A novel AIE-active camphor-based fluorescent probe for simultaneous detection of Al³⁺ and Zn²⁺ at dual channels in living cells and zebrafish

Abstract

A novel dual-functional probe N′-(2-hydroxy-5-((4,7,7-trimethyl-3-oxobicyclo[2.2.1] heptan-2-ylidene)methyl) benzylidene)picolinohydrazide (PSH) was constructed from natural camphor. This probe showed strong yellow-green fluorescence at 535 nm due to its aggregation-induced emission (AIE) feature. Interestingly, the probe PSH displayed a significant turn-on fluorescence response towards Al³⁺ (green fluorescence at 500 nm) and Zn²⁺ (orange fluorescence at 555 nm) at two different emissive channels. The detection limits of PSH towards Al³⁺ and Zn²⁺ were found to be 12.1 nM and 14.2 nM, respectively. PSH exhibited excellent selectivity and anti-interference performance and could distinguish between Al³⁺/Zn²⁺ and identify whether Zn²⁺ exists in the PSH-Al³⁺ complex by adding ATP. The binding mechanisms between PSH and Al³⁺/Zn²⁺ ions were supported by ¹H NMR, HRMS analysis, and density functional theory (DFT) calculations. Based on its outstanding sensing properties, the probe PSH was used to establish molecular logic function gates. Moreover, the probe PSH could be applied to detect Al³⁺ and Zn²⁺ in real environmental water, and fluorescence detection was well demonstrated by test strips. Furthermore, the probe PSH was employed for imaging Al³⁺ and Zn²⁺ in HeLa cells and zebrafish.

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Introduction

Metal ions have a great impact on human life, which causes biological denaturation by coordinating with bioactive molecules.^1–3 For example, zinc ions are not only an essential trace element for the human body but also for all living organisms. They play a key role in regulating cell apoptosis, nucleic acid and protein metabolism, neural signal transmission, the immune system, and many other molecular mechanisms. However, the metabolic disorder of zinc ions is related to neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, epilepsy,^4–7etc. Aluminum is the most abundant metal element in the Earth's crust, and it is also an essential element for the human body.^8–10 Aluminum is widely present in our lives, so we don't have to worry about insufficient intake, but excessive intake can cause some diseases such as Alzheimer's disease, Parkinson's disease, osteoporosis, nervous system problems, etc.^11–13 Therefore, it is very important to design and develop a biosensor that can effectively detect Zn²⁺ and Al³⁺ in biological systems and environments.

Nowadays, several methods such as electrochemical analysis, atomic absorption spectroscopy, isotope chromatography, and chromatography have been reported for trace metal element detection.^14–17 However, these traditional detection methods have issues such as complex pretreatment, expensive equipment, cumbersome operation, and time-consuming detection, which make them unsuitable for real-time detection of on-site samples.^18–20 Compared with traditional detection methods, fluorescent probe detection technology has the advantages of simple sample preparation, fast response, high sensitivity, good selectivity, and easy operation.^21–23 It has been widely used in chemistry, medicine, environmental science, life science, and other fields. Especially in recent years, the fluorescent probe has seen gradual development in high efficiency and trace, microscopic, and real-time analysis.

Aggregation-induced emission (AIE), a special photophysical phenomenon, was first reported by Tang and co-workers.²⁴ AIE is characterized by molecules in a dilute solution that have no fluorescence, but their fluorescence would be enhanced in an aggregated state. AIE luminogens have the advantage of superior biological imaging and excellent photo-stability compared with aggregation-caused quenching (ACQ).^25,26 Therefore, it is of great significance to construct fluorescent probes with AIE properties.

In recent years, a great number of organic small-molecule fluorescent probes for detecting metal ions have been developed. However, some of these fluorescent probes had poor water solubility and limits to their applicability.^27,28 Although many of the reported probes have high selectivity for a single analyte, differential response probes that can detect multiple analytes would be more effective and are needed for practical applications. Compared to one-to-one type probes, one probe for multiple analyses is more advantageous in regards to efficiency, simplicity, time consumption, and cost, which has attracted more attention for metal ion detection.^29–33 Hence, it was important to develop fluorescent probes with high water solubility and simultaneous detection of multiple targets.

Camphor, traditionally acquired through the distillation of the wood of Lauraceae trees, is an important and renewable source of natural terpenoids, and can also be chemically synthesized mainly with turpentine as a raw material. Camphor plays a crucial medicinal role with insecticidal, antimicrobial, antiviral, anticancer and antitussive activities.^34–37 Furthermore, the rigid structure of the camphor molecule could effectively reduce the energy consumption during a molecular collision, which provides camphor with good photophysical properties.^38–40 Therefore, based on the physical properties and medicinal value of camphor, it was very meaningful to synthesize fluorescent probes from camphor.

Herein, we present a novel camphor-derived fluorescent turn-on probe PSH with AIE properties for the dual detection of Al³⁺/Zn²⁺. The probe PSH contains imino, carbonyl, and phenolic hydroxyl groups and can provide multiple sites for binding with Al³⁺ and Zn²⁺. This probe PSH shows excellent sensitivity and selectivity and a low detection limit toward Al³⁺ and Zn²⁺. After binding with Al³⁺, the PSH-Al³⁺ complex was responsive to ATP and released the probe free. The complexation mechanism of PSH towards Zn²⁺/Al³⁺ was confirmed by ¹H NMR, HRMS analysis, and density functional theory (DFT) calculations. Based on its outstanding sensing properties, the probe PSH was used to establish molecular logic function gates. Moreover, the probe PSH could be applied to detect Al³⁺ and Zn²⁺ in real environmental water, and fluorescence detection was well demonstrated by test strips. Furthermore, the probe PSH was used to detect Al³⁺ and Zn²⁺ in HeLa cells and zebrafish by bioimaging.

