A simple quinoline-derived fluorescent sensor for the selective and sequential detection of copper(II) and sulfide ions and its application in living-cell imaging

Abstract

A simple amide compound (QLBA), prepared from quinoline-2-carboxylic acid and 2-(1H-benzo[d]imidazol-2-yl)aniline, was synthesized and evaluated as an efficient fluorescence chemosensor for the selective recognition of copper ions (Cu²⁺). This sensor could detect Cu²⁺ in CH₃OH/HEPPES buffer in a wide pH range (4.0–10.0), with obvious fluorescence quenching. The obtained QLBA–Cu²⁺ complex could be used as a new cascade sensor for detecting sulfide anions (S²⁻). The corresponding detection limits were found to be 2.2 × 10⁻⁷ M for Cu²⁺ and 4.6 × 10⁻⁷ M for S²⁻. The sensing mechanisms of QLBA toward Cu²⁺ were systematically investigated in detail by NMR, HR-MS analysis, and density functional theory (DFT) calculations. In addition, this sensor was verified to be of low cytotoxicity and to have good imaging characteristics for the detection of Cu²⁺ and further for the recognition of S²⁻ in living cells, suggesting that QLBA was a useful tool for tracking Cu²⁺ and S²⁻ ions in vivo.

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Introduction

Copper(II) ions (Cu²⁺) rank as one of the vital trace metal elements for human life and play essential roles in various biological processes, including the detoxification of reactive oxygen species and neurotransmitter biosynthesis and degradation, as well as roles in the functioning and maintenance of the structural stability of proteins.^1–3 However, alterations in the cellular homeostasis of Cu²⁺ are connected to serious neurodegenerative diseases, such as Alzheimer's, Menkes, and Wilson diseases.^2–4 Similarly, continual exposure to high concentrations of sulfide anions (S²⁻) can cause harm to human health, including cumulative damage, pain, loss of consciousness, and irritation of mucous membranes.⁵ The sulfide anion is present in numerous products and is used in the manufacture of sulfuric acid, chemical fertilizers, dyes, and cosmetics, for example.⁶ Biosystems also contain sulfide from the microbial reduction of sulfate by anaerobic bacteria and sulfide generation from sulfur-containing amino acids in meat proteins.⁷ Therefore, the rapid and efficient detection of Cu²⁺ and S²⁻ is crucial to control their concentration levels in the biosphere and thus their direct impact on human health.

In the past few decades, the detection and imaging of relevant biological molecules via fluorescent chemical sensor strategies in living systems have proven to be important in the chemistry–biology interface, owing to the significance in biomedical implications.⁸ Until now, numerous chemosensors have been developed to monitor copper and sulfide ions due to the simplicity, high sensitivity, selectivity, and real-time analysis of fluorescence technology.^6b,9–11 However, there are only a few reports on fluorescent chemosensors for selectively detecting copper and sulfide ions in biological systems with a single sensor.¹² However, the detection of multiple targets with one single receptor is desirable as it would be more effective and economic than a one-to-one analysis process. Therefore, the development of single sensor systems with multiple targets has attracted significant attention.

The sulfide ion is known to react with a copper ion to form very stable CuS with a very low-solubility product (constant K_sp = 6.3 × 10⁻³⁶).^12c Considering this strategy of the higher affinity of Cu²⁺ toward S²⁻, low-cytotoxic and high-selectivity coordinated sensors for Cu²⁺ detection could be used for the further detection of S²⁻ in living systems based on CuS precipitation. Recently, some fluorescent sensors for Cu²⁺ and S²⁻ detection based on a copper sulfide precipitation strategy have been reported.^{10b,12d,13,14} However, most of them have not been applied in biological recognition research, which is highly important and necessary to study various health diseases.

In view of the recent literature, quinoline-based and benzimidazole-based derivatives are commonly utilized as receptors for the recognition of metal ions and anions due to their good photostability.^14–16 In the molecular structure of these derivatives, the N atoms of the quinoline and benzimidazole units generally act as chelating sites toward a metal center, leading to significant fluorescence signal changes. Based on this idea, herein, a simple amide compound prepared from quinoline-2-carboxylic acid and 2-(1H-benzo[d]imidazol-2-yl)aniline was designed, synthesized, and evaluated as a target chemosensor (QLBA) to selectively detect Cu²⁺ with obvious fluorescence quenching (Scheme 1). In addition, the obtained QLBA–Cu²⁺ complex could be used as a new cascade sensor to further detect S²⁻ through a Cu²⁺ displacement approach. Furthermore, we studied application of the sensor in fluorescence imaging in HeLa cells by confocal fluorescence microscopy, with the results suggesting that QLBA is a useful tool for tracking Cu²⁺ and S²⁻ in vivo.

