Mitochondria-targeted Fluorescent Probe Research Articles

An ultra-fast, NIR, mitochondria-targeted fluorescent probe for sulfur dioxide based on benzopyrylium and its imaging of in living cells

Near-infrared emission, is a significant influencing contributor to the detection and visualization of biomolecules and their associated target analytes by fluorescent probes in vivo. SO2 is a critical component of reactive sulfur species (RSS), playing an irreplaceable influence towards various physiological activities. Excessive inhalation of SO2 can cause irreparable respiratory damage, which also is associated with cardiovascular disease, lung cancer and neurological disorders. In this work, we have rationally developed a NIR (690 nm) fluorescence probe 1 based on benzopyrylium towards SO2. The probe 1 can ratiometricly detect SO2 in ultra-fast time (10 s) and overcome physiological interferences from long-term exposure of biothiols. The probe 1 displayed a large blue-shift of 210 nm along with the obvious fluorescence transition from red to blue. The sensing mechanism was proved to be the π-conjugated system of benzopyrylium unit in probe 1 was broken duo to the active CC double bond attacked by SO2. Fortunately, the probe 1 can serve as an effective cell-permeable molecular tool to detect and visualize dynamic equilibrium of SO2 in mitochondria of living cells.

Mitochondria-targeting turn-on fluorescent probe for HClO detection and imaging in living cells.

Hypochlorous/hypochlorite (HClO/ClO-), one of the most important signal molecule, plays a crucial role in many cellular signaling pathways. It is reported that the HClO/ClO- level in mitochondria is important to maintain the normal mitochondrial function. Herein, we present two simple fluorescent probes BAC and mitochondria-targeting fluorescent probe TACB for the detection of ClO-. Probes BAC &TACB could be sensitively and selectivity detecting ClO- at the nanomolar levels with the detection limit of 1.64×10-9M and 9.86×10-8M, respectively. Additionally, probes BAC &TACB with the response unit of CO moiety could selectively detect ClO- over other various analytes such as anions, metal ions and OH, 1O2, H2O2. The response time of probe TACB for ClO- (<20s), implying that it could offer a real-time analytical assay of ClO-. Finally, probe BAC was used for monitoring the ClO- in HEK293T cells and probe TACB could be utilized to track the fluctuations of exogenous ClO- levels in the mitochondria of Hela cells.

A new mitochondria-targeting fluorescent probe for ratiometric detection of H2O2 in live cells

With this research we presented a ratiometric and mitochondria-target fluorescent probe (Mito-HT) for detection of H2O2 both in vitro and in live cells. Mito-HT was constructed by direct conjugation of aryl boronate to fluorophore with three synthetic steps. The borate group is cleaved from Mito-HT in the presence of H2O2, resulting in the exposure of the hydroxyl group of the electron donating group. Then the ICT mechanism was turned on, and the fluorescence emission of Mito-HT at 493 nm was red-shifted to 562 nm, thereby achieving radiometric detection of H2O2. Mito-HT exhibited a highly selectivity towards H2O2, and this interaction can be completed within 40 min. Mito-HT could be used for quantitative detection of H2O2 (0–200 μM) through ratiometric fluorescence signal readout. And limit of detection (LOD) is approximately 0.33 μM. The relatively high stability and medium fluorescence quantum yield of Mito-HT (0.39) and Mito-HT-OH (0.43) enable clear mitochondria localization and dual-channel fluorescence imaging of H2O2 in live cells with confocal microscopy.

MitoCLox: A Novel Mitochondria-Targeted Fluorescent Probe for Tracing Lipid Peroxidation.