Results and discussion

AIE performance of PSH

The fluorescence feature of PSH was evaluated in the state of solid powder that showed strong yellow fluorescence at 535 nm (Fig. S1†). Moreover, to investigate the AIE properties of PSH, acetonitrile was used as a decent solvent system while water was used as a poor solvent system. As shown in Fig. 1(a and b), the PSH showed a very weak fluorescence intensity in the decent solvent system(f_w = 0 vol%); however, a strong fluorescence emission intensity was observed in the poor solvent system (f_w = 99.9 vol%). As presented in Fig. 1c, the fluorescence color change of the probe in different water contents was obtained under a 365 nm UV light. It was observed that the yellow fluorescence gradually enhances with a water content of more than 80% and reached maximum fluorescence intensity at 90% water content, which indicated the formation of an aggregated status in a poor solvent system and that the probe PSH has good AIE performance. Moreover, the fluorescence quantum yields of PSH in the solution system (ACN/water = 3/7) and solution system (ACN/water = 1/9) were calculated as 0.58% and 15.18%, respectively, which also proved the emission enhancement effect. Therefore, PSH as the AIE fluorescent material has a potential application prospect in fluorescence detection.

Fig. 1 (a) The fluorescence spectra of PSH (10 μM) in ACN/water solutions with the changed water volume fractions. λ_ex = 365 nm. (b) The fluorescence emission intensity of PSH (10 μM) at 535 nm in ACN/water solutions with the changed water volume fractions. (c) The color change photographs of PSH with the changed water volume fractions under a 365 nm UV lamp.

The AIE properties of probe PSH was also confirmed by dynamic light scattering (DLS) and scanning electron microscopy (SEM) studies. As shown in Fig. 2a, the average size of probe PSH was 256.6 nm in the 70% water volume fraction solutions. As expected, the average size of probe PSH was 705.8 nm in the 90% water volume fraction solutions, which is significantly higher than the former and confirms that molecular aggregation has occurred (Fig. 2b). In addition, scanning electron microscopy (SEM) analysis visually reveals that the probe PSH forms larger aggregated nanoparticles in a solution with a higher water content.

Fig. 2 DLS size distribution of probe PSH (10 μM) in ACN/water solutions with (a) 3/7 (v/v)and (b) 1/9 (v/v). SEM images of probe PSH (50 μM) in ACN/water solutions with (c) 3/7 (v/v)and (d) 1/9 (v/v).

Spectroscopic responses of PSH towards Al³⁺ and Zn²⁺

The probe PSH, which has multiple oxygen and nitrogen sites, should be able to coordinate with metal ions. The spectroscopic responses of the probe PSH to Al³⁺/Zn²⁺ are shown in Fig. 3. In the UV-vis absorption spectra (Fig. 3a), the probe PSH emerged with a strong absorption peak at 302 nm. After the addition of Al³⁺/Zn²⁺ to the PSH solutions, the initial maximum absorption peak was bathochromic-shifted to 321 nm/375 nm, respectively, and the color of the solutions changed from colorless to faint yellow to the naked eye. Moreover, as shown in Fig. 3b, the probe PSH showed a faint emission at about 550 nm (λ_ex = 365 nm) in the fluorescence spectra. Interestingly, after adding Al³⁺ or Zn²⁺, a remarkable “turn on” fluorescence enhancement with the two different wavelength responses (500 nm and 555 nm) was observed and accompanied with the fluorescent color exclusively changing from colorless to green and yellow respectively. When the probe was coordinated with Al³⁺ and Zn²⁺, the intramolecular charge density was transferred, which led to fluorescence emission changes. Based on the above fact and owing to different charge densities and radii between Al³⁺ and Zn²⁺, the Al³⁺/Zn²⁺-complexes exhibited a different charge density transfer thereby emitting two different emission wavelengths. As shown in Fig. S11,† we found that there was a greater enhancement of fluorescence intensity in the ACN/HEPES buffer (3/7, v/v) solution than in other ACN/HEPES buffer solvent mixtures, indicating that the ACN/HEPES buffer (3/7, v/v) solution is more suitable to investigate the recognition behavior of probe PSH towards Al³⁺ and Zn²⁺. Moreover, the fluorescence quantum yield of PSH increased from 0.58% to 12.87% and 10.37% in the presence of Al³⁺ and Zn²⁺, respectively. Therefore, after the addition of Al³⁺ or Zn²⁺, the PSH fluorescence enhancement phenomenon may be explained by the stable complexes formed between PSH and Al³⁺/Zn²⁺, leading to the chelation-enhanced fluorescence (CHEF) effect by a PSH complexation with metal ions.^41,42 Besides, the fluorescence emission enhancement of PSH is because the –CH=N– isomerization and PET process can be restrained.^43,44

Fig. 3 (a) UV-vis absorption spectra of PSH (10 μM) with or without Al³⁺/Zn²⁺ (15 μM) in ACN/HEPES buffer (v/v, 3/7, 10 mM, pH = 7.4). Inset: the color change of PSH after the addition of Al³⁺/Zn²⁺ under sunlight. (b) The fluorescence spectra of PSH (10 μM) in the presence or absence of 15 μM Al³⁺/Zn²⁺ in ACN/HEPES buffer (v/v, 3/7, 10 mM, pH = 7.4). Inset: the color change photographs of PSH after the addition of Al³⁺/Zn²⁺ under a 365 nm UV lamp.

Sensitivity of PSH for Zn²⁺ and Al³⁺

To assess the sensitivity of probe PSH toward Al³⁺/Zn²⁺, the fluorescence titration of PSH was investigated for each ion, respectively. The fluorescence spectra emission peak at 500 nm showed a 25-fold enhancement for emission peak intensity with the continued addition of Al³⁺ (0–15 μM) to the PSH solution (Fig. 4a). As shown in Fig. 4b, a similar phenomenon was observed with the addition of Zn²⁺ (0–15 μM), where the emission peak at 555 nm was accompanied by a 20-fold enhancement of the emission intensity. Two good linear relationships of probe PSH toward Al³⁺ (R² = 0.995) or Zn²⁺ (R² = 0.992) between the fluorescence intensity and the ion concentrations (0–14 μM or 0–12 μM) were formed (Fig. 5). The limit of detection (LOD) was calculated to be 12.1 nM for Al³⁺ and 14.2 nM for Zn²⁺ based on the formula 3σ/m where σ is the standard deviation of the control sample and m is the slope of the linearity of the equation. The results prove that PSH could effectively distinguish between Al³⁺ and Zn²⁺ with superior sensitivity.

