D‑Penicillamine‑Stabilized Gold Nanoclusters as a Selective Fluorescent Sensor for Tetracycline

D‑Penicillamine‑Stabilized Gold Nanoclusters as a Selective Fluorescent Sensor for Tetracycline | 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 D‑Penicillamine‑Stabilized Gold Nanoclusters as a Selective Fluorescent Sensor for Tetracycline Luis Marco-Sabater, Elena Zaballos-García, Jorge Escorihuela, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9405870/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 11 You are reading this latest preprint version Abstract The development of simple, rapid, and cost‑effective sensing methods for monitoring antibiotic residues in water is crucial due to the widespread use of antibiotics in livestock farming and the consequent risk of environmental contamination. In this study, we present a facile synthesis of gold nanoclusters (AuNCs) stabilized with D‑penicillamine (AuNC@D‑Pen), a thiolated ligand that provides excellent colloidal stability and supports strong photoluminescent properties. The synthetic protocol affords highly uniform nanoclusters whose fluorescence emission, centered at approximately 650 nm, is both intense and stable under typical environmental conditions.The fluorescence of AuNC@D‑Pen undergoes progressive quenching in the presence of tetracycline, enabling a sensitive and reliable optical detection strategy. The detection limit (LOD) was determined to be 0.9 ppm, with a linear response range extending from 0.5 to 220 µM, which positions this system as a promising tool for practical analytical applications, including routine water‑quality monitoring. In addition, the sensor exhibits excellent selectivity for tetracycline over common potentially interfering species such as anions, metal ions, and amino acids, highlighting the robustness of the sensing mechanism. Overall, the AuNC‑based probe represents an efficient platform for the rapid, selective, and affordable detection of antibiotic contaminants in aqueous environments. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction In today’s world, antibiotics remain fundamental to global health, from routine medical care to complex surgical procedures and the management of infectious diseases. [1,2] Among the different families of antibiotics, tetracyclines (TCs) are a group of broad-spectrum antibiotics widely used in veterinary medicine, particularly in poultry, cattle, and swine farming. [3] This family of antibiotics characterized by a rigid tetracyclic fused nucleus, labeled as A, B, C, and D, possesses various functional groups such as hydroxyl, carbonyl, and dimethylamino (Scheme 1 ). Since their discovery in 1948, their widespread use has raised growing concerns about antimicrobial resistance and environmental contamination, [4] as large proportions of these antibiotics are excreted unmetabolized and enter aquatic systems. [5] In this regard, tetracycline pollution is considered a global environmental threat due to its extensive use in aquaculture, livestock, and human medicine. [6] Large fractions are excreted unmetabolized (up to 70–90%), subsequently entering soils and aquatic environments, where they are highly persistent because of their hydrophilic nature and resistance to natural degradation. [7] Studies across Europe have detected tetracycline concentrations in surface waters ranging from 0 to 0.02 ppb, but can reach higher concentrations up to 0.54 ppb in municipal wastewater treatment plant effluents. [8] The growing concern over antibiotic residues and resistance highlights the need for precise analytical monitoring. In this regard, several analytical methods are used to detect tetracyclines, [9] being chromatographic methods, such as HPLC or LC–MS, the most widely employed because of their high precision and sensitivity; however they require expensive instrumentation, long analysis times, and specialized operation, limiting their routine use. [10,11] On the other hand, electrochemical methods, including those modified with nanomaterials, provide fast response, high sensitivity, and low cost, making them suitable for on‑site analysis, but they generally suffer from matrix interferences, and limited long‑term stability. [12,13] Electrophoresis methods suffer from limited separation capability and require strict pH control, which hampers their practical applicability. [14,15] Immunoassay‑based techniques offer high selectivity through antibody recognition, yet they often present higher detection limits and longer assay times compared with emerging sensor technologies. [16–18] Chemiluminescence techniques typically respond to a broad range of compounds, making them suitable only for high-purity samples like pharmaceutical formulations. [19,20] In contrast, fluorescence analysis has gained significant attention in recent years due to its advantages: low cost, ease of operation, high sensitivity and stability, rapid signal response, real-time detection capability, excellent reproducibility, and minimal sample damage. [21] These attributes make fluorescence-based methods highly promising for the selective and accurate detection of TCs. In recent years, a wide range of fluorescent nanomaterials, such as quantum dots, carbon-based nanomaterials, rare earth-doped nanoparticles, and metallic nanoclusters, have gained significant attention in fluorescence sensing. [22] Among them, metallic nanoclusters (NCs) have emerged as particularly promising nanomaterials due to their exceptional properties, including high photostability, strong photoluminescence, large Stokes shifts, low toxicity, high quantum yields, excellent water solubility, and biocompatibility. [23] Over the past decade, considerable research has focused on the synthesis of silver (AgNC) and gold nanoclusters (AuNC), which have been widely employed as luminescent probes across various interdisciplinary fields. Among the diverse types of metal NCs, gold, silver, and copper nanoclusters have been extensively studied. [24–26] AuNC have been stablished as highly effective fluorescent probes for the detection of tetracyclines due to their unique optical properties and strong interactions with antibiotic molecules. [27] These ultrasmall clusters exhibit size‑dependent fluorescence that can be selectively quenched or enhanced when tetracyclines interact with the nanomaterial, enabling sensitive, rapid, and label‑free detection. Their excellent biocompatibility, high quantum yield, and tunable surface chemistry make gold nanoclusters particularly suitable for applications in food‑safety monitoring and environmental analysis, where low detection limits and reliable performance in complex matrices are essential. [28] Along the last decade, different fluorescent AuNC-based sensors have been reported for the detection of tetracyclines. Among them, AuNCs capped with thiolated ligands, such as glutathione [29] and N-acetyl-L-cysteine [30] , have shown limits of detection (LOD) of 2.4 and 0.8 ppm, respectively. Lower LODs can be achieved using rare-earth metals such as Eu(III) salts. In this regard, systems involving Eu(III) complexes of L-histidine-caped AuNCs [31] or BSA‑stabilized AuNCs [32] allowed the detection of TC with a detection limit of 2 ppb in both systems. More complex systems using microfluidic chip with ovalbumin‑stabilized AuNCs in a have achieved the detection of TC in chicken muscle with a LOD of 90 ppb. [33] In this study, we describe the preparation of D-penicillamine-capped AuNC ( AuNC@D-Pen ) via a simple protocol which avoids the use of additional chemicals and complicated synthetic and laborious purification steps. The synthesized AuNC@D-Pen demonstrated strong sensitivity for detecting tetracycline (TC) at low concentrations. Specifically, the characteristic emission at 652 nm showed a clear reduction in intensity upon increasing concentrations of TC, demonstrating strong sensitivity even at low analyte levels. This quenching behaviour is attributed to intermolecular interactions between TC and the ligand‑protected nanocluster surface. The detection method for TC has the advantages of high sensitivity and selectivity towards other interfering analytes, simple operation, and applicability to environmental water samples. Experimental Materials . All the chemicals and solvents used in this thesis degree were of analytical grade and used without any additional purification. Gold(III) chloride (HAuCl 4 ·3H 2 O), sodium chloride (NaCl), potassium chloride (KCl), magnesium chloride (MgCl 2 ), calcium chloride (CaCl 2 ), aluminium chloride (AlCl 3 ), sodium nitrate (NaNO 3 ), sodium carbonate (Na 2 CO 3 ), copper sulphate (CuSO 4 ), L-phenylalanine (L-Phe), L-valine (L-Val), L-histidine (L-His), tetracycline and ampicillin were purchased from Sigma-Aldrich. D-penicillamine was purchased from BLD Pharmatech. For all aqueous solutions, high purity deionized water from a Millipore system was used. Equipment. The following technical instruments were used for analysis and characterization. Centrifugation was performed on a Beckman Coulter's Microfuge 16 benchtop centrifuge. UV-vis absorption spectra were recorded on a PerkinElmer 1050 + UV/vis/NIR spectrometer. All the measurements were performed using 1cm×1cm path length quartz cuvettes. Fluorescence spectra were recorded on a FLS1000 photoluminescence spectrometer from Edinburgh Instruments. The quantum yield was measured with a Hamamatsu C9920-02 absolute PL Quantum Yield Measurement