A turn-on fluorescent chemosensor based on acylhydrazone for sensing of Mg²⁺ with a low detection limit

Abstract

A novel highly selective chemosensor for Mg²⁺ ions based on the naphthalene group as the fluorophore has been designed and synthesized, which shows a fluorescence turn-on response from colorless to green for Mg²⁺ ions in DMSO–H₂O solutions. L could perform as an “OFF–ON” molecular switch, which was cyclically controlled by Mg²⁺ and EDTA. Moreover, further study demonstrated that the detection limit for the fluorescence response of L to Mg²⁺ was down to 9.2 × 10⁻¹⁰ M, which was lower than with other sensors for Mg²⁺. Test strips based on L were also fabricated, which could act as a convenient and efficient Mg²⁺ test kit. The sensor L could detect Mg²⁺ in two different sources of water, distilled and tap water.

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Introduction

The magnesium ion (Mg²⁺) plays vital roles in biological systems.^1–3 For plants, magnesium is one of the ingredients for forming chlorophyll and is involved with forming phosphate compounds and phosphate metabolism.^4,5 In the cells of the human body, Mg²⁺ is one of the most abundant cations, and participates in numerous basic biochemical reactions. Magnesium also plays a crucial role in skeletal development and bone remodeling.^6,7 Magnesium ion can influence nervous impulses and tension development in muscle, and modulates, amongst others, ion transport in nerves and mitochondria.^8–10 If the concentrations of magnesium in the cytosol and subcellular regions are abnormal, there is a possibility of the occurrence of a disease, such as diabetes, hypertension, epilepsy and Alzheimer's disease.^11–13 In particular, detection of Mg²⁺ has been of considerable interest and a great amount of effort has been devoted to the design and synthesis of sensitive and selective sensors for Mg²⁺ ion.

Nonetheless, reports about fluorescent sensors for Mg²⁺ are still few compared with those on other important ions, such as Zn²⁺, Al³⁺, Hg²⁺ and CN⁻.^14–27 Even so, some methodologies for detecting Mg²⁺ are often interfered with by Zn²⁺ and Ca²⁺. To date, as far as we know, β-diketone, crown ether derivatives, and nanoparticles have been employed for selective detection of Mg²⁺ ions.^28–30 It is worth mentioning that their synthetic processes are intricate. Hence, there is interest in designing a highly selective, sensitive and simple chemosensor that recognizes Mg²⁺ without the interference of other metal ions.^31,32

Our research group has a longstanding interest in molecular recognition.^33–38 We have successfully developed a simple and efficient optical fluorescent acylhydrazone chemosensor L that can detect Mg²⁺ in DMSO–H₂O solution. To the best of our knowledge, acylhydrazone is easy and inexpensive to prepare. The naphthalene group acts as the fluorophore, while the hydroxyl and imine groups in the sensor molecule L enhance the coordination capacity. As a result, the addition of Mg²⁺ inhibits the ESIPT process and increases its structural rigidity, which leads to a strong green emission. The mechanism of this process was verified by spectroscopic methods, including ¹H NMR, DFT and mass spectrometry. The fluorescent detection limit of the sensor for Mg²⁺ was 9.2 × 10⁻¹⁰ M, while the corresponding detection limit by the naked eye was determined to be 5.0 × 10⁻⁸ M under a UV lamp at 365 nm.

Results and discussion

The molecule L was conveniently synthesized via the condensation of 3,4,5-trihydroxybenzhydrazide and 2-hydroxy-1-napthaldehyde. The recognition abilities of L were investigated by adding perchlorate salts (Fe³⁺, Hg²⁺, Ag⁺, Ca²⁺, Cu²⁺, Co²⁺, Ni²⁺, Cd²⁺, Pb²⁺, Zn²⁺, Cr³⁺ and Mg²⁺) into DMSO–H₂O solutions (Scheme 1).

Scheme 1 Synthesis of receptor L.

Receptor L was found to have limited solubility in water, and this compelled us to use this sensor in mixed solvents. The fluorescence emission was examined upon adding various metal ions: Fe³⁺, Hg²⁺, Ag⁺, Ca²⁺, Cu²⁺, Co²⁺, Ni²⁺, Cd²⁺, Pb²⁺, Zn²⁺, Cr³⁺ and Mg²⁺ ions in DMSO/H₂O (7 : 3, v/v, 0.01 M HEPES, pH = 8.5). Only compound L displayed a weak, single fluorescence emission band at 480 nm when excited at 378 nm in DMSO–H₂O media. Changes in the spectral pattern were observed only with the addition of 20 equivalents of Mg²⁺, when sensor L produced a strong fluorescence band at 480 nm and responded with a dramatic color change, from colorless to green. However, upon addition of other metal ions no significant changes in color were observed (Fig. 1). In addition, the fluorescence emission was also examined upon adding MnCl₂ and CaCl₂ (Fig S3†). The addition of Mn²⁺ caused no significant spectral change. The absorption spectra of L in the presence of other ions were also tested. The results suggested that sensor L could display an excellent selectivity for Mg²⁺ over all other ions tested (Fig S4†).

