Raspberry-derived carbon dots for specific detection of intracellular copper ions

Raspberry-derived carbon dots for specific detection of intracellular copper ions | 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 Raspberry-derived carbon dots for specific detection of intracellular copper ions Yalan Xu, Yiwei Liu, Liping Feng, Muhua Wang, Yueyi Xia, Luoxing Yang, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5841683/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The detection of intracellular copper ions is of significant importance in biomedical research and disease diagnosis. In this study, blue emissive carbon dots (B-CDs) were successfully synthesized using raspberries as a carbon source through a simple hydrothermal method. Transmission electron microscopy (TEM) analysis revealed that the B-CDs had an average particle size of 9.05 nm. Characterization techniques combined with theoretical calculations, confirmed that the fundamental structural unit of B-CDs is a twelve-membered aromatic ring rich in oxygen and nitrogen functional groups. The B-CDs exhibited high selectivity for Cu²⁺ ions, showing a strong linear response in the concentration range of 0 to 150 µM, with a detection limit of 0.39 µM. Zeta potential and hydrodynamic size measurements indicated that the B-CDs interact with Cu²⁺ ions via electrostatic forces. Further studies revealed that the fluorescence quenching of B-CDs in the presence of Cu²⁺ is primarily due to a dynamic quenching process. Moreover, B-CDs were successfully applied to detect intracellular Cu²⁺. These findings not only show significant potential of B-CDs in fluorescence sensing but also provide valuable insights for the design of efficient carbon-based sensors. Raspberry Blue fluorescence Carbon dots Copper ion detection Cell imaging Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Copper is one of the essential minor metal elements in the human body, playing an indispensable role in various biological activities, including cellular respiration, neurotransmitter synthesis, and antioxidant defense mechanisms[1]. Moderate levels of Cu 2+ maintain the normal metabolism, while imbalances in copper homeostasis may lead to a variety of physiological disorders, such as jaundice, liver necrosis, gastric ulcers, and Wilson's disease [2, 3]. Therefore, the detection of Cu 2+ is of great significance in the diagnosis of diseases. Although many traditional techniques are employed to determining the content of Cu 2+ , such as inductively coupled plasma optical emission spectrometry (ICP-OES), atomic absorption spectroscopy (AAS), membrane filtration and liquid/ion-exchange chromatography[4-6]. However, these analytical techniques generally require expensive instruments, time-consuming operations and complicated sample preparation processes. Especially the poor spatial resolution makes these traditional methods not suitable for the detection of Cu 2+ in living cells. The fluorescent sensors have the advantages of simplicity, low cost, high selectivity and fast response, which are widespread used in many areas. Up to now, a large number of fluorescent sensors have been reported based on organic small molecules to detect Cu 2+ ions[7-9], nevertheless, most of them require masking agents to eliminate interference from other metals, and they may also have high biological toxicity, poor optical stability and water solubility, which is not conducive to biological detection. In recent years, with the development of nanotechnology, carbon dots (CDs), as a class of promising fluorescent nanomaterials, have drawn tremendous attention, owning to their unique properties of good water solubility, resistance to photobleaching, low toxicity, and tunable and stable fluorescence emission[10-12]. Various carbon sources, including aliphatic small molecules, aromatic organic compounds, polymers, and natural biomass, have been explored for the synthesis of CDs[13, 14]. Among them, natural biomass raspberry ( Rubus idaeus L.) has garnered our particular interest due to its unique sensory and nutritional properties[15]. They contain various active compounds, including sugars, vitamin C, terpene, flavonoids, phenols, fatty acids, and alkaloids, etc[16]. These components not only could enhance the optical and chemical properties of CDs during synthesis but also align with green chemistry principles by enabling resource recycling and contributing to sustainable development. As shown in Scheme 1, in this work, blue fluorescent CDs (B-CDs) were synthesized using raspberry as the carbon source through a simple hydrothermal method, and successfully applied in the detection of intracellular Cu 2+ . Through a combination of comprehensive experimental characterization and theoretical simulations, the structural units of raspberry-derived B-CDs will be determined. The interaction mechanism between B-CDs and Cu 2+ was explored using techniques such as zeta potential measurements, fluorescence lifetime analysis, and, ultraviolet-visible absorption spectrum, etc. This study not only provides a novel strategy for the specific detection of intracellular Cu 2+ , but also contributes to the conversion of biomass into valuable carbon nanomaterials, promoting the sustainable recycling of natural resources. 2. Experimental Section 2.1 Materials Raspberries ( Rubus idaeus L.) were brought from Lishui Benrun Agriculture Co., Ltd. Inorganic salts and amino acids were of analytical grade, obtained directly from China Pharmaceutical Group Shanghai Chemical Reagent Co., Ltd. 2.2 Synthesis of the B-CDs Fresh raspberries were subjected to freeze-drying and subsequently ground into powders. A suitable amount of the raspberry powders was dispersed in 10 mL deionized water, and the suspension was then transferred into a 20 mL hydrothermal reactor, which carried out at 180°C for 5 h. After cooling to room temperature, the crude products were filtered through a 0.22 µm microporous membrane to remove any excessively carbonized materials. The filtrate was placed in a 1000 Da dialysis bag to remove unreacted small molecules. Following dialysis, the solution inside the bag was freeze-dried to obtain the B-CDs powders. 2.3 Detection of Cu 2+ with B-CDs 26.28 mg/mL B-CDs stock solution was dispersed in acetic acid and sodium acetate buffer pH (6.0). Subsequently, different concentration of CuCl 2 solution were added into the B-CDs (0.8 mg/mL) aqueous solution, and their fluorescence intensity was measured using a fluorescence spectrometer (FluoroMax-4) with an excitation wavelength of 440 nm. The selective response of B-CDs to Cu 2+ was tested in the coexistence of various metal cation (Na + , K + , Mg 2+ , Ca 2+ , Fe 2+ , Co 2+ Zn 2+ , Mn 2+ , Cu 2+ , Fe 3+ , Al 3+ or Cr 3+ ), anion (Cl ‒ , HCO 3 ‒ , HPO 4 2‒ , or SO 4 2‒ ), and amino acid, including, leucine (Leu), phenylalanine (Phe), methionine (Met), proline (Pro), glycine (Gly), glutamic acid (Glu), valine (Val), arginine (Arg), lysine (Lys), or tyrosine (Tyr). 