A highly sensitive and selective fluorescent probe for quantitative detection of Al³⁺ in food, water, and living cells

Abstract

Three novel β-pinene-based fluorescent probes 2a–2c were designed and synthesized for the selective detection of Al³⁺. Probe 2a showed higher fluorescence intensity toward Al³⁺ than the other two compounds. Probe 2a determined the concentration of Al³⁺ with a rapid response time (45 s), wide pH range (pH = 1–9), excellent sensitivity (LOD = 8.1 × 10⁻⁸ M) and good selectivity. The recognition mechanism of probe 2a toward Al³⁺ was confirmed by ¹H NMR, HRMS and DFT analysis. Probe 2a was successfully used as a signal tool to quantitatively detect Al³⁺ in food samples and environmental water samples. Furthermore, probe 2a was successfully utilized to label intracellular Al³⁺, indicating its promising applications in living cells.

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1. Introduction

As the third most abundant element and the most widely used metal ion on Earth, aluminum is extensively used in a variety of fields, but is harmful to the environment and living systems.^1–4 The increasing concentration of Al³⁺ in pollutants can deeply influence the growth of plants, and lead to soil acidification^5,6 and underground water contamination.⁷ Moreover, the accumulation of Al³⁺ in the body can greatly affect the absorption of calcium in bone tissue and induce several diseases such as Alzheimer's, Parkinson's epilepsy, seizures, and renal and liver damage.^8–11 In 1989, the World Health Organization (WHO) and the United Nations Food and Agriculture Organization (UNFAO) identified Al³⁺ as a food pollutant to be controlled. In 2011, WHO/UNFAO revised the weekly allowable intake of Al³⁺ from 7 mg kg⁻¹ to 2 mg kg⁻¹.¹² Therefore, it is significant to detect and control the Al³⁺ concentration in water and food samples.

Compared to traditional detection methods, a fluorescent probe has become one of the most widely used tools for detecting metal ions.^13–15 In the past few years, many fluorescent probes for Al³⁺ detection have been reported.^16–25 However, there are still some shortcomings in reported Al³⁺ fluorescent probes, including a complex synthesis process, poor selectivity and sensitivity, easy interference by other metal ions, Zn²⁺, Cu²⁺, Cr³⁺, Hg²⁺, and even F⁻,^26–30 and lack of applicability to water samples and food samples.^31–35 Therefore, developing a fluorescence probe with high sensitivity, good selectivity, rapid response and low toxicity for detecting Al³⁺ is particularly meaningful.

Nopinone is obtained by the oxidation of β-pinene, which is a primary ingredient in natural turpentine. It is often used in the production of medicine and perfume. In addition, the rigid structure of the nopinone molecule can reduce the energy loss of non-radiative transitions in the fluorescence emission process. Furthermore, molecules with a nopinone skeleton structure have good biological compatibility and low cytotoxicity.³⁶ Thus, the development of novel fluorescence probes from nopinone is very promising.

In this paper, we synthesized three new indazole derivatives for the specific detection of Al³⁺, using natural nopinone as the starting material. Probes 2a–2c could be synthesized by a simple two-step reaction and displayed a rapid ratiometric fluorescence toward Al³⁺ in aqueous solution (pH = 7.4). In addition, probe 2a had a high selectivity for Al³⁺ over other metal ions and a rapid response time. The detection limit of probe 2a was found to be 8.1 × 10⁻⁸ M, which is lower than that of many reported Al³⁺ fluorescence probes. The ¹H NMR, HRMS and theoretical calculations showed the detection mechanism. Furthermore, probe 2a was successfully proved for the quantitative detection of Al³⁺ in food and environmental water samples. More importantly, cell experiments also demonstrated that probe 2a could be used as a signal tool to detect the concentration of Al³⁺ in living cells.

