A novel ‘‘donor-π-acceptor’’ type fluorescence probe for sensing pH: mechanism and application in vivo
Graphical abstract
Introduction
Intracellular pH (pHi) [1] plays many critical roles in cell, enzyme, and tissue activities, including proliferation and apoptosis [2], [3], multidrug resistance (MDR) [4], ion transport [5], endocytosis [6], and muscle contraction [7]. In diverse prokaryotic species and different subcellular compartments of eukaryotic cells, the pHi values can vary from highly acidic to basic values [8]. Abnormal pHi values can cause cardiopulmonary and neurologic problems (such as cancer and Alzheimer's) [9], [10], [11]. Hence, sensing and monitoring pHi fluctuations is essentially important for understanding physiological and pathological processes and exploring cellular metabolisms.
Fluorescent probes had attracted considerable attention over many other methods for pH detection due to its high sensitivity, noninvasiveness, and fast response time. Moreover, combining with confocal laser scanning microscopy and flow cytometry, this technique can particularly provide high spatial and temporal observation of pHi changes [11], [12]. Up to now, some pH fluorescence probes have been reported [13], [14], [15], [16], [17]. In general, there are two types of synthetic pH probes, one type for cytosol that work at pH 6.80–7.40, and another type for the acidic organelles (for example, lysosomes) functioning in the pH range 4.50–6.00 [18], [19], [20]. Unfortunately, little research is reported on the development of extreme-acidity pH probes (pH < 4.00) and extreme-alkaline (pH > 9.00) [8], [21]. Despite the fact that most of the living species could hardly live in highly acidic or alkaline conditions, a great number of microorganisms such as “acidophiles” and Helicobacter pylori particularly favour harsh environments [22], [23]. Moreover, in some eukaryotic cells, acidic and alkaline pH has important effect on organelles along the secretory and endocytic pathways [24]. For another example, enteric pathogens are acidic organelles able to reach small intestine by passing through the highly acidic mammalian stomach, resulting in life-threatening infections [21], [25]. Even for mammals, there are some parts with very low pH value, such as gastric juice, the pH level of which can also influence their physiological process remarkably [21], [26]. Because of lacking effective ways to detect such acidic pH in living species, the precise pH values in these cellular compartments remain elusive [21]. Therefore, it is necessary to develop new types of probes that can be used for efficiently accessing the intracellular pH value under both extremely acidic and alkaline conditions.
Qualitative measurements of pHi can be achieved using fluorescent indicators that switch on or off at sharply defined pH values. However, such measurements may be influenced by many factors, including optical path length, changes of temperature, altered excitation intensities, and varied emission collection efficiencies [27]. In contrast, ratiometric probes could normalize these interferences, and thus allow accurate quantitation [28], [29]. Many ratiometric pH sensors have been developed utilizing pH sensitive fluorophores along with reference dyes. For example, fluorescein derivatives have been paired with quantum dots, pyrene, and other dyes to fabricate pH sensors [30], [31], [32].
Pyrene shows photophysical properties that make it appropriate for constructing chemosensor due to its high fluorescence quantum yield, chemical stability, and long fluorescence lifetime [33], [34]. Additionally, pyrene shows monomer-excimer dual fluorescence [35], and the fluorescence intensity ratio of the excimer to monomer emission (IEx/IEM) is sensitive to conformational changes of the pyrene-functionalized system [33], [36]. Consequently, pyrene is an ideal fluorophore for designing ratiometric chemosensor. Up to date, many pyrene-based ratiometric chemosensors have been designed [31], [35], [37], [38], [39], [40], [41], [42]
In this present work, we report a novel fluorescent probe (1-(pyren-1-yl) – 3-(6-methoxypridin-3—yl)-acrylketone, PMPA) for detection pH with D–π–A by connecting pyrene as an electron donor (D) and methoxypyridine as an electron acceptor (A). The D–π–A structure type is frequently adopted as a fluorophore to construct intramolecular charge transfer (ICT) fluorescent probes displaying a large Stokes shift [43]. PMPA features a remarkably large Stokes shift, good stability and rapid response to pH, which are highly desirable for a fluorescent probe to achieve reliable and sensitive fluorescence detection. Furthermore, the probe PMPA is also successfully applied to fluorescence imaging in living cells, which indicates that the probe could noninvasively image acid and alkaline pH values fluctuations in biological systems.
Section snippets
Chemical reagents
PMPA was synthesized in our own laboratory. All reactions were magnetically stirred and monitored by thin-layer chromatography (TLC). All other reagents and solvents were purchased from Sinopharm Chemical Reagent Beijing Co., Ltd. Redistilled water was used throughout all experiments. Unless otherwise stated, all chemicals and solvents were analytical grade and used without further purification.
UV−vis spectra were recorded on a Shimadzu UV-2450 spectrophotometer. Fluorescence spectra were
Investigation of spectral properties of PMPA
The absorption and fluorescence pH titration experiments of PMPA (15.0 µM) were performed in DMSO/water (3/200, V/V). The absorption exhibited two obvious absorption bands at 330 nm (ε = 34,667 M−1 cm−1) and 410 nm (ε = 25,333 M−1 cm−1) and the absorption peaks of PMPA significantly diminished upon increasing the acidity of solution from pH 7.30 to 1.10 (Fig. 1a). However, a blue-shift of the absorption maximum from 330 nm (ε = 33,333 M−1 cm−1) to 297 nm (ε = 5333 M−1 cm−1) was observed upon increasing the
Conclusion
In this paper, we have developed a new ratiometric fluorescence probe PMPA for the selective detection of pH. The pH titrations indicated that PMPA displayed a remarkable emission enhancement with a pKa of 2.70 and responded linearly to minor pH fluctuations within the extremely acidic range of 1.26–3.97. Interestingly, PMPA also exhibited strong pH-dependent characteristics with pKa 9.32 and linear response to extreme-alkalinity range of 8.54–10.36. In addition, this probe PMPA had the
Acknowledgements
The work was supported by the National Natural Science Foundation of China (Nos. 21472118 and 21672131), the Program for the Top Young and Middle-aged Innovative Talents of Higher Learning Institutions of Shanxi (No. 2013802), Talents Support Program of Shanxi Province (No. 2014401), Shanxi Province Outstanding Youth Fund (No. 2014021002).
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