2.4 Cellular toxicity test The cytotoxicity of B-CDs to HepG2 cells was evaluated using the CCK-8 assay kit. Briefly, dulbecco’s modified eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) was added to a 96-well plate and incubated in a cell culture incubator at 37°C with 5% CO 2 for 12 h. The plate was divided into control and sample groups, in the sample group, 100 µL of the B-CDs solution with concentrations of 2, 10, 20, 30, 40, 50, 60, 80, and 100 µg/mL was added to each well. HepG2 cells in the logarithmic growth phase were counted, and the cell density was adjusted to 2×10 4 cells per well before seeding into the 96-well plate. After the cells were further cultured for 12 h, the medium was removed and washed three times with PBS. Subsequently, 100 µL of culture medium containing 10% CCK-8 was added to each well, and the plate was incubated for 2 h. The absorbance at 450 nm was measured using a microcoder. 2.5 Cell imaging HepG2 cells were cultured with 100 µg/mL B-CDs for 6 hours, followed by the addition of 10 µmol/L Cu²⁺ for a further 30 min incubation. After washing with PBS, cell images were captured using a confocal laser scanning microscope (λex = 405 nm). The fluorescence signals from the B-CDs were recorded within the emission range of 450–500 nm. 3. Results and Discussion 3.1 Physicochemical characterization The morphology of raspberry-derived B-CDs was investigated by transmission electron microscopy (TEM), as presented in Fig. 1 A, B-CDs are uniformly dispersed with an average particle size of 9.05 nm. The X-ray powder diffraction (XRD) pattern in Fig. 1 B display a broad peak around 20°, which confirmed the amorphous carbon (002) crystal planes in B-CDs[ 17 ]. The Fourier transform infrared (FT-IR) spectra were used to identify the surface functional groups of B-CDs, as shown in Fig. 1 C. A strong absorption peak around 3300 cm⁻¹ is attributed to the stretching vibrations of O‒H and N‒H groups[ 18 ]. The sharp peaks at 1670 cm⁻¹ and 1017 cm⁻¹ correspond to C = O and C–O stretching vibrations, respectively[ 19 ]. Additional FT-IR signals at 1531, 1397, 1189, and 765 cm⁻¹ are consistent with the stretching vibrations of C = C, C = N, C–N, and aromatic C-H (Ar-H) bonds[ 20 ]. The surface properties and compositions of B-CDs were further analyzed using X-ray photoelectron spectroscopy (XPS). As depicted in Figure S1 , the XPS survey spectrum of B-CDs revealed three major peaks corresponding to C1s, N1s, and O1s. The C1s spectrum, shown in Fig. 1 D, indicates the presence of three distinct carbon species: C‒C/C = C at 284.0 eV, C‒O/C‒N at 286.2 eV, and C = O at 288.9 eV[ 21 – 23 ]. The high-resolution N1s spectrum (Fig. 1 E) can be resolved into two peaks at 400.2 eV and 401.9 eV, attributed to C‒N‒C and N‒H, respectively[ 24 ]. The O1s spectrum (Fig. 1 F) further confirms the presence of C = O (530.6 eV), C‒O (531.7 eV), and C‒OH (532.5 eV) groups on the B-CD surface[ 25 , 26 ]. H-nuclear magnetic resonance ( 1 H NMR) spectroscopy offers valuable insights into the hydrogen components of B-CDs. As shown in Figure S2, a prominent peak at 12 ppm corresponds to the proton in the − COOH group[ 27 ]. Further analysis in the same figure reveals peaks between 7 and 8 ppm, indicative of aromatic hydrogens (Ar − H)[ 28 ]. Signals within the 4 to 6 ppm range are attributed to the − OH groups in the benzene structure, while the peak at 6.5 ppm is associated with hydrogen on the pyridine ring[ 19 ]. Figure S3 provides additional structural details of B-CDs, showing distinct − O−C = O signals ranging from 160 to 180 ppm, which correspond to carboxylate groups[ 29 ]. Moreover, the aromatic carbon signals in the 100 to 140 ppm range further support the presence of aromatic structures[ 30 ]. These NMR and XPS results align with the FT-IR data, confirming the presence of hydrophilic oxygen and nitrogen functional groups, which are conducive to improve the stability of B-CDs in aqueous solutions. 3.2 Theoretical calculation The structure of B-CDs derived from raspberry remains different to determine due to the inherent complexity of the raw materials. To address this solution, Beijing density functional (BDF) calculations were systematically applied to explore potential structural units of B-CDs[ 31 – 34 ]. As shown in Fig. 2 , energy gaps (Eg) of the simulated structures are mainly depended on the number of conjugated aromatic rings. Specifically, as the number of conjugated aromatic rings increases from 1 to 12, the Eg of the optimized models decreases gradually from 6.79 eV to 1.66 eV. Subsequently, varying numbers of pyridine nitrogen atoms were incorporated into the stimulated structures containing 12 conjugated aromatic rings. Among these configurations, the polycyclic aromatic compound with three pyridine nitrogen atoms exhibited the lowest Eg value, at 1.64 eV. Complementary FT-IR and XPS analyses indicated the presence of functional groups, including amino, hydroxyl, and carboxyl groups, on the surface of the B-CDs. Based on these findings, these functional groups were introduced into the polycyclic aromatic structure and optimized their position and quantity. Finally, the optimized model, labeled “24” showed excellent agreement with the experimentally measured Eg value of B-CDs (1.52 eV, Figure S4). Therefore, the model structure marked “24”, featuring a twelve-membered aromatic ring enriched with oxygen and nitrogen functional groups, is proposed as the structural unit of B-CDs. 3.3 Fluorescence response of B-CDs to Cu 2+ The optical properties of B-CDs were investigated using fluorescence spectroscopy. As the excitation wavelength increased from 360 nm to 460 nm, the fluorescence emissive center of B-CDs progressively redshifted, exhibiting obvious excitation-dependent behavior (Figure S5), which is primarily attributed to different surface states on the B-CDs[ 35 ]. The optimal excitation wavelength for B-CDs was identified as 440 nm, and their maximum fluorescence emission at 506 nm with a quantum yield of 12.4%. As the concentration of Cu 2+ increased, the fluorescence intensity of B-CDs gradually decreased (Fig. 3 A). The fluorescence intensity of B-CDs exhibits a good linear correlation with Cu 2+ concentrations ranging from 0 to 150 µM (Fig. 3 B). Their corresponding detection limit is determined to be 0.39 µM, based on a signal-to-noise ratio of 3. As can be seen in the inset of Fig. 3 A, under daylight illumination, the addition of 150 µM Cu 2+ to the B-CDs solution results in a color change from yellow to reddish-brown. When exposed to UV light, B-CDs emit a bright blue fluorescence, which is noticeably quenched upon the addition of Cu 2+ . The fluorescence stability of B-CDs was investigated under different pH conditions, as shown in Figure S6, the fluorescence intensity of B-CDs was lower in both acidic and basic environments compared to neutral conditions (pH 7.0). This decrease in fluorescence can be attributed to the protonation and deprotonation of surface functional groups, such as hydroxyl, amino, and carboxyl groups. In acidic conditions, these groups become protonated (e.g., –OH 2 + , –NH 3 + ), which enhances intermolecular interactions and leads to fluorescence quenching[ 36 ]. In contrast, in