Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 151 (2015) 218–224
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A water-soluble pH fluorescence probe based on quaternary ammonium salt for bioanalytical applications Xuan-Xuan Zhao a, Di Ge b,c, Xi Dai a, Wen-Li Wu a, Jun-Ying Miao b,⇑, Bao-Xiang Zhao a,⇑ a
Institute of Organic Chemistry, School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100, PR China Institute of Developmental Biology, School of Life Science, Shandong University, Jinan 250100, PR China c School of Biological Science and Technology, University of Jinan, Jinan 250022, PR China b
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
A novel pH probe based on
quaternary ammonium salt soluble in water was developed. The charge-induced effect can avoid the ‘‘alkalizing effect’’ and improve the sensitivity. The probe responds linearly to pH 4.6–5.8 and is reversible between pH 4.2–7.2. The probe with excellent anti-interference capability was well used for monitoring the pH fluctuations in lysosome.
a r t i c l e
i n f o
Article history: Received 28 March 2015 Received in revised form 15 May 2015 Accepted 28 June 2015 Available online 29 June 2015 Keywords: Fluorescence probe H+ Quaternary ammonium salt Alkalizing effect Lysosome
a b s t r a c t A novel fluorescence probe Rhodamine–Ethanediamine–Iodomethane (REI) was successfully prepared to serve as an efficient sensing platform for H+ with fully reversibility mainly between the pH 4.2 and 7.2 in simple buffer solution. The introduction of quaternary ammonium salt with positive charge can not only manage to increase the solubility and sensitivity of probe REI, but also avoid the ‘‘alkalizing effect’’ due to charge-induced effect compared to the reference probe Rhodamine–Ethanediamine (RE). In particular, probe REI was well used for monitoring the weak acid pH fluctuations in lysosome of the live HeLa cells due to its excellent biological properties, including low cytotoxicity, high selectivity, good sensitivity and membrane permeability. Ó 2015 Elsevier B.V. All rights reserved.
1. Introduction On the one hand, intracellular H+ plays a pivotal role in many cellular events, including cell growth [1], apoptosis, endocytosis [2,3], homeostasis, ion transport [4], cell adhesion [5], and other cellular processes [6–9], on the other hand, the disorder of the H+ within different organelles may lead to the dysfunction of the ⇑ Corresponding authors. E-mail addresses: [email protected] (D. Ge), [email protected] (J.-Y. Miao), [email protected] (B.-X. Zhao). http://dx.doi.org/10.1016/j.saa.2015.06.111 1386-1425/Ó 2015 Elsevier B.V. All rights reserved.
organization and even to a diseased state [10–16], which have made the detection of intracellular pH be particularly important. In the past decades, quite a few fluorescence probes have been developed for various bio-related targets based on target-triggered fluorescence intensity changes [17–25] due to their high sensitivity, fast analysis with spatial resolution for providing in situ and real-time information and nondestructive sample preparation. Moreover, fluorescence microscopic imaging technique can map the spatial and temporal distribution of analytes within living cells [26–32].
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Currently, lots of novel fluorescence probes were reported based on the philosophy that the combination between the proton and the lone pair electrons within nitrogen, which could change the optical properties of fluorescent probes [33–38], besides that, pH-induced ring-opening reaction of the rhodamine is broadly used by varying the conjugated system [39–54]. Even though lots of rhodamine-based fluorescence probes have been widely applied in both solution and biosensing systems to discriminate the analytes from other biological abundant species so far, poor solubility of these probes in biological environments might result in decreased detection sensitivity, or even lose the response signal, which is a research bottleneck as regards progress in some areas of cell biology or medicine [55–58]. To increase the water solubility of the probes, several water soluble groups have also been introduced for intracellular pH measurements such as 1,2,3-triazole, hydroxyl and other methods [59–62]. To our curiosity, the positive charge in the probe may be not only beneficial to the solubility of the fluorescence probe, but also conductive to the sensitivity of it. Given that, we reported REI as a novel pH platform for both water samples and bioanalytical applications (Scheme 1). Comparing with the reference RE, probe REI showed a faster response, higher selectivity, and significant increase of fluorescence intensity. The probe is reversible fluorescence response to pH over the pH range of 4.0–7.4 in buffer solution. Moreover, the larger pKa of probe REI (5.2) compared to RE (5.0) made it possible to detect the intramolecular pH. Since the quaternary ammonium salt within probe REI could lead to the charge-induced effect and avoid of ‘‘alkalizing effect’’ [63], our design philosophy might find wide applications in designing of water-soluble probes for the detection of various bio-related targets. 