A Thiosemicarbazone-Derived Fluorescent Probe For The Detection Of Silver Ions And Bioimaging Application

A Thiosemicarbazone-Derived Fluorescent Probe For The Detection Of Silver Ions And Bioimaging Application | 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 Thiosemicarbazone-Derived Fluorescent Probe For The Detection Of Silver Ions And Bioimaging Application Arumugam Sreedevi, Gunasekaran Prabhakaran, Nattamai Bhuvanesh, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8086522/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 27 Mar, 2026 Read the published version in Journal of Fluorescence → Version 1 posted 14 You are reading this latest preprint version Abstract A simple thiosemicarbazone-derived ( E )-2-((2-Hydroxynaphthalen-1-yl)Methylene)-N-Methyl-N-Phenylhydrazinecarbothioamide was prepared and characterized using diverse analytical methods and spectroscopic techniques. The definitive elucidation of its crystal structure was achieved through single-crystal X-ray diffraction analysis. The synthesized compound (2NP) was found to be highly specific and sensitive towards sensing silver ions. Time-dependent fluorescence studies of 2NP showed selectivity towards Ag + ion with a detection limit of 5.8995 x 10 − 5 M with a binding constant of 1.7x10 2 M − 1 . The binding stoichiometry of 2NP + Ag complex was further confirmed by Jobs Plot analysis, Benesi-Hidebrand analysis. 1H NMR, ESI-TOF and DFT studies confirmed the formation of [Ag(2NP) 2 ] complex(2:1). Further, the compound 2NP has been tested for its cytotoxicity and bioimaging studies was also performed in Mouse Fibroblast Cell Lines (L929). 2-hydroxy naphthaldehyde N-Phenyl-N-methyl thiosemicarbazone silver ion sensing DFT Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Though naturally occurring silver ions exhibit important properties such as safe green germicide, excellent antibiotic, and a good sterilizer [ 1 , 2 ], excess of silver ion becomes toxic to human health. It is known that photographic, electronic, pharmaceutical industries release an enormous amount of silver in the form of wastes, polluting water sources and agricultural fields [ 3 , 4 ]. The permissible limit of the total silver in water is 900nM [ 5 ] and any increase in silver ion concentration affects the re-productivity of aquatic animals since it is known to produce reactive oxygen species, namely free radicals. Silver build up in humans results in growth retardation, liver degenerative changes, and enlarged hearts [ 6 ] and deposits of silver precipitates in skin and eyes [ 7 ]. Separation and removal of silver ions is quite difficult due to the fact that silver ions possess only a moderate coordination ability. This has forced one to design and develop sensitive and selective methods to determine the trace quantities of silver. Among the various physicochemical methods used to sense the metal ions using some probe materials, the optical method has become unique due to its advantages of rapid signaling, low cost, straightforward operating modes, and great selectivity and sensitivity [ 8 – 12 ]. The following categories mostly comprise optical approaches for metal ion sensing: (1) The fluorescence approach [ 13 , 14 ], which uses fluorescence amplification, quenching, or intensity fluctuation to identify metal ions (2) The colorimetric approach [ 15 , 16 ], which uses colorimetric changes or variations in the UV absorption spectrum to identify metal ions selectively; (3) Surface enhanced Raman scattering spectroscopy and circular dichroism spectroscopy [ 17 , 18 ]. From the point of view of the probes employed for sensing of metal ions in such studies, thiosemicarbazone based Schiff bases have been found to be apt and perfect compounds for the detection of different analytes because they possess fluorescence turn-on-off properties and of strong donor atoms O, N and S. In the search for a better Schiff base for the sensing of Ag + ions in trace quantities, we carried out the synthesis, characterization and investigated the efficacy of the thiosemicarbazone based Schiff base (2NP) towards selective and sensitive tracking of Ag + ions in polar solvent medium. Through chelation enhanced fluorescence (CHEF), the probe 2NP showed turn on response towards silver ions. The coordination behavior of the Schiff base with Ag + ions has been investigated through several techniques including density functional theory calculations. Materials and methods 2-Hydroxy naphthaldehyde was procured from Sigma-Aldrich, then meticulously synthesized N-Phenyl-N-methyl thiosemicarbazone following a previously established protocol [ 19 ]. All other essential chemicals were sourced commercially and utilized directly, eliminating the need for further purification. 1 H NMR and 13 C NMR spectra were meticulously recorded on a 400 MHz Bruker spectrometer. We used DMSO-d 6 as the solvent and TMS as the internal reference for these analyses. Infrared spectra were subsequently captured using a Shimadzu spectrophotometer, providing further structural insights. Optical measurements were recorded on a Shimadzu UV-240 spectrophotometer and Jasco FP-8200 spectrofluorometer, utilizing a 1 cm quartz cuvette. Throughout these optical experiments, excitation and emission slit widths were consistently emission spectra were collected at a controlled temperature of 24 ± 1°C. Elemental analysis (C, H, N) was performed using a Vario EL III elemental analyzer. Synthesis of Chemosensor 2NP Equimolar amount of 2-hydroxy-1-naphthaldehyde (1.0 mmol) and N-phenyl-N-methyl thiosemicarbazone (1.0 mmol) were dissolved in 20 mL of ethanol. The mixture was then heated under reflux for 4 hours to accelerate the reaction. Upon cooling, a yellow solid precipitated from the solution which was subsequently filtered and recrystallized from mixture of dichloromethane/ethanol (1:1), yielding yellow-coloured crystals suitable for X-ray diffraction studies. Yield (88%); m.p. 212–213 ᵒ C. IR (KBr): νmax: 3447, 3370, 1581, 1487 cm − 1 . 1 H NMR (400 MHz, DMSO-d 6 ): Chemical shifts (δ) observed at 12.76 (s, -OH), 10.90 (s, NH), 9.34 (s, 1H, CH = N), 7.98 (d, 1H, J = 7.6J = 7.6J = 7.6 Hz, Q-C8-H), 7.88–7.84 (t, 2H, Ar-H), 7.56–7.52 (m, 3H, Ar-H), 7.08–7.16 (m, Ar-H), 7.51–7.42 (m, 4H, Ar-H), 7.41–7.40 (d, J = 2.8J = 2.8J = 2.8 Hz, 1H), 3.64 (s, 3H, CH 3 _33) 13 C-NMR (100 MHz, DMSO-d 6 ):δ 179.20, 157.05, 144.91, 143.85, 132, 131.57,130.04, 128.80, 127.77, 127.61, 127.28, 126.77, 123.27, 120.33, 119.09, 108.52, 43.88. 