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A simple acidic ‘turn-on’ fluorescent pH probe based on BOPYIN and its visual detection and cellular imaging
Journal Pre-proof A simple acidic ‘turn-on’ fluorescent pH probe based on BOPYIN and its visual detection and cellular imaging Xiaohui Yuan, Tingting Zhang, Jiaying Yan, Xi Chen, Long Wang, Xiang Liu, Kaibo Zheng, Nuonuo Zhang PII:
S0143-7208(19)32500-8
DOI:
https://doi.org/10.1016/j.dyepig.2020.108318
Reference:
DYPI 108318
To appear in:
Dyes and Pigments
Received Date: 28 October 2019 Revised Date:
13 February 2020
Accepted Date: 28 February 2020
Please cite this article as: Yuan X, Zhang T, Yan J, Chen X, Wang L, Liu X, Zheng K, Zhang N, A simple acidic ‘turn-on’ fluorescent pH probe based on BOPYIN and its visual detection and cellular imaging, Dyes and Pigments (2020), doi: https://doi.org/10.1016/j.dyepig.2020.108318. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd.
Xiaohui Yuan:, Writing - Original Draft, Investigation, Data Curation Tingting Zhang: Methodology, Writing - Original Draft Jiaying Yan: Software, Validation, Resources Xi Chen: Visualization Long Wang: Supervision Xiang Liu: Writing - Review & Editing Kaibo Zheng: Conceptualization, Resources Nuonuo Zhang: Conceptualization, Funding acquisition, Project administration
A simple acidic ‘turn-on’ fluorescent pH probe based on BOPYIN and its visual detection and cellular imaging
Xiaohui Yuan,a Tingting Zhang,a Jiaying Yan,a,b Xi Chen,a Long Wang,a Xiang Liu,a,c Kaibo Zheng,a* and Nuonuo Zhanga* a
College of Materials and Chemical Engineering, Key Laboratory of Inorganic Nonmetallic Crystalline and Energy Conversion Materials, China Three Gorges University, Hubei, Yichang 443002, PR China. E-mail address: [email protected] and [email protected] b State Key Laboratory of Coordination Chemistry, Nanjing University, Jiangsu, Nanjing 210093, PR China c Material Analysis and Testing Center, China Three Gorges University, Hubei, Yichang 443002, PR China
Abstract A simple high selective and sensitive fluorescent pH probe based on a new seven-membered
BOPYIN
(BOrondifluoride-3,3-dimethyl-2-[2-(2-PYrrolyl)
ethenyl]INdole, Probe-NH2, Scheme 1) has been designed and synthesized. With the increase of acidity (pH from 5.51 to 2.47), the emission intensity dramatically enhanced to strong fluorescence. The linear response range is from 5.5 to 3.0 with pKa = 3.63, which is suitable for pH detection in biological system. Moreover, this probe has excellent selectivity response to H+, short response time (less than 10 seconds), reversibility and low cytotoxicity. Possible sensing mechanism studies demonstrated that the linear response of the probes to acidity of solution is the conjugation of H+ with response site N atom in primary amino group on BOPYIN. Besides, the fluorescent change of the Probe-NH2 relies on PET and/or ICT process which is proved by experiment and further confirmed by DFT theoretical calculation. Finally, the probe has been successfully applied for visual detection and cellular imaging.
Keywords: BOPYIN; fluorescent pH probe; Cellular imaging; Visual detection; Extreme acidity
1 Introduction Hydronium ion plays a crucial role in several essential processes, such as biochemical, environmental monitoring and clinical analysis. In biology process, the reduction of hydronium ion may induce cellular dysfunction and lead to some diseases including Alzheimer's disease and cancer[1-4]. Therefore, the monitor of pH change in cells, tissues and organisms is very important. Nowadays, electrochemistry[5], nuclear magnetic resonance (NMR)[6], absorption, and emission spectroscopy[7] measure pH value as the mainstream pH detection method. However, fluorescent pH probe is one of the utilized method due to its operational simplicity, high sensitivity, excellent spatial and real-time detection for monitoring intracellular concentration of hydronium ion.
Scheme 1 The synthetic route for Probe-NH2
Fluorescent pH sensors are developed based on diverse fluorescent molecules, nanoparticles and so on. In the past decades, fluorescent small molecules have drawn increasing attention as fluorescent probes for pH detection due to their designability, easy modification and excellent photophysical properties, which mainly based on excited-state intramolecular proton transfer (ESIPT), fluorescence resonance energy transfer (FRET), intramolecular charge transfer (ICT) and photoinduced electron transfer (PET) process. Traditional single-atom electron donor N acts as reaction position for pH detecting process based on the mechanism mentioned above. The existence form of N atom in neutral molecules are classified as three categories, primary amino, secondary amino and tertiary amino form. Therein, tertiary amino groups have been widely studied in pH detection, such as pyridine[8-11],
indole[12-14], benzothiazole[15, 16], benzimidazole[17] and other tertiary amino groups in or out of the ring[18-26]. The research on the primary amino groups is limited due to their instability or destructive inactivation under extreme pH condition[27-30]. Thus, it is particularly urgent to develop stable molecular fluorescent dyes to tolerate the harsh pH milieu in the fields of medical, biological and environmental science. BODIPY is an fascinating kind of popular fluorescent dye and applied in various fields[31-35]. In our previous work[36-40], BOPYIN as an analogue of BODIPY, and their derivatives are reported with a broad range of biocompatibility, excellent fluorescence properties, large Stokes shifts, good molar absorptivity and simple synthesis via peripheral modification to adjust the electronic structure. However, there is no available information about BOPYINs derivatives which are suitable for pH detection, especially for strong acidic condition (pH < 4). There is a high demand in the applications such as waste-water evaluation, environmental protection and disease monitor. As a subsequent work on BOPYIN and their derivatives, a new BOPYIN probe with amino responding group is designed and synthesized (Scheme 1, Probe-NH2). Interestingly, Probe-NH2 decorated with primary amino group has fast response to hydronium ion in strong acidic environments. Therefore, Probe-NH2 as a pH sensor for strong acidic condition is investigated in this work. Meanwhile, PET and/or ICT process of the emission change is proved by experiment and calculation.
2. Experimental section 2.1 Materials and apparatus All reagents and solvents were obtained from commercial sources and used without further purification, unless otherwise noted. All chromatographic separations were carried out on silica gel (300-400 mesh).
13
C NMR spectra were recorded on Bruker
400 MHz spectrometer at 298 K. DMSO-d6 was used as solvent and TMS as internal reference. The chemical shifts were reported in parts per million (δ) relative to the appropriate reference signal: residual DMSO (the quintet centered at 2.50 ppm). High
resolution mass spectra were measured on Bruker APCI instrument. UV-Vis spectra in various solvents were detected on Shimadzu UV-2600 in 10 mm quartz cell spectrometer. Fluorescence spectra were obtained using a Hitachi FL-4600 spectrometer. Fluorescent images were taken on a Carl Zeiss LSM 710 confocal laser scanning microscope (Jena, Germany). pH values were measured using an INESA PHS-25 pH meter. Gaussian 09 programs were used in theoretical calculations. 2.2 Synthesis and characterization of the fluorescent probe Herein, a novel pH-sensor Probe-NH2 was synthesized by the reaction of 5-nitro substituted trimethyl indole hydrochloride and 2-formylpyrrole based on Knoevenagel condensation, then coordination with BF3 OEt2 and reduction by SnCl2 2H2O (Scheme 1). This probe contains primary amine unit as electron donor and parent core of BOPYIN as fluorescence moieties. Synthesis of BOPYIN-NO2 A mixture of 5-nitro trimethyl indole hydrochloride 1 (410 mg, 2.0 mmol) and 2-formylpyrrole 2 (190 mg, 2.0 mmol) in EtOH (30 mL) was stirred under reflux for 1 h. After completion of reaction confirmed by TLC, this crude product was purified by flash chromatography (silica gel) to afford compound 3 with trans and cis isomer. Then, a mixture of compound 3 (280 mg, 1.0 mmol), Et3N (1.5 mL, 11.0 mmol) in toluene was added BF3•OEt2 (9.5 mmol, 1.2 mL). This mixture was stirred under reflux for 1 h. After completion of reaction confirmed by TLC, the reaction mixture was washed with water (30 mL × 3), dried over anhydrous Na2SO4 and concentrated under vacuum. The crude product was purified by chromatography (silica gel, hexanes/CH2Cl2 3:2) to afford BOPYIN-NO2 as a solid in 35% yield. 1H NMR (400 MHz, DMSO) δ 8.59 (d, J = 2.4 Hz, 1H), 8.36 (dd, J = 9.0, 2.4 Hz, 1H), 8.12 (d, J = 9.0 Hz, 1H), 7.76 (s, 1H), 7.74 (d, J = 11.4 Hz, 1H), 7.23 (d, J = 2.9 Hz, 1H), 6.66 (dd, J = 3.6, 2.2 Hz, 1H), 6.42 (d, J = 11.3 Hz, 1H), 1.56 (s, 6H).
