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A New Dual-modal Colorimetric/Fluorescence Molecular Probe for Ratiometric Sensing of pH and Its Application Luling Wu, Xiaolin Li, Chusen Huang, and Nengqin Jia Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b02398 • Publication Date (Web): 19 Jul 2016 Downloaded from http://pubs.acs.org on July 20, 2016

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Analytical Chemistry

A New Dual-modal Colorimetric/Fluorescence Molecular Probe for Ratiometric Sensing of pH and Its Application Luling Wu, Xiaolin Li, Chusen Huang*, Nengqin Jia* The Education Ministry Key Laboratory of Resource Chemistry, Shanghai Key Laboratory of Rare Earth Functional Materials, and Shanghai Municipal Education Committee Key Laboratory of Molecular Imaging Probes and Sensors, Department of Chemistry, Shanghai Normal University, 100 Guilin Road, Shanghai 200234, China. E-mail: [email protected]; [email protected]. Fax: 86-21-64321833 ABSTRACT: As traditional pH meters cannot work well for minute regions (such as subcellular organelles) and in harsh media, molecular pH-sensitive devices for monitoring pH changes in diverse local heterogeneous environments are urgently needed. Here we report a new dual-modal colorimetric/fluorescence merocyanine-based molecular probe (CPH) for ratiometric sensing of pH. Compared with previously reported pH probes, CPH bearing the benzyl group at the nitrogen position of indolium group and the phenol which is used as the acceptor for proton could response to pH changes immediately through both the ratiometric fluorescence signal readout and naked-eye colorimetric observation. The sensing process exhibited highly stable and reversible. Most importantly, the suitable pKa value (6.44) allows CPH to presumably accumulate in lysosomes and become a lysosome-target fluorescent probe. By using CPH, the intralysosomal pH fluctuation stimulated by antimalaria drug chloroquine was successfully tracked in live cells through the ratiometric fluorescence images. Additionally, CPH could be immobilized on test papers, which exhibited a rapid and reversible colorimetric response to acid/base vapor through the naked-eye colorimetric analysis. This proof-of-concept study presents the potential application of CPH as the molecular tool for monitoring intralysosomal pH fluctuation in live cells, as well as paving a way for developing the economic, re-usable, and fast-response optical pH meters for colorimetric sensing acid/base vapor with direct naked-eye observation.

INTRODUCTION As traditional pH meters cannot work well for minute regions and in harsh media, molecular pH-sensitive devices for monitoring pH changes in diverse local heterogeneous environments are urgently needed. For instance, intracellular pH can powerfully influence the cellular homeostasis and many, probably most, physiological processes.1 The pH fluctuation of lysosome, one of the acid subcellular compartments, might influence the protein degradation, plasma membrane repair and even the cell death.2,3 Similarly, the abnormal cellular pH values are considered closely related to many diseases, such as cancer cells have a lower intracellular pH value (6.7-7.1) than extracellular pH (above 7.4), while the normal extracellular pH (about 7.4) is commonly higher than the intracellular pH value (about 7.2). This dysregulated pH in cancer cells and their microenvironment could be responsible for promoting the cancer cell proliferation, metabolic adaptation, cell migration and invasion.4 Thus development of new technology for determining the pH fluctuation in minute regions, such as lysosome in cells, could provide us an insight into investigating and understanding the pH-dependent cell behaviors that could be beneficial for designing pH-related disease therapeutics. On the other side, the pH value also plays crucial role in monitoring the environment changes. Specifically, a low-cost, easy-totake and portable test paper for detect pH changes in harsh environmental conditions (such as for testing the acid vapor or base vapor in industrial plants) is an urgent need. The last decades have witnessed the great advance in optical chemical probes for pH detection in exploiting the pH fluctua-