40.13. (TOF-ES+) m/z [(M-1)] 334.42 (335.42). Anal. Calced. For C 19 H 17 N 2 OS (335.42) C, 68.03; H, 5.11; N, 12.53; Found: C, 67.71; H, 4.99; N, 12.36%. In Vitro Cytotoxicity analysis: The MTT assay for comp-1 was done as per the method followed by subashini et al.,[ 20 ]. Briefly, the Mouse Fibroblast Cell Line (L929) was treated with comp-1 at various concentrations (5, 25, 50, 75 and 100 µM), and DMSO was used as a control. After treatment, 1mg/mL of MTT solution was added to L929 cells and further incubated for 3–4 h. After incubation, the absorbance was taken at 570 nm, and an image was captured in an inverted phase contrast microscope. Theoretical Studies Theoretical calculations of the probe 2NP was performed using Gaussian 16 W software and the GaussView 6.0 molecular visualization package [ 21 , 22 ]. These calculations were carried out under B3LYP 6–311 + + g(d,p) basis set level of theory. Electronic properties such as the HOMO-LUMO gap were also determined at the same theoretical level [ 23 , 24 ]. Initially optimization calcluation of the silver ion alone completed at the CAM-B3LYP-D3/6-31G(d) level of theory. Results and discussion Synthesis of Schiff base, 2NP Condensation of 2-hydroxy naphthaldehyde with N-phenyl-N-methyl thiosemicarbazone in ethanol yielded a yellow fluorescent compound 2NP synthesized with good yields (Scheme 1 ). Its identity was confirmed through advanced techniques, including FT-IR, 1 HNMR, 13 C NMR, HRMS (Fig. S1 -S4) and single crystal X-ray analysis. A clear ORTEP representation of 2NP is showcased in Fig. 1 . Relevant X-ray data collection and structure refinement are meticulously presented in Table 1 . Crystallographic data for 2NP has been deposited with the Cambridge Crystallographic Data Centre under the supplementary publication number CCDC 2193495. Copies Of the data can be obtained free of charge on application to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK [fax: +44 (0) 1223336033 or email: [email protected] ]. Absorption and Emission studies The UV-Vis absorption spectrum of 2NP reveals a prominent absorption peak at 520 nm, attributed to an intramolecular charge transfer transition (Fig. S5). Using this wavelength as the excitation source, fluorescence studies were conducted, and 2NP displayed maxima at 550 nm in a DMSO-H₂O mixture (1:1 v/v) buffered with 50 mM HEPES at pH 7.4. On introducing Ag⁺ ions to the same solution, a significant increase in emission intensity was observed. In contrast, the addition of other metal ions under identical conditions did not result in a comparable enhancement, suggesting that 2NP exhibits a high selectivity for Ag⁺ ions. This enhancement likely results from a specific interaction or binding between 2NP and Ag⁺, which could stabilize the excited state or inhibit non-radiative decay pathways. These findings point to the potential application of 2NP as a fluorescent probe for detecting silver ions in aqueous systems. Further exploration of binding mechanisms and structural interactions could provide a deeper understanding of this selective response. The emission spectra recorded for treating 2NP with different metals Such as K + , Na + , Mg 2+ , Ca 2+ , Li + , Sr 2+ , Ba 2+ , Pb 2+ , Al 3+ , Bi 2+ , Zn 2+ , Cu 2+ , Fe 2+ , Fe 3+ , Co 2+ , Mn 2+ , Ni 2+ , Cr 3+ , Hg 2+ , Cd 2+ and Zr 4+ "No significant variations in the intensity of the emission spectra were observed for 2NP in the presence of 100 equivalents of each component (Fig. 2 )." Which indicated the binding of Ag + ion only with 2NP. Further, when the fluorescence emission spectra of 2NP were recorded with increasing concentration of Ag + , there was a regular increment in the intensity of the emission showing clearly the binding of 2NP with Ag + ions [Fig. 3 ] Antijamming studies In order to conclusively say that the probe 2NP is only binding Ag + ions, further studies are needed and antijamming study is one among them. Hence, we carried out the fluorescence emission spectra of 2NP with various metal ions and also the emission spectra of 2NP with Ag + ions to which different metal ions were added (Fig. 4 ). The green bar illustrates the probe 2NP fluorescence when interacting with distinct metal ions, while orange bar displays the fluorescence alteration of probe 2NP with Ag + ion with other metal ions. Based on Fig. 4 , the fluorescence intensity of the orange bars appeared to stay nearly the same, indicating that other metal ions did not significantly affect the fluorescence, it is concluded that the probe responses only to Ag + ions and not to other metal ions added. Further, it proved that the other metal ions do not have any influence on the selectivity and sensitivity of the probe 2NP with Ag + ions. pH and Time effect studies Since the biological pH is in the range 7.4 to 7.6, we carried out the interaction of 2NP with Ag + ions at 7.4 pH. However, it is necessary to investigate whether there is any effect on the fluorescence behavior under various pH values. In this connection, we carried out the fluorescence emission studies from pH 1 to 14 using dilute HCl and NaOH. Free 2NP didn’t show any significant change in fluorescent intensity though there was a slight increase in fluorescence intensity in the pH range of 6 to 8. A striking contrast emerged when the same experiment was performed with 2NP in the presence of Ag + ion, steady enhancement of fluorescent intensity was observed with an abrupt change at pH 7. The results clearly revealed that the probe is absolutely suitable for sensing at biological pH in Fig. 5 . To find out the time taken for the time-dependent fluorescence measurements of 2NP were carried out in the presence of Ag + in a 1:1 v/v DMSO-H 2 O solution with 50 mM HEPES buffer at pH 7.4 to assess the maximum fluorescence intensity enhancement. The findings revealed complexation of 2NP with Ag + completed within 3 min (Fig.S6). From the above results, a time limit of 3 min was set as a standard for all fluorescent-based experiments. Reversibility studies To study the reversibility of the fluorescent probe 2NP to Ag + ion, EDTA titration experiments were carried out. On the addition of 100 equiv. of Ag + ion to the fluorescent Probe, there was an appreciable enhancement in the fluorescence intensity. Further addition of sodium salt of EDTA (100 equiv.) to the above solution quenched the fluorescence intensity and finally reached that of 2NP indicating that EDTA replaced 2NP completely from the complex formed. The same experiment was repeated several times for repeatability and reproducibility and the results clearly supported that Ag + ion recognition is a reversible process with respect to 2NP (Fig. 6 .) Job’s plot and association constant analysis To determine the stoichiometric ratio between the fluorescent probe 2NP and Ag + ion, Job’s plot analysis was conducted, as shown in Fig. S7. The results indicated a maximum interaction at a mole fraction of 0.7 at 560 nm, corresponding to a 2:1 ratio of 2NP to Ag + ion. HRMS analysis of 2NP and 2NP + Ag (Fig. S8) further confirms the formation of 2:1 complexation of 2NP with Ag + ions where 2NP + Ag showed a molecular ion peak at 777.1238. This stoichiometry was further validated using the Benesi-Hildebrand nonlinear curve fitting method [ 25 ]. The formation constant (K a ) was Found to be 1.7x10 2 M − 1 with R 2 = 0.97221 (Fig.S9) and the detection limit (LoD) was found to be 5.8995x10 − 5 M using the formula 3δ/S (26). 1 H NMR Titration To confirm the binding interaction of 2NP with Ag + ions, 1 H NMR titration experiments were performed in DMSO/D 2 O mixture. The chemical shifts of probe 2NP in the absence and presence of Ag + (0–4 equiv) ions are shown in Fig.S10. Upon the addition of Ag + ions, vivid changes were observed in the chemical shift value of naphthalene –OH proton and -CH = N proton. On increasing the Ag + ion concentration, OH proton peak gradually diminishes indicating the complexation of Ag + ion with 2NP through deprotonation of OH group of 2NP and neutral coordination through N of -CH = N group which shows characteristic changes in the proton signal values. Theoretical calculation In order to further confirm the mode of binding and the number of 2NP molecules around Ag + ion, we carried out a detailed DFT studies and the results obtained are discussed as follows. The structures of the compounds investigated and their energies obtained from DFT calculations are given in Fig. 7 . It’s particularly insightful to observe the stark contrast in energy difference between mono coordinated silver complex and the free ligand is only 49 eV while the same is 20128.832 eV between the ligand and