Fluorescent paper sensor fabricated by carbazole-based probes for dual visual detection of Cu²⁺ and gaseous H₂S

Abstract

We report a fluorescent carbazole-based probe to achieve visual detection of Cu²⁺ and gaseous H₂S with paper sensors in a fluorescent “on–off–on” mode. A common carbazole mother molecule is reconstructed by dual substitute and hydrazine reactions to obtain triple dentates to a single Cu²⁺ ion, and thus the fluorescence of the carbazole ring is strongly quenched by the captured Cu²⁺. Subsequently, the coordinative Cu²⁺ can be snatched by S²⁻ via the formation of CuS, leading to the recovery of carbazole fluorescence. The above reactions provide the sensitive and prompt detection of Cu²⁺ and S²⁻ with the limits of 65 nM and 0.29 μM, respectively. The reusability of the “on–off–on” fluorescent switch is well demonstrated by a recycling experiment with alternate additions of Cu²⁺ and S²⁻. Moreover, paper sensors are fabricated and used for the visual detection of Cu²⁺ and gaseous H₂S with the limits of 10⁻⁶ M and 15 ppm.

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Introduction

The sulfide anion (S²⁻) as a toxic traditional pollutant is widespread in the environment, and has a deleterious effect on human health, especially in the respiratory system.¹ Sulfide anions can hydrolyze to more toxic and caustic species including hydrogen sulfide (H₂S) and hydrosulfide (HS⁻) in buffer solution at physiological pH.² H₂S has not only been known as an environmental hazard for many decades but also it has been regarded as the third gaseous transmitter after NO and CO.^3–5 Physiological levels of H₂S can regulate intracellular redox status and fundamental signalling processes, including relaxation of vascular smooth muscles, mediation of neurotransmission, inhibition of insulin signaling, regulation of inflammation.⁶ However, abnormal levels of H₂S in cells are associated with many diseases, such as Alzheimer's disease, liver cirrhosis, diabetes and Down's syndrome.^7,8

Fluorescence probes with the advantages of real-time measurement, high sensitivity and selectivity for the detection of H₂S are remarkable over other detection methods, such as simple colorimetric assays, electrochemical sensor, monobromobimane, high pressure liquid chromatography and gas chromatography.⁹ The H₂S-sensitive fluorescent probes are usually comprised of reaction-site modifying fluorophore and metal complex to achieve irreversible or reversible H₂S-specific reactions.^4,10,11 Time-consuming process and strictly reactive condition required in the former probe limit its applications. In contrast, the latter one with fast-response capability is more practical in the analysis of environment pollution.

Meanwhile, paper sensors have attracted widespread interest in environmental monitoring and explosive analysis,^11,12 because they have the advantages of visual detection, portability, low cost, and feasibility of on-site assay. Some traditional methods often require tedious sample-preparation steps to indirectly detect H₂S, so it is very necessary to develop a paper sensor for direct detection of H₂S. Organic dyes are usually employed to fabricate test papers for visual detection of analytes owing to their high fluorescence quantum yield and easily modified properties. Carbazole and their derivatives not only have fine optical properties and high chemical stability but also possess strong emission and absorption properties.¹³ Although some studies on carbazole-based compounds in devices have been reported,^14,15 their strong emission properties utilized as a chemosensor have attract attention just in recent years.^16,17

Herein, fluorescence probe 3-N,N-(diacethydrazide)-9-ethylcarbazole (CAH) comprised of a carbazole signaling handle and diacetyl hydrazine binding moiety was reconstructed by dual substitute and hydrazine reactions to obtain triple dentates, which could easily coordinate with metal ion due to its enough nitrogen and oxygen atoms. Probe CAH exhibited highly selective response to Cu²⁺, and its fluorescence could be strongly quenched by Cu²⁺. It is well known that Cu²⁺ can react with sulfide anion to form a very stable species CuS (K_sp = 1.27 × 10⁻³⁶).¹⁸ On basis of the fact, the coordinative Cu²⁺ can be snatched by H₂S via the formation of CuS, leading to the recovery of carbazole fluorescence. The reusability of the “on–off–on” fluorescent switch is well demonstrated by a recycling experiment. Additionally, the application in the aspect of visually detecting Cu²⁺ and gaseous H₂S with the paper sensors is clearly demonstrated.

