Skip to main content
NCBI home page
ACS Omega logoLink to ACS Omega
. 2020 Aug 3;5(31):19896–19904. doi: 10.1021/acsomega.0c02963

Nanomolar Detection of H2S in an Aqueous Medium: Application in Endogenous and Exogenous Imaging of HeLa Cells and Zebrafish

Sanay Naha †, Natesan Thirumalaivasan ‡, Somenath Garai †, Shu-Pao Wu ‡,*, Sivan Velmathi †,*
PMCID: PMC7424736  PMID: 32803086

Abstract

graphic file with name ao0c02963_0010.jpg

The homeostasis of short-lived reactive species such as hydrogen sulfide/hypochlorous acid (H2S/HOCl) in biological systems is essential for maintaining intercellular balance. An unchecked increase in biological H2S concentrations impedes homeostasis. In this report, we present a molecular probe pyrene-based sulfonyl hydrazone derived from pyrene for the selective detection of H2S endogenously as well as exogenously through a “turn-off” response in water. The structure of the receptor is confirmed by Fourier-transform infrared spectroscopy, 1H and 13C nuclear magnetic resonance spectroscopy, electrospray ionization mass spectrometry, and single-crystal X-ray diffraction studies. The receptor shows excellent green emission in both the aqueous phase and solid state. Quenching of green emission of the receptor is observed only when H2S is present in water with a detection limit of 18 nM. Other competing anions and cations do not have any influence on the receptor’s optical properties. The efficiency of H2S detection is not negatively impacted by other reactive sulfur species too. The sensing mechanism of H2S follows a chemodosimetric reductive elimination of sulfur dioxide, which is supported by product isolation. The receptor is found to be biocompatible, as evident by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and its utility is extended to endogenous and exogenous fluorescence imaging of HeLa cells and zebrafish.

Introduction

A typical unpleasant rotten egg smell is the odor characteristic of hydrogen sulfide (H2S), a compound commonly found in terrestrial and biological ecosystems.1−3 An exact redox balance between reactive oxygen species (ROS) and reactive sulfur species (RSS) is essential for the active maintenance of cellular processes such as cell proliferation,4 differentiation, and apoptosis.5 H2S is a RSS as well as a reducing agent. Therefore, any imbalance in biological H2S levels is detrimental to the immunological well-being of organisms.6 Common consequences of this imbalance include peroxidation, DNA damage,7 carcinogenesis,8 Alzheimer’s disease,9 gastric mucosal injury,10 stroke, diabetes,11 Down’s syndrome,12 liver cirrhosis,13 and even death.14 H2S is known as the third most abundant gas transmitter following nitrous oxide (NO) and carbon monoxide (CO).15,16 The presence of H2S in vivo and in vitro is essential for the smooth conductance of a diverse array of imperative biological processes such as regulation of vascular tone via the adenosine triphosphate-sensitive potassium channel, myocardial contractility, neurotransmission by the integration of antioxidant and insulin secretion.17−20 H2S also has an obligatory role in the modulation of the nervous, respiratory, gastrointestinal, and endocrine systems.21−24 The proliferation of H2S inside living cells is due to the following three enzymes: 3-mercaptopyruvate sulfurtransferase, cystathionine-b-synthase, and cystathionine-b-lyase, which catalyze cysteine and homocysteine to produce H2S, and due to some nonenzymatic processes.25−28 Though H2S is an important compound in biological processes, at higher levels, it is quite harmful.29 The estimated tolerable concentration of H2S in physiology lies within the nanomolar–millimolar range.30 Apart from biological applications, there is the widespread use of H2S in industries such as in the production of sulfur (element) and sulfuric acid, inorganic sulfides for dyes, rubber, pesticides, plastic additives, polymers, and leather manufacturing. Besides, H2S is also used in the purification of transition metals and inorganic acids, treatment of metallic surfaces, production of heavy water, and utilized as an additive in lubricants. There is a growing need to develop molecular probes for the qualitative and quantitative detection of H2S at the macroscopic and microscopic levels.

Most of the institutional methods possess some major disadvantages such as troublesome sample preparation procedures, sophisticated instrumentation, and high maintenance cost. In contrast, optical chemosensors, precisely fluorescent chemosensors have monumental advantages such as easy usage, simple synthetic procedure, naked-eye visualization, cost-effectiveness, real-time applicability, fastness, mostly noninstrumental, and portability.28−30

In recent times, Wang and Liu have reported the significance of the aggregation-induced emission (AIE) probe for bioimaging, drug delivery, and cancer theragnostics.31 Though fluorescent materials received ample attention in biomedical research, the aggregation-caused quenching (ACQ) effect at high concentrations or in the aggregated state of the fluorophore has greatly limited their biomedical applications. Fluorescent materials with the AIE effect show exactly the opposite effect of the ACQ and exhibited significant advantages in terms of tunable emission, excellent photostability, and biocompatibility. Therefore, the effect of solvent in sensing or imaging application is omnipresent. Numerous reports have been listed on H2S detection based on various metal–organic frameworks, luminophores, nanoparticles, biomolecules, metal complexes, and hetero-organic receptors which mostly followed a chemodosimetric mechanism.32−41

On this note, here, we report an organic sulfone (pyrene-based sulfonyl hydrazone, PBSH), which is selective and specific for H2S in an aqueous medium with optimum sensing efficiency without any external interference from competing analytes, ROS, and RSS. The receptor PBSH has significant advancement over recently published receptors (Table S1).

Results and Discussion

The receptor PBSH was synthesized in 90% yield and characterized by infrared (IR) spectroscopy, 1H and 13C nuclear magnetic resonance (NMR) spectroscopy, electrospray ionization–mass spectrometry (ESI-MS), and single-crystal X-ray diffraction (XRD) spectroscopy methods (Scheme 1). The synthesized receptor PBSH has an interesting molecular framework that consists of an intense signaling unit like pyrene moiety and a detecting site for analytes such as sulfonamide, which is susceptible to reduction. The receptor was examined for anion, cation, and reactive species selectivity in aqueous media using ultraviolet (UV)–visible, fluorescence, and 1H NMR spectroscopic techniques. The obtained results are documented in subsequent sections.

