A chemosensor with a paddle structure based on a BODIPY chromophore for sequential recognition of Cu²⁺ and HSO₃⁻

Abstract

In this study, a highly selective chemosensor ML based on a BODIPY fluorescent chromophore was synthesized for sequential recognition of Cu²⁺ and HSO₃⁻ in a CH₃OH/H₂O (99 : 1 v/v) system, which contained three recognition sites and its structure characterized by ¹H NMR, ¹³C NMR and ESI-HR-MS. The sensor ML showed an obvious “on–off” fluorescence quenching response toward Cu²⁺ and the ML-Cu²⁺ complex showed an “off–on” fluorescence enhancement response toward HSO₃⁻. The detection limit of the sensor ML was 0.36 μM to Cu²⁺ and 1.4 μM to HSO₃⁻. In addition, the sensor ML showed a 1 : 3 binding stoichiometry to Cu²⁺ and the recovery rate of ML-Cu²⁺ complex identifying HSO₃⁻ could be over 70%. Sensor ML showed remarkable detection ability in a pH range of 4–8.

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Introduction

Copper is an essential trace element for organisms and plays an important role in various biological processes such as catalysis of enzymes, gene expression and protein synthesis.^1–5 Under normal conditions, the allowable limit of Cu²⁺ is 15.7–23.6 μM in blood and 20–30 μM in drinking water, respectively.^6,7 A moderate concentration of Cu²⁺ is helpful to maintain the normal function of the body, but an excess concentration of Cu²⁺ may cause many neurological complications such as Wilson's disease and Alzheimer's disease.^8–11 Therefore, it is imperative to develop a simple, rapid and sensitive analytical method for detecting Cu²⁺. Some fluorescent sensors can identify metal ions selectively and colorimetrically^12,13 and have many advantages such as high selectivity and sensitivity, simple operation and low cost, making them attractive in the detection of Cu²⁺.^14–19

SO₂ is the main component of acid rain as it can be dissolved in neutral aqueous solution to form a mixed system with a HSO₃⁻ to SO₃²⁻ molar ratio of about 3 : 1.^20–23 HSO₃⁻ plays an important role in both enzymatic and non-enzymatic reactions, and it is widely used as preservatives in foods and beverages.^24,25 However, excessive intake of HSO₃⁻ can not only cause damage to tissues, cells and biological macromolecules, but also can cause respiratory disease and many neurological disorders.^26–28 The acceptable daily intake of HSO₃⁻ should be less than 0.7 mg kg⁻¹ of body weight²⁹ and the sulfite content in foods and beverages should be less than 125 μM.²⁴ In recent years, fluorescent sensors have been developed using different approaches for detecting HSO₃⁻, such as coordination to metal ions,³⁰ selective reaction with aldehyde,^31,32 selective deprotection of levulinate group^33,34 and Michael-type addition.^35–37 However, there are few reports on the development of chemical sensors that can sequentially identify metal ions and HSO₃⁻ and simultaneously avoid interference by other sulfur-containing compounds. Clearly, it is more efficient and cost effective to sequentially identify metal ions and anions in practical applications.

BODIPY fluorescent dye is often used as a chromophore for the design and synthesis of fluorescent sensors because of its high molar extinction coefficient, high fluorescence quantum yield, good photothermal stability, easy structural modification and adjustable absorption and emission wavelengths to the infrared visible region.^38–42 In this study, we synthesized a chemosensor ML with a paddle structure^43,44 and three metal ion recognition sites by introducing three BODIPY fluorescent chromophores. This sensor showed an “on–off–on” fluorescence response to Cu²⁺ and HSO₃⁻ in CH₃OH/H₂O (99 : 1 v/v) system. Importantly, the colour of the test solution changed obviously, indicating that the sensor ML could be used for naked eye detection of Cu²⁺ and HSO₃⁻ (Scheme 1).

Scheme 1 Synthesis of sensor molecule ML.