Scheme 1 QLBA as a fluorescent chemosensor for Cu²⁺ detection, and its corresponding complex as a cascade sensor for the recognition of S²⁻ in living cells (SC denotes Solution Circumstance).

Results and discussion

As illustrated in Scheme 2, the target compound, QLBA, was facilely synthesized via the reaction of quinaldic acid chloride and 2-(1H-benzo[d]imidazol-2-yl)aniline (BMA) (BMA was first synthesized according to the literature procedure.¹⁷). In the structure of this chemosensor, the N atoms of the benzimidazole unit, quinolone unit, and –NHCO– bonded coordinated Cu²⁺ and generated a stable complex composed of five- and six-membered rings. The center copper(II) effectively quenched the fluorescence of QLBA, leading to formation of the non-fluorescence complex. Based on this copper sulfide precipitation strategy, the obtained complex could perform as a new cascade sensor for further recognizing S²⁻.

Scheme 2 Schematic for the synthesis of the fluorescent sensor QLBA.

Absorbance and fluorescence of QLBA for Cu²⁺

The detection selectivity of QLBA toward other metal cations, including Cu²⁺, Na⁺, K⁺, Mg²⁺, Ca²⁺, Co²⁺, Zn²⁺, Mn²⁺, Fe³⁺, Al³⁺, Cr³⁺, Pb²⁺, Hg²⁺, and Cd²⁺, was initially investigated in CH₃OH–HEPES buffer (1/9, v/v, pH 7.2). Free chemosensor QLBA (10 μM) showed two absorption bands at 241 nm and 294 nm, respectively. Upon the addition of 3 equiv. of other metal ions, only Cu²⁺ induced apparent spectral changes (Fig. S5†). The absorption band centered at 241 nm shifted to 246 nm, while the bands at 294 nm decreased, and two new peaks at 309 and 374 nm appeared simultaneously accompanied by a visible color change of the solution from colorless to pale orange. However, no clear changes in the absorption spectra of QLBA were observed with the other tested metal cations. In the fluorescence test (Fig. 1), the addition of 3 equiv. of other metal ions did not significantly disturb the fluorescence spectrum of QLBA solution. When 3 equiv. of Cu²⁺ was added to the solution of QLBA, dramatic fluorescence quenching (quenching efficiency of 86%) was observed, suggesting that QLBA showed a specific response to Cu²⁺ due to the chelation-enhanced fluorescence quenching (CHEQ) effect.^18a,b The CHEQ behavior of QLBA in the presence of Cu(II) could be attributed to the paramagnetic effect from the spin–orbit coupling of Cu²⁺ inducing the fluorescence quenching.^12d,18

Fig. 1 (a) Fluorescence emission spectra and (b) fluorescent intensity at 435 nm of QLBA (10 μM) in the presence of various metal ions (30 μM) in CH₃OH–HEPES buffer (1/9, v/v, pH 7.2). 1, only QLBA; then with the addition of 2, Cu²⁺; 3, Na⁺; 4, K⁺; 5, Mg²⁺; 6, Ca²⁺; 7, Co²⁺; 8, Zn²⁺; 9, Mn²⁺; 10, Fe³⁺; 11, Al³⁺; 12, Cr³⁺; 13, Pb²⁺; 14, Hg²⁺; 15, Cd²⁺ (λ_ex = 370 nm).

To further estimate the specificity of QLBA toward Cu²⁺, competitive experiments were carried out (Fig. 2a and S6†). The results showed that this fluorescence quenching was not interfered by other metal ions, which revealed that the chemosensor QLBA achieved high selectivity for the recognition of Cu²⁺ ion.