the bis coordinated complex. This observation clearly indicates that two molecules of 2NP are coordinated to Ag + ion which is also in conformity with the results obtained from Job’s plot analysis. The bond lengths calculated for the uncoordinated ligand 2NP and that of the complex, [Ag(2NP) 2 ] were compared to find out the coordination sites of the ligand (Table S1 ). The bond lengths found for uncoordinated ligand are: C11-C14 (1.404 Å), C14- O40(1.392 Å,), C11-C17 (1.486 Å), C17-N19 (1.310 Å,), N19-N20 (1.394 Å), N20-C22 (1.397 Å), C22-S23 (1.729 Å,), C22-N24 (1.374 Å) and N24-C25 (1.482 Å).The bond lengths calculated for the complex showed elongations for C11-C14 (1.438 Å), C17- N19(1.327Å), N19-N20(1.440 Å ), N20-C22 ( 1.400 Å ), C22-S23 ( 1.7350 Å)bonds while there were reductions in bond lengths for C14-O40 (1.326 Å ), C11-C17 (1.4320 Å), C22- N24 ( 1.365 Å) and N24-C25 (1.480 Å). The C22-S23 bond found for the ligand 1.729 Å has increased to 1.7350 Å indicating the coordination of S atom of the ligand. On the other hand the C-O bond length found in the ligand, C14-O40(1.392 Å,) has decreased to C14-O40 (1.326 Å ) revealing the second coordination through O atom of the hydroxyl group. Here, we need look at the bond length of C11-N1 and N1-N2. C11 = N1 bond length should decrease after coordination through N1. The results of the DFT calculations of energy level diagrams are shown in Fig. 7 . It is noteworthy that energy separation between HOMO and LUMO of the free ligand (2NP) is 2.90 eV whereas the same is 1.60eV for the bis coordinated silver complex indicative of a stabilized [Ag(2NP) 2 ] complex (Fig. 8 ) In Vitro Cytotoxicity analysis and Bio imaging Studies The potential of the 2NP fluorescent probe as a tool for Ag + ion detection in a cellular environment was studied. Notably before live cell imaging, the in vitro cytotoxicity analysis of compound 2NP against Mouse Fibroblast Cell Line (L929) was done and shown in Table S2. The fluorescent probe 2NP showed only 17% cytotoxicity even at a higher concentration of 100 µM, and 83% of the cells were viable at the same concentration. This showed that compound 2NP is less toxic and can be safely used for bio imaging. To evaluate the intracellular sensing ability of 2NP, live cell imaging was performed using confocal laser scanning microscopy. Live cells were incubated with 2NP (20 µM) both in the absence and presence of Ag⁺ ions (10 µM) and subjected to fluorescence imaging [ 27 ]. As shown in Fig. 9 , cells treated with 2NP alone exhibited negligible fluorescence, indicating that 2NP remains non-fluorescent under normal cellular conditions. However, upon the addition of Ag⁺ ions, a remarkable enhancement in fluorescence intensity was observed, which can be attributed to the formation of the 2NP–Ag⁺ complex within the cellular environment. This significant fluorescence enhancement within the intracellular environment underscores the efficacy of 2NP as a selective and sensitive bioimaging agent for detecting Ag⁺ ions in living cells. Plausible Mechanism A mechanistic pathway is illustrated in Scheme 2 based on the experimental results obtained from fluorescence study, 1 NMR titration, HRMS-ESI-TOF and DFT studie s. . The fluorescent probe 2NP shows weak absorbance and fluorescence in the free state which shows a turn-off response, due to free C = N bond rotation. Upon complexation with Ag + ions, rigidity of the structure increases and suppresses the free rotation and charge transfer possibility. This is more pronounced in the fluorescence intensity enhancement on the probe 2NP complexation with Ag + ions. The probe 2NP was compared with other probes that detect silver ions and the same is tabulated in Table 1 . Conclusion In this study, we successfully developed and characterized a new thiosemicarbazone (2NP) probe employing physicochemical techniques. Its precise molecular arrangement was unequivocally determined through single-crystal X-ray diffraction studies. The investigations of absorption and emission studies demonstrated exceptional selectivity of 2NP towards silver ions even in the presence of numerous other metal ions. The binding stoichiometry of 2NP + Ag complex was further confirmed by Jobs Plot analysis, Benesi-Hidebrand analysis. 1H NMR, ESI-TOF and DFT studies confirmed the formation exact binding sites and confirmed that two molecules of 2NP coordinate to each silver ion in the resulting [Ag(2NP)] 2 complex with a detection limit of 5.8995 x 10 − 5 M with a binding constant of 1.7x10 2 M − 1 . The low cytotoxicity and intracellular imaging shows the potential ability of the fluorescent probe 2NP as a bio imaging agent. Declarations Acknowledgement Authors acknowledge the Department of Science and Technology, Govt. of India, New Delhi for having provided the infrastructure through DST FIST program. Author Statement Arumugam Sreedevi: Conceptualization, Data curation, Formal analysis, Writing – original draft, Writing – review & editing. Gunasekaran Prabhakaran: Data curation, Formal analysis, Writing – original draft, Writing – review & editing. Nattamai Bhuvanesh: Conceptualization, Data curation, Formal analysis, Writing – original draft, Writing – review & editing. Malathi Mahalingam: Data curation, Formal analysis, Writing – original draft, Raju Nandhakumar: Conceptualization, Data curation, Formal analysis, Writing – original draft, Writing – review & editing. Gopalan Subashini: Conceptualization, Data curation, Formal analysis, Funding acquisition, Project administration, Writing – original draft, Writing – review & editing. Funding: The authors did not receive any funds or grants from any organization for the submitted work. 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Supplementary Files SupportingInformation.docx Schem1.png Scheme 1 Synthesis of ( E )-2-((2-Hydroxynaphthalen-1-Yl)Methylene)-N-Methyl-N-Phenylhydrazinecarbothioamide. Schem2.png Scheme.2 Plausible Mechanism for the detection of Ag + ion by Fluorescent probe 2NP. Cite Share Download PDF Status: Published Journal Publication published 27 Mar, 2026 Read the published version in Journal of Fluorescence → Version 1 posted Editorial decision: Revision requested 24 Dec, 2025 Reviews received at journal 20 Dec, 2025 Reviews received at journal 19 Dec, 2025 Reviews received at journal 17 Dec, 2025 Reviews received at journal 15 Dec, 2025 Reviewers agreed at journal 11 Dec, 2025 Reviewers agreed at journal 11 Dec, 2025 Reviewers agreed at journal 09 Dec, 2025 Reviewers agreed at journal 05 Dec, 2025 Reviewers agreed at journal 04 Dec, 2025 Reviewers invited by journal 02 Dec, 2025 Editor assigned by journal 20 Nov, 2025 Submission checks completed at journal 20 Nov, 2025 First submitted to journal 11 Nov, 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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2","display":"","copyAsset":false,"role":"figure","size":506213,"visible":true,"origin":"","legend":"\u003cp\u003eFluorescence response of receptor 2NP (4 × 10\u003csup\u003e-6\u003c/sup\u003e M) in the presence of 100 equivalents of various metal ions in DMSO-H\u003csub\u003e2\u003c/sub\u003eO mixture. (λem = 520 nm)\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8086522/v1/3db1b11dc7e13aa7e394efb2.png"},{"id":97439646,"identity":"aa0420cf-2c6a-45a8-9a99-0290826ff538","added_by":"auto","created_at":"2025-12-04 11:43:55","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":463067,"visible":true,"origin":"","legend":"\u003cp\u003eFluorescence emission titration spectra of receptor 2NP (4 ×10\u003csup\u003e-6\u003c/sup\u003e M) in the incremental progression of Ag\u003csup\u003e+ \u003c/sup\u003eions (0–80 equivalents) in HEPES (50 mM) buffered DMSO-H2O, 1:1 (v/v) solution (pH = 7.4) at 520 nm.