Fig. 4 The fluorescence spectra of (a) 0–15 μM Al³⁺ and (b) 0–15 μM Zn²⁺ added to 10 μM PSH in ACN/HEPES buffer (v/v, 3/7, 10 mM, pH = 7.4). λ_ex = 365 nm.

Fig. 5 The linear curve of the emission intensity of PSH with Al³⁺ (a) and Zn²⁺ (b) concentrations (0–14 μM or 0–12 μM) in ACN/HEPES buffer (v/v, 3/7, 10 mM, pH = 7.4).

Selectivity and interference of PSH for Zn²⁺ and Al³⁺

The selectivity of PSH toward Al³⁺/Zn²⁺ was measured by fluorescence. PSH was treated with various metal ions (Co₂⁺, Hg²⁺, Ag⁺, Cr³⁺, Pb²⁺, Na⁺, Ni²⁺, Mg²⁺, Ce⁴⁺, Cd²⁺, K⁺, Cr⁶⁺, Cu²⁺, Mn²⁺, Fe³⁺, La³⁺, Sn²⁺, Ca²⁺, Fe²⁺, Cu⁺, Al³⁺ and Zn²⁺). As depicted in Fig. 6a, the probe PSH was non-fluorescent in the fluorescence spectra. Upon the addition of Al³⁺ and Zn²⁺, the fluorescence spectra signal intensity increased dramatically (500 nm and 555 nm, respectively). After adding various other metal ions, the fluorescence spectra did not show significant changes.

Fig. 6 (a) Fluorescence spectra of PSH (10 μM) upon addition of 15 μM Al³⁺ and Zn²⁺ and other metal ions (30 μM) in ACN/HEPES buffer (v/v, 3/7, 10 mM, pH = 7.4). The fluorescence intensity (λ_em = 460 nm) of PSH (10 μM) with (b) Al³⁺ (15 μM) and (c) Zn²⁺ (15 μM) mixed with different competitive anions (30 μM) including (1) Co₂⁺, (2) Hg²⁺, (3) Ag⁺, (4) Cr³⁺, (5) Pb²⁺, (6) Na⁺, (7) Ni²⁺, (8) Mg²⁺, (9) Ce⁴⁺, (10) Cd²⁺, (11) K⁺, (12) Cr⁶⁺, (13) Cu²⁺, (14) Mn²⁺, (15) Fe³⁺, (16) La³⁺, (17) Sn²⁺, (18) Ca²⁺, (19) Fe²⁺, and (20) Cu⁺ in ACN/HEPES buffer (v/v, 3/7, 10 mM, pH = 7.4). (d) Fluorescence emission spectra of PSH-Zn²⁺ after the addition of Al³⁺ in ACN/HEPES buffer (v/v, 3/7, 10 mM, pH = 7.4).

To further evaluate the PSH anti-interference ability for Al³⁺/Zn²⁺, the specificity experiment was tested and replicated 5 times with other competing metal ions. As displayed in Fig. 6b and c, after the addition of the other metal ions into the PSH-Al³⁺ and PSH-Zn²⁺ solutions, respectively, there was negligible change in the fluorescence spectra. Interestingly, after gradually adding Al³⁺ to the PSH-Zn²⁺ solution, the fluorescence emission peak shifted from 555 nm to 500 nm and was accompanied by a fluorescence color transformation from orange to green (Fig. 6d), which could indicate that the PSH had better selectivity for Al³⁺ than Zn²⁺. This phenomenon could be attributed to a ligand-to-ligand transfer¹³ and that the PSH-Al³⁺ complex is more stable than the PSH-Zn²⁺ complex. The above results suggest that the probe PSH can selectively detect Al³⁺/Zn²⁺ over other metal ions and specifically identify Al³⁺/Zn²⁺ in the presence of other competing metal ions.

Furthermore, identifying whether Zn²⁺ exists in the PSH-Al³⁺ solution is also very important. Given the chelation of Al³⁺ for ATP,⁴⁵ as shown in Fig. 7, when Zn²⁺ and Al³⁺ exist in the PSH solution at the same time, the addition of ATP will lead to the production of an Al³⁺−ATP complex, releasing free the probe PSH and allowing it to chelate with Zn²⁺, which is accompanied by a significant enhancement of the fluorescence emission intensity at 555 nm. Upon the addition of ATP into the PSH and PSH-Zn²⁺ solution, there was no change in the fluorescence spectrum. The above results indicate that the Al³⁺−ATP complex is more stable than the Al³⁺−PSH complex and that the PSH can selectively detect Al³⁺ and Zn²⁺.

Fig. 7 Fluorescence emission spectra of PSH + Al³⁺, PSH + Al³⁺ + Zn²⁺, PSH + Zn²⁺, and the addition of ATP in ACN/HEPES buffer (v/v, 3/7, 10 mM, pH = 7.4).

Response time and pH effect

The time-dependent response of probe PSH toward the Al³⁺ and Zn²⁺ experiment was further investigated. The fluorescence emission intensity of PSH at 500 nm heightened quickly and reached its highest intensity within 10 min after adding Al³⁺ to the solution (Fig. 8a). Similarly, the response time of PSH to Zn²⁺ reached an equilibrium value within 13 min.

Fig. 8 Fluorescence intensity changes at 500 nm and 555 nm for PSH (10 μM) at (a) different times and (b) different pH values with or without Al³⁺ and Zn²⁺.

To explore the potential practical application of PSH, the change in the fluorescence spectra was recorded in different ranges of pH. As illustrated in Fig. 8b, compared with the probe PSH in a pH of 7.4, its fluorescence intensity showed little change in the pH range of 1.0–9.0, which verified that the PSH was stable in this pH range. Under strong alkaline conditions, the fluorescence enhancement phenomenon of PSH can be attributed to the deprotonation of –OH and –NH groups, leading to the PET effect being inhibited. As the blue line shows, after the addition of Al³⁺, the fluorescence intensity increased at pH 2.0 and reached its maximum at pH 6.0, and the most suitable condition for detection was found in the pH range from 6.0 to 12.0. Similarly, after adding the Zn²⁺ to the PSH solution, the fluorescence intensity showed its best enhancement in the pH range of 6.0–10.0. Therefore, the results show that the probe PSH has the capability of detecting Al³⁺ and Zn²⁺ in a wide pH range and is also suitable for the physiological pH range. When the pH value was greater than 10.0, the binding of Al³⁺/Zn²⁺ to hydroxide ions reduced the coordination of Al³⁺/Zn²⁺ with the probe; therefore, the fluorescence intensity of the PSH-Al³⁺/Zn²⁺ complexes decreased sharply. In summary, PSH can detect Al³⁺ and Zn²⁺ in real-time and is conducive to biological application detection.