2. Experimental

2.1. General information

All reagents and solvents were of analytical grade and bought from commercial sources. UV-Vis absorption spectra were recorded by a PerkinElmer Lambda 950. PL emission spectra were tested using PerkinElmer LS55. The ¹H NMR and ¹³C NMR spectra were recorded in CDCl₃ solutions on a Bruker AV 400 spectrometer. The purity of the synthesized compounds was recorded by America Agilent 1260 Infinity liquid chromatography. All pH measurements were recorded using a Sartorius Basic pH-Meter PB-20. High-resolution mass spectra (HRMS) were tested by an America Agilent 5975c mass spectrometer. Melting points were recorded using an X-6 microscopic melting point apparatus. All measurements were performed at room temperature. The 5.0 × 10⁻⁵ M solutions of various metal ions (Ag⁺, Hg²⁺, Na⁺, Mg²⁺, K⁺, Ca²⁺, Fe²⁺, Fe³⁺, Co²⁺, Ni²⁺, Cu²⁺, Zn²⁺, Ba²⁺, Sn²⁺, Pb²⁺, Cr²⁺, Pb²⁺, Bi³⁺ and Al³⁺) were prepared in deionized water from their nitrate, chloride or sulfate, and stored at room temperature.

2.2. Synthesis

2.2.1. Synthesis of compounds 1a–1c. Under nitrogen atmosphere, nopinone (2 mol) was aroylated with a–c (3 mol) catalyzed by NaH (6 mol) in 1,2-dimethoxyethane. After cooling to room temperature, 20 ml of distilled water was added to the reaction mixture, and it was extracted with ethyl acetate. The ethyl acetate phase was washed with distilled water to neutrality. After recovering the solvent, the obtained crude 1a was purified by column chromatography (100–200 mesh silica gel, eluent: PE : EA = 10 : 1, v/v) to obtain compounds 1a–1c.

Compound 1a was a pale yellow grease, yield: 71.3%; ¹H NMR (400 MHz, CDCl₃) δ: 15.58 (s, 1H), 8.68 (dd, J = 4.9, 1.7 Hz, 1H), 8.00 (d, J = 8.0 Hz, 1H), 7.88–7.72 (m, 1H), 7.35–7.30 (m, 1H), 3.28–2.95 (m, 2H), 2.59 (dt, J = 22.5, 5.4 Hz, 2H), 2.32 (qt, J = 5.9, 2.6 Hz, 1H), 1.49 (d, J = 9.9 Hz, 1H), 1.35 (s, 3H), 0.96 (s, 3H); ¹³C NMR (100 MHz, DMSO) δ: 153.35, 148.51, 137.18, 127.72, 124.90, 123.41, 121.44, 105.43, 57.40, 54.31, 48.29, 28.34, 27.26, 25.41, 21.18; HRMS (m/z): [M + H]⁺ calculated for C₁₅H₁₇NO₂ + H⁺, 244.1341; found, 244.1335.

Compound 1b was a pale yellow grease, yield: 73.8%; ¹H NMR (400 MHz, CDCl₃) δ: 15.41 (s, 1H), 8.95 (dd, J = 2.2, 0.9 Hz, 1H), 8.67 (dd, J = 4.9, 1.7 Hz, 1H), 8.03 (dt, J = 8.0, 2.0 Hz, 1H), 7.40 (ddd, J = 7.9, 4.8, 0.9 Hz, 1H), 2.71 (dd, J = 4.0, 3.0 Hz, 2H), 2.65–2.53 (m, 2H), 2.32 (tt, J = 5.9, 3.1 Hz, 1H), 1.47 (d, J = 9.5 Hz, 1H), 1.36 (s, 3H), 0.97 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ: 209.55, 169.51, 150.69, 149.01, 135.67, 131.29, 123.24, 104.89, 54.84, 39.77, 39.61, 28.07, 27.66, 25.79, 21.54; HRMS (m/z): [M + H]⁺ calculated for C₁₅H₁₇NO₂ + H⁺, 244.1338; found, 244.1331.

