A Novel Ferrocene-Derivative-Based Dual-Response Chemosensor for Selective Al³⁺ Detection: Fluorescence and Electrochemical Signaling

A Novel Ferrocene-Derivative-Based Dual-Response Chemosensor for Selective Al³⁺ Detection: Fluorescence and Electrochemical Signaling | 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 Novel Ferrocene-Derivative-Based Dual-Response Chemosensor for Selective Al³⁺ Detection: Fluorescence and Electrochemical Signaling Wenqin Liu, Yanping Liu, Zenghui Li, Santai Zou, Pingnan Wan, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7514239/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 16 Dec, 2025 Read the published version in Journal of Fluorescence → Version 1 posted 12 You are reading this latest preprint version Abstract A new Chemosensor, 5-ferrocenylsalicylaldehyde-2-pyridinehydrazone ( SP-Fc ), was designed and synthesized by condensing 5-ferrocenylsalicylaldehyde with 2-hydrazinopyridine. Its structure was characterized by 1 H NMR, 13 C NMR and HRMS. SP-Fc could be used to identify Al 3+ through dual optical and electrochemical responses. Upon addition of Al 3+ , the sensor exhibited a significant fluorescence enhancement attributed to CHEF and the inhibition of both PET and ESIPT processes. The sensor showed a high selectivity for Al 3+ in DMSO solution at pH = 6 and was not disturbed by other competing metal ions. In additon, the detection limit for Al 3+ was observed as 1.30 × 10 − 7 M. Job’s plot analysis and NMR titration experiment confirmed 1:1 stiochiometry between Al 3+ and probe through phenolic oxygen, imine nitrogen and pyridine nitrogen atoms. Moreover, the electrochemical signals of SP-Fc in the presence of the Al 3+ was shifted significantly compared with those of the other metal cations tested, indicating SP-Fc could be used to be a dual-response chemosensor for Al 3+ with excellent sensitivity and selectivity both in optical and electrochemical ways. Also, Confocal fluorescence microscopy imaging demonstrated that SP-Fc can monitor Al 3+ in living MCF-7 cells with low cytotoxicity. Chemosensor ferrocene derivative fluorescence electrochemistry dual-response Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Aluminum is the third most abundant element (8.3wt%) in the Earth’s crust, after oxygen and silicon. As the most abundant metal, aluminum is characterized by good ductility and is ubiquitous in daily applications such as food additives, aluminum-based pharmaceuticals and cooking utensils [ 1 – 3 ]. These widespread applications lead to frequent human exposure to aluminum. According to the World Health Organization (WHO), the average daily human intake of aluminum is approximately 3–10 mg, while the permissible limit in drinking wateris 7.4 × 10 − 6 M [ 4 ]. Although aluminum is generally considered less toxic than heavy metals, excess intake in the human body is linked to an increased risk of several diseases like Parkinson’s disease and Alzheimer’s dementia. Additionally, it is well-known that 40% of the world’s acidic soils are contaminated with aluminum ions, which impair plant growth and development [ 5 – 6 ]. Given the close association of Al 3+ with human health and environmental protection, the development of reliable methods for the rapid and sensitive detection of Al 3+ in the biosphere is of considerable importance. In the past decades, a variety of modern techniques for sensing metal ions has been developed such as atomic absorption spectrometry [ 7 ], atomic emission spectrometry [ 8 ], and inductively coupled plasma mass spectrometry [ 9 ] etc. However, most of these methods require the high cost instruments, large quantity of sample and are not appropriate in many situations. Thus, the effective colorimetric, fluorescent and electrochemical chemosensors have received much attention due to their distinct advantages, including high sensitivity, low cost and real-time detection capability. But some of these single-channel sensors may be restricted by interfering substance in the actual samples, such as turbidity and color [ 10 ]. In contrast, multi-channel sensors which could detecte analytes through multiple physical responses, provide better selectivity, higher sensitivity and self-calibration ability through variable detecting methods with low background in complex systems [ 11 ]. Consequently, construction and development of multichannel sensors for detecting Al 3+ would be highly desirable from the viewpoint of the practical applications. Recent research shows that ferrocene derivatives are a good platform for multichannel sensing of various bioactive and toxic analytes on account of its excellent redox properties, good solubility, structural stability, and ease of derivatization, which can be effectively integrated with fluorogenic/chromogenic units [ 12 – 13 ]. When interacting with an analyte, ferrocene-based chemosensors not only express a significant potential shift of the Fe 3+ /Fe 2+ redox couple, but also show other optical signal changes, which allow a single sensor to be applied in various experimental conditions. There are numerous ferrocene-based multichannel probes for metal ions have been developed, these multichannel probes were constructed by incorporating rhodamine, triazole, selenide, and schiff base scaffolds into one or two cyclopentadienyl rings of ferrocene [ 14 ]. Most of the reported ferrocene-based multichannel probes have been utilized for detecting metal ions such as Hg 2+ [ 15 – 17 ], Cu 2+ [ 18 ], Pb 2+ [ 19 – 20 ], Fe 3+ [ 21 – 22 ], etc. While the detection of Al 3+ has received comparatively less attention [ 23 ]. This disparity may arise from the poor coordination ability and strong hydration ability [ 24 ]. Therefore, a ration design and preparation of novel ferrocene-based multichannel chemosensors for Al 3+ is still challenging. In general, Al 3+ being a hard acid, prefers hard donor sites like N and O in its coordination sphere. Thus, various Schiff base derivatives of salicylaldehyde have been reported for recognition of Al 3+ owning to their generally one step synthesis and mixed N, O-donor sites [ 24 – 28 ]. Additionally, an N-containing pyridal unit with remarkable binding ability to metal ions has good optical properties. Therefore, salicylaldehyde pyridinehydrazone derivatives are a good platform for Al 3+ on account of the strong coordination ability between the nitrogen atom of imine, the oxygen atom of hydroxyl and metal ions, which may inhibit Photoinduced Electron Transfer (PET) and Excited State Intramolecular Proton Transfer (ESIPT) process as well as result in the Chelation-enhanced Fluorescence (CHEF). Based on this consideration, we decided to combine the redox activity of the ferrocenyl group with the turn-on fluorescent behavior and selective ability of the salicylaldehyde hydrazones as binding sites to construct a novel mutichannel chemosensors that specifically recognizing Al 3+ (scheme 1 ). Therefore, the target probe 5-ferrocenylsalicylaldehyde-2-pyridinehydrazone ( SP-Fc ) has been designed by strategically introducing ferrocene into the salicylaldehyde skeleton. To the best of our knowledge, our probe is the first example of ferrocene-appended salicylaldehyde hydrazone moiety connected through carbon carbon single bond. Herein, we report the synthesis and characterization of 5-ferrocenylsalicylaldehyde-2-pyridinehydrazone ( SP-Fc ) as a new mutichannel chemosensor for selective Al 3+ ions detection via optical and electrometric readouts. As expected, the sensor SP-Fc exhibited a weak fluoresence based on PET and ESIPT process. After binding with Al 3+ , a new complex, SP-Fc- Al 3+ , was formed and displayed a strong fluoresence due to CHEF effect and the inhibition of both PET and ESIPT processes. Furthermore, the electrochemical signals of SP-Fc in the presence of the Al 3+ was shifted significantly, showing efficient electrochemical sensors. In a word, SP-Fc could be used to be a dual-response chemosensor for Al 3+ with excellent sensitivity and selectivity both in optical and electrochemical ways. 2. Experimental 2.1 Reagents and Apparatus The fine chemicals 4-aminophenol and ferrocene were purchased from Aladdin co. (Shanghai, China) and were used without further purification. DMSO and 10 mM different pH Tris-HCl buffer were purchased from Beyotime Biotechnology. All the chlorinated salts were purchased from Macklin and Aladdin co. (Shanghai, China). All salts were all dissolved in Tris-HCl buffer at 10 mM for analysis. 1 H NMR and 13 C NMR spectra were recorded on a AVANCE Ⅲ HD 600 MHz spectrometer with chemical shifts reported in ppm (in CDCl 3 ; TMS as internal standard). High resolution mass (HRMS) spectrum were recorded in AB SCIEX high-resolution time-of-flight mass spectrometer (TripleTOF5600+) using HPLC acetonitrile as solvent. Melting points were determined on an X4 Digital Micro Melting Point apparatus. The measurements of pH were done in a digital pH meter (Merck). UV-vis spectra were acquired on an Evolution One UV-vis spectrophotometer (Thermo Scientific). Fluorescence spectra and absorption of MTT experiments were obtained on the full-wavelength multifunctional microplate reader (Tecan Infinite M1000 Pro). Electrochemical measurements were conducted with a Guangzhou IGS4030 Electrochemical Workstation. MTT experiments were carried out on 96-well plates (Corning). Fluorescence images were taken by the confocal laser scanning microscope (Olympus FV3000). The deionized water was prepared on a Milli-Q water purification system and used throughout all experiments. 