Fig. 1 Fluorescence spectra response of L (2.0 × 10⁻⁵ M) in DMSO/H₂O (7 : 3, v/v, 0.01 M HEPES, pH = 8.5) upon addition of Mg²⁺. Inset: photograph in the presence of various cations under UV-lamp (365 nm).

It was well known that fluorescent probes for Mg²⁺ might encounter interference from other cations, particularly Zn²⁺ and Cd²⁺. Thus, competitive behavior was tested to further elucidate whether the coexistence of competing metal cations interfered with the detection of Mg²⁺. In the solutions of sensor L with Mg²⁺, when solutions of L were added to solutions of other competing cations, it was clear that the receptor L was highly selective for the detection of Mg²⁺ in DMSO/H₂O (7 : 3, v/v, 0.01 M HEPES, pH = 8.5) (Fig. 2).

Fig. 2 Fluorescence spectra response of L (2.0 × 10⁻⁵ M) in the presence of various cations in DMSO/H₂O (7 : 3, v/v, 0.01 M HEPES, pH = 8.5) in response to Mg²⁺.

As shown in Fig. 3, the fluorescence titration of Mg²⁺ was performed using a 20 μM solution of L in DMSO/H₂O (7 : 3, v/v, 0.01 M HEPES, pH = 8.5). With the addition of Mg²⁺ (0.2 M), a gradual increase in the fluorescence intensity of chemosensor L at 480 nm was observed as the Mg²⁺ volume increased from 0 to 1.7 equivalents. The solution containing Mg²⁺ also showed a change in fluorescence color from colorless to grass green.

Fig. 3 Fluorescence titration spectra of L (2.0 × 10⁻⁵ M) in DMSO/H₂O (7 : 3, v/v, 0.01 M HEPES, pH = 8.5) upon adding of an increasing concentration of Mg²⁺ (λ_ex = 378 nm). Inset: photograph of L upon adding of Mg²⁺ under UV-lamp (365 nm).

The detection limit is one of the most important parameters in ion sensing. For various practical purposes, it is very important to detect analytes at low concentrations. The naked eye detection limit for Mg²⁺ was determined to be 5.0 × 10⁻⁸ M, through use of a UV lamp at 365 nm (Fig. 4). The detection limit for fluorescent spectrum changes was 9.2 × 10⁻¹⁰ M for Mg²⁺, as calculated on the basis of 3δ/S (where δ is the standard deviation of the blank solution and S is the slope of the calibration curve), which indicates the high detection sensitivity (Fig. 5). The result of the analysis is as follows: linear calibration equation: I = 1.30432C + 53.84714, R² = 0.997.

Fig. 4 Naked-eye detection limit under UV light at 365 nm. From left to right the concentration of Mg²⁺: 10⁻⁵ M, 5 × 10⁻⁶ M, 5 × 10⁻⁷ M, 5 × 10⁻⁸ M, 5 × 10⁻⁹ M.

Fig. 5 Fluorescence detection limit spectra of L (2.0 × 10⁻⁵ M) in DMSO/H₂O (7 : 3, v/v, 0.01 M HEPES, pH = 8.5) solution upon adding of a concentration of Mg²⁺.

We also made comparisons with other reported magnesium selective sensors, and the detection limit of L was much lower.^29–31 Some did not explain the detection limits. This indicated that probe L could be used to quantitatively detect Mg²⁺ at extremely low concentrations and in a relatively wide range (Table 1).

Table 1 Comparison of detection limits

Journal	Structures	Detection limits (fluorometric)	Solvent
This work		9.2 × 10^−10 M	DMSO/H2O (7 : 3, v/v)
39		5.16 × 10^−7 M	Ethanol
40		1.44 × 10^−6 M	Acetonitrile
41		2.37 × 10^−8 M	Deionized water

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The pH dependence of the probe L system in DMSO/H₂O (7 : 3, v/v, 0.01 M HEPES, pH = 8.5) was also checked by fluorescence emission spectroscopy. Mg²⁺ ion was added to buffered solutions of L at different pH values (1–12). The probe L was insensitive to H⁺ and OH⁻. However, the results with the L–Mg²⁺ system in DMSO–H₂O media indicated that the reaction of L (2.0 × 10⁻⁵ M) with Mg²⁺ only occurred effectively in the pH range between 8 and 11, where the fluorescence intensity at 480 nm was enhanced more significantly, which indicated that the sensor L and Mg²⁺ ions and formed a magnesium complex in weak alkaline solutions (Fig. S5†).

According to ¹H NMR spectra (Fig. 6), the addition of Mg²⁺ made the two proton signals of –OH and –NH at 12.96 (s, 1H) and 11.92 (s, 1H) ppm gradually disappear. Due to coordination of the O atom of the carbonyl group in L, the signal of the two hydroxyl atoms at 9.29 (s, 2H) ppm in the benzene ring showed a significant downfield shift. To further investigate the interaction between the sensor L and Mg²⁺, IR spectra were collected, and the stretching vibration peak of (–C=O) in L at 1605 cm⁻¹ shifted to 1619 cm⁻¹ when L interacted with Mg²⁺ (Fig. S6†). These phenomena indicate that in L–Mg²⁺, the Mg²⁺ coordinated with the oxygen atoms on the acylhydrazone group.