13
C NMR (101 MHz,
DMSO) δ 183.85, 149.92, 145.23, 143.58, 140.84, 135.41, 135.13, 127.46, 125.10, 118.50, 118.06, 117.95, 117.84, 115.53, 101.87, 51.88, 24.55. HRMS (ESI) Calcd. for C16H14N3O2BF2 [M+H]+: 330.1223, found 330.1219. Synthesis of Probe-NH2
A mixture of BOPYIN-NO2 (658 mg, 2.0 mmol) and SnCl2•2H2O in DMF (48 mL) was stirred at room temperature for 1 h. After completion of reaction confirmed by TLC, water was added. The residue was filtrated and collected. The crude product was purified by chromatography (silica gel, hexanes/CH2Cl2 1:1) to afford Probe-NH2 as a solid in 28% yield. 1H NMR (400 MHz, DMSO-d6) δ 7.69 (d, J = 8.7 Hz, 1H), 7.44 (s, 1H), 7.41 (d, J = 11.7 Hz, 1H), 6.88 (d, J = 2.5 Hz, 1H), 6.71 (d, J = 2.2 Hz, 1H), 6.60 (dd, J = 8.8, 2.3 Hz, 1H), 6.47 (dd, J = 3.5, 2.4 Hz, 1H), 6.26 (d, J = 11.7 Hz, 1H), 5.55 (s, 2H), 1.39 (s, 6H).
13
C NMR (101 MHz, DMSO) δ 177.83,
149.00, 145.01, 136.51, 134.52, 134.13, 130.59, 122.32, 119.58, 119.49, 113.86, 113.55, 107.92, 103.60, 51.80, 25.08. HRMS (ESI) Calcd. for C16H16N3BF2 [M+H]+: 300.1481, found 300.1478.
3 Results and discussion 3.1 Spectroscopic properties and optical responses to pH The absorption and fluorescence spectra of Probe-NH2 (10.0 µM) were collected in 0.01 mol/L PBS buffer (PBS/CH3CN = 6: 1, v/v) with various pH (Fig.1, S1). Absorption spectra of the Probe-NH2 were examined in the pH ranging from 2.01 to 7.49 (Fig. S1). With the increase in the acidity of the solution from pH = 7.49 to 4.02, no significant change was detected in absorption spectra. With the acidity further increased from pH = 3.5 to 2.01, the increasing absorption split into two obvious peaks from a sole maximum absorbance peak around 480 nm, and the color of the solution changed from orange to lemon. The characteristic changes in absorption spectra with pH ranging from 2.01 to 7.49 indicates that this feature results from the PET and/or ICT effect. Furthermore, fluorescence properties were also studied. As shown in Fig. 1, the fluorescent intensity of Probe-NH2 in aqueous solution is heavily dependent on the acidity of the solution. The emission of Probe-NH2 is almost non-fluorescent in neutral solution. When the acidity increases (pH range: 2.47-5.01), the fluorescence intensity gradually enhances. That’s because protonation of the primary amino-group led to the recovery of the very strong fluorescence with a violet color at pH = 2.47.
The fluorescence intensity decreased slightly (pH range: 0.50-2.47) probably due to decomposition under strong acidic condition.
Fig. 1 Change of fluorescence spectra of Probe-NH2 (10 µM) in 0.01 mol/L PBS buffer (PBS/CH3CN = 6: 1, v/v) at various pH (0.52-7.49) (λex = 478 nm, slits: 5 nm/5 nm).
3.2 Possible sensing mechanism Binding Behavior of Probe with H+ The Probe-NH2 was designed to show the sensing ability of aromatic amine with H+ in acidic conditions. The pH sensitivity is introduced via the amine moiety upon either protonation or deprotonation. The probe exists in two forms, as either the orange nonfluorescent base form or the yellow fluorescent acid form (Scheme 2). 1H NMR experiments were carried out to prove the structure of probe after binding with H+. As shown in Table S2 and Fig. S3, when 15 equiv. of HCl is added to Probe-NH2 solution in DMSO-d6, an upfield shift is observed for the chemical shift values of the indole protons H1, H2, H3, double bond protons H4, H5 and pyrrole protons H6, H7, H8. The upfield chemical shift of these protons is obviously due to H+ binding with amine N resulting in the decrease of electron density around these protons. Thus, it is clear that H+ binds with amine N in Probe-NH2 causing the significant optical response to acidic pH. It’s well known that the lone pair of electrons on the nitrogen atom can induce fluorescence quenching via PET and/or ICT process[41-43]. Additionally, ICT process is easily occurred in conjugated D-π-A system. Probe-NH2 contains aromatic amine as donor and conjugated BOPYIN as acceptor. Hence, the sensing mechanism are speculated to PET and/or ICT process. 3.3 Principle of Operation and the Basis of Quantitative Assay According to the fluorescence spectra, sigmoidal fitting of fluorescence intensity at an
excitation wavelength of 478 nm versus various pH values and the linear response ranging from pH 3.0 to 5.5 were concluded in Fig. 2, which is connected with the ability of the Probe-NH2 to recognize proton. The relationship between the pH value and fluorescence intensity at λem = 478 nm can be expressed quantitatively using a Henderson-Hasselbalch type equation (Eq. 1)[27, 44]:
I −I log max = pH − pKa I − I min
(Eq. 1)
Where I is the observed fluorescence intensity; Imax and Imin represent the maximum and minimum of the fluorescence intensity of the Probe-NH2 in its acid (pH 2.47) and conjugate base (pH 7.49) form, respectively. Therefore, the linear response in Fig. 2b can be expressed by the following Eq. 2 of the calibration line:
I −I log max = 1.054 pH − 3.630( R = 0.992) I − I min
(Eq. 2)
where R is the linear correlation coefficient. According to the Eq. 2, the calculated pKa value of Probe-NH2 was 3.63, which is suitable for studying on acidic organelles. This result confirmed that Probe-NH2 is not only highly sensitive to the pH changes, but also linearly correlated to the pH in the range of 3.0-5.5 (R2 = 0.992) (Fig. 2b).
Fig. 2 pH dependence of the fluorescence emission intensity of Probe-NH2 (a); Plot of log[(Imax-I) / (I-Imin)] as a function of the pH (b).
3.4 Solvent-dependent properties Absorption and emission spectra were collected in different solvents (Fig. 3 and Table S1). Absorption spectra were similar in different solvents with a small peak at 374-386 nm and a sharp peak at 482-498 nm. Although the emission intensity was
weak in those organic solvents, the fluorescence intensity decreased and the emission peak red-shifted as the increase of solvent polarity. A large Stokes shift of 3490-4773 cm-1 was detected in various solvents. It indicated the influence of solvent effect on its optical properties. Based on this result, we chose CH3CN as solvent due to its low toxicity and water solubility in which it had large Stokes shift of 4405 cm-1 and non-fluorescence. Then, the spectra were measured using mixed solvents in different ratios of PBS/CH3CN (1:1, 3:1, 6:1, 9:1) as shown in Fig. S2. According to this result, the ratio of PBS/CH3CN (6:1) was chosen due to its low toxicity and solubility of probe compared to 1:1, 3:1 and 9:1, respectively.
Fig. 3 Absorption (a) and emission (b) spectra of Probe-NH2 in Toluene, DCM, THF, DMF, DMSO, CH3CN, C2H5OH and MeOH.
3.5 Theoretical calculation To gain insight into the mechanism of Probe-NH2, the PET and/or ICT effects were studied by using theory calculation method. The geometries of the ground states and the excited-state structures were optimized by the density functional theory (DFT) methodology with a B3LYP hybrid function method, and the 6-311G(d) basis set was used for all atoms[45]. Without considering the influence of solvent, toluene has been selected as the solvent in calculations based on the polarizable continuum model (PCM)[46, 47]. All calculations of the present work were performed using Gaussian 09 program package[48]. Probe-NH2 has two absorption peaks at 376, 491 nm and weak emission at 595 nm with moderate Stokes shift around 100 nm in the absorption and emission spectra in toluene, which is in good agreement with the calculation. (Fig. 4)
Fig. 4 TD-DFT (B3LYP/6-311G(d)) simulated stick spectra (Absorption: black column; Emission: red column) along with their normalized steady-state absorption spectrum (black solid line), emission spectrum (red solid line) of Probe-NH2 in toluene.
From the view of the geometric constructions, Probe-NH2 in the ground state (S0) and the lowest excited state (S1) are similar. The dihedral angle of indole unit and pyrrole unit slightly decreased (Fig. S4). However, the plane is obviously distorted from S1 to S0 of Probe-NH3+. The dihedral angle of indole unit and pyrrole unit of Probe-NH3+ in S1 state showed drastically decrease to S0 state. The geometric structures change between Probe-NH2 and Probe-NH3+ in S0 and S1 states is related to the electron distribution in frontier molecular orbitals. The frontier molecular orbitals, absorptions and emission related parameters are concluded in Fig. 5, Tables S3-5, which is related to the absorption and emission change between Probe-NH2 and Probe-NH3+.
Fig. 5 The frontier molecular orbitals involved in the vertical excitation and emission of Probe-NH2 (a) and its protonated from Probe-NH3+ (b) based on TD-DFT calculation in toluene.