Compound 1c was a pale yellow grease, yield: 76.5%; ¹H NMR (400 MHz, CDCl₃) δ: 15.26 (s, 1H), 8.78–8.67 (m, 2H), 7.63–7.52 (m, 2H), 2.77–2.64 (m, 2H), 2.68–2.54 (m, 2H), 2.32 (tt, J = 6.0, 3.1 Hz, 1H), 1.47 (d, J = 9.8 Hz, 1H), 1.36 (s, 3H), 0.97 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ: 209.98, 168.74, 150.06, 142.47, 122.14, 105.30, 54.92, 39.65, 39.62, 27.95, 27.60, 25.78, 21.55; HRMS (m/z): [M + H]⁺ calculated for C₁₅H₁₇NO₂ + H⁺, 244.1339; found, 244.1332.

2.2.2. Synthesis of 2a–2c. 1a–1c (0.5 mmol), 60% N₂H₄·H₂O (1 mmol), and 10 ml of absolute ethanol were added into a round-bottom flask equipped with a thermometer and reflux condenser. The cyclization reaction was carried out at reflux for 4 h. After recovering the ethanol, the obtained crude 2a–2c was purified by column chromatography (100–200 mesh silica gel, eluent: PE : EA = 15 : 1, v/v) to obtain compounds 2a–2c.

Compound 2a was a white solid powder, yield: 85.6%, mp: 146.2–147.2 °C; ¹H NMR (400 MHz, DMSO) δ: 12.59 (s, 1H), 8.61 (d, J = 4.8 Hz, 1H), 7.96–7.58 (m, 2H), 7.29 (t, J = 6.3 Hz, 1H), 3.00 (dd, J = 16.3, 3.0 Hz, 1H), 2.89 (dd, J = 16.3, 2.6 Hz, 1H), 2.80 (t, J = 5.2 Hz, 1H), 2.71 (dt, J = 9.3, 5.8 Hz, 1H), 2.29 (tt, J = 5.6, 2.8 Hz, 1H), 1.39 (s, 3H), 1.27 (d, J = 9.3 Hz, 1H), 0.65 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ: 160.79, 149.58, 149.04, 137.13, 136.77, 122.19, 120.15, 110.67, 41.96, 41.30, 41.07, 32.52, 26.36, 26.32, 21.49; HRMS (m/z): [M + H]⁺ calculated for C₁₅H₁₇N₃ + H⁺, 240.1501; found, 240.1494.

Compound 2b was a white solid powder, yield: 82.4%, mp: 150.5–151.5 °C; ¹H NMR (400 MHz, DMSO) δ: 12.61 (s, 1H), 8.94 (d, J = 2.3 Hz, 1H), 8.51 (dd, J = 4.8, 1.6 Hz, 1H), 8.07 (dt, J = 8.0, 2.0 Hz, 1H), 7.48 (dd, J = 8.0, 4.8 Hz, 1H), 2.98 (dd, J = 15.8, 3.1 Hz, 1H), 2.91–2.80 (m, 2H), 2.71 (dt, J = 9.3, 5.8 Hz, 1H), 2.31 (dt, J = 5.7, 2.9 Hz, 1H), 1.39 (s, 3H), 1.29 (d, J = 9.3 Hz, 1H), 0.66 (s, 3H); ¹³C NMR (100 MHz, CHCl₃) δ: 157.91, 148.11, 146.80, 137.91, 132.80, 128.07, 123.70, 110.22, 58.08, 41.41, 41.14, 32.37, 26.23, 26.01, 21.46; HRMS (m/z): [M + H]⁺ calculated for C₁₅H₁₇NO₂ + H⁺, 240.1504; found, 240.1497.