2.2 Synthesis of sensor SP-Fc 2.2.1 Synthesis of 5-ferrocenylsalicylaldehyde ( 2 ) 4-ferrocenylphenol (1.20 g, 4.3 mmol) and 15 mL glacial acetic acid were placed into a 100 mL three-neck flask and heated to 90°C in an oil bath. Then hexamethylenetetramine (1.20 g, 8.6 mmol) was dissolved in 15 mL of glacial acetic acid. The solution was slowly added to the three-necked flask via a constant-pressure dropping funnel and continued to stir for 2.5 h. After cooling to room temperature, the substrate was treated with 1M hydrochloric acid (20 mL) and reacted for another 1h at ambient temperature, then extracted with dichloromethane (50 mL) three times. The combined organic phases were dried with anhydrous sodium sulphate and evaporated under in vacuo. The residue was purified by column chromatography over silica gel eluted with petroleum ether : ethyl acetate = 20:1. Yield: 40% (0.52 g), m.p.:132.5-133.2℃, 1 H NMR (600 MHz, CDCl 3 ) : δ 10.94 (s, 1H), 9.95 (s, 1H), 7.71 (dd, J = 5.4 Hz、1.8 Hz, 1H), 7.62 (d, J = 2.4 Hz, 1H), 6.99 (d, J = 9.0 Hz, 1H), 4.62 (t, J = 1.8 Hz, 2H), 4.35 (t, J = 1.8 Hz, 2H), 4.08 (s, 5H); 13 C NMR (150 MHz, CDCl 3 ): δ 196.6, 159.9, 135.0, 131.3, 130.1, 120.55, 117.8, 84.2, 69.6, 69.0, 66.1; HR MS (ESI-TOF) m/z: [M + H] + for C 17 H 14 FeO 2 : Calcd 307.0423, found 307.0394. 2.2.2 Synthesis of 5-ferrocenylsalicylaldehyde-2-pyridinehydrazone ( SP-Fc ) 5-ferrocenylsalicylaldehyde (0.153 g, 0.5 mmol) and 2-hydrazinopyridine (0.054 g, 0.5 mmol) were dissolved in 15 mL absolute ethanol under nitrogen atomsphere. The mixture was refluxed for 4 h. The resulting precipitate was filtrated and washed with 20 mL of absolute ethanol. Recrystallized with ethanol, dried under vacuum and obtained the orange solid SP-Fc . Yield: 58% (0.23 g), m.p.:198.6-199.7℃, 1 H NMR (600 MHz, CDCl 3 ) : δ 10.95 (s, 1H, -OH), 10.56 (s, 1H, -NH-), 8.30 (s, 1H, -CH = N-), 8.14 (dd, J = 7.2 Hz、1.2 Hz, 1H), 7.67–7.70 (m, 2H, ph-H), 7.40 (dd, J = 12.6 Hz、7.8 Hz, 1H), 7.08 (d, J = 12.6 Hz, 1H), 6.85 (d, J = 12.6 Hz, 1H), 6.80 (dd, J = 10.2 Hz、1.8 Hz, 1H), 4.71 (t, J = 1.8 Hz, 2H, Fc-H), 4.30 (t, J = 1.8 Hz, 2H, Fc-H), 4.03 (s, 5H, Fc-H); 13 C NMR (150 MHz, CDCl 3 ) : δ 155.6, 155.4, 147.9, 142.9, 138.6, 130.5, 128.5, 126.9, 118.0, 116.8, 116.5, 106.6, 85.3, 69.5, 68.4, 66.0; HR MS(ESI-TOF) m/z: [M + H] + for C 22 H 19 FeN 3 O: calced 398.0958, found 398.0943. 2.3 Standard solution The stock solutions (0.01 M) of chlorinated salts (Na + , Li + , Cu 2+ , Ni 2+ , Fe 2+ , Zn 2+ , Mg 2+ , Pb 2+ , Mn 2+ , Cd 2+ , Hg 2+ , Cr 3+ , Al 3+ , Fe 3+ ) were prepared with 10 mM Tris-HCl buffer solution (pH 6.0). The 5-ferrocenylsalicylaldehyde-2-pyridinehydrazone stock solution (0.001 M) was prepared in DMSO. Test solutions were prepared by placing calculation amount of 5-ferrocenylsalicylaldehyde-2-pyridinehydrazone stock solution and each metal solution into a test tube, and then diluted to 0.2 mL with Tris-HCl buffer solution (10 mM, pH 6.0). The resulting solution was mixed well and recorded 10 min after the addition of determinand at room temperature. The excitation wavelength used in the fluorescence examination was 370 nm, and the slits width of excitation and emission were all 10 nm. 2.4 Electrochemical testing method A solution of probe SP-Fc (1×10 − 4 M) in acetonitrile was prepared using 0.1 M tetrabutylammonium hexafluorophosphate (Bu₄NPF₆) as the supporting electrolyte. A 3 mL aliquot of the SP-Fc solution was combined with 1 equivalent (1 equivalent = 30 µL of 1×10 − 4 mol/L solution) of the analyte metal ion solution using a micropipette. Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) measurements were subsequently performed. All measurements employed a standard three-electrode configuration: a glassy carbon electrode (GCE) served as the working electrode, a platinum wire as the counter electrode, and an Ag/AgCl electrode as the reference electrode. The scan rate was set to 0.1 V/s. Prior to use, the glassy carbon electrode was polished with 0.05 µm Al₂O₃ slurry and thoroughly rinsed via sonication in ultrapure water. 3. Results and discussion 3.1 Synthesis The target probe SP-Fc was efficiently synthesized with a high yield by condensing 5-ferrocenylsalicylaldehyde ( 2 ) with 2-hydrazinopyridine, as illustrated in scheme 2 . 5-ferrocenylsalicylaldehyde was the key intermediate which was obtained by formylating 4-ferrocenylphenol ( 1 ) through the Duff reaction. The material 4-ferrocenylphenol ( 1 ) was synthesized using a previously reported method [ 29 ]. Then, The obtained ferrocene derivative SP-Fc underwent characterization through 1 H NMR and 13 C NMR as well as ESI-TOF mass spectrometry (Fig. S1 -S3). To evaluate the metal recognition capabilities of sensor SP-Fc , various studies, including optical, electrochemical, and 1 H NMR titrations, were conducted. 3.2 The optical properties of SP-Fc toward various metal ions To assess the recognizing properties of the probe SP-Fc to metal ions, the fluorescence response of SP-Fc (10 µM) in the presence of various metal ions (100 µM) such as Na + , Li + , Cu 2+ , Ni 2+ , Fe 2+ , Zn 2+ , Mg 2+ , Pb 2+ , Mn 2+ , Cd 2+ , Hg 2+ , Cr 3+ , Al 3+ , Fe 3+ (chlorinated salts) were tested in a Tris-HCl buffer (10 mM, pH = 6.0). The fluorescence emission spectra were recorded within 10 min after the addition of various metal ions. As shown in Fig. 1 a, free probe SP-Fc exhibited a weak fluorescence emission among the range of 400–600 nm when excited with UV lamp at 365 nm, which can be attributed to two fluorescent quenching mechnisms including the photo-induced electron transfer (PET) of ferrocene to the pyridyl group and the excited-state intramolecular proton transfer (ESIPT) between the phenolic hydroxyl group and the imine nitrogen [ 30 ]. when the sensor was treated with a variety of metal cations, only Al 3+ generated outstanding fluoresence enhancement at 445 nm, which displayed strong “turn-on” fluorescent emission. Enhancement of fluorescence emission might be, owing to the prevention of the PET and ESIPT process as well as the chelation-enhanced fluorescence (CHEF) effect when SP-Fc was complexed with Al 3+ . Under the same experimental conditions, other competing metal ions had no effect on the fluorescence. Meanwhile, The UV-Vis absorption spectra of probe SP-Fc was also recorded in the absence and presence of one equivalent of the Al 3+ ions in DMSO solution (Fig. S4). The solution of free SP-Fc exhibited two intense absorption band in the UV region (300 nm and 348 nm) and a relatively weak band in the visible region. The high energy (HE) bands can be ascribed to π-π* and L-π* electronic transitions, and the low energy (LE) band originated from the localized excitation either from an Fe (II) d-d transition or a metal-ligand charge transfer (MLCT) process (dπ-π*) (visible region) [ 31 ], which was responsible for the orange color of the sensors. However, the intensity of absorption bands (348 nm) decreased with a concomitant appearance of two clear isosbestic points at 309 nm and 372 nm after addition of Al 3+ ions. The generation of isosbestic points clearly indicated the formation of SP-Fc-Al 3+ complex. Hence, it could be concluded that probe SP-Fc was a highly selective fluorescence “turn-on” sensor for Al 3+ . To further investigation of the specificity of SP-Fc , competitive experiments were carried out in the presence of Al 3+ ions (100 µM) mixed with other competitive metal ions (100 µM). Their fluorescence intensities were recorded and Fig. 2 depicts the fluorescence changes observed. As can be clearly seen, the fluorescence emission intensity of SP-Fc /Al 3+ solution showed no pronounced interference by Na + , Li + , Zn 2+ , Mg 2+ , Pb 2+ , Mn 2+ , Cd 2+ , Hg 2+ , Fe 3+ . While the fluoresence response of SP-Fc to Al 3+ in the presence of Cu 2+ , Ni 2+ and Fe 2+ is slightly decreased but clearly detectable, although these ions showed no significant effect on the fluorescence intensity of SP-Fc individually (Fig. 1 b). The above-results on the changes of fluorescence spectra demonstrated that SP-Fc can be used as a highly effective fluorescent chemosensor for the detection of Al 3+ over other common metal ions. 3.3 pH effect on sensor SP-Fc. The performance characteristic of the sensor was monitored for its coordination ability toward Al center under different pH conditions. In this work, all the fluorescence measurements were conducted in 10 mM Tris-HCl buffer solvent. The variation of fluorescence intensity for SP-Fc against different pH values in the absence and presence of Al 3+ were recorded and the results were shown in Fig. S5. The aqueous solutions of the free SP-Fc showed no obvious changes in the fluorescence intensity at 445 nm over the pH range of 4.0–9.0, which meaning that SP-Fc was stable over the wide pH range. In acidic environments (pH < 6.8), the emission intensity of SP-Fc-Al 3+ complex was dramatically increased. However, in the neutral conditions (pH 6.8–7.4), the fluorescence intensity of SP-Fc -Al 3+ was gradually decreased. While in the basic conditions (pH > 7.4), almost no changes in the fluorescence intensity were found after Al 3+ addition with the reason of the formation of Al(Ⅲ) hydroxide complex under this condition. The pH study shows that SP-Fc could work in a broad pH range with very low background fluorescence. pH 6 was chosen in the next study because it is the most remarkable fluorescence intensity changes under this condition. 