Materials Science and Engineering C 52 (2015) 97–102
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Fluorescent probe based on heteroatom containing styrylcyanine: pH-sensitive properties and bioimaging in vivo Xiaodong Yang a, Ya Gao c, Zhibing Huang c, Xiaohui Chen c, Zhiyong Ke c, Peiliang Zhao b, Yichen Yan b, Ruiyuan Liu b,⁎, Jinqing Qu a,⁎ a b c
School of Chemistry and Chemical Engineering, South China University of Technology, Guangzhou 510640, PR China Department of Organic Pharmaceutical Chemistry, School of Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515, PR China School of Basic Medical Science, Southern Medical University, Guangzhou 510515, PR China
a r t i c l e
i n f o
Article history: Received 7 October 2014 Received in revised form 11 January 2015 Accepted 22 March 2015 Available online 24 March 2015 Keywords: Fluorescent pH sensor Styrylcyanine Bioimaging Chemosensor
a b s t r a c t A novel fluorescent probe based on heteroatom containing styrylcyanine is synthesized. The fluorescence of probe is bright green in basic and neutral media but dark orange in strong acidic environments, which could be reversibly switched. Such behavior enables it to work as a fluorescent pH sensor in the solution state and a chemosensor for detecting acidic and basic volatile organic compounds. Analyses by NMR spectroscopy confirm that the protonation or deprotonation of pyridinyl moiety is responsible for the sensing process. In addition, the fluorescent microscopic images of probe in live cells and zebrafish are achieved successfully, suggesting that the probe has good cell membrane permeability and low cytotoxicity. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Application of fluorescent compounds to monitoring pH change has attracted considerable attention. On the one hand, pH plays important roles in biological process such as cellular proliferation and apoptosis, enzymatic activity, ion transport, and muscle contraction [1–6]. Abnormal pHi values have been linked to cell dysfunction and some common diseases such as cancer and Alzheimer's disease [7,8]. On the other hand, fluorescence microscopic imaging techniques employing fluorescent probes can provide a spatial and temporal observation of pH changes [9–14]. Thus, in the last decade, a series of fluorescent pH probes containing heteroatom groups have been developed [15–22]. Protonation of heteroatom-containing groups such as pyridinyl and amino could significantly change their electron-withdrawing property, resulting in change of the emission color or fluorescent intensity [23–25]. However, a majority of pH fluorescent probes respond to neutral pH ranging from 6 to 8 [26–29] and weakly acid pH ranging from 4 to 6 [30–33]. Very few probes are sensitive to pH value below 4 [34,35] and suitable for ⁎ Corresponding authors. E-mail addresses: [email protected] (R. Liu), [email protected] (J. Qu).
http://dx.doi.org/10.1016/j.msec.2015.03.042 0928-4931/© 2015 Elsevier B.V. All rights reserved.
extremely acid conditions [36,37]. Therefore, the detection of the strong acid (pH value below 2) conditions is still a difficult problem to be solved. Another issue is that most sensors have no selectivity in recognition of different cells except quite a few fluorescent biosensor for targeted imaging of cancer cells [38] and specific mitochondrial imaging to identify differentiating brown adipose cells [39]. In this paper, we prepared styrylcyanine (probe 1, Scheme 1) containing pyridinyl groups via reaction of 4-pyridinecarboxaldehyde with 1,1,2-trimethylbenz[e]indole. The biosensor can monitor pH gradients in a pH range of 2.0–4.0 with different fluorescent colors, which means high selectivity and good sensitivity. It quenched immediately when pH value is below 2 indicating an extremely sensitive response to strong acid. In addition, another strength of the probe is to make a dye plate to detect some acidic or alkaline volatile organic compounds, such as hydrochloric acid, trifluoroacetic acid, triethylamine, and so forth. This may be a useful alarm apparatus in the industrial application. Compared with some complicated synthesis routes [26,37,40] and sensor of metal complexes [26,38], the luminescent molecule we designed with easily synthetic method and simple structure is more attractive. Most importantly, probe 1 exhibits bright fluorescence in living cells and living organism showing low-cytotoxicity and long-term stability. However, an issue we have to face is that the pH probe lacks selectivity
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CHO N
N
EtOH, piperidine + N
reflux, 12 h
N
1
Scheme 1. Synthesis of probe 1.
in recognition of different cells which is a development orientation of the fluorescent probes in the application of biological aspect. Thus, our work should be taken into consideration to design new probes and expect that they can solve this problem to some extent in the future.
2. Experimental section 2.1. Reagents CH3CN was purchased from Acros. Other chemicals were purchased from Sigma-Aldrich and used as received without further purification. A buffer solution of pH 1–14 was prepared by diluting HCl (1 M) and NaOH (1 M) and confirmed by a pH meter. Mixtures of probe 1 in water with various pH values were prepared by adding water CH3CN solutions into buffer solutions with specific pH values.
2.2. Apparatus Absorption spectra were measured at room temperature on a JASCO J-820 with 1.0 cm glass cells. Fluorescence emission spectra were obtained at room temperature on a FLS-920 Edinburgh Fluorescence Spectrophotometer, with a Xenon lamp and 1.0 cm quartz cells. The 1 H NMR and 13C NMR spectra were recorded at 400 and 100 MHz, respectively. Elemental analysis was performed on an Eager 300 elemental microanalyzer. Particle sizes of the nano-aggregates were determined on a Zeta-plus potential Analyzer. pH measurements were performed on a Beckman 340 pH/Temp meter. Morphologies of nanoparticles were studied by a JEOL 100CX transmission electron microscope.
2.3. Fluorescence quantum yield of probe 1 Quantum yields of probe 1 were determined using quinine sulfate in 0.1 N H2SO4 as standards according to a published method.
Fig. 1. Absorption and emission spectra of probe 1 in H2O [1] = 30 μM.
2.4. Procedures for sample preparation in pH-switching Experimental details are shown in Table S1. To avoid dilution effect, paralleled samples were prepared. The “Cycle no. 0” means that the dye molecules are first put in deionized water without any acid and base. This aqueous mixture is prepared by adding an aliquot of concentrated DMSO stock solution of probe 1 into a large volume of water. From no. 0.5 to no. 4, base and acid were added alternately to adjust the pH. Additional water, if needed, is added to ensure that the final dye concentrations of all samples are identical.
2.5. Cell culture and fluorescence imaging The HeLa cell, 5–8F cell, and LoVo cell line were provided by School of Basic Medical Science, Southern Medical University Guangzhou (China). Cells were grown in RPMI 1640 medium supplemented with 10% FBS (Fetal Bovine Serum) and 1% antibiotics at 37 °C in humidified environment of 5% CO2. Cells were plated on 6-well plate at 5 × 104 cells per well and allowed to adhere for 12 h. Fluorescence imaging was performed with a inverted fluorescence microscope (IX71, Olympus). Before the experiments, cells were washed with PBS and then incubated with 1 (10 μM) in PBS for 30 min at 37 °C. Cell imaging was then carried out after washing cells with physiological saline. Emission was collected at 510–550 nm for green channel.