The absorption peak with oscillator strengths over 0.61 originates from the electron transition from HOMO to LUMO. The emission peak with oscillator strengths over 0.45 originates from the electron transition from LUMO to HOMO for Probe-NH2 and LUMO to HOMO-2 for Probe-NH3+, respectively. According to the distribution of electrons, the electrons on HOMO orbits of Probe-NH2 mainly localized on the amino donor and then transferred to the BOPYIN acceptor (LUMO orbits), indicating a strong PET and/or ICT effects. Electrons smoothly transferred from primary amino (free receptor) to BOPYIN (fluorophore) in Probe-NH2, then PET and/or ICT process occurs. Meanwhile, the electron distributions of HOMO, LUMO orbits in ground state, HOMO-2 and LUMO orbits in excited state of Probe-NH3+ are similar, which almost located on BOPYIN fluorophores. Thus, PET and/or ICT process is prohibited. The energy level of free receptor, fluorophore and bound receptor are calculated for further discussing the mechanism of this fluorescence “turn-on” process (Scheme 2). Before the protonation, the HOMO energy level of free receptor (-5.685 eV) is between the HOMO (-5.785 eV) and LUMO (-2.564 eV) energy level of fluorophore. Therefore, the emission of Probe-NH2 is quenched by a PET process from free receptor to fluorophore skeleton. After protonation, the HOMO energy of bound receptor (-9.417 eV) is much lower than that of fluorophore, which prohibited the PET process, resulting in the strong fluorescent signal in Probe-NH3+. Additionally, the HOMO-LUMO energy gap of Probe-NH3+ (3.15 eV) is higher than Probe-NH2 (2.95 eV) confirming that the ICT process through a donor-π-acceptor system in this compound is disrupted.
Scheme 2 Reaction of Probe-NH2 in neutral and acid solution, HOMO energy level of free receptor, bound receptor and HOMO, LUMO energy level of fluorophore.
3.6 Real-time Response and Reversibility Study For practical application, it’s very important for the real-time determination[17, 49, 50]. Thus, the reaction-time profile of Probe-NH2 in the PBS/CH3CN solution of pH 7.31, 3.51 and 2.50 was measured. As shown in Fig. 6a, Probe-NH2 responds to the pH rapidly, the emission intensity enhances markedly within 10 seconds and then reaches a steady state. Therefore, Probe-NH2 could be applied to monitor the pH variation in real time. If the fluorescent pH probe is reversible, it will increase greatly for the potential application in real-time pH monitoring [51]. To examine the reversibility of this pH-dependent emission enhancement, the pH value of PBS/CH3CN solution was adjusted back and forth between 7.40 and 2.51 by using hydrochloric acid and sodium hydroxide (Fig. 6a). As shown in Fig. 6b, the results clearly reveal that these processes are reversible well. This suggests that reversibility of this probe between the protonated and deprotonated forms occurred. Thus, the cycle of pH-mediated fluorescence change (enhancement and elimination) and the real-time pH monitoring by Probe-NH2 are feasible.
Fig. 6 (a) Reaction-time profile of Probe-NH2 (10 µM in CH3CN/PBS, v/v, 1/6) at different pH values; (b) Reversibility (520 nm) of Probe-NH2 (10 µM in CH3CN/PBS, v/v, 1/6) between pH 7.40 and 2.51.
3.7 Selectivity studies
Fig. 7 Fluorescence intensity change at 478 nm of Probe-NH2 (10µM) in the presence of some common bio-related metal cations and anions in buffer solution at pH 2.5 and pH 7.5, λex = 478 nm, slit: 5.0 nm/5.0 nm. 1. blank; 2. Cr3+ (2 mM); 3. Mg2+ (2 mM); 4. Na+ (2 mM); 5. K+ (2 mM); 6. Zn2+ (2 mM); 7. Cu2+ (2 mM); 8. Co2+ (2 mM); 9. Fe2+ (2 mM); 10. Al3+ (2 mM). (a) pH =2.5, (b) pH = 7.5.
Selectivity is very important parameter to evaluate the properties of probe for the potential application in more complicated environments. Various metal ions including Cr3+, Mg2+, Na+, K+, Zn2+, Cu2+, Co2+, Fe2+ and Al3+ were introduced as competitive ions under the same test conditions with pHs of 2.5 and 7.4 for detecting the fluorescent response of Probe-NH2. As shown in Fig. 7, no significant changes were detected in emission spectra with the addition of the different metal ions, which means the high selectivity of this probe for hydronium ion. 3.8 Application
Fig. 8 Visual photographs of (a, b) Probe-NH2 (10 µM in CH3CN/PBS, v/v, 1/6) and (c) change in color of Probe-NH2 test strips (1 neutral, 2 exposed in HCl gas, 3 exposed in HCl, then Et3N gas) under 365 nm UV light. HCl (aq, 12 M), Et3N (7.2 M).
Visual detection of pH by the method of solution and test paper was also carried out. In solution, the change in color of Probe-NH2 was dramatic under 365 nm UV light in Fig. 8a,b. After the test papers were dipped in Probe-NH2 solutions (100 µM
in CH3CN) for 0.5 min, the observed color changes of the test strips were recorded as shown in Fig. 8c. Under irradiation with 365 nm UV light, the fluorescence of neutral test strip is weak; the acidic test strip exposed in HCl gas turns on and that then exposed in Et3N gas turns off in Fig. 8c. Therefore, the test strip experiment demonstrates that Probe-NH2 can be conveniently and efficiently used as a pH probe. We also studied whether Probe-NH2 could image the intracellular pH by conducting live (T24) cell imaging (Fig. 9, S11). We incubated cells in probe solution of 10 µM CH3CN/PBS. In T24 cells, Probe-NH2 shows strong fluorescence at pH 4.0 and non-emissive fluorescence at pH 7.4 with 10 µM concentration excitation at 480 nm.
Fig. 9 Fluorescence images of T24 cells incubated with 10 µM of Probe-NH2 in pH =4.0 and 7.4. Images were acquired using the confocal fluorescence microscope. Scale Bar: 25 µm.
4 Conclusion In summary, a new BOPYIN derivative as a ‘turn-on’ fluorescent pH probe was simply designed and synthesized with high selectivity, sensitivity, stability, short response time and low cytotoxicity, which applied in strongly acidic environments. With a unique response site (primary amino group) to hydronium ion, Probe-NH2 showed non-fluorescent to strong fluorescent with the increase acidity of solution at the pH from 5.51 to 2.47. The linear response curve is formed from pH 3.0 to 5.5, and the evaluated pKa value is 3.63. The fluorescence change of this probe is based on the PET and/or ICT process, which is proved by experiment and theoretical calculation. Furthermore, the probe has been successfully applied for visual detection and cellular imaging, with a ‘turn-on’ fluorescence in acidic environments. In a word, Probe-NH2
with the response site of amino group is first reported with excellent ability for detecting hydronium ion in strong acidic environments and applied in visual detection and biological imaging.
Acknowledgements This work was supported by National Natural Science Foundation of China (No. 21606145, 21805166), The 111 Project (D20015), State Key Laboratory of Coordination Chemistry Foundation of Nanjing University (No. SKLCC1811).
References [1] Davies TA, Fine RE, Johnson RJ, Levesque CA, Rathbun WH, Seetoo KF, et al. Non-age Related Differences in Thrombin Responses by Platelets from Male Patients with Advanced Alzheimer′s Disease. Biochemical and Biophysical Research Communications. 1993;194(1):537-43. [2] Bhuniya S, Maiti S, Kim E-J, Lee H, Sessler JL, Hong KS, et al. An Activatable Theranostic for Targeted Cancer Therapy and Imaging. Angewandte Chemie International Edition. 2014;53(17):4469-74. [3] Vegesna GK, Janjanam J, Bi J, Luo F-T, Zhang J, Olds C, et al. pH-activatable near-infrared fluorescent probes for detection of lysosomal pH inside living cells. Journal of Materials Chemistry B. 2014;2(28):4500-8. [4] Yue X, Yanez CO, Yao S, Belfield KD. Selective cell death by photochemically induced pH imbalance in cancer cells. Journal of the American Chemical Society. 2013;135(6):2112-5. [5] Ellis D, Thomas RC. Microelectrode measurement of the intracellular pH of mammalian heart cells. Nature. 1976;262(5565):224-5. [6] Sehgal AA, Duma L, Bodenhausen G, Pelupessy P. Fast Proton Exchange in Histidine: Measurement of Rate Constants through Indirect Detection by NMR Spectroscopy. Chemistry – A European Journal. 2014;20(21):6332-8. [7] Han
J,
Burgess K.
Fluorescent
Indicators for
Intracellular
pH.
Chemical Reviews.
2010;110(5):2709-28. [8] Chao J, Wang H, Zhang Y, Yin C, Huo F, Song K, et al. A novel ‘‘donor-π-acceptor’’ type fluorescence probe for sensing pH: mechanism and application in vivo. Talanta. 2017;174:468-76. [9] Chao J, Wang H, Song K, Li Z, Zhang Y, Yin C, et al. A highly sensitive acidic pH fluorescent probe and its application to E. coli cells. Tetrahedron. 2016;72(50):8342-9. [10] Xu Z, Ren A-M, Wang D, Guo J-F, Feng J-K, Yu X. A theoretical investigation on two latest two-photon pH fluorescent probes. Journal of Photochemistry and Photobiology A: Chemistry. 2014;293:50-6. [11] Yuan C, Li J, Xi H, Li Y. A sensitive pyridine-containing turn-off fluorescent probe for pH detection. Materials Letters. 2019;236:9-12. [12] Shen S-L, Zhang X-F, Ge Y-Q, Zhu Y, Lang X-Q, Cao X-Q. A near-infrared lysosomal pH probe based on rhodamine derivative. Sensors and Actuators B: Chemical. 2018;256:261-7. [13] Li J, Zhuge X, Yan X, Li Y, Yuan C. Two pH-responsive fluorescence probes based on indole derivatives. Optical Materials. 2019;90:257-63.
