Mitochondria-targeted Ratiometric Fluorescent Probe Research Articles

Revealing the elevation of Zn2+ in the brain of depressed mice by a ratiometric fluorescent probe with dual near-infrared emissions.

A mitochondria-targeted ratiometric fluorescent probe (Mito-Zn) was first designed and synthesized with dual emissions both located in the near-infrared region, for Zn2+ detection with high sensitivity and selectivity. By using the developed Mito-Zn, a high level of Zn2+ in the depressed mouse brain was discovered for the first time.

A mitochondria-targeted ratiometric NIR fluorescent probe for simultaneously monitoring viscosity and ONOO− based on two different channels in living HepG2 cells

Mitochondrial viscosity and ONOO− are involved in various pathological and physiological processes. Abnormal viscosity or irregular ONOO− concentration would disrupt the normal function of cells. Hence, we skillfully designed and synthesized a dual-responsive probe Mito-HC-TZ for the simultaneous detection of ONOO− and viscosity by different channels. 3-ethylbenzothiazolium was acted as fluorescent rotor to regulate the twisted intramolecular charge transfer (TICT) process; the C = C of 3-ethylbenzothiazolium linked to the xanthene core could be easily broken by ONOO−. Thus, the probe responded to viscosity and ONOO− by red and orange fluorescence channels, respectively. Mito-HC-TZ had high selectivity, low cytotoxicity and wide range of pH values. The results of cell imaging experiments showed the probe could be used to respectively monitor the viscosity and ONOO− level in HepG2 cells.

A mitochondrial suitable ratiometric fluorescent probe for tracking pH change during mitophagy

Intracellular pH is one of the important parameters of cellular metabolism. The function of mitochondria is highly dependent on pH. Developing ratiometric fluorescent probes with suitable alkaline pKa for the detection of mitochondrial pH is urgently demanded. Herein, a mitochondria-targeted ratiometric pH fluorescent probe SNRF was designed based on 1,6-dihydroxynaphthalene and 4-diethylaminoketo acid. SNRF contains a hydroxyl electron donor and a nitrogen positron acceptor with excellent molecular charge transfer (ICT) performance. The hydroxyl group is a pH-sensitive group, which is prone to be protonated under acidic conditions and deprotonated under alkaline conditions. The ratiometric performance of SNRF can avoid the interference from instrumental factors, concentration of probe and microenvironment around the probe. SNRF exhibits a suitable pKa (8.30) with a linear variation between pH 7.4–9.0, making it ideal for monitoring mitochondrial pH changes. And, SNRF can be used to detect pH changes in HeLa cells and zebrafish without interference from other biologically active molecules. Furthermore, SNRF showed excellent mitochondrial targeting and was successfully used to track pH changes during rapamycin-induced mitophagy.

Engineering a mitochondria-targeted ratiometric fluorescent probe with a large Stokes shift for H2S-specific assaying in foodstuffs and living cells

Hydrogen sulfide (H2S) is a toxic gas found in foods. On-site monitoring hydrogen sulfide level in foodstuffs is desired. Hence, a mitochondria-targeted ratiometric fluorescent sensor (NBP-HS) with a large Stokes shift for H2S detection was constructed based on assembling 2-thiophenecarbonyl group and merocyanine (T-B). The fluorescence ratio (I590/I520) of NBP-HS displayed a good linear relationship related to H2S concentration (0−60 μM) with the detection limit of 5.4 × 10−8 M in HEPES buffer. The sensing mechanism was determined by HRMS analysis and DFT calculation. Interestingly, the H2S-response ratiometric probe was successfully used to on-site measure the freshness of meat by recording the color change. The analysis results of test strips were completely consistent with the analysis results of the colonies count. The probe shows an excellent response to H2S in ratiometric fluorescence in living cells, illustrating the broad applicability of NBP-HS for imaging in biosystems.

Construction of a mitochondria-targeted ratiometric fluorescent probe for monitoring hydrazine in soil samples and culture cells

Isoniazid and its major metabolite, hydrazine (N2H4), may interfere with mitochondrial function and have negative effects on cells. Consequently, an understanding of the role of N2H4 in mitochondria is highly desirable for protecting human health. Herein, we report a novel mitochondria-targeted ratiometric fluorescent probe (Mitro-N2H4) for N2H4 detection. Mitro-N2H4 exhibited an attenuation of green emission at 521 nm and an enhancement of yellow emission at 590 nm in the presence of N2H4 because of hydrazinolysis, indicating that it can be used as a ratiometric chemosensor for N2H4 with high selectivity and sensitivity. Such on-site monitoring of N2H4 vapour using test strips and N2H4-moistened soil analysis demonstrated its advantages in potential application for the convenient sensing of N2H4. Moreover, the rationally designed probe has many potential applications for imaging N2H4 produced in situ during the metabolism of isoniazid in living cells based on the ratio of the fluorescent signal.