Peroxidation of cardiolipin (CL) in the inner mitochondrial membrane plays a key role in the development of various pathologies and, probably, aging. The four fatty acid tails of CL are usually polyunsaturated, which makes CL particularly sensitive to peroxidation. Peroxidation of CL is involved in the initiation of apoptosis, as well as in some other important cellular signaling chains. However, the studies of CL peroxidation are strongly limited by the lack of methods for its tracing in living cells. We have synthesized a new mitochondria-targeted fluorescent probe sensitive to lipid peroxidation (dubbed MitoCLox), where the BODIPY fluorophore, carrying a diene-containing moiety (as in the C11-BODIPY (581/591) probe), is conjugated with a triphenylphosphonium cation (TPP+) via a long flexible linker that contains two amide bonds. The oxidation of MitoCLox could be measured either as a decrease of absorbance at 588 nm or as an increase of fluorescence in the ratiometric mode at 520/590 nm (emission). In CL-containing liposomes, MitoCLox oxidation was induced by cytochrome c and developed in parallel with cardiolipin oxidation. TPP+-based mitochondria-targeted antioxidant SkQ1, in its reduced form, inhibited oxidation of MitoCLox concurrently with the peroxidation of cardiolipin. Molecular dynamic simulations of MitoCLox in a cardiolipin-containing membrane showed affinity of positively charged MitoCLox to negatively charged CL molecules; the oxidizable diene moiety of MitoCLox resided on the same depth as the cardiolipin lipid peroxides. We suggest that MitoCLox could be used for monitoring CL oxidation in vivo and, owing to its flexible linker, also serve as a platform for producing peroxidation sensors with affinity to particular lipids.

Fluorescence Imaging of Mitochondria with Three Different Sets of Signals Based on Fluorene Cation Fluorescent Probe.

Herein, we develop a novel mitochondria-targetable fluorescence probe (FVPI) based on fluorene cation derivative. This mitochondria-targetable fluorescence probe exhibits large Stokes shift in various solutions. In addition, FVPI displays numerous advantages, including high photostability, low cytotoxicity, and two-photon properties. View of the above features, FVPI is successfully capable of imaging mitochondria in biological systems with three different sets of signals. This finding will provide a new platform for the constructing mitochondria-targetable fluorescent probes with excellent optical properties.

A mitochondria-targeting highly specific fluorescent probe for fast sensing of endogenous peroxynitrite in living cells

Peroxynitrite (ONOO−), an endogenous reactive oxygen species, acts an important role in various physiological and pathological processes. Herein, we designed a mitochondria targeting fluorescent probe MBTBE for sensitive determination of ONOO−. MBTBE exhibits rapid response (< 1 min) and high selectivity towards peroxynitrite with distinct color changing (colorless to orange). The fluorescence intensity at 569 nm was linearly correlated with the concentration of ONOO− (0–10 μM) with a detection limit of 16 nM. Additionally, MBTBE possesses good biocompatibility and mitochondria-targeting. Furthermore, MBTBE was successfully applied to monitor fluctuations in ONOO-levels and H2S-induced ONOO− clearing in HeLa cells, indicating excellent potential applications in bioanalysis analysis.

Detecting and imaging of SO2 derivatives in living cells with zero cross-talk colorimetric mitochondria-targeted fluorescent probe

It is well known that excessive levels of sulfur dioxide and its derivatives are connected to diverse diseases. Therefore, developing highly sensitive probes to detect and monitor sulfite in living cells is important for the diagnosis of disease and the study of biochemical processes in vivo. In this report, two zero cross-talk ratiometric fluorescent probes were synthesized (CA-ID-MC and CA-BI-MC), which were derived from carbazole-indolenine π-conjugated system for effective detection of sulfite in living cells. Observably, CA-BI-MC exhibited the largest emission shift of 157 nm from 617 to 460 nm with the addition of various concentrations of sulfite, which is beneficial for high-resolution imaging of the sulfite. CA-BI-MC also exhibits high sensitivity and low cytotoxicity. More importantly, this probe successfully located mitochondria and sensed the sulfite in HeLa cells caused by exogenous stimulation.

