A single fluorescent probe for multiple analyte sensing: efficient and selective detection of CN⁻, HSO₃⁻ and extremely alkaline pH

Abstract

A simple tailor-made water-soluble broadly emitting (500–650 nm) fluorescent probe 3-methyl-2-(N-ethylcarbazole-3vinyl)-benzothiazolium iodide (IECBT) for the selective and sensitive detection of multiple analytes, including CN⁻, HSO₃⁻ and extremely alkaline pH, is designed and synthesized via the ethylene bridging of 3-formyl-N-ethylcarbazole and 2,3-dimethyl benzothizolium iodide, which is a useful fluorescent probe for monitoring these analytes at extremely low concentrations quantitatively. CN⁻ and HSO₃⁻ are expected to undergo 1,4-addition reactions with the C-4 atom in the ethylene group of IECBT. This water-soluble fluorescent probe exhibits excellent sensitivity and selectivity toward CN⁻ in DMSO, which also results in a prominent fluorescence ratiometric change and a color change. The high selectivity and sensitivity of IECBT toward HSO₃⁻ and extremely alkaline pH over other coexisting species in water are also observed. The titration experiments show that it features a remarkably large Stokes shift and good stability towards CN⁻, HSO₃⁻ or extremely alkaline pH with a significant fluorescence turn-off response. Importantly, we demonstrate that IECBT can be used for the real-time sensing and bioimaging of CN⁻, HSO₃⁻ or extremely alkaline pH in living samples.

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1. Introduction

The development of new synthetic chromophores/probes for the efficient detection of various chemically, environmentally, and biologically significant species has witnessed a surge over the past few years.¹ Especially, the recognition and sensing of anions have been of considerable interest because of their critical impact on physiological and environmental systems.² Various anions have different roles and importance.³ Among various anions, cyanide (CN⁻) is one of the most concerning anions because it is an extremely toxic anion and can directly lead to the death of organisms.⁴ However, cyanide salts are still used as industrial materials in plastics production, gold mining, and other fields.⁵ Bisulfite (HSO₃⁻) is another important ion which has been widely used as an antimicrobial agent, an enzyme inhibitor, and an antioxidant in food, beverages, and pharmaceutical products.⁶ Unfortunately, extensive intake of HSO₃⁻ would lead to harmful effects in tissue, cells, and biomacromolecules, causing asthmatic attacks and allergic reactions in some individuals.⁷ Many reports have appeared in the literature describing the detection of either CN⁻ or HSO₃⁻ ions,⁸ but examples of a sensor which can detect both anions are relatively less known. Notably, intracellular pH plays a vital role in cell biology including receptor-mediated signal transduction, calcium regulation, ion transport, and homeostasis.⁹ Therefore, monitoring pH in live cells is highly desirable for better understanding of physiological and pathological processes.¹⁰ Recently, many excellent pH-dependent fluorescent probes with near neutral¹¹ or acidic response behavior¹² have been exploited for applications in biological systems. Nevertheless, little research is reported on the development of extremely alkaline pH probes.¹³ Undoubtedly, design of multiple analyte responsive probes for the rapid, efficient, and selective detection of CN⁻, HSO₃⁻, and extremely alkaline pH is important and attractive because of their wide range of applications.

Fluorometry has attracted much more attention over many other methods for CN⁻, HSO₃⁻, and pH detection due to its simplicity, noninvasiveness, high sensitivity, excellent specificity, and fast response time.¹⁴ In particular, fluorescence imaging provides high spatial and temporal observation of CN⁻, HSO₃⁻, and pH changes in live samples¹⁵ with the combination of fluorescent probes and confocal laser scanning microscopy. As a versatile probe technique, a ratiometric sensor for CN⁻ detection has attracted significant attention as an alternative to first-generation intensity probes due to its sensitivity and the built-in correction for avoiding environmental effects. Moreover, to the best of our knowledge, few fluorescent probes have been reported for rapid detection of HSO₃⁻ in 100% aqueous media¹⁶ and little attention is paid to the fluorescent pH probes functioning in the extremely alkaline pH range (pH > 9). Hence, a pressing need exists to develop a fluorescent probe satisfying the multiple criteria of multiple analyte response, performing in 100% aqueous media and showing live-cell permeability for monitoring CN⁻, HSO₃⁻, and extremely alkaline pH in biological systems.

In the present work, we report a single water-soluble broadly emitting (500–650 nm) fluorescent probe (IECBT), which can respond to CN⁻, HSO₃⁻ and extremely alkaline pH, via the ethylene bridging of 3-formyl-N-ethylcarbazole as an electron donor (D) and 2,3-dimethyl benzothizolium iodide as an electron acceptor (A). The D–π–A structure type is frequently adopted as a fluorophore to construct intramolecular charge transfer (ICT) fluorescent probes displaying a large Stokes shift.¹⁷ Here, CN⁻ and HSO₃⁻ are expected to undergo 1,4-addition reactions with the C-4 atom in the ethylene group, which is activated by the electron-withdrawing feature of the positively-charged benzothizolium fragment. In DMSO media, the ratiometric probe IECBT not only displays high sensitivity and selectivity toward CN⁻ ions, but also exhibits two well-separated emission peaks before and after CN⁻ addition (Δλ = 199 nm), which favors fluorescence resolution. On the other hand, the supernal selectivity and sensitivity of IECBT are achieved toward HSO₃⁻ ions and alkaline pH in water. Importantly, IECBT features a remarkably large Stokes shift, good stability and rapid response to CN⁻, HSO₃⁻ and extremely alkaline pH, which are highly desirable for a fluorescent probe to achieve reliable and sensitive fluorescence detection. Fluorescence CN⁻ and HSO₃⁻ imaging in living cells by IECBT is also successfully applied. Last but not least, laser scanning confocal microscopy of Escherichia coli cells indicates that IECBT could image alkaline pH value fluctuations in biological systems.