2.6. Cytotoxicity assays HeLa cells, 5–8F cells, and Lovo cells were grown in RPMI 1640 medium supplemented with 10% FBS (Fetal Bovine Serum) and 1% antibiotic at 37 °C in humidified environment of 5% CO2 and 95% air. Immediately before the experiment, the cells well placed in a 96-well plate, followed by addition of increasing concentrations of probe 1. The final concentrations of the probe were kept from 0 to 20 μM. The cells were then incubated at 37 °C in an atmosphere of 5% CO2 and
Fig. 2. Variations of fluorescent spectra of probe 1 (30 μM) with pH in water.
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N
99
H
N
H
N
N
probe 1+
probe 1
Scheme 2. Proposed protonation process of probe 1 in strong acidic condition.
Fig. 3. Partial 1H NMR spectra of probe 1 upon addition of trifluoroacetic acid (TFA) in DMSO-d6 containing (a) 0 and (b) 0.01 mL of trifluoroacetic acid (TFA). The spectrum in (c) is obtained by adding 0.02 mL of triethylamine into (b).
95% air for 24 h, followed by MTT assays (n = 5). Untreated assay with RPMI 1640 (n = 5) was also conducted under the same conditions.
ROI was calculated pixel-by-pixel. All data were expressed as mean ± standard deviation.
2.7. Fluorescence imaging in zebrafishes
2.8. Synthesis of probe 1
Zebrafish eggs, 12 hpf, were incubated in respective 25 mL petri dishes with probe 1 (20 mL fish water, 10 μM). These embryos were then kept under standard conditions at 28 °C for 5 days in agreement with Home Office regulations on a 12 h light/12 h dark cycle. Tricaine methane sulphonate (MS222) was used to anesthetise the fish before any fluorescence microscopy. Fluorescence imaging experiments were performed on a FV 1000IX81 confocal laser scanning microscope (Olympus, Japan) with FV5LAMAR for excitation at 559 nm through a 100× 0.4 NA objective. Optical sections were acquired at 0.8 μm. The fluorescence was collected in the ranges of 510–550 nm (probe 1) Region of interest (ROI) was selected based on peripheries of zebrafishes. Image processing and analysis were performed on Olympus software (FV10-ASW), and the ratio of
A mixture of 2.09 g (10 mmol) of 1,1,2-trimethyl-1H-benzo[e]indole and 1.07 g (10 mmol) 4-pyridinecarboxaldehyde was refluxed in 50 ml of anhydride ethanol with one drop of piperidine for 12 h. After the solvent was removed under reduced pressure, the yellow solid was purified by column chromatography using methylene chloride as the eluent. 1.91 g of yellow product was obtained. Yield: 64%. 1 H NMR (400 MHz, DMSO-d6) δ (ppm) = 8.65 (d, J = 4.7, 2H), 8.19 (d, J = 8.4, 1H), 8.04 (d, J = 8.2, 1H), 7.97 (d, J = 8.5, 1H), 7.82 (dd, J = 13.2, 7.7, 4H), 7.69 (s, 1H), 7.67–7.60 (m, 1H), 7.52 (t, J = 7.5, 1H), 1.65 (s, 6H). 13 C NMR (100 MHz, DMSO-d6) δ = 184.29, 150.74, 150.20, 143.00, 139.83, 134.40, 132.20, 129.46, 129.05, 128.04, 126.75, 124.89, 123.59, 123.05, 121.72, 120.18, 54.22, 21.85.
Fig. 4. (a) Emission spectra of probe 1 in acidic, neutral, and basic conditions. Reversible switching of the (b) emission intensity of probe 1 by repeated adjustment of its solution to acidic and basic environments. For experimental details see Table S1, ESI.† [1] = 30 μM; λex = 350 nm.
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characterized by NMR, mass spectroscopies and elemental analysis, which gave satisfactory data corresponding to its molecular structure (see ESI for details). 3.2. pH-dependent optical properties The absorption and emission spectra of probe 1 were measured in water (Fig. 1). Two absorption bands appeared around 350 and 300 nm. The emission band relative to the corresponding absorption band with the maximum wavelength in the region of 510 nm was observed. The fluorescent quantum yield, Φf, of probe 1 was 0.16. To explore the pH-dependent optical properties of probe 1, the fluorescent pH titrations of probe 1 was performed. As shown in Fig. 2, probe 1 was green luminescent at high pH values and its PL intensity decreased slightly when the pH was N 5. At pH b 5, the green emission faded gradually. However, as the pH decreased to 3, a new orange emission peak emerged at 600 nm and the PL intensity decreased with pH. When the pH exceeded 1, the fluorescence was quenched. 3.3. Mechanism of the pH response Fig. 5. Fluorescent color of probe 1 deposited on a TLC plate by repeated fuming with TFA and TEA vapors.
IR(v−1, LiBr): 3050, 2973, 2927, 2836, 1595, 1545, 1502, 1461, 1415, 1217, 970, 823, 744. MS (MALDI-Cl): C21H18N2 m/z 298.1470 for [M + H]+ 299.1544. Elemental analysis: Calcd C, 84.53; H, 6.08; N, 9.39. Found C, 85.04; H, 6.11; N, 8.85. mp: 246.5–247.0 °C. 2.9. Absorption and fluorescence analysis A typical experimental procedure was described as follows: Stock solutions of probe 1 (CH3CN, 30 mM) were prepared in flasks equipped with stopcocks. The probe 1 solution (10 μL) was transferred to a vial, and then the resulting mixture was diluted to 10 mL with DI water to give the sample solution, of which [1] was adjusted to be 30 μM. The concentration of probe 1 was 30 μM throughout the analysis experiments except that otherwise pointed out. The fluorescent intensity was measured with the excitation wavelength 350 nm except as otherwise noted, and the excitation and emission slits were set to 1 and 1 nm, respectively. 3. Results and discussion 3.1. Synthesis Probe 1 was prepared by reaction 4-pyridinecarboxaldehyde with 1,1,2-trimethylbenz[e]indole in EtOH (Scheme 1). Probe 1 was
Probe 1 is a hemicyanine dye containing pyridine moeties, offering nitrogen atom for protonation. Addition of a drop of trifluoroacetic acid (TFA) into a DMSO solution of probe 1 protonates its pyridine unit and generates probe 1 + (Scheme 2). This is proved by the 1H NMR spectra shown in Fig. 3. The pyridine protons shifted downfield after protonation because of the transformation of the pyridine group in probe 1 to an electron-deficient pyridinium unit in probe 1+. When the solvent mixture was adjusted to a basic environment by injection of an excess amount of Et3N, all the proton signals were quickly shifted back to their original positions (Fig. 3c), showing a high reversibility. The fluorescent transitions were reversible upon pH change. Starting from neutral aqueous conditions, alternative addition of NaOH and HCl adjusted the resultant solution to basic (pH ~ 13) or acidic (pH ~ 3) environments. From the basic to acidic conditions, the corresponding emission maxima were switched from 510 nm to 600 nm, and vice versa (Fig. 4). Such response was very fast and could be repeated for many cycles without any change in the spectral profiles. Since the fluorescence of probe 1 was pH-sensitive in solution state, we wondered whether probe 1 could detect volatile organic compounds with high acidity and basicity in solid state. Due to its strong mechanical strength, we utilized a thin-layer chromatography (TLC) plate as a solid support. The probe-loaded plate emitted a weak fluorescence due to the partial protonation of probe 1 by the weak acidity of the silica gel (Figs. 5 and S5). After fuming with TFA vapor, the dye plate turned dark yellow and emitted yellow fluorescent light. It, however, converted back to its green emissive form when treated with TEA vapor. The switch between
Fig. 6. (a) Fluorescent imaging of LoVo cells incubated with probe 1 (10 μM) from green channel; (b) bright-field imaging of (b).
