Ratiometric Emission Fluorescent pH Probe for Imaging of Living Cells

Feb 9, 2015 - ... ratiometric fluorescence emission (F522nm/F630nm) characteristics with pKa 3.27 and linear response to extreme-acidity range of 3.8â...
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Ratiometric Emission Fluorescent pH Probe for Imaging of Living Cells in Extreme Acidity Weifen Niu,†,‡ Li Fan,† Ming Nan,† Zengbo Li,† Dongtao Lu,† Man Shing Wong,†,§ Shaomin Shuang,† and Chuan Dong*,† †

Institute of Environmental Science, College of Chemistry and Chemical Engineering, Shanxi University, Taiyuan 030006, People’s Republic of China ‡ Department of Chemistry and Chemical Engineering, Yangtze Normal University, Chongqing, Fuling 408100, People’s Republic of China § Department of Chemistry and Institute of Advanced Materials, Hong Kong Baptist University, Hong Kong SAR, People’s Republic of China S Supporting Information *

ABSTRACT: A novel ratiometric emission fluorescent probe, 1,1dimethyl-2-[2-(quinolin-4-yl)vinyl]-1H-benzo[e]indole (QVBI), is facilely synthesized via ethylene bridging of benzoindole and quinoline. The probe exhibits ratiometric fluorescence emission (F522nm/F630nm) characteristics with pKa 3.27 and linear response to extreme-acidity range of 3.8−2.0. Also, its high fluorescence quantum yield (Φ = 0.89) and large Stokes shift (110 nm) are favorable. Moreover, QVBI possesses highly selective response to H+ over metal ions and some bioactive molecules, good photostability, and excellent reversibility. The probe has excellent cell membrane permeability and is further applied successfully to monitor pH fluctuations in live cells and imaging extreme acidity in Escherichia coli cells without influence of autofluorescence and native cellular species in biological systems.

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mammalian stomach.6,7 Thus, the precise detrmination of intracellular pH value under extreme-acidity conditions still faces considerable challenges. Ma and co-workers21 first developed a fluorescent probe, 1,9-dihydro-3-phenyl-4Hpyrazolo[3,4-b]quinolin-4-one, with a sensitive response under extremely acidic and extremely alkaline conditions, which made the probe feasible for further applications in the cell. Another promising finding has been reported by Chen and co-workers,22 namely, that a protein-based pH probe HdeA58DMN was prepared and expanded for measurement of extreme acidity in E. coli cells. This probe with the genetically encoded nature may provide a useful tool for monitoring the intracellular pH in diverse prokaryotic and eukaryotic species. More recently, a turn-off fluorescent probe based on coumarin and imidazole derivative has been developed for detection of extreme acidity (pKa = 2.1) in bacteria.23 However, the probe required ultraviolet excitation and may somewhat cause cellular damage due to lengthy irradiation. Meanwhile, it showed blue fluorescence emission where cells may have strong autofluorescence interference. Cells can emit broad autofluorescence covering the entire visible spectrum. Cellular autofluorescence can mask the signals

ntracellular pH plays many pivotal roles in a mass of cellular events, such as cell growth and apoptosis,1 endocytosis,2 receptor-mediated signal transduction,3 ion transport and homeostasis,4,5 and other cellular processes. In diverse prokaryotic species and different subcellular compartments of eukaryotic cells, there exist different pH homeostases.6,7 Abnormal pH values can cause cardiopulmonary and neurologic problems (e.g., cancer and Alzheimer’s disease).8,9 Therefore, monitoring pH in live cells is highly desirable for better understanding of physiological and pathological processes. Fluorometry has attracted much more attention over many other methods for pH detection due to its noninvasiveness, high sensitivity, excellent specificity, and fast response time. In particular, fluorescent imaging provides high spatial and temporal observation of pH changes in live cells10 with the combination of fluorescent probes and confocal laser scanning microscopy. Currently, many excellent pH-dependent fluorescent probes with near neutral11−16 or weak acidic response behavior17−19 have been exploited for applications in biological systems. Unfortunately, little research is reported on the development of extreme-acidity pH probes (pH < 4). Normally, extreme acidity is fatal for the majority of living organisms. However, some microorganisms such as acidophiles and Helicobacter pylori can live under such extreme conditions.6,20 Furthermore, enteric bacteria such as Escherichia coli and Salmonella species survive through the highly acidic © XXXX American Chemical Society

