H2S In HeLa Cells Research Articles

A highly selective fluorescent probe for hydrogen sulfide detection in food, water, and living cells

Hydrogen sulfide (H2S)–an endogenously produced vapor–is essential for various physiological and neurological functions. However, abnormal H2S levels can negatively impact both the environment and food quality. Herein, a novel fluorescent probe, 2-(2-benzothiazol-2-ylvinyl)-5-diethylaminophenyl-2,4-dinitrobenzenesulfonate (BDD), has been designed and synthesized for the detection of H2S. BDD exhibits high selectivity, rapid response to H2S within 6minutes, and a low limit of detection (LOD = 0.87μM), with stable emission at pH range between 6 and 12. Moreover, BDD effectively recovers H2S in water samples with recovery rates ranging from 98.8% to 102.1% and monitors both internal and external H2S in HeLa cells. Additionally, BDD-loaded test strips are promising as a tool for measuring the freshness of raw meat on-site. These findings establish the foundation for significant advancements in detecting H2S in various environments, food sources, and biological systems.

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.

A 1,3,4-thiadiazole based turn-on probe with large Stoke’s shift and ESIPT effect for fluorescent detection of H2S and its applications

Based on a useful 1,3,4-thiadiazole scaffold in our lab, a turn-on fluorescent probe for sensing H2S in environment, biological imaging and food analysis was rationally designed and synthesized. In this probe, 1,3,4-thiadiazole unit was used as fluorophore with ESIPT effect and 2,4-dinitrophenyl moiety was used as recognition fragment and fluorescent bursting agent to inhibt ESIPT. Thus, a PET process was formed and an “off–on” procedure arose after added target analyte. These brought the probe could specifically detect H2S without being interfered by other biological thiols. It showed high sensitivity (LOD=60.8 nM), large Stoke’s shift (133 nm) and had a stable performance over pH range of 6.0–9.0 in H2S monitoring system. More importantly, probe could be used for detection of H2S in real water, three beer samples and red wine sample. Furthermore, probe was successfully utilized for visualizing H2S in HeLa cells and applied for real-time monitoring of H2S during food spoilage by “naked eye” and smartphone colorimetric analysis.

Benzylic Trifluoromethyl Accelerates 1,6-Elimination Toward Rapid Probe Activation.

Activity-based detection of hydrogen sulfide in live cells can expand our understanding of its reactivity and complex physiological effects. We have discovered a highly efficient method for fluorescent probe activation, which is driven by H2S-triggered 1,6-elimination of an α-CF3-benzyl to release resorufin. In detecting intracellular H2S, 4-azido-(α-CF3)-benzyl resorufin offers significantly faster signal generation and improved sensitivity compared to 4-azidobenzyl resorufin. Computed free energy profiles for the 1,6-elimination process support the hypothesis that a benzylic CF3 group can reduce the activation energy barrier toward probe activation. This novel probe design allows for near-real-time detection of H2S in HeLa cells under stimulation conditions.

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

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

A novel iridium(III) complex-based ratiometric luminescence probe for monitoring hydrogen sulfide in living cells and zebrafish

Hydrogen sulfide exhibits crucial functions in many biological and physiological processes. The abnormal levels of H2S have been revealed to be associated with numerous human diseases. The majority of existing fluorescent probes toward H2S may still need to be improved in terms of single output signal, water solubility, biotoxicity and photostability. The construction of a ratiometric fluorescent probe based on metal complex is one effective strategy for avoiding the mentioned problems for precisely detecting H2S. Herein, we report an iridium(III) complex-based ratiometric luminescence probe (Ir-PNBD), which is designed by coupling the 7-nitro-2,1,3-benzoxadiazoles (NBD) to one of the bipyridine ligands of Ir (III) complex luminophore through a piperazition moiety. Ir-PNBD owns high selectivity and sensitivity toward H2S, and an excellent ability to target mitochondria. Moreover, Ir-PNBD was further successfully utilized to visualize exogenous and endogenous H2S in HeLa cells and zebrafish. Our work offers new opportunities to gain deeper insights into the construction of transition metal complex-based ratiometric luminescent probes and expands their applications in biomedical imaging and disease diagnosis.