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8086522/v1/5d2983be2b3c23b30f783f3b.png"},{"id":97668636,"identity":"a67ad61d-a865-4d62-ae1d-0531ff5f8e4d","added_by":"auto","created_at":"2025-12-08 09:25:54","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":261794,"visible":true,"origin":"","legend":"\u003cp\u003eAnti-jamming experiment of receptor 2NP (4 × 10\u003csup\u003e-6\u003c/sup\u003e M) in DMSO-H2O solution. Green bar: Fluorescent emission of receptor 2NP upon addition of various interfering metal ions (100 equivalents). Orange bar: Fluorescent emission of receptor 2NP in presence of other tested metal ions (100 equivalents) and Ag\u003csup\u003e+\u003c/sup\u003e ions (100 equivalents).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8086522/v1/38f6bf25c0a5187a03335298.png"},{"id":97667784,"identity":"79b933ff-c60d-4709-9575-01c3514d5583","added_by":"auto","created_at":"2025-12-08 09:24:15","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":124396,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of pH on 2NP and 2NP+Ag\u003csup\u003e+ \u003c/sup\u003eions.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8086522/v1/672045e6030d5543883e104c.png"},{"id":97667060,"identity":"360daf0a-572b-432c-9004-9debad73aef5","added_by":"auto","created_at":"2025-12-08 09:22:42","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":260635,"visible":true,"origin":"","legend":"\u003cp\u003eReversibility studies of the fluorescent probe 2NP to Ag\u003csup\u003e+\u003c/sup\u003e ion upon EDTA complexation studies.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8086522/v1/91ebe7815ae9eb88582c9f59.png"},{"id":97439665,"identity":"891a6f8d-fadd-4a4b-a36e-52cf5d6de9a3","added_by":"auto","created_at":"2025-12-04 11:43:55","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":203296,"visible":true,"origin":"","legend":"\u003cp\u003eOptimized Geometry of 2NP, 2NP+Ag and (2NP)\u003csub\u003e2\u003c/sub\u003eAg.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-8086522/v1/f5e3c28ff4e898ca9aa76948.png"},{"id":97439653,"identity":"de68d1aa-1cab-4912-8afc-73e1c2f5deeb","added_by":"auto","created_at":"2025-12-04 11:43:55","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":959774,"visible":true,"origin":"","legend":"\u003cp\u003eMolecular energy level diagrams of (2NP)\u003csub\u003e2 \u003c/sub\u003eAg\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-8086522/v1/a53224b087e9a716daac4aa9.png"},{"id":97668455,"identity":"fea87845-72f7-477e-ba7c-d9102aab9000","added_by":"auto","created_at":"2025-12-08 09:25:35","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":136345,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBio imaging experiments with \u003c/strong\u003eMouse Fibroblast Cell Line (L929) \u003cstrong\u003ea)\u003c/strong\u003e Bright field image of L929 cell line \u003cstrong\u003eb)\u003c/strong\u003e fluorescent image of L929 cell line incubated with 2NP \u003cstrong\u003ec)\u003c/strong\u003e fluorescent image of L929 cell line incubated with 2NP and Ag\u003csup\u003e+\u003c/sup\u003e ions.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-8086522/v1/8dc519e7a12028a0c1e1608a.png"},{"id":105756064,"identity":"c45817ed-b9cb-4750-a543-d2c9b6cc6d54","added_by":"auto","created_at":"2026-03-30 16:35:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4689133,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8086522/v1/94c485b9-c523-4351-bd06-ffd7683e282e.pdf"},{"id":97667601,"identity":"c4dafda4-7251-44fe-b457-f2d8bf24425f","added_by":"auto","created_at":"2025-12-08 09:23:52","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2534180,"visible":true,"origin":"","legend":"","description":"","filename":"SupportingInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-8086522/v1/46942932d7b391bc3ff33a19.docx"},{"id":97439650,"identity":"e560788e-6966-48f4-8beb-28bd87d10181","added_by":"auto","created_at":"2025-12-04 11:43:55","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":80102,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScheme 1\u003c/strong\u003e Synthesis of (\u003cem\u003eE\u003c/em\u003e)-2-((2-Hydroxynaphthalen-1-Yl)Methylene)-N-Methyl-N-Phenylhydrazinecarbothioamide.\u003c/p\u003e","description":"","filename":"Schem1.png","url":"https://assets-eu.researchsquare.com/files/rs-8086522/v1/f4c20ea3e2946821bcff6deb.png"},{"id":97439652,"identity":"1095e75a-83b7-4d6b-82fd-465930073691","added_by":"auto","created_at":"2025-12-04 11:43:55","extension":"png","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":599107,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScheme.2 \u003c/strong\u003ePlausible Mechanism for the detection of Ag\u003csup\u003e+\u003c/sup\u003e ion by Fluorescent probe 2NP.\u003c/p\u003e","description":"","filename":"Schem2.png","url":"https://assets-eu.researchsquare.com/files/rs-8086522/v1/5bebe71ae072c188212f7a71.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"A Thiosemicarbazone-Derived Fluorescent Probe For The Detection Of Silver Ions And Bioimaging Application","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThough naturally occurring silver ions exhibit important properties such as safe green germicide, excellent antibiotic, and a good sterilizer [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], excess of silver ion becomes toxic to human health. It is known that photographic, electronic, pharmaceutical industries release an enormous amount of silver in the form of wastes, polluting water sources and agricultural fields [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The permissible limit of the total silver in water is 900nM [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] and any increase in silver ion concentration affects the re-productivity of aquatic animals since it is known to produce reactive oxygen species, namely free radicals. Silver build up in humans results in growth retardation, liver degenerative changes, and enlarged hearts [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] and deposits of silver precipitates in skin and eyes [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Separation and removal of silver ions is quite difficult due to the fact that silver ions possess only a moderate coordination ability. This has forced one to design and develop sensitive and selective methods to determine the trace quantities of silver.\u003c/p\u003e\u003cp\u003eAmong the various physicochemical methods used to sense the metal ions using some probe materials, the optical method has become unique due to its advantages of rapid signaling, low cost, straightforward operating modes, and great selectivity and sensitivity [\u003cspan additionalcitationids=\"CR9 CR10 CR11\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The following categories mostly comprise optical approaches for metal ion sensing: (1) The fluorescence approach [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], which uses fluorescence amplification, quenching, or intensity fluctuation to identify metal ions (2) The colorimetric approach [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], which uses colorimetric changes or variations in the UV absorption spectrum to identify metal ions selectively; (3) Surface enhanced Raman scattering spectroscopy and circular dichroism spectroscopy [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. From the point of view of the probes employed for sensing of metal ions in such studies, thiosemicarbazone based Schiff bases have been found to be apt and perfect compounds for the detection of different analytes because they possess fluorescence turn-on-off properties and of strong donor atoms O, N and S. In the search for a better Schiff base for the sensing of Ag\u003csup\u003e+\u003c/sup\u003e ions in trace quantities, we carried out the synthesis, characterization and investigated the efficacy of the thiosemicarbazone based Schiff base (2NP) towards selective and sensitive tracking of Ag\u003csup\u003e+\u003c/sup\u003e ions in polar solvent medium. Through chelation enhanced fluorescence (CHEF), the probe 2NP showed turn on response towards silver ions. The coordination behavior of the Schiff base with Ag\u003csup\u003e+\u003c/sup\u003e ions has been investigated through several techniques including density functional theory calculations.