Received: November 4, 2014 Accepted: January 23, 2015

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Analytical Chemistry from probe molecules, making it difficult to distinguish the probe molecule form the background.24 To overcome this problem, two approaches are usually followed. One is to increase the fluorescence intensity of the probe. By increasing the fluorescence intensity, the target molecules will be highlighted from the background. The other approach is to shift the wavelength to the red or near-infrared region, where cells have weaker autofluorescence.25 Therefore, a pressing need exists to develop a fluorescent probe satisfying the multiple criteria of long-wavelength excitation/emission, high fluorescence quantum yield, and live-cell permeability for monitoring extreme acidity in living cells. Herein, we aim to design a pH probe with high quantum yield and near-infrared fluorescent emission for extreme-acidity response in living cells. Considering the inherent complexity and constant evolvement of cells, it will be desirable for the probe to possess ratiometric fluorescence emission characteristics. Ratiometric measurement is superior to single emission intensity measurement since it not only exhibits large spectral shifts but also provides ratiometric intensity at two different emission wavelengths, which can cancel out most possible effects of environmental variations, probe distribution, and instrumental performance and thus offer a more accurate analysis.26,27 The new fabricated probe by ethylene bridging of benzoindole and quinoline displays favorable optical properties for extreme-acidity pH detection in living cells.

Scheme 1. Synthetic Scheme for QVBI

temperature and filtered to remove NaOAc, and then filtrate was concentrated by evaporation under reduced pressure. The resultant product was purified by column chromatography with methylene chloride and methyl alcohol (40/3 v/v) as eluent to give a yellow solid, yield 0.66 g (38%). 1H NMR (DMSO-d6, 300 MHz), δ (ppm) 1.72 (s, 6H), 7.55−7.57 (t, 1H), 7.66− 7.73 (t, 1H), 7.73−7.78 (t, 2H), 7.78−7.91 (m, 2H), 8.0−8.13 (m, 4H), 8.22−8.24 (d, 1H), 8.41−8.44 (d, 1H), 8.59−8.64 (d, 1H), 8.99−9.01 (d, 1H). 13C NMR (DMSO-d6, 300 MHz), δ (ppm) 21.333, 53.759, 117.350, 119.760, 122.580, 123.001, 124.437, 124.879, 126.266, 126.669, 127.542, 128.580, 129.075, 129.886, 131.728, 139.378, 140.001, 147.796, 149.778, 150.235, 183.790. MS (MALDI-TOF) m/z 350.1608 for [M + H]+. UV−Vis and Fluorescence pH Titrations. Stock solution of QVBI (1.0 mM) was prepared in ethanol. The solution for spectroscopic determination was obtained by diluting the stock solution to 5 μM in ethanol/water (2/1 v/v) medium. In the pH titration experiments, 3 mL of solution was poured into a quartz optical cell of 1 cm optical path length, and slight pH variations of the solution were achieved by adding the minimum volumes of HCl (1.0 M). Spectral data were recorded after each addition. Excitation and emission bandwidths were both set at 2 nm, and the excitation wavelengths were 412 nm. All spectroscopic experiments were carried out at room temperature. Human Renal Carcinoma Cell Culture. Human renal carcinoma cells 7860 were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), and the cells were seeded in the culture dish and cultured at 37 °C in a 5% CO2 atmosphere for 48 h. Then the culture medium was removed, and the cells were washed with phosphate-buffered saline (PBS). QVBI dissolved in DMSO was added into different PBS buffers (pH 7.4 and 4.2) and incubated with cells for an additional 30 min at 37 °C. The concentration of QVBI in the buffer solutions was controlled at 30 μM. After that, the cells were washed three times in PBS, pH 7.4 or 4.2, to remove excess QVBI. Fluorescence images were collected on a confocal laser scanning microscope. E. coli Cell Culture. In light of the method reported in literature,23 E. coli cells were incubated at 37 °C in Luria− Bertani (LB) culture medium (tryptone 10 g/L, yeast extract 5 g/L, and NaCl 10 g/L) for 17 h in a table concentrator at 180 rpm. Then the culture was centrifuged at 5000 rpm for 5 min to collect E. coli cells. The sediment was washed with sterile water and then resuspended in solutions of different pH (1.8 and 7.4). Five minutes after resuspension, the probe QVBI dissolved in DMSO was added into every tube to a final probe concentration of 30 μM. E. coli cells with the probe were incubated in a table concentrator for 2 h and then smeared on slides and observed by confocal laser scanning microscope.

