Information Fluorescent pH sensor constructed from a heteroatom-containing luminogen with tunable AIE and ICT characteristics

a HKUST Shenzhen Research Institute, No. 9 Yuexing 1st RD, South Area, Hi-tech Park, Nanshan, Shenzhen, China 518057. E-mail: tangbenz@ust.hk b Department of Chemistry, Division of Biomedical Engineering, Institute for Advanced Study and Institute of Molecular Functional Materials, The Hong Kong University of Science & Technology, Clear Water Bay, Kowloon, Hong Kong, China. c Guangdong Innovative Research Team, SCUT-HKUST Joint Research Laboratory, State Key Laboratory of Luminescent Materials and Devices, South China University of Technology, Guangzhou, 510640, China.


Introduction
pH is critical to all life forms. Only a small change in environmental pH can devastate a number of plant and animal lives. Acidication of soils, streams, lakes and seawater caused by acid rain and excessive human activities and untreated sewage can lead to ecological disaster. The catastrophic consequences of such pollution are the depletion of individual species, reduction in biodiversity, the elimination of specic strains, etc. Monitoring the pH level is thus crucial to the preservation of our living environment. Among various analytical techniques, uorescence (FL)-based methods have attracted special interest because they enjoy superb sensitivity, selectivity, rapidity, portability, etc. 1 The main chemical analytes contributing to pH are ionic species existing or functioning in aqueous media. Incongruously, however, most luminescent materials are hydrophobic aromatics that are barely soluble in water. Although their water solubility can be improved by the incorporation of hydrophilic groups, the resulting amphiphilic molecules tend to aggregate when dissolved in water. Aggregation can partially or even completely quench light emission and this aggregationcaused quenching (ACQ) effect has limited the real world applications of many lead luminophores identied by the laboratory solution-screening process in an engineering robust form. 2 We observed a phenomenon of aggregation-induced emission (AIE) in some propeller-like molecules such as siloles and tetraphenylethenes. 3 Instead of quenching, aggregate formation enhances their light emission, turning them from weak luminophores in solution to strong emitters in the aggregated state. The restriction of intramolecular rotation (IMR) has been found to be the main cause for the AIE effect. Since then, many scientists around the world have worked on the design of new AIE luminogens and explored their practical applications in optoelectronics and sensory systems. 3b,3c,4,5 Our research team has utilized AIE uorogens to develop uorescent pH sensors that sensitively respond to subtle pH changes. For example, a hexaphenylsilole derivative 1,1-bis-[4-(diethylaminomethyl)phenyl]-2,3,4,5-tetraphenylsilole can dissolve readily in acidic aqueous media due to the transformation of its amino groups to ammonium salts. 6 The resulting non-emissive aqueous solution is turned on by increasing the pH value. The working principle involved in the pH sensing process is the dissolution (deaggregation) and aggregation of the AIE luminogen at the appropriate pH value. Following this mechanism, a uorescent pH sensor with the opposite response can be readily designed. Thus, the emission of 1-[2-(4-hydroxyphenyl)ethynyl]pentaphenylsilole, a hydroxylated silole, is switched on and off in the low and high pH regions, as the luminogenic molecules are aggregated and deaggregated (or dissolved) in the acidic and basic aqueous media, respectively. 7 By taking advantage of the AIE effect and chemical reactivity towards OH À and H + ions, a red emissive zwitterionic hemicyanine dye constructed from tetraphenylethene and N-alkylated indolium shows different emission colors and intensities at various pH values, and thus works as a uorescent sensor to follow pH changes in a wide range. 8 Protonation of some heteroatom-containing groups such as pyridinyl, amino and phenol will signicantly change their electron-withdrawing properties. This will have an obvious effect on the emission color or intensity of uorophores with these pH-sensitive groups. Some uorescent pH sensors based on the above process have been designed and synthesized but the detection is normally carried out in the solution state due to the ACQ effect. 9 In the pursuit of new AIE materials for application as uorescent pH sensors, in this edge article, we designed and constructed a heteroatom-containing luminogen (CP 3 E, Scheme 1) from diphenylethene, carbazole and pyridine building blocks. CP 3 E is AIE-active and displays the feature of intramolecular charge transfer (ICT) 10,11 due to the donor (D)acceptor (A) interaction between its carbazolyl and pyridinyl units. Its emission can be reversibly switched between blue and dark state by repeated protonation and deprotonation, thus enabling it to work as a uorescent pH sensor in both the solution and solid states as well as a probe for acidic and basic organic vapors.

