Fluorescent Probe For H2S Research Articles

An aggregation-induced fluorescence probe for H2S and its application in living cells

Hydrogen sulphide (H2S) plays an important role in the regulation of a number of important physiological and pathological processes. Therefore, the development of new fluorescent probes for H2S is of great importance and has recently attracted much attention. In this paper, a novel coumarin-based fluorescent probe named (1E)-1-(1-(6-chloro-2-oxo-2H-chromen-3-yl)ethylidene)thiosemicarbazide (1), which contained a CN moiety as a specific reaction site for nucleophilic addition of H2S, was prepared. Upon addition of H2S, probe 1 exhibited significant fluorescence enhancement with a detection limit as low as 0.54 μM, accompanied by a colour change from cyan to colourless. The mechanism was confirmed by ESI-MS, Job’s plot, DFT and the probe 1 was successfully applied to the imaging of H2S in HeLa cells.

The Application of Hydrogen Sulfide Fluorescent Probe in Food Preservation, Detection and Evaluation.

This work primarily reviewed the response mechanism of fluorescent probes for H2S detection in foodstuffs in recent years, as well as the methodologies employed for detecting foodstuffs. Firstly, the significance of studying H2S gas as an important signaling molecule is introduced. Subsequently, a review of the response mechanism of the scientific community on how to detect H2S in foodstuffs samples by fluorescent probe technology is carried out. Secondly, the methods commonly used for detecting foodstuffs samples are discussed, including the test strip method and the spiking recovery methods. Nevertheless, despite the significant advancements in this field, there remain some research gaps. Finally, the article identifies the remaining issues that require further attention in current research and proposes avenues for future investigation. More importantly, this work identifies the current limitations of research in this field and proposes future applications of fluorescent probes for H2S in assessing food freshness and determining food spoilage. Therefore, this review will provide robust technical support for the protection of consumer health and the advancement of the sustainable development of the food industry and also put forward some new ideas and suggestions for future research.

Activity-Based Fluorescent Probes for Hydrogen Sulfide and Related Reactive Sulfur Species.

Hydrogen sulfide (H2S) is not only a well-established toxic gas but also an important small molecule bioregulator in all kingdoms of life. In contemporary biology, H2S is often classified as a "gasotransmitter," meaning that it is an endogenously produced membrane permeable gas that carries out essential cellular processes. Fluorescent probes for H2S and related reactive sulfur species (RSS) detection provide an important cornerstone for investigating the multifaceted roles of these important small molecules in complex biological systems. A now common approach to develop such tools is to develop "activity-based probes" that couple a specific H2S-mediated chemical reaction to a fluorescent output. This Review covers the different types of such probes and also highlights the chemical mechanisms by which each probe type is activated by specific RSS. Common examples include reduction of oxidized nitrogen motifs, disulfide exchange, electrophilic reactions, metal precipitation, and metal coordination. In addition, we also outline complementary activity-based probes for imaging reductant-labile and sulfane sulfur species, including persulfides and polysulfides. For probes highlighted in this Review, we focus on small molecule systems with demonstrated compatibility in cellular systems or related applications. Building from breadth of reported activity-based strategies and application, we also highlight key unmet challenges and future opportunities for advancing activity-based probes for H2S and related RSS.

Modulation of Near-Infrared Mitochondria-Targetable fluorescent probe for H2S bioimaging through the modification of heavy atom iodine

H2S is correlated with mitochondrial dysfunction, which results in the death of cells. Two near-infrared fluorescent probes, Mito-HS-1 and Mito-HS-2, were designed for mitochondrial H2S imaging. Initially, the synthesis protocol of expensive IR-780-based hemicyanine (HXPI) was optimized with an appreciate yield of 80 % as compared with 14–56 % previously reported. Iodine atom was introduced to HXPI to obtain iodine-HXPI whose Stokes shift was increased to be 90 nm. On account of the rapid and fast nucleophilic attack of H2S, HXPI-based Mito-HS-1 could be applied for the real time imaging of mitochondrial H2S. Besides some similar optical properties with Mito-HS-1, iodine-HXPI-based Mito-HS-2 exhibited wider linear range (3–150 μM), more stable fluorescent imaging and more favorable specificity in vitro. Both Mito-HS-1 and Mito-HS-2 could be used to image exogenous H2S in cells, with Mito-HS-2 showing fairly better signal-to-noise. Additionally, the Pearson correlation coefficient of two probes demonstrated that they could successfully monitor mitochondrial H2S in A549 cells and Hela cells.