Cell membrane-targeted surface enhanced Raman scattering nanoprobes for the monitoring of hydrogen sulfide secreted from living cells

Hydrogen sulfide (H2S), an important gas signal molecule, participates in intercellular signal transmission and plays a considerable role in physiology and pathology. However, in-situ monitoring of H2S level during the processes of material transport between cells remains considerably challenging. Herein, a cell membrane-targeted surface-enhanced Raman scattering (SERS) nanoprobe was designed to quantitatively detect H2S secreted from living cells. The nanoprobes were fabricated by assembling cholesterol-functionalized DNA strands and dithiobis(phenylazide) (DTBPA) molecules on core-shell gold nanostars embedded with 4-mercaptoacetonitrile (4-MBN) (AuNPs@4-MBN@Au). Thus, three functions including cell-membrane targeted via cholesterol, internal standard calibration, and responsiveness to H2S through reduction of azide group in DTBPA molecules were integrated into the nanoprobes. In addition, the nanoprobes can quickly respond to H2S within 90 s and sensitively, selectively, and reliably detect H2S with a limit of detection as low as 37 nM due to internal standard-assisted calibration and reaction specificity. Moreover, the nanoprobes can effectively target on cell membrane and realize SERS visualization of dynamic H2S released from HeLa cells. By employing the proposed approach, an intriguing phenomenon was observed: the other two major endogenous gas transmitters, carbon monoxide (CO) and nitric oxide (NO), exhibited opposite effect on H2S production in living cells stimulated by related gas release molecules. In particular, the introduction of CO inhibited the generation of H2S in HeLa cells, while NO promoted its output. Thus, the nanoprobes can provide a robust method for investigating H2S-related extracellular metabolism and intercellular signaling transmission.

Design and synthesis of two iridium(Ⅲ) complex probes for rapid sensing of hydrogen sulfide

Hydrogen sulfide (H2S) is an important sulfur-containing signaling molecule in the human body, as well as a highly toxic gas that is soluble in water. A variety of pathological processes are associated with abnormal metabolism of hydrogen sulfide, so it is very important to develop an assay that can rapidly and specifically detect H2S. In this work, two iridium(Ⅲ) complexes were designed and synthesized using ester groups as reaction sites for recognition of H2S. As the H2S concentration increased, the photoluminescence of the probe was rapidly quenched within 5seconds, with a detection limit as low as 0.45 μM. Among them, the probe Ir1, whose luminescence was located in the visible region, had been successfully used for the detection of trace amounts of H2S in water samples with a recovery rate of 92.5 ~ 104.0% and a relative standard deviation (RSD, n=5) of < 2.8%. The luminescence of probe Ir2 was located in the near-infrared region, which could avoid the interference of biological autofluorescence and be successfully used for luminescence imaging of H2S in HeLa cells. This study provided a promising method for rapid detection of trace H2S in water samples and cellular microenvironments.

A novel fluorescent probe for imaging endogenous hydrogen sulfide in living cells and mice models of acute liver injury

Hydrogen sulfide (H2S) performs essential functions in several physiological and pathological procedures, such as liver cirrhosis, diabetes mellitus, and cancer. To optimize the H2S detection in practical applications, herein, a new probe YG-H2S for the fluorescent imaging of H2S was developed. Compared with the current H2S probes, the main advantages of YG-H2S were a wide pH range (5.0–9.0), a relatively fast response (within 10 min), and high selectivity from the competitive species (including biological mercaptan). Owing to the low cytotoxicity of YG-H2S, it was good for the biological depth imaging in biological events. YG-H2S could detect endogenous H2S in HeLa cells. In addition, YG-H2S could monitor the level of H2S in 4 T1-xenograft and acute liver injury model mice as well as in separate tissues. Consequently, YG-H2S might be a potential method for detecting H2S in preliminary diagnosis.

A simple benzothiazole-based turn-on fluorescent probe for detecting H2S in HeLa cells

H2S is an endogenously produced gaseous signaling molecule that plays a vital role in many biological processes. Small molecule fluorescent probes as a powerful tool can be used to in vivo and in vitro visualize the H2S with many advantages. At present, many fluorescence probes were reported based on the nucleophilicity of H2S, which were susceptible to interference from other nucleophilic species and possessed poor selectivity. In this work, we designed a simple benzothiazole-based turn-on fluorescence probe HBT-PBA that was modified with pyridyl disulfide as a recognition group and had good selectivity towards H2S over biothiols. Additionally, HBT-PBA was utilized for fluorescence images of the H2S in the lysosomes and endoplasmic reticulum of HeLa cells.