An ICT-based fluorescent probe for ratiometric monitoring the fluctuations of peroxynitrite in mitochondria

Peroxynitrite (ONOO−) is a well-known reactive oxygen species (ROS) which closely involves in various physiological and pathological processes. It is reported that mitochondria are the major site of ONOO− production. Normal level of ONOO− benefits mitochondria biological functions, while its overproduction will induce oxidative stress, resulting in an imbalance of mitochondria homeostasis. Therefore, accurate monitoring of ONOO− in mitochondria will be beneficial for elucidating the physiological metabolism and fluctuations of ONOO−. Herein, a novel mitochondria-targeted ratiometric fluorescent probe Mito-NA for ONOO− was designed rationally by regulating its intramolecular charge transfer (ICT) effect. Once reaction with ONOO−, Mito-NA manifested remarkable ratiometric fluorescence changes (18-fold) with a large red-shift (∼104 nm) emission, which was ascribed to the enhanced ICT process caused by the elimination of the electron-withdrawing recognition group 3-(trifluoromethyl) cinnamic acid and the exposure of electron-donating hydroxyl on Mito-NA structure. Furthermore, Mito-NA exhibited satisfactory performances for ONOO− such as good sensitivity (LOD =0.12 μM), high selectivity to ONOO− over other ROS, fast response and good mitochondrial targeting. More importantly, Mito-NA was successfully applied for ratiometric imaging and monitoring the fluctuations of mitochondrial ONOO− in living cells.

A new FRET-based ratiometric fluorescent probe for the detection of SO2 derivatives in mitochondria of living cells

We developed a new mitochondria-targeted ratiometric fluorescent probe (named PPA) for SO2 derivatives (HSO3−/SO32−) with a large (pseudo) Stokes shift (209 nm) based on FRET platform in which water-soluble (E)-4-(4-(dimethylamino)styryl)-1-alkylpyridin-1-ium was chosen as acceptor moiety and phenothiazine oxide as donor moiety. Probe PPA showed specific response toward HSO3−/SO32− over H2S, GSH, Cys and Hcy. The recognition group underwent a nucleophilic addition reaction with HSO3−/SO32− to give rise to clear dual-emission signal changes in enhanced blue channel and subdued red channel under a single wavelength excitation. Moreover, probe PPA was successfully used for the detection of endogenous HSO3−/SO32− and bioimaging of mitochondria-targeted in living cells.

A mitochondria-targeting ratiometric fluorescent probe for the detection of sulfur dioxide in living cells

A mitochondria-targeting ratiometric fluorescent probe was developed for the detection of sulfur dioxide in living cells.

A novel mitochondria-targeted ratiometric fluorescent probe for endogenous sulfur dioxide derivatives as a cancer-detecting tool.

A new mitochondria-targeted fluorescent probe RBC, constructed using a coumarin moiety which was selected as the donor and a benzothiazole derivative as the acceptor, for SO2 derivatives (HSO3-/SO32-) was presented. The probe designed on a new FRET platform showed high selectivity and a low detection limit. Importantly, the probe could respond to HSO3-/SO32- within 35 s. Furthermore, the probe could target mitochondria and was successfully used for fluorescence imaging of endogenous bisulfite in HepG2 with low cytotoxicity, which significantly assisted in cancer diagnosis.

A mitochondria-targeted ratiometric fluorescent probe for endogenous cyanide in biological samples

Various fluorescence probes for cyanide anions in water solution have been reported, but the investigation for endogenous cyanide recognition in plant tissues and mammalian cells is still limited. Here, a mitochondria-targeted cyanide fluorescent probe Mito-CP with colorimetric and ratiometric response was developed. The detection for cyanide was performed via the nucleophilic addition of cyanide to carbon-carbon double bond between coumarin moiety and pyridine moiety of the probe, which resulted in a color change from red to colorless and fluorescence change from red to blue. The probe showed high sensitivity (0.17 nM) and selectivity for cyanide over other common anion species and biothiols and could detect cyanide in real water samples with quantitative detection. Probe Mito-CP was successfully applied to detect endogenous cyanide in cassava root plant tissues. In addition, colocalization experiment showed that the probe has the property of targeting mitochondria with overlap coefficient being 0.93. A549 and HeLa living cells imaging experiment demonstrated the practical value of the probe in tracing CN− in biological systems. The probe also can detect endogenous cyanide change in mitochondria of rat pheochromocytoma (PC12) cells.