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Fig. 7. (a) Fluorescent imaging of zebrafish (five days old) incubated with probe 1 (30 μM) from bright-field; (b) green channel imaging of (a), (c) overlap of (a) and (b).
the yellow weak emissive and bright green state could be repeated many times. The selectivity of probe 1 to H+ over metal ions was investigated by competition experiments. The results showed that physiologically important metal ions (K+, Na+, Ca2 +, and Mg2 +) did not give any notable emission change to probe 1 (300 μM) at their physiological concentration. Other heavy and transition metal ions (300 μM) also had no interference (Fig. S6). 3.4. Bioimaging for living cells and organism We also explored the application of probe 1 in other field. We used LoVo Colon cancer and HeLa cells as model cell to study the subcellular distribution of probe 1 by a inverted fluorescence microscope. Firstly, to evaluate the cytotoxicity of probe 1, we performed standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays using HeLa, 5–8F, and LoVo cells at concentration from 0–20 μM of probe 1 for 24 h, respectively. The result clearly showed that probe 1 was nontoxic to the cultured cells under the experimental conditions (Fig. S7). Herein cultured LoVo cells were incubated with probe 1 (10 μM) for 30 min at 37 °C. As predicted, fluorescent images showed that probe 1 was cell membrane permeable and localized in the cytoplasm region (Figs. 6 and S8). These results clearly mean that probe 1 has good cell membrane permeability and could image organelles in living cells. Encouraged by the living cell experiments, we evaluated the effectiveness of probe 1 for fluorescent imaging in a living vertebrate organism, zebrafish. Five day old zebrafish were incubated in solutions of probe 1 at 10 μM and the distribution of fluorescence within the zebrafish was monitored (Fig. 7). Probe 1 showed significant uptake within the fish with clear regions of concentrated fluorescence in the yolk sac and alimentary canal, including the intestinal tract and biliary system (gall bladder, liver, pancreas, solitary islet and bile ducts). In addition, the probe itself was found to be rather stable in the body of zebrafish, because no noticeable change in the averaged ratio value was observed at least within 1 h. These in vivo studies showed that probe 1 can enter zebrafish, and image in living organism. 4. Conclusions In summary, a novel styrylcyanine-based fluorescent pH probe 1 was synthesized via facile methods. Probe 1 was green fluorescent in basic and neutral media but dark orange in strong acidic environments. Its emission could be reversibly switched between bright green and dark orange by repeated protonation and deprotonation. Such behavior enables it to work as a chemosensor for detecting volatile organic
compounds with high acidity and basicity in solid state. Subsequently, the fluorescent microscopic images of probe 1 in live cells and in the whole body of zebrafish were achieved successfully, suggesting that probe 1 has good cell membrane permeability and potential application for imaging in live cells and living organism. Acknowledgments This work was partially supported by the National Natural Science Foundation of China (Nos. 21244007, 81271642). Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.msec.2015.03.042. References [1] L. Fan, Y.J. Fu, Q.L. Liu, D.T. Lu, C. Dong, S.M. Shuang, Novel far-visible and near-infrared pH probes based on styrylcyanine for imaging intracellular pH in live cells, Chem. Commun. 48 (2012) 11202–11204. [2] A. Roos, W.F. Boron, Intracellular pH, Physiol. Rev. 61 (1981) 296–434. [3] J.L. Fan, M.M. Hu, P. Zhan, X.J. Peng, Energy transfer cassettes based on organic fluorophores: construction and applications in ratiometric sensing, Chem. Soc. Rev. 42 (2013) 29–43. [4] R.T. Kennedy, L. Huang, C.A. Aspenwall, Extracellular pH is required for rapid release of insulin from Zn-insulin precipitates in β-cell secretory vesicles during exocytosis, J. Am. Chem. Soc. 118 (1996) 1795–1796. [5] K.R. Hoyt, I.J. Reynolds, Alkalinization prolongs recovery from glutamate-induced increases in intracellular Ca2+ concentration by enhancing Ca2+ efflux through the mitochondrial Na+/Ca2+ exchanger in cultured rat forebrain neurons, J. Neurochem. 71 (1998) 1051–1058. [6] E.R. Chin, D.G. Alllen, The contribution of pH-dependent mechanisms to fatigue at different intensities in mammalian single muscle fibres, J. Physiol. 512 (1998) 831–840. [7] H. Izumi, T. Torigoe, H. Ishiguchi, H. Uramoto, Y. Yoshida, M. Tanabe, T. Ise, T. Murakami, T. Yoshida, M. Nomoto, K. Kohno, Cellular pH regulators: potentially promising molecular targets for cancer chemotherapy, Cancer Treat. Rev. 29 (2003) 541–549. [8] T.A. Davies, R.E. Fine, R.J. Johnson, C.A. Levesque, W.H. Rathbun, K.F. Seetoo, S.J. Smith, G. Strohmeier, L. Volicer, L. Delva, Non-age related differences in thrombin responses by platelets from male patients with advanced Alzheimer's disease, Biochem. Biophys. Res. Commun. 194 (1993) 537–543. [9] Z.G. Song, Y.N. Hong, R.T.K. Kwok, J.W.Y. Lam, B. Liu, B.Z. Tang, A dual-mode fluorescence “turn-on” biosensor based on an aggregation-induced emission luminogen, J. Mater. Chem. B 2 (2014) 1717–1723. [10] N. Kumari, S. Jha, S. Bhattacharya, An efficient probe for rapid detection of cyanide in water at parts per billion levels and naked-eye detection of endogenous cyanide, Chem. Asian. J. 9 (2014) 830–837. [11] R.A. Gottlieb, H.A. Giesing, J.Y. Zhu, R.L. Engler, B.M. Babior, Cell acidification in apoptosis: granulocyte colony-stimulating factor delays programmed cell death in neutrophils by up-regulating the vacuolar H(+)-ATPase, Proc. Natl. Acad. Sci. U. S. A. 92 (1995) 5965–5968.