Experimental

Reagents and instrumentation

3-Amino-9-ethylcarbazole was purchased from J & K Chemical Technology. Ethyl bromoacetate and 4-(2-hydroxyerhyl)piperazine-1-erhanesulfonic acid (HEPES) was provided by Sigma-Aldrich. Other reagents were received from Shanghai Chemicals Ltd. Commercially available compounds were used without further purification. Solvents were dried according to standard procedures. Ultrapure water (18 MΩ) was used for the preparation of all solutions. All reactions were magnetically stirred and monitored by thin-layer chromatography (TLC) using Spectrochem GF254 silica gel-coated plates. Column chromatography was performed using 200–300 mesh silica gel.

¹H NMR and ¹³C NMR spectra were recorded on Bruker 400 MHz Ultrashield spectrometer for using TMS as an internal standard. High-resolution mass spectra (HR-MS) were obtained using an Agilent Q-TOF 6540 mass spectrometer. UV-visible absorption spectra were measured by a Shimadu UV-2550 spectrometer. Fluorescence spectra were performed on Cary Eclipse fluorescence spectrophotometer. The Fourier transform infrared (FT-IR) spectra were obtained with a Thermo Scientific Nicolet iS10 spectrometer.

Synthesis of intermediate 3-N,N-(diethylacetate)-9-ethylcarbazole (2)

3-Amino-9-ethylcarbazole (0.5 g, 2.38 mmol), anhydrous potassium carbonate (1.25 g, 9.52 mmol) and potassium iodide (0.790 g, 4.76 mmol) were dissolved in 20 mL of anhydrous acetonitrile. Ethyl bromoacetate (0.795 g, 4.76 mmol) in 10 mL anhydrous acetonitrile was added dropwise and the mixture was refluxed for 12 h. The solid potassium carbonate was removed by filtration and the solvent was evaporated to give a dark brown oily liquid. The liquid was purified by column chromatography (silica gel, petroleum ether : ethyl acetate = 8 : 1) to obtained compound 3-N,N-(diethylacetate)-9-ethylcarbazole (0.66 g, 1.86 mmol, y. 73%). ¹H NMR (400 MHz, CDCl₃): δ 8.01 (d, J = 7.8 Hz, 1H), 7.42 (d, J = 2.6 Hz, 1H), 7.40 (dd, J = 7.0, 1.2 Hz, 1H), 7.33 (d, J = 8.2 Hz, 1H), 7.27 (d, J = 8.8 Hz, 1H), 7.18–7.12 (m, 1H), 6.96 (dd, J = 8.8, 2.6 Hz, 1H), 4.31–4.19 (m, 10H), 1.38 (t, J = 7.2 Hz, 3H), 1.28 (t, J = 7.1 Hz, 6H). ¹³C NMR (100 MHz, CDCl₃): 171.46, 142.01, 140.52, 134.63, 125.48, 123.58, 122.16, 118.09, 114.53, 108.96, 108.36, 105.69, 60.92, 54.78, 37.53, 14.27, 13.82. HR-MS (m/z, ESI): calculated for C₂₂H₂₆N₂NaO₄ m/z = 405.1755 [M + Na]⁺. Found m/z = 405.1790.

Synthesis of compound 3-N,N-(diacethydrazide)-9-ethylcarbazole (CAH)

3-N,N-(Diethylacetate)-9-ethylcarbazole (0.5 g, 1.31 mmol) was dissolved in anhydrous ethanol (20 mL), and then excess hydrazine hydrate (2 mL) was added to the system. The mixture was refluxed for 8 h. After cooling and standing overnight, a pale-grey solid was obtained. The solid was separately washed with n-hexane and ethanol, and dried under vacuum to gain CAH in 80% yield. ¹H NMR (400 MHz, d₆-DMSO): δ 10.05 (s, 2H), 7.96 (d, J = 7.6 Hz, 1H), 7.50 (d, J = 8.2 Hz, 1H), 7.44 (d, J = 8.9 Hz, 1H), 7.42–7.35 (m, 1H), 7.18 (d, J = 2.4 Hz, 1H), 7.11 (dd, J = 10.9, 3.9 Hz, 1H), 6.68 (dd, J = 8.9, 2.5 Hz, 1H), 4.58–4.29 (m, 6H), 4.14 (s, 4H), 1.25 (s, 3H). ¹³C NMR (100 MHz, d₆-DMSO): 170.23, 139.94, 133.05, 125.42, 121.87, 120.04, 117.74, 111.76, 109.59, 108.88, 101.80, 55.67, 13.66. HR-MS (m/z, ESI): calculated for C₁₈H₂₃N₆O₂ m/z = 355.1928 [M + H]⁺. Found m/z = 355.1882.