Results and discussion
Synthesis CP 3 E was synthesized in a moderate yield according to the synthetic procedures shown in Scheme 1. Briey, addition of 4-cyanopyridine (1) into a lithiated THF solution of bromobenzene followed by hydrolysis in water formed 4-pyridophenone (2). Wittig-Horner reaction of 2 with diethyl 4-iodobenzylphosphonate (3) 12 in the presence of potassium tert-butoxide generated 1-(4-pyridinyl)-1-phenyl-2-(4-iodophenyl)ethene (4), which reacted with carbazole to give the designed product as a white powder. CP 3 E was characterized by NMR and mass spectroscopies and elemental analysis, which gave satisfactory data corresponding to its molecular structure (see ESI † for details). Single crystals of CP 3 E were grown from its hexaneethyl acetate mixture (3 : 1, v/v) and analyzed crystallographically. The data are provided in Table S1 and S2. †

Tunable optical properties
Albeit weak, CP 3 E exhibits the ICT phenomenon owing to the presence of electron-donating and accepting units in the molecular structure. As shown in Fig. 1A, the UV spectrum has a peak at 340 nm in hexane, which changes little when the measurement is performed in other solvents with increasing polarity. The solvent polarity also does not exert much inuence on its photoluminescence (PL) properties, with the emission maximum and intensity in hexane being 13 nm blue-shied and half, respectively, from those in acetonitrile, the most polar solvent used for the measurement (Fig. 1B). A Lippert-Mataga plot of Stokes shi against the orientation polarizability of the solvent gives an upward straight line with a small slope, indicative of a weak ICT feature.
Addition of a drop of triuoroacetic acid (TFA) into a chloroform solution of CP 3 E protonates its pyridine unit and generates CP 3 EH + (Scheme 2). This is proved by the 1 H NMR spectra shown in Fig. 2. The pyridinyl protons (a and b) shi downeld aer protonation because of the transformation of the pyridine ring in CP 3 E to an electron-decient pyridinium unit in CP 3 EH + . The resonances of phenyl A and olen (labeled with "c") protons also move to lower elds for the same reason. The 1 H NMR spectrum is fully restored when an excess of triethylamine (TEA) is injected into the solution, suggesting that the transformation between CP 3 E and CP 3 EH + is completely reversible and non-destructive in nature. Since the 1 H NMR analysis suggests that the pyridinium unit in CP 3 EH + is a strong electron-accepting group, it is thus anticipated that CP 3 EH + exhibits a stronger ICT effect but weaker PL than CP 3 E in the same solvent. This is indeed the case. As depicted in Fig. S1, † the optical properties of CP 3 EH + , particularly the PL process, are Scheme 1 Synthetic route to CP 3 E.
, y F ¼ emission wavenumber and Df ¼ orientation polarizability ¼ (3 À 1)/ (23 + 1) À (n 2 À 1)/(2n 2 + 1), where 3 ¼ dielectric constant and n ¼ refractive index. more susceptible to the change in solvent polarity. For example, the UV spectrum has a peak at 380 nm in chloroform, which moves to 391 nm when the measurement is carried out in acetonitrile. While CP 3 EH + emits a yellow PL at 558 nm in chloroform, it becomes a red uorophore and luminescences weakly at $640 nm in acetonitrile. Its Stokes shi increases more rapidly on increasing the solvent polarity, further substantiating the discussion above. It is noteworthy that in the same solvent, CP 3 E shows redder absorption, which is indicative of its higher conjugation.