Construction, photophysical properties, structure-activity relationship, and applications of fluorescein analogs

The investigation on the construction strategy, relationship between the structure and photophysical properties of fluorescein analogs plays an important role in the rational design of specific fluorescent dyes. In this work, a fluorescein analog Fc-1, containing a reactive aldehyde group was prepared using 2′, 7′-dichlorofluorescein (DCF) as the raw material and Vilsmeier–Haack reaction mechanism. Meanwhile, two more fluorescein analogs Fc-2 and Fc-3 were synthesized based on Fc-1 and the Knoevenagel reaction mechanism. The results of Fc-1 indicated that the yield of Fc-1 was higher than 95% after a step of the low-temperature hydrolysis (40 °C) from an intermediate imine salt DCF-3. The fluorescence quantum yield (Φ) of Fc-1 was relatively high in polar protic solvents, even in H2O (Φ = 0.25). Fc-1 had large Stokes shifts in different solvents (≧ 123 nm), which was indentified by the Gaussian calculation of Fc-1. Meanwhile, Fc-1 was also used as a ratio and colorimetric fluorescent probe for H2S in vitro, and the dianion ([Fc-1-2H]2-) mechanism for detecting H2S was proposed by nuclear magnetic resonance (NMR), pH titration, and Gaussian calculation. Additionally, Fc-1 can be used in leukocyte classification because of its relatively high Φ in water, cell permeability, and cytotoxicity. All the data of dyes Fc-2 and Fc-3 indicated that the derivatization of the carboxyl group for Fc-2 did not affect the photophysical properties of Fc-2. Both of them had relatively large Stokes shifts and long emission wavelengths. The large Stokes shift of Fc-2 and Fc-3 was due to the distribution of electron clouds in the excited states of Fc-2 and Fc-3. The results of pH titration proved that dyes Fc-2 and Fc-3 were sensitive to the changes in pH and exhibited their potentials as pH probes. Thus, the strategy of constructing fluorescent dyes based on Vilsmeier–Haack and Knoevenagel reactions and the relationship between structure and properties will be beneficial to developing more dyes with large Stokes shift.

The coumarin-pyrazole dye for detection of hydrogen sulfide in cells

Fluorescent probes for H2S are often interfered by other thiols. In this work, a coumarin-pyrazole dye with 2,4-dinitrosulfonyl group was designed for the detection of H2S. The probe exhibits weak fluorescence in water due to the photo induced electron transfer (PET) by 2,4-dinitrosulfonyl. After the sulfonyl group is cleaved off by H2S, strong fluorescence appears. The probe can specifically detect H2S without being interfered by other biological thiols, and shows a wide applicable pH range, low detection and wide detection range. The excellent detection properties of the probe can also be used to detect endogenous and exogenous H2S in cells. In addition, the probes can be made into portable test paper for the detection of H2S in solutions and can detect H2S in different water samples.

Sequential Accumulation of ‘Driver’ Pathway Mutations Induces the Upregulation of Hydrogen-Sulfide-Producing Enzymes in Human Colonic Epithelial Cell Organoids

