A selective pyridinedicarbohydrazide-based “naked-eye” fluorescent probe for detecting Cu 2+ ions in aqueous solutions

A selective pyridinedicarbohydrazide-based “naked-eye” fluorescent probe for detecting Cu 2+ ions in aqueous solutions | 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 selective pyridinedicarbohydrazide-based “naked-eye” fluorescent probe for detecting Cu 2+ ions in aqueous solutions Ali Zamani, Mahmood Tajbakhsh, Yaghoub Sarrafi This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6740853/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 02 Sep, 2025 Read the published version in Journal of Fluorescence → Version 1 posted 11 You are reading this latest preprint version Abstract A novel fluorescent and colorimetric probe (L3), based on 2,6-pyridinedicarbohydrazide, was rationally designed and synthesized for the highly selective and reversible detection of Cu²⁺ ions. The structural and optical properties of the probe were comprehensively characterized by FT-IR, ¹H NMR, UV-Vis spectroscopy, fluorescence analysis, ESI⁺-MS, and elemental analysis. Upon interaction with Cu²⁺, probe L3 exhibited distinct optical responses, including significant fluorescence quenching, a notable redshift in the UV-Vis spectrum, and a rapid, visible color change from colorless to greenish-yellow under aqueous conditions and across a broad pH range. The detection limit was calculated to be as low as 2.28 × 10⁻⁹ M, with an association constant (Ka) of 2.2 × 10¹¹ M⁻², indicating strong binding affinity. Job's plot and ¹H NMR titration confirmed a 1:2 stoichiometric complex formation between L3 and Cu²⁺ ions. The probe also exhibited excellent reversibility, as confirmed by fluorescence recovery upon adding Na₂EDTA, suggesting its suitability for real-time and reusable sensing applications. Furthermore, the practical utility of L3 was demonstrated using test strips for Cu²⁺ detection in aqueous environments, and its effectiveness was further validated in real water samples, including tap water, drinking water, and seawater, achieving recovery rates up to 99.2%. These findings suggest that L3 is promising for the sensitive, rapid, and cost-effective monitoring of copper ions in environmental and drinking water systems. Copper ions Colorimetric Reversible probe Pyridine-based sensor Naked-eye detection Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Introduction The pervasive influence of transition and heavy metal ions across biological systems and environmental matrices has positioned their accurate detection as a central challenge in modern analytical chemistry [1–6]. Fluorescence-based detection systems have emerged as a preferred analytical tool due to their exceptional sensitivity, straightforward operation, and ability to provide immediate and real-time responses [7–10]. These advantages have facilitated their extensive adoption in various fields, including environmental monitoring, medical diagnostics, pollution control, biomedical research, and quality assessment in the industrial and food sectors [11–19]. Recently, increasing attention has been devoted to engineering fluorescent probes capable of selectively recognizing biologically and environmentally relevant metal ions [20–24]. Notably, copper (II) a redox-active trace element, plays indispensable roles in vital physiological processes such as mitochondrial respiration, connective tissue maturation, and neurological function [22,25–30]. Despite its essentiality, sustained exposure to elevated copper levels can disturb cellular homeostasis and has been linked to hepatic and renal toxicity, as well as the progression of neurodegenerative disorders, including Wilson’s, Menkes, and Alzheimer’s diseases [31–35]. In light of these concerns, the World Health Organization has established 2 ppm as the maximum permissible level of copper in drinking water [36,37]. Given its dual role as both a critical micronutrient and a potential toxicant, the reliable quantification of Cu²⁺ ions in complex matrices remains an urgent analytical imperative [38–40]. Although several 2,6-pyridinedicarbohydrazide (PDH)-based probes have been explored for Cu²⁺ detection, many still face limitations in terms of detection limit, association constant, reversibility, or practical applicability in aqueous media. For example, PDH functionalized with salicylaldehyde demonstrated turn-off fluorescence with a detection limit of 3.11 × 10⁻⁵ M and a binding constant of 1.978 × 10⁵ M⁻¹, yet lacked applicability in test strip formats and real water samples [41]. Similarly, PDH functionalized with a 2-hydroxy-1-naphthaldehyde ligand showed a 1:2 stoichiometry and a detection limit of 0.097 μM, but exhibited a lower binding constant (Ka = 11.145) and limited practical demonstration beyond laboratory conditions [42]. More recently, PDH functionalized with indole-3-carboxaldehyde exhibited strong Cu²⁺ binding (Ka = 2.9–3.5 × 10¹¹ M⁻², LOD = 4.2 × 10⁻⁹ M) and rapid visual detection using test strips; however, it did not demonstrate fluorescence reversibility or performance in real-world matrices such as seawater [43]. In this context, we have developed a novel dual-mode (fluorescent/colorimetric) probe, L3, based on a functionalized 2,6-pyridinedicarbohydrazide core. L3 combines ultra-trace detection capability (LOD = 2.28 × 10⁻⁹ M), very high binding affinity (Ka = 2.2 × 10¹¹ M⁻²), excellent selectivity, and rapid signal response. Notably, it exhibits full reversibility using EDTA, strong performance over a broad pH range (5–9), and robust applicability in real water samples. These features distinguish L3 as a high-performance, field-adaptable sensor for practical, low-cost Cu²⁺ monitoring in both laboratory and environmental settings. Experimental 2.1 Apparatus Tetramethylsilane (TMS) is used as the internal standard, and CDCl 3 or DMSO-d6 is used as the solvent in the 1 H and 13 C NMR spectrometer on a Bruker Ultrashield 400 MHz Avance III spectrometer. A JASCO FP-8300 fluorescence spectrophotometer was employed to record the fluorescence spectra. With the use of a Unico 4802 UV/Vis double beam spectrophotometer, the UV–visible absorption spectra were recorded. A Costech-ECS 4010 CHNSO Analyzer was utilized to evaluate the outcomes of the elemental analyses. MSD Agilent 5975C was used to obtain mass spectra. A Fourier-transform infrared (FT-IR) spectrum of the compounds was obtained using a Bruker TENSOR 27 device. The melting point was measured using an Electrothermal Engineering IA9100 melting point apparatus. All pH measurements were conducted using a Metrohm 744 digital pH meter. 2.2 Materials All solvents and chemicals were analytical grades, obtained from Sigma-Aldrich, and utilized without additional purification. Metal ions (Ba 2 + , K + , Pb 2+ , Hg 2+ , Al 3+ , Zn 2+ , Fe 2+ , Tl + , Mg 2+ , Ni 2+ , Nd 3+ , Fe 3+ , Ag + , Ca 2+ , Cs + , Co 2+ , Cd 2+ , Sr 2+ , Mn 2+ , and Cu 2+ ions) were used in the form of their acetate salts. 2.3 Synthesis 2.3.1 Synthesis of dimethyl pyridine-2,6-dicarboxylate (L1) [44]: A solution of the 2,6-pyridinedicarboxylic acid (250 mg, 1.5 mmol) and 95% H 2 SO 4 (2.93 g, 34.9 mmol, 1.59 mL) in MeOH (25 mL) was heated under reflux for 14 h. After extraction with dichloromethane (2 × 25 mL), the reaction mixture was neutralized with a saturated NaHCO₃ solution, and the organic layer was dried over anhydrous Na₂SO₄. The organic solvent was evaporated under reduced pressure to afford a white solid (0.292 g, %100). m.p. 121 °C; 13 CNMR (100 MHz, CDCl 3 ) δ ppm 165.1, 148.2, 138.4, 128.0, 53.2 1 HNMR (400 MHz, DMSO-d6) δ ppm 8.32 (2H, d, J = 7.6 Hz, Ar-H), 8.03 (1H, t, J = 7.6 Hz, Ar-H), 4.03 (6H, s, OCH 3 ); (Figs. S1-S3). 2.3.2 Synthesis of pyridine-2,6-dicarbohydrazide (L2) [45]: A solution of pyridine-2,6-dimethylester (0.23 g, 1.11 mmol) and hydrazine monohydrate (1.18 g, 23.5 mmol, 1.14 mL) in 25 mL MeOH was refluxed for 12 h. The reaction mixture was filtered to yield a white solid, which was subsequently washed with methanol (3 × 20 mL) to afford pure white crystals (0.19 g, 86%). m.p. 298 °C; 13 CNMR (100 MHz DMSO-d6) δ ppm 162.4, 148.9, 139.8, 124.1; 1 H NMR (400 MHz, DMSO-d6) δ ppm 10.63 (2H, s, NH), 8.13 (3H, s, Ar-H), 4.63 (4H, s, NH 2 ); (Figs. S4-S6). 2.3.3 Synthesis of N,N' -((((pyridine-2,6-dicarbonyl)bis(hydrazin-2-yl-1-ylidene))bis(methaneylylidene))bis(4,1-phenylene))diacetamide (L3) A mixture of compound L2 (0.5 g, 2.56 mmol), and N -(4-formylphenyl)acetamide (0.85 g, 5.2 mmol) was dissolved in ethanol (35 mL), in a round-bottomed flask. The reaction mixture was refluxed and agitated for seven hours under an argon atmosphere. The resultant white solid was separated by filtration and cleansed using hot ethanol. After drying, a white solid (probe L3) was obtained (1.16 g, 94%). m.p. 312°c; Anal. calc. for C 25 H 23 N 7 O 4 : C, 61.85; H, 4.78; N, 20.20, Found: C, 61.69; H, 4.91; N, 20.03; 13 C NMR (100 MHz, DMSO-d6) δ ppm 169.1, 159.7, 150.4, 148.8, 141.8, 140.4, 129.1, 128.5, 125.8, 119.4, 24.6; 1 H NMR (400MHz, DMSO-d6) δ ppm 12.25 (2H, s, NH), 10.19 (2H, s, NH), 8.70 (2H, S, C=NH), 8.37(2H, m, ArH), 8.28(1H, m, ArH), 7.77 (4H, m, ArH), 7.72 (4H, m, ArH), 2.08 (6H, s, CH 3 ); 1 H NMR (400MHz, D 2 O) δ ppm 8.37(2H, m, ArH), 8.28(1H, m, ArH), 7.77 (4H, m, ArH), 7.72 (4H, m, ArH), 2.08 (6H, s, CH 3 ); FT-IR spectrum (cm -1 ): 3511, 3258, 3106, 3061, 2969, 1674, 1662, 1616, 1592, 1512, 1452, 1410, 1370, 1320, 1262, 1174, 1078,1004, 963, 844; The mass spectrum reveals a molecular ion at m/z = 485, which aligns with the probe L3; (Figs. S7-S11). Results and Discussion The colorimetric probe L3 for the recognition of Cu 2+ ions was synthesized by reacting 2,6-pyridinedicarbohydrazide with 4-acetamidobenzaldehyde in ethanol and isolated as a white solid with a high yield (Scheme 1) and characterized by 1 H NMR, 13 C NMR, ESI + -MS, FT-IR, and elemental analysis (Figs S1–S11 in supporting information). The optical properties were assessed using UV-visible and fluorescence spectroscopy. 