alkaline conditions, deprotonation (e.g., –NH 2 , –COO⁻) alters the surface charge and hydrophilicity of B-CDs, further affecting their fluorescence properties[ 35 ]. In neutral pH, the surface functional groups remain in a more stable charge state, preserving the optimal electronic structure and resulting in the strongest fluorescence emission. Therefore, B-CDs exhibit superior fluorescence stability and intensity under neutral pH, making them particularly suitable for Cu²⁺ ion detection in cells, where the intracellular pH typically remains close to neutral[ 29 ]. To investigate the selectivity of B-CDs for Cu 2+ in the presence of other common metal cations, non-metal anions, and small biological molecules typically found in cells, B-CDs showed strong selectivity for Cu 2+ , as shown in Fig. 2 C. Further analysis revealed that the fluorescence response of B-CDs is closely associated with Cu²⁺ binding, while the presence of other ions caused only minimal changes in fluorescence intensity. This suggests that the selectivity of B-CDs for Cu 2+ is not significantly influenced by the presence of other substances. This selective characteristic enhances B-CDs potential for applications in biological imaging and Cu 2+ sensing. 3.4 Mechanism of B-CDs response to Cu 2+ The fluorescence response mechanism of B-CDs to Cu²⁺ was explored in depth using zeta potential characterization. As shown in Fig. 4 A, B-CDs possess negative surface charges, with an average zeta potential of -9.84 mV. When positively charged 50 µM Cu 2+ ions were introduced, the overall zeta potential of the B-CDs solution decreases significantly to -5.94 mV, showing a marked difference compared to the solution without Cu 2+ . This phenomenon is likely due to the electrostatic interaction between Cu 2+ and the negatively charged surface of B-CDs[ 20 ]. Moreover, the addition of Cu 2+ also caused an increase in the hydrated particle size of B-CDs (Fig. 4 B). Cu 2+ facilitates the aggregation of B-CDs via electrostatic attraction, which leads to aggregation-induced fluorescence quenching[ 37 ]. Based on these observations, it can be inferred that the introduction of Cu 2+ not only alters the surface charge distribution of B-CDs through electrostatic adsorption but also promotes their aggregation, thereby suppressing their fluorescence emission. Dynamic quenching and static quenching are the two primary mechanisms of fluorescence quenching[ 38 , 39 ]. Dynamic quenching occurs when the quencher interacts with the excited-state molecules of the fluorophore, transferring or dissipating the excitation energy through collisions, reducing the fluorescence lifetime and intensity[ 40 ]. This process is dependent on the collision frequency between molecules and is reversible. In contrast, static quenching involves the formation of a stable complex between the quencher and the ground-state molecules of the fluorophore, preventing these molecules from effectively participating in excitation and emission, which leads to a decrease in fluorescence intensity[ 41 ]. Static quenching is irreversible and independent of the excited-state lifetime. Thus, time-resolved photoluminescence measurements were conducted on B-CDs to investigate the deactivation dynamics of excited-state electrons. The photoluminescence decay curves of B-CDs were fitted using a dual-exponential formula, and the average fluorescence lifetime is 4.50 ns. With the addition of Cu 2+ (50 µM), lifetime of B-CDs decreased slightly to 4.37 ns (Fig. 4 C). The almost constant fluorescence lifetime indicated that Cu 2+ primarily ascribed to static quenching in B-CDs. Further evidence for this mechanism is provided by UV-Vis absorption spectroscopy, which shows a significant increase in the absorbance of B-CDs following the addition of Cu 2+ (Fig. 4 D), suggested that Cu²⁺ form a complex with the ground-state B-CDs, reducing the number of available photons for excitation and suppressing fluorescence emission. Consequently, Cu 2+ quenching primarily results from static quenching through ground-state complexation, leading to a decrease in fluorescence intensity. 3.5 Cytotoxicity and Cellular Imaging To evaluate the potential of B-CDs for living cell imaging, their cytotoxicity was first assessed using the CCK-8 assay in HepG2 cells. As shown in Figure S7, after 24 hours of incubation with B-CDs at concentrations ranging from 5 to 100 µg/mL, nearly 100% of the cells remained viable, indicating that B-CDs exhibit negligible or very low cytotoxicity. Fluorescence imaging was then performed to observe the cellular uptake of B-CDs. HepG2 cells treated with 0.1 mg/mL B-CDs displayed strong blue fluorescence under 405 nm excitation (Fig. 5 ). The small size and favorable surface functional groups of the B-CDs, which facilitate efficient cellular uptake. However, when the cells were co-incubated with 10 µM Cu²⁺, the fluorescence intensity in the blue channel was significantly weakened, demonstrating that B-CDs can successfully detect Cu 2+ in living cells through a fluorescence turn-off mode. 4. Conclusions In summary, B-CDs were successfully synthesized using raspberries as a carbon source through a simple hydrothermal method. The resulting B-CDs demonstrated excellent water solubility, good biocompatibility, and outstanding photoluminescence properties. Comprehensive characterization and theoretical calculations revealed that the fundamental building block of the B-CDs is a twelve-membered aromatic ring structure rich in oxygen and nitrogen functional groups. The obtained B-CDs exhibited high selectivity toward Cu 2+ ions, with a strong linear response over the concentration range of 0 to 150 µM and a detection limit of 0.39 µM. Zeta potential and hydrodynamic size measurements indicated that the B-CDs interact with Cu²⁺ through electrostatic forces. Furthermore, UV-visible spectra and fluorescence lifetime studies suggested that the fluorescence quenching of B-CDs in the presence of Cu²⁺ primarily results from a dynamic quenching process. The excellent fluorescence properties of B-CDs enabled their successful application in the detection of intracellular Cu²⁺. These findings not only underscore the significant potential of B-CDs in fluorescence sensing but also provide valuable insights for designing efficient carbon-dot-based sensors. In the future, B-CDs are expected to have broad applications in environmental monitoring, intracellular ion detection, and disease diagnostics, positioning them as a promising platform for fluorescence-based sensing. Declarations Acknowledgments This research is supported by the National Natural Science Foundation of China (NO. 22076072, and NO 22306082). 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Yang (2021) Green synthesis of biomass-derived carbon quantum dots as fluorescent probe for Fe 3+ detection, Inorg. Chem. Commun. 