A hemicyanine fluorescent probe with intramolecular charge transfer (ICT) mechanism for highly sensitive and selective detection of acidic pH and its application in living cells

Analytica Chimica Acta xxx (xxxx) xxx

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A hemicyanine fluorescent probe with intramolecular charge transfer (ICT) mechanism for highly sensitive and selective detection of acidic pH and its application in living cells Yingying Zhang a, Fanqiang Bu a, Yanliang Zhao b, Bing Zhao a, Liyan Wang a, Bo Song a, * a b

College of Chemistry and Chemical Engineering, Qiqihar University, Qiqihar, 161006, China School of Physics and Optoelectronics Engineering, Ludong University, Yantai, 264025, China

h i g h l i g h t s

g r a p h i c a l a b s t r a c t


Benzindole-based fluorescent probe BiDD was developed.
BiDD was applied to detect pH changes in cells with good fluorescence photostability and used for staining lysosome.

a r t i c l e i n f o

a b s t r a c t

Article history: Received 15 August 2019 Received in revised form 13 November 2019 Accepted 15 November 2019 Available online xxx

Intracellular pH (pHi) plays an essential role in organelles. Fluorescent probe combined with fluorescence imaging analytical approach has been used for detection pH fluctuation due to high sensitivity and good photostability. Herein, a benzoindole-based colorimetric and naked-eye hemicyanine fluorescent probe 2,3-trimethy-3-[2,4-(dihyoxyl-4-yl)vinyl]-3H-benzo[e]indole (BiDD) was developed in one step. Upon the decreasing of pH, BiDD exhibited strong a pH-dependent characteristic with pKa 4.98 and responded linearly within the pH range of 4.4e6.2. BiDD also showed high sensitivity and selectivity, colorimetric and fluorometric dual-modal response, high photostability, low cytotoxicity as well as good cell membrane permeability. More importantly, the probe was applied to sense and visualize the pH fluctuations in HeLa cells successfully by the fluorescence confocal microscope. © 2019 Elsevier B.V. All rights reserved.

Keywords: Hemicyanine Fluorescent probe Colorimetric Benzoindole pH Living cells

1. Introduction Intracellular pH (pHi) plays a significant role in varieties of biological processes, including cell growth, proliferation, apoptosis

* Corresponding author. E-mail address: [email protected] (B. Song).

[1,2], endocytosis [3], muscle contraction [4], ion transport, homeostasis and signal transduction [5,6]. The pH value of extracellular fluid is nearly basic (7.4) under the normal physiological condition, whereas it is significantly lower than (6.2e6.9) in tumor formation circumstance [7]. As abnormal pH variations in pH can lead to cellular dysfunction, which further brings about numerous serious diseases, including Alzheimer’s disease and cancer [8,9], maintaining a stable pH value is essential for all organisms. The

https://doi.org/10.1016/j.aca.2019.11.040 0003-2670/© 2019 Elsevier B.V. All rights reserved.

Please cite this article as: Y. Zhang et al., A hemicyanine fluorescent probe with intramolecular charge transfer (ICT) mechanism for highly sensitive and selective detection of acidic pH and its application in living cells, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2019.11.040

