A turn-on fluorescent chemical sensor based on imidazole-Schiff base structure for highly selective and accurate detection of Al3+ ions in living cells

A turn-on fluorescent chemical sensor based on imidazole-Schiff base structure for highly selective and accurate detection of Al3+ ions in living cells | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article A turn-on fluorescent chemical sensor based on imidazole-Schiff base structure for highly selective and accurate detection of Al3+ ions in living cells Jing Wang, Lu Ren, Yanqi Liu, Wanru Jia, Huihong Zhang, Dawei Zhang, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6114045/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract An imidazole-Schiff base fluorescent probe EBI was designed and synthesized for the sensitive and selective detection of Al 3+ . The probe EBI exhibits good anti-interference performance against Al 3+ in the presence of multiple metal ions. The reaction reached the platform in about 30 min after adding Al 3+ , and the fluorescence detection limit was 1.62 nM. The coordination ratio of probe EBI to Al 3+ is 1:1, and the coordination reaction between carbonyl group, hydroxyl group, –HN–N=C– and Al 3+ limits the molecular distortion and fluorescence quenching effect of photoelectron transfer, thus inducing the enhancement of green fluorescence. The probe can be recovered and used alternately by EDTA in the detection of Al 3+ . In addition, the fluorescence quenching of EBI–Al 3+ by PO 4 3− was also evident in the selected anion range. The probe was also applied to the successful monitoring Al 3+ and PO 4 3− in living cells. Turn-on Al3+ ions Imidazole-Schiff Fluorescence probe Cell imaging Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 1. Introduction Due to the rapid development of modern industry, the serious pollution of the environment by heavy metals is a significant issue. The threat posed to both the ecological environment and human health should not be underestimated [ 1 ]. As the most abundant metallic element in the Earth's crust, aluminum exhibits several advantages, including ease of processing, corrosion resistance, and user convenience [ 2 – 3 ]. Currently, it is widely utilized in various fields such as textiles, medicine, papermaking, food additives, and daily life [ 4 ]. However, as a non-essential element in the human body, aluminum exerts gradual and cumulative adverse effects on human health [ 5 ]. Excessive intake of aluminum ions can cause detrimental effects on multiple organ systems, including the central nervous system, kidneys, skeletal muscles, blood, and cardiovascular system [ 6 – 7 ]. It can also contribute to a spectrum of diseases, including Parkinson's disease, Alzheimer's disease, and osteoporosis [ 8 – 10 ]. Therefore, developing methods for the detection of aluminum ions in both environmental and biological systems is considered critically important [ 11 – 12 ]. With the introduction of chemical sensors, fluorescent chemical sensors based on the specific response of metal ions have been widely studied in the fields of chemistry, materials, biology, and environmental science. This is attributed to their advantages of low cost, ease of operation, high sensitivity and specificity, robust real-time monitoring capabilities, and rapid response times [ 13 – 15 ]. The strategy for qualitative and quantitative detection of metal ions, which involves the interaction between chemical sensors and metal ions leading to altered fluorescence intensity of the sensors, is considered an effective approach [ 16 ]. In recent years, there has been significant attention on the construction of fluorescent probes for detecting aluminum ions [ 17 ]. However, existing aluminum ion fluorescence sensors frequently fail to simultaneously achieve all ideal characteristics, including high sensitivity, high selectivity, strong anti-interference capability, and a significant Stokes shift [ 18 – 20 ]. Therefore, the development of new aluminum ion fluorescent probes that possess the adaptability, water solubility, sensitivity, and accuracy required in complex environments presents a significant challenge [ 21 – 22 ]. Extensive attention has been devoted to frameworks incorporating imidazole and Schiff base structures in numerous chemical sensors, attributed to their remarkable biological activity, catalytic performance, and fluorescence characteristics [ 23 – 25 ]. Due to the ease of adjusting their stereoelectronic structure, Schiff bases can readily coordinate with most transition metals. Consequently, they have found widespread application in constructing small-molecule fluorescence sensors, as well as in developing agents for molecular imaging and tools for biological studies [ 26 – 28 ]. On the other hand, imidazole is a planar, electron-deficient aromatic compound containing two nitrogen atoms, which exhibit distinct charge characteristics [ 29 – 30 ]. The ring and donor group with significant electron-withdrawing ability can facilitate intramolecular charge transfer (ICT) [ 31 – 32 ]. Excellent luminescent properties have been exhibited by most compounds based on the imidazole structure, and they have found widespread applications in the fields of fluorescence sensing and luminescent materials [ 33 – 35 ]. In this paper, imidazole-containing derivatives were used as fluorophores and Schiff base was introduced as a recognition group, based on the application of imidazole and Schiff base derivatives in fluorescent probes. The N' -(4-(diethylamino)-2-hydroxybenzylidene)-4- (4,5-diphenyl-1 H -imidazol-2-yl)benzohydrazide (EBI) sensor, which is a fluorescent molecular probe for aluminum ions at physiological pH, has been designed and synthesized. The fluorescence characteristics, including selectivity, anti-interference, detection limit, recycling, and application conditions were systematically studied. Simultaneously, theoretical calculations and fluorescence imaging experiments were conducted to investigate the mechanism of aluminum ion recognition. 2. Experimental 2.1 Materials and methods Methyl 4-acetylbenzoate, benzoyl, N , N -diethylsalicylaldehyde are all analytical grade reagents provided by Aladdin Reagent Co. Ltd. and they can be used without further purification. The nitrates of various metal ions (Cu 2+ , Ag + , Co 2+ , Zn 2+ , Pb 2+ , Al 3+ , Cr 3+ , K + , Ni 2+ , Mn 2+ , Mg 2+ , Fe 3+ , Fe 2+ , Cd 2+ , Hg 2+ , Na + , Ca 2+ , Ba 2+ , Sr 2+ ) are provided by Tianjin Damao Chemical Reagent Factory. The other solvents and reagents were supplied by Tianjin Fuyu Fine Chemical Co. Ltd. Melting point was determined with a WRS-2 melting point apparatus and was uncorrected. 1 H NMR and 13 C NMR spectra were recorded on Bruker-AVANCE-600 MHz spectrometer using TMS as an internal standard. The IR spectra were recorded on an IRAfnity-1 instrument with KBr disks. Mass spectra were determined on US Agilent1290-micrOTOF QII mass instrument. The UV-vis absorption spectra were measured on models TU-1100. The fluorescence spectra were recorded on F97XP fluorescence spectrophotometer. 2.2 General procedure for synthesis of probe EBI 4-(4,5-Diphenyl-1H-imidazol-2-yl)benzohydrazide (0.708 g, 2.0 mmol) and N , N diethyl salicylaldehyde (0.386 g, 2.0 mmol) were added to 10 ml of anhydrous ethanol. After the reaction was completed, it was cooled to room temperature. The crude product was obtained by extraction and filtration, followed by recrystallization with anhydrous ethanol to yield a yellow solid. This pure target product had a yield of 82%. Light yellow solid, m.p. = 290.2–291.8 o C; IR (KBr) ( v /cm − 1 ) 3447.6, 2972.4, 2832.0, 2716.3, 1592.6, 1362.3, 1240.5, 1135.4, 1065.6, 774.3; 1 H NMR (400 MHz, DMSO) δ (ppm) 11.86 (s, 1H), 11.48 (s, 1H), 11.19 (s, 1H), 8.70 (s, 1H), 8.22 (d, J = 8.3 Hz, 1H), 8.03 (d, J = 8.5 Hz, 1H), 7.74 (dd, J = 7.3, 2.2 Hz, 2H), 7.55 (dd, J = 14.9, 7.3 Hz, 3H), 7.47 (q, J = 5.8 Hz, 5H), 7.26–7.21 (m, 2H), 6.27 (td, J = 8.5, 2.1 Hz, 2H), 5.92 (d, J = 2.4 Hz, 1H), 3.41 (s, 4H), 1.12 (t, J = 7.0 Hz, 6H); 13 C NMR (151 MHz, DMSO) δ (ppm) 162.11, 161.58, 160.13, 150.58, 144.96, 135.34, 133.45, 132.74, 132.01, 129.50, 129.39, 129.11, 128.99, 128.91, 128.63, 128.41, 127.96, 127.51, 127.27, 125.32, 106.88, 104.07, 97.92, 44.20, 12.95; HRMS (EI): calcd for C 33 H 32 N 5 O 2 [M + H] + 530.2556, found 530.2472 (Fig. S1 –S4). 