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e2-Hydroxy naphthaldehyde was procured from Sigma-Aldrich, then meticulously synthesized N-Phenyl-N-methyl thiosemicarbazone following a previously established protocol [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. All other essential chemicals were sourced commercially and utilized directly, eliminating the need for further purification. \u003csup\u003e1\u003c/sup\u003eH NMR and \u003csup\u003e13\u003c/sup\u003eC NMR spectra were meticulously recorded on a 400 MHz Bruker spectrometer. We used DMSO-d\u003csup\u003e6\u003c/sup\u003e as the solvent and TMS as the internal reference for these analyses. Infrared spectra were subsequently captured using a Shimadzu spectrophotometer, providing further structural insights. Optical measurements were recorded on a Shimadzu UV-240 spectrophotometer and Jasco FP-8200 spectrofluorometer, utilizing a 1 cm quartz cuvette. Throughout these optical experiments, excitation and emission slit widths were consistently emission spectra were collected at a controlled temperature of 24\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C. Elemental analysis (C, H, N) was performed using a Vario EL III elemental analyzer.\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eSynthesis of Chemosensor 2NP\u003c/h2\u003e\u003cp\u003eEquimolar amount of 2-hydroxy-1-naphthaldehyde (1.0 mmol) and N-phenyl-N-methyl thiosemicarbazone (1.0 mmol) were dissolved in 20 mL of ethanol. The mixture was then heated under reflux for 4 hours to accelerate the reaction. Upon cooling, a yellow solid precipitated from the solution which was subsequently filtered and recrystallized from mixture of dichloromethane/ethanol (1:1), yielding yellow-coloured crystals suitable for X-ray diffraction studies. Yield (88%); m.p. 212\u0026ndash;213 \u003csup\u003eᵒ\u003c/sup\u003eC. IR (KBr): νmax: 3447, 3370, 1581, 1487 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-d\u003csup\u003e6\u003c/sup\u003e): Chemical shifts (δ) observed at 12.76 (s, -OH), 10.90 (s, NH), 9.34 (s, 1H, CH\u0026thinsp;=\u0026thinsp;N), 7.98 (d, 1H, J\u0026thinsp;=\u0026thinsp;7.6J\u0026thinsp;=\u0026thinsp;7.6J\u0026thinsp;=\u0026thinsp;7.6 Hz, Q-C8-H), 7.88\u0026ndash;7.84 (t, 2H, Ar-H), 7.56\u0026ndash;7.52 (m, 3H, Ar-H), 7.08\u0026ndash;7.16 (m, Ar-H), 7.51\u0026ndash;7.42 (m, 4H, Ar-H), 7.41\u0026ndash;7.40 (d, J\u0026thinsp;=\u0026thinsp;2.8J\u0026thinsp;=\u0026thinsp;2.8J\u0026thinsp;=\u0026thinsp;2.8 Hz, 1H), 3.64 (s, 3H, CH\u003csub\u003e3\u003c/sub\u003e_33) \u003csup\u003e13\u003c/sup\u003eC-NMR (100 MHz, DMSO-d\u003csup\u003e6\u003c/sup\u003e):δ 179.20, 157.05, 144.91, 143.85, 132, 131.57,130.04, 128.80, 127.77, 127.61, 127.28, 126.77, 123.27, 120.33, 119.09, 108.52, 43.88. 40.13. (TOF-ES+) m/z [(M-1)] 334.42 (335.42). Anal. Calced. For C\u003csub\u003e19\u003c/sub\u003eH\u003csub\u003e17\u003c/sub\u003eN\u003csub\u003e2\u003c/sub\u003eOS (335.42) C, 68.03; H, 5.11; N, 12.53; Found: C, 67.71; H, 4.99; N, 12.36%.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eIn Vitro Cytotoxicity analysis:\u003c/h3\u003e\n\u003cp\u003eThe MTT assay for comp-1 was done as per the method followed by subashini et al.,[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Briefly, the Mouse Fibroblast Cell Line (L929) was treated with comp-1 at various concentrations (5, 25, 50, 75 and 100 \u0026micro;M), and DMSO was used as a control. After treatment, 1mg/mL of MTT solution was added to L929 cells and further incubated for 3\u0026ndash;4 h. After incubation, the absorbance was taken at 570 nm, and an image was captured in an inverted phase contrast microscope.\u003c/p\u003e\n\u003ch3\u003eTheoretical Studies\u003c/h3\u003e\n\u003cp\u003eTheoretical calculations of the probe 2NP was performed using Gaussian 16 W software and the GaussView 6.0 molecular visualization package [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. These calculations were carried out under B3LYP 6\u0026ndash;311\u0026thinsp;+\u0026thinsp;+\u0026thinsp;g(d,p) basis set level of theory. Electronic properties such as the HOMO-LUMO gap were also determined at the same theoretical level [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Initially optimization calcluation of the silver ion alone completed at the CAM-B3LYP-D3/6-31G(d) level of theory.\u003c/p\u003e"},{"header":"Results and discussion","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003eSynthesis of Schiff base, 2NP\u003c/h2\u003e\u003cp\u003eCondensation of 2-hydroxy naphthaldehyde with N-phenyl-N-methyl thiosemicarbazone in ethanol yielded a yellow fluorescent compound 2NP synthesized with good yields (Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Its identity was confirmed through advanced techniques, including FT-IR, \u003csup\u003e1\u003c/sup\u003eHNMR, \u003csup\u003e13\u003c/sup\u003eC NMR, HRMS (Fig.\u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e-S4) and single crystal X-ray analysis. A clear ORTEP representation of 2NP is showcased in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Relevant X-ray data collection and structure refinement are meticulously presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Crystallographic data for 2NP has been deposited with the Cambridge Crystallographic Data Centre under the supplementary publication number CCDC 2193495. Copies Of the data can be obtained free of charge on application to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK [fax: +44 (0) 1223336033 or \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eemail:[email protected]\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eAbsorption and Emission studies\u003c/h2\u003e\u003cp\u003eThe UV-Vis absorption spectrum of 2NP reveals a prominent absorption peak at 520 nm, attributed to an intramolecular charge transfer transition (Fig. S5). Using this wavelength as the excitation source, fluorescence studies were conducted, and 2NP displayed maxima at 550 nm in a DMSO-H₂O mixture (1:1 v/v) buffered with 50 mM HEPES at pH 7.4. On introducing Ag⁺ ions to the same solution, a significant increase in emission intensity was observed. In contrast, the addition of other metal ions under identical conditions did not result in a comparable enhancement, suggesting that 2NP exhibits a high selectivity for Ag⁺ ions. This enhancement likely results from a specific interaction or binding between 2NP and Ag⁺, which could stabilize the excited state or inhibit non-radiative decay pathways. These findings point to the potential application of 2NP as a fluorescent probe for detecting silver ions in aqueous systems. Further exploration of binding mechanisms and structural interactions could provide a deeper understanding of this selective response.