Switching-Type Fluorescence Probe Based on Carbon Dot Nanocomposites for the Visual Detection of Superoxide Anions

Switching-Type Fluorescence Probe Based on Carbon Dot Nanocomposites for the Visual Detection of Superoxide Anions | 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 Switching-Type Fluorescence Probe Based on Carbon Dot Nanocomposites for the Visual Detection of Superoxide Anions Jiao Zhang, Bin Liao, Yuxin Pei, Yuxin Huang, Zhiyan Liu, Yuanqiang Wang, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6467425/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 11 Jul, 2025 Read the published version in Journal of Fluorescence → Version 1 posted 7 You are reading this latest preprint version Abstract Superoxide anion (O₂⁻), as a crucial member of reactive oxygen species (ROS), plays a pivotal role in cellular function regulation within biological systems and has been extensively implicated in the pathogenesis and progression of numerous diseases. In this paper, we successfully prepare a switching fluorescent probe (O-QD@Ag) for visual monitoring of superoxide anion (O₂⁻) by coating silver nanolayer on the surface of carbon quantum dots(CQDs). The experimental results show that the fluorescence signal of the composite is quenched in the presence of silver nanolayers. However, in the presence of O₂⁻, the fluorescence signal recovers due to the oxidation etching process of silver nanolayer, and there is a good linear relationship between the fluorescence intensity of the probe and the O₂⁻ concentration. In addition, O-QD@Ag composite exhibits excellent biocompatibility and low toxicity, which is characterized by low toxicity in normal MCF-10A and MDA-MB-436 cancer cells, and is suitable for selective detection of intracellular O₂⁻. To sum up, the probe not only provides an effective means to monitor the pathological process of chronic diseases such as cancer, cardiovascular diseases and neurodegenerative diseases but also shows the potential value of application in disease prognosis evaluation. Carbon dots Fluorescence sensor Superoxide Anions Nanoprobe Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Superoxide anion (O₂⁻) is a critical reactive oxygen species (ROS) that plays a significant role in various physiological and pathological processes 1 , 2 . O₂⁻ is mainly generated through biochemical pathways such as the mitochondrial respiratory chain and NADPH oxidase, and its normal levels are essential for maintaining cellular signaling, immune responses, and redox homeostasis 3 . However, excessive O₂⁻ production can trigger oxidative stress, leading to cellular damage and a series of pathological changes. Oxidative stress has been implicated in the development of major diseases, including cancer, cardiovascular diseases, neurodegenerative disorders, and chronic inflammation 4 – 6 . Specifically, in the cardiovascular system, overproduction of O₂⁻ can result in endothelial dysfunction and accelerate the progression of atherosclerosis 7 . In the nervous system, excessive accumulation of O₂⁻ is associated with the pathogenesis of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases 8 . Moreover, O₂⁻ can promote tumor cell adhesion by inducing endothelial cell apoptosis and enhancing the expression of various adhesion molecules, potentially contributing to tumor recurrence 9 , 10 . Therefore, studying the dynamics of intracellular oxygen concentration is beneficial to early diagnosis and disease prognosis monitoring. Despite the promising potential of O₂⁻ detection in biomedical research and clinical diagnostics, quantifying O₂⁻ remains challenging due to its high reactivity, short half-life, and low concentration in biological systems 11 , 12 . Traditional methods for detecting O₂⁻ primarily include chemiluminescence, electrochemical techniques, ultraviolet-visible absorption spectroscopy, and chromatography 13 . Chemiluminescence typically relies on the reaction of O₂⁻ with luminescent substrates such as persulfates or luminol to generate light signals 14 . Although this method offers high sensitivity, it is often complicated by the need for sophisticated equipment and cumbersome procedures. Electrochemical methods measure current or voltage changes resulting from the reduction or oxidation reactions of O₂⁻, offering good selectivity 15 . However, they are susceptible to interference from other substances and require specialized instruments. Ultraviolet-visible absorption spectroscopy involves the reaction of O₂⁻ with substrates like Nitrotetrazolium Blue chloride, which alters absorbance and can be used for detection 16 , 17 . While this method is straightforward, its sensitivity is relatively low and it is prone to interference from solvents and other components. Chromatography, while capable of quantitative analysis, often involves lengthy and complex procedures 18 . Consequently, although these traditional methods can be effective in certain applications, they present limitations in terms of sensitivity, real-time monitoring, and operational convenience. In recent years, the rapid advancement of fluorescent probe technology has revolutionized the detection of O₂⁻, with fluorescence signal changes emerging as a highly promising and sensitive alternative for their detection 19 , 20 . Fluorescent probes interact specifically with target substances, altering their fluorescence properties (such as intensity or wavelength) to enable both qualitative and quantitative analysis 21 . Compared to traditional methods, fluorescent probes offer several advantages, including high sensitivity, real-time monitoring, low cost, and ease of use. These probes can detect O₂⁻ across a wide concentration range through changes in fluorescence, allowing for non-invasive detection within biological systems. However, traditional small-molecule fluorescent probes also face several challenges 22 , 23 . Firstly, there are solubility and safety problems. Some small molecular groups are difficult to dissolve in the sample to be tested, and the dissociated products may have certain chemical toxicity to organisms. Secondly, in a complex disease environment, such as the acid environment of tumor, the introduced fluorophore may miss the target, resulting in an inaccurate fluorescence signal and affecting its stability. In addition, small molecular fluorescent probes need to find suitable fluorophores and constantly optimize the molecular structure, which leads to increased cost and time cost. To address the aforementioned issues, carbon dots (CDs), as a novel fluorescent probe material, have attracted considerable attention in recent years 24 . CDs are a class of nanomaterials with unique optical, electrical, and chemical properties, typically with sizes below 10 nm 25 . CDs inherit the inherent stability and durability of carbon materials and can be synthesized via simple chemical methods, offering a low-cost solution 26 , 27 . More importantly, CDs possess excellent optical properties, good water solubility, low toxicity, and favorable biocompatibility, making them ideal fluorescent probe materials for bioimaging, sensing, and therapeutic applications. 