2.3. Preparation and spectroscopic determination of mixed ligands and metal ions The stock solution of various metal ions (1.0×10 − 3 mol/L chloride or nitrate salts) was prepared in deionized water. The stock solution of EBI (1.0×10 − 4 mol/L) was prepared in anhydrous ethanol and then diluted to the desired concentration with an EtOH/H 2 O solution (v/v = 1/1). In the titration experiment, 3.0 mL of EBI reserve solution is added to a 10 mL centrifuge tube, and an appropriate proportion of the ionic solution is added. The test solution required for detection is then thoroughly shaken. The test solution was then transferred to a quartz colorimetric dish, and UV-vis absorption and fluorescence spectra were determined at room temperature using 3.0 mL of the solution. The UV-vis absorption spectra of all analytical solutions were recorded in the range of 250–650 nm, while the fluorescence emission spectra were recorded in the range of 415–700 nm under excitation at 395 nm. 2.4. MTT assay and cell imaging In vitro cytotoxicity of probe EBI to HepG-2 cells was tested by MTT assay. HepG-2 cells were cultured in 96-well microplates using Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum. The culture was maintained at 37°C with 95% air humidity and 5% CO 2 for 24 h. Then the medium was replaced with fresh medium containing different concentrations of EBI (0, 10, 20, 30, 40, and 50 µM), and it continued to be cultured for 24 h. After the incubation period of 24 h, the cells were washed twice with PBS and subsequently incubated with MTT reagent at a concentration of 0.5 mg/mL for a duration of 4 h. The HepG-2 cells were incubated with DMEM solutions containing 50 µM EBI and EBI–Al 3+ for 30 min in a 37°C environment, which contained 95% air and 5% CO 2 . Then, the culture medium was removed, and the cells were washed three times with PBS to eliminate any drugs attached to the cell surface. Subsequently, cell fluorescence imaging was performed. 3. Results and discussion 3.1 Synthesis and characterizations The synthetic route of probe EBI is outlined in Scheme 1 . Compound 3 was synthesized according to previously similar procedures [ 36 ]. 4-(4,5-Diphenyl-1 H -imidazol-2- yl)benzohydrazide ( 4 ) is obtained by hydrazine hydrolysis of compound 3 and hydrazine hydrate, and then compound 4 and 4-(diethylamino)-2-hydroxybenzaldehyde ( 5 ) are stirred with ethanol at room temperature to form compound N' -(4-(diethylamino)-2-hydroxybenzylidene)-4-(4,5- diphenyl- 1 H -imidazol-2-yl)benzohydrazide (EBI). The synthesized probe EBI was confirmed by FT-IR, HR-MS, 1 H and 13 C NMR (See Supporting Information). 3.2 Selective recognition of probe EBI To explore the selective recognition performance of probe EBI for metal ions, experiments were carried out in an EtOH/H 2 O (1:1, v/v) using a UV-vis spectrophotometer and fluorescence spectrophotometer. Nineteen common metal ions were added to the solution of probe EBI. Figure 1 shows that after adding Al 3+ , the fluorescence intensity of EBI increased significantly. Correspondingly, with the exception of a slight enhancement of the fluorescence intensity by Zn 2+ and a blue shift by Cu 2+ , the fluorescence intensity of the EBI is almost unchanged with the addition of other ions. This phenomenon that can also be clearly observed with the naked eye under a 365 nm UV lamp. The addition of Al 3+ enhances the fluorescence of EBI, possibly due to a molecular structure wherein the N − N bond is rotated and the cavity formed by − C = O coordinates with Al 3+ . According to previous studies, probes containing amide and imide groups can coordinate with metal ions, especially Al 3+ [ 37 ]. The above experimental results show that the probe EBI has excellent selective recognition of Al 3+ . 3.3 Effects of coexisting metal ions on the selective recognition of probe EBI The presence of various metal ions in the environment may affect the accuracy of the probe. To investigate the effect of other metal ions on the recognition of Al 3+ by the probe EBI, interference experiments with coexisting metal ions were explored. The concentration of coexisting metal ions and Al 3+ is five equivalents higher than that of the probe EBI. As can be seen from Fig. 2 , the presence of Al 3+ significantly enhanced the fluorescence intensity of probe EBI, while the coexistence of other metal ions except Zn 2+ had no significant effect on the fluorescence intensity of probe EBI. While Zn 2+ can enhance the fluorescence intensity of the EBI probe, the fluorescence intensity decreases to a Zn 2+ absence state in the presence of Fe 3+ or Cr 3+ . This phenomenon can be used to easily distinguish between Al 3+ and Zn 2+ . The above experimental results show that the recognition ability of the probe EBI for Al 3+ is significantly higher than that of other metals, and the presence of other metals does not affect its recognition selectivity. 3.4. Al 3+ recognition sensitivity study of probe EBI Recognition sensitivity is an important parameter to determine whether a probe is suitable for use in real-world scenarios. In order to study the sensitivity of the probe EBI to identify Al 3+ , the relationship between different Al 3+ concentrations and the UV-vis and fluorescence intensities of the probe was studied. As shown in the Fig. 3 A, with the increasing Al 3+ concentration (0–100 µM), the fluorescence peak at 520 nm increases uniformly. The green fluorescence of the solution gradually brightens under a 365 nm UV lamp, and the intensity of fluorescence is increase by 29 times compared to that before the addition of aluminum ions. Ultraviolet-visible titration of probe EBI in ethanol/water with Al 3+ was performed, as shown in Fig. 3 B. The UV-vis spectrum of probe EBI has a distinct absorption band at 395 nm. With the increasing Al 3+ concentration, the absorption peak at 395 nm red-shifts to 418 nm, and two well-defined isoelectric points appear in the UV-vis spectrum, around 400 and 462 nm, respectively. The results showed that Al 3+ formed new species under the probe EBI. A Job plot was generated by recording the continuous change in fluorescence intensity during titration to determine the binding ratio between the probe EBI and Al 3+ . During the titration, Job’s plot of the probe EBI with Al 3+ was recorded from a continuous change in the intensity of the fluorescence peak at 520 nm (Fig. 4 A) to determine the binding ratio of the EBI to Al 3+ . The results confirmed that when the addition of Al 3+ was 100 µM, there was an inflection point in the Job’s plot, indicating that the coordination ratio of the probe EBI to Al 3+ was 1:1. The binding rate between the probe EBI and Al 3+ is shown in Fig. S5. According to the titration data, a good linear line is obtained by fitting the Benesi-Hildebrand equation, and the binding constants K a of the probe EBI and Al 3+ is 1.79×10 4 M − 1 (R 2 = 0.9997, Fig. S5). The detection limit for EBI selective identification of Al 3+ is calculated. As shown in Fig. 4 B, the titration curve is linear when the concentrations of Al 3+ are in the range of 0 − 50 µM. According to the formula LOD = 3 σ / k , the detection limit of the probe EBI for Al 3+ is calculated to be 1.62 nM (R 2 = 0.9934), which proves the high sensitivity of the probe EBI and its potential application in the detection of Al 3+ . 3.5. Study on the stability and applicable pH range of probe The fluorescence of EBI was quickly detected after adding 2 equivalents of Al 3+ , and it was found that the fluorescence intensity of the solution was rapidly enhanced and the reaction reached a stable state within 30 min and remained unchanged for at least 300 min (Fig. 5 A and Fig. S6). According to the kinetic equation Y = I-exp (A*x), the kinetic constant of EBI is 0.9882 min − 1 . The effect of pH value on the identification of Al 3+ by probe EBI was investigated. The fluorescence intensity of EBI in EtOH/H 2 O solution (1:1, v/v, 100 µM, pH 7.4, λ em = 520 nm) has little change in the range of pH = 1 − 12. After adding 2 equivalents of Al 3+ , the fluorescence has large enhancement in pH range 5.0–8.0, indicating that EBI has excellent recognition performance for Al 3+ in the physiological pH range. 3.6. Study on circularity and competition of EBI Reusability is one of the important indexes to evaluate the performance of coordinated sensors. EDTA is a common ligand used to evaluate the recyclability of EBI to detect Al 3+ . After adding Al 3+ , the fluorescence intensity of EBI increased at λ em = 520 nm. Then, after adding EDTA, the fluorescence emission intensity significantly returned to the original value, and the prepared probe could be easily regenerated by EDTA (Fig. 6 A). The above phenomenon can be repeated for at least 5 cycles, indicating that the probe EBI exhibits excellent reversibility in recognizing Al 3+ (Fig. S7). The influence of anions on the detection of Al 3+ by probe EBI was also evaluated. The fluorescence response intensity of EBI–Al 3+ was measured by adding various anions (PO 4 3− , HPO 4 2− , H 2 PO 4 − , SO 4 2− , ClO − , F − , Cl − , Br − , I − , Ac − , NO 3 − , CO 3 2− , HCO 3 − , S 2 O 3 2− and S 2− ) into the system (Fig. 6 B). First, none of the anions in the above test range significantly changed the fluorescence intensity of EBI in the absence of Al 3+ . In the presence of PO 4 3− , the fluorescence intensity of EBI–Al 3+ shows obvious quenching, while other anions have no obvious quenching phenomenon (Fig. 6 C). In particular, the presence of F − leads to the enhancement of fluorescence intensity of EBI–Al 3+ , which may be the formation of hydrogen bonding complexes between F − and the –NH group of EBI imidazole ring [ 38 ]. 