Compound 2c was a white solid powder, yield: 87.1%, mp: 157.1–158.1 °C; ¹H NMR (400 MHz, DMSO) δ: 12.78 (s, 1H), 8.61 (d, J = 5.1 Hz, 2H), 7.65 (d, J = 5.2 Hz, 2H), 3.00 (dd, J = 15.9, 3.1 Hz, 1H), 2.93–2.80 (m, 2H), 2.72 (dt, J = 9.4, 5.8 Hz, 1H), 2.32 (dt, J = 5.7, 2.9 Hz, 1H), 1.40 (s, 3H), 1.28 (d, J = 9.4 Hz, 1H), 0.64 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ: 157.96, 150.07, 139.12, 119.83, 111.60, 41.39, 41.33, 41.14, 32.36, 26.25, 26.21, 21.46, 18.39; HRMS (m/z): [M + H]⁺ calculated for C₁₅H₁₇NO₂ + H⁺, 240.1498; found, 240.1496.

2.3. Cell culture and imaging

HeLa cells were incubated with probe 2a (5.0 × 10⁻⁶ M) in Dulbecco's Modified Eagle's Medium (DMEM) for 24 h at 37 °C. The cells were treated with aluminum chloride (5.0 × 10⁻⁵ M). After incubation for 1 h, the cells were washed with phosphate-buffered saline (PBS) three times, and the treated cells were used for cell imaging. The cell images were obtained using a confocal microscope at an excitation wavelength between 300 and 340 nm. Subsequently, in the control experiment, HeLa cells were only incubated with 2a (5.0 × 10⁻⁶ M), and then washed with PBS three times for cell imaging. The cell images were obtained with a confocal microscope at an excitation wavelength between 300 and 340 nm.

3. Results and discussion

3.1. Synthesis and structural characteristics of probes 2a–2c

The synthetic procedure and molecular structure of probes 2a–2c are shown in Scheme 1. The probes 2a–2c were synthesized by two steps including the Claisen condensation of a–c with the β-pinene derivative nopinone catalyzed by NaH in a solution of 1,2-dimethoxyethane at reflux to obtain 1a–1c, and 1a–1c was further condensed with 60% N₂H₄·H₂O at reflux to obtain 2a–2c.

Scheme 1 Synthesis of probe 2a–2c.

3.2. Fluorescence properties of 2a–2c toward different metals

The selectivity behavior of probes 2a–2c toward various metal ions, including K⁺, Ba²⁺, Na⁺, Cr²⁺, Co²⁺, Mg²⁺, Fe²⁺, Fe³⁺, Mn²⁺, Ca²⁺, Pb²⁺, Cu²⁺, Ag⁺, Pb²⁺, Zn²⁺, Bi³⁺, Ni²⁺, Sn²⁺, and Al³⁺ was studied by fluorescence spectroscopy. The changes in the fluorescence spectroscopy of 2a–2c before and after the addition of various metal ions are shown in Fig. 1. This shows that compound 2a exhibited the best selectivity and highest fluorescence intensity toward Al³⁺, which implies a strong coordination ability between 2a and Al³⁺. The changes in the UV-Vis absorption spectra of 2a before and after the addition of various metal ions are shown in Fig. S1.† Upon addition of Al³⁺ (10 equiv.) to a solution of probe 2a, the absorption peak at 280 nm almost disappears and the peak at 330 nm is greatly enhanced. These results show that probe 2a could be used as a fluorescent probe to selectively detect the presence of Al³⁺.

Fig. 1 Fluorescence emission spectra of 2a–2c (5.0 × 10⁻⁶ M) upon the addition of 10 equiv. of various metal ions in C₂H₅OH solution.