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EXPERIMENTAL SECTION Materials and Apparatus. All chemicals and solvents were of analytical grade and used without further purification. 1,1,2Trimethyl-1H-benzo[e]indole was purchased from Sigma− Aldrich. 4-Quinolinecarboxaldehyde was purchased from Alfa Aesar. Human renal carcinoma cells 7860 were kindly provided by Shanxi Medical University (China). All other chemicals were commercially available from Beijing Chemical Reagent Co. 1 H NMR spectra were recorded on a Bruker 300 MHz NMR spectrometer (Bruker biospin, Switzerland) in the solvent deuterated dimethyl sulfoxide (DMSO-d6) with tetramethylsilane (TMS) as an internal standard. Mass spectrometric data were obtained with a Bruker Autoflex II matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometer (Bruker Daltonics, Germany). Absorption spectra were taken on a TU-1901 double-beam UV−vis spectrophotometer (Beijing Purkinje General Instrument Co., Ltc., Beijing, China). Fluorescence spectra measurements were performed on an FLS-920 Edinburgh fluorescence spectrophotometer (Edinburgh Co., Ltd.) equipped with a xenon discharge lamp and a 1 mL fluor micro cell. Fluorescent images were taken on an FV1000 confocal laser scanning microscope (Olympus Co., Ltd. Japan) with an objective lens. E. coli was incubated in a table concentrator (Shanghai Yiheng Instruments Co., Ltd.) and centrifuged (Shanghai Anting Scientific Instrument Factory). Deionized water was obtained from a Milli-Q water purification system (Millipore). pH values were measured with a Beckman Φ 50 pH meter (Shanghai LeiCi Device Works, Shanghai, China). Synthesis and Characterization of Fluorescent Probe. The synthetic route of QVBI is depicted in Scheme 1. A mixture of 1,1,2-trimethyl-1H-benzo[e]indole (1.05 g, 5.0 mmol), 4-quinolinecarboxaldehyde (0.943g, 6.0 mmol), and NaOAc (0.493 g, 6.0 mmol) in Ac2O (30 mL) was stirred overnight at 80 °C under an nitrogen atmosphere. After completion, the reaction mixture was cooled to room

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RESULTS AND DISCUSSION Optical Response to pH. To study the optical responses of QVBI to pH, standard pH titrations of absorption spectra and fluorescence emission spectra were performed. Figure 1a shows the UV−vis absorption spectral change of QVBI at various pH values. As the pH is decreased from 6.7 to 1.5, the absorbance B

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Figure 1. (a) Change of absorption spectra of QVBI as pH decreased from 6.7 to 1.5. (Inset) Sigmoidal fitting of pH-dependent absorbance at 374 nm. (b) Change of fluorescence spectra of QVBI as pH decreased from 6.7 to 1.5 (λex = 412 nm). (Inset) Sigmoidal fitting of pH-dependent fluorescence intensity at 522 nm.