Recently, a CRISPR-Cas9 genome-editing system was developed with introduced sequential ‘driver’ mutations in the WNT, MAPK, TGF-β, TP53 and PI3K pathways into organoids derived from normal human intestinal epithelial cells. Prior studies have demonstrated that isogenic organoids harboring mutations in the tumor suppressor genes APC, SMAD4 and TP53, as well as the oncogene KRAS, assumed more proliferative and invasive properties in vitro and in vivo. A separate body of studies implicates the role of various hydrogen sulfide (H2S)-producing enzymes in the pathogenesis of colon cancer. The current study was designed to determine if the sequential mutations in the above pathway affect the expression of various H2S producing enzymes. Western blotting was used to detect the expression of the H2S-producing enzymes cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3-MST), as well as several key enzymes involved in H2S degradation such as thiosulfate sulfurtransferase/rhodanese (TST), ethylmalonic encephalopathy 1 protein/persulfide dioxygenase (ETHE1) and sulfide-quinone oxidoreductase (SQR). H2S levels were detected by live-cell imaging using a fluorescent H2S probe. Bioenergetic parameters were assessed by Extracellular Flux Analysis; markers of epithelial-mesenchymal transition (EMT) were assessed by Western blotting. The results show that the consecutive mutations produced gradual upregulations in CBS expression—in particular in its truncated (45 kDa) form—as well as in CSE and 3-MST expression. In more advanced organoids, when the upregulation of H2S-producing enzymes coincided with the downregulation of the H2S-degrading enzyme SQR, increased H2S generation was also detected. This effect coincided with the upregulation of cellular bioenergetics (mitochondrial respiration and/or glycolysis) and an upregulation of the Wnt/β-catenin pathway, a key effector of EMT. Thus sequential mutations in colon epithelial cells according to the Vogelstein sequence are associated with a gradual upregulation of multiple H2S generating pathways, which, in turn, translates into functional changes in cellular bioenergetics and dedifferentiation, producing more aggressive and more invasive colon cancer phenotypes.

A highly sensitive and selective fluorescent probe for tracking hydrogen sulfide in red wine

AbstractA novel fluorescent H2S probe (LODNBS) composed of 2,4‐dinitrobenzenesulfonyl (DNBS) units was designed and synthesized. The DNBS units not only blocked PET and ICT effects, but also triggered H2S‐mediated thiolysis to cause the fluorescence changes. Probe LODNBS performed excellent sensitivity and selectivity for H2S, strong fluorescence enhancement (65‐fold), low detection limit (72 nM) and real‐time response (within 5 min). The sensing mechanism of LODNBS towards H2S was proposed and verified by ESI‐MS and 1H NMR analysis. Under a UV lamp, the fluorescence color changes could be easily detected by the naked eye. In addition, the probe LODNBS could track H2S in red wine samples with high accuracy and good recovery, and has practical value in food analysis.

Mechanism of cystathionine-β-synthase inhibition by disulfiram: The role of bis(N,N-diethyldithiocarbamate)-copper(II)

BackgroundHydrogen sulfide (H2S) is an endogenous mammalian gasotransmitter. Cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3-MST) are the principal enzymes responsible for its biogenesis. A recent yeast screen suggested that disulfiram (a well-known inhibitor of aldehyde dehydrogenase and a clinically used drug in the treatment of alcoholism) may inhibit CBS in a cell-based environment. However, prior studies have not observed any direct inhibition of CBS by disulfiram. We investigated the potential role of bioconversion of disulfiram to bis(N,N-diethyldithiocarbamate)-copper(II) complex (CuDDC) in the inhibitory effect of disulfiram on H2S production and assessed its effect in two human cell types with high CBS expression: HCT116 colon cancer cells and Down syndrome (DS) fibroblasts. MethodsH2S production from recombinant human CBS, CSE and 3-MST was measured using the fluorescent H2S probe AzMC. Mouse liver homogenate (a rich source of CBS) was also employed to measure H2S biosynthesis. The interaction of copper with accessible protein cysteine residues was evaluated using the DTNB method. Cell proliferation and viability were measured using the BrdU and MTT methods. Cellular bioenergetics was evaluated by Extracellular Flux Analysis. ResultsWhile disulfiram did not exert any significant direct inhibitory effect on any of the H2S-producing enzymes, its metabolite, CuDDC was a potent inhibitor of CBS and CSE. The mode of its action is likely related to the complexed copper molecule. In cell-based systems, the effects of disulfiram were variable. In colon cancer cells, no significant effect of disulfiram was observed on H2S production or proliferation or viability. In contrast, in DS fibroblasts, disulfiram inhibited H2S production and improved proliferation and viability. Copper, on its own, failed to have any effects on either cell type, likely due to its low cell penetration. CuDDC inhibited H2S production in both cell types studied and exerted the functional effects that would be expected from a CBS inhibitor: inhibition of cell proliferation of cancer cells and a bell-shaped effect (stimulation of proliferation at low concentration and inhibition of these responses at higher concentration) in DS cells. Control experiments using a chemical H2S donor showed that, in addition to inhibiting CBS and CSE, part of the biological effects of CuDDC relates to a direct reaction with H2S, which occurs through its complexed copper. ConclusionsDisulfiram, via its metabolite CuDDC acts as an inhibitor of CBS and a scavenger of H2S, which, in turn, potently suppresses H2S levels in various cell types. Inhibition of H2S biosynthesis may explain some of the previously reported actions of disulfiram and CuDDC in vitro and in vivo. Disulfiram or CuDDC may be considered as potential agents for the experimental therapy of various pathophysiological conditions associated with H2S overproduction.