\u003c/p\u003e\u003cp\u003eThe emission spectra recorded for treating 2NP with different metals Such as K\u003csup\u003e+\u003c/sup\u003e, Na\u003csup\u003e+\u003c/sup\u003e, Mg\u003csup\u003e2+\u003c/sup\u003e, Ca\u003csup\u003e2+\u003c/sup\u003e, Li\u003csup\u003e+\u003c/sup\u003e, Sr\u003csup\u003e2+\u003c/sup\u003e, Ba\u003csup\u003e2+\u003c/sup\u003e, Pb\u003csup\u003e2+\u003c/sup\u003e, Al\u003csup\u003e3+\u003c/sup\u003e, Bi\u003csup\u003e2+\u003c/sup\u003e, Zn\u003csup\u003e2+\u003c/sup\u003e, Cu\u003csup\u003e2+\u003c/sup\u003e, Fe\u003csup\u003e2+\u003c/sup\u003e, Fe\u003csup\u003e3+\u003c/sup\u003e, Co\u003csup\u003e2+\u003c/sup\u003e, Mn\u003csup\u003e2+\u003c/sup\u003e, Ni\u003csup\u003e2+\u003c/sup\u003e, Cr\u003csup\u003e3+\u003c/sup\u003e, Hg\u003csup\u003e2+\u003c/sup\u003e, Cd\u003csup\u003e2+\u003c/sup\u003e and Zr\u003csup\u003e4+\u003c/sup\u003e \"No significant variations in the intensity of the emission spectra were observed for 2NP in the presence of 100 equivalents of each component (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\" Which indicated the binding of Ag\u003csup\u003e+\u003c/sup\u003e ion only with 2NP. Further, when the fluorescence emission spectra of 2NP were recorded with increasing concentration of Ag\u003csup\u003e+\u003c/sup\u003e, there was a regular increment in the intensity of the emission showing clearly the binding of 2NP with Ag\u003csup\u003e+\u003c/sup\u003e ions [Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e]\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eAntijamming studies\u003c/h3\u003e\n\u003cp\u003eIn order to conclusively say that the probe 2NP is only binding Ag\u003csup\u003e+\u003c/sup\u003e ions, further studies are needed and antijamming study is one among them. Hence, we carried out the fluorescence emission spectra of 2NP with various metal ions and also the emission spectra of 2NP with Ag\u003csup\u003e+\u003c/sup\u003e ions to which different metal ions were added (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The green bar illustrates the probe 2NP fluorescence when interacting with distinct metal ions, while orange bar displays the fluorescence alteration of probe 2NP with Ag\u003csup\u003e+\u003c/sup\u003e ion with other metal ions. Based on Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, the fluorescence intensity of the orange bars appeared to stay nearly the same, indicating that other metal ions did not significantly affect the fluorescence, it is concluded that the probe responses only to Ag\u003csup\u003e+\u003c/sup\u003e ions and not to other metal ions added. Further, it proved that the other metal ions do not have any influence on the selectivity and sensitivity of the probe 2NP with Ag\u003csup\u003e+\u003c/sup\u003e ions.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003epH and Time effect studies\u003c/h3\u003e\n\u003cp\u003eSince the biological pH is in the range 7.4 to 7.6, we carried out the interaction of 2NP with Ag\u003csup\u003e+\u003c/sup\u003e ions at 7.4 pH. However, it is necessary to investigate whether there is any effect on the fluorescence behavior under various pH values. In this connection, we carried out the fluorescence emission studies from pH 1 to 14 using dilute HCl and NaOH. Free 2NP didn\u0026rsquo;t show any significant change in fluorescent intensity though there was a slight increase in fluorescence intensity in the pH range of 6 to 8. A striking contrast emerged when the same experiment was performed with 2NP in the presence of Ag\u003csup\u003e+\u003c/sup\u003e ion, steady enhancement of fluorescent intensity was observed with an abrupt change at pH 7. The results clearly revealed that the probe is absolutely suitable for sensing at biological pH in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. To find out the time taken for the time-dependent fluorescence measurements of 2NP were carried out in the presence of Ag\u003csup\u003e+\u003c/sup\u003e in a 1:1 v/v DMSO-H\u003csub\u003e2\u003c/sub\u003eO solution with 50 mM HEPES buffer at pH 7.4 to assess the maximum fluorescence intensity enhancement. The findings revealed complexation of 2NP with Ag\u003csup\u003e+\u003c/sup\u003e completed within 3 min (Fig.S6). From the above results, a time limit of 3 min was set as a standard for all fluorescent-based experiments.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eReversibility studies\u003c/h2\u003e\u003cp\u003eTo study the reversibility of the fluorescent probe 2NP to Ag\u003csup\u003e+\u003c/sup\u003e ion, EDTA titration experiments were carried out. On the addition of 100 equiv. of Ag\u003csup\u003e+\u003c/sup\u003e ion to the fluorescent Probe, there was an appreciable enhancement in the fluorescence intensity. Further addition of sodium salt of EDTA (100 equiv.) to the above solution quenched the fluorescence intensity and finally reached that of 2NP indicating that EDTA replaced 2NP completely from the complex formed. The same experiment was repeated several times for repeatability and reproducibility and the results clearly supported that Ag\u003csup\u003e+\u003c/sup\u003e ion recognition is a reversible process with respect to 2NP (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e.)\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eJob\u0026rsquo;s plot and association constant analysis\u003c/h2\u003e\u003cp\u003eTo determine the stoichiometric ratio between the fluorescent probe 2NP and Ag\u003csup\u003e+\u003c/sup\u003e ion, Job\u0026rsquo;s plot analysis was conducted, as shown in Fig. S7. The results indicated a maximum interaction at a mole fraction of 0.7 at 560 nm, corresponding to a 2:1 ratio of 2NP to Ag\u003csup\u003e+\u003c/sup\u003e ion. HRMS analysis of 2NP and 2NP\u0026thinsp;+\u0026thinsp;Ag (Fig. S8) further confirms the formation of 2:1 complexation of 2NP with Ag\u003csup\u003e+\u003c/sup\u003e ions where 2NP\u0026thinsp;+\u0026thinsp;Ag showed a molecular ion peak at 777.1238. This stoichiometry was further validated using the Benesi-Hildebrand nonlinear curve fitting method [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The formation constant (K\u003csub\u003ea\u003c/sub\u003e) was Found to be 1.7x10\u003csup\u003e2\u003c/sup\u003e M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e with R\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.97221 (Fig.S9) and the detection limit (LoD) was found to be 5.8995x10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e M using the formula 3δ/S (26).\u003c/p\u003e\u003cp\u003e\u003csup\u003e\u003cb\u003e1\u003c/b\u003e\u003c/sup\u003e\u003cb\u003eH NMR Titration\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo confirm the binding interaction of 2NP with Ag\u003csup\u003e+\u003c/sup\u003e ions, \u003csup\u003e1\u003c/sup\u003eH NMR titration experiments were performed in DMSO/D\u003csub\u003e2\u003c/sub\u003eO mixture. The chemical shifts of probe 2NP in the absence and presence of Ag\u003csup\u003e+\u003c/sup\u003e (0\u0026ndash;4 equiv) ions are shown in \u003cb\u003eFig.S10.\u003c/b\u003e Upon the addition of Ag\u003csup\u003e+\u003c/sup\u003e ions, vivid changes were observed in the chemical shift value of naphthalene \u0026ndash;OH proton and -CH\u0026thinsp;=\u0026thinsp;N proton. On increasing the Ag\u003csup\u003e+\u003c/sup\u003e ion concentration, OH proton peak gradually diminishes indicating the complexation of Ag\u003csup\u003e+\u003c/sup\u003e ion with 2NP through deprotonation of OH group of 2NP and neutral coordination through N of -CH\u0026thinsp;=\u0026thinsp;N group which shows characteristic changes in the proton signal values.