Fig. 2 (a) The fluorescence intensity at 435 nm of QLBA (10 μM) with other metal ions (30 μM) in CH₃OH–HEPES buffer (1/9, v/v, pH 7.2), and then with addition of 30 μM of Cu²⁺. 1, QLBA; 2, Cu²⁺; 3, Cu²⁺ + Na⁺; 4, Cu²⁺ + K⁺; 5, Cu²⁺ + Mg²⁺; 6, Cu²⁺ + Ca²⁺; 7, Cu²⁺ + Co²⁺; 8, Cu²⁺ + Zn²⁺; 9, Cu²⁺ + Mn²⁺; 10, Cu²⁺ + Fe³⁺; 11, Cu²⁺ + Al³⁺; 12, Cu²⁺ + Cr³⁺; 13, Cu²⁺ + Pb²⁺; 14, Cu²⁺ + Hg²⁺; 15, Cu²⁺ + Cd²⁺ (λ_ex = 370 nm). (b) Fluorescence emission spectra recorded for QLBA (10 μM) upon the gradual addition of Cu²⁺ (0–30 μM), and (inset) calibration curve obtained for fluorescence intensity at 435 nm versus the concentration changes of Cu²⁺ ion.

The pH effect on the fluorescence response of QLBA to Cu²⁺ was also investigated in a pH range of 3.0–12.0 (Fig. S6†). It was found that there were no obvious changes in the fluorescence intensity at 435 nm of free QLBA in the pH range from 4.0 to 10.0, suggesting that this chemosensor was quite stable (Fig. S7–S9†). However, the addition of Cu²⁺ led to the fluorescent quenching, especially in the pH range of 4.0–10.0. It is worth mentioning that the fluorescence intensity of QLBA–Cu²⁺ increased to some extent at pH > 10.0, due to the formation of copper(II) hydroxide complex in a high pH environment.¹⁹ These results indicate that QLBA could be used as an efficient sensor for detecting Cu²⁺ ions over a considerably wide pH range (4.0–10.0) compared to the numerous previously reported rhodamine-based and Schiff base chemosensors, which are sensitive to acid environments.^{11e,16d,20–22}

In order to quantitatively evaluate the sensing capability of QLBA toward Cu²⁺, fluorescence titration analysis was further investigated. As shown in Fig. 2b, the fluorescence emission of QLBA (10 μM) was gradually quenched upon the addition of Cu²⁺ (0–30 μM). The efficient fluorescence quenching of QLBA was attributed to the coordination to a paramagnetic Cu(II) center. The plot of the fluorescence intensity versus Cu²⁺ concentration (0–1.0 equiv.) exhibited a good linear relationship with R² = 0.993 (Fig. S10†). The limit of detection was found to be 2.2 × 10⁻⁷ M from the equation LOD = 3 × S_b/S,²³ which is quite a bit lower than the recommended maximum contaminant level (MCL) for Cu²⁺ in drinking water (31 μM), as defined by the World Health Organization (WHO).²⁴ To evaluate the application of QLBA in monitoring Cu²⁺ in drinking water, this sensor was then applied to the detection of Cu(II) in methanol/tap water (1 : 9, v/v). As shown in Fig. S11,† the fluorescence intensity decreased gradually upon the addition of Cu²⁺ (0–10 μM). The results indicated that this easy-to-synthesize chemosensor is suitable for the detection of Cu²⁺ in a natural water sample.

Sensing mechanism of QLBA to Cu²⁺

In order to directly confirm the binding stoichiometry between QLBA and copper ions, high-resolution mass spectrometry spectra (HR-MS) analysis of QLBA–Cu²⁺ was performed (Fig. 3). The cluster peaks at m/z = 365.1389 (calcd = 365.1397), corresponding to [QLBA + H]⁺, and m/z = 426.0533 (calcd = 426.0536), corresponding to [QLBA + Cu – H]⁺, could be clearly observed when 2 equiv. of Cu²⁺ was added to the solution of QLBA, suggesting a 1 : 1 QLBA–Cu²⁺ binding ratio. Based on this ESI-MS analysis, the association constants (K_a) were calculated to be 1.16 × 10⁵ M⁻¹ for QLBA–Cu²⁺ from nonlinear curve fitting of the fluorescence titration data (Fig. S10†), according to the Benesi–Hildebrand equation.²⁵

Fig. 3 ESI mass spectra of QLBA in the presence of CuCl₂ (2 equiv.), indicating the formation of a 1 : 1 QLBA–Cu²⁺ complex.