Sensing mechanism of PSH for Al³⁺ and Zn²⁺

To clarify the reaction mechanism, the stoichiometric ratio of probe PSH toward Al³⁺ and Zn²⁺ was confirmed first by Job's plot studies. As presented in Fig. S2,† the inflection point occurred when the concentration of Al³⁺/Zn²⁺ accounts for 50% of the PSH + Al³⁺/Zn²⁺ total concentration, which indicated that the complex-ratios were 1 : 1. The complex ratios were also definitively proven by HRMS analysis (Fig. S3†) and the HRMS of PSH-Al³⁺ shows peaks at 405.26 and 430.46 corresponding to [PSH+ 2H⁺]⁺ and [PSH+ Al³⁺]⁺ respectively. In the HRMS of PSH-Zn²⁺, the peaks at 436.68 and 468.11 were attributed to [PSH+ MeOH + H⁺]⁺ and [PSH+ Zn²⁺]⁺ respectively. In addition, according to the Benesi–Hildebrand equation, the binding constants of PSH toward Al³⁺ and Zn²⁺ were figured to be 3.77 × 10⁴ M⁻¹ and 6.58 × 10⁴ M⁻¹ respectively (Fig. S4†).⁴⁶

The ¹H NMR titration experiment was performed in DMSO-d₆ to further investigate the sensing mechanism. As shown in Fig. 9, the ¹H NMR spectra of probe PSH showed a proton peak at 12.56, 11.75, and 8.89 ppm, which corresponded to –NH, –OH, and HC=N respectively. After adding Al³⁺ or Zn²⁺, both –NH and –OH peaks at 12.56 and 11.75 ppm disappeared, which indicates that the binding sites of Al³⁺/Zn²⁺ to PSH are on the carbonyl and the phenolic hydroxyl groups. Furthermore, the signal of HC=N at 8.89 ppm shifted. The analysis shows that the Al³⁺ and Zn²⁺ coordinate with phenolic hydroxyl, imine, and carbonyl. The above results verified the previous hypothesis that the C=N in the PSH molecule inhibits the emission of fluorescence by isomerization. Otherwise, once Al³⁺/Zn²⁺ is complexed with the PSH, the C=N structure is destroyed and the isomerization is restrained, which is why the fluorescence increases significantly.

Fig. 9 Partial ¹H NMR spectra of PSH with or without 1 equiv. (a) Al³⁺ and (b) Zn²⁺ in DMSO-d₆, respectively.

To further explore the reaction mechanism of probe PSH toward Al³⁺/Zn²⁺, the density functional theory (DFT) computation was conducted by using the Gaussian 09 program with rb3lyp/6-311g (d,p) basis. The optimized structures of probe PSH, PSH-Al³⁺ and PSH-Zn²⁺ are presented in Fig. 10. For PSH-Al³⁺/Zn²⁺, Al³⁺/Zn²⁺ chelates with the phenolic hydroxyl, imine, and carboxide of the PSH. After the completion of structural optimization, the distance of the Al³⁺ from the phenolic hydroxyl, imine, and carbonyl was 1.721, 1.842, and 1.718 Å, and that of Zn²⁺ was 1.843, 2.021, and 1.905 Å, respectively, which were in line with the coordination bond distance range.^47,48 These results indicate that the PSH-Al³⁺/Zn²⁺ forms a stable complex and verifies that the complex-ratios are 1 : 1.

Fig. 10 The frontier molecular orbitals and optimized geometry configurations of PSH, PSH-Al³⁺, and PSH-Zn²⁺ complexes.

The highest occupied molecular orbitals (HOMO) and the lowest occupied molecular orbitals (LUMO) of PSH-Al³⁺/Zn²⁺ are also shown in Fig. 10. When the probe PSH is in a free state, in the transition of the HOMO to LUMO process, the free electrons on the detection group (donor) can be transferred to the HOMO of the excited fluorophore (acceptor) through the PET effect, which leads to fluorescence quenching. After Al³⁺/Zn²⁺ complex with the detection group of PSH, it causes the HOMO orbital energy level of the detection group to drop below the HOMO energy level of the excited state fluorophore (decreases from −5.73 eV to −15.31/−12.02 eV, respectively). The HOMO orbital energy level of the detection group (donor) is reduced, and the electrons cannot pass to the HOMO of the fluorophore (acceptor), so the probe will fluoresce, which is consistent with the previously reported PET mechanism of metal complexation. Furthermore, the HOMO–LUMO energy gap (ΔE) of PSH (3.80 eV) is higher than that of PSH-Al³⁺ (1.34 eV) and PSH-Zn²⁺ (1.61 eV), which also verifies that the complexation of PSH with Al³⁺/Zn²⁺ is more likely to occur, causing a dramatic enhancement of fluorescence intensity. According to Job's plot, HRMS analysis, the ¹H NMR titration experiment, and DFT computation, the sensing mechanism of PSH with Al³⁺ or Zn²⁺ is presented in Fig. 11.

Fig. 11 The proposed sensing mechanisms of PSH to Al³⁺ and Zn²⁺.