130: 108636. A. Iqbal, Y. Tian, X. Wang, D. Gong, Y. Guo, K. Iqbal, Z. Wang, W. Liu, W. Qin (2016) Carbon dots prepared by solid state method via citric acid and 1,10-phenanthroline for selective and sensing detection of Fe 2+ and Fe 3+ , Sens. Actuators, B 237:408-415. D. Gu, L. Hong, L. Zhang, H. Liu, S. Shang (2018) Nitrogen and sulfur co-doped highly luminescent carbon dots for sensitive detection of Cd (II) ions and living cell imaging applications, J. Photochem. Photobiol. B, 186:144-151. Scheme 1 Scheme 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files floatimage1.png Scheme1. 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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-5841683","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":405617045,"identity":"dabb7259-a733-4da0-b0c3-e4b3e2edb81e","order_by":0,"name":"Yalan Xu","email":"","orcid":"","institution":"Lishui University","correspondingAuthor":false,"prefix":"","firstName":"Yalan","middleName":"","lastName":"Xu","suffix":""},{"id":405617046,"identity":"7d33df5f-9bf5-406a-842b-030c70708624","order_by":1,"name":"Yiwei Liu","email":"","orcid":"","institution":"Lishui University","correspondingAuthor":false,"prefix":"","firstName":"Yiwei","middleName":"","lastName":"Liu","suffix":""},{"id":405617049,"identity":"8d26220e-6e1c-4360-8ac8-b62d920d026d","order_by":2,"name":"Liping Feng","email":"","orcid":"","institution":"Lishui University","correspondingAuthor":false,"prefix":"","firstName":"Liping","middleName":"","lastName":"Feng","suffix":""},{"id":405617052,"identity":"f6ad16b8-15da-4894-9dc2-a2c68c1f3a06","order_by":3,"name":"Muhua Wang","email":"","orcid":"","institution":"Lishui University","correspondingAuthor":false,"prefix":"","firstName":"Muhua","middleName":"","lastName":"Wang","suffix":""},{"id":405617055,"identity":"fc556d50-5fc9-40fa-ae7d-0f7c87a80f62","order_by":4,"name":"Yueyi Xia","email":"","orcid":"","institution":"Lishui University","correspondingAuthor":false,"prefix":"","firstName":"Yueyi","middleName":"","lastName":"Xia","suffix":""},{"id":405617056,"identity":"9da57181-0771-433e-8a1c-e1278b79d544","order_by":5,"name":"Luoxing Yang","email":"","orcid":"","institution":"Lishui University","correspondingAuthor":false,"prefix":"","firstName":"Luoxing","middleName":"","lastName":"Yang","suffix":""},{"id":405617057,"identity":"b7be92a3-4d2b-4abf-b84e-3519155b8430","order_by":6,"name":"Junjie Yuwang","email":"","orcid":"","institution":"Lishui University","correspondingAuthor":false,"prefix":"","firstName":"Junjie","middleName":"","lastName":"Yuwang","suffix":""},{"id":405617058,"identity":"7c5c4802-1c98-4201-8a3d-cdf1d10bc25e","order_by":7,"name":"Xiaoli Sun","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1ElEQVRIiWNgGAWjYFAC5sYHCQb/5PjAHDaitDA2G3woOGDMRoqWNskZHw4kthGtRX5GYoM0j8Gd9Db+MwYMH8oOM/DPbsCvxeDMwQZjHoNnuW0SOQaMM84dZpC4c4CAFvbGhmQeA2agFiDJ23aYwUAigYDDmhkbDgMVp7MBHcb8lxgtDMcbGxtnGBxOYGPIMWBmJEYL0C/NDB8M0gzbJNIKDvacS+eRuEHIYTOSj/9I+GMjz89/eOODH2XWcvwzCDkMGRwAYh4S1I+CUTAKRsEowAUALchBm42OoCMAAAAASUVORK5CYII=","orcid":"","institution":"Lishui University","correspondingAuthor":true,"prefix":"","firstName":"Xiaoli","middleName":"","lastName":"Sun","suffix":""}],"badges":[],"createdAt":"2025-01-16 12:08:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5841683/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5841683/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":74645926,"identity":"d2cf058d-bdb8-4e3f-8c08-86b776271a53","added_by":"auto","created_at":"2025-01-24 09:54:13","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1388022,"visible":true,"origin":"","legend":"\u003cp\u003e(A) TEM image of B-CDs, inset: the size distribution histograms of B-CDs. The scale is 50 nm. (B) The XRD spectra of B-CDs. (C) The FT-IR spectrum of B-CDs.\u003c/p\u003e\n\u003cp\u003e(D-F) High-resolution C1s, N1s and O1s XPS spectra for B-CDs.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-5841683/v1/510f22f407931b71b584f6ee.png"},{"id":74645935,"identity":"95b03048-5f79-4ef1-ba97-9b059ca23ccf","added_by":"auto","created_at":"2025-01-24 09:54:14","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2141927,"visible":true,"origin":"","legend":"\u003cp\u003eThe optimized basic structures range from one to twelve conjugated rings based on Beijing density function (BDF) calculations\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-5841683/v1/0ad625861f6dd54e9b7c2296.png"},{"id":74645929,"identity":"712070ba-f9aa-4a96-b8db-fec08d26586c","added_by":"auto","created_at":"2025-01-24 09:54:14","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":664681,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Fluorescence emission spectra for 0.80 mg/mL B-CDs in the presence of various Cu\u003csup\u003e2+\u003c/sup\u003e concentration (0–150 μM), (inset: photographs of B-CDs with and without Cu\u003csup\u003e2+\u003c/sup\u003e under daylight and 365 nm UV light). (B) The variation in fluorescence intensity of the B-CDs solution as a function of Cu\u003csup\u003e2+\u003c/sup\u003e concentrations, ranging from 0 to 150 μM. (C) The influence of interfering substances on the fluorescence intensity of 0.80 mg/mL B-CDs. The test substances include 50 μM concentrations of metal cations, nonmetallic anions or amino acids.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-5841683/v1/22b45a1e0b10c099fa633453.png"},{"id":74645933,"identity":"72d09ac9-1426-4a35-a67d-b67d03df031c","added_by":"auto","created_at":"2025-01-24 09:54:14","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":540170,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in the (A) Zeta potential, (B) hydrodynamic size, (C) fluorescence lifetime, and (D) UV-Vis absorption spectra of B-CDs in the presence and absence of Cu\u003csup\u003e2+\u003c/sup\u003e. The symbols “*” (P \u0026lt; 0.05), and “**” (P \u0026lt; 0.01), indicate significant differences from B-CDs, respectively.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-5841683/v1/f79a71bd5fe3675f3bef538b.png"},{"id":74645932,"identity":"554b13c1-872e-4532-a608-ea36adea9382","added_by":"auto","created_at":"2025-01-24 09:54:14","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2135694,"visible":true,"origin":"","legend":"\u003cp\u003eConfocal fluorescence images of HepG2 cells. The scale bar is 50 μm, the concentration of B-CDs is 0.10 mg/mL.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-5841683/v1/066d0ce6b450c72ea63dfa6a.png"},{"id":74670047,"identity":"9262215f-c812-4015-a25a-e10400b3be8d","added_by":"auto","created_at":"2025-01-24 14:02:21","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7420047,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5841683/v1/15f67f99-c1e3-4a04-8734-0843fcc16455.pdf"},{"id":74645922,"identity":"e3f47155-d2fb-42e4-ab5b-0f07d2e876ba","added_by":"auto","created_at":"2025-01-24 09:54:13","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":338337,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScheme1.\u003c/strong\u003e The preparation of B-CDs and response to intracellular Cu\u003csup\u003e2+\u003c/sup\u003e\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-5841683/v1/83009a66ba947c2df36b0e97.png"},{"id":74645931,"identity":"20e71a4a-16f3-4a10-a68b-f9cf6fce6b76","added_by":"auto","created_at":"2025-01-24 09:54:14","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":5978884,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Supportinginformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-5841683/v1/249a8c6ad0e357488e0edcec.