Endogenous HClO Research Articles

Rational construction of an AIE-active fluorescent probe bearing three reaction sites for individual and continuous detection of H2S and HClO with single-wavelength excitation

Endogenous H2S and HClO play vital roles in regulating the redox status in vivo, and are involved in many pathophysiological events. Accordingly, accurate sensing and imaging of intracellular H2S and HClO have become greatly in demand. However, conventional double-response fluorescent probes for these two substances suffered from the notorious aggregation-caused quenching effect and required multiple excitations to achieve discrimination determination. In this study, we firstly constructed an AIE-active fluorescent probe with three potential reaction sites for individual and continuous detection of H2S and HClO using a single excitation wavelength. The multiple-site probe exhibited distinctive fluorescence behaviors to H2S and HClO with fast response (H2S: 12 min; HClO: 1.8 min), well-separated emission peaks (88 nm), high sensitivity and good selectivity due to H2S-induced thiolysis of 2,4-dinitrophenyl ether assisted by an adjacent aldehyde and HClO-triggered sulfur oxidation. The corresponding recognition mechanisms were fully confirmed by HRMS, 1H NMR and control experiments. Moreover, the probe was successfully utilized for separate and continuous imaging of intracellular H2S and HClO in different fluorescent channels, indicating its potential for further study the biological functions of H2S and HClO in living systems.

Elevated hypochlorous acid levels in asthmatic mice were disclosed by a near-infrared fluorescence probe

Asthma is a respiratory inflammatory disease that seriously threatens human health. A growing body of evidence suggests that hypochlorous acid (HClO) plays an instrumental role in inflammation-related diseases, and therefore we hypothesize that it may be associated with asthma. Unfortunately, tracking HClO levels in asthma remains challenging due to the lack of effective measures for in vivo imaging. Herein, we exquisitely designed a near-infrared fluorescence probe dicyanomethylene-4H-pyran-dimethylthiocarbamyl (DCM-DMTC) for exploring the relationship between HClO and asthma, which has high sensitivity (about 76 times), a low limit of detection (44 nM), and great selectivity for HClO. In addition, the probe DCM-DMTC was successfully employed in tracing exogenous and endogenous HClO in living cells. Notably, the higher levels of HClO in the lungs of asthmatic mice than in normal mice were visualized by fluorescence imaging for the first time, indicating a remarkably intimate association between asthma and the overproduction of HClO.

A novel near-infrared fluorescent probe for rapid sensing of HClO in living cells and zebrafish.

Reactive oxygen species (ROS) are significant active species in living organisms, and their coordination maintains the function of organelles to resist the invasion of foreign substances. Hypochlorous acid (HClO) is not only an eventful signaling species but also a kind of ROS, which plays an irreplaceable role in the immune system. However, its abnormal levels can cause cell damage or even apoptosis, which in turn leads to the onset of a series of diseases such as inflammation, neurological diseases, and even cancer. Based on this, we designed a near-infrared fluorescent probe with a large Stokes shift for ultrafast response to HClO. Furthermore, the probe exhibits excellent sensitivity and selectivity toward HClO over other species. The probe was successfully applied to visualize endogenous and exogenous HClO in living cells and in zebrafish. This unique study is the key to providing a trustworthy tool for imaging based on the in vitro and in vivo imaging of endogenous HClO, which possesses great potential for the use in future studies of HClO-related biology and pathology.

An ESIPT fluorescent probe for ultrarapid HClO detection during reagent-stimulated oxidative stress in cells and zebrafish

As a reliable signaling biomolecule for oxidative stress, the accurate hypochlorous acid (HClO) detection during agent-stimulated oxidative stress plays a vital role in pathological and physiological mechanism exploration for disease theranostics. Therefore, the development of robust analytical tools for HClO is highly significant. Herein we presented an ESIPT fluorescent probe (MEBTA-Cl) for HClO detection. MEBTA-Cl exhibited ultrarapid-response (around 6 s), high sensitivity (42-folds) and low detection limit (7.8 nM) toward HClO titration in solution. MEBTA-Cl was capable to detect both exo-/endogenous HClO in living cells. Moreover, MEBTA-Cl was successfully employed to monitor HClO level in acetaminophen (APAP), disulfiram (DSF) and doxorubicin (DOX)-stimulated oxidative stress. Importantly, using this useful probe, it was available to detect HClO in lipopolysaccharides (LPS) and APAP treated zebrafish.