Molecular logic gate behavior of PSH

In recent years, due to the good practicability of molecular logic gates, the exploration of its development has gradually increased. The characteristics of PSH toward Al³⁺, Zn²⁺, and ATP showed that it can be applied to IMPLICATION logic gate applications, in which the Al³⁺ (input 1), Zn²⁺ (input 2), and ATP (input 3) are used as three chemical inputs and the fluorescence emission signal at 500 nm (output 1) and 555 nm (output 2) served as two outputs (Fig. 12 and Table 1). Meanwhile, the presence and absence of the chemical input were assigned “1” and “0”, respectively. In the above logic gates, when the fluorescence emission intensity is enhanced at 500 nm (output 1) or 555 nm (output 2) it is defined as “1” (on), otherwise it is defined as “0” (off). In the “Output 1 (500 nm)” logic gates, when the “Al³⁺” and “Al³⁺ + Zn²⁺” are present, the “Output 1” signal is “1” (500 nm fluorescence emission “turn-on”). In the “Output 2 (555 nm)” logic gates, either of the inputs “Zn²⁺”, “Zn²⁺ + ATP” and “Zn²⁺ + Al³⁺ + ATP” is defined with “1” (555 nm fluorescence emission “turn-on”), and all of the other “Input” combinations are defined as “0” (no fluorescence). The study of logic gates shows that the single fluorescent probe PSH has potential application prospects in molecular devices.

Fig. 12 (a) The corresponding visualization figure, (b) the molecular logic circuit with three inputs (input 1: Al³⁺, input 2: Zn²⁺, input 3: ATP) and two outputs (output 1: 500 nm, output 2: 555 nm).

Table 1 The corresponding truth table for the molecular logic gate

Input 1 (Al^3+)	Input 2 (Zn^2+)	Input 3 (ATP)	Output 1500 nm	Output 2555 nm
0	0	0	0	0
1	0	0	1	0
0	1	0	0	1
0	0	1	0	0
1	1	0	1	0
1	0	1	0	0
0	1	1	0	1
1	1	1	0	1

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Applications in real water and test strips

To further evaluate the applicability of the probe PSH toward Al³⁺/Zn²⁺, the specific recognition of probe PSH to Al³⁺/Zn²⁺ was evaluated in distilled water, tap water, and Xuanwu Lake water. As we all know, Al³⁺ and Zn²⁺ widely exist in daily life and have a great impact on human health. The fluorescence intensity of the PSH was enhanced after adding different concentrations of Al³⁺ (0, 2, 5, 10, 15μM) into the ACN/water samples (v/v = 3/7) solution system (Fig. 13). As shown in Table 2, the recovery of different concentrations of Al³⁺ (5, 10, 15 μM) was measured in three different kinds of water. From these results, the probe PSH could detect the Al³⁺ concentration in the three water samples with good recovery ranging from 92.2% to 102.2%, from 92.5% to 108.6%, and from 96.6% to 113.8%, respectively. The probe PSH toward Zn²⁺ is also presented in Fig. S5 and Table S1,† which also suggests that PSH had a good recovery percentage for Zn²⁺. Therefore, the probe PSH could be used to detect Al³⁺ with high sensitivity and good recovery in environmental water samples.

Fig. 13 (a) The fluorescence emission intensity of PSH towards different concentrations of Al³⁺ in different water samples; the linear plots between the PSH-Al³⁺ fluorescence intensity and different concentrations of Al³⁺ in (b) distilled water, (c) tap water, and (d) Xuanwu Lake water.

Table 2 Determination results of PSH towards Al³⁺ in three kinds of water samples

Water sample	Added Al^3+ (μM)	Found Al^3+ (μM)	Recovery (%)
Distilled water	0	Not detected	—
	2	2.04 ± 0.04	102.0
	5	4.61 ± 0.19	92.2
	10	10.03 ± 0.29	100.3
	15	15.33 ± 0.37	102.2
Tap water	0	0.06 ± 0.05	0.0
	2	1.85 ± 0.43	92.5
	5	5.43 ± 0.55	108.6
	10	9.93 ± 0.63	99.3
	15	14.97 ± 0.82	99.8
Xuanwu Lake water	0	0.22 ± 0.12	0.0
	2	2.18 ± 0.23	109.0
	5	5.69 ± 0.45	113.8
	10	10.07 ± 0.54	100.7
	15	14.49 ± 0.71	96.6

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The test strip experiment was performed to further investigate its practical application. First, the test paper strips were immersed in an ethanol solution containing probe PSH (50 μM) and dried at room temperature, which showed a faint yellow color. Then the probe PSH-loaded test strips were exposed to different concentrations of Al³⁺ or Zn²⁺ (0, 20, 50, 80, 100, 120, 150, 200 μM); the results are shown in Fig. 14. It was observed that the color of the test strips changed from faint yellow to green or golden yellow and gradually enhanced as the Al³⁺/Zn²⁺ concentration increased under a 365 nm UV lamp. Hence, the probe PSH could be applied to the convenient and fast detection of Al³⁺/Zn²⁺.

Fig. 14 Photographs of test strips containing probe PSH with increasing concentrations of (a) Al³⁺ and (b) Zn²⁺ (0–200 μM).

Bioimaging in HeLa cells and zebrafish

Inspired by the abovementioned responsive performance of probe PSH for Al³⁺/Zn²⁺, HeLa cell and zebrafish imaging experiments were performed to investigate the biological application. The MTT assay was first conducted to estimate the cytotoxicity of PSH for HeLa cells. As displayed in Fig. S6,† the HeLa cell viability exceeded 90% after incubation with 0–30 μM probe PSH at 37 °C for 24 h, which confirmed that the probe PSH has weak cytotoxicity and provides the possibility of bioimaging detection of Al³⁺/Zn²⁺ in HeLa cells. Fig. 15 shows the HeLa cell confocal fluorescence images (λ_ex = 405 nm, collected fluorescence wavelength: 480–580 nm) of PSH and PSH that was treated with Al³⁺/Zn²⁺. In the HeLa cells where only the probe PSH (10 μM) was added, there was almost no fluorescence intensity (Fig. S7†). After adding different concentrations of Al³⁺/Zn²⁺(10, 20, 30 μM) into the PSH-loaded cells and incubating for 1 h, it exhibited a gradient-enhanced fluorescence change in the green or orange channel, which suggests that the probe PSH has good biocompatibility and can detect Al³⁺/Zn²⁺ in cells with high sensitivity. The 3D surface plot and Fig. S7† vividly and intuitively show the change of fluorescence intensity in HeLa cells.

Fig. 15 Fluorescence microscopy images and 3D surface plot of HeLa cells incubated with PSH for 2 h and then cultured with different concentrations of (a) Al³⁺ and (b) Zn²⁺ for 0.5 h.