102
X. Yang et al. / Materials Science and Engineering C 52 (2015) 97–102
[12] S. Ohkuma, B. Poole, Fluorescence probe measurement of the intralysosomal pH in living cells and the perturbation of pH by various agents, Proc. Natl. Acad. Sci. U. S. A. 75 (1978) 3327–3331. [13] J. Han, K. Burgess, Fluorescent indicators for intracellular pH, Chem. Rev. 110 (2010) 2709–2728. [14] R.R. Hu, R.Q. Ye, J.W.Y. Lam, M. Li, C.W.T. Leung, B.Z. Tang, Conjugated polyelectrolytes with aggregation-enhanced emission characteristics: synthesis and their biological applications, Chem. Asian. J. 8 (2013) 2436–2445. [15] L. Fan, Q.L. Liu, D.T. Lu, H.P. Shi, Y.F. Yang, Y.F. Li, C. Dong, S.M. Shuang, A novel far-visible and near-infrared pH probe for monitoring near-neutral physiological pH changes: imaging in live cells, J. Mater. Chem. B 1 (2013) 4281–4288. [16] L.C. Ho, C.M. Ou, C.L. Li, S.Y. Chen, H.W. Li, H.T. Chang, Sensitive pH probes of retroself-quenching fluorescent nanoparticles, J. Mater. Chem. B 1 (2013) 2425–2432. [17] F. Galindo, M.I. Burguete, L. Vigara, S.V. Luis, N. Kabir, J. Gavrilovic, D.A. Russell, Synthetic macrocyclic peptidomimetics as tunable ph probes for the fluorescence imaging of acidic organelles in live cells, Angew. Chem. Int. Ed. 44 (2005) 6504–6508. [18] R.A. Bissell, A.J. Bryan, A. Prasanna de Silva, C.P. McCoy, Fluorescent PET (photoinduced electron transfer) sensors with targeting/anchoring modules as molecular versions of submarine periscopes for mapping membrane-bounded protons, J. Chem. Soc. Chem. Commun. 4 (1994) 405–407. [19] N. Marcotte, A.M. Brouwer, Carboxy SNARF-4 F as a fluorescent ph probe for ensemble and fluorescence correlation spectroscopies, J. Phys. Chem. B 109 (2005) 11819–11828. [20] J. Han, A. Loudet, R. Barhoumi, R.C. Burghardt, K. Burgess, A ratiometric ph reporter for imaging protein-dye conjugates in living cells, J. Am. Chem. Soc. 131 (2009) 1642–1643. [21] Y.Q. Tian, F.Y. Su, W. Weber, V. Nandakumar, B.R. Shumway, Y.G. Jin, X.F. Zhou, M.R. Holl, R.H. Johnson, D.R. Meldrum, A series of naphthalimide derivatives as intra and extracellular pH sensors, Biomaterials 31 (2010) 7411–7422. [22] X.F. Zhou, F.Y. Su, H.G. Lu, P. Senechal-Willis, Y.Q. Tian, R.H. Johnson, D.R. Meldrum, An FRET-based ratiometric chemosensor for in vitro cellular fluorescence analyses of pH, Biomaterials 33 (2012) 171–180. [23] Z.Y. Yang, W. Qin, J.W.Y. Lam, S.J. Chen, H.H.Y. Sung, I.D. Williams, B.Z. Tang, Fluorescent pH sensor constructed from a heteroatom-containing luminogen with tunable AIE and ICT characteristics, Chem. Sci. 4 (2013) 3725–3730. [24] S.J. Chen, J.Z. Liu, Y. Liu, H.M. Su, Y.N. Hong, C.K.W. Jim, R.T.K. Kwok, N. Zhao, W. Qin, J.W.Y. Lam, K.S. Wong, B.Z. Tang, An AIE-active hemicyanine fluorogen with stimuliresponsive red/blue emission: extending the pH sensing range by “switch + knob” effect, Chem. Sci. 3 (2012) 1804–1809. [25] E. Galbraith, T.D. James, Boron based anion receptors as sensors, Chem. Soc. Rev. 39 (2010) 3831–3842.
[26] Q.Q. Zhang, M. Zhou, A profluorescent ratiometric probe for intracellular pH imaging, Talanta 131 (2015) 666–671. [27] R. Gui, X. An, W. Huang, An improved method for ratiometric fluorescence detection of pH and Cd2+ using fluorescein isothiocyanate-quantum dots conjugates, Anal. Chim. Acta 767 (2013) 134–140. [28] B.K. McMahon, R. Pal, D. Parker, A bright and responsive europium probe for determination of pH change within the endoplasmic reticulum of living cells, Chem. Commun. 49 (2013) 5363–5365. [29] T. Jokic, S.M. Borisov, R. Saf, D.A. Nielsen, M. Kuhl, I. Klimant, Highly photostable near-infrared fluorescent pH indicators and sensors based on BF2-chelated tetraarylazadipyrromethene dyes, Anal. Chem. 84 (2012) 6723–6730. [30] X. Du, N.Y. Lei, P. Hu, Z. Lei, D.H. Ong, X. Ge, Z. Zhang, M.H. Lam, In vivo imaging of the morphology and changes in pH along the gastrointestinal tract of Japanese medaka by photonic band-gap hydrogel microspheres, Anal. Chim. Acta 787 (2013) 193–202. [31] H.S. Lv, S.Y. Huang, B.X. Zhao, J.Y. Miao, A new rhodamine B-based lysosomal pH fluorescent indicator, Anal. Chim. Acta 788 (2013) 177–182. [32] H.S. Lv, S.Y. Huang, Y. Xu, X. Dai, J.Y. Miao, B.X. Zhao, A new fluorescent pH probe for imaging lysosomes in living cells, Bioorg. Med. Chem. Lett. 24 (2014) 535–538. [33] H.S. Lv, J. Liu, J. Zhao, B.X. Zhao, J.Y. Miao, Highly selective and sensitive pHresponsive fluorescent probe in living Hela and HUVE Ccells, Sensors Actuators B Chem. 177 (2013) 956–963. [34] M. Yang, Y. Song, M. Zhang, S. Lin, Z. Hao, Y. Liang, D. Zhang, P.R. Chen, Converting a solvatochromic fluorophore into a protein-based pH indicator for extreme acidity, Angew. Chem. 51 (2012) 7674–7679. [35] X. Zhang, S.Y. Jing, S.Y. Huang, X.W. Zhou, J.M. Bai, B.X. Zhao, New fluorescent pH probes for acid conditions, Sensors Actuators B Chem. 206 (2015) 663–670. [36] H. Li, H. Guan, X. Duan, J. Hu, G. Wang, Q. Wang, An acid catalyzed reversible ring-opening/ring-closure reaction involving a cyano-rhodamine spirolactam, Org. Biomol. Chem. 11 (2013) 1805–1809. [37] D.Y. Kim, H.J. Kim, pH-sensitive optode membrane covalently incorporated with an ICT dye forlow pH values, Sensors Actuators B Chem. 206 (2015) 508–515. [38] C.Q. Ding, Y. Tian, Gold nanocluster-based fluorescence biosensor for targeted imaging in cancer cells and ratiometric determination of intracellular pH, Biosens. Bioelectron. 65 (2015) 183–190. [39] M. Gao, C.K. Sim, C.W.T. Leung, Q.L. Hu, G.X. Feng, F. Xu, B.Z. T., B. Liu, A fluorescent light-up probe with AIE characteristics for specific mitochondrial imaging to identify differentiating brown adipose cells, Chem. Commun. 50 (2014) 8312–8315. [40] J.I.A. Rashid, N.A. Yusof, J. Abdullah, U. Hashimc, R. Hajian, The utilization of SiNWs/ AuNPs-modified indium tin oxide (ITO) in fabrication of electrochemical DNA sensor, Mater. Sci. Eng. C 45 (2014) 270–276.
