Endogenous HOCl Research Articles

A Novel Phenoxazine‐based Fluorescent Probe for the Detection of HOCl in Living Cells

Hypochlorous acid (HOCl), one of the reactive oxygen species (ROS), is a key microbicidal agent which is used for natural defense. However, it is also linked to varieties of human diseases owing to the overproduction of HOCl. Much effort has been made to exploit selective fluorescent sensors for the detection of HOCl, but most of them have some disadvantages such as short excitation wavelength, low selectivity, and slow response and so on. These restrict the biological application of the probes. In this work, BR‐O was designed and synthesized on the base of phenoxazine for the detection of HOCl. BR‐O exhibited a violent fluorescence enhancement in the presence of HOCl, showing excellent selectivity and high sensitivity. More importantly, the probe BR‐O was capable of detecting exogenous and endogenous HOCl in living cells.

A two-photon fluorescent probe for turn-on monitoring HOCl level in endoplasmic reticulum

To understand the relationship between endoplasmic reticulum (ER) stress and the level of hypochlorous acid (HOCl), it is highly desired to develop an ER-targetable HOCl probe to monitoring the HOCl level. In this study, a new turn-on type of two-photon ER-targetable fluorescent probe (ER-NPA) is designed for fluorescent sensing and imaging HOCl in ER using 4-hydroxy-1,8-naphthalimide as the fluorophore, p-aminophenyl ether as the recognition receptor, and methyl sulfonamide as ER-targetable group. The probe ER-NPA exhibits some intrinsic features such as excellent water solubility, good biocompatibility, significantly fast and enhanced fluorescence response to HOCl within 60 s, and detection limit as low as 6.2 nM for HOCl determination. The probe ER-NPA is successfully applied to real-time monitoring of exogenous and endogenous HOCl fluctuations in HeLa cells and zebrafish during ER stress. The developed probe ER-NPA may provide a new strategy to reveal the chemical biology roles of HOCl in ER stress-related diseases.

The near-infrared fluorescent probes based on phenoxazine for the rapid detection of hypochlorous acid

Hypochlorous acid (HOCl) is tightly related with a series of diseases, and plays an important part in biological processes. Herein, we designed and synthesized two “turn-on” near-infrared (NIR) probes (BC-2 and BC-3) based on phenoxazine to sense and image HOCl both in vitro and in vivo. In the presence of HOCl, the fluorophore phenoxazine was released and the fluorescence intensity significantly increased. The probes show excellent selectivity, high sensitivity, and rapid response towards HOCl. Moreover, the probe BC-3 is capable of rapidly detecting endogenous HOCl in living cells and in the inflammation area from the mouse models of arthritis.

A general strategy for selective detection of hypochlorous acid based on triazolopyridine formation

Triazolopyridines are an important kind of fused-ring compounds. A HOCl-promoted triazolopyridine formation strategy is reported here for the first time in which hypochlorous acid (HOCl) mildly and efficiently promotes the formation of 1,2,4-triazolo[4,3-a]pyridines NT1-NT6 from various 2-pyridylhydrazones N1-N6. N6, a rhodol-pyridylhydrazone hybrid, was developed into a fluorescent probe for the selective detection of HOCl, and successfully applied to probe endogenous HOCl in living cells and zebrafish in situ and in real time. The present intramolecular cyclization reaction is selective and atom-economical, thereby not only providing an important approach for the convenient synthesis of triazolopyridines, but also offering a general strategy for sensitive, selective and biocompatible detection of endogenous HOCl in complex biosystems.

Selective Detection and Visualization of Exogenous/endogenous Hypochlorous Acid in Living Cells using a BODIPY-based Red-emitting Fluorescent Probe.

Herein, a red-emitting fluorescent probe DM-BDP-OCl containing a para-DMTC benzyl pyridinium moiety at the meso position of BODIPY as self-immolative portion for the detection of HOCl was designed and synthesized. DM-BDP-OCl exhibited excellent specificity and a fast response for HOCl beyond other ROS/RNS. It was used for the accurately measurable detection of HOCl with a linear range from 0 μM to 50 μM, and the detection limit for HOCl reached 60 nM. Moreover, the probe could directly monitor fluctuations of exogenous and endogenous HOCl in living HeLa and RAW 264.7 cells. This work provided a powerful and convenient imaging tool for probing pathological and physiological actions of HOCl.