Scheme 1. Schematic Synthesis of Receptor PBSH.

Scheme 1

Open in a new tab

Single-Crystal Studies

The compound PBSH was characterized by single-crystal XRD which confirms that the compound crystallizes in monoclinic space group P21/c with a unit cell volume of 2184(10) Å3 having four molecules within. The molecular structure is depicted in Figure 1a, which indicates the near-planar arrangement of the pyrene ring with the sulfonylhydrazide plane (the torsion angle is only 10.2(4)°) containing sp2-hybridized nitrogen sites. On the contrary, the normal of the toluene sulfonyl moiety assumes a near-perpendicular arrangement with that of the pyrene ring plane with an angle of 105.0(3)°, which in turn leads to the overall “L” shape of the molecule. The overall packing of the molecules in the unit cell involves an interesting head-to-tail overlap among two of such molecules, which are stabilized by an extensive but incomplete π–π stacking interaction of only the two six-membered rings of the four annulenes (Figure S1); this originated from the overall rather skewed parallel orientation of the pyrene rings (with a dihedral angle of 0.0(0)° and an inter-ring twist angle of 0.0(14)°; however, pyrene rings are substantially shifted by 1.75(1) Å with an overall centroid–centroid distance of 3.97(1) Å) (Figure 1b). However, two of the six-membered rings of four annulenes are strongly involved in two major types of π–π stacking interactions, as enunciated by Figure 1c (the ring centroid–centroid distance is 3.67(2) Å with an inter-ring shift distance of 0.78(2) and 0.82(2) Å, respectively) and Figure 1d (the ring centroid–centroid distance is 3.95(2) Å with an inter-ring shift distance of 1.82(2) and 1.64(2) Å, respectively). The bimolecular π–π assembly is further stabilized by intermolecular π–π stacking interactions involving the parallelly oriented toluene moieties which represent the shorter arm of “L” (with a dihedral angle of 0.0(3)° and an inter-ring twist angle of 0.0(11)°; however, pyrene rings are substantially shifted by 1.86(2) Å with an overall centroid–centroid distance of 4.22(1) Å). Therefore, there is an effective π–π stacking interaction network that is extended to two dimensions to generate alternative hydrophobic layers that sandwich a lesser intense hydrophilic layer comprising sulfonylhydrazide moieties (Figure S2). The hydrophilic layer is stabilized by intermolecular hydrogen bonding interactions of the sulfonyl groups with the hydrazide groups (medium-strength hydrogen bonding with a D–A distance of 2.89(1) Å). The detailed crystal structure can be obtained free of cost from CCDC by quoting the depository number, CCDC—1848349. These mutual intralayer stabilizations of the hydrophobic and hydrophilic moiety assemblies of the molecule lead to a very interesting “hydrophilic–hydrophobic–hydrophilic”-type double-layered packing structures, as depicted in Figure 1b. These pertinent orientations of the double-layered structure bear surprising similarity to the biological cell wall (Figure S3). The compound exhibits strong fluorescence in the solid state, which can be attributed to the very intense hydrophobic assembly of the pyrene rings, segregated by the hydrophilic layers.

Figure 1.

Figure 1

Open in a new tab

(a) Ellipsoidal representation of the molecular structure of PBSH; (b) crystal packing of the molecule resembling the bilayered structure, with the view of the (3 × 3) supercell along the (011) direction; (c,d) pictorial depiction of two different modes of π–π stacking interactions in between the pyrene moieties of PBSH.

Solvent Effect

The chemical and optical behavior of organic compounds is intrinsically dependent on the polarity of the medium. To ascertain the effect of polarity of the solvents on PBSH, solutions of 3 mL of 25 μM in various solvents such as water, dimethyl sulfoxide (DMSO), dimethylformamide (DMF), acetonitrile (ACN), ethanol (EtOH), methanol (MeOH), acetone, tetrahydrofuran (THF), dichloromethane (DCM), and toluene were prepared (Table S2). Naked-eye observation and emission under a long-wave UV light is depicted in Figure 2. It is obvious from naked-eye observations that a discernible light yellow color is produced only in DMSO and DMF. As far as emission is concerned, an intense green fluorescence is observed only in water. Other solvents produce a blue fluorescence.

Figure 2.

Figure 2

Open in a new tab

Images under a long-wave UV light and the naked-eye observation of various solvent interaction with PBSH in the order of decreasing polarity (A. water, B. DMSO, C. DMF, D. ACN, E. EtOH, F. MeOH, G. Acetone, H. THF, I. DCM, and J. Toluene). Photograph courtesy of “Sanay Naha”. Copyright 2020.

From Figure 2, it is evident that the optical properties of PBSH differ in water in comparison to the other solvents. The electronic and fluorescence responses were recorded for all the solutions of PBSH. In the UV–visible spectrum, the absorption maxima shift by about 20 nm as the polarity is gradually increased (from toluene, λmax at 360 nm, to −water, λmax at 382 nm). In the emission spectrum, a large red shift of 100 nm is observed as the polarity is increased (from toluene, λem at 427 nm, to water, λem at 522 nm) (Figure S4). Polarity plays an important role in the emission output of the receptor PBSH. As the highest polarity solvent (water) resulted in green emission while other polarities resulted in blue emission, it is of keen interest to examine the effect of an aqueous–organic mixed solvent system on the emission of PBSH. A series of glass vials containing 50 μM of PBSH have been filled with various ratios of water and DMSO solvent ranging from 100% water to 100% DMSO. The increment factor for DMSO is 10% in the successive stage. From the naked-eye image, it is observed that the solution of PBSH in 100% DMSO shows a yellowish color, which disappeared after 40% water content in the solution. In the image under UV light, it is self-explanatory, as upon increasing the water content in DMSO, the emission color, as well as the intensity, gets altered, and in 100% water, the emission is intense green (Figure 3a). Electronic spectra of the solutions followed a pattern in shifting the λmax from 360 to 382 nm for 100% DMSO to 100% water (Figure 3b). In the emission spectrum, a periodic change in λem is observed with a 100 nm Stokes shift (Figure 3c) (Figure S5). As a large fluorescence enhancement is observed in water, it is the most desirable solvent for sensing applicability; all photophysical studies were carried out in 100% water medium.