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intracellular pH value would fall to 6.0 during the ischemia processes [10,11]. Hence, establishing effective methods for real-time monitor and precise detection of intracellular pH is important for understanding the cell behavior in biological systems. Numerous methods have been developed to detect intracellular pH change recently. Among those approaches, absorbance spectroscopy, microelectrodes and nuclear magnetic resonance have drawn more attention [12e17]. Compared to these methods, fluorescent probe has been extensively used and become an essential analytical tool for pH monitoring in intact and subcellular regions due to its outstanding characteristics, high sensitivity, good selectivity, fast response and simple operation [18,19]. Moreover, fluorescent probe integrated with confocal laser scanning microscopy for the most part provide a high spatial and temporal-spatial for obsering pH fluctuation. Recently, lots of fluorescent probes have been developed for detection of pH change [20e25], unfortunately, most of them have potential damage to cells and affected by the cellular auto-fluorescence due to the short absorption and emission wavelengths [26,27]. Some current probes have the disadvantages of complicated synthesis processes, poor photostability and working in a large amount of organic solvent, which is expected to be improved. Therefore, it is essential to use a simple synthesis method and develop a fluorescent probe with large Stokes shift to detect pH fluctuation. Hemicyanine dyes with donor-p-acceptor structure usually exhibit intramolecular charge transfer (ICT) effect and produce large Stokes shift [28]. Lots of hemicyanine based fluorescent dyes were developed for organ staining and monitoring of intercellular material changes due to a wide range of emissions, excellent fluorescent properties, structure stability on molecular label and high membrane permeability, which made them suitable for imaging in living cells. Herein, a hemicyanine fluorescent probe was designed and synthesized by one step, namely, 2,3-trimethy-3-[2,4(dihyoxyl-4-yl) vinyl]-3H -benzo[e]indole (BiDD), using the benzoindole and aromatic compound as starting material. The acidic pH change was successfully detected, and the lysosomal pH was monitored by confocal fluorescence microscopy. Under the acidic conditions, the aromatic benzaldehyde motif acts as an electrondonating (D) group and the protonation of benzoindole N atom acts as an electron-accepting (A) moiety, which further leads to a red-shift in both absorption and emission spectra, which was attributed to a significant enhancement of the electronwithdrawing ability of protonated benzoindole moiety and then further enhanced the conjugated system of the molecular. BiDD not only showed high selectivity to Hþ in the presence of different ions and biologically active substances but also can quantitatively detect pH value in the range of 4.4e6.2. What’s important, the probe exhibited large Stokes Shift under neutral (124 nm) and acidic conditions (162 nm) (Table S1, ESy), respectively, which can reduce excitation interference and overcome the auto-fluorescence of biological samples. The confocal fluorescent images of BiDD monitor the acidic pH fluctuation in living cells were conducted and changes in acidic pH were also examined.

By using the Brucker AVANCE-NMR spectrometer (600 MHz) equipment, the 1H NMR and 13C NMR characterization of the probe were received, respectively. HRMS spectra were obtained by using the Waters LCT Premier XE spectrometer in the ESI model. Absorption spectra of the optical experiment were tested under the TU-1901 double-beam UVevis. Fluorescence emission spectra were measured by using the Varian Cary Eclipse spectrophotometer. pH values of the experiment solution were tested with the Beckman F 50 pH meter. Fluorescence images were taken on a FV1000 confocal laser scanning microscope (Olympus Co., Ltd. Japan).

2. Experiment section

The MTT assay was used to detect the compatibility of BiDD with HeLa cells. More details were shown in Supporting information (ESy).

2.1. Materials and instruments All reagents of this paper were purchased from local commercial shops and used without further purification. 2,3,3-trimethyl-3H-benzo[e]indole and 2,4dihyoxybenzaldehyde were purchased from Aladdin Chemical Reagent Ltd. Twice-distilled water was used during the process of experiment. Lyso-Tracker Red (DND-99) and HeLa cells were achieved from Beijing Biotech Co., Ltd.

