An intermolecular pyrene excimer-based ratiometric fluorescent probes for extremely acidic pH and its applications

Journal Pre-proof An intermolecular pyrene excimer-based ratiometric fluorescent probes for extremely acidic pH and its applications Tian Liu, Zijie Huang, Ruihong Feng, Zhiqi Ou, Sha Wang, Liting Yang, Li-Jun Ma PII:

S0143-7208(19)32399-X

DOI:

https://doi.org/10.1016/j.dyepig.2019.108102

Reference:

DYPI 108102

To appear in:

Dyes and Pigments

Received Date: 12 October 2019 Revised Date:

27 November 2019

Accepted Date: 2 December 2019

Please cite this article as: Liu T, Huang Z, Feng R, Ou Z, Wang S, Yang L, Ma L-J, An intermolecular pyrene excimer-based ratiometric fluorescent probes for extremely acidic pH and its applications, Dyes and Pigments (2020), doi: https://doi.org/10.1016/j.dyepig.2019.108102. 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. © 2019 Published by Elsevier Ltd.

Author Contribution Statement An intermolecular pyrene excimer-based ratiometric fluorescent probes for extremely acidic pH and its applications

Tian Liua, Zijie Huanga, Ruihong Fenga, Zhiqi Oua, Sha Wanga, Liting Yanga, Li-Jun Maa,b,c*

a

School of Chemistry, South China Normal University, Shipai, Guangzhou, P. R.

China. b

Guangzhou Key Laboratory of Analytical Chemistry for Biomedicine, South China

Normal University, Guangzhou, P. R. China. c

Key Laboratory of Theoretical Chemistry of Environment Ministry of Education,

Guangzhou, P. R. China.; Tian Liu: investigation Zijie Huang: verification Ruihong Feng: visualization/data presentation Zhiqi Ou: verification Sha Wang: Development or design of methodology Liting Yang: Supervision Li-Jun Ma: Project administration

Graphical abstract

The ratiometric fluorescent probes based on intermolecular pyrene excimer, 1, 2 and 3, can detect pH value of the extremely acidic water environment (distilled water, tap water and Zhujiang River water) with high sensitivity, fast response time, good photostability, strong anti-ion interference ability and good repeatability.

O O

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+N 1 H H R2 +

pH = 1.0

N R 2

pH = 3.0

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O

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N R 1 R2

probe R1

probe in extremely acidic water

R 1=R 2=H , probe 1 R 1=H , R 2=CH2CH2OH , probe 2 R 1=R 2= CH2CH 2OH , probe 3

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An intermolecular pyrene excimer-based ratiometric fluorescent probes for extremely acidic pH and its applications

Tian Liua, Zijie Huanga, Ruihong Fenga, Zhiqi Oua, Sha Wanga, Liting Yanga, Li-Jun Maa,b,c*

a

School of Chemistry, South China Normal University, Shipai, Guangzhou, P. R.

China. b

Guangzhou Key Laboratory of Analytical Chemistry for Biomedicine, South China

Normal University, Guangzhou, P. R. China. c

Key Laboratory of Theoretical Chemistry of Environment Ministry of Education,

Guangzhou, P. R. China.;

1

Abstract

Fluorescent ratiometric probes based on monomer/excimer of pyrene group are often used to detect cations. However, only a few pyrene-containing ratiometric pH probes have been reported so far, and almost all of them produce intramolecular pyrene excimer. In this work, three new ratiometric fluorescent pH probes based on intermolecular pyrene excimer, pyrene-1-carboxamide (1), N-(2-hydroxyethyl)pyrene -1-carboxamide (2) and N,N-bis(2-hydroxyethyl) pyrene-1-carboxamide (3), were synthesized. These probes, 1, 2, and 3, have pKa value of 1.93, 1.85 and 2.07, respectively, and can fluorescently determine the pH value of the extremely acidic aqueous solution with a detection range of about 1.0-2.5. When the pH value was decreased from 2.5 to 1.0, these probes showed a ratiometric fluorescent response signal due to the conversion of monomer/excimer of pyrene group. Therefore, intermolecular excimer of the pyrene group from these probes can be produced in extremely acidic environment. Further tests show that these probes have potential as excellent pH fluorescent probes, with high sensitivity, fast response time, good photostability, strong anti-ion interference ability and high repeatability, etc. The applicability of these probes was demonstrated with environment water samples. In addition, fluorescent pH test paper was successfully developed, which can monitor pH values in the solution through pH-related change of fluorescent color of the probes. Keywords Ratiometric, pH probe, pyrene, extremely acidic, water samples 2