Figure 3.

Figure 3

Open in a new tab

(a) Naked-eye color change and the change under a long-wave UV light, (b) electronic spectra of all solutions starting from 100% water to 100% DMSO, and (c) fluorescence intensity comparison of all solutions at two different λem 427 and 522 nm. Photograph courtesy of “Sanay Naha”. Copyright 2020.

Visual Selectivity for H2S

For an optical receptor, an observable fluorescence response must be produced by either a “switch on” or “switch off” process. This response demonstrates the selectivity and specificity of the receptor toward an analyte. To examine the anion selectivity, first 2 equiv of all anionic species such as CN–, F–, Cl–, Br–, I–, AcO–, HSO4–, HPO42–, NO2–, NO3–, OH–, S2O3, CO32–, SCN–, SO32–, O2–, and IO3– were added to 3 mL of 25 μM solution of PBSH in the water medium. There were no observable color change and emission property. Hence, it can be concluded that anions do not influence the optical properties of PBSH (Figure S6).

In the case of cations, to 3 mL of 25 μM solution of PBSH, 2 equiv of cations such as Ca2+, Mg2+, Ag+, Fe3+, Al3+, Hg2+, Co2+, Cd2+, Cr3+, Cu2+, Fe2+, Ni2+, Au3+, Pb2+, Zn2+, and Mn2+ were added. Cations too did not show any interaction with PBSH. However, as soon as H2S/(HS–) was added to PBSH, the emission was quenched by 7-fold. It can, therefore, be said that the interaction of the receptor with H2S produces fluorescence quenching (Figure S7).

As PBSH interacts with H2S via fluorescence quenching, the effect of other RSS on the optical properties of the receptor was examined. To ascertain RSS selectivity, 3 mL of 25 μM solution of PBSH was treated with 2 equiv of RSS such as H2S, citrate, cysteine, homocysteine, and glutathione (GSH), and ROS such as hydrogen peroxide (H2O2), HOCl, and tert-butyl hydroperoxide (t-BuOOH). Among all the added reactive species, H2S is the only analyte that can specifically interact with PBSH (Figure 4). Therefore, it is recognizable that PBSH is specific and selective for H2S.

Figure 4.

Figure 4

Open in a new tab

Image of the selectivity experiment of PBSH for H2S among all RSS (i.e., H2S, citrate, cysteine, homocysteine, GSH, H2O2, HOCl, and t-BuOOH) via turn-off the response of green fluorescence. Photograph courtesy of “Sanay Naha”. Copyright 2020.

Electronic and Fluorescence Response

The electronic spectrum of PBSH with RSS was recorded. The spectra for PBSH and PBSH + RSS appear similar, and there is hardly any observable change in the electronic spectrum. However, in the emission spectrum, a 7-fold quenching of green fluorescence of PBSH along with a 30 nm blue shift of λem by H2S is observed in water, which is supported by density functional theory calculations (Figure S8). The quenching of fluorescence can be attributed to the cleavage of the molecular framework to its smallest nonemissive material. A sequential fluorometric titration of PBSH by H2S shows a saturation of the receptor at 1 equiv. During titration, the blue shift of λem is observed. Interference studies also reaffirm the selectivity and specificity of the receptor toward H2S. Similarly, an interference experiment was carried out to justify the selectivity of the receptor PBSH toward H2S even in the presence of other competing RSS, which is evident from the plot (Figure 5).

Figure 5.

Figure 5

Open in a new tab

Spectroscopic responses of PBSH in the presence of RSS. (a) UV–vis response of PBSH with all RSS in the water medium, (b) emission of PBSH in the presence of various RSS on excitation at 382 nm and a slit width of 5 nm for both excitation and emission, (c) incremental titration of PBSH by H2S aqueous solution, (d) plot of concentration of H2S in sequential addition vs fluorescence intensity at 522 nm, and (e) interference study of PBSH selectivity for H2S over other RSS (1. only PBSH, 2. citrate, 3. cysteine, 4. homocysteine, 5. GSH, 6. H2O2hydrogen peroxide, 7. HOCl, and 8. t-BuOOH).

pH Effect in Sensing

The sensitivity and effectiveness of a receptor at various pH values are some of the prime aspects to look at. Biological, as well as real-time, applicability of a receptor invariably depends on the pH of the governing medium. Detection and bioimaging of H2S in acidic pH are some of the challenging aspects. To examine the pH effect, 3 mL of 25 μM solution of PBSH at various pH values was prepared using phosphate-buffered saline (PBS) buffer, and emission of each pH solution was recorded before and after the addition of H2S in all solutions. PBSH shows an intense green emission in the pH range from 4 to 10, but as the pH goes beyond 10, instead of the green emission a bluish emission is observed (Figure S9). In the emission spectrum of PBSH at various pHs before and after the addition of H2S, a blue shift of 45 nm has been observed with a decrease in intensity within the pH range 4–9. Therefore, PBSH is efficient within a wide range of pH 4–9 and can be used for in vivo cellular imaging for H2S (Figure 6).

Figure 6.

Figure 6

Open in a new tab

(a) Fluorometric responses of only PBSH and PBSH + H2S at various pHs. (b) Comparison of fluorescence intensity before and after the addition of H2S in respective pH solutions of PBSH.