[14] Jin D, Wang B, Hou Y, Du Y, Li X, Chen L. Novel near-infrared pH-sensitive cyanine-based fluorescent probes for intracellular pH monitoring. Dyes and Pigments. 2019;170:107612. [15] Chao J, Liu Y, Sun J, Fan L, Zhang Y, Tong H, et al. A ratiometric pH probe for intracellular pH imaging. Sensors and Actuators B: Chemical. 2015;221:427-33. [16] Sun M, Du L, Yu H, Zhang K, Liu Y, Wang S. An intramolecular charge transfer process based fluorescent probe for monitoring subtle pH fluctuation in living cells. Talanta. 2017;162:180-6. [17] Wu Y-C, You J-Y, Jiang K, Wu H-Q, Xiong J-F, Wang Z-Y. Novel benzimidazole-based ratiometric fluorescent probes for acidic pH. Dyes and Pigments. 2018;149:1-7. [18] Xiao H, Zhang R, Wu C, Li P, Zhang W, Tang B. A new pH-sensitive fluorescent probe for visualization of endoplasmic reticulum acidification during stress. Sensors and Actuators B: Chemical. 2018;273:1754-61. [19] Georgiev NI, Said AI, Toshkova RA, Tzoneva RD, Bojinov VB. A novel water-soluble perylenetetracarboxylic diimide as a fluorescent pH probe: Chemosensing, biocompatibility and cell imaging. Dyes and Pigments. 2019;160:28-36. [20] Georgiev NI, Krasteva PV, Bojinov VB. A ratiometric 4-amido-1,8-naphthalimide fluorescent probe based on excimer-monomer emission for determination of pH and water content in organic solvents. Journal of Luminescence. 2019;212:271-8. [21] Xia S, Fang M, Wang J, Bi J, Mazi W, Zhang Y, et al. Near-infrared fluorescent probes with BODIPY donors and rhodamine and merocyanine acceptors for ratiometric determination of lysosomal pH variance. Sensors and Actuators B: Chemical. 2019;294:1-13. [22] Niu G, Zhang P, Liu W, Wang M, Zhang H, Wu J, et al. Near-Infrared Probe Based on Rhodamine Derivative for Highly Sensitive and Selective Lysosomal pH Tracking. Analytical Chemistry. 2017;89(3):1922-9. [23] Ye F, Liang X-M, Wu N, Li P, Chai Q, Fu Y. A new perylene-based fluorescent pH chemosensor for strongly acidic condition. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy. 2019;216:359-64. [24] Cao X-J, Chen L-N, Zhang X, Liu J-T, Chen M-Y, Wu Q-R, et al. A NBD-based simple but effective fluorescent pH probe for imaging of lysosomes in living cells. Analytica Chimica Acta. 2016;920:86-93. [25] Chao J, Song K, Zhang Y, Yin C, Huo F, Wang J, et al. A pyrene-based colorimetric and fluorescent pH probe with large stokes shift and its application in bioimaging. Talanta. 2018;189:150-6. [26] Liu Z, Li G, Wang Y, Li J, Mi Y, Zou D, et al. Quinoline-based ratiometric fluorescent probe for detection of physiological pH changes in aqueous solution and living cells. Talanta. 2019;192:6-13. [27] Ma Q, Li X, Feng S, Liang B, Zhou T, Xu M, et al. A novel acidic pH fluorescent probe based on a benzothiazole derivative. Spectrochimica acta Part A, Molecular and biomolecular spectroscopy. 2017;177:6-13. [28] Tan Y, Yu J, Gao J, Cui Y, Wang Z, Yang Y, et al. A fluorescent pH chemosensor for strongly acidic conditions based on the intramolecular charge transfer (ICT) effect. RSC Advances. 2013;3(15):4872. [29] Urano Y, Asanuma D, Hama Y, Koyama Y, Barrett T, Kamiya M, et al. Selective molecular imaging of viable cancer cells with pH-activatable fluorescence probes. Nature Medicine. 2008;15(1):104-9. [30] Wang Z, Zhang Y, Li M, Yang Y, Xu X, Xu H, et al. Two D-π-A type fluorescent probes based on isolongifolanone for sensing acidic pH with large Stokes shifts. Tetrahedron. 2018;74(24):3030-7. [31] Ding Y, Tang Y, Zhu W, Xie Y. Fluorescent and colorimetric ion probes based on conjugated oligopyrroles. Chemical Society Reviews. 2015;44(5):1101-12. [32] Ding Y, Zhu W-H, Xie Y. Development of Ion Chemosensors Based on Porphyrin Analogues.
Chemical Reviews. 2017;117(4):2203-56. [33] Wang Q, Wei X, Li C, Xie Y. A novel p-aminophenylthio- and cyano- substituted BODIPY as a fluorescence turn-on probe for distinguishing cysteine and homocysteine from glutathione. Dyes and Pigments. 2018;148:212-8. [34] Ma F, Zhou L, Liu Q, Li C, Xie Y. Selective Photocatalysis Approach for Introducing ArS Units into BODIPYs through Thiyl Radicals. Organic Letters. 2019;21(3):733-6. [35] Lv F, Tang B, Hao E, Liu Q, Wang H, Jiao L. Transition-metal-free regioselective cross-coupling of BODIPYs with thiols. Chemical Communications. 2019;55(11):1639-42. [36] Zhang T, Yan J, Hu Y, Liu X, Wen L, Zheng K, et al. A simple central seven-membered BOPYIN: synthesis, structural, spectroscopic properties and cellular imaging application. Chemistry – A European Journal. 2019;25(39):9266-71. [37] Zhang T, Wen L, Liu G, Yan J, Liu X, Zheng K, et al. A stable AIEgen cis-diarylethene-Based ‘ESIPT’ benchmark. Chemical Communication. 2019;55:13713-6. [38] Zhang N, Wen L, Liu X, Chen W, Yan J. One pot synthesis N-confused porphyrin-dipyrrin conjugates and their optical properties. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy. 2019:117661. [39] Li M, Zheng K, Chen H, Liu X, Xiao S, Yan J, et al. A novel 2,5-bis(benzo[d]thiazol-2-yl)phenol scaffold-based ratiometric fluorescent probe for sensing cysteine in aqueous solution and serum. Spectrochim Acta Part A: Molecular and Biomolecular Spectroscopy. 2019;217:1-7. [40] Liu X, Hamon J-R. Recent developments in penta-, hexa- and heptadentate Schiff base ligands and their metal complexes. Coordination Chemistry Reviews. 2019;389:94-118. [41] Wu J, Jiang L, Verwilst P, An J, Zeng H, Zeng L, et al. A colorimetric and fluorescent lighting-up sensor based on ICT coupled with PET for rapid, specific and sensitive detection of nitrite in food. Chemical Communications. 2019;55(67):9947-50. [42] Zhu D, Yan X, Ren A, Cai W, Duan Z, Luo Y. Modulation of ICT and PET processes in boranil derivatives: a ratiometric fluorescent probe for imaging of cysteine. Analytical Methods. 2019;11(19):2579-84. [43] Hu Q, Duan C, Wu J, Su D, Zeng L, Sheng R. Colorimetric and Ratiometric Chemosensor for Visual Detection of Gaseous Phosgene Based on Anthracene Carboxyimide Membrane. Analytical Chemistry. 2018;90(14):8686-91. [44] Ma Q-J, Li H-P, Yang F, Zhang J, Wu X-F, Bai Y, et al. A fluorescent sensor for low pH values based on a covalently immobilized rhodamine–napthalimide conjugate. Sensors and Actuators B: Chemical. 2012;166-167:68-74. [45] Hehre WJ, Ditchfield R, Pople JA. Self-Consistent Molecular Orbital Methods. XII. Further Extensions of Gaussian-Type Basis Sets for Use in Molecular Orbital Studies of Organic Molecules. The Journal of Chemical Physics. 1972;56(5):2257-61. [46] Miertuš S, Scrocco E, Tomasi J. Electrostatic interaction of a solute with a continuum. A direct utilizaion of AB initio molecular potentials for the prevision of solvent effects. Chem Phys. 1981;55(1):117-29. [47] Cammi R, Tomasi J. Remarks on the use of the apparent surface charges (ASC) methods in solvation problems: Iterative versus matrix-inversion procedures and the renormalization of the apparent charges. Journal of Computational Chemistry. 1995;16(12):1449-58. [48] Frisch MJ, Trucks GW, Schlegel HB, Scuseria GE, Robb MA, Cheeseman JR, et al. Gaussian 09, revision A.02. Wallingford CT: Gaussian Inc; 2009
[49] Long L, Li X, Zhang D, Meng S, Zhang J, Sun X, et al. Amino-coumarin based fluorescence ratiometric sensors for acidic pH and their application for living cells imaging. RSC Advances. 2013;3(30):12204-9. [50] Zhang X-F, Zhang T, Shen S-L, Miao J-Y, Zhao B-X. A ratiometric lysosomal pH probe based on the naphthalimide–rhodamine system. Journal of Materials Chemistry B. 2015;3(16):3260-6. [51] Hu J, Wu F, Feng S, Xu J, Xu Z, Chen Y, et al. A convenient ratiomeric pH probe and its application for monitoring pH change in living cells. Sensors and Actuators B: Chemical. 2014;196:194-202.