1. Introduction As we all know, monitoring of the pH value plays an significant role in wide range of fields, especially in chemical reactions, environmental analysis and biological systems[1-6]. Hence, the accurate measurement of pH value in these fields is extremely important[7-8]. Many techniques have been reported for the detection of pH value, such as acid-base indicator titration[9-10], microelectrodes[11], potentiometric titration[12-13], absorption spectroscopy, electrochemical, and nuclear magnetic resonance (NMR)[14-16]. Compared with the traditional methods, fluorescent probes for pH determination have many advantages, such as high selectivity and sensitivity, operational simplicity, low cost and

real-time

monitoring[17-21]. Therefore, fluorescent probe technique has become one of the most powerful tools for monitoring the pH value in environmental analysis and biological systems. During the past few years, numerous fluorescent probes have been developed for specific and highly sensitive determination of pH value[18-23]. However, most of them can only monitor pH values under weak acid or weak alkaline conditions[24-26], but only few can measure pH under extremely acidic conditions. Even fewer probes can specifically determine pH value in a fluorescent ratiometric manner[19,23,27-28]. In spite of the pH values in most parts of human body is around 7.0 under normal physiological conditions, there are still a few places where the pH value is acidic. For example, the pH value is around 4.5 in lysosomes, around 1.0 in gastric juice[29]. As is known to all, the ratiometric fluorescent probe can avoid data distortions caused by 3

variable probe concentration, instrument sensitivity, and environmental conditions through the self-calibration of two emission bands[30-33]. Therefore, it is meaningful to design and synthesize ratiometric pH fluorescent probe capable of monitoring the pH values under extremely acidic conditions. Pyrene group has been frequently used as a fluorophore [34-36], due to its large absorption coefficient, long singlet lifetime, high fluorescence quantum yield and chemical stability, etc. In addition, pyrene group shows monomer/excimer dual fluorescence, which provides a valued strategy for the design of ratiometric fluorescent probe [37]. Such pyrene-based ratiometric fluorescent probes have been developed

for

the

detection

metal

phosphate-containing biomolecules

ions,

proteins

nucleic [37-38].

acids,

phosphates

However,

or

pyrene-based

ratiometric pH fluorescent probes are rare, and they are all based on the fluorescent emission of intramolecular excimer of the pyrene group. For example, Shiraishi et al. reported a pH- and H2O-driven pyrene-based ratiometric fluorescent probe that show a pH-controlled bending movement of the polyamine chain leading to a formation of an intramolecular excimer of the pyrene fragments when the dimer solve in H2O [39]. Zhang et al. reported a bispyrene-fluorescein hybrid FRET cassette that shows a ratiometric time-resolved response for pH value using a bispyrene moiety was served as the energy donor and fluorescein was chosen as the energy acceptor [40]. In this paper, three simple pyrene-based ratiometric pH fluorescent probes, pyrene-1-carboxamide (1), N-(2-hydroxyethyl)pyrene-1-carboxamide (2), and N,Nbis(2-hydroxyethyl)pyrene-1-carboxamide (3), were designed and synthesized. All of 4