Experimental

Materials and methods

Compound 1 and 2 were synthesized as described in previous studies.^45,46 Other solvents and starting materials were purchased from Aladdin and Energy Chemical Reagents Ltd. Ultrapure water was used through all the tests. The pH of all solutions was adjusted on a PHS-3C pH meter (Shanghai, China). The ¹H NMR (400 M) and ¹³C NMR (100 M) spectra were recorded on a spectrometer (Switzerland) in CDCl₃-d and DMSO-d₆ solutions. The SEI-MS spectra of sensor ML was recorded on a Bruker Solarix XR Fourier transform-ion cyclotron resonance (FT-ICR) mass spectrometer. The UV-spectras of all samples were recorded on a UV-2602 spectrophotometer (Shanghai, China), and all fluorescence spectras were recorded on a Hitachi F-2500 spectrophotometer (Japan).

Synthesis

Preparation of sensor ML. A 50 mL round-bottomed flask containing compound 1 (0.2 mmol, 42 mg), compound 2 (0.62 mmol, 210 mg), 20 mL absolute ethanol and a drop of glacial acetic acid was heated to reflux for 12 h and TLC monitoring. The mixture was cooled to room temperature, filtered, and washed with ice ethanol, and the filter cake was purified by silica gel column chromatography (petroleum ether : ethyl acetate = 5 : 1–1 : 1) to afford an orange solid (172 mg, 73%). ¹H NMR (CDCl₃-d, 400 MHz, TMS): δ (ppm) 13.45–13.53 (m, 2H), 13.10–13.14 (m, 1H), 8.81–8.92 (m, 3H), 7.45–7.50 (m, 6H), 7.35–7.40 (m, 6H), 6.01 (s, 6H), 2.57 (s, 18H), 1.46 (s, 18H). ¹³C NMR (CDCl₃-d, 100 MHz, TMS): δ (ppm): 185.5, 155.9, 149.2, 142.8, 140.2, 139.7, 132.4, 131.4, 130.0, 121.5, 118.3, 107.2, 14.7, 14.6. The NMR spectra of sensor ML were shown in Fig. S1 and S2.† HRMS (ESI): m/z calcd for C₆₆H₆₁B₃F₆N₉O₃ [(M + H)⁺]: 1174.5083, found 1174.5103 (Fig. S3†).

General procedure for fluorescence spectra experiments

In this study, sixteen kinds of metal cations and thirteen kinds of anions were selected. Cations included: K⁺, Ca²⁺, Na⁺, Li⁺, Mg²⁺, Zn²⁺, Fe³⁺, Cu²⁺, Al³⁺, Ag⁺, Hg²⁺, Cs⁺, Pb²⁺, Cd²⁺, Ba²⁺ and Ni²⁺; anions included: F⁻, Cl⁻, Br⁻, I⁻, OAc⁻, HCO₃⁻, CO₃²⁻, HSO₃⁻, SO₃²⁻, SO₄²⁻, S²⁻, NO₃⁻ and NO₂⁻. All tests were carried out at room temperature (25 °C) in CH₃OH/H₂O (99 : 1 v/v) system. The UV-vis absorption spectra and fluorescence spectra of sensor ML at a concentration of 5 × 10⁻⁶ M were recorded and the excitation wavelength was 475 nm, the slits of emission and excitation were 5 nm in all experiments. The stock solutions of various metal ions and anions (1 × 10⁻² M) were prepared from chlorine and sodium salts in ultrapure water, respectively.

Results and discussion

UV and fluorescence response of sensor ML towards Cu²⁺

The UV-vis spectra of sensor ML and 10 equiv. different metal ions responses were shown in Fig. 1. The sensor ML showed no response to metal ions except Cu²⁺. The maximum absorption wavelength showed a significant red shift from 499 nm to 510 nm with a red shift of 11 nm, and the absorbance was also significantly reduced. The color of the solution changed from yellow to pink, indicating that the sensor ML could be used for naked eye recognition of Cu²⁺.

Fig. 1 UV-vis spectra of sensor ML in CH₃OH/H₂O (99 : 1 v/v) system with the addition of 10 equiv. of metal ions and blank.

