Accepted Manuscript Novel benzimidazole-based ratiometric fluorescent probes for acidic pH Yan-Cheng Wu, Jia-Yi You, Kai Jiang, Han-Qing Wu, Jin-Feng Xiong, Zhao-Yang Wang PII:
S0143-7208(17)31504-8
DOI:
10.1016/j.dyepig.2017.09.043
Reference:
DYPI 6268
To appear in:
Dyes and Pigments
Received Date: 9 July 2017 Revised Date:
2 September 2017
Accepted Date: 17 September 2017
Please cite this article as: 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 (2017), doi: 10.1016/j.dyepig.2017.09.043. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. 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.
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Novel benzimidazole-based ratiometric fluorescent probes for acidic pH
Yan-Cheng Wuab, Jia-Yi Youa, Kai Jianga, Han-Qing Wua, Jin-Feng Xiong*c and Zhao-Yang
a
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Wang*a
School of Chemistry and Environment, South China Normal University; Key Laboratory of Theoretical Chemistry of
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Environment, Ministry of Education, Guangzhou 510006, China
College of Materials Science and Engineering, South China University of Technology, Guangzhou 510640, China
c
HEC Pharm R&D Center, Dongguan 523871, China
* Corresponding Author
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b
E-mail:
[email protected]. Tel: 8620-39310258.
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E-mail:
[email protected].
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ABSTRACT: 2,6-Bis(1-alkyl-1H-benzo[d]imidazol-2-yl)naphthalene (2a-2c) as three new benzimidazole molecules have been designed, synthesized and applied as ratiometric fluorescent pH probes in
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the acidic pH region with high-efficiency and fast response. For probe 2a, upon decreasing the pH from 7.5 to 2.5, the emission spectra exhibit a large red shift from 402 to 472 nm, and the emission ratio (I472/I402) changes dramatically from 0.10 to 3.42 with a pKa value of 2.92. No
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significant interference on emission behavior is observed in the presence of various metal ions.
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Similar results are obtained for probes 2b and 2c. 1H NMR and DFT calculations studies demonstrate that the ratiometric response of the probes to acidic pH is due to H+ binding with one N atom in C=N of benzimidazole ring and the subsequent enhancement of the intramolecular charger transfer (ICT) process. In addition, these probes can be used in real-time and reversible
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pH sensing. Finally, the probes have been successfully applied to the visual detection of pH not
Keywords:
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only in solution, but also in test paper.
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Benzimidazole; Fluorescent probe; pH sensing, Ratiometric response; ICT
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1. Introduction Since pH is a key parameter in a wide range of fields, such as chemical process control, biological systems and environmental monitoring, the measurement of pH is of great significance
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to the protection of the environment and life [1-6]. Owing to many advantages of the fluorescent detection technique, such as high selectivity and sensitivity, operational simplicity, low cost and real-time monitoring, the design and synthesis of fluorescent pH probes with high-efficiency and
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fast response have become a highly popular topic of scientists' interests [7-12].
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Recently, a number of fluorescent probes have been used to the measurement of pH [13-23]. Amongst them, most fluorescent probes only have an intensity change in single-emission window with the response to the variation of pH [18-23]. However, fluorescence signals are easily influenced by environmental factors, such as temperature, solvent polarity and excitation
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wavelength [24,25]. It is well-known that, the ratiometric fluorescent probe can encompass a built-in correction for the above-mentioned environmental effects to improve the selectivity and sensitivity since the ratiometric measurements are based on the change in the ratio of the
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emission intensity at two wavelengths [26-30]. Therefore, there are some reports about
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ratiometric fluorescent pH probes in recent year, and the typical examples include polymers [31-33], carbon nanodots [34-36], metal-organic frameworks [37,38], and organic small molecules [39-43]. In these efforts, organic small molecules-based fluorescent ratiometric probes are easier to be designed and prepared. However, some deficiencies still exist in organic small molecule-based ratiometric fluorescent pH probes. For instance, more steps are necessary in the synthesis [39-41]. Utilizing that the N-H unit and the N atom in C=Ν of benzimidazole ring can interact with
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[25,45,49]. In addition, only a few benzimidazole-based probes can be applicable for strong acidic conditions with pH below 4 [45,48,49]. Therefore, it is important to develop benzimidazole-based ratiometric pH probes for extreme acidity with high-efficiency and fast
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response.