2. Experimental 2.1. Materials and equipment for the synthesis and optical analysis of REI All reagents and solvents were purchased from commercial sources and used without further purification. All solvents used in spectroscopic analysis are spectroscopic grade. Deionized water was employed throughout the process of absorption and fluorescence determination. Britton–Robinson (B–R) buffer was mixed by 40 mM acetic acid, boric acid, and phosphoric acid. Dilute hydrochloric acid and sodium hydroxide were used for tuning pH values. The solutions of metal ions were obtained from nitrate salts which were dissolved in deionized water. All samples were prepared at room temperature, shaken for 10 s and stood for 15 min before measurement. LysoSensorTM Green DND-189 was used as lysosome tracker (Invitrogen, America). Melting points were determined on an XD-4 digital micro melting point apparatus. 1H NMR and 13C NMR spectra were recorded on a Bruker Avance 300 spectrometer using d6-DMSO as solvent and tetramethylsilane (TMS) as internal standard. IR spectra were recorded with an IR spectrophotometer VERTEX 70 FT-IR (Bruker Optics). High-resolution mass spectrometry (HRMS) spectra were recorded on a Q-TOF6510 spectrograph (Agilent). Fluorescent measurements were performed on
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a Perkin Elmer LS-55 luminescence spectrophotometer and UV– vis spectra were carried out on a U-4100 UV–vis-NIR Spectrometer (Hitachi). The pH values were measured by a PHS-3C digital pH-meter (YouKe, Shanghai, China). The images were obtained using confocal fluorescence microscopy (LSM700) with eye piece 10 magnification and objective 20 magnification. Light absorption was measured using a SpectraMAX190 microplate spectrophotometer (GMI Co, USA). The software ImageJ was used for acquiring the fluorescence values and the data were analyzed by software SPSS 17.0. 2.2. Synthesis of 2-(30 ,60 -bis(diethylamino)-3-oxospiro[isoindoline1,90 -xanthen]-2-yl)-N,N,N-trimethylethanaminium (REI) Sodium hydride (60%) (80 mg, 2 mmol) was added to a solution of RE (484 mg, 1 mmol) in acetonitrile (MeCN, 30 mL) at room temperature. Thereafter a solution of iodomethane in MeCN (30 mL) was added dropwise in 5 min then the mixture was stirred at room temperature for 5 h until the reaction was finished by TLC monitoring (MeOH). After removal of the solvent, 20 mL water was added and extracted with ethyl acetate (3 portions of 30 mL). The combined organic layer was washed with water, brine, and dried over anhydrous Na2SO4. After filtration, the solvent was evaporated and the crude product was recrystallized from PE/EA (1:1, v/v, 20 mL) to give the target compound REI as pink powder in 98.7% yield (521 mg). mp: 193–195 °C. IR (KBr), t: 3443, 3082, 2970, 2928, 1691, 1614, 1515, 1470, 1382, 1267, 1224, 1118, 821, 791 cm1; 1 H NMR (d6-DMSO, 300 MHz), d (ppm): 7.79–7.85 (1H, m, ArH), 7.50–7.59 (2H, m, ArH), 7.02–7.08 (1H, m, ArH), 6.35–6.42 (6H, m, ArH), 3.43 (2H, t, J = 7.20 Hz, NCH2CH2N+), 3.34 (8H, q, J = 7.05 Hz, CH2CH3), 3.05 (2H, t, J = 7.20 Hz, NCH2CH2N+), 2.93 (9H, s, N(CH3)3), 1.09 (12H, t, J = 7.05 Hz, CH2CH3); 13C NMR (d6-DMSO, 75 MHz), d (ppm):167.71, 153.85, 153.26, 149.10, 133.72, 129.97, 129.03, 128.68, 124.19, 122.92, 108.88, 104.57, 97.91, 64.97, 63.35, 52.69, 44.19, 34.03, 12.86; HRMS: calcd for [M]+ C33H43N4O+2, 527.3386, found: 527.3339. 2.3. Fluorescence imaging of HeLa cells with probe REI HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (HyClone) at 37 °C in a humidified incubator containing 5% CO2 in air. The cells were passaged every two days at a ratio 1:5. 2 104 mL1 HeLa cells were seeded on uncoated glass bottomed dishes (U = 2 mm) for 24 h. After removing the medium, the cells were rinsed twice with 1 PBS (pH = 7.2) and then treated with different concentrations (0.5, 1, 2.5, 5 lM) of probe. Subsequently the cells were rinsed twice with 1 PBS and fluorescence images were captured by confocal fluorescence microscope at different time intervals. 2.4. Co-localization of the probe with lysosome in HeLa cells HeLa cells were incubated with the probe (5 lM) for 1 h and then washed twice with 1 PBS and loaded with 1 lM lysosome tracker (LysoSensorTM Green DND-189) for 1 h. Then the cells were
Scheme 1. The synthesis of probe REI.