3.1 Colorimetric recognition The selective detection of environmentally relevant metal ions by probe L3 was evaluated through its interaction with 20 different metal ions, including Ba²⁺, K⁺, Pb²⁺, Hg²⁺, Al³⁺, Zn²⁺, Fe²⁺, Tl⁺, Mg²⁺, Ni²⁺, Nd³⁺, Fe³⁺, Ag⁺, Ca²⁺, Cs⁺, Co²⁺, Cd²⁺, Sr²⁺, Mn²⁺, and Cu²⁺, in a DMSO/H₂O (7:3, v/v) solution. The selectivity was assessed visually and spectroscopically using UV-Vis and fluorescence techniques. As shown in Fig. 1, the addition of 2.0 equivalents of these metal ions to a 1.0 × 10⁻⁵ M solution of L3 revealed that only Cu²⁺ induced a noticeable color change from colorless to greenish-yellow. The probe L3 is colorless in solution and exhibits an absorption band at 330 nm, which is attributed to an intermolecular π–π* charge transfer transition, in agreement with previous reports [46,47]. Upon the addition of 2.0 equivalents of Cu²⁺ ions, a clear bathochromic shift of approximately 30 nm and a decrease in the intensity of the 330 nm band was observed. Simultaneously, a new absorption band emerged at 360 nm, along with a broad band extending into the visible region (Fig. 2), suggesting the occurrence of an intramolecular charge transfer (ICT) process [41,46,48–50]. In contrast, none of the other tested metal ions caused significant changes in the absorption spectrum of L3, further confirming its high selectivity toward Cu²⁺ ions. To gain deeper insight into the binding behavior, UV-Vis titration of L3 (5.0 × 10⁻⁶ M) with increasing amounts of Cu²⁺ (0–2.0 eq) was conducted in DMSO/H₂O (7:3, v/v). The absorbance at 330 nm gradually decreased, accompanied by the growth of a new absorption band between 370 and 480 nm (Fig. 3). A well-defined isosbestic point at 369 nm was observed during the titration, indicating a clean interconversion between the free and complexed forms of L3 and confirming the formation of a stable L3+Cu²⁺ complex. 3.2 Fluorescent recognition of Cu 2+ The fluorescence response of L3 (5.0 µM) in DMSO/H 2 O (7:3, v/v) solution towards various metal ions (2.0 eq) was tested to further investigate the sensing capabilities of L3. As shown in Fig. 4, probe L3 demonstrated a significant emission at 472 nm when excited at 330 nm, which might be attributable to the ICT process. The addition of various metal ions, including Ba 2+ , K + , Pb 2+ , Al 3+ , Hg 2+ , Zn 2+ , Fe 2+ , Tl + , Mg 2+ , Ni 2+ , Nd 3+ , Fe 3+ , Ag + , Ca 2+ , Cs + , Co 2+ , Cd 2+ , Sr 2+ , and Mn 2+ ions to L3 solution caused almost no discernible change in fluorescence spectra. However, the addition of Cu 2+ to probe L3 caused a significant change in fluorescence and its quenching. According to the previous results, after adding Cu 2 + ions, the L3 probe shows complete fluorescence quenching, which could be due to the formation of the L3+Cu 2+ complex, thus confirming the ICT process. This selectivity can be attributed to the inability of other metal ions to form comparable interactions with probe L3, thereby failing to induce significant fluorescence changes. The high affinity of L3 for Cu²⁺ may be explained by soft-soft interactions between the Cu²⁺ ions and the nitrogen donor atoms within the probe structure [51]. The involvement of nitrogen atoms in coordinating with Cu²⁺ enhances molecular conjugation and promotes electron delocalization, consistent with the ICT process. Supporting evidence includes the observed redshift in the UV-Vis absorption spectrum, a rapid and distinct color change upon Cu²⁺ addition, and pronounced fluorescence quenching—all of which confirm complex formation via an ICT-driven binding mechanism [41,46,49,52–54]. 3.3 Binding constants Fluorescence quenching was seen upon the gradual addition of Cu 2+ ions to the probe L3 solution (Fig. 5), indicating complexation between the probe L3 and Cu 2+ ions. The association constant Ka of L3+ Cu 2+ complex was calculated from the fluorescence titration to be 2.2 × (Fig. S12). Additionally, the probe's detection limit for Cu 2+ ions was calculated using 3d/S, as , indicating that it is capable of detecting extremely low copper concentrations in DMSO/H 2 O (7:3, v/v) (Fig. S13). Using Job plot's continuous variation techniques, the stoichiometric ratio of probe L3 and copper was calculated (Fig. S14). According to the results, the concentration of the probe-guest complex reaches a maximum when the mole fraction of the probe is around 0.67, which could indicate the formation of a 1:2 complex between probe L3 and copper ions. 3.4 Interference studies High selectivity is one of the essential qualities of the probe L3 responding to Cu 2+ ions. Fluorescence interference investigations were conducted under the same lab conditions in the presence of various competing metal ions to further support the probe L3's great selectivity for Cu 2+ ions. As seen in Fig. 6, the fluorescence intensity at 472 nm for L3 (5.0 µM) with Cu 2+ (2.0 eq) was barely affected by the presence of the other metal cations (2.0 eq). The distinctive selectivity of L3 for Cu 2+ ions may be ascribed to the soft-soft metal interactions between the Cu 2+ and nitrogen atoms of the probe L3. These results clearly show that L3 might be a highly responsive sensor for the Cu 2+ ions with good anti-interference performance among the other relevant metal ions. 3. 5 Binding mode of probe L3 towards Cu 2+ ions The binding of copper ions with probe L3 is supported by the 1 H NMR titration and the FT-IR spectra. The results of the 1 H NMR titration experiment allow for the identification of a binding mode between the probe L3 and the copper ions. The 1 H NMR spectra of probe L3 with different amounts of Cu 2+ ions (0, 1.0, and 2.0 eq) were recorded and shown in Fig. 7. Upon addition of Cu 2+ , PDH amide protons become broadened and shifted from 12.25 to 12.78 ppm. A new broad peak at 10.52 appeared next to the amide protons of phenylacetamide, and the amide peak disappeared. The N=CH protons were also broadened and shifted from 8.71 to 8.89 ppm. There was a slight up-field shift in other peaks (approx. 0.06 ppm). The spectra indicate that amide protons and N = CH protons are involved in Cu 2+ ions binding with L3. The results showed that adding two equivalents of copper causes significant changes in the spectra. These results indicate the formation of a complex with stoichiometry beyond a simple 1:1 ratio, likely a 1:2 (L3:Cu²⁺) complex. The Job's plot illustrates that L3 forms a 1:2 complex with Cu 2+ , which was further confirmed by UV–Vis and NMR titrations. There are some significant differences between the results of the probe L3 and L3+Cu 2+ in IR analysis (Fig. S15). The metal-nitrogen linkages are confirmed by the IR spectra of L3 and L3+Cu 2+ . The distinct absorption bands at 987 and 933 cm -1 lead to the Cu-N stretching and bending vibration bands. In addition, bands at 3511, and 3258 cm -1 for –NH, 1674, and 1662 cm -1 for C=O, and 1592 cm -1 for C=N in the L3 spectrum disappeared on the complexation with Cu 2+ ions. At 3298, 1615, 1566, and 1392 cm -1 , a few novel bands that support the complexation occurring with C=N, -NH appeared. The changes described above in the IR spectrum of probe L3 after the addition of the Cu 2+ ions, and the results of the 1 H NMR titration, indicate that NH protons and C=N are important in the binding of Cu 2+ ions with prob L3. 3.6 pH effect To evaluate the pH-dependent fluorescence response of probe L3, measurements were conducted in a DMSO/H₂O (7:3, v/v) solution across a broad pH range (2–12). As illustrated in Fig. 8, the fluorescence intensity of L3 remained stable and relatively strong within the pH range from 4 to 10, with an optimal performance between pH 5 and 9. Below pH 4, fluorescence was significantly diminished, likely due to the protonation of donor nitrogen atoms in the ligand framework, which may inhibit coordination to Cu²⁺ ions. Conversely, a marked decrease in fluorescence above pH 10 may be attributed to potential deprotonation-induced structural perturbations of the probe. Therefore, the pH range of 5–9 was identified as the most suitable operational conditions for Cu²⁺ detection using L3, which was adopted for subsequent sensing experiments. 3.7 Time The sensitivity of probe L3 was further evaluated by examining its fluorescence response time upon exposure to Cu²⁺ ions. To this end, a 5.0 µM solution of L3 was treated with 2.0 equivalents of Cu²⁺ in a DMSO/H₂O (7:3, v/v), and the fluorescence intensity was monitored over time. As illustrated in Fig. 9, the addition of Cu²⁺ ions led to a sharp and immediate decrease in fluorescence intensity, which then remained nearly constant for up to 30 minutes. This rapid and stable quenching behavior suggests a fast complexation process between L3 and Cu²⁺ ions, highlighting the probe’s potential for real-time detection of copper ions in aqueous media. 3.8 Reversibility test Reversibility is a key feature of an efficient probe, especially in applications like metal ion detection. The reversibility of probe L3 was investigated using disodium ethylenediaminetetraacetate (Na 2 EDTA) well-known metal chelator [55,56]. Upon sequential addition of Na₂EDTA to a preformed L3+Cu²⁺ complex, a marked restoration of fluorescence emission at 472 nm was observed, closely resembling the emission profile of the unbound probe. This fluorescence recovery implies that Na₂EDTA effectively competes for Cu²⁺ ions, displacing them from the coordination sphere of L3 and regenerating its emissive state (Fig. S16). Such reversible binding behavior is a critical feature for real-time monitoring applications, indicating that L3 retains its sensing functionality over multiple detection–regeneration cycles. 3.9 Practical application Filter papers were used to study the primary applicability of probe L3. Filter papers were dipped into a solution containing (100 µM) L3 to create test strips, which were then allowed to air dry. Afterward, the test strips were sequentially immersed in different concentrations of copper (0-2 eq) for 1 min, then dried, and observed clearly under sunlight and 365 nm UV light. The filter paper color changes (Fig. 10) demonstrated that as the concentration of copper was raised, the color gradually changed from white to dark yellow under daylight, and from greenish yellow to dark gray under a 365 nm UV lamp. These findings demonstrate the practical potential of probe L3 for detecting Cu²⁺ ions at varying concentrations in aqueous environments, utilizing test strips observable under both natural light and UV illumination. 