3.3. Optimization studies of probe toward Al³⁺

The detection conditions for probe 2a toward Al³⁺ were optimized by investigating the influence of the concentration of 2a, pH range, response time, and EtOH/HEPES buffer. The fluorescent intensity can reach a steady state after adding Al³⁺ into a solution of 2a for 45 s (Fig. S2C†). Further tests for determining the selectivity and sensitivity of probe 2a toward Al³⁺ were performed in aqueous buffer solution (EtOH/HEPES buffer, 10 mM, v/v = 6/4, pH = 7) (Fig. S2†); the concentration of probe 2a was 5.0 × 10⁻⁶ M (Fig. S2D†). As shown in Fig. S2A,† significant enhancement in fluorescence intensity was observed in the pH range 1–9, which indicated that it is suitable for application in living systems. We studied the photo-stability of the 2a–Al³⁺ complex in aqueous buffer solution (EtOH/HEPES buffer, 10 mM, v/v = 6/4, pH = 7). After continuous illumination for 60 h, the fluorescence intensity of the 2a–Al³⁺ complex did not show any obvious change in fluorescence (Fig. S3†). There was good photo-stability of the 2a–Al³⁺ complex, indicating that probe 2a could be identified as a practical method for Al³⁺ discrimination.

3.4. Competitive selectivity of the 2a toward Al³⁺

To further investigate the selectivity of 2a as a fluorescence probe for Al³⁺, competition experiments were carried out in the presence of Al³⁺ mixed with other metal ions, such as K⁺, Ba²⁺, Na⁺, Cr²⁺, Co²⁺, Mg²⁺, Fe²⁺, Fe³⁺, Mn²⁺, Ca²⁺, Pb²⁺, Cu²⁺, Ag⁺, Pb²⁺, Zn²⁺, Bi³⁺, Ni²⁺, and Sn²⁺. As shown in Fig. 2, other metal ions had very little influence on the fluorescence intensity of the 2a–Al³⁺ complex. Combined with the data in Fig. 1, this demonstrates that probe 2a has very high selectivity toward Al³⁺.

Fig. 2 Fluorescence intensity of 2a (5.0 × 10⁻⁶ M) in buffer solution (EtOH/HEPES buffer, v/v = 6/4, 10 mM HEPES, pH = 7.4) and its complexes with Al³⁺ (5.0 × 10⁻⁵ M) in the presence of various metal ions (5.0 × 10⁻⁵ M), λ_ex = 330 nm.

3.5. Sensitivity behavior of 2a toward Al³⁺

The sensitivity of probe 2a toward Al³⁺ was examined with a fluorescence titration method, and the fluorescence titration spectra of probe 2a toward Al³⁺ are shown in Fig. 3. As shown in Fig. 4B, the 5.0 × 10⁻⁶ M probe 2a solution (EtOH/HEPES buffer, v/v = 6/4, 10 mM HEPES, pH = 7.4) exhibited non-fluorescence. Upon the addition of Al³⁺ into the probe 2a solution (5.0 × 10⁻⁶ M), a green fluorescence dramatically appeared. Fig. 4A reveals that the fluorescence intensities at 495 nm increased linearly between the fluorescence intensity and the low Al³⁺ concentration in the range 0–1.2 × 10⁻⁵ M, y = 63.43x + 50.82, R² = 0.9908 (fluorescence quantum yield Φ = 0.49, when the concentration of Al³⁺ was 1.5 × 10⁻⁵ M). The detection limit (LOD) for Al³⁺ was found to be 8.1 × 10⁻⁸ M by using DL = 3σ/k (where DL is the detection limit, σ is the standard deviation of the blank solution and k is the slope of the calibration plot). The association constant (K_B) of probe 2a with Al³⁺ was determined to be 1.89 × 10³ M⁻¹via the Benesi–Hildebrand equation^37–39 (see Fig. 4C). Table S1† summarizes the detection limits of recently reported Al³⁺ fluorescent sensors and highlights their applications in food samples.^40–46 The detection limit of 2a toward Al³⁺ is the lowest among these reported probes, implying that probe 2a can straightforwardly detect the concentration of Al³⁺ in water samples and food samples.

Fig. 3 Fluorescence spectral changes in 2a (5.0 × 10⁻⁶ M) upon addition of Al³⁺ (0–1.5 × 10⁻⁵ M) in solution (EtOH/HEPES buffer, v/v = 6/4, 10 mM HEPES, pH = 7.4), λ_ex = 330 nm.