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Raspberry-derived carbon dots for specific detection of intracellular copper ions","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eCopper is one of the essential minor metal elements in the human body, playing an indispensable role in various biological activities, including cellular respiration, neurotransmitter synthesis, and antioxidant defense mechanisms[1]. Moderate levels of Cu\u003csup\u003e2+\u003c/sup\u003e maintain the normal metabolism, while imbalances in copper homeostasis may lead to a variety of physiological disorders, such as jaundice, liver necrosis, gastric ulcers, and Wilson\u0026apos;s disease\u0026nbsp;[2, 3].\u0026nbsp;Therefore, the detection of\u0026nbsp;Cu\u003csup\u003e2+\u003c/sup\u003e is of great significance in the diagnosis of diseases.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAlthough many traditional\u0026nbsp;techniques\u0026nbsp;are employed to determining the content of\u0026nbsp;Cu\u003csup\u003e2+\u003c/sup\u003e, such as\u0026nbsp;inductively coupled plasma optical emission spectrometry (ICP-OES), atomic absorption spectroscopy (AAS),\u0026nbsp;membrane filtration and liquid/ion-exchange chromatography[4-6]. However, these analytical techniques generally require expensive instruments, time-consuming operations and complicated sample preparation processes. Especially the poor spatial resolution makes these traditional methods not suitable for the detection of Cu\u003csup\u003e2+\u003c/sup\u003e in living cells. The fluorescent sensors have the advantages of simplicity, low cost, high selectivity and fast response, which are widespread used in many areas. Up to now, a large number of fluorescent sensors have been reported based on organic small molecules to detect Cu\u003csup\u003e2+\u003c/sup\u003e ions[7-9], nevertheless, most of them require masking agents to eliminate interference from other metals, and they may also have high biological toxicity, poor optical stability and water solubility, which is not conducive to biological detection.\u003c/p\u003e\n\u003cp\u003eIn recent years, with the development of nanotechnology, carbon dots (CDs), as a class of promising fluorescent nanomaterials, have drawn tremendous attention, owning to their unique properties of good water solubility, resistance to photobleaching, low toxicity, and tunable and stable fluorescence emission[10-12]. Various carbon sources, including aliphatic small molecules, aromatic organic compounds, polymers, and natural biomass, have been explored for the synthesis of CDs[13, 14]. Among them, natural biomass raspberry (\u003cem\u003eRubus idaeus\u003c/em\u003e L.) has garnered our particular interest due to its unique sensory and nutritional properties[15]. They contain various active compounds, including sugars, vitamin C, terpene, flavonoids, phenols, fatty acids, and alkaloids, etc[16]. These components not only could enhance the optical and chemical properties of CDs during synthesis but also align with green chemistry principles by enabling resource recycling and contributing to sustainable development.\u003c/p\u003e\n\u003cp\u003eAs shown in Scheme 1, in this work, blue fluorescent CDs (B-CDs) were synthesized using raspberry as the carbon source through a simple hydrothermal method, and successfully applied in the detection of intracellular Cu\u003csup\u003e2+\u003c/sup\u003e. Through a combination of comprehensive experimental characterization and theoretical simulations, the structural units of raspberry-derived B-CDs will be determined. The interaction mechanism between B-CDs and Cu\u003csup\u003e2+\u003c/sup\u003e was explored using techniques such as zeta potential measurements, fluorescence lifetime analysis, and, ultraviolet-visible absorption spectrum, etc. This study not only provides a novel strategy for the specific detection of intracellular Cu\u003csup\u003e2+\u003c/sup\u003e, but also contributes to the conversion of biomass into valuable carbon nanomaterials, promoting the sustainable recycling of natural resources.\u003c/p\u003e"},{"header":"2. Experimental Section","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Materials\u003c/h2\u003e \u003cp\u003eRaspberries (\u003cem\u003eRubus idaeus\u003c/em\u003e L.) were brought from Lishui Benrun Agriculture Co., Ltd. Inorganic salts and amino acids were of analytical grade, obtained directly from China Pharmaceutical Group Shanghai Chemical Reagent Co., Ltd.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Synthesis of the B-CDs\u003c/h2\u003e \u003cp\u003eFresh raspberries were subjected to freeze-drying and subsequently ground into powders. A suitable amount of the raspberry powders was dispersed in 10 mL deionized water, and the suspension was then transferred into a 20 mL hydrothermal reactor, which carried out at 180\u0026deg;C for 5 h. After cooling to room temperature, the crude products were filtered through a 0.22 \u0026micro;m microporous membrane to remove any excessively carbonized materials. The filtrate was placed in a 1000 Da dialysis bag to remove unreacted small molecules. Following dialysis, the solution inside the bag was freeze-dried to obtain the B-CDs powders.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Detection of Cu\u003csup\u003e2+\u003c/sup\u003e with B-CDs\u003c/h2\u003e \u003cp\u003e26.28 mg/mL B-CDs stock solution was dispersed in acetic acid and sodium acetate buffer pH (6.0). Subsequently, different concentration of CuCl\u003csub\u003e2\u003c/sub\u003e solution were added into the B-CDs (0.8 mg/mL) aqueous solution, and their fluorescence intensity was measured using a fluorescence spectrometer (FluoroMax-4) with an excitation wavelength of 440 nm. The selective response of B-CDs to Cu\u003csup\u003e2+\u003c/sup\u003e was tested in the coexistence of various metal cation (Na\u003csup\u003e+\u003c/sup\u003e, K\u003csup\u003e+\u003c/sup\u003e, Mg\u003csup\u003e2+\u003c/sup\u003e, Ca\u003csup\u003e2+\u003c/sup\u003e, Fe\u003csup\u003e2+\u003c/sup\u003e, Co\u003csup\u003e2+\u003c/sup\u003e Zn\u003csup\u003e2+\u003c/sup\u003e, Mn\u003csup\u003e2+\u003c/sup\u003e, Cu\u003csup\u003e2+\u003c/sup\u003e, Fe\u003csup\u003e3+\u003c/sup\u003e, Al\u003csup\u003e3+\u003c/sup\u003e or Cr\u003csup\u003e3+\u003c/sup\u003e), anion (Cl\u003csup\u003e‒\u003c/sup\u003e, HCO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e‒\u003c/sup\u003e, HPO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e2‒\u003c/sup\u003e, or SO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e2‒\u003c/sup\u003e), and amino acid, including, leucine (Leu), phenylalanine (Phe), methionine (Met), proline (Pro), glycine (Gly), glutamic acid (Glu), valine (Val), arginine (Arg), lysine (Lys), or tyrosine (Tyr).