Highlight A novel BOPYIN based ‘turn-on’ fluorescent probe for acidic pH was developed. The PET/ICT mechanism is confirmed by spectra and DFT calculations studies. There is no significant interference caused by common metal ions to this pH probe. This probe can be used in real-time and reversible pH sensing. Applications were carried out for visual detection and cellular imaging.
Declaration of interests ☒ 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. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
S0143-7208(19)32500-8
DOI:
https://doi.org/10.1016/j.dyepig.2020.108318
Reference:
DYPI 108318
To appear in:
Dyes and Pigments
Received Date: 28 October 2019 Revised Date:
13 February 2020
Accepted Date: 28 February 2020
Please cite this article as: Yuan X, Zhang T, Yan J, Chen X, Wang L, Liu X, Zheng K, Zhang N, A simple acidic ‘turn-on’ fluorescent pH probe based on BOPYIN and its visual detection and cellular imaging, Dyes and Pigments (2020), doi: https://doi.org/10.1016/j.dyepig.2020.108318. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd.
Xiaohui Yuan:, Writing - Original Draft, Investigation, Data Curation Tingting Zhang: Methodology, Writing - Original Draft Jiaying Yan: Software, Validation, Resources Xi Chen: Visualization Long Wang: Supervision Xiang Liu: Writing - Review & Editing Kaibo Zheng: Conceptualization, Resources Nuonuo Zhang: Conceptualization, Funding acquisition, Project administration
A simple acidic ‘turn-on’ fluorescent pH probe based on BOPYIN and its visual detection and cellular imaging
Xiaohui Yuan,a Tingting Zhang,a Jiaying Yan,a,b Xi Chen,a Long Wang,a Xiang Liu,a,c Kaibo Zheng,a* and Nuonuo Zhanga* a
College of Materials and Chemical Engineering, Key Laboratory of Inorganic Nonmetallic Crystalline and Energy Conversion Materials, China Three Gorges University, Hubei, Yichang 443002, PR China. E-mail address: [email protected] and [email protected] b State Key Laboratory of Coordination Chemistry, Nanjing University, Jiangsu, Nanjing 210093, PR China c Material Analysis and Testing Center, China Three Gorges University, Hubei, Yichang 443002, PR China
Abstract A simple high selective and sensitive fluorescent pH probe based on a new seven-membered
BOPYIN
(BOrondifluoride-3,3-dimethyl-2-[2-(2-PYrrolyl)
ethenyl]INdole, Probe-NH2, Scheme 1) has been designed and synthesized. With the increase of acidity (pH from 5.51 to 2.47), the emission intensity dramatically enhanced to strong fluorescence. The linear response range is from 5.5 to 3.0 with pKa = 3.63, which is suitable for pH detection in biological system. Moreover, this probe has excellent selectivity response to H+, short response time (less than 10 seconds), reversibility and low cytotoxicity. Possible sensing mechanism studies demonstrated that the linear response of the probes to acidity of solution is the conjugation of H+ with response site N atom in primary amino group on BOPYIN. Besides, the fluorescent change of the Probe-NH2 relies on PET and/or ICT process which is proved by experiment and further confirmed by DFT theoretical calculation. Finally, the probe has been successfully applied for visual detection and cellular imaging.
Keywords: BOPYIN; fluorescent pH probe; Cellular imaging; Visual detection; Extreme acidity
1 Introduction Hydronium ion plays a crucial role in several essential processes, such as biochemical, environmental monitoring and clinical analysis. In biology process, the reduction of hydronium ion may induce cellular dysfunction and lead to some diseases including Alzheimer's disease and cancer[1-4]. Therefore, the monitor of pH change in cells, tissues and organisms is very important. Nowadays, electrochemistry[5], nuclear magnetic resonance (NMR)[6], absorption, and emission spectroscopy[7] measure pH value as the mainstream pH detection method. However, fluorescent pH probe is one of the utilized method due to its operational simplicity, high sensitivity, excellent spatial and real-time detection for monitoring intracellular concentration of hydronium ion.
Scheme 1 The synthetic route for Probe-NH2
Fluorescent pH sensors are developed based on diverse fluorescent molecules, nanoparticles and so on. In the past decades, fluorescent small molecules have drawn increasing attention as fluorescent probes for pH detection due to their designability, easy modification and excellent photophysical properties, which mainly based on excited-state intramolecular proton transfer (ESIPT), fluorescence resonance energy transfer (FRET), intramolecular charge transfer (ICT) and photoinduced electron transfer (PET) process. Traditional single-atom electron donor N acts as reaction position for pH detecting process based on the mechanism mentioned above. The existence form of N atom in neutral molecules are classified as three categories, primary amino, secondary amino and tertiary amino form. Therein, tertiary amino groups have been widely studied in pH detection, such as pyridine[8-11],
indole[12-14], benzothiazole[15, 16], benzimidazole[17] and other tertiary amino groups in or out of the ring[18-26]. The research on the primary amino groups is limited due to their instability or destructive inactivation under extreme pH condition[27-30]. Thus, it is particularly urgent to develop stable molecular fluorescent dyes to tolerate the harsh pH milieu in the fields of medical, biological and environmental science. BODIPY is an fascinating kind of popular fluorescent dye and applied in various fields[31-35]. In our previous work[36-40], BOPYIN as an analogue of BODIPY, and their derivatives are reported with a broad range of biocompatibility, excellent fluorescence properties, large Stokes shifts, good molar absorptivity and simple synthesis via peripheral modification to adjust the electronic structure. However, there is no available information about BOPYINs derivatives which are suitable for pH detection, especially for strong acidic condition (pH < 4). There is a high demand in the applications such as waste-water evaluation, environmental protection and disease monitor. As a subsequent work on BOPYIN and their derivatives, a new BOPYIN probe with amino responding group is designed and synthesized (Scheme 1, Probe-NH2). Interestingly, Probe-NH2 decorated with primary amino group has fast response to hydronium ion in strong acidic environments. Therefore, Probe-NH2 as a pH sensor for strong acidic condition is investigated in this work. Meanwhile, PET and/or ICT process of the emission change is proved by experiment and calculation.
2. Experimental section 2.1 Materials and apparatus All reagents and solvents were obtained from commercial sources and used without further purification, unless otherwise noted. All chromatographic separations were carried out on silica gel (300-400 mesh).
13
C NMR spectra were recorded on Bruker
400 MHz spectrometer at 298 K. DMSO-d6 was used as solvent and TMS as internal reference. The chemical shifts were reported in parts per million (δ) relative to the appropriate reference signal: residual DMSO (the quintet centered at 2.50 ppm). High
resolution mass spectra were measured on Bruker APCI instrument. UV-Vis spectra in various solvents were detected on Shimadzu UV-2600 in 10 mm quartz cell spectrometer. Fluorescence spectra were obtained using a Hitachi FL-4600 spectrometer. Fluorescent images were taken on a Carl Zeiss LSM 710 confocal laser scanning microscope (Jena, Germany). pH values were measured using an INESA PHS-25 pH meter. Gaussian 09 programs were used in theoretical calculations. 2.2 Synthesis and characterization of the fluorescent probe Herein, a novel pH-sensor Probe-NH2 was synthesized by the reaction of 5-nitro substituted trimethyl indole hydrochloride and 2-formylpyrrole based on Knoevenagel condensation, then coordination with BF3 OEt2 and reduction by SnCl2 2H2O (Scheme 1). This probe contains primary amine unit as electron donor and parent core of BOPYIN as fluorescence moieties. Synthesis of BOPYIN-NO2 A mixture of 5-nitro trimethyl indole hydrochloride 1 (410 mg, 2.0 mmol) and 2-formylpyrrole 2 (190 mg, 2.0 mmol) in EtOH (30 mL) was stirred under reflux for 1 h. After completion of reaction confirmed by TLC, this crude product was purified by flash chromatography (silica gel) to afford compound 3 with trans and cis isomer. Then, a mixture of compound 3 (280 mg, 1.0 mmol), Et3N (1.5 mL, 11.0 mmol) in toluene was added BF3•OEt2 (9.5 mmol, 1.2 mL). This mixture was stirred under reflux for 1 h. After completion of reaction confirmed by TLC, the reaction mixture was washed with water (30 mL × 3), dried over anhydrous Na2SO4 and concentrated under vacuum. The crude product was purified by chromatography (silica gel, hexanes/CH2Cl2 3:2) to afford BOPYIN-NO2 as a solid in 35% yield. 1H NMR (400 MHz, DMSO) δ 8.59 (d, J = 2.4 Hz, 1H), 8.36 (dd, J = 9.0, 2.4 Hz, 1H), 8.12 (d, J = 9.0 Hz, 1H), 7.76 (s, 1H), 7.74 (d, J = 11.4 Hz, 1H), 7.23 (d, J = 2.9 Hz, 1H), 6.66 (dd, J = 3.6, 2.2 Hz, 1H), 6.42 (d, J = 11.3 Hz, 1H), 1.56 (s, 6H).