3.7. Binding model and theoretical calculation Here, in order to clarify the detection method of probe EBI for Al 3+ , the reaction products of EBI were determined by FT-IR and 1 H NMR titration (Fig. S8 and Fig. S9). After Al 3+ is added to the solution of EBI, the hydrogen proton signals at 11.19 ppm and 11.48 ppm attributed to hydroxyl and amino groups are disappeared, which indicates that hydroxyl and amino groups participate in coordination with Al 3+ . In addition, the proton of –N–N = CH– changes from 8.70 ppm to 8.22 ppm, and the aromatic proton also shows a certain chemical shift change. The complexation ratio of probe EBI to Al 3+ was 1:1 based on the Job’s plot data. In summary, the reaction mechanism of EBI and Al 3+ is speculated as follows: carbonyl group, hydroxyl group and –HN–N = CH– of EBI are coordinated with Al 3+ , supporting the hypothesis that EBI is an "O–N–O" type ligand (Fig. 7 ) [ 39 ]. The fluorescence of probe EBI was enhanced by 29 times after ion recognition. To have a deeper insight into the relationship between the structural changes of probe EBI on its complexation with Al 3+ and the optical response of EBI to Al 3+ , time-dependent density functional theory (TD-DFT) calculations with the B3LYP/6-31G** methods are carried out using the Gaussian 16 program. The optimized geometry and the highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) of the probes EBI and EBI–Al 3+ are shown in Fig. 8 . The molecular optimization of TD-DFT shows that the carbonyl phenylimidazole of EBI has a dihedral Angle with the amine hydroxyl group. The molecule of EBI mainly shows charge transfer from the phenylimidazole ring to the N , N diethylaminosalicylaldehyde. The main energy transfer is from HOMO to LUMO with a 3.455 eV of energy gap ( Δ ). To EBI–Al 3+ , the molecule exhibits high planarity because of the complex. EBI–Al 3+ exhibits main energy transfer from HOMO to LUMO + 1 with 1.676 eV of energy gap ( Δ ). The calculated energy gap between HOMO and LUMO of EBI is higher (3.455 eV) than EBI–Al 3+ (1.357 eV) (Fig. S10, Table S1 ). Therefore, the complexation of EBI with Al 3+ is energetically advantageous. These results show that the interaction between Al 3+ and probe EBI effectively reduces the HOMO-LUMO bandgap of EBI–Al 3+ , and the proposed binding mode of probe EBI and Al 3+ is confirmed by theoretical calculations. 3.8. Test strips experiment In order to explore the practical application of probe EBI, strip tests based on filter paper were carried out. Firstly, the test paper was immersed in EBI (1×10 − 3 mol/L) ethanol solution, and then naturally dried to make a variety of metal ion detection test paper. The test strip showed a weak blue-green fluorescence emission under 365 nm light irradiation. After the addition of excessive Al 3+ , the fluorescence color of the test strip showed bright green. Meanwhile, the fluorescent color of the test strips treated with excessive Zn 2+ was dark green, while the fluorescent color of the test strips treated with other ions did not change significantly (Fig. 8 A). Figure 8 B shows the test strips with different concentrations of Al 3+ under 365 nm UV lamp. It can be observed that with the increase of Al 3+ concentration, the bright green fluorescence of the test strip becomes more obvious. The results show that the probe EBI can detect Al 3+ by a simple dipping paper method with only a portable UV lamp. 3.9. Cell imaging It is of great significance to study the biological application of probe systems. Firstly, the cytotoxicity of probe EBI was measured by MTT method. As shown in Fig. S11, the viability of HepG-2 cells was greater than 90% at an experimental concentration of 50 µM, indicating that EBI has low cytotoxicity and can be used for biological imaging. Subsequently, the feasibility of EBI and EBI–Al 3+ for live cell imaging was evaluated using confocal fluorescence microscopy (Fig. 9 ). After incubating with probe EBI at 37°C for 30 min, the HepG-2 cells emit extremely weak green fluorescence under a fluorescence microscope. However, when the HepG-2 cells were incubated with EBI and Al 3+ at 37°C for 30 min, they showed significant green fluorescence under a fluorescence microscope. Then PO 4 3− was added to the HepG-2 cell system of EBI–Al 3+ and incubated at 37°C for 30 min. The quenching of green fluorescence was obviously observed under fluorescence microscope. These results suggest that probe EBI can penetrate cell membranes to image Al 3+ and PO 4 3− in living cells. 4. Conclusions In summary, a turn-on fluorescence sensing material EBI based on the imidazole-Schiff base structure has been designed and synthesized. EBI has high sensitivity, selectivity and anti-interference to Al 3+ , which is superior to other competing metal ions. EBI reaction reached the platform about 30 min after adding Al 3+ , and the fluorescence detection limit was 1.62 nM. The induction mechanism of probe EBI to Al 3+ is thought to be due to the coordination between carbonyl group, hydroxyl group, –HN–N = C– and Al 3+ . In addition, the probe EBI can be made into test paper to quickly identify Al 3+ . Cytotoxicity and cell imaging results showed that EBI probes have low toxicity and can penetrate cell membranes to image Al 3+ in living cells. Declarations Acknowledgements The work was supported by "the State Key Laboratory of Fine Chemicals, Dalian University of Technology" (KF2212). The authors acknowledge the assistance of JLICT Center of Characterization and Analysis Authors' contributions JW done investigation; validation; and writing-original draft preparation. YQL done the synthesis and characterization of compounds. WRJ and HHZ done metal ion detection. LR performed fluorescence imaging of metal ions in living cells of the compound. DWZ did project administration, supervised the work, revised, and completed the manuscript. YLZ was responsible for the time-dependent density functional theory (TD-DFT) calculations of the compound and the revision of the manuscript. All authors reviewed the manuscript. Funding Not applicable. Availability of data and materials The data and materials that support the findings of this study are available in the supplementary material of this article. Confict of interest The authors declare that they have no known competing fnancial interests or personal relationships that could have appeared to infuence the work reported in this paper. Ethical approval This declaration is not applicable. References G. Asaithambi, V. Periasamy, Res. Chem. Intermediat. 45, 1295–1308 (2019) H. Peng, Y. Han, N. Lin, H. Liu, Opt. Mater. 95, 109210 (2019) X. Pan, J. Jiang, J. Li, W. Wu, J. Zhang, Inorg. Chem. 58, 12618–12627 (2019) G. Yang, P. Li, Y. Han, L. Tang, Y. Liu, H. Xin et al., Mater. Chem. Phys. 295, 127145 (2023) A.A. Unar, T.G. Kazi, H.I. Afridi, J.A. Baig, A.A. Lashari, Talanta 273, 125847 (2024) P. Yin, Q. Niu, J. Liu, T. Wei, T. Hu, T. Li et al., Sens. Actuators B Chem. 331, 129418 (2021) F.F. Guo, B.B. Wang, W.N. Wu, W.Y. 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Phys. 295, 127145 (2023) Schemes Scheme 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Onlinefloatimage3.png Scheme 1. The synthetic route of probe EBI. Onlinefloatimage2.png Graphical Abstract: An imidazole-Schiff base fluorescent probe EBI was designed and synthesized for the sensitive and selective detection of Al 3+ . The probe EBI exhibits good anti-interference performance against Al 3+ in the presence of multiple metal ions. the fluorescence quenching of EBI–Al 3+ by PO 4 3− was also evident in the selected anion range. The probe was applied to the successful monitoring Al 3+ and PO 4 3− in living cells. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6114045","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":422152298,"identity":"a43bd5c8-7a5c-4f35-b49e-aa26160275e6","order_by":0,"name":"Jing Wang","email":"","orcid":"","institution":"Jilin Institute of Chemical Technology","correspondingAuthor":false,"prefix":"","firstName":"Jing","middleName":"","lastName":"Wang","suffix":""},{"id":422152299,"identity":"fef429e1-ec9a-46f0-a539-8e83ad3709e5","order_by":1,"name":"Lu Ren","email":"","orcid":"","institution":"Jilin Institute of Chemical Technology","correspondingAuthor":false,"prefix":"","firstName":"Lu","middleName":"","lastName":"Ren","suffix":""},{"id":422152300,"identity":"0890a544-4910-4e34-a23b-9137899b7965","order_by":2,"name":"Yanqi Liu","email":"","orcid":"","institution":"Jilin Institute of Chemical Technology","correspondingAuthor":false,"prefix":"","firstName":"Yanqi","middleName":"","lastName":"Liu","suffix":""},{"id":422152301,"identity":"79cafcd0-a5d1-4365-9901-de33c45c3196","order_by":3,"name":"Wanru Jia","email":"","orcid":"","institution":"Jilin Institute of Chemical Technology","correspondingAuthor":false,"prefix":"","firstName":"Wanru","middleName":"","lastName":"Jia","suffix":""},{"id":422152302,"identity":"2d7e4407-b241-46b3-8df2-d9e245144129","order_by":4,"name":"Huihong Zhang","email":"","orcid":"","institution":"Jilin Institute of Chemical