Scheme 2. Acid−Base Form Equilibrium

Figure 3. Fluorescence intensity of 5 μM QVBI in ethanol/water (2/1 v/v) at different pH in the presence of various species: (1) blank; (2) 30 mM Na+; (3) 30 mM K+; (4) 20 mM Mg2+; (5) 20 mM Ca2+; (6) 20 mM Ba2+; (7) 0.1 mM Fe3+; (8) 1 mM Fe2+; (9) 1.2 mM Al3+; (10) 3 mM Cu2+; (11) 20 mM Zn2+; (12) 20 mM Mn2+; (13) 15 mM Ni2+; (14) 1 mM Co2+; (15) 0.1 mM Cr3+; (16) 0.5 mM Pb2+; (17) 5 mM Cd2+; (18) 5 mM Hg2+; (19) 0.5 mM Ag+; (20) 2 mM glucose; (21) 2 mM leucine; (22) 2 mM DL-methionine; (23) 2 mM valine; (24) 2 mM L-threonine; (25) 2 mM cysteine; (26) 2 mM glycine; (27) 2 mM arginine; (28) 2 mM serine; (29) 2 mM histidine; (30) 5 mM vitamin C. λex = 412 nm.

Figure 2. 1H NMR (300 MHz) spectra of QVBI in DMSO at pH 6.7 (top), 3.6 (middle), and 2.0 (bottom).

at 374 nm is reduced and, concomitantly, a new peak at 434 nm appears and increases dramatically. A well-defined isosbestic point at 412 nm indicates that the binding of quinoline with H+ is the only reaction process (implying that no other reaction processes have occurred). The red shift in the absorption spectra confirms that the intramolecular charge transfer (ICT) effect of the probe is enhanced with decreasing pH because of the H+ binding-induced enhancement of the electron-withdrawing ability of quinoline. The inset in Figure 1a shows the results of sigmoidal fitting of the pH-dependent absorbance at 374 nm, affording a pKa value of 3.25. A further observation is that the fluorescent green of the solution fades away. Moreover, the molar extinction coefficient is an important parameter for evaluation of a fluorescent probe. According to Figure 1a, the molar extinction coefficient of QVBI could be obtained by use of the Lambert−Beer law in base form, ε1 = 92 000 L·mol−1· cm−1 (λmax = 374 nm, pH 6.7), and acid form, ε2 = 79800 L· mol−1·cm−1 (λmax = 434 nm, pH 1.5).

Figure 4. Changes in fluorescence intensity of QVBI between pH 6.7 (λem = 522 nm) and 1.8 (λem = 630 nm). λex = 412 nm.

As shown in Figure 1b, when the pH value is higher than 5.0, the QVBI solution exhibits an intense emission band centered at 522 nm (λex = 412 nm) with a large Stokes shift of 110 nm. The large Stokes shift could help to reduce the excitation interference. Then its fluorescence quantum yield (Φ) is 0.89 relative to quinine sulfate solution. This quantum yield is very high and can effectively overcome the autofluorescence of cells. When the pH is decreased from 5.0 to 1.5, the fluorescence C