Fluorescence response to Cu²⁺ and H₂S

In the process of testing, the spectral determinations used the 1 cm quartz cuvettes at room temperature. The stock solution of CAH (1 and 0.5 mM) was prepared in DMSO, which was separately diluted to 30 and 5 μM with HEPES buffer (10 mM, pH = 7.4). The absorption was scanned from 250 nm to 450 nm, and the Cu²⁺ solutions (10 mM) were added to the 30 μM CAH solution (2 mL) in portions (total volumes of 0, 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 μL). The fluorescence spectra were scanned from 370 nm to 600 nm, and the Cu²⁺ solutions (1 mM) were added to the 5 μM CAH solution (2 mL) in portions (total volumes of 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24 μL). When the resultant solution was mixed thoroughly, the optical spectra were measured. The fluorescence changes of the probe in the absence/presence of Cu²⁺ were observed under a 365 nm UV lamp and the corresponding photographs were recorded with a digital camera.

CAH–Cu²⁺ prepared in situ by adding 10 mM Cu²⁺ (2 μL) to 5 μM CAH solution (2 mL) was used as the probe for next detection of H₂S. Appropriate amount of Na₂S stock solution (1 mM) was added into the above-mentioned probe solution and mixed thoroughly. The final concentrations of H₂S were calculated to be 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 20 and 22 μM, and fluorescence spectra were recorded. The fluorescence changes of the probe in the absence/presence of H₂S were observed under a 365 nm UV lamp and the corresponding photographs were recorded with a digital camera.

Recycling experiment

The fluorescence intensity was successively recorded when 0 and 2 μL of 10 mM Cu²⁺ was added to 2 mL of 5 μM CAH buffer solution, and then was recorded upon the addition of 10 mM S²⁻ (4 μL) to the same solution. The experimental process by sequentially adding Cu²⁺ and S²⁻ to the same solution was repeated five times, and the volume ratios of Cu²⁺ and S²⁻ were 0 : 0, 2 : 0, 2 : 4, 6 : 4, 6 : 8, 10 : 8, 10 : 12, 14 : 12, 14 : 16, 18 : 20 and 18 : 24 μL. The corresponding photographs of fluorescence changes of the probe were observed under a 365 nm UV lamp and recorded with a digital camera.

Preparation of paper sensor for the visual detection of Cu²⁺ and gaseous H₂S

First, several circular pieces of paper about 1 cm in diameter were separated from a common piece of filter paper by a perforator, which would be used as the substrate for immobilizing the probe. Then, 0.1 mM of the probe solution was carefully dropped onto one piece of these papers, where the solution was able to uniform distribution. After the solution was dried in air, the fluorescence indicating paper was obtained. Finally, 5 μL different concentrations of Cu²⁺ (0, 10⁻⁶, 10⁻⁵, 10⁻⁴ and 10⁻³ M) solution were separately dropped on these paper sensors. After the solution was dried, the fluorescence changes of test paper were recorded by digital camera under a 365 nm UV lamp.

A wild-mouth bottle of H₂S gas was collected, which was prepared by a reaction between Na₂S and diluted H₂SO₄. Rectangular paper strips were obtained from one common piece of filter paper by means of a sharp knife and straightedge, which were used as the substrate for immobilizing the probe. When each 10 μL of CAH–Cu²⁺ solution (0.5 mM) was separately dropped onto these pieces of paper strip, paper sensors were obtained. The prepared paper sensors were then placed in clear glass containers (300 mL in volume) for visual detection of gaseous H₂S in air. Different volumes (0, 3, 8, 13 and 24 μL) of the gas sample sucked by a micro syringe were separately injected into the same volume of containers with the paper sensors. The fluorescence changes of paper sensors were recorded by digital camera under a 365 nm UV lamp.