A near-infrared fluorescent probe for H2S based on tandem reaction to construct iminocoumarin-benzothiazole and its application in food, water, living cells

With the merits of non-destructive, high penetration ability and minimizing autofluorescence, near-infrared (NIR) fluorescent probes have attracted much attention. In this paper, a NIR emission fluorescent turn-on probe THQ-L for H2S was synthesized by the knoevenagel condensation between tetrahydroquinoxaline-6- formaldehyde derivative and 2-benzothiazoleacetonitrile. THQ-L can recognize H2S through tandem reaction triggered by HS− to construct 1,4-diethylpiperazine-modified iminocoumarin-benzothiazole, which produces a strong red fluorescent signal. THQ-L displayed an excellent selectivity toward H2S, a large stokes shift (126 nm), a high signal-to-noise ratio (200-fold), the detection limits of 38.3 nM in PBS (10 mM, pH 7.4, 30% THF). The application study indicates that THQ-L can sensitively detect H2S in red wine, natural waters, living cells and can be prepared for a test paper strip for the qualitative detection of H2S.

Linker-Regulated H2S Release from Aromatic Peptide Amphiphile Hydrogels.

Controlled release is an essential requirement for delivery of hydrogen sulfide (H2S) because of its reactive nature, short half-life in biological fluids, and toxicity at high concentrations. In this context, H2S delivery via hydrogels may be beneficial as they can deliver H2S locally at the site of interest. Herein, we employed hydrogels based on aromatic peptide amphiphiles (APAs) with tunable mechanical properties to modulate the rates of H2S release. The APAs contained an aromatic S-aroylthiooxime (SATO) H2S donor attached with a linker to a short IAVEEE hexapeptide. Linker units included carbonyl, substituted O-methylenes, alkenyl, and alkyl segments with the goal of evaluating the role of linker structure on self-assembly, capacity for hydrogelation, and H2S release rate. We studied each peptide by transmission electron microscopy, circular dichroism spectroscopy, and rheology, and we measured H2S release rates from each gel, triggering SATO decomposition and release of H2S by addition of cysteine (Cys). Using an H2S-selective electrode probe as well as a turn-on fluorescent H2S probe in the presence of H9C2 cardiomyocytes, we found that the rate of H2S release from the hydrogels depended on the rate of Cys penetration into the nanofiber core with stiffer gels showing longer overall release.