Mitochondria-targeted fluorescent probes for oxidative stress imaging

The excessive accumulation of hypochlorite could cause tissue damage and a range of diseases. However, the exact role of hypochlorite in biological activity remains elusive. In many of the reported probes for hypochlorite, DMSO which is an efficient HOCl and •OH scavenger was used as solvent. Besides, no fluorescent probes have been reported to quantify the accumulation of HOCl in living cells and in vivo. In order to reach this purpose, in this study, we report two mitochondria-targeted probes (L1 and L2) for detecting ClO−. These probes which were easy to be synthesized, shown high sensitivity and fast response (within 3 min) to ClO−. Fluorescent imaging of probes in MCF-7 cells and in zebrafish indicated that L1/L2 could be used for detecting ClO− in living cells and organisms. ClO− could also be used as a dual input logic gate for L1 and L2. Probe L2 could be used to monitor oxidative stress-related biological processes in nude mice.

Visualization of Mitochondrial Viscosity in Inflammation, Fatty Liver, and Cancer Living Mice by a Robust Fluorescent Probe.

Elucidating the internal relationship between diseases and mitochondrial viscosity remains a great challenge due to the lack of the studies on multidisease living animals. So far, demonstrating abnormal mitochondria viscosity in the fatty liver and tumor living mice has not been achieved. To address this critical challenge, the powerful two-photon and mitochondria-targeted fluorescence probe CBI-V was designed herein for viscosity detection with deep red emission. Utilizing the new probe CBI-V, the mitochondrial viscosity alteration in living systems caused by monensin or nystatin has been monitored sensitively and selectively in real time. Moreover, the probe is capable of imaging mitochondrial viscosity in the inflammatory models at the cell, zebrafish, and mice level. Importantly, the visualization of mitochondrial viscosity has been achieved in both fatty liver and tumor living mice for the first time. This work not only advances the study of the relationship between disease and mitochondrial viscosity but also opens up an efficient way for diagnosing mitochondrial viscosity related diseases.

A mitochondria targetable and viscosity sensitive fluorescent probe and its applications for distinguishing cancerous cells

The determination of mitochondrial viscosity is of great importance owing to its crucial roles in the diffusion-mediated processes. It has demonstrated that the viscosity in cancer cells is higher than normal cells. Developing of viscosity sensitive fluorescent probes for distinguishing normal cells and cancer cells is necessary. Herein, we present a mitochondrial-targeting fluorescent probe PFV for the detection of viscosity in live cells. It exhibited outstanding sensitivity to viscosity, free from disturbing by ROS/sulfuret even at high concentrations. Equipped with mitochondria targeting ability, PFV was then applied to mitochondrial viscosity detection. Most importantly, by viscosity monitoring PFV successfully differentiated normal hepatic cells and cancerous hepatic cells. The present study would offer a broadly applicable for the determination of viscosity in complex systems.

A mitochondrial-targeted fluorescent probe to sense pH and HOCl in living cells

A series of rosamine dyes were designed and synthesized through one-step reaction. Among them, bifunctional fluorescent probe RosPO(OH)2 showed excellent water solubility and large molar extinction coefficient. RosPO(OH)2 underwent a two-step ionization process as the pH value of the HEPES buffer increased, accompanied a blue shift of UV absorption and an enhancement of fluorescence intensity. This means it can be used as a pH sensor. After the addition of HOCl, the fluorescence of RosPO(OH)2 was quenched and the color of the solution changed from pink to colorless which permitted "naked eye" detection. RosPO(OH)2 could be used as a sensor for detecting HOCl with high selectivity, rapid response, low detection limit (52 nM) and a wide range of pH application (pH 1–10). Colocalization results showed that RosPO(OH)2 could specifically stain in mitochondria. Due to the low toxicity and the excellent water solubility of RosPO(OH)2, it had been successfully applied to image pH and HOCl in living cells.