Quantification of H2S

The limit of quantification (LoQ) and limit of detection (LoD) stand to describe the lowest concentration of an analyte that can be reliably measured by an analytical method. As the sensing method is found to be chemodosimetric, binding constant calculation is not applicable as no stable binding takes place. The LoD and LoQ for H2S are found to be 18 and 68 nM, respectively. The quantum yield of the entire process has been calculated with respect to rhodamine B as a reference. The quantum yield (ϕ) for only PBSH is 14-fold greater than PBSH + H2S, which are 0.42 and 0.03, respectively (Figure S10).

Sensing Mechanism

The presence of a sulfonamide group imparts solubility to the PBSH molecules in water, alongside the π–π stacking of the pyrene ring, which is the driving force for the intense green emission in water. Upon reaction with H2S, the sulfonylhydrazide moiety beaks to the hydrazide moiety which leads to the release of toluene and SO2; subsequently, the intermolecular π–π stacking in between the bimolecular π–π assembly breaks down, which in effect leads to the quenching to the fluorescence intensity, which is further supported by the lifetime experiment of H2S detection (Figure S11). To ascertain the sensing mechanism of PBSH, the ACN–water solvent mixture has been used over DMSO–water, as the reaction time, product separation and isolation, and workup of the reaction are economical and easy to do in the ACN–water mixture. The reaction between PBSH and H2S was carried out in the ACN–water mixed solvent maintained at a 1:1 ratio. The progress of the reaction was monitored using thin-layer chromatography. Upon standing at room temperature for another 1 h to allow for complete precipitation, a bright yellow color precipitate was observed and filtered. The dried precipitate was characterized using 1H NMR and 13C NMR spectroscopy (Figures S12 and S13). On comparing the NMR data of the receptor PBSH and H2S-treated PBSH, we could see obvious differences, that is, the peaks at 2.36 ppm (for the methyl functional group) and 8.89 ppm (for secondary −NH) do not appear in the 1H NMR spectra of PBSH + H2S, but a new peak at 7.03 ppm (for primary −NH2) is observed. The peaks, as well as the proton counts in the aromatic region, got decreased because of the removal of the toluene group. These data are well supported by 13C NMR data. Hence, the sensing mechanism is confirmed as the reductive removal of SO2 from sulfonamide, which results in toluene and pyrene hydrazide (Figure 7).

Figure 7.

Figure 7

Open in a new tab

Proposed reductive chemodosimetric sensing mechanism of H2S.

Application of PBSH

The utility of a receptor is justified until and unless it is reliably applicable in real-time conditions. To prove PBSH authenticity in H2S detection and estimation, PBSH has been applied for endogenous as well as for exogenous confocal fluorescence imaging of HeLa cells, followed by fluorescence imaging of zebrafish. The results are as follows.

Endogenous Imaging of HeLa Cells and Zebrafish

HeLa cells were obtained from the Food Industry Research and Development Institute (Taiwan). The cell culture condition is maintained using Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum at 37 °C under 5% CO2 and 18 mm glass coverslips for plating. After 24 h, the cells were washed with PBS and thereafter kept for 30 min incubated with 20 μM R in 2 mL DMEM. After washing the excess “PBSH” with PBS, the treated cells were incubated with 150 μM GSH for endogenous H2S production and 40 μM H2S solution for exogenous detection separately and allowed another 30 min incubation. To examine H2S uptake, the cells were treated with 2 mL of 1.0 mM H2S (10 μM) in sterilized PBS (pH 7.4) and incubated at 37 °C under 5% CO2 environment for 30 min. After washing excess H2S from the treated cells, culture medium (2 mL) was added to the cultured cells, followed by a 10 mM solution of “PBSH” in water and incubated for another 30 min. The treated cells were washed with PBS 3 times and examined. From the image (Figure 8a), it is evident that “PBSH” has excellent permeability through the HeLa cell membrane and has intense green fluorescence. PBSH is capable of showing intense green emission in cellular conditions, which are concluded from the sections Solvent Effect and pH Effect. The endogenous formation of H2S leads to the quenching of green emission of PBSH, which is repeated by the external addition of H2S. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay result is strongly supported because nontoxicity of PBSH to 20 μM concentration of PBSH in water cell viability is observed to be nearly 90% (Figure S14).

Figure 8.

Figure 8

Open in a new tab

Bioapplicability of PBSH for H2S detection. (a) Endogenous and exogenous HeLa cell imaging and (b) zebrafish imaging. Photograph courtesy of “Natesan Thirumalaivasan”. Copyright 2020.

The receptor PBSH is also applied for fluorescence zebrafish imaging. The eggs of zebrafish were subjected to a 3-day maturation period and subsequently used for confocal fluorescence imaging experiments. First, the zebrafish were placed in 2 mL of 20 μM solution of the receptor PBSH to allow for smooth intake and then rested for 30 min at room temperature. After the standby period, the school of fish was washed 2–5 times with deionized water, and fluorescence images were recorded. The receptor is inherently emissive in the fish with an intense green emission. The cleaned fish were treated with 2 mL of 15 μM solution of H2S and allowed for another 30 min. Then again, after cleaning the fish, their fluorescence images were recorded. The fish show no emission, and the intensity of the green emission is quenched. These tests show that the receptor can be used as a “turn-off” H2S marker for in vivo applications and can precisely indicate the presence of H2S (Figure 8b).

Conclusions

In summary, the synthesized receptor “PBSH” shows selectivity toward H2S among all RSS and ROS in 100% water. The sensing mechanism is proved to be chemodosimetric reductive elimination of SO2, which is well supported by the identification of separated products after the reaction. The utility of the receptor is extended for endogenous as well as for exogenous detection of H2S in HeLa cells and applicable in zebrafish imaging.