tion in subcellular compartments,5,6 extracellular matrix,7 whole cells8 and environment science.9 On the construction of these optical chemical probes, a variety of dyes including fluorescein, naphthalimide, cyanine and BODIPY etc. were introduced because the dye based optical pH probes enable us to dynamically monitor the pH changes through the visible fluorescence and/or color changes of the dyes, and thus enables the pH detection with a high sensitivity and spatiotemporal resolution.10-12 However, most of the dye based pH probes work through the single fluorescence intensity changes,10,11,13,14 which will be interfered by dye aggregation and excitation. Thus, to measure the pH, especially for the quantitative determination, with these single-wavelength probes requires a preliminary calibration. Such obstacles can be overcome by a ratiometric detection mechanism. Most recently, some pioneering works have been carried out for ratiometric pH sensing, and makes the intracellular pH sensing and imaging more accurate and high resolution through the ratiometric fluorescence signal readout.8,11,15-21 But, the majority of these ratiometric probes focus on fluorescence mapping of the cellular pH changes, and much of these probes can not work well for colorimetric detection of pH. This is particularly severe in the environment monitoring, as the naked-eye colorimetric analysis combined with test papers is low-cost, easyto-take and the most convenient tools for monitoring the environmental pH changes. Especially, it is great demand for the acid/base vapor detection, where the traditional pH test can not work well.

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Cyanine and hemicyanine dyes with high molar extinction coefficients at the maximum absorption wavelength and water solubility, as well as the low cell toxicity have been widely explored for colorimetric and/or fluorescence sensing.21-23 However, most of cyanine and hemicynaine based pH indicators work with the reversible protonation of amine nitrogen atom of indolium group of the cyanine dyes, which could induce the reversible “off-on” fluorescence changes, but no ratiometric fluorescence detection could be realized. The conjugation between hemicyanine with another dyes such as aggregation-induced emission (AIE) dyes,24,25 Rhodamine dyes26, spirocyanine,27 quinoline17 and benzothiazole,28 as well as the FRET donor (or acceptor)23 can address this obstacles.11,21,23 But, all above conjugations need elegant design and/or complex synthesis. Additionally, there is still rarely cyanine and merocyanine-based naked-eye probes reported for colorimetric detection of pH. Herein, we presented a new merocyaninebased molecular probe (CPH, scheme 1) for ratiometric detection of pH. It can work well in both the colorimetric and fluorescence modal. The working principle of CPH is illustrated in Scheme 1. The probe was constructed based on merocynaine bearing two major moieties, the first one is the benzyl group which was introduced at the nitrogen position of indolium group on merocyanine dye. Another moiety is the phenol, which is used as the acceptor for proton. Compared to previously reported merocyanine based pH indicators, CPH has following properties: i) The N-benzyl substituent on indole nitrogen will make this merocyanine more stable than the reported merocynaine containing N-alkyl substituents, which will hinder the nucleophilic addition of OH- to C=N bond of indole group. ii) The bearing phenol moiety will be response to pH changes through the reversible phenol and phenolate change. Based on above structure-property relationships, CPH can work well for dual-modal colorimetric/fluorescence ratiometric sensing of the fluctuation of pH. iii) CPH could be used as lysosome probe in live cell imaging and sensing the pH changes in lysosomes through the ratiometric fluorescence signal readout. iv) CPH also could be immobilized on test papers, which could exhibit a rapid and reversible colorimetric response to acid/base vapor through the naked-eye colorimetric analysis.