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Herein, for a continuation of our ongoing program aimed at developing novel fluorescent probes for different analytes [50-53], we have synthesized and characterized three benzimidazole-based fluorescent probes as new ratiometric fluorescent pH probes (Scheme 1). These probes display excellent performance and potential application as anticipated. Their
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sensing mechanism via the enhanced intramolecular charger transfer (ICT) effect has been
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established with the help of 1H NMR and DFT calculations studies.
Scheme 1.
Synthesis of probes 2.
2. Experimental 2.1. Materials and instruments 4
ACCEPTED MANUSCRIPT Melting point was performed on an X-5 digital melting point apparatus without correcting. 1
H and 13C NMR spectra were collected on a BRUKER DRX-400 spectrometer in CDCl3 using
TMS as an internal standard. Mass spectra (MS) were recorded on a Thermo LCQ DECA XP
UV-vis
spectra
were
measured
by using
a
Shimazu
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MAX mass spectrometer. Elemental analysis was obtained with Perkin Elmer Series II 2400. UV-2550
ultraviolet
visible
spectrophotometer at room temperature. The fluorescence spectra were recorded with a Hitachi
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F-2700 spectrophotometer at room temperature; and the slit width was 5 nm for both excitation
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and emission. pH values were recorded on a LIDA Model PHS-25C Bench pH/MV meter. All reagents were purchased from commercial suppliers and used without purification. 2,6-Bis(1H-benzo[d]imidazol-2-yl)naphthalene 1 was prepared according to our literature [52].
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2.2. Synthesis
According to the literature [50], a 100 mL round-bottom flask was charged with compound 1 (3 mmol, 1.080 g), the selected bromoalkane (6.2 mmol) and solid NaOH (12 mmol, 0.480 g)
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in CH3CN (30 mL). The reaction mixture was stirred at 80 °C for 2-4 h under the monitoring of
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TLC. After the solvent was removed, the residue was dissolved in dichloromethane. The organic layers were washed with water three times and then dried over anhydrous MgSO4. The concentration under vacuum gave a crude product, which was purified by column chromatography on silica gel with gradient eluents of petroleum ether and ethyl acetate to afford pure compound 2. 2,6-Bis(1-decyl-1H-benzo[d]imidazol-2-yl)naphthalene (2a). White solid, 1.702 g, yield 88.5%, m.p. 97.2-98.5
; UV-vis (THF) λmax: 327.0 nm; 1H NMR (CDCl3-TMS, 400 MHz): δ =
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31.8, 45.0, 110.2, 120.1, 122.5, 122.9, 127.1, 129.0, 129.1, 129.4, 133.2, 135.8, 143.3, 153.2; IR (film), ν, cm-1: 3055, 2955, 2922, 2855, 1616, 1489, 1454, 1329, 893, 824, 746; ESI-MS, m/z (%):
C 82.45, H 8.81, N 8.74, Found: C 82.58, H 9.01, N 8.72.
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Calcd for C44H57N4+ ([M+H]+): 641.46 (100%), Found: 641.50 (100%); Anal. Calcd for C44H56N4:
83.7%, m.p. 93.3-94.2
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2,6-Bis(1-dodecyl-1H-benzo[d]imidazol-2-yl)naphthalene (2b). White solid, 1.750 g, yield ; UV-vis (THF) λmax: 327.0 nm; 1H NMR (CDCl3-TMS, 400 MHz): δ =
0.85 (t, J = 8.0 Hz, 6H), 1.06-1.41 (m, 36H), 1.85-1.95 (m, 4H), 4.34 (t, J = 8.0 Hz, 4H), 7.31-7.39 (m, 4H), 7.44-7.50 (m, 2H), 7.84-7.90 (m, 2H), 7.92 (d, J = 8.0 Hz, 2H), 8.09 (d, J = 8.0 Hz, 2H),
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8.31 (s, 2H); 13C NMR (CDCl3-TMS, 100 MHz): δ = 14.1, 22.7, 26.8, 29.1, 29.3, 29.4, 29.5, 29.6, 29.9, 31.9, 45.0, 110.2, 120.1, 122.5, 122.9, 127.2, 129.1, 129.4, 133.2, 135.8, 143.3, 153.2; IR
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(film), ν, cm-1: 3063, 2930, 2855, 1616, 1489, 1458, 1329, 893, 819, 746; ESI-MS, m/z (%): Calcd for C48H65N4+ ([M+H]+): 697.52 (100%), Found: 697.47 (100%); Anal. Calcd for C48H64N4: C
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82.71, H 9.25, N 8.04, Found: C 82.78, H 9.34, N 7.96.