3.4 Sensitivity of SP-Fc toward Al 3+ The sensing capability of SP-Fc toward Al 3+ ion was further investigated in detail by fluorescence titration analysis. The fluorescent spectra were recorded after addition with different concentrations from 0 µM to 200 µM Al 3+ (Fig. 3 a). As Al 3+ ion was gradually added, the fluorescence intensity (at 445 nm) of the chemosensor gradually enhanced and reached the maximum after addition of 10 equivalent of Al 3+ . Subsequently, upon further addition of Al 3+ , the fluorescence spectrum remained at a plate au (Fig. 3 b). These observations indicated that the conversion of SP-Fc to the corresponding aluminum-probe complex was implemented in the detecting process. As shown in Fig. 3 b, a linear relationship (R 2 = 0.9903) between the fluorescence intensity and Al 3+ concentration (0–10 µM) demonstrated that Al 3+ concentration could be quantitatively detected by SP-Fc . The limits of detection (LOD) were calculated as 1.30 × 10 − 7 M for Al 3+ using thee quation LOD = 3*S b /S [ 32 ], where S b is the standard deviation of 10 replicate measurements of blank SP-Fc solution and S is the slope of the calibration curve. The result suggests that the sensor has great potential for sensitive detection of Al 3+ in biological samples. 3.5 Electrochemical sensing properties of sensor SP-Fc Taking advantages of the well-behaved electrochemically reversible nature of ferrocene derivatives, the electrochemical characteristics of sensor SP-Fc for metal-recognition properties were investigated by Cyclic voltammetry (CV) and differential pulse voltammetry (DPV). The reversibility and relative oxidation potential of the redox process were determined in a CH 3 CN solution (1×10 − 4 M) containing 0.1 M tetrabutylammonium hexafluorophosphate (Bu₄NPF₆) as the supporting electrolyte. As can be seen from Fig. 4 , the sensor SP-Fc exhibits a reversible single-electron oxidation process at E 1/2 = 0.477 V, due to the ferrocene/ferrocenium redox couple. No perturbation of the cyclic and differential pulse voltammograms of sensor SP-Fc was observed in the presence of several metal cations such as Li + , Na + , Mg 2+ , Fe 2+ , Fe 3+ , Cu 2+ , Zn 2+ , Cd 2+ , Cr 3+ , Mn 2+ , Hg 2+ , Pb 2+ ions, even in large excess. However, a significant cathodic change of the oxidation peak (E 1/2 ) versus Fc + /Fc was shifted from 0.477 V to 0.135 V (∆E 1/2 = 0.342 V) upon addition of 1 equiv. of Al 3+ , similar phenomena displayed in DPV of SP-Fc . These shifts were attributed to the formation of the corresponding SP-Fc-Al 3+ . So the receptor SP-Fc was an excellent electrochemical sensor for recognition of Al 3+ ions with specific selectivity. 3.6 Mechanism of SP-Fc in sensing Al 3+ To determine the binding stoichiometry of between SP-Fc and responding metal ions (Al 3+ ), Job’s method for fluorescence titration was carried out (Fig. 5 ). The maximum fluorescence band was reached at a molar fraction of 0.5, indicating a 1:1 ratio for SP-Fc - Al 3+ complex. To further confirm the results of spectroscopic and electrochemical experiments, 1 H NMR titration experiments of SP-Fc with Al 3+ were conducted in DMSO-d 6 (Fig. 6 ). In the free sensor spectrum, phenolic hydroxyl (OHa) resonated as a singlet at δ 10.95 ppm. The imine proton (-CHb = N-) and hydrazinic N-H proton (-NHc-) resonated at 8.30 ppm and 10.56 ppm, respectively. Upon addition of 1.0 equiv. Al 3+ ions to the SP-Fc solution, the signal of phenolic hydroxyl (OHa) was disappeared, indicating deprotonation and coordination of the phenolic oxygen to Al 3+ . The imine proton peak (Hb) was shifted downfield by ca. 0.18 ppm, such downfield shift suggested that Al 3+ was chelated by nitrogen atom of the imine. While the hydrazinic N-H proton exhibited upfield shift from 10.56 ppm to 10.47 ppm, suggesting non-involvement of the hydrazinic nitrogen in metal binding. These chemical shift changes confirm adduct formation between Al 3+ and SP-Fc . According to the above discussions and take into account the Job’s plot, a plausible binding mode and fluorescence sensing mechnism are proposed in Scheme 3 . Wherein Al 3+ coordinates via the phenolic oxygen, imine nitrogen and pyridine nitrogen atoms. 3.7 Cell imaging To explore its application in cell imaging, the cytotoxicity of the sensor SP-Fc was studied. We performed MTT assay in MDA-MB-231cells treated with a series of concentrations (5, 10, 20, and 50 µM, respectively) of SP-Fc for 24 h. As shown in Fig. S3, 10 µM SP-Fc showed no obvious cytotoxicity with cell viabilities greater than 90%. The results indicate that SP-Fc has low toxicit for MCF-7 cells. To further investigate the potential biological application of SP-Fc in living cells, the intracellular Al 3+ imaging of human breast cancer cells was performed by fluorescence microscopy. For this purpose, MDA-MB-231 cells were incubated with various concentrations of aqueous Al 3+ solutions (0, 10, and 50 µM) in growth media for 2 h at 37 ℃, and then treated with 10 µM SP-Fc for 6 h before imaging. SP-Fc was found to be cell membrane permeable and to recognize intracellular aluminumion, resulting in bright blue intracellular fluorescence could clearly be observed by confocal fluorescence microscopy (Fig. S6) With an increase in Al 3+ concentration from 0 to 50 µM, the fluorescence intensity of the cell swith SP-Fc increased gradually, which could be attributed to the formation of the intracellular SP-Fc -Al 3+ complex. Meanwhile, the simple cells (with nothing treated) and the cells with 10 µM SP-Fc in absence of Al 3+ were also incubated in growth media as the control samples. Both of them exhibited no fluorescence. The result of this experiment showed that the SP-Fc sensor has the potential for live cell imaging and could be used for the detection of additional and intrinsic Al 3+ ion of the living cells. Conclusion A new chemosensor 5-ferrocenylsalicylaldehyde-2-pyridinehydrazone ( SP-Fc ) was successfully synthesized by combining the redox-active ferrocene unit with the metal-binding salicylaldehyde hydrazone scaffold. Its binding properties towards various metal ions were evaluated through optical and electrochemical analyses. SP-Fc exhibited selective“turn-on”fluorescence toward Al 3+ , attributed to efficient inhibition of both PET and ESIPT process upon Al 3+ binding and CHEF. The 1:1 binding stoichiometry was confirmed, with a detection limit of 1.30 × 10 − 7 M based on fluoresence detection. Notably, Al 3+ addition induced a significant anodic shift (∆E 1/2 = 0.342 V) in the ferrocene redox potential, providing a complementary electrochemical detection channel. Moreover, the sensor SP-Fc also could be used to determine Al 3+ in living cells, making it a promising chemosensor for detection of Al 3+ in the biological system. Declarations Competing Interests The authors have no relevant financial or non-financial interests to disclose. Funding This work was supported by Science and Technology Research Project of Jiangxi Provincial Department of Education (Grant numbers [GJJ2200953] ) ; Jiangxi Provincial Health and Family Planning Commission-Traditional Chinese Medicine Research Project (Grant numbers [2024B0017] ) ; College Students' Innovation and Entrepreneurship Training Program (2024). Jiangxi Province 2024 Graduate Innovation Special Fund Project (YC2024-S750). Author Contribution Author ContributionsWenqin Liu: Methodology, Investigation, Writing-original draft, Data curation, Writing-review & editing, Conceptualization. Yanping Liu : Formal analysis, Investigation. Zenghui Li : Data curation. Santai Zou: Data curation. 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Tetrahedron 120:132878. https://doi.org/10.1016/j.tet.2022.132878 Zhang B, Liu H, Wu F, Hao G, Chen Y, Tan C, Tan Y, Jiang Y (2017) A dual-response quinoline-based fluorescent sensor for the detection of Copper (II) and Iron (III) ions in aqueous medium. Sens Actuators B 243:765–774. https://doi.org/10.1016/j.snb.2016.12.067 Schemes Schemes 1 to 3 are available in the Supplementary Files section Additional Declarations No competing interests reported. Supplementary Files Scheme1.png Scheme 1 molecule design of the chemosensor SP-Fc Scheme2.png Scheme 2 Synthetic route of chemosensor SP-Fc. SupportingInformation.doc Scheme3.png Scheme 3. Proposed binding mode for SP-Fc with Al 3+ and the mechanism of fluorescence enhancement for the chemosenor SP-Fc chelating with Al 3+ . Cite Share Download PDF Status: Published Journal Publication published 16 Dec, 2025 Read the published version in Journal of Fluorescence → Version 1 posted Editorial decision: Revision requested 25 Sep, 2025 Reviews received at journal 24 Sep, 2025 Reviews received at journal 20 Sep, 2025 Reviewers agreed at journal 17 Sep, 2025 Reviewers agreed at journal 17 Sep, 2025 Reviews received at journal 16 Sep, 2025 Reviewers agreed at journal 15 Sep, 2025 Reviewers agreed at journal 15 Sep, 2025 Reviewers invited by journal 15 Sep, 2025 Editor assigned by journal 04 Sep, 2025 Submission checks completed at journal 04 Sep, 2025 First submitted to journal 02 Sep, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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15:33:08","extension":"html","order_by":23,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":109447,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7514239/v1/cc6a88b5dea0f31c7c7070fb.html"},{"id":92010703,"identity":"2f089b58-8706-488c-a898-9b8455953ec2","added_by":"auto","created_at":"2025-09-23 15:41:08","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":402873,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Fluorescence emission spectra of \u003cstrong\u003eSP-Fc\u003c/strong\u003e (10 μM) in the presence of 10 equiv. of various metal ions in Tris-HCl buffer (10 mM, pH 6.0). Insert: photograph of a solution of \u003cstrong\u003eSP-Fc\u003c/strong\u003e (10 μM) in the presence of Al\u003csup\u003e3+ \u003c/sup\u003eions under UV irradiation (λ\u003csub\u003eex\u003c/sub\u003e = 365 nm). (b) The fluorescent intensity at 445 nm of \u003cstrong\u003eSP-Fc\u003c/strong\u003e (10 μM) with 100 μM of various metal ions. 1, Free; 2, Na\u003csup\u003e+\u003c/sup\u003e; 3, Li\u003csup\u003e+\u003c/sup\u003e; 4, Cu\u003csup\u003e2+\u003c/sup\u003e; 5, Ni\u003csup\u003e2+\u003c/sup\u003e; 6, Fe\u003csup\u003e2+\u003c/sup\u003e; 7, Zn\u003csup\u003e2+\u003c/sup\u003e; 8, Mg\u003csup\u003e2+\u003c/sup\u003e; 9, Pb\u003csup\u003e2+\u003c/sup\u003e; 10, Mn\u003csup\u003e2+\u003c/sup\u003e; 11, Cd\u003csup\u003e2+\u003c/sup\u003e; 12, Hg\u003csup\u003e2+\u003c/sup\u003e; 13, Cr\u003csup\u003e3+\u003c/sup\u003e; 14,Al\u003csup\u003e3+\u003c/sup\u003e; 15, Fe\u003csup\u003e3+ \u003c/sup\u003e(λ\u003csub\u003eex\u003c/sub\u003e = 370 nm, λ\u003csub\u003eem\u003c/sub\u003e = 445 nm).