2.2. Synthesis and characterization of fluorescent probe The probe was synthesized by ethylene bridging 2,3,3trimethyl-3H-benzo[e]indole and 2,4-dihyoxybenzaldehyde. The synthetic route and the characterization of the target product can be found in Supporting information (ESy) (see Scheme 1). 2.3. General procedure for spectroscopic measurements Stock solution of BiDD (2 mM) was prepared in EtOH solution. The probe (20 mM) solution for optical experiments was obtained by diluting the stock solution in the water/EtOH solution (v/ v ¼ 1:1). The HCl (0.1 M) and NaOH (0.1 M) solution were used to adjust the minor change of pH values. During all optical experiments, excitation slit width was set at 10 nm, whereas emission slit width was 12 nm. For the fluorescence optical experiments, lex ¼ 393 nm. All spectroscopic experiments were conducted at room temperature. 2.4. Theoretical calculations of the probe combined with Hþ All the electronic structures of the probe were calculated by using the Gaussian 09 program suite [29], which plays a critical role in understanding the combination model between this probe and Hþ further. The ground states and excited states of the target molecules were calculated by density functional theory (DFT) and linear-response time-dependent density (TDDFT) were employed to calculate the ground and excited states of the target molecule [30]. By using the B3LYP-DJ functional with the 6-31G (d), the ground state (S0) and the first (S1) excited-state with natural population analysis (NPA) charge of the probe structure were optimized and calculated, respectively. Under the basic setting of 6311 þ G (d, p), the energies of all molecules were successfully evaluated. In order to keep consistent with experimental test solution of the probe, the solution of water/ethanol (v/v ¼ 1:1) was selected, including the integral equation formalism (IEF) [31] version of the polarizable continu considered um (PCM) model [32]. 2.5. Cell cytotoxicity assay

Scheme 1. The synthesis scheme of BiDD.

Please cite this article as: Y. Zhang et al., A hemicyanine fluorescent probe with intramolecular charge transfer (ICT) mechanism for highly sensitive and selective detection of acidic pH and its application in living cells, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2019.11.040

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2.6. Cell culture and fluorescence imaging According to the previous studies, HeLa cells were fostered in 96-well microplate culture dish and cultured in DMEM with 10% FBS at 37  C, 5% CO2 atmosphere for a night [33,34]. The probe BiDD (20 mM) for cell imaging was dissolved in EtOH solution. Then the probe solution was added into the HeLa cells and co-cultured for 30 min at 37  C, the excess BiDD solution was removed. Finally, different pH PBS buffers (pH 7.00, 4.98 and 2.50) were stained on the above cells for another 30 min [35], respectively, then the fluorescence images were collected on a fluorescence confocal microscope. To investigate the probe’s application in lysosome, after HeLa cells were washed with PBS (pH ¼ 7.4) three times, the probe BiDD (20 mM) was added into the cells co-cultured for 30 min. Then, the Lyso-Tracker Red (pH ¼ 7.4) was added into the above-mentioned cells for 1 h at 37  C and collect the fluorescence images. 3. Results and discussion 3.1. Optical properties of BiDD in aqueous media To further investigate the optical properties of BiDD in the aggregated characteristics, a solvent-poor fluorescence experiment was performed firstly, which was commonly used for investigation of the AIE (Aggregation-Induced Emission) behavior of probes. In

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this experiment, ethanol is a good solvent for BiDD, while water is a poor solvent for it. Therefore, adding water into the ethanol solution can dissolve the probe better and produce aggregation behavior, then in turns induces an enhancement in the fluorescence intensity of BiDD. The absorbance and fluorescence response of BiDD with different volume fractions of water (fw) (0%e90%) in ethanol solution were researched. As shown in Fig. 1a, BiDD showed weak fluorescence in pure ethanol (EtOH) whereas strong fluorescence in the solvent of water/EtOH under UV-lamp (365 nm). It worth noting that when the fw value in the mixed system was up to 50%, the probe showed strong fluorescence than in pure ethanol solution. The absorbance change of BiDD as the increasing of water fractions was investigated in Fig. S1 (ESy). The UVeVis absorption band at 393 nm blue-shifted gradually and the e of BiDD solution declined as fw increased. However, the fluorescence intensity of the probe was influenced by varying extents (Fig. 1c). When water was added into EtOH solution, the fluorescence intensity increased along with the increase of fw, reaching 50%. The reason for this phenomenon is that when a poor solvent (water herein) was added into good solvent (EtOH), the soluble BiDD molecules was formed through crystal stacking. The phenomenon suggested that the probe is an aggregation-induced emission active (AIE-active) compound with an increase in water fractions (10%e50%). However, an opposite behavior was observed when the fw increased (50e90%), the fluorescence intensity was decreased due to the