them displayed excellent pH-dependent fluorescent properties and a high sensitively response in the pH range below 2.5. Compared with previously reported pH fluorescent probes, these probes 1-3 have the following favored properties: (1) probes 1-3 show the fluorescent ratiometric response signal for pH value from intermolecular excimer of pyrene group, (2) probes 1-3 can fluorescently monitor pH value of the extremely acidic environment (in the range of about 1.0-2.5), (3) probes 1-3 show high sensitivity, fast response time, good photostability, strong anti-ion interference ability and good repeatability during the application, (4) probes 1-3 can be used to determine pH value of environment water samples, and (5) probes 1-3 can be used in the development of pH test paper, respectively. 2. Experimental 2.1. Reagents and apparatus 1-Pyrenecarboxaldehyde was purchased from J&K Scientific LTD. Others chemicals used were all of analytical grade. Distilled-deionized water was used to prepare all aqueous solutions. All chemicals were purchased from commercial sources and used without further purification. Mass spectra were carried out on an Orbitrap-Fusion-Lumos mass spectrometer (ThermoFisher). FTIR spectra were recorded on the spectrometer (Nicolet 330) equipped with attenuated total reflection (ATR) mode. 1H NMR spectra were recorded on a Varian NMR Systems 600 MHz spectrometer with deuterated dimethyl sulfoxide (DMSO-d6) as the solvent and tetramethylsilane (TMS) as the internal

5

standard. All fluorescent emission spectra were recorded on a Hitachi F-4600 fluorescence spectrophotometer. The fluorescent quantum yields were measured on a Hamamatsu C9920 ‐ 02G absolute PL quantum yield spectrometer. UV-vis absorption spectra were obtained on a Shimadzu UV-2700 spectrophotometer at room temperature. All the pH values of aqueous solutions were measured with a PHS-3C digital pH meter. 2.2. Synthesis of fluorescent probes As shown in Scheme 1, fluorescent probes 1, 2 and 3 were prepared from the condensation

of

pyrene-1-carbonyl

chloride

with

ammonium

hydroxide,

2-aminoethanol and diethanol amine, respectively. These probes were characterized by 1H NMR,

13

C NMR and HRMS, and the corresponding spectra (Figs. S1-S6) are

included in the Supporting Information (SI). All properties of the three probes are tabulated in Table 1.

Scheme 1. The synthetic route to three probes 1-3. 1-Pyrenecarboxylic Acid 6

To a solution of 1-pyrenecarboxaldehyde (1.5 g, 6.5 mmol) in dry acetone (20.0 mL), potassium permanganate (4.0 g) dissolved in distilled water (20.0 mL) was added. The resulting mixture was refluxed at 80 °C for 3 h, under the monitoring of thin-layer chromatography (TLC). Thereafter, the mixture was cooled to room temperature and filtered. The filtrate was then adjusted to a pH of ~8.5 with sodium carbonate solution, and washed with dichloromethane three times to remove unreacted raw material. The aqueous solution was adjusted a pH of ~2.0 with dilute hydrochloric acid, and solid precipitate appeared. The precipitate was collected and vaccum-dried to obtain light yellow solids (1.0 g, 62.6%). Pyrene-1-carbonyl chloride 1-Pyrenecarboxylic acid (0.25 g, 1.0 mmol) was dissolved in 15.0 mL dichloromethane, followed by the addition of SOCl2 (1.0 mL). The resulting mixture was stirred at 40 °C for 7 h under the monitoring of TLC. After the reaction, the solvent was removed under vacuum and the crude product (pyrene-1-carbonyl chloride) was ready for next step. Pyrene-1-carboxamide (probe 1) The prepared crude pyrene-1-carbonyl chloride was dissolved in 10.0 mL dichloromethane, followed by gradual addition of a solution of 30% ammonium hydroxide (4.0 mL) in dichloromethane. The mixture was stirred for 1.5 h at room temperature under the monitoring of TLC. Thereafter, the resulting solution was washed with deionized water for three times; the organic layer was collected and 7