To further investigate the biological application of probe PSH, zebrafish, classic laboratory model organisms, were selected as the research objects to verify the feasibility of the probe to detect Al³⁺/Zn²⁺ in biological tissues. The two-day-old zebrafish were fed in the embryo medium and then the PSH (10 μM) was added for 20 min to ensure that the zebrafish absorbed the probe into their tissues and organs. As presented in Fig. 16, the control group that only contained PSH had almost no fluorescence emission intensity (λ_ex = 405 nm). Upon the addition of Al³⁺ or Zn²⁺ (20 μM) in the PSH-loaded embryo medium for 10 min and rinsed twice with pure water, the abdomen of zebrafish showed strong green or orange fluorescence (Fig. 16b and c), which indicates that the PSH can detect Al³⁺ or Zn²⁺ in zebrafish. The above results suggest that the PSH has good tissue penetration and can be applied to visually detect Al³⁺ or Zn²⁺ in HeLa cells and zebrafish.

Fig. 16 Fluorescence microscopy images of zebrafish: (a, d and f) incubated with PSH for 1 h; incubated with PSH for 1 h and then incubation with (b, d and g) Zn²⁺ and (c, e and h) Al³⁺ for 0.5 h.

Experimental

Materials and instruments

The chemicals and biological reagents were purchased from Shanghai Haohong Scientific Co., Ltd, Titan, and Sigma-Aldrich without purification. ¹H NMR and ¹³C NMR spectra were recorded in DMSO-d₆ solutions with a Bruker AV 500 Spectrometer. The UV-vis absorption spectra were recorded on a Shimadzu UV-2450 spectrophotometer. The fluorescence spectrum was recorded using a PerkinElmer LS55 spectrophotometer. High-resolution mass spectra (HRMS) were recorded using an Agilent 5975c mass spectrometer. Fluorescence images were captured using a confocal laser scanning microscope (Zeiss LSM 710, Germany).

Synthesis of compound PSH

Compound PSH was synthesized as shown in Scheme 1. The intermediates 1 and 2 were prepared according to the literature.³⁹ Compound 2 (0.284 g, 1 mmol), picolinohydrazide (0.137 g, 1 mmol), glacial acetic acid (0.12 g, 2 mmol), and ethanol (10 mL) were stirred and refluxed in a three-necked flask for 12 h until the conversion ratio of compound 1 exceeded 95% (monitored by TLC). After ending the reaction, the reaction mixture was evaporated under vacuum to remove excess solvents. The residue was extracted three times with 200 mL ethyl acetate and washed with saturated brine until neutral. The organic layer was dried with anhydrous sodium sulfate, filtered and concentrated to obtain a crude product which was purified by recrystallization in absolute ethanol to afford the white solid product PSH, yield 75%. ¹H NMR (600 MHz, DMSO-d₆) δ: 12.56 (s, 1H), 11.75 (s, 1H), 8.89 (s, 1H), 8.73 (d, J = 4.7 Hz, 1H), 8.14 (d, J = 7.8 Hz, 1H), 8.10–8.04 (m, 1H), 7.68 (q, J = 5.1 Hz, 2H), 7.55 (dd, J = 8.6, 2.2 Hz, 1H), 7.09 (s, 1H), 7.03 (d, J = 8.5 Hz, 1H), 3.14 (d, J = 4.1 Hz, 1H), 2.16 (tt, J = 11.7, 9.2, 4.5 Hz, 1H), 1.78 (ddd, J = 13.5, 11.2, 3.7 Hz, 1H), 1.48 (ddd, J = 12.4, 9.1, 3.7 Hz, 1H), 1.37 (ddd, J = 13.6, 9.1, 4.7 Hz, 1H), 0.97 (s, 3H), 0.92 (s, 3H), 0.73 (s, 3H). ¹³C NMR (150 MHz, DMSO-d₆) δ: 207.11, 160.94, 158.76, 150.00, 149.55, 149.09, 140.50, 138.53, 132.98, 132.29, 127.68, 126.94, 126.54, 123.31, 119.46, 117.65, 56.80, 48.97, 46.79, 40.53, 30.65, 26.03, 20.64, 18.46, 9.76. HRMS (m/z): [M + H]⁺ calcd for C₂₄H₂₅N₃O₃ + H⁺, 404.1977; found, 404.1974.

Scheme 1 Synthesis of probe PSH.

Spectral measurements

The probe PSH was dissolved in acetonitrile to obtain its stock solution (1 mM). The stock solutions (10 mM) of various metal ions (Co₂⁺, Hg²⁺, Ag⁺, Cr³⁺, Pb²⁺, Na⁺, Ni²⁺, Mg²⁺, Ce⁴⁺, Cd²⁺, K⁺, Cr⁶⁺, Cu²⁺, Mn²⁺, Fe³⁺, La³⁺, Sn²⁺, Ca²⁺, Fe²⁺, Cu⁺, Al³⁺ and Zn²⁺) were prepared in deionized water. The absorption and fluorescence spectral data were measured in ACN/HEPES buffer (v/v, 3/7, 10 mM, pH = 7.4). The fluorescence spectral data were obtained at an excitation of 365 nm and the slit widths were set at 10 nm and 5 nm.

DFT calculations

Geometry optimization of PSH, [PSH-Al³⁺], and [BDNOL-Zn²⁺] was performed by the opt rb3lyp/6-311g (d,p) method using Gaussian 09 software.⁴⁹

Detection Al³⁺/Zn²⁺ in real water samples

Distilled water, tap water, and Xuanwu Lake water were used as the water samples without purification after standing for 10 h. The probe PSH (10 μM) was added to the ACN/water samples (v/v = 3/7) and then different concentrations of Al³⁺ and Zn²⁺ (0–15 μM) were added.

Detection of Al³⁺/Zn²⁺ on filter paper

The filter paper strips were first immersed in the solution of probe PSH (50 μM). After that, the test strips were taken out and dried in air and the treated strips were put into solutions of different concentrations of Al³⁺ or Zn²⁺ (0, 20, 50, 80, 100, 120, 150, 200 μM).