Development of dual-fluorophore and dual-site multifunctional fluorescent probe for detecting HClO and H2S based on rhodamine-coumarin units

HClO and H2S commonly coexist in organisms and act synergistically to maintain the redox dynamic balance in vivo; however, it is difficult to trace precisely and simultaneously HClO and H2S by single recognition-type fluorescent probes. In this paper, a novel probe (MTRN) was designed for detecting specifically HClO and H2S with dual-emission property by using an acrylate and a phenyl isothiocyanate as recognition sites. MTRN possessed outstanding performances including ultrasensitivity (LOD = 3.39 nM), high specificity and short response time (<2 min) for HClO under excitation of 530 nm with the fluorescence’s color change from dark to red. Moreover, upon treating with H2S, the fluorescence peak centered at 488 nm of MTRN enhanced obviously within 1 min and color changed from colorless to blue, the LOD was found to be 0.13 µM. The possible reaction mechanism for HClO and H2S in CH3CN/PBS (9/1, v/v) was substantiated via 1H NMR and HRMS. Above all, MTRN was successfully used to detect HClO and H2S in HeLa cells and made into test strips to detect HClO and H2S.

A Novel Fast and Sensitive Fluorescent Hydrogen Sulfide Probe with Large Stokes Shift for Imaging Living Cells and Zebrafish

As an important physiological gas transmitter, hydrogen sulfide (H2S) plays an important role in the immune system and is closely related to the occurrence of many diseases, such as cirrhosis, arterial disease, down syndrome, heart disease, and Alzheimer’s disease. In recent years, H2S probes have been reported frequently, but they still have some shortcomings, such as slow response time, small Stokes shift and insufficient sensitivity. Herein, a novel H2S probe NM–H2S was designed and synthesized. The probe NM–H2S emitted red fluorescence at 625 nm, and had a large Stokes shift (130 nm), which significantly reduces the background interference of the probe NM–H2S in biological imaging. The probe NM–H2S showed remarkable fluorescence color change, rapid response (within 10 min) and high sensitivity (0.4 μM detection limit) to the analyte H2S. Not only that, the probe NM–H2S showed good selectivity in a series of chemical analytes. In addition, the probe NM–H2S was also capable of detecting H2S in Hela cells and zebrafish by turning on fluorescence in biological applications.

BODIPY-NBD dyad for highly selective and sensitive detection of hydrogen sulfide in cells and zebrafish

Hydrogen sulfide (H2S) has been regarded as the third endogenous gas signaling molecule. The development of suitable tools for H2S detection in vitro and in vivo has always been a focus of research. In this work, three BODIPY-NBD dyads (o/m/p-BNP) were designed and synthesized using BODIPY and NBD as the fluorophore and quencher, respectively. The position of the NBD moiety in the probe showed different fluorescence quenching abilities. All probes showed highly selective to H2S. Probe o-BNP displayed the maximum fluorescence enhancement (c.a. 1300-fold) and the lowest detection limit (105 nM). Probe o-BNP can visualize the production of endogenous H2S in HeLa cells and zebrafish.

An Endoplasmic Reticulum-Targeted Ratiometric Fluorescent Probe for the Sensing of Hydrogen Sulfide in Living Cells and Zebrafish.

Hydrogen sulfide (H2S) is an endogenous gaseous signaling molecule in many physiological processes. Relevant investigations indicated that H2S plays a cytoprotective effect under endoplasmic reticulum stress. Currently, it is still a challenge to design effective methods for ratio detection of endoplasmic reticulum H2S. Herein we are the first to construct a ratiometric near-infrared fluorescent probe (M-H2S) for sensing H2S in the endoplasmic reticulum. M-H2S has high selectivity and sensitivity toward H2S (LOD = 39.1 nM). Additionally, M-H2S possessed an excellent endoplasmic reticulum targeting ability. Moreover, M-H2S was successfully utilized to visualize exogenous/endogenous H2S in HeLa cells and zebrafish. Particularly important, endogenously produced H2S was observed under endoplasmic reticulum stress induced by tunicamycin.

