Mitochondria-targeted ratiometric fluorescent probe for real time monitoring of pH in living cells
Introduction
As a vital energy supplying organelle [1], mitochondria play significant roles in metabolism of cells, organs, tissues, as well as the whole living body including cell signaling via reactive oxygen species production [2], regulation of Ca2+ homeostasis [3], [4], and the triggering of cell death [5]. The microenvironment of mitochondria is closely related to its function, especially pH which can influence all biochemical processes directly. For example, under physiological conditions, mitochondria maintain an alkaline matrix (pH ∼ 8), the proton-motive potential across the inner membrane drives ATP synthesis and ion and metabolite uptake into the matrix [6]. Meanwhile, the proton motive force further acts to regulate Ca2+ homeostasis [7], [8], which in turn modulates dehydrogenase activity associated with the tricarboxylic acid (TCA) cycle [9], as well as adenine nucleotide translocase [10] and ATP synthase [11]. It is no doubt that small changes of mitochondrial pH will significantly influence its biologic functions. On the other side, production of excess reactive oxygen species (ROS) lead to mitochondrial autophagy and apoptosis which can further induce mitochondria acidification [12]. Moreover, abnormal levels of mitophagy are closely related to various pathological conditions, including cardiovascular diseases [13], neurodegenerative diseases [14], and Reye's syndrome [15]. Therefore, real-time detailed monitoring of mitochondrial pH changes is urgently necessary, which have significant effect on mitochondrial biology and associated diseases.
As an excellent detection technique, fluorescent probes have attracted increasing attention due to their high selectivity, excellent sensitivity and real-time and high spatial resolution imaging in living cells or organisms. As far as we know, there are only a few examples reported for the real-time monitoring of mitochondrial pH changes in intact cells including green fluorescent proteins (GFPs) modified with mitochondrial targeting peptides [6], [16], [17]. Compared with these protein probes, small fluorescent probes are expected to be more readily applicable for use in native cells with less interference from the organism. Early this year, Tang reported the first single wavelength near-infrared-emitting fluorescent probe for monitoring mitochondrial pH [18]. More recently, Kim reported a mitochondria-immobilized pH-sensitive off-on fluorescent probe [19]. Compared to these intensity-based fluorescence sensors, ratiometric fluorescent probes can efficiently exclude interfere with signal output by concentration, instrumental efficiency, and environmental conditions through ratiometric self-calibration of the two emission bands to allow an accurate and quantitative measurement [20]. Disappointedly, no ratiometric fluorescents probe for monitoring mitochondrial pH have been reported yet. Herein, we would like to present the first ratiometric fluorescent probe for real-time monitoring mitochondrial pH.
Previously, we described a water-soluble near-infrared probe for ratiometric sensing of SO2 based on the 7-hydroxy coumarin and TCF [21]. The phenolic hydroxyl group is a pH sensitive group: in acidic or neutral conditions, the hydroxyl group is a weak electron-donating group; however the deprotonated O− is a much stronger electron-donating group under alkaline conditions. Protonation or deprotonation of the phenolic hydroxyl group can result in the fluorescent emission spectrum blue-shift or red-shift, respectively [22], [23]. As a double membrane organelle, the membrane electrical potential (ψm) of mitochondria can reach up to −180 mv [24]. So some positively charged species can enrich in the mitochondria like Rhodamine 123, triphenylphosphonium (TPP) salts, pyridinium salts and peptides with arginine [25], [26], [27], [28], [29], [30], [31], [32], [33], [34], [35]. We also reported a mitochondria-targeted colorimetric and fluorescent probe for hypochlorite based on the pyridine moiety this year [36]. Due to the brilliant electron-withdrawing property as well as the conjugated system combined with coumarin, the pyridinium salt was chosen as a strong electron-withdrawing group as well as the mitochondria-targeting group. Herein, we proposed a pH-sensitive mitochondrial-targeted fluorescent probe CP consisting of 7-hydroxy coumarin and pyridinium moieties linked via a double bond.
Section snippets
Materials and general instruments
Mito Tracker Deep Red and LysoTracker Deep Red were purchased from Invitrogen. Mitochondrial membrane potential assay kit (TMRE, ab113852) was purchased from abcam®. Unless otherwise noted, materials were obtained from commercial suppliers and were used without further purification. All the solvents were dried according to the standard methods prior to use. All of the solvents were either HPLC or spectroscopic grade in the optical spectroscopic studies. TLC analyses were performed on silica gel
Synthesis and characterization of the fluorescent probe CP
Through the condensation of 4-methyl pyridinium with 7-hydroxy coumarin-3-carbaldehyde in EtOH with the catalyst of piperidine (Scheme 1) for 12 h, CP was obtained via filtration in 63.9% yield. The structure was confirmed by 1H NMR, 13C NMR and HRMS (see supporting information).