3.10 Analysis of Real Samples The applicability of probe L3 for detecting Cu 2+ ions in real samples such as tap water, drinking water, and the Caspian Sea water was investigated employing the fluorescence method. First, the Cu 2+ ions concentration in each sample was determined using atomic absorption spectroscopy, and then three concentrations of the Cu 2+ ions (10, 20, and 30 μ M) were prepared for each sample by the addition of Cu 2+ to the samples employing a spiking technique. All the samples were titrated against the probe L3, and the results exhibited an excellent recovery of up to 99.2% (Table 1). These findings underscore the probe L3’s potential as a reliable tool for the detection of Cu²⁺ ions in diverse aqueous environments, thus making it a promising candidate for monitoring copper contamination in environmental water sources. Table 1 Quantification of Cu 2+ ions in real aqueous samples Water samples Amount of standard Cu 2+ ion added ( μ M) Total Cu 2+ ion found ( μ M) (n =3) a Recovery of Cu 2 + ions added (%) Tap water 10.0 9.81 0.981 20.0 19.76 0.988 30.0 29.63 0.987 Drinking water 10.0 9.90 0.990 20.0 19.84 0.992 30.0 29.71 0.990 Caspian Sea water 10.0 9.61 0.961 20.0 19.49 0.974 30.0 29.11 0.97 a: All experiments were repeated three times, and the mean values were reported. 3.11 Comparison To assess the performance of the synthesized probe L3 for Cu²⁺ detection, a comparative analysis was conducted with several previously reported fluorescent receptors, as summarized in Table 2. While all listed probes exhibit useful sensing capabilities, L3 demonstrates several notable advantages in aqueous environments. These include a distinct fluorescence turn-off response upon Cu²⁺ binding, a higher association constant, a lower limit of detection (LOD), and superior selectivity. Furthermore, L3 operates effectively across a broad pH range (5–9), showing excellent performance in real water samples, and is compatible with test strip-based analyses. Collectively, these attributes highlight the practical potential of L3 as a versatile and robust sensor for copper ions detection. Table 2 Comparison of the reported probes for detecting the Cu 2+ ions Ref Detection method LOD (M) Association constant pH Reversible Application (yes or no) [41] Fluorescence, UV-Vis 1.978 × 10 -5 M 3.11× 10 5 M − 1 4-8 NR NR [46] UV-Vis 1.2 × 10 -7 M 9.08 × 10 4 M -1 NR NR NR [57] Fluorescence, UV-Vis 1.49 × 10 -6 M 8.89 × 10 3 M -1 4-10 Yes NR [58] Fluorescence, UV-Vis 5 × 10 -9 M 2.3 × 10 5 M − 1 NR NR NR [59] Fluorescence, UV-Vis 1.89 × 10 -7 M 1.5 × NR NR Yes [60] Fluorescence 0.48 × 10 -6 M 6.04 × 10 7 M -2 5-9 NR Yes [61] Fluorescence, UV-Vis 2.7 × 10 -7 M 4.86 × 10 4 M -1 4-9 NR Yes [62] Fluorescence, UV-Vis 1.39 × 10 − 7 M 2.1 × 10 5 M − 1 NR Yes Yes [63] Fluorescence 1.21 × 10 − 8 M 1.79 × 10 6 M − 1 3-11 Yes NR [64] Fluorescence 4.5× 10 -8 M 9.9× 10 9 M -2 NR Yes Yes [65] Fluorescence 1.64 × 10 -8 M 1.22 × 10 3 M -1 3-11 No Yes [66] Fluorescence 1.82 ×10 -8 M 6.59 × 10 5 M − 1 NR NR Yes [43] Fluorescence, UV-Vis 4.2 ×10 -9 M 2.9-3.5 ×10 11 M -2 4.5-11.5 NR Yes This work Fluorescence, UV-Vis M 2.2 × 5-9 Yes Yes NR: Not reported Conclusion In conclusion, we have developed a highly efficient fluorescent and colorimetric probe (L3) for the selective detection of Cu²⁺ ions, based on a 2,6-pyridinedicarbohydrazide scaffold. The probe exhibited reliable and distinct optical responses, including fluorescence quenching and redshifted absorption upon coordination with Cu²⁺ ions. Spectroscopic analysis and ¹H NMR titration confirmed the formation of a well-defined 1:2 (L3:Cu²⁺) complex, while the high association constant and low detection limit (2.28 × 10⁻⁹ M) demonstrated its strong binding affinity and sensitivity. L3 functioned effectively across a pH range of 5.0 to 9.0 and displayed excellent reversibility, as evidenced by fluorescence restoration upon EDTA treatment, confirming its potential for reusable and real-time monitoring. The practical utility of L3 was further enhanced by the successful fabrication of test strips capable of visually detecting Cu²⁺ under both daylight and UV light. Moreover, the probe maintained high accuracy in real water samples, with recovery rates reaching up to 99.2%. These results underscore the applicability of L3 as a robust and sensitive platform for environmental and analytical monitoring of copper ions. These findings support the potential of L3 for the future development of portable Cu²⁺ detection systems for environmental and biological applications. Declarations The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Ethics Approval This is an observational study. The University of Mazandaran Research Ethics Committee has confirmed that no ethical approval is required. Competing Interests The authors have no relevant financial or non-financial interests to disclose. Author contributions Ali Zamani: Methodology, Formal analysis, Validation, Investigation, Resources, Writing - original draft. Mahmood Tajbakhsh: Conceptualization, Validation, Investigation, Writing - review & editing. Yaghoub Sarrafi: Validation, Conceptualization, Investigation. Funding Partial financial support was received from the Research Council of the University of Mazandaran. Data Availability The datasets generated during and analyzed during the current study are not publicly available due to [According to policies of the University of Mazandaran that all analysis devices are not connected to the Internet, the output of all devices is printed as a sheet or available on CD.] but are available from the Journal or the corresponding authors. Acknowledgments Financial support for this work from the Research Council of the University of Mazandaran is gratefully acknowledged. References Valeur B, Leray I. No Title. Coord Chem Rev 2000;205:3. de Silva AP, Fox DB, Huxley AJM, Moody TS. Combining luminescence, coordination and electron transfer for signalling purposes. Coord Chem Rev 2000;205:41–57. https://doi.org/https://doi.org/10.1016/S0010-8545(00)00238-1. 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Supplementary Files supportingmaterial.docx Cite Share Download PDF Status: Published Journal Publication published 02 Sep, 2025 Read the published version in Journal of Fluorescence → Version 1 posted Editorial decision: Revision requested 15 Jun, 2025 Reviews received at journal 15 Jun, 2025 Reviews received at journal 14 Jun, 2025 Reviews received at journal 11 Jun, 2025 Reviewers agreed at journal 10 Jun, 2025 Reviewers agreed at journal 05 Jun, 2025 Reviewers agreed at journal 05 Jun, 2025 Reviewers invited by journal 05 Jun, 2025 Editor assigned by journal 27 May, 2025 Submission checks completed at journal 27 May, 2025 First submitted to journal 24 May, 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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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-6740853","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":467300496,"identity":"3d3dcc9a-67f9-4fa4-b885-c6ae56fcf7db","order_by":0,"name":"Ali Zamani","email":"","orcid":"","institution":"University of Mazandaran","correspondingAuthor":false,"prefix":"","firstName":"Ali","middleName":"","lastName":"Zamani","suffix":""},{"id":467300497,"identity":"1043e3f9-f074-4206-8cde-92287649f1c7","order_by":1,"name":"Mahmood Tajbakhsh","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1ElEQVRIiWNgGAWjYHCCNAaGAwwJ/DAuGyH1PDAtkg0kaGEDazE4QKyr7NkPPHvw40xdnvGN7OQPDDV2DHzSBDTz8CSkG/bcOFxsdiN3gwHDsWQGNr4EQg5LSJPg+XAgcRtQSwID2wEGNh5CfuF/kCb550Nd4uYZuRsOMPwjRotEQpo0zw3mxA0SuRsbGNuI0XLjQbqxzJnDiTPOvN3MkNiXzENQC3t/TtrDN8fqEvvbczd/+PDNTk6+h4AWoD0JCHYCOKIIAvYDRCgaBaNgFIyCEQ0ABZBDFnIOJ+gAAAAASUVORK5CYII=","orcid":"","institution":"University of Mazandaran","correspondingAuthor":true,"prefix":"","firstName":"Mahmood","middleName":"","lastName":"Tajbakhsh","suffix":""},{"id":467300499,"identity":"ac7bf36c-0b9d-421c-ac2b-165ddb087a98","order_by":2,"name":"Yaghoub Sarrafi","email":"","orcid":"","institution":"University of Mazandaran","correspondingAuthor":false,"prefix":"","firstName":"Yaghoub","middleName":"","lastName":"Sarrafi","suffix":""}],"badges":[],"createdAt":"2025-05-24 20:38:26","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6740853/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6740853/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10895-025-04473-x","type":"published","date":"2025-09-02T15:57:58+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":84313679,"identity":"63e4c10f-13cf-4164-aae0-7f3d44b8280b","added_by":"auto","created_at":"2025-06-10 13:02:07","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":91022,"visible":true,"origin":"","legend":"\u003cp\u003eThe change in the color of (1.0 × 10\u003csup\u003e-5\u003c/sup\u003e M) L3 in DMSO/H\u003csub\u003e2\u003c/sub\u003eO (7:3, v/v) with (2.0 eq) various metal ions\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6740853/v1/7f69d915a21847c97919ebb4.jpg"},{"id":84313681,"identity":"775692a3-1eb3-48f9-be8d-752b40929650","added_by":"auto","created_at":"2025-06-10 13:02:07","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":70748,"visible":true,"origin":"","legend":"\u003cp\u003eAbsorbance spectra of L3 (5.0 μM) in the absence and presence of 2.0 eq various metal\u003cbr\u003e\nions in DMSO/H\u003csub\u003e2\u003c/sub\u003eO (7/3, v/v) solution\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6740853/v1/26b546a34996c981503e1222.jpg"},{"id":84316100,"identity":"8223975c-e482-4a80-8efc-ad3f4ede89d5","added_by":"auto","created_at":"2025-06-10 13:26:07","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":68405,"visible":true,"origin":"","legend":"\u003cp\u003eAbsorption spectra of L3 (5.0 M) in DMSO/H\u003csub\u003e2\u003c/sub\u003eO (7/3, v/v) upon addition of various amounts of Cu\u003csup\u003e2+ \u003c/sup\u003eions\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6740853/v1/d576b06fd787b9f06ced7510.jpg"},{"id":84313682,"identity":"4a278863-7764-486b-bfa8-998eba610ebd","added_by":"auto","created_at":"2025-06-10 