Fig. 1. (a)The color change of BiDD under different amounts of water under the UV-lamp (365 nm). (b)The Tyndall effect of the probe under different amounts of water via the laser pen under room temperature. (c)The change of the fluorescence emission spectra of BiDD (20 mM) with different amounts of the water systems (water/EtOH (v/v) ¼ 1:9e9:1), lex ¼ 393 nm, silt 10/12 nm. (d)The scatter plot of the fluorescence intensity and the fluorescence images of the probe BiDD under different amounts of water. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Please cite this article as: Y. Zhang et al., A hemicyanine fluorescent probe with intramolecular charge transfer (ICT) mechanism for highly sensitive and selective detection of acidic pH and its application in living cells, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2019.11.040

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excessive aggregation behavior of probe BiDD via Tyndall effect (Fig. 1b) which caused the ACQ (Aggregation-caused quenching) behavior as shown in Fig. 1d. This was a reasonable that when the fw value was higher than 50%, BiDD aggregated excessive in water, the precipitate formed more quickly and an amorphous aggregate of nanoparticles was appeared, so the amounts of effective chromophores in per unit volume were decreased and the emission was quenched. We also investigated the effect of fw on the maximum emission intensity and emission wavelengths of BiDD (Fig. S2, ESy). The scanning electron microscope (SEM) analysis method was also used to research the morphology of the precipitates when the water fraction was 0%, 50% and 90% in the water/EtOH solutions of BiDD, respectively, as shown in Fig. S3 (ESy). The aggregation of BiDD gradually enhanced with the increasing of the water fraction. We can clearly see that the nanoparticles of BiDD showed irregular granular in shape and the probe exhibited planar nature without molecular aggregation in pure EtOH (Fig. S3a, b). What’s more, BiDD changed from random particle state to a pile of spherical morphology in a large area dispersion of the case of the 50% water in EtOH mixture as shown Fig. S3 c, d. Those signs indicated that the probe BiDD began to aggregate when fw up to 50%. It worth noting that the aggregation state of BiDD changed to independent circular-like when water volume fraction was 90% in the mixture solution system. The planarity of BiDD (fw ¼ 50%) was slightly higher than 90%. When BiDD was added into water/EtOH solutions (fw > 50%), the molecules aggregated through intermolecular hydrogen bonds along two dimensions, further formed precipitate as we previously reported. 3.2. Optical properties of pH The temperature optimization experiments of the probe BiDD (20 mM) were performed firstly at neutral condition. As shown in Fig. S3 (ESy), the absorbance and fluorescence intensity of the probe have excellent stability in a temperature range from 10  C to 100  C. For the convenience of the experimental operation, all spectroscopic experiments were carried out at room temperature. The UVevis and fluorescence pH titration experiments of BiDD (20 mM) were examined in water/ethanol (1/1, v/v) solutions. The UVevis absorption spectral of BiDD upon the pH change were shown in Fig. S5 (ESy). Two absorption bands were (393 nm and 482 nm) exhibited in the spectra. As the solution pH was decreased from 7.00 to 2.59, the absorption peaks at 393 nm (e1 ¼ 2.45  104 L mol1 cm1) was reduced, and a new red-shifted absorption band was appeared at 487 nm (e2 ¼ 3.56  104 L mol1 cm1) with a isosbestic point existed at 425 nm, indicating that the protonation of benzoindole moiety was the only reaction process during the experiment. And the redshifted of the spectra from 393 nm to 482 nm suggested that the ICT effect from electron-donor aromatic aldehyde (D) group to electron-acceptor benzoindole moiety (A) has been enhanced owning to the protonation of benzoindole moiety. Besides, the obvious color changed from pale yellow to orange with decreasing pH (Fig. S5 inset, ESy) indicated that BiDD could serve as a “nakedeye” probe for determination of acidic pH in aqueous solution. As shown in Fig. 2, the pH titration experiments provided a good indication of BiDD exhibited pH-dependent behavior in fluorescence emission spectra with an intense emission band centered at 517 nm (F ¼ 0.77) with large Stokes shift of 124 nm at neutral conditions (lex ¼ 393 nm) (Fig. S6, ESy). When pH value was decreased to 4.98, a new emission band was centered at 555 nm (F ¼ 0.32) and fluorescence intensity declined gradually with large Stokes shift (162 nm) under acidic conditions (Fig. S7, ESy). The results of fluorescence emission spectra showed that the redshifted from 517 nm to 555 nm may be attributed to the