¹H NMR spectra in DMSO-d₆ were also collected for QLBA and the QLBA–Cu²⁺ complex to investigate the bonding mechanism (Fig. 4). Upon the addition of Cu²⁺ ions (0–2.0 equiv.) to QLBA, the chemical shift of the amino (–NH–) H_e at 14.52 ppm shifted down to 13.76 ppm, whereby the chemical signal gradually weakened (but did not disappear), while the amide (–CONH–) H_j disappeared. At the same time, a chemical shift of the quinolone unit protons (H_k, δ 8.68; H_l, δ 9.07; H_m, δ 8.41; H_n, δ 8.18; H_o, δ 8.18; H_p, δ 8.35 ppm) also shifted down to some extent (H_k, δ 8.62; H_l, δ 8.84; H_m, δ 8.28; H_n, δ 8.10; H_o, δ 8.10; H_p, δ 8.28 ppm). The above changes in the chemical shift signals suggest that the N atoms of the quinolone and benzimidazole units and the N atom of the –CONH– bond might be involved in the coordination toward the copper(II) center, which is similar to previous reports.^14,26

Fig. 4 ¹H NMR titration analysis of (1) QLBA, (2) QLBA + 0.5 equiv. Cu²⁺, (3) QLBA + 1.0 equiv. Cu²⁺, and QLBA + 2.0 equiv. Cu²⁺ in DMSO-d₆.

To better understand the mode of interactions of Cu²⁺ with QLBA, density functional theory (DFT) calculations with Becke's three-parameter Lee–Yang–Parr (B3LYP) correlation functional were carried out using the Gaussian 09 package. The global minimal structures for QLBA and the QLBA–Cu²⁺ complex are shown in Fig. 5. The distances of Cu²⁺ ion from the quinolone unit N atom (N₁) and amide N (N₂) are 2.12 Å and 1.98 Å, respectively, and 2.02 Å from the benzimidazole unit N atom (N₃). These data indicate that this spatial structure could effectively provide adequate space to accommodate the corresponding Cu²⁺ (Fig. 5). The DFT calculation data were supported by ¹H NMR and HR-MS studies, which were also consistent with Kim, Anand, and Hou's research.²⁷

Fig. 5 Frontier molecular orbital profiles of QLBA and QLBA–Cu²⁺ based on DFT (B3LYP/6-31G*) calculations.

The frontier molecular orbitals (FMOs) plots of QLBA and QLBA–Cu²⁺ were next analyzed (Fig. 5). The energy band gap between the HOMO–LUMO of QLBA and QLBA–Cu²⁺ were 3.81 eV and 3.48 eV, respectively. The energy decrease in the LUMO level was more remarkable than that in the HOMO level, indicating that the LUMO was more stabilized than the HOMO. The energies of both the HOMO and the LUMO of QLBA were less stabilized than QLBA–Cu²⁺, therefore, the conversion of QLBA to QLBA–Cu²⁺ becomes easier by the narrowing of the energy gap between the HOMO and LUMO due to the formation of a stable QLBA–Cu²⁺ complex.

QLBA–Cu²⁺ complex to detect anions

We also investigated the binding behavior of the metal-based chemosensor (QLBA–Cu²⁺ complex) toward various anions and sulfur species, including F⁻, Cl⁻, Br⁻, NO₂⁻, NO₃⁻, OAc⁻, H₂PO₄⁻, ClO₄⁻, SO₄²⁻, SO₃²⁻, lysine, and cysteine (Fig. 6a). Among these ions and amino acids, only S²⁻ could recover the fluorescence intensity of the QLBA–Cu²⁺ system. The addition of S²⁻ to the QLBA–Cu²⁺ complex solution led to fluorescence recovery at 435 nm, which could be attributed to the displacement strategy of Cu²⁺ chelating ligands. In this strategy, sulfide ions can coordinate with copper ions to form a very stable species copper sulfide precipitation, resulting in the release of the free QLBA.