Cytotoxicity assay

The cytotoxicity of PSH to HeLa cells was obtained by the standard MTT method. HeLa cells were cultured in 96-well plates containing DMEM complete medium (containing 10% fetal bovine serum and 1% penicillin–streptomycin) under an environment of 37 °C and 5% carbon dioxide. After 24 h, different concentrations of compound PSH (0, 10, 20, 30 μM) were added to the 96-well plates and incubated for 24 h. The cells in each well were then treated with MTT solution (10 μL). Then the solutions in the well plates was taken out and 150 mL DMSO was added to each well. The OD value of each well was recorded with a microplate reader at a wavelength of 540 nm.

Cellular imaging

The probe PSH was dissolved in DMSO, and diluted with DMEM to obtain a final concentration of 10 μM. The HeLa cells were cultured in a 12-well plate containing 10 μM PSH. After 2 h, the HeLa cells were washed with PBS to wipe off the PSH on the cells’ surface, and then HeLa cells were incubated with Al³⁺ and Zn²⁺ (0, 10, 20, 30 μM) for 0.5 h. The treated cells were washed with PBS three times. The fluorescence imaging photos were acquired using a confocal laser scanning microscope (ZEISS LSM 710).

Bioimaging in zebrafish

Zebrafish experiments were conducted in accordance with the National Care and Use of Laboratory Animals by the National Animal Research Authority (China) and guidelines of Animal Care and Use issued by the Medical School of Southeast University Institutional Animal Care and Use Committee, and the experiments were approved by the Institutional Animal Care and Use Committee of the Medical School of Southeast University. The two-day-old zebrafish were obtained from Shanghai FishBio Co., Ltd. The zebrafish were fed in the embryo medium containing PSH (10 μM) to ensure that the zebrafish absorbed the probe into their tissues and organs. After 1 h, the zebrafish were washed with PBS and then incubated with 20 μM Al³⁺ and Zn²⁺ for 0.5 h. The fluorescence imaging photos of zebrafish were observed using a confocal laser scanning microscope from the blue channel (λ_ex = 405 nm, collected fluorescence wavelength: 480–580 nm).

Conclusion

In summary, a simple Schiff base probe PSH with the AIE feature was synthesized and characterized based on natural camphor. This probe showed strong yellow-green fluorescence at 535 nm due to its aggregation-induced emission (AIE) feature. The probe could detect both Al³⁺ and Zn²⁺ with high sensitivity and two different wavelength responses (500 nm and 555 nm) accompanied by the fluorescence intensity enhancement. The detection limits of PSH towards Al³⁺ and Zn²⁺ were calculated to be 12.1 nM and 14.2 nM, respectively. PSH exhibited excellent selectivity and anti-interference performance and could distinguish between Al³⁺/Zn²⁺ and identify whether Zn²⁺ existed in the PSH-Al³⁺ complex by adding ATP. The complexation mechanism of the PSH for Al³⁺ and Zn²⁺ based on the CHEF effect and the inhibition of the PET process was strongly proven by ¹H NMR, HRMS analysis, and density functional theory (DFT) calculations. Based on its outstanding sensing properties, the probe PSH was used to establish molecular logic function gates by using Al³⁺, Zn²⁺, and ATP as three chemical inputs. Moreover, probe PSH can be applied to detect Al³⁺ and Zn²⁺ in real environmental water, and the fluorescent detection was well demonstrated by test strips. Furthermore, probe PSH had good biocompatibility which was used for sensitive fluorescence images of Al³⁺ and Zn²⁺ in HeLa cells. The fluorescent probe PSH was also applied in bioimaging in zebrafish.

Author contributions

Shuai Gong: investigation, methodology, formal analysis, writing–original draft. Yan Zhang: investigation, methodology. Ahui Qin: investigation, formal analysis. Mingxin Li: formal analysis. Yu Gao: formal analysis. Chenglong Zhang: formal analysis. Jie Song: computational chemistry. Xu: resources. Zhonglong Wang: supervision, writing-reviewing and editing. Shifa Wang: funding acquisition, writing-review and editing.

Conflicts of interest

The authors declare no competing financial interest.

Acknowledgements

We acknowledge the financial assistance from the National Natural Science Foundation of China (No. 32071707).