17 , 28 . CDs can be easily functionalized and modified on the surface, allowing for the targeted detection of specific molecules 29 . Despite the potential of CDs in superoxide anion detection, their application is limited by issues such as low fluorescence quantum yields, challenges in surface modification, potential biotoxicity, and fluorescence quenching 30 . Therefore, there is a need to develop new carbon dot probes or optimize existing ones to enhance sensitivity, stability, and biocompatibility. In this study, we successfully synthesized a carbon dot-silver nanocomposite with yellow-green fluorescence, termed O-QD@Ag. By encapsulating the carbon dots (O-QD) with a silver nanoparticle layer, the fluorescence of the CDs was effectively quenched. Interestingly, in the presence of O₂⁻, the silver nanoparticle layer was oxidatively etched, releasing the carbon dots and restoring fluorescence, thereby constructing a "turn-on" fluorescent probe(Scheme 1 ). O-QD@Ag enables real-time monitoring of intracellular O₂⁻ dynamics, facilitating a deeper understanding of the role of O₂⁻ in oxidative stress-related diseases. 2. Experimental Section2.1 Reagents and Materials The citric acid (CA)、O-Phenylenediamine (OPD)、Silver nitrate (AgNO 3 )、Sodium borohydride (NaBH 4 )、 L-Ascorbic acid、 6-Hydroxypurine、xanthine oxidase (XO)、glucose (Glu)、uric acid (UA)、hydrogen peroxide (H 2 O 2 )、glutathione (GSH)、Sodium chloride (NaCL)、Phosphate-buffered saline (PBS)、MTT、Fetal bovine serum (FBS)、EBSS-Earle’s salts. All reagents were of analytical grade and were used as received without further purification. Deionized (DI) water was used throughout this study. All aqueous solutions were prepared with ultrapure water (≥ 18.25 MΩ cm) from a Milli-Q Plus system (Millipore, Bedford, MA, USA).2.2 Synthesis of O-QD and O-QD@Ag 0.2 g of citric acid and 0.33 g of o-phenylenediamine were dissolved in 15 mL of ultrapure water and transferred to a flask. The solution was then subjected to a condensation reflux reaction at 100°C in an oil bath for 12 hours. After the reaction was completed, the reaction mixture was collected and centrifuged at 10000 rpm for 10 min, repeated three times. The supernatant was filtered by 0.2µm microporous membrane and the excess impurities were removed by dialysis. The dialysate was then freeze-dried at -60°C for 48 hours to yield the O-QD solid powder. 3 mg of O-QD and 2.8 mg of L-ascorbic acid were dissolved in 15 mL of ultrapure water with magnetic stirring for 5 min to ensure complete dissolution. Freshly prepared 4 mM AgNO₃ solution was then added dropwise while stirring continuously for 30 min, during which the solution color gradually changed from pale yellow to black. The resulting solution was collected and centrifuged at 10000 rpm for 10 min, repeated three times, after which the precipitate was freeze-dried at -60°C to obtain the O-QD@Ag solid powder.2.3 Stability of O-QD and O-QD@Ag To evaluate the stability of O-QD and O-QD@Ag, 10 µL of O-QD and O-QD@Ag samples at a concentration of 100 µg/mL were added to 2 mL of NaCl solutions with varying concentrations (0-0.5 M) and exposed to environments with different pH values (5.5–8.5). The mixtures were then incubated at a constant temperature of 25°C for 10 min. The stability was assessed by monitoring changes in fluorescence intensity. 2.4 Detection of Superoxide Anions Based on previous studies, the enzymatic reaction between xanthine oxidase (XO, 200 mU/mL) and hypoxanthine (HX, 100 µM) can stably generate 2 mM/L of O₂⁻ 31 , 32 . To prepare O₂⁻ samples at different concentrations, the concentration of XO was adjusted within the range of 0-200 mU/mL, using phosphate buffer as the reaction medium. O-QD@Ag (1 mg/mL) was added to the O₂⁻-generating reaction solutions and incubated at 37°C for 10 min. The fluorescence spectra of the reaction solutions were measured using an RF-6000 fluorescence spectrophotometer, with an excitation wavelength of 380 nm and an emission wavelength of 570 nm. The recorded fluorescence intensity was denoted as F₀, while the fluorescence intensity after oxidation etching of O-QD@Ag by O₂⁻ at various concentrations was denoted as F. A correlation curve between the fluorescence intensity ratio (F/F₀) and the O₂⁻ concentration was subsequently established. 2.5 The binding of the O-QD@Ag composite to the Superoxide anion Given the complexity of physiological systems, selectivity is a crucial criterion for evaluating the performance of fluorescent probes. Therefore, we selected potential interfering analytes to assess their impact on the fluorescence of O-QD@Ag. Specifically, a range of bioactive molecules and metal ions (Na⁺, Ga²⁺, Mg²⁺, K⁺, Mn²⁺, CO₃²⁻, Fe³⁺, UA, DA, Glu, H₂O₂, NO₃²⁻, OH⁻, NO, ¹O₂, O₂⁻) were chosen to evaluate their effects on fluorescence intensity. In the experiment, inorganic ions at a concentration of 500 µM and ROS at 100 µM were separately added to the O-QD@Ag solution, followed by fluorescence spectrum measurement after incubation at 25°C for 10 min. The selectivity of O-QD@Ag was verified at the cellular level. First, MDA-MB-436 cells were seeded into 12-well plates at a density of 1×10⁴ cells per well and incubated for 1 hour to allow cell adhesion. Then, 500 µL of culture medium containing 10 µL of O-QD@Ag solution (100 µg/mL) was added to the cells and incubated at 37°C for 30 min. After incubation, the MDA-MB-436 cells were washed three times with phosphate-buffered saline (PBS, pH 7.4) to remove residual nanoprobes and prevent interference from probes adhering to the extracellular matrix. Next, endogenous interferents such as H₂O₂ (5 µM), UA (5 µM), DA (5 µM), and glucose (5 µM) were separately added to the cells incubated in 0.1 M phosphate buffer and incubated for another 30 min. The cells were then washed three more times with phosphate-buffered saline. Finally, cellular fluorescence imaging was performed using a fluorescence microscope with an excitation wavelength of 380 nm.2.6 In vitro cytotoxicity study MDA-MB-436 and MCF-10A cells were seeded into 96-well plates at a density of 3×10⁵ cells per well, with 100 µL of culture medium added to each well, and incubated for 24 hours. After incubation, the original culture medium was removed, and 100 µL of fresh medium containing varying concentrations of the test substances (0, 10, 20, 40, 60, 80, 100 µg/mL) was added to each well. After a further 24 hours of incubation, 5 µL of MTT solution (5 mg/mL) was added to each well along with 100 µL of fresh culture medium, and the plates were incubated for 4 hours. Following incubation, the MTT-containing medium was discarded, and 150 µL of dimethyl sulfoxide (DMSO) was added to each well. The cells were lysed at 37°C in a dry incubator for 30 min. Finally, the absorbance of each well at 490 nm was measured using a microplate reader. Cell viability was calculated using the following formula: Cell viability (%) = \(\:\frac{\text{t}\text{e}\text{s}\text{t}\:\text{s}\text{a}\text{m}\text{p}\text{l}\text{e}}{\text{b}\text{l}\text{a}\text{n}\text{k}\:\text{s}\text{a}\text{m}\text{p}\text{l}\text{e}}\) × 100% 2.7 Detecting intracellular O₂⁻ levels using O-QD@Ag in MDA-MB-436 Cancer Cell O-QD@Ag was used as a fluorescent nanoprobe for imaging. MDA-MB-436 cells were seeded into 24-well plates at a density of 1×10⁴ cells per well and incubated for 24 hours before treatment. The cells were then incubated in 200 µL of culture medium containing 10 µL of O-QD@Ag solution (100 µg/mL) at 37°C for 6 hours. After incubation, the MDA-MB-436 cells were washed three times with phosphate-buffered saline (PBS, pH 7.4) to remove residual nanoprobes. Subsequently, fresh medium containing different concentrations of O₂⁻ (0 mM, 0.5 mM, 1 mM, 1.5 mM, 2 mM) was added, and fluorescence imaging was performed using a fluorescence microscope with excitation at 380 nm. To evaluate the in vivo imaging capability of O-QD@Ag, LPS was added to induce autophagy in live cells to generate O₂⁻and fluorescence microscopy images were obtained after O₂⁻ scavenging by Tiron. 