and GSH.  Figure 7A). The fluorescence intensity of probe 13 was shown to be stable across a wide pH range ( Figure 7B). As shown in Figure 7C, it took up to 3 min for a maximum detection of fluorescence to be detected. Probe 13 was reported to have high specificity to H 2 S, as supported by Figure 7D; signals were negligible for a dozen other analytes, such as cysteine, SO 3 2− , and GSH. With these studies, it was proven that probe 13 has a high specificity, sensitivity, and selectivity toward H 2 S [64].  Figure 7A). The fluore cence intensity of probe 13 was shown to be stable across a wide pH range ( Figure 7B). A shown in Figure 7C, it took up to 3 min for a maximum detection of fluorescence to detected. Probe 13 was reported to have high specificity to H2S, as supported by Figu 7D; signals were negligible for a dozen other analytes, such as cysteine, SO3 2− , and GS With these studies, it was proven that probe 13 has a high specificity, sensitivity, and s lectivity toward H2S [64].   Figure 8A). As shown in Figure 8B, the limit of detection was as low as 0.17 µM. As shown in Figure 8C, with various interference chemicals including H 2 O 2 , NO 2 − , and Ca 2+ , there was no obvious fluorescence change of probe 16, and, after 25 min, the fluorescence reached its maximum value ( Figure 8D). These results suggest the probe 16 has high selectivity toward H 2 S [65].
Yongru Zhang et al. examined the sensitivity of probe 18 (Scheme 7) toward H 2 S. Na 2 S was used for the source of H 2 S. As outlined in Figure 9A, in HEPES buffer, probe 18 responded to Na 2 S with different fluorescence signals at 537 nm, which indicated an excellent linear relationship (R 2 = 0.9860) at 537 nm. Moreover, a detection limit of 0.15 µM H 2 S was reported for probe 18 ( Figure 9B). These results showed that probe 18 was a good monitoring system for H 2 S [66]. Songjiao Li et al. investigated the fluorescence response of probe 16 (Scheme 6) t in PBS ( Figure 8A). As shown in Figure 8B, the limit of detection was as low as 0.17 As shown in Figure 8C, with various interference chemicals including H2O2, NO2 Ca 2+ , there was no obvious fluorescence change of probe 16, and, after 25 min, the flu cence reached its maximum value ( Figure 8D). These results suggest the probe 16 ha selectivity toward H2S [65].  Figure 9A, in HEPES buffer, probe sponded to Na2S with different fluorescence signals at 537 nm, which indicated an lent linear relationship (R 2 = 0.9860) at 537 nm. Moreover, a detection limit of 0.15 μM was reported for probe 18 ( Figure 9B). These results showed that probe 18 was a monitoring system for H2S [66].  Applications of H 2 S Probes Based on TICT Probe 13 (Scheme 5) was used to detect H 2 S in HeLa cells. As shown in Figure 10B, a weak fluorescence signal was associated with the cells being only incubated with the probe. In contrast, after probe 18 was treated with Na 2 S for 30 min, the HeLa cells had strong fluorescence signals ( Figure 10E). The overlaid image ( Figure 10F) clearly showed the green fluorescence in the HeLa cells. These results prove the utility of probe 13 as a H 2 S sensor [64].
Probe 13 (Scheme 5) was used to detect H2S in HeLa cells. As shown in Figure 10B, a weak fluorescence signal was associated with the cells being only incubated with the probe. In contrast, after probe 18 was treated with Na2S for 30 min, the HeLa cells had strong fluorescence signals ( Figure 10E). The overlaid image ( Figure 10F) clearly showed the green fluorescence in the HeLa cells. These results prove the utility of probe 13 as a H2S sensor [64]. Probe 16 (Scheme 6) was used to image H2S in HeLa cells, as shown in Figure 11. There was only a weak fluorescence signal when HeLa cells treated by the probe alone ( Figure 11A). With the addition of Na2S to the cells, the green fluorescence signal increased ( Figure 11B). However, when the H2S inhibitor PAG was added, the fluorescence signal was weak again ( Figure 11C). This indicates that probe 16 could be useful in monitoring exogenous H2S in living cells. In contrast, the use of cystine, which could induce the production of endogenous H2S, leads to an increase in green signal once more ( Figure 11D). Probe 16 (Scheme 6) was used to image H 2 S in HeLa cells, as shown in Figure 11. There was only a weak fluorescence signal when HeLa cells treated by the probe alone ( Figure 11A). With the addition of Na 2 S to the cells, the green fluorescence signal increased ( Figure 11B). However, when the H 2 S inhibitor PAG was added, the fluorescence signal was weak again ( Figure 11C). This indicates that probe 16 could be useful in monitoring exogenous H 2 S in living cells. In contrast, the use of cystine, which could induce the production of endogenous H 2 S, leads to an increase in green signal once more ( Figure 11D). When PAG and cystine were added concurrently, the fluorescence intensity in the HeLa cells was weak. This was due to the inhibition of H 2 S ( Figure 11E,F) [66]. When PAG and cystine were added concurrently, the fluorescence intensity in the HeLa cells was weak. This was due to the inhibition of H2S ( Figure 11E, F) [66].