Experimental Section

Materials and Instruments

1-Pyrenecarboxaldehyde and p-toluenesulfonylhydrazide were purchased from Sigma-Aldrich and used as received. The solvents used for synthesis and photophysical studies were of spectroscopic grade. For photophysical studies, double-distilled water was used, and the pH of water was 6.7 and conductivity was 30 dS/m. For the stock solution of all anions, tetrabutylammonium salts were purchased, and for all cations, chloride salts were used. Sodium sulfide was used as an H2S donor, purchased from Sigma-Aldrich, and was used as received. A Bruker DRX-300, an Agilent Unity INOVA-500, and a Bruker Ascend 500 NMR spectrometer were used for NMR characterization. UV–vis spectra were obtained using an Agilent 8453 UV–vis spectrometer. Fluorescence spectra measurements were obtained from the Hitachi F-7000 fluorescence spectrophotometer. Lifetime measurement studies were done using the Horiba DeltaTime instrument coupled with a time-correlated single-photon counting system. Fluorescence images were taken on a Leica TCS SP5 X AOBS and a Leica TCS SP5 II confocal fluorescence microscope.

Spectroscopic Procedures

The stock solution of the receptor “PBSH” (1.0 × 10–3 M in DMSO) was prepared and kept at room temperature. Then, the stock solution was diluted with 100% H2O to a final concentration of 2.5 × 10–5 M for spectral analysis. All anions, cations, and RSS stock solutions were prepared at the concentration of 5 × 10–3 M in water.

Synthesis of PBSH

In a hot-dry 100 mL, round-bottom flask, 15 mL of ethanolic solution of 1-pyrenecarboxaldehyde (460 mg, 2 mM) was prepared and a catalytic amount of glacial acetic acid (100 μL) was added. The resultant acidic mixture was stirred for 10 min and then 5 mL ethanolic solution of p-toluenesulfonylhydrazide (400 mg, 2.1 mM) was added dropwise over a time period of 10 min. The resultant mixture was set for reflux for 3 h. The reaction mixture was cooled to room temperature, followed by the addition of 20 mL of diethyl ether and kept at room temperature for 12 h. A greenish-yellow precipitate was obtained, which was separated through filtration and washed with hot water and hot ethanol, and then the product was dried in a vacuum oven. The compound was further purified by recrystallization in the methanol–ACN medium. The yield of the reaction is 357 mg (90%) (Scheme 1).

Melting point: (189–190) °C.

FT-IR (ν/cm–1): 3449 (−S–OH), 3025(−NH), 1594 (C=N, azomethine) (Figure S15).

1H (500 MHz; DMSO-d6; Me4Si): δ 8.89 (s, 1H), 8.65 (d, 1H), 8.3–8.09 (m, 8H), 7.9 (d, 2H), 7.4 (d, 2H), 2.3 (s, 3H) (Figure S16).

13C ppm (120 MHz; DMSO-d6; Me4Si): 146.69, 144.05, 136.68, 132.35, 131.27, 130.54, 130.22, 129.24, 128.94, 128.69, 127.86, 127.8, 127.1, 126.73, 126.61, 126.26, 125.77, 125.6, 124.5, 124.14, 122.96 and 21.47 (Figure S17).

LCMS (ESI-MS) m/z: calcd for C24H18N2O2S, 398; found, 399 [M + H] (Figure S18).

Acknowledgments

S.N. is highly thankful to the Department of Science and Technology, INDIA (DST-INDIA), for providing financial support through the INSPIRE Fellowship (IF150881), the TEEP internship program, and NCTU, Taiwan, for research infrastructure.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.0c02963.

  • Spectroscopic characterization details of the receptor PBSH; various experimental procedures; comparison table of recent publications; polarity index of various solvents; and unit cell and supercell packing of PBSH (PDF)

Author Present Address

§ Department of Chemistry, Banaras Hindu University, Varanasi, India.

The authors declare no competing financial interest.

Supplementary Material

ao0c02963_si_001.pdf (1.6MB, pdf)