13:02:07","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":74635,"visible":true,"origin":"","legend":"\u003cp\u003eFluorescence spectra of probe L3 (5.0 M) in DMSO/H\u003csub\u003e2\u003c/sub\u003eO (7/3, v/v) solution in the presence of various metal ions (2.0 eq). Excited at 330 nm\u003c/p\u003e","description":"","filename":"Picture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6740853/v1/080f56731d0eb836cb2d6080.jpg"},{"id":84315220,"identity":"5dbc9d95-b124-4437-a07b-3df23c631779","added_by":"auto","created_at":"2025-06-10 13:18:07","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":74986,"visible":true,"origin":"","legend":"\u003cp\u003eFluorescence spectra of probe L3 (5.0 M) upon the addition of Cu\u003csup\u003e2+\u003c/sup\u003e ions (0–2 eq). Excited at 330 nm\u003c/p\u003e","description":"","filename":"Picture5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6740853/v1/681a14d632613233df099f6e.jpg"},{"id":84313689,"identity":"7acbca99-fda1-43ba-a854-b301f0d57143","added_by":"auto","created_at":"2025-06-10 13:02:07","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":93150,"visible":true,"origin":"","legend":"\u003cp\u003eThe variations in the fluorescence intensity of L3 (5.0× 10\u003csup\u003e-6\u003c/sup\u003e M) in DMSO/H2O (7:3 v/v) with the addition of 2.0 eq of the specified cations (ex = 330 nm, em = 472 nm)\u003c/p\u003e","description":"","filename":"Picture6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6740853/v1/cf3fb7a591486bc6c0b6db06.jpg"},{"id":84313684,"identity":"b84a5434-7a15-41ed-8432-a44ff8c3ec9b","added_by":"auto","created_at":"2025-06-10 13:02:07","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":155750,"visible":true,"origin":"","legend":"\u003cp\u003eL3+Cu\u003csup\u003e2+\u003c/sup\u003e \u003csup\u003e1\u003c/sup\u003eH NMR titration spectra upon the addition of Cu\u003csup\u003e2+\u003c/sup\u003e ions in DMSO-d6 solution\u003c/p\u003e","description":"","filename":"Picture7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6740853/v1/834a0b2059278ff43c4cfe6e.jpg"},{"id":84313686,"identity":"b23ec7ff-52cd-46aa-b014-dad3c1e20e33","added_by":"auto","created_at":"2025-06-10 13:02:07","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":38448,"visible":true,"origin":"","legend":"\u003cp\u003eVariation of fluorescence (472 nm) of free probe L3 \u003cstrong\u003e(\u003c/strong\u003e5.0 M) and in the presence of (2.0 eq) Cu\u003csup\u003e2+ \u003c/sup\u003ein DMSO/H\u003csub\u003e2\u003c/sub\u003eO (7/3, v/v) at different pH (ex = 330 nm, em = 472 nm)\u003c/p\u003e","description":"","filename":"Picture8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6740853/v1/84811578d75e9f73bfef5ad2.jpg"},{"id":84313687,"identity":"39278579-0b03-4339-ba89-936a670c0548","added_by":"auto","created_at":"2025-06-10 13:02:07","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":67805,"visible":true,"origin":"","legend":"\u003cp\u003eTime-dependent fluorescence response of L3 (5.0 × 10\u003csup\u003e-6\u003c/sup\u003e M) in DMSO/H2O (7/3 V/V) in the presence of copper ions (ex = 330 nm, em = 472 nm)\u003c/p\u003e","description":"","filename":"Picture9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6740853/v1/d896cb333b0721376996678c.jpg"},{"id":84314850,"identity":"4a35f2f9-f02c-40ce-a678-44ee8cc05f1a","added_by":"auto","created_at":"2025-06-10 13:10:07","extension":"jpg","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":77883,"visible":true,"origin":"","legend":"\u003cp\u003eThe test strips' color changes under a UV lamp and daylight for detecting various concentrations of Cu\u003csup\u003e2+\u003c/sup\u003e ions (0, 0.5, 1, 1.5, 1.75, and 2 eq) in DMSO/H\u003csub\u003e2\u003c/sub\u003eO solution (7/3, v/v)\u003c/p\u003e","description":"","filename":"Picture10.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6740853/v1/f2d2549ec1c0e2f4ca0fcb2b.jpg"},{"id":90827985,"identity":"5ec842eb-9a29-420f-a072-540925ab76bf","added_by":"auto","created_at":"2025-09-08 16:04:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1661236,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6740853/v1/6ed0d699-1387-4f21-95d6-75f9ee235d1a.pdf"},{"id":84313688,"identity":"9c9cae73-9c02-4423-8bfa-0c070ca1ea78","added_by":"auto","created_at":"2025-06-10 13:02:07","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":945739,"visible":true,"origin":"","legend":"","description":"","filename":"supportingmaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-6740853/v1/27d89ed3b1c5f4c1cc8bc8cd.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"A selective pyridinedicarbohydrazide-based “naked-eye” fluorescent probe for detecting Cu 2+ ions in aqueous solutions","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe pervasive influence of transition and heavy metal ions across biological systems and environmental matrices has positioned their accurate detection as a central challenge in modern analytical chemistry [1\u0026ndash;6]. Fluorescence-based detection systems have emerged as a preferred analytical tool due to their exceptional sensitivity, straightforward operation, and ability to provide immediate and real-time responses [7\u0026ndash;10]. These advantages have facilitated their extensive adoption in various fields, including environmental monitoring, medical diagnostics, pollution control, biomedical research, and quality assessment in the industrial and food sectors [11\u0026ndash;19]. Recently, increasing attention has been devoted to engineering fluorescent probes capable of selectively recognizing biologically and environmentally relevant metal ions [20\u0026ndash;24]. Notably, copper (II) a redox-active trace element, plays indispensable roles in vital physiological processes such as mitochondrial respiration, connective tissue maturation, and neurological function [22,25\u0026ndash;30]. Despite its essentiality, sustained exposure to elevated copper levels can disturb cellular homeostasis and has been linked to hepatic and renal toxicity, as well as the progression of neurodegenerative disorders, including Wilson\u0026rsquo;s, Menkes, and Alzheimer\u0026rsquo;s diseases [31\u0026ndash;35]. In light of these concerns, the World Health Organization has established 2 ppm as the maximum permissible level of copper in drinking water [36,37]. Given its dual role as both a critical micronutrient and a potential toxicant, the reliable quantification of Cu\u0026sup2;⁺ ions in complex matrices remains an urgent analytical imperative [38\u0026ndash;40]. Although several 2,6-pyridinedicarbohydrazide (PDH)-based probes have been explored for Cu\u0026sup2;⁺ detection, many still face limitations in terms of detection limit, association constant, reversibility, or practical applicability in aqueous media. For example, PDH functionalized with salicylaldehyde demonstrated turn-off fluorescence with a detection limit of 3.11 \u0026times; 10⁻⁵ M and a binding constant of 1.978 \u0026times; 10⁵ M⁻\u0026sup1;, yet lacked applicability in test strip formats and real water samples [41]. Similarly, PDH functionalized with a 2-hydroxy-1-naphthaldehyde ligand showed a 1:2 stoichiometry and a detection limit of 0.097 \u0026mu;M, but exhibited a lower binding constant (Ka = 11.145) and limited practical demonstration beyond laboratory conditions [42]. More recently, PDH functionalized with indole-3-carboxaldehyde exhibited strong Cu\u0026sup2;⁺ binding (Ka = 2.9\u0026ndash;3.5 \u0026times; 10\u0026sup1;\u0026sup1; M⁻\u0026sup2;, LOD = 4.2 \u0026times; 10⁻⁹ M) and rapid visual detection using test strips; however, it did not demonstrate fluorescence reversibility or performance in real-world matrices such as seawater [43]. In this context, we have developed a novel dual-mode (fluorescent/colorimetric) probe, L3, based on a functionalized 2,6-pyridinedicarbohydrazide core. L3 combines ultra-trace detection capability (LOD = 2.28 \u0026times; 10⁻⁹ M), very high binding affinity (Ka = 2.2 \u0026times; 10\u0026sup1;\u0026sup1; M⁻\u0026sup2;), excellent selectivity, and rapid signal response. Notably, it exhibits full reversibility using EDTA, strong performance over a broad pH range (5\u0026ndash;9), and robust applicability in real water samples. These features distinguish L3 as a high-performance, field-adaptable sensor for practical, low-cost Cu\u0026sup2;⁺ monitoring in both laboratory and environmental settings.\u003c/p\u003e"},{"header":"Experimental","content":"\u003cp\u003e\u003cstrong\u003e2.1 Apparatus\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTetramethylsilane (TMS) is used as the internal standard, and CDCl\u003csub\u003e3\u003c/sub\u003e or DMSO-d6 is used as the solvent in the \u003csup\u003e1\u003c/sup\u003eH and \u003csup\u003e13\u003c/sup\u003eC NMR spectrometer on a Bruker Ultrashield 400 MHz Avance III spectrometer. A JASCO FP-8300 fluorescence spectrophotometer was employed to record the fluorescence spectra. With the use of a Unico 4802 UV/Vis double beam spectrophotometer, the UV\u0026ndash;visible absorption spectra were recorded.\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003eA Costech-ECS 4010 CHNSO Analyzer was utilized to evaluate the outcomes of the elemental analyses. MSD Agilent 5975C was used to obtain mass spectra. A Fourier-transform infrared (FT-IR) spectrum of the compounds was obtained using a Bruker TENSOR 27 device. The melting point was measured using an Electrothermal Engineering IA9100 melting point apparatus. All pH measurements were conducted using a Metrohm 744 digital pH meter.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2 Materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll solvents and chemicals were analytical grades, obtained from Sigma-Aldrich, and utilized without additional purification. Metal ions (Ba\u003csup\u003e2\u003c/sup\u003e\u003csup\u003e+\u003c/sup\u003e, K\u003csup\u003e+\u003c/sup\u003e, Pb\u003csup\u003e2+\u003c/sup\u003e, Hg\u003csup\u003e2+\u003c/sup\u003e, Al\u003csup\u003e3+\u003c/sup\u003e, Zn\u003csup\u003e2+\u003c/sup\u003e, Fe\u003csup\u003e2+\u003c/sup\u003e, Tl\u003csup\u003e+\u003c/sup\u003e, Mg\u003csup\u003e2+\u003c/sup\u003e, Ni\u003csup\u003e2+\u003c/sup\u003e, Nd\u003csup\u003e3+\u003c/sup\u003e, Fe\u003csup\u003e3+\u003c/sup\u003e, Ag\u003csup\u003e+\u003c/sup\u003e, Ca\u003csup\u003e2+\u003c/sup\u003e, Cs\u003csup\u003e+\u003c/sup\u003e, Co\u003csup\u003e2+\u003c/sup\u003e, Cd\u003csup\u003e2+\u003c/sup\u003e, Sr\u003csup\u003e2+\u003c/sup\u003e, Mn\u003csup\u003e2+\u003c/sup\u003e,\u003csup\u003e\u0026nbsp;\u003c/sup\u003eand Cu\u003csup\u003e2+\u003c/sup\u003e ions) were used in the form of their acetate salts.