3 Results and Discussion 3.1 Synthesis of O-QD and O-QD@Ag composite O-QD@Ag was successfully prepared to be used as a turn-on fluorescent probe to detect O₂⁻. Initially, O-QD was prepared using a hydrothermal synthesis method, and through the in-situ reduction of a silver nanolayer on its surface, the O-QD@Ag composite was successfully obtained 33 . The morphology and average particle size of both O-QD and O-QD@Ag were characterized by transmission electron microscopy (TEM). As shown in Fig. 1 a, O-QD exhibited a spherical shape with a smooth surface and an average diameter of 3.42 ± 1.17 nm. Similarly, O-QD@Ag (Fig. 1 b) also displayed a spherical shape with uniform distribution, with an average diameter of 4.47 ± 0.12 nm. These results indicate that the silver nanolayer was successfully deposited on the surface of O-QD as the particle size increased. Furthermore, the successful synthesis of O-QD@Ag was confirmed by mapping images (Figs. 1 c and S1). To evaluate the electrophoretic mobility and stability of the nanoparticles, both O-QD and O-QD@Ag were dissolved in PBS buffer, and their zeta (ζ) potentials were measured. As shown in Fig. 1 d, the ζ potentials of O-QD and O-QD@Ag were − 6.88 mV and 12.91 mV, respectively. Additionally, Fourier-transform infrared spectroscopy (FTIR) analysis further confirmed the functional group structures of O-QD and O-QD@Ag. As presented in Fig. 1 e, the peaks at 3364.46 cm⁻¹ and 3215.98 cm⁻¹ for O-QD are attributed to the stretching vibrations of -O-H and -N-H groups, respectively, while the peak at 2602.55 cm⁻¹ corresponds to the stretching of -C-H 34 . The absorption band around 1583.65 cm⁻¹ is assigned to -C = O, -C = N, and -C = C groups 35 . Furthermore, absorption bands of -C-O and -C-H are also observed in the spectrum. Compared to O-QD, the vibration intensity of the peak near 1583.65 cm⁻¹ in O-QD@Ag is reduced, which may be due to the reduction of -C = O or -C = N to -C-OH or -C-NH 36 . X-ray photoelectron spectroscopy (XPS) was employed to further analyze the surface elemental composition of O-QD and O-QD@Ag. As shown in Figs. 1 f-i and S2, O-QD exhibited strong peaks at 284.80 eV, 398.49 eV, and 531.24 eV, corresponding to C1s, N1s, and O1s, respectively. In contrast, changes in binding energy for O-QD@Ag were primarily attributed to the reduction of functional groups during the in situ reduction of the silver nanolayer. The Ag spectrum of O-QD@Ag displayed two distinct peaks at 367.20 eV and 373.21 eV, corresponding to Ag3d5/2 and Ag3d3/2, which are characteristic of metallic Ag(0) 37 . The appearance of the Ag3d peaks confirms the successful formation of a silver nanolayer in the final O-QD@Ag composite. 3.2 Optical properties of O-QD and O-QD@Ag We subsequently characterized the fluorescence properties of the samples using UV-visible spectroscopy and a fluorescence spectrophotometer. As illustrated in Fig. 2 a, no significant absorption peak was observed for the standalone CA, whereas a characteristic peak at 206 nm was present for OPD, which was also retained in O-QD. In Fig. 2 b, as the excitation wavelength varied from 360 nm to 460 nm (in 20 nm increments), the fluorescence emission of O-QD exhibited excitation dependence, confirming an optimal excitation wavelength of 380 nm and an emission wavelength of 570 nm. The stability of CDs is critical for real-time environmental and bioanalytical applications 38 . To assess the stability of O-QD and O-QD@Ag, we investigated the fluorescence response under different concentrations of NaCl and varying pH levels. The results indicated that, when the NaCl concentration ranged from 0 to 0.5 M, the fluorescence intensity of the CDs remained relatively unchanged (Fig. 2 c). Furthermore, the pH stability was evaluated by adjusting the pH of the solution from 5.5 to 8.5 using phosphoric acid and NaOH. The findings showed that fluorescence intensity decreased in both acidic and basic conditions but remained stable in a neutral environment (pH 6–8), demonstrating excellent stability under physiological conditions (pH 7). In conclusion, O-QD@Ag holds great potential as a turn-on fluorescent probe for detecting O₂⁻ in biological systems. 3.3 The specific binding of O-QD@Ag to the superoxide anion To preliminarily assess the ability of O-QD@Ag to detect O₂⁻ in vitro, we measured the fluorescence changes of the O-QD@Ag solution before and after the addition of 2 mM O₂⁻. As shown in Figs. 2 e and 2 f, the fluorescence of O-QD@Ag was effectively quenched under excitation at 380 nm, likely due to the plasmon resonance of the Ag nanolayer on the surface 39 . After oxidation by O₂⁻, the fluorescence was restored, aligning with the emission peak of O-QD, and exhibiting a rapid response to O₂⁻. Next, the specific binding capability of O-QD@Ag to O₂⁻ was investigated. To determine the selectivity of the O-QD@Ag-based fluorescence assay for O₂⁻, we examined the fluorescence spectra of O-QD@Ag before and after the addition of O₂⁻ and other interfering substances. Compared to the other substances, O₂⁻ induced a 10-fold increase in fluorescence intensity, demonstrating the excellent selectivity of O-QD@Ag for O₂⁻ (Fig. 3 a). To further confirm this result at the cellular level, O-QD@Ag was co-incubated with several endogenous substances, as shown in Figure S3, where fluorescence was restored in the presence of O₂⁻, further verifying the high specificity of O-QD@Ag for O₂⁻. Subsequently, detailed fluorescence titration experiments were conducted with O-QD@Ag and varying concentrations of O₂⁻. As shown in Fig. 3 b, the fluorescence intensity increased systematically as the O₂⁻ concentration rose from 1 mM to 4 mM. Additionally, the linear relationship between fluorescence intensity and O₂⁻ concentration was investigated. As shown in Fig. 3 c, a strong linear correlation (R 2 = 0.994) was observed between fluorescence intensity and O₂⁻ concentration in the range of 0.2 mM to 1.6 mM. These findings suggest that O-QD@Ag holds the potential for dynamic in vitro monitoring of O₂⁻ concentrations. 