2,6-Bis(1-tetradecyl-1H-benzo[d]imidazol-2-yl)naphthalene (2c). White solid, 1.625 g, yield 71.9%, m.p. 88.6-89.3
; UV-vis (THF) λmax: 328.0 nm; 1H NMR (CDCl3-TMS, 400 MHz):
δ = 0.86 (t, J = 8.0 Hz, 6H), 1.07-1.39 (m, 44H), 1.85-1.95 (m, 4H), 4.35 (t, J = 8.0 Hz, 4H), 7.30-7.39 (m, 4H), 7.44- 7.50 (m, 2H), 7.84-7.90 (m, 2H), 7.92 (d, J = 8.0 Hz, 2H), 8.09 (d, J = 8.0 Hz, 2H), 8.31 (s, 2H);
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C NMR (CDCl3-TMS, 100 MHz): δ = 14.1, 22.7, 26.7, 29.4, 29.5,
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Calcd for C52H72N4: C 82.93, H 9.64, N 7.44, Found: C 82.88, H 9.75, N 7.52.
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2.3. Computational methods
According to the literature [24,54], all of the calculations were obtained using density
3. Results and discussion
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functional theory (DFT) with the B3LYP/6-31G (d) level of the Gaussian 09 program.
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3.1. Synthesis and Photophysical Properties of 2
As shown in Scheme 1, the target molecules 2a-2c were synthesized via a simple and efficient N-alkylation reaction of compound 1 and the selected 1-bromoalkane with the yields of 13
C NMR, ESI-MS [the corresponding
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71.9-88.5%. Their structures were characterized by 1H,
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spectra (Figs. S1-S9) can be seen in Supporting Information (SI)], IR, and elemental analysis. In an effort to gain insight into the photophysical process of the target molecules, we firstly investigated the absorption and emission behaviors of compounds 2a-2c in different non-polar or polar solvents. They show similar absorption spectra, and there is a slight blueshift with the increase of the solvent polarity (Figs. S10a-S12a). The emission spectra of compounds 2a-2c show two shoulder peaks in less polar solvents, but merging into a single peak in polar solvents (Figs. S10b-S12b). It's worth noting that the fluorescence intensity of 2 is relatively high in THF,
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Fig. 1.
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studies on the fluorescent properties of compounds 2a-2c.
The fluorescence spectra (λex = 325 nm) of compound 2c (10 µM) in H2O/THF system
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with different water fractions.
The emission spectra of compound 2c in THF-H2O mixed solvents had also been
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investigated. As shown in Fig. 1, compound 2c exhibits a strong emission at 400 nm with a shoulder peak at 389 nm in THF solution. When the water fraction (fw) is less than 60%, there is a slight change in fluorescence intensity. Further increasing fw may lead to a dramatic decline in the emission intensity, indicating that the maximum water content is 60%. Perhaps, the probes may aggregate in the presence of a large amount of water, resulting in fluorescence quenching (aggregation-caused quenching effect). However, all the aqueous mixtures are homogenous without precipitates, suggesting that the aggregates are of nano-dimensions. This is proved by the 8
ACCEPTED MANUSCRIPT level-off tail observed in the UV spectrum (Fig. S13) in the longer wave-length region when the fw values exceeded 60% [53,55]. These results show that compounds 2 has a high tolerance to water. Thus, we chose the binary system with an fw of 60% (H2O/THF, v/v, 3/2) as the solvent for
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studying the pH responsive properties of the probes.
3.2. Optical Response to pH
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To study the optical responses of compounds 2 to pH, standard pH titrations of absorption
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and emission spectra were performed. The spectroscopic properties of probe 2a under different pH conditions in H2O/THF (v/v, 3/2) solution were first selected to be examined (Fig. 2). As the pH is decreased from 7.5 to 2.5, the absorbance at 282 and 325 nm is gradually decreased accompanying by the formation of a new peak at 245 nm and a shoulder peak at around 370 nm
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(Fig. 2a). Further decrease in pH does not induce any change in the absorption spectrum.
(a) Fig. 2.
(b)
(a) UV-vis absorption spectra and (b) emission spectra of probe 2a (10 µM in H2O/THF, v/v, 3/2) with the different pH (Inset: plot of ratio of I472/I402 as a function of pH).