Fig. 2. The change of the fluorescence emission spectra of BiDD (20 mM) with decreasing pH from 7.00 to 2.59 (lex ¼ 393 nm, silt 10/12 nm). Inset: The fluorescent color of the solution changed from green to red with decreasing pH. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

enhanced ICT effects. Meanwhile, the visual fluorescence of BiDD solution changes from green to red under UV light (365 nm) irradiation at pH 7.0 and 2.59 indicates that the probe can be used as a colorimetric probe. The pH sensitivity of the probe attributed to the N moiety of benzoindole upon either protonation or deprotonation (Scheme 2). What’s more, the sigmoidal fitting of pH-dependent fluorescence intensity provided an apparent pKa value of 4.98 (Fig. 3a), and the fluorescence intensity showed good linearity to pH values in the range 4.6e5.6 (Fig. 3b), the linear regression equation was F517nm ¼ 132.5354 pH-320.2283 (R2 ¼ 0.9928). This result indicated that this probe has potential application value for the quantitative determination of acidic pH values by the fluorescence methods. 3.3. Binding behavior and theoretical calculations of BiDD with Hþ The naked-eye change of the solution’s color and the spectral properties are both attributed to the ICT effect enhancement from the aromatic aldehyde (D) group to benzoindole moiety (A). The pH sensitivity of the probe on account of the benzoindole N moiety upon either protonation or deprotonation. To confirm this hypothesis, 1H NMR experiments of BiDD were carried out at two pH values (pH 7.0, 4.5) in DMSO‑d6 (Fig. S8, ESy). When pH values of the solution were decreased from 7.0 to 4.5, the chemical shift values of the benzoindole proton (H1) and vinyl protons (H2, H3) a significant lower field shift, indicating that Hþ had combined with the benzoindole N atom, which further resulted the decreasing of electron density around these protons and induced the optical response of probe to acidic pH. It worth noting that the chemical shift values of other protons around aromatic aldehyde (H4, H5) remain almost unchanged, which further proved that the benzoindole N atom was protonated under acidic conditions. What’s more, we also adopted the DFT and TDDFT calculations to further prove the benzoindole N atom of the molecular was protonated under acidic conditions and the detailed molecular properties of BiDD (deprotonation) and BiDD þ Hþ (protonation) were calculated. A battery of theory properties about BiDD, including optimized structures, the highest occupied molecular orbital (HOMO) and the lowest unoccupied (LUMO) orbital plots, the transition energy DE (eV) and oscillator strength (f) were calculated in the S0 and S1 states, respectively. All calculation results are shown in Fig. 4. As for BiDD, the electrons were distributed

Please cite this article as: Y. Zhang et al., A hemicyanine fluorescent probe with intramolecular charge transfer (ICT) mechanism for highly sensitive and selective detection of acidic pH and its application in living cells, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2019.11.040

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Scheme 2. Acid-Base form equilibrium of BiDD.

Fig. 3. (a) The sigmoidal fitting of the pH-dependent with I/Imax of the probe BiDD (lex ¼ 393 nm, silt 10/12 nm). (b) The good linearity in the pH range of 4.6e5.6 of the probe BiDD. Data are expressed as mean values standard error of the mean of three independent experiments.