calibrated with pH 4 and pH 9.18 buffers before use. 1H and 13 C NMR spectra were recorded employing a Bruker AV-400 spectrometer with chemical shifts expressed in parts per million (Me4Si as internal standard). Electrosprayionization (ESI) mass spectrometry was performed in a HP 1100LC-MS spectrometer. Fluorescence measurements were determined on a Hitachi Fluorescence spectrophotometer F-7000. Excitation and emission slit widths were modified to adjust the fluorescence intensity to a suitable range. Absorption spectra were measured on a Hitachi U-3900 UV/VIS spectrophotometer. Synthesis of target probe CPH (detailed synthetic procedures please see the supporting information). To a solution of 2 (1.23g, 3.29 mmol, 1.0 eq, detailed s) and in anhydrous ethanol (EtOH), 4-hydroxybenzaldehyde (0.48 g, 3.95 mmol) was added under nitrogen atmosphere. The reaction mixture was continued to stirring for about 10 h at 80 oC. TLC showed the reaction was completed, filtrated. The solvent was evaporated in vacuo and the residue was purified by flash column chromatography eluting with CH2Cl2/CH3OH (12:1) to afford product as kermesinus solid (1.04 g, 66% yield). 1H NMR (400 MHz, DMSO-d6) δ 8.52 (d, J = 15.8 Hz, 1H), 8.11 (t, J = 8.7 Hz, 2H), 7.96 (d, J = 8.3 Hz, 2H), 7.89 (dd, J = 6.2, 2.3 Hz, 1H), 7.68 (dd, J = 6.5, 2.0 Hz, 1H), 7.58 (d, J = 15.9 Hz, 1H), 7.54 (t, J = 2.6 Hz, 1H), 7.46 (d, J = 8.3 Hz, 2H), 6.94 (s, 1H), 6.92 (s, 1H), 6.04 (s, 2H), 1.86 (s, 6H); 13C NMR (100 MHz, DMSO-d6) δ 183.41, 167.46, 164.96, 156.85, 143.98, 141.56, 139.40, 135.03, 131.40, 130.82, 129.62, 129.48129.39, 127.64, 126.66, 123.82, 117.37, 115.51, 109.24 , 52.71, 49.46, 26.93. HRMS (ES+): calc. for C26H24NO3 [MBr]+ 398.1756, found 398.1759. Cell culture. HeLa cells were obtained from American Type Culture collection, and grown in DMEM (High glucose) medium supplemented with 10% FBS. Cells were incubated in a 5% CO2 humidified incubator at 37 oC and typically passaged with sub-cultivation ratio of 1:4 every two days. Intracellular fluorescence imaging with CPH. The Hela cells were seeded into confocal petri dwash in complete medium (90% DMEM and 10% FBS), and then incubated for 12 h under standard culture conditions (atmosphere of 5% CO2 and 95% air at 37°C) to allow the cells attach. The cells were washed three times with DMEM, and then were incubated with 2 mL of probe CPH (50 µM) for 30 min under standard culture conditions. Then the cells were washed once with DMEM, and loaded with fresh DMEM for imaging. Fluorescence images were collected by Leica TCS SP5 II confocal laser scanning microscopy using HC× PLAPO 63X oil objective (NA: 1.40), probe CPH excited at 488 nm and it's emissions were collected in the range of 500-550 nm (channel 1, green) and 570-630 nm (channel 2, yellow).

Scheme 1. Design strategy and proposed mechanism for ratiometric detection of pH changes through the acid-base equilibrium and resonance structure of probe CPH.

RESULTS AND DISCUSSION

EXPERIMENTAL SECTION Materials and methods. All chemical reagents and solvents were purchased from commercial sources and used without further purification. Thin-layer chromatography (TLC) was performed on silica gel plates and visualized by UV. Column chromatography was performed using silica gel (Hailang, Qingdao) 300-400 mesh. Cell Counting Kit-8 (CCK-8) was purchased from SIGMA-ALDRICH. All pH measurements were carried out using a pH meter (Leici PHB-4) which was

Design strategy and synthesis. Our investigation begins with the design of a colorimetric and fluorometric dual-modal chemical probe for ratiometric detection of pH fluctuation. Since the merocyanine dyes have a typical D-π-A structure and high molar extinction coefficients at maximum absorption wavelength,29 many colorimetric and fluorometric dual-modal merocynaine-based probes have been developed for cyanide,30 mercury ion31,32 and fluoride detection.33 The mecrocyanine based pH probes have also been designed for live cell imaging. But, most of these merocyanine based pH probes work through the reversible nucleophilic addition of OH- to the


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C=N bond in the indole group of merocyanine or based on photoinduced electron transfer with conjugation with another proton-sensitive nitrogen-containing modulators.11 Consequently, many of these probes are fluorescence “off-on” probe for pH changes and can not work with ratiometric analysis. By the elegant design and complex synthesis procedures, some merocyanine-based derivatives through the conjugation with AIE dyes,24,25 Rhodamine dyes26 and spirocyanine,27 as well as the FRET donor (or acceptor)23 have been prepared as ratiometric fluorescent pH probe for live cell imaging. Nonetheless, these probes still can not realize the colorimetric detection of pH changes through the naked-eye observation, especially for detecting the acid/based vapor. Inspired by the photophysical property of merocyanine, we designed and synthesized target probe CPH, which has N-benzyl substituent on indole nitrogen to hinder the nucleophilic addition of OH- to C=N bond of indole group of CPH and phenol moiety that was taken as the selective proton acceptor. Consequently, CPH will exhibit a typical D-π-A structure and displayed a dual-modal colorimetric and fluorometric response to pH fluctuation through the intramolecular charge transfer (ICT) effect. Meanwhile, compared with the synthetic procedures for other cyanime and merocynaine based pH probes, the extreme convenience of synthesis is another special attraction of the target probe CPH. As shown in Scheme S1, the commercially available phenylhydrazine is easily converted to 2,3,3-trimethyl3H-indole, which can be further treated with 4(bromomethyl)benzoic acid and 4-hydroxybenzaldehyde, respectively, in two synthetic steps to afford the final compound CPH. Characterization of CPH performance towards pH. Next, we explored the colorimetric and fluorometric response of CPH towards pH changes. Initially, the pH titration was conducted in water. The maximum absorption of CPH gradually decreased at 439 nm and concomitantly increased at 535 nm with pH values increased from 3 to 11. An isosbestic point appears at 471 nm (Figure 1A). Then through the naked-eye observation, the color of CPH solution changes from yellow to red with the