System. The pH measurements were carried out by using a Crison GLP 21 pH meter. Transmission electron microscopy (TEM) images were acquired using a HITACHI HT7800 microscope with a filament of LaB6 operating at 100 kV. Fourier transform infrared (FT-IR) spectra were recorded on a Thermo Scientific Nicolet iS10. X-ray photoelectron spectroscopy (XPS) spectra were acquired with VG-Microtech Multilab 3000 equipment. The 1 H spectrum were registered at room temperature in a Bruker AvanceIII 300 spectrometer, with a 300 MHz Bruker magnet. The chemical shifts (δ) are reported in ppm using deuterium oxide, 99.9% atom (D 2 O) as solvent. Preparation of AuNC@D-Pen. A freshly prepared aqueous solution of HAuCl 4 (50 µL, 50 mM) was diluted in water (1 mL), and an aqueous solution of D-penicillamine (53 µL, 1 M) was added. The mixture was set for 5 days at room temperature, obtaining a white precipitated (fluorescent under UV-light, 365 nm) and a colourless solution. The precipitate was isolated by centrifugation at 10000 rev/min for 15 min. The supernatant was slowly removed without disturbing the precipitate, which had been washed two times by dispersion in water and precipitation by centrifugation at 10000 rev/min for 15 min. After purification, the AuNC@D-Pen were dispersed in water or the solutions were diluted with buffer solution, meanwhile, to avoid the effect of the pH change. Fluorescence detection of tetracycline. Fluorescence experiments were conducted at room temperature using an excitation wavelength of 310 nm. Tetracycline solutions of varying concentrations were freshly prepared and sequentially added to 3 mL of AuNC@D-Pen solution (citrate buffer, pH 4.3, with an absorbance of 0.4 at 305 nm). Photoluminescence spectra were recorded at room temperature immediately after each addition of tetracycline. The concentration of TC was plotted on the x-axis, while the corresponding photoluminescence intensity was plotted on the y-axis. Finally, a linear correlation curve was generated to determine the concentration of tetracycline. Selectivity studies of AuNC@D-Pen. To evaluate the selectivity of the prepared AuNC@D-Pen , a series of selective experiments were performed. Initially, 5 µL of a 0.05 M TC solution was added to a solution of AuNC@D-Pen to establish the baseline photoluminescence (PL) intensity as a control. Subsequently, 5 µL of 0.05 M solutions of different potential interfering analytes, such as common anions and cations (NaCl, KCl, MgCl 2 , AlCl 3 , Na 2 CO 3 , MnCl 2 , CaCl 2 and MgSO 4 ), amino acids (L-Phe, L-Val, and L-His), and ampicillin, were added to the AuNC@D-Pen solution. Fluorescence intensity values were measured after an incubation time of 5 minutes at room temperature. The fluorescence intensity response of AuNC@D-Pen to each analyte was measured at an excitation wavelength of 310 nm. Detection of TC in water samples. We performed recovery experiments using both tap water and lake water. Tap water without pretreatment was tested for TC. For TC detection in lake water, the sample was initially centrifuged to remove suspended particles. Next, water samples were adjusted at pH around 4.0 by adding HCl solution. Known concentrations of TC (0.8, 1.2, 2.5, and 5.0 µM) were added to each water sample, and the resulting solutions were analysed under the same optimized conditions used for the calibration curve. For each concentration level, three independent measurements were carried out, allowing the calculation of both the recovery percentage and the corresponding relative standard deviation (RSD). Results and discussion Synthesis and characterization of AuNC@D-Pen. For the preparation of AuNC@D-Pen , we used the bottom-up approach that involves assembling atoms or molecules into nanoclusters with atomic precision. [34] The advantage of this method is that it offers excellent control over size, composition, and surface chemistry, making it ideal for producing atomically precise gold nanoclusters, often with sizes around 2 nm. To this purpose, a freshly prepared 1 mL of an aqueous solution of HAuCl 4 (50 µL, 50 mM) and another aqueous solution of D-penicillamine (53 µL, 1 M). Both solutions were mixed in a 1.5 mL Eppendorf tube. After a few days, we monitored the formation of gold nanoclusters by checking their fluorescence with a UV lamp (365 nm). After 5 days, a white precipitate appeared and was isolated by centrifugation, washed with water, and isolated, again, by centrifugation. After purification, the water suspension was stored at room temperature. A schematic representation of the synthesis is shown in Fig. 1 . The photoluminescence properties of AuNC@D‑Pen were investigated by recording their emission spectrum upon excitation at 310 nm. As shown in Fig. 2 (red line), the nanoclusters exhibited a broad emission band with an emission maximum centred at 652 nm. This wide spectral profile is characteristic of ligand‑protected gold nanoclusters, whose electronic transitions arise from discrete energy levels rather than the band‑like structure typical of larger nanoparticles. Importantly, the emission spectrum remained unchanged when the excitation wavelength varied between 300 and 420 nm, demonstrating that the luminescence originates from intrinsic, relaxed excited states of the nanoclusters. The absence of excitation‑dependent shifts indicates that the observed signal is true photoluminescence rather than an artefact from scattering, surface defects, or heterogeneous emissive species. The AuNC@D‑Pen sample displayed a photoluminescence quantum yield (φₚₗ) of 1%. Although modest, this quantum yield reflects efficient relaxation pathways within the metal–ligand framework and confirms that D‑penicillamine provides a stable surface environment supporting radiative recombination. The molar ratio of gold:ligand is a critical parameter in the bottom-up synthesis of gold nanoclusters, as it strongly influences the size, monodispersity, stability, and surface chemistry of the nanoclusters. [35] The molar ratio was investigated for D-penicillamine to obtain high quality luminescent gold nanoclusters. Under the described conditions, the concentration of HAuCl 4 was kept constant and different HAuCl 4 /D-Pen molar ratios were assayed: 1:1, 1:2, 1:5, 1:10, 1:20. After recording to the fluorescent spectrum of the nanocluster under different molar ratios, the highest fluorescence intensity gradually decreased with the HAuCl 4 /D-Pen molar ratio with a maximum of intensity for the nanocluster with the 1:20 HAuCl 4 /D-Pen molar ratio ( Fig. S1 ). Based on these observations, we conclude that the optimal molar ratio for synthesizing strongly luminescent AuNC@D‑Pen is 1:20, ensuring both efficient surface stabilization and maximized fluorescence. The influence of reaction time using a HAuCl 4 /D-Pen molar ratios of 1:20. was also investigated. A progressive increase in the fluorescence intensity was observed during the first several days of incubation, indicating ongoing structural formation of the AuNC@D‑Pen system. The fluorescence reached its maximum after approximately five days ( Fig. S2 ), suggesting that this period is required for the nanoclusters to fully develop their optimal luminescent properties. The 1 H NMR spectrum of D‑penicillamine displayed two characteristic peaks at 1.41 and 1.49 ppm, corresponding to the protons of the methyl groups, along with a signal at 3.62 ppm assigned to the chiral methine (CH) proton. Upon coordination to gold and formation of AuNC@D‑Pen , both methyl peaks experience a slight downfield shift to 1.43 and 1.51 ppm, respectively, while the methine proton shifts to 3.82 ppm ( Fig. S3 ). These changes in chemical shift are indicative of ligand–metal interactions and support the successful incorporation of D‑penicillamine onto the AuNC surface. The morphology of the AuNC@D-Pen was characterized using transmission electron microscopy (TEM). As shown in Fig. 3 , the micrographs of the prepared AuNC@D-Pen exhibited uniform dispersion and predominantly spherical morphology. Importantly, the particles appear well isolated from one another, with no evidence of large agglomerates or significant clustering. This lack of aggregation indicates that D‑penicillamine provides effective surface stabilization, preventing particle–particle fusion and maintaining colloidal stability during synthesis and imaging. Detailed quantitative analysis of the TEM images further confirms the presence of spherical gold nanoclusters with an average diameter of 2.5 ± 0.4 nm, a size range characteristic of well‑defined, ligand‑protected Au nanoclusters. This particle size is consistent with previously reported penicillamine-capped AuNCs. [36–38] This narrow size distribution is consistent with controlled nucleation and growth processes typically observed in thiolate‑stabilized gold nanocluster systems, supporting the successful formation of small, monodisperse AuNCs. The functional groups on the surface of AuNC@D-Pen were characterized by Fourier Transform Infrared (FTIR) spectroscopy. FTIR spectroscopy is a powerful tool for characterizing gold nanoclusters, especially when they are functionalized with thiolated ligands. [39] D-penicillamine exhibits the S–H stretching band in the region of 2550–2600 cm⁻¹, and upon binding to gold, this band disappears or is significantly reduced, indicating formation of Au–S bonds. As shown in Fig. 4 , the band corresponding to the S-H bond in D-penicillamine disappears when the