Fig. 6 (a) Fluorescence spectra recorded for QLBA–Cu²⁺ (10 μM) upon the addition of various anions (30 μM), and (b) the fluorescent intensity at 435 nm for QLBA–Cu²⁺ (10 μM) upon the addition of various anions (1, QLBA; 2, QLBA + Cu²⁺; 3, QLBA + Cu²⁺ + S²⁻; then addition of 4, F⁻; 5, Cl⁻; 6, Br⁻; 7, NO₃⁻; 8, NO₂⁻; 9, SO₄²⁻; 10, SO₃²⁻; 11, OAc⁻; 12, H₂PO₄⁻; 13, ClO₄⁻; 14, lysine; 15, cysteine.) in CH₃OH–HEPES buffer (1/9, v/v, pH 7.2). (λ_ex = 370 nm).

A competitive experiment with the QLBA–Cu²⁺ system was also carried out to estimate the specificity of QLBA–Cu²⁺ toward sulfide ions in the presence of other anions. As shown in Fig. 6b, very low interference in the fluorescence spectra was observed both in the absence and presence of other anions. The results demonstrate that the obtained QLBA–Cu²⁺ complex can be used as an efficient fluorescent sensor for recognizing S²⁻. The anion sensing capability of the obtained complex was further investigated via fluorescence titration analysis (Fig. 7). As the concentration of S²⁻ increased, the fluorescence of the QLBA–Cu²⁺ system was recovered gradually by a copper sulfide precipitation strategy, leading to prominent fluorescence OFF–ON switching. Based on the fluorescence titration analysis, the limit of detection for S²⁻ was found to be 0.46 μM using the equation LOD = 3 × S_b/S (Fig. 7b).

Fig. 7 (a) Fluorescence emission spectra for QLBA–Cu²⁺ (10 μM) upon the gradual addition of S²⁻ (0–25 μM) in MeOH–HEPES (v/v, 1/9, pH 7.2). (b) Variation and (inset) calibration curve obtained for fluorescence intensity at 435 nm recorded for QLBA–Cu²⁺ (10 μM) upon the gradual addition of S²⁻ in MeOH–HEPES buffer solution (λ_ex = 370 nm).

Cell imaging

To test the cytotoxicity of this chemosensor in living cells, MTT assay was performed in HeLa cells treated with various concentrations (2.0, 5.0, 10, 20, and 50 μM) of QLBA for 24 h. As shown in Fig. S12,† HeLa cells incubated with 20 μM QLBA showed no significant cytotoxicity.

Inspired by the interesting photophysical properties of QLBA for its high selectivity and sensitivity detection of Cu²⁺ and subsequent recognition for S²⁻, we extended our study to recognize Cu²⁺ and S²⁻ ions in biological systems by living-cell imaging. To perform this experiment, HeLa cells were incubated with 20 μM QLBA in growth media at 37 °C for 4 h; here, blue intracellular fluorescence could be observed by confocal fluorescence microscopy (Fig. 8b). In contrast, HeLa cells were supplemented with 20 μM Cu²⁺ ions for 0.5 h at 37 °C and exhibited almost no fluorescence (Fig. 8c). Then, S²⁻ ions were introduced into the growth media, where the initial blue fluorescence of QLBA was gradually recovered with the increasing S²⁻ concentration (Fig. 8c–e). The above experiments indicate that the QLBA sensor is reversible, highly cell membrane-permeable, and can be used as a potential tool for tracking intracellular Cu²⁺ and S²⁻ ions in living cells.

Fig. 8 Confocal fluorescence microscopy images of HeLa cells. (a) HeLa cells incubated without QLBA; (b) HeLa cells incubated with only 20 μM QLBA for 4 h; (c) HeLa cells incubated with only 20 μM QLBA for 4 h, and then 20 μM Cu²⁺; and then (d) 10 μM S²⁻, and then (e) 20 μM S²⁻. Images were obtained after extensive washing of cells with PBS buffer (10 mM, pH 7.4).