Figure 1. (A) Absorption spectra of target probe CPH (10 µM) at different pH values in water. (B) Visual pictures of 20 µM CPH (40mM HEPES buffer containing 1% DMSO as co-solvent) at pH 4.0 to 10.0 under natural light. (C) Emission spectra of CPH (10 µM) at different pH in water (λex = 471 nm). (D) Emission intensity ratio changes (F522 /F559) over the pH range 3–11. The red line represents the non-linear fitting of the experimental data by origin software (version 8.0).

increment in pH values (Figure 1B), it is interesting that the colorimetric transition was reversible upon pH fluctuation. The fluorescence behavior of emission spectra was similar when excited at 471 nm. The maximum emission at 522 nm decreased with concomitant ingrowth of fluorescence emission at 559 nm (Figure 1C) upon the enhancement of pH values. Meanwhile, fluorescence changes of the test solution from bright green to magenta (Figure 1C, inset) was visible with UV lamp irradiation (365 nm). Additionally, fluorescence intensity ratio between maximum emission at 522 and 559 nm was recorded in Figure 1D, which displayed that the intensity ratio changed gradually decreased with the enhancement of pH values. And the pKa value was calculated to 6.44 by using Hasselbach-type mass action equation (detailed calculation please see the supporting information). In order to elucidate the proposed mechanism, 1H-NMR titration was conducted in DMSO-d6 solution of CPH with addition of Et3N, then the spectrum was reinstalled by addition of CF3COOD. As shown in Figure 2 and Figure S1, compared with spectra of CF3COOD reinstalled and no Et3N treated CPH solution, the addition of Et3N could induce the up-field shift for protons at c, d, j, l, k position, which were assigned to vinylene protons, benzyl protons and aromatic protons of benzyl moiety. However, it is interesting that no chemical shift and splitting was observed for peak e, which was assigned to methyl protons on the indolium moiety (Figure S1). Meanwhile, there was also no splitting for peak j, assigned to benzyl protons. These results were different from previous reported literatures that suggested the peak e and j were split into two groups because of the breakage of the symmetry of the structure of merocyanine through the nucleophilic addition of OHto C=N bond of indole group.24,34,35 Thus, we deduced that Nbenzyl substituent on indole nitrogen will prevent the nucleophilic addition of OH- to C=N bond of indole group of CPH, leading to the different pH-sensing mechanism from reported merocyanine based pH probes, whereby the fluorescence of merocyanine part will be quenched upon destroying the large p-electron conjugation system of merocyanine through the addition of OH- to C=N bond of indole group. Herein, the upfield shift of c, d, j, l, k

Figure 2. 1H NMR spectra of probe in 0.5 mL of DMSO-d6, (A) before and (B) after addition of Et3N. (C) The spectrum was reinstalled by addition of CF3COOD.

peaks were ascribed to the deprotonation of phenol and carboxyl moiety of CPH (Scheme 1). The splitting for protons at



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f, g, h, i positions disappeared and hard to be assigned because of the spectral overlaps (Figure 2B), due to that CPH exists in the base form. All these results indicated the mechanism for colorimetric and fluorometric response of CPH towards pH changes is mainly based on ICT effect of CPH through the reversible phenol and phenolate change. This deduced mechanism can be also supported by the red shift in absorption and emission of CPH spectra with increased pH values (Figure 1A, 1C). Then pH reversibility study of CPH in water between pH 3.0 and 11.0 was conducted. Initially, the pH value was modulated at 3.0, the ratio of the fluorescence intensity at 522 and 559 nm was recorded. Then the pH values increased to 11.0 led to a decrease in fluorescence intensity ratio, which could be quickly recovered by decreasing the pH values to 3.0. This sensing process could be reversibly performed at least 6 cycles (Figure 3), indicating CPH was a highly reversible probe for pH sensing. Meanwhile, the stability of CPH was also studied by measuring the fluorescence intensity ratio changes over 120 min. There were no remarkable ratio changes at pH 4.0, 7.8 and 10.4, respectively, demonstrating the sensing process was stable.

