A ratiometric fluorescent sensor for pH fluctuation and its application in living cells with low dark toxicity
Graphical abstract
With a ratiometric fluorescent pH sensor, stimulated pH fluctuation has been successfully tracked in a ratiometric manner via fluorescence imaging with low dark toxicity.
Introduction
As a crucial physiological parameter, pH, an abbreviation for “power of hydrogen” initially, plays a critical role in both intracellular (pHi) and extracellular (pHe) milieu, which is influenced by diverse physiological and pathological processes. Hence quantitatively measuring pH is useful for cellular analysis or diagnosis [1]. For example, an acidic environment may be associated with inflammation or tumor [2]. Different from the acidic organelles, mitochondria display a slightly basic pH value [3], [4], and plays critical roles in many physiological activities including energy production through the respiratory chain [5], [6], cell signalling via reactive oxygen species production [7], regulation of Ca2+ homeostasis [8], [9], [10], and the triggering of cell death [11]. The unique function of mitochondria depends on the mitochondrial pH, and the physiological pHm deviation is of great significance for the understanding of mitochondria physiology although slight difference exists [12], [13]. Fortunately, this minor pHm deviation can be concealed in the case of using turn-on fluorescent sensors since the fluorescence intensity can be affected by local sensor concentration, microenvironment and imaging parameters, and so on. Therefore, the development of facile and reliable methods to monitor pHi especially the basic pHi in living cells is highly demanded in understanding the physiology and pathology of mitochondria.
Fluorescence imaging serves as an essential tool in the study of biological molecules, pathways, and processes in living cells owing to its ability in providing spatial-temporal information at microscopic, mesoscopic, and macroscopic levels [14], [15], [16], [17], [18]. So far, many organic molecular fluorescent pH sensors have been reported [19], [20], [21], [22], [23], but most of them are designed for acidic pH, while sensors for basic pH are rare and their design remains challengeable.
It is well known that the emission intensity may be influenced by many factors, such as optical path length and the illumination intensity. In addition, when turn-on mode fluorescent sensors are used in biosystem for minor pHi deviation, which can be concealed since the fluorescence intensity can be affected by local sensor concentration, microenvironment and imaging parameters, etc. A ratiometric approach can eliminate the effects of these factors and realize more effective detection via the ratio of fluorescence intensities at two different wavelengths [24], [25]. Up to now, the reported ratiometric fluorescent sensors toward basic pH are very rare [26], [27]. Therefore, it is of great interest to design ratiometric fluorescent sensors for basic pH, especially with the delicate detection range.
Compared with the nanoparticle-based ratiometric pHi sensors, small-molecule ratiometric pHi sensors are more appealing due to their simple staining procedures, good reproducibility, and a great number of alterative emission wavelengths for imaging [28], [29]. Moreover, small-molecule sensors can be more readily endowed with pH sensitivity. Given this, small-molecule sensors are among the most promising approaches to ratiometric pHi imaging and tracking [30].
As a very important kind of organic functional dyes, cyanine derivatives have many merits such as much bigger conjugate plane, cationic quaternary ammonium salt, high molar absorption coefficient, wide spectral range, and high fluorescence quantum yield. On the other hand, the spectral wavelength of these compounds can belong to near-infrared range, can effectively avoid the self-fluorescence of biological systems, and improve the sensitivity and avoid interference. Therefore, cyanine-based fluorescent chemosensors have been widely and deeply studied [31], [32], [33], [34], [35].
In this paper, we used a hemicyanine derivative as the fluorophore and synthesized a fluorescent molecule 1 (Scheme 1), for ratiometric pH sensing and imaging [36], [37]. It exhibits reversible pH sensing ability from pH 6.0 to 9.0 with a linear response from pH 7.0 to 8.8. Ratiometric pH imaging via confocal microscopy has also been effectively demonstrated.