Fig. 2 showed that as the Cu²⁺ concentration increased, the absorbance decreased gradually and the absorption peak became wider. The maximum absorption wavelength shifted from 499 nm to 510 nm, and the color of the solution changed gradually from yellow to pink. The sensor ML coordinated with Cu²⁺ to form a stable complex, resulting in a decrease in the intensity of its UV-vis spectrum and a red shift. The five equal concentration points were observed at 339 nm, 378 nm, 444 nm, 460 nm and 509 nm, respectively, indicating that the sensor ML and the complex ML-Cu²⁺ were in a dynamic equilibrium.

Fig. 2 UV-vis spectra of sensor ML of various concentrations [0 μM, 0.5 μM, 1.0 μM, 2.0 μM, 3.0 μM, 4.0 μM, 5.0 μM, 10 μM, 15 μM, 20 μM Cu²⁺] in CH₃OH/H₂O (99 : 1 v/v) system.

Fig. 3 showed that the addition of Cu²⁺ resulted in almost complete quenching of the fluorescence intensity of the sensor ML, but only a slight change in the fluorescence intensity of other metal ions. The maximum emission wavelength was 515 nm and the Stokes shift was 40 nm, and thus the sensor ML showed good selectivity to Cu²⁺ in CH₃OH/H₂O (99 : 1 v/v) system at an excitation of 475 nm. As shown in Fig. S4,† the fluorescence spectras with the presence of other metal cations were almost the same as that with the presence of only Cu²⁺, which also indicated that the detection of Cu²⁺ would not be affected by the presence of other metal cations.

Fig. 3 Fluorescence spectra of sensor ML in CH₃OH/H₂O (99 : 1 v/v) system at an excitation of 475 nm with the addition of 10 equiv. of metal ions and blank.

Fig. 4 showed that as the Cu²⁺ concentration increased, the fluorescence intensity of the sensor ML decreased gradually. At a Cu²⁺ concentration of 8 equiv., the fluorescence intensity was approximately zero and the quenching rate was above 90%, indicating that the sensor ML was highly sensitive to Cu²⁺. The binding constant K_a was determined to be 1.70 × 10⁴ M⁻¹ (R² = 0.99675, Fig. S5†) and the detection limit was calculated to be 0.36 μM by LOD = 3σ/m (Fig. S6†). The Job curve at the upper right corner showed that the inflection point was observed at a Cu²⁺ concentration of 0.76 equiv., indicating that the coordination ratio of the sensor ML and Cu²⁺ was 1 : 3.

Fig. 4 Fluorescence spectra of 5 μM sensor ML with the addition of various concentrations of Cu²⁺ in CH₃OH/H₂O (99 : 1 v/v) system at an excitation of 475 nm. The inset plots showed that sensor ML formed a 1 : 3 complex with Cu²⁺.

Fluorescence response of ML-Cu²⁺ complex towards HSO₃⁻

The complex formed by the sensor ML and Cu²⁺ was used as a new sensor ML-Cu²⁺ for sequential recognition of anions. As shown in Fig. 5, the fluorescence intensity was enhanced after the addition of anions containing HSO₃⁻, but remained unchanged after the addition of other anions. The fluorescence was recovered to a large extent after adding HSO₃⁻ with a response rate of 70%, indicating that the new sensor could identify HSO₃⁻ specifically. As shown in Fig. S7,† the addition of HSO₃⁻ to ML-Cu²⁺ containing other anions still resulted in an obvious enhance in the fluorescence response. Thus, the sensor ML-Cu²⁺ was not interfered by other anions, and the interference of other sulfur-containing anions was also excluded, which further indicated that the ML-Cu²⁺ had good selectivity to HSO₃⁻.

Fig. 5 Fluorescence spectra of ML-Cu²⁺ (5 μM) complex with the addition of 10 equiv. different anions in CH₃OH/H₂O (99 : 1 v/v) system.

It could be seen from Fig. 6 that the intensity of the strongest fluorescence emission peak gradually increased with the increase of HSO₃⁻ concentration to 44 equiv., after which the intensity remained largely unchanged. Furthermore, the detection limit was 1.4 μM according to the formula LOD = 3σ/m (Fig. S8†).