Nucleophilic Addition Based on FRET
Fluorescence resonance energy transfer (FRET) occurs between an excited donor and an acceptor at the ground state. Donors emit in a shorter wavelength that overlaps with the acceptor's absorbance. A nonradioactive energy transfer occurs, i.e., the FRET phenomenon, such that the fluorescence intensity of the donor is much lower than when it exists alone, while the fluorescence emitted by the acceptor is greatly enhanced. This energy transfer occurs without the appearance of a photon and results from long-range dipole-dipole interactions between the donor and acceptor. Fluorescence of excited species is quenched in the presence of the analyte (H 2 S), which transduces a signal [67][68][69]. As an example, in Scheme 8, the coumarin is excited and transfers energy to the indolium, which fluoresces. Upon H 2 S reacted with the indolium, the FRET process is interrupted, and the coumarin fluoresces. Yunzhen Yang et al. designed a ratiometric fluorescent probe working via both the ICT and the FRET mechanisms. Scheme 9 illustrates the coupling reaction between the carboxylic acid group of 21 and the secondary cyclic amine of 22 with EDCI, HOBT, and DIEA as coupling conditions. The new carbon-carbon bond resulted in probe 23 with a yield of 70%. In response to H2S, probe 23 could exhibit different signals depending on the concentration of the analyte. At higher concentrations, the azide group was reduced to a terminal amine whose lone pair contributed to the conjugated system. The ICT was activated, and this was revealed by a signal with more red character. However, at lower concentrations, FRET was the indicator, with a signal of more blue character [68].

Synthesis of H 2 S Probes Based on FRET
Xiao Feng et al. synthesized an NIR fluorescent probe operating according to the FRET principle. The coumarin derivative 20 was obtained via a single-step condensation reaction (Scheme 8). The aldehyde of compound 19 was allowed to react with the Nmethyl indolium salt under reflux to form a new carbon-carbon bond. A good yield of 70% was reported, and the induced conjugation provided a means of detection through spectroscopy. During the detection of H 2 S, the indolium moiety undergoes nucleophilic attack. Conjugation was lost, thereby quenching the FRET process. It was noted that, under a single-wavelength excitation, this probe could show different signal changes with the H 2 S concentration [67].
Yunzhen Yang et al. designed a ratiometric fluorescent probe working via both the ICT and the FRET mechanisms. Scheme 9 illustrates the coupling reaction between the carboxylic acid group of 21 and the secondary cyclic amine of 22 with EDCI, HOBT, and DIEA as coupling conditions. The new carbon-carbon bond resulted in probe 23 with a yield of 70%. In response to H 2 S, probe 23 could exhibit different signals depending on the concentration of the analyte. At higher concentrations, the azide group was reduced to a terminal amine whose lone pair contributed to the conjugated system. The ICT was activated, and this was revealed by a signal with more red character. However, at lower concentrations, FRET was the indicator, with a signal of more blue character [68]. Molecules 2023, 28, x FOR PEER REVIEW 16 Scheme 9. Synthetic route and detection mechanism of probe 23.
Yuming Zhang et al. developed two ratiometric fluorescent H2S sensors. Both formed following condensation reactions between the carboxylic acid group of the marin derivative merocyanine fluorophore 24 and the amine groups of the two gr emitting fluorophores 25 and 27. Probes 26 and 28, respectively, were obtained as sh in Scheme 10 using DCC chemistry and a HOSu catalyst. Low yields of 35% were repo In detection of the H2S analyte, the indolium moieties of the probes underwent nu philic attack by H2S. FRET was turned off in the process, thereby transducing the si [69].  Figure 12, the fluorescence titration demonstrated that probe 20 had two distinct emission bands. Interestingly, as the concentration of H 2 S was increased, the ratio of these two peaks changed; the 587 nm signal became more intense while the 474 nm signal became less intense as H 2 S was added. The opposite was also found to be true; lower concentrations increased the lower wavelength signal and reduced the higher one. These findings prove that probe 20 could be potentially used to quantitatively determine the concentration of H 2 S in a sample [67].  Figure 12, the fluorescence titration demonstrated that probe 20 had two distinct emission bands. Interestingly, as the concentration of H2S was increased, the ratio of these two peaks changed; the 587 nm signal became more intense while the 474 nm signal became less intense as H2S was added. The opposite was also found to be true; lower concentrations increased the lower wavelength signal and reduced the higher one. These findings prove that probe 20 could be potentially used to quantitatively determine the concentration of H2S in a sample [67].  Figure 13A); however, with increasing concentrations of H2S, this peak at 650 nm decreased in favor of the emerging peak at 540 nm ( Figure  13B). At concentrations below 200 uM, 650 nm was the major peak, whereas 550 nm was the major peak between a concentration of 200 uM and 2 mM. This phenomenon revealed Yunzhen Yang et al. studied the optical properties of probe 23 (Scheme 9). The free probe was shown to emit at 650 nm ( Figure 13A); however, with increasing concentrations of H 2 S, this peak at 650 nm decreased in favor of the emerging peak at 540 nm ( Figure 13B). At concentrations below 200 uM, 650 nm was the major peak, whereas 550 nm was the major peak between a concentration of 200 uM and 2 mM. This phenomenon revealed an isosbestic point in Figure 13B at 620 nm. The color of emission varied depending on the concentration of H 2 S as illustrated in Figure 13C; more blue character was correlated to higher concentrations of the analyte. As shown in Figure 13D, concentrations of H 2 S below 200 µm gave peak ratios overwhelmingly favoring the peak at 650 nm. This agreed with the rest of the data because lower concentrations resulted in signals with redder intensity [68].