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7514239/v1/c5e8053cbbf5bedb12587839.png"},{"id":92008895,"identity":"7cea332a-8efb-44a5-9644-5a22748dc4c0","added_by":"auto","created_at":"2025-09-23 15:33:07","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":54745,"visible":true,"origin":"","legend":"\u003cp\u003eThe fluorescent intensity at 445 nm of \u003cstrong\u003eSP-Fc\u003c/strong\u003e (10 μM) with 100 μM of Al\u003csup\u003e3+\u003c/sup\u003e ions, and then added 100 μM of various metal ions in buffer solution. 1, Free + Al\u003csup\u003e3+\u003c/sup\u003e; 2, Na\u003csup\u003e+ \u003c/sup\u003e+ Al\u003csup\u003e3+\u003c/sup\u003e; 3, Li\u003csup\u003e+ \u003c/sup\u003e+ Al\u003csup\u003e3+\u003c/sup\u003e; 4, Cu\u003csup\u003e2+ \u003c/sup\u003e+ Al\u003csup\u003e3+\u003c/sup\u003e; 5, Ni\u003csup\u003e2+ \u003c/sup\u003e+ Al\u003csup\u003e3+\u003c/sup\u003e; 6, Fe\u003csup\u003e2+ \u003c/sup\u003e+ Al\u003csup\u003e3+\u003c/sup\u003e; 7, Zn\u003csup\u003e2+ \u003c/sup\u003e+ Al\u003csup\u003e3+\u003c/sup\u003e; 8, Mg\u003csup\u003e2+ \u003c/sup\u003e+ Al\u003csup\u003e3+\u003c/sup\u003e; 9, Pb\u003csup\u003e2+ \u003c/sup\u003e+ Al\u003csup\u003e3+\u003c/sup\u003e; 10, Mn\u003csup\u003e2+ \u003c/sup\u003e+ Al\u003csup\u003e3+\u003c/sup\u003e; 11, Cd\u003csup\u003e2+ \u003c/sup\u003e+ Al\u003csup\u003e3+\u003c/sup\u003e; 12, Hg\u003csup\u003e2+ \u003c/sup\u003e+ Al\u003csup\u003e3+\u003c/sup\u003e; 13, Cr\u003csup\u003e3+ \u003c/sup\u003e+ Al\u003csup\u003e3+\u003c/sup\u003e; 14, Fe\u003csup\u003e3+ \u003c/sup\u003e+ Al\u003csup\u003e3+ \u003c/sup\u003e(λ\u003csub\u003eex\u003c/sub\u003e = 370 nm, λ\u003csub\u003eem\u003c/sub\u003e = 445 nm).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7514239/v1/1b80712a19e9ab089318edc9.png"},{"id":92011756,"identity":"b1df0ac0-7f9f-4430-939b-9fd4590ba1a5","added_by":"auto","created_at":"2025-09-23 15:49:08","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":121166,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Fluorescence emission spectra of 10 μM \u003cstrong\u003eSP-Fc\u003c/strong\u003e in the presence of different concentration of Al\u003csup\u003e3+ \u003c/sup\u003ein Tris-HCl buffer (10 mM, pH 6.0). (b) The plot of the fluorescence intensity of \u003cstrong\u003eSP-Fc\u003c/strong\u003e (10 μM) at 445 nm versus the concentration changes of Al\u003csup\u003e3+ \u003c/sup\u003eions (0-200 μM) in Tris-HCl buffer, the inset shows the linear range of the curve. The spectra were obtained 10 min after Al\u003csup\u003e3+\u003c/sup\u003e addition (λ\u003csub\u003eex\u003c/sub\u003e = 370 nm, λ\u003csub\u003eem\u003c/sub\u003e = 445 nm).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7514239/v1/b3c260897904e937070d572e.png"},{"id":92011757,"identity":"96baa66b-98c5-4a9e-9b33-000a8fe7cdfa","added_by":"auto","created_at":"2025-09-23 15:49:08","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":111925,"visible":true,"origin":"","legend":"\u003cp\u003eThe CV and DPV curves of the \u003cstrong\u003eSP-Fc\u003c/strong\u003e in CH\u003csub\u003e3\u003c/sub\u003eCN (100 μM) containing 0.1 M tetrabutyl ammonium hexafluorophosphate as electrolyte with the addition of 1 equiv. of each metal ions.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7514239/v1/918bfab87c97cc8cfa63b46d.png"},{"id":92008900,"identity":"eda6dcb4-b158-4e0e-b30a-5287fcb38443","added_by":"auto","created_at":"2025-09-23 15:33:08","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":50159,"visible":true,"origin":"","legend":"\u003cp\u003eJob’s plot obtained for \u003cstrong\u003eSP-Fc\u003c/strong\u003e and Al\u003csup\u003e3+\u003c/sup\u003e ion in Tris-HCl buffer (10 mM, pH 6.0). The total concentration of \u003cstrong\u003eSP-Fc\u003c/strong\u003e and Al\u003csup\u003e3+\u003c/sup\u003e was fixed at 20 μM (λ\u003csub\u003eex\u003c/sub\u003e = 370 nm, λ\u003csub\u003eem\u003c/sub\u003e = 445 nm).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7514239/v1/7d2501a1f50a26cffb4d0f95.png"},{"id":92010705,"identity":"21204f0b-589b-4ac5-8e2f-cf47d1c042b8","added_by":"auto","created_at":"2025-09-23 15:41:08","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":17837,"visible":true,"origin":"","legend":"\u003cp\u003eEvolution of the \u003csup\u003e1\u003c/sup\u003eH NMR spectra of \u003cstrong\u003eSP-Fc\u003c/strong\u003e upon addition of 1.0 equiv. Al\u003csup\u003e3+\u003c/sup\u003e in DMSO.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7514239/v1/2110289e333612286ab67e6b.png"},{"id":98814327,"identity":"a621f6df-e2ab-4fdb-9ed9-b4ecbbd776a8","added_by":"auto","created_at":"2025-12-22 16:12:21","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1786957,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7514239/v1/5723bed0-15c5-4a39-89b0-57dd9954b2db.pdf"},{"id":92008896,"identity":"d13b636f-f19d-404c-b056-f218693c0d77","added_by":"auto","created_at":"2025-09-23 15:33:08","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":36376,"visible":true,"origin":"","legend":"\u003cp\u003eScheme 1 molecule design of the chemosensor \u003cstrong\u003eSP-Fc\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Scheme1.png","url":"https://assets-eu.researchsquare.com/files/rs-7514239/v1/eab95371068a757ffcdbdfb4.png"},{"id":92008904,"identity":"94d6b1e7-a652-464c-b160-3aa9321a55cd","added_by":"auto","created_at":"2025-09-23 15:33:08","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":30237,"visible":true,"origin":"","legend":"\u003cp\u003eScheme 2 Synthetic route of chemosensor \u003cstrong\u003eSP-Fc.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Scheme2.png","url":"https://assets-eu.researchsquare.com/files/rs-7514239/v1/7b007a30f9197623f76119c9.png"},{"id":92008915,"identity":"dc7e5a33-5e87-48e8-b737-d1cb979a213e","added_by":"auto","created_at":"2025-09-23 15:33:08","extension":"doc","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":4630016,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"SupportingInformation.doc","url":"https://assets-eu.researchsquare.com/files/rs-7514239/v1/de88d552e01edebcc5bb8b0f.doc"},{"id":92008926,"identity":"5ec52cc3-db59-4d25-830a-a389b6d579ea","added_by":"auto","created_at":"2025-09-23 15:33:08","extension":"png","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":81046,"visible":true,"origin":"","legend":"\u003cp\u003eScheme 3. Proposed binding mode for \u003cstrong\u003eSP-Fc \u003c/strong\u003ewith\u003cstrong\u003e \u003c/strong\u003eAl\u003csup\u003e3+ \u003c/sup\u003eand the mechanism of fluorescence enhancement for the chemosenor \u003cstrong\u003eSP-Fc \u003c/strong\u003echelating\u003cstrong\u003e \u003c/strong\u003ewith\u003cstrong\u003e \u003c/strong\u003eAl\u003csup\u003e3+ \u003c/sup\u003e.\u003c/p\u003e","description":"","filename":"Scheme3.png","url":"https://assets-eu.researchsquare.com/files/rs-7514239/v1/f1a3787ac4d062162aa838fd.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"A Novel Ferrocene-Derivative-Based Dual-Response Chemosensor for Selective Al³⁺ Detection: Fluorescence and Electrochemical Signaling","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eAluminum is the third most abundant element (8.3wt%) in the Earth\u0026rsquo;s crust, after oxygen and silicon. As the most abundant metal, aluminum is characterized by good ductility and is ubiquitous in daily applications such as food additives, aluminum-based pharmaceuticals and cooking utensils [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. These widespread applications lead to frequent human exposure to aluminum. According to the World Health Organization (WHO), the average daily human intake of aluminum is approximately 3\u0026ndash;10 mg, while the permissible limit in drinking wateris 7.4 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e M [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Although aluminum is generally considered less toxic than heavy metals, excess intake in the human body is linked to an increased risk of several diseases like Parkinson\u0026rsquo;s disease and Alzheimer\u0026rsquo;s dementia. Additionally, it is well-known that 40% of the world\u0026rsquo;s acidic soils are contaminated with aluminum ions, which impair plant growth and development [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Given the close association of Al\u003csup\u003e3+\u003c/sup\u003e with human health and environmental protection, the development of reliable methods for the rapid and sensitive detection of Al\u003csup\u003e3+\u003c/sup\u003e in the biosphere is of considerable importance.