Results and discussion

Synthetic route and proposed mechanism

The fluorescent probe CAH was synthesized through a two-step reaction as shown in Scheme 1A. First, compound 1 was refluxed with ethyl bromoacetate in the presence of anhydrous potassium carbonate and potassium iodide for 12 h to form intermediate 2 characterized by ¹H NMR, ¹³C NMR and HR-MS (see ESI†). Subsequently, the target compound CAH was achieved by refluxing intermediate 2 with hydrazine hydrate for 8 h. The obtained compound CAH was silver-grey solid, and its structure was characterized by ¹H NMR, ¹³C NMR, FT-IR and HR-MS (see ESI†). CAH–Cu²⁺ prepared in situ by the addition of 10 μM Cu²⁺ to the solution of CAH (5 μM) was used as the probe for the next detection of S²⁻.

Scheme 1 (A) Synthetic route of probe CAH; (B) the proposed mechanism of CAH to successively detect Cu²⁺ and S²⁻.

Fluorescent probe CAH by reconstruction of fluorophore carbazole to obtain triple dentates was able to efficiently trap Cu²⁺, and its fluorescence was strongly quenched by Cu²⁺. Subsequently, Cu²⁺ could be easily snatched by S²⁻ via the formation of an extremely stable species CuS to achieve the release and fluorescence recovery of probe. Interestingly, the fluorescent “on–off–on” process could repeatedly occur by the alternate addition of Cu²⁺ and S²⁻ to the solution of probe. The most possible sensing mechanism for the detection of Cu²⁺ and S²⁻ was proposed and described in Scheme 1B.

Characterization of the probe structure

In the FT-IR spectrum of CAH (Fig. S1A†), the peak at 3432 cm⁻¹ indicated the existence of N–H bond from the acethydrazide. The overlapped broad peaks at 1650 and 1640–1618 cm⁻¹ could be attributed to the stretching vibration of C=O bond and bending vibrations of N–H bond in the acethydrazide, respectively. The peak at 1401 cm⁻¹ originated from the stretching vibration of C–N bond. Upon the formation of complex CAH–Cu²⁺, N–H bond vibration of CAH at 3432 cm⁻¹ broadened and shifted to 3347 cm⁻¹, which might due to the stretching vibration of O–H bond (Fig. S1B†). At the same time, a new peak was found at 660 cm⁻¹ which ascribed to the out-of-plane ring bend of O–H bond. Compared with the vibration of C=O at 1650 cm⁻¹, the corresponding peak of complex CAH–Cu²⁺ became weaker and shifted to 1625 cm⁻¹. The emerged strong peak at 1148 cm⁻¹ was originated from the stretching vibration of C–O bond.¹⁹ The results implied the structure changes from keto to enol form, and the oxygen atom of enol form involved in the formation of coordination bond. In addition, the new peaks at 602 and 423 cm⁻¹ confirmed the formation of Cu–O and Cu–N bond,²⁰ which further manifested compound CAH coordinating with Cu²⁺ by the nitrogen and oxygen atom. The HR-MS spectrum of CAH showed a molecular-ion peak [M + H]⁺ at m/z 355.19 (Fig. S2†). Adding Cu²⁺ into the solution, the fluorescence of CAH was rapidly quenched due to the formation of non-fluorescent complex, CAH–Cu²⁺. The mass spectrum of complex CAH–Cu²⁺ showed a prominent peak at m/z = 417.21, corresponding to [CAH–Cu²⁺] (Fig. S3A†). Successively, the addition of Na₂S to the CAH–Cu²⁺ solution was able to promptly recover its fluorescence through removal of Cu²⁺ and regeneration of probe CAH. The parent peak [(CAH–Cu²⁺) − Cu²⁺ + H]⁺ of CAH–Cu²⁺/Na₂S solution was located at m/z 355.19 (Fig. S3B†), which was equivalent to that of probe CAH. Furthermore, CAH–Cu²⁺ formed between CAH and Cu²⁺ was found to be 1 : 1 in stoichiometry, which was proved by Job's plot (Fig. S4†). All the results were able to well elucidate the mechanism of probe for the successive detection of Cu²⁺ and S²⁻.