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Fluorescent probe based on heteroatom containing styrylcyanine: pH-sensitive properties and bioimaging in vivo Xiaodong Yang a, Ya Gao c, Zhibing Huang c, Xiaohui Chen c, Zhiyong Ke c, Peiliang Zhao b, Yichen Yan b, Ruiyuan Liu b,⁎, Jinqing Qu a,⁎ a b c
School of Chemistry and Chemical Engineering, South China University of Technology, Guangzhou 510640, PR China Department of Organic Pharmaceutical Chemistry, School of Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515, PR China School of Basic Medical Science, Southern Medical University, Guangzhou 510515, PR China
a r t i c l e
i n f o
Article history: Received 7 October 2014 Received in revised form 11 January 2015 Accepted 22 March 2015 Available online 24 March 2015 Keywords: Fluorescent pH sensor Styrylcyanine Bioimaging Chemosensor
a b s t r a c t A novel fluorescent probe based on heteroatom containing styrylcyanine is synthesized. The fluorescence of probe is bright green in basic and neutral media but dark orange in strong acidic environments, which could be reversibly switched. Such behavior enables it to work as a fluorescent pH sensor in the solution state and a chemosensor for detecting acidic and basic volatile organic compounds. Analyses by NMR spectroscopy confirm that the protonation or deprotonation of pyridinyl moiety is responsible for the sensing process. In addition, the fluorescent microscopic images of probe in live cells and zebrafish are achieved successfully, suggesting that the probe has good cell membrane permeability and low cytotoxicity. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Application of fluorescent compounds to monitoring pH change has attracted considerable attention. On the one hand, pH plays important roles in biological process such as cellular proliferation and apoptosis, enzymatic activity, ion transport, and muscle contraction [1–6]. Abnormal pHi values have been linked to cell dysfunction and some common diseases such as cancer and Alzheimer's disease [7,8]. On the other hand, fluorescence microscopic imaging techniques employing fluorescent probes can provide a spatial and temporal observation of pH changes [9–14]. Thus, in the last decade, a series of fluorescent pH probes containing heteroatom groups have been developed [15–22]. Protonation of heteroatom-containing groups such as pyridinyl and amino could significantly change their electron-withdrawing property, resulting in change of the emission color or fluorescent intensity [23–25]. However, a majority of pH fluorescent probes respond to neutral pH ranging from 6 to 8 [26–29] and weakly acid pH ranging from 4 to 6 [30–33]. Very few probes are sensitive to pH value below 4 [34,35] and suitable for ⁎ Corresponding authors. E-mail addresses: [email protected] (R. Liu), [email protected] (J. Qu).
http://dx.doi.org/10.1016/j.msec.2015.03.042 0928-4931/© 2015 Elsevier B.V. All rights reserved.
extremely acid conditions [36,37]. Therefore, the detection of the strong acid (pH value below 2) conditions is still a difficult problem to be solved. Another issue is that most sensors have no selectivity in recognition of different cells except quite a few fluorescent biosensor for targeted imaging of cancer cells [38] and specific mitochondrial imaging to identify differentiating brown adipose cells [39]. In this paper, we prepared styrylcyanine (probe 1, Scheme 1) containing pyridinyl groups via reaction of 4-pyridinecarboxaldehyde with 1,1,2-trimethylbenz[e]indole. The biosensor can monitor pH gradients in a pH range of 2.0–4.0 with different fluorescent colors, which means high selectivity and good sensitivity. It quenched immediately when pH value is below 2 indicating an extremely sensitive response to strong acid. In addition, another strength of the probe is to make a dye plate to detect some acidic or alkaline volatile organic compounds, such as hydrochloric acid, trifluoroacetic acid, triethylamine, and so forth. This may be a useful alarm apparatus in the industrial application. Compared with some complicated synthesis routes [26,37,40] and sensor of metal complexes [26,38], the luminescent molecule we designed with easily synthetic method and simple structure is more attractive. Most importantly, probe 1 exhibits bright fluorescence in living cells and living organism showing low-cytotoxicity and long-term stability. However, an issue we have to face is that the pH probe lacks selectivity
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CHO N
N
EtOH, piperidine + N
reflux, 12 h
N
1
Scheme 1. Synthesis of probe 1.
in recognition of different cells which is a development orientation of the fluorescent probes in the application of biological aspect. Thus, our work should be taken into consideration to design new probes and expect that they can solve this problem to some extent in the future.
2. Experimental section 2.1. Reagents CH3CN was purchased from Acros. Other chemicals were purchased from Sigma-Aldrich and used as received without further purification. A buffer solution of pH 1–14 was prepared by diluting HCl (1 M) and NaOH (1 M) and confirmed by a pH meter. Mixtures of probe 1 in water with various pH values were prepared by adding water CH3CN solutions into buffer solutions with specific pH values.
2.2. Apparatus Absorption spectra were measured at room temperature on a JASCO J-820 with 1.0 cm glass cells. Fluorescence emission spectra were obtained at room temperature on a FLS-920 Edinburgh Fluorescence Spectrophotometer, with a Xenon lamp and 1.0 cm quartz cells. The 1 H NMR and 13C NMR spectra were recorded at 400 and 100 MHz, respectively. Elemental analysis was performed on an Eager 300 elemental microanalyzer. Particle sizes of the nano-aggregates were determined on a Zeta-plus potential Analyzer. pH measurements were performed on a Beckman 340 pH/Temp meter. Morphologies of nanoparticles were studied by a JEOL 100CX transmission electron microscope.
2.3. Fluorescence quantum yield of probe 1 Quantum yields of probe 1 were determined using quinine sulfate in 0.1 N H2SO4 as standards according to a published method.
Fig. 1. Absorption and emission spectra of probe 1 in H2O [1] = 30 μM.
2.4. Procedures for sample preparation in pH-switching Experimental details are shown in Table S1. To avoid dilution effect, paralleled samples were prepared. The “Cycle no. 0” means that the dye molecules are first put in deionized water without any acid and base. This aqueous mixture is prepared by adding an aliquot of concentrated DMSO stock solution of probe 1 into a large volume of water. From no. 0.5 to no. 4, base and acid were added alternately to adjust the pH. Additional water, if needed, is added to ensure that the final dye concentrations of all samples are identical.
2.5. Cell culture and fluorescence imaging The HeLa cell, 5–8F cell, and LoVo cell line were provided by School of Basic Medical Science, Southern Medical University Guangzhou (China). Cells were grown in RPMI 1640 medium supplemented with 10% FBS (Fetal Bovine Serum) and 1% antibiotics at 37 °C in humidified environment of 5% CO2. Cells were plated on 6-well plate at 5 × 104 cells per well and allowed to adhere for 12 h. Fluorescence imaging was performed with a inverted fluorescence microscope (IX71, Olympus). Before the experiments, cells were washed with PBS and then incubated with 1 (10 μM) in PBS for 30 min at 37 °C. Cell imaging was then carried out after washing cells with physiological saline. Emission was collected at 510–550 nm for green channel.