\u003c/p\u003e\u003cp\u003eIn the past decades, a variety of modern techniques for sensing metal ions has been developed such as atomic absorption spectrometry [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], atomic emission spectrometry [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], and inductively coupled plasma mass spectrometry [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] etc. However, most of these methods require the high cost instruments, large quantity of sample and are not appropriate in many situations. Thus, the effective colorimetric, fluorescent and electrochemical chemosensors have received much attention due to their distinct advantages, including high sensitivity, low cost and real-time detection capability. But some of these single-channel sensors may be restricted by interfering substance in the actual samples, such as turbidity and color [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. In contrast, multi-channel sensors which could detecte analytes through multiple physical responses, provide better selectivity, higher sensitivity and self-calibration ability through variable detecting methods with low background in complex systems [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Consequently, construction and development of multichannel sensors for detecting Al\u003csup\u003e3+\u003c/sup\u003e would be highly desirable from the viewpoint of the practical applications.\u003c/p\u003e\u003cp\u003eRecent research shows that ferrocene derivatives are a good platform for multichannel sensing of various bioactive and toxic analytes on account of its excellent redox properties, good solubility, structural stability, and ease of derivatization, which can be effectively integrated with fluorogenic/chromogenic units [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. When interacting with an analyte, ferrocene-based chemosensors not only express a significant potential shift of the Fe\u003csup\u003e3+\u003c/sup\u003e/Fe\u003csup\u003e2+\u003c/sup\u003e redox couple, but also show other optical signal changes, which allow a single sensor to be applied in various experimental conditions. There are numerous ferrocene-based multichannel probes for metal ions have been developed, these multichannel probes were constructed by incorporating rhodamine, triazole, selenide, and schiff base scaffolds into one or two cyclopentadienyl rings of ferrocene [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Most of the reported ferrocene-based multichannel probes have been utilized for detecting metal ions such as Hg\u003csup\u003e2+\u003c/sup\u003e [\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], Cu\u003csup\u003e2+\u003c/sup\u003e [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], Pb\u003csup\u003e2+\u003c/sup\u003e [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], Fe\u003csup\u003e3+\u003c/sup\u003e [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], etc. While the detection of Al\u003csup\u003e3+\u003c/sup\u003e has received comparatively less attention [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. This disparity may arise from the poor coordination ability and strong hydration ability [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Therefore, a ration design and preparation of novel ferrocene-based multichannel chemosensors for Al\u003csup\u003e3+\u003c/sup\u003e is still challenging.\u003c/p\u003e\u003cp\u003eIn general, Al\u003csup\u003e3+\u003c/sup\u003e being a hard acid, prefers hard donor sites like N and O in its coordination sphere. Thus, various Schiff base derivatives of salicylaldehyde have been reported for recognition of Al\u003csup\u003e3+\u003c/sup\u003e owning to their generally one step synthesis and mixed N, O-donor sites [\u003cspan additionalcitationids=\"CR25 CR26 CR27\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Additionally, an N-containing pyridal unit with remarkable binding ability to metal ions has good optical properties. Therefore, salicylaldehyde pyridinehydrazone derivatives are a good platform for Al\u003csup\u003e3+\u003c/sup\u003e on account of the strong coordination ability between the nitrogen atom of imine, the oxygen atom of hydroxyl and metal ions, which may inhibit Photoinduced Electron Transfer (PET) and Excited State Intramolecular Proton Transfer (ESIPT) process as well as result in the Chelation-enhanced Fluorescence (CHEF). Based on this consideration, we decided to combine the redox activity of the ferrocenyl group with the turn-on fluorescent behavior and selective ability of the salicylaldehyde hydrazones as binding sites to construct a novel mutichannel chemosensors that specifically recognizing Al\u003csup\u003e3+\u003c/sup\u003e (scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Therefore, the target probe 5-ferrocenylsalicylaldehyde-2-pyridinehydrazone (\u003cb\u003eSP-Fc\u003c/b\u003e) has been designed by strategically introducing ferrocene into the salicylaldehyde skeleton. To the best of our knowledge, our probe is the first example of ferrocene-appended salicylaldehyde hydrazone moiety connected through carbon carbon single bond. Herein, we report the synthesis and characterization of 5-ferrocenylsalicylaldehyde-2-pyridinehydrazone (\u003cb\u003eSP-Fc\u003c/b\u003e) as a new mutichannel chemosensor for selective Al\u003csup\u003e3+\u003c/sup\u003e ions detection via optical and electrometric readouts. As expected, the sensor \u003cb\u003eSP-Fc\u003c/b\u003e exhibited a weak fluoresence based on PET and ESIPT process. After binding with Al\u003csup\u003e3+\u003c/sup\u003e, a new complex, \u003cb\u003eSP-Fc-\u003c/b\u003eAl\u003csup\u003e3+\u003c/sup\u003e, was formed and displayed a strong fluoresence due to CHEF effect and the inhibition of both PET and ESIPT processes. Furthermore, the electrochemical signals of \u003cb\u003eSP-Fc\u003c/b\u003e in the presence of the Al\u003csup\u003e3+\u003c/sup\u003e was shifted significantly, showing efficient electrochemical sensors. In a word, \u003cb\u003eSP-Fc\u003c/b\u003e could be used to be a dual-response chemosensor for Al\u003csup\u003e3+\u003c/sup\u003e with excellent sensitivity and selectivity both in optical and electrochemical ways.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"2. Experimental","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Reagents and Apparatus\u003c/h2\u003e\u003cp\u003eThe fine chemicals 4-aminophenol and ferrocene were purchased from Aladdin co. (Shanghai, China) and were used without further purification. DMSO and 10 mM different pH Tris-HCl buffer were purchased from Beyotime Biotechnology. All the chlorinated salts were purchased from Macklin and Aladdin co. (Shanghai, China). All salts were all dissolved in Tris-HCl buffer at 10 mM for analysis.\u003c/p\u003e\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eH NMR and \u003csup\u003e13\u003c/sup\u003eC NMR spectra were recorded on a AVANCE Ⅲ HD 600 MHz spectrometer with chemical shifts reported in ppm (in CDCl\u003csub\u003e3\u003c/sub\u003e; TMS as internal standard). High resolution mass (HRMS) spectrum were recorded in AB SCIEX high-resolution time-of-flight mass spectrometer (TripleTOF5600+) using HPLC acetonitrile as solvent. Melting points were determined on an X4 Digital Micro Melting Point apparatus. The measurements of pH were done in a digital pH meter (Merck). UV-vis spectra were acquired on an Evolution One UV-vis spectrophotometer (Thermo Scientific). Fluorescence spectra and absorption of MTT experiments were obtained on the full-wavelength multifunctional microplate reader (Tecan Infinite M1000 Pro). Electrochemical measurements were conducted with a Guangzhou IGS4030 Electrochemical Workstation. MTT experiments were carried out on 96-well plates (Corning). Fluorescence images were taken by the confocal laser scanning microscope (Olympus FV3000). The deionized water was prepared on a Milli-Q water purification system and used throughout all experiments.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Synthesis of sensor SP-Fc\u003c/h2\u003e\u003cdiv id=\"Sec5\" class=\"Section3\"\u003e\u003ch2\u003e\u003cb\u003e2.2.1 Synthesis of 5-ferrocenylsalicylaldehyde\u003c/b\u003e (\u003cb\u003e2\u003c/b\u003e)\u003c/h2\u003e\u003cp\u003e4-ferrocenylphenol (1.20 g, 4.3 mmol) and 15 mL glacial acetic acid were placed into a 100 mL three-neck flask and heated to 90\u0026deg;C in an oil bath. Then hexamethylenetetramine (1.20 g, 8.6 mmol) was dissolved in 15 mL of glacial acetic acid. The solution was slowly added to the three-necked flask via a constant-pressure dropping funnel and continued to stir for 2.5 h. After cooling to room temperature, the substrate was treated with 1M hydrochloric acid (20 mL) and reacted for another 1h at ambient temperature, then extracted with dichloromethane (50 mL) three times. The combined organic phases were dried with anhydrous sodium sulphate and evaporated under in vacuo. The residue was purified by column chromatography over silica gel eluted with petroleum ether : ethyl acetate\u0026thinsp;=\u0026thinsp;20:1. Yield: 40% (0.52 g), m.p.:132.5-133.2℃, \u003csup\u003e1\u003c/sup\u003eH NMR (600 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) : \u003cem\u003eδ\u003c/em\u003e 10.94 (s, 1H), 9.95 (s, 1H), 7.71 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.4 Hz、1.8 Hz, 1H), 7.62 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2.4 Hz, 1H), 6.99 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;9.0 Hz, 1H), 4.62 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1.8 Hz, 2H), 4.35 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1.8 Hz, 2H), 4.08 (s, 5H); \u003csup\u003e13\u003c/sup\u003eC NMR (150 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e): \u003cem\u003eδ\u003c/em\u003e 196.6, 159.9, 135.0, 131.3, 130.1, 120.55, 117.8, 84.2, 69.6, 69.0, 66.1; HR MS (ESI-TOF) m/z: [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e for C\u003csub\u003e17\u003c/sub\u003eH\u003csub\u003e14\u003c/sub\u003eFeO\u003csub\u003e2\u003c/sub\u003e: Calcd 307.0423, found 307.0394.