A mitochondria-targeted fluorescence probe for ratiometric detection of endogenous hypochlorite in the living cells

doi:10.1016/j.aca.2016.11.019

Analytica Chimica Acta

Volume 950, 15 January 2017, Pages 178-183

https://doi.org/10.1016/j.aca.2016.11.019 Get rights and content

Highlights

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A colorimetric and ratiometric fluorescent probe CPBT for hypochlorite was developed.
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CPBT can target mitochondria of cells.
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CPBT was designed based on FRET mechanism and displayed larger gap between two emission peaks.
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CPBT was applied to detect exogenous and endogenous hypochlorite in living RAW264.7 cells.

Abstract

A mitochondria-targeted fluorescence probe (CPBT) for ratiometric detection of endogenous hypochlorite in the living cells was developed. CPBT could detect hypochlorite with high selectivity and sensitivity in a ratiometric manner based on FRET mechanism. In absence of hypochlorite, when CPBT was excited with absorption maximum wavelength of the donor moiety, it showed the emission of acceptor moiety because of FRET process. However, in the presence of hypochlorite, the reaction of CC double bond with hypochlorite interrupted the conjugation system resulting in the inhibition of FRET process and the emission of the donor moiety. The two well-resolved emission bands can ensure accurate detection of hypochlorite. A good linear relationship between the fluorescence intensity ratios of the two emissions and the ClO⁻ concentrations in the range from 41.8 nM (detection limit) to 12.5 μM was established. Importantly, CPBT could localize mainly in the mitochondria of RAW264.7 cells. CPBT was successfully used to fluorescence ratiometric imaging of endogenous hypochlorite in RAW264.7 cells.

Graphical abstract

Introduction

Hypochlorite (ClO⁻), one of the important reactive oxygen species (ROS), is a crucial intermediate of a wide variety of biological processes in living system. Endogenous hypochlorite is mainly produced from the reaction of chloride ion and hydrogen peroxide catalyzed by the enzyme myeloperoxidase (MPO) in leukocytes including macrophages, monocytes, and neutrophils [1], [2], [3]. And hypochlorite functions as a microbicidal agent in living organisms and plays a crucial role in the immune system [4], [5]. However, deficient or excessive level of endogenous hypochlorite is harmful to the human health [6], [7], [8], [9]. Therefore, the rapid, sensitive and selective detection of hypochlorite in biological samples is of significant interest. Fluorescent probes that can quickly respond to the change in concentration of hypochlorite could be used to study the distribution of hypochlorite in living cells [10], [11], [12], [13], [14].

As mitochondria are believed to be the main source of intracellular ROS [15], monitoring of hypochlorite in mitochondria is crucial to study effects of hypochlorite on living cells. Even though many fluorescent probes have been developed for in vitro and in vivo imaging of hypochlorite [13], [14], [16], [17], [18], only a limited number of mitochondria-targeted fluorescent probes have been reported [19], [20], [21], [22]. Nevertheless, most of the reported mitochondria-targeted fluorescent probes for hypochlorite are the “turn-on” type which may be influenced by many factors such as probe concentration, environmental conditions, instrument efficiency and photobleaching. Therefore, it is highly desirable to develop mitochondria-targeted ratiometric fluorescent probes for endogenous hypochlorite detection.

Förster resonance energy transfer (FRET), defined as an excited-state energy interaction from an excited donor to a suitable ground-state acceptor fluorophore, has been widely adopted to design fluorescent probes [23], [24], [25], [26]. For an effective FRET, two requirements are necessary: a substantial spectral overlap between the donor emission and the acceptor absorption; the distance between donor and acceptor in the range of 10–100 Å. Moreover, FRET can achieve large Stokes shifts and two well-separated emission bands with comparable intensities, which could afford a built-in correction for environmental effects. Notably, so far only a few FRET-based probes for detection of hypochlorite have been reported [27], [28], [29].

On the continuation of our previous work [30], [31], [32], [33], we develop a mitochondria-targeted fluorescence probe (CPBT) for ratiometric detection of endogenous hypochlorite in the living cells. CPBT could detect hypochlorite with high selectivity and sensitivity in a ratiometric manner based on the oxidation of hypochlorite toward CC double bond.