Aggregation-induced emission
We then investigated the photophysical properties of both dyes in the aggregated state. The diluted THF solution (10 mM) of CP 3 E emits a blue PL at $455 nm (Fig. 3A). Gradual addition of water into the THF solution slightly red-shis the PL spectrum as the ICT effect becomes stronger in solvent mixtures with higher polarity. The emission intensity, on the other hand, remains low even when up to 70% of water is added to the THF solution. Aerwards, it rises swily. The higher the water fraction (f w ), the stronger is the light emission. From the dilute THF solution to 99% aqueous mixture, the PL intensity increases by $22-fold. Since CP 3 E is insoluble in water, its molecules must have been aggregated in the presence of a large amount of water. However, all the aqueous mixtures are homogenous without precipitates, suggesting that the aggregates are of nano-dimensions. This is proved by the level-off tail observed in the UV spectrum in the longer wavelength region in aqueous mixtures with high f w values (Fig. S2A †). Analyses by particle size analyzer and transmission electronic microscopy also reveal the formation of nanoaggregates with diameters in the range of 100-200 nm (Fig. S2B †). Clearly, aggregate formation has enhanced the PL of CP 3 E or, in other words, it is AIE-active. As discussed before, CP 3 E is a weak ICT dye. Thus, even as the polarity of the solvent mixture becomes higher with gradual addition of water into the THF solution, its photophysical properties are little affected. On the other hand, the active rotation of the periphery aryl rings has consumed the energy of the excitons. Thus, although CP 3 E is emissive in THF and THF-water mixtures with low water fractions, the intensity is relatively weak. At f w > 70%, aggregates form, which restricts the IMR process and blocks the nonradiative relaxation channels 13 due to the stabilization of the molecular conformation by multiple C-H/p hydrogen bonds (Fig. S3 †). Meanwhile, the twisted conformation of the dye molecule has effectively hampered the formation of detrimental species such as excimers by strong intermolecular interaction. 14 All these collectively make CP 3 E a strong emitter in the aggregated state.
CP 3 EH + , on the other hand, displays distinctly different emission behaviour. From the disappearance of the absorption peak at $350 nm, complete protonation of CP 3 E to CP 3 EH + was achieved only in acidied THF-water mixtures with 50% or higher water content (Fig. S4A †). Thus, we investigated the PL of CP 3 EH + in aqueous mixtures with f w values larger than 50%. As the polarity of the aqueous mixture and hence the ICT effect become higher and stronger with progressive addition of water Scheme 2 Reversible transformation between CP 3 E and CP 3 EH + by repeated protonation and deprotonation.  into the 50% aqueous mixture, the PL of CP 3 EH + is weakened gradually (Fig. 3C). At f w > 85%, aggregates form, which activates the AIE process and hence makes the solvent mixture emissive again. The magnitude of emission induction is, however, limited as the PL intensity in 99% aqueous mixture is basically the same as that at 50% water content (Fig. 3D). The PL maximum is located at shorter wavelength, presumably due to the decrease in the solvent effect on the photophysical properties of the aggregates. The size of the nanoaggregates determined by zeta potential analyzer is 30-50 nm (Fig. S4B †), which is much smaller than that of CP 3 E as CP 3 EH + is less hydrophobic and thus can still dissolve in aqueous mixtures with high water contents.

pH sensing
The AIE effect of CP 3 E and its reversible transformation between the bright neutral state and dim cationic form by repeated protonation and deprotonation encouraged us to explore its application as a uorescent pH sensor. As shown in Fig. 4A, the PL spectrum of CP 3 E in THF-buffer mixture (3 : 7, v/v) at pH 1 is almost a at line parallel to the abscissa because of its transformation to the weakly emissive CP 3 EH + under such acidic conditions. The spectrum was intensied when the pH was increased from 1 to 4 because of the progressive decrease in the population of CP 3 EH + in solution (Fig. 4B). At pH > 4, the CP 3 E molecules are less likely to undergo protonation and its emission thus remains unaltered. The uorescent photos of the solutions shown in the insets also demonstrate the same phenomenon. The extent of transformation of CP 3 E to CP 3 EH + at different pH values can also be followed by UV analysis (Fig. S5A †). At pH 1-2, CP 3 EH + is predominant in solution and absorbs at $400 nm. The absorption is weakened progressively with increasing pH. Meanwhile, a new peak owing to the absorption of CP 3 E appears at $350 nm. At pH 4, a weak absorption tail is still observed at the longer wavelength region but disappears completely at pH 5, suggesting that no protonation occurs at such a pH value. The spectra measured at higher pH are basically the same as that at pH 5.
The detection can also be performed in the aggregated state. Since CP 3 E is hydrophobic in nature, it will form aggregates in a THF-buffer mixture with 90% water content. At pH 1-2, it will protonate to form CP 3 EH + that possesses lower hydrophobicity but a stronger ICT effect, thus rendering the solution almost non-emissive (Fig. 4C). The emission was turned on again with increasing magnitude when the pH was increased progressively from 2 to 7 because most of the CP 3 E molecules are not protonated, as demonstrated by the appearance of a strong absorption peak at $350 nm in the UV spectrum (Fig. S5B †). Aerwards, the PL intensity remains almost constant. From pH 1 to 12, the emission is enhanced by more than 80-fold, which is much higher than that in the solution state (Fig. 4D). Evidently, the nanoaggregates work as a more sensitive pH sensor than their isolated species in solution, thanks to their AIE feature.
For real-world applications, it is preferable to perform the detection on solid supports because this requires no complex and expensive equipment and is thus simple, quick and convenient. 15 In such regard, we deposited CP 3 E on lter paper and checked its PL aer addition of several drops of buffer solutions of different pH. The untreated strip emits bright blue PL upon photoexcitation (inset in Fig. 4E). Addition of acidic buffer solutions (pH 1 or 2) weakens the PL and changes the emission color to yellow. In contrast, the paper strips give blue light at pH 3 and 4 but the intensity is not as strong as that of the untreated one. At pH > 4, no difference in the emission intensity was basically observed between the CP 3 E-loaded strips as the dye exists predominantly in its neutral form. Instead of visual observation, we also measured their PL using a spectrophotometer and obtained results similar to those obtained in the solution and aggregated states ( Fig. 4E and F).
Since the PL of CP 3 E is pH-sensitive, we can make use of such a property to utilize it as a uorescent sensor for the detection of volatile organic compounds with high acidity and basicity. 16 Due to its strong mechanical strength, we utilized a thin-layer chromatography (TLC) plate as a solid support. The dye-loaded plate emits a greenish-yellow light due to the partial transformation of CP 3 E to CP 3 EH + by the weak acidity of the silica gel (Fig. 5A). Aer fuming with TFA vapour, the dye spot turns red and emits weakly. It, however, converts back to its blue emissive neutral form when treated with TEA vapour. The switch between the weakly emissive and bright blue states can be repeated