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eTheoretical calculation\u003c/h2\u003e\u003cp\u003eIn order to further confirm the mode of binding and the number of 2NP molecules around Ag\u003csup\u003e+\u003c/sup\u003e ion, we carried out a detailed DFT studies and the results obtained are discussed as follows. The structures of the compounds investigated and their energies obtained from DFT calculations are given in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e. It\u0026rsquo;s particularly insightful to observe the stark contrast in energy difference between mono coordinated silver complex and the free ligand is only 49 eV while the same is 20128.832 eV between the ligand and the bis coordinated complex. This observation clearly indicates that two molecules of 2NP are coordinated to Ag\u003csup\u003e+\u003c/sup\u003e ion which is also in conformity with the results obtained from Job\u0026rsquo;s plot analysis.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe bond lengths calculated for the uncoordinated ligand 2NP and that of the complex, [Ag(2NP)\u003csub\u003e2\u003c/sub\u003e] were compared to find out the coordination sites of the ligand (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The bond lengths found for uncoordinated ligand are: C11-C14 (1.404 \u0026Aring;), C14- O40(1.392 \u0026Aring;,), C11-C17 (1.486 \u0026Aring;), C17-N19 (1.310 \u0026Aring;,), N19-N20 (1.394 \u0026Aring;), N20-C22 (1.397 \u0026Aring;), C22-S23 (1.729 \u0026Aring;,), C22-N24 (1.374 \u0026Aring;) and N24-C25 (1.482 \u0026Aring;).The bond lengths calculated for the complex showed elongations for C11-C14 (1.438 \u0026Aring;), C17- N19(1.327\u0026Aring;), N19-N20(1.440 \u0026Aring; ), N20-C22 ( 1.400 \u0026Aring; ), C22-S23 ( 1.7350 \u0026Aring;)bonds while there were reductions in bond lengths for C14-O40 (1.326 \u0026Aring; ), C11-C17 (1.4320 \u0026Aring;), C22- N24 ( 1.365 \u0026Aring;) and N24-C25 (1.480 \u0026Aring;). The C22-S23 bond found for the ligand 1.729 \u0026Aring; has increased to 1.7350 \u0026Aring; indicating the coordination of S atom of the ligand. On the other hand the C-O bond length found in the ligand, C14-O40(1.392 \u0026Aring;,) has decreased to C14-O40 (1.326 \u0026Aring; ) revealing the second coordination through O atom of the hydroxyl group. Here, we need look at the bond length of C11-N1 and N1-N2. C11\u0026thinsp;=\u0026thinsp;N1 bond length should decrease after coordination through N1. The results of the DFT calculations of energy level diagrams are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e. It is noteworthy that energy separation between HOMO and LUMO of the free ligand (2NP) is 2.90 eV whereas the same is 1.60eV for the bis coordinated silver complex indicative of a stabilized [Ag(2NP)\u003csub\u003e2\u003c/sub\u003e] complex (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e)\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eIn Vitro Cytotoxicity analysis and Bio imaging Studies\u003c/h2\u003e\u003cp\u003eThe potential of the 2NP fluorescent probe as a tool for Ag\u003csup\u003e+\u003c/sup\u003e ion detection in a cellular environment was studied. Notably before live cell imaging, the in vitro cytotoxicity analysis of compound 2NP against Mouse Fibroblast Cell Line (L929) was done and shown in Table S2. The fluorescent probe 2NP showed only 17% cytotoxicity even at a higher concentration of 100 \u0026micro;M, and 83% of the cells were viable at the same concentration. This showed that compound 2NP is less toxic and can be safely used for bio imaging.\u003c/p\u003e\u003cp\u003eTo evaluate the intracellular sensing ability of 2NP, live cell imaging was performed using confocal laser scanning microscopy. Live cells were incubated with 2NP (20 \u0026micro;M) both in the absence and presence of Ag⁺ ions (10 \u0026micro;M) and subjected to fluorescence imaging [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e, cells treated with 2NP alone exhibited negligible fluorescence, indicating that 2NP remains non-fluorescent under normal cellular conditions. However, upon the addition of Ag⁺ ions, a remarkable enhancement in fluorescence intensity was observed, which can be attributed to the formation of the 2NP\u0026ndash;Ag⁺ complex within the cellular environment. This significant fluorescence enhancement within the intracellular environment underscores the efficacy of 2NP as a selective and sensitive bioimaging agent for detecting Ag⁺ ions in living cells.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003ePlausible Mechanism\u003c/h2\u003e\u003cp\u003eA mechanistic pathway is illustrated in Scheme \u003cspan refid=\"Sch2\" class=\"InternalRef\"\u003e2\u003c/span\u003e based on the experimental results obtained from fluorescence study, \u003csup\u003e1\u003c/sup\u003eNMR titration, HRMS-ESI-TOF and DFT studie\u003cb\u003es.\u003c/b\u003e. The fluorescent probe 2NP shows weak absorbance and fluorescence in the free state which shows a turn-off response, due to free C\u0026thinsp;=\u0026thinsp;N bond rotation. Upon complexation with Ag\u003csup\u003e+\u003c/sup\u003e ions, rigidity of the structure increases and suppresses the free rotation and charge transfer possibility. This is more pronounced in the fluorescence intensity enhancement on the probe 2NP complexation with Ag\u003csup\u003e+\u003c/sup\u003e ions. The probe 2NP was compared with other probes that detect silver ions and the same is tabulated in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this study, we successfully developed and characterized a new thiosemicarbazone (2NP) probe employing physicochemical techniques. Its precise molecular arrangement was unequivocally determined through single-crystal X-ray diffraction studies. The investigations of absorption and emission studies demonstrated exceptional selectivity of 2NP towards silver ions even in the presence of numerous other metal ions. The binding stoichiometry of 2NP\u0026thinsp;+\u0026thinsp;Ag complex was further confirmed by Jobs Plot analysis, Benesi-Hidebrand analysis. 1H NMR, ESI-TOF and DFT studies confirmed the formation exact binding sites and confirmed that two molecules of 2NP coordinate to each silver ion in the resulting [Ag(2NP)]\u003csub\u003e2\u003c/sub\u003e complex with a detection limit of 5.8995 x 10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e M with a binding constant of 1.7x10\u003csup\u003e2\u003c/sup\u003e M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The low cytotoxicity and intracellular imaging shows the potential ability of the fluorescent probe 2NP as a bio imaging agent.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors acknowledge the Department of Science and Technology, Govt. of India, New Delhi for having provided the infrastructure through DST FIST program.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eArumugam Sreedevi: Conceptualization, Data curation, Formal analysis, Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing. Gunasekaran Prabhakaran: Data curation, Formal analysis, Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing. Nattamai Bhuvanesh: Conceptualization, Data curation, Formal analysis, Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing. Malathi Mahalingam: Data curation, Formal analysis, Writing \u0026ndash; original draft, Raju Nandhakumar: Conceptualization, Data curation, Formal analysis, Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing. Gopalan Subashini: Conceptualization, Data curation, Formal analysis, Funding acquisition, Project administration, Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eThe authors did not receive any funds or grants from any organization for the submitted work.