Technology","correspondingAuthor":false,"prefix":"","firstName":"Huihong","middleName":"","lastName":"Zhang","suffix":""},{"id":422152303,"identity":"0e137021-e1fb-4f14-842f-2226a2f36984","order_by":5,"name":"Dawei Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/0lEQVRIiWNgGAWjYDACCRDBBsTMzAcMHhiAxQyI1cKWYJBgAOYTq4WBx4AhgYEILfKzm589/FJml9jPzvOhIKHAro6BvXmbBEPNHZxaGOccMzeWOZecOLOZdwPQYckSDDzHyiQYjj3DqYVZIsFMWrKNOXfDYbCWAxIMEjlmEowNh3FqYZNI/wbUUp+7/zDPA4gW+Tf4tfAAzZT82HY4dwMzDwPUFh78WiQkcsqkGc4dr59xmM0A5BfJNp60YouEY7i1yM9I3yb5o6zamL//8DODD3/s+PnZD2+88aEGtxZwEPBA/QWOD3AcJeDVAAzoH1CtDwgoHAWjYBSMghEKAGjGTEdfPwUOAAAAAElFTkSuQmCC","orcid":"","institution":"Jilin Institute of Chemical Technology","correspondingAuthor":true,"prefix":"","firstName":"Dawei","middleName":"","lastName":"Zhang","suffix":""},{"id":422152304,"identity":"ed1152dd-ff01-4172-8669-7a948fb6b860","order_by":6,"name":"Yiliang Zhang","email":"","orcid":"","institution":"Jilin University","correspondingAuthor":false,"prefix":"","firstName":"Yiliang","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2025-02-26 14:23:24","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6114045/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6114045/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":77632173,"identity":"c29930d2-1b56-4f08-a69f-ff1cb66743ee","added_by":"auto","created_at":"2025-03-03 17:49:44","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":662682,"visible":true,"origin":"","legend":"\u003cp\u003eThe fluorescence (A) and UV-vis (B) responses of EBI (100 µM) upon addition of various metal ions (100 µM) in EtOH/H\u003csub\u003e2\u003c/sub\u003eO (1:1, v/v) solution (λ\u003csub\u003eex\u003c/sub\u003e=395 nm), (C) The color change of various metal ions after being added to probe EBI (100 µM) EtOH/H\u003csub\u003e2\u003c/sub\u003eO (1/1, v/v) solution under 365 nm UV lamp.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6114045/v1/5c1f9ebf9c206f4e09cb047c.jpeg"},{"id":77631958,"identity":"e1523715-4134-48a8-8b17-e50ff9b38226","added_by":"auto","created_at":"2025-03-03 17:41:44","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":16115,"visible":true,"origin":"","legend":"\u003cp\u003eThe fluorescence responses of EBI (100 µM) upon addition of various metal ions (100 µM) in EtOH/H\u003csub\u003e2\u003c/sub\u003eO (1:1, v/v) solution (λ\u003csub\u003eex\u003c/sub\u003e=395 nm).\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-6114045/v1/d77af67f1873680cff6f8339.png"},{"id":77631959,"identity":"a88ffb40-7fbb-4e9b-ae5e-52dd6d3bbb8b","added_by":"auto","created_at":"2025-03-03 17:41:44","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":459595,"visible":true,"origin":"","legend":"\u003cp\u003eThe fluorescence (A) and UV-vis (B) responses of EBI (100 µM) in EtOH/H\u003csub\u003e2\u003c/sub\u003eO (1:1, v/v) upon the addition of Al\u003csup\u003e3+\u003c/sup\u003e (λ\u003csub\u003eex\u003c/sub\u003e=395 nm).\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6114045/v1/6a41a401fb5e4fac86666312.jpeg"},{"id":77633477,"identity":"8e8cbd96-6239-4ea3-a5e1-7e2a1539c257","added_by":"auto","created_at":"2025-03-03 17:57:47","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":193581,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Job’s plot for probe EBI (100 μM) and Al\u003csup\u003e3+\u003c/sup\u003e complexation in EtOH/H\u003csub\u003e2\u003c/sub\u003eO (1/1, v/v); (B) The linear fit between EBI with Al\u003csup\u003e3+\u003c/sup\u003e.\u003c/p\u003e","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6114045/v1/30e5eac215b2f109bbed81a1.jpeg"},{"id":77632178,"identity":"7ec57f15-ab9d-4625-b862-b48e335b582f","added_by":"auto","created_at":"2025-03-03 17:49:44","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":248185,"visible":true,"origin":"","legend":"\u003cp\u003eSpectral response, fluorescence response kinetic curve (A), and pH dependence curve (B) of probe EBI (100 μM) to Al\u003csup\u003e3+\u003c/sup\u003e.\u003c/p\u003e","description":"","filename":"floatimage8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6114045/v1/f7d6ae6790f8b76321e6ac62.jpeg"},{"id":77631963,"identity":"ef0eea3e-cb6c-428c-8b9b-a7913f459554","added_by":"auto","created_at":"2025-03-03 17:41:44","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":678419,"visible":true,"origin":"","legend":"\u003cp\u003eFluorescence response of ethanol solution of EBI (100 μM) and Al\u003csup\u003e3+\u003c/sup\u003e (100 μM) upon addition of EDTA (100 μM) (A) and various anions (100 μM) (B) with an excitation at 395 nm, and color change under 365 nm UV lamp (C).\u003c/p\u003e","description":"","filename":"floatimage9.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6114045/v1/8c56f8289ebd534975ede84e.jpeg"},{"id":77632175,"identity":"dbe21a99-8d62-476f-83e1-759a3a8a8b00","added_by":"auto","created_at":"2025-03-03 17:49:44","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":18206,"visible":true,"origin":"","legend":"\u003cp\u003eProposed structures of EBI–Al\u003csup\u003e3+\u003c/sup\u003e complexes\u003c/p\u003e","description":"","filename":"floatimage10.png","url":"https://assets-eu.researchsquare.com/files/rs-6114045/v1/d9c673cd18cb9a561feb0b69.png"},{"id":77632176,"identity":"c9f6bc6b-dc32-4b79-992f-02cbb0bf5893","added_by":"auto","created_at":"2025-03-03 17:49:44","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":157554,"visible":true,"origin":"","legend":"\u003cp\u003eOptimized molecular configuration and frontier orbitals of EBI and EBI–Al\u003csup\u003e3+\u003c/sup\u003e.\u003c/p\u003e","description":"","filename":"floatimage11.png","url":"https://assets-eu.researchsquare.com/files/rs-6114045/v1/b74c5b32117eb261785d54ef.png"},{"id":77631962,"identity":"f460ed1e-a64a-409b-8926-7d54ada16342","added_by":"auto","created_at":"2025-03-03 17:41:44","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":109348,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. 8 \u003c/strong\u003eThe photographs of EBI based test strips upon addition of Al\u003csup\u003e3+\u003c/sup\u003e and other ions under UV lamp at 365 nm (A) and different concentrations of Al\u003csup\u003e3+\u003c/sup\u003e under 365 nm UV lamp (B).\u003c/p\u003e","description":"","filename":"floatimage12.png","url":"https://assets-eu.researchsquare.com/files/rs-6114045/v1/dc5127122f07b87992a8b108.png"},{"id":77632177,"identity":"eeece54f-bb9d-4b7d-8019-c806270aef61","added_by":"auto","created_at":"2025-03-03 17:49:44","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":271241,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. 9\u003c/strong\u003e Fluorescence microscope image of HepG-2 cells. (A) Fluorescent image of HepG-2 cells with 50 μM EBI for 30 min; (B) Fluorescence images of HepG-2 cells incubated with 50 μM EBI–Al\u003csup\u003e3+\u003c/sup\u003e for 30 min at 37°C; (C) Fluorescence images of HepG-2 cells incubated with 50 μM EBI–Al\u003csup\u003e3+\u003c/sup\u003e–PO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e3−\u003c/sup\u003e for 30 min at 37°C.\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-6114045/v1/f55b425b8ff1017b9a0ff3fd.png"},{"id":80821432,"identity":"51b50e44-00de-40fe-822b-7cfea57d0c7b","added_by":"auto","created_at":"2025-04-17 12:17:06","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3629287,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6114045/v1/78ec8bbe-e2ac-43c6-88dd-a92c9f4712ae.pdf"},{"id":77631955,"identity":"5e6bdc1a-7582-4cec-a181-12cb4604c69e","added_by":"auto","created_at":"2025-03-03 17:41:43","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":29348,"visible":true,"origin":"","legend":"\u003cp\u003eScheme 1. The synthetic route of probe EBI.\u003c/p\u003e","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6114045/v1/60630a961f48b0f4156eee40.png"},{"id":77631954,"identity":"6b91a128-5082-4719-89f5-ae68bebfa911","added_by":"auto","created_at":"2025-03-03 17:41:43","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":24510,"visible":true,"origin":"","legend":"\u003cp\u003eGraphical Abstract:\u003c/p\u003e\n\u003cp\u003eAn imidazole-Schiff base fluorescent probe EBI was designed and synthesized for the sensitive and selective detection of Al\u003csup\u003e3+\u003c/sup\u003e. The probe EBI exhibits good anti-interference performance against Al\u003csup\u003e3+\u003c/sup\u003e in the presence of multiple metal ions. the fluorescence quenching of EBI–Al\u003csup\u003e3+\u003c/sup\u003e by PO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e3−\u003c/sup\u003e was also evident in the selected anion range. The probe was applied to the successful monitoring Al\u003csup\u003e3+\u003c/sup\u003e and PO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e3−\u003c/sup\u003e in living cells.