Miscellaneous applications
Instead of a uorescent pH sensor, we also explored the hightech applications of CP 3 E in other elds. We found that CP 3 E can work as a uorescent visualizer for intracellular imaging. As depicted in Fig. 6, the AIE-active CP 3 E selectively stains the cytoplasmic regions of living HeLa cells. Bright blue light was observed from the lipid droplets, presumably due to the hydrophobic nature of the CP 3 E molecules, which favors their accumulation on these biomacromolecules. We are currently utilizing CP 3 E to sense pH inside the living cells and the details will be presented in the future.

Mechanism
The above discussion states that CP 3 E and CP 3 EH + possess distinctly different photophysical properties. While it is proven that the different electron-accepting abilities of the pyridine and pyridinium units contribute to different ICT effects in CP 3 E and CP 3 EH + , what causes the change in the AIE feature? We believe the molecular conformation plays a crucial role. The single-crystal structure of CP 3 E provided in Fig. 7 shows that the molecule adopts a twisted conformation due to the steric effect between the aryl rings. Aer protonation, its conformation becomes more planar, as revealed by the optimized structure of CP 3 EH + obtained by theoretical calculations. The selected bond lengths and dihedral angles summarized in Table S3 † also support this speculation. Whereas the pyridine and phenyl A rings twist from the vinyl core by 51.7 and 40.8 , respectively, the amplitude becomes smaller in CP 3 EH + (34.2 and 26.6 ). This implies better electronic communication in CP 3 EH + , thus shortening the single bonds connecting the components and imparting CP 3 EH + with a high conjugation and hence a smaller energy band gap (Fig. S6 †). Previous studies have shown that molecules with a more at conformation enjoy higher electronic conjugation and lower potential energy. 3c Thus, they are less likely to undergo intramolecular rotation even when they contain rotatable aromatic rings. As the restriction of the IMR process is mainly responsible for the AIE phenomenon, the relatively low tendency of intramolecular rotation in CP 3 EH + due to its more planar conformation should, in most cases, lead to a weaker AIE effect.

Conclusions
In this work, a heteroatom-containing organic uorophore (CP 3 E) was designed and synthesized from carbazole, pyridine and diphenylethene building blocks. CP 3 E exhibits an ICT effect caused by the D-A interaction between its carbazole and pyridine units. Whereas it emits faintly in solution, it becomes highly emissive in the aggregated state, demonstrating the AIE phenomenon. Its emission can be reversibly switched between blue and dark states by repeated protonation and deprotonation. Such behaviour enables it to work as a uorescent pH sensor in both the solution and solid states and a chemosensor for detecting volatile organic compounds with high acidity and basicity. CP 3 E can selectively stain the cytoplasmic regions of living cells. Analyses by NMR spectroscopy, single-crystal X-ray diffraction and theoretical calculations suggest that the change in electron affinity of the pyridinyl moiety and molecular conformation upon protonation/deprotonation is responsible for the sensing process. Thus, introduction of an AIE unit to the traditional pH sensitive uorophores is an efficient route to expand their practical useful application from solely solution to both solution and solid states. Such results also provide valuable information on the future design of new uorescent probes, whose light-emitting behaviours can be readily tuned by simply varying the environmental conditions.