Conclusion

In summary, we successfully developed a simple and high-efficiency quinoline–benzimidazole conjugate fluorescence chemosensor, QLBA, for Cu²⁺ detection in MeOH–HEPES buffer (1/9, v/v, pH 7.2) over various common metal ions. This sensor is capable of detecting Cu²⁺ with a 1 : 1 stoichiometry in a wide pH range (4.0–10.0) and with obvious fluorescence quenching. The obtained QLBA–Cu²⁺ complex revealed excellent sensitivity for the prominent detection of S²⁻ over other anions based on the copper sulfide precipitation strategy, leading to prominent fluorescence OFF–ON switching. The detection limits were determined to be 2.2 × 10⁻⁷ M for Cu²⁺ and 4.6 × 10⁻⁷ M for S²⁻ in the optimized testing system. In addition, this biosensor was verified to be of low cytotoxicity and displayed good imaging characteristics for the detection of Cu²⁺ and for the recognition of S²⁻ in living cells. We believe that this simple, low-cost, and high-efficiency biosensor has great potential for the detection of multiple analytes in actual living biosystems.

Experimental

Materials and general methods

All the metal nitrate salts (Aladdin), quinoline-2-carboxylic acid (98%, Aladdin), Anthranilic acid (98%, Aladdin), 1,2-diaminobenzene (98%, Aladdin), and oxalyl chloride (98%, Sinopharm Chemical Reagent Co.) were used as received without further purification. All the other reagents and solvents were purchased from Sinopharm Chemical Reagent Co. and used as received. All the solutions were prepared using ultrapurified water (18.4 MΩ cm), deionized by a Milli-QSP reagent water system (Millipore). All the nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Ultrashield TM 400 PLUS spectrometer. UV-Vis spectroscopy measurements were acquired on a SCINCO S-4100 UV/Vis spectrophotometer. Fluorescence spectra were recorded using a Jobin Yvon FluoroLog-3-TCSPC spectrofluorometer. Fluorescence images were obtained by an Olympus FV1000 Inverted Confocal IX81 Microscope. The optical density was measured with a TECAN Infinite M1000PRO Microplate Reader. Mass spectra (MS) were obtained with a Waters Q-TOF premier Mass Spectrometer. IR spectra were taken in KBr disks on a Bruker VERTEX 70 spectrometer. Melting points were determined on a Thomas-Hoover capillary melting point apparatus.

Synthesis of QLBA

The fluorescent chemosensor QLBA was synthesized by stirring quinaldic acid chloride and 2-(1H-benzo[d]imidazol-2-yl)aniline (BMA) in anhydrous dichloromethane at room temperature, as shown in Scheme 2. First, 2-(1H-benzo[d]imidazol-2-yl)aniline (BMA) was synthesized according to the literature procedure (for ¹H NMR, see ESI Fig. S1†).¹⁷ Quinaldic acid chloride was freshly prepared according to our previous literature.^16b Into a 125 mL two-necked flask equipped with a magnetic stirring bar, 1.05 g (5 mmol) of BMA and 35 mL anhydrous dichloromethane were added. The mixture was stirred in an ice bath for 10 min, and then equivalent freshly prepared quinaldic acid chloride, which was dissolved in 10 mL anhydrous dichloromethane, was added dropwise into the mixture. After the end of the addition, the mixture was stirred at room temperature for 3 h. The solvent was removed under reduced pressure and the residue was then subjected to column chromatography (neutral alumina gel, EtOAc/hexane 1 : 2), affording QLBA as a white powder 1.12 g, yield: 62%. Mp: 231–233 °C. ¹H NMR (400 MHz, DMSO-d₆) d (ppm): 14.52 (s, 1H), 13.22 (s, 1H), 9.07 (d, J = 8.2 Hz, 1H), 8.68 (d, J = 8.0, 1H), 8.41 (d, J = 8.0 Hz, 1H), 8.35 (d, J = 8.0 Hz, 1H), 8.18 (m, 2H), 8.06 (t, J = 8.0 Hz, 1H), 7.96 (d, J = 8.0 Hz, 1H), 7.82 (t, J = 8.0, 1H), 7.64 (m, 2H), 7.39 (m, 3H) (Fig. S1†). ¹³C NMR (100 MHz, DMSO-d₆) d (ppm): 164.03, 151.14, 150.87, 146.61, 143.30, 138.71, 138.14, 134.43, 131.45, 131.13, 129.71, 129.52, 128.98, 128.79, 128.38, 124.06, 123.85, 122.84, 121.08, 119.48, 119.01, 117.74, 112.17 (Fig. S2†). HR-MS calcd for C₂₃H₁₆N₄O [QLBA + H]⁺: 365.1397, found: 365.1389 (Fig. S3†). Elemental analysis calcd for C₂₃H₁₆N₄O: C, 75.81; H, 4.43; N, 15.38%. Found: C, 75.74; H, 4.57; N, 15.29%.