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Cellular toxicity test\u003c/h2\u003e \u003cp\u003eThe cytotoxicity of B-CDs to HepG2 cells was evaluated using the CCK-8 assay kit. Briefly, dulbecco\u0026rsquo;s modified eagle\u0026rsquo;s medium (DMEM) containing 10% fetal bovine serum (FBS) was added to a 96-well plate and incubated in a cell culture incubator at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e for 12 h. The plate was divided into control and sample groups, in the sample group, 100 \u0026micro;L of the B-CDs solution with concentrations of 2, 10, 20, 30, 40, 50, 60, 80, and 100 \u0026micro;g/mL was added to each well. HepG2 cells in the logarithmic growth phase were counted, and the cell density was adjusted to 2\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells per well before seeding into the 96-well plate. After the cells were further cultured for 12 h, the medium was removed and washed three times with PBS. Subsequently, 100 \u0026micro;L of culture medium containing 10% CCK-8 was added to each well, and the plate was incubated for 2 h. The absorbance at 450 nm was measured using a microcoder.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Cell imaging\u003c/h2\u003e \u003cp\u003eHepG2 cells were cultured with 100 \u0026micro;g/mL B-CDs for 6 hours, followed by the addition of 10 \u0026micro;mol/L Cu\u0026sup2;⁺ for a further 30 min incubation. After washing with PBS, cell images were captured using a confocal laser scanning microscope (λex\u0026thinsp;=\u0026thinsp;405 nm). The fluorescence signals from the B-CDs were recorded within the emission range of 450\u0026ndash;500 nm.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and Discussion","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n \u003ch2\u003e3.1 Physicochemical characterization\u003c/h2\u003e\n \u003cp\u003eThe morphology of raspberry-derived B-CDs was investigated by transmission electron microscopy (TEM), as presented in Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA, B-CDs are uniformly dispersed with an average particle size of 9.05 nm. The X-ray powder diffraction (XRD) pattern in Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB display a broad peak around 20\u0026deg;, which confirmed the amorphous carbon (002) crystal planes in B-CDs[\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e]. The Fourier transform infrared (FT-IR) spectra were used to identify the surface functional groups of B-CDs, as shown in Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC. A strong absorption peak around 3300 cm⁻\u0026sup1; is attributed to the stretching vibrations of O‒H and N‒H groups[\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e]. The sharp peaks at 1670 cm⁻\u0026sup1; and 1017 cm⁻\u0026sup1; correspond to C\u0026thinsp;=\u0026thinsp;O and C\u0026ndash;O stretching vibrations, respectively[\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e]. Additional FT-IR signals at 1531, 1397, 1189, and 765 cm⁻\u0026sup1; are consistent with the stretching vibrations of C\u0026thinsp;=\u0026thinsp;C, C\u0026thinsp;=\u0026thinsp;N, C\u0026ndash;N, and aromatic C-H (Ar-H) bonds[\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e]. The surface properties and compositions of B-CDs were further analyzed using X-ray photoelectron spectroscopy (XPS). As depicted in Figure \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e, the XPS survey spectrum of B-CDs revealed three major peaks corresponding to C1s, N1s, and O1s. The C1s spectrum, shown in Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eD, indicates the presence of three distinct carbon species: C‒C/C\u0026thinsp;=\u0026thinsp;C at 284.0 eV, C‒O/C‒N at 286.2 eV, and C\u0026thinsp;=\u0026thinsp;O at 288.9 eV[\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e]. The high-resolution N1s spectrum (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eE) can be resolved into two peaks at 400.2 eV and 401.9 eV, attributed to C‒N‒C and N‒H, respectively[\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e]. The O1s spectrum (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eF) further confirms the presence of C\u0026thinsp;=\u0026thinsp;O (530.6 eV), C‒O (531.7 eV), and C‒OH (532.5 eV) groups on the B-CD surface[\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e\n \u003cp\u003eH-nuclear magnetic resonance (\u003csup\u003e1\u003c/sup\u003eH NMR) spectroscopy offers valuable insights into the hydrogen components of B-CDs. As shown in Figure S2, a prominent peak at 12 ppm corresponds to the proton in the \u0026minus;\u0026thinsp;COOH group[\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e]. Further analysis in the same figure reveals peaks between 7 and 8 ppm, indicative of aromatic hydrogens (Ar\u0026thinsp;\u0026minus;\u0026thinsp;H)[\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e]. Signals within the 4 to 6 ppm range are attributed to the \u0026minus;\u0026thinsp;OH groups in the benzene structure, while the peak at 6.5 ppm is associated with hydrogen on the pyridine ring[\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e]. Figure S3 provides additional structural details of B-CDs, showing distinct\u0026thinsp;\u0026minus;\u0026thinsp;O\u0026minus;C\u0026thinsp;=\u0026thinsp;O signals ranging from 160 to 180 ppm, which correspond to carboxylate groups[\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e]. Moreover, the aromatic carbon signals in the 100 to 140 ppm range further support the presence of aromatic structures[\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e]. These NMR and XPS results align with the FT-IR data, confirming the presence of hydrophilic oxygen and nitrogen functional groups, which are conducive to improve the stability of B-CDs in aqueous solutions.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003ch2\u003e3.2 Theoretical calculation\u003c/h2\u003e\n \u003cp\u003eThe structure of B-CDs derived from raspberry remains different to determine due to the inherent complexity of the raw materials. To address this solution, Beijing density functional (BDF) calculations were systematically applied to explore potential structural units of B-CDs[\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e]. As shown in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e, energy gaps (Eg) of the simulated structures are mainly depended on the number of conjugated aromatic rings. Specifically, as the number of conjugated aromatic rings increases from 1 to 12, the Eg of the optimized models decreases gradually from 6.79 eV to 1.66 eV. Subsequently, varying numbers of pyridine nitrogen atoms were incorporated into the stimulated structures containing 12 conjugated aromatic rings. Among these configurations, the polycyclic aromatic compound with three pyridine nitrogen atoms exhibited the lowest Eg value, at 1.64 eV. Complementary FT-IR and XPS analyses indicated the presence of functional groups, including amino, hydroxyl, and carboxyl groups, on the surface of the B-CDs. Based on these findings, these functional groups were introduced into the polycyclic aromatic structure and optimized their position and quantity. Finally, the optimized model, labeled \u0026ldquo;24\u0026rdquo; showed excellent agreement with the experimentally measured Eg value of B-CDs (1.52 eV, Figure S4). Therefore, the model structure marked \u0026ldquo;24\u0026rdquo;, featuring a twelve-membered aromatic ring enriched with oxygen and nitrogen functional groups, is proposed as the structural unit of B-CDs.