Fig. 4. Using TDDFT calculated the HOMO-LUMO distributions of the probe BiDD and the optimized structures of protonation and deprotonation forms of BiDD (lex ¼ 393 nm).

deposition uniformity in the molecule’s conjugate system both at the HOMO and LUMO level. On the contrary, as for BiDD þ Hþ, the electrons of the HOMO level were spread in the benzoindole moiety for the most part while the LUMO level were mostly concentrated in the ethylene bond and the aromatic aldehyde moiety. Above distribution of electrons provided a good condition to approve the ICT effect occurred in this molecular when the probe was

deprotonated and protonated. It is noteworthy that the probe BiDD had DE of 2.949 ev and 2.210 ev at the HOMO and LUMO levels, respectively, while the DE of BiDDþHþ was 2.616 ev and 1.995 ev, respectively. The energy band gap’s decreasing illustrated that ICT effect has happened when BiDD combined with Hþ and put the kindest interpretation of the red-shifted phenomenon both in absorption and emission

Please cite this article as: Y. Zhang et al., A hemicyanine fluorescent probe with intramolecular charge transfer (ICT) mechanism for highly sensitive and selective detection of acidic pH and its application in living cells, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2019.11.040

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spectra of the probe. 3.4. Selectivity studies Considering the complexity of intracellular environment, the fluorescence selectivity experiment of the probe BiDD toward Hþ over other some competitive species (metal ions, anions and bioactive species) was investigated at pH 7.0, 6.3, 4.98 and 2.52, respectively. As shown in Fig. 5, there were no visible effects on the fluorescence intensity of the probe at four different pH values toward various ions and bioactive species. These results suggested that BiDD showed an excellent selectivity response to acidic pH without inferenced by other substances and has a good potential to be applied in detecting pH values in living cells. 3.5. Time-stability and reversibility of BiDD To prove that the stability of this probe BiDD is not affected by the preservation time, we investigated the time-stability of the probe within a period of 2 h. The fluorescence intensity of BiDD (20 mM) at pH 7.0, 4.99 and 2.60 were monitored and recorded, respectively, which revealed that the probe has good time-stability among the tested pH range and response rapidly to Hþ (Fig. 6a). Reversibility of probe to pH change was also investigated. The pH values of the solution were adjusted repeatedly between 7.1 and 4.0 for seven times (Fig. 6b). The response and recovery times of BiDD in different pH solutions was fully reversible. At the same time, the solution color changed repeatedly between green (pH 7.10) and red (pH 4.90). It is clear that BiDD had great potential to be used for rapid monitoring of pH values. 3.6. Cell cytotoxicity assay It is possible for the probe, BiDD, to be used for intracellular pH imaging due to its high selectivity and great sensitivity to Hþ. Hence, it is important to assess the cytotoxicity of BiDD to living cells by the MTT assay (Fig. S9, ESy). The good viability (more than 80%) of HeLa cells under different concentrations of probe (10 mMe100 mM) revealed that the probe has low toxicity to cells