Sample preparation and measurements

Stock solutions (0.001 M) of chlorinated salts (Na⁺, K⁺, Mg²⁺, Ca²⁺, Cu²⁺, Co²⁺, Zn²⁺, Fe²⁺, Al³⁺, Fe³⁺, Cr³⁺, Hg²⁺, Pb²⁺, Cd²⁺) were prepared with 10 mM HEPES buffer solution (pH 7.2). The QLBA stock solution was prepared in methanol. Test solutions were prepared by placing a calculated amount of the QLBA stock solution into a test tube, adding an appropriate aliquot of each metal, and then diluting the solution to 3.0 mL with methanol and buffer solution (V_MeOH/V_{HEPES buffer}, 1/9, pH 7.2). QLBA–Cu²⁺ solution for the detection of anions were prepared via adding the chemical reaction amount of Cu²⁺ ions into QLBA solution, and these were then used to recognize the anions (F⁻, Cl⁻, Br⁻, NO₂⁻, NO₃⁻, OAc⁻, H₂PO₄⁻, ClO₄⁻, SO₄²⁻, SO₃²⁻, lysine, and cysteine). All the fluorescence spectra were recorded 10 min after the addition of the determinant at 25 °C, with the excitation wavelength set at 370 nm (excitation/emission slit widths: 2 nm).

Calculation of the association constant

The association constant (K_a) of QLBA–Cu²⁺ was obtained from nonlinear curve fitting of the fluorescence titration data according to Benesie–Hildebrand equation (eqn (1)),²⁵ where F₀, F, and F_min are the fluorescence intensity of QLBA in the absence of Cu²⁺, at a certain concentration of Cu²⁺ ion, and the minimum fluorescence intensity of [QLBA–Cu²⁺] in the linear range, [M] is the Cu²⁺ concentration, and n is the binding stoichiometry.

Cell cytotoxicity assay

To test the cytotoxicity of QLBA, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed. After treatment with QLBA (2.0, 5.0, 10, 20, and 50 μM) for 24 h, 10 μL of a MTT solution (5 mg mL⁻¹ PBS) was added into each well of a 96-well culture plate and incubated continuously at 37 °C for 4 h. Then, all the media were removed from the wells and 100 μL dimethyl sulfoxide was added into each well. The optical density was measured at 570 nm (OD₅₇₀) wavelength with a Microplate Reader (TECAN Infinite M1000PRO). The cell viability was expressed as the optical density (OD) ratio of the treatment to the control.

Cell viability and confocal imaging

HeLa cells were cultured in RPMI-1640 medium with 10% Fetal Bovine Serum (FBS), 10 IU mL⁻¹ of penicillin, and 10 μg mL⁻¹ of streptomycin under a humidified atmosphere of 5% CO₂ in air. The concentrations of the counted cells were adjusted to 1 × 10⁶ cells per mL for confocal imaging in RPMI-1640 medium. Cells were incubated with 20 μM QLBA in the culture medium for 4 h at 37 °C and then incubated with 0 and 20 μM of Cu²⁺ for 0.5 h at 37 °C. Then, the cells were washed three times with PBS and incubated with 10 and 20 μM of sulfide ions in PBS at 37 °C for 0.5 h in a CO₂ incubator. After that, the cells were washed with PBS another three times prior to the fluorescence imaging. All of the specimens were photographed using an Olympus FV1000-IX81 confocal laser scanning microscope. Fluorescence images were obtained by excitation with a multiline Hg laser and analyzed using ImageJ software (Wayne Rasband, National Institutes of Health, Bethesda, MD).

Acknowledgements

We thank the advice of Dr Lele Zhang (the Analytical & Testing Center of Graduate School at Shenzhen, Peking University). This work was supported by the National Natural Science Foundation of China (No. 21302108), Shenzhen Municipal government SZSITIC (No. CXB201104210014A, JCYJ20140509151735023), and Shenzhen Reform Commission (Disciplinary Development Program for Chemical Biology).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra15938a

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