Mitochondria-Targeted Ratiometric Chemdosimeter to Detect Hypochlorite Acid for Monitoring the Drug-Damaged Liver and Kidney.

Liver and kidney injury caused by drug toxicity is a serious threat to human health. Acetaminophenol (APAP), as a common antipyretic and analgesic drug, inevitably causes injury. When it is overused, hypochlorous acid (HClO) is excessively generated due to metabolic abnormalities, resulting in the accumulation of HClO in the mitochondria of liver and kidney tissues and causing damage. In this study, we designed a series of HClO responsive ratiometric chemdosimeter NRH-X (NRH-O, NRH-S, and NRH-C) to evaluate liver and kidney injury, and found that NRH-O has a specific sensitive response to HClO. NRH-O can not only monitor the variations of endogenous HClO content of living cells by fluorescence ratio changes in the mitochondria but also detect the upregulation of HClO induced by APAP. In addition, NRH-O can also be used for anatomic diagnosis of liver and kidney injury by fluorescence ratio imaging of HClO in the tissues of inflammatory mice.

A new and fast-response two-photon fluorescent probe based on (p-Nitrophenylsulfonyl) hydrazine for detecting endogenous HClO and its application in zebrafish imaging

Hypochlorous acid (HClO), as an important reactive oxygen species (ROS), plays a vital role in life activities. When invading bacteria and pathogens, HClO can play a destructive role, which can also regulate inflammation and maintain the balance of apoptosis. Therefore, the detection of HClO in the living body is very necessary. In this paper, a novel and modified two-photon probe Cou-ClO was designed and synthesized by (p-Nitrophenylsulfonyl) hydrazine and 7-(diethylaMino)-2-oxo-2H-chroMene-3-carbaldehyde. The probe Cou-ClO exhibited fast response (30 s), good stability (60 min) high selectivity and low cytotoxicity toward HClO. The probe Cou-ClO had been successfully used for two-photon imaging to detect endogenous HClO in living cells. In particular, the penetration depth of the probe Cou-ClO for HClO two-photon imaging in mouse tissues could reach 55 µm. The probe Cou-ClO could also be used for live zebrafish imaging.

A red-emissive benzothiazole-based luminophore with ESIPT and AIE natures and its application for detecting and imaging hypochlorous acid

A new “ESIPT + AIE” based dye of benzothiazole with red emission and a large Stokes shift was constructed by combining 2-(2′-hydroxyphenyl)benzothiazole as the ESIPT unit and α-cyanostilbene as the AIE unit. The compound BACN was found to be a ideal HClO chemosensor, and presented palpable fluorescence and colorimetric responses toward HClO via the HClO-trigged oxidation cleavage of the ethylene bridge activated by the electron withdrawing cyano group. BACN was capable of recognizing HClO rapidly (12 s) and sensitively under physiological conditions, with good selectivity over other biologically pertinent substances. Thanks to strong red emission (λem = 606 nm) and large Stokes shift (213 nm) resulted from the combination of ESIPT and AIE effects, it was successfully utilized for the recognition of exogenous and endogenous HClO in living HeLa cells.

An acid-enhanced OFF-ON fluorescent probe for the detection of hypochlorous acid in rheumatoid arthritis

Excessive production of hypochlorite acid (HClO) and lactic acid are general hallmarks in the microenvironment of rheumatoid arthritis (RA). Here, we, for the first time, report an acid-enhanced “OFF-ON” fluorescent probe PPS for the detection of HClO in vivo. The probe PPS showed good water solubility, large Stokes shift (143 nm), and fast response toward HClO within 100 s. In the presence of HClO, the sulfur atom in the core of phenothiazine would be oxidized into sulfoxide, triggering intense fluorescence at 580 nm, whose fluorescence intensity could be further enhanced under acidic conditions. Moreover, the exogenous and endogenous HClO in living cells could be sensitively and selectively detected by PPS with a low LOD of 24 nM. Notably, PPS was able to rapidly visualize endogenous HClO generation in a RA mouse model, exhibiting a 2.3-fold higher fluorescence intensity than it in normal joint and 4.1-fold enhanced fluorescence intensity at acidic pH.