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intensity is reduced gradually and the fluorescence emission spectra exhibit a red shift from 522 to 630 nm (pH < 2), a pronounced shift of 108 nm. The large red shift of the signals is attributed to the enhanced ICT effect. Meanwhile, the visual fluorescence of QVBI solution changes from green to red at pH 6.7 and 1.8; the photo is taken under illumination with a UV lamp (Figure 1b). The pKa value could also be calculated from the pH dependence of total integrated fluorescence emission at 522 nm with excitation at 412 nm. Sigmoidal fitting yields a pKa value of 3.27 (inset of Figure 1b), which was very close to that from absorption measurements. More importantly, the marked red shift of emission spectra provides a good opportunity to achieve ratiometric detection. When the pH decreases from 6.7 to 1.5, the emission ratio (F522nm/F630nm) also changes dramatically from 6.67 to 0.14 (Figure S1a, Supporting Information). From analysis of the emission ratio, a pKa of 3.18 is calculated. The emission ratio also shows good linearity with pH in the range 3.8−2.0, according to the linear regression equation F522nm/F630nm = −5.65 + 3.20pH, with a linear coefficient of 0.9944. Thus, this probe could be potentially useful for quantitative determination of extreme-acidity pH values by both normal and ratiometric fluorescence methods. Moreover, we investigated the fluorescence of QVBI at pH 6.7 and 1.5 with an excitation wavelength of 488 nm, and the result is shown in Figure S1b (Supporting Information). The fluorescence intensity of 630 nm at pH 1.5 is higher than that at pH 6.7 (λex = 488 nm). Considering the application of QVBI in the living cells, fluorescence pH titrations were carried out in the presence of 10% cell medium (DMEM) (Figure S2, Supporting Information). The properties of the probe in the presence of cell medium are consistent with Figure 1b. It demonstrates that the complex environment of the cell medium should not affect the probe response. Binding Behavior of Probe with H+. We designed the probe for binding of N of quinoline with H+ to be responsible for the probe ability to determine extreme-acidity pH, because the quinoline N favors H+ binding in acidic conditions. The pH sensitivity is introduced via the quinoline N moiety upon either protonation or deprotonation. The probe exists in two forms, as either the green fluorescent base form or the red fluorescent acid form (Scheme 2), and the absorption and fluorescence emission properties are due to the ICT effect between the two nitrogen atoms of benzoindole and quinoline. To confirm this, 1H NMR experiments were carried out. As shown in Figure 2, when 1 μL of DCl (1 M) is added to QVBI solution in DMSO-d6 (pH 3.6), a downfield shift is observed

Figure 5. Dynamic fluorescence imaging of human renal carcinoma cells 7860. (a−g) Image in green channel; (h) bright-field image of panel a; (i) temporal profile of average fluorescence intensity in regions of interest 1−8 shown in panels a−g. Excitation wavelength was 405 nm, and emission was collected in the green channel (500− 550 nm). Scale bar, 20 μm.

Figure 6. Confocal fluorescence imaging of human renal carcinoma cells 7860 incubated with 30 μM QVBI for 30 min at pH 7.4 (a) and 4.2 (d). (b) Bright-field image of panel a; (e) bright-field image of panel d; (c) merged image of panels a and b; (f) merged image of panels d and e. Excitation wavelength was 405 nm, and emission was collected in the green channel (500−550 nm). Scale bar, 20 μm.

Figure 7. Visualization of pH changes in E. coli by confocal laser scanning microscopy at pH 7.4 (a−e) and pH 1.8 (f−j). (a, f) Image in green channel (500−550 nm); (b, g) image in red channel (610−660 nm); (c, h) bright-field image of panels a and f; (d) merged image of panels a and c; (e) merged image of panels b and c; (i) merged image of panels f and h; (j) merged image of panels g and h. The excitation wavelengths were 405 and 488 nm for green and red fluorescence, respectively. Scale bar, 5 μm. D