Fluorescence turn-off detection of Cu²⁺

The fluorescent spectra of probe CAH centered at 425 nm were recorded upon the excitation wavelength of 350 nm in HEPES buffer (10 mM HEPES, 1% DMF as cosolvent, pH = 7.2) at ambient temperature. Fluorescence intensity (I₄₂₅ nm) changes of CAH (5 μM) treated with different concentrations of Cu²⁺ were monitored over time. As shown in Fig. S5,† the intensity of CAH rapidly decreased and nearly reached the minimum value within 10 min upon the addition of 5.0, 7.5 and 10 μM Cu²⁺. The fluorescence intensity of CAH did not change for 30 min, demonstrating Cu²⁺ could efficiently quench the fluorescence of probe. Therefore, the subsequent fluorescence studies were carried out after an equilibration of 10 min.

The interaction between probe CAH and Cu²⁺ was examined by UV-vis absorption spectra. As shown in Fig. 1, free CAH (30 μM) in HEPES buffer showed three absorption bands centered at 275, 308 and 375 nm, which could be assigned as n–π* and π–π* transitions.²¹ Upon the addition of Cu²⁺ (0–60 μM), the absorption peak at 308 nm was gradually decreased, and the peaks at 275 and 375 nm separately shifted to shorter wavelengths 265 and 355 nm. Meanwhile, a new absorption peak around 400 nm was observed, indicating the formation of complex CAH–Cu²⁺.

Fig. 1 UV-vis spectra of CAH (30 μM) upon the addition of Cu²⁺ (0–100 μM) in HEPES buffer solution (10 mM HEPES, 1% DMF, pH = 7.4).

Fluorescent spectra of probe CAH centered at 425 nm were recorded upon excitation wavelength of 350 nm in HEPES buffer (10 mM HEPES, 1% DMF as cosolvent, pH = 7.4) at ambient temperature. In terms of fluorescence emission, CAH emitted a bright blue fluorescence with a quantum yield of 0.40 (Φ₁, Fig. S6 and Table S1†).²² In order to evaluate Cu²⁺-responsive nature of CAH, fluorescence titration with Cu²⁺ in varying concentrations was conducted. As shown in Fig. 2A, upon the addition of Cu²⁺ with increasing amounts (0–10 μM), the fluorescence intensity of CAH was gradually diminished by the formation of complex CAH–Cu²⁺ (Φ₂ = 0.003). When 10 μM of Cu²⁺ was added to the solution of CAH, dramatic fluorescent quenching (quenching efficiency (I₀ − I)/I₀ × 100 = 98%, I₄₂₅ nm) was observed, meaning that Cu²⁺ could effectively quenched the fluorescence of CAH. Fluorescence quenching of the probe may occur by the excitation energy transfer from the ligand to the metal d-orbital and/or ligand to metal charge transfer (LMCT).²³ The decrease in emission band at 425 nm was in a good linear relationship (R² = 0.9967) between fluorescence intensity and Cu²⁺ concentration in the range from 1.0 × 10⁻⁶ to 8.0 × 10⁻⁶ M with 5 μM probe (Fig. 2B). The binding constant (K) derived from the fluorescence titration data was found to be 2.8 × 10² (R² = 0.9971, Fig. S7†) using Benesi–Hildebrand plot.²⁴ The limit of detection (LOD) for Cu²⁺ was estimated to be 65 nM in HEPES buffer based on LOD = 3δ/k (Fig. S8†),²⁵ which is below the maximum permissive level of Cu²⁺ in drinking water (20 μM) set by the U.S. Environmental Protection Agency.²⁶

Fig. 2 (A) Fluorescence titration of CAH (5 μM) with Cu²⁺ in HEPES buffer solution (10 mM HEPES, 1% DMF, pH = 7.4). The insets are the corresponding photographs of CAH in the absence and presence of Cu²⁺ under a 365 nm UV lamp. (B) The linear relationship between the fluorescence intensity and the concentration of Cu²⁺ (λ_ex = 350 nm, λ_em = 425 nm).