\u003c/p\u003e","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6114045/v1/4a1b749a369692a0b0222c7b.png"},{"id":77631969,"identity":"67a4c342-9e1c-4627-a142-0ff020fca170","added_by":"auto","created_at":"2025-03-03 17:41:44","extension":"doc","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":5632512,"visible":true,"origin":"","legend":"","description":"","filename":"EBISupportingInformationResearchonChemicalIntermediates.doc","url":"https://assets-eu.researchsquare.com/files/rs-6114045/v1/3ffad3c3d26ba294edb7b5d5.doc"}],"financialInterests":"No competing interests reported.","formattedTitle":"A turn-on fluorescent chemical sensor based on imidazole-Schiff base structure for highly selective and accurate detection of Al3+ ions in living cells","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eDue to the rapid development of modern industry, the serious pollution of the environment by heavy metals is a significant issue. The threat posed to both the ecological environment and human health should not be underestimated [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. As the most abundant metallic element in the Earth's crust, aluminum exhibits several advantages, including ease of processing, corrosion resistance, and user convenience [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Currently, it is widely utilized in various fields such as textiles, medicine, papermaking, food additives, and daily life [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. However, as a non-essential element in the human body, aluminum exerts gradual and cumulative adverse effects on human health [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Excessive intake of aluminum ions can cause detrimental effects on multiple organ systems, including the central nervous system, kidneys, skeletal muscles, blood, and cardiovascular system [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. It can also contribute to a spectrum of diseases, including Parkinson's disease, Alzheimer's disease, and osteoporosis [\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Therefore, developing methods for the detection of aluminum ions in both environmental and biological systems is considered critically important [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWith the introduction of chemical sensors, fluorescent chemical sensors based on the specific response of metal ions have been widely studied in the fields of chemistry, materials, biology, and environmental science. This is attributed to their advantages of low cost, ease of operation, high sensitivity and specificity, robust real-time monitoring capabilities, and rapid response times [\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The strategy for qualitative and quantitative detection of metal ions, which involves the interaction between chemical sensors and metal ions leading to altered fluorescence intensity of the sensors, is considered an effective approach [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. In recent years, there has been significant attention on the construction of fluorescent probes for detecting aluminum ions [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. However, existing aluminum ion fluorescence sensors frequently fail to simultaneously achieve all ideal characteristics, including high sensitivity, high selectivity, strong anti-interference capability, and a significant Stokes shift [\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Therefore, the development of new aluminum ion fluorescent probes that possess the adaptability, water solubility, sensitivity, and accuracy required in complex environments presents a significant challenge [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Extensive attention has been devoted to frameworks incorporating imidazole and Schiff base structures in numerous chemical sensors, attributed to their remarkable biological activity, catalytic performance, and fluorescence characteristics [\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Due to the ease of adjusting their stereoelectronic structure, Schiff bases can readily coordinate with most transition metals. Consequently, they have found widespread application in constructing small-molecule fluorescence sensors, as well as in developing agents for molecular imaging and tools for biological studies [\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. On the other hand, imidazole is a planar, electron-deficient aromatic compound containing two nitrogen atoms, which exhibit distinct charge characteristics [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The ring and donor group with significant electron-withdrawing ability can facilitate intramolecular charge transfer (ICT) [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Excellent luminescent properties have been exhibited by most compounds based on the imidazole structure, and they have found widespread applications in the fields of fluorescence sensing and luminescent materials [\u003cspan additionalcitationids=\"CR34\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this paper, imidazole-containing derivatives were used as fluorophores and Schiff base was introduced as a recognition group, based on the application of imidazole and Schiff base derivatives in fluorescent probes. The \u003cem\u003eN'\u003c/em\u003e-(4-(diethylamino)-2-hydroxybenzylidene)-4- (4,5-diphenyl-1\u003cem\u003eH\u003c/em\u003e-imidazol-2-yl)benzohydrazide (EBI) sensor, which is a fluorescent molecular probe for aluminum ions at physiological pH, has been designed and synthesized. The fluorescence characteristics, including selectivity, anti-interference, detection limit, recycling, and application conditions were systematically studied. Simultaneously, theoretical calculations and fluorescence imaging experiments were conducted to investigate the mechanism of aluminum ion recognition.\u003c/p\u003e"},{"header":"2. Experimental","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Materials and methods\u003c/h2\u003e \u003cp\u003eMethyl 4-acetylbenzoate, benzoyl, \u003cem\u003eN\u003c/em\u003e, \u003cem\u003eN\u003c/em\u003e-diethylsalicylaldehyde are all analytical grade reagents provided by Aladdin Reagent Co. Ltd. and they can be used without further purification. The nitrates of various metal ions (Cu\u003csup\u003e2+\u003c/sup\u003e, Ag\u003csup\u003e+\u003c/sup\u003e, Co\u003csup\u003e2+\u003c/sup\u003e, Zn\u003csup\u003e2+\u003c/sup\u003e, Pb\u003csup\u003e2+\u003c/sup\u003e, Al \u003csup\u003e3+\u003c/sup\u003e, Cr \u003csup\u003e3+\u003c/sup\u003e, K\u003csup\u003e+\u003c/sup\u003e, Ni \u003csup\u003e2+\u003c/sup\u003e, Mn\u003csup\u003e2+\u003c/sup\u003e, Mg\u003csup\u003e2+\u003c/sup\u003e, Fe\u003csup\u003e3+\u003c/sup\u003e, Fe\u003csup\u003e2+\u003c/sup\u003e, Cd\u003csup\u003e2+\u003c/sup\u003e, Hg\u003csup\u003e2+\u003c/sup\u003e, Na\u003csup\u003e+\u003c/sup\u003e, Ca\u003csup\u003e2+\u003c/sup\u003e, Ba\u003csup\u003e2+\u003c/sup\u003e, Sr\u003csup\u003e2+\u003c/sup\u003e) are provided by Tianjin Damao Chemical Reagent Factory. The other solvents and reagents were supplied by Tianjin Fuyu Fine Chemical Co. Ltd. Melting point was determined with a WRS-2 melting point apparatus and was uncorrected. \u003csup\u003e1\u003c/sup\u003eH NMR and \u003csup\u003e13\u003c/sup\u003eC NMR spectra were recorded on Bruker-AVANCE-600 MHz spectrometer using TMS as an internal standard. The IR spectra were recorded on an IRAfnity-1 instrument with KBr disks. Mass spectra were determined on US Agilent1290-micrOTOF QII mass instrument. The UV-vis absorption spectra were measured on models TU-1100. The fluorescence spectra were recorded on F97XP fluorescence spectrophotometer.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 General procedure for synthesis of probe EBI\u003c/b\u003e\u003c/h2\u003e \u003cp\u003e4-(4,5-Diphenyl-1H-imidazol-2-yl)benzohydrazide (0.708 g, 2.0 mmol) and \u003cem\u003eN\u003c/em\u003e, \u003cem\u003eN\u003c/em\u003e diethyl salicylaldehyde (0.386 g, 2.0 mmol) were added to 10 ml of anhydrous ethanol. After the reaction was completed, it was cooled to room temperature. The crude product was obtained by extraction and filtration, followed by recrystallization with anhydrous ethanol to yield a yellow solid. This pure target product had a yield of 82%.\u003c/p\u003e \u003cp\u003eLight yellow solid, m.p. = 290.2\u0026ndash;291.8 \u003csup\u003eo\u003c/sup\u003eC; IR (KBr) (\u003cem\u003ev\u003c/em\u003e/cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) 3447.6, 2972.4, 2832.0, 2716.3, 1592.6, 1362.3, 1240.5, 1135.4, 1065.6, 774.3; \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO) \u003cem\u003eδ\u003c/em\u003e (ppm) 11.86 (s, 1H), 11.48 (s, 1H), 11.19 (s, 1H), 8.70 (s, 1H), 8.22 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.3 Hz, 1H), 8.03 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.5 Hz, 1H), 7.74 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.3, 2.2 Hz, 2H), 7.55 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;14.9, 7.3 Hz, 3H), 7.47 (q, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.8 Hz, 5H), 7.26\u0026ndash;7.21 (m, 2H), 6.27 (td, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.5, 2.1 Hz, 2H), 5.92 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2.4 Hz, 1H), 3.41 (s, 4H), 1.12 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.0 Hz, 6H); \u003csup\u003e13\u003c/sup\u003eC NMR (151 MHz, DMSO) \u003cem\u003eδ\u003c/em\u003e (ppm) 162.11, 161.58, 160.13, 150.58, 144.96, 135.34, 133.45, 132.74, 132.01, 129.50, 129.39, 129.11, 128.99, 128.91, 128.63, 128.41, 127.96, 127.51, 127.27, 125.32, 106.88, 104.07, 97.92, 44.20, 12.95; HRMS (EI): calcd for C\u003csub\u003e33\u003c/sub\u003eH\u003csub\u003e32\u003c/sub\u003eN\u003csub\u003e5\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e 530.2556, found 530.2472 (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u0026ndash;S4).