Figure 12.
Fluorescence spectra of the probe 20 with the addition of Na2S in PBS buffer. Reprinted with permission from Ref. [67]. Copyright ©2016 Royal Society of Chemistry.
Yunzhen Yang et al. studied the optical properties of probe 23 (Scheme 9). The free probe was shown to emit at 650 nm ( Figure 13A); however, with increasing concentrations of H2S, this peak at 650 nm decreased in favor of the emerging peak at 540 nm ( Figure  13B). At concentrations below 200 uM, 650 nm was the major peak, whereas 550 nm was the major peak between a concentration of 200 uM and 2 mM. This phenomenon revealed an isosbestic point in Figure 13B at 620 nm. The color of emission varied depending on the concentration of H2S as illustrated in Figure 13C; more blue character was correlated to higher concentrations of the analyte. As shown in Figure 13D, concentrations of H2S below 200 μm gave peak ratios overwhelmingly favoring the peak at 650 nm. This agreed with the rest of the data because lower concentrations resulted in signals with redder intensity [68].  Yuming Zhang et al. studied the optical properties of probe 26 (Scheme 10) and its ability to sense H 2 S. As shown in Figure 14A, when HS − was added, the signal at 530 nm increased significantly, while the signal at 665 nm showed a decrease. Probe 28 was shown to behave similarly in Figure 14B. The isosbestic point resulted around 625 nm ( Figure 14A) and 640 nm ( Figure 14B). These results prove that both probes 26 and 28 could potentially serve as radiometric sensing probes for H 2 S. increased significantly, while the signal at 665 nm showed a decrease. Probe 28 was shown to behave similarly in Figure 14B. The isosbestic point resulted around 625 nm ( Figure  14A) and 640 nm ( Figure 14B). These results prove that both probes 26 and 28 could potentially serve as radiometric sensing probes for H2S. Probe 20, the first mentioned sensor operating by FRET, was used to detect H2S in HeLa cells (Scheme 8). Figure 15 shows the probe colocalization in the cells both with ( Figure 15E-H) and without ( Figure 15A-D) the H2S analyte. When H2S was added, probe

The Application of H 2 S Based on FRET Probes
Probe 20, the first mentioned sensor operating by FRET, was used to detect H 2 S in HeLa cells (Scheme 8). Figure 15 shows the probe colocalization in the cells both with ( Figure 15E-H) and without ( Figure 15A-D) the H 2 S analyte. When H 2 S was added, probe 20 was found to preferentially distribute in the mitochondrion, where it remained even after sensing for 1 h. This quality is displayed by the merged images of the cells under brightfield and deep-red wavelengths ( Figure 15C,G). Furthermore, the probe's photostability was evaluated by the HeLa cell images which were exposed for different lengths of time. Coefficients for quantitation of colocalization with the cells were reported as 0.924 and 0.891 ( Figure 15D,H). These images suggest that probe 20 has use as a ratiometric tracker of H 2 S levels within the mitochondrion [67].  [69]. Copyright ©2020 Royal Society of Chemistry.

The Application of H2S Based on FRET Probes
Probe 20, the first mentioned sensor operating by FRET, was used to detect H2S in HeLa cells (Scheme 8). Figure 15 shows the probe colocalization in the cells both with ( Figure 15E-H) and without ( Figure 15A-D) the H2S analyte. When H2S was added, probe 20 was found to preferentially distribute in the mitochondrion, where it remained even after sensing for 1 h. This quality is displayed by the merged images of the cells under bright-field and deep-red wavelengths ( Figure 15C, G). Furthermore, the probe's photostability was evaluated by the HeLa cell images which were exposed for different lengths of time. Coefficients for quantitation of colocalization with the cells were reported as 0.924 and 0.891 ( Figure 15D, H). These images suggest that probe 20 has use as a ratiometric tracker of H2S levels within the mitochondrion [67]. Probe 23 (Scheme 9) was also used to detect H2S in the HeLa cells; however, unlike probe 20, this compound was able to distinguish between high and low levels of Probe 23 (Scheme 9) was also used to detect H 2 S in the HeLa cells; however, unlike probe 20, this compound was able to distinguish between high and low levels of concentration, with a cutoff at 50 µM ( Figure 16I). In the control group, only weak fluorescence was shown ( Figure 16A). As shown in Figure 16B-D, the intensities of fluorescence signals increased in both the green and the red channels at concentrations from 10 to 50 µM. As the concentration of NaHS was increased from 100 to 500 µM, the emission in the green channel maintained this enhancement (Figure 16(E2,F2,G2)); meanwhile, the emission in the red channel was weakened conspicuously (Figure 16(E3,F3,G3)). The ratios between the two signals were marked by a significant increase ( Figure 16E-G). As shown in Figure 16H, the normalized fluorescence intensity of the green and red channels first exhibited a synergistic change (increased together) and then an antagonistic change (green channel increased while red channel decreased). Moreover, as shown in Figure 16I, the ratio of the signals first exhibited a slow increase; then, in the high range of the H 2 S concentrations, it increased significantly, which could help in identifying the cutoff point [68].
tios between the two signals were marked by a significant increase ( Figure 16E-G). As shown in Figure 16H, the normalized fluorescence intensity of the green and red channels first exhibited a synergistic change (increased together) and then an antagonistic change (green channel increased while red channel decreased). Moreover, as shown in Figure 16I, the ratio of the signals first exhibited a slow increase; then, in the high range of the H2S concentrations, it increased significantly, which could help in identifying the cutoff point [68]. The third FRET-based H2S probe, 26, was used to detect intracellular levels of the H2S analyte (Scheme 10). In Figure 17A, the green channel shows medium-intensity fluorescence while Figure 17B shows the red channel with strong emission. The combinatory image of Figure 17D indicates low intracellular H2S level in HepG-2 cells. When the cells (I) normalized ratio of the fluorescence signals from the green channel and red channel in the panels (A4-G4). Reprinted with permission from Ref. [68]. Copyright ©2019 Royal Society of Chemistry.
The third FRET-based H 2 S probe, 26, was used to detect intracellular levels of the H 2 S analyte (Scheme 10). In Figure 17A, the green channel shows medium-intensity fluorescence while Figure 17B shows the red channel with strong emission. The combinatory image of Figure 17D indicates low intracellular H 2 S level in HepG-2 cells. When the cells were treated with NaHS for 30 min, the green channel was increased ( Figure 17E), while the red channel was significantly reduced ( Figure 17F), resulting in enhancement of the ratio image ( Figure 17H). These results showed that probe 26 could detect the intracellular H 2 S [69].