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ACCEPTED MANUSCRIPT As shown in Fig. 2b, upon decreasing the pH from 7.5 to 2.5, the emission spectra of probe 2a exhibits a shift from 402 to 472 nm with an obvious red shift of 70 nm. The large red shift of the signals may be attributed to the enhanced ICT effect because of the H+ binding-induced
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enhancement of the electron-withdrawing ability of benzimidazole ring. A well-defined isoemission point is found at 460 nm, indicating that only one benzimidazole ring could bind with H+ [24]. The large red shift makes probe 2a suitable for ratiometric pH sensing in the acidic
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region. When the pH is decreased from 7.5 to 2.5, the emission intensity ratio (I472/I402) changes
as a function of pH (Fig. S14).
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from 0.10 to 3.42 (Inset Fig. 2b). The pKa value of 2.92 can be calculated from the emission ratio
Similar behaviors were also observed for both 2b (Fig. S15) and 2c (Fig. S16). As the pH is decreased, the emission spectra of both 2b (Fig. S15b) and 2c (Fig. S16b) exhibit an apparent
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red shift from 402 to 472 nm, with the emission ratio (I472/I402) changing from 0.11 and 0.10 to 3.13 and 3.53 respectively (Inset Figs. S15b and S16b). Similarly, the pKa value of 2b and 2c can
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be calculated to be 2.90 and 2.51 respectively (Fig. S17).
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3.3. Possible Sensing Mechanism
In order to get insight into the sensing mechanism of probes 2 for acidic pH, 1H NMR experiments were carried out according to the literature [24,56]. As shown in Fig. 3, with the addition of incremental amounts of CF3COOD (1-10 equiv.) to the solution of probe 2a (20 mM in CDCl3), a downfield shift is observed for the chemical shift values of the benzimidazole protons (Hd, He, Hf) and naphthalene protons (Ha, Hb). When 20 equiv. of CF3COOD is added to above solution, the chemical shift values of these protons remain almost constant. The downfield
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ACCEPTED MANUSCRIPT chemical shift of these protons is obviously due to the H+ binding with the N atom in C=Ν of benzimidazole ring, which results in the decrease of electron density around these protons [24,56]. In addition, the chemical shift values of the benzimidazole protons (Hg) and naphthalene
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protons (Hc) are almost unchanged, indicating that the alkylated N atom in benzimidazole ring does not bind with H+ [24]. Therefore, it is clear that the H+ binding with the N atom in C=Ν of
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benzimidazole ring in probes 2 results in the significant optical response to acidic pH.
Fig. 3.
Partial 1H NMR spectra of probe 2a (20 mM in CDCl3) and probe 2a with the different equiv. CF3COOD.
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Fig. 4.
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Calculated (DFT B3LYP/6-31G (d) level) HOMO-LUMO energy levels and the
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interfacial plots of the orbitals for probe 2a, the 2a-H+ complex and the 2a-2H+ complex.
To better understand the optical responses of probes 2 upon binding with H+, we further
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carried out DFT calculations using probe 2a as an example (Fig. 4). The DFT calculations study shows a HOMO→LUMO charge transfer in both 2a and 2a-H+. For probe 2a, the LUMO is
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mainly localized on the central naphthalene core and partly distributed on both benzimidazole rings, whereas the HOMO is evenly distributed on the central naphthalene core and both benzimidazole rings. For 2a-H+ complex, the LUMO is mainly localized on the central naphthalene core and benzimidazolium moiety, whereas the HOMO is mainly distributed on another benzimidazole ring without the appendage of H+. These results indicate that the ICT process in 2a-H+ is enhanced comparing with that in 2a [24]. In addition, the energy gap (△E) between the HOMO and LUMO of the 2a-H+ complex (2.65583 eV) is smaller than that of probe 12
ACCEPTED MANUSCRIPT 2a (3.84089 eV), indicating that the 2a-H+ complex is more stable than probe 2a. The result is in good agreement with the red shift in emission spectra of probe 2a upon binding with H+. For bis-protonation species (2a-2H+), the LUMO is mainly localized on the central
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naphthalene core and partly distributed on both benzimidazole rings, whereas the HOMO is mainly distributed on one N-alkyl chain. It is worth noting that the ∆E of 2a-2H+ is increased (3.20387 eV) comparing with that of 2a-H+. These results further confirm that the
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mono-protonation is difficult to further formation of bis-protonation [54]. What is more, the
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results of DFT calculations for probe 2b and its protonated species (Fig. S18) are basically similar. Thus, the above conclusions are further confirmed.
Based on the above research results, the sensing mechanism for pH can be concluded in the following (Scheme 2): When proton is added into the solution of probes 2, H+ can bind with one
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N atom in C=Ν of benzimidazole ring [57], and H+ binding induces an enhancement of the electron-withdrawing ability of benzimidazole. Therefore, the fluorescence changes via
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enhancing the ICT process in 2-H+ complex [45,56].