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rinsed three times with 1 PBS. Finally the images were obtained by confocal fluorescence microscope. 2.5. Imaging of pH changes within the lysosome 2 104 mL1 HeLa cells were seeded on uncoated glass bottomed dishes (U = 2 mm) for 24 h and then pre-incubated with different concentrations of Chloroquine (CQ) for 6 h and then treated with REI (5 lM) for 1 h or REI (2.5 lM) for 2 h. Finally the cells were rinsed twice with 1 PBS and images were captured by confocal fluorescence microscope. 2.6. Cytotoxicity assay As previously reported, HeLa cells were seeded onto 96-well plates, then treated with 0.1% DMSO (as control) or the probe at 5 lM for 24 h. Cell viability assay was performed by sulforhodamine B (SRB) protocol. 3. Results and discussion 3.1. Synthetic scheme In our newly designed pH probe REI (Scheme 1), a rhodamine unit was designed as a fluorophore, because its spiro structure with some excellent optical properties is sensitive to environmental pH. Moreover, it was optimized in a salt form because the positive charge may not only help to increase the water solubility of the probe, but also promote the spiro-opening under the acid condition, which could efficiently increase the sensitivity of REI to pH, including response time, increased multiple of fluorescence intensity and the liner relationship. Additionally, the pH-sensitivity and the appropriate pKa made it possible for probe REI to detect the intramolecular pH. The synthetic route of probe REI is shown in Scheme 1. REI was first synthesized and characterized using NMR, IR and HRMS analytical spectroscopic techniques, which agreed well with the proposed structure. 3.2. UV–vis and fluorescence sensing performance of probe REI The UV–vis absorption spectra of REI (10 lM) were investigated at pH values of 4.0–7.4 (Figure S1, ESI). Under neutral condition, probe REI showed almost no absorption in the range of 500– 600 nm, which indicated that the rhodamine cyclolactam in probe
REI was the dominant species. With decreasing of pH from 6.0 to 4.0, the absorbance centered at 560 nm increased significantly due to the ring-opening of the rhodamine cyclolactam. Accordingly, the distinct color changes of the solution from colorless to pink could serve probe REI as a ‘‘naked-eye’’ indicator for acidic pH (Figure S1). Consistently, the fluorescence titration of probe REI (1 lM) was performed (Fig. 1a). The probe was non-fluorescence (pH > 6.0), however, decreasing pH values gave rise to both the increase of fluorescence intensity at 580 nm (I580nm) and a discernable fluorescence color change concomitantly within a few seconds, which should be due to the ring-opening induced by the increase of the H+. What’s worth mentioning is that with a large increase in the emission intensity (kem = 580 nm, Fig. 1b) within the pH range of 6.0–4.0, REI is the most sensitive pH probe to date (U4.0 = 0.27, rhodamine B in EtOH as the standard [64]). Moreover, the inset of Fig. 1b also demonstrated a good linearity (R2 = 0.98905) between fluorescent intensity (I580nm) of REI (1 lM) and pH ranging from 4.6 to 5.8. The analysis of fluorescence intensity changes as a function of pH using the Henderson–Hasselbalch equation yielded a pKa value of 5.2 (Figure S2, ESI), which implied that probe REI could serve as a functional pH probe for weak acidic organelles in vivo like lysosome. To achieve potential application in biosystems, response to the H+ selectively over other potentially competing species coexisting in the sample is quite essential for a fluorescence probe. Therefore, the interference experiments of probe REI were performed to estimate the influence caused by essential metal ions, environmental or biological abundant species both in acidic and neutral conditions, which led almost no appreciable changes in relative fluorescence intensity of REI, even upon the addition of significantly higher competing species (Figure S3, ESI). These results corroborated that probe REI, as a highly efficient probe, could be practically applied for the detection of pH even with the potential interferences coexisting from environments. The response time of REI to different H+ concentrations was tested by measuring the fluorescent response about 30 min at room temperature (Fig. 2a). Probe REI could instantly respond to the change of H+ (pH = 4.2) once they were placed in the buffer solution, and the rapid increase of fluorescence intensity was observed within seconds and peaked in about 2 min then remained stable, which indicated that the spirocyclic part of the rhodamine structure was sensitive to such H+ concentration. Meanwhile, pH 5.2 showed smaller fluorescence changes and no significant
Fig. 1. (a) Change of the fluorescence spectra of REI (1 lM) in aqueous solution containing DMSO (B–R buffer: DMSO = 95: 5, v/v) with decreasing pH from 7.4 to 4.0 (kex = 560 nm). (b) Sigmoidal fitting of the pH-dependent fluorescence intensity at 580 nm. Inset: the good linearity in the pH range of 4.6–5.8.
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changes were observed at pH 7.2. It should be noticed that the stable fluorescence platform at acid condition supported the deduction that probe REI reached dissociation equilibrium and was insusceptible to the medium, light and air. Interestingly, even though the reference RE could respond to H+ under the same condition (pH = 4.2), it needed longer response time (about 5 times of REI) to reach dissociation equilibrium and the final intensity was weaker than that of REI (Fig. 2b). Hence, it could be speculated that the quaternary ammonium salt played important roles in the structural change from the spirocyclic (non-fluorescent) to the ring-opening (fluorescent) forms in response to H+ with the charge-induced effect, which also helped to avoid the ‘‘alkalizing effect’’ and improve the sensitivity of probe REI. Excellent reversibility of pH chemosensor is particularly encouraging in practical application. So, the solution pH was circularly modulated between 4.2 and 7.2 for several times and the fluorescence intensities of REI were recorded subsequently (Figure S4,
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ESI). The results showed that after the solution was prepared alternately into an acidic solution of pH = 4.2, approximately 91% of the original fluorescence signal was successfully restored after 6 cycles. Contrastly, spirocyclic structure of the probe was associated with quenching of the fluorescence under neutral solution (pH = 7.2). A good reversibility of the probe toward pH enabled us to propose a mechanism for the equilibrium of probe REI between pH = 4.2 and pH = 7.2 (Scheme 1). A possible interaction between probe REI and H+ is also suitable for the similar phenomenon of other pH probes with the same functional fluorophore as probe REI [39,59,65,66]. 3.3. Application study of bio-imagine Since the lumenal environment of lysosomes was at a pH range from 4.6 to 5.0, the cell imaging assay was performed to investigate the application of the probe in sensing H+ in biological systems.