Fig. 6 Fluorescence spectra of 5 μM ML-Cu²⁺ in the presence of various concentrations of HSO₃⁻ in CH₃OH/H₂O (99 : 1 v/v) system at an excitation of 475 nm.

Effect of pH

Fig. 7 showed that the fluorescence of sensor ML was quenched after the addition of Cu²⁺ at pH = 4–11, and sensor ML could sequentially recognize Cu²⁺ and HSO₃⁻ at pH = 4–8. It might be that the disruption of the structure of sensor ML under strong acid and alkali conditions affected the coordination ability of sensor ML with ions. Furthermore, HSO₃⁻ and OH⁻ could not coexist in a large amount under alkaline conditions, so sensor ML had no recognition response to HSO₃⁻ at pH = 9–12.

Fig. 7 Changes of the fluorescence intensity of sensor ML towards Cu²⁺ (10 equiv.) and HSO₃⁻ (10 equiv.) over a wide range of pH in CH₃OH/H₂O (99 : 1 v/v) system at room temperature.

Reversibility of sensor ML for Cu²⁺ and HSO₃⁻

The stability of sensor ML in identifying Cu²⁺ and HSO₃⁻ was discussed with Cu²⁺ and HSO₃⁻ as input signals and fluorescence intensity as output signals. As shown in Fig. 8, the fluorescence intensity quenched by Cu²⁺ still remained stable after four cycles; the recovery rate of the fluorescence intensity after the first addition of HSO₃⁻ was above 70%, and then the three recovery rates gradually decreased. The figure at the upper right corner showed that the sensor ML could be used to sequentially identify Cu²⁺ and HSO₃⁻ in practical applications.

Fig. 8 The sequential recognition of sensor ML (10 μM) upon alternate addition of Cu²⁺ and HSO₃⁻ in CH₃OH/H₂O (99 : 1 v/v) system (λ_ex = 475 nm).

The possible mechanism of sensor ML for Cu²⁺ and HSO₃⁻

The Job curve showed that the coordination ratio of sensor ML to Cu²⁺ was 1 : 3. The sensor ML provided three coordination points through the three O atoms on the carbonyl group and the three N atoms at the –NH-position to form a metal copper by 1 : 3 bonding with Cu²⁺. This sensor ML showed an “on–off–on” fluorescence response to Cu²⁺ and HSO₃⁻ in CH₃OH/H₂O (99 : 1 v/v) system.

In order to better understand the binding mode between sensor ML and Cu²⁺, the ¹H NMR titration experiments were conducted as showed in Fig. S9.† As the Cu²⁺ concentration increased, the N–H signals gradually disappeared with respect to sensor ML. The broadening of signals could also be affected by coordination. The molecular ion peak (ESI-MS) at 1713.14284 [sensor ML + 3Cu²⁺ + 3ClO₄⁻ + 3H₂O; m/z calcd for 1713.16792] (see Fig. S10†) indicated an approximately 1 : 3 complexation between sensor ML and Cu²⁺. Based on the analysis of Job's plot, mass spectrometry and ¹H NMR, the possible coordination mode was shown in Scheme 2.

Scheme 2 The possible mechanism of ML-Cu²⁺ with HSO₃⁻.

Conclusions

In this study, sensor ML was successfully synthesized for sequential recognition of Cu²⁺ and HSO₃⁻. Sensor ML showed an obvious “on–off” fluorescence quenching response toward Cu²⁺ with a quenching efficiency of above 90%, and the colour of the solution changed from yellow to pink. The ML-Cu²⁺ complex could be used to identify HSO₃⁻ selectively with a recovery rate of above 70%. Sensor ML showed a 1 : 3 binding stoichiometry to Cu²⁺ with a complexation constant of 1.70 × 10⁴ M⁻¹. The detection limits for Cu²⁺ and HSO₃⁻ were calculated to be 0.36 μM and 1.4 μM, respectively. The stable pH range of sensor ML to Cu²⁺ and ML-Cu²⁺ to HSO₃⁻ was from 4 to 8.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

We gratefully acknowledge the financial support from the Science Foundation of North University of China (No. 110121).

Notes and references

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c9ra08345f

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