3.4 Detecting Superoxide Anion Levels using O-QD@Ag in MDA-MB-436 cancer cells To further evaluate the ability of O-QD@Ag to detect O₂⁻ levels, MDA-MB-436 cancer cells, and MCF-10A normal cells were chosen as models. Initially, MTT assays were conducted to assess the biocompatibility and safety of O-QD@Ag. As shown in Fig. 4 , when the concentrations of O-QD and O-QD@Ag ranged from 10 µg/mL to 100 µg/mL, the cell viability remained above 80%, indicating good biocompatibility. Based on the fluorescence intensity data, a concentration of 100 µg/mL was selected for subsequent experiments. Next, the ability of O-QD@Ag to detect O₂⁻ at the cellular level was assessed as a fluorescence probe. MDA-MB-436 cells were treated with O-QD@Ag, followed by the addition of different concentrations of O₂⁻ (0 mM, 0.5 mM, 1 mM, 1.5 mM, 2 mM), with the O₂⁻-free group serving as a control. The results showed that the fluorescence intensity of O-QD@Ag gradually recovered with increasing O₂⁻ concentrations, and this recovery was concentration-dependent (Fig. 5 a), which was consistent with the quantitative analysis (Fig. 5 b). To further investigate the application of this probe for detecting endogenous O₂⁻, lipopolysaccharide (LPS) was used to induce oxidative stress in the cells, thereby increasing the intracellular ROS levels 40 . MDA-MB-436 cells were pretreated with LPS for 60 min to elevate the O₂⁻ content, followed by 30 min of incubation with the probe and imaging, which showed fluorescence signal recovery. To confirm that this signal change was indeed caused by O₂⁻, Tiron, a specific scavenger of O₂⁻ 41 , 42 , was used to incubate the LPS-pretreated cells for 30 min, after which the probe was added for incubation and imaging (Fig. 6 ). The results demonstrated that Tiron could reverse the fluorescence signal changes induced by LPS. In conclusion, the cell imaging experiments confirmed that the O-QD@Ag probe is capable of effectively detecting endogenous O₂⁻ in cells. 4 Conclusion In summary, we successfully developed an O-QD@Ag fluorescent probe with yellow-green fluorescence properties. The probe exhibits a remarkable fluorescence recovery pattern in response to varying concentrations of superoxide anion (O₂⁻) solutions, with a strong linear correlation between the normalized fluorescence intensity and O₂⁻ concentration (R² = 0.994). The effective detection range of the probe is 0.2-2 µM. Therefore, due to the high specificity between O-QD@Ag and superoxide anions, this probe allows for dynamic monitoring of intracellular O₂⁻ levels. We believe that this probe holds significant potential for further applications in the detection and prognosis of pathological processes in chronic diseases such as cancer, cardiovascular disorders, and neurodegenerative diseases. Declarations Conflicts of interest “The authors have no relevant financial or non-financial interests to disclose.” Ethics approval This is a critical study, so no ethical approval was involved in this study. Funding This work was financially supported by the Chongqing Bureau of Science and Technology (CSTB2024NSCQ-KJFZMSX0046). Funding results of the Action plan for high-quality development of graduate students of Chongqing University of Technology(gzlcx20243224). Author Contribution Jiao Zhang: Data curation, Writing – original draft. Bin Liao：Investigation, Resources, Formal analysis. YuXin Pei: Investigation, Formal analysis. ZhiYan Liu: Investigation, Formal analysis. YuXin Huang: Investigation, Resources. YuanQiang Wang: Investigation, Resources. Qinghua Yu: Conceptualization, Funding acquisition, validation, Writing – review & editing, Supervision. Wanyi Chen: Conceptualization, Writing – review & editing, Supervision, Project administration. Data availability The raw data supporting the conclusions of this article will be made available by the authors on request. 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Additional Declarations No competing interests reported. Supplementary Files Scheme1.png Scheme 1 Preparation of O-QD and O-QD@Ag, and schematic diagram of O₂⁻ detection using the "turn-on" fluorescent probe O-QD@Ag Supportinginformation.docx Cite Share Download PDF Status: Published Journal Publication published 11 Jul, 2025 Read the published version in Journal of Fluorescence → Version 1 posted Editorial decision: Revision requested 22 May, 2025 Reviews received at journal 14 May, 2025 Reviewers agreed at journal 05 May, 2025 Reviewers invited by journal 05 May, 2025 Editor assigned by journal 24 Apr, 2025 Submission checks completed at journal 24 Apr, 2025 First submitted to journal 16 Apr, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6467425","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":452281396,"identity":"ea811603-743b-4de5-babe-02de16e203cb","order_by":0,"name":"Jiao Zhang","email":"","orcid":"","institution":"Chongqing University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Jiao","middleName":"","lastName":"Zhang","suffix":""},{"id":452281397,"identity":"11b68710-c220-4041-bc6d-4d01260c1052","order_by":1,"name":"Bin Liao","email":"","orcid":"","institution":"Chongqing University Cancer Hospital","correspondingAuthor":false,"prefix":"","firstName":"Bin","middleName":"","lastName":"Liao","suffix":""},{"id":452281398,"identity":"4ea11526-e56a-463a-aff1-f2d7cff5e27f","order_by":2,"name":"Yuxin Pei","email":"","orcid":"","institution":"Chongqing University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Yuxin","middleName":"","lastName":"Pei","suffix":""},{"id":452281399,"identity":"ddae6e03-08ae-41eb-a6ec-bf4a981661f9","order_by":3,"name":"Yuxin Huang","email":"","orcid":"","institution":"Chongqing University","correspondingAuthor":false,"prefix":"","firstName":"Yuxin","middleName":"","lastName":"Huang","suffix":""},{"id":452281400,"identity":"d05702c2-129c-424a-bf30-b08dd3d4b69e","order_by":4,"name":"Zhiyan Liu","email":"","orcid":"","institution":"Chongqing University","correspondingAuthor":false,"prefix":"","firstName":"Zhiyan","middleName":"","lastName":"Liu","suffix":""},{"id":452281401,"identity":"fd3a40f5-5a8e-484a-930e-e7e9eb3ec58b","order_by":5,"name":"Yuanqiang Wang","email":"","orcid":"","institution":"Chongqing University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Yuanqiang","middleName":"","lastName":"Wang","suffix":""},{"id":452281402,"identity":"70ad8d4a-c0c7-4cfa-b5e4-55beb0b365fe","order_by":6,"name":"Qinghua Yu","email":"","orcid":"","institution":"Chongqing University Cancer Hospital","correspondingAuthor":false,"prefix":"","firstName":"Qinghua","middleName":"","lastName":"Yu","suffix":""},{"id":452281403,"identity":"1172fa3f-b4af-45a5-bb03-44de8a7f44a9","order_by":7,"name":"Wanyi Chen","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3UlEQVRIiWNgGAWjYDACCSjNxt4AphkbiNfCc4BULQwSCURq4Z/dfOwxT82dxD7Jxw9v8zDYyG44wPzsAV5L7hxLN+Y59iyxTTrN2JqHIc14wwE2cwN8Wgwkcsykc9gOA7XksEnzMBxO3HCAh00Cv5b8b9I5/4BaJM+AtPwnRgvQ8Nw2oBYJHpCWA4S1SNxIM5P+23fYuI0nzdhyjkGy8czDbGZ4tfDPSH4mOePbYdn57Ycf3nhTYSfbd7z5GV4tMODYALKSARRUzMSoBwJ7sCuJVDwKRsEoGAUjDAAAdHxELrSM4zkAAAAASUVORK5CYII=","orcid":"","institution":"Chongqing University Cancer Hospital","correspondingAuthor":true,"prefix":"","firstName":"Wanyi","middleName":"","lastName":"Chen","suffix":""}],"badges":[],"createdAt":"2025-04-17 03:08:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6467425/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6467425/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10895-025-04414-8","type":"published","date":"2025-07-11T15:57:17+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":82261935,"identity":"ae652c62-0d72-46bc-8171-58eddf87ff4e","added_by":"auto","created_at":"2025-05-08 12:19:23","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2034661,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Transmission electron microscopy (scale = 20nm) of O-QD, illustrated by the size distribution of O-QD statistically measured by TEM results; (b) A projection electron microscope (scale =20nm) of O-QD@Ag, illustrated by the size distribution of O-QD@Ag counted by TEM; (c) elemental analysis diagram of O-QD@Ag; (d) Zeta potential of O-QD, O-QD@Ag; (e) FT-IR spectra of O-QD, O-QD@Ag; (f) XPS high-resolution Ag3d for O-QD@Ag; (g) O-QD@Ag XPS high-resolution C 1s; (h)O-QD@Ag XPS high-resolution N1s; (i) XPS high-resolution O1s spectrum of O-QD@Ag.