ligand coordinates gold atoms. The loss of the S–H band therefore confirms ligand and highlights the high affinity of sulfur for gold, a well‑known feature that explains the stability of thiolate‑protected gold nanoclusters. X-ray photoelectron spectroscopy (XPS) measurements were performed to investigate the electronic structure and surface chemical composition of the gold nanoclusters. The XPS analysis provides insights into the oxidation states of gold and the nature of the chemical interactions between gold atoms and stabilizing ligands. [40] In particular, analysis of the C 1s core-level spectra showed multiple components (Fig. 5 a), typically associated with different carbon bonding environments such as C–C/C–H (~ 284.8 eV), C–N/C–O (~ 286.2 eV), C–S (~ 287.4 eV)and O–C = O (~ 288.5 eV), which are indicative of the different carbon environments present in penicillamine ligand used to stabilize the AuNCs. The high-resolution S 2p spectra provides information of the bonding environment of sulphur atoms originating from thiol-based ligands. The S 2p region exhibited characteristic doublet peaks, corresponding to the spin-orbit components S 2p₃/₂ and S 2p₁/₂, typically observed around 162–164 eV (Fig. 5 b). Peaks near ~ 162 eV are indicative of sulphur atoms covalently bonded to gold (Au–S), confirming thiolate-gold interactions. [41,42] Finally, the Au 4f spectra is generally used to confirm the metallic state of gold within the clusters. As shown in Fig. 5 c, the high-resolution Au 4f spectra revealed characteristic doublets corresponding to Au 0 and the oxidized species (e.g., Au⁺), as inferred from the peaks at 84.7 and 88.4 eV for Au 0 , and 85.3 and 89.0 eV for Au + . [43–45] We evaluated the stability of AuNC@D-Pen at different pH values ( Fig. S4 ). The stability of gold nanoclusters is highly dependent on the pH of the surrounding environment, as pH can influence their surface charge, ligand conformation, aggregation behaviour, among other effects. [46] For these experiments, were prepared AuNC@D-Pen solutions with similar value for the absorbance at 310 nm around, and then small amounts of a stock solution of NaOH 0.1 M was added to adjust the pH to the desired value. After shaking and allowing the sample resting at room temperature for 5 minutes, the fluorescence at 650 nm was measured following excitation at 305 nm. The results indicate that the fluorescence intensity of AuNC@D-Pen at 650 nm was kept constant and near the maximum value in the pH range from 2.1 until 4.9. However, at pH = 6.2 the fluorescence decreased dramatically until pH = 8.9. This effect may be attributed to the degree of deprotonation of the organic ligand with the pH. This behaviour was irreversible as when reaching pH = 8.9, we added acid (HCl 0.5 M) until pH = 3.8, and the original value of fluorescence was not recovered. [47] AuNC@D-Pen for fluorimetric detection of tetracycline. Under the optimal experimental conditions described previously, we evaluated the analytical performance and sensitivity of the ratiometric fluorescence sensor for tetracycline (TC) detection. As shown in Fig. 6 a, the fluorescence intensity of AuNC@D-Pen at 652 nm progressively decreased as the concentration of TC increased. Because of this well-defined fluorescence response, the intensity ratio F/F 0 exhibited a clear and proportional dependence on TC concentration. When fitting the data, a strong linear correlation was obtained for concentrations ranging from 0.5 to 220 µM (Fig. 6 b). The calibration curve followed the equation: F/F 0 = 0.9682 − 0.0031 [TC]/µM, with an excellent correlation coefficient (R² = 0.99344), demonstrating high analytical reliability. Based on the 3σ criterion, the limit of detection (LOD) was determined to be 0.9 ppm, highlighting the good sensitivity of the sensing nanomaterial. We believe that the interaction between TC and AuNC@D-Pen might involve intermolecular hydrogen bonds between the hydroxyl (–OH) and carbonyl (C = O) groups located on one of the aromatic rings of the tetracycline scaffold as illustrated in Fig. 7 . To support this hypothesis, we performed density functional theory (DFT) calculations using Gaussian 16 [48] at the B3LYP‑D3/def2‑TZVP level of theory, [49,50] employing the SDD pseudopotential for gold. [51] To this end, we used a model consisting of 18 gold atoms and 14 D-penicillamine ligands. The optimized structure obtained from the computational study reveals the formation of hydrogen bonds between tetracycline’s OH and C = O groups and the functional groups of the D-penicillamine ligand shell. These results support the proposed interaction model and align well with previously reported systems, where hydrogen bonding plays a major role in adsorption, orientation, and stabilization. [30] Sensor selectivity is a critical parameter, particularly when detecting specific analytes within complex matrices. High selectivity ensures that the sensor responds primarily to the target compound, minimizing interference from other substances that may be present in the sample. [52] This is especially important in real-world applications, such as food safety, environmental monitoring, or clinical diagnostics, where samples often contain a multitude of species which can interact increasing or decreasing the signal. Thus, we evaluated the selectivity of the AuNC@D-Pen sensor towards tetracycline by exposing the nanomaterial to a selection of potentially interfering ions and molecules. For this purpose, we prepared stock solutions containing cations such as Na + , K + , Mg 2+ , Ca 2+ , Al 3+ , anions such as Cl − , NO 3 − , CO 3 2− ; natural amino acids such as phenylalanine (L-Phe), valine (L-Val), or histidine (L-His), and another antibiotic, Ampiciline. The fluorescence intensity response of AuNC@D-Pen to each interfering analyte was measured at an excitation wavelength of 310 nm. As shown in Fig. 8 , the fluorescence intensity ratio remained almost unchanged (a slight decrease was observed in some cases) in the presence of these interfering species, indicating minimal non-specific interactions. These potential interferents have a negligible impact on the fluorescence signal, which remains largely unaffected in their presence. In contrast, the addition of a solution of TC induced a quenching in the fluorescence, confirming the sensor's strong and specific interaction with tetracycline. These results clearly demonstrate that the AuNC@D-Pen sensor possesses high selectivity for tetracycline over other coexisting substances, making it a reliable and robust platform for the selective detection of tetracycline in complex sample environments. Tetracycline detection in real samples. To evaluate the practical applicability of the proposed method for tetracycline detection in real samples, we conducted assays in tap water and lake water. To this end, different amounts of TCs (0.8, 1.2, 2.5 and 5.0 µM) were added into water samples, analysed using the obtained calibration curve and recovery tests in three repeated measurements were performed and the relative standard deviation (RSD) was obtained. As shown in Table 1 , the recovery values ranged from 96.2% to 104.4%, demonstrating excellent accuracy and minimal matrix interference. The low RSD values further confirm the good repeatability of the measurements. These results indicate that the AuNC@D-Pen sensor possesses acceptable results for TC determination in a water samples. Table 1 TC determination in real samples. Sample Spiked (mM) Detected ± SD RSD (%) Recovery (%) Tap water 0.8 0.82 ± 0.05 6.1 102.5 1.2 1.24 ± 0.09 7.3 103.3 2.5 2.43 ± 0.19 7.8 97.2 5.0 5.14 ± 0.11 2.1 102.8 Lake water 0.8 0.77 ± 0.06 7.8 96.3 1.2 1.25 ± 0.11 8.8 104.2 2.5 2.61 ± 0.21 8.0 104.4 5.0 5.21 ± 0.22 4.2 104.2 Conclusions In conclusion, the findings presented in this work demonstrate the development of a rapid and moderately sensitive assay for detecting tetracycline, based on the fluorescence quenching of gold nanoclusters stabilized with D-penicillamine. The AuNC@D-Pen system displayed a fluorescence peak at 652, attributed to the intrinsic emission of D-Pen-stabilized AuNCs. Upon the addition of tetracycline, the fluorescence intensity at 652 was quenched, offering a linear detection range from 0.5 to 220 µM and a low limit of detection (LOD) of 0.9 ppm. The sensor was successfully validated in real water samples, demonstrating its practical applicability. The AuNC@D-Pen system offers distinct advantages, including straightforward preparation, rapid response time, broad linear range, good sensitivity and selectivity. This work may inspire the development of additional fluorescence-based antibiotic sensors utilizing gold nanoclusters. Based on these findings, ongoing research in our laboratory is focused on engineering other nanostructures that combine AuNCs with thiolated ligands to enhance sensitivity and broaden the detection spectrum. Declarations Author contributions Conceptualization: J.E. and E.Z.G. Data curation: L.M.S. and J.E. Formal analysis: L.M.S. and J.E. Funding acquisition: J. P. P. Investigation: L.M.S. and J.E. Methodology: J. E. and E. Z. G. Project administration: J. P. P. Resources: J. P. P. Supervision: J. E. and E. Z. G. Writing – original draft: J.E. and E.Z.G. Writing – review & editing: J. E., E. Z. G. and J. P. P. All authors have given approval to the final version of the manuscript. Conflicts of interest There are no conflicts to declare. Data availability All the experimental data are presented in the main text and supplementary information (SI). Supplementary information is available. See DOI: Acknowledgements The present research was financed by Generalitat Valenciana under Prometeo program (CIPROM/2022/57). This study forms part of the Advanced Materials program and was supported by MCIU with funding from the European Union NextGenerationEU (PRTR-C17.I1) and by Generalitat Valenciana (MFA/2022/051). This work was also supported by the Horizon Europe Marie Curie call for Staff Exchanges 2022 (AMRAMR 101131231). The computational resources from the Servei d'Informàtica de la Universitat de Valencia (SIUV) are gratefully acknowledged. The authors are grateful to SCSIE of University of Valencia for providing TEM and HRMS facilities. 