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3 Synthesis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e2.3.1 Synthesis of dimethyl pyridine-2,6-dicarboxylate (L1) [44]:\u003c/p\u003e\n\u003cp\u003eA solution of the 2,6-pyridinedicarboxylic acid (250 mg, 1.5 mmol) and 95% H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e (2.93 g, 34.9 mmol, 1.59 mL) in MeOH (25 mL) was heated under reflux for 14 h. After extraction with dichloromethane (2 \u0026times; 25 mL), the reaction mixture was neutralized with a saturated NaHCO₃ solution, and the organic layer was dried over anhydrous Na₂SO₄. The organic solvent was evaporated under reduced pressure to afford a white solid (0.292 g, %100). m.p. 121 \u0026deg;C; \u003csup\u003e13\u003c/sup\u003eCNMR (100 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) \u0026delta; ppm 165.1, 148.2, 138.4, 128.0, 53.2 \u003csup\u003e1\u003c/sup\u003eHNMR (400 MHz, DMSO-d6) \u0026delta; ppm 8.32 (2H, d, J = 7.6 Hz, Ar-H), 8.03 (1H, t, J = 7.6 Hz, Ar-H), 4.03 (6H, s, OCH\u003csub\u003e3\u003c/sub\u003e); (Figs. S1-S3).\u003c/p\u003e\n\u003cp\u003e2.3.2\u0026nbsp;Synthesis of pyridine-2,6-dicarbohydrazide (L2) [45]:\u003c/p\u003e\n\u003cp\u003eA solution of pyridine-2,6-dimethylester (0.23 g, 1.11 mmol) and hydrazine monohydrate (1.18 g, 23.5 mmol, 1.14 mL) in 25 mL MeOH was refluxed for 12 h. The reaction mixture was filtered to yield a white solid, which was subsequently washed with methanol (3 \u0026times; 20 mL) to afford pure white crystals (0.19 g, 86%). m.p. 298 \u0026deg;C; \u003csup\u003e13\u003c/sup\u003eCNMR (100 MHz DMSO-d6) \u0026delta; ppm 162.4, 148.9, 139.8, 124.1; \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-d6) \u0026delta; ppm 10.63 (2H, s, NH), 8.13 (3H, s, Ar-H), 4.63 (4H, s, NH\u003csub\u003e2\u003c/sub\u003e); (Figs. S4-S6).\u003c/p\u003e\n\u003cp\u003e2.3.3 Synthesis of \u003cem\u003eN,N\u0026apos;\u003c/em\u003e-((((pyridine-2,6-dicarbonyl)bis(hydrazin-2-yl-1-ylidene))bis(methaneylylidene))bis(4,1-phenylene))diacetamide (L3)\u003c/p\u003e\n\u003cp\u003eA mixture of compound L2 (0.5 g, 2.56 mmol), and \u003cem\u003eN\u003c/em\u003e-(4-formylphenyl)acetamide (0.85 g, 5.2 mmol) was dissolved\u0026nbsp;in ethanol (35 mL), in a round-bottomed flask. The reaction mixture was refluxed and agitated for seven hours under an argon atmosphere. The resultant white solid was separated by filtration and cleansed using hot ethanol. After drying, a white solid (probe L3) was obtained (1.16 g, 94%).\u0026nbsp;m.p. 312\u0026deg;c; Anal. calc. for C\u003csub\u003e25\u003c/sub\u003eH\u003csub\u003e23\u003c/sub\u003eN\u003csub\u003e7\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e: C, 61.85; H, 4.78; N, 20.20, Found: C, 61.69; H, 4.91; N, 20.03; \u003csup\u003e13\u003c/sup\u003eC NMR (100 MHz, DMSO-d6) \u0026delta; ppm 169.1, 159.7, 150.4, 148.8, 141.8, 140.4, 129.1, 128.5, 125.8, 119.4, 24.6; \u003csup\u003e1\u003c/sup\u003eH NMR (400MHz, DMSO-d6) \u0026delta; ppm 12.25 (2H, s, NH), 10.19 (2H, s, NH), 8.70 (2H, S, C=NH), 8.37(2H, m, ArH), 8.28(1H, m, ArH), 7.77 (4H, m, ArH), 7.72 (4H, m, ArH), 2.08 (6H, s, CH\u003csub\u003e3\u003c/sub\u003e ); \u003csup\u003e1\u003c/sup\u003eH NMR (400MHz, D\u003csub\u003e2\u003c/sub\u003eO) \u0026delta; ppm 8.37(2H, m, ArH), 8.28(1H, m, ArH), 7.77 (4H, m, ArH), 7.72 (4H, m, ArH), 2.08 (6H, s, CH\u003csub\u003e3\u003c/sub\u003e ); FT-IR spectrum (cm\u003csup\u003e-1\u003c/sup\u003e): 3511, 3258, 3106, 3061, 2969, 1674, 1662, 1616, 1592, 1512, 1452, 1410, 1370, 1320, 1262, 1174, 1078,1004, 963, 844; The mass spectrum reveals a molecular ion at m/z = 485, which aligns with the probe L3; (Figs. S7-S11).\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003eThe colorimetric probe L3 for the recognition of Cu\u003csup\u003e2+\u003c/sup\u003e ions was synthesized by reacting 2,6-pyridinedicarbohydrazide with 4-acetamidobenzaldehyde in ethanol and isolated as a white solid with a high yield (Scheme 1) and characterized by \u0026nbsp;\u003csup\u003e1\u003c/sup\u003eH NMR, \u003csup\u003e13\u003c/sup\u003eC NMR,\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eESI\u003csup\u003e+\u003c/sup\u003e-MS, FT-IR, and elemental analysis (Figs S1\u0026ndash;S11 in supporting information). The optical properties were assessed using UV-visible and fluorescence spectroscopy.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.1 Colorimetric recognition\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe selective detection of environmentally relevant metal ions by probe L3 was evaluated through its interaction with 20 different metal ions, including Ba\u0026sup2;⁺, K⁺, Pb\u0026sup2;⁺, Hg\u0026sup2;⁺, Al\u0026sup3;⁺, Zn\u0026sup2;⁺, Fe\u0026sup2;⁺, Tl⁺, Mg\u0026sup2;⁺, Ni\u0026sup2;⁺, Nd\u0026sup3;⁺, Fe\u0026sup3;⁺, Ag⁺, Ca\u0026sup2;⁺, Cs⁺, Co\u0026sup2;⁺, Cd\u0026sup2;⁺, Sr\u0026sup2;⁺, Mn\u0026sup2;⁺, and Cu\u0026sup2;⁺, in a DMSO/H₂O (7:3, v/v) solution. The selectivity was assessed visually and spectroscopically using UV-Vis and fluorescence techniques. As shown in Fig. 1, the addition of 2.0 equivalents of these metal ions to a 1.0 \u0026times; 10⁻⁵ M solution of L3 revealed that only Cu\u0026sup2;⁺ induced a noticeable color change from colorless to greenish-yellow. The probe L3 is colorless in solution and exhibits an absorption band at 330 nm, which is attributed to an intermolecular \u0026pi;\u0026ndash;\u0026pi;* charge transfer transition, in agreement with previous reports [46,47]. Upon the addition of 2.0 equivalents of Cu\u0026sup2;⁺ ions, a clear bathochromic shift of approximately 30 nm and a decrease in the intensity of the 330 nm band was observed. Simultaneously, a new absorption band emerged at 360 nm, along with a broad band extending into the visible region (Fig. 2), suggesting the occurrence of an intramolecular charge transfer (ICT) process [41,46,48\u0026ndash;50]. In contrast, none of the other tested metal ions caused significant changes in the absorption spectrum of L3, further confirming its high selectivity toward Cu\u0026sup2;⁺ ions. To gain deeper insight into the binding behavior, UV-Vis titration of L3 (5.0 \u0026times; 10⁻⁶ M) with increasing amounts of Cu\u0026sup2;⁺ (0\u0026ndash;2.0 eq) was conducted in DMSO/H₂O (7:3, v/v). The absorbance at 330 nm gradually decreased, accompanied by the growth of a new absorption band between 370 and 480 nm (Fig. 3). A well-defined isosbestic point at 369 nm was observed during the titration, indicating a clean interconversion between the free and complexed forms of L3 and confirming the formation of a stable L3+Cu\u0026sup2;⁺ complex.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2 Fluorescent recognition of Cu\u003csup\u003e2+\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe fluorescence response of L3 (5.0 \u0026micro;M) in DMSO/H\u003csub\u003e2\u003c/sub\u003eO (7:3, v/v) solution towards various metal ions (2.0 eq) was tested to further investigate the sensing capabilities of L3.\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003eAs shown in Fig. 4, probe L3 demonstrated a significant emission at 472 nm when excited at 330 nm, which might be attributable to the ICT process.\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003eThe addition of various metal ions, including Ba\u003csup\u003e2+\u003c/sup\u003e, K\u003csup\u003e+\u003c/sup\u003e, Pb\u003csup\u003e2+\u003c/sup\u003e, Al\u003csup\u003e3+\u003c/sup\u003e, Hg\u003csup\u003e2+\u003c/sup\u003e, Zn\u003csup\u003e2+\u003c/sup\u003e, Fe\u003csup\u003e2+\u003c/sup\u003e, Tl\u003csup\u003e+\u003c/sup\u003e, Mg\u003csup\u003e2+\u003c/sup\u003e, Ni\u003csup\u003e2+\u003c/sup\u003e, Nd\u003csup\u003e3+\u003c/sup\u003e, Fe\u003csup\u003e3+\u003c/sup\u003e, Ag\u003csup\u003e+\u003c/sup\u003e, Ca\u003csup\u003e2+\u003c/sup\u003e, Cs\u003csup\u003e+\u003c/sup\u003e, Co\u003csup\u003e2+\u003c/sup\u003e, Cd\u003csup\u003e2+\u003c/sup\u003e, Sr\u003csup\u003e2+\u003c/sup\u003e, and Mn\u003csup\u003e2+\u0026nbsp;\u003c/sup\u003eions to L3 solution caused almost no discernible change in fluorescence spectra. However, the addition of Cu\u003csup\u003e2+\u003c/sup\u003e to probe L3 caused a significant change in fluorescence and its quenching. According to the previous results, after adding Cu\u003csup\u003e2\u003c/sup\u003e\u003csup\u003e\u003cspan dir=\"RTL\"\u003e+\u003c/span\u003e\u003c/sup\u003e ions, the L3 probe shows complete fluorescence quenching, which could be due to the formation of the L3+Cu\u003csup\u003e2+\u003c/sup\u003e complex, thus confirming the ICT process. This selectivity can be attributed to the inability of other metal ions to form comparable interactions with probe L3, thereby failing to induce significant fluorescence changes. The high affinity of L3 for Cu\u0026sup2;⁺ may be explained by soft-soft interactions between the Cu\u0026sup2;⁺ ions and the nitrogen donor atoms within the probe structure [51]. The involvement of nitrogen atoms in coordinating with Cu\u0026sup2;⁺ enhances molecular conjugation and promotes electron delocalization, consistent with the ICT process. Supporting evidence includes the observed redshift in the UV-Vis absorption spectrum, a rapid and distinct color change upon Cu\u0026sup2;⁺ addition, and pronounced fluorescence quenching\u0026mdash;all of which confirm complex formation via an ICT-driven binding mechanism [41,46,49,52\u0026ndash;54].