Selectivity of probe CAH for Cu²⁺

For a fluorescent sensor, it is very important to possess high sensitivity for Cu²⁺ over other metal ions, and still retain good selective response to Cu²⁺ even in the presence of disruptors. The fluorescence spectra of CAH (5 μM) response to various cations were examined as shown in Fig. 2 (black bars). Results demonstrated that the addition of other ions had negligible influences on the fluorescence intensity of CAH, such as Li⁺, Na⁺, K⁺, Ag⁺, Zn²⁺, Mn²⁺, Cd²⁺, Hg²⁺, Mg²⁺, Ca²⁺, Ba²⁺, Co²⁺, Al³⁺, Pb²⁺, Ni²⁺, Fe²⁺ and Fe³⁺, even if their concentrations reached up to 50 μM. In contrast, dramatic fluorescence quenching of CAH was observed upon the addition of 10 μM Cu²⁺, demonstrating that the probe had a great sensitivity and selectivity for Cu²⁺. Further interference experiments were performed to investigate whether other coexistent metal ions had significant effects on the selectivity of CAH for Cu²⁺. The results of Fig. 2 (black bars) showed no significant influence on the fluorescence intensity of probe in the presence of other cations. However, the addition of 10 μM Cu²⁺ to the above solutions of CAH with 50 μM various ions, the fluorescence intensity was almost completely quenched, as shown in Fig. 2 (red bars). It was consistent with the phenomenon generated by the separate addition of 10 μM Cu²⁺, implying that CAH had a better sensitivity for Cu²⁺ than other ions. Therefore, the results mentioned-above demonstrated that CAH was able to serve as a potential chemsensor for Cu²⁺ with high selectivity and anti-interference performance (Fig. 3).

Fig. 3 Fluorescence responses of CAH (5 μM) to various cations in HEPES buffer solution (10 mM HEPES, 1% DMF, pH = 7.4). Black bars represent intensity changes of CAH upon the addition of Cu²⁺ (10 μM) and various cations (50 μM), and red bars represent intensity changes of the above solution upon the subsequent addition of 10 μM Cu²⁺ (λ_ex = 350 nm, λ_em = 425 nm).

Fluorescent turn-on detection of S²⁻

Time-dependent changes in the fluorescence intensity (I₄₂₅ nm) of CAH–Cu²⁺ (5 μM) were separately monitored in the presence of 10, 15 and 20 μM S²⁻ as shown in Fig. 4. After the addition of S²⁻, the fluorescence of probe immediately recovered and reached a maximum within 1 min. The recovered fluorescence intensity at 425 nm still kept stable and constant for 30 min. Together with the stability of CAH–Cu²⁺ and CAH in HEPES buffer solution, it is feasible and applicable for probe to detect S²⁻ in the environment.

Fig. 4 Time-dependent fluorescence intensity (λ_ex = 350 nm, λ_em = 425 nm) change of CAH–Cu²⁺ (5 μM) with S²⁻ (5, 10 and 20 μM) in HEPES buffer solution (10 mM HEPES, 1% DMF, pH = 7.4).

The fluorescence spectra of CAH–Cu²⁺ upon the addition of different concentrations of S²⁻ were obtained. As the increasing concentrations of S²⁻ (0–20 μM) were added, the fluorescence intensity (I₄₂₅ nm) of probe was gradually recovered from dark to blue (Fig. 5A). The fluorescence intensity increased in a S²⁻ concentration-dependent way as displayed in Fig. 5B. The enhancement of intensity (I₄₂₅ nm) was in a good linear relationship (R² = 0.9984) between fluorescence intensity and the concentrations of S²⁻ in the range from 0 to 1.5 × 10⁻⁵ M with the detection limit of 0.29 μM in HEPES buffer (Fig. S9†). The results proved that the probe had a high sensitivity for the detection of S²⁻.