Fig. 2. (a) Time courses of fluorescence intensity of REI at various pH values. (b) The comparison of the time response of probe REI and RE at the pH of 4.2. (kem = 580 nm).
Fig. 3. Fluorescence microscope images of living HeLa cells with different concentrations of REI for 0.5, 1, 2 h at 37 °C. ⁄P < 0.05; ⁄⁄P < 0.01, n = 3. Images are representative of 3 independent experiments.
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After incubated with different concentrations of REI for 0.5–2 h, the fluorescence microscope images of the processed cells were obtained by confocal fluorescence microscopy (Fig. 3). The different fluorescence intensity changes not only demonstrated the time and dose-depended characteristics of probe REI, but also implied its excellent membrane-permeable.
To further determine the subcellular distribution of probe REI, the LysosensorTM Green DND-189, an excellent lysosome tracker, was used to stain the cells with probe REI. The strong green signals were collected after HeLa cells incubated with lysosome tracker (1 lM) for 1 h (Fig. 4b). In contrast, the fluorescence of the cells incubated with REI (5 lM) was observed in the red channel
Fig. 4. Confocal fluorescence imaging of living HeLa cells co-stained with REI (5 lM) and LysoSensorÒGreen (1 lM) for 1 h. (a) Red emission from REI; (b) Green emission from LysoSensorÒGreen; (c) Overlay of (a) and (b). Areas of co-localization appeared in yellow; (d) The correlation of LysoSensorÒGreen and REI intensities. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 5. Fluorescence microscope imaging of living HeLa cells pretreated with Chloroquine for 6 h, and then treated with (A, B) 5 lM REI for 1 h; (C, D) 2.5 lM REI for 2 h.
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(Fig. 4a). Fortunately, the merged image (Fig. 4c) of red and green channels exhibited distinct pH dependent patterns with yellow fluorescence. The overlap coefficient (0.77) also suggested the probe REI could specifically label lysosomes in other cells. Monitoring pH changes in lysosome efficiently has great significance since pH can affect endocytosis, exocytosis, autophagy and other cellular processes [3,6–9,67]. For one thing, the weakly basic substances can cause an increase of lysosomal pH by leaking protons out of lysosomes, for another, the concentration of chloroquine required to cause such effect is considerably lower than that of other weak bases [67]. In this study, the fluorescence intensities within the cells were gradually weakened accompanied with increased concentration of chloroquine from 0 to 20 lM, which exhibited the well performance of REI as an indicator in the monitoring pH changes of lysosomal in living cells with faster response and more significant results compared with other rhodamine-based pH fluorescence probes [39,65] (Fig. 5). The excellent results suggested that REI was favorable for real-time tracking of the pH in both real solutions and biological samples compared with other pH probes (Table S1). SRB assays protocol revealed that the cell viabilities of HeLa cells were not affected by incubated with 5 lM REI for 24 h at the experimental conditions (Figure S5, ESI). 4. Conclusion In summary, a novel pH fluorescence cassette REI based on the introduction of quaternary ammonium salt was successfully developed to serve both in simple buffer solution and complex cell media applications. The introduction of the positive charge can not only improve the solubility in water and reactivity of probe REI but also avoid the ‘‘alkalizing effect’’ with respect to the reference probe RE. Moreover, probe REI, with fully reversibility mainly within the pH range from 4.2 to 7.2, could also be applied for monitoring pH changes at the range of pH from 4.6–5.8 with satisfying results. In particular, probe REI was well used for monitoring the weak acid pH fluctuations in lysosome of the live HeLa cells due to its excellent biological properties. Since numerous rhodamine-based small molecular probes have been developed to recognize a wide range of targets, our strategy provides a new sensing platform for wide applications in biological samples and other fields. Acknowledgments This study was supported by the Natural Science Foundation of Shandong Province (ZR2014BM004). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.saa.2015.06.111. References [1] R. Martinez-Zaguilan, B.F. Chinnock, S. Wald-Hopkins, M. Bernas, D. Way, M. Weinand, M.H. Witte, R.J. Gillies, Cell. Physiol. Biochem. 6 (1996) 169–184. [2] J.K. Laihia, J.P. Kallio, P. Taimen, H. Kujari, V.M. Kähäri, L. Leino, J. Invest. Dermatol. 130 (2010) 2431–2439. [3] C. Nilsson, K. Kågedal, U. Johansson, K. Öllinger, Cancer Treat. Rev. 25 (2004) 185–194. [4] K.R. Hoyt, I.J. Reynolds, J. Neurochem. 71 (1998) 1051–1058. [5] Y. Shimizu, S.W. Hont III, Immunol. Today 17 (1996) 565–573. [6] H. Izumi, T. Torigoe, H. Ishiguchi, H. Uramoto, Y. Yoshida, M. Tanabe, T. Ise, T. Murakami, T. Yoshida, M. Nomoto, Cancer Treat. Rev. 29 (2003) 541–549. [7] M. Chesler, Physiol. Rev. 83 (2003) 1183–1221. [8] A.M. Paradiso, R.Y. Tsien, T.E. Machen, Nature 325 (1987) 447–450. [9] I. Yuli, A. Oplatka, Science 235 (1987) 340–342.