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e3.3 Binding constants\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFluorescence quenching was seen upon the gradual addition of Cu\u003csup\u003e2+\u003c/sup\u003e ions to the probe L3 solution (Fig. 5), indicating complexation between the probe L3 and Cu\u003csup\u003e2+\u003c/sup\u003e ions. The association constant Ka of L3+ Cu\u003csup\u003e2+\u003c/sup\u003e complex was calculated from the fluorescence titration to be 2.2 \u0026times; \u0026nbsp; \u0026nbsp; (Fig. S12). Additionally, the probe\u0026apos;s detection limit for Cu\u003csup\u003e2+\u003c/sup\u003e ions was calculated using 3d/S, as \u0026nbsp;, indicating that it is capable of detecting extremely low copper concentrations in DMSO/H\u003csub\u003e2\u003c/sub\u003eO (7:3, v/v) (Fig. S13). Using Job plot\u0026apos;s continuous variation techniques, the stoichiometric ratio of probe L3 and copper was calculated\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003e(Fig. S14). According to the results, the concentration of the probe-guest complex reaches a maximum when the mole fraction of the probe is around 0.67, which could indicate the formation of a 1:2 complex between probe L3 and copper ions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4 Interference studies\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHigh selectivity is one of the essential qualities of the probe L3 responding to Cu\u003csup\u003e2+\u003c/sup\u003e ions. Fluorescence interference investigations were conducted under the same lab conditions in the presence of various competing metal ions to further support the probe L3\u0026apos;s great selectivity for Cu\u003csup\u003e2+\u003c/sup\u003e ions. As seen in Fig. 6, the fluorescence intensity at 472 nm for L3 (5.0 \u0026micro;M) with Cu\u003csup\u003e2+\u003c/sup\u003e (2.0 eq) was barely affected by the presence of the other metal cations (2.0 eq). The distinctive selectivity of L3 for Cu\u003csup\u003e2+\u003c/sup\u003e ions may be ascribed to the soft-soft metal interactions between the Cu\u003csup\u003e2+\u003c/sup\u003e and nitrogen atoms\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003eof the probe L3. These results clearly show that L3 might be a highly responsive sensor for the Cu\u003csup\u003e2+\u003c/sup\u003e ions with good anti-interference performance among the other relevant metal ions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3. 5\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eBinding mode of probe L3 towards Cu\u003csup\u003e2+\u003c/sup\u003e ions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe binding of copper ions with probe L3 is supported by the \u003csup\u003e1\u003c/sup\u003eH NMR titration and the FT-IR spectra. The results of the \u003csup\u003e1\u003c/sup\u003eH NMR titration experiment allow for the identification of a binding mode between the probe L3 and the copper ions. The \u003csup\u003e1\u003c/sup\u003eH NMR spectra of probe L3 with different amounts of Cu\u003csup\u003e2+\u003c/sup\u003e ions (0, 1.0, and 2.0 eq) were recorded and shown in Fig. 7.\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003eUpon addition of Cu\u003csup\u003e2+\u003c/sup\u003e, PDH amide protons become broadened and shifted from 12.25 to 12.78 ppm. A new broad peak at 10.52 appeared next to the amide protons of phenylacetamide, and the amide peak disappeared. The N=CH protons were also broadened and shifted from 8.71 to 8.89 ppm. There was a slight up-field shift in other peaks (approx. 0.06 ppm). The spectra indicate that amide protons and N = CH protons are involved in Cu\u003csup\u003e2+\u003c/sup\u003e ions binding with L3. The results showed that adding two equivalents of copper causes significant changes in the spectra. These results indicate the formation of a complex with stoichiometry beyond a simple 1:1 ratio, likely a 1:2 (L3:Cu\u0026sup2;⁺) complex. The Job\u0026apos;s plot illustrates that L3 forms a 1:2 complex with Cu\u003csup\u003e2+\u003c/sup\u003e, which was further confirmed by UV\u0026ndash;Vis and NMR titrations.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;There are some significant differences between the results of the probe L3 and L3+Cu\u003csup\u003e2+\u003c/sup\u003e in IR analysis (Fig. S15). The metal-nitrogen linkages are confirmed by the IR spectra of L3 and L3+Cu\u003csup\u003e2+\u003c/sup\u003e. The distinct absorption bands at 987 and 933 cm\u003csup\u003e-1\u003c/sup\u003e lead to the Cu-N stretching and bending vibration bands. In addition, bands at 3511, and 3258 cm\u003csup\u003e-1\u003c/sup\u003e for \u0026ndash;NH, 1674, and 1662 cm\u003csup\u003e-1\u003c/sup\u003e for C=O, and 1592 cm\u003csup\u003e-1\u003c/sup\u003e for C=N in the L3 spectrum disappeared on the complexation with Cu\u003csup\u003e2+\u003c/sup\u003e ions. At 3298, 1615, 1566, and 1392 cm\u003csup\u003e-1\u003c/sup\u003e, a few novel bands that support the complexation occurring with C=N, -NH appeared. The changes described above in the IR spectrum of probe L3 after the addition of the Cu\u003csup\u003e2+\u003c/sup\u003e ions, and the results of the \u003csup\u003e1\u003c/sup\u003eH NMR titration, indicate that NH protons and C=N are important in the binding of Cu\u003csup\u003e2+\u003c/sup\u003e ions with prob L3.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.6 pH effect\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate the pH-dependent fluorescence response of probe L3, measurements were conducted in a DMSO/H₂O (7:3, v/v) solution across a broad pH range (2\u0026ndash;12). As illustrated in Fig. 8, the fluorescence intensity of L3 remained stable and relatively strong within the pH range from 4 to 10, with an optimal performance between pH 5 and 9. Below pH 4, fluorescence was significantly diminished, likely due to the protonation of\u0026nbsp;donor nitrogen atoms in the ligand framework, which may inhibit coordination to Cu\u0026sup2;⁺ ions. Conversely, a marked decrease in fluorescence above pH 10 may be attributed to potential deprotonation-induced structural perturbations of the probe. Therefore, the pH range of 5\u0026ndash;9 was identified as the most suitable operational conditions for Cu\u0026sup2;⁺ detection using L3, which was adopted for subsequent sensing experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.7 Time\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe sensitivity of probe L3 was further evaluated by examining its fluorescence response time upon exposure to Cu\u0026sup2;⁺ ions. To this end, a 5.0 \u0026micro;M solution of L3 was treated with 2.0 equivalents of Cu\u0026sup2;⁺ in a DMSO/H₂O (7:3, v/v), and the fluorescence intensity was monitored over time. As illustrated in Fig. 9, the addition of Cu\u0026sup2;⁺ ions led to a sharp and immediate decrease in fluorescence intensity, which then remained nearly constant for up to 30 minutes. This rapid and stable quenching behavior suggests a fast complexation process between L3 and Cu\u0026sup2;⁺ ions, highlighting the probe\u0026rsquo;s potential for real-time detection of copper ions in aqueous media.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e3.8 Reversibility test\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eReversibility is a key feature of an efficient probe, especially in applications like metal ion detection. The reversibility of probe L3 was investigated using disodium ethylenediaminetetraacetate (Na\u003csub\u003e2\u003c/sub\u003eEDTA) well-known metal chelator [55,56]. Upon sequential addition of Na₂EDTA to a preformed L3+Cu\u0026sup2;⁺ complex, a marked restoration of fluorescence emission at 472 nm was observed, closely resembling the emission profile of the unbound probe. This fluorescence recovery implies that Na₂EDTA effectively competes for Cu\u0026sup2;⁺ ions, displacing them from the coordination sphere of L3 and regenerating its emissive state (Fig. S16). Such reversible binding behavior is a critical feature for real-time monitoring applications, indicating that L3 retains its sensing functionality over multiple detection\u0026ndash;regeneration cycles.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.9 Practical application\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFilter papers were used to study the primary applicability of probe L3. Filter papers were dipped into a solution containing (100 \u0026micro;M) L3 to create test strips, which were then allowed to air dry. Afterward, the test strips were sequentially immersed in different concentrations of copper (0-2 eq) for 1 min, then dried, and observed clearly under sunlight and 365 nm UV light. The filter paper color changes (Fig. 10) demonstrated that as the concentration of copper was raised, the color gradually changed from white to dark yellow under daylight, and from greenish yellow to dark gray under a 365 nm UV lamp. These findings demonstrate the practical potential of probe L3 for detecting Cu\u0026sup2;⁺ ions at varying concentrations in aqueous environments, utilizing test strips observable under both natural light and UV illumination.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.10 Analysis of Real Samples\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe applicability of probe L3 for detecting Cu\u003csup\u003e2+\u003c/sup\u003e ions in real samples such as tap water, drinking water, and the Caspian Sea water was investigated employing the fluorescence method. First, the Cu\u003csup\u003e2+\u003c/sup\u003e ions concentration in each sample was determined using atomic absorption spectroscopy, and then three concentrations of the Cu\u003csup\u003e2+\u003c/sup\u003e ions (10, 20, and 30 \u003cem\u003e\u0026mu;\u003c/em\u003eM) were prepared for each sample by the addition of Cu\u003csup\u003e2+\u003c/sup\u003e to the samples employing a spiking technique. \u0026nbsp;All the samples were titrated against the probe L3, and the results exhibited an excellent recovery of up to 99.2% (Table 1). These findings underscore the probe L3\u0026rsquo;s potential as a reliable tool for the detection of Cu\u0026sup2;⁺ ions in diverse aqueous environments, thus making it a promising candidate for monitoring copper contamination in environmental water sources.