A ratiometric two-photon fluorescence probe for monitoring mitochondrial HOCl produced during the traumatic brain injury process

Mitochondrial hypochlorous acid (HOCl) is closely related to the redox balance in mitochondria. Abnormal HOCl levels in mitochondria can induce mitochondrial inactivation and further cause cell apoptosis. Consequently, developing a reliable method for detecting HOCl in mitochondria is essential. A mitochondria-targeting two-photon fluorescence probe (Mito-P-OCl) with zero cross-talk ratiometric emission was synthesized and applied to study the traumatic brain injury process in mice. The probe gives a considerable variation (up to 58.5-fold) in its fluorescence intensity ratio (I595/I453) upon reacting with HOCl for 150 s. Mito-P-OCl exhibited excellent properties such as fast response, high sensitivity with a low detection limit of 55.4 nM, high selectivity and deep tissue penetration (270 μm). Moreover, the probe possesses the capability of monitoring endogenous HOCl in living cells and tissues using dual-emission channels and two-photon microscopy.

A simple lysosome-targeted probe for detection of hypochlorous acid in living cells

Since HOCl plays an indispensable role in a variety of physiological and pathological processes, monitoring the level of hypochlorous acid (HOCl) in living cells is critical. Based on this, it is necessary to develop a convenient and simple technique to detect HOCl in living cells. In this contribution, a simple 2-hydroxy-6-morpholino-1H-benzo[de]isoquinoline-1,3(2 H)-dione (NDS) was developed as fluorescent probe for monitoring HOCl in live cells. Probe NDS exhibited preeminent specificity towards HOCl over other various bioactive substances. In addition, the probe could respond to HOCl in solutions within 16 s and the limit of detection was as low as 105.24 nM. NDS has good lysosomal targeting and has been successfully applied to the expression of exogenous and endogenous lysosomal HOCl in HeLa cells. This study provides a promising tool for studying physiological or pathological related processes.

HKOCl-4: a rhodol-based yellow fluorescent probe for the detection of hypochlorous acid in living cells and tissues

Highly sensitive and selective yellow probes, HKOCl-4 and its derivatives, have been developed for detecting endogenous HOCl in cytosol and mitochondria of living cells. In addition, visualization of HOCl production in ischemic stroke model has been achieved with HKOCl-4r.

Development of iridium(III) phosphorescent probe for hypochlorous acid detection in macrophages cells and cancer cells co-culture system and application in inflamed mouse model

Macrophages infiltrated in tumor microenvironment (TME) were found to play vital roles in immune responses and tumor development. In the process of immune, macrophages could produce pro-inflammatory mediators including ROS. Thus, targeting macrophages related ROS and distinguishing macrophages and cancer cells in one or more types of tumor and tumor microenvironments is extremely important and necessary to better understand tumor-associated macrophages (TAMs) functions and provide new insights into macrophage-centred therapeutic. Herein, we developed two unique Ir(III) probes, Ir1 and Ir2, for HOCl detection. Because of the dual protection of C^N ligand, Ir2 exhibits high sensitivity and selectivity in the present of HOCl. Therefore, Ir2 can be employed to detect HOCl in flow cytometry and image exogenous and endogenous HOCl in mitochondria. Importantly, Ir2 was applied to distinguish macrophages cells and cancer cells in TAMs by flow cytometry. Moreover, Ir2 is applicable in staining five kinds of organs of mouse tissues and monitoring HOCl generation in the LPS-stimulated inflammation of mouse model with two-photon microscopy (TPM).

Highly Selective Detection of Hypochlorous Acid by a Bis-heteroleptic Ru(II) Complex of Pyridyl-1,2,3-triazole Ligand via C(sp2)-H Hydroxylation.