Mitochondria-Targeted Ratiometric Fluorescent Probe Based on Diketopyrrolopyrrole for Detecting and Imaging of Endogenous Superoxide Anion in Vitro and in Vivo.

Intracellular reactive oxygen species is involved in a wide variety of physiological and pathological processes. In this work, we have developed a new mitochondria-targeting probe (DPP-S) for superoxide anion detection with ratiometric fluorescence response. DPP-S exhibited an obvious color change from violet to orange along with a distinct fluorescence change with maximum emission peak from 652 to 545 nm in response to superoxide anion. The limit of detection of DPP-S for superoxide anion was calculated to be 20.5 nM. Imaging studies taken in MCF-7 and RAW264.7 cells showed that DPP-S could be employed as a ratiometric fluorescent probe for endogenous superoxide anion detection and imaging in living cells with a large emission shift. Furthermore, the colocalization study indicated that DPP-S can localize in mitochondria specifically. Finally, the fluorescent probe was successfully applied for superoxide anion imaging in mice.

A Mitochondria-Targeted Ratiometric Fluorescent pH Probe.

The pH of mitochondria (pHm) is crucial in physiological processes. Here, we report a new mitochondria-targeted ratiometric pH-sensitive fluorescent probe for monitoring the mitochondrial pH change. This probe, IR-ANNA, was constructed by coupling a pH-sensitive naphthalimide derivative ANNA and a mitochondria-targeted pH-insensitive cyanine dye. IR-ANNA showed good mitochondria-targeted ability, excellent pH response, and low cytotoxicity, which makes IR-ANNA suitable for monitoring pHm in living cells. Moreover, IR-ANNA has been successfully applied for monitoring the pH change of mitochondria under the stimuli of FCCP, NAC, and H2O2 in living cells.

A colorimetric and ratiometric fluorescent probe for cyanide sensing in aqueous media and live cells.

A novel probe, 3-benzyl-2-(N-ethylcarbazole-3-vinyl)-benzothiazolium bromide (L), was synthesized and utilized for the specific detection of cyanide ions in neutral aqueous solution (HEPES buffer, pH 7.4). This probe displays a fast response through visible colorimetric and fluorogenic changes toward CN-, allowing for ratiometric sensing of cyanide in aqueous media. The ratiometric response is due to the breaking of the extensive π-conjugation and the intramolecular charge transfer (ICT) by the nucleophilic conjugation addition of cyanide to L, which is ascertained by NMR and ESI-MS analysis as well as density functional theory (DFT) calculations. The detection limit of CN- (3.39 × 10-7 M) is well below the limit recommended in the World Health Organization (WHO) guidelines for drinking water. Moreover, fluorescence co-localization studies demonstrate that L is a specific mitochondria-targeting fluorescent probe. As far as we are aware, compound L is the first mitochondria-targeting probe for cyanide detection and can be used for sensing CN- in living cells with dual channel ratiometric fluorescence imaging.

A mitochondria-targeted and FRET-based ratiometric fluorescent probe for detection of SO2 derivatives in water

A mitochondria-targeted ratiometric fluorescent probe based on the fluorophore pyrido[1,2-a]benzimidazole was developed. It showed high sensitivity and selectivity toward SO2 derivatives in water. The probe was also successfully applied to detect intrinsically generated SO2 derivatives in living glioma cells, which may be helpful in ascertaining the biological roles of SO2 in mitochondria.

A mitochondria-targeted ratiometric fluorescent probe for detection of SO2 derivatives in living cells and in vivo

A new near-infrared (NIR) fluorescent probe for colorimetric and ratiometric detection of SO2 derivatives was developed based on conjugated hybrid coumarin-hemicyanine. The probe can detect HSO3− / SO32− in HEPES buffer (10 mM, pH 7.4, with 10% DMF, v/v) with a large emission shift (259 nm). Importantly, it was successfully used for fluorescence imaging of endogenous bisulfite in BT-474 cells and zebrafish.