Section snippets

Apparatus and chemicals

Unless otherwise stated, all reagents were purchased from commercial suppliers and used without further purification. Double-distilled water was used throughout all experiments. Melting points were measured on an XD-4 digital micro-melting point apparatus. ¹H NMR and ¹³C NMR spectra were recorded on a Bruker Avance 300 MHz spectrometer. HRMS spectra were obtained on a Q-TOF6510 spectrograph (Agilent). UV–vis spectra were measured by using a Hitachi U-4100 spectrophotometer. All the pH

Design and synthesis of CPBT

It is well-known that many cationic moieties, such as triphenylphosphonium [35], [36], rhodamine [37], [38], pyridium [39], [40] or cyanine moiety [41], [42] possess a positive charge, which can easily accumulate in the mitochondria of living cells [43]. Otherwise, CC double bond can be oxidized by hypochlorite under mild conditions to various products including relatively unstable chlorinated species that can undergo further oxidation to more stable non-chlorinated species such as aldehyde and

Conclusion

We developed a ratiometric fluorescence probe based on coumarin-piperazine-styrylbenzothiazolium conjugate platform for detecting hypochlorite selectively and sensitively. The FRET-based probe displayed larger gap between two emission peaks. The probe could target mitochondria of cells and successfully applied to detect exogenous and endogenous hypochlorite in living RAW264.7 cells.

Acknowledgements

This study was supported by the Natural Science Foundation of Shandong Province (ZR2014BM004) and Major Project of Science and Technology of Shandong Province (NO. 2015ZDJS04001 and 2015ZDJS04003).

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Hypochlorous acid (HClO) is a key signal molecule involved in various physiological and pathological processes as well as the immune system. Recent study demonstrated that excess HClO in the body is closely related to diabetes since it can induce stress in the endoplasmic reticulum (ER). Unfortunately, up to now, developing a near-infrared (NIR) fluorescent probe that can detect ER HClO remains a challenge. Herein, we designed and synthesized NIR-ER-HClO, which stains ER with high selectivity, sensitivity, and hypotoxicity. The probe could accurately monitor exogenous and endogenous HClO variations in living cells. Besides, NIR-ER-HClO was successfully used to image HClO in a diabetic mice model. Compared with the control group, NIR-ER-HClO showed a stronger NIR fluorescence signal in diabetic mice and in the blood of people with diabetes. Thus, NIR-ER-HClO provides an effective method for diagnosing ER HClO in related diseases.
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Hypochlorite (ClO⁻) and antioxidants have been reported closely correlated with oxidative stress and related diseases. In recent years, many probes, especially fluorescent and colorimetric probes, have been developed to detect ClO⁻ and antioxidants. It is meaningful to develop probes which can be used to detect them in these two modes. Herein, a novel dual mode carbon dots (DM-CDs) which can rapidly respond to ClO⁻ and antioxidants by both fluorescent and colorimetric mode were synthesized. GSH was chosen as model antioxidants to evaluate its detection performance. The fluorescence of DM-CDs can be quenched by ClO⁻ and then recovered by GSH, so sensitive and accurate detection of ClO⁻ and GSH can be achieved. Meanwhile, the colour variation of this system can be observed by naked eye and detected quantitatively by UV–Vis spectra. The excellent analytical performance of DM-CDs together with their good biocompatibility endow them with great feasibility in cell imaging and real-time detection of intracellular ClO⁻ and GSH.
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Citation Excerpt :
Within this context, it is particularly important to develop sensitive and selective methods for real-time monitoring of ClO− in environmental and biological systems. Among various analytical methods, fluorescent sensor technique is undoubtedly one of the most convenient and efficient tools for the sensing and imaging of hypochlorite in vitro and in vivo owing to high sensitivity, rapid response time, simplicity of implementation and excellent real time bio-imaging [14–18]. So far, a wide variety of fluorescent probes with various reaction sites have been developed for selective detection of hypochlorite [19–23], some showed great potential in biological applications.
A novel dicyanoisophorone-based red-emissive fluorescence probe (YT) with large Stokes shift (230 nm) was synthesized for rapid (<20 s) and selective detection of hypochlorite ions in nearly 100% aqueous medium. YT responded to hypochlorite ions via the ClO⁻-promoted oxidative deprotection of thioacetal, leading to a red shift in its fluorescence maximum from 590 nm to 640 nm accompanied by naked-eye color change from orange to red. The emission response of the probe toward ClO⁻ presented a good linear relationship in the 5–160 μM concentration range, with the LOD of 4.64 μM. Further, the probe YT was successfully employed in exogenous and LPS-induced endogenous imaging of ClO⁻ in live cells and zebrafish, demonstrating its potential applications in biological science.
A tosylhydrazone-based probe for the ratiometric fluorescent detection of hypochlorite in endoplasmic reticulum of living cells
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The endoplasmic reticulum (ER) is the largest organelle in the cell and is associated with the synthesis of biological proteins, while the abnormal accumulation of HClO may damage such proteins. Herein, a relatively simple tosylhydrazone probe (1) was designed for the detection of ClO⁻ via ratiometric fluorescent signals in a semi-aqueous solution. The free probe displays a blue emission at 475 nm, while a new fluorescent band at 525 nm occurred upon the addition of ClO⁻. This ratiometric signal F₅₂₅/F₄₇₅ is highly selective and sensitive toward ClO⁻, with a limit of detection being 10.1 nM. Finally, the probe can be used for imaging exogenous and endogenous ClO⁻in ER of HeLa cells.
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A spectroscopic probe CMBT was synthesized and characterized. CMBT showed the specific recognition for HClO based on the turn-on blue fluorescence and naked-eye change from pink to colorless. NMR, IR, HRMS-ESI, and spectral analysis suggested that colorimetric and fluorescent change of CMBT to HClO originated from the conversion of CMBT to starting material coumarin-aldehyde 1 caused by the oxidization of HClO, which was responsible for the fluorescence recovery. The detection limit was calculated to be 1.61 μM and 6.58 μM for fluorescence and UV–vis analysis with a range up to 1 mM. HClO's fluorescence detection was successfully achieved in tap and river water samples. The prepared convenient paper test strips showed a distinct color change in varying concentrations of HClO. A multi-input molecular logic circuit was constructed.