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6467425/v1/8ae875588b3266a7086a35a6.png"},{"id":82261934,"identity":"a49cdbf1-3ef1-4d0a-9c96-10ceed26b9ae","added_by":"auto","created_at":"2025-05-08 12:19:23","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1334281,"visible":true,"origin":"","legend":"\u003cp\u003e(a) UV-VIS absorption spectra of CA, OPD and O-QD; (b) Fluorescence spectra of O-QD aqueous solution at different excitation wavelengths; (c) Fluorescence intensity of O-QD and O-QD@Ag at different concentrations of NaCl; (d) Fluorescence intensity of O-QD and O-QD@Ag at different pH; (e) Fluorescence spectra of O-QD@Ag and O-QD@Ag+O₂⁻-; (f) the time response of O-QD@Ag+O₂⁻\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6467425/v1/32672cf196edc46c1e86c4c9.png"},{"id":82261932,"identity":"7fcf72e3-84e1-46db-b563-97cac5a7c914","added_by":"auto","created_at":"2025-05-08 12:19:23","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":528970,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Fluorescence intensity of the probe O-QD@Ag when various analytes were added to PBS: 1: blank,2:Na⁺, 3:Ga²⁺, 4:Mg²⁺,5: K⁺, 6:Mn²⁺,7: CO₃²⁻,8: Fe³⁺, 9:UA,1110: DA, 11:Glu, 12: H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2,\u003c/sub\u003e13: No\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e2-\u003c/sup\u003e,14: OH,\u0026nbsp; 15: \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2,\u003c/sub\u003e16: NO,17:O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e; (b) The fluorescence spectra of O-QD@Ag combined with different concentrations of O₂⁻, the excitation wavelength is 380 nm; (c) O-QD@Ag Fluorescence intensity in response to O₂⁻ concentration. Illustration: Standard curve of fluorescence intensity and O2- concentration.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6467425/v1/94e57f3715d0bb99fb43cf78.png"},{"id":82261957,"identity":"0447745d-6ab8-42c4-b7a8-2b6c005d8012","added_by":"auto","created_at":"2025-05-08 12:19:24","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":409096,"visible":true,"origin":"","legend":"\u003cp\u003eThe MTT results used MDA-MB-436 and MCF-10A cells.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6467425/v1/7c4f57371452eb18b79e1d43.png"},{"id":82261941,"identity":"b4dbe225-22e0-4253-a842-bebf0c2a00db","added_by":"auto","created_at":"2025-05-08 12:19:23","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":554235,"visible":true,"origin":"","legend":"\u003cp\u003eExogenous response of O-QD@Ag to O₂⁻. (a) Intracellular O₂⁻levels were measured in MDA-MB-436 cancer cells using O-QD@Ag (scale =200 µm). (b) Quantitative analysis of exogenous responses.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6467425/v1/a0bad527374b949fa5224685.png"},{"id":82262947,"identity":"a3366398-5891-4a80-bcba-74ac328680ce","added_by":"auto","created_at":"2025-05-08 12:27:23","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":491272,"visible":true,"origin":"","legend":"\u003cp\u003eThe endogenous response of O-QD@Ag to O₂⁻. (a) Confocal images of the O₂⁻response of O-QD@Ag in the control group (O-QD@Ag), LPS-induced, LPS-induced MDA-MB-436 cells treated with Trion or Tiron alone (scale = 200 μm); (b) Quantitative analysis of endogenous responses.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6467425/v1/8fd4fc9f3df58781c034d617.png"},{"id":86699462,"identity":"d6e4a701-84b8-4f28-9035-4f5ac85dfcf3","added_by":"auto","created_at":"2025-07-14 16:10:04","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5328648,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6467425/v1/2b5b01dd-fed3-4fc9-8e05-8466aeec72cf.pdf"},{"id":82261931,"identity":"8f5d9305-9302-4ba2-9feb-e90e297eca52","added_by":"auto","created_at":"2025-05-08 12:19:23","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1378036,"visible":true,"origin":"","legend":"\u003cp\u003eScheme 1 Preparation of O-QD and O-QD@Ag, and schematic diagram of O₂⁻ detection using the \"turn-on\" fluorescent probe O-QD@Ag\u003c/p\u003e","description":"","filename":"Scheme1.png","url":"https://assets-eu.researchsquare.com/files/rs-6467425/v1/6551578da437f1d22ad58850.png"},{"id":82261938,"identity":"f74b6e17-49e3-41a0-bcc0-30c2cf3df800","added_by":"auto","created_at":"2025-05-08 12:19:23","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":574112,"visible":true,"origin":"","legend":"","description":"","filename":"Supportinginformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-6467425/v1/279b899cfdbdca502aaa0218.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Switching-Type Fluorescence Probe Based on Carbon Dot Nanocomposites for the Visual Detection of Superoxide Anions","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eSuperoxide anion (O₂⁻) is a critical reactive oxygen species (ROS) that plays a significant role in various physiological and pathological processes\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. O₂⁻ is mainly generated through biochemical pathways such as the mitochondrial respiratory chain and NADPH oxidase, and its normal levels are essential for maintaining cellular signaling, immune responses, and redox homeostasis\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. However, excessive O₂⁻ production can trigger oxidative stress, leading to cellular damage and a series of pathological changes. Oxidative stress has been implicated in the development of major diseases, including cancer, cardiovascular diseases, neurodegenerative disorders, and chronic inflammation\u003csup\u003e\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Specifically, in the cardiovascular system, overproduction of O₂⁻ can result in endothelial dysfunction and accelerate the progression of atherosclerosis\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. In the nervous system, excessive accumulation of O₂⁻ is associated with the pathogenesis of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Moreover, O₂⁻ can promote tumor cell adhesion by inducing endothelial cell apoptosis and enhancing the expression of various adhesion molecules, potentially contributing to tumor recurrence\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Therefore, studying the dynamics of intracellular oxygen concentration is beneficial to early diagnosis and disease prognosis monitoring.