A novel and fast-responsive two-photon fluorescent probe with modified group for monitoring endogenous HClO accompanied by a large turn-on signal and its application in zebrafish imaging

Hypochlorous acid (HClO) plays a critical role in physiological activities of maintaining the stable oxidation balance of organisms, which was proved to relate to some serious diseases. In this work, 4-nitrobenzenesulfonylhydrazide based fast-responsive two-photon fluorescent probe CoPh-ClO was designed and synthesized reasonably, which possessed low cytotoxicity, good anti-interference characteristics, a large Stokes shift (85 nm), and good two-photon performance. In addition, probe CoPh-ClO was successfully applied to detect exogenous HClO in living HeLa cells and endogenous HClO in living RAW264.7 cells respectively. Moreover, we successfully achieved tissues imaging with a deep penetration depth of 65 µm and zebrafish imaging accompanied with a high contrast (about 45-fold). Interestingly, the introduce of benzene ring between fluorophore and reaction site made probe CoPh-ClO more sensitive (only 20 s) with a large turn-on signal. The probe CoPh-ClO was modified and possessed better stability (more than 10 mins) even in excessive HClO. All of mentioned above merits demonstrated that CoPh-ClO could be a promising imaging tool for monitoring HClO in various physiological processes, and the introduction of benzene ring would provide a new perspective for the development of multi-function probes.

An AIE based fluorescent chemosensor for ratiometric detection of hypochlorous acid and its application

Detecting and imaging intracellular hypochlorous acid (HClO) is of great importance owning to its prominent role in numerous pathological and physiological processes. In this contribution, a novel AIE-based fluorescent chemosensor has been developed by employing a benzothiazole derivative. The synthesized probe displayed remarkable ratiometric fluorescent response to HClO with a large emission shift (139 nm), resulting in naked-eye fluorescence changes from red to blue. Under the optimal conditions, this probe was capable of quantitatively detecting HClO within 10 s, and possessed good sensitivity and high selectivity toward HClO over other biologically relevant species. Moreover, it has been successfully utilized to image the exogenous and endogenous HClO in living cells through dual channels, and conveniently detect hypochlorous acid solution on test strips with better accuracy, demonstrating its potential for monitoring HClO in biological and environment fields.

Bioimaging of hypochlorous acid using a near-infrared fluorescent probe derived from rhodamine dye with a large Stokes shift

Hypochlorous acid (HClO) is a bioactive molecule that bears an important function in various physiological and pathological processes such as cell signal transduction, pathogen invasion, and redox balance regulation. The near-infrared (NIR) fluorescent probe with a large Stokes shift for bioimaging of HClO is envisioned to outperform other fluorescence probes with following advantages: greater penetration depth, smaller overlap between emission and absorption spectra, and greater reduction in false results caused by the excitation light and the scattered light, aiming to achieve selective and sensitive bioimaging of HClO in vivo. Herein, a NIR fluorescent probe, TJM, was synthesized by modifying the structure of rhodamine dye to make it water soluble and biocompatible for living cells. Under being excited at 585 nm, TJM exhibits an emission peak at 730 nm with a large Stokes shift of 145 nm. TJM also provides a fast NIR fluorescent response to HClO with excellent selectivity, high sensitivity, and low detection limit (0.11 μM). The fluorescence intensity of TJM is triggered by HClO based on HClO-induced oxidization of dibenzoyl hydrazine to dibenzoyl diimide, allowing imaging of HClO in living cells, zebrafish, and different disease mice models. TJM has been demonstrated a suitable NIR probe for imaging endogenous HClO in biological systems.

Rational design of a facile camphor-based fluorescence turn-on probe for real-time tracking of hypochlorous acid in vivo and in vitro.

A simple yet highly effective camphor-derived fluorescent probe named 3-(anthracen-9-ylmethylene)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-one oxime (ATHO) was developed based on an oxime recognition receptor and anthracene fluorophore. The probe ATHO exhibited a remarkably enhanced fluorescence response to HClO (∼7.2 fold). Meanwhile, this probe exhibited a low detection limit (0.118 μM), ultrafast response time (within seconds), excellent photostability (>260 min), wide linear range (0-180 μM), low probe concentration (2 μM), and high selectivity toward HClO over various interfering species. The sensing mechanism of the probe ATHO for HClO was supported by HRMS analysis and theoretical calculations. Furthermore, the probe ATHO was utilized to quantitatively determine HClO levels in environmental water samples. Additionally, the biological imaging of the probe ATHO for exogenous and endogenous HClO was successfully demonstrated in vitro and in vivo.

A lysosome-targetable fluorescent probe based on HClO-mediated cyclization reaction for imaging of hypochlorous acid.