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terference reagents, respectively. The results suggested the high selectivity of CPH towards pH. Fluorescence imaging with CPH in live cells. All the above results promoted us to investigate the fluorescence response of CPH to intracellular pH in live cells. Firstly, the cytotoxicity of CPH was evaluated by CCK-8 assay. About 90% cell viability could be observed after the Hela cells were treated with CPH for 12 h at the concentration between 0 and 70 µM. There were still no remarkable influence on cell viability when the incubation times extends to 24 h. (Figure S2). Then the ratiometric fluorescence imaging with CPH in live Hela cells was performed. The cells were treated with CPH and incubated for 30 min, followed by washing with DMEM for one time. After the fresh DMEM was loaded, the fluorescence images were taken by confocal microscopy. A strong fluorescence signal in channel 1 (green, 500-550 nm) but nearly background fluorescence signal in channel 2 (yellow, 570630 nm) was observed from intracellular zone (Figure S3). It is notably that the fluorescence signal from channel 1 is predominantly localized in the small and spherical zone, which indicated the lysosomal localization. This phenomena could be attributed to the suitable pKa value of CPH (approximately 6.44 according to Figure 1). Generally, the pH in lysosomes is about 3.5-6.0,1,36,37 which makes CPH exist in the acid form. Thereby the strong fluorescence from channel 1 (green, Figure S3) while negligible fluorescence from channel 2 (yellow, Figure S3) was observed. Additionally, compared with the acid microenvironment in lysosome, CPH with pKa value of 6.44 is a relatively weak base. As results, CPH would presumably accumulate in the lysosomes.38,39

Figure 3. (A) pH reversibility study of CPH (10 µM) in water (λex = 471 nm) between pH 11.0 and 3.0. (B) The time courses of fluorescence intensity ratio of CPH (10 µM) in water at different pH values (4.0, 7.8 and 10.4, respectively). (C-D) Fluorescence intensity ratio changes of 10 µM CPH in 40 mM HEPES buffer at pH 6.80 (c) and 8.00 (d) in the presence of diverse metal ions and bioactive small molecules, respectively. 1, blank; 2, Al3+ (50 µM); 3, Zn2+ (50 µM); 4, Ca2+ (50 µM) ; 5, Cu2+ (50 µM); 6, Fe2+ (50 µM); 7, Fe3+ (50 µM); 8, Mg2+ (50 µM); 9, NH4+ (50 µM); 10, F(50 µM); 11, Cl- (50 µM); 12, Br- (50 µM); 13, I- (50 µM); 14, SO42- (50 µM); 15, H2S (50 µM); 16, Cysteine (50 µM); 17, Homocysteine (50 µM); 18, Glycine (50 µM); 19, Arginine (50 µM); 20, Histidine (50 µM); 21, Glutathione (50 µM); 22, Glucose (50 µM); 23, ATP (Adenosine triphosphate, 50 µM). λex = 471nm. Error bars represent s.d..

To further explore if CPH could still work well in complex environments, some metal ions and bioactive small molecules were taken as the interference reagents to test the selective response of CPH to proton. The assays were conducted by adding 5-fold higher concentration of different interference reagents into the CPH solution at pH 6.8 and 8.0, respectively. As displayed in Figure 3C-D, there were no significant changes in fluorescence intensity ratios when the CPH solutions were treated with 5-fold higher concentration of different in-

Figure 4. Lysosome-targeting properties of probe CPH in Hela cells. (A-B) The co-localization images of CPH and Lyso-Tracker Red co-stained living Hela cells. Green image: CPH stained signal collected from channel 1 (500-550 nm); Red image: LysoTracker labelled signal collected at 650-800 nm (Pearson's correlation Rr = 0.835 and overlap coefficient R = 0.875). Excited at 488 nm. Scale bar is 5 µm. (D) Intensity profile of ROI (regions


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of interest) analysis (green line, CPH location; red line, LysoTracker Red location) across HeLa cells. (C, E-G) 3D surface plot analysis of the co-localization images of CPH and Lyso-Tracker Red co-stained living Hela cells with interactive 3D surface plot function in the Image J software.