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Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
A water-soluble pH fluorescence probe based on quaternary ammonium salt for bioanalytical applications Xuan-Xuan Zhao a, Di Ge b,c, Xi Dai a, Wen-Li Wu a, Jun-Ying Miao b,⇑, Bao-Xiang Zhao a,⇑ a
Institute of Organic Chemistry, School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100, PR China Institute of Developmental Biology, School of Life Science, Shandong University, Jinan 250100, PR China c School of Biological Science and Technology, University of Jinan, Jinan 250022, PR China b
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
A novel pH probe based on
quaternary ammonium salt soluble in water was developed. The charge-induced effect can avoid the ‘‘alkalizing effect’’ and improve the sensitivity. The probe responds linearly to pH 4.6–5.8 and is reversible between pH 4.2–7.2. The probe with excellent anti-interference capability was well used for monitoring the pH fluctuations in lysosome.
a r t i c l e
i n f o
Article history: Received 28 March 2015 Received in revised form 15 May 2015 Accepted 28 June 2015 Available online 29 June 2015 Keywords: Fluorescence probe H+ Quaternary ammonium salt Alkalizing effect Lysosome
a b s t r a c t A novel fluorescence probe Rhodamine–Ethanediamine–Iodomethane (REI) was successfully prepared to serve as an efficient sensing platform for H+ with fully reversibility mainly between the pH 4.2 and 7.2 in simple buffer solution. The introduction of quaternary ammonium salt with positive charge can not only manage to increase the solubility and sensitivity of probe REI, but also avoid the ‘‘alkalizing effect’’ due to charge-induced effect compared to the reference probe Rhodamine–Ethanediamine (RE). In particular, probe REI was well used for monitoring the weak acid pH fluctuations in lysosome of the live HeLa cells due to its excellent biological properties, including low cytotoxicity, high selectivity, good sensitivity and membrane permeability. Ó 2015 Elsevier B.V. All rights reserved.
1. Introduction On the one hand, intracellular H+ plays a pivotal role in many cellular events, including cell growth [1], apoptosis, endocytosis [2,3], homeostasis, ion transport [4], cell adhesion [5], and other cellular processes [6–9], on the other hand, the disorder of the H+ within different organelles may lead to the dysfunction of the ⇑ Corresponding authors. E-mail addresses: [email protected] (D. Ge), [email protected] (J.-Y. Miao), [email protected] (B.-X. Zhao). http://dx.doi.org/10.1016/j.saa.2015.06.111 1386-1425/Ó 2015 Elsevier B.V. All rights reserved.
organization and even to a diseased state [10–16], which have made the detection of intracellular pH be particularly important. In the past decades, quite a few fluorescence probes have been developed for various bio-related targets based on target-triggered fluorescence intensity changes [17–25] due to their high sensitivity, fast analysis with spatial resolution for providing in situ and real-time information and nondestructive sample preparation. Moreover, fluorescence microscopic imaging technique can map the spatial and temporal distribution of analytes within living cells [26–32].
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Currently, lots of novel fluorescence probes were reported based on the philosophy that the combination between the proton and the lone pair electrons within nitrogen, which could change the optical properties of fluorescent probes [33–38], besides that, pH-induced ring-opening reaction of the rhodamine is broadly used by varying the conjugated system [39–54]. Even though lots of rhodamine-based fluorescence probes have been widely applied in both solution and biosensing systems to discriminate the analytes from other biological abundant species so far, poor solubility of these probes in biological environments might result in decreased detection sensitivity, or even lose the response signal, which is a research bottleneck as regards progress in some areas of cell biology or medicine [55–58]. To increase the water solubility of the probes, several water soluble groups have also been introduced for intracellular pH measurements such as 1,2,3-triazole, hydroxyl and other methods [59–62]. To our curiosity, the positive charge in the probe may be not only beneficial to the solubility of the fluorescence probe, but also conductive to the sensitivity of it. Given that, we reported REI as a novel pH platform for both water samples and bioanalytical applications (Scheme 1). Comparing with the reference RE, probe REI showed a faster response, higher selectivity, and significant increase of fluorescence intensity. The probe is reversible fluorescence response to pH over the pH range of 4.0–7.4 in buffer solution. Moreover, the larger pKa of probe REI (5.2) compared to RE (5.0) made it possible to detect the intramolecular pH. Since the quaternary ammonium salt within probe REI could lead to the charge-induced effect and avoid of ‘‘alkalizing effect’’ [63], our design philosophy might find wide applications in designing of water-soluble probes for the detection of various bio-related targets. 