2.6. Cytotoxicity assays HeLa cells, 5–8F cells, and Lovo cells were grown in RPMI 1640 medium supplemented with 10% FBS (Fetal Bovine Serum) and 1% antibiotic at 37 °C in humidified environment of 5% CO2 and 95% air. Immediately before the experiment, the cells well placed in a 96-well plate, followed by addition of increasing concentrations of probe 1. The final concentrations of the probe were kept from 0 to 20 μM. The cells were then incubated at 37 °C in an atmosphere of 5% CO2 and
Fig. 2. Variations of fluorescent spectra of probe 1 (30 μM) with pH in water.
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N
99
H
N
H
N
N
probe 1+
probe 1
Scheme 2. Proposed protonation process of probe 1 in strong acidic condition.
Fig. 3. Partial 1H NMR spectra of probe 1 upon addition of trifluoroacetic acid (TFA) in DMSO-d6 containing (a) 0 and (b) 0.01 mL of trifluoroacetic acid (TFA). The spectrum in (c) is obtained by adding 0.02 mL of triethylamine into (b).
95% air for 24 h, followed by MTT assays (n = 5). Untreated assay with RPMI 1640 (n = 5) was also conducted under the same conditions.
ROI was calculated pixel-by-pixel. All data were expressed as mean ± standard deviation.
2.7. Fluorescence imaging in zebrafishes
2.8. Synthesis of probe 1
Zebrafish eggs, 12 hpf, were incubated in respective 25 mL petri dishes with probe 1 (20 mL fish water, 10 μM). These embryos were then kept under standard conditions at 28 °C for 5 days in agreement with Home Office regulations on a 12 h light/12 h dark cycle. Tricaine methane sulphonate (MS222) was used to anesthetise the fish before any fluorescence microscopy. Fluorescence imaging experiments were performed on a FV 1000IX81 confocal laser scanning microscope (Olympus, Japan) with FV5LAMAR for excitation at 559 nm through a 100× 0.4 NA objective. Optical sections were acquired at 0.8 μm. The fluorescence was collected in the ranges of 510–550 nm (probe 1) Region of interest (ROI) was selected based on peripheries of zebrafishes. Image processing and analysis were performed on Olympus software (FV10-ASW), and the ratio of
A mixture of 2.09 g (10 mmol) of 1,1,2-trimethyl-1H-benzo[e]indole and 1.07 g (10 mmol) 4-pyridinecarboxaldehyde was refluxed in 50 ml of anhydride ethanol with one drop of piperidine for 12 h. After the solvent was removed under reduced pressure, the yellow solid was purified by column chromatography using methylene chloride as the eluent. 1.91 g of yellow product was obtained. Yield: 64%. 1 H NMR (400 MHz, DMSO-d6) δ (ppm) = 8.65 (d, J = 4.7, 2H), 8.19 (d, J = 8.4, 1H), 8.04 (d, J = 8.2, 1H), 7.97 (d, J = 8.5, 1H), 7.82 (dd, J = 13.2, 7.7, 4H), 7.69 (s, 1H), 7.67–7.60 (m, 1H), 7.52 (t, J = 7.5, 1H), 1.65 (s, 6H). 13 C NMR (100 MHz, DMSO-d6) δ = 184.29, 150.74, 150.20, 143.00, 139.83, 134.40, 132.20, 129.46, 129.05, 128.04, 126.75, 124.89, 123.59, 123.05, 121.72, 120.18, 54.22, 21.85.
Fig. 4. (a) Emission spectra of probe 1 in acidic, neutral, and basic conditions. Reversible switching of the (b) emission intensity of probe 1 by repeated adjustment of its solution to acidic and basic environments. For experimental details see Table S1, ESI.† [1] = 30 μM; λex = 350 nm.
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characterized by NMR, mass spectroscopies and elemental analysis, which gave satisfactory data corresponding to its molecular structure (see ESI for details). 3.2. pH-dependent optical properties The absorption and emission spectra of probe 1 were measured in water (Fig. 1). Two absorption bands appeared around 350 and 300 nm. The emission band relative to the corresponding absorption band with the maximum wavelength in the region of 510 nm was observed. The fluorescent quantum yield, Φf, of probe 1 was 0.16. To explore the pH-dependent optical properties of probe 1, the fluorescent pH titrations of probe 1 was performed. As shown in Fig. 2, probe 1 was green luminescent at high pH values and its PL intensity decreased slightly when the pH was N 5. At pH b 5, the green emission faded gradually. However, as the pH decreased to 3, a new orange emission peak emerged at 600 nm and the PL intensity decreased with pH. When the pH exceeded 1, the fluorescence was quenched. 3.3. Mechanism of the pH response Fig. 5. Fluorescent color of probe 1 deposited on a TLC plate by repeated fuming with TFA and TEA vapors.
IR(v−1, LiBr): 3050, 2973, 2927, 2836, 1595, 1545, 1502, 1461, 1415, 1217, 970, 823, 744. MS (MALDI-Cl): C21H18N2 m/z 298.1470 for [M + H]+ 299.1544. Elemental analysis: Calcd C, 84.53; H, 6.08; N, 9.39. Found C, 85.04; H, 6.11; N, 8.85. mp: 246.5–247.0 °C. 2.9. Absorption and fluorescence analysis A typical experimental procedure was described as follows: Stock solutions of probe 1 (CH3CN, 30 mM) were prepared in flasks equipped with stopcocks. The probe 1 solution (10 μL) was transferred to a vial, and then the resulting mixture was diluted to 10 mL with DI water to give the sample solution, of which [1] was adjusted to be 30 μM. The concentration of probe 1 was 30 μM throughout the analysis experiments except that otherwise pointed out. The fluorescent intensity was measured with the excitation wavelength 350 nm except as otherwise noted, and the excitation and emission slits were set to 1 and 1 nm, respectively. 3. Results and discussion 3.1. Synthesis Probe 1 was prepared by reaction 4-pyridinecarboxaldehyde with 1,1,2-trimethylbenz[e]indole in EtOH (Scheme 1). Probe 1 was
Probe 1 is a hemicyanine dye containing pyridine moeties, offering nitrogen atom for protonation. Addition of a drop of trifluoroacetic acid (TFA) into a DMSO solution of probe 1 protonates its pyridine unit and generates probe 1 + (Scheme 2). This is proved by the 1H NMR spectra shown in Fig. 3. The pyridine protons shifted downfield after protonation because of the transformation of the pyridine group in probe 1 to an electron-deficient pyridinium unit in probe 1+. When the solvent mixture was adjusted to a basic environment by injection of an excess amount of Et3N, all the proton signals were quickly shifted back to their original positions (Fig. 3c), showing a high reversibility. The fluorescent transitions were reversible upon pH change. Starting from neutral aqueous conditions, alternative addition of NaOH and HCl adjusted the resultant solution to basic (pH ~ 13) or acidic (pH ~ 3) environments. From the basic to acidic conditions, the corresponding emission maxima were switched from 510 nm to 600 nm, and vice versa (Fig. 4). Such response was very fast and could be repeated for many cycles without any change in the spectral profiles. Since the fluorescence of probe 1 was pH-sensitive in solution state, we wondered whether probe 1 could detect volatile organic compounds with high acidity and basicity in solid state. Due to its strong mechanical strength, we utilized a thin-layer chromatography (TLC) plate as a solid support. The probe-loaded plate emitted a weak fluorescence due to the partial protonation of probe 1 by the weak acidity of the silica gel (Figs. 5 and S5). After fuming with TFA vapor, the dye plate turned dark yellow and emitted yellow fluorescent light. It, however, converted back to its green emissive form when treated with TEA vapor. The switch between
Fig. 6. (a) Fluorescent imaging of LoVo cells incubated with probe 1 (10 μM) from green channel; (b) bright-field imaging of (b).