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Phys Chem Chem Phys 7:3297–3305. https://doi.org/10.1039/B508541A Ehlers AW, Böhme M, Dapprich S, Gobbi A, Höllwarth A, Jonas V, Köhler KF, Stegmann R, Veldkamp A, Frenking G (1993) A Set of f-Polarization Functions for Pseudo-Potential Basis Sets of the Transition Metals SC-Cu, Y-Ag and La-Au. Chem Phys Lett 208:111–114. http://dx.doi.org/10.1016/0009-2614(93)80086-5 Peveler WJ, Yazdani M, Rotello VM (2016) electivity and Specificity: Pros and Cons in Sensing. ACS Sens 1:1282–1285. https://doi.org/10.1021/acssensors.6b00564 Additional Declarations No competing interests reported. Supplementary Files SupportingInformation.pdf Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 04 May, 2026 Reviews received at journal 02 May, 2026 Reviewers agreed at journal 26 Apr, 2026 Reviews received at journal 22 Apr, 2026 Reviewers agreed at journal 22 Apr, 2026 Reviewers agreed at journal 22 Apr, 2026 Reviewers agreed at journal 21 Apr, 2026 Reviewers invited by journal 21 Apr, 2026 Editor assigned by journal 13 Apr, 2026 Submission checks completed at journal 13 Apr, 2026 First submitted to journal 13 Apr, 2026 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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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-9405870","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":629751620,"identity":"bb188ac5-2210-4439-8422-9d74724c50bc","order_by":0,"name":"Luis Marco-Sabater","email":"","orcid":"","institution":"University of Valencia","correspondingAuthor":false,"prefix":"","firstName":"Luis","middleName":"","lastName":"Marco-Sabater","suffix":""},{"id":629751621,"identity":"ee9858b0-5a2a-4454-9b20-c3e85a2edb49","order_by":1,"name":"Elena Zaballos-García","email":"","orcid":"","institution":"University of Valencia","correspondingAuthor":false,"prefix":"","firstName":"Elena","middleName":"","lastName":"Zaballos-García","suffix":""},{"id":629751622,"identity":"85cb6550-ae95-4cf8-b2d4-13f7e8e6aedb","order_by":2,"name":"Jorge Escorihuela","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/0lEQVRIiWNgGAWjYBACxgYkzmEglmFgb8CuFKcWHgaeAyRYyQzWIpFAQFV77+MXHxhs7Pnbex8eLmyz4eGf+cZMgqGiDrfDeo6bWc5gSEuccea4weGZbWk8ErdzgFrOHMatZUYamzEPw+EEA4k0hsO8bYd5GG7nGBswtuH2EFjLH4b/9lAt/3nkb54BavmHx2Ez0pgfMzAcYNwA0XKAx+AGj+EDxgZmPH45xsbYY5AM9MsxhsM855J5DM+kFT5IOIbbL4btbcwfflTYAUOsjfkzT5mdnNzxwxsOfKjB7TDDBgY2CQYDdOEEnBoYGOSBUfMBj/woGAWjYBSMAgYGAIfxT+CHNH//AAAAAElFTkSuQmCC","orcid":"","institution":"University of Valencia","correspondingAuthor":true,"prefix":"","firstName":"Jorge","middleName":"","lastName":"Escorihuela","suffix":""},{"id":629751623,"identity":"2eb30907-b6b3-4b19-ac68-db4e76388fce","order_by":3,"name":"Julia Perez-Prieto","email":"","orcid":"","institution":"Instituto de Ciencial Molecular","correspondingAuthor":false,"prefix":"","firstName":"Julia","middleName":"","lastName":"Perez-Prieto","suffix":""}],"badges":[],"createdAt":"2026-04-13 15:09:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9405870/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9405870/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108155555,"identity":"da15629d-2ee9-45ed-bf41-cc16b2817552","added_by":"auto","created_at":"2026-04-30 02:36:21","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":74497,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic representation of the synthesis of \u003cstrong\u003eAuNC@D-Pen\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-9405870/v1/ad82a10365ac45dadc1da2f6.png"},{"id":108155562,"identity":"7ab0231a-13ab-407a-9068-67e1ec45530c","added_by":"auto","created_at":"2026-04-30 02:36:21","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":16296,"visible":true,"origin":"","legend":"\u003cp\u003eEmission (red) and excitation (blue) spectra of AuNC@D-Pen.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-9405870/v1/7f1b54a600788f2bcb05731a.png"},{"id":108182625,"identity":"4bedf9d8-78a0-4641-8e50-e6427ebe381e","added_by":"auto","created_at":"2026-04-30 08:59:28","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":136671,"visible":true,"origin":"","legend":"\u003cp\u003e(a) SEM image of \u003cstrong\u003eAuNC@D-Pen\u003c/strong\u003e. (b) Representative histogram of particle size.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-9405870/v1/af3c4acf285a0f16430c3331.png"},{"id":108155556,"identity":"cd083d03-fc8c-4d46-8250-559cfea3a381","added_by":"auto","created_at":"2026-04-30 02:36:21","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":51482,"visible":true,"origin":"","legend":"\u003cp\u003eFTIR spectra of D-penicillamine (black line) and AuNC@D-Pen (red line).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-9405870/v1/1d33c948f3381832397d8b55.png"},{"id":108182877,"identity":"42ca9cc3-a922-4d53-bc45-341440adb048","added_by":"auto","created_at":"2026-04-30 08:59:39","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":87412,"visible":true,"origin":"","legend":"\u003cp\u003eXPS high-resolution spectra of (a) C 1s, (b) S2p and (c) Au 4f of AuNC@D-Pen\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-9405870/v1/7c64b9a83535e615af5be998.png"},{"id":108155558,"identity":"e716cfa1-a454-42be-9927-1155ed4de745","added_by":"auto","created_at":"2026-04-30 02:36:21","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":74365,"visible":true,"origin":"","legend":"\u003cp\u003e(a) PL spectra of AuNC@D-Pen upon addition of [TC] from 0 to 250 μM. (b) Relationship between the relative PL intensity F/F\u003csub\u003e0\u003c/sub\u003e and the concentration of TC in the range 0.5–250 μM, where F\u003csub\u003e0\u003c/sub\u003e represents the PL intensity in the absence of TC and F represents the PL upon addition of TC.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-9405870/v1/edf64eb0df3f77d24c163fc9.png"},{"id":108183247,"identity":"eea6f237-14e9-4418-b6c5-712b99ee5419","added_by":"auto","created_at":"2026-04-30 09:00:01","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":218726,"visible":true,"origin":"","legend":"\u003cp\u003ePlausible interaction between AuNC@D-Pen and TC.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-9405870/v1/d4a73630955cc9a876d652bf.png"},{"id":108155559,"identity":"9198d18e-26f0-485c-a28c-631c05b4e92d","added_by":"auto","created_at":"2026-04-30 02:36:21","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":624472,"visible":true,"origin":"","legend":"\u003cp\u003eSelectivity of AuNC@D-Pen against other compounds.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-9405870/v1/57b67af42d58576255f01f7d.png"},{"id":108183916,"identity":"460cf634-318d-4495-84ce-83899b4731e0","added_by":"auto","created_at":"2026-04-30 09:03:02","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1273775,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9405870/v1/bc916cc5-64bb-4fbc-8745-ad86ff345b02.pdf"},{"id":108155554,"identity":"2fc2fde6-7e90-4eb4-9854-cfbba26aa9cf","added_by":"auto","created_at":"2026-04-30 02:36:21","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":369540,"visible":true,"origin":"","legend":"","description":"","filename":"SupportingInformation.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9405870/v1/fd7e6062ab11bcb37ca1d418.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"D‑Penicillamine‑Stabilized Gold Nanoclusters as a Selective Fluorescent Sensor for Tetracycline","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIn today\u0026rsquo;s world, antibiotics remain fundamental to global health, from routine medical care to complex surgical procedures and the management of infectious diseases.\u003csup\u003e[1,2]\u003c/sup\u003e Among the different families of antibiotics, tetracyclines (TCs) are a group of broad-spectrum antibiotics widely used in veterinary medicine, particularly in poultry, cattle, and swine farming.\u003csup\u003e[3]\u003c/sup\u003e This family of antibiotics characterized by a rigid tetracyclic fused nucleus, labeled as A, B, C, and D, possesses various functional groups such as hydroxyl, carbonyl, and dimethylamino (Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Since their discovery in 1948, their widespread use has raised growing concerns about antimicrobial resistance and environmental contamination,\u003csup\u003e[4]\u003c/sup\u003e as large proportions of these antibiotics are excreted unmetabolized and enter aquatic systems.\u003csup\u003e[5]\u003c/sup\u003e In this regard, tetracycline pollution is considered a global environmental threat due to its extensive use in aquaculture, livestock, and human medicine.