\u003c/p\u003e \u003cp\u003eDespite the promising potential of O₂⁻ detection in biomedical research and clinical diagnostics, quantifying O₂⁻ remains challenging due to its high reactivity, short half-life, and low concentration in biological systems\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Traditional methods for detecting O₂⁻ primarily include chemiluminescence, electrochemical techniques, ultraviolet-visible absorption spectroscopy, and chromatography\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Chemiluminescence typically relies on the reaction of O₂⁻ with luminescent substrates such as persulfates or luminol to generate light signals\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Although this method offers high sensitivity, it is often complicated by the need for sophisticated equipment and cumbersome procedures. Electrochemical methods measure current or voltage changes resulting from the reduction or oxidation reactions of O₂⁻, offering good selectivity\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. However, they are susceptible to interference from other substances and require specialized instruments. Ultraviolet-visible absorption spectroscopy involves the reaction of O₂⁻ with substrates like Nitrotetrazolium Blue chloride, which alters absorbance and can be used for detection\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. While this method is straightforward, its sensitivity is relatively low and it is prone to interference from solvents and other components. Chromatography, while capable of quantitative analysis, often involves lengthy and complex procedures\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Consequently, although these traditional methods can be effective in certain applications, they present limitations in terms of sensitivity, real-time monitoring, and operational convenience.\u003c/p\u003e \u003cp\u003eIn recent years, the rapid advancement of fluorescent probe technology has revolutionized the detection of O₂⁻, with fluorescence signal changes emerging as a highly promising and sensitive alternative for their detection\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Fluorescent probes interact specifically with target substances, altering their fluorescence properties (such as intensity or wavelength) to enable both qualitative and quantitative analysis\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Compared to traditional methods, fluorescent probes offer several advantages, including high sensitivity, real-time monitoring, low cost, and ease of use. These probes can detect O₂⁻ across a wide concentration range through changes in fluorescence, allowing for non-invasive detection within biological systems. However, traditional small-molecule fluorescent probes also face several challenges\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Firstly, there are solubility and safety problems. Some small molecular groups are difficult to dissolve in the sample to be tested, and the dissociated products may have certain chemical toxicity to organisms. Secondly, in a complex disease environment, such as the acid environment of tumor, the introduced fluorophore may miss the target, resulting in an inaccurate fluorescence signal and affecting its stability. In addition, small molecular fluorescent probes need to find suitable fluorophores and constantly optimize the molecular structure, which leads to increased cost and time cost.\u003c/p\u003e \u003cp\u003eTo address the aforementioned issues, carbon dots (CDs), as a novel fluorescent probe material, have attracted considerable attention in recent years\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. CDs are a class of nanomaterials with unique optical, electrical, and chemical properties, typically with sizes below 10 nm\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. CDs inherit the inherent stability and durability of carbon materials and can be synthesized via simple chemical methods, offering a low-cost solution\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. More importantly, CDs possess excellent optical properties, good water solubility, low toxicity, and favorable biocompatibility, making them ideal fluorescent probe materials for bioimaging, sensing, and therapeutic applications.\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. CDs can be easily functionalized and modified on the surface, allowing for the targeted detection of specific molecules\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Despite the potential of CDs in superoxide anion detection, their application is limited by issues such as low fluorescence quantum yields, challenges in surface modification, potential biotoxicity, and fluorescence quenching\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Therefore, there is a need to develop new carbon dot probes or optimize existing ones to enhance sensitivity, stability, and biocompatibility.\u003c/p\u003e \u003cp\u003eIn this study, we successfully synthesized a carbon dot-silver nanocomposite with yellow-green fluorescence, termed O-QD@Ag. By encapsulating the carbon dots (O-QD) with a silver nanoparticle layer, the fluorescence of the CDs was effectively quenched. Interestingly, in the presence of O₂⁻, the silver nanoparticle layer was oxidatively etched, releasing the carbon dots and restoring fluorescence, thereby constructing a \"turn-on\" fluorescent probe(Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). O-QD@Ag enables real-time monitoring of intracellular O₂⁻ dynamics, facilitating a deeper understanding of the role of O₂⁻ in oxidative stress-related diseases.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"2. Experimental Section2.1 Reagents and Materials","content":"\u003cp\u003eThe citric acid (CA)、O-Phenylenediamine (OPD)、Silver nitrate (AgNO\u003csub\u003e3\u003c/sub\u003e)、Sodium borohydride (NaBH\u003csub\u003e4\u003c/sub\u003e)、 L-Ascorbic acid、 6-Hydroxypurine、xanthine oxidase (XO)、glucose (Glu)、uric acid (UA)、hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e)、glutathione (GSH)、Sodium chloride (NaCL)、Phosphate-buffered saline (PBS)、MTT、Fetal bovine serum (FBS)、EBSS-Earle\u0026rsquo;s salts. All reagents were of analytical grade and were used as received without further purification. Deionized (DI) water was used throughout this study. All aqueous solutions were prepared with ultrapure water (\u0026ge;\u0026thinsp;18.25 MΩ cm) from a Milli-Q Plus system (Millipore, Bedford, MA, USA).2.2 Synthesis of O-QD and O-QD@Ag\u003c/p\u003e \u003cp\u003e0.2 g of citric acid and 0.33 g of o-phenylenediamine were dissolved in 15 mL of ultrapure water and transferred to a flask. The solution was then subjected to a condensation reflux reaction at 100\u0026deg;C in an oil bath for 12 hours. After the reaction was completed, the reaction mixture was collected and centrifuged at 10000 rpm for 10 min, repeated three times. The supernatant was filtered by 0.2\u0026micro;m microporous membrane and the excess impurities were removed by dialysis. The dialysate was then freeze-dried at -60\u0026deg;C for 48 hours to yield the O-QD solid powder.