Fig. 6 ¹H NMR spectra of free L and in the presence of Mg²⁺.

An obtained mass spectrum confirmed that the sensor L ion peak was detected at m/z 339.08, which corresponded to [L + H]⁺, and the magnesium complex ion peak appeared at m/z 439.09, which indicated that after deprotonation, probe L reacted with Mg²⁺ and one DMSO to form a stable complex [L − 2H⁺ + Mg²⁺ + 1DMSO +1H⁺]⁺ (Fig. S7†). A job plot analysis showed a 1 : 1 stoichiometry for the L–Mg²⁺ complex (Fig. 7). Based on the data in the job plot, ¹H NMR spectra and IR spectra, a stable complex between L and Mg²⁺ was proposed.

Fig. 7 Job plot of L with Mg²⁺.

The sensing mechanism was estimated based on the above experiments. In solution, L exhibited weak fluorescence owing to ESIPT. The addition of Mg²⁺ broke the intramolecular hydrogen bond and deprotonated the secondary amine. Hence, excited state intramolecular proton transfer was inhibited. The sensor L showed strong green fluorescence (Scheme 2).

Scheme 2 A possible sensing mechanism of the sensor L with Mg²⁺.

In order to investigate the reversibility of the sensor L, the addition of Mg²⁺ to a solution of sensor L that had no fluorescence showed increasing fluorescence (That was “ON”). After adding of EDTA to the solution of L–Mg²⁺ complex, there was no fluorescence (That was “OFF”). In fact, this “OFF–ON–OFF” switching process could be repeated several times with little fluorescent efficiency loss (Fig. 8).

Fig. 8 Fluorescence experiment showing the reversibility and reusability of the L for sensing Mg²⁺. Emission intensity changes at 480 nm for L upon addition of 10 equiv. Mg²⁺ before and after addition of 10 equiv. EDTA.

To further confirm the proposed mechanism of sensor L with Mg²⁺, we performed DFT calculations at the B3LYP/6-311g (2d, p) level of theory. The highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) of L and the L + Mg²⁺ complex were investigated and are shown in Table 2. The HOMO–LUMO energy band gap of L + Mg²⁺ complex was 0.12157 au, which was lower than that of L (0.13522 au).

Table 2 Density functional theory results for L and L + Mg^2+a

	L	L + Mg^2+
a Blue ball is nitrogen, grey ball is carbon, red ball is oxygen, yellow ball is magnesium, and white ball is hydrogen.
Optimized structure
Diagrams of LUMOs
Diagrams of HOMOs
Energy gaps	0.13522 au	0.12157 au

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To investigate the practical application of chemosensor L, test strips were prepared by immersing filter papers into a DMSO/H₂O (7 : 3, v/v, 0.01 M HEPES, pH = 8.5) solution of L (0.1 M) and then drying in air. The test strips containing L were utilized to sense Mg²⁺ and other metal ions. As shown in Fig. 9, when the test strips were added to solutions of Mg²⁺ and the other ions, a significant color change was observed only with Mg²⁺ solution under a 365 nm UV lamp. Moreover, potentially competitive ions exerted no influence on the detection of Mg²⁺ by the test strips. Therefore, the test strips based on L could quickly and conveniently detect Mg²⁺ in solutions. The practical application of sensor L for selective sensing of Mg²⁺ in two different sources of water, distilled and tap water, has also been demonstrated. The results showed that sensor L was a sensitive sensor and could be applied in environmental analysis (Fig. 10).

Fig. 9 Photographs of L on test strips (a) only L, (b) after immersion into solutions with Mg²⁺, (c) after immersion into DMSO solutions with other cations, (d) after immersion into solutions with Mg²⁺ and other cations under irradiation at 365 nm.

Fig. 10 Images of Mg²⁺ induced turn-on fluorescence in different sources of water.

Conclusions

In summary, we have developed a sensor that can detect Mg²⁺ ions in a weakly alkaline medium of DMSO/H₂O (7 : 3, v/v, 0.01 M HEPES, pH = 8.5) with high selectivity and special sensitivity. Moreover, the fluorescence detection limit was low 9.2 × 10⁻¹⁰ M for Mg²⁺. In addition, L could be used as a novel NOR logic gate triggered by Mg²⁺ and EDTA. Test strips based on L were also fabricated, which could serve as a practical fluorescent sensor to detect Mg²⁺ in field measurements or in test kits. Thus, the probe could have potential applications in both environmental and biological systems for the monitoring of magnesium.

Acknowledgements

This study was supported by the National Nature Science Foundation of China (No. 21467012).

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Footnote

† Electronic supplementary information (ESI) available: Complete experimental procedures and some of the spectroscopic. See DOI: 10.1039/c7ra04462c

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