\u003csup\u003e[6]\u003c/sup\u003e Large fractions are excreted unmetabolized (up to 70\u0026ndash;90%), subsequently entering soils and aquatic environments, where they are highly persistent because of their hydrophilic nature and resistance to natural degradation.\u003csup\u003e[7]\u003c/sup\u003e Studies across Europe have detected tetracycline concentrations in surface waters ranging from 0 to 0.02 ppb, but can reach higher concentrations up to 0.54 ppb in municipal wastewater treatment plant effluents.\u003csup\u003e[8]\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe growing concern over antibiotic residues and resistance highlights the need for precise analytical monitoring. In this regard, several analytical methods are used to detect tetracyclines,\u003csup\u003e[9]\u003c/sup\u003e being chromatographic methods, such as HPLC or LC\u0026ndash;MS, the most widely employed because of their high precision and sensitivity; however they require expensive instrumentation, long analysis times, and specialized operation, limiting their routine use.\u003csup\u003e[10,11]\u003c/sup\u003e On the other hand, electrochemical methods, including those modified with nanomaterials, provide fast response, high sensitivity, and low cost, making them suitable for on‑site analysis, but they generally suffer from matrix interferences, and limited long‑term stability.\u003csup\u003e[12,13]\u003c/sup\u003e Electrophoresis methods suffer from limited separation capability and require strict pH control, which hampers their practical applicability.\u003csup\u003e[14,15]\u003c/sup\u003e Immunoassay‑based techniques offer high selectivity through antibody recognition, yet they often present higher detection limits and longer assay times compared with emerging sensor technologies.\u003csup\u003e[16\u0026ndash;18]\u003c/sup\u003e Chemiluminescence techniques typically respond to a broad range of compounds, making them suitable only for high-purity samples like pharmaceutical formulations.\u003csup\u003e[19,20]\u003c/sup\u003e In contrast, fluorescence analysis has gained significant attention in recent years due to its advantages: low cost, ease of operation, high sensitivity and stability, rapid signal response, real-time detection capability, excellent reproducibility, and minimal sample damage.\u003csup\u003e[21]\u003c/sup\u003e These attributes make fluorescence-based methods highly promising for the selective and accurate detection of TCs.\u003c/p\u003e \u003cp\u003eIn recent years, a wide range of fluorescent nanomaterials, such as quantum dots, carbon-based nanomaterials, rare earth-doped nanoparticles, and metallic nanoclusters, have gained significant attention in fluorescence sensing.\u003csup\u003e[22]\u003c/sup\u003e Among them, metallic nanoclusters (NCs) have emerged as particularly promising nanomaterials due to their exceptional properties, including high photostability, strong photoluminescence, large Stokes shifts, low toxicity, high quantum yields, excellent water solubility, and biocompatibility.\u003csup\u003e[23]\u003c/sup\u003e Over the past decade, considerable research has focused on the synthesis of silver (AgNC) and gold nanoclusters (AuNC), which have been widely employed as luminescent probes across various interdisciplinary fields. Among the diverse types of metal NCs, gold, silver, and copper nanoclusters have been extensively studied.\u003csup\u003e[24\u0026ndash;26]\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eAuNC have been stablished as highly effective fluorescent probes for the detection of tetracyclines due to their unique optical properties and strong interactions with antibiotic molecules.\u003csup\u003e[27]\u003c/sup\u003e These ultrasmall clusters exhibit size‑dependent fluorescence that can be selectively quenched or enhanced when tetracyclines interact with the nanomaterial, enabling sensitive, rapid, and label‑free detection. Their excellent biocompatibility, high quantum yield, and tunable surface chemistry make gold nanoclusters particularly suitable for applications in food‑safety monitoring and environmental analysis, where low detection limits and reliable performance in complex matrices are essential.\u003csup\u003e[28]\u003c/sup\u003e Along the last decade, different fluorescent AuNC-based sensors have been reported for the detection of tetracyclines. Among them, AuNCs capped with thiolated ligands, such as glutathione\u003csup\u003e[29]\u003c/sup\u003e and N-acetyl-L-cysteine\u003csup\u003e[30]\u003c/sup\u003e, have shown limits of detection (LOD) of 2.4 and 0.8 ppm, respectively. Lower LODs can be achieved using rare-earth metals such as Eu(III) salts. In this regard, systems involving Eu(III) complexes of L-histidine-caped AuNCs\u003csup\u003e[31]\u003c/sup\u003e or BSA‑stabilized AuNCs\u003csup\u003e[32]\u003c/sup\u003e allowed the detection of TC with a detection limit of 2 ppb in both systems. More complex systems using microfluidic chip with ovalbumin‑stabilized AuNCs in a have achieved the detection of TC in chicken muscle with a LOD of 90 ppb.\u003csup\u003e[33]\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eIn this study, we describe the preparation of D-penicillamine-capped AuNC (\u003cb\u003eAuNC@D-Pen\u003c/b\u003e) via a simple protocol which avoids the use of additional chemicals and complicated synthetic and laborious purification steps. The synthesized \u003cb\u003eAuNC@D-Pen\u003c/b\u003e demonstrated strong sensitivity for detecting tetracycline (TC) at low concentrations. Specifically, the characteristic emission at 652 nm showed a clear reduction in intensity upon increasing concentrations of TC, demonstrating strong sensitivity even at low analyte levels. This quenching behaviour is attributed to intermolecular interactions between TC and the ligand‑protected nanocluster surface. The detection method for TC has the advantages of high sensitivity and selectivity towards other interfering analytes, simple operation, and applicability to environmental water samples.\u003c/p\u003e"},{"header":"Experimental","content":"\u003cp\u003e \u003cb\u003eMaterials\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eAll the chemicals and solvents used in this thesis degree were of analytical grade and used without any additional purification. Gold(III) chloride (HAuCl\u003csub\u003e4\u003c/sub\u003e\u0026middot;3H\u003csub\u003e2\u003c/sub\u003eO), sodium chloride (NaCl), potassium chloride (KCl), magnesium chloride (MgCl\u003csub\u003e2\u003c/sub\u003e), calcium chloride (CaCl\u003csub\u003e2\u003c/sub\u003e), aluminium chloride (AlCl\u003csub\u003e3\u003c/sub\u003e), sodium nitrate (NaNO\u003csub\u003e3\u003c/sub\u003e), sodium carbonate (Na\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e), copper sulphate (CuSO\u003csub\u003e4\u003c/sub\u003e), L-phenylalanine (L-Phe), L-valine (L-Val), L-histidine (L-His), tetracycline and ampicillin were purchased from Sigma-Aldrich. D-penicillamine was purchased from BLD Pharmatech. For all aqueous solutions, high purity deionized water from a Millipore system was used.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEquipment.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe following technical instruments were used for analysis and characterization. Centrifugation was performed on a Beckman Coulter's Microfuge 16 benchtop centrifuge. UV-vis absorption spectra were recorded on a PerkinElmer 1050\u0026thinsp;+\u0026thinsp;UV/vis/NIR spectrometer. All the measurements were performed using 1cm\u0026times;1cm path length quartz cuvettes. Fluorescence spectra were recorded on a FLS1000 photoluminescence spectrometer from Edinburgh Instruments. The quantum yield was measured with a Hamamatsu C9920-02 absolute PL Quantum Yield Measurement System. The pH measurements were carried out by using a Crison GLP 21 pH meter. Transmission electron microscopy (TEM) images were acquired using a HITACHI HT7800 microscope with a filament of LaB6 operating at 100 kV. Fourier transform infrared (FT-IR) spectra were recorded on a Thermo Scientific Nicolet iS10. X-ray photoelectron spectroscopy (XPS) spectra were acquired with VG-Microtech Multilab 3000 equipment. The \u003csup\u003e1\u003c/sup\u003eH spectrum were registered at room temperature in a Bruker AvanceIII 300 spectrometer, with a 300 MHz Bruker magnet. The chemical shifts (δ) are reported in ppm using deuterium oxide, 99.9% atom (D\u003csub\u003e2\u003c/sub\u003eO) as solvent.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePreparation of AuNC@D-Pen.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eA freshly prepared aqueous solution of HAuCl\u003csub\u003e4\u003c/sub\u003e (50 \u0026micro;L, 50 mM) was diluted in water (1 mL), and an aqueous solution of D-penicillamine (53 \u0026micro;L, 1 M) was added. The mixture was set for 5 days at room temperature, obtaining a white precipitated (fluorescent under UV-light, 365 nm) and a colourless solution. The precipitate was isolated by centrifugation at 10000 rev/min for 15 min. The supernatant was slowly removed without disturbing the precipitate, which had been washed two times by dispersion in water and precipitation by centrifugation at 10000 rev/min for 15 min. After purification, the \u003cb\u003eAuNC@D-Pen\u003c/b\u003e were dispersed in water or the solutions were diluted with buffer solution, meanwhile, to avoid the effect of the pH change.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFluorescence detection of tetracycline.