Photoinduced Electron Transfer (PET)
In PET fluorescent molecular probes, there is a photoinduced electron transfer between the fluorophore and the acceptor unit, which has a very strong quenching effect on fluorescence; usually, electrons are transferred from the donor to the excited fluorophore. Therefore, before binding to the analyte, the probe does not emit fluorescence or the fluorescence is very weak. Once the receptor is bound to the analyte, the photoinduced electron transfer is inhibited, which causes the fluorophore to emit fluorescence [72][73][74].
were treated with NaHS for 30 min, the green channel was increased ( Figure 17E), while the red channel was significantly reduced (Figure 17F), resulting in enhancement of the ratio image ( Figure 17H). These results showed that probe 26 could detect the intracellular H2S [69].  [69]. Copyright ©2020 Royal Society of Chemistry.

Photoinduced Electron Transfer (PET)
In PET fluorescent molecular probes, there is a photoinduced electron transfer between the fluorophore and the acceptor unit, which has a very strong quenching effect on fluorescence; usually, electrons are transferred from the donor to the excited fluorophore. Therefore, before binding to the analyte, the probe does not emit fluorescence or the fluorescence is very weak. Once the receptor is bound to the analyte, the photoinduced electron transfer is inhibited, which causes the fluorophore to emit fluorescence [72][73][74].

Synthesis and Mechanism of H2S Probes Based on PET
Hanchuang Zhu et al. designed probe 31 according to the PET mechanism (Scheme 11). The chloro substituent of compound 30 was displaced under basic conditions, and its adjacent carbon was attacked by the hydroxy group of compound 29. The resulting probe 31 served as the H2S sensor, and it achieved a yield of 70%. In the presence of H2S, nucleophilic attack occurred at the C-O bond para to the nitro group. As a result, probe 29 was formed again along with a thiol derivative of compound 32. The PET was turned off in the process, and the H2S analyte was detected [72]. The chloro substituent of compound 30 was displaced under basic conditions, and its adjacent carbon was attacked by the hydroxy group of compound 29. The resulting probe 31 served as the H 2 S sensor, and it achieved a yield of 70%. In the presence of H 2 S, nucleophilic attack occurred at the C-O bond para to the nitro group. As a result, probe 29 was formed again along with a thiol derivative of compound 32. The PET was turned off in the process, and the H 2 S analyte was detected [72].  [69]. Copyright ©2020 Royal Society of Chemistry.

Photoinduced Electron Transfer (PET)
In PET fluorescent molecular probes, there is a photoinduced electron transfer between the fluorophore and the acceptor unit, which has a very strong quenching effect on fluorescence; usually, electrons are transferred from the donor to the excited fluorophore. Therefore, before binding to the analyte, the probe does not emit fluorescence or the fluorescence is very weak. Once the receptor is bound to the analyte, the photoinduced electron transfer is inhibited, which causes the fluorophore to emit fluorescence [72][73][74].

Synthesis and Mechanism of H2S Probes Based on PET
Hanchuang Zhu et al. designed probe 31 according to the PET mechanism (Scheme 11). The chloro substituent of compound 30 was displaced under basic conditions, and its adjacent carbon was attacked by the hydroxy group of compound 29. The resulting probe 31 served as the H2S sensor, and it achieved a yield of 70%. In the presence of H2S, nucleophilic attack occurred at the C-O bond para to the nitro group. As a result, probe 29 was formed again along with a thiol derivative of compound 32. The PET was turned off in the process, and the H2S analyte was detected [72]. Scheme 11. Synthetic route and detection mechanism of probe 31. Similar to the previous example, Jinshuai Lan et al. also produced an NIR probe capable of detecting H 2 S by PET (Scheme 12). Probe 34 was obtained through a coupling reaction between the chloro substituent of compound 30 and the hydroxyl group of compound 33. This reaction produced probe 34 with a yield of 50%. In the presence of the H 2 S analyte, the NBD C-O bond was cleaved as indicated in Scheme 12, and the PET process was turned off [73]. Similar to the previous example, Jinshuai Lan et al. also produced an NIR probe capable of detecting H2S by PET (Scheme 12). Probe 34 was obtained through a coupling reaction between the chloro substituent of compound 30 and the hydroxyl group of compound 33. This reaction produced probe 34 with a yield of 50%. In the presence of the H2S analyte, the NBD C-O bond was cleaved as indicated in Scheme 12, and the PET process was turned off [73]. . This coupling reaction produced compound 37 with a yield of 40%. The C-O bond of probe 37 underwent a nucleophilic attack by the H2S analyte, resulting in its cleavage. A species identical to staring material 35 and a thiol derivative of 36 were left as products. This reduction in conjugation quenches the PET, allowing for a signal to be detected [74]. Scheme 13. Synthetic route and detection mechanism of probe 37.