upon experimental conditions at the concentration of 20 mM, which suggested that the probe has good potential to be used in intracellular imaging of living cells. 3.7. Fluorescence imaging of living cells As mentioned earlier, the probe shows great time-stability during 2 h at the room temperature and low toxicity to living cells. Hence, the photostability of the probe with Hela cells by fluorescence confocal microscope was further investigated. BiDD (20 mM) (pH ¼ 7.4) (F ¼ 0.77) was cultivated with HeLa cells at the environment of 5% CO2, 37  C for 30 min. Then the fluorescence images were collected at different periods of time (0 s, 10 s, 20 s, 40 s, 60 s, 80 s, 100 s and 120 s), respectively, as shown in Fig. 7. We can clearly see that with the increasing of incubation time, the relative fluorescence intensity of the probe with Hela cells remained above 85% after 40 s, indicating that the probe has good fluorescence photostability and can be used for long-term application in cells. Then, the probe was applied to detect pH fluctuation in living cells. Hela cells were washed with PBS buffer solution at different pH conditions (pH ¼ 7.00, 4.98, and 2.50), respectively. As shown in Fig. 8, the bright-field images confirmed the viability of the coincubated BiDD-HeLa cells (Fig. 8aec). Those cells exhibited strong green fluorescence at pH 7.0 (Fig. 8d), whereas the fluorescence brightness obviously declined upon the pH decreasing to pH 4.98 and 2.50 (Fig. 8e and f), which have good consistency with the fluorescent titration experiments in different pH solutions. Those results demonstrated that the probe could detect acidic pH fluctuations in living cells. The probe BiDD has good ability in detecting acidic pH variations, and lysosome is the typical acidic organelle in cells. To prove the probe has good potential application in the lysosome, the probe was compared with commercial Lyso-Tracker Red (a well-known red-missive tracker for lysosome). As shown in Fig. 9b, the bright green fluorescence mainly distributed in cytoplasm, indicating that BiDD has excellent cells membrane permeability. What’s important, the subcellular regions co-incubated with BiDD matched well with Lyso-Tracker Red (Fig. 9d, h). These results indicated that BiDD can selectively stain lysosome in cells. The reactive oxygen species (ROS) such as H2O2 could cause a redistribution of Hþ driving from the acidified organelles to cytosolic compartments [36,37]. Thus, different reactive oxygen species were used to induce changes in the pH of the lysosome. BiDD-HeLa cells were treated with NAC (0.1 mM) for 30 min and the fluorescence images were collected. We can clearly see that the adding of NAC resulted in a slight increasing of pH (Fig. 10h). However, after an addition of 0.1 mM of H2O2 onto the BiDD-HeLa cells for 30 min, the fluorescence intensity obviously declined compared with nontreated cells (Fig. 10f), revealing that the decreasing of lysosomal pH (Fig. 10i). The results implied that the probe has respond rapidly to the acidic pH changes which induced by ROS and can be used for lysosomal pH imaging. 4. Conclusion

Fig. 5. The fluorescence intensity of BiDD (20 mM) at pH 7.10, 6.30, 4.98 and 2.52, respectively in the presence of various ions and bioactive species. (1) blank; (2) Kþ (150 mM); (3) Naþ (150 mM); (4) Mn2þ (0.2 mM); (5) Zn2þ (0.2 mM); (6) Ca2þ (0.2 mM); (7) Cd2þ (0.2 mM); (8) Co2þ (0.2 mM); (9) Pb2þ (0.2 mM); (10) Ni2þ (0.2 mM); (11) Mg2þ (0.2 mM); (12) Hg2þ (0.2 mM); (13) Fe2þ (0.2 mM); (14)Ba2þ (0.2 mM); (15) Al3þ (0.2 mM); (16) F (0.2 mM); (17) Cl(0.2 mM); (18) Br (0.2 mM); (19) I (0.2 mM); (20) HCO 3 (0.2 mM); (21) Cys (0.2 mM); (22) Gsh (0.2 mM); (23) Hcy (0.2 mM). (lex ¼ 393 nm, silt 10/12 nm). Data are expressed as mean values standard error of the mean of three independent experiments.