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section3\"\u003e\u003ch2\u003e\u003cb\u003e2.2.2 Synthesis of 5-ferrocenylsalicylaldehyde-2-pyridinehydrazone\u003c/b\u003e (\u003cb\u003eSP-Fc\u003c/b\u003e)\u003c/h2\u003e\u003cp\u003e5-ferrocenylsalicylaldehyde (0.153 g, 0.5 mmol) and 2-hydrazinopyridine (0.054 g, 0.5 mmol) were dissolved in 15 mL absolute ethanol under nitrogen atomsphere. The mixture was refluxed for 4 h. The resulting precipitate was filtrated and washed with 20 mL of absolute ethanol. Recrystallized with ethanol, dried under vacuum and obtained the orange solid \u003cb\u003eSP-Fc\u003c/b\u003e. Yield: 58% (0.23 g), m.p.:198.6-199.7℃, \u003csup\u003e1\u003c/sup\u003eH NMR (600 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) : \u003cem\u003eδ\u003c/em\u003e 10.95 (s, 1H, -OH), 10.56 (s, 1H, -NH-), 8.30 (s, 1H, -CH\u0026thinsp;=\u0026thinsp;N-), 8.14 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.2 Hz、1.2 Hz, 1H), 7.67\u0026ndash;7.70 (m, 2H, ph-H), 7.40 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;12.6 Hz、7.8 Hz, 1H), 7.08 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;12.6 Hz, 1H), 6.85 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;12.6 Hz, 1H), 6.80 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;10.2 Hz、1.8 Hz, 1H), 4.71 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1.8 Hz, 2H, Fc-H), 4.30 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1.8 Hz, 2H, Fc-H), 4.03 (s, 5H, Fc-H); \u003csup\u003e13\u003c/sup\u003eC NMR (150 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) : \u003cem\u003eδ\u003c/em\u003e 155.6, 155.4, 147.9, 142.9, 138.6, 130.5, 128.5, 126.9, 118.0, 116.8, 116.5, 106.6, 85.3, 69.5, 68.4, 66.0; HR MS(ESI-TOF) m/z: [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e for C\u003csub\u003e22\u003c/sub\u003eH\u003csub\u003e19\u003c/sub\u003eFeN\u003csub\u003e3\u003c/sub\u003eO: calced 398.0958, found 398.0943.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Standard solution\u003c/h2\u003e\u003cp\u003eThe stock solutions (0.01 M) of chlorinated salts (Na\u003csup\u003e+\u003c/sup\u003e, Li\u003csup\u003e+\u003c/sup\u003e, Cu\u003csup\u003e2+\u003c/sup\u003e, Ni\u003csup\u003e2+\u003c/sup\u003e, Fe\u003csup\u003e2+\u003c/sup\u003e, Zn\u003csup\u003e2+\u003c/sup\u003e, Mg\u003csup\u003e2+\u003c/sup\u003e, Pb\u003csup\u003e2+\u003c/sup\u003e, Mn\u003csup\u003e2+\u003c/sup\u003e, Cd\u003csup\u003e2+\u003c/sup\u003e, Hg\u003csup\u003e2+\u003c/sup\u003e, Cr\u003csup\u003e3+\u003c/sup\u003e, Al\u003csup\u003e3+\u003c/sup\u003e, Fe\u003csup\u003e3+\u003c/sup\u003e) were prepared with 10 mM Tris-HCl buffer solution (pH 6.0). The 5-ferrocenylsalicylaldehyde-2-pyridinehydrazone stock solution (0.001 M) was prepared in DMSO. Test solutions were prepared by placing calculation amount of 5-ferrocenylsalicylaldehyde-2-pyridinehydrazone stock solution and each metal solution into a test tube, and then diluted to 0.2 mL with Tris-HCl buffer solution (10 mM, pH 6.0). The resulting solution was mixed well and recorded 10 min after the addition of determinand at room temperature. The excitation wavelength used in the fluorescence examination was 370 nm, and the slits width of excitation and emission were all 10 nm.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Electrochemical testing method\u003c/h2\u003e\u003cp\u003eA solution of probe \u003cb\u003eSP-Fc\u003c/b\u003e (1\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e M) in acetonitrile was prepared using 0.1 M tetrabutylammonium hexafluorophosphate (Bu₄NPF₆) as the supporting electrolyte. A 3 mL aliquot of the \u003cb\u003eSP-Fc\u003c/b\u003e solution was combined with 1 equivalent (1 equivalent\u0026thinsp;=\u0026thinsp;30 \u0026micro;L of 1\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e mol/L solution) of the analyte metal ion solution using a micropipette. Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) measurements were subsequently performed. All measurements employed a standard three-electrode configuration: a glassy carbon electrode (GCE) served as the working electrode, a platinum wire as the counter electrode, and an Ag/AgCl electrode as the reference electrode. The scan rate was set to 0.1 V/s. Prior to use, the glassy carbon electrode was polished with 0.05 \u0026micro;m Al₂O₃ slurry and thoroughly rinsed via sonication in ultrapure water.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results and discussion","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Synthesis\u003c/h2\u003e\u003cp\u003eThe target probe \u003cb\u003eSP-Fc\u003c/b\u003e was efficiently synthesized with a high yield by condensing 5-ferrocenylsalicylaldehyde (\u003cb\u003e2\u003c/b\u003e) with 2-hydrazinopyridine, as illustrated in scheme \u003cspan refid=\"Sch2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. 5-ferrocenylsalicylaldehyde was the key intermediate which was obtained by formylating 4-ferrocenylphenol (\u003cb\u003e1\u003c/b\u003e) through the Duff reaction. The material 4-ferrocenylphenol (\u003cb\u003e1\u003c/b\u003e) was synthesized using a previously reported method [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Then, The obtained ferrocene derivative \u003cb\u003eSP-Fc\u003c/b\u003e underwent characterization through \u003csup\u003e1\u003c/sup\u003eH NMR and \u003csup\u003e13\u003c/sup\u003eC NMR as well as ESI-TOF mass spectrometry (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e-S3). To evaluate the metal recognition capabilities of sensor \u003cb\u003eSP-Fc\u003c/b\u003e, various studies, including optical, electrochemical, and \u003csup\u003e1\u003c/sup\u003eH NMR titrations, were conducted.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e3.2 The optical properties of SP-Fc toward various metal ions\u003c/h2\u003e\u003cp\u003eTo assess the recognizing properties of the probe \u003cb\u003eSP-Fc\u003c/b\u003e to metal ions, the fluorescence response of \u003cb\u003eSP-Fc\u003c/b\u003e (10 \u0026micro;M) in the presence of various metal ions (100 \u0026micro;M) such as Na\u003csup\u003e+\u003c/sup\u003e, Li\u003csup\u003e+\u003c/sup\u003e, Cu\u003csup\u003e2+\u003c/sup\u003e, Ni\u003csup\u003e2+\u003c/sup\u003e, Fe\u003csup\u003e2+\u003c/sup\u003e, Zn\u003csup\u003e2+\u003c/sup\u003e, Mg\u003csup\u003e2+\u003c/sup\u003e, Pb\u003csup\u003e2+\u003c/sup\u003e, Mn\u003csup\u003e2+\u003c/sup\u003e, Cd\u003csup\u003e2+\u003c/sup\u003e, Hg\u003csup\u003e2+\u003c/sup\u003e, Cr\u003csup\u003e3+\u003c/sup\u003e, Al\u003csup\u003e3+\u003c/sup\u003e, Fe\u003csup\u003e3+\u003c/sup\u003e (chlorinated salts) were tested in a Tris-HCl buffer (10 mM, pH\u0026thinsp;=\u0026thinsp;6.0). The fluorescence emission spectra were recorded within 10 min after the addition of various metal ions. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, free probe \u003cb\u003eSP-Fc\u003c/b\u003e exhibited a weak fluorescence emission among the range of 400\u0026ndash;600 nm when excited with UV lamp at 365 nm, which can be attributed to two fluorescent quenching mechnisms including the photo-induced electron transfer (PET) of ferrocene to the pyridyl group and the excited-state intramolecular proton transfer (ESIPT) between the phenolic hydroxyl group and the imine nitrogen [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. when the sensor was treated with a variety of metal cations, only Al\u003csup\u003e3+\u003c/sup\u003e generated outstanding fluoresence enhancement at 445 nm, which displayed strong \u0026ldquo;turn-on\u0026rdquo; fluorescent emission. Enhancement of fluorescence emission might be, owing to the prevention of the PET and ESIPT process as well as the chelation-enhanced fluorescence (CHEF) effect when \u003cb\u003eSP-Fc\u003c/b\u003e was complexed with Al\u003csup\u003e3+\u003c/sup\u003e. Under the same experimental conditions, other competing metal ions had no effect on the fluorescence. Meanwhile, The UV-Vis absorption spectra of probe \u003cb\u003eSP-Fc\u003c/b\u003e was also recorded in the absence and presence of one equivalent of the Al\u003csup\u003e3+\u003c/sup\u003e ions in DMSO solution (Fig. S4). The solution of free \u003cb\u003eSP-Fc\u003c/b\u003e exhibited two intense absorption band in the UV region (300 nm and 348 nm) and a relatively weak band in the visible region. The high energy (HE) bands can be ascribed to π-π* and L-π* electronic transitions, and the low energy (LE) band originated from the localized excitation either from an Fe (II) d-d transition or a metal-ligand charge transfer (MLCT) process (dπ-π*) (visible region) [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], which was responsible for the orange color of the sensors. However, the intensity of absorption bands (348 nm) decreased with a concomitant appearance of two clear isosbestic points at 309 nm and 372 nm after addition of Al\u003csup\u003e3+\u003c/sup\u003e ions. The generation of isosbestic points clearly indicated the formation of \u003cb\u003eSP-Fc-Al\u003c/b\u003e\u003csup\u003e\u003cb\u003e3+\u003c/b\u003e\u003c/sup\u003e complex. Hence, it could be concluded that probe \u003cb\u003eSP-Fc\u003c/b\u003e was a highly selective fluorescence \u0026ldquo;turn-on\u0026rdquo; sensor for Al\u003csup\u003e3+\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo further investigation of the specificity of \u003cb\u003eSP-Fc\u003c/b\u003e, competitive experiments were carried out in the presence of Al\u003csup\u003e3+\u003c/sup\u003e ions (100 \u0026micro;M) mixed with other competitive metal ions (100 \u0026micro;M). Their fluorescence intensities were recorded and Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e depicts the fluorescence changes observed. As can be clearly seen, the fluorescence emission intensity of \u003cb\u003eSP-Fc\u003c/b\u003e/Al\u003csup\u003e3+\u003c/sup\u003e solution showed no pronounced interference by Na\u003csup\u003e+\u003c/sup\u003e, Li\u003csup\u003e+\u003c/sup\u003e, Zn\u003csup\u003e2+\u003c/sup\u003e, Mg\u003csup\u003e2+\u003c/sup\u003e, Pb\u003csup\u003e2+\u003c/sup\u003e, Mn\u003csup\u003e2+\u003c/sup\u003e, Cd\u003csup\u003e2+\u003c/sup\u003e, Hg\u003csup\u003e2+\u003c/sup\u003e, Fe\u003csup\u003e3+\u003c/sup\u003e. While the fluoresence response of \u003cb\u003eSP-Fc\u003c/b\u003e to Al\u003csup\u003e3+\u003c/sup\u003e in the presence of Cu\u003csup\u003e2+\u003c/sup\u003e, Ni\u003csup\u003e2+\u003c/sup\u003e and Fe\u003csup\u003e2+\u003c/sup\u003e is slightly decreased but clearly detectable, although these ions showed no significant effect on the fluorescence intensity of \u003cb\u003eSP-Fc\u003c/b\u003e individually (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). The above-results on the changes of fluorescence spectra demonstrated that \u003cb\u003eSP-Fc\u003c/b\u003e can be used as a highly effective fluorescent chemosensor for the detection of Al\u003csup\u003e3+\u003c/sup\u003e over other common metal ions.