A FRET-based ratiometric fluorescent probe for detection of intrinsically generated SO2 derivatives in mitochondria

A mitochondria-targeted ratiometric fluorescent probe based on hemicyanine and pyrido[1,2-a]benzimidazole was presented. It shows high sensitivity and selectivity toward SO2 in pure water. The limit of detection (LOD) was as low as 26.7 nM, which is superior to most reported probes. Most importantly, the probe was successfully used for fluorescence imaging of endogenous bisulfite in mitochondria in Glioma cells.

Development of a mitochondrial-targeted ratiometric probe for the detection of SO2 in living cells and zebrafishes.

Sulfur dioxide (SO2), as the fourth gas signal molecule, is closely related to many diseases including respiratory diseases, nervous system diseases, cardiovascular diseases and cancers. Herein, we present a novel mitochondria-targeted ratiometric fluorescent probe (CS) for the detection of SO2 in living cells and zebrafishes. The probe CS was constructed on the base of coumarin and benzopyranium fluorophores, and exhibited intense blue and red fluorescence simultaneously. After the response to SO2, the fluorescence at 433 nm enhanced significantly while the fluorescence at 683 nm decreased obviously. The probe CS has a high sensitivity and selectivity to SO2. Moreover, the probe CS has been successfully applied to the detection of mitochondrial SO2 in living cells and zebrafishes.

A mitochondria-targeting ratiometric fluorescent probe for imaging hydrogen peroxide with long-wavelength emission and large Stokes shift

Hydrogen peroxide, one of the curial reactive oxygen species, is believed to involve in a wide range of physiological processes. In this work, we designed and synthesized a new fluorescent probe, HKB, for the detection of hydrogen peroxide, by taking advantage of the combination of ICT and ESIPT mechanisms. Different from other benzothiazole-based ratiometric probes, the intramolecular ESIPT process is preserved in probe HKB and the large Stokes shift is remained. Probe HKB displayed significant ratiometric fluorescence variations (I594nm/ I666nm) upon the addition of 50 equiv. of hydrogen peroxide under a single-wavelength excitation at 440 nm in PBS buffer (pH = 7.0, 10 mM, 30% CH3CN) and showed a large Stokes shift change from 102 nm to 254 nm. Moreover, this probe exhibited excellent mitochondria targeting property with a 0.94 Pearson’s colocalization coefficient using Rh123 as a reference. Gratifyingly, preliminary biological experiments indicated that probe HKB could sense both the exogenous and endogenous hydrogen peroxide in living HeLa cells.

A new mitochondria-targeted ratiometric fluorescent probe based on diketopyrrolopyrrole for imaging endogenous HOCl in living cells.

Hypochlorous acid (HOCl) is a highly effective oxidant produced during phagocytosis and plays an important role in a variety of physiological and pathological processes. In this work, a new ratiometric fluorescent probe (PTZ-TDPP) based on a diketopyrrolopyrrole (DPP) and phenothiazine platform, was firstly designed and synthesized for the selective detection of HOCl. PTZ-TDPP showed an obvious color change from purple to pink along with a distinct fluorescence change from bright red to yellow in response to HOCl. The limit of detection towards HOCl was calculated to be 190 nM. For biological applications, PTZ-TDPP is of great development potential in detecting endogenous HOCl in living RAW 264.7 cells. A co-localization study employing MitoTracker Green showed that PTZ-TDPP was specifically located in the mitochondria of RAW 264.7 cells. Therefore, PTZ-TDPP has great potential for the detection of endogenous HOCl in living cells.

An ESIPT-based mitochondria-targeted ratiometric and NIR-emitting fluorescent probe for hydrogen peroxide and its bioimaging in living cells

A mitochondria-targetable ratiometric fluorescent probe, HBTPB, has been developed for the detection of H2O2 with a near infrared (NIR) emission and a large Stokes shift (357 nm). Probe HBTPB displays high selectivity toward H2O2 over other reactive oxygen/nitrogen species (ROS/RNS), biological thiols and anions. The detection limit of this probe is as low as 5.9 × 10−7 M. The sensing process confirmed by NMR and mass spectral analysis shows that the addition of H2O2 to the solution of HBTPB triggers an oxidative hydrolysis and a subsequent elimination reaction undergoes to release an ESIPT (excited-state intramolecular proton transfer) featured fluorescent dye. Probe HBTPB is cell permeable and has been applied to sense exogenous and endogenous H2O2 in living A549 cells.