2. Experimental 2.1. Materials and equipment for the synthesis and optical analysis of REI All reagents and solvents were purchased from commercial sources and used without further purification. All solvents used in spectroscopic analysis are spectroscopic grade. Deionized water was employed throughout the process of absorption and fluorescence determination. Britton–Robinson (B–R) buffer was mixed by 40 mM acetic acid, boric acid, and phosphoric acid. Dilute hydrochloric acid and sodium hydroxide were used for tuning pH values. The solutions of metal ions were obtained from nitrate salts which were dissolved in deionized water. All samples were prepared at room temperature, shaken for 10 s and stood for 15 min before measurement. LysoSensorTM Green DND-189 was used as lysosome tracker (Invitrogen, America). Melting points were determined on an XD-4 digital micro melting point apparatus. 1H NMR and 13C NMR spectra were recorded on a Bruker Avance 300 spectrometer using d6-DMSO as solvent and tetramethylsilane (TMS) as internal standard. IR spectra were recorded with an IR spectrophotometer VERTEX 70 FT-IR (Bruker Optics). High-resolution mass spectrometry (HRMS) spectra were recorded on a Q-TOF6510 spectrograph (Agilent). Fluorescent measurements were performed on
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a Perkin Elmer LS-55 luminescence spectrophotometer and UV– vis spectra were carried out on a U-4100 UV–vis-NIR Spectrometer (Hitachi). The pH values were measured by a PHS-3C digital pH-meter (YouKe, Shanghai, China). The images were obtained using confocal fluorescence microscopy (LSM700) with eye piece 10 magnification and objective 20 magnification. Light absorption was measured using a SpectraMAX190 microplate spectrophotometer (GMI Co, USA). The software ImageJ was used for acquiring the fluorescence values and the data were analyzed by software SPSS 17.0. 2.2. Synthesis of 2-(30 ,60 -bis(diethylamino)-3-oxospiro[isoindoline1,90 -xanthen]-2-yl)-N,N,N-trimethylethanaminium (REI) Sodium hydride (60%) (80 mg, 2 mmol) was added to a solution of RE (484 mg, 1 mmol) in acetonitrile (MeCN, 30 mL) at room temperature. Thereafter a solution of iodomethane in MeCN (30 mL) was added dropwise in 5 min then the mixture was stirred at room temperature for 5 h until the reaction was finished by TLC monitoring (MeOH). After removal of the solvent, 20 mL water was added and extracted with ethyl acetate (3 portions of 30 mL). The combined organic layer was washed with water, brine, and dried over anhydrous Na2SO4. After filtration, the solvent was evaporated and the crude product was recrystallized from PE/EA (1:1, v/v, 20 mL) to give the target compound REI as pink powder in 98.7% yield (521 mg). mp: 193–195 °C. IR (KBr), t: 3443, 3082, 2970, 2928, 1691, 1614, 1515, 1470, 1382, 1267, 1224, 1118, 821, 791 cm1; 1 H NMR (d6-DMSO, 300 MHz), d (ppm): 7.79–7.85 (1H, m, ArH), 7.50–7.59 (2H, m, ArH), 7.02–7.08 (1H, m, ArH), 6.35–6.42 (6H, m, ArH), 3.43 (2H, t, J = 7.20 Hz, NCH2CH2N+), 3.34 (8H, q, J = 7.05 Hz, CH2CH3), 3.05 (2H, t, J = 7.20 Hz, NCH2CH2N+), 2.93 (9H, s, N(CH3)3), 1.09 (12H, t, J = 7.05 Hz, CH2CH3); 13C NMR (d6-DMSO, 75 MHz), d (ppm):167.71, 153.85, 153.26, 149.10, 133.72, 129.97, 129.03, 128.68, 124.19, 122.92, 108.88, 104.57, 97.91, 64.97, 63.35, 52.69, 44.19, 34.03, 12.86; HRMS: calcd for [M]+ C33H43N4O+2, 527.3386, found: 527.3339. 2.3. Fluorescence imaging of HeLa cells with probe REI HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (HyClone) at 37 °C in a humidified incubator containing 5% CO2 in air. The cells were passaged every two days at a ratio 1:5. 2 104 mL1 HeLa cells were seeded on uncoated glass bottomed dishes (U = 2 mm) for 24 h. After removing the medium, the cells were rinsed twice with 1 PBS (pH = 7.2) and then treated with different concentrations (0.5, 1, 2.5, 5 lM) of probe. Subsequently the cells were rinsed twice with 1 PBS and fluorescence images were captured by confocal fluorescence microscope at different time intervals. 2.4. Co-localization of the probe with lysosome in HeLa cells HeLa cells were incubated with the probe (5 lM) for 1 h and then washed twice with 1 PBS and loaded with 1 lM lysosome tracker (LysoSensorTM Green DND-189) for 1 h. Then the cells were
Scheme 1. The synthesis of probe REI.