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003e3.3 Fluorescence response of B-CDs to Cu\u003csup\u003e2+\u003c/sup\u003e\u003c/h2\u003e\n \u003cp\u003eThe optical properties of B-CDs were investigated using fluorescence spectroscopy. As the excitation wavelength increased from 360 nm to 460 nm, the fluorescence emissive center of B-CDs progressively redshifted, exhibiting obvious excitation-dependent behavior (Figure S5), which is primarily attributed to different surface states on the B-CDs[\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e]. The optimal excitation wavelength for B-CDs was identified as 440 nm, and their maximum fluorescence emission at 506 nm with a quantum yield of 12.4%. As the concentration of Cu\u003csup\u003e2+\u003c/sup\u003e increased, the fluorescence intensity of B-CDs gradually decreased (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA). The fluorescence intensity of B-CDs exhibits a good linear correlation with Cu\u003csup\u003e2+\u003c/sup\u003e concentrations ranging from 0 to 150 \u0026micro;M (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB). Their corresponding detection limit is determined to be 0.39 \u0026micro;M, based on a signal-to-noise ratio of 3. As can be seen in the inset of Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA, under daylight illumination, the addition of 150 \u0026micro;M Cu\u003csup\u003e2+\u003c/sup\u003e to the B-CDs solution results in a color change from yellow to reddish-brown. When exposed to UV light, B-CDs emit a bright blue fluorescence, which is noticeably quenched upon the addition of Cu\u003csup\u003e2+\u003c/sup\u003e. The fluorescence stability of B-CDs was investigated under different pH conditions, as shown in Figure S6, the fluorescence intensity of B-CDs was lower in both acidic and basic environments compared to neutral conditions (pH 7.0). This decrease in fluorescence can be attributed to the protonation and deprotonation of surface functional groups, such as hydroxyl, amino, and carboxyl groups. In acidic conditions, these groups become protonated (e.g., \u0026ndash;OH\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e, \u0026ndash;NH\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e), which enhances intermolecular interactions and leads to fluorescence quenching[\u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e]. In contrast, in alkaline conditions, deprotonation (e.g., \u0026ndash;NH\u003csub\u003e2\u003c/sub\u003e, \u0026ndash;COO⁻) alters the surface charge and hydrophilicity of B-CDs, further affecting their fluorescence properties[\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e]. In neutral pH, the surface functional groups remain in a more stable charge state, preserving the optimal electronic structure and resulting in the strongest fluorescence emission. Therefore, B-CDs exhibit superior fluorescence stability and intensity under neutral pH, making them particularly suitable for Cu\u0026sup2;⁺ ion detection in cells, where the intracellular pH typically remains close to neutral[\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e]. To investigate the selectivity of B-CDs for Cu\u003csup\u003e2+\u003c/sup\u003e in the presence of other common metal cations, non-metal anions, and small biological molecules typically found in cells, B-CDs showed strong selectivity for Cu\u003csup\u003e2+\u003c/sup\u003e, as shown in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC. Further analysis revealed that the fluorescence response of B-CDs is closely associated with Cu\u0026sup2;⁺ binding, while the presence of other ions caused only minimal changes in fluorescence intensity. This suggests that the selectivity of B-CDs for Cu\u003csup\u003e2+\u003c/sup\u003e is not significantly influenced by the presence of other substances. This selective characteristic enhances B-CDs potential for applications in biological imaging and Cu\u003csup\u003e2+\u003c/sup\u003e sensing.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003e3.4 Mechanism of B-CDs response to Cu\u003csup\u003e2+\u003c/sup\u003e\u003c/h2\u003e\n \u003cp\u003eThe fluorescence response mechanism of B-CDs to Cu\u0026sup2;⁺ was explored in depth using zeta potential characterization. As shown in Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA, B-CDs possess negative surface charges, with an average zeta potential of -9.84 mV. When positively charged 50 \u0026micro;M Cu\u003csup\u003e2+\u003c/sup\u003e ions were introduced, the overall zeta potential of the B-CDs solution decreases significantly to -5.94 mV, showing a marked difference compared to the solution without Cu\u003csup\u003e2+\u003c/sup\u003e. This phenomenon is likely due to the electrostatic interaction between Cu\u003csup\u003e2+\u003c/sup\u003e and the negatively charged surface of B-CDs[\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e]. Moreover, the addition of Cu\u003csup\u003e2+\u003c/sup\u003e also caused an increase in the hydrated particle size of B-CDs (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB). Cu\u003csup\u003e2+\u003c/sup\u003e facilitates the aggregation of B-CDs via electrostatic attraction, which leads to aggregation-induced fluorescence quenching[\u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e]. Based on these observations, it can be inferred that the introduction of Cu\u003csup\u003e2+\u003c/sup\u003e not only alters the surface charge distribution of B-CDs through electrostatic adsorption but also promotes their aggregation, thereby suppressing their fluorescence emission.\u003c/p\u003e\n \u003cp\u003eDynamic quenching and static quenching are the two primary mechanisms of fluorescence quenching[\u003cspan class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e39\u003c/span\u003e]. Dynamic quenching occurs when the quencher interacts with the excited-state molecules of the fluorophore, transferring or dissipating the excitation energy through collisions, reducing the fluorescence lifetime and intensity[\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e]. This process is dependent on the collision frequency between molecules and is reversible. In contrast, static quenching involves the formation of a stable complex between the quencher and the ground-state molecules of the fluorophore, preventing these molecules from effectively participating in excitation and emission, which leads to a decrease in fluorescence intensity[\u003cspan class=\"CitationRef\"\u003e41\u003c/span\u003e]. Static quenching is irreversible and independent of the excited-state lifetime. Thus, time-resolved photoluminescence measurements were conducted on B-CDs to investigate the deactivation dynamics of excited-state electrons. The photoluminescence decay curves of B-CDs were fitted using a dual-exponential formula, and the average fluorescence lifetime is 4.50 ns. With the addition of Cu\u003csup\u003e2+\u003c/sup\u003e (50 \u0026micro;M), lifetime of B-CDs decreased slightly to 4.37 ns (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC). The almost constant fluorescence lifetime indicated that Cu\u003csup\u003e2+\u003c/sup\u003e primarily ascribed to static quenching in B-CDs. Further evidence for this mechanism is provided by UV-Vis absorption spectroscopy, which shows a significant increase in the absorbance of B-CDs following the addition of Cu\u003csup\u003e2+\u003c/sup\u003e (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eD), suggested that Cu\u0026sup2;⁺ form a complex with the ground-state B-CDs, reducing the number of available photons for excitation and suppressing fluorescence emission. Consequently, Cu\u003csup\u003e2+\u003c/sup\u003e quenching primarily results from static quenching through ground-state complexation, leading to a decrease in fluorescence intensity.