dried with addition of anhydrous MgSO4. Solvents in the dried organic solution was removed by vacuum, and probe 1 was obtained as a gray solid (0.20 g, 82.2%). N-(2-hydroxyethyl)pyrene-1-carboxamide (probe 2) The prepared crude pyrene-1-carbonyl chloride was dissolved in 10.0 mL dichloromethane, followed by gradual addition of a solution of 2-Aminoethanol (2.0 mL) in dichloromethane. The resulting mixture was stirred for 3 h at room temperature under the monitoring of TLC. Thereafter, the resulting solution was washed with deionized water for three times; the organic layer was collected and dried with addition of anhydrous MgSO4. Solvents in the dried organic solution was removed by vacuum, and probe 2 was obtained as a light yellow solid (0.21 g, 73.0%). N,N-bis(2-hydroxyethyl)pyrene-1-carboxamide (probe 3) The prepared crude pyrene-1-carbonyl chloride was dissolved in 10.0 mL dichloromethane, followed by gradual addition of a solution of diethanol amine (2.0 mL) in dichloromethane. The resulting mixture was stirred for 2 h at room temperature under the monitoring of TLC. Thereafter, the resulting solution was washed with deionized water for three times; the organic layer was collected and dried with addition of anhydrous MgSO4. Solvent in the dried organic solution was removed by vacuum. The crude product obtained was further purified by column chromatography with ethyl acetate and methyl alcohol (10/1, v/v) as eluent to give the probe 3 as a yellow solid (0.17 g, 51.2%). 8

Table. 1 All properties of probes 1-3. Probe 1

Probe 2

Probe 3

265-267 246.09145; calcd for [1+H+], 246.09134

175-177 290.11768; calcd for [2+H+] 290.11756

172-174 334.14389; calcd for [3+H+] 334.14377

8.62(d,J=9.3Hz,1H),8.378.32(m,3H),8.27(dd,J=9.1, 5.0Hz,2H),8.23(d,J=8.9 Hz,1H),8.19(t,J=6.9Hz, 2H),8.13(t,J=7.6Hz,1H), 7.79(s,1H) 171.37,132.30,132.06,131.19, 130.66,128.72,128.47,128.23, 127.69,127.02,126.24,126.04, 125.74,125.33,124.85,124.32, 124.14

8.29(d,J=8.9Hz,1H), 8.25-8.16(m,3H), 8.16-7.97(m,5H), 4.41-4.36(m,2H),3.65(t, J=5.5Hz, 2H),3.49(d, J=5.3Hz,2H) 169.45,132.60,131.99,131.20, 130.69,128.70,128.50,128.23, 127.70,127.04,126.23,126.04, 125.75,125.28,124.86,124.26, 124.13,60.34,42.80

3352,3181,1606,1595,1510, 1452,1433,1371,1264,1198, 1065,848,725,678 0.97 (pH=∼2.5) 0.92 (pH=∼1.0) 8.69 (pH=∼3.0) 3.72 (pH=∼1.0) 1.93

3335,3268,2924,2852,1660, 1624,1596,1535,1420,1295, 1047,853,843,709,668 0.81 (pH=∼2.5) 0.68 (pH=∼1.0) 17.68 (pH=∼3.0) 4.23 (pH=∼1.0) 1.85

8.33-8.22(m,3H), 8.21-8.09(m,3H), 8.09-8.01(m,1H), 8.00-7.89(m,2H),4.37(d, J=1.8Hz,2H),3.86(s,3H), 3.66(s,1H),3.25(t, 4H) 170.81,132.88,131.21,131.14, 130.81,128.82,128.32,127.70, 127.07,127.00,126.21,126.01, 125.20,124.79,124.62,124.16, 124.10,59.20,59.09,51.88, 47.97 3268,2922,2853,1630,1596, 1534,1421,1295,1047,843, 709,667 0.13 (pH=∼2.5) 0.22 (pH=∼1.0) 11.17 (pH=∼3.0) 3.57 (pH=∼1.0) 2.07

m.p. HRMS(m/z) 1

H NMR

(600 MHz, DMSO-d6, δ(ppm)) 13

C NMR

(151 MHz, DMSO-d6, δ(ppm)) IR

The quantum yields Fluorescent lifetimes (ns) pKa

2.3. Analysis Stock solutions of probes 1, 2 and 3 (5.0 mM) were prepared in DMSO, ethanol and ethanol, respectively. Organic solvents can improve the dispersion of aromatic pyrene-containing probes (original in solid form) during the preparation of test solution. The test solution of probe 1 was obtained by diluting the stock solution to 10.0 µM in water containing 5% DMSO at room temperature. And the test solutions of probe 2 and 3 were obtained by diluting the stock solution to 10.0 µM in water. The pH values of the aqueous solution were adjusted with HCl (1.0 M). All spectroscopic experiments were carried out at room temperature. The excitation wavelength was 350 nm in all fluorescent spectra. 9