Optical Properties of H2S Based on PET Probes
The optical properties of H2S probe 31 (Scheme 11) were summarized by Hanchuang Zhu et al. as shown in Figure 18. Compound 31 displayed a fluorescence wavelength at 643 nm upon addition of H2S up to 50 μM ( Figure 18A), demonstrating a high sensitivity toward the analyte. The definite linear relationship between the concentration of H2S and fluorescence intensity indicated that H2S could be detected accurately in levels as low as 6.2 × 10 −8 mol·L −1 ( Figure 18B). These results indicate that probe 31 has potential as a H2S sensor and deserves further research [72]. This reduction in conjugation quenches the PET, allowing for a signal to be detected [74]. pable of detecting H2S by PET (Scheme 12). Probe 34 was obtained through a coupling reaction between the chloro substituent of compound 30 and the hydroxyl group of compound 33. This reaction produced probe 34 with a yield of 50%. In the presence of the H2S analyte, the NBD C-O bond was cleaved as indicated in Scheme 12, and the PET process was turned off [73]. . This coupling reaction produced compound 37 with a yield of 40%. The C-O bond of probe 37 underwent a nucleophilic attack by the H2S analyte, resulting in its cleavage. A species identical to staring material 35 and a thiol derivative of 36 were left as products. This reduction in conjugation quenches the PET, allowing for a signal to be detected [74]. Scheme 13. Synthetic route and detection mechanism of probe 37.

Optical Properties of H2S Based on PET Probes
The optical properties of H2S probe 31 (Scheme 11) were summarized by Hanchuang Zhu et al. as shown in Figure 18. Compound 31 displayed a fluorescence wavelength at 643 nm upon addition of H2S up to 50 μM ( Figure 18A), demonstrating a high sensitivity toward the analyte. The definite linear relationship between the concentration of H2S and fluorescence intensity indicated that H2S could be detected accurately in levels as low as 6.2 × 10 −8 mol·L −1 ( Figure 18B). These results indicate that probe 31 has potential as a H2S sensor and deserves further research [72]. Scheme 13. Synthetic route and detection mechanism of probe 37.

Optical Properties of H 2 S Based on PET Probes
The optical properties of H 2 S probe 31 (Scheme 11) were summarized by Hanchuang Zhu et al. as shown in Figure 18. Compound 31 displayed a fluorescence wavelength at 643 nm upon addition of H 2 S up to 50 µM (Figure 18A), demonstrating a high sensitivity toward the analyte. The definite linear relationship between the concentration of H 2 S and fluorescence intensity indicated that H 2 S could be detected accurately in levels as low as 6.2 × 10 −8 mol·L −1 ( Figure 18B). These results indicate that probe 31 has potential as a H 2 S sensor and deserves further research [72].
Jinshuai Lan et al. presented the optical properties of probe 34 (Scheme 12; Figure 19A). The fluorescence signal at 650 nm increased as the concentration of Na 2 S increased from 0 to 500 µm. As such, a distinct linear relationship was reported between the concentration of the H 2 S analyte and the fluorescence intensity. The best fit line was used to report that S 2− concentrations under 60 µm could be accurately detected by probe 34 ( Figure 19B) [73].
The optical properties of probe 37 in response to H 2 S (Scheme 13) were presented by Xinying Jing et al. in Figure 20A. Although the free probe did fluoresce weakly at 460 nm, this signal increased significantly upon addition of the H 2 S analyte. The strong linear relationship between the concentration of S 2− and fluorescence intensity at 460 nm ( Figure 20B) suggests that the limit of detection of the probe could be as low as 2.07 × 10 −7 M. As shown in Figure 20C, D, the fluorescence signal approached its maximum intensity after 15 min [74].  Figure  19A). The fluorescence signal at 650 nm increased as the concentration of Na2S increased from 0 to 500 μm. As such, a distinct linear relationship was reported between the concentration of the H2S analyte and the fluorescence intensity. The best fit line was used to report that S 2− concentrations under 60 μm could be accurately detected by probe 34 ( Figure  19B) [73]. Figure 19. Fluorescence spectra changes of the probe 34 upon addition of increasing amount of Na2S. Reprinted with permission from Ref. [73]. Copyright ©2019 Royal Society of Chemistry.
The optical properties of probe 37 in response to H2S (Scheme 13) were presented by Xinying Jing et al. in Figure 20A. Although the free probe did fluoresce weakly at 460 nm, this signal increased significantly upon addition of the H2S analyte. The strong linear relationship between the concentration of S 2− and fluorescence intensity at 460 nm ( Figure  20B) suggests that the limit of detection of the probe could be as low as 2.07 × 10 −7 M. As shown in Figure 20C, D, the fluorescence signal approached its maximum intensity after 15 min [74].   Figure  19A). The fluorescence signal at 650 nm increased as the concentration of Na2S increased from 0 to 500 μm. As such, a distinct linear relationship was reported between the concentration of the H2S analyte and the fluorescence intensity. The best fit line was used to report that S 2− concentrations under 60 μm could be accurately detected by probe 34 ( Figure  19B) [73]. Figure 19. Fluorescence spectra changes of the probe 34 upon addition of increasing amount of Na2S. Reprinted with permission from Ref. [73]. Copyright ©2019 Royal Society of Chemistry.
The optical properties of probe 37 in response to H2S (Scheme 13) were presented by Xinying Jing et al. in Figure 20A. Although the free probe did fluoresce weakly at 460 nm, this signal increased significantly upon addition of the H2S analyte. The strong linear relationship between the concentration of S 2− and fluorescence intensity at 460 nm ( Figure  20B) suggests that the limit of detection of the probe could be as low as 2.07 × 10 −7 M. As shown in Figure 20C, D, the fluorescence signal approached its maximum intensity after 15 min [74]. Figure 19. Fluorescence spectra changes of the probe 34 upon addition of increasing amount of Na 2 S. Reprinted with permission from Ref. [73]. Copyright ©2019 Royal Society of Chemistry.