\u003c/p\u003e \u003cp\u003e3 mg of O-QD and 2.8 mg of L-ascorbic acid were dissolved in 15 mL of ultrapure water with magnetic stirring for 5 min to ensure complete dissolution. Freshly prepared 4 mM AgNO₃ solution was then added dropwise while stirring continuously for 30 min, during which the solution color gradually changed from pale yellow to black. The resulting solution was collected and centrifuged at 10000 rpm for 10 min, repeated three times, after which the precipitate was freeze-dried at -60\u0026deg;C to obtain the O-QD@Ag solid powder.2.3 Stability of O-QD and O-QD@Ag\u003c/p\u003e \u003cp\u003eTo evaluate the stability of O-QD and O-QD@Ag, 10 \u0026micro;L of O-QD and O-QD@Ag samples at a concentration of 100 \u0026micro;g/mL were added to 2 mL of NaCl solutions with varying concentrations (0-0.5 M) and exposed to environments with different pH values (5.5\u0026ndash;8.5). The mixtures were then incubated at a constant temperature of 25\u0026deg;C for 10 min. The stability was assessed by monitoring changes in fluorescence intensity.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Detection of Superoxide Anions\u003c/h2\u003e \u003cp\u003eBased on previous studies, the enzymatic reaction between xanthine oxidase (XO, 200 mU/mL) and hypoxanthine (HX, 100 \u0026micro;M) can stably generate 2 mM/L of O₂⁻\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. To prepare O₂⁻ samples at different concentrations, the concentration of XO was adjusted within the range of 0-200 mU/mL, using phosphate buffer as the reaction medium. O-QD@Ag (1 mg/mL) was added to the O₂⁻-generating reaction solutions and incubated at 37\u0026deg;C for 10 min. The fluorescence spectra of the reaction solutions were measured using an RF-6000 fluorescence spectrophotometer, with an excitation wavelength of 380 nm and an emission wavelength of 570 nm. The recorded fluorescence intensity was denoted as F₀, while the fluorescence intensity after oxidation etching of O-QD@Ag by O₂⁻ at various concentrations was denoted as F. A correlation curve between the fluorescence intensity ratio (F/F₀) and the O₂⁻ concentration was subsequently established.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.5 The binding of the O-QD@Ag composite to the Superoxide anion\u003c/h2\u003e \u003cp\u003eGiven the complexity of physiological systems, selectivity is a crucial criterion for evaluating the performance of fluorescent probes. Therefore, we selected potential interfering analytes to assess their impact on the fluorescence of O-QD@Ag. Specifically, a range of bioactive molecules and metal ions (Na⁺, Ga\u0026sup2;⁺, Mg\u0026sup2;⁺, K⁺, Mn\u0026sup2;⁺, CO₃\u0026sup2;⁻, Fe\u0026sup3;⁺, UA, DA, Glu, H₂O₂, NO₃\u0026sup2;⁻, OH⁻, NO, \u0026sup1;O₂, O₂⁻) were chosen to evaluate their effects on fluorescence intensity. In the experiment, inorganic ions at a concentration of 500 \u0026micro;M and ROS at 100 \u0026micro;M were separately added to the O-QD@Ag solution, followed by fluorescence spectrum measurement after incubation at 25\u0026deg;C for 10 min. The selectivity of O-QD@Ag was verified at the cellular level. First, MDA-MB-436 cells were seeded into 12-well plates at a density of 1\u0026times;10⁴ cells per well and incubated for 1 hour to allow cell adhesion. Then, 500 \u0026micro;L of culture medium containing 10 \u0026micro;L of O-QD@Ag solution (100 \u0026micro;g/mL) was added to the cells and incubated at 37\u0026deg;C for 30 min. After incubation, the MDA-MB-436 cells were washed three times with phosphate-buffered saline (PBS, pH 7.4) to remove residual nanoprobes and prevent interference from probes adhering to the extracellular matrix. Next, endogenous interferents such as H₂O₂ (5 \u0026micro;M), UA (5 \u0026micro;M), DA (5 \u0026micro;M), and glucose (5 \u0026micro;M) were separately added to the cells incubated in 0.1 M phosphate buffer and incubated for another 30 min. The cells were then washed three more times with phosphate-buffered saline. Finally, cellular fluorescence imaging was performed using a fluorescence microscope with an excitation wavelength of 380 nm.2.6 In vitro cytotoxicity study\u003c/p\u003e \u003cp\u003eMDA-MB-436 and MCF-10A cells were seeded into 96-well plates at a density of 3\u0026times;10⁵ cells per well, with 100 \u0026micro;L of culture medium added to each well, and incubated for 24 hours. After incubation, the original culture medium was removed, and 100 \u0026micro;L of fresh medium containing varying concentrations of the test substances (0, 10, 20, 40, 60, 80, 100 \u0026micro;g/mL) was added to each well. After a further 24 hours of incubation, 5 \u0026micro;L of MTT solution (5 mg/mL) was added to each well along with 100 \u0026micro;L of fresh culture medium, and the plates were incubated for 4 hours. Following incubation, the MTT-containing medium was discarded, and 150 \u0026micro;L of dimethyl sulfoxide (DMSO) was added to each well. The cells were lysed at 37\u0026deg;C in a dry incubator for 30 min. Finally, the absorbance of each well at 490 nm was measured using a microplate reader. Cell viability was calculated using the following formula: Cell viability (%) = \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\frac{\\text{t}\\text{e}\\text{s}\\text{t}\\:\\text{s}\\text{a}\\text{m}\\text{p}\\text{l}\\text{e}}{\\text{b}\\text{l}\\text{a}\\text{n}\\text{k}\\:\\text{s}\\text{a}\\text{m}\\text{p}\\text{l}\\text{e}}\\)\u003c/span\u003e\u003c/span\u003e\u0026times; 100%\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Detecting intracellular O₂⁻ levels using O-QD@Ag in MDA-MB-436 Cancer Cell\u003c/h2\u003e \u003cp\u003eO-QD@Ag was used as a fluorescent nanoprobe for imaging. MDA-MB-436 cells were seeded into 24-well plates at a density of 1\u0026times;10⁴ cells per well and incubated for 24 hours before treatment. The cells were then incubated in 200 \u0026micro;L of culture medium containing 10 \u0026micro;L of O-QD@Ag solution (100 \u0026micro;g/mL) at 37\u0026deg;C for 6 hours. After incubation, the MDA-MB-436 cells were washed three times with phosphate-buffered saline (PBS, pH 7.4) to remove residual nanoprobes. Subsequently, fresh medium containing different concentrations of O₂⁻ (0 mM, 0.5 mM, 1 mM, 1.5 mM, 2 mM) was added, and fluorescence imaging was performed using a fluorescence microscope with excitation at 380 nm. To evaluate the in vivo imaging capability of O-QD@Ag, LPS was added to induce autophagy in live cells to generate O₂⁻and fluorescence microscopy images were obtained after O₂⁻ scavenging by Tiron.\u003c/p\u003e"},{"header":"3 Results and Discussion","content":"\u003cp\u003e3.1 Synthesis of O-QD and O-QD@Ag composite\u003c/p\u003e \u003cp\u003eO-QD@Ag was successfully prepared to be used as a turn-on fluorescent probe to detect O₂⁻. Initially, O-QD was prepared using a hydrothermal synthesis method, and through the in-situ reduction of a silver nanolayer on its surface, the O-QD@Ag composite was successfully obtained\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. The morphology and average particle size of both O-QD and O-QD@Ag were characterized by transmission electron microscopy (TEM).