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1\u0026nbsp;\u003c/strong\u003eQuantification of Cu\u003csup\u003e2+\u003c/sup\u003e ions in real aqueous samples\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003eWater samples\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAmount of standard Cu\u003csup\u003e2+\u003c/sup\u003e ion added (\u003cem\u003e\u0026mu;\u003c/em\u003eM)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTotal Cu\u003csup\u003e2+\u003c/sup\u003e ion found (\u003cem\u003e\u0026mu;\u003c/em\u003eM) (n =3)\u003csup\u003ea\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003eRecovery of Cu\u003csup\u003e2\u003c/sup\u003e\u003cstrong\u003e\u003csup\u003e+\u003c/sup\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eions added (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003eTap water\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003e10.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003e9.81\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003e0.981\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003e20.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003e19.76\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003e0.988\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003e30.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003e29.63\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003e0.987\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003eDrinking water\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003e10.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003e9.90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003e0.990\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003e20.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003e19.84\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003e0.992\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003e30.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003e29.71\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003e0.990\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003eCaspian Sea water\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003e10.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003e9.61\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003e0.961\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003e20.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003e19.49\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003e0.974\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003e30.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003e29.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003e0.97\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003csup\u003ea: All experiments were repeated three times, and the mean values were reported.\u0026nbsp;\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.11 \u0026nbsp;Comparison\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo assess the performance of the synthesized probe L3 for Cu\u0026sup2;⁺ detection, a comparative analysis was conducted with several previously reported fluorescent receptors, as summarized in Table 2. While all listed probes exhibit useful sensing capabilities, L3 demonstrates several notable advantages in aqueous environments. These include a distinct fluorescence turn-off response upon Cu\u0026sup2;⁺ binding, a higher association constant, a lower limit of detection (LOD), and superior selectivity. Furthermore, L3 operates effectively across a broad pH range (5\u0026ndash;9), showing excellent performance in real water samples, and is compatible with test strip-based analyses. Collectively, these attributes highlight the practical potential of L3 as a versatile and robust sensor for copper ions detection.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;Table 2\u0026nbsp;\u003c/strong\u003eComparison of the reported probes for detecting the Cu\u003csup\u003e2+\u003c/sup\u003e ions\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"609\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 51px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRef\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 109px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDetection method\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 110px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eLOD (M)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 116px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAssociation constant\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e\u003cstrong\u003epH\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eReversible\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 80px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eApplication (yes or no)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 51px;\"\u003e\n \u003cp\u003e[41]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 109px;\"\u003e\n \u003cp\u003eFluorescence, UV-Vis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 110px;\"\u003e\n \u003cp\u003e1.978 \u0026times; 10\u003csup\u003e-5\u003c/sup\u003e M\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 116px;\"\u003e\n \u003cp\u003e3.11\u0026times; 10\u003csup\u003e5\u003c/sup\u003e M\u003csup\u003e\u0026minus; 1\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e4-8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eNR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 80px;\"\u003e\n \u003cp\u003eNR\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 51px;\"\u003e\n \u003cp\u003e[46]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 109px;\"\u003e\n \u003cp\u003eUV-Vis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 110px;\"\u003e\n \u003cp\u003e1.2 \u0026times; 10\u003csup\u003e-7\u003c/sup\u003e M\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 116px;\"\u003e\n \u003cp\u003e9.08 \u0026times; 10\u003csup\u003e4\u0026nbsp;\u003c/sup\u003eM\u003csup\u003e-1\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003eNR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eNR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 80px;\"\u003e\n \u003cp\u003eNR\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 51px;\"\u003e\n \u003cp\u003e[57]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 109px;\"\u003e\n \u003cp\u003eFluorescence, UV-Vis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 110px;\"\u003e\n \u003cp\u003e1.49 \u0026times; 10\u003csup\u003e-6\u003c/sup\u003e M\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 116px;\"\u003e\n \u003cp\u003e8.89 \u0026times; 10\u003csup\u003e3\u0026nbsp;\u003c/sup\u003eM\u003csup\u003e-1\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e4-10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 80px;\"\u003e\n \u003cp\u003eNR\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 51px;\"\u003e\n \u003cp\u003e[58]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 109px;\"\u003e\n \u003cp\u003eFluorescence, UV-Vis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 110px;\"\u003e\n \u003cp\u003e5 \u0026times; 10\u003csup\u003e-9\u0026nbsp;\u003c/sup\u003eM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 116px;\"\u003e\n \u003cp\u003e2.3 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e M\u003csup\u003e\u0026minus; 1\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003eNR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eNR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 80px;\"\u003e\n \u003cp\u003eNR\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 51px;\"\u003e\n \u003cp\u003e[59]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 109px;\"\u003e\n \u003cp\u003eFluorescence, UV-Vis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 110px;\"\u003e\n \u003cp\u003e1.89 \u0026times; 10\u003csup\u003e-7\u003c/sup\u003e M\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 116px;\"\u003e\n \u003cp\u003e1.5 \u0026times; \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003eNR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eNR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 80px;\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 51px;\"\u003e\n \u003cp\u003e[60]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 109px;\"\u003e\n \u003cp\u003eFluorescence\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 110px;\"\u003e\n \u003cp\u003e0.48 \u0026times; 10\u003csup\u003e-6\u003c/sup\u003e\u003csub\u003e\u0026nbsp;\u003c/sub\u003eM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 116px;\"\u003e\n \u003cp\u003e6.04 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e M\u003csup\u003e-2\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e5-9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eNR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 80px;\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 51px;\"\u003e\n \u003cp\u003e[61]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 109px;\"\u003e\n \u003cp\u003eFluorescence, UV-Vis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 110px;\"\u003e\n \u003cp\u003e2.7 \u0026times; 10\u003csup\u003e-7\u003c/sup\u003e M\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 116px;\"\u003e\n \u003cp\u003e4.86 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e M\u003csup\u003e-1\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e4-9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eNR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 80px;\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 51px;\"\u003e\n \u003cp\u003e[62]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 109px;\"\u003e\n \u003cp\u003eFluorescence, UV-Vis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 110px;\"\u003e\n \u003cp\u003e1.39 \u0026times; 10\u003csup\u003e\u0026minus; 7\u003c/sup\u003e M\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 116px;\"\u003e\n \u003cp\u003e2.1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e M\u003csup\u003e\u0026minus; 1\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003eNR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 80px;\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 51px;\"\u003e\n \u003cp\u003e[63]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 109px;\"\u003e\n \u003cp\u003eFluorescence\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 110px;\"\u003e\n \u003cp\u003e1.21 \u0026times; 10\u003csup\u003e\u0026minus; 8\u0026nbsp;\u003c/sup\u003eM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 116px;\"\u003e\n \u003cp\u003e1.79 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e