Optical Properties of CP
The UV-Vis absorption and fluorescent emission spectroscopy of CP (10 μM)was investigated in B–R buffer solution (40 mM acetic acid, phosphoric acid, and boric acid) at various pH values (Fig. 1). The probe displayed
Conclusion
In summary, we have presented a novel water-soluble fluorescent probe (CP) that combines the 7-hydroxy coumarin and pyridinium platforms. This probe exhibits excellent pH sensitivity and extraordinary anti-interference capability with biologically-relevant cations, anions, reactive sulfur species and oxidative-stress-associated redox chemicals. Meanwhile, with good biocompatibility, CP was further used for ratiometric fluorescence imaging in living cells with excellent mitochondrial targeting
Acknowledgment
This work was financially supported by the National Program on Key Basic Research Project of China (973 Program, 2012CB720603 and 2013CB328900), the National Science Foundation of China (No. 21232005, 21472131, J1310008 and J1103315), the Specialized Research Fund for the Doctoral Program of Higher Education in China (20120181130006) and State Key Lab of Oral Diseases (Sichuan University) (SKLODOF2014OF01). We also thank Professor Jason J. Chruma (Sichuan University) for assistance with
References (47)
- et al.
Old players in a new role: mitochondria-associated membranes, VDAC, and ryanodine receptors as contributors to calcium signal propagation from endoplasmic reticulum to the mitochondria
Cell Calcium
(2002) - et al.
Mitochondrial calcium signalling and cell death: approaches for assessing the role of mitochondrial Ca2+ uptake in apoptosis
Cell Calcium
(2006) Apoptotic pathways: the roads to ruin
Cell
(1998)- et al.
Mitochondrial cation transport systems
Methods Enzymol
(1995) Inhibition of oxidative phosphorylation by a Ca2+-induced diminution of the adenine nucleotide translocator
Biochim Biophys Acta
(1983)- et al.
Mitochondrial phospholipid hydroperoxide glutathione peroxidase suppresses apoptosis mediated by a mitochondrial death pathway
J Biol Chem
(1999) - et al.
Mitochondrial pH monitored by a new engineered green fluorescent protein mutant
J Biol Chem
(2004) - et al.
pH difference across the outer mitochondrial membrane measured with a green fluorescent protein mutant
Biochem Biophys Res Commun
(2005) - et al.
Role of an energized inner membrane in mitochondrial protein import. Delta psi drives the movement of presequences
J Biol Chem
(1991) - et al.
Mitochondrial-targeted fluorescent probes for reactive oxygen species
Curr Opin Chem Biol
(2010)
Control of mitochondrial shape
Curr Opin Cell Biol
Selective degradation of mitochondria by mitophagy
Arch Biochem Biophys
Mitochondria: a historical review
J Cell Biol
Fundamentals of Biochemistry
Measurement of cytosolic, mitochondrial, and Golgi pH in single living cells with green fluorescent proteins
Proc Natl Acad Sci U S A
Eur J Biochem
Role of calcium ions in regulation of mammalian intramitochondrial metabolism
Physiol Rev
Calcium-binding ATPase inhibitor protein of bovine heart mitochondria. Role in ATP synthesis and effect of Ca2+
Biochemistry
Mitochondria and mitophagy: the yin and yang of cell death control
Circ Res
Mitophagy: mechanisms, pathophysiological roles, and analysis
Biol Chem
Mitochondrial ultrastructure in Reye's syndrome (encephalopathy and fatty degeneration of the viscera)
N Engl J Med
A near-infrared-emitting fluorescent probe for monitoring mitochondrial pH
Chem Commun
Mitochondria-immobilized pH-sensitive off–on fluorescent probe
J Am Soc Chem
Cited by (147)
From cell membrane to mitochondria: Time-dependent AIE photosensitizer for fluorescence imaging and photodynamic anticancer therapy
2023, Sensors and Actuators B: ChemicalMolecular engineering to construct thieno[3,2-c]pyridinium based photosensitizers for mitochondrial polarity imaging and photodynamic anticancer therapy
2023, Sensors and Actuators B: Chemical