2. Experimental section

2.1 Materials and methods

IECBT was synthesized in our own laboratory. Unless otherwise stated, all reagents were purchased from commercial suppliers and used without further purification. Twice-distilled water was used throughout all experiments. Reactions were carried out using the magnetic stirrers and monitored by thin layer chromatography (TLC). Absorption spectra were measured using a UV-757CRT spectrophotometer (Shanghai Precision & Scientific Instrument Co., Shanghai, China) in a 4.5 mL (1 cm in diameter) cuvette with 2 mL of solution. Fluorescence measurements were conducted on a Hitachi F-7000 fluorescence spectrophotometer. NMR spectra were recorded on a Bruker instrument (AVANCE III HD) with TMS as the internal standard in DMSO-d₆, with 600 MHz for ¹H NMR and 150 MHz for ¹³C NMR, respectively. Mass spectra were acquired using an Agilent Accurate-Mass-Q-TOF MS 6520 system equipped with an electrospray ionization (ESI) source (Agilent, USA).

2.2 Synthesis of 3-methyl-2-(N-ethylcarbazole-3vinyl)-benzothiazolium iodide (IECBT)

3-Formyl-N-ethylcarbazole (0.141 g, 0.5 mmol) and 2,3-dimethyl benzothizolium iodide (0.167 g, 0.5 mmol) were heated in dry ethanol (5 mL) at 150 °C for 6 h under reflux with vigorous stirring. The reaction was cooled to room temperature and the precipitate was collected by filtration, washed with diethyl ether, and dried in vacuo. IECBT was obtained as an orange solid in 55% yield (0.169 g). As depicted in Scheme 1, IECBT was easily synthesized in a one-step reaction, and the purification was simple. ¹H NMR (DMSO-d₆, δ/ppm): 8.947 (s, 1H), 8.430 (m, 2H), 8.243 (m, 3H), 8.063 (d, J = 16.2 Hz, 1H), 7.876 (m, 2H), 7.785 (t, J = 7.2 Hz, 1H), 7.741 (t, J = 7.8 Hz, 1H), 7.581 (t, J = 7.2 Hz, 1H), 7.367 (t, J = 7.2 Hz, 1H), 4.547 (d, J = 7.2 Hz, 2H), 4.377 (s, 3H), 1.386 (t, J = 7.2 Hz, 3H). ¹³C NMR (DMSO-d₆, δ/ppm): 172.46, 151.06, 142.70, 142.51, 140.80, 129.65, 128.58, 127.86, 127.34, 125.67, 124.96, 124.58, 124.19, 123.42, 122.70, 121.09, 120.76, 116.98, 110.60, 40.54, 37.91, 36.61, 14.32. HRMS (ESI) calcd for [M]⁺ 355.1264, found 355.1252 (Fig. S1, ESI†).

Scheme 1 Synthesis of IECBT and sensing mechanisms for CN⁻ and HSO₃⁻.

2.3 General procedure for spectral measurements

IECBT was dissolved in acetonitrile/water (4/6, v/v) medium to obtain 1.0 mM stock solutions. CN⁻ and HSO₃⁻ solutions were prepared by dissolving potassium cyanide or sodium bisulfite in deionized water. The aqueous solutions of cations and anions for selectivity experiments and titration experiments were prepared from the corresponding hydrochloride salts or sodium salts in deionized water at an appropriate concentration. In the pH titration experiments, the slight pH variations of the solutions were achieved by adding the minimum volumes of HCl (0.5 M) or NaOH (0.25 M, 0.5 M and 1.0 M). All spectroscopic experiments were carried out at room temperature and the excitation and emission slit widths were 10 nm and 5 nm, respectively.

2.4 U251 cell culture and imaging

U251 cells were cultured in DMEM containing 10% FBS (Fetal Bovine Serum) and 1% antibiotic–antimycotic solution at 37 °C in a 5% CO₂/95% air incubator. One day before imaging, the cells were plated on 35 mm diameter round glass Petri dishes and allowed to adhere for 24 h. Subsequently, the cells were incubated with IECBT (10 μM) dissolved in acetonitrile/water (4/6, v/v) for 30 min at 37 °C and then washed with pre-warmed PBS (pH = 7.4) three times. Then cells were stimulated with CN⁻ (30 μM) and HSO₃⁻ (20 μM) in their culture dish, respectively, and images were collected on a FV1000 confocal laser scanning microscope (Olympus Co., Ltd, Japan) with a 200× objective lens over 2 min.

2.5 E. coli cell culture and imaging

In light of the method reported in the literature,¹⁸E. coli cells were incubated at 37 °C in Luria-Bertani (LB) culture medium (tryptone 10 g L⁻¹, yeast extract 5 g L⁻¹, and NaCl 10 g L⁻¹) for 17 h in a table concentrator at 180 rpm. Then the culture was centrifuged at 5000 rpm for 5 min to collect E. coli cells. The sediment was washed with sterile water and then resuspended in solutions of different pH values (8.00, 9.89 and 11.98). Five minutes after resuspension, the probe IECBT dissolved in acetonitrile/water (4/6, v/v) was added into every tube to a final probe concentration of 20 μM. E. coli cells with the probe were incubated in a table concentrator for 2 h and then smeared on slides and observed using a ZEISS LSM 880 confocal laser scanning microscope with a 10× objective lens.

3. Results and discussion

3.1 Proposed mechanism

The visible color and strong fluorescence of IECBT were due to its outstanding ICT effect from the electron-donor carbazole group to the electron-acceptor benzothiazole moiety. As illustrated in Fig. 1, ¹H NMR analysis was firstly carried out to demonstrate the proposed 1,4-addition mechanism. After addition of CN⁻ or HSO₃⁻ into the solution of IECBT, respectively, all of the ¹H NMR signals shifted upfield because the nucleophilic attack of CN⁻ or HSO₃⁻ toward C-4 disturbed the π-conjugation and weakened its electron withdrawing characteristic. Fig. 1b illustrates that the proton signals (H-a, at δ 4.377) of the methyl group connected with N⁺ were significantly shifted up-field to δ 3.223 after addition of CN⁻. And the vinyl protons at δ 8.063 (H-b) and δ 8.430 (H-c) were dramatically upfield shifted to δ 4.939 (H-b and H-c) upon cyanide addition at room temperature due to the 1,4-addition reaction. This observation clearly indicated that the cyanide anion was added to the C-4 atom in the ethylene group (Fig. 1). In addition, the formation of the IECBT–CN adduct was also confirmed by mass spectrometry analysis (ESIMS), and the peak at m/z 396.1517 (calc. = 396.1528) corresponding to [IECBT–CN + H]⁺ was clearly observed (Fig. S2, ESI†). Notably, from Fig. 1c, we could breezily find that the proton signals (H-b, at δ 8.063 and H-c, at δ 8.430) which prominently appeared at δ 4.484–4.375 after HSO₃⁻ addition confirmed the 1,4-addition reaction between IECBT and HSO₃⁻, undoubtedly. Furthermore, this adduct was further characterized by MS analysis (Fig. S3, ESI†), where the peak at m/z 451.1129 (calc. = 451.1144) corresponding to [IECBT–HSO₃ + H]⁺ was observed.

Fig. 1 ¹H NMR spectra of IECBT in DMSO-d₆ before (a) and after the addition of (b) KCN or (c) NaHSO₃ (portion ¹H NMR spectra).

3.2 Investigation of spectral properties of IECBT

We first decided to examine the absorption spectra of IECBT (15 μM) after addition of various amounts of CN⁻ (0–20 μM). Before that, the effect of different solvent conditions on the fluorescence properties of the system was investigated (see ESI†) (Fig. S4, ESI†). IECBT exhibited an obvious absorption peak at 468 nm (Fig. 2a). Upon addition of CN⁻, the absorption peak at 468 nm was gradually attenuated and a new broad band from around 260 to 300 nm was increased, suggesting that the π-conjugation was broken. As a consequence, an obvious color change in the solution from orange to colorless occurred, which could be observed easily by the naked eye (inset of Fig. 2a). Meanwhile, a clear isosbestic point was also noted at 330 nm, indicating the formation of the IECBT–CN adduct. The fluorescence response of IECBT (10 μM) toward CN⁻ (0–45 μM) in DMSO was investigated as demonstrated in Fig. 2b. In the absence of CN⁻, only one peak was clearly observed at 575 nm (λ_ex = 340 nm) with a large Stokes shift of 235 nm, which was highly desirable for a fluorescent probe because the large gap between the excitation wavelength and the emission wavelength can effectively diminish the measurement error caused by the excitation light and scattered light. However, the addition of CN⁻ resulted in a significant decrease in emission at 575 nm and an excellent increase of the emission band centered at 376 nm, which also implied that the nucleophilic attack of CN⁻ interrupted the π-conjugation of IECBT. Accordingly, a well-defined isoemission point at 534 nm was noticed and the fluorescence color changed from orange to deep blue (inset of Fig. 2b). Then a titration profile of the ratio of the emission intensities at 376 and 575 nm (I₃₇₆/I₅₇₅) to the CN⁻ concentrations from 0 to 45 μM could be observed (Fig. 2c). The ratio value increased dramatically by >270-fold (from 0.21 to 57) with the addition of CN⁻. Moreover, as plotted in the inset of Fig. 2c, the signal ratio I₃₇₆/I₅₇₅ showed a good linearity with CN⁻ concentration in the range of 0–10 μM [Y = 0.18X + 0.16 (R² = 0.9895)]. The detection limit was calculated to be 0.94 μM (S/N = 3), which was below the WHO detection level. It was also worth mentioning that the shift of the two emission wavelengths was very large (ΔF = 199 nm), which contributed to the accurate measurement of the intensities of the two emission peaks, as well as resulted in a huge ratiometric value. These findings showed that IECBT was indeed a sensitive and quantitative detector of CN⁻ ions in DMSO media. Time-dependent modulations in the fluorescence of the probe (10 μM) in the presence of CN⁻ (17 μM) suggested that the reaction could be completed within 10 min under the experimental conditions (Fig. S5, ESI†). Thus, the detection was delayed for 10 min to ensure that the reaction between IECBT and CN⁻ was accomplished completely.

Fig. 2 (a) Absorption spectra of IECBT (15 μM) upon the addition of CN⁻ in DMSO. CN⁻ concentrations were varied in the following order: 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, and 20 μM. Inset: The photograph of IECBT solution (15 μM) in the absence or presence of CN⁻ (20 μM). (b) Fluorescence spectra of IECBT (10 μM) upon addition of CN⁻ in DMSO. CN⁻ concentrations were varied in the following order: 0, 2.5, 5, 7.5, 10, 12.5, 15, 17.5, 20, 22.5, 25, 27.5, 30, 32.5, 35, 40, and 45 μM. λ_ex = 340 nm. Slits: 10 nm/5 nm. Voltage = 500 V. Inset: The photograph of IECBT solution (10 μM) in the absence or presence of CN⁻ (35 μM) under a UV light. (c) The plot of I₃₇₆/I₅₇₅versus the concentration of CN⁻ (0–45 μM). Inset: The linearity of the concentration of CN⁻ in the range of 0–10 μM.

To gain insight into the degree of selectivity toward CN⁻ ions exhibited by IECBT, we investigated the fluorescence behavior of IECBT (10 μM) in the presence of various typical potential interfering anions, such as F⁻, Cl⁻, Br⁻, I⁻, CO₃²⁻, HCO₃⁻, NO₃⁻, SO₄²⁻, AcO⁻, SCN⁻, HSO₃⁻, SO₃²⁻, S₂O₃²⁻, S²⁻ and HS⁻ (1 equiv., respectively). Remarkably, as shown in Fig. S6 (ESI†), an unperturbed effect on the intensity ratio (I₃₇₆/I₅₇₅) of IECBT was observed for anions, compared with that obtained for CN⁻. Moreover, competitive assays indicated that the coexistence of all these typical analytes (100 equiv.) had little impact on the ability of IECBT to detect CN⁻-ions (Fig. 3). These experiments showed that IECBT had high selectivity and strong anti-jamming capacity for CN⁻ determination against all kinds of anions.

Fig. 3 Fluorescence responses of IECBT (10 μM) in DMSO toward the competing anions (100 equiv. respectively). The black bars represented the addition of 35 μM CN⁻ to the probe solution. The red bars displayed the subsequent addition of different competing anions to the fluorescent probe. λ_ex = 340 nm. Slits: 10 nm/5 nm. Voltage = 500 V.

The sensing ability of IECBT for HSO₃⁻ was then studied. The reaction media, pH value, and response time were investigated to optimize the sensing conditions. The fluorescence spectral response of IECBT (10 μM) in the absence and presence of HSO₃⁻ (20 μM) at different pH values was evaluated as shown in Fig. S7 (ESI†). IECBT was stable in aqueous solution and displayed the best response for HSO₃⁻. Thus, the distilled water was selected for the spectral studies. The reaction time between IECBT and HSO₃⁻ was estimated within 2 min to ensure complete reaction between IECBT (10 μM) and HSO₃⁻ (10 μM) (Fig. S8, ESI†). The absorption spectrum of IECBT (10 μM) showed a strong band at 450 nm (Fig. 4a). With increasing concentration of HSO₃⁻ (0–28 μM), the absorption peak at 450 nm was gradually reduced and a new absorption peak appeared at 276 nm with an isosbestic point at 281 nm. This phenomenon led to a distinguishable solution color change from croci to colorless (inset of Fig. 4a). The fluorescence spectra of IECBT (10 μM) after addition of various amounts of HSO₃⁻ (0–28 μM) are shown in Fig. 4b. As anticipated, IECBT exhibited a remarkable emission at 568 nm (λ_ex = 465 nm) with a large Stokes shift of 103 nm. The addition of HSO₃⁻ caused a gradual decrease in emission at 568 nm with a fluorescence color change from orange to nonfluorescence (inset of Fig. 4b). A titration profile of the responses of the relative emission intensity (F₀ − F) at 568 nm to the HSO₃⁻ concentrations from 0 to 28 μM could be observed (Fig. 4c). As plotted in the inset of Fig. 4c, the signal (F₀ − F) at 568 nm showed a good linearity with HSO₃⁻ concentration in the range of 0.5–9 μM [Y = 115.28X + 135.20 (R² = 0.9907)]. The detection limit was calculated to be 0.53 μM (S/N = 3). These results suggested that IECBT might be an excellent candidate for practical HSO₃⁻ analysis in aqueous media.

Fig. 4 (a) Absorption spectra of IECBT (10 μM) upon addition of HSO₃⁻ in aqueous solution. HSO₃⁻ concentrations were varied in the following order: 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, and 28 μM. Inset: The photograph of IECBT solution (10 μM) in the absence or presence of HSO₃⁻ (30 μM). (b) Fluorescence spectra of IECBT (10 μM) upon addition of HSO₃⁻ in aqueous solution. HSO₃⁻ concentrations were varied in the following order: 0, 0.5, 1, 2, 3, 4, 5, 7, 9, 11, 13, 17, 21, and 28 μM. λ_ex = 465 nm. Slits: 10 nm/5 nm. Voltage = 600 V. Inset: The photograph of IECBT solution (10 μM) in the absence or presence of HSO₃⁻ (30 μM) under a UV light. (c) The plot of F₀ − F (568 nm) versus the concentration of HSO₃⁻ (0–28 μM). Inset: The linearity of the concentration of HSO₃⁻ in the range of 0.5–9 μM.

The selectivity experiments were carried out by monitoring the relative emission intensity ratio (F₀ − F)/F₀ of the probe in the presence of various typical interfering anions including F⁻, Cl⁻, Br⁻, I⁻, CO₃²⁻, HCO₃⁻, NO₃⁻, SO₄²⁻, AcO⁻, SCN⁻, CN⁻, S₂O₃²⁻, S²⁻ and HS⁻ (1 equiv., respectively) as shown in Fig. S9 (ESI†). No significant responses were obtained for these anions and the (F₀ − F)/F₀ values varied in a limited range between −0.005 and 0.2. By contrast, treatment of IECBT with HSO₃⁻ resulted in an obvious (F₀ − F)/F₀ response. In addition, competition experiments of IECBT were also ascertained by various anions mentioned previously in the same environment. The signaling of IECBT toward HSO₃⁻ was not affected by the presence of 20 equiv. of coexisting species (Fig. 5). These phenomenon ulteriorly proved that IECBT could be used as an efficient signaling probe for HSO₃⁻ in aqueous media.

Fig. 5 Fluorescence responses of IECBT (10 μM) in aqueous solution toward the competing anions (20 equiv. respectively). The black bars represented the addition of different competing anions to the probe solution. The red bars displayed the subsequent addition of 25 μM HSO₃⁻ to the fluorescent probe. λ_ex = 465 nm. Slits: 10 nm/5 nm. Voltage = 600 V.

To study the optical responses of IECBT to pH, the UV-Vis absorption and fluorescence emission spectra of IECBT (10 μM) were further investigated in pure water at various pH values (pH 6.44–11.21). IECBT displayed highly sensitive responses to the pH changes. As depicted in Fig. 6a, when the pH increased from 6.44 to 11.21, the maximum absorption band at 450 nm of IECBT in aqueous media was significantly quenched. A further observation was a notable color change from orange-to-colorless with increasing pH (inset of Fig. 6a), which indicated that IECBT could serve as a visual probe for the determination of pH fluctuations. The fluorescence properties of IECBT as a function of pH are depicted in Fig. 6b. When the pH value was lower than 6.44, the IECBT solution exhibited an intense emission band centered at 568 nm (λ_ex = 465 nm) with a large Stokes shift of 103 nm. The large Stokes shift could help to reduce the excitation interference. However, very weak red fluorescence emission was observed as the pH was increased to 11.21. Concomitantly, a notable color change from orange to nonfluorescence under ultraviolet light (inset of Fig. 6b) was observed. Fig. 6c shows the results of sigmoidal fitting of the pH-dependent emission at 568 nm, affording a pK_a value of 9.75. The fluorescence intensity at 568 nm also displayed good linearity with pH in the range 9.48–10.07, according to the linear regression equation F = 19715.56 − 1930.65 × pH, with a linear coefficient of 0.9852 (inset of Fig. 6c). The stability of IECBT was tested by measuring the fluorescence response over 2 h. Fig. S10 (ESI†) depicts the time course of fluorescence intensity of the probe (10 μM) at pH 6.44, 6.66, and 9.70, respectively, which proved that IECBT could respond to the pH fluctuations within 10 min and the media were stable to light and air.

Fig. 6 (a) Absorption spectra of IECBT (10 μM) as pH increased from 6.44 to 11.21. Inset: The photograph of IECBT solution (10 μM) as pH increased from 6.44 to 11.21. (b) Fluorescence spectra of IECBT (10 μM) as pH increased from 6.44 to 11.21. Inset: The photograph of IECBT solution (10 μM) as pH increased from 6.44 to 11.21 under a UV light. λ_ex = 465 nm. Slits: 10 nm/5 nm. Voltage = 600 V. (c) Sigmoidal fitting of the pH-dependent fluorescence intensity at 568 nm. Inset: The linearity in the pH range of 9.48–10.07.

For a good pH fluorescent probe, excellent anti-interference capacity is a key factor. To confirm that IECBT could measure the pH value efficiently, the selectivity of IECBT (10 μM) to alkaline pH over metal ions was investigated at pH 7.11 and 8.09 by competition experiments, respectively. As shown in Fig. 7, physiologically ubiquitous metal ions (K⁺, Na⁺, Ca²⁺ and Mg²⁺) over their physiological concentrations did not show any obvious emission change in pH measurement. Also in the case of heavy and transition metal ions, such as Ni²⁺, Cr³⁺, Co²⁺, Mn²⁺, Zn²⁺, Cd²⁺, Al³⁺, Cu²⁺, Hg²⁺, Fe³⁺, Fe²⁺ and Ag⁺ (0.2 mM) together with IECBT, almost no adverse effect on the intensity was observed. These results revealed that IECBT showed an excellent selectivity response to alkaline pH in the presence of background metal ions.

Fig. 7 Fluorescence responses (568 nm) of IECBT (10 μM) in aqueous solution at pH 7.11 and 8.09 in the presence of diverse metal ions. 1, blank; 2, K⁺ (150 mM); 3, Na⁺ (150 mM); 4, Ca²⁺ (10 mM); 5, Ni²⁺ (0.2 mM); 6, Cr³⁺ (0.2 mM); 7, Co²⁺ (0.2 mM); 8, Mn²⁺ (0.2 mM); 9, Zn²⁺ (0.2 mM); 10, Cd²⁺ (0.2 mM); 11, Al³⁺ (0.2 mM); 12, Mg²⁺ (0.2 mM); 13, Cu²⁺ (0.2 mM); 14, Hg²⁺ (0.2 mM); 15, Fe³⁺ (0.2 mM); 16, Fe²⁺ (0.2 mM); 17, Ag⁺ (0.2 mM). λ_ex = 465 nm. Slits: 10 nm/5 nm. Voltage = 600 V.

3.3 Cell cytotoxicity assay

Since IECBT features large Stokes shifts, high selectivity, excellent sensitivity and good stability for CN⁻, HSO₃⁻ or extremely alkaline pH sensing, it is possible to explore its use in intracellular imaging. Before that, it is crucial to evaluate the cytotoxicity of IECBT as well as the cell viability after the addition of CN⁻ in living cells by MTT assay (see ESI†). As shown in Fig. S11 (ESI†), the percentage of viable HeLa cells after treatment with IECBT (0, 0.1 1, 10 μM) for 24 hours was over 90%, which demonstrated that IECBT displayed low cytotoxicity to living cells. Fig. S12 (ESI†) depicts the viability of HeLa cells incubated with IECBT (10 μM) at various CN⁻ concentrations from 0 to 30 μM. These results demonstrated that more than 80% of the cells are viable for 90 minutes, showing the low toxicity of IECBT (10 μM) after the addition of CN⁻ (0 to 30 μM) to cultured cells under experimental conditions and inferring its potential use in the intracellular imaging of living cells.

3.4 Imaging of living cells

Encouraged by the aforementioned results, we further investigated the applicability of IECBT for intracellular CN⁻ or HSO₃⁻ detection and imaging with U251 cells, respectively. Dynamic fluorescence imaging was performed to observe the reaction process between IECBT and these anions mentioned previously in real time. We used the confocal laser scanning microscope to directly visualize the fluorescence change of U251 cells incubated with IECBT at a certain concentration of CN⁻ (30 μM) in its culture dish over time (0, 30 s, 1 min, and 2 min) (Fig. 8). The red fluorescence (500–600 nm) and blue fluorescence (425–475 nm) were captured by 488 and 405 nm lasers, respectively, and the fluorescence intensity was calculated by use of commercial software. It was obvious that the U251 cells displayed bright red fluorescence (Fig. 8a) and very faint blue fluorescence emission (Fig. 8e), initially. With extended times, the red fluorescence intensity decreased sharply (Fig. 8a–d); by contrast, the blue fluorescence intensity increased pronouncedly (Fig. 8e–h). We also explored the fluorescence change of IECBT after adding HSO₃⁻ (20 μM) in the living U251 cells over time (0, 10 s, 30 s, 1 min, and 2 min) (Fig. 9). Using excitation wavelengths at 488 nm, the images of the red channel were obtained in the 500–600 nm detection range. Fig. 9a shows that a marked red fluorescence was observed. However, extended times caused a gradual quenching (Fig. 9a–e). These cell experiments showed excellent cell membrane permeability of IECBT, and it could be used to detect CN⁻ or HSO₃⁻ in living cells, respectively.

Fig. 8 Dynamic fluorescence imaging of U251 cells incubated with IECBT (10 μM) at a certain concentration of CN⁻ (30 μM) in its culture dish. (a–d) Image in the red channel (500–600 nm); (e–h) the image in the blue channel (425–475 nm); (i) the bright-field image of panel a; the excitation wavelengths were 488 and 405 nm for red and blue fluorescence, respectively.

Fig. 9 Dynamic fluorescence imaging of U251 cells incubated with IECBT (10 μM) at a certain concentration of HSO₃⁻ (20 μM) in its culture dish. (a–e) Image in the red channel; (f) the bright-field image of panel a; the excitation wavelength was 488 nm, and emission was collected in the red channel (500–600 nm).

Meanwhile, to further confirm the subcellular distributions of IECBT, a blue nucleus specific staining probe 4,6-diamidino-2-phenylindole (DAPI) was used to co-stain HeLa cells with IECBT. As shown in Fig. 10, the blue emissions were from the nuclei specific staining probe DAPI (Fig. 10b), and the red emissions which were well-dispersed in the entire cell region were generated from our probe IECBT (Fig. 10a). According to the clear cell morphology (Fig. 10c) and dual-color fluorescence imaging (Fig. 10d), we could also see very clearly that the red emissions from IECBT were well-dispersed in the entire cell region.

Fig. 10 Confocal microscopy images of 10 μM IECBT in HeLa cells co-stained with 1 mg mL⁻¹ DAPI. (a) Image in the red channel (500–600 nm); (b) the image in the blue channel (425–475 nm); (c) the bright-field image of panel a; (d) the merged image of a and b. The excitation wavelengths were 488 and 405 nm for red and blue fluorescence, respectively. (ZEISS LSM 880 confocal laser scanning microscope, 200× objective lens.)

3.5 pH bioimaging in E. coli

The imaging of E. coli incubated with IECBT under various pH conditions was further studied to investigate its ability to monitor pH changes in living samples. To simulate the extremely alkaline environment in E. coli, buffer solutions with pH at 8.00, 9.89 and 11.98, respectively, were used to incubate bacteria. The optical window at the red channel (500–600 nm) was chosen as a signal output. Under selective excitation at 488 nm, the E. coli cells displayed bright red fluorescence (Fig. 11a) at pH 8.00. The red fluorescence intensity dropped sharply with the pH increasing to 9.89 (Fig. 11b), and almost no fluorescence was observed when the pH increased to 11.98 (Fig. 11c). These results confirmed that IECBT was able to image pH fluctuations under extreme alkalinity in E. coli cells.

Fig. 11 Confocal microscopy images of 20 μM IECBT in E. coli cells clamped at pH 8.00 (a and d), 9.89 (b and e), 11.98 (c and f). (a–c) Images in the red channel; (d) the bright-field image of panel a; (e) the bright-field image of panel b; (f) the bright-field image of panel c; the excitation wavelength was 488 nm, and the red channel images were collected at 500–600 nm (63× oil immersion lens).

4. Conclusion

In summary, we have described the facile synthesis, optical properties, and living cell or bacterial imaging applications of IECBT which is synthesized by ethylene bridging of 3-formyl-N-ethylcarbazole and 2,3-dimethyl benzothizolium iodide, a unique type of a single fluorescent probe that can detect CN⁻, HSO₃⁻ or extremely alkaline pH. IECBT is found to be an excellent ratiometric probe for the selective sensing of CN⁻ in DMSO via 1,4-addition reaction. At the same time, this probe shows high selectivity and sensitivity toward HSO₃⁻ in aqueous solution based on the 1,4-addition mechanism. Also, pH titration indicates that IECBT exhibited a remarkable pH-dependent behavior with a pK_a value of 9.75. Moreover, other advantageous properties of IECBT include low detection limits, fine stability, good water-solubility and large Stokes shifts towards CN⁻, HSO₃⁻ and pH, all of which are favorable for intracellular CN⁻, HSO₃⁻ or pH imaging. The application of IECBT to CN⁻ and HSO₃⁻ imaging in live U251 cells is also achieved successfully, indicating that the probe has good cell membrane permeability and can be used to rapidly detect CN⁻ or HSO₃⁻ in living cells, respectively. Since IECBT holds perfect properties, it is used to noninvasively measure extremely alkaline pH in E. coli cells. To our knowledge, IECBT represents the first fluorescent probe for monitoring multiple analytes such as CN⁻, HSO₃⁻ or extremely alkaline pH in living samples. Therefore, this work provides a promising probe for the effective detection and quantification of the extremely toxic CN⁻ found in biological and environmental samples and HSO₃⁻ for food safety and quality control, clinical and environmental applications. What is more, it is also anticipated that IECBT is a hopeful candidate for real-time tracking of pH changes, especially under extremely alkaline conditions in the biomedical and biological fields.

Acknowledgements

The work was supported by the National Natural Science Foundation of China (No. 21472118), the Shanxi Province Science Foundation for Youths (No. 2012021009-4 and 2013011011-1), the Shanxi Province Foundation for Returnee (No. 2012-007), the Taiyuan Technology Star Special (No. 12024703), the Program for the Top Young and Middle-aged Innovative Talents of Higher Learning Institutions of Shanxi (TYMIT, No. 2013802), the Talents Support Program of Shanxi Province (No. 2014401) and the Shanxi Province Outstanding Youth Fund (No. 2014021002), the Scientific and Technological Innovation Programs of Higher Education Institutions in Shanxi (20111001), and the Shanxi Province Foundation for College Students of Science and Technology Innovation projects (No. 2013328).

Notes and references

1. (a) G. Aragay, J. Pons and A. Merkoci, Chem. Rev., 2011, 111, 3433–3458; (b) K. P. Carter, A. M. Young and A. E. Palmer, Chem. Rev., 2014, 114, 4564–4601; (c) D.-G. Cho and J. L. Sessler, Chem. Soc. Rev., 2009, 38, 1647–1662; (d) M. Formica, V. Fusi, L. Giorgi and M. Micheloni, Coord. Chem. Rev., 2012, 256, 170–192; (e) L. E. Santos-Figueroa, M. E. Moragues, E. Climent, A. Agostini, R. Martinez-Manez and F. Sancenon, Chem. Soc. Rev., 2013, 42, 3489–3613; (f) D. Sareen, P. Kaur and K. Singh, Coord. Chem. Rev., 2014, 265, 125–154; (g) L. Yuan, W. Y. Lin, Y. N. Xie, B. Chen and S. Zhu, J. Am. Chem. Soc., 2012, 134, 1305–1315; (h) Y. Y. Han, C. Q. Ding, J. Zhou and Y. Tian, Anal. Chem., 2015, 87, 5333–5339; (i) A. Balamurugan and H. Lee, Macromolecules, 2015, 48, 3934–3940.
2. (a) H. J. Kim, S. Bhuniya, R. K. Mahajan, R. Puri, H. Liu, K. C. Ko, J. Y. Lee and J. S. Kim, Chem. Commun., 2009, 7128–7130; (b) J. P. Hill, N. K. Subbaiyan, F. D'Souza, Y. S. Xie, S. Sahu, N. M. Sanchez-Ballester, G. J. Richards, T. Morif and K. Ariga, Chem. Commun., 2012, 48, 3951–3953; (c) G. Sivaraman and D. Chellappa, J. Mater. Chem. B, 2013, 1, 5768–5772; (d) Q. L. Xu, C. H. Heo, G. Kim, H. W. Lee, H. M. Kim and J. Yoon, Angew. Chem., Int. Ed., 2015, 54, 1–6; (e) H. X. Zhang, J. Liu, Y. Q. Sun, Y. Y. Huo, Y. H. Li, W. Z. Liu, X. Wu, N. S. Zhu, Y. W. Shi and W. Guo, Chem. Commun., 2015, 51, 2721–2724; (f) S. Chen, P. Huo and X. Z. Song, Sens. Actuators, B, 2015, 221, 951–955.
3. (a) M. Breugst and H. Mayr, J. Am. Chem. Soc., 2010, 132, 15380–15389; (b) M. G. Fete, Z. Havlas and J. Michl, J. Am. Chem. Soc., 2011, 133, 4123–4131; (c) J. J. Hettige, H. K. Kashyap, H. V. R. Annapureddy and C. J. Margulis, J. Phys. Chem. Lett., 2013, 4, 105–110; (d) T. D. Ashton, K. A. Jolliffe and F. M. Pfeffer, Chem. Soc. Rev., 2015, 44, 4547–4595.
4. (a) Cyanide in Biology, ed. B. Vennesland, E. F. Comm, J. Knownles, J. Westly and F. Wissing, Academic, London, 1981; (b) K. W. Kulig, Cyanide Toxicity, U. S. Department of Health and Human Services, Atlanta, GA, 1991; (c) Guidelines for Drinking-Water Quality, World Health Organization, Geneva, 1996.
5. (a) H. Sun, Y. Y. Zhang, S. H. Si, D. R. Zhu and Y. S. Fung, Sens. Actuators, B, 2005, 108, 925–932; (b) S. K. Kwon, S. Kou, H. N. Kim, X. Q. Chen, H. Hwang, S. W. Nam, S. H. Kim, K. M. K. Swamy, S. Park and J. Yoon, Tetrahedron Lett., 2008, 49, 4102–4105; (c) S. Vallejos, P. Estévez, F. C. García, F. Serna, J. L. de la Peña and J. M. García, Chem. Commun., 2010, 46, 7951–7953.
6. (a) A. Isaac, A. J. Wain, R. G. Compton, C. Livingstone and J. Davis, Analyst, 2005, 130, 1343–1344; (b) J. Xu, K. Liu, D. Di, S. Shao and Y. Guo, Inorg. Chem. Commun., 2007, 10, 681–684; (c) L. C. de Azevedo, M. M. Reis, L. F. Motta, G. O. da Rocha, L. A. Silva and J. B. De Andeade, J. Agric. Food Chem., 2007, 55, 8670–8680.
7. (a) S. L. Taylor, N. A. Higle and R. K. Bush, Adv. Food Res., 1986, 30, 1–76; (b) R. C. Claudia and J. C. Francisco, Food Chem., 2009, 112, 487–491; (c) P. D. Tzanavaras, E. Thiakouli and D. G. Themelis, Talanta, 2009, 77, 1614–1619; (d) M. Koch, R. Köppen, D. Siegel, A. Witt and I. Nehls, J. Agric. Food Chem., 2010, 58, 9463–9467; (e) National Standards of the People's Republic of China, GB 2760-2007, 16.
8. (a) N. Kumari, S. Jha and S. Bhattacharya, J. Org. Chem., 2011, 76, 8215–8222; (b) P. B. Pati, Sens. Actuators, B, 2016, 222, 374–390; (c) W. Xu, C. L. Teoh, J. J. Peng, D. D. Su, L. Yuan and Y. T. Chang, Biomaterials, 2015, 56, 1–9; (d) P. Jayasudha, R. Manivannan and K. P. Elango, Sens. Actuators, B, 2015, 221, 1441–1448; (e) Y. Q. Sun, P. Wang, J. Liu, J. Y. Zhang and W. Guo, Analyst, 2012, 137, 3430–3433.
9. (a) R. Martinez-Zaguilln and R. J. Gillies, Cell. Physiol. Biochem., 1996, 6, 169–184; (b) M. Lakadamyali, M. J. Rust, H. P. Babcock and X. Zhuang, Proc. Natl. Acad. Sci. U. S. A., 2003, 100, 9280–9285; (c) X. Liu, Y. Xu, R. Sun, Y. Xu, J. Lu and J. Ge, Analyst, 2013, 138, 6542–6550; (d) K. R. Hoyt and I. J. Reynolds, J. Neurochem., 1998, 7, 1051–1058; (e) R. G. W. Anderson and L. Orci, J. Cell Biol., 1988, 106, 539–543.
10. (a) E. S. Trombetta, M. Ebersold, W. Garrett, M. Pypaert and I. Mellman, Science, 2003, 299, 1400–1403; (b) Y. Urano, D. Asanuma, Y. Hama, Y. Koyama, T. Barrett, M. Kamiya, T. Nagano, T. Watanabe, A. Hasegawa, P. L. Choyke and H. Kobayashi, Nat. Med., 2009, 15, 104–109; (c) T. A. Krulwich, G. Sachs and E. Padan, Nat. Rev. Microbiol., 2011, 9, 330–343.
11. (a) L. Fan, Q. L. Liu, D. T. Lu, H. P. Shi, Y. F. Yang, Y. F. Li, C. Dong and S. M. Shuang, J. Mater. Chem. B, 2013, 1, 4281–4288; (b) G. K. Vegesna, J. Janjanam, J. Bi, F. T. Luo, J. T. Zhang, C. Olds, A. Tiwari and H. Y. Liu, J. Mater. Chem. B, 2014, 2, 4500–4508; (c) Y. H. Li, Y. J. Wang, S. Yang, Y. R. Zhao, L. Yuan, J. Zheng and R. H. Yang, Anal. Chem., 2015, 87, 2495–2503.
12. (a) W. F. Niu, L. Fan, M. Nan, Z. B. Li, D. T. Lu, M. S. Wong, S. M. Shuang and C. Dong, Anal. Chem., 2015, 87, 2788–2793; (b) J. B. Chao, Y. H. Liu, J. Y. Sun, Y. B. Zhang, H. B. Tong and Z. Q. Li, Sens. Actuators, B, 2015, 221, 427–433; (c) L. Fan, S. Q. Gao, Z. B. Li, W. F. Niu, W. J. Zhang, S. M. Shuang and C. Dong, Sens. Actuators, B, 2015, 221, 1069–1076.
13. M. H. Su, Y. Liu, H. M. Ma, Q. L. Ma, Z. H. Wang, J. L. Yang and M. X. Wang, Chem. Commun., 2001, 960–961.
14. (a) J. Chan, S. Dodani and C. Chang, Nat. Chem., 2012, 4, 973–984; (b) M. Schäferling, Angew. Chem., Int. Ed., 2012, 51, 3532–3554; (c) A. P. de Silva, H. Q. Nimal Gunaratne, T. Gunnlaugsson, A. J. M. Huxley, C. P. McCoy, J. T. Rademacher and T. E. Rice, Chem. Rev., 1997, 97, 1515–1566.
15. J. Han and K. Burgess, Chem. Rev., 2010, 110, 2709–2728.
16. (a) Y. Sun, D. Zhao, S. W. Fan, L. Duan and R. F. Li, J. Agric. Food Chem., 2014, 62, 3405–3409; (b) W. Q. Chen, Q. Fang, D. L. Yang, H. Y. Zhang, X. Z. Song and J. Foley, Anal. Chem., 2015, 87, 609–616.
17. (a) L. L. Hao, J. F. Li, P. H. Lin, R. F. Dong, D. X. Li, S. M. Shuang and C. Dong, Chin. Chem. Lett., 2010, 21, 9–12; (b) S. L. Zhang, B. Zhao, L. Ran, D. B. Qin and J. W. Luo, Huaxue Shiji, 2014, 36, 925–927; (c) J. Oriou, F. Ng, G. Hadziioannou, C. Brochon and E. Cloutet, J. Polym. Sci., Part A: Polym. Chem., 2015, 53, 2059–2068.
18. (a) M. Y. Yang, Y. Q. Song, M. Zhang, S. X. Lin, Z. Y. Hao, Y. Liang, D. M. Zhang and P. R. Chen, Angew. Chem., Int. Ed., 2012, 51, 7674–7679; (b) Y. Xu, Z. Jiang, Y. Xiao, F. Z. Bi, J. Y. Miao and B. X. Zhao, Anal. Chim. Acta, 2014, 820, 146–151.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6tb00119j

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