Misplaced or excessive hypochlorous acid in lysosomes has a close association with lots of diseases, so monitoring hypochlorous acid in lysosomes is particularly necessary. In the present work, a novel lysosome-targetable fluorescent probe (Lyso-R-HClO) for hypochlorous acid based on a HClO-mediated cyclization reaction was developed. In the fluorescent probe, the morpholine unit and the site of a HClO-mediated cyclization reaction were, respectively, used as the lysosome-targetable group and the response group. The probe has high selectivity and high sensitivity to hypochlorous acid, with a linear range from 5.0 × 10-8 to 3.0 × 10-6M and a detection limit of 15nM; it was successfully used to image endogenous and exogenous lysosomal HClO. Finally, Lyso-R-HClO was further applied to image lysosomal HClO produced in bacteria-infected macrophage with satisfactory results, which indicate that it is an useful tool for studies of lysosomal HClO and the role of lysosome.

A specifically triggered turn-on fluorescent probe platform and its visual imaging of HClO in cells, arthritis and tumors

Understanding disease-related processes at the molecular level is of great importance for the prevention and treatment of diseases. However, due to the lack of effective analytical tools, it is challenging to gain insight into the relationships between a specific bioactive molecule and the associated disease. Herein, a rapid turn-on resorufin-based fluorescent probe platform utilizing the HClO-specific oxidative cleavage of the amide was constructed, allowing the visualization of HClO in vitro and in vivo. These probes could quickly respond to HClO (< 50 s) with high selectivity and sensitivity (12–153 nM). The probe REClO-6 had the fastest response (30 s) and the highest sensitivity (12 nM), and was successfully used for the imaging of endogenous and exogenous HClO in cells and zebrafish. Notably, it was also successfully applied to the imaging of HClO in mouse arthritis and solid tumors. This study provided a rapid imaging analysis tool, which would be used to investigate the relationship between HClO and the disease-related physiological processes.

De Novo Design of a Robust Fluorescent Probe for Basal HClO Imaging in a Mouse Parkinson's Disease Model.

Elevated HClO gets involved in the pathogenesis of Parkinson's disease (PD). Herein, a novel fluorescent probe NUU-1 was designed and synthesized. Distinct from the general strategies, NUU-1 features two distinct HClO reactive sites, a HClO-specific reaction site and a HClO-nonspecific reactive site, which in turn endows NUU-1 with the "0 + 1 > 1" amplification effect, that thus dramatically promotes the selectivity. NUU-1 displayed a fast response rate (within 15 s), remarkable fluorescence enhancement (about 538-fold), and excellent sensitivity (LOD = 25.8 nM) in response to HClO while the remaining fluorescence silence toward other common ROS (H2O2, •OH, ONOO-, O2•-, and 1O2) even at high concentrations (up to 0.5 mM). NUU-1 allows for the imaging of both exogenous and endogenous HClO in living dopaminergic cells (SH-SY5Y). Moreover, by employing NUU-1 as the probe, the image of HClO in C. elegans and zebrafish was successfully achieved. Significantly, in the first trial, NUU-1 was successfully utilized for the brain basal HClO imaging in PD mice models and distinguished PD brain tissues from normal control, thereby holding great potential for in-depth biological applications.

A Fast-Responsive OFF-ON Near-Infrared-II Fluorescent Probe for In Vivo Detection of Hypochlorous Acid in Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is a common chronic autoimmune inflammatory disease, and its etiology is closely related to the overproduction of hypochlorous acid (HClO). However, early detection of RA using an activatable near-infrared-II (NIR-II, 1000-1700 nm) fluorescent probe remains challenging. Herein, we first report an "OFF-ON" NIR-II fluorescent probe named PTA (phenothiazine triphenylamine) for imaging HClO in deep-seated early RA. Electron-rich phenothiazine in the core of PTA was utilized as both an HClO-recognition moiety and a precursor of electron acceptors, displaying a typical donor-acceptor-donor structure with excellent NIR-II emission at 936/1237 nm once reacted with HClO. The probe PTA exhibited good water solubility, high photostability, and rapid response capability toward HClO within 30 s. Moreover, it was able to sensitively and specifically detect exogenous and endogenous HClO in living cells in both visible and NIR-II windows. Notably, PTA enabled the sensitive and rapid visualization of HClO generation in an inflammatory RA mouse model, showing a 4.3-fold higher NIR-II fluorescence intensity than that in normal hindlimb joints. These results demonstrate that PTA holds great promise as a robust platform for diagnosis of HOCl-mediated inflammatory disorders.

Selective Imaging of HClO in the Liver Tissue In Vivo Using a Near-infrared Hepatocyte-specific Fluorescent Probe.