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Preparation and spectroscopic determination of mixed ligands and metal ions\u003c/h2\u003e \u003cp\u003eThe stock solution of various metal ions (1.0\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e mol/L chloride or nitrate salts) was prepared in deionized water. The stock solution of EBI (1.0\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e mol/L) was prepared in anhydrous ethanol and then diluted to the desired concentration with an EtOH/H\u003csub\u003e2\u003c/sub\u003eO solution (v/v\u0026thinsp;=\u0026thinsp;1/1). In the titration experiment, 3.0 mL of EBI reserve solution is added to a 10 mL centrifuge tube, and an appropriate proportion of the ionic solution is added. The test solution required for detection is then thoroughly shaken. The test solution was then transferred to a quartz colorimetric dish, and UV-vis absorption and fluorescence spectra were determined at room temperature using 3.0 mL of the solution. The UV-vis absorption spectra of all analytical solutions were recorded in the range of 250\u0026ndash;650 nm, while the fluorescence emission spectra were recorded in the range of 415\u0026ndash;700 nm under excitation at 395 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. MTT assay and cell imaging\u003c/h2\u003e \u003cp\u003eIn vitro cytotoxicity of probe EBI to HepG-2 cells was tested by MTT assay. HepG-2 cells were cultured in 96-well microplates using Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum. The culture was maintained at 37\u0026deg;C with 95% air humidity and 5% CO\u003csub\u003e2\u003c/sub\u003e for 24 h. Then the medium was replaced with fresh medium containing different concentrations of EBI (0, 10, 20, 30, 40, and 50 \u0026micro;M), and it continued to be cultured for 24 h. After the incubation period of 24 h, the cells were washed twice with PBS and subsequently incubated with MTT reagent at a concentration of 0.5 mg/mL for a duration of 4 h.\u003c/p\u003e \u003cp\u003eThe HepG-2 cells were incubated with DMEM solutions containing 50 \u0026micro;M EBI and EBI\u0026ndash;Al\u003csup\u003e3+\u003c/sup\u003e for 30 min in a 37\u0026deg;C environment, which contained 95% air and 5% CO\u003csub\u003e2\u003c/sub\u003e. Then, the culture medium was removed, and the cells were washed three times with PBS to eliminate any drugs attached to the cell surface. Subsequently, cell fluorescence imaging was performed.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and discussion","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Synthesis and characterizations\u003c/h2\u003e \u003cp\u003eThe synthetic route of probe EBI is outlined in Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Compound \u003cb\u003e3\u003c/b\u003e was synthesized according to previously similar procedures [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. 4-(4,5-Diphenyl-1\u003cem\u003eH\u003c/em\u003e-imidazol-2- yl)benzohydrazide (\u003cb\u003e4\u003c/b\u003e) is obtained by hydrazine hydrolysis of compound \u003cb\u003e3\u003c/b\u003e and hydrazine hydrate, and then compound 4 and 4-(diethylamino)-2-hydroxybenzaldehyde (\u003cb\u003e5\u003c/b\u003e) are stirred with ethanol at room temperature to form compound \u003cem\u003eN'\u003c/em\u003e-(4-(diethylamino)-2-hydroxybenzylidene)-4-(4,5- diphenyl- 1\u003cem\u003eH\u003c/em\u003e-imidazol-2-yl)benzohydrazide (EBI). The synthesized probe EBI was confirmed by FT-IR, HR-MS, \u003csup\u003e1\u003c/sup\u003eH and \u003csup\u003e13\u003c/sup\u003eC NMR (See Supporting Information).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Selective recognition of probe EBI\u003c/h2\u003e \u003cp\u003eTo explore the selective recognition performance of probe EBI for metal ions, experiments were carried out in an EtOH/H\u003csub\u003e2\u003c/sub\u003eO (1:1, v/v) using a UV-vis spectrophotometer and fluorescence spectrophotometer. Nineteen common metal ions were added to the solution of probe EBI. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e shows that after adding Al\u003csup\u003e3+\u003c/sup\u003e, the fluorescence intensity of EBI increased significantly. Correspondingly, with the exception of a slight enhancement of the fluorescence intensity by Zn\u003csup\u003e2+\u003c/sup\u003e and a blue shift by Cu\u003csup\u003e2+\u003c/sup\u003e, the fluorescence intensity of the EBI is almost unchanged with the addition of other ions. This phenomenon that can also be clearly observed with the naked eye under a 365 nm UV lamp. The addition of Al\u003csup\u003e3+\u003c/sup\u003e enhances the fluorescence of EBI, possibly due to a molecular structure wherein the N\u0026thinsp;\u0026minus;\u0026thinsp;N bond is rotated and the cavity formed by \u0026minus;\u0026thinsp;C\u0026thinsp;=\u0026thinsp;O coordinates with Al\u003csup\u003e3+\u003c/sup\u003e. According to previous studies, probes containing amide and imide groups can coordinate with metal ions, especially Al\u003csup\u003e3+\u003c/sup\u003e [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. The above experimental results show that the probe EBI has excellent selective recognition of Al\u003csup\u003e3+\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Effects of coexisting metal ions on the selective recognition of probe EBI\u003c/h2\u003e \u003cp\u003eThe presence of various metal ions in the environment may affect the accuracy of the probe. To investigate the effect of other metal ions on the recognition of Al\u003csup\u003e3+\u003c/sup\u003e by the probe EBI, interference experiments with coexisting metal ions were explored. The concentration of coexisting metal ions and Al\u003csup\u003e3+\u003c/sup\u003e is five equivalents higher than that of the probe EBI. As can be seen from Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, the presence of Al\u003csup\u003e3+\u003c/sup\u003e significantly enhanced the fluorescence intensity of probe EBI, while the coexistence of other metal ions except Zn\u003csup\u003e2+\u003c/sup\u003e had no significant effect on the fluorescence intensity of probe EBI. While Zn\u003csup\u003e2+\u003c/sup\u003e can enhance the fluorescence intensity of the EBI probe, the fluorescence intensity decreases to a Zn\u003csup\u003e2+\u003c/sup\u003e absence state in the presence of Fe\u003csup\u003e3+\u003c/sup\u003e or Cr\u003csup\u003e3+\u003c/sup\u003e. This phenomenon can be used to easily distinguish between Al\u003csup\u003e3+\u003c/sup\u003e and Zn\u003csup\u003e2+\u003c/sup\u003e. The above experimental results show that the recognition ability of the probe EBI for Al\u003csup\u003e3+\u003c/sup\u003e is significantly higher than that of other metals, and the presence of other metals does not affect its recognition selectivity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Al\u003csup\u003e3+\u003c/sup\u003e recognition sensitivity study of probe EBI\u003c/h2\u003e \u003cp\u003eRecognition sensitivity is an important parameter to determine whether a probe is suitable for use in real-world scenarios. In order to study the sensitivity of the probe EBI to identify Al\u003csup\u003e3+\u003c/sup\u003e, the relationship between different Al\u003csup\u003e3+\u003c/sup\u003e concentrations and the UV-vis and fluorescence intensities of the probe was studied. As shown in the Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, with the increasing Al\u003csup\u003e3+\u003c/sup\u003e concentration (0\u0026ndash;100 \u0026micro;M), the fluorescence peak at 520 nm increases uniformly. The green fluorescence of the solution gradually brightens under a 365 nm UV lamp, and the intensity of fluorescence is increase by 29 times compared to that before the addition of aluminum ions. Ultraviolet-visible titration of probe EBI in ethanol/water with Al\u003csup\u003e3+\u003c/sup\u003e was performed, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB. The UV-vis spectrum of probe EBI has a distinct absorption band at 395 nm. With the increasing Al\u003csup\u003e3+\u003c/sup\u003e concentration, the absorption peak at 395 nm red-shifts to 418 nm, and two well-defined isoelectric points appear in the UV-vis spectrum, around 400 and 462 nm, respectively. The results showed that Al\u003csup\u003e3+\u003c/sup\u003e formed new species under the probe EBI. A Job plot was generated by recording the continuous change in fluorescence intensity during titration to determine the binding ratio between the probe EBI and Al\u003csup\u003e3+\u003c/sup\u003e. During the titration, Job\u0026rsquo;s plot of the probe EBI with Al\u003csup\u003e3+\u003c/sup\u003e was recorded from a continuous change in the intensity of the fluorescence peak at 520 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA) to determine the binding ratio of the EBI to Al\u003csup\u003e3+\u003c/sup\u003e. The results confirmed that when the addition of Al\u003csup\u003e3+\u003c/sup\u003e was 100 \u0026micro;M, there was an inflection point in the Job\u0026rsquo;s plot, indicating that the coordination ratio of the probe EBI to Al\u003csup\u003e3+\u003c/sup\u003e was 1:1. The binding rate between the probe EBI and Al\u003csup\u003e3+\u003c/sup\u003e is shown in Fig. S5. According to the titration data, a good linear line is obtained by fitting the Benesi-Hildebrand equation, and the binding constants \u003cem\u003eK\u003c/em\u003e\u003csub\u003ea\u003c/sub\u003e of the probe EBI and Al\u003csup\u003e3+\u003c/sup\u003e is 1.79\u0026times;10\u003csup\u003e4\u003c/sup\u003e M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.9997, Fig. S5). The detection limit for EBI selective identification of Al\u003csup\u003e3+\u003c/sup\u003e is calculated. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, the titration curve is linear when the concentrations of Al\u003csup\u003e3+\u003c/sup\u003e are in the range of 0\u0026thinsp;\u0026minus;\u0026thinsp;50 \u0026micro;M. According to the formula LOD\u0026thinsp;=\u0026thinsp;3\u003cem\u003eσ\u003c/em\u003e/\u003cem\u003ek\u003c/em\u003e, the detection limit of the probe EBI for Al\u003csup\u003e3+\u003c/sup\u003e is calculated to be 1.62 nM (R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.9934), which proves the high sensitivity of the probe EBI and its potential application in the detection of Al\u003csup\u003e3+\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Study on the stability and applicable pH range of probe\u003c/h2\u003e \u003cp\u003eThe fluorescence of EBI was quickly detected after adding 2 equivalents of Al\u003csup\u003e3+\u003c/sup\u003e, and it was found that the fluorescence intensity of the solution was rapidly enhanced and the reaction reached a stable state within 30 min and remained unchanged for at least 300 min (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and Fig. S6). According to the kinetic equation Y\u0026thinsp;=\u0026thinsp;I-exp (A*x), the kinetic constant of EBI is 0.9882 min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The effect of pH value on the identification of Al\u003csup\u003e3+\u003c/sup\u003e by probe EBI was investigated. The fluorescence intensity of EBI in EtOH/H\u003csub\u003e2\u003c/sub\u003eO solution (1:1, v/v, 100 \u0026micro;M, pH 7.4, λ\u003csub\u003eem\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;520 nm) has little change in the range of pH\u0026thinsp;=\u0026thinsp;1\u0026thinsp;\u0026minus;\u0026thinsp;12. After adding 2 equivalents of Al\u003csup\u003e3+\u003c/sup\u003e, the fluorescence has large enhancement in pH range 5.0\u0026ndash;8.0, indicating that EBI has excellent recognition performance for Al\u003csup\u003e3+\u003c/sup\u003e in the physiological pH range.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.6. Study on circularity and competition of EBI\u003c/h2\u003e \u003cp\u003eReusability is one of the important indexes to evaluate the performance of coordinated sensors. EDTA is a common ligand used to evaluate the recyclability of EBI to detect Al\u003csup\u003e3+\u003c/sup\u003e. After adding Al\u003csup\u003e3+\u003c/sup\u003e, the fluorescence intensity of EBI increased at λ\u003csub\u003eem\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;520 nm. Then, after adding EDTA, the fluorescence emission intensity significantly returned to the original value, and the prepared probe could be easily regenerated by EDTA (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). The above phenomenon can be repeated for at least 5 cycles, indicating that the probe EBI exhibits excellent reversibility in recognizing Al\u003csup\u003e3+\u003c/sup\u003e (Fig. S7). The influence of anions on the detection of Al\u003csup\u003e3+\u003c/sup\u003e by probe EBI was also evaluated. The fluorescence response intensity of EBI\u0026ndash;Al\u003csup\u003e3+\u003c/sup\u003e was measured by adding various anions (PO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e3\u0026minus;\u003c/sup\u003e, HPO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e2\u0026minus;\u003c/sup\u003e, H\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e, SO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e2\u0026minus;\u003c/sup\u003e, ClO\u003csup\u003e\u0026minus;\u003c/sup\u003e, F\u003csup\u003e\u0026minus;\u003c/sup\u003e, Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e, Br\u003csup\u003e\u0026minus;\u003c/sup\u003e, I\u003csup\u003e\u0026minus;\u003c/sup\u003e, Ac\u003csup\u003e\u0026minus;\u003c/sup\u003e, NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e, CO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e2\u0026minus;\u003c/sup\u003e, HCO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e, S\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e2\u0026minus;\u003c/sup\u003e and S\u003csup\u003e2\u0026minus;\u003c/sup\u003e) into the system (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). First, none of the anions in the above test range significantly changed the fluorescence intensity of EBI in the absence of Al\u003csup\u003e3+\u003c/sup\u003e. In the presence of PO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e3\u0026minus;\u003c/sup\u003e, the fluorescence intensity of EBI\u0026ndash;Al\u003csup\u003e3+\u003c/sup\u003e shows obvious quenching, while other anions have no obvious quenching phenomenon (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). In particular, the presence of F\u003csup\u003e\u0026minus;\u003c/sup\u003e leads to the enhancement of fluorescence intensity of EBI\u0026ndash;Al\u003csup\u003e3+\u003c/sup\u003e, which may be the formation of hydrogen bonding complexes between F\u003csup\u003e\u0026minus;\u003c/sup\u003e and the \u0026ndash;NH group of EBI imidazole ring [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.7. Binding model and theoretical calculation\u003c/h2\u003e \u003cp\u003eHere, in order to clarify the detection method of probe EBI for Al\u003csup\u003e3+\u003c/sup\u003e, the reaction products of EBI were determined by FT-IR and \u003csup\u003e1\u003c/sup\u003eH NMR titration (Fig. S8 and Fig. S9). After Al\u003csup\u003e3+\u003c/sup\u003e is added to the solution of EBI, the hydrogen proton signals at 11.19 ppm and 11.48 ppm attributed to hydroxyl and amino groups are disappeared, which indicates that hydroxyl and amino groups participate in coordination with Al\u003csup\u003e3+\u003c/sup\u003e. In addition, the proton of \u0026ndash;N\u0026ndash;N\u0026thinsp;=\u0026thinsp;CH\u0026ndash; changes from 8.70 ppm to 8.22 ppm, and the aromatic proton also shows a certain chemical shift change. The complexation ratio of probe EBI to Al\u003csup\u003e3+\u003c/sup\u003e was 1:1 based on the Job\u0026rsquo;s plot data. In summary, the reaction mechanism of EBI and Al\u003csup\u003e3+\u003c/sup\u003e is speculated as follows: carbonyl group, hydroxyl group and \u0026ndash;HN\u0026ndash;N\u0026thinsp;=\u0026thinsp;CH\u0026ndash; of EBI are coordinated with Al\u003csup\u003e3+\u003c/sup\u003e, supporting the hypothesis that EBI is an \"O\u0026ndash;N\u0026ndash;O\" type ligand (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e) [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. The fluorescence of probe EBI was enhanced by 29 times after ion recognition.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo have a deeper insight into the relationship between the structural changes of probe EBI on its complexation with Al\u003csup\u003e3+\u003c/sup\u003e and the optical response of EBI to Al\u003csup\u003e3+\u003c/sup\u003e, time-dependent density functional theory (TD-DFT) calculations with the B3LYP/6-31G** methods are carried out using the Gaussian 16 program. The optimized geometry and the highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) of the probes EBI and EBI\u0026ndash;Al\u003csup\u003e3+\u003c/sup\u003e are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e8\u003c/span\u003e. The molecular optimization of TD-DFT shows that the carbonyl phenylimidazole of EBI has a dihedral Angle with the amine hydroxyl group. The molecule of EBI mainly shows charge transfer from the phenylimidazole ring to the \u003cem\u003eN\u003c/em\u003e, \u003cem\u003eN\u003c/em\u003e diethylaminosalicylaldehyde. The main energy transfer is from HOMO to LUMO with a 3.455 eV of energy gap (\u003cem\u003eΔ\u003c/em\u003e). To EBI\u0026ndash;Al\u003csup\u003e3+\u003c/sup\u003e, the molecule exhibits high planarity because of the complex. EBI\u0026ndash;Al\u003csup\u003e3+\u003c/sup\u003e exhibits main energy transfer from HOMO to LUMO\u0026thinsp;+\u0026thinsp;1 with 1.676 eV of energy gap (\u003cem\u003eΔ\u003c/em\u003e). The calculated energy gap between HOMO and LUMO of EBI is higher (3.455 eV) than EBI\u0026ndash;Al\u003csup\u003e3+\u003c/sup\u003e (1.357 eV) (Fig. S10, Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Therefore, the complexation of EBI with Al\u003csup\u003e3+\u003c/sup\u003e is energetically advantageous. These results show that the interaction between Al\u003csup\u003e3+\u003c/sup\u003e and probe EBI effectively reduces the HOMO-LUMO bandgap of EBI\u0026ndash;Al\u003csup\u003e3+\u003c/sup\u003e, and the proposed binding mode of probe EBI and Al\u003csup\u003e3+\u003c/sup\u003e is confirmed by theoretical calculations.