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μM. We select eight cells within the red region of the visual field (Figure 5), and the fluorescence intensity is calculated by use of commercial software. As shown in Figure 5b−g,i, after 10 s, green fluorescence emission of cells is observed. Most notably, QVBI can easily and quickly diffuse into cells. With extended times, the fluorescence intensity of the cells increases gradually. After about 80 s, the fluorescence intensity is basically stable but also no fluorescence quenching is observed until 650 s. This dynamic fluorescence imaging experiment demonstrates that the probe has excellent cell membrane permeability. Moreover, we used BIU-87 cells as a model to study the photostability under laser (Figures S5 and S6, Supporting Information). The experimental results indicate that the fluorescence intensity of most cells shows no obvious quenching within 10 min. After that, the fluorescence intensity of the cells is reduced gradually, and after about 30 min, it is basically stable until 180 min. The green fluorescence is still clearly observed. In view of the excellent cell membrane permeability, we are likely to study pH-dependent fluorescence imaging in living cells. Human renal carcinoma cells 7860 are incubated with QVBI for 30 min at 37 °C in different PBS buffers (pH 7.4 and 4.2). The concentration of QVBI in the medium is controlled at 30 μM. Fluorescence images are collected on a confocal laser scanning microscope. QVBI is excited at 405 nm and its green emission is collected in the green channel (500−550 nm). As shown in Figure 6, the cells exhibit strong green fluorescence emission (Figure 6a) at pH 7.4, and at pH 4.2 the fluorescence intensity clearly decreases (Figure 6d). This indicates that QVBI is capable of imaging pH fluctuations in living cells. The unique behavior of QVBI for ratio measurement of extreme acidity prompts us to apply it for monitoring pH changes in living samples. Next, we use the confocal laser scanning microscope to directly visualize the fluorescence change of E. coli cells incubated with QVBI under various pH conditions in its periplasmic space (Figure 7). The green fluorescence (500−550 nm) and red fluorescence (610−660 nm) are captured by 405 and 488 nm laser, respectively. It is obvious that the E. coli cells display bright green fluorescence (Figure 7a) and very weak red fluorescence (Figure 7b) emission at pH 7.4. When the extracellular pH decreases to pH 1.8, the green fluorescence intensity drops sharply (Figure 7f); by contrast, the red fluorescence intensity increases slightly (Figure 7g). The results are consistent with Figure 1b and Figure S1b (Supporting Information) and confirm that E. coli cells could live under such highly acidic conditions, which may serve as a source to cause infections. Moreover, the probe is able to image under extreme acidity in E. coli cells and we believe that it can also play an important role in other biological systems with extreme-acidity conditions. Furthermore, we quantify the average fluorescence intensity of the selected seven E. coli cells (Figure 7) by use of commercial software and calculate the ratio of the fluorescence intensity at green channel and red channel (Fgreen/Fred). Figure S7 (Supporting Information) depicts the ratio by histogram. It is seen that over 12-fold ratio decrease is observed when the extracellular pH drops from 7.4 to 1.8, which is perfectly in agreement with the ratiometric fluorescence behavior in Figure S1a (Supporting Information). This result demonstrates that the ratiometric measurement is very reliable. QVBI, to our knowledge, is the first ratiometric emission fluorescent pH probe for noninvasively monitoring extremely acidic extracellular pH.