References

1. M. B. Gumpu, S. Sethuraman, U. M. Krishnan and J. B. B. Rayappan, Sens. Actuators, B, 2015, 213, 515–533.
2. D. Li, K. Morimoto, T. Takeshita and Y. Lu, Environ. Health Prev. Med., 2001, 6, 27–32.
3. J. Labuda, K. Bubnicova, L. Kovalova, M. Vanickova, J. Mattusch and R. Wennrich, Sensors, 2005, 5, 411–423.
4. L. M. Plum, L. Rink and H. Haase, Int. J. Environ. Res. Public Health, 2010, 7, 1342–1365.
5. S. Erdemir and S. Malkondu, Sens. Actuators, B, 2013, 188, 1225–1229.
6. E. L. Que, D. W. Domaille and C. J. Chang, Chem. Rev., 2008, 108, 1517–1549.
7. C. Migliorini, E. Porciatti, M. Luczkowski and D. Valensin, Coord. Chem. Rev., 2012, 256, 352–368.
8. M. Jaishankar, T. Tseten, N. Anbalagan, B. B. Mathew and K. N. Beeregowda, Interdiscip. Toxicol., 2014, 7, 60–72.
9. J. Y. Jung, S. J. Han, J. Chun, C. Lee and J. Yoon, Dyes Pigm., 2012, 94, 423–426.
10. H. Liu, T. Liu, J. Li, Y. Zhang, J. Li, J. Song, J. Qu, W. Y. Wong and J. Mater, Chem. B, 2018, 6, 5435–5442.
11. G. Zhao, G. Wei, Z. Yan, B. Guo, S. Guang, R. Wu and H. Xu, Anal. Chim. Acta, 2020, 1095, 185–196.
12. J. Fu, Y. Chang, B. Li, H. Mei, L. Yang and K. Xu, Analyst, 2019, 144, 5706–5716.
13. R. Rahman and H. Upadhyaya, J. Plant Biol., 2020, 64, 101–121.
14. M. Ghaedi, A. Shokrollahi, K. Niknam and M. Soylak, Sep. Sci. Technol., 2009, 44, 773–786.
15. J. Heilmann, S. F. Boulyga and K. G. Heumann, J. Anal. At. Spectrom., 2009, 24, 385–390.
16. R. J. Cassella, O. I. Magalhaes, M. T. Couto, E. L. Lima, M. A. Neves and F. M. Coutinho, Talanta, 2005, 67, 121–128.
17. D. Omanović, C. Garnier, K. Gibbon-Walsh and I. Pižeta, Electrochem. Commun., 2015, 61, 78–83.
18. W. W. Zhao, J. J. Xu and H. Y. Chen, Analyst, 2016, 141, 4262–4271.
19. G. March, T. D. Nguyen and B. Piro, Biosensors, 2015, 5, 241–275.
20. C. F. Harrington, R. Clough, H. R. Hansen, S. J. Hill and J. F. Tyson, J. Anal. At. Spectrom., 2010, 25, 1185–1216.
21. Q. F. Niu, T. Sun, T. D. Li, Z. R. Guo and H. Pang, Sens. Actuators, B, 2018, 266, 730–743.
22. D. Udhayakumari, S. Velmathi, Y. M. Sung and S. P. Wu, Sens. Actuators, B, 2014, 198, 285–293.
23. Z. Chen, Y. Niu, G. Cheng, L. Tong, G. Zhang, F. Cai, T. Chen, B. Liu and B. Tang, Analyst, 2017, 142, 2781–2785.
24. J. Luo, Z. Xie, J. W. Lam, L. Cheng, H. Chen, C. Qiu, H. S. Kwok, X. Zhan, Y. Liu, D. Zhu and B. Z. Tang, Chem. Commun., 2001, 18, 1740–1741.
25. Y. Wang, Y. Qiu, A. Sun, Y. Xiong, H. Tan, Y. Shi, P. Yu, G. Roy, L. Zhang and J. Yan, Anal. Chim. Acta, 2020, 1133, 109–118.
26. S. M. Sayed, X.-F. Li, H.-R. Jia, S. Durrani, F.-G. Wu and X. Lu, Sens. Actuators, B, 2021, 343, 130128.
27. X. J. Sun, T. T. Liu, N. N. Li, S. Zeng and Z. Y. Xing, Spectrochim. Acta, Part A, 2020, 228, 117786.
28. T. T. Liu, J. Xu, C. G. Liu, S. Zeng, Z. Y. Xing, X. J. Sun and J. L. Li, J. Mol. Liq., 2020, 300, 112250.
29. K. Boonkitpatarakul, J. Wang, N. Niamnont, B. Liu, L. McDonald, Y. Pang and M. Sukwattanasinitt, ACS Sens., 2015, 1, 144–150.
30. J. Sun, B. Ye, G. Xia and H. Wang, Sens. Actuators, B, 2017, 249, 386–394.
31. S. Zeng, S. J. Li, X. J. Sun, T. T. Liu and Z. Y. Xing, Dyes Pigm., 2019, 170, 107642.
32. H. Zhang, T. Sun, Q. Ruan, J.-L. Zhao, L. Mu, X. Zeng, Z. Jin, S. Su, Q. Luo, Y. Yan and C. Redshaw, Dyes Pigm., 2019, 162, 257–265.
33. X. J. He, Q. Xie, J. Y. Fan, C. C. Xu, W. Xu, Y. H. Li, F. Ding, H. Deng, H. Chen and J. L. Shen, Dyes Pigm., 2020, 177, 108255.
34. W. Chen, I. Vermaak and A. Viljoen, Molecules, 2013, 18, 5434–5454.
35. F. Juteau, V. Masotti, J. M. Bessiere, M. Dherbomez and J. Viano, Fitoterapia, 2002, 73, 532–535.
36. B. Tirillini, E. R. Velasquez and R. Pellegrino, Planta Med., 1996, 62, 372–373.
37. A. Viljoen, S. van Vuuren, E. Ernst, M. Klepser, B. Demirci, K. H. C. Baser and B. E. van Wyk, J. Ethnopharmacol., 2003, 88, 137–143.
38. Y. Zhang, Z. L. Wang, J. Song, M. X. Li, Y. Q. Yang, X. Xu, H. J. Xu and S. F. Wang, Anal. Methods, 2019, 11, 3958–3965.
39. Z. L. Wang, Y. Zhang, J. Yin, M. X. Li, H. Luo, Y. Q. Yang, X. Xu, Q. Yong and S. F. Wang, Sens. Actuators, B, 2020, 320, 128249.
40. Z. Wang, Y. Zhang, J. Yin, Y. Yang, H. Luo, J. Song, X. Xu and S. Wang, ACS Sustainable Chem. Eng., 2020, 8, 12348–12359.
41. Y. Jeong and J. Yoon, Inorg. Chim. Acta, 2012, 381, 2–14.
42. S. Goswami, S. Das, K. Aich, D. Sarkar, T. K. Mondal, C. K. Quah and H. K. Fun, Dalton Trans., 2013, 42, 15113–15119.
43. L. Wang, W. Qin, X. Tang, W. Dou and W. Liu, J. Phys. Chem. A, 2011, 115, 1609–1616.
44. H. L. Lu, W. K. Wang, X. X. Tan, X. F. Luo, M. L. Zhang, M. Zhang and S. Q. Zang, Dalton Trans., 2016, 45, 8174–8181.
45. G. E. Jackson and K. V. Voyi, Polyhedron, 1987, 6, 2095–2098.
46. S. Gharami, K. Aich, L. Patra and T. K. Mondal, New J. Chem., 2018, 42, 8646–8652.
47. L. J. Daumann, K. E. Dalle, G. Schenk, R. P. McGeary, P. V. Bernhardt, D. L. Ollis and L. R. Gahan, Dalton Trans., 2012, 41, 1695–1708.
48. F. Wang, J. H. Moon, R. Nandhakumar, B. Kang, D. Kim, K. M. Kim, J. Y. Lee and J. Yoon, Chem. Commun., 2013, 49, 7228–7230.
49. M. J. Frisch, Gaussian 09, Revision A.1, Gaussian Incorp, Wallingford, CT, 2009.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/d1an01733k

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