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eFluorescence experiments were conducted at room temperature using an excitation wavelength of 310 nm. Tetracycline solutions of varying concentrations were freshly prepared and sequentially added to 3 mL of \u003cb\u003eAuNC@D-Pen\u003c/b\u003e solution (citrate buffer, pH 4.3, with an absorbance of 0.4 at 305 nm). Photoluminescence spectra were recorded at room temperature immediately after each addition of tetracycline. The concentration of TC was plotted on the x-axis, while the corresponding photoluminescence intensity was plotted on the y-axis. Finally, a linear correlation curve was generated to determine the concentration of tetracycline.\u003c/p\u003e \u003cp\u003e \u003cb\u003eSelectivity studies of AuNC@D-Pen.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo evaluate the selectivity of the prepared \u003cb\u003eAuNC@D-Pen\u003c/b\u003e, a series of selective experiments were performed. Initially, 5 \u0026micro;L of a 0.05 M TC solution was added to a solution of \u003cb\u003eAuNC@D-Pen\u003c/b\u003e to establish the baseline photoluminescence (PL) intensity as a control. Subsequently, 5 \u0026micro;L of 0.05 M solutions of different potential interfering analytes, such as common anions and cations (NaCl, KCl, MgCl\u003csub\u003e2\u003c/sub\u003e, AlCl\u003csub\u003e3\u003c/sub\u003e, Na\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e, MnCl\u003csub\u003e2\u003c/sub\u003e, CaCl\u003csub\u003e2\u003c/sub\u003e and MgSO\u003csub\u003e4\u003c/sub\u003e), amino acids (L-Phe, L-Val, and L-His), and ampicillin, were added to the \u003cb\u003eAuNC@D-Pen\u003c/b\u003e solution. Fluorescence intensity values were measured after an incubation time of 5 minutes at room temperature. The fluorescence intensity response of \u003cb\u003eAuNC@D-Pen\u003c/b\u003e to each analyte was measured at an excitation wavelength of 310 nm.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDetection of TC in water samples.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe performed recovery experiments using both tap water and lake water. Tap water without pretreatment was tested for TC. For TC detection in lake water, the sample was initially centrifuged to remove suspended particles. Next, water samples were adjusted at pH around 4.0 by adding HCl solution. Known concentrations of TC (0.8, 1.2, 2.5, and 5.0 \u0026micro;M) were added to each water sample, and the resulting solutions were analysed under the same optimized conditions used for the calibration curve. For each concentration level, three independent measurements were carried out, allowing the calculation of both the recovery percentage and the corresponding relative standard deviation (RSD).\u003c/p\u003e"},{"header":"Results and discussion","content":"\u003cp\u003e \u003cb\u003eSynthesis and characterization of AuNC@D-Pen.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eFor the preparation of \u003cb\u003eAuNC@D-Pen\u003c/b\u003e, we used the bottom-up approach that involves assembling atoms or molecules into nanoclusters with atomic precision.\u003csup\u003e[34]\u003c/sup\u003e The advantage of this method is that it offers excellent control over size, composition, and surface chemistry, making it ideal for producing atomically precise gold nanoclusters, often with sizes around 2 nm. To this purpose, a freshly prepared 1 mL of an aqueous solution of HAuCl\u003csub\u003e4\u003c/sub\u003e (50 \u0026micro;L, 50 mM) and another aqueous solution of D-penicillamine (53 \u0026micro;L, 1 M). Both solutions were mixed in a 1.5 mL Eppendorf tube. After a few days, we monitored the formation of gold nanoclusters by checking their fluorescence with a UV lamp (365 nm). After 5 days, a white precipitate appeared and was isolated by centrifugation, washed with water, and isolated, again, by centrifugation. After purification, the water suspension was stored at room temperature. A schematic representation of the synthesis is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe photoluminescence properties of \u003cb\u003eAuNC@D‑Pen\u003c/b\u003e were investigated by recording their emission spectrum upon excitation at 310 nm. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e (red line), the nanoclusters exhibited a broad emission band with an emission maximum centred at 652 nm. This wide spectral profile is characteristic of ligand‑protected gold nanoclusters, whose electronic transitions arise from discrete energy levels rather than the band‑like structure typical of larger nanoparticles. Importantly, the emission spectrum remained unchanged when the excitation wavelength varied between 300 and 420 nm, demonstrating that the luminescence originates from intrinsic, relaxed excited states of the nanoclusters. The absence of excitation‑dependent shifts indicates that the observed signal is true photoluminescence rather than an artefact from scattering, surface defects, or heterogeneous emissive species.\u003c/p\u003e \u003cp\u003eThe \u003cb\u003eAuNC@D‑Pen\u003c/b\u003e sample displayed a photoluminescence quantum yield (φₚₗ) of 1%. Although modest, this quantum yield reflects efficient relaxation pathways within the metal\u0026ndash;ligand framework and confirms that D‑penicillamine provides a stable surface environment supporting radiative recombination.\u003c/p\u003e \u003cp\u003eThe molar ratio of gold:ligand is a critical parameter in the bottom-up synthesis of gold nanoclusters, as it strongly influences the size, monodispersity, stability, and surface chemistry of the nanoclusters.\u003csup\u003e[35]\u003c/sup\u003e The molar ratio was investigated for D-penicillamine to obtain high quality luminescent gold nanoclusters. Under the described conditions, the concentration of HAuCl\u003csub\u003e4\u003c/sub\u003e was kept constant and different HAuCl\u003csub\u003e4\u003c/sub\u003e/D-Pen molar ratios were assayed: 1:1, 1:2, 1:5, 1:10, 1:20. After recording to the fluorescent spectrum of the nanocluster under different molar ratios, the highest fluorescence intensity gradually decreased with the HAuCl\u003csub\u003e4\u003c/sub\u003e/D-Pen molar ratio with a maximum of intensity for the nanocluster with the 1:20 HAuCl\u003csub\u003e4\u003c/sub\u003e/D-Pen molar ratio (\u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e). Based on these observations, we conclude that the optimal molar ratio for synthesizing strongly luminescent \u003cb\u003eAuNC@D‑Pen\u003c/b\u003e is 1:20, ensuring both efficient surface stabilization and maximized fluorescence.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe influence of reaction time using a HAuCl\u003csub\u003e4\u003c/sub\u003e/D-Pen molar ratios of 1:20. was also investigated. A progressive increase in the fluorescence intensity was observed during the first several days of incubation, indicating ongoing structural formation of the \u003cb\u003eAuNC@D‑Pen\u003c/b\u003e system. The fluorescence reached its maximum after approximately five days (\u003cb\u003eFig. S2\u003c/b\u003e), suggesting that this period is required for the nanoclusters to fully develop their optimal luminescent properties.\u003c/p\u003e \u003cp\u003eThe \u003csup\u003e1\u003c/sup\u003eH NMR spectrum of D‑penicillamine displayed two characteristic peaks at 1.41 and 1.49 ppm, corresponding to the protons of the methyl groups, along with a signal at 3.62 ppm assigned to the chiral methine (CH) proton. Upon coordination to gold and formation of \u003cb\u003eAuNC@D‑Pen\u003c/b\u003e, both methyl peaks experience a slight downfield shift to 1.43 and 1.51 ppm, respectively, while the methine proton shifts to 3.82 ppm (\u003cb\u003eFig. S3\u003c/b\u003e). These changes in chemical shift are indicative of ligand\u0026ndash;metal interactions and support the successful incorporation of D‑penicillamine onto the AuNC surface.\u003c/p\u003e \u003cp\u003eThe morphology of the \u003cb\u003eAuNC@D-Pen\u003c/b\u003e was characterized using transmission electron microscopy (TEM). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, the micrographs of the prepared \u003cb\u003eAuNC@D-Pen\u003c/b\u003e exhibited uniform dispersion and predominantly spherical morphology. Importantly, the particles appear well isolated from one another, with no evidence of large agglomerates or significant clustering. This lack of aggregation indicates that D‑penicillamine provides effective surface stabilization, preventing particle\u0026ndash;particle fusion and maintaining colloidal stability during synthesis and imaging. Detailed quantitative analysis of the TEM images further confirms the presence of spherical gold nanoclusters with an average diameter of 2.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4 nm, a size range characteristic of well‑defined, ligand‑protected Au nanoclusters. This particle size is consistent with previously reported penicillamine-capped AuNCs.\u003csup\u003e[36\u0026ndash;38]\u003c/sup\u003e This narrow size distribution is consistent with controlled nucleation and growth processes typically observed in thiolate‑stabilized gold nanocluster systems, supporting the successful formation of small, monodisperse AuNCs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe functional groups on the surface of \u003cb\u003eAuNC@D-Pen\u003c/b\u003e were characterized by Fourier Transform Infrared (FTIR) spectroscopy. FTIR spectroscopy is a powerful tool for characterizing gold nanoclusters, especially when they are functionalized with thiolated ligands.