Applications of H 2 S PET Probes
Zebrafish were used as living models to demonstrate the biological applications of probe 31 (Scheme 11). As shown in Figure 21A, when the zebrafish were only incubated with probe 31, the images showed fluorescence in the green and red channels due to inherent biothiols within the cells. After treatment with the probe plus N-ethylmaleimide (NEM), a scavenger of biothiols, the zebrafish showed no fluorescence signal in either the green or the red channel ( Figure 21B). Lastly, H 2 S and probe 31 were added ( Figure 21C), which resulted in red and green fluorescence signals, as seen in Figure 21A. This strongly supports the potential use of probe 31 to detect both intracellular and extracellular H 2 S in zebrafish. Future research might discover applications of this compounds in other animal models [72].
The biological applications of probe 37 (Scheme 13) were also demonstrated using zebrafish ( Figure 22). Half an hour after being treated with only probe 37, a fluorescence signal was seen, albeit dim, but this was solely due to intracellular biothiols ( Figure 22B). Upon the addition of Na 2 S at 100 µM, the blue fluorescence was enhanced significantly by 20 min (Figure 22E). This proves that probe 37 is useful in detecting H 2 S in a living organism [74]. Reprinted with permission from Ref. [74]. Copyright ©2019 Royal Society of Chemistry.

Applications of H2S PET Probes
Zebrafish were used as living models to demonstrate the biological applications of probe 31 (Scheme 11). As shown in Figure 21A, when the zebrafish were only incubated with probe 31, the images showed fluorescence in the green and red channels due to inherent biothiols within the cells. After treatment with the probe plus N-ethylmaleimide (NEM), a scavenger of biothiols, the zebrafish showed no fluorescence signal in either the green or the red channel ( Figure 21B). Lastly, H2S and probe 31 were added ( Figure 21C), which resulted in red and green fluorescence signals, as seen in Figure 21A. This strongly supports the potential use of probe 31 to detect both intracellular and extracellular H2S in zebrafish. Future research might discover applications of this compounds in other animal models [72].   Reprinted with permission from Ref. [74]. Copyright ©2019 Royal Society of Chemistry.

Applications of H2S PET Probes
Zebrafish were used as living models to demonstrate the biological applications of probe 31 (Scheme 11). As shown in Figure 21A, when the zebrafish were only incubated with probe 31, the images showed fluorescence in the green and red channels due to inherent biothiols within the cells. After treatment with the probe plus N-ethylmaleimide (NEM), a scavenger of biothiols, the zebrafish showed no fluorescence signal in either the green or the red channel ( Figure 21B). Lastly, H2S and probe 31 were added ( Figure 21C), which resulted in red and green fluorescence signals, as seen in Figure 21A. This strongly supports the potential use of probe 31 to detect both intracellular and extracellular H2S in zebrafish. Future research might discover applications of this compounds in other animal models [72].

H 2 S Probes Based on Azide Group Reduction
In addition to nucleophilic addition, the reduction of azide groups is also a useful method for detecting H 2 S [75][76][77][78][79][80]. Typically, this involves a unit of hydrogen sulfide attacking an azide and reducing it to an amine via a reduction reaction, as opposed to nucleophilic attack of the H 2 S analyte to quench the fluorescence reaction. In the process, the overall electron distribution of the probe is altered, which can lead to an increase in fluorescence under certain conditions. These probes have been applied for both in vivo and in vitro testing by chemists to target the lysosomes and mitochondria of cells [79,80]. signal was seen, albeit dim, but this was solely due to intracellular biothiols ( Figure 22B). Upon the addition of Na2S at 100 μM, the blue fluorescence was enhanced significantly by 20 min (Figure 22E). This proves that probe 37 is useful in detecting H2S in a living organism [74].