Design, synthesis and evaluation of a novel hepatocyte-targeting fluorescent probe to accurately detect H2S in mitochondria

A novel hepatocyte-targeting and mitochondria-targeting fluorescent probe Dye-H2S-Gal was designed and synthesized. Dye-H2S-Gal with low toxicity and short response time (within 1 min) shows good selectivity toward H2S, which is not disturbed by anions (CO32−, SO42−, HCO32−, T-BuO-), reactive oxygen and biothiols (Cys, GSH, H2O2, T-BuO-) and the representative cations (Ag+, Ba2+, Ca2+, Cd2+, Ce2+, Co2+, Cr2+, Cu2+, Eu3+, Fe2+, Fe3+, Hg2+, K+, Li+, Mg2+, Mn2+, Ni2+, NH4+, Na+). The OFF-ON switch of Dye-H2S-Gal is triggered by H2S via removing the thiophenecarboxylate group. Good mitochondria-targeting capacity of Dye-H2S-Gal is driven by the electrostatic interaction between a quaternary amine group of Dye-H2S-Gal with positive charge and the mitochondria membrane with negative charges, and its good hepatocyte-targeting capacity is driven by the specific recognition of the galactose group on the asialoglycoprotein receptor (ASGPR) overexpressed on liver cells. On the whole, Dye-H2S-Gal can precisely monitor mitochondria H2S level in liver cells and is of great significance for the early diagnosis and precise treatment of liver diseases.

A two-photon mitochondria-targeted fluorescent probe for the detection of pH fluctuation in tumor and living cells

Developing small-molecular fluorescent sensors for the detection of pH fluctuation is highly demanded yet challenging. Herein, a novel pH sensor, stilbazolium-based compound L1, was rational designed and developed. Notably, the two-photon activity responds strongly to protonation due to the increased extent of symmetrical charge transfer within molecule. Benefiting from the considerable two-photon activity and biocompatibility, it was observed that L1 could certainly sensor pH changes in living cells by using two-photon confocal microscopy and stimulated emission depletion nanoscopy methods.

Phosphinate-based mitochondria-targeted fluorescent probe for imaging and detection of endogenous superoxide in live cells and in vivo

Monitoring superoxide anion radical (O2•-) in live cells and in vivo is of great significance since O2•- is the precursor of other reactive oxygen species and has been closely associated with various physiological and pathological processes. Herein, we developed a novel mitochondria-targeted fluorescent probe PF-MitoSOX Green in which the phosphinate moiety is utilized to recognize O2•- with high sensitivity and selectivity. Confocal imaging results illustrated that PF-MitoSOX Green can not only detect intracellular O2•-, but also can conveniently visualize its production in cells and Caenorhabditis elegans. The present study illustrates that PF-MitoSOX Green provides a novel approach for imaging and detecting O2•- in live cells and in vivo.

A near-infrared and mitochondria-targeted fluorescence probe for ratiometric monitoring of sulfur dioxide derivatives in living cells.

A promising near-infrared emissive and mitochondria-targeted fluorescence probe (SNB) for the ratiometric detection of sulfur dioxide derivatives with a novel reaction mechanism was developed on the basis of FRET and the ICT platform. Probe SNB showed favorable fluorescence properties, including an active NIR emission signal (671 nm), ultra-large Stokes shift (251 nm) and an ultra-broad emission band gap (193 nm). Furthermore, SNB was applied to detect sulfur dioxide derivatives with a low detection limit (17 nM), excellent sensitivity and exceptional selectivity. Of greater significance, SNB also successfully implemented the real-time and ratiometric surveillance for exogenous or endogenous sulfur dioxide derivatives in living HeLa, L-O2 and HepG2 cells. Interestingly, SNB possessed a subcellular organelle targeting ability with a preferred co-localization coefficient of 0.91 for mitochondria, which revealed its promising potential in the detection of subcellular sulfur dioxide derivatives.

Development of a mitochondria-targeted fluorescent probe for the ratiometric visualization of sulfur dioxide in living cells and zebrafish

A new mitochondria-targeted fluorescent probe was engineered for the ratiometric visualization of sulfur dioxide in living cells and zebrafish.

Discriminating normal and inflammatory models by viscosity changes with a mitochondria-targetable fluorescent probe.