13
C NMR (101 MHz,
DMSO) δ 183.85, 149.92, 145.23, 143.58, 140.84, 135.41, 135.13, 127.46, 125.10, 118.50, 118.06, 117.95, 117.84, 115.53, 101.87, 51.88, 24.55. HRMS (ESI) Calcd. for C16H14N3O2BF2 [M+H]+: 330.1223, found 330.1219. Synthesis of Probe-NH2
A mixture of BOPYIN-NO2 (658 mg, 2.0 mmol) and SnCl2•2H2O in DMF (48 mL) was stirred at room temperature for 1 h. After completion of reaction confirmed by TLC, water was added. The residue was filtrated and collected. The crude product was purified by chromatography (silica gel, hexanes/CH2Cl2 1:1) to afford Probe-NH2 as a solid in 28% yield. 1H NMR (400 MHz, DMSO-d6) δ 7.69 (d, J = 8.7 Hz, 1H), 7.44 (s, 1H), 7.41 (d, J = 11.7 Hz, 1H), 6.88 (d, J = 2.5 Hz, 1H), 6.71 (d, J = 2.2 Hz, 1H), 6.60 (dd, J = 8.8, 2.3 Hz, 1H), 6.47 (dd, J = 3.5, 2.4 Hz, 1H), 6.26 (d, J = 11.7 Hz, 1H), 5.55 (s, 2H), 1.39 (s, 6H).
13
C NMR (101 MHz, DMSO) δ 177.83,
149.00, 145.01, 136.51, 134.52, 134.13, 130.59, 122.32, 119.58, 119.49, 113.86, 113.55, 107.92, 103.60, 51.80, 25.08. HRMS (ESI) Calcd. for C16H16N3BF2 [M+H]+: 300.1481, found 300.1478.
3 Results and discussion 3.1 Spectroscopic properties and optical responses to pH The absorption and fluorescence spectra of Probe-NH2 (10.0 µM) were collected in 0.01 mol/L PBS buffer (PBS/CH3CN = 6: 1, v/v) with various pH (Fig.1, S1). Absorption spectra of the Probe-NH2 were examined in the pH ranging from 2.01 to 7.49 (Fig. S1). With the increase in the acidity of the solution from pH = 7.49 to 4.02, no significant change was detected in absorption spectra. With the acidity further increased from pH = 3.5 to 2.01, the increasing absorption split into two obvious peaks from a sole maximum absorbance peak around 480 nm, and the color of the solution changed from orange to lemon. The characteristic changes in absorption spectra with pH ranging from 2.01 to 7.49 indicates that this feature results from the PET and/or ICT effect. Furthermore, fluorescence properties were also studied. As shown in Fig. 1, the fluorescent intensity of Probe-NH2 in aqueous solution is heavily dependent on the acidity of the solution. The emission of Probe-NH2 is almost non-fluorescent in neutral solution. When the acidity increases (pH range: 2.47-5.01), the fluorescence intensity gradually enhances. That’s because protonation of the primary amino-group led to the recovery of the very strong fluorescence with a violet color at pH = 2.47.
The fluorescence intensity decreased slightly (pH range: 0.50-2.47) probably due to decomposition under strong acidic condition.
Fig. 1 Change of fluorescence spectra of Probe-NH2 (10 µM) in 0.01 mol/L PBS buffer (PBS/CH3CN = 6: 1, v/v) at various pH (0.52-7.49) (λex = 478 nm, slits: 5 nm/5 nm).
3.2 Possible sensing mechanism Binding Behavior of Probe with H+ The Probe-NH2 was designed to show the sensing ability of aromatic amine with H+ in acidic conditions. The pH sensitivity is introduced via the amine moiety upon either protonation or deprotonation. The probe exists in two forms, as either the orange nonfluorescent base form or the yellow fluorescent acid form (Scheme 2). 1H NMR experiments were carried out to prove the structure of probe after binding with H+. As shown in Table S2 and Fig. S3, when 15 equiv. of HCl is added to Probe-NH2 solution in DMSO-d6, an upfield shift is observed for the chemical shift values of the indole protons H1, H2, H3, double bond protons H4, H5 and pyrrole protons H6, H7, H8. The upfield chemical shift of these protons is obviously due to H+ binding with amine N resulting in the decrease of electron density around these protons. Thus, it is clear that H+ binds with amine N in Probe-NH2 causing the significant optical response to acidic pH. It’s well known that the lone pair of electrons on the nitrogen atom can induce fluorescence quenching via PET and/or ICT process[41-43]. Additionally, ICT process is easily occurred in conjugated D-π-A system. Probe-NH2 contains aromatic amine as donor and conjugated BOPYIN as acceptor. Hence, the sensing mechanism are speculated to PET and/or ICT process. 3.3 Principle of Operation and the Basis of Quantitative Assay According to the fluorescence spectra, sigmoidal fitting of fluorescence intensity at an
excitation wavelength of 478 nm versus various pH values and the linear response ranging from pH 3.0 to 5.5 were concluded in Fig. 2, which is connected with the ability of the Probe-NH2 to recognize proton. The relationship between the pH value and fluorescence intensity at λem = 478 nm can be expressed quantitatively using a Henderson-Hasselbalch type equation (Eq. 1)[27, 44]:
I −I log max = pH − pKa I − I min
(Eq. 1)
Where I is the observed fluorescence intensity; Imax and Imin represent the maximum and minimum of the fluorescence intensity of the Probe-NH2 in its acid (pH 2.47) and conjugate base (pH 7.49) form, respectively. Therefore, the linear response in Fig. 2b can be expressed by the following Eq. 2 of the calibration line:
I −I log max = 1.054 pH − 3.630( R = 0.992) I − I min
(Eq. 2)
where R is the linear correlation coefficient. According to the Eq. 2, the calculated pKa value of Probe-NH2 was 3.63, which is suitable for studying on acidic organelles. This result confirmed that Probe-NH2 is not only highly sensitive to the pH changes, but also linearly correlated to the pH in the range of 3.0-5.5 (R2 = 0.992) (Fig. 2b).
Fig. 2 pH dependence of the fluorescence emission intensity of Probe-NH2 (a); Plot of log[(Imax-I) / (I-Imin)] as a function of the pH (b).
3.4 Solvent-dependent properties Absorption and emission spectra were collected in different solvents (Fig. 3 and Table S1). Absorption spectra were similar in different solvents with a small peak at 374-386 nm and a sharp peak at 482-498 nm. Although the emission intensity was
weak in those organic solvents, the fluorescence intensity decreased and the emission peak red-shifted as the increase of solvent polarity. A large Stokes shift of 3490-4773 cm-1 was detected in various solvents. It indicated the influence of solvent effect on its optical properties. Based on this result, we chose CH3CN as solvent due to its low toxicity and water solubility in which it had large Stokes shift of 4405 cm-1 and non-fluorescence. Then, the spectra were measured using mixed solvents in different ratios of PBS/CH3CN (1:1, 3:1, 6:1, 9:1) as shown in Fig. S2. According to this result, the ratio of PBS/CH3CN (6:1) was chosen due to its low toxicity and solubility of probe compared to 1:1, 3:1 and 9:1, respectively.
Fig. 3 Absorption (a) and emission (b) spectra of Probe-NH2 in Toluene, DCM, THF, DMF, DMSO, CH3CN, C2H5OH and MeOH.
3.5 Theoretical calculation To gain insight into the mechanism of Probe-NH2, the PET and/or ICT effects were studied by using theory calculation method. The geometries of the ground states and the excited-state structures were optimized by the density functional theory (DFT) methodology with a B3LYP hybrid function method, and the 6-311G(d) basis set was used for all atoms[45]. Without considering the influence of solvent, toluene has been selected as the solvent in calculations based on the polarizable continuum model (PCM)[46, 47]. All calculations of the present work were performed using Gaussian 09 program package[48]. Probe-NH2 has two absorption peaks at 376, 491 nm and weak emission at 595 nm with moderate Stokes shift around 100 nm in the absorption and emission spectra in toluene, which is in good agreement with the calculation. (Fig. 4)
Fig. 4 TD-DFT (B3LYP/6-311G(d)) simulated stick spectra (Absorption: black column; Emission: red column) along with their normalized steady-state absorption spectrum (black solid line), emission spectrum (red solid line) of Probe-NH2 in toluene.
From the view of the geometric constructions, Probe-NH2 in the ground state (S0) and the lowest excited state (S1) are similar. The dihedral angle of indole unit and pyrrole unit slightly decreased (Fig. S4). However, the plane is obviously distorted from S1 to S0 of Probe-NH3+. The dihedral angle of indole unit and pyrrole unit of Probe-NH3+ in S1 state showed drastically decrease to S0 state. The geometric structures change between Probe-NH2 and Probe-NH3+ in S0 and S1 states is related to the electron distribution in frontier molecular orbitals. The frontier molecular orbitals, absorptions and emission related parameters are concluded in Fig. 5, Tables S3-5, which is related to the absorption and emission change between Probe-NH2 and Probe-NH3+.
Fig. 5 The frontier molecular orbitals involved in the vertical excitation and emission of Probe-NH2 (a) and its protonated from Probe-NH3+ (b) based on TD-DFT calculation in toluene.