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.8. Test strips experiment\u003c/h2\u003e \u003cp\u003eIn order to explore the practical application of probe EBI, strip tests based on filter paper were carried out. Firstly, the test paper was immersed in EBI (1\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e mol/L) ethanol solution, and then naturally dried to make a variety of metal ion detection test paper. The test strip showed a weak blue-green fluorescence emission under 365 nm light irradiation. After the addition of excessive Al\u003csup\u003e3+\u003c/sup\u003e, the fluorescence color of the test strip showed bright green. Meanwhile, the fluorescent color of the test strips treated with excessive Zn\u003csup\u003e2+\u003c/sup\u003e was dark green, while the fluorescent color of the test strips treated with other ions did not change significantly (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). Figure\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e8\u003c/span\u003eB shows the test strips with different concentrations of Al\u003csup\u003e3+\u003c/sup\u003e under 365 nm UV lamp. It can be observed that with the increase of Al\u003csup\u003e3+\u003c/sup\u003e concentration, the bright green fluorescence of the test strip becomes more obvious. The results show that the probe EBI can detect Al\u003csup\u003e3+\u003c/sup\u003e by a simple dipping paper method with only a portable UV lamp.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.9. Cell imaging\u003c/h2\u003e \u003cp\u003eIt is of great significance to study the biological application of probe systems. Firstly, the cytotoxicity of probe EBI was measured by MTT method. As shown in Fig. S11, the viability of HepG-2 cells was greater than 90% at an experimental concentration of 50 \u0026micro;M, indicating that EBI has low cytotoxicity and can be used for biological imaging. Subsequently, the feasibility of EBI and EBI\u0026ndash;Al\u003csup\u003e3+\u003c/sup\u003e for live cell imaging was evaluated using confocal fluorescence microscopy (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e9\u003c/span\u003e). After incubating with probe EBI at 37\u0026deg;C for 30 min, the HepG-2 cells emit extremely weak green fluorescence under a fluorescence microscope. However, when the HepG-2 cells were incubated with EBI and Al\u003csup\u003e3+\u003c/sup\u003e at 37\u0026deg;C for 30 min, they showed significant green fluorescence under a fluorescence microscope. Then PO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e3\u0026minus;\u003c/sup\u003e was added to the HepG-2 cell system of EBI\u0026ndash;Al\u003csup\u003e3+\u003c/sup\u003e and incubated at 37\u0026deg;C for 30 min. The quenching of green fluorescence was obviously observed under fluorescence microscope. These results suggest that probe EBI can penetrate cell membranes to image Al\u003csup\u003e3+\u003c/sup\u003e and PO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e3\u0026minus;\u003c/sup\u003e in living cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Conclusions","content":"\u003cp\u003eIn summary, a turn-on fluorescence sensing material EBI based on the imidazole-Schiff base structure has been designed and synthesized. EBI has high sensitivity, selectivity and anti-interference to Al\u003csup\u003e3+\u003c/sup\u003e, which is superior to other competing metal ions. EBI reaction reached the platform about 30 min after adding Al\u003csup\u003e3+\u003c/sup\u003e, and the fluorescence detection limit was 1.62 nM. The induction mechanism of probe EBI to Al\u003csup\u003e3+\u003c/sup\u003e is thought to be due to the coordination between carbonyl group, hydroxyl group, \u0026ndash;HN\u0026ndash;N\u0026thinsp;=\u0026thinsp;C\u0026ndash; and Al\u003csup\u003e3+\u003c/sup\u003e. In addition, the probe EBI can be made into test paper to quickly identify Al\u003csup\u003e3+\u003c/sup\u003e. Cytotoxicity and cell imaging results showed that EBI probes have low toxicity and can penetrate cell membranes to image Al\u003csup\u003e3+\u003c/sup\u003e in living cells.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe work was supported by \u0026quot;the State Key Laboratory of Fine Chemicals, Dalian University of Technology\u0026quot; (KF2212). The authors acknowledge the assistance of JLICT Center of Characterization and Analysis\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u0026nbsp;\u003c/strong\u003eJW done investigation; validation; and writing-original draft preparation. YQL done the synthesis and characterization of compounds. WRJ and HHZ done metal ion detection. LR performed fluorescence imaging of metal ions in living cells of the compound. DWZ did project administration, supervised the work, revised, and completed the manuscript. YLZ was responsible for the time-dependent density functional theory (TD-DFT) calculations of the compound and the revision of the manuscript. All authors reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data and materials that support the findings of this study are available in the supplementary material of this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConfict of interest\u0026nbsp;\u003c/strong\u003eThe authors declare that they have no known competing fnancial interests or personal relationships that could have appeared to infuence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u0026nbsp;\u003c/strong\u003eThis declaration is not applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eG. Asaithambi, V. Periasamy, Res. Chem. 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Liq. 300, 112250 (2020)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eK. Krishnaveni, M. Iniya, D. Jeyanthi, A. Siva, D. Chellappa, Spectrochim. Acta A Mol. Biomol. Spectrosc. 205, 557\u0026ndash;567 (2018)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eG. Yang, P. Li, Y. Han, L. Tang, Y. Liu, H. Xin et al., Mater. Chem. Phys. 295, 127145 (2023)\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Schemes","content":"\u003cp\u003eScheme 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Turn-on, Al3+ ions, Imidazole-Schiff, Fluorescence probe, Cell imaging","lastPublishedDoi":"10.21203/rs.3.rs-6114045/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6114045/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAn imidazole-Schiff base fluorescent probe EBI was designed and synthesized for the sensitive and selective detection of Al\u003csup\u003e3+\u003c/sup\u003e. The probe EBI exhibits good anti-interference performance against Al\u003csup\u003e3+\u003c/sup\u003e in the presence of multiple metal ions. The reaction reached the platform in about 30 min after adding Al\u003csup\u003e3+\u003c/sup\u003e, and the fluorescence detection limit was 1.62 nM. The coordination ratio of probe EBI to Al\u003csup\u003e3+\u003c/sup\u003e is 1:1, and the coordination reaction between carbonyl group, hydroxyl group, –HN–N=C– and Al\u003csup\u003e3+\u003c/sup\u003e limits the molecular distortion and fluorescence quenching effect of photoelectron transfer, thus inducing the enhancement of green fluorescence. The probe can be recovered and used alternately by EDTA in the detection of Al\u003csup\u003e3+\u003c/sup\u003e. In addition, the fluorescence quenching of EBI–Al\u003csup\u003e3+\u003c/sup\u003e by PO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e3−\u003c/sup\u003e was also evident in the selected anion range. The probe was also applied to the successful monitoring Al\u003csup\u003e3+\u003c/sup\u003e and PO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e3−\u003c/sup\u003e in living cells.\u003c/p\u003e","manuscriptTitle":"A turn-on fluorescent chemical sensor based on imidazole-Schiff base structure for highly selective and accurate detection of Al3+ ions in living cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-03 17:41:39","doi":"10.21203/rs.3.rs-6114045/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9fba7ee0-d817-49f2-99b4-049dde4fde2a","owner":[],"postedDate":"March 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-04-17T12:08:48+00:00","versionOfRecord":[],"versionCreatedAt":"2025-03-03 17:41:39","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6114045","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6114045","identity":"rs-6114045","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}