Fig. 4 (A) A linear increase in intensity at 495 nm of probe 2a (5.0 × 10⁻⁶ M) with increasing concentrations of Al³⁺ from 0 to 1.2 × 10⁻⁵ M, λ_ex = 330 nm. (B) A plot of the fluorescence intensity versus the concentrations of Al³⁺ (0 to 1.5 × 10⁻⁵ M) in buffer solution (EtOH/HEPES buffer, v/v = 6/4, pH = 7.4). (C) Benesi–Hildebrand analysis of the emission changes for the complexation between 2a and Al³⁺.

3.6. Binding ratio and detection mechanism between probe 2a and Al³⁺

A Job plot experiment was carried out to determine the stoichiometry between 2a and Al³⁺. As shown in Fig. S4,† the stoichiometry ratio of 2a to Al³⁺ was found to be 1 : 1. The binding mode of probe 2a toward Al³⁺ was confirmed by ¹H NMR experiments in DMSO, as shown in Fig. 5. In the presence of 1.0 equiv. of Al³⁺, the proton signal of pyrazole (H₁) disappeared and the proton signal of the pyridine moiety shifted upfield. So, the sensing mechanism of probe 2a toward Al³⁺ could be the result of the synergistic complexation of the N atom in pyrazole and pyridine rings to Al³⁺ with a 1 : 1 stoichiometry. From the HRMS spectra (Fig. S5†), the mass peak at m/z 359.1464 corresponds to [2a + Al³⁺ + 2Cl⁻ + Na]⁺ (calculated at 359.2094). The proposed coordination mechanism is shown in Scheme 2. Furthermore, the energies of both the HOMO and LUMO of 2a and the 2a–Al³⁺ complex were calculated (Fig. S6†). The decrement in energy band gap confirms that there are obvious intramolecular charge-transfer (ICT) phenomena in 2a–Al³⁺ complexes. Therefore, the calculated results were in good agreement with the emission wavelengths of 2a and 2a–Al³⁺.

Fig. 5 ¹H NMR spectra changes in 2a with the addition of Al³⁺.

Scheme 2 Proposed coordination mechanism of 2a with Al³⁺.

3.7. Preparation of the test strips

As shown in Fig. S7,† the test strips showed no fluorescence under 365 nm UV-lamp when they were prepared by soaking filter papers in an ethanol solution of 2a (2.0 × 10⁻⁴ M) and dried in air. When immersed in an aqueous solution of Al³⁺ (2.0 × 10⁻⁴ M), the test strips showed green fluorescence. Therefore, the 2a-based test strips show promising application for the detection of Al³⁺ in water by fluorimetric changes.

3.8. Determination in different water samples

Novel probe 2a (5 × 10⁻⁵ M) was used for the detection of the concentration of Al³⁺ in tap water, distilled water, and lake water samples. All the water samples were collected and simply filtered. As shown in Fig. S8,† a good linear relationship was obtained between the fluorescence intensity at 495 nm and the concentration of Al³⁺ (0, 2, 5, 10, 15 × 10⁻⁶ M) in various water samples. The results listed in Table 1 show that in all the water samples recovery was higher than 95%. Therefore, the novel Al³⁺ fluorescent probe can be used for detection of the concentration of Al³⁺ in real water samples.

Table 1 Application of 2a in the determination of Al³⁺ in various water samples

Samples	Add (1 × 10^−6 M)	Detected (1 × 10^−6 M)	Recovery (%)
Tap water	0	0.62	0
	2	2.04	102.80
	5	5.15	103.20
	10	9.85	98.46
	15	14.93	99.54
Distilled water	0	0.17	0.00
	2	1.95	97.50
	5	5.11	102.20
	10	10.13	101.30
	15	14.69	97.96
Lake water	0	0.65	0.00
	2	1.92	96
	5	4.91	98.2
	10	10.06	100.64
	15	14.31	95.4

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3.9. Application in food samples