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Fig. 7. (a) Fluorescent imaging of zebrafish (five days old) incubated with probe 1 (30 μM) from bright-field; (b) green channel imaging of (a), (c) overlap of (a) and (b).
the yellow weak emissive and bright green state could be repeated many times. The selectivity of probe 1 to H+ over metal ions was investigated by competition experiments. The results showed that physiologically important metal ions (K+, Na+, Ca2 +, and Mg2 +) did not give any notable emission change to probe 1 (300 μM) at their physiological concentration. Other heavy and transition metal ions (300 μM) also had no interference (Fig. S6). 3.4. Bioimaging for living cells and organism We also explored the application of probe 1 in other field. We used LoVo Colon cancer and HeLa cells as model cell to study the subcellular distribution of probe 1 by a inverted fluorescence microscope. Firstly, to evaluate the cytotoxicity of probe 1, we performed standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays using HeLa, 5–8F, and LoVo cells at concentration from 0–20 μM of probe 1 for 24 h, respectively. The result clearly showed that probe 1 was nontoxic to the cultured cells under the experimental conditions (Fig. S7). Herein cultured LoVo cells were incubated with probe 1 (10 μM) for 30 min at 37 °C. As predicted, fluorescent images showed that probe 1 was cell membrane permeable and localized in the cytoplasm region (Figs. 6 and S8). These results clearly mean that probe 1 has good cell membrane permeability and could image organelles in living cells. Encouraged by the living cell experiments, we evaluated the effectiveness of probe 1 for fluorescent imaging in a living vertebrate organism, zebrafish. Five day old zebrafish were incubated in solutions of probe 1 at 10 μM and the distribution of fluorescence within the zebrafish was monitored (Fig. 7). Probe 1 showed significant uptake within the fish with clear regions of concentrated fluorescence in the yolk sac and alimentary canal, including the intestinal tract and biliary system (gall bladder, liver, pancreas, solitary islet and bile ducts). In addition, the probe itself was found to be rather stable in the body of zebrafish, because no noticeable change in the averaged ratio value was observed at least within 1 h. These in vivo studies showed that probe 1 can enter zebrafish, and image in living organism. 4. Conclusions In summary, a novel styrylcyanine-based fluorescent pH probe 1 was synthesized via facile methods. Probe 1 was green fluorescent in basic and neutral media but dark orange in strong acidic environments. Its emission could be reversibly switched between bright green and dark orange by repeated protonation and deprotonation. Such behavior enables it to work as a chemosensor for detecting volatile organic
compounds with high acidity and basicity in solid state. Subsequently, the fluorescent microscopic images of probe 1 in live cells and in the whole body of zebrafish were achieved successfully, suggesting that probe 1 has good cell membrane permeability and potential application for imaging in live cells and living organism. Acknowledgments This work was partially supported by the National Natural Science Foundation of China (Nos. 21244007, 81271642). Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.msec.2015.03.042. References [1] L. Fan, Y.J. Fu, Q.L. Liu, D.T. Lu, C. Dong, S.M. Shuang, Novel far-visible and near-infrared pH probes based on styrylcyanine for imaging intracellular pH in live cells, Chem. Commun. 48 (2012) 11202–11204. [2] A. Roos, W.F. Boron, Intracellular pH, Physiol. Rev. 61 (1981) 296–434. [3] J.L. Fan, M.M. Hu, P. Zhan, X.J. Peng, Energy transfer cassettes based on organic fluorophores: construction and applications in ratiometric sensing, Chem. Soc. Rev. 42 (2013) 29–43. [4] R.T. Kennedy, L. Huang, C.A. Aspenwall, Extracellular pH is required for rapid release of insulin from Zn-insulin precipitates in β-cell secretory vesicles during exocytosis, J. Am. Chem. Soc. 118 (1996) 1795–1796. [5] K.R. Hoyt, I.J. Reynolds, Alkalinization prolongs recovery from glutamate-induced increases in intracellular Ca2+ concentration by enhancing Ca2+ efflux through the mitochondrial Na+/Ca2+ exchanger in cultured rat forebrain neurons, J. Neurochem. 71 (1998) 1051–1058. [6] E.R. Chin, D.G. Alllen, The contribution of pH-dependent mechanisms to fatigue at different intensities in mammalian single muscle fibres, J. Physiol. 512 (1998) 831–840. [7] H. Izumi, T. Torigoe, H. Ishiguchi, H. Uramoto, Y. Yoshida, M. Tanabe, T. Ise, T. Murakami, T. Yoshida, M. Nomoto, K. Kohno, Cellular pH regulators: potentially promising molecular targets for cancer chemotherapy, Cancer Treat. Rev. 29 (2003) 541–549. [8] T.A. Davies, R.E. Fine, R.J. Johnson, C.A. Levesque, W.H. Rathbun, K.F. Seetoo, S.J. Smith, G. Strohmeier, L. Volicer, L. Delva, Non-age related differences in thrombin responses by platelets from male patients with advanced Alzheimer's disease, Biochem. Biophys. Res. Commun. 194 (1993) 537–543. [9] Z.G. Song, Y.N. Hong, R.T.K. Kwok, J.W.Y. Lam, B. Liu, B.Z. Tang, A dual-mode fluorescence “turn-on” biosensor based on an aggregation-induced emission luminogen, J. Mater. Chem. B 2 (2014) 1717–1723. [10] N. Kumari, S. Jha, S. Bhattacharya, An efficient probe for rapid detection of cyanide in water at parts per billion levels and naked-eye detection of endogenous cyanide, Chem. Asian. J. 9 (2014) 830–837. [11] R.A. Gottlieb, H.A. Giesing, J.Y. Zhu, R.L. Engler, B.M. Babior, Cell acidification in apoptosis: granulocyte colony-stimulating factor delays programmed cell death in neutrophils by up-regulating the vacuolar H(+)-ATPase, Proc. Natl. Acad. Sci. U. S. A. 92 (1995) 5965–5968.