Scheme 2.
Proposed mechanism of fluorescence change for probes 2 upon addition of H+.
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of the probes 2 within the additives except for Fe3+ causing a slight increase of I472/I402. Meanwhile, the addition of different metal ions has no obvious influence on the emission ratio of the probes 2 at pH = 3.0. These results demonstrate that probes 2 can be considered as selective
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fluorescent probes for acidic pH sensing.
Fig. 5.
The ratio of fluorescence intensity of probe 2a (10 µM) at I472/I402 containing different
metal ions at pH 7.0 (black bar) and 3.0 (blue bar). All the concentration of the metal ions is 500 µM (λex = 325 nm).
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values was measured. As shown in Fig. 6a, the probe 2a can respond to the variation of pH rapidly, the emission ratios change markedly within 5 seconds and then reach a steady state.
Fig. 6.
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is very much shorter than the reported before [30,58].
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Therefore, the probes 2 can be used to monitor the pH variation in real time. Their response time
(a) Reaction-time profile of probe 2a (10 µM in H2O/THF, v/v, 3/2) at different pH
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values; (b) Reversibility of the emission ratio (I472/I407) of probe 2a (10 µM in H2O/THF, v/v, 3/2) between pH 7.4 and 2.5.
If the fluorescence change of pH probe is reversible, the potential application in real-time pH monitoring will be increased greatly [59]. To examine the reversibility of these pH-dependent emission ratio changes, the pH value of the solution was adjusted back and forth between 7.4 and
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pH-mediated fluorescence conversion and the real-time pH monitoring by the probes is feasible.
3.6. Visual Detection of pH
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In order to further investigate the application of probes 2, we studied the visualization of
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fluorescent color change by using probe 2a at H2O/THF solution (v/v, 3/2, 10 µM) under different pH conditions (Fig. 7a) [11,22]. Under irradiation with a 365 nm UV light, the color of the solution is changed from blue-violet to cyan with the decrease of pH value, which is consistent with the change of fluorescence spectra. Therefore, probe 2a can be used as a
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naked-eye.
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convenient probe to distinguish different pH value through fluorescent color change by
Fig. 7.
Visual photographs of (a) probe 2a (10 µM in H2O/THF, v/v, 3/2) and (b) change in
color of probe 2a test strips with different pH solutions under 365 nm UV light.
Visual detection of pH by the method of test paper was also carried out according to the 16
ACCEPTED MANUSCRIPT literature [16,60]. After the test paper were dipped in neutral (pH = 7.0) and acidic (pH = 2.5) solutions containing probe 2a (10 µM in H2O/THF, v/v, 3/2) for 1 min and then dried in air, the observed colour changes of the test strips were recorded as shown in Fig. 7b. Under irradiation
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with 365 nm UV light, the neutral (pH = 7.0) test strip is blue-violet, and the acidic (pH = 2.5) test strip is cyan. Therefore, the test strip experiment demonstrates that probe 2a can be
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conveniently and efficiently used as a pH probe.
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4. Conclusions
In summary, we have designed and synthesized three novel benzimidazole molecules as ratiometric fluorescent pH probes. The emission spectra of the probes exhibit marked red shifts, resulting in large changes in the emission ratios. The ratiometric response of the probes to acidic
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pH is due to the H+ binding with one N atom in C=Ν of benzimidazole ring and the induced enhancement of the ICT process, which is confirmed by 1H NMR experiments and DFT calculations. In addition, there is no significant interference caused by common metal ions to
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these pH probes. Finally, they display potential applications to monitor the pH variation in real
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time and by naked-eyes.
Acknowledgements
We are grateful to Guangzhou Science and Technology Project Scientific Special (General Items, No. 201607010251), Guangdong Provincial Science and Technology Project (No. 2017A010103016) and the NSF of Guangdong Province (No. 2014A030313429) for financial
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Appendix A. Supplementary data
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Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/
Han JY, Burgess K. Fluorescent indicators for intracellular pH. Chem Rev 2010; 110:
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1. Three novel ratiometric fluorescent probes for acidic pH are developed. 2. The enhanced ICT mechanism is confirmed by 1H NMR and DFT calculations studies.
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3. There is no significant interference caused by common metal ions to these pH probes.
4. These probes can be used in real-time and reversible pH sensing.
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5. There is a potential application of monitoring the pH variation by
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naked-eyes.