In summary, a simple fluorescent probe BiDD was facilely synthesized via one step based on ethylene bridging of benzoindole and 2,4-dihyoxybenzaldehyde. BiDD displayed a strong pHdependent behavior as the pH values range from 7.00 to 2.59 with a pKa value of 4.98. The large Stokes shift of BiDD under medium (124 nm) and acidic condition (162 nm), which can reduce the excitation interference. The probe displayed characteristics of low cytotoxicity and good photostability, which provided a good condition for the probe successfully applied to detect intracellular pH

Please cite this article as: Y. Zhang et al., A hemicyanine fluorescent probe with intramolecular charge transfer (ICT) mechanism for highly sensitive and selective detection of acidic pH and its application in living cells, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2019.11.040

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Fig. 6. (a) The change in the fluorescence intensity of BiDD (20 mM) with times at pH 7.00, 4.99, 2.60, respectively. (b) The change in the fluorescence intensity of BiDD (20 mM) between pH 7.10 and 4.90 (lex ¼ 393 nm). Data are expressed as mean values standard error of the mean of three independent experiments.

Fig. 7. (I) The fluorescence microscopy images of BiDD (20 mM) in HeLa cells during different periods of time (0, 10s, 20s, 40s, 60s, 80s, 100s, 120 s) under neutral condition (pH 7.0) at room temperature, lex ¼ 393 nm. Scale bar, 50 mm. (II) The scatter diagram of the relative intensity of the probe fluorescence images during different periods of time. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 8. (I) The fluorescence microscopy images of BiDD (20 mM) in HeLa cells clamped at pH 7.40 (a, d, j), 4.98 (b, e, h) and 2.50 (c, f, i) at room temperature, respectively. The first row (a, b, c) shows the corresponding bright-field transmission images. The second row (d, e, f) shows the green channel images at lex ¼ 393 nm. The merge images (h, i, j) generated by image software (the third row). Scale bar, 50 mm. (II) The column chart of the relative intensity of the probe fluorescence images under different pH values. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Please cite this article as: Y. Zhang et al., A hemicyanine fluorescent probe with intramolecular charge transfer (ICT) mechanism for highly sensitive and selective detection of acidic pH and its application in living cells, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2019.11.040

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Fig. 9. The confocal fluorescence images of BiDD (20 mM) (b, f) and imaging of co-labeled with 0.1 mM Lyso Tracker Red (c, g) in HeLa cells at room temperature. Merge images (d, h) of rank two and three. (a, e) Bright field. (b, f) Green channel for probe BiDD, lem ¼ 500e550 nm, lex ¼ 393. (c, g) Red channel for Lyso Tracker Red, lem ¼ 570e620 nm (d, h) Merge images from (a, e, b, f, c, g). The first row (a, b, c, d) shows the original picture of BiDD with Lyso Tracker Red in HeLa cells. The second row (e, f, g, h) shows the enlarged view of BiDD with Lyso Tracker Red in HeLa cells. Scale bar, 50 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 10. (I). The fluorescence microscopy images of probe BiDD cultured with HeLa cells then treated with NAC (0.1 mM) (e) and H2O2 (0.1 mM) (f) at room temperature, respectively. The first row (a, b, c) shows the bright-field transmission images. The second row (d, e, f) shows the green channel images at lex ¼ 393 nm. The images were collected at 500e550 nm with lex ¼ 393 nm. (II)The column chart of the relative intensity of the probe fluorescence images under different ROS. Scale bar, 50 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

change and monitor the lysosome pH under acidic conditions in living cells, indicating that the probe has a good application potential for pH monitoring in biomedical and biological fields.

Gene Line Bioscience company provides cell imaging services for us. Appendix A. Supplementary data

Author contribution statement Yingying Zhang: Conceptualization; Methodology; Writing Original Draft; Writing - Review & Editing. Fanqiang Bu: Investigation. Yanliang Zhao: Software; Resources. Bing Zhao: Funding acquisition. Liyan Wang: Funding acquisition. Bo Song: Supervision; Project administration. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements The authors would like to thank the Natural Science Foundation of China (21506106) and Natural Science Foundation of Heilongjiang Province (LC2017004, LH2019B021). We also thank the

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Please cite this article as: Y. Zhang et al., A hemicyanine fluorescent probe with intramolecular charge transfer (ICT) mechanism for highly sensitive and selective detection of acidic pH and its application in living cells, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2019.11.040