Styrylcyanine-based ratiometric and tunable fluorescent pH sensors

Abstract

Styrylcyanine derivatives based on indolenine and salicylaldehydes act as excellent fluorescent sensors with ratiometric changes in absorption and emission profiles in response to pH variation.

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Maintaining the right pH window is critical for many biological and industrial processes and hence there is significant interest to develop efficient sensors for monitoring pH variations. Apart from the electrochemical method that has been used generally, recent years have witnessed an upsurge in the development of fluorescent sensors for reporting pH changes.^1–6 Compared to the former category, fluorescent pH sensors offer high sensitivity, selectivity, fast response, spatial resolution, real time monitoring and noninvasiveness.⁷ Sensors based on emission changes in a single window are generally influenced by environmental interferences like fluctuations in source intensity, photobleaching of the sensor, analyte concentration etc., leading to errors in quantification. Ratiometric fluorescent sensors use the ratio of fluorescence emission intensity at two different wavelengths to report the information about the analyte, and provide an inherent correction mechanism for environmental interferences.^8–13 They generally exhibit either an ‘on–off’ (or ‘off–on’) or an ‘off–on–off’ (or ‘on–off–on’) response to pH changes. While the ‘on–off’ (or ‘off–on’) fluorescent sensors have been extensively studied, there are only limited number of reports on ‘off–on–off’ sensors which is primarily due to complicated molecular design strategies needed to meet the structural and electronic requirements.^14–18 Moreover, changing the pH response status from ‘off–on’ to ‘off–on–off’ through simple substituent manipulation without modifying the molecular scaffold is difficult and rarely explored.¹⁹ Most of the known sensors operate in acidic-near neutral pH but those which respond to pH change in the basic region are not many.²⁰ As part of our interest to develop tunable pH sensors with detection limit spanning to basic region as well, we synthesized and systematically studied a group of styrylcyanine-based probes and the results are delineated below.

A number of pH sensitive units like indole, thiazole etc., connected to substituted aryl units through ethylene spacers are known to respond to pH variation through clear change in their absorption emission profiles.^21–23 Majority of such styrylcyanine derivatives respond to acidic to neutral pH ranges only, which is directly linked to protonation–deprotonation equilibria of hetero atoms present.^24–26 In order to expand the response range, we considered the combination of N-nonalkylated indolenine ring with a phenolic unit to get analogues of the type 1–3 shown in Fig. 1a. Such ‘two-centre’ systems could in principle generate charged or neutral species depending upon the pH, having different electronic characteristics (Fig. 1b). These compounds were prepared in one step through condensation of 2,3,3-trimethylindolenine with corresponding salicylaldehydes in presence of DIPEA following a literature procedure with a slight modification.²⁷ The experimental details and spectral data of these compounds are provided in the ESI.† Their olefins had trans geometry, as evidenced by large ³J_H,H values (∼16 Hz) for associated protons. We were able to crystallize 2 and 3 by slow evaporation of their solutions in ethyl acetate–hexane. Their X-ray structures and relevant parameters are presented in the Fig. S1 and Table S1.†

Fig. 1 (a) Structures of styrylcyanine derivatives used in this study; (b) pH dependent-ionization of these compounds.

The absorption–emission profiles of 1, 2 and 3 were recorded in Britton–Robinson (BR) buffer at room temperature at different pH. They showed large Stokes shifts (1: 151 nm; 2: 130 nm; 3: 176 nm) and high molar absorptivities (Table S2, ESI†). Distinct effects of substituents on their electronic properties were evident from their solvatochromic behaviour. Compound 1 gave negative solvatochromism whereas it was positive for 2 and 3. Solvent polarity-dependent variation in emission intensity was also noticed in all the three compounds (Fig. S2†). The absorption spectrum of 1 had a maximum at 409 nm at pH 2.6 (Fig. 2a). The λ_max shifted gradually to 355 nm (blue shift of 54 nm) upon increasing the pH from 2.6 to 6.3 with a concomitant decrease in the absorption at 409 nm with a clear isosbestic point at 380 nm. A further increase in pH from 7.5 to 11.5 resulted in the decrease of absorption at 355 nm with the emergence of a new peak at 416 nm through an isosbestic point at 392 nm (Fig. 2b). The three absorption maxima at different pH (409, 355 and 416 nm) are likely due to protonated amine, neutral compound and phenoxide entities, respectively (Fig. 1b). Changes in absorption profile followed clear ratiometric behaviour and the calibration curves exhibited considerable pH response in the range of 4.5–6 and 8.1–11.5 (Fig. S3a and b†).