Fig. 5 (A) Fluorescence titration of CAH–Cu²⁺ (5 μM) with S²⁻ in HEPES buffer solution (10 mM HEPES, 1% DMF, pH = 7.4). The insets are the corresponding photographs of CAH–Cu²⁺ in the absence and presence of S²⁻ under a 365 nm UV lamp. (B) The linear relationship between the fluorescence intensity and the concentration of S²⁻ (λ_ex = 350 nm, λ_em = 425 nm).

Selectivity of probe CAH–Cu²⁺ for S²⁻

The fluorescence responses of CAH–Cu²⁺ to other anions and biothiol, such as F⁻, Cl⁻, SCN⁻, NO₂⁻, H₂PO₄⁻, NO₃⁻, SO₄²⁻, HPO₄²⁻, Br⁻, S₂O₃²⁻, I⁻, HCO₃⁻, AcO⁻, PPi, GSH, Cys and Hcy were also examined (Fig. 6, black bars). Obviously, other anions had slight influences on the fluorescence intensity compared with S²⁻ even if the concentrations of these anions reached up to 50 μM, indicating that probe CAH–Cu²⁺ possessed a predominant selectivity for S²⁻ over other anions. Although probe CAH–Cu²⁺ separately gave about 2-fold, 5-fold, 8-fold and 7-fold fluorescence enhancement for PPi, GSH, Cys and Hcy, there were more than 31-fold fluorescence enhancements when the system was treated with S²⁻. The result manifested probe CAH–Cu²⁺ was capable of distinguishing S²⁻ from PPi, GSH, Cys and Hcy. Additionally, it was very important to explore whether CAH–Cu²⁺ could still retain highly sensitive response to S²⁻ in the presence of other interfering species. The interference experiments were carried out in the presence of other potential coexisting ions at relatively high concentration. Fig. 6 (black bars) showed that the probe was non-responsive to other anions, and just a minor fluorescence enhancement was induced in the presence of PPi, GSH, Cys and Hcy. Whereafter, the fluorescence of probe recovered immediately upon the addition of S²⁻ to the above solution, which is consistent with the result generated by the separate addition of S²⁻ (Fig. 6, red bars). The results implied that the probe CAH–Cu²⁺ could identify S²⁻ over other anions and biothiols with high sensitivity and selectivity in assay conditions.

Fig. 6 Fluorescence responses of CAH–Cu²⁺ (5 μM) to various specieses in HEPES buffer solution (10 mM HEPES, 1% DMF as cosolvent, pH = 7.4). Black bars represent fluorescence intensity of CAH–Cu²⁺ upon the addition of 20 μM S²⁻ and various specieses (20 μM PPi, GSH, Cys and Hcy; 50 μM other anions); red bars represent intensity changes of the above solution that occur upon the subsequent addition of 20 μM S²⁻ (λ_ex = 350 nm, λ_em = 425 nm).

Effect of pH

The fluorescence intensity of probes CAH and CAH–Cu²⁺ in various pH values was measured, because a good stability of probe in suitable pH values is very important for its practical applications. As shown in Fig. 7, the fluorescence of CAH was weak in the acidic environment due to the protonation of amino group, leading to a weak coordination ability of Cu²⁺. When the pH was increased from 6 to 9, satisfactory fluorescence intensity of CAH and Cu²⁺-quenching abilities were exhibited. At pH about 7, the fluorescence intensity of CAH–Cu²⁺ reached its minimum value, indicating probe possessed the highest sensing ability in physiological pH. Probe CAH–Cu²⁺ in weak acidic and near neutral media is favorable for sensing assay of S²⁻ in environment.

Fig. 7 Fluorescence intensity of 5 μM CAH (), CAH + 10 μM Cu²⁺ () and CAH–Cu²⁺ + 20 μM S²⁻ () at various pH in HEPES buffer solution (10 mM HEPES, 1% DMF as cosolvent).

Reusability and application of probe

On the basis of the fact that the fluorescence of probe CAH was quenched and recovered by adding Cu²⁺ and S²⁻, the reusability of fluorescence system was investigated by repeating the processes of binding and release. As shown in Fig. 8, the fluorescence could be turn off and on with alternate addition of Cu²⁺ and S²⁻, the recovered fluorescence intensity could roughly keep consistent even recycled for 5 times. The fluorescence changes of probe in the absence and presence of Cu²⁺/S²⁻ were separately observed, and the corresponding photographs (Fig. 8) were recorded. The mentioned-above results implied that the fluorescent switch could be developed as a regeneratable fluorescence sensor: “on–off” type for Cu²⁺and “off–on” type for S²⁻.