H2S Probes Based on Azide Group Reduction
In addition to nucleophilic addition, the reduction of azide groups is also a useful method for detecting H2S [75][76][77][78][79][80]. Typically, this involves a unit of hydrogen sulfide attacking an azide and reducing it to an amine via a reduction reaction, as opposed to nucleophilic attack of the H2S analyte to quench the fluorescence reaction. In the process, the overall electron distribution of the probe is altered, which can lead to an increase in fluorescence under certain conditions. These probes have been applied for both in vivo and in vitro testing by chemists to target the lysosomes and mitochondria of cells [79,80]  In the presence of H2S, the azido group was reduced to an amino group, thereby transducing a signal in the blue region upon fluorescence [76].
Nithya Velusamy et al. reported a separate on/off probe, compound 43, which could monitor H 2 S in living cells. With HATU and DiPEA in THF as coupling conditions, the carboxylic acid of compound 41 reacted with the terminal amine of compound 42 to form probe 43 at a yield of 40% (Scheme 15). In the presence of H 2 S, the azido group was reduced to an amino group, thereby transducing a signal in the blue region upon fluorescence [76]. Scheme 14. Synthetic route and detection mechanism of probe 40.
Nithya Velusamy et al. reported a separate on/off probe, compound 43, which could monitor H2S in living cells. With HATU and DiPEA in THF as coupling conditions, the carboxylic acid of compound 41 reacted with the terminal amine of compound 42 to form probe 43 at a yield of 40% (Scheme 15). In the presence of H2S, the azido group was reduced to an amino group, thereby transducing a signal in the blue region upon fluorescence [76].  Figure 23A shows that the fluorescence signal at 422 nm increased conspicuously while the concentration of H2S was raised to 180 μM. Manipulation of the fluorescence spectra provided a definite linear relationship between the fluorescence intensity and the concentration of Na2S [75]. Scheme 16. Synthetic route and detection mechanism of probe 47.

Optical Properties of Probes Based on Azide Group Reduction
The probe developed by Qian Fang et al. (Scheme 14) was investigated, and its optical properties were determined. Figure 23A shows that the fluorescence signal at 422 nm increased conspicuously while the concentration of H 2 S was raised to 180 µM. Manipulation of the fluorescence spectra provided a definite linear relationship between the fluorescence intensity and the concentration of Na 2 S [75].
Nithya Velusamy et al. reported the optical properties of probe 43 (Scheme 15; Figure 24). Absorption spectra showed a bathochromic shift as concentration of the H 2 S analyte was increased ( Figure 24A), and fluorescence spectra indicated that the detection limit was as low as 10 µM ( Figure 24B). As shown in Figure 24C, the fluorescence intensity reached a plateau after 70 min. Many sulfur-containing biomarkers were added to the probe, but only Na 2 S produced a strong signal ( Figure 24D). These data show that probe 40 was very useful as a H 2 S probe because of its high selectivity and sensitivity [76].

Optical Properties of Probes Based on Azide Group Reduction
The probe developed by Qian Fang et al. (Scheme 14) was investigated, and its optic properties were determined. Figure 23A shows that the fluorescence signal at 422 nm in creased conspicuously while the concentration of H2S was raised to 180 μM. Manipulatio of the fluorescence spectra provided a definite linear relationship between the fluore cence intensity and the concentration of Na2S [75]. . Absorption spectra showed a bathochromic shift as concentration of the H2S analy was increased ( Figure 24A), and fluorescence spectra indicated that the detection limit wa as low as 10 μM ( Figure 24B). As shown in Figure 24C, the fluorescence intensity reache a plateau after 70 min. Many sulfur-containing biomarkers were added to the probe, bu only Na2S produced a strong signal ( Figure 24D). These data show that probe 40 was ver useful as a H2S probe because of its high selectivity and sensitivity [76].   Figure 25A indicates a strong response to the addition of H2S and no response to other selected bioanalytes such as cysteine, glutamine, and glycine. The shift in absorption wavelength from 375 to 440 nm could even be detected by the naked eye. Increasing the concentration of H2S from 0 eq. to 25 eq. (equivalent volume of the probe), the absorption intensity of the peak at 375 nm gave way to another at 440 nm ( Figure 25B). The isosbestic point shown in Figure 25B was around 400 nm. This selectivity shows that probe 47 has potential to be used as a H2S sensor [77].   Figure 25A indicates a strong response to the addition of H 2 S and no response to other selected bioanalytes such as cysteine, glutamine, and glycine. The shift in absorption wavelength from 375 to 440 nm could even be detected by the naked eye. Increasing the concentration of H 2 S from 0 eq. to 25 eq. (equivalent volume of the probe), the absorption intensity of the peak at 375 nm gave way to another at 440 nm ( Figure 25B). The isosbestic point shown in Figure 25B was around 400 nm. This selectivity shows that probe 47 has potential to be used as a H 2 S sensor [77]. response to other selected bioanalytes such as cysteine, glutamine, and glycine. The shift in absorption wavelength from 375 to 440 nm could even be detected by the naked eye. Increasing the concentration of H2S from 0 eq. to 25 eq. (equivalent volume of the probe), the absorption intensity of the peak at 375 nm gave way to another at 440 nm ( Figure 25B). The isosbestic point shown in Figure 25B was around 400 nm. This selectivity shows that probe 47 has potential to be used as a H2S sensor [77].   [77]. Copyright ©2020 Royal Society of Chemistry.