References

  1. Jeroschewski P.; Steuckart C.; Kühl M. An Amperometric Microsensor for the Determination of H2S in Aquatic Environments. Anal. Chem. 1996, 68, 4351–4357. 10.1021/ac960091b. [DOI] [Google Scholar]
  2. Cardoso A.; Liu H.; Dasgupta P. K. Fluorometric fiber optic drop sensor for atmospheric hydrogen sulfide. Talanta 1997, 44, 1099–1106. 10.1016/s0039-9140(96)02202-3. [DOI] [PubMed] [Google Scholar]
  3. Zhang Z.; Jiang H.; Xing Z.; Zhang X. A highly selective chemiluminescent H2S sensor. Sens. Actuators, B 2004, 102, 155–161. 10.1016/j.snb.2004.04.015. [DOI] [Google Scholar]
  4. Kapse V. D.; Ghosh S. A.; Chaudhari G. N.; Raghuwanshi F. C. Nanocrystalline In2O3-based H2S sensors operable at low temperatures. Talanta 2008, 76, 610–616. 10.1016/j.talanta.2008.03.050. [DOI] [PubMed] [Google Scholar]
  5. Yao K.; Caruntu D.; Zeng Z.; Chen J.; O’Connor C. J.; Zhou W. Parts per Billion-Level H2S Detection at Room Temperature Based on Self-Assembled In2O3 Nanoparticles. J. Phys. Chem. C 2009, 113, 14812–14817. 10.1021/jp905189f. [DOI] [Google Scholar]
  6. Chen X.; Zhou Y.; Peng X.; Yoon J. Fluorescent and colorimetric probes for detection of thiols. Chem. Soc. Rev. 2010, 39, 2120–2135. 10.1039/b925092a. [DOI] [PubMed] [Google Scholar]
  7. Peng H.; Cheng Y.; Dai C.; King A. L.; Predmore B. L.; Lefer D. J.; Wang B. A fluorescent probe for fast and quantitative detection of hydrogen sulfide in blood. Angew. Chem., Int. Ed. 2011, 50, 9672–9675. 10.1002/anie.201104236. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Yu F.; Li P.; Song P.; Wang B.; Zhao J.; Han K. An ICT-based strategy to a colorimetric and ratiometric fluorescence probe for hydrogen sulfide in living cells. Chem. Commun. 2012, 48, 2852–2854. 10.1039/c2cc17658k. [DOI] [PubMed] [Google Scholar]
  9. Zang W.; Nie Y.; Zhu D.; Deng P.; Xing L.; Xue X. Core-Shell In2O3/ZnO Nanoarray Nanogenerator as a Self-Powered Active Gas Sensor with High H2S Sensitivity and Selectivity at Room Temperature. J. Phys. Chem. C 2014, 118, 9209–9216. 10.1021/jp500516t. [DOI] [Google Scholar]
  10. Lin V. S.; Chen W.; Xian M.; Chang C. J. Chemical probes for molecular imaging and detection of hydrogen sulfide and reactive sulfur species in biological systems. Chem. Soc. Rev. 2015, 44, 4596–4618. 10.1039/c4cs00298a. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Sarfraz J.; Ihalainen P.; Määttänen A.; Gulin T.; Koskela J.; Wilén C.-E.; Kilpelä A.; Peltonen J. A printed H2S sensor with electro-optical response. Sens. Actuators, B 2014, 191, 821–827. 10.1016/j.snb.2013.10.011. [DOI] [Google Scholar]
  12. Yu F.; Han X.; Chen L. Fluorescent probes for hydrogen sulfide detection and bioimaging. Chem. Commun. 2014, 50, 12234–12249. 10.1039/c4cc03312d. [DOI] [PubMed] [Google Scholar]
  13. Li Z.; Wang N.; Lin Z.; Wang J.; Liu W.; Sun K.; Fu Y. Q.; Wang Z. Room-Temperature High-Performance H2S Sensor Based on Porous CuO Nanosheets Prepared by Hydrothermal Method. ACS Appl. Mater. Interfaces 2016, 8, 20962–20968. 10.1021/acsami.6b02893. [DOI] [PubMed] [Google Scholar]
  14. Kumar A.; Samanta S.; Singh A.; Roy M.; Singh S.; Basu S.; Chehimi M. M.; Roy K.; Ramgir N.; Navaneethan M.; Hayakawa Y.; Debnath A. K.; Aswal D. K.; Gupta S. K. Fast Response and High Sensitivity of ZnO Nanowires Cobalt Phthalocyanine Heterojunction Based H2S Sensor. ACS Appl. Mater. Interfaces 2015, 7, 17713–17724. 10.1021/acsami.5b03652. [DOI] [PubMed] [Google Scholar]
  15. Zhang H.; Xie Y.; Wang P.; Chen G.; Liu R.; Lam Y.-W.; Hu Y.; Zhu Q.; Sun H. An iminocoumarin benzothiazole-based fluorescent probe for imaging hydrogen sulfide in living cells. Talanta 2015, 135, 149–154. 10.1016/j.talanta.2014.12.044. [DOI] [PubMed] [Google Scholar]
  16. Rosolina S. M.; Carpenter T. S.; Xue Z.-L. Bismuth-based, disposable sensor for the detection of hydrogen sulfide gas. Anal. Chem. 2016, 88, 1553–1558. 10.1021/acs.analchem.5b04489. [DOI] [PubMed] [Google Scholar]
  17. Wang Y.; Liu B.; Xiao S.; Wang X.; Sun L.; Li H.; Xie W.; Li Q.; Zhang Q.; Wang T. Low-Temperature H2S Detection with Hierarchical Cr-Doped WO3 Microspheres. ACS Appl. Mater. Interfaces 2016, 8, 9674–9683. 10.1021/acsami.5b12857. [DOI] [PubMed] [Google Scholar]
  18. He L.; Dong B.; Liu Y.; Lin W. Fluorescent chemosensors manipulated by dual/triple interplaying sensing mechanisms. Chem. Soc. Rev. 2016, 45, 6449–6461. 10.1039/c6cs00413j. [DOI] [PubMed] [Google Scholar]
  19. Zhang P.; Nie X.; Gao M.; Zeng F.; Qin A.; Wu S.; Tang B. Z. A highly selective fluorescent nanoprobe based on AIE and ESIPT for imaging hydrogen sulfide in live cells and zebrafish. Mater. Chem. Front. 2017, 1, 838–845. 10.1039/c6qm00223d. [DOI] [Google Scholar]
  20. Palanisamy S.; Lee L.-Y.; Wang Y.-L.; Chen Y.-J.; Chen C.-Y.; Wang Y.-M. A water-soluble and fast response fluorescent turn-on copper complex probe for H2S detection in zebrafish. Talanta 2016, 147, 445–452. 10.1016/j.talanta.2015.10.019. [DOI] [PubMed] [Google Scholar]