\u003csup\u003e[39]\u003c/sup\u003e D-penicillamine exhibits the S\u0026ndash;H stretching band in the region of 2550\u0026ndash;2600 cm⁻\u0026sup1;, and upon binding to gold, this band disappears or is significantly reduced, indicating formation of Au\u0026ndash;S bonds. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, the band corresponding to the S-H bond in D-penicillamine disappears when the ligand coordinates gold atoms. The loss of the S\u0026ndash;H band therefore confirms ligand and highlights the high affinity of sulfur for gold, a well‑known feature that explains the stability of thiolate‑protected gold nanoclusters.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eX-ray photoelectron spectroscopy (XPS) measurements were performed to investigate the electronic structure and surface chemical composition of the gold nanoclusters. The XPS analysis provides insights into the oxidation states of gold and the nature of the chemical interactions between gold atoms and stabilizing ligands.\u003csup\u003e[40]\u003c/sup\u003e In particular, analysis of the C 1s core-level spectra showed multiple components (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea), typically associated with different carbon bonding environments such as C\u0026ndash;C/C\u0026ndash;H (~\u0026thinsp;284.8 eV), C\u0026ndash;N/C\u0026ndash;O (~\u0026thinsp;286.2 eV), C\u0026ndash;S (~\u0026thinsp;287.4 eV)and O\u0026ndash;C\u0026thinsp;=\u0026thinsp;O (~\u0026thinsp;288.5 eV), which are indicative of the different carbon environments present in penicillamine ligand used to stabilize the AuNCs.\u003c/p\u003e \u003cp\u003eThe high-resolution S 2p spectra provides information of the bonding environment of sulphur atoms originating from thiol-based ligands. The S 2p region exhibited characteristic doublet peaks, corresponding to the spin-orbit components S 2p₃/₂ and S 2p₁/₂, typically observed around 162\u0026ndash;164 eV (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). Peaks near ~\u0026thinsp;162 eV are indicative of sulphur atoms covalently bonded to gold (Au\u0026ndash;S), confirming thiolate-gold interactions.\u003csup\u003e[41,42]\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eFinally, the Au 4f spectra is generally used to confirm the metallic state of gold within the clusters. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec, the high-resolution Au 4f spectra revealed characteristic doublets corresponding to Au\u003csup\u003e0\u003c/sup\u003e and the oxidized species (e.g., Au⁺), as inferred from the peaks at 84.7 and 88.4 eV for Au\u003csup\u003e0\u003c/sup\u003e, and 85.3 and 89.0 eV for Au\u003csup\u003e+\u003c/sup\u003e.\u003csup\u003e[43\u0026ndash;45]\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe evaluated the stability of \u003cb\u003eAuNC@D-Pen\u003c/b\u003e at different pH values (\u003cb\u003eFig. S4\u003c/b\u003e). The stability of gold nanoclusters is highly dependent on the pH of the surrounding environment, as pH can influence their surface charge, ligand conformation, aggregation behaviour, among other effects.\u003csup\u003e[46]\u003c/sup\u003e For these experiments, were prepared \u003cb\u003eAuNC@D-Pen\u003c/b\u003e solutions with similar value for the absorbance at 310 nm around, and then small amounts of a stock solution of NaOH 0.1 M was added to adjust the pH to the desired value. After shaking and allowing the sample resting at room temperature for 5 minutes, the fluorescence at 650 nm was measured following excitation at 305 nm. The results indicate that the fluorescence intensity of \u003cb\u003eAuNC@D-Pen\u003c/b\u003e at 650 nm was kept constant and near the maximum value in the pH range from 2.1 until 4.9. However, at pH\u0026thinsp;=\u0026thinsp;6.2 the fluorescence decreased dramatically until pH\u0026thinsp;=\u0026thinsp;8.9. This effect may be attributed to the degree of deprotonation of the organic ligand with the pH. This behaviour was irreversible as when reaching pH\u0026thinsp;=\u0026thinsp;8.9, we added acid (HCl 0.5 M) until pH\u0026thinsp;=\u0026thinsp;3.8, and the original value of fluorescence was not recovered.\u003csup\u003e[47]\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e \u003cb\u003eAuNC@D-Pen for fluorimetric detection of tetracycline.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eUnder the optimal experimental conditions described previously, we evaluated the analytical performance and sensitivity of the ratiometric fluorescence sensor for tetracycline (TC) detection. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea, the fluorescence intensity of \u003cb\u003eAuNC@D-Pen\u003c/b\u003e at 652 nm progressively decreased as the concentration of TC increased. Because of this well-defined fluorescence response, the intensity ratio F/F\u003csub\u003e0\u003c/sub\u003e exhibited a clear and proportional dependence on TC concentration. When fitting the data, a strong linear correlation was obtained for concentrations ranging from 0.5 to 220 \u0026micro;M (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). The calibration curve followed the equation: F/F\u003csub\u003e0\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.9682 \u0026minus; 0.0031 [TC]/\u0026micro;M, with an excellent correlation coefficient (R\u0026sup2; = 0.99344), demonstrating high analytical reliability. Based on the 3σ criterion, the limit of detection (LOD) was determined to be 0.9 ppm, highlighting the good sensitivity of the sensing nanomaterial.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe believe that the interaction between TC and \u003cb\u003eAuNC@D-Pen\u003c/b\u003e might involve intermolecular hydrogen bonds between the hydroxyl (\u0026ndash;OH) and carbonyl (C\u0026thinsp;=\u0026thinsp;O) groups located on one of the aromatic rings of the tetracycline scaffold as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e. To support this hypothesis, we performed density functional theory (DFT) calculations using Gaussian 16\u003csup\u003e[48]\u003c/sup\u003e at the B3LYP‑D3/def2‑TZVP level of theory,\u003csup\u003e[49,50]\u003c/sup\u003e employing the SDD pseudopotential for gold.\u003csup\u003e[51]\u003c/sup\u003e To this end, we used a model consisting of 18 gold atoms and 14 D-penicillamine ligands. The optimized structure obtained from the computational study reveals the formation of hydrogen bonds between tetracycline\u0026rsquo;s OH and C\u0026thinsp;=\u0026thinsp;O groups and the functional groups of the D-penicillamine ligand shell. These results support the proposed interaction model and align well with previously reported systems, where hydrogen bonding plays a major role in adsorption, orientation, and stabilization.\u003csup\u003e[30]\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSensor selectivity is a critical parameter, particularly when detecting specific analytes within complex matrices. High selectivity ensures that the sensor responds primarily to the target compound, minimizing interference from other substances that may be present in the sample.\u003csup\u003e[52]\u003c/sup\u003e This is especially important in real-world applications, such as food safety, environmental monitoring, or clinical diagnostics, where samples often contain a multitude of species which can interact increasing or decreasing the signal.\u003c/p\u003e \u003cp\u003eThus, we evaluated the selectivity of the \u003cb\u003eAuNC@D-Pen\u003c/b\u003e sensor towards tetracycline by exposing the nanomaterial to a selection of potentially interfering ions and molecules. For this purpose, we prepared stock solutions containing cations such as Na\u003csup\u003e+\u003c/sup\u003e, K\u003csup\u003e+\u003c/sup\u003e, Mg\u003csup\u003e2+\u003c/sup\u003e, Ca\u003csup\u003e2+\u003c/sup\u003e, Al\u003csup\u003e3+\u003c/sup\u003e, anions such as Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e, NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e, CO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e2\u0026minus;\u003c/sup\u003e; natural amino acids such as phenylalanine (L-Phe), valine (L-Val), or histidine (L-His), and another antibiotic, Ampiciline. The fluorescence intensity response of \u003cb\u003eAuNC@D-Pen\u003c/b\u003e to each interfering analyte was measured at an excitation wavelength of 310 nm. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e, the fluorescence intensity ratio remained almost unchanged (a slight decrease was observed in some cases) in the presence of these interfering species, indicating minimal non-specific interactions. These potential interferents have a negligible impact on the fluorescence signal, which remains largely unaffected in their presence. In contrast, the addition of a solution of TC induced a quenching in the fluorescence, confirming the sensor's strong and specific interaction with tetracycline. These results clearly demonstrate that the \u003cb\u003eAuNC@D-Pen\u003c/b\u003e sensor possesses high selectivity for tetracycline over other coexisting substances, making it a reliable and robust platform for the selective detection of tetracycline in complex sample environments.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eTetracycline detection in real samples.