The Application of H 2 S Probe Based on Azide Group Reduction
Compound 40 (Scheme 14) was used to detect H 2 S in living zebrafish. Following the treatment with probe 40 only, weak fluorescence was seen. This was solely due to the H 2 S which would normally be found within cells ( Figure 26D,F). However, after adding H 2 S and incubating with probe 40 for another 30 min, a strong blue fluorescence signal was observed ( Figure 26A,C). These results show that probe 40 can be useful to detect H 2 S in living zebrafish [75]. Compound 40 (Scheme 14) was used to detect H2S in living zebrafish. Following the treatment with probe 40 only, weak fluorescence was seen. This was solely due to the H2S which would normally be found within cells ( Figure 26D, F). However, after adding H2S and incubating with probe 40 for another 30 min, a strong blue fluorescence signal was observed ( Figure 26A, C). These results show that probe 40 can be useful to detect H2S in living zebrafish [75]. The presence of H2S was detected in four different cell lines by probe 43 (Scheme 15). HeLa ( Figure 27A-D), MDA-MB-231 (E-H), DU145 (I-L), and 3T3-L1 cells (M-P) were all used to demonstrate this ability. As shown in all control groups, the fluorescence signals were all very weak without the probe present. This is because endogenous H2S levels are insufficient to elicit a signal. However, adding H2S increased the signal dramatically in most cases ( Figure 27D, H, L). The 3T3-L1 cell line displayed the last pronounced response to H2S by probe 43 ( Figure 27P). There is strong evidence that probe 43 is a useful probe for H2S in living cells [76]. The presence of H 2 S was detected in four different cell lines by probe 43 (Scheme 15). HeLa ( Figure 27A-D), MDA-MB-231 (E-H), DU145 (I-L), and 3T3-L1 cells (M-P) were all used to demonstrate this ability. As shown in all control groups, the fluorescence signals were all very weak without the probe present. This is because endogenous H 2 S levels are insufficient to elicit a signal. However, adding H 2 S increased the signal dramatically in most cases ( Figure 27D,H,L). The 3T3-L1 cell line displayed the last pronounced response to H 2 S by probe 43 ( Figure 27P). There is strong evidence that probe 43 is a useful probe for H 2 S in living cells [76]. Another compound was investigated as a potential sensor for H2S in HeLa cells. Probe 47 (Scheme 16) displayed only weak fluorescence intensity within the cells by themselves ( Figure 28 (A1-3)). However, when the cells were treated with H2S, an obvious green fluorescence was seen ( Figure 28 (A4-9)). Varying concentrations of H2S were used, and their quantitative fluorescence intensities are plotted in Figure 28B. Together, this study shows that probe 47 has potential in detecting elevated levels of H2S within HeLa cells [77]. Reprinted with permission from Ref. [76]. Copyright ©2017 Royal Society of Chemistry.
Another compound was investigated as a potential sensor for H 2 S in HeLa cells. Probe 47 (Scheme 16) displayed only weak fluorescence intensity within the cells by themselves ( Figure 28(A1-3)). However, when the cells were treated with H 2 S, an obvious green fluorescence was seen ( Figure 28(A4-9)). Varying concentrations of H 2 S were used, and their quantitative fluorescence intensities are plotted in Figure 28B. Together, this study shows that probe 47 has potential in detecting elevated levels of H 2 S within HeLa cells [77].  [76]. Copyright ©2017 Royal Society of Chemistry.
Another compound was investigated as a potential sensor for H2S in HeLa cells. Probe 47 (Scheme 16) displayed only weak fluorescence intensity within the cells by themselves ( Figure 28 (A1-3)). However, when the cells were treated with H2S, an obvious green fluorescence was seen ( Figure 28 (A4-9)). Varying concentrations of H2S were used, and their quantitative fluorescence intensities are plotted in Figure 28B. Together, this study shows that probe 47 has potential in detecting elevated levels of H2S within HeLa cells [77]. Ref. [77]. Copyright ©2020 Royal Society of Chemistry.

Summary and Outlook
Hydrogen sulfide is one of the most important gases in living organisms, and any abnormal changes in the concentration of it in vivo have been associated with many kinds of diseases [81]. Therefore, the development of highly sensitive H 2 S probes for use in living cells has become an important goal for research groups all around the world. Chemists have developed an assortment of on/off sensors which can be visualized with readily available NIR cameras. In this review, we exhibited many hydrogen sulfide probes based on several detection mechanisms, discussed their optical properties, and provided examples of their biological applications. In summary, most of the characteristic absorption peaks of fluorescent probes were in the visible and near-infrared range. The probes have exhibited promising, applicable prospects both in vivo and in vitro. These probes show excellent characteristics, such as good selectivity, low toxicity, and high sensitivity.
The diverse roles played by H 2 S in the body provide myriad avenues of scientific discovery. Already, it has been demonstrated that H 2 S is an important signaling gas in the nervous system [82]. For example, neurodegenerative diseases such as Parkinson's disease have been linked to a reduction in its levels, and targeted supplementation of H 2 S to affected structures has proven to be therapeutical [81,83]. Not only certain diseases but also aging itself is connected to endogenous H 2 S levels because it has powerful properties against oxidative stress [84]. For this reason, future work should go into improving the applicability of these probes and perhaps integrating them with therapeutic techniques. The sensitivity of these probes needs to be improved to allow for more precise quantitative analysis, i.e., a better map of the H 2 S concentration gradient in the body. Additionally, by further extending the excitation wavelengths of the probes, signaling could improve due to less background interference. Lastly, applying H 2 S sensors to living models such as rodents would allow researchers to observe how these sensors behave under more variables. Perhaps one day, novel H 2 S-based treatments will be added to our pharmacopeia to slow, halt, or even reverse some of the most unavoidable afflictions we face.