Next, the co-localization with commercially available lysosome indicator (LysoTracker® Deep DND-99) in live Hela cells was further studied to confirm if CPH could be a lysosome-target probe. As indicated in Figure 4A-B, the convincing or orange fluorescence was obtained by overlaying of images of CPH stained signal collected from channel 1 (green, 500-550 nm) and images of Lyso-Tracker labelled signal collected at 650-800 nm (red), implying the colocalization of CPH with lysosomes. Similar to the results in Figure S3, the fluorescence from channel 2 was also negligible through analyzing the enlarged images in Figure S4. In addition, by using the plot analysis of regions of interest (ROI) across the HeLa cells in Figure 4b, we can observe a large overlap between signals of CPH stained location (green line in Figure 4D) and Lyso-Tracker stained lysosomes (red line in Figure 4D). Finally, 3D surface plot analysis of images in Figure 4B further clearly displayed the co-localization images of CPH and Lyso-Tracker Red co-stained living Hela cells. All these results suggested that CPH can accumulate in lysosomes and potentially become a highly selective fluorescent probe for sensing pH fluctuation in lysosomes. Unlike the majority of previously reported lysosome target probes that are designed by conjugation with lysosome target group such as the weakly basic amines morpholine, aniline etc..5,6,40-42 Herein, the relatively low pKa value of CPH (approximately 6.44 according to Figure 1) makes it become a weak base relative to the acid environment of lysosome, allowing the accumulation of CPH in lysosomes. This results were also consistent with previously studies that weakly basic compounds could accumulate in intracellular lysosomes.11,43 Fluorescence ratiometric detection of the pH fluctuation in lysosome with CPH. We next sought to investigate the ratiometric sensing performance of CPH in monitoring the pH fluctuation in lysosome. The antimalaria drug chloroquine, a lysosomal inhibitor that could change the lysosomal pH, was used for stimulate the Hela cells. Before the cholorquine stimulation, the Hela cells were initially co-incubated with CPH and washed with fresh DMEM. As depicted in Figure 5A, the CPH-stained cells exhibited a strong fluorescence signal in channel 1 (green, 500-550 nm) but nearly background fluorescence signal in channel 2 (yellow, 570-630 nm) before treatment with chloroquine. Then the fluorescence signal from channel 2 (yellow) gradually increased with a concomitant decrease in the fluorescence signal from channel 1 (green) after the cells were treated with enhanced concentration of chloroquine (50, 65 and 80 µM respectively). This phenomena could be ascribed to the increased pH values in lysosomes after the cells were stimulated by chloroquine, which was also supported by previous study that chloroquine can cause an increase in intralysosomal pH.38,39,41,44,45 Next, the semi-quantitative calculation of ratio of channel 2 to channel 1 (yellow/green) at averaged emission intensity was further conducted. Similarly, the emission ratio values increased from about 0.4 to 1.6 after the chloroquine stimulation (Figure 5B). Meanwhile, the ratiometric images were constructed according to the fluorescence signal of channel 1 (green) and channel 2 (yellow) with ImageJ software. Notably, the treatment of in-

creasing concentration of chloroquine resulted in an increase in the emission ratio value according to the color bar representing the channel 2 / channel 1 (yellow/green) emission ratio. Intriguingly, compared with the ratio images stimulated with different concentration of chloroquine, an uneven distribution of the ratio values in intralysosomes was observed. This may due to the non-uniform concentration of chloroquine distributed in lysosomes. Consequently, the pH values in intralysosomes exhibited an uneven distribution. All these results suggested that CPH is a sensitive ratiometric fluorescence probe for pH and could reveal the pH fluctuation in lysosomes through the ratiomeric fluorescence signal readout. In addition, CPH could also be as a fluorescent tool for directly tracing the subcellular distribution of chloroquine after the cellular uptake of this antimalaria drug, which is beneficial for further studying the mechanism of chloroquine in regulating the cellular process. Thus, the visualization of stimulation process with CPH will present a convenient tool for the new lysosomerelated drugs discovery.