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rinsed three times with 1 PBS. Finally the images were obtained by confocal fluorescence microscope. 2.5. Imaging of pH changes within the lysosome 2 104 mL1 HeLa cells were seeded on uncoated glass bottomed dishes (U = 2 mm) for 24 h and then pre-incubated with different concentrations of Chloroquine (CQ) for 6 h and then treated with REI (5 lM) for 1 h or REI (2.5 lM) for 2 h. Finally the cells were rinsed twice with 1 PBS and images were captured by confocal fluorescence microscope. 2.6. Cytotoxicity assay As previously reported, HeLa cells were seeded onto 96-well plates, then treated with 0.1% DMSO (as control) or the probe at 5 lM for 24 h. Cell viability assay was performed by sulforhodamine B (SRB) protocol. 3. Results and discussion 3.1. Synthetic scheme In our newly designed pH probe REI (Scheme 1), a rhodamine unit was designed as a fluorophore, because its spiro structure with some excellent optical properties is sensitive to environmental pH. Moreover, it was optimized in a salt form because the positive charge may not only help to increase the water solubility of the probe, but also promote the spiro-opening under the acid condition, which could efficiently increase the sensitivity of REI to pH, including response time, increased multiple of fluorescence intensity and the liner relationship. Additionally, the pH-sensitivity and the appropriate pKa made it possible for probe REI to detect the intramolecular pH. The synthetic route of probe REI is shown in Scheme 1. REI was first synthesized and characterized using NMR, IR and HRMS analytical spectroscopic techniques, which agreed well with the proposed structure. 3.2. UV–vis and fluorescence sensing performance of probe REI The UV–vis absorption spectra of REI (10 lM) were investigated at pH values of 4.0–7.4 (Figure S1, ESI). Under neutral condition, probe REI showed almost no absorption in the range of 500– 600 nm, which indicated that the rhodamine cyclolactam in probe
REI was the dominant species. With decreasing of pH from 6.0 to 4.0, the absorbance centered at 560 nm increased significantly due to the ring-opening of the rhodamine cyclolactam. Accordingly, the distinct color changes of the solution from colorless to pink could serve probe REI as a ‘‘naked-eye’’ indicator for acidic pH (Figure S1). Consistently, the fluorescence titration of probe REI (1 lM) was performed (Fig. 1a). The probe was non-fluorescence (pH > 6.0), however, decreasing pH values gave rise to both the increase of fluorescence intensity at 580 nm (I580nm) and a discernable fluorescence color change concomitantly within a few seconds, which should be due to the ring-opening induced by the increase of the H+. What’s worth mentioning is that with a large increase in the emission intensity (kem = 580 nm, Fig. 1b) within the pH range of 6.0–4.0, REI is the most sensitive pH probe to date (U4.0 = 0.27, rhodamine B in EtOH as the standard [64]). Moreover, the inset of Fig. 1b also demonstrated a good linearity (R2 = 0.98905) between fluorescent intensity (I580nm) of REI (1 lM) and pH ranging from 4.6 to 5.8. The analysis of fluorescence intensity changes as a function of pH using the Henderson–Hasselbalch equation yielded a pKa value of 5.2 (Figure S2, ESI), which implied that probe REI could serve as a functional pH probe for weak acidic organelles in vivo like lysosome. To achieve potential application in biosystems, response to the H+ selectively over other potentially competing species coexisting in the sample is quite essential for a fluorescence probe. Therefore, the interference experiments of probe REI were performed to estimate the influence caused by essential metal ions, environmental or biological abundant species both in acidic and neutral conditions, which led almost no appreciable changes in relative fluorescence intensity of REI, even upon the addition of significantly higher competing species (Figure S3, ESI). These results corroborated that probe REI, as a highly efficient probe, could be practically applied for the detection of pH even with the potential interferences coexisting from environments. The response time of REI to different H+ concentrations was tested by measuring the fluorescent response about 30 min at room temperature (Fig. 2a). Probe REI could instantly respond to the change of H+ (pH = 4.2) once they were placed in the buffer solution, and the rapid increase of fluorescence intensity was observed within seconds and peaked in about 2 min then remained stable, which indicated that the spirocyclic part of the rhodamine structure was sensitive to such H+ concentration. Meanwhile, pH 5.2 showed smaller fluorescence changes and no significant
Fig. 1. (a) Change of the fluorescence spectra of REI (1 lM) in aqueous solution containing DMSO (B–R buffer: DMSO = 95: 5, v/v) with decreasing pH from 7.4 to 4.0 (kex = 560 nm). (b) Sigmoidal fitting of the pH-dependent fluorescence intensity at 580 nm. Inset: the good linearity in the pH range of 4.6–5.8.
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changes were observed at pH 7.2. It should be noticed that the stable fluorescence platform at acid condition supported the deduction that probe REI reached dissociation equilibrium and was insusceptible to the medium, light and air. Interestingly, even though the reference RE could respond to H+ under the same condition (pH = 4.2), it needed longer response time (about 5 times of REI) to reach dissociation equilibrium and the final intensity was weaker than that of REI (Fig. 2b). Hence, it could be speculated that the quaternary ammonium salt played important roles in the structural change from the spirocyclic (non-fluorescent) to the ring-opening (fluorescent) forms in response to H+ with the charge-induced effect, which also helped to avoid the ‘‘alkalizing effect’’ and improve the sensitivity of probe REI. Excellent reversibility of pH chemosensor is particularly encouraging in practical application. So, the solution pH was circularly modulated between 4.2 and 7.2 for several times and the fluorescence intensities of REI were recorded subsequently (Figure S4,
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ESI). The results showed that after the solution was prepared alternately into an acidic solution of pH = 4.2, approximately 91% of the original fluorescence signal was successfully restored after 6 cycles. Contrastly, spirocyclic structure of the probe was associated with quenching of the fluorescence under neutral solution (pH = 7.2). A good reversibility of the probe toward pH enabled us to propose a mechanism for the equilibrium of probe REI between pH = 4.2 and pH = 7.2 (Scheme 1). A possible interaction between probe REI and H+ is also suitable for the similar phenomenon of other pH probes with the same functional fluorophore as probe REI [39,59,65,66]. 3.3. Application study of bio-imagine Since the lumenal environment of lysosomes was at a pH range from 4.6 to 5.0, the cell imaging assay was performed to investigate the application of the probe in sensing H+ in biological systems.