A Ru(II) complex (Ru-1) of a substituted pyridyl-1,2,3-triazole ligand (BtPT) for highly selective "light-up" detection of hypochlorous acid is presented. An unusual anti-Markovnikov HOCl addition to the C═C bond of 1,2,3-triazole and a highly specific C(sp2)-H hydroxylation over epoxidation made Ru-1 a highly selective luminescent HOCl probe. The abnormal regio- and stereoselective HOCl addition and subsequent hydroxylation mechanism in detail is supported by the combination of ESI-MS, 1H/13C NMR spectroscopy, and 1H NMR titration. The hydroxylation at the C5 center in 1,2,3-triazole increases the electron density and makes BtPT a better σ-donor as well as π-donor, which in turn increases the 3MC-3MLCT energy gap and inhibits the nonradiative decay from the excited state of Ru-1 and is the key reason for luminescence light-up. Most importantly, the exogenous and endogenous HOCl imaging in the living HEK293T cells is also demonstrated. The probe showed low cytotoxicity and efficiently permeated the cell membrane. The cell-imaging experiments revealed rapid staining of the extranuclear region of HEK293T cells which clearly indicates the presence of cytoplasmic HOCl. The endogenous HOCl generation and imaging, stimulated by lipopolysaccharides (LPS) and paraquat in the HEK293T cells, is also demonstrated.

Development of a Concise Rhodamine-Formylhydrazine Type Fluorescent Probe for Highly Specific and Ultrasensitive Tracing of Basal HOCl in Live Cells and Zebrafish.

Hypochlorous acid (HOCl) has received special attention by virtue of its pivotal antimicrobial nature, and the appropriate amount of HOCl is beneficial to innate immunity of host to cope with microbial invasion. However, the uncontrollable accumulation of HOCl is implicated in many human diseases and even cancers. Thus, to determine its deeper biological functions, it is significantly important to specifically monitor intracellular HOCl in biosystems. Herein, we rationally designed a simple fluorescent probe FH-HA on the basis of the formylhydrazine recognition receptor and rhodamine B fluorophore. It is worth noting that the formylhydrazine moiety for the first time is adopted as the recognition receptor for specifically recognizing HOCl. Additionally, probe FH-HA also exhibited excellent performance in many areas including satisfactory water-solubility, high specificity, and excellent sensitivity. Notably, probe FH-HA could quickly respond to HOCl (within 3 s), which facilitates the tracing of transient HOCl. More importantly, probe FH-HA was capable of specifically tracing the fluctuations of endogenous HOCl in living cells and zebrafish, and it could monitor basal HOCl in cancer cells to distinguish cancer cells from normal ones.

A new ratiometric fluorescent probe for sensing lysosomal HOCl based on fluorescence resonance energy transfer strategy.

In this paper, ratiometric imaging of lysosomal HOCl was realized with a molecular probe (CR-Ly) based on fluorescence resonance energy transfer by using coumarin as the donor and rhodamine as acceptor. CR-Ly showed high sensitivity and fast response to HOCl. Moreover, CR-Ly exhibited excellent selectivity and sensitivity for HOCl over other biologically relevant species. Furthermore, it was successfully utilized to image the endogenous HOCl with low cytotoxity. And CR-Ly was capable of targeting lysosomes and monitoring lysosomal hypochlorous acid changes owing to the presence of the morpholine moiety. We believe that probe CR-Ly would be helpful to further research on the HOCl-associated diseases in lysosomes.

A simple sensor based on 1,8-naphthalimide with large Stokes shift for detection of hypochlorous acid in living cells.