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003e3.5 Cytotoxicity and Cellular Imaging\u003c/h2\u003e\n \u003cp\u003eTo evaluate the potential of B-CDs for living cell imaging, their cytotoxicity was first assessed using the CCK-8 assay in HepG2 cells. As shown in Figure S7, after 24 hours of incubation with B-CDs at concentrations ranging from 5 to 100 \u0026micro;g/mL, nearly 100% of the cells remained viable, indicating that B-CDs exhibit negligible or very low cytotoxicity. Fluorescence imaging was then performed to observe the cellular uptake of B-CDs. HepG2 cells treated with 0.1 mg/mL B-CDs displayed strong blue fluorescence under 405 nm excitation (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e). The small size and favorable surface functional groups of the B-CDs, which facilitate efficient cellular uptake. However, when the cells were co-incubated with 10 \u0026micro;M Cu\u0026sup2;⁺, the fluorescence intensity in the blue channel was significantly weakened, demonstrating that B-CDs can successfully detect Cu\u003csup\u003e2+\u003c/sup\u003e in living cells through a fluorescence turn-off mode.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"4. Conclusions","content":"\u003cp\u003eIn summary, B-CDs were successfully synthesized using raspberries as a carbon source through a simple hydrothermal method. The resulting B-CDs demonstrated excellent water solubility, good biocompatibility, and outstanding photoluminescence properties. Comprehensive characterization and theoretical calculations revealed that the fundamental building block of the B-CDs is a twelve-membered aromatic ring structure rich in oxygen and nitrogen functional groups. The obtained B-CDs exhibited high selectivity toward Cu\u003csup\u003e2+\u003c/sup\u003e ions, with a strong linear response over the concentration range of 0 to 150 \u0026micro;M and a detection limit of 0.39 \u0026micro;M. Zeta potential and hydrodynamic size measurements indicated that the B-CDs interact with Cu\u0026sup2;⁺ through electrostatic forces. Furthermore, UV-visible spectra and fluorescence lifetime studies suggested that the fluorescence quenching of B-CDs in the presence of Cu\u0026sup2;⁺ primarily results from a dynamic quenching process. The excellent fluorescence properties of B-CDs enabled their successful application in the detection of intracellular Cu\u0026sup2;⁺. These findings not only underscore the significant potential of B-CDs in fluorescence sensing but also provide valuable insights for designing efficient carbon-dot-based sensors. In the future, B-CDs are expected to have broad applications in environmental monitoring, intracellular ion detection, and disease diagnostics, positioning them as a promising platform for fluorescence-based sensing.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research is supported by the National Natural Science Foundation of China (NO. 22076072, and NO 22306082). We gratefully acknowledge HZWTECH for providing computation facilities.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work has been conducted by financial support from National Natural Science Foundation of China (NO. 22076072, and NO 22306082).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding author\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCorrespondence to Xiaoli Sun, [email protected]\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eN. Dey, N. Kumari, D. Biswakarma, S. Jha, S. Bhattacharya (2019) Colorimetric indicators for specific recognition of Cu\u003csup\u003e2+\u003c/sup\u003e and Hg\u003csup\u003e2+\u003c/sup\u003e in physiological media: Effect of variations of signaling unit on optical response, Inorg. Chim. Acta 487:50-57.\u003c/li\u003e\n\u003cli\u003eY. Hao, L. Liu, Y. Long, J. Wang, Y.N. Liu, F. 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Photobiol. B, 186:144-151.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Scheme 1","content":"\u003cp\u003eScheme 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Raspberry, Blue fluorescence, Carbon dots, Copper ion detection, Cell imaging","lastPublishedDoi":"10.21203/rs.3.rs-5841683/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5841683/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe detection of intracellular copper ions is of significant importance in biomedical research and disease diagnosis. In this study, blue emissive carbon dots (B-CDs) were successfully synthesized using raspberries as a carbon source through a simple hydrothermal method. Transmission electron microscopy (TEM) analysis revealed that the B-CDs had an average particle size of 9.05 nm. Characterization techniques combined with theoretical calculations, confirmed that the fundamental structural unit of B-CDs is a twelve-membered aromatic ring rich in oxygen and nitrogen functional groups. The B-CDs exhibited high selectivity for Cu\u0026sup2;⁺ ions, showing a strong linear response in the concentration range of 0 to 150 \u0026micro;M, with a detection limit of 0.39 \u0026micro;M. Zeta potential and hydrodynamic size measurements indicated that the B-CDs interact with Cu\u0026sup2;⁺ ions via electrostatic forces. Further studies revealed that the fluorescence quenching of B-CDs in the presence of Cu\u0026sup2;⁺ is primarily due to a dynamic quenching process. Moreover, B-CDs were successfully applied to detect intracellular Cu\u0026sup2;⁺. These findings not only show significant potential of B-CDs in fluorescence sensing but also provide valuable insights for the design of efficient carbon-based sensors.\u003c/p\u003e","manuscriptTitle":"Raspberry-derived carbon dots for specific detection of intracellular copper ions","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-24 09:54:05","doi":"10.21203/rs.3.rs-5841683/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"2c607f60-0eb6-4714-a6d0-a9f372dbe014","owner":[],"postedDate":"January 24th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-01-24T13:53:59+00:00","versionOfRecord":[],"versionCreatedAt":"2025-01-24 09:54:05","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5841683","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5841683","identity":"rs-5841683","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}