Fig. 2. (a) Time courses of fluorescence intensity of REI at various pH values. (b) The comparison of the time response of probe REI and RE at the pH of 4.2. (kem = 580 nm).
Fig. 3. Fluorescence microscope images of living HeLa cells with different concentrations of REI for 0.5, 1, 2 h at 37 °C. ⁄P < 0.05; ⁄⁄P < 0.01, n = 3. Images are representative of 3 independent experiments.
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After incubated with different concentrations of REI for 0.5–2 h, the fluorescence microscope images of the processed cells were obtained by confocal fluorescence microscopy (Fig. 3). The different fluorescence intensity changes not only demonstrated the time and dose-depended characteristics of probe REI, but also implied its excellent membrane-permeable.
To further determine the subcellular distribution of probe REI, the LysosensorTM Green DND-189, an excellent lysosome tracker, was used to stain the cells with probe REI. The strong green signals were collected after HeLa cells incubated with lysosome tracker (1 lM) for 1 h (Fig. 4b). In contrast, the fluorescence of the cells incubated with REI (5 lM) was observed in the red channel
Fig. 4. Confocal fluorescence imaging of living HeLa cells co-stained with REI (5 lM) and LysoSensorÒGreen (1 lM) for 1 h. (a) Red emission from REI; (b) Green emission from LysoSensorÒGreen; (c) Overlay of (a) and (b). Areas of co-localization appeared in yellow; (d) The correlation of LysoSensorÒGreen and REI intensities. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 5. Fluorescence microscope imaging of living HeLa cells pretreated with Chloroquine for 6 h, and then treated with (A, B) 5 lM REI for 1 h; (C, D) 2.5 lM REI for 2 h.
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(Fig. 4a). Fortunately, the merged image (Fig. 4c) of red and green channels exhibited distinct pH dependent patterns with yellow fluorescence. The overlap coefficient (0.77) also suggested the probe REI could specifically label lysosomes in other cells. Monitoring pH changes in lysosome efficiently has great significance since pH can affect endocytosis, exocytosis, autophagy and other cellular processes [3,6–9,67]. For one thing, the weakly basic substances can cause an increase of lysosomal pH by leaking protons out of lysosomes, for another, the concentration of chloroquine required to cause such effect is considerably lower than that of other weak bases [67]. In this study, the fluorescence intensities within the cells were gradually weakened accompanied with increased concentration of chloroquine from 0 to 20 lM, which exhibited the well performance of REI as an indicator in the monitoring pH changes of lysosomal in living cells with faster response and more significant results compared with other rhodamine-based pH fluorescence probes [39,65] (Fig. 5). The excellent results suggested that REI was favorable for real-time tracking of the pH in both real solutions and biological samples compared with other pH probes (Table S1). SRB assays protocol revealed that the cell viabilities of HeLa cells were not affected by incubated with 5 lM REI for 24 h at the experimental conditions (Figure S5, ESI). 4. Conclusion In summary, a novel pH fluorescence cassette REI based on the introduction of quaternary ammonium salt was successfully developed to serve both in simple buffer solution and complex cell media applications. The introduction of the positive charge can not only improve the solubility in water and reactivity of probe REI but also avoid the ‘‘alkalizing effect’’ with respect to the reference probe RE. Moreover, probe REI, with fully reversibility mainly within the pH range from 4.2 to 7.2, could also be applied for monitoring pH changes at the range of pH from 4.6–5.8 with satisfying results. In particular, probe REI was well used for monitoring the weak acid pH fluctuations in lysosome of the live HeLa cells due to its excellent biological properties. Since numerous rhodamine-based small molecular probes have been developed to recognize a wide range of targets, our strategy provides a new sensing platform for wide applications in biological samples and other fields. Acknowledgments This study was supported by the Natural Science Foundation of Shandong Province (ZR2014BM004). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.saa.2015.06.111. References [1] R. Martinez-Zaguilan, B.F. Chinnock, S. Wald-Hopkins, M. Bernas, D. Way, M. Weinand, M.H. Witte, R.J. Gillies, Cell. Physiol. Biochem. 6 (1996) 169–184. [2] J.K. Laihia, J.P. Kallio, P. Taimen, H. Kujari, V.M. Kähäri, L. Leino, J. Invest. Dermatol. 130 (2010) 2431–2439. [3] C. Nilsson, K. Kågedal, U. Johansson, K. Öllinger, Cancer Treat. Rev. 25 (2004) 185–194. [4] K.R. Hoyt, I.J. Reynolds, J. Neurochem. 71 (1998) 1051–1058. [5] Y. Shimizu, S.W. Hont III, Immunol. Today 17 (1996) 565–573. [6] H. Izumi, T. Torigoe, H. Ishiguchi, H. Uramoto, Y. Yoshida, M. Tanabe, T. Ise, T. Murakami, T. Yoshida, M. Nomoto, Cancer Treat. Rev. 29 (2003) 541–549. [7] M. Chesler, Physiol. Rev. 83 (2003) 1183–1221. [8] A.M. Paradiso, R.Y. Tsien, T.E. Machen, Nature 325 (1987) 447–450. [9] I. Yuli, A. Oplatka, Science 235 (1987) 340–342.
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