Hypochlorous acid (HOCl), one of the most reactive and deleterious reactive oxygen species (ROS), plays a vital role in many pathological and physiological processes. However, as a result of the highly reactive and diffusible nature of HOCl, its uncontrolled production may lead to an adverse effect on host physiology. Because of its biological importance, many efforts have been focused on developing selective fluorescent probes to image HOCl. However, it is still challenging to design a fluorescent probe with exclusive selectivity towards HOCl. In this study, a novel fluorescent probe for HOCl, Probe 1 was rationally designed based on 1,8-naphthalimide. As the concentration of HOCl increased, the fluorescence intensity of the probe gradually decreased, and the solution color changed from yellow-green to colorless, indicating this is a “naked-eye sensor”. Probe 1 has a large Stokes shift (120 nm), which can effectively avoid fluorescence self-absorption. In addition, Probe 1 shows excellent selectivity to HOCl among different ions including common ROS, high sensitivity, fast response (<2 min), high fluorescence quantum yield (Φ = 0.93) and low detection limit (0.237 μM). Finally, the imaging results in HeLa cells showed that the probe could be used for the detection of exogenous and endogenous HOCl, and proved the potential of the probe as a biosensor for the detection of HOCl.

A highly specific and ultrasensitive probe for the imaging of inflammation-induced endogenous hypochlorous acid.

A turn-on two-photon fluorescent probe HCA-Green for hypochlorous acid (HOCl) was synthesized using 4-methylamino-1,8-naphthalimide (MNA) as a two-photon fluorophore and p-hydroxyaniline as a leaving-recognition domain. Both the probe and the fluorophore were investigated under one- and two-photon excitation modes. The fluorescence intensity of the probe was enhanced by ∼229-fold and ∼193-fold under one-photon and two-photon excitation, respectively, after reacting with HOCl. A maximal two-photon action cross-section of 50 GM was obtained under excitation at 810 nm. The probe exhibited high sensitivity with a detection limit of 42.3 nM, as well as high selectivity, low cytotoxicity, and good photostability. Two-photon microscopy (TPM) was conducted to visualize HOCl levels in living cells and tissues. The production of endogenous HOCl induced by lipopolysaccharide-mediated inflammation was successfully monitored with this probe.

A far-red to NIR emitting ultra-sensitive probe for the detection of endogenous HOCl in zebrafish and the RAW 264.7 cell line.

Fluorescence imaging is a dynamic tool for monitoring the functions of biomolecules in living systems. Probes with emission in the far-red to near-infrared range have been found to demonstrate great application prospects in bioimaging due to their deep tissue penetration, low background fluorescence, minimum photodamage to the tissue and high sensitivity. The present study aimed to construct a far-red to near-infrared emitting probe (PI) bearing dicyanoisophorone coupled with a phenothiazine moiety. The probe exhibits an instant response towards HOCl with a colour change from red to yellow with a 100 fold fluorescence enhancement at 620 nm with a low detection limit (42 nM). The electron-rich sulfur atom can be oxidised by HOCl; this endows the probe with distinct selectivity over other anions as well as other reactive oxygen species. The probe demonstrates great cell permeability with low toxicity to the cell. Therefore, the probe can be effectively applied in biological systems to monitor the endogenous level of HOCl produced by PMA in living systems and in the fluorescence imaging of endogenous HOCl in zebrafish and the RAW 264.7 cell line.

A mitochondria-targeted near-infrared fluorescent probe with a large Stokes shift for real-time detection of hypochlorous acid.

Hypochlorous acid (HOCl) has been known to be intertwined with various pathophysiological processes. In this paper, a novel mitochondria-targeted near-infrared (NIR) fluorescent probe L based on a chromenylium-phenothiazine conjugate has been designed and synthesized for the detection of hypochlorous acid. Due to the HOCl-promoted intramolecular charge transfer processes, the probe L shows near-infrared fluorescence emission at 672 nm, large Stokes shifts of 97 nm, fast response, and high selectivity and sensitivity. Furthermore, the probe L is biocompatible, cell-membrane permeable and mitochondria-targetable, and can be used for endogenous HOCl imaging in living cells.

Rational Design of a Hepatoma-Specific Fluorescent Probe for HOCl and Its Bioimaging Applications in Living HepG2 Cells.