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo evaluate the practical applicability of the proposed method for tetracycline detection in real samples, we conducted assays in tap water and lake water. To this end, different amounts of TCs (0.8, 1.2, 2.5 and 5.0 \u0026micro;M) were added into water samples, analysed using the obtained calibration curve and recovery tests in three repeated measurements were performed and the relative standard deviation (RSD) was obtained. As shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, the recovery values ranged from 96.2% to 104.4%, demonstrating excellent accuracy and minimal matrix interference. The low RSD values further confirm the good repeatability of the measurements. These results indicate that the \u003cb\u003eAuNC@D-Pen\u003c/b\u003e sensor possesses acceptable results for TC determination in a water samples.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eTC determination in real samples.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSpiked (mM)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDetected\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eRSD (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRecovery (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eTap water\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.82\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e6.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e102.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e7.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e103.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e2.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e7.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e97.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e5.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e102.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eLake water\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.77\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e7.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e96.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e8.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e104.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e2.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e8.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e104.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e5.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e104.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn conclusion, the findings presented in this work demonstrate the development of a rapid and moderately sensitive assay for detecting tetracycline, based on the fluorescence quenching of gold nanoclusters stabilized with D-penicillamine. The \u003cb\u003eAuNC@D-Pen\u003c/b\u003e system displayed a fluorescence peak at 652, attributed to the intrinsic emission of D-Pen-stabilized AuNCs. Upon the addition of tetracycline, the fluorescence intensity at 652 was quenched, offering a linear detection range from 0.5 to 220 \u0026micro;M and a low limit of detection (LOD) of 0.9 ppm. The sensor was successfully validated in real water samples, demonstrating its practical applicability. The \u003cb\u003eAuNC@D-Pen\u003c/b\u003e system offers distinct advantages, including straightforward preparation, rapid response time, broad linear range, good sensitivity and selectivity. This work may inspire the development of additional fluorescence-based antibiotic sensors utilizing gold nanoclusters. Based on these findings, ongoing research in our laboratory is focused on engineering other nanostructures that combine AuNCs with thiolated ligands to enhance sensitivity and broaden the detection spectrum.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eAuthor contributions\u003c/p\u003e\n\u003cp\u003eConceptualization: J.E. and E.Z.G. Data curation: L.M.S. and J.E. Formal analysis: L.M.S. and J.E. Funding acquisition: J. P. P. Investigation: L.M.S. and J.E. Methodology: J. E. and E. Z. G. Project administration: J. P. P. Resources: J. P. P. Supervision: J. E. and E. Z. G. Writing \u0026ndash; original draft: J.E. and E.Z.G. Writing \u0026ndash; review \u0026amp; editing: J. E., E. Z. G. and J. P. P. All authors have given approval to the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003eConflicts of interest\u003c/p\u003e\n\u003cp\u003eThere are no conflicts to declare.\u003c/p\u003e\n\u003cp\u003eData availability\u003c/p\u003e\n\u003cp\u003eAll the experimental data are presented in the main text and supplementary information (SI). Supplementary information is available. See DOI:\u003c/p\u003e\n\u003cp\u003eAcknowledgements\u003c/p\u003e\n\u003cp\u003eThe present research was financed by Generalitat Valenciana under Prometeo program (CIPROM/2022/57). This study forms part of the Advanced Materials program and was supported by MCIU with funding from the European Union NextGenerationEU (PRTR-C17.I1) and by Generalitat Valenciana (MFA/2022/051). This work was also supported by the Horizon Europe Marie Curie call for Staff Exchanges 2022 (AMRAMR 101131231). The computational resources from the Servei d\u0026apos;Inform\u0026agrave;tica de la Universitat de Valencia (SIUV) are gratefully acknowledged. The authors are grateful to SCSIE of University of Valencia for providing TEM and HRMS facilities.\u003c/p\u003e"},{"header":"References","content":"\n\u003col\u003e\n \u003cli\u003eMuteeb G, Rehman MT, Shahwan M, Aatif M (2023) Origin of Antibiotics and Antibiotic Resistance, and Their Impacts on Drug Development: A Narrative Review. Pharmaceuticals 16:1615. https://doi.org/10.3390/ph16111615\u003c/li\u003e\n \u003cli\u003eAhmed S, Ning J, Peng D, Chen T, Ahmad I, Ali A, Lei Z, Shabbir MAB, Cheng G, Yuan Z (2020) Current advances in immunoassays for the detection of antibiotics residues: a review. Food Agric Immunol 31:268–290. https://doi.org/10.1080/09540105.2019.1707171\u003c/li\u003e\n \u003cli\u003ePearson JC, Gillett E, Gadri ND, Dionne B (2025) Tetracyclines, the old and the new: A narrative review. 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ACS Sens 1:1282–1285. https://doi.org/10.1021/acssensors.6b00564\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"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":"","lastPublishedDoi":"10.21203/rs.3.rs-9405870/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9405870/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe development of simple, rapid, and cost‑effective sensing methods for monitoring antibiotic residues in water is crucial due to the widespread use of antibiotics in livestock farming and the consequent risk of environmental contamination. In this study, we present a facile synthesis of gold nanoclusters (AuNCs) stabilized with D‑penicillamine (AuNC@D‑Pen), a thiolated ligand that provides excellent colloidal stability and supports strong photoluminescent properties. The synthetic protocol affords highly uniform nanoclusters whose fluorescence emission, centered at approximately 650 nm, is both intense and stable under typical environmental conditions.The fluorescence of AuNC@D‑Pen undergoes progressive quenching in the presence of tetracycline, enabling a sensitive and reliable optical detection strategy. The detection limit (LOD) was determined to be 0.9 ppm, with a linear response range extending from 0.5 to 220 \u0026micro;M, which positions this system as a promising tool for practical analytical applications, including routine water‑quality monitoring. In addition, the sensor exhibits excellent selectivity for tetracycline over common potentially interfering species such as anions, metal ions, and amino acids, highlighting the robustness of the sensing mechanism. Overall, the AuNC‑based probe represents an efficient platform for the rapid, selective, and affordable detection of antibiotic contaminants in aqueous environments.\u003c/p\u003e","manuscriptTitle":"D‑Penicillamine‑Stabilized Gold Nanoclusters as a Selective Fluorescent Sensor for Tetracycline","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-30 02:36:16","doi":"10.21203/rs.3.rs-9405870/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-05-04T16:15:44+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-03T00:33:01+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"290528730890309375609592148191666907533","date":"2026-04-26T13:26:55+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-22T19:51:30+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"224458534633995245783754830544440322423","date":"2026-04-22T12:37:00+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"283546874568744272553943536554489489764","date":"2026-04-22T07:12:14+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"88950225946913203073436171776114517434","date":"2026-04-22T00:36:18+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-21T11:35:01+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-14T00:44:41+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-14T00:43:58+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Fluorescence","date":"2026-04-13T14:58:55+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":"ede3e67f-8bb0-4572-bf55-63349180b6c0","owner":[],"postedDate":"April 30th, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Revision requested","date":"2026-05-04T16:15:44+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-03T00:33:01+00:00","index":20,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-12T12:36:22+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-30 02:36:16","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9405870","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9405870","identity":"rs-9405870","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}