Fig. 2 pH-dependent absorption spectra of 1 (3.3 × 10⁻⁵ M in BR buffer): (a) the band at 409 nm decreases with the emergence of a new one at 355 nm as the pH changes from 2.6 to 6.3; (b) decrease of 355 nm band and formation of one at 416 nm with pH change from 7.5–11.5.

The emission behaviour of 1 as a function of pH was also encouraging. On excitation at 409 nm, it gave an emission maximum at 524 nm at pH 2.5. The emission intensity gradually enhanced with a blue shift on increasing the pH, reaching 475 nm at pH 6.8 (Fig. S4†). In the pH range of 6.8–10.5, a new red shifted emission peak at 560 nm evolved with concomitant decrease in intensity of the band at 475 nm (Fig. 3a). The intensity of emission at 560 nm increased 15-fold in this pH range (Fig. S5a†). The ratiometric behaviour of 1 was evident from isoemission point at 500 nm, and the overall response (I₅₆₀/I₄₇₅ between pH 7–10.5) was an ‘off–on’ type (Fig. 3b). It was also reversible and the output over 4 cycles is presented in Fig. S6a.† From the plot of normalized fluorescence vs. pH, an apparent pK_a of 8.46 was predicted for 1 (Fig. S5b†). There was no change in the emission intensity when samples of 1 at different pH (2.7, 6 and 9) were monitored for 140 min, which confirmed its excellent stability (Fig. S6b†).

Fig. 3 pH-dependent change in the fluorescence emission of 1 (λ_ex: 409 nm, 3.3 × 10⁻⁵ M in BR buffer); disappearance of band at 475 nm and formation of a new one at 560 nm on increasing the pH from 6.8 to 10.5 is shown. Inset: ratiometric response curve showing the variation of I₅₆₀/I₄₇₅ with pH.

The pH-dependent absorption profile of 2 was similar to that of compound 1. On increasing the pH from 2.6 to 5, the intensity of absorption at 430 nm decreased with the formation of a new peak at 366 nm which remained steady till pH 8. Further increase of pH (from 8.1 to 11.5) resulted in a decrease in intensity of absorption peak at 366 nm and a new peak emerged at 434 nm as shown in Fig. 4a and b. As can be seen, the absorption spectra showed two well-defined isosbestic points at 382 nm and 409 nm. The ratiometric calibration curves for this absorption (A₃₆₆/A₄₃₀ and A₄₃₄/A₃₆₆) as a function of pH exhibited good linearity between pH 4–6 and 8–11 (Fig. S7a and b†). The fluorescence emission spectrum of 2 displayed a distinct emission peak at 518 nm. Intensity of this emission increased steadily on increasing pH, and there was 55-fold emission enhancement on changing pH from 2.5 to 6.2. However, the intensity started decreasing subsequently till it became minimum at pH 11.5 (Fig. 4c and d). Importantly, the emission intensity was nearly steady between pH 6–8 making it useful in cellular studies (Fig. 4e).

Fig. 4 pH-dependent absorption spectra of 2 (3.3 × 10⁻⁵ M in BR buffer): (a) the band at 430 nm decreases with the emergence of a new one at 366 nm as the pH changes from 2.6 to 5.0; (b) decrease of 366 nm band and formation of one at 434 nm with pH change from 8.1–11.5; (c) & (d) emission profile in the pH range 2.5–12; (e) bell-shaped curve showing off–on–off behaviour; (f) response-curve showing variation of I₅₉₉/I₅₁₈ with pH.

A plot of emission intensity at 518 nm vs. pH revealed a highly symmetrical bell-shaped curve representing an ‘off–on–off’ behaviour (Fig. 4e). Interestingly, there was appearance of a small emission band at 599 nm as the pH changed from 10.5–12 which was ratiometric with respect to the decrease of emission at 518 nm. Response-curve showing variation of I₅₉₉/I₅₁₈ with pH is given in Fig. 4f. Apparent pK_a values of 4.21 and 9.51 were obtained for 2 from the plot of normalized fluorescence vs. pH (Fig. S8†). The fluorescence emission intensity at 518 nm remained unaltered for 140 min at pH 3.6, 6.5 and 9 confirming the stability of the molecule under the experimental conditions (Fig. S9†). Thus, 2 behaved as an ‘off–on–off’ fluorescent sensor in the range pH 2–12 with maximum emission at 518 nm between pH 6–8, and additionally as an ‘off–on’ sensor in more basic range (pH 10.5–12) with a new emission at 599 nm. Enhanced intramolecular electron/charge transfer from the electron rich p-hydroxy anisole ring to the protonated indolenine ring in pH range 6–8 is likely responsible for the emission at 518 nm.