A new peptide-based chemosensor for selective imaging of copper ion and hydrogen sulfide in living cells

Hydrogen sulfide (H2S), like CO and NO, is an endogenous gas signal molecule that can play a vital role in many physiological processes. Therefore, it's of great significance to study the efficient and sensitive imaging technology of H2S in vivo. Copper is an essential trace element in the human body that is very important for many cellular functions and related metabolic processes. In this study we report the design of a peptide L (FITC-Ahx-His-Glu-Phe-His-NH2) that can act as an “On-Off-On” fluorescent chemical sensor to detect Cu2+ and S2−, and cysteine (Cys) and glutathione (GSH) can also replace Cu2+ from L-Cu system. The capability of this peptide fluorescent probe to detect H2S in HeLa cells is also evaluated.

Perylenequinone-based "turn on" fluorescent probe for hydrogen sulfide with high sensitivity in living cells.

Hydrogen sulfide (H2S) is a kind of gaseous signal molecule in many physiological processes. In order to detect H2S, a novel "turn on" fluorescent probe 6,12-dihydroxyperylene-1,7-dione (DPD) was designed and synthesized. The probe DPD is fluorescence silence, while the addition of H2S induces an obvious green fluorescence with an obvious color change from dark blue to yellow-green. The probe shows excellent selectivity, fast response (2.5min) and linear curve (0-90μM) in wide effective pH range (4-10). Competition experiments are also revealed in corresponding studies and the detection limit is 3.6μM. The response mechanism is proved to be the reduction of the probe by H2S, which is confirmed by 1H NMR. Furthermore, through the fluorescence turn-on signal toward H2S in Hela cells, probe DPD was successfully applied to monitor H2S in living Hela cells.

A ratiometric fluorescent probe for detection of endogenous and exogenous hydrogen sulfide in living cells

A ratiometric visualized fluorescent probe of H2S of intramolecular charge transfer (ICT) and excited intramolecular proton transfer (ESIPT) mechanisms due to solvation effects has been designed and synthesized. This chemosensor shows the distinct signal changes with dual-emission in blue and green fluorescence spectral channel (from 495 nm to 525 nm) upon the addition of H2S in a single wavelength excitation. This chemosensor exhibits the low detection limit (91 nM) and high sensitivity and selectivity. Based on this, this chemosensor was successfully used not only to monitor H2S exogenously but also used to detect and image the endogenously generated H2S in HeLa cells with excellent performance.

A Lysosome-Targetable Fluorescent Probe for Simultaneously Sensing Cys/Hcy, GSH, and H2S from Different Signal Patterns.

Biothiols, a vital branch of reactive sulfur species (RSS) family, are indispensable in human physiology. However, the exact functional roles of each biothiol involved in complicated physiological activities are still not fully clarified. A critical barrier is a lack of robust molecular tools which can simultaneously visualize different biothiols with distinct emission signals. Herein, the first lysosome-targetable fluorescent probe, Lyso-RC, which could respond to Cys/Hcy, GSH, and H2S with different sets of signal patterns was developed. Lyso-RC responds to Cys/Hcy, GSH, and H2S with the fluorescence signal patterns of blue-red, green-red, and red, respectively. Significantly, Lyso-RC is capable of discriminating lysosomal Cys/Hcy, GSH, and H2S in HeLa cells.

A near-infrared fluorescent probe based on nucleophilic substitution–cyclization for selective detection of hydrogen sulfide and bioimaging

Endogenous hydrogen sulfide (H2S) has been acknowledged as the third important gasotransmitter and plays a vital role to human health and diseases. A "turn-on" near-infrared fluorescent probe DCM-PBA based on nucleophilic substitution–cyclization has been developed for the detection of hydrogen sulfide. The probe showed high selectivity and sensitivity toward H2S and was successfully applied to imaging H2S in HeLa cells.

Two-photon fluorescent probe for hydrogen sulfide based on a red-emitting benzocoumarin dye

Hydrogen sulfide (H2S) is an endogenous gasotransmitter and plays intriguing biological roles. To study the biological role of H2S, efficient fluorescent probes are in great demand. For imaging of H2S in deep-tissue, a two-photon probe that emits in the red wavelength region is of choice to avoid the autofluorescence from intrinsic biomolecules. Here, we disclose such a probe, which, developed based on an acetyl benzocoumarin fluorophore, can be excited at 900 nm under two-photon excitation and emit in the red region. The probe shows high reactivity, selectivity, and sensitivity in in vitro assays. Two-photon microscopic imaging of H2S in HeLa cells aided by the probe demonstrates that it is potentially useful to study H2S level changes in cells and tissues influenced by external stimuli.