It is well known that Al³⁺ is widely used in food products. But, excessive ingested Al³⁺ may cause several diseases. Therefore, it is practically important to detect Al³⁺ in food products using this novel fluorescent probe. Some food samples containing Al³⁺, such as chips, fried chicken, tea, sausage, biscuit and baby biscuit were chosen to examine the application of probe 2a (5 × 10⁻⁵ M) in food samples. These food samples were first crushed and 20% (v/v) HCl aqueous solution was added. Then it was stirred for one day until the solution became turbid. The mixture was filtered to obtain the Al³⁺-containing filtrate. The fluorescence intensity at 495 nm displayed a good linear relationship with the concentration of Al³⁺ (0, 2, 5, 10, 15 × 10⁻⁶ M) (R² = 0.992, Fig. S9†). The results listed in Table 2 show that probe 2a can detect the concentration of Al³⁺ in different food solutions with good recovery, ranging from 96 to 103%. Therefore, the novel Al³⁺ probe 2a can be applied as a simple method to detect the concentration of Al³⁺ in various food samples.

Table 2 Results for the determination of Al³⁺ in various food samples

Samples	Al^3+ (1 × 10^−6 M)	Added (1 × 10^−6 M)	Found (1 × 10^−6 M)	Recovery (%)
Chips	6.15	3	8.99	98.25
		6	12.22	100.57
Fried chicken	5.13	3	8.32	102.33
		6	10.96	98.47
Sausage	2.89	3	5.78	98.13
		6	8.57	96.40
Tea	1.47	3	4.29	95.97
		6	7.18	96.12
Biscuit	0.55	3	3.49	98.30
		6	6.33	96.64
Baby biscuit	0	3	3.11	103.67
		6	6.12	102

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3.10. Cellular imaging

Fluorescence imaging experiments of 2a were performed to study the utility of probe 2a in living cells. Cytotoxicity assays results showed that compound 2a had low cytotoxicity to HeLa cells (Fig. S10†). HeLa cells were incubated with 2a (5.0 × 10⁻⁶ M) at 37 °C for 24 h. And no obvious fluorescence was observed. However, after HeLa cells were incubated with AlCl₃ for 1 h, remarkable fluorescence enhancement can clearly be detected (Fig. 6). The fluorescence imaging experiments show that the novel Al³⁺ fluorescent probe 2a has potential application in living cells.

Fig. 6 (a) Fluorescent image of HeLa cells treated with probe 2a (5.0 × 10⁻⁶ M) in the absence of Al³⁺; (b) microscope image of HeLa cells treated with probe 2a (5.0 × 10⁻⁶ M) in the absence of Al³⁺; (c) merged image of frames (a) and (b); (d) microscope image of HeLa cells treated with Al³⁺ (5.0 × 10⁻⁵ M) and probe 2a (5.0 × 10⁻⁶ M); (e) fluorescence image of HeLa cells treated with Al³⁺ (5.0 × 10⁻⁵ M) and probe 2a (5.0 × 10⁻⁶ M); (f) merged image of frames (d) and (e).

4. Conclusions

In summary, we developed a β-pinene-based fluorescent probe 2a from natural β-pinene derivative nopinone for the detection of Al³⁺. The new fluorescent probe 2a exhibits advantages, such as clearer change in fluorescence, wider pH range, higher sensitivity, better selectivity, lower detection limit, and simpler synthetic procedures. The sensing mechanism of 2a with Al³⁺ was studied by ¹H NMR, HRMS, and DFT analysis. Fluorescence probe 2a can be used as fluorescent sensing tool for the real-time detection of Al³⁺ in aqueous media, food samples, and living cells.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

The research was supported by the National Natural Science Foundation of China (No. 31470592), Jiangsu Provincial Key Lab for the Chemistry and Utilization of Agro-Forest Biomass, the open Fund of Jiangsu Key Laboratory for Biomass Energy and Material (JSBEM201702), and Priority Academic Program Development of Jiangsu Higher Education Institutions, China.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c9ra00447e

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