Liver injury is typified by an inflammatory response. Hypochlorous acid (HClO), an important endogenous reactive oxygen species, is regarded as a biomarker associated with liver injury. Near-infrared (NIR) fluorescent probes with the advantage of deep tissue penetrating and low auto-fluorescence interference are more suitable for bioimaging in vivo. Thus, in this work, we designed and synthesized a novel NIR hepatocyte-specific fluorescent probe named NHF. The probe NHF showed fast response (<3 s), large spectral variation, and good selectivity to trace HClO in buffer solution. By employing N-acetylgalactosamine (GalNAc) as the targeting ligand, probe NHF can be actively delivered to the liver tissue of zebrafish and mice. It is important that probe NHF is the first NIR hepatocyte-specific fluorescent probe, which successfully visualized the up-regulation of endogenous HClO in the oxygen-glucose deprivation/reperfusion (OGD/R) model HepG2 cells and dynamically monitored APAP-induced endogenous HClO in the liver tissue of zebrafish and mice.

A Sensitive and Selective Fluorescent Probe for Real-Time Detection and Imaging of Hypochlorous Acid in Living Cells.

Hypochlorous acid (HClO), a reactive oxygen species, plays an essential role in the processes of physiology and pathology via reacting with most biological molecules. The abnormal level of HClO may cause inflammation, especially arthritis. To further understand its key role in inflammation, in situ detection of HClO is necessary. Herein, a water-soluble small molecule fluorescent probe (HDI-HClO) is employed to monitor and identify trace amounts of HClO in the biological system. In the presence of HClO, the probe releases a hydroxyl group emitting strong fluorescence because of the restoration of the intramolecular charge transfer process. Furthermore, this probe displays a 150-fold fluorescence enhancement accompanied by a large Stokes shift and a lower detection limit (8.3 nM). Moreover, the probe can make a rapid response to HClO within 8 s, which provides the possibility of real-time monitoring of intracellular HClO. Based on the advantages of rapid dynamics, good water solubility, and excellent biocompatibility, this probe could effectively monitor the fluctuations of exogenous and endogenous HClO in living cells. The fluorescence imaging of HDI-HClO indicated that it is an excellent potential approach for comprehending the relationship between inflammation and HClO.

Facile and highly selective sensing of hypochlorous acid in aqueous solution and living cells by using as-prepared WSe2 quantum dots

Abstract The monitoring of biological hypochlorous acid (HClO) in living cells is vital in clarifying its physiological functions. Generally, HClO fluorescent probes that can be used for intracellular detection are small organic molecules that require complex synthesis steps. Herein, the as-prepared WSe2 quantum dots (QDs) without any further modification was verified to be a HClO fluorescent probe due to the unique chemical property of selenium. Compared with molecular probes, WSe2 QDs displayed improved performance in HClO detection, such as the low limit of detection (1.1 nM), high selectivity, fast response, good aqueous solubility, friendly operation, excellent photostability, and pH-independent response. Furthermore, the most outstanding advantage of as-prepared WSe2 QDs was their ready availability with up to the gram-large scale. WSe2 QDs were successfully used for the detection of exogenous and endogenous HClO in living cells. Meanwhile, this probe was applied to discriminate normal cells and cancerous cells on the basal of their different HClO levels. The sensing mechanism of WSe2 QDs to HClO was further carefully demonstrated and proposed to be HClO induced the oxidation and degradation of WSe2 QDs into non-fluorescent products. We anticipate that the as-prepared WSe2 QDs should have broad applications in chemical analysis and revealing the physiological function of HClO in vivo.

A POSS-assisted fluorescent probe for the rapid detection of HClO in mitochondria with a large emission wavelength in dual channels.

Hypochlorous acid (HClO) is closely related to many diseases and is an inevitable part of the physiological processes. It is significant to detect HClO in mitochondria for getting meaningful physiological and pathological information. However, adequate tools to detect HClO with emissions in two channels are rarely reported. To achieve this target, in this work, a "turn-off" visual and near infrared (NIR) fluorescent dual emission probe D6 based on polyhedral oligomeric silsesquioxanes (POSS) was successfully designed and synthesized. D6 showed high selectivity and sensitivity to HClO. Notably, the emission wavelength of D6 reached 820 nm due to the assistance of the POSS cage. In addition, bioimaging experiments clearly showed that probe D6 promoted the visualization of exogenous and endogenous HClO in living HepG2 cells and zebrafish models.