3. Results and discussion 3.1. Fluorescent response to pH The fluorescent spectra of three probes 1-3 under different pH conditions in water solution were measured. As shown in Fig. 1, probes 1-3 displayed the strong characteristic fluorescent emission bands in the range of 382-420 nm, originated from the monomer of pyrene group at pH higher than ∼2.5 [41]. The quantum yields of probes 1-3 are 0.97, 0.81 and 0.13, respectively, indicating that these probes exhibit strong fluorescent emission. It is feasible that the hydrogen bond between carbonyl group on pyrene ring and water molecule can shield the fluorescent quenching of dissolved oxygen in water. At the same time, the introduction of flexible chain on pyrene group will reduce the rigidity of probe molecule, increase the vibration and rotation in molecule, consume the energy of excited state and increase the nonradiation deactivation, so the fluorescent quantum yield of probe 2 and 3 is lower than that of probe 1. Such high quantum yield can facilitate fluorescent measurement and imaging, and greatly reduce the excitation interference. When the pH value was decreased from about 2.5 to about 1.0, the fluorescent emission band of pyrene monomer decreased gradually, while the fluorescent emission band of pyrene excimer increased apparently. The results show that probes 1-3 have fluorescent response to the pH value in extremely acidic aqueous environment, using the ratio of the fluorescent bands between monomer and excimer. As far as we know, the study is the first report that the change of pH value can produce intermolecular excimer of pyrene group. When the pH value is about 1.0, the quantum yields of probes 1-3 are 0.92, 10

0.68 and 0.22, respectively. The results show that pyrene groups in three fluorescent probes molecules have excellent fluorescent emission properties in the form of monomers and excimers. As a result, these probes show varied fluorescent colors under different pH values, as shown in the illustration of Fig. 1. In addition, the time-resolved fluorescent decay spectra of probes 1-3 in water solution were measured at pH 3.0 and 1.0 as shown in Figs. S7-S9. When pH value was decreased from 3.0 to 1.0, fluorescent lifetimes of probes 1-3 change from 8.69, 17.68 and 11.17 to 3.72, 4.23 and 3.57 ns, respectively. The long fluorescent lifetime can be attributed to pyrene monomer species, while short fluorescent lifetime will be attributed to pyrene excimer species [39, 42-43]. The result is in accord with stable fluorescent spectra, and also indicates the conversion of monomer/excimer of probes 1-3 in extremely acidic aqueous environment.

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Fig. 1 The fluorescent emission spectra of probes 1 (A), 2 (B) and 3 (C) under different pH conditions in water solution. Inset: The fluorescent color of water solution of three probes 1-3 under different pH values. Illumination wavelength: 365 nm UV light. When the pH value was decreased from 4.0 to 0.5, the plots of the fluorescent intensity ratio of excimer and monomer (Iexcimer/Imonomer) as a function of pH value

11

were showed in Fig. 2. The fluorescent intensity changes of probes 1-3 was fitted as a function of pH by the Henderson-Hasselbach-type mass action equation [44-45], which obtained three pKa values of 1.93, 1.85 and 2.07. The results indicate that probes 1-3 are suitable for ratiometric pH sensing in the extremely acidic environment [46]. When pH value was decreased from about 2.5 to about 1.0, the values (Iexcimer/Imonomer) of probes 1-3 change from 0.35, 0.25 and 0.21 to 4.37, 3.61 and 3.55, respectively. The result indicated that compounds 1-3 are highly sensitive probes to pH value within the range of pH value 1.0-2.5. In addition, UV-vis absorption spectra of probes 1-3 at the different pH values were also measured in supporting information. As the pH value is decreased from 2.5 to 1.0, there was only slight variation in the

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absorbance band (Figs. S10-S12).