for the chemical shift values of the quinoline protons H1, H2, H3, and H6 and vinyl protons H8. When 10 μL of DCl (1 M) is added to above solution (pH 2.0), the chemical shift values of quinoline protons H1, H2, H3, H4, H5, and H6 and vinyl protons H8 shift downfield, and the chemical shift values of benzoindole protons remain almost constant. The downfield chemical shift of these protons is obviously due to H+ binding with quinoline N, which results in the decrease of electron density around these protons. Thus, it is clear that H+ binding with quinoline N in probe QVBI causes the significant optical response to acidic pH. Selectivity Studies. It is noteworthy that QVBI shows high selectivity toward H+ over competitive species such as metal ions and some bioactive molecules at pH 6.7, 3.5, and 1.8, respectively. As shown in Figure 3, high concentrations (over their physiological concentrations) of metal ions and some bioactive molecules cause no visible effect on the fluorescence intensity of the probe at pH 6.7, 3.5, and 1.8. These results reveal that QVBI shows an excellent selectivity response to H+ in the presence of metal ions and some bioactive molecules. Photostability and Reversibility of QVBI. The stability of the probe is tested by measuring the fluorescent response during 3 h. Figure S3 (Supporting Information) shows the time course of fluorescence intensity of the probe (5 μM) at pH 6.7, 3.7, and 1.8 at room temperature. The fluorescence intensity is continuously monitored and recorded. The experimental results indicate that the probe can instantly respond to the change of H+ concentration and the probe solution possesses good photostability. Thus, the probe can be used to monitor pH variation in real time. Reversible response is another very important parameter to assess the performance of a new fluorescent probe. The pH value of the solution is switched back and forth between 6.7 and 1.8 by using concentrated hydrochloric acid and aqueous sodium hydroxide. As shown in Figure 4, the results clearly reveal that these processes are fully reversible. The response and recovery times in different pH solutions are rapid, within seconds. Meanwhile, the visual fluorescence color of solution changes between green (pH 6.7) and red (pH 1.8). It is clear that QVBI exhibits a highly reversible response to pH. Cell Cytotoxicity Assay. Since QVBI shows excellent selectivity to H+ and high photostability, it is possible to explore its use in intracellular pH imaging. Before that, it is crucial to evaluate the cytotoxicity of QVBI to living cells by MTT assay (see Supporting Information). Figure S4 depicts the viability of human renal carcinoma cells 7860 under various probe concentrations from 0.01 μM to 50 μM. The results demonstrate that more than 82% of cells are viable, showing the low toxicity of the probe to cultured cells under experimental conditions and inferring their potential use in intracellular imaging of living cells. Imaging of Living Cells. In order to explore potential applications in imaging of living cells, primarily, QVBI is employed for cell membrane permeability. Dynamic fluorescence imaging is performed to observe the process of QVBI diffusing into cells in real time by confocal laser scanning microscope. Cultured human renal carcinoma cells 7860 in culture dish with PBS are placed under the lens of a confocal laser scanning microscope, and immediately recording begins of fluorescence imaging in the green channel (500−550 nm) by a 405 nm laser. Figure 5a shows that no fluorescence is observed. Then a certain volume of QVBI is directly added into the culture dish. The concentration of QVBI is controlled at 30 E