Figure 5. Ratiometric fluorescence images and cellular response of CPH-stained Hela cells stimulated with chloroquine. (A) Ratiometric fluorescence images of the CPH-stained cells before and after the stimulation with various concentrations of chloroquine (50, 65 and 80 µM respectively) for 30 min. Excitation wavelength for CPH: 488 nm; Emission collection, channel 1 (green): 500-550 nm; Channel 2 (yellow): 570-630 nm. Scale bar is 25 µm. (B) Semi-quantitative determination of CPH-stained Hela cells according to the ratio of averaged fluorescence intensity of channel 2 (yellow, 570-630 nm) to channel 1 (green, 500-550 nm). The semi-quantitative calculation was conducted by ImageJ software. Error bars represent s.d.



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Colorimetric detection of acid/base vapor with CPH based portable test papers. The use of CPH as the ratiometric pH probe is not limited to solutions. A commonly available light grey background paper was cut into strips which were written with the solution of acid/base form of CPH, respectively. After drying in air, we can observe the different color and solidstate fluorescence on the treated strips (Figure 6A). Inspired by the favorable features of this CPH based test strips and that to address the urgent need for simple, economic tools for dynamic monitoring of the acid/base vapor detection in environment, especially for where the traditional pH test papers can not work well, we exploited the dynamic colorimetric sensing performance of the CPH based test strips for acid/base vapor. After sensing the base vapor, the characters written with CPH solution on the portable test paper changed immediately from yellow to red, which could be quickly recovered to yellow by treating the strip with acid vapor (Figure 6B, Movie S1 in the supporting information). Significantly, this dynamic colorimetric sensing process could be reversibly performed at least 5 cycles, suggesting the high reversibility and stability of the CPH based test papers for colorimetric detection of acid/base vapor. Thus, this sensing performance of CPH based strips will pave a way for preparing the economic, re-usable, and fast-response pH test papers for the colorimetric and accurate detection of acid/base vapor through the naked-eye observation.

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dual-modal colorimetric and fluorometric response to pH fluctuation through the intramolecular charge transfer (ICT) effect. Meanwhile, the N-benzyl substituent on indole nitrogen of CPH makes the reversible pH sensing process more stable. Furthermore, the pKa value of CPH (6.44) allows CPH to presumably accumulate in the lysosomes and become a lysosome-target probe. By using CPH, we have developed a new ratiometric method for determining pH changes in both aqueous solution and solid-state test papers. Notably, fluorescence ratiometric images for tracing the intralysosomal pH fluctuation stimulated by chloroquine through the ratiomeric fluorescence signal readout were successfully achieved. An economic, stable and fast-response CPH based test strip was prepared for the colorimetric detection of acid/base vapor through the naked-eye observation. This proof-of-concept study presents the potential application of CPH in live cell imaging for ratiometric monitoring of intralysosomal pH fluctuation in live cells, as well as in paving a way for developing the economic, re-usable, and fast-response optical pH meters for colorimetric sensing acid/base vapor through the direct naked-eye observation.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Detailed synthetic procedures, characterization of probes, cell cultures, protocols for live cell fluorescent imaging, preparation of CPH based test strips and the solid-state based sensing performance (PDF) The Probe CPH immobilized portable test paper for naked-eye visible and reversible detection of acid/base vapor. The reversibility of the colorimetric response of this portable test paper for acid/base vapor is more than 5 cycles (Movie)

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected]; [email protected]. Fax: 86-2164321833

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT

Figure 6. (A) The different color and solid-state fluorescence of the portable paper written with the solution of acid/base form of CPH, respectively. Photographs were taken at room temperature under ambient light (top) and UV light (bottom). (B) Probe CPH immobilized portable test paper for naked-eye visible and reversible detection of acid/base vapor. The reversibility of the colorimetric response of this portable test paper for acid/base vapor is more than 5 cycles.

CONCLUSIONS In summary, a new dual-modal colorimetric/fluorescence merocyanine-based molecular probe (CPH) was designed and synthesized for ratiometric detection of pH. CPH has Nbenzyl substituent on indole nitrogen to hinder the nucleophilic addition of OH- to C=N bond of indole group of CPH and phenol moiety that was taken as the selective proton acceptor. This typical D-π-A structure enables CPH to exhibit a

We thank National Natural Science Foundation of China (Grants 21302125, 21373138), Doctoral Fund of Ministry of Education of China (Grant No. 20133127120005), Program for Shanghai Sci. & Tech. Committee (Grants 13ZR1458800), Shanghai “Chenguang” Program (14CG42).

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For TOC only


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Fluorescence Molecular Probe for

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