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Fig. 2 The plots of the fluorescent intensity ratio of excimer and monomer (Iexcimer/Imononer) of 1 (A), 2 (B) and 3 (C) as a function of pH value. 3.2. Real-time response and reversibility study For practical application, the response time and photostability are very important parameter to assess the performance of a new fluorescent probe. The fluorescent

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response time profiles of probes 1-3 in aqueous solutions with a pH of 1.06 and 2.48 were obtained. As shown in Fig. 3, the fluorescent intensity ratio of excimer and monomer (Iexcimer/Imonomer) of probes 1-3 responded to these two pH values rapidly and then remained stable for at least 10 minutes. The results indicate that probes 1-3 can be used as a real-time and fast fluorescent probe to detect the pH value in aqueous

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Fig. 3 Reaction-time profile of probes 1 (A), 2 (B) and 3 (C) at different pH values 1.06 and 2.48. The reversibility of the fluorescent response signal of probes 1-3 between pH value about 1.0 and 2.5 was further investigated. As shown in Fig. 4, the cycle of pH-dependent fluorescent signal conversion could last at least four times. This indicates that probes 1-3 have good reversibility in measuring an extremely acidic pH value between pH about 1.0 and 2.5. In addition, similar fluorescent responses to pH value were found when pH value of the aqueous solution containing probes 1-3 was adjusted with hydrochloric acid, sulfuric acid or nitric acid solutions. These results imply that high concentration of hydrogen ions can induce the transition of pyrene monomer to excimer.

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Fig. 4 Reversibility of the fluorescent response of probes 1 (A), 2 (B) and 3 (C) between two different pH values. 3.3. Interference studies In order to test the practical application of probes 1-3 in the fluorescent detection for pH value, interference experiments were performed to estimate the influence caused by other ions, which may be present in the systems being analyzed. The fluorescent intensity ratio of excimer and monomer (Iexcimer/Imonomer) of probes 1-3 in the absence or presence of an excess of Al3+, Ba2+, Cd2+, Co2+, Cr3+, Cu2+, Fe3+, Hg2+, Mg2+, Mn2+, Ni2+, Pb2+, Ag+, K+, Na+, F-, Cl-, NO3-,SO42- and PO43- (100.0 µM) at pH 1.38 were shown in Fig. 5. The results indicate that these common ions have negligible impact on the fluorescent response signal of probes 1-3. Thus, probes 1-3 possess an excellent selectivity response to extremely acidic pH value even in the presence of metal ions. 3.0

1.8

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_

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SO

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3

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Cl

Na +

Ag +

+

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C o 2+

C r 3+ C u 2+

B a 2+ C d 2+

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0.0

only probe 2

2.5

Cr 3+ C u 2+

probe 1 +other ions

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1.8

Fig. 5 The fluorescent intensity ratio of excimer and monomer (Iexcimer/Imonomer) of probes 1-3 in water solution in the presence of 100.0 µM metal ions and common 14

anions, respectively. 3.4. Application in environmental water samples Encouraged by the desirable pH-dependent spectral properties of probes 1-3, we then examined their potential application for monitoring pH changes in environmental water samples. Tap water, Zhujiang River water and distilled water were used to prepare aqueous solution of the different pH values water solutions, respectively. The fluorescent response signals of probes 1-3 in these three water samples were compared. As shown in Fig. 6, the variation of fluorescent response signal in the three water samples was very similar. The results imply that probes 1-3 can be used to measure extremely acidic pH in environment water samples. 7

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7

distilled water tap water Zhujiang River

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Fig. 6 The plots of fluorescent response signal of probes 1 (A), 2 (B) and 3 (C) at different pH values in three different water samples. 3.5. Application of pH test paper In order to further investigate the application of probes 1, 2 and 3 in pH detection, a test strip experiment was carried out. First, test papers were conveniently prepared by dip-coating a solution of these three probes and then drying them in air [47]. When the test papers were immersed in an aqueous solution at pH value 1.06 and 2.48, the

15

test strips developed different color changes, as shown in Fig. 7. Under 365 nm UV light, the pH value 1.06 test strip developed blue-green at a pH value of 1.06, and a blue-violet at a pH value of 2.48. The result demonstrates that probes 1-3 can be conveniently and efficiently used as a pH probe.