Fig. 8 Fluorescence intensity of 5 μM CAH (2 mL) upon the alternate addition of Cu²⁺–S²⁻ solution (10 mM) with several volume ratios (0 : 0, 2 : 0, 2 : 4, 6 : 4, 6 : 8, 10 : 8, 10 : 12, 14 : 12, 14 : 16, 18 : 20 and 18 : 24 μL) in HEPES buffer solution (10 mM HEPES, 1% DMF as cosolvent, pH = 7.4). The insets are the corresponding photographs taken at a 365 nm UV lamp.

To achieve visual detection of Cu²⁺ in aqueous solution, a paper-based sensor made of filter paper has been developed. First, 2.0 μL of probe solution (0.1 mM) was carefully dropped onto a piece of filter paper. After the solution was dried in air, the fluorescence indicating paper was obtained, forming a spot about 1 cm in diameter. Then, different concentrations of Cu²⁺ were dropped on the test paper. As shown in Fig. 9A, we could see an obvious brightness change from blue to dark under a UV lamp. The detection limit for Cu²⁺ was about 10⁻⁶ M. Probe CAH–Cu²⁺ was capable of fast response to S²⁻, which prompted us to expect feasibility of directly detecting gaseous H₂S. To conveniently and efficiently detect gaseous H₂S, a paper sensor was fabricated by dropping 10 μL of 0.5 mM probe solution on a piece of paper. Such prepared paper sensors were exposed to gas samples containing different amounts of H₂S for 1 min, and then illuminated under a UV lamp. It was obvious that the fluorescence brightness changes gradually became clear with increasing concentrations of H₂S, and the visual detection limit was about 15 ppm as shown in Fig. 9B. The result demonstrated that the paper sensor immobilized with probe CAH–Cu²⁺ was able to serve as a simple way to direct and visual detection of gaseous H₂S. Furthermore, the reusability of paper sensor was verified by successively adding Cu²⁺ (10⁻³ M) and being exposed in gaseous H₂S (120 ppm) for 2 times (Fig. S10†). The result implied that the fluorescent switch could also be developed as a regeneratable paper sensor: “on–off” type for Cu²⁺ and “off–on” type for gaseous H₂S.

Fig. 9 Visual detection of Cu²⁺ and gaseous H₂S under illumination of a 365 nm UV lamp in the dark. (A) The fluorescence image set of CAH spot upon the addition of different concentrations of Cu²⁺. (B) The paper sensors were exposed in gaseous H₂S with different concentrations.

Conclusions

In summary, a carbazole-based fluorescence probe to present an “on–off–on” type fluorescence response mode for sequential detection of Cu²⁺ and S²⁻ has been developed. High selectivity and sensitivity of probe response to Cu²⁺ are barely affected by coexistence of other interfering analytes. The subsequent addition of S²⁻ can effectively snatch Cu²⁺ from the complex CAH–Cu²⁺ to immediately recover the quenched fluorescence by releasing free probe. The detection limits of Cu²⁺ and S²⁻ can reach 65 and 290 nM, which are lower than that of most probes. The good reusability of the fluorescent sensor for cyclical detection of Cu²⁺ and S²⁻ is fully demonstrated by its recycling experiment. Additionally, paper sensors were applied to visually detect Cu²⁺ and gaseous H₂S with the limit of 10⁻⁶ M and 15 ppm.

Acknowledgements

This work was supported by National Basic Research Program of China (2015CB932002), China-Singapore Joint Project (2015DFG92510), National Natural Science Foundation of China (No. 21335006, 21475135, 21375131 and 21277145), Science and Technology Service Network Initiative (KFJ-SW-STS-172) and Natural Science Foundation of Anhui Province (1408085MKL52).

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: UV/Vis absorption, detection limit, fluorescence quantum yield and other additional data, ¹H NMR, ¹³C NMR, and HR-MS. See DOI: 10.1039/c6ra10293j

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