Liver cancer is a kind of high mortality cancer due to the difficulty of early diagnosis. And according to the reports, the concentration of reactive oxygen species (ROS) was higher in cancer cells than normal cells. Therefore, developing an effective fluorescent probe for hepatoma-selective imaging of hypochlorous acid (HOCl) which is one of the vital ROS is of great importance for understanding the role of HOCl in liver cancer pathogenesis. However, the cell-selective fluorescent probe still remains a difficult task among current reports. Herein, a galactose-appended naphthalimide (Gal-NPA) with p-aminophenylether as a new receptor and galactose moiety as hepatoma targeting unit was synthesized and employed to detect endogenous HOCl in living HepG2 cells. This probe was proved to possess good water solubility and could respond specifically to HOCl. In addition, probe Gal-NPA could completely react to HOCl within 3 s meanwhile accompanied by tremendous fluorescence enhancement. The quantitative linear range between fluorescence intensities and the HOCl concentrations was 0 to 1 μM (detection limit = 0.46 nM). More importantly, fluorescence confocal imaging experiments showed that probe Gal-NPA could discriminate hepatoma cells over other cancer cells and simultaneously trace endogenous HOCl levels in living HepG2 cells. And we thus anticipate that probe Gal-NPA has the potential application for revealing the functions of HOCl in hepatoma cells.

A smart approach toward rhodamine spiro-ring derivatives sensing platform for lysosome-targetable imaging applications

As acidic organelles (pH 4.0–5.5) in living cells, lysosomes play key roles in numerous cellular biological processes, and associate with diverse diseases. Small molecular probes based fluorescence imaging techniques capable of providing information on target analytes in lysosomes are highly appreciated. Herein, for the first time we propose a new strategy of constructing pH-independent rhodamine based spiro-ring platform for lysosomal imaging applications. As a proof of concept, by incorporating lysosome specific moiety morpholine, a thio-lactone rhodamine analogue HN4MS for HOCl with high sensitivity, high selectivity and fast response at lysosomal pH value was developed. Moreover, possessing ability of imaging in lysosome organelles, the new probe was successfully applied to visualize exogenous and endogenous HOCl in living cells and tissues, which verified the present avenue for expansion of organelle targetable imaging tools.

Endoplasmic Reticulum-Targeted Ratiometric N-Heterocyclic Carbene Borane Probe for Two-Photon Microscopic Imaging of Hypochlorous Acid.

The naphthoimidazolium borane 4 is shown to be a selective probe for HOCl over other reactive oxygen species. Unlike other boronate-reactive oxygen species (ROS) fluorogenic probes that are oxidized by HOCl through a nucleophilic borono-Dakin oxidation mechanism, probe 4 is distinguished by its electrophilic oxidation mechanism involving B-H bond cleavage. Two-photon microscopy experiments in living cells and tissues with the probe 4 demonstrate the monitoring of endogenous HOCl generation and changes in HOCl concentrations generated in the endoplasmic reticulum during oxidative stress situations.

Synthesis of an ultrasensitive BODIPY-derived fluorescent probe for detecting HOCl in live cells.

Hypochlorous acid (HOCl) is a critical member of the reactive oxygen species (ROS) produced by immune cells to fight infections. On the other hand, HOCl in homeostasis causes oxidative damage to biomolecules and is linked to many diseases, including inflammatory, neurodegenerative, and cardiovascular diseases. Herein, we detail a procedure for the preparation of a boron-dipyrromethene (BODIPY)-derived fluorescent probe for HOCl (BClO) and its application as an imaging reagent in living cells. BClO is synthesized in one pot through a four-step procedure that is nearly the same as that for conventional BODIPY dye preparation, except for the ratio of starting materials. BClO has an extremely rapid response (saturated within seconds) and is ultrasensitive to HOCl. The detection limit of BClO reaches the subnanomolar range, which is the highest HOCl sensitivity to date. Taking advantage of the ultrasensitive character of BClO, we have previously demonstrated its ability to detect endogenous HOCl generated by macrophages and shown that it can also be used to discriminate cancer cell lines (which show high HOCl production) from non-cancer cell lines (which show low HOCl production). The protocol requires ~2 d for probe synthesis and up to ~18 h for fluorescence imaging and flow cytometry assays.