Fig. 7 Color changes of test papers based on three probes at two different pH values under 360 nm UV light. 3.6 The mechanism of pH detection In order to obtain more detailed information on the sensing mechanism, 1H NMR titrations of the probes under acidic and alkaline conditions were performed in DMSO-d6/D2O (1/1, v/v). As depicted in Fig.S13, the protons peaks gave the clearest signal in the 1H NMR titration spectra of probe 2. However, the chemical shift of protons showed negligible variation at acidic and alkaline conditions. Such experimental results may be due to the high concentration of probes in NMR spectra measurement, which results in more pyrene groups existing in aggregates. In addition, the presence of a large number of organic solvents is required for NMR spectroscopy. However, the presence of high concentration of organic solvents may affect the aggregation behavior of pyrene groups of the probes. The electrostatic repulsion 16

between protonated amino or imino groups may prevent pyrene groups from approaching each other to form aggregates when the pH value is 2.5 [39, 42]. When the pH value is less than 2.5, the high concentration of hydrogen protons in the solution reduces the effect of electrostatic repulsion between protonated amino or imino groups, which may induce hydrogen bond interaction between the carbonyl group on one pyrene group and the hydrogen (or protonated hydrogen) on the amino or imino group on the other pyrene. Subsequently, the strong hydrophobicity of pyrene groups resulted in pyrene-specific intermolecular excimer. Therefore, the high concentration of hydrogen protons and strong hydrophobic interaction between water molecules with pyrene groups lead to aggregation of probes in extremely acidic aqueous solutions, which contributed to the fluorescent response signaling. The proposed mechanism of probes 1-3 for pH detection in extremely acidic solution is showed in Scheme 2. In the extremely acidic environment, the high concentration of hydrogen protons in the solution may induce hydrogen bond interaction which can make two probe molecules close to each other. Thus, the difficulty degree in forming the hydrogen bond interaction leads to a small difference in pKa of probes 1-3.

O O N R 2

R1

R

+N 1 H H R2 +

pH = 1.0 pH = 3.0 O

R1

R2

N R 1 R2

probe R1

R 1=R 2=H , probe 1 R 1=H , R 2=CH2CH2OH , probe 2 R 1=R 2= CH2CH 2OH , probe 3

probe in extremely acidic water

17

R2

Scheme 2 Proposed mechanism of probes 1-3 for pH measurements in extremely acidic solution. 4. Conclusions In summary, three new simple ratiometric fluorescent pH probes were designed and synthesized to detect pH value in extremely acid conditions. When the pH is changed from 2.5 to 1.0, probes 1-3 show a ratiometric fluorescent response signal from intermolecular excimer of the pyrene group. And probes 1-3 show high sensitivity, fast response time, good photostability, strong anti-ion interference ability and the multiple repeatable measurement performance for pH monitoring. Furthermore, probes 1-3 can be used to detect the pH value of environmental water system. Finally, the three probes were successfully used to prepare fluorescent pH test paper. Acknowledgments This work was partially supported by the project Science and Technology Program of Guangzhou (No. 201804010465), the project of Guangdong Natural Science Foundation, China (No. 2015A030313392). References [1] Wu YC, You JY, Jiang K, Wu HQ, Xiong JF, Wang ZY. Novel benzimidazole-based ratiometric fluorescent probes for acidic pH. Dyes Pigments 2018;149:1-7. [2] Liu X, Han J, Zhang Y, Yang X, Cui Y, Sun G. A novel pH probe based on ratiometric fluorescent properties of dicyanomethylene-4H-chromene platform.

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HIGHLIGHTS •Probes 1-3 can ratiometric fluorescent measure pH value in the range of about 1.0-2.5 •Probes 1-3 show high sensitivity, fast response time and good photostability •Probes 1-3 show strong anti-ion interference ability and good repeatability •Probes 1-3 can be used in the detection pH value of the environmental water samples •Probes 1-3 can be used in the production of pH test paper

Conflict of interest The authors declared that they have no conflicts of interest to this work. We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted Li-Jun Ma