\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eFegade U, Sharma H, Tayade K, Attarde S, Singh N, Kuwar A (2013) An amide based dipodal Zn\u003csup\u003e2+\u003c/sup\u003e complex: nano-molar detection of HSO\u003csub\u003e4\u003c/sub\u003e \u003csup\u003e\u0026ndash;\u003c/sup\u003e in a semi-aqueous system. Org Biomol Chem (11): 6824\u0026ndash;6828\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKlasen HJ (2000) Historical review of the use of silver in the treatment of burns. I Early uses Burns. 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Fox, Gaussian 09Gaussian, Inc., Wallingford CT\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDennington R, Keith T, Millam J (2009) Gauss View, Version 5. Semichem Inc., Shawnee Mission\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eConners KA (1987) Binding Constants; Wiley: New York, ; (b) B. Valeur, Molecular Fluorescence. Principles and Applications; Wiley-VCH: Weinheim, 2002\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eShortreed M, Kopelman R, Kuhn M, Hoyland B (1996) l Fluorescent fiber-optic calcium sensor for physiological measurements. Analytical chemistry, 68(8), 1414\u0026ndash;1418\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSubashini G, Shankar R, Arasakumar T, Mohan PS (2017) Quinoline appended pyrazoline based Ni sensor and its application towards live cell imaging and environmental monitoring. Sens Actuat B: Chem, (243): 549\u0026ndash;556\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eVelmurugan K, Suresh S, Santhoshkumar S, Saranya M, Nandhakumar R (2016) A simple Chalcone \u0026ndash;based rationmetric chemosensor for silver ion. Luminescence, (3): 22\u0026ndash;727\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBhuvanesh N, Suresh S, Ram kumar P, Mothi EM, Kannan K, Rajesh Kannan V, Nandhakumar R (2018) Small molecule turn on fluorescence Propbe for silver ion and application to bioimaging. J Photochem Photobiol A: Chem. (360): 6\u0026ndash;12\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBhuvanesh N, Suresh S, Prabhu J, Kannan K, Rajesh Kannan V, Nandhakumar R (2018) Ratiometric fluorescent chemosensor for silver ion and its bacterial cell imaging. Opt Mater (82): 123\u0026ndash;129\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChen Z, Zhou H, Gu W, Liu T, Xie Z, Yang L, Ma LJ (2019) A medium \u0026ndash; controlled fluorescent enhancement probe for Ag\u003csup\u003e+\u003c/sup\u003e and Cu\u003csup\u003e2+\u003c/sup\u003e derived from pyrene \u0026ndash; containing Schiffbase. J Photochem Photobiol A: Chem (379): 5\u0026ndash;10\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYang B, Zhu D, Zhang X, Zhang W, Liu J, Xue Y, Wei C, Bi Y, Fan A (2021) Bifunctional, Off-On Fluorescence Probe Based on Naphthalene for the Detection of Ag\u003csup\u003e+\u003c/sup\u003e and Al\u003csup\u003e3+\u003c/sup\u003e and Its Application in Practical Water Samples, as a Logic gate and as Test Paper. Chem Select. (6): 8830\u0026ndash;8838\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Table 1","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e"},{"header":"Schemes ","content":"\u003cp\u003eSchemes are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-fluorescence","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jofl","sideBox":"Learn more about [Journal of Fluorescence](https://www.springer.com/journal/10895)","snPcode":"10895","submissionUrl":"https://submission.nature.com/new-submission/10895/3","title":"Journal of Fluorescence","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"2-hydroxy naphthaldehyde, N-Phenyl-N-methyl thiosemicarbazone, silver ion sensing, DFT","lastPublishedDoi":"10.21203/rs.3.rs-8086522/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8086522/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eA simple thiosemicarbazone-derived (\u003cem\u003eE\u003c/em\u003e)-2-((2-Hydroxynaphthalen-1-yl)Methylene)-N-Methyl-N-Phenylhydrazinecarbothioamide was prepared and characterized using diverse analytical methods and spectroscopic techniques. The definitive elucidation of its crystal structure was achieved through single-crystal X-ray diffraction analysis. The synthesized compound (2NP) was found to be highly specific and sensitive towards sensing silver ions. Time-dependent fluorescence studies of 2NP showed selectivity towards Ag\u003csup\u003e+\u003c/sup\u003e ion with a detection limit of 5.8995 x 10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e M with a binding constant of 1.7x10\u003csup\u003e2\u003c/sup\u003e M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The binding stoichiometry of 2NP\u0026thinsp;+\u0026thinsp;Ag complex was further confirmed by Jobs Plot analysis, Benesi-Hidebrand analysis. 1H NMR, ESI-TOF and DFT studies confirmed the formation of [Ag(2NP)\u003csub\u003e2\u003c/sub\u003e] complex(2:1). Further, the compound 2NP has been tested for its cytotoxicity and bioimaging studies was also performed in Mouse Fibroblast Cell Lines (L929).\u003c/p\u003e","manuscriptTitle":"A Thiosemicarbazone-Derived Fluorescent Probe For The Detection Of Silver Ions And Bioimaging Application","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-04 11:43:50","doi":"10.21203/rs.3.rs-8086522/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-12-24T20:15:05+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-20T07:18:14+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-20T00:26:55+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-17T09:11:24+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-15T15:12:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"189430553357213152323645186173610735862","date":"2025-12-11T14:29:25+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"244628942827928434535889700207560954225","date":"2025-12-11T14:09:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"294614630883327039186426183005217415828","date":"2025-12-09T08:48:10+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"123739579642269988421491652011506012124","date":"2025-12-05T08:35:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"95732209307762341730870498457853820515","date":"2025-12-05T01:33:41+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-02T17:06:24+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-20T06:42:28+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-11-20T06:41:34+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Fluorescence","date":"2025-11-11T11:42:05+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":"9ea3be28-e95e-4ff8-9d89-ac72a5210c05","owner":[],"postedDate":"December 4th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-03-30T16:31:42+00:00","versionOfRecord":{"articleIdentity":"rs-8086522","link":"https://doi.org/10.1007/s10895-026-04734-3","journal":{"identity":"journal-of-fluorescence","isVorOnly":false,"title":"Journal of Fluorescence"},"publishedOn":"2026-03-27 16:09:21","publishedOnDateReadable":"March 27th, 2026"},"versionCreatedAt":"2025-12-04 11:43:50","video":"","vorDoi":"10.1007/s10895-026-04734-3","vorDoiUrl":"https://doi.org/10.1007/s10895-026-04734-3","workflowStages":[]},"version":"v1","identity":"rs-8086522","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8086522","identity":"rs-8086522","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}