M\u003csup\u003e\u0026minus; 1\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e3-11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 80px;\"\u003e\n \u003cp\u003eNR\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 51px;\"\u003e\n \u003cp\u003e[64]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 109px;\"\u003e\n \u003cp\u003eFluorescence\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 110px;\"\u003e\n \u003cp\u003e4.5\u0026times; 10\u003csup\u003e-8\u0026nbsp;\u003c/sup\u003eM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 116px;\"\u003e\n \u003cp\u003e9.9\u0026times; 10\u003csup\u003e9\u003c/sup\u003e M\u003csup\u003e-2\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003eNR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 80px;\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 51px;\"\u003e\n \u003cp\u003e[65]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 109px;\"\u003e\n \u003cp\u003eFluorescence\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 110px;\"\u003e\n \u003cp\u003e1.64 \u0026times; 10\u003csup\u003e-8\u0026nbsp;\u003c/sup\u003eM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 116px;\"\u003e\n \u003cp\u003e1.22 \u0026times; 10\u003csup\u003e3\u0026nbsp;\u003c/sup\u003eM\u003csup\u003e-1\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e3-11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 80px;\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 51px;\"\u003e\n \u003cp\u003e[66]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 109px;\"\u003e\n \u003cp\u003eFluorescence\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 110px;\"\u003e\n \u003cp\u003e1.82 \u0026times;10\u003csup\u003e-8\u0026nbsp;\u003c/sup\u003eM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 116px;\"\u003e\n \u003cp\u003e6.59 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e M\u003csup\u003e\u0026minus; 1\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003eNR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eNR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 80px;\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 51px;\"\u003e\n \u003cp\u003e[43]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 109px;\"\u003e\n \u003cp\u003eFluorescence, UV-Vis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 110px;\"\u003e\n \u003cp\u003e4.2 \u0026times;10\u003csup\u003e-9\u003c/sup\u003e M\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 116px;\"\u003e\n \u003cp\u003e2.9-3.5 \u0026times;10\u003csup\u003e11\u003c/sup\u003e M\u003csup\u003e-2\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e4.5-11.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eNR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 80px;\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 51px;\"\u003e\n \u003cp\u003eThis work\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 109px;\"\u003e\n \u003cp\u003eFluorescence, UV-Vis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 110px;\"\u003e\n \u003cp\u003e\u0026nbsp;M\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 116px;\"\u003e\n \u003cp\u003e2.2 \u0026times; \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e5-9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eYes\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 80px;\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eNR: Not reported\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, we have developed a highly efficient fluorescent and colorimetric probe (L3) for the selective detection of Cu²⁺ ions, based on a 2,6-pyridinedicarbohydrazide scaffold. The probe exhibited reliable and distinct optical responses, including fluorescence quenching and redshifted absorption upon coordination with Cu²⁺ ions. Spectroscopic analysis and ¹H NMR titration confirmed the formation of a well-defined 1:2 (L3:Cu²⁺) complex, while the high association constant and low detection limit (2.28 × 10⁻⁹ M) demonstrated its strong binding affinity and sensitivity. L3 functioned effectively across a pH range of 5.0 to 9.0 and displayed excellent reversibility, as evidenced by fluorescence restoration upon EDTA treatment, confirming its potential for reusable and real-time monitoring. The practical utility of L3 was further enhanced by the successful fabrication of test strips capable of visually detecting Cu²⁺ under both daylight and UV light. Moreover, the probe maintained high accuracy in real water samples, with recovery rates reaching up to 99.2%. These results underscore the applicability of L3 as a robust and sensitive platform for environmental and analytical monitoring of copper ions. These findings support the potential of L3 for the future development of portable Cu²⁺ detection systems for environmental and biological applications.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis is an observational study. The University of Mazandaran Research Ethics Committee has confirmed that no ethical approval is required.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAli Zamani:\u003c/strong\u003e Methodology, Formal analysis, Validation, Investigation, Resources, Writing - original draft. \u003cstrong\u003eMahmood Tajbakhsh:\u003c/strong\u003e Conceptualization, Validation, Investigation, Writing - review \u0026amp; editing. \u003cstrong\u003eYaghoub Sarrafi:\u003c/strong\u003e Validation, Conceptualization, Investigation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePartial financial support was received from the Research Council of the University of Mazandaran.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during and analyzed during the current study are not publicly available due to [According to policies of the University of Mazandaran that all analysis devices are not connected to the Internet, the output of all devices is printed as a sheet or available on CD.] but are available from the Journal or the corresponding authors.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFinancial support for this work from the Research Council of the University of Mazandaran is gratefully acknowledged.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eValeur B, Leray I. 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Sensors Actuators B Chem 2012;173:862\u0026ndash;7. https://doi.org/https://doi.org/10.1016/j.snb.2012.07.112.\u003c/li\u003e\n\u003c/ol\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":"Copper ions, Colorimetric, Reversible probe, Pyridine-based sensor, Naked-eye detection","lastPublishedDoi":"10.21203/rs.3.rs-6740853/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6740853/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"A novel fluorescent and colorimetric probe (L3), based on 2,6-pyridinedicarbohydrazide, was rationally designed and synthesized for the highly selective and reversible detection of Cu²⁺ ions. The structural and optical properties of the probe were comprehensively characterized by FT-IR, ¹H NMR, UV-Vis spectroscopy, fluorescence analysis, ESI⁺-MS, and elemental analysis. Upon interaction with Cu²⁺, probe L3 exhibited distinct optical responses, including significant fluorescence quenching, a notable redshift in the UV-Vis spectrum, and a rapid, visible color change from colorless to greenish-yellow under aqueous conditions and across a broad pH range. The detection limit was calculated to be as low as 2.28 × 10⁻⁹ M, with an association constant (Ka) of 2.2 × 10¹¹ M⁻², indicating strong binding affinity. Job's plot and ¹H NMR titration confirmed a 1:2 stoichiometric complex formation between L3 and Cu²⁺ ions. The probe also exhibited excellent reversibility, as confirmed by fluorescence recovery upon adding Na₂EDTA, suggesting its suitability for real-time and reusable sensing applications. Furthermore, the practical utility of L3 was demonstrated using test strips for Cu²⁺ detection in aqueous environments, and its effectiveness was further validated in real water samples, including tap water, drinking water, and seawater, achieving recovery rates up to 99.2%. These findings suggest that L3 is promising for the sensitive, rapid, and cost-effective monitoring of copper ions in environmental and drinking water systems.","manuscriptTitle":"A selective pyridinedicarbohydrazide-based “naked-eye” fluorescent probe for detecting Cu 2+ ions in aqueous solutions","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-10 13:02:02","doi":"10.21203/rs.3.rs-6740853/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-06-15T13:37:56+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-15T12:23:01+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-14T04:07:51+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-11T15:34:25+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"76678282823454917776411580938351398060","date":"2025-06-10T06:17:21+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"115750858040457449790137670270771260937","date":"2025-06-06T03:08:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"206413583596158351214937619023586299155","date":"2025-06-05T18:33:44+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-06-05T10:30:47+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-05-27T20:21:36+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-05-27T20:20:27+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Fluorescence","date":"2025-05-24T20:33:17+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":"9fd35b79-c7bd-48dd-9092-b0493437fc7e","owner":[],"postedDate":"June 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-09-08T16:01:25+00:00","versionOfRecord":{"articleIdentity":"rs-6740853","link":"https://doi.org/10.1007/s10895-025-04473-x","journal":{"identity":"journal-of-fluorescence","isVorOnly":false,"title":"Journal of Fluorescence"},"publishedOn":"2025-09-02 15:57:58","publishedOnDateReadable":"September 2nd, 2025"},"versionCreatedAt":"2025-06-10 13:02:02","video":"","vorDoi":"10.1007/s10895-025-04473-x","vorDoiUrl":"https://doi.org/10.1007/s10895-025-04473-x","workflowStages":[]},"version":"v1","identity":"rs-6740853","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6740853","identity":"rs-6740853","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}