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e3.3 pH effect on sensor SP-Fc.\u003c/h2\u003e\u003cp\u003eThe performance characteristic of the sensor was monitored for its coordination ability toward Al center under different pH conditions. In this work, all the fluorescence measurements were conducted in 10 mM Tris-HCl buffer solvent. The variation of fluorescence intensity for \u003cb\u003eSP-Fc\u003c/b\u003e against different pH values in the absence and presence of Al\u003csup\u003e3+\u003c/sup\u003e were recorded and the results were shown in Fig. S5. The aqueous solutions of the free \u003cb\u003eSP-Fc\u003c/b\u003e showed no obvious changes in the fluorescence intensity at 445 nm over the pH range of 4.0\u0026ndash;9.0, which meaning that \u003cb\u003eSP-Fc\u003c/b\u003e was stable over the wide pH range. In acidic environments (pH\u0026thinsp;\u0026lt;\u0026thinsp;6.8), the emission intensity of \u003cb\u003eSP-Fc-Al\u003c/b\u003e\u003csup\u003e\u003cb\u003e3+\u003c/b\u003e\u003c/sup\u003e complex was dramatically increased. However, in the neutral conditions (pH 6.8\u0026ndash;7.4), the fluorescence intensity of \u003cb\u003eSP-Fc\u003c/b\u003e-Al\u003csup\u003e3+\u003c/sup\u003e was gradually decreased. While in the basic conditions (pH\u0026thinsp;\u0026gt;\u0026thinsp;7.4), almost no changes in the fluorescence intensity were found after Al\u003csup\u003e3+\u003c/sup\u003e addition with the reason of the formation of Al(Ⅲ) hydroxide complex under this condition. The pH study shows that \u003cb\u003eSP-Fc\u003c/b\u003e could work in a broad pH range with very low background fluorescence. pH 6 was chosen in the next study because it is the most remarkable fluorescence intensity changes under this condition.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e3.4 Sensitivity of SP-Fc toward Al\u003csup\u003e3+\u003c/sup\u003e\u003c/h2\u003e\u003cp\u003eThe sensing capability of \u003cb\u003eSP-Fc\u003c/b\u003e toward Al\u003csup\u003e3+\u003c/sup\u003e ion was further investigated in detail by fluorescence titration analysis. The fluorescent spectra were recorded after addition with different concentrations from 0 \u0026micro;M to 200 \u0026micro;M Al\u003csup\u003e3+\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). As Al\u003csup\u003e3+\u003c/sup\u003e ion was gradually added, the fluorescence intensity (at 445 nm) of the chemosensor gradually enhanced and reached the maximum after addition of 10 equivalent of Al\u003csup\u003e3+\u003c/sup\u003e. Subsequently, upon further addition of Al\u003csup\u003e3+\u003c/sup\u003e, the fluorescence spectrum remained at a plate au (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). These observations indicated that the conversion of \u003cb\u003eSP-Fc\u003c/b\u003e to the corresponding aluminum-probe complex was implemented in the detecting process. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb, a linear relationship (R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.9903) between the fluorescence intensity and Al\u003csup\u003e3+\u003c/sup\u003e concentration (0\u0026ndash;10 \u0026micro;M) demonstrated that Al\u003csup\u003e3+\u003c/sup\u003e concentration could be quantitatively detected by \u003cb\u003eSP-Fc\u003c/b\u003e. The limits of detection (LOD) were calculated as 1.30 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;7\u003c/sup\u003e M for Al\u003csup\u003e3+\u003c/sup\u003e using thee quation LOD\u0026thinsp;=\u0026thinsp;3*S\u003csub\u003eb\u003c/sub\u003e/S [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], where S\u003csub\u003eb\u003c/sub\u003e is the standard deviation of 10 replicate measurements of blank \u003cb\u003eSP-Fc\u003c/b\u003e solution and S is the slope of the calibration curve. The result suggests that the sensor has great potential for sensitive detection of Al\u003csup\u003e3+\u003c/sup\u003e in biological samples.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e3.5 Electrochemical sensing properties of sensor SP-Fc\u003c/h2\u003e\u003cp\u003eTaking advantages of the well-behaved electrochemically reversible nature of ferrocene derivatives, the electrochemical characteristics of sensor \u003cb\u003eSP-Fc\u003c/b\u003e for metal-recognition properties were investigated by Cyclic voltammetry (CV) and differential pulse voltammetry (DPV). The reversibility and relative oxidation potential of the redox process were determined in a CH\u003csub\u003e3\u003c/sub\u003eCN solution (1\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e M) containing 0.1 M tetrabutylammonium hexafluorophosphate (Bu₄NPF₆) as the supporting electrolyte. As can be seen from Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, the sensor \u003cb\u003eSP-Fc\u003c/b\u003e exhibits a reversible single-electron oxidation process at E\u003csub\u003e1/2\u003c/sub\u003e = 0.477 V, due to the ferrocene/ferrocenium redox couple. No perturbation of the cyclic and differential pulse voltammograms of sensor \u003cb\u003eSP-Fc\u003c/b\u003e was observed in the presence of several metal cations such as Li\u003csup\u003e+\u003c/sup\u003e, Na\u003csup\u003e+\u003c/sup\u003e, Mg\u003csup\u003e2+\u003c/sup\u003e, Fe\u003csup\u003e2+\u003c/sup\u003e, Fe\u003csup\u003e3+\u003c/sup\u003e, Cu\u003csup\u003e2+\u003c/sup\u003e, Zn\u003csup\u003e2+\u003c/sup\u003e, Cd\u003csup\u003e2+\u003c/sup\u003e, Cr\u003csup\u003e3+\u003c/sup\u003e, Mn\u003csup\u003e2+\u003c/sup\u003e, Hg\u003csup\u003e2+\u003c/sup\u003e, Pb\u003csup\u003e2+\u003c/sup\u003e ions, even in large excess. However, a significant cathodic change of the oxidation peak (E\u003csub\u003e1/2\u003c/sub\u003e) versus Fc\u003csup\u003e+\u003c/sup\u003e/Fc was shifted from 0.477 V to 0.135 V (∆E\u003csub\u003e1/2\u003c/sub\u003e = 0.342 V) upon addition of 1 equiv. of Al\u003csup\u003e3+\u003c/sup\u003e, similar phenomena displayed in DPV of \u003cb\u003eSP-Fc\u003c/b\u003e. These shifts were attributed to the formation of the corresponding \u003cb\u003eSP-Fc-Al\u003c/b\u003e\u003csup\u003e3+\u003c/sup\u003e. So the receptor \u003cb\u003eSP-Fc\u003c/b\u003e was an excellent electrochemical sensor for recognition of Al\u003csup\u003e3+\u003c/sup\u003e ions with specific selectivity.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e3.6 Mechanism of SP-Fc in sensing Al\u003csup\u003e3+\u003c/sup\u003e\u003c/h2\u003e\u003cp\u003eTo determine the binding stoichiometry of between \u003cb\u003eSP-Fc\u003c/b\u003e and responding metal ions (Al\u003csup\u003e3+\u003c/sup\u003e), Job\u0026rsquo;s method for fluorescence titration was carried out (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The maximum fluorescence band was reached at a molar fraction of 0.5, indicating a 1:1 ratio for \u003cb\u003eSP-Fc\u003c/b\u003e-\u003cb\u003eAl\u003c/b\u003e\u003csup\u003e\u003cb\u003e3+\u003c/b\u003e\u003c/sup\u003e complex.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo further confirm the results of spectroscopic and electrochemical experiments, \u003csup\u003e1\u003c/sup\u003eH NMR titration experiments of \u003cb\u003eSP-Fc\u003c/b\u003e with Al\u003csup\u003e3+\u003c/sup\u003e were conducted in DMSO-d\u003csub\u003e6\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). In the free sensor spectrum, phenolic hydroxyl (OHa) resonated as a singlet at δ 10.95 ppm. The imine proton (-CHb\u0026thinsp;=\u0026thinsp;N-) and hydrazinic N-H proton (-NHc-) resonated at 8.30 ppm and 10.56 ppm, respectively. Upon addition of 1.0 equiv. Al\u003csup\u003e3+\u003c/sup\u003e ions to the \u003cb\u003eSP-Fc\u003c/b\u003e solution, the signal of phenolic hydroxyl (OHa) was disappeared, indicating deprotonation and coordination of the phenolic oxygen to Al\u003csup\u003e3+\u003c/sup\u003e. The imine proton peak (Hb) was shifted downfield by ca. 0.18 ppm, such downfield shift suggested that Al\u003csup\u003e3+\u003c/sup\u003e was chelated by nitrogen atom of the imine. While the hydrazinic N-H proton exhibited upfield shift from 10.56 ppm to 10.47 ppm, suggesting non-involvement of the hydrazinic nitrogen in metal binding. These chemical shift changes confirm adduct formation between Al\u003csup\u003e3+\u003c/sup\u003e and \u003cb\u003eSP-Fc\u003c/b\u003e. According to the above discussions and take into account the Job\u0026rsquo;s plot, a plausible binding mode and fluorescence sensing mechnism are proposed in Scheme \u003cspan refid=\"Sch3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. Wherein Al\u003csup\u003e3+\u003c/sup\u003e coordinates via the phenolic oxygen, imine nitrogen and pyridine nitrogen atoms.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e3.7 Cell imaging\u003c/h2\u003e\u003cp\u003eTo explore its application in cell imaging, the cytotoxicity of the sensor \u003cb\u003eSP-Fc\u003c/b\u003e was studied. We performed MTT assay in MDA-MB-231cells treated with a series of concentrations (5, 10, 20, and 50 \u0026micro;M, respectively) of \u003cb\u003eSP-Fc\u003c/b\u003e for 24 h. As shown in Fig. S3, 10 \u0026micro;M \u003cb\u003eSP-Fc\u003c/b\u003e showed no obvious cytotoxicity with cell viabilities greater than 90%. The results indicate that \u003cb\u003eSP-Fc\u003c/b\u003e has low toxicit for MCF-7 cells.