  21. Zhang P.; Hong Y.; Wang H.; Yu M.; Gao Y.; Zeng R.; Longa Y.; Long J. Selective visualization of endogenous hydrogen sulfide in lysosomes using aggregation induced emission dots. Polym. Chem. 2017, 8, 7271–7278. 10.1039/c7py01696d. [DOI] [Google Scholar]
  22. Somboot W.; Jakmunee J.; Kanyanee T. An exploiting of cost-effective direct current conductivity detector in gas diffusion flow injection system. Talanta 2017, 170, 298–305. 10.1016/j.talanta.2017.04.015. [DOI] [PubMed] [Google Scholar]
  23. a Abu-Hani A. F. S.; Greish Y. E.; Mahmoud S. T.; Awwad F.; Ayesh A. I. Low-temperature and fast response H2S gas sensor using semiconducting chitosan film. Sens. Actuators, B 2017, 253, 677–684. 10.1016/j.snb.2017.06.103. [DOI] [Google Scholar]; b Xu Q.-c.; Gu Z.-y.; Xing G.-w. Design and synthesis of a Cu (II)-complex-based carbazole-hemocyanin hybrid for fluorescent sensing of H2S in SDS micellar solution. Tetrahedron 2017, 73, 2123–2130. 10.1016/j.tet.2017.02.060. [DOI] [Google Scholar]
  24. a Fu Q.; Chen G.; Liu Y.; Cao Z.; Zhao X.; Li G.; Yu F.; Chen L.; Wang H.; You J. In situ quantification and evaluation of ClO–/H2S homeostasis in inflammatory gastric tissue by applying a rationally designed dual-response fluorescence probe featuring a novel H+-activated mechanism. Analyst 2017, 142, 1619–1627. 10.1039/c7an00244k. [DOI] [PubMed] [Google Scholar]; b Ding S.; Feng W.; Feng G. Rapid and highly selective detection of H2S by nitrobenzofurazan (NBD) ether-based fluorescent probes with an aldehyde group. Sens. Actuators, B 2017, 238, 619–625. 10.1016/j.snb.2016.07.117. [DOI] [Google Scholar]; c Lv J.; Wang F.; Qiang J.; Ren X.; Chen Y.; Zhang Z.; Wang Y.; Zhang W.; Chen X. Enhanced response speed and selectivity of fluorescein-based H2S probe via the cleavage of nitrobenzene sulfonyl ester assisted by ortho aldehyde groups. Biosens. Bioelectron. 2017, 87, 96–100. 10.1016/j.bios.2016.08.018. [DOI] [PubMed] [Google Scholar]
  25. a Wang S.; Ding H.; Wang Y.; Fan C.; Tu Y.; Liu G.; Pu S. An “off–on–off” sensor for sequential detection of Cu2+ and hydrogen sulfide based on a naphthalimide–rhodamine B derivative and its application in dual-channel cell imaging. RSC Adv. 2018, 8, 33121–33128. 10.1039/c8ra05963b. [DOI] [PMC free article] [PubMed] [Google Scholar]; b Wang W.; Wu C.; Yang C.; Li G.; Han Q.-B.; Li S.; Lee S. M.-Y.; Leung C.-H.; Ma D.-L. A dual-functional luminescent probe for imaging H2S in living zebrafish and discrimination hypoxic cells from normoxic cells. Sens. Actuators, B 2018, 255, 1953–1959. 10.1016/j.snb.2017.08.222. [DOI] [Google Scholar]
  26. a Thirumalaivasan N.; Venkatesan P.; Wu S.-P. Highly selective turn-on probe for H2S with imaging applications in vitro and in vivo. New J. Chem. 2017, 41, 13510–13515. 10.1039/c7nj02869e. [DOI] [Google Scholar]; b Kang J.; Huo F.; Ning P.; Meng X.; Chao J.; Yin C. Two red-emission single and double ‘arms’ fluorescent materials stemmed from ‘one-pot’ reaction for hydrogen sulfide vivo imaging. Sens. Actuators, B 2017, 250, 342–350. 10.1016/j.snb.2017.04.180. [DOI] [Google Scholar]
  27. a Eom N. S. A.; Cho H.-B.; Lim H.-R.; Hwang T.-Y.; Song Y.; Choa Y.-H. Ultrasensitive detection of low-ppm H2S gases based on palladium-doped porous silicon sensors. RSC Adv. 2018, 8, 29995–30001. 10.1039/c8ra05520c. [DOI] [PMC free article] [PubMed] [Google Scholar]; b Su P.; Zhu Z.; Wang J.; Cheng B.; Wu W.; Iqbal K.; Tang Y. A biomolecule-based fluorescence chemosensor for sequential detection of Ag+ and H2S in 100% aqueous solution and living cells. Sens. Actuators, B 2018, 273, 93–100. 10.1016/j.snb.2018.06.037. [DOI] [Google Scholar]; c Das S.; Sahoo P. A colorimetric sensor for hydrogen sulfide: Detection from biogas and quantitative estimation in water. Sens. Actuators, B 2019, 291, 287–292. 10.1016/j.snb.2019.04.089. [DOI] [Google Scholar]
  28. a da Silva C. B.; Gil E. S.; da Silveira Santos F.; Moras A. M.; Steffens L.; Goncalves P. F. B.; Moura D. J.; Lüdtke D. S.; Rodembusch F. S. Proton-Transfer-Based Azides with Fluorescence Off–On Response for Detection of Hydrogen Sulfide: An Experimental, Theoretical, and Bioimaging Study. J. Org. Chem. 2018, 83, 15210–15224. 10.1021/acs.joc.8b02489. [DOI] [PubMed] [Google Scholar]; b Hong J.; Feng W.; Feng G. Highly selective near-infrared fluorescent probe with rapid response, remarkable large Stokes shift and bright fluorescence for H2S detection in living cells and animals. Sens. Actuators, B 2018, 262, 837–844. 10.1016/j.snb.2018.02.070. [DOI] [Google Scholar]
  29. a Kwon N.; Hu Y.; Yoon J. Fluorescent Chemosensors for Various Analytes Including Reactive Oxygen Species, Biothiol, Metal Ions, and Toxic Gases. ACS Omega 2018, 3, 13731–13751. 10.1021/acsomega.8b01717. [DOI] [PMC free article] [PubMed] [Google Scholar]; b Rajalakshmi K.; Nam Y.-S.; Youg S.; Selvaraj M.; Lee K.-B. Biocompatible silica nanoparticles conjugated with azidocoumarin for trace level detection and visualization of endogenous H2S in PC3 cells. Sens. Actuators, B 2018, 259, 307–315. 10.1016/j.snb.2017.12.078. [DOI] [Google Scholar]