Section snippets
Methods and materials
1H and 13C NMR spectra were recorded on a Bruker 400 NMR spectrometer. Chemical shifts are reported in parts per million using tetramethylsilane (TMS) as the internal standard. Mass spectra were obtained on a high resolution mass spectrometer (IonSpec4.7 T FTMS-MALDI/DHB).
All of the chemicals were purchased from commercial suppliers and used without further purification. All of the reactions were performed under an argon atmosphere using solvents purified by standard methods. Compound 1 was
Spectroscopic study and pH-sensing behaviour of sensor 1
Due to the good solubility of compound 1 in water, a HEPES solution was utilized as the medium to determine the absorption and emission spectra of 1 (HEPES solution, 1.0 × 10−5 M) at different pH values. As shown in Fig. 1, with the increasing of pH value from 5.0 to 9.6, the peak at 430 nm in the UV-vis spectra decreased gradually while a new band developed at 535 nm until the maxima absorbance reached pH 9.6 (Fig. 1a). Meanwhile, one apparent isosbestic point was observed at about 463 nm,
Conclusion
A colorimetric and ratiometric fluorescent organic molecular sensor with single excitation wavelength was firstly synthesized for sensing both acidic and basic pH with high sensitivity. The linear ratiometric pH response range from pH 7.0 to 8.8, the reversible pH sensing ability, and the cell membrane permeability makes this sensor especially suitable for the practical tracking of pHi fluctuation in live cells via ratiometric imaging.
Acknowledgements
We are grateful for the financial supports from National Natural Science Foundation of China (U1504203 and J1210060), Key Laboratory of Photochemical Conversion and Optoelectronic Materials and Zhengzhou University.
References (37)
- et al.
Mitochondrial dismissal in mammals, from protein degradation to mitophagy
Biochim Biophys Acta
(2014) - et al.
Switch from ER-mitochondrial to SR-mitochondrial calcium coupling during muscle differentiation
Cell Calcium
(2012) - et al.
An ultrasensitive near-infrared ratiometric fluorescent probe for imaging mitochondrial polarity in live cells and in vivo
Chem Sci
(2016) - et al.
Mitochondria-targeted ratiometric fluorescent probe for real time monitoring of pH in living cells
Biomaterials
(2015) - et al.
Highly sensitive cell imaging “Off-On” fluorescent probe for mitochondria and ATP
Biosens Bioelectron
(2015) - et al.
In vivo real-time visualization of tissue blood flow and angiogenesis using Ag2S quantum dots in the NIR-II window
Biomaterials
(2014) - et al.
Ratiometric detection of pH fluctuation in mitochondria with a new fluorescein/cyanine hybrid sensor
Chem Sci
(2015) - et al.
2-Styrylindolium based fluorescent probes visualize neurofibrillary tangles in Alzheimer's disease
Bioorg Med Chem Lett
(2012) - et al.
Acidity generated by the tumor microenvironment drives local invasion
Cancer Res
(2013) - et al.
Characterization of very acidic phagosomes in breast cancer cells and their association with invasion
J Cell Sci
(1994)
Sensors and regulators of intracellular pH
Nat Rev Mol Cell Bio
Full-range intracellular pH sensing by an aggregation-induced emission-active two-channel ratiometric fluorogen
J Am Chem Soc
The mitochondrial pathways of apoptosis
Adv Mitochondr Med
Targeting mitochondrial reactive oxygen species as novel therapy for inflammatory diseases and cancers
J Hematol Oncol
MICU1 encodes a mitochondrial EF hand protein required for Ca2+ uptake
Nature
Mitochondria-immobilized pH-sensitive off-on fluorescent probe
J Am Chem Soc
Far-red and near-IR AIE-active fluorescent organic nanoprobes with enhanced tumor-targeting efficacy: shape-specific effects
Angew Chem Int Ed
Managing diabetes with nanomedicine: challenges and opportunities
Nat Rev Drug Discov
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