Objective of synthesizing 3 was to have a pH sensor for the acidic range. Presence of nitro group on the phenolic ring lowers the pK_a of the molecule and was expected to shift its pH response to acidic range. The emission spectrum of 3 at pH 2.5 showed λ_{max, em} at 605 nm. However, its intensity was found to decrease with increasing pH, reaching nearly zero at pH 7 (Fig. S10†). Time evolved absorption and emission spectra of 3 in acidic pH showed gradual decrease in the absorption and emission intensity (Fig. S11†) suggestive of chemical instability and its unsuitability for sensing pH variations in the acidic region. Decomposition of merocyanine derivatives in aqueous media to aldehyde and indolenine precursors has been reported and similar pathway is likely operating in the case of 3 after protonation.²⁸ Solvatochromic behaviour, sensitivity of this compound to lower pH, and its degradation in presence and absence of light were studied in detail and the results are included in the ESI.†

Good stability and emission profile of 2 in biologically relevant pH range motivated us to investigate its performance in cellular environment. Its emission was unaffected by metal ions such as Mg²⁺, Ca²⁺, Zn²⁺, Cd²⁺, Hg²⁺, Cu²⁺, Fe²⁺ etc. which was also encouraging (Fig. S13†). To make sure that it is not cytotoxic in the concentration range used for imaging, its effect on L-929 mouse fibroblast cells (CCL-1) was first studied by MTT assay (ESI†). It was found non-cytotoxic below 0.1 mg mL⁻¹ and had an IC₅₀ of 0.328 mg mL⁻¹. Its uptake and distribution was subsequently studied in human liver hepatocellular cells (HepG2) using a laser scanning confocal microscope. After incubating with 2 (at dose of 0.1 mg mL⁻¹ for 12 h), the cells were fixed in buffered formalin solution for 30 min and cell nucleus was counter stained with PI (1 μg mL⁻¹ in PBS) for 1 min. Uptake was then imaged by illuminating with 405 blue diode laser and emission was observed at various band pass ranges such as 420–480 nm, 470–500 nm and 505–530 nm. Excitation at 405 nm was found enough to give a broad range of emission from 420–530 nm; the signal from 420–480 nm emission filter was sufficient to clearly visualize the cellular uptake. Images obtained: (a) in the bright field, (b) detected at 420–480 nm from emission of 2, (c) detected with long pass 560 nm from the emission of propidium iodide, and (d) an overlay of later two are presented in Fig. 5. Images from 2 h incubation and other experimental details are given in the ESI.† Distinct advantages of the sensors presented here include simple synthesis, modulation of pH response window through substituent variation, and ratiometric behaviour in both absorption and emission profiles.

Fig. 5 Confocal microscopic images of HepG2 cells after incubating with 2 (0.1 mg mL⁻¹) for 12 h: (a) transmission image; (b) localization of 2 within the cells imaged at 420–480 nm from emission of 2; (c) red portions denotes emission from PI used to counter stain the cell nucleus and (d) merge of (b) and (c).

In summary, the results outlined here highlights the ability of styrylcyanine derivatives based on indolenine and salicylaldehydes to act as excellent fluorescent sensors with ratiometric response to pH changes. The design makes use of differences in protonation–deprotonation equilibria in response to change in the substitution on the phenolic moiety in these compounds. When the ring was unsubstituted (1), the compound exhibited an ‘off–on’ response for the emission at 560 nm on changing the pH from 7–11. Remarkably, introduction of –OMe group at the para position allowed the molecule (2) to give an ‘off–on–off’ response for the pH change from 2–12, with the emission (518 nm) intensity maximum between pH 6–8. It was successfully used for imaging HepG2 cells and had no toxicity in the concentration range used in confocal microscopic examination.

Acknowledgements

The authors gratefully acknowledge Department of Science and Technology (DST), New Delhi (project no. SR/FT/CS-80/2010) for financial support. SAIF Kochi is acknowledged for single crystal X-ray analysis. ATP thanks National Institute of Technology Calicut for research fellowship.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: Experimental details, crystallographic and spectral data, additional pH dependent absorption–emission profiles, cytotoxicity studies and cell imaging protocols. CCDC 1025091 and 1025092. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c4ra10959g

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