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¹: Equal contribution.

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A mitochondria-targeted fluorescence probe for ratiometric detection of endogenous hypochlorite in the living cells

Highlights

Abstract

Graphical abstract

Introduction

Section snippets

Apparatus and chemicals

Design and synthesis of CPBT

Conclusion

Acknowledgements

Arch. Biochem. Biophys.

Dyes Pigments

Sens. Actuat. B

Biomaterials

Free Radic. Biol. Med.

Sens. Actuat. B

Sens. Actuat. B

Dyes Pigments

Biosens. Bioelectron.

Curr. Opin. Chem. Biol.

Sens. Actuat. B

Galloylated catechins as potent inhibitors of hypochlorous acid-induced DNA damage

Chem. Res. Toxicol.

Identification of products formed by reaction of 3',5'-Di-0-acetyl-2'-deoxyguanosine with hypochlorous acid or a myeloperoxidase-H2O2-Cl-System

Chem. Res. Toxicol.

Development of a functional ruthenium(II) complex for probing hypochlorous acid in living cells

Dalton Trans.

A ratiometric fluorescent probe for hypochlorite based on a deoximation reaction

Chem. Eur. J.

Hypochlorous acid and its pharmacological antagonism an update picture

Gen. Pharmac.

Evidence for rapid inter- and intramolecular chlorine transfer reactions of histamine and carnosine hloramines: implications for the prevention of hypochlorous-acid-mediated damage

Biochemistry

Myeloperoxidase-generated oxidants and atherosclerosis

Free Radic. Biol. Med.

Myeloperoxidase, a catalyst for lipoprotein oxidation, is expressed in human atherosclerotic lesions

J. Clin. Invest.

Fluorescent hydroxylamine derived from the fragmentation of pamam dendrimers for intracellular hypochlorite recognition

Chem. Eur. J.

An “Enhanced PET”-based fluorescent probe with ultrasensitivity for imaging basal and elesclomol-induced HClO in cancer cells

J. Am. Chem. Soc.

Recent progress in chromogenic and fluorogenic chemosensors for hypochlorous acid

Analyst

A highly specific BODIPY-Based probe localized in mitochondria for HClO maging

Analyst

Mitochondria-targeted colorimetric and fluorescent probes for hypochlorite and their applications for in vivo imaging

Chem. Commun.

Identification of products formed by reaction of 3',5'-Di-0-acetyl-2'-deoxyguanosine with hypochlorous acid or a myeloperoxidase-H₂O₂-Cl-System