\u003c/p\u003e\u003cp\u003eTo further investigate the potential biological application of \u003cb\u003eSP-Fc\u003c/b\u003e in living cells, the intracellular Al\u003csup\u003e3+\u003c/sup\u003e imaging of human breast cancer cells was performed by fluorescence microscopy. For this purpose, MDA-MB-231 cells were incubated with various concentrations of aqueous Al\u003csup\u003e3+\u003c/sup\u003e solutions (0, 10, and 50 \u0026micro;M) in growth media for 2 h at 37 ℃, and then treated with 10 \u0026micro;M \u003cb\u003eSP-Fc\u003c/b\u003e for 6 h before imaging. \u003cb\u003eSP-Fc\u003c/b\u003e was found to be cell membrane permeable and to recognize intracellular aluminumion, resulting in bright blue intracellular fluorescence could clearly be observed by confocal fluorescence microscopy (Fig. S6) With an increase in Al\u003csup\u003e3+\u003c/sup\u003e concentration from 0 to 50 \u0026micro;M, the fluorescence intensity of the cell swith \u003cb\u003eSP-Fc\u003c/b\u003e increased gradually, which could be attributed to the formation of the intracellular \u003cb\u003eSP-Fc\u003c/b\u003e-Al\u003csup\u003e3+\u003c/sup\u003e complex. Meanwhile, the simple cells (with nothing treated) and the cells with 10 \u0026micro;M \u003cb\u003eSP-Fc\u003c/b\u003e in absence of Al\u003csup\u003e3+\u003c/sup\u003e were also incubated in growth media as the control samples. Both of them exhibited no fluorescence. The result of this experiment showed that the \u003cb\u003eSP-Fc\u003c/b\u003e sensor has the potential for live cell imaging and could be used for the detection of additional and intrinsic Al\u003csup\u003e3+\u003c/sup\u003e ion of the living cells.\u003c/p\u003e\u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eA new chemosensor 5-ferrocenylsalicylaldehyde-2-pyridinehydrazone (\u003cb\u003eSP-Fc\u003c/b\u003e) was successfully synthesized by combining the redox-active ferrocene unit with the metal-binding salicylaldehyde hydrazone scaffold. Its binding properties towards various metal ions were evaluated through optical and electrochemical analyses. \u003cb\u003eSP-Fc\u003c/b\u003e exhibited selective\u0026ldquo;turn-on\u0026rdquo;fluorescence toward Al\u003csup\u003e3+\u003c/sup\u003e, attributed to efficient inhibition of both PET and ESIPT process upon Al\u003csup\u003e3+\u003c/sup\u003e binding and CHEF. The 1:1 binding stoichiometry was confirmed, with a detection limit of 1.30 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;7\u003c/sup\u003e M based on fluoresence detection. Notably, Al\u003csup\u003e3+\u003c/sup\u003eaddition induced a significant anodic shift (∆E\u003csub\u003e1/2\u003c/sub\u003e = 0.342 V) in the ferrocene redox potential, providing a complementary electrochemical detection channel. Moreover, the sensor \u003cb\u003eSP-Fc\u003c/b\u003e also could be used to determine Al\u003csup\u003e3+\u003c/sup\u003e in living cells, making it a promising chemosensor for detection of Al\u003csup\u003e3+\u003c/sup\u003e in the biological system.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eCompeting Interests\u003c/h2\u003e\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis work was supported by Science and Technology Research Project of Jiangxi Provincial Department of Education (Grant numbers [GJJ2200953] ) ; Jiangxi Provincial Health and Family Planning Commission-Traditional Chinese Medicine Research Project (Grant numbers [2024B0017] ) ; College Students' Innovation and Entrepreneurship Training Program (2024). Jiangxi Province 2024 Graduate Innovation Special Fund Project (YC2024-S750).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAuthor ContributionsWenqin Liu: Methodology, Investigation, Writing-original draft, Data curation, Writing-review \u0026amp; editing, Conceptualization. Yanping Liu : Formal analysis, Investigation. Zenghui Li : Data curation. Santai Zou: Data curation. Pingnan Wan:Investigation. Yan Lin: Funding acquisition, Resources, Methodology, Project administration, Supervision, Writing-review \u0026amp; editing. All authors reviewed the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBurgess J (1996) Man and the elements of Group 3 and 13. 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Sens Actuators B 243:765\u0026ndash;774. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.snb.2016.12.067\u003c/span\u003e\u003cspan address=\"10.1016/j.snb.2016.12.067\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Schemes","content":"\u003cp\u003eSchemes 1 to 3 are available in the Supplementary Files section\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-fluorescence","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jofl","sideBox":"Learn more about [Journal of Fluorescence](https://www.springer.com/journal/10895)","snPcode":"10895","submissionUrl":"https://submission.nature.com/new-submission/10895/3","title":"Journal of Fluorescence","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Chemosensor, ferrocene derivative, fluorescence, electrochemistry, dual-response","lastPublishedDoi":"10.21203/rs.3.rs-7514239/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7514239/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eA new Chemosensor, 5-ferrocenylsalicylaldehyde-2-pyridinehydrazone (\u003cb\u003eSP-Fc\u003c/b\u003e), was designed and synthesized by condensing 5-ferrocenylsalicylaldehyde with 2-hydrazinopyridine. Its structure was characterized by \u003csup\u003e1\u003c/sup\u003eH NMR, \u003csup\u003e13\u003c/sup\u003eC NMR and HRMS. \u003cb\u003eSP-Fc\u003c/b\u003e could be used to identify Al\u003csup\u003e3+\u003c/sup\u003e through dual optical and electrochemical responses. Upon addition of Al\u003csup\u003e3+\u003c/sup\u003e, the sensor exhibited a significant fluorescence enhancement attributed to CHEF and the inhibition of both PET and ESIPT processes. The sensor showed a high selectivity for Al\u003csup\u003e3+\u003c/sup\u003e in DMSO solution at pH\u0026thinsp;=\u0026thinsp;6 and was not disturbed by other competing metal ions. In additon, the detection limit for Al\u003csup\u003e3+\u003c/sup\u003e was observed as 1.30 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;7\u003c/sup\u003e M. Job\u0026rsquo;s plot analysis and NMR titration experiment confirmed 1:1 stiochiometry between Al\u003csup\u003e3+\u003c/sup\u003e and probe through phenolic oxygen, imine nitrogen and pyridine nitrogen atoms. Moreover, the electrochemical signals of \u003cb\u003eSP-Fc\u003c/b\u003e in the presence of the Al\u003csup\u003e3+\u003c/sup\u003e was shifted significantly compared with those of the other metal cations tested, indicating \u003cb\u003eSP-Fc\u003c/b\u003e could be used to be a dual-response chemosensor for Al\u003csup\u003e3+\u003c/sup\u003e with excellent sensitivity and selectivity both in optical and electrochemical ways. Also, Confocal fluorescence microscopy imaging demonstrated that \u003cb\u003eSP-Fc\u003c/b\u003e can monitor Al\u003csup\u003e3+\u003c/sup\u003e in living MCF-7 cells with low cytotoxicity.\u003c/p\u003e","manuscriptTitle":"A Novel Ferrocene-Derivative-Based Dual-Response Chemosensor for Selective Al³⁺ Detection: Fluorescence and Electrochemical Signaling","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-23 15:33:03","doi":"10.21203/rs.3.rs-7514239/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-09-25T16:38:13+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-24T11:31:22+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-20T14:15:18+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"275745185030246442817640703029690788778","date":"2025-09-17T22:18:03+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"216458658248570671718643026761642407986","date":"2025-09-17T14:10:35+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-16T07:46:35+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"76678282823454917776411580938351398060","date":"2025-09-16T00:55:57+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"54893096038485909716345180391919324506","date":"2025-09-15T13:09:17+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-15T12:04:55+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-04T07:52:51+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-04T07:51:53+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Fluorescence","date":"2025-09-02T06:49:20+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-fluorescence","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jofl","sideBox":"Learn more about [Journal of Fluorescence](https://www.springer.com/journal/10895)","snPcode":"10895","submissionUrl":"https://submission.nature.com/new-submission/10895/3","title":"Journal of Fluorescence","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"2d4ac921-0c4f-46ed-8163-d9bded1d7173","owner":[],"postedDate":"September 23rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-12-22T16:07:56+00:00","versionOfRecord":{"articleIdentity":"rs-7514239","link":"https://doi.org/10.1007/s10895-025-04627-x","journal":{"identity":"journal-of-fluorescence","isVorOnly":false,"title":"Journal of Fluorescence"},"publishedOn":"2025-12-16 15:57:40","publishedOnDateReadable":"December 16th, 2025"},"versionCreatedAt":"2025-09-23 15:33:03","video":"","vorDoi":"10.1007/s10895-025-04627-x","vorDoiUrl":"https://doi.org/10.1007/s10895-025-04627-x","workflowStages":[]},"version":"v1","identity":"rs-7514239","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7514239","identity":"rs-7514239","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}