Intracellular viscosity abnormalities can lead to diabetes, neurodegenerative diseases and cancer. In this work, we developed a mitochondria-targetable fluorescent probe (EIMV) for discriminating normal and inflammatory models by viscosity changes. It was found that EIMV showed excellent properties, including high photostability, low cytotoxicity, red emission and favorable biocompatibility. In view of these unique features, this probe could successfully identify normal and cancer cells via viscosity changes. Furthermore, the EIMV probe successfully identified zebrafish with different viscosities by the same method. Moreover, EIMV exhibited different fluorescence signals in normal and inflammatory mice due to changes in viscosity. Therefore, the probe provides a new method to study the relationship between diseases and viscosity in the fields of biology and medicine.

A mitochondria-targeted fluorescent probe based on coumarin-pyridine derivatives for hypochlorite imaging in living cells and zebrafish.

Hypochlorite plays a critical role in various physiological processes and is involved in many diseases. Thus, real-time, rapid, and accurate monitoring of hypochlorite has important medical and physiological significance. Herein, a novel coumarin-pyridine derivative (CPD) probe was designed and synthesized, which exhibited fantastic advantages, such as a rapid response (within 10 s), naked eye recognition, large Stokes shift (185 nm), dual-channel detection, and high selectivity and sensitivity toward OCl- (detection limit 0.012 μM, S/N = 3). Furthermore, the current CPD probe was successfully used to image OCl- in the mitochondria of both A549 cells and zebrafish, which further demonstrated its suitability for practical applications in biological systems.

Enhanced fluorescence sensing of hypochlorous acid using serum albumin as a signal amplifier.

Hypochlorous acid (HOCl/ClO-) is well-known as a pivotal member of reactive oxygen species (ROS) and acts as a vital regulator of cell activities including cell differentiation, migration, and immune response. However, overexpressed ClO- may cause tissue damage; thus, significant efforts have been devoted to ClO- sensing at the molecular level. Herein, a mitochondria-targeting fluorescent probe for ClO- was developed based on oxidation by ClO-, releasing a green emissive luminogen under physiological conditions. In particular, a "signal amplifier", bovine serum albumin (BSA), was utilized to promote the fluorescence response, which effectively converted the probe from a "turn-off" mode to a ratiometric fluorescence system. This analytical strategy reduced the limit of detection to as low as 1.5 nM and was also applicable to live cells, accompanied by significant fluorescence change from red to green with a modified signal-to-noise ratio. Moreover, the probe exhibited good performance for targeting mitochondria and monitoring endogenous ClO-. Based on the results of this study, the well-designed probe and analytical method could be promising for intracellular ClO- detection.

Selective visualization of live-cell mitochondrial thiophenols and their induced oxidative stress process by a rationally designed rhodol-based fluorescent probe

Mitochondria as cellular powerhouses are the preferential targets affected by thiophenols, an important class of highly toxic environmental pollutants, and are linked to the production of pathogenic reactive oxygen species (ROS) induced by trace thiophenol residues. For real-time and accurate sensing, it is critically important to develop highly sensitive fluorescent probes for the specific detection of mitochondrial thiophenols. Herein, we report the first mitochondria-targeted fluorescent probe (ROAL) to image thiophenols in living cells. The development of ROAL was based on a novel red-emitting rhodol derivative (ROAP). ROAL proved to be highly selective to thiophenols among various analytes including aliphatic thiols, and renders an ultrasensitive off-on fluorescence response to thiophenols with a remarkable detection limit (8.1 nM). The probe efficiently stains mitochondria with a high Pearson’s co-localization coefficient (0.95) using Mito Tracker Green FM as reference, thereby ensuring the specific detection of mitochondrial thiophenols in living HepG2 and HeLa cells. In particular, using this probe we for the first time proved that endogenous reactive oxygen species have the capacity to eliminate thiophenols in living cells, suggesting that thiophenols might induce cellular oxidative stress.