Nitrobenzoxadiazole Ether-Based Near-Infrared Fluorescent Probe with Unexpected High Selectivity for H2S Imaging in Living Cells and Mice

H2S is an important endogenous gasotransmitter, and its detection in living systems is of great significance. Especially, selective and sensitive near-infrared (NIR) fluorescent H2S probes with rapid response and large Stokes shift are highly desirable because of their superiority for in vivo detection. Probes with nitrobenzoxadiazole (NBD) ether as reaction sites have been well-explored recently to detect biothiols or H2S/biothiols simultaneously, rather than to detect H2S selectively. In this work, a new NBD ether-based NIR fluorescent probe was developed, which was unexpectedly found to show high selectivity for H2S over various other analytes including biothiols, making it practical for specific detection of H2S both in vitro and in vivo. Upon response to H2S, this probe showed rapid and significant turn-on NIR emission changes centered at 744 nm within 3 min, together with a remarkable large Stokes shift (166 nm) and high sensitivity (LOD: 26 nM). Moreover, imaging exogenous and endogenous H2S in living cells and rapid imaging of H2S in living mice with this probe was successfully applied with excellent performance.

Modulation of fluorescence sensing properties of coumarin-based fluorescent probe for H2S and its application in cell imaging.

A coumarin-based turn on probe, named 3-((E)-3-(1H-indol-3-yl)acryloyl)-2H-chromen-2-one(IAC) was designed and synthesized for the detection of H2S in aqueous medium. IAC showed no obvious red fluorescence in Tris-HCl(pH = 7.2, 60% DMF), a 28.2-fold fluorescence enhancement was found when 40 equiv. H2S was added. Other analytes such as anions, metal ions and GSH, Cys, Gly did no significant fluorescence enhancement at 580 nm to IAC. The red fluorescence enhancement mechanism between IAC and H2S was considered by DFT and ESI-MS. Overall, IAC was successfully applied in cell imaging.

A highly selective ratiometric fluorescent probe for H2S based on new heterocyclic ring formation and detection in live cells

ABSTRACTA 3-indolylacrylate derivative, 3-IA, prepared by connecting an ethyl acrylate in 3-position of indole has been synthesised and characterised. Ethyl acrylate moiety acts as the Michael acceptor towards H2S, and the resultant addition product then participates in intramolecular cyclisation with the ester group at 2-position to form another new heterocyclic ring. Blue fluorescence of 3-IA turned into green in presence of H2S, leading to ratiometric behaviour of the fluorescent sensor with large stokes shift of 55 nm. Probe 3-IA has excellent selectivity towards H2S over other biothiols and other competing anions. Density function theory/time-dependent density function theory calculations were carried out to validate the reaction mechanism and the electronic properties of 3-IA. Importantly, the ratiometric probe 3-IA shows great promise in H2S detection by simple visual fluorescent inspection in filter paper-based protocol. The probe shows its excellent ability to detect H2S in different natural water samples. Furthermore, we have employed our probe to detect H2S for ratiometric imaging in live Vero cell.

Two-photon fluorescence probe for precisely detecting endogenous H2S in lysosome by employing a dual lock system

Hydrogen sulfide (H2S) is involved in various pathological and physiological processes as well as regulation of lysosomal functions. Thus, it is essential to develop precise method for the detection of H2S in lysosome. Although a large number of fluorescent probes for H2S have been developed, the probe for lysosomal H2S assay still remains rare. Herein, a two-photon fluorescent probe, named Lyso-Nap, is designed and synthesized for detecting H2S in lysosome by employing a dual lock system (dual PET process and dual reaction sites). The fluorescence of Lyso-Nap with dual lock system is opened by H2S only when the probe is localized within acidic lososomes, which can avoid the disturbance from neutral cytosol and other organelles. Moreover, two-photon fluorescent probes with weak auto-fluorescence, large penetration depth and reduced photodamage, are particularly favored for bioimaging. Accordingly, Lyso-Nap can be successfully used for precisely tracking and detecting endogenous H2S in lysosome of living cells and tissue.

A turn-on endoplasmic reticulum-targeted two-photon fluorescent probe for hydrogen sulfide and bio-imaging applications in living cells, tissues, and zebrafish

As one of the important gas signal molecules, hydrogen sulfide (H2S) is associated with many important physiological processes in living organisms. Organelles, especially endoplasmic reticulum (ER), play a crucial role in the cell metabolism. Accordingly, the detection of H2S in the ER is of high interest. Toward this goal, we have described the development of the first ER-targeted fluorescent H2S probe (Na-H2S-ER). The new probe exhibited favorable features, such as a large turn-on fluorescence signal (45-fold fluorescence enhancement), high sensitivity and selectivity. The probe was successfully employed for imaging exogenous and endogenous H2S in the living HeLa cells. Significantly, the new probe Na-H2S-ER was employed to visualize H2S in the ER of living cells for the first time. In addition, the probe was also successfully used for imaging H2S in the living tissues up to a depth of 100 μm and in the living zebrafish.