102
X. Yang et al. / Materials Science and Engineering C 52 (2015) 97–102
[12] S. Ohkuma, B. Poole, Fluorescence probe measurement of the intralysosomal pH in living cells and the perturbation of pH by various agents, Proc. Natl. Acad. Sci. U. S. A. 75 (1978) 3327–3331. [13] J. Han, K. Burgess, Fluorescent indicators for intracellular pH, Chem. Rev. 110 (2010) 2709–2728. [14] R.R. Hu, R.Q. Ye, J.W.Y. Lam, M. Li, C.W.T. Leung, B.Z. Tang, Conjugated polyelectrolytes with aggregation-enhanced emission characteristics: synthesis and their biological applications, Chem. Asian. J. 8 (2013) 2436–2445. [15] L. Fan, Q.L. Liu, D.T. Lu, H.P. Shi, Y.F. Yang, Y.F. Li, C. Dong, S.M. Shuang, A novel far-visible and near-infrared pH probe for monitoring near-neutral physiological pH changes: imaging in live cells, J. Mater. Chem. B 1 (2013) 4281–4288. [16] L.C. Ho, C.M. Ou, C.L. Li, S.Y. Chen, H.W. Li, H.T. Chang, Sensitive pH probes of retroself-quenching fluorescent nanoparticles, J. Mater. Chem. B 1 (2013) 2425–2432. [17] F. Galindo, M.I. Burguete, L. Vigara, S.V. Luis, N. Kabir, J. Gavrilovic, D.A. Russell, Synthetic macrocyclic peptidomimetics as tunable ph probes for the fluorescence imaging of acidic organelles in live cells, Angew. Chem. Int. Ed. 44 (2005) 6504–6508. [18] R.A. Bissell, A.J. Bryan, A. Prasanna de Silva, C.P. McCoy, Fluorescent PET (photoinduced electron transfer) sensors with targeting/anchoring modules as molecular versions of submarine periscopes for mapping membrane-bounded protons, J. Chem. Soc. Chem. Commun. 4 (1994) 405–407. [19] N. Marcotte, A.M. Brouwer, Carboxy SNARF-4 F as a fluorescent ph probe for ensemble and fluorescence correlation spectroscopies, J. Phys. Chem. B 109 (2005) 11819–11828. [20] J. Han, A. Loudet, R. Barhoumi, R.C. Burghardt, K. Burgess, A ratiometric ph reporter for imaging protein-dye conjugates in living cells, J. Am. Chem. Soc. 131 (2009) 1642–1643. [21] Y.Q. Tian, F.Y. Su, W. Weber, V. Nandakumar, B.R. Shumway, Y.G. Jin, X.F. Zhou, M.R. Holl, R.H. Johnson, D.R. Meldrum, A series of naphthalimide derivatives as intra and extracellular pH sensors, Biomaterials 31 (2010) 7411–7422. [22] X.F. Zhou, F.Y. Su, H.G. Lu, P. Senechal-Willis, Y.Q. Tian, R.H. Johnson, D.R. Meldrum, An FRET-based ratiometric chemosensor for in vitro cellular fluorescence analyses of pH, Biomaterials 33 (2012) 171–180. [23] Z.Y. Yang, W. Qin, J.W.Y. Lam, S.J. Chen, H.H.Y. Sung, I.D. Williams, B.Z. Tang, Fluorescent pH sensor constructed from a heteroatom-containing luminogen with tunable AIE and ICT characteristics, Chem. Sci. 4 (2013) 3725–3730. [24] S.J. Chen, J.Z. Liu, Y. Liu, H.M. Su, Y.N. Hong, C.K.W. Jim, R.T.K. Kwok, N. Zhao, W. Qin, J.W.Y. Lam, K.S. Wong, B.Z. Tang, An AIE-active hemicyanine fluorogen with stimuliresponsive red/blue emission: extending the pH sensing range by “switch + knob” effect, Chem. Sci. 3 (2012) 1804–1809. [25] E. Galbraith, T.D. James, Boron based anion receptors as sensors, Chem. Soc. Rev. 39 (2010) 3831–3842.
[26] Q.Q. Zhang, M. Zhou, A profluorescent ratiometric probe for intracellular pH imaging, Talanta 131 (2015) 666–671. [27] R. Gui, X. An, W. Huang, An improved method for ratiometric fluorescence detection of pH and Cd2+ using fluorescein isothiocyanate-quantum dots conjugates, Anal. Chim. Acta 767 (2013) 134–140. [28] B.K. McMahon, R. Pal, D. Parker, A bright and responsive europium probe for determination of pH change within the endoplasmic reticulum of living cells, Chem. Commun. 49 (2013) 5363–5365. [29] T. Jokic, S.M. Borisov, R. Saf, D.A. Nielsen, M. Kuhl, I. Klimant, Highly photostable near-infrared fluorescent pH indicators and sensors based on BF2-chelated tetraarylazadipyrromethene dyes, Anal. Chem. 84 (2012) 6723–6730. [30] X. Du, N.Y. Lei, P. Hu, Z. Lei, D.H. Ong, X. Ge, Z. Zhang, M.H. Lam, In vivo imaging of the morphology and changes in pH along the gastrointestinal tract of Japanese medaka by photonic band-gap hydrogel microspheres, Anal. Chim. Acta 787 (2013) 193–202. [31] H.S. Lv, S.Y. Huang, B.X. Zhao, J.Y. Miao, A new rhodamine B-based lysosomal pH fluorescent indicator, Anal. Chim. Acta 788 (2013) 177–182. [32] H.S. Lv, S.Y. Huang, Y. Xu, X. Dai, J.Y. Miao, B.X. Zhao, A new fluorescent pH probe for imaging lysosomes in living cells, Bioorg. Med. Chem. Lett. 24 (2014) 535–538. [33] H.S. Lv, J. Liu, J. Zhao, B.X. Zhao, J.Y. Miao, Highly selective and sensitive pHresponsive fluorescent probe in living Hela and HUVE Ccells, Sensors Actuators B Chem. 177 (2013) 956–963. [34] M. Yang, Y. Song, M. Zhang, S. Lin, Z. Hao, Y. Liang, D. Zhang, P.R. Chen, Converting a solvatochromic fluorophore into a protein-based pH indicator for extreme acidity, Angew. Chem. 51 (2012) 7674–7679. [35] X. Zhang, S.Y. Jing, S.Y. Huang, X.W. Zhou, J.M. Bai, B.X. Zhao, New fluorescent pH probes for acid conditions, Sensors Actuators B Chem. 206 (2015) 663–670. [36] H. Li, H. Guan, X. Duan, J. Hu, G. Wang, Q. Wang, An acid catalyzed reversible ring-opening/ring-closure reaction involving a cyano-rhodamine spirolactam, Org. Biomol. Chem. 11 (2013) 1805–1809. [37] D.Y. Kim, H.J. Kim, pH-sensitive optode membrane covalently incorporated with an ICT dye forlow pH values, Sensors Actuators B Chem. 206 (2015) 508–515. [38] C.Q. Ding, Y. Tian, Gold nanocluster-based fluorescence biosensor for targeted imaging in cancer cells and ratiometric determination of intracellular pH, Biosens. Bioelectron. 65 (2015) 183–190. [39] M. Gao, C.K. Sim, C.W.T. Leung, Q.L. Hu, G.X. Feng, F. Xu, B.Z. T., B. Liu, A fluorescent light-up probe with AIE characteristics for specific mitochondrial imaging to identify differentiating brown adipose cells, Chem. Commun. 50 (2014) 8312–8315. [40] J.I.A. Rashid, N.A. Yusof, J. Abdullah, U. Hashimc, R. Hajian, The utilization of SiNWs/ AuNPs-modified indium tin oxide (ITO) in fabrication of electrochemical DNA sensor, Mater. Sci. Eng. C 45 (2014) 270–276.