The absorption peak with oscillator strengths over 0.61 originates from the electron transition from HOMO to LUMO. The emission peak with oscillator strengths over 0.45 originates from the electron transition from LUMO to HOMO for Probe-NH2 and LUMO to HOMO-2 for Probe-NH3+, respectively. According to the distribution of electrons, the electrons on HOMO orbits of Probe-NH2 mainly localized on the amino donor and then transferred to the BOPYIN acceptor (LUMO orbits), indicating a strong PET and/or ICT effects. Electrons smoothly transferred from primary amino (free receptor) to BOPYIN (fluorophore) in Probe-NH2, then PET and/or ICT process occurs. Meanwhile, the electron distributions of HOMO, LUMO orbits in ground state, HOMO-2 and LUMO orbits in excited state of Probe-NH3+ are similar, which almost located on BOPYIN fluorophores. Thus, PET and/or ICT process is prohibited. The energy level of free receptor, fluorophore and bound receptor are calculated for further discussing the mechanism of this fluorescence “turn-on” process (Scheme 2). Before the protonation, the HOMO energy level of free receptor (-5.685 eV) is between the HOMO (-5.785 eV) and LUMO (-2.564 eV) energy level of fluorophore. Therefore, the emission of Probe-NH2 is quenched by a PET process from free receptor to fluorophore skeleton. After protonation, the HOMO energy of bound receptor (-9.417 eV) is much lower than that of fluorophore, which prohibited the PET process, resulting in the strong fluorescent signal in Probe-NH3+. Additionally, the HOMO-LUMO energy gap of Probe-NH3+ (3.15 eV) is higher than Probe-NH2 (2.95 eV) confirming that the ICT process through a donor-π-acceptor system in this compound is disrupted.
Scheme 2 Reaction of Probe-NH2 in neutral and acid solution, HOMO energy level of free receptor, bound receptor and HOMO, LUMO energy level of fluorophore.
3.6 Real-time Response and Reversibility Study For practical application, it’s very important for the real-time determination[17, 49, 50]. Thus, the reaction-time profile of Probe-NH2 in the PBS/CH3CN solution of pH 7.31, 3.51 and 2.50 was measured. As shown in Fig. 6a, Probe-NH2 responds to the pH rapidly, the emission intensity enhances markedly within 10 seconds and then reaches a steady state. Therefore, Probe-NH2 could be applied to monitor the pH variation in real time. If the fluorescent pH probe is reversible, it will increase greatly for the potential application in real-time pH monitoring [51]. To examine the reversibility of this pH-dependent emission enhancement, the pH value of PBS/CH3CN solution was adjusted back and forth between 7.40 and 2.51 by using hydrochloric acid and sodium hydroxide (Fig. 6a). As shown in Fig. 6b, the results clearly reveal that these processes are reversible well. This suggests that reversibility of this probe between the protonated and deprotonated forms occurred. Thus, the cycle of pH-mediated fluorescence change (enhancement and elimination) and the real-time pH monitoring by Probe-NH2 are feasible.
Fig. 6 (a) Reaction-time profile of Probe-NH2 (10 µM in CH3CN/PBS, v/v, 1/6) at different pH values; (b) Reversibility (520 nm) of Probe-NH2 (10 µM in CH3CN/PBS, v/v, 1/6) between pH 7.40 and 2.51.
3.7 Selectivity studies
Fig. 7 Fluorescence intensity change at 478 nm of Probe-NH2 (10µM) in the presence of some common bio-related metal cations and anions in buffer solution at pH 2.5 and pH 7.5, λex = 478 nm, slit: 5.0 nm/5.0 nm. 1. blank; 2. Cr3+ (2 mM); 3. Mg2+ (2 mM); 4. Na+ (2 mM); 5. K+ (2 mM); 6. Zn2+ (2 mM); 7. Cu2+ (2 mM); 8. Co2+ (2 mM); 9. Fe2+ (2 mM); 10. Al3+ (2 mM). (a) pH =2.5, (b) pH = 7.5.
Selectivity is very important parameter to evaluate the properties of probe for the potential application in more complicated environments. Various metal ions including Cr3+, Mg2+, Na+, K+, Zn2+, Cu2+, Co2+, Fe2+ and Al3+ were introduced as competitive ions under the same test conditions with pHs of 2.5 and 7.4 for detecting the fluorescent response of Probe-NH2. As shown in Fig. 7, no significant changes were detected in emission spectra with the addition of the different metal ions, which means the high selectivity of this probe for hydronium ion. 3.8 Application
Fig. 8 Visual photographs of (a, b) Probe-NH2 (10 µM in CH3CN/PBS, v/v, 1/6) and (c) change in color of Probe-NH2 test strips (1 neutral, 2 exposed in HCl gas, 3 exposed in HCl, then Et3N gas) under 365 nm UV light. HCl (aq, 12 M), Et3N (7.2 M).
Visual detection of pH by the method of solution and test paper was also carried out. In solution, the change in color of Probe-NH2 was dramatic under 365 nm UV light in Fig. 8a,b. After the test papers were dipped in Probe-NH2 solutions (100 µM
in CH3CN) for 0.5 min, the observed color changes of the test strips were recorded as shown in Fig. 8c. Under irradiation with 365 nm UV light, the fluorescence of neutral test strip is weak; the acidic test strip exposed in HCl gas turns on and that then exposed in Et3N gas turns off in Fig. 8c. Therefore, the test strip experiment demonstrates that Probe-NH2 can be conveniently and efficiently used as a pH probe. We also studied whether Probe-NH2 could image the intracellular pH by conducting live (T24) cell imaging (Fig. 9, S11). We incubated cells in probe solution of 10 µM CH3CN/PBS. In T24 cells, Probe-NH2 shows strong fluorescence at pH 4.0 and non-emissive fluorescence at pH 7.4 with 10 µM concentration excitation at 480 nm.
Fig. 9 Fluorescence images of T24 cells incubated with 10 µM of Probe-NH2 in pH =4.0 and 7.4. Images were acquired using the confocal fluorescence microscope. Scale Bar: 25 µm.
4 Conclusion In summary, a new BOPYIN derivative as a ‘turn-on’ fluorescent pH probe was simply designed and synthesized with high selectivity, sensitivity, stability, short response time and low cytotoxicity, which applied in strongly acidic environments. With a unique response site (primary amino group) to hydronium ion, Probe-NH2 showed non-fluorescent to strong fluorescent with the increase acidity of solution at the pH from 5.51 to 2.47. The linear response curve is formed from pH 3.0 to 5.5, and the evaluated pKa value is 3.63. The fluorescence change of this probe is based on the PET and/or ICT process, which is proved by experiment and theoretical calculation. Furthermore, the probe has been successfully applied for visual detection and cellular imaging, with a ‘turn-on’ fluorescence in acidic environments. In a word, Probe-NH2
with the response site of amino group is first reported with excellent ability for detecting hydronium ion in strong acidic environments and applied in visual detection and biological imaging.
Acknowledgements This work was supported by National Natural Science Foundation of China (No. 21606145, 21805166), The 111 Project (D20015), State Key Laboratory of Coordination Chemistry Foundation of Nanjing University (No. SKLCC1811).
References [1] Davies TA, Fine RE, Johnson RJ, Levesque CA, Rathbun WH, Seetoo KF, et al. Non-age Related Differences in Thrombin Responses by Platelets from Male Patients with Advanced Alzheimer′s Disease. Biochemical and Biophysical Research Communications. 1993;194(1):537-43. [2] Bhuniya S, Maiti S, Kim E-J, Lee H, Sessler JL, Hong KS, et al. An Activatable Theranostic for Targeted Cancer Therapy and Imaging. Angewandte Chemie International Edition. 2014;53(17):4469-74. [3] Vegesna GK, Janjanam J, Bi J, Luo F-T, Zhang J, Olds C, et al. pH-activatable near-infrared fluorescent probes for detection of lysosomal pH inside living cells. Journal of Materials Chemistry B. 2014;2(28):4500-8. [4] Yue X, Yanez CO, Yao S, Belfield KD. Selective cell death by photochemically induced pH imbalance in cancer cells. Journal of the American Chemical Society. 2013;135(6):2112-5. [5] Ellis D, Thomas RC. Microelectrode measurement of the intracellular pH of mammalian heart cells. Nature. 1976;262(5565):224-5. [6] Sehgal AA, Duma L, Bodenhausen G, Pelupessy P. Fast Proton Exchange in Histidine: Measurement of Rate Constants through Indirect Detection by NMR Spectroscopy. Chemistry – A European Journal. 2014;20(21):6332-8. [7] Han
J,
Burgess K.
Fluorescent
Indicators for
Intracellular
pH.
Chemical Reviews.
2010;110(5):2709-28. [8] Chao J, Wang H, Zhang Y, Yin C, Huo F, Song K, et al. A novel ‘‘donor-π-acceptor’’ type fluorescence probe for sensing pH: mechanism and application in vivo. Talanta. 2017;174:468-76. [9] Chao J, Wang H, Song K, Li Z, Zhang Y, Yin C, et al. A highly sensitive acidic pH fluorescent probe and its application to E. coli cells. Tetrahedron. 2016;72(50):8342-9. [10] Xu Z, Ren A-M, Wang D, Guo J-F, Feng J-K, Yu X. A theoretical investigation on two latest two-photon pH fluorescent probes. Journal of Photochemistry and Photobiology A: Chemistry. 2014;293:50-6. [11] Yuan C, Li J, Xi H, Li Y. A sensitive pyridine-containing turn-off fluorescent probe for pH detection. Materials Letters. 2019;236:9-12. [12] Shen S-L, Zhang X-F, Ge Y-Q, Zhu Y, Lang X-Q, Cao X-Q. A near-infrared lysosomal pH probe based on rhodamine derivative. Sensors and Actuators B: Chemical. 2018;256:261-7. [13] Li J, Zhuge X, Yan X, Li Y, Yuan C. Two pH-responsive fluorescence probes based on indole derivatives. Optical Materials. 2019;90:257-63.