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(9) Davies, T. A.; Fine, R. E.; Johnson, R. J.; Levesque, C. A.; Rathbun, W. H.; Seetoo, K. F.; Smith, S. J.; Strohmeier, G.; Volicer, L.; et al. Biochem. Biophys. Res. Commun. 1993, 194, 537−543. (10) Han, J.; Burgess, K. Chem. Rev. 2010, 110, 2709−2728. (11) Tang, B.; Yu, F. B.; Li, P.; Tong, L.; Duan, X.; Xie, T.; Wang, X. J. Am. Chem. Soc. 2009, 131, 3016−3023. (12) Fan, L.; Liu, Q. L.; Lu, D. T.; Shi, H. P.; Yang, Y. F.; Li, Y. F.; Dong, C.; Shuang, S. M. J. Mater. Chem. B 2013, 1, 4281−4288. (13) Han, J.; Loudet, A.; Barhoumi, R.; Burghardt, R. C.; Burgess, K. J. Am. Chem. Soc. 2009, 131, 1642−1643. (14) Nakata, E.; Yukimachi, Y.; Nazumi, Y.; Uto, Y.; Maezawa, H.; Hashimoto, T.; Okamoto, Y.; Hori, H. Chem. Commun. 2010, 46, 3526−3528. (15) Saha, U. C.; Dhara, K.; Chattopadhyay, B.; Mandal, S. K.; Mondal, S.; Sen, S.; Mukherjee, M.; Smaalen, S. V.; Chattopadhyay, P. Org. Lett. 2011, 13, 4510−4513. (16) Zhou, J.; Fang, C. L.; Chang, T. J.; Liu, X. J.; Shangguan, D. H. J. Mater. Chem. B 2013, 1, 661−667. (17) Fan, L.; Fu, Y. J.; Liu, Q. L.; Lu, D. T.; Dong, C.; Shuang, S. M. Chem. Commun. 2012, 48, 11202−11204. (18) Zhu, H.; Fan, J. L.; Xu, Q. L.; Li, H. L.; Wang, J. Y.; Gao, P.; Peng, X. J. Chem. Commun. 2012, 48, 11766−11768. (19) Li, G. P.; Zhu, D. J.; Xue, L.; Jiang, H. Org. Lett. 2013, 15, 5020−5023. (20) Merrell, D. S.; Camilli, A. Curr. Opin. Microbiol. 2002, 5, 51−55. (21) Su, M. H.; Liu, Y.; Ma, H. M.; Ma, Q. L.; Wang, Z. H.; Yang, J. L.; Wang, M. X. Chem. Commun. 2001, 960−961. (22) Yang, M. Y.; Song, Y. Y.; Zhang, M.; Lin, S. X.; Hao, Z. Y.; Liang, Y.; Zhang, D. M.; Chen, P. R. Angew. Chem., Int. Ed. 2012, 51, 7674−7679. (23) Xu, Y.; Jiang, Z.; Xiao, Y.; Bi, F. Z.; Miao, J. Y.; Zhao, B. X. Anal. Chim. Acta 2014, 820, 146−151. (24) Monici, M. Biotechnol. Annu. Rev. 2005, 11, 227−256. (25) Wu, W. W.; Li, A. DQ. Nanomedicine 2007, 2, 523−531. (26) Komatsu, K.; Urano, Y.; Kojima, H.; Nagano, T. J. Am. Chem. Soc. 2007, 129, 13447−13454. (27) Long, L. L.; Li, X. F.; Zhang, D. D.; Meng, S. C.; Zhang, J. F.; Sun, X. L.; Zhang, C.; Zhou, L. P.; Wang, L. RSC Adv. 2013, 3, 12204−12209.

CONCLUSION In summary, we have facilely synthesized a ratiometric emission fluorescent probe QVBI, based on ethylene bridging of benzoindole and quinoline. QVBI displays strong pH-dependent behavior, and the emission ratio is highly sensitive within the extremely acidic range. QVBI also shares some other desired properties, such as high fluorescence quantum yield, which contributes to overcome the autofluorescence of cells; large Stokes shift, which can reduce the excitation interference; as well as good selectivity and excellent photostability and cell membrane permeability. Since QVBI holds the perfect properties, it is used to noninvasively measure extremely acidic pH in E. coli cells as a ratiometric emission fluorescent probe. To our knowledge, QVBI represents the first ratiometric emission fluorescent probe for monitoring extreme acidity in living cells. Moreover, QVBI also is applied to monitor intracellular pH changes of human renal carcinoma cells successfully. It is anticipated that QVBI is a promising candidate for real-time tracking of pH changes, especially under extremely acidic conditions in the biomedical and biological fields.

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ASSOCIATED CONTENT

S Supporting Information *

Additional text, describing calculation of quantum yield and cell cytotoxicity assay, and 10 figures, showing fluorescence spectra and fitting of intensity, cell cytotoxic effects, photostability measurements, comparison of red and green fluorescence intensity, 1H and 13C NMR spectra of QVBI, and MALDI-TOF MS spectra. This material is available free of charge via the Internet at http://pubs.acs.org/.

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AUTHOR INFORMATION

Corresponding Author

*E-mail [email protected]; fax +86-351-7018613. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (21175086, 21175087, and 21475080), the Shanxi International S&T Cooperation Program of China (2011081017), and Natural Science Foundation of Chongqing Municipal Education Commission (KJ1401210). We appreciate Shanxi Medical University (Taiyuan, China) for kindly providing us with the living cells. M.S.W. thanks the Hundred Talent Program of Shanxi Province for the financial support.

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REFERENCES

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