  30. Sedgwick A. C.; Wu L.; Han H.-H.; Bull S. D.; He X.-P.; James T. D.; Sessler J. L.; Tang B. Z.; Tian H.; Yoon J. Excited-state intramolecular proton-transfer (ESIPT) based fluorescence sensors and imaging agents. Chem. Soc. Rev. 2018, 47, 8842–8880. 10.1039/c8cs00185e. [DOI] [PubMed] [Google Scholar]
  31. Wang H.; Liu G. Advances in luminescent materials with aggregation-induced emission (AIE) properties for biomedical applications. J. Mater. Chem. B 2018, 6, 4029–4042. 10.1039/c8tb00674a. [DOI] [PubMed] [Google Scholar]
  32. Wang H.; Lustig W. P.; Li J. Sensing and capture of toxic and hazardous gases and vapors by metal-organic frameworks. Chem. Soc. Rev. 2018, 47, 4729–4756. 10.1039/c7cs00885f. [DOI] [PubMed] [Google Scholar]
  33. Lee Y. H.; Kweon O. Y.; Kim H.; Yoo J. H.; Han S. G.; Oh J. H. Recent advances in organic sensors for health self-monitoring systems. J. Mater. Chem. C 2018, 6, 8569–8612. 10.1039/c8tc02230e. [DOI] [Google Scholar]
  34. Ovsianytskyi O.; Nam Y.-S.; Tsymbalenko O.; Lan P.-T.; Moon M.-W.; Lee K.-B. Highly sensitive chemiresistive H2S gas sensor based on graphene decorated with Ag nanoparticles and charged impurities. Sens. Actuators, B 2018, 257, 278–285. 10.1016/j.snb.2017.10.128. [DOI] [Google Scholar]
  35. Zhao B.; Yang Y.; Wu Y.; Yang B.; Chai J.; Hu X.; Liu B. To re-evaluate the emission mechanism, AIE activity of 5-azidofluorescein and its reaction with H2S and NO. Sens. Actuators, B 2018, 256, 79–88. 10.1016/j.snb.2017.10.020. [DOI] [Google Scholar]
  36. El-Maghrabey M. H.; Watanabe R.; Kishikawa N.; Kuroda N. Detection of hydrogen sulfide in water samples with 2-(4-hydroxyphenyl)-4,5-di(2-pyridyl)imidazole-copper(II) complex using environmentally green microplate fluorescence assay method. Anal. Chim. Acta 2019, 1057, 123–131. 10.1016/j.aca.2019.01.006. [DOI] [PubMed] [Google Scholar]
  37. a Liu L.; Shang X.; He R.; Li J.; Chen Y.; Chen H.; Wang T. Anion binding ability and cytotoxicity of a selective colorimetric chemosensor for H2S based on Zn(II) complex. Inorg. Chim. Acta 2019, 495, 118994–119000. 10.1016/j.ica.2019.118994. [DOI] [Google Scholar]; b Liu F.; Wang J.; You R.; Yang Z.; He J.; Wang C.; Liu A. YSZ-based solid electrolyte type sensor utilizing ZnMoO4 sensing electrode for fast detection of ppb-level H2S. Sens. Actuators, B 2020, 302, 127205–127212. 10.1016/j.snb.2019.127205. [DOI] [Google Scholar]
  38. Asaithambi G.; Periasamy V. Hydrogen sulfide detection by ESIPT based fluorescent sensor: Potential in living cells imaging. J. Photochem. Photobiol., A 2019, 369, 97–105. 10.1016/j.jphotochem.2018.10.013. [DOI] [Google Scholar]
  39. Yang J.; Yang B.; Wen G.; Liu B. Dual sites fluorescence probe for H2S and Hg2+ with “AIE Transformers” function. Sens. Actuators, B 2019, 296, 126670–126678. 10.1016/j.snb.2019.126670. [DOI] [Google Scholar]
  40. a Mittapelli L. L.; Nawale G. N.; Gholap S. P.; Varghese O. P.; Gore K. R. A turn-on fluorescent GFP chromophore analog for highly selective and efficient detection of H2S in aqueous and in living cells. Sens. Actuators, B 2019, 298, 126875–126882. 10.1016/j.snb.2019.126875. [DOI] [Google Scholar]; b Cao T.; Teng Z.; Gong D.; Qian J.; Liu W.; Iqbal K.; Qin W.; Guo H. A ratiometric fluorescent probe for detection of endogenous and exogenous hydrogen sulfide in living cells. Talanta 2019, 198, 185–192. 10.1016/j.talanta.2019.02.017. [DOI] [PubMed] [Google Scholar]
  41. a Kaushik R.; Ghosh A.; Amilan Jose D. Recent progress in hydrogen sulphide (H2S) sensors by metal displacement approach. Coord. Chem. Rev. 2017, 347, 141–157. 10.1016/j.ccr.2017.07.003. [DOI] [Google Scholar]; b Kaushik R.; Ghosh A.; Singh A.; Jose D. A. Colorimetric sensor for the detection of H2S and its application in molecular half-subtractor. Anal. Chim. Acta 2018, 1040, 177–186. 10.1016/j.aca.2018.08.028. [DOI] [PubMed] [Google Scholar]; c Kaushik R.; Sakla R.; Ghosh A.; Selvan G. T.; Selvakumar P. M.; Jose D. A. Selective detection of H2S by copper complex embedded in vesicles through metal indicator displacement approach. ACS Sens. 2018, 3, 1142–1148. 10.1021/acssensors.8b00174. [DOI] [PubMed] [Google Scholar]; d Jeyasingh V.; Murugesan K.; Lakshminarayanan S.; Selvapalam N.; Das G.; Piramuthu L. A molecular phototropic system for cyanide: Detection and sunlight driven harvesting of cyanide with molecular sunflower. Spectrochim. Acta, Part A 2020, 234, 118207–118213. 10.1016/j.saa.2020.118207. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

ao0c02963_si_001.pdf (1.6MB, pdf)

Articles from ACS Omega are provided here courtesy of American Chemical Society

RESOURCES