[14] Jin D, Wang B, Hou Y, Du Y, Li X, Chen L. Novel near-infrared pH-sensitive cyanine-based fluorescent probes for intracellular pH monitoring. Dyes and Pigments. 2019;170:107612. [15] Chao J, Liu Y, Sun J, Fan L, Zhang Y, Tong H, et al. A ratiometric pH probe for intracellular pH imaging. Sensors and Actuators B: Chemical. 2015;221:427-33. [16] Sun M, Du L, Yu H, Zhang K, Liu Y, Wang S. An intramolecular charge transfer process based fluorescent probe for monitoring subtle pH fluctuation in living cells. Talanta. 2017;162:180-6. [17] Wu Y-C, You J-Y, Jiang K, Wu H-Q, Xiong J-F, Wang Z-Y. Novel benzimidazole-based ratiometric fluorescent probes for acidic pH. Dyes and Pigments. 2018;149:1-7. [18] Xiao H, Zhang R, Wu C, Li P, Zhang W, Tang B. A new pH-sensitive fluorescent probe for visualization of endoplasmic reticulum acidification during stress. Sensors and Actuators B: Chemical. 2018;273:1754-61. [19] Georgiev NI, Said AI, Toshkova RA, Tzoneva RD, Bojinov VB. A novel water-soluble perylenetetracarboxylic diimide as a fluorescent pH probe: Chemosensing, biocompatibility and cell imaging. Dyes and Pigments. 2019;160:28-36. [20] Georgiev NI, Krasteva PV, Bojinov VB. A ratiometric 4-amido-1,8-naphthalimide fluorescent probe based on excimer-monomer emission for determination of pH and water content in organic solvents. Journal of Luminescence. 2019;212:271-8. [21] Xia S, Fang M, Wang J, Bi J, Mazi W, Zhang Y, et al. Near-infrared fluorescent probes with BODIPY donors and rhodamine and merocyanine acceptors for ratiometric determination of lysosomal pH variance. Sensors and Actuators B: Chemical. 2019;294:1-13. [22] Niu G, Zhang P, Liu W, Wang M, Zhang H, Wu J, et al. Near-Infrared Probe Based on Rhodamine Derivative for Highly Sensitive and Selective Lysosomal pH Tracking. Analytical Chemistry. 2017;89(3):1922-9. [23] Ye F, Liang X-M, Wu N, Li P, Chai Q, Fu Y. A new perylene-based fluorescent pH chemosensor for strongly acidic condition. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy. 2019;216:359-64. [24] Cao X-J, Chen L-N, Zhang X, Liu J-T, Chen M-Y, Wu Q-R, et al. A NBD-based simple but effective fluorescent pH probe for imaging of lysosomes in living cells. Analytica Chimica Acta. 2016;920:86-93. [25] Chao J, Song K, Zhang Y, Yin C, Huo F, Wang J, et al. A pyrene-based colorimetric and fluorescent pH probe with large stokes shift and its application in bioimaging. Talanta. 2018;189:150-6. [26] Liu Z, Li G, Wang Y, Li J, Mi Y, Zou D, et al. Quinoline-based ratiometric fluorescent probe for detection of physiological pH changes in aqueous solution and living cells. Talanta. 2019;192:6-13. [27] Ma Q, Li X, Feng S, Liang B, Zhou T, Xu M, et al. A novel acidic pH fluorescent probe based on a benzothiazole derivative. Spectrochimica acta Part A, Molecular and biomolecular spectroscopy. 2017;177:6-13. [28] Tan Y, Yu J, Gao J, Cui Y, Wang Z, Yang Y, et al. A fluorescent pH chemosensor for strongly acidic conditions based on the intramolecular charge transfer (ICT) effect. RSC Advances. 2013;3(15):4872. [29] Urano Y, Asanuma D, Hama Y, Koyama Y, Barrett T, Kamiya M, et al. Selective molecular imaging of viable cancer cells with pH-activatable fluorescence probes. Nature Medicine. 2008;15(1):104-9. [30] Wang Z, Zhang Y, Li M, Yang Y, Xu X, Xu H, et al. Two D-π-A type fluorescent probes based on isolongifolanone for sensing acidic pH with large Stokes shifts. Tetrahedron. 2018;74(24):3030-7. [31] Ding Y, Tang Y, Zhu W, Xie Y. Fluorescent and colorimetric ion probes based on conjugated oligopyrroles. Chemical Society Reviews. 2015;44(5):1101-12. [32] Ding Y, Zhu W-H, Xie Y. Development of Ion Chemosensors Based on Porphyrin Analogues.
Chemical Reviews. 2017;117(4):2203-56. [33] Wang Q, Wei X, Li C, Xie Y. A novel p-aminophenylthio- and cyano- substituted BODIPY as a fluorescence turn-on probe for distinguishing cysteine and homocysteine from glutathione. Dyes and Pigments. 2018;148:212-8. [34] Ma F, Zhou L, Liu Q, Li C, Xie Y. Selective Photocatalysis Approach for Introducing ArS Units into BODIPYs through Thiyl Radicals. Organic Letters. 2019;21(3):733-6. [35] Lv F, Tang B, Hao E, Liu Q, Wang H, Jiao L. Transition-metal-free regioselective cross-coupling of BODIPYs with thiols. Chemical Communications. 2019;55(11):1639-42. [36] Zhang T, Yan J, Hu Y, Liu X, Wen L, Zheng K, et al. A simple central seven-membered BOPYIN: synthesis, structural, spectroscopic properties and cellular imaging application. Chemistry – A European Journal. 2019;25(39):9266-71. [37] Zhang T, Wen L, Liu G, Yan J, Liu X, Zheng K, et al. A stable AIEgen cis-diarylethene-Based ‘ESIPT’ benchmark. Chemical Communication. 2019;55:13713-6. [38] Zhang N, Wen L, Liu X, Chen W, Yan J. One pot synthesis N-confused porphyrin-dipyrrin conjugates and their optical properties. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy. 2019:117661. [39] Li M, Zheng K, Chen H, Liu X, Xiao S, Yan J, et al. A novel 2,5-bis(benzo[d]thiazol-2-yl)phenol scaffold-based ratiometric fluorescent probe for sensing cysteine in aqueous solution and serum. Spectrochim Acta Part A: Molecular and Biomolecular Spectroscopy. 2019;217:1-7. [40] Liu X, Hamon J-R. Recent developments in penta-, hexa- and heptadentate Schiff base ligands and their metal complexes. Coordination Chemistry Reviews. 2019;389:94-118. [41] Wu J, Jiang L, Verwilst P, An J, Zeng H, Zeng L, et al. A colorimetric and fluorescent lighting-up sensor based on ICT coupled with PET for rapid, specific and sensitive detection of nitrite in food. Chemical Communications. 2019;55(67):9947-50. [42] Zhu D, Yan X, Ren A, Cai W, Duan Z, Luo Y. Modulation of ICT and PET processes in boranil derivatives: a ratiometric fluorescent probe for imaging of cysteine. Analytical Methods. 2019;11(19):2579-84. [43] Hu Q, Duan C, Wu J, Su D, Zeng L, Sheng R. Colorimetric and Ratiometric Chemosensor for Visual Detection of Gaseous Phosgene Based on Anthracene Carboxyimide Membrane. Analytical Chemistry. 2018;90(14):8686-91. [44] Ma Q-J, Li H-P, Yang F, Zhang J, Wu X-F, Bai Y, et al. A fluorescent sensor for low pH values based on a covalently immobilized rhodamine–napthalimide conjugate. Sensors and Actuators B: Chemical. 2012;166-167:68-74. [45] Hehre WJ, Ditchfield R, Pople JA. Self-Consistent Molecular Orbital Methods. XII. Further Extensions of Gaussian-Type Basis Sets for Use in Molecular Orbital Studies of Organic Molecules. The Journal of Chemical Physics. 1972;56(5):2257-61. [46] Miertuš S, Scrocco E, Tomasi J. Electrostatic interaction of a solute with a continuum. A direct utilizaion of AB initio molecular potentials for the prevision of solvent effects. Chem Phys. 1981;55(1):117-29. [47] Cammi R, Tomasi J. Remarks on the use of the apparent surface charges (ASC) methods in solvation problems: Iterative versus matrix-inversion procedures and the renormalization of the apparent charges. Journal of Computational Chemistry. 1995;16(12):1449-58. [48] Frisch MJ, Trucks GW, Schlegel HB, Scuseria GE, Robb MA, Cheeseman JR, et al. Gaussian 09, revision A.02. Wallingford CT: Gaussian Inc; 2009
[49] Long L, Li X, Zhang D, Meng S, Zhang J, Sun X, et al. Amino-coumarin based fluorescence ratiometric sensors for acidic pH and their application for living cells imaging. RSC Advances. 2013;3(30):12204-9. [50] Zhang X-F, Zhang T, Shen S-L, Miao J-Y, Zhao B-X. A ratiometric lysosomal pH probe based on the naphthalimide–rhodamine system. Journal of Materials Chemistry B. 2015;3(16):3260-6. [51] Hu J, Wu F, Feng S, Xu J, Xu Z, Chen Y, et al. A convenient ratiomeric pH probe and its application for monitoring pH change in living cells. Sensors and Actuators B: Chemical. 2014;196:194-202.
Highlight A novel BOPYIN based ‘turn-on’ fluorescent probe for acidic pH was developed. The PET/ICT mechanism is confirmed by spectra and DFT calculations studies. There is no significant interference caused by common metal ions to this pH probe. This probe can be used in real-time and reversible pH sensing. Applications were carried out for visual detection and cellular imaging.
Declaration of interests ☒ 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. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: