A benzimidazole-derived fluorescent chemosensor for Cu(II)-selective turn-off and Zn(II)-selective ratiometric turn-on detection in aqueous solutions

Abstract

A 2,6-dibenzoimidazolyl-4-methoxyphenol conjugate (BBMP) was designed and synthesized to develop a novel fluorescent chemosensor for Cu²⁺ and Zn²⁺. The as-fabricated BBMP showed an excited-state intramolecular proton-transfer (ESIPT) fluorescence emission at 542 nm that was selectively and efficiently quenched by Cu²⁺ in a Tris-HCl solution (70% THF, pH = 7.4) due to the inhibition of ESIPT upon binding to metal ions. In contrast, the addition of Zn²⁺ not only resulted in a substantial decline in the original ESIPT fluorescence of BBMP but was also accompanied by the appearance of a new blue fluorescence emission at 462 nm. This indicated the potential for the present BBMP as a novel fluorescent probe for Cu²⁺ (turn-off) and Zn²⁺ (ratiometric turn-on) detection in aqueous solutions. The linear range and detection limit were found to be 0–5 μM and 0.16 μM for Cu²⁺, as well as 0–10 μM and 0.1 μM for Zn²⁺, respectively. A 2 : 1/1 : 1 coordination mode between BBMP and Cu²⁺/Zn²⁺ was confirmed by Job plots and mass analysis. Potential practical applications were also demonstrated by the analysis of real water samples and living cell imaging.

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1. Introduction

The design and development of organic small-molecule fluorescent chemosensors for the detection of various metal ions has attracted extensive attention.^1–4 Zn²⁺ and Cu²⁺ are known to be important trace elements that are involved in many physiological processes.^5,6 However, it has also been noted that an imbalance of Zn²⁺ and Cu²⁺ concentrations can lead to various diseases, such as metabolic disorders, Parkinson's disease, and coronary heart disease.^7–10 Hence, it would be of great significance to develop a highly selective and sensitive fluorescent chemosensor to quantitatively detect Cu²⁺ and Zn²⁺ in biological and environmental fields. In this regard, a number of fluorescent sensors for Cu²⁺ and Zn²⁺ detection have been designed based on various coordination scaffolds.^9–27 For example, a naphthalene Schiff-base system was designed as a fluorescent sensor for Cu²⁺ and Zn²⁺, where the fluorescence of the sensor was quenched by Cu²⁺or enhanced by Zn²⁺ over other metal cations in DMSO, respectively.¹¹ Kim et al. reported a phenanthrene-based multifunctional chemosensor that showed a slightly enhanced and blue-shifted (45 nm) fluorescence emission in the presence of Zn²⁺, whereas Cu²⁺ caused a serious quenching of the original fluorescence.¹⁸ Recently, a dual-functional Schiff-base fluorescent sensor containing the coumarin fluorophore was developed for Zn²⁺ and Cu²⁺ detection, in which Zn²⁺ induced a red-shift (46 nm) of the emission wavelength, allowing it to be used for the construction of a molecular logic gate with metal ion inputs and fluorescence signal outputs.²² Even the dual recognition of different Cu²⁺ and Zn²⁺ ions by a single sensor has been achieved, but most of their fluorescence emission showed no distinct or just a slight shift from the original one over binding events. It is practically undesirable for a dual-functional fluorescent chemosensor to be used for two different metal ions due to the discrimination being more difficult to as ascertain from a fluorescence intensity change than from a color change. Moreover, it has been considered that the fluorescence intensity is usually dependent on the measurement conditions, such as the concentration, excitation light intensity, and instrument efficiency, and that a ratiometric fluorescent sensor could overcome such shortcomings.^3,28,29 Therefore, a dual-functional fluorescent sensor for Cu²⁺ and Zn²⁺ that could exhibit a large emission wavelength shift from the original one upon binding with metal ions would be promising for a ratiometric sensor, but would be challenging to achieve.³⁰

Recently, benzimidazole-containing fluorescent sensors have attracted much interest due to their nice coordination ability toward metal ions.^31–36 Anbu et al. reported a benzimidazole-based Schiff-base-type fluorescent sensor that could differentially detect Cu²⁺ and Zn²⁺ through a fluorescence on–off and enhancement manner, respectively.³⁶ However, in the case of Zn²⁺ detection, the fluorescence emission was only blue-shifted by 10 nm. In this work, we designed and developed the novel benzimidazole-derived fluorescent sensor BBMP (Scheme 1) for Cu²⁺ and Zn²⁺. The present BBMP exhibited an excited-state intramolecular proton-transfer (ESIPT) emission at 542 nm and fluorescence quenching upon binding to Cu²⁺ and Zn²⁺ due to the inhibition of ESIPT. Notably, the BBMP also showed an 82 nm blue-shifted fluorescence emission upon binding to Zn²⁺, allowing ratiometric detection. Thus a benzimidazole-derived fluorescent sensor was developed for Cu²⁺-selective turn-off and Zn²⁺-selective ratiometric turn-on detection in aqueous solution. Furthermore, this was successfully used for Cu²⁺ and Zn²⁺ detection in real water samples and for fluorescence imaging in living cells, respectively.

Scheme 1 Synthesis of BBMP.

2. Experimental

2.1. Materials and methods

4-Nethoxyphenol, hexamethylenetetramine, o-phenylenediamine, trifluoroacetic, hydrochloride acid, and all organic solvents were analytical grade and purchased from Sinopharm Chemical Reagents Co. (Shanghai, China). The solutions of metal cations were prepared by dissolving the corresponding perchlorates (Mg²⁺, Hg²⁺), chlorides (Ca²⁺, Al³⁺, Mn²⁺, Co²⁺, Pd²⁺), sulfates (Fe²⁺, Cu²⁺), or nitrates (Fe³⁺, Ni²⁺, Zn²⁺, Ag⁺, Cd²⁺, Pb²⁺) salts with deionized water (10 mM). The stock solution of the BBMP sensor (1 mM) was dissolved in DMSO and the working solution was obtained by dilution with Tris-HCl buffer solution (10 mM, pH 7.4). The prepared solution was used for spectral testing and cellular imaging.

An Edinburgh FS5 spectrofluorometer was employed to measure the fluorescence spectra at room temperature (the slit width was set to 5 nm for both excitation and emission). Absolute fluorescence quantum yield measurements were carried out on an Edinburgh FS5 spectrofluorometer equipped with an integrating sphere (EI-FS5-SC-30). The UV-vis absorption spectra were collected on a Shimadzu UV-1800 spectrophotometer. The ¹H and ¹³C NMR spectra were recorded on a Bruker AVANCE III 400 MHz spectrometer with TMS as the internal standard. The high-resolution mass spectra were obtained on Micro TOFII 10257, Agilent 7250, and JEOL-JMS-T100LP AccuTOF mass spectrometers, respectively.

For analysis of real water samples, lake water and drinking water samples were collected from Songjiang Campus of Donghua University and a water dispenser, respectively. Zinc gluconate oral liquid (Harbin Pharm Group Sanjing Pharmaceutical Co. Ltd, China) was purchased from a local medical store. Fluorescence imaging in living HeLa cells was observed by an Axio Vert, A1 Carl Zeiss fluorescence microscopy system (Jena, Germany).

2.2. Synthesis of BBMP

The BBMP sensor was synthesized by a concise two-step process, as summarized in Scheme 1. First, 2,6-diformyl-4-methoxyphenol was synthesized according to our previously works.^37–39 Specifically, under a N₂ atmosphere, 4-methoxyphenol (15 mmol) and hexamethylenetetramine (45 mmol) were dissolved in trifluoroacetic acid (25 mL) and refluxed at 110 °C for 72 h. The mixture was cooled to 90 °C and 1 M HCl solution was added and stirring was continued for 24 h. The mixture was filtered and the filtrate was extracted with dichloromethane (25 mL × 3). Then the filtered solid and the extract were combined and washed with water until neutral pH. The organic phase was dried over anhydrous Na₂SO₄ for half an hour and concentrated under reduced pressure evaporation. The crude product was further purified by column chromatography (silica gel, ethyl acetate : petroleum ether = 1 : 10). Finally, a yellow flocculent solid was obtained. Yield, 30%. ¹H NMR (400 MHz, chloroform-d), δ (ppm): 11.13 (s, 1H), 10.22 (s, 2H), 7.51 (s, 2H), 3.86 (s, 3H).

Subsequently, under a N₂ atmosphere, 2,6-diformyl-4-methoxyphenol (1 mmol) and o-phenylenediamine (2.4 mmol) were dissolved in dichloromethane (15 mL) and refluxed for 63 h. After the reaction was completed, the mixture was cooled to room temperature, and the crude product was obtained by filtration, and washed with dichloromethane three times to obtain the target BBMP as an orange solid. Yield, 16.7%. ¹H NMR (400 MHz, DMSO-d₆), δ (ppm): 13.50 (s, 3H), 7.95 (s, 2H), 7.71 (dd, J = 6.0, 3.0 Hz, 4H), 7.29 (dd, J = 6.0, 3.0 Hz, 4H), 3.92 (s, 3H); ¹³C NMR (101 MHz, DMSO-d₆), δ (ppm): 152.26, 150.86, 150.37, 138.01, 123.20, 116.38, 115.57, 115.08, 56.37. MS: m/z calcd for C₂₁H₁₆N₄O₂ 357.1347 (M + H)⁺; found 357.1352 (M + H)⁺.

3. Results and discussion

3.1. Photophysical properties of BBMP

The absorption and fluorescence spectra of BBMP were first studied in various solvents (Fig. 1). It could be seen that BBMP showed a yellowish-green fluorescence emission at 548 nm and two absorption peaks around 300 nm as well as 375 nm in the weak polar solvents DEE, CHCl₃, and THF, respectively. Notably, the fluorescence emission of BBMP at 548 nm was slightly blue-shifted several nanometers in the strong polar solvents DMSO, DMF, and MeOH, accompanied by the appearance of weak fluorescence around 470 nm, respectively (Fig. 1a). Meanwhile, a new and broad absorption peak was discernable at 400–500 nm in these polar solvents (Fig. 1b). The large Stokes shift (ca. 8400 cm⁻¹) and similar molecular structure feature to 2-(2-hydroxyphenyl)benzimidazole most probably indicated an ESIPT emission nature.⁴⁰ Therefore, the solvent-dependent photophysical properties could be tentatively rationalized according to a subtle solvent effect on the intramolecular hydrogen-bond interaction between the imidazole N atom and phenolic OH in BBMP. The weak polar solvents DEE, CHCl₃, and THF had little effect on the intramolecular hydrogen bonds in BBMP, and thus normal ESIPT emission was observed at 548 nm, without disturbance to the absorption spectra. In contrast, the strong polar solvents DMSO, DMF, and MeOH, could cause competition between the intermolecular and intramolecular hydrogen-bond interactions, as confirmed by the observation of a substantial change in the absorption and fluorescence spectra in these polar solvents. The more prominent change in DMF and MeOH could be related to their basic and protic nature, respectively. ESIPT emission only underwent a slight disturbance in the polar solvents, indicating that strong intramolecular hydrogen-bond interactions existed in BBMP. Interestingly, when NaOH was introduced into the THF solution of BBMP, a largely blue-shifted fluorescence emission at 490 nm and a red-shifted absorption band at 407 nm were observed (Fig. 1). It could be ascribed to the ESIPT being completely inhibited due to the anionic BBMP formed by the deprotonation in the presence of a strong base, as has previously been observed in 2-(2-hydroxyphenyl)benzothiazole derivatives.⁴¹ In addition, the absolute fluorescence quantum yields of BBMP were measured to be in the range of 0.6–0.9 in various solvents (Table S1†).

Fig. 1 Fluorescence (a) and absorption (b) spectra of BBMP (10 μM) in various solvents. The excitation wavelength was 370 nm.

3.2. Selectivity toward Zn²⁺ and Cu²⁺ detection

Then the selectivity of BBMP toward various metal ions was investigated in aqueous solution (pH 7.4 Tris-HCl buffer containing 70% THF) as shown in Fig. 2. Without metal ions, the BBMP sensor showed a strong yellowish-green ESIPT fluorescence at 542 nm under excitation at 370 nm. Significant fluorescence quenching was observed in the presence of Cu²⁺, whereas Zn²⁺ selectively induced only a moderate quenching of the original ESIPT emission accompanied by the appearance of new blue fluorescence at 462 nm (Fig. 2a). In contrast, the fluorescence of BBMP showed almost no obvious change in the presence of other metal ions (Ca²⁺, Mg²⁺, Al³⁺, Mn²⁺, Fe³⁺, Fe²⁺, Ni²⁺, Pd²⁺, Ag⁺, Cd²⁺, Hg²⁺, Pb²⁺), except for Co²⁺, which caused a slight quenching effect. This demonstrated that the present BBMP could serve as a novel fluorescent sensor for Cu(II)-selective turn-off and Zn(II)-selective ratiometric turn-on detection in aqueous solution. Fig. 2b shows the absorption spectra of BBMP in the absence and presence of various metal ions. Obviously red-shifted absorption was observed upon the addition of Cu²⁺, Zn²⁺, and Co²⁺ respectively, suggesting that BBMP could substantially coordinate with these metal ions. Combined with the photophysical properties of BBMP in various solvents (Fig. 1), it could tentatively be concluded that the ESIPT emission was inhibited upon binding to the metal ions.

Fig. 2 Fluorescence (a) and absorption (b) spectra of BBMP (10 μM) upon the addition of 1 equiv. of various metal ions in Tris-HCl aqueous solution (10 mM, pH 7.4, containing 70% THF). The excitation wavelength was 370 nm.

To further examine the potential interference of others metal ions toward Cu²⁺ and Zn²⁺ detection, competition experiments were also conducted by measuring the fluorescence intensity at 542 nm and the intensity ratio at 462/541 nm. Fig. 3 shows that no significant interference was observed in the turn-off detection of Cu²⁺ and ratiometric turn-on detection of Zn²⁺, respectively. Thus, the present BBMP sensor could selectively and differentially detect Cu²⁺ and Zn²⁺ in aqueous solution.

Fig. 3 Competition experiments for Cu²⁺ (a) and Zn²⁺ (b) in aqueous Tris-HCl solution (10 mM, pH 7.4, 70% THF). The excitation wavelength was 370 nm.

3.3. Response time and effect of the pH

To obtain the optimal detection conditions, the response time and effect of the pH were investigated. As shown in Fig. 4a, upon the addition of 1 equiv. Cu²⁺, the fluorescence of BBMP (10 μM) at 542 nm rapidly declined but became stable within 2 min. In the case of Zn²⁺, the fluorescence intensity ratio of 465/541 nm increased to the maximum within 3 min (Fig. 4b). This indicated that the response of BBMP toward Cu²⁺ and Zn²⁺ was fast, and all the solution measurements were therefore performed after standing for 3 min.

Fig. 4 Change in the fluorescence intensity of BBMP (10 μM) with time upon the addition of 1 equiv. Cu²⁺ (a) and Zn²⁺ (b) in aqueous Tris-HCl solution (10 mM, pH 7.4, 70% THF), respectively. The excitation wavelength was 370 nm.

Subsequently, the fluorescence of BBMP in the absence and presence of 1 equiv. Cu²⁺ and Zn²⁺ was tested in the pH range of 2–11, respectively. It was found that the yellowish-green fluorescence of BBMP almost remained constant at pH <9, while the intensity at 542 nm slightly declined between pH 9–10, and a blue fluorescence emission appeared at 489 nm at pH >10 (Fig. 5a and S1a†). This could be attributed to deprotonation of the phenolic OH in BBMP and as anionic BBMP was produced at pH >10. In the presence of Cu²⁺, the fluorescence of BBMP was drastically quenched at pH <10, and also a blue fluorescence at 489 nm was observed at pH >10 originating from the anionic BBMP (Fig. 5a and S1b†). Notably, upon the addition of 1 equiv. Zn²⁺, the yellowish-green fluorescence of BBMP was slightly changed at pH <6 and moderate quenching was observed between pH 7–10, accompanied by the appearance of new blue fluorescence emission at 465 nm (Fig. 5b and S1c†). This allowed the ratiometric fluorescence detection of Zn²⁺. Similarly, blue fluorescence at 489 nm from the anionic BBMP was also observed at pH >10 (Fig. S1c†). In this work, pH 7.4 Tris-HCl buffer solution was then selected to meet the requirements for physiological environment applications.

Fig. 5 Change in the fluorescence intensity of BBMP (10 μM) at different pH 2–11 upon the addition of 1 equiv. Cu²⁺ (a) and Zn²⁺ (b) in aqueous Tris-HCl solution (10 mM, pH 7.4, 70% THF), respectively. The excitation wavelength was 370 nm.

3.4. Fluorescence and absorption titrations

Under the optimal experimental conditions, fluorescence and absorption titrations were performed to evaluate the sensing performance. Fig. 6 shows the fluorescence spectra and titration curves with the continuous increase in the concentration of Cu²⁺ and Zn²⁺ in Tris-HCl buffer (10 mM, pH 7.4, 70% THF), respectively. It could be seen that the fluorescence intensity of the BBMP sensor at 542 nm gradually decreased with increasing the concentration of Cu²⁺, and a good linear relationship was obtained (R² = 0.99027) within the concentration range 0–5 μM (Fig. 6a and b), with a detection limit of 0.16 μM (S/N = 3). As shown in Fig. 6c and d, upon the continuous addition of Zn²⁺, the fluorescence intensity decreased at 541 nm and was enhanced at 465 nm, and a good linear relationship (R² = 0.99037) was obtained between the ratio of the fluorescence intensity (F_465nm/F_541nm) and the Zn²⁺ concentration within the concentration range 0–10 μM, with a detection limit of 0.1 μM (S/N = 3). When compared against the previously reported sensing systems, the present sensor showed a comparable and even better performance (Table S2†).

Fig. 6 Fluorescence titration spectra of BBMP (10 μM) upon the addition of different concentrations of Cu²⁺ (a) and Zn²⁺ (c) in aqueous Tris-HCl solution (10 mM, pH 7.4, 70% THF), as well as a corresponding linear correlation between the fluorescence intensity and concentrations of Cu²⁺ (b) and Zn²⁺ (d), respectively. The excitation wavelength was 370 nm.

In addition, absorption titrations of Cu²⁺ and Zn²⁺ were also performed in Tris-HCl buffer (10 mM, pH 7.4, 70% THF), respectively. It was found that the absorption gradually decreased at 375 nm and increased at 420 nm with increasing the concentration of Cu²⁺ and Zn²⁺, respectively (Fig. S2†). A well-defined isosbestic point was observed at 386 nm, suggesting a complex was formed between the BBMP sensor and metal ions.

3.5. Binding reversibility and modes of BBMP to Cu²⁺ and Zn²⁺

Binding reversibility was investigated by the addition of ethylene diamine tetraacetic acid (EDTA) into the mixed solution of BBMP with Cu²⁺ or Zn²⁺. It was observed that the introduction of EDTA could almost recover the fluorescence of BBMP at 542 nm, indicting the competitive chelation between EDTA and Cu²⁺ or Zn²⁺ would release the free BBMP and thus restore its fluorescence. The further addition of Cu²⁺ or Zn²⁺ could then cause fluorescence quenching again, confirming the binding between BBMP and Cu²⁺ or Zn²⁺ was reversible (Fig. S3†).

The coordination stoichiometric ratio between BBMP and Cu²⁺ or Zn²⁺ was investigated by the Job's plot method, with the absorption spectra and results shown in Fig. 7. Obviously, a 2 : 1 or 1 : 1 binding mode between BBMP and Cu²⁺ or Zn²⁺ was obtained. This was further confirmed by ESI-MS analysis of the mixture solution of BBMP with Cu²⁺ or Zn²⁺ in MeOH, respectively. A prominent peak was found at m/z 837.08774, which could be readily assigned to [2BBMP–2H + Cu²⁺ + 2MeOH]⁺ species (Fig. S4a and c†). In addition, the mixture solution of BBMP with Zn²⁺ exhibited a complex peak at m/z 609.22409, which was attributed to [BBMP + Zn(NO₃)₂ + 2MeOH]⁺ species (Fig. S4b and d†). This demonstrated that BBMP formed a 2 : 1 complex with Cu²⁺ and a 1 : 1 complex with Zn²⁺. To further elucidate the binding mode, the ground-state molecular geometries of BBMP and its complexes with Cu²⁺ or Zn(NO₃)₂ were optimized by DFT calculations using the Gaussian 09 program package at the B3LYP/6-31 + G(d) level,⁴² and the results are displayed in Fig. 8. The optimized BBMP showed that one benzimidazole unit was near coplanar with the phenol ring and the distance of the imidazole N⋯HO was 1.7199 Å, indicating the existence of intramolecular hydrogen-bonding that facilitated the ESIPT (Fig. 8a). However, the other benzimidazole unit in BBMP was found to be twisted about 53.8° from the phenol plane. Regarding the optimized complexes, both the imidazole N and phenolic O atoms were involved in coordination with metal ions, where the distances of O⋯Cu, N⋯Cu, O⋯Zn, and N⋯Zn were found to be 2.2186, 1.9812, 1.9968, and 2.0880 Å, respectively (Fig. 8b and c). Notably, the benzimidazole units involved in coordination were twisted 22.4° (Cu complex) or 16.2° (Zn complex) from the phenol plane. However, the non-coordinated benzimidazole units were almost planar with the phenol ring, where the distance of the phenolic O⋯HN (imine) was 1.8285 Å (Cu complex) or 1.9075 Å (Zn complex), suggesting intramolecular hydrogen-bonding was most probably involved in the complexes (Fig. 8b and c).

Fig. 7 Job plots for BBMP + Cu²⁺ (a) and BBMP + Zn²⁺ (b) complexes in aqueous Tris-HCl solution (10 mM, pH 7.4, 70% THF), respectively. Both the total concentrations of [BBMP] + [Cu²⁺] = 20 μM and [BBMP] + [Zn²⁺] = 20 μM.

Fig. 8 Calculated molecular structures of BBMP (a) and its complexes with Cu²⁺ (b) and Zn(NO₃)₂ (c). Gray, blue, red, pink, light blue, and white colors denote C, N, O, Cu, Zn, and H atoms, respectively. All the H atoms except imine NH in (b) and (c) were omitted for clarity.

Therefore, the sensing mechanism could be understood by considering both the coordination interaction and natural property of the metal cation. Upon the addition of Cu²⁺/Zn²⁺, the complex formed between BBMP and the metal ion via the imidazole N atom and deprotonated phenolic group coordination could inhibit the ESIPT effect.¹⁶ A turn-off response was observed in the presence Cu²⁺ due to its paramagnetic character, whereas a blue-shifted and turn-on fluorescence response with Zn²⁺ was observed, possibly resulting from a chelation-enhanced fluorescence effect.

3.6. Fluorescence detection of Cu²⁺ and Zn²⁺ in real samples

In order to investigate the practical application of the BBMP sensor, the fluorescence detection of Cu²⁺ and Zn²⁺ was performed in real water samples and zinc gluconate oral liquid, respectively. The lake water samples were collected from Songjiang Campus of Donghua University and filtrated to remove solid impurities before use. A standard addition method was used and five measurements were collected to estimate the relative standard deviation (RSD) and recovery rate. The analysis results are summarized in Tables 1 and 2. It was found that the recovery rate and RSD were 106–127.5% and 0.41–1.76% for Cu²⁺ detection in the real water samples (Table 1). The Zn²⁺ amount in zinc gluconate oral liquid was determined to be 4.12 μM, which matched well with the real value of 4 μM. The recovery rate and RSD were 99.5–108.14% and 1.19–6.02% for Zn²⁺ detection in the real samples (Table 2). Therefore, this demonstrated that the present BBMP sensor can be used as a promising fluorescent sensor for the rapid detection of Cu²⁺ and Zn²⁺ in practical water samples.

Table 1 Detection of Cu²⁺ in real water samples (n = 5)

Samples	Added Cu^2+ (μM)	Detected Cu^2+ (μM)	Recovery (%)	RSD (%)
Lake water	2	2.55 ± 0.09	127.50	1.76
	4	4.31 ± 0.05	107.75	0.41
Drinking water	2	2.48 ± 0.04	124.00	0.59
	4	4.24 ± 0.08	106.00	0.74

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Table 2 Detection of Zn²⁺ in real samples (n = 5)

Samples	Added Zn^2+ (μM)	Detected Zn^2+ (μM)	Recovery (%)	RSD (%)
Lake water	4	4.65 ± 0.42	108.14	3.02
	7	7.60 ± 0.43	104.11	2.07
Zinc gluconate oral liquid	0	4.12 ± 0.41	—	3.90
	2	6.09 ± 0.21	99.50	1.19

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3.7. Fluorescence imaging in living cells

The application for Cu²⁺ and Zn²⁺ imaging in living cells was also investigated by confocal fluorescence microscopy. HeLa cells were first incubated with BBMP (10 μM) at 37 °C for 30 min, and subsequently incubated with 20 μM Cu²⁺ or Zn²⁺ and then observed under the confocal fluorescence microscope. Bright green fluorescence was observed in living cells incubated only with BBMP (Fig. 9a–c). Further incubation with Cu²⁺ caused a quenching of the green fluorescence (Fig. 9d–f), whereas a brilliant blue fluorescence was observed in living cells with further Zn²⁺ incubation (Fig. 9g–i). Thus the BBMP sensor is a promising sensor for Cu²⁺ and Zn²⁺ imaging in living cells.

Fig. 9 Bright-field (a, d and g), dark-field (b, e and h), and merged (c, f and i) images of living HeLa cells. The HeLa cells were incubated with BBMP (10 μM) (a–c), BBMP and further with Cu²⁺ (20 μM) (d–f), and BBMP and further with Zn²⁺ (20 μM) (g–i) at 37 °C for 30 min, respectively.

4. Conclusions

We successfully developed a new benzimidazole-derived fluorescence chemosensor BBMP for Cu²⁺ and Zn²⁺ detection in aqueous solution. The BBMP exhibited an ESIPT fluorescence emission, which was quenched upon binding to Cu²⁺ and Zn²⁺ due to an inhibition of ESIPT. Notably, the BBMP also showed an 82 nm blue-shifted fluorescence from the original ESIPT emission upon binding Zn²⁺, allowing the ratiometric detection. A Cu²⁺-selective turn-off and Zn²⁺-selective ratiometric turn-on fluorescent sensor was thus achieved. The binding mode was studied with Job's plot, mass analysis, and DFT calculation, where a 2 : 1 or 1 : 1 complex was formed between BBMP and Cu²⁺ or Zn²⁺ with imidazole N and phenolic O atoms coordination. The linear range and detection limit were found to be 0–5 μM and 0.16 μM for Cu²⁺, and 0–10 μM and 0.1 μM for and Zn²⁺, respectively. Furthermore, the sensor was also successfully used for Cu²⁺ and Zn²⁺ detection in real water samples and fluorescence imaging in living cells.

Author contributions

Wan-Yu Zhu: investigation, data curation, and writing – original draft. Kai Liu: writing – review & editing. Xuan Zhang: conceptualization, methodology, writing – review & editing, supervision, funding acquisition.

Conflicts of interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work was financially supported by the Research Foundation from National Innovation Center of Advanced Dyeing & Finishing Technology (No. 2022GCJJ10).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d3sd00020f

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