Design of BODIPY-based near-infrared fluorescent probes for H2S

Fluorescent probes with near-infrared (NIR) emission are of great interest in biology imaging, owing to their minimal autofluorescence from biological samples and deep tissue penetration. Here, we reported two NIR fluorescence probes by masking the free hydroxyl group in a NIR fluorescent dye. We demonstrated that the designed probes showed highly selective and sensitive responsiveness to H2S. Owing to H2S triggered light-up NIR emission, the probes were suitable for applications in living cells imaging.

A novel ratiometric fluorescent H2S probe based on tandem nucleophilic substitution/cyclization reaction and its bioimaging

The third gasotransmitter hydrogen sulfide (H2S) plays a vital role to human health and diseases. A water-soluble ratiometric fluorescent probe (HS-1) for hydrogen sulfide (H2S) has been developed, which resulted in an increase of the emission at 403 nm along with a concomitant decrease of fluorescence peak at 519 nm, the ratios of emission intensities at 403 and 519 nm (I403/I519) displayed an increase from 0.27 to 8.54. This probe showed excellent selectivity toward H2S over other common anions and biothiols. Furthermore, these successes of intracellular imaging experiments in A549 cells indicated that this probe is suitable for imaging of ex-/endogenous hydrogen sulfide in living cells.

Assessment of H2S in vivo using the newly developed mitochondria-targeted mass spectrometry probe MitoA

Hydrogen sulfide (H2S) is produced endogenously in vivo and has multiple effects on signaling pathways and cell function. Mitochondria can be both an H2S source and sink, and many of the biological effects of H2S relate to its interactions with mitochondria. However, the significance of mitochondrial H2S is uncertain, in part due to the difficulty of assessing changes in its concentration in vivo. Although a number of fluorescent H2S probes have been developed these are best suited to cells in culture and cannot be used in vivo. To address this unmet need we have developed a mitochondria-targeted H2S probe, MitoA, which can be used to assess relative changes in mitochondrial H2S levels in vivo. MitoA comprises a lipophilic triphenylphosphonium (TPP) cation coupled to an aryl azide. The TPP cation leads to the accumulation of MitoA inside mitochondria within tissues in vivo. There, the aryl azido group reacts with H2S to form an aryl amine (MitoN). The extent of conversion of MitoA to MitoN thus gives an indication of the levels of mitochondrial H2S in vivo. Both compounds can be detected sensitively by liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of the tissues, and quantified relative to deuterated internal standards. Here we describe the synthesis and characterization of MitoA and show that it can be used to assess changes in mitochondrial H2S levels in vivo. As a proof of principle we used MitoA to show that H2S levels increase in vivo during myocardial ischemia.

A mitochondrial-targeted two-photon fluorescent probe for imaging hydrogen sulfide in the living cells and mouse liver tissues

Hydrogen sulfide (H2S), as signaling molecule, plays a crucial role in many biological processes in living organisms. At the organelle level, we need to study tools to explain the complexity of physiological H2S in mitochondria. Toward this goal, we have developed a new example of a fast responsive and mitochondrial-targeted two-photon fluorescent H2S probe (MT-HS) with a large turn-on fluorescence signal (40-fold fluorescence enhancement). The addition of H2S to MT-HS results a dramatic fluorescence enhancement around 540nm. The probe could image exogenous and endogenous H2S in living cells and the probe was located in mitochondria with high colocalization coefficient compared with Mitochondrial-Tracker. The large fluorescence enhancement of the two-photon probe MT-HS renders it attractive for imaging H2S in living tissues with deep tissue penetration.