\u003c/p\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, O-QD exhibited a spherical shape with a smooth surface and an average diameter of 3.42\u0026thinsp;\u0026plusmn;\u0026thinsp;1.17 nm. Similarly, O-QD@Ag (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb) also displayed a spherical shape with uniform distribution, with an average diameter of 4.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12 nm. These results indicate that the silver nanolayer was successfully deposited on the surface of O-QD as the particle size increased. Furthermore, the successful synthesis of O-QD@Ag was confirmed by mapping images (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec and S1). To evaluate the electrophoretic mobility and stability of the nanoparticles, both O-QD and O-QD@Ag were dissolved in PBS buffer, and their zeta (ζ) potentials were measured. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed, the ζ potentials of O-QD and O-QD@Ag were \u0026minus;\u0026thinsp;6.88 mV and 12.91 mV, respectively. Additionally, Fourier-transform infrared spectroscopy (FTIR) analysis further confirmed the functional group structures of O-QD and O-QD@Ag. As presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee, the peaks at 3364.46 cm⁻\u0026sup1; and 3215.98 cm⁻\u0026sup1; for O-QD are attributed to the stretching vibrations of -O-H and -N-H groups, respectively, while the peak at 2602.55 cm⁻\u0026sup1; corresponds to the stretching of -C-H\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. The absorption band around 1583.65 cm⁻\u0026sup1; is assigned to -C\u0026thinsp;=\u0026thinsp;O, -C\u0026thinsp;=\u0026thinsp;N, and -C\u0026thinsp;=\u0026thinsp;C groups\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Furthermore, absorption bands of -C-O and -C-H are also observed in the spectrum. Compared to O-QD, the vibration intensity of the peak near 1583.65 cm⁻\u0026sup1; in O-QD@Ag is reduced, which may be due to the reduction of -C\u0026thinsp;=\u0026thinsp;O or -C\u0026thinsp;=\u0026thinsp;N to -C-OH or -C-NH\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. X-ray photoelectron spectroscopy (XPS) was employed to further analyze the surface elemental composition of O-QD and O-QD@Ag. As shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef-i and S2, O-QD exhibited strong peaks at 284.80 eV, 398.49 eV, and 531.24 eV, corresponding to C1s, N1s, and O1s, respectively. In contrast, changes in binding energy for O-QD@Ag were primarily attributed to the reduction of functional groups during the in situ reduction of the silver nanolayer. The Ag spectrum of O-QD@Ag displayed two distinct peaks at 367.20 eV and 373.21 eV, corresponding to Ag3d5/2 and Ag3d3/2, which are characteristic of metallic Ag(0)\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. The appearance of the Ag3d peaks confirms the successful formation of a silver nanolayer in the final O-QD@Ag composite.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Optical properties of O-QD and O-QD@Ag\u003c/h2\u003e \u003cp\u003eWe subsequently characterized the fluorescence properties of the samples using UV-visible spectroscopy and a fluorescence spectrophotometer. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, no significant absorption peak was observed for the standalone CA, whereas a characteristic peak at 206 nm was present for OPD, which was also retained in O-QD. In Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, as the excitation wavelength varied from 360 nm to 460 nm (in 20 nm increments), the fluorescence emission of O-QD exhibited excitation dependence, confirming an optimal excitation wavelength of 380 nm and an emission wavelength of 570 nm.\u003c/p\u003e \u003cp\u003eThe stability of CDs is critical for real-time environmental and bioanalytical applications\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. To assess the stability of O-QD and O-QD@Ag, we investigated the fluorescence response under different concentrations of NaCl and varying pH levels. The results indicated that, when the NaCl concentration ranged from 0 to 0.5 M, the fluorescence intensity of the CDs remained relatively unchanged (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). Furthermore, the pH stability was evaluated by adjusting the pH of the solution from 5.5 to 8.5 using phosphoric acid and NaOH. The findings showed that fluorescence intensity decreased in both acidic and basic conditions but remained stable in a neutral environment (pH 6\u0026ndash;8), demonstrating excellent stability under physiological conditions (pH 7). In conclusion, O-QD@Ag holds great potential as a turn-on fluorescent probe for detecting O₂⁻ in biological systems.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e3.3 The specific binding of O-QD@Ag to the superoxide anion\u003c/h2\u003e \u003cp\u003eTo preliminarily assess the ability of O-QD@Ag to detect O₂⁻ in vitro, we measured the fluorescence changes of the O-QD@Ag solution before and after the addition of 2 mM O₂⁻. As shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef, the fluorescence of O-QD@Ag was effectively quenched under excitation at 380 nm, likely due to the plasmon resonance of the Ag nanolayer on the surface\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. After oxidation by O₂⁻, the fluorescence was restored, aligning with the emission peak of O-QD, and exhibiting a rapid response to O₂⁻. Next, the specific binding capability of O-QD@Ag to O₂⁻ was investigated. To determine the selectivity of the O-QD@Ag-based fluorescence assay for O₂⁻, we examined the fluorescence spectra of O-QD@Ag before and after the addition of O₂⁻ and other interfering substances. Compared to the other substances, O₂⁻ induced a 10-fold increase in fluorescence intensity, demonstrating the excellent selectivity of O-QD@Ag for O₂⁻ (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). To further confirm this result at the cellular level, O-QD@Ag was co-incubated with several endogenous substances, as shown in Figure S3, where fluorescence was restored in the presence of O₂⁻, further verifying the high specificity of O-QD@Ag for O₂⁻. Subsequently, detailed fluorescence titration experiments were conducted with O-QD@Ag and varying concentrations of O₂⁻. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb, the fluorescence intensity increased systematically as the O₂⁻ concentration rose from 1 mM to 4 mM. Additionally, the linear relationship between fluorescence intensity and O₂⁻ concentration was investigated. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec, a strong linear correlation (R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.994) was observed between fluorescence intensity and O₂⁻ concentration in the range of 0.2 mM to 1.6 mM. These findings suggest that O-QD@Ag holds the potential for dynamic in vitro monitoring of O₂⁻ concentrations.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Detecting Superoxide Anion Levels using O-QD@Ag in MDA-MB-436 cancer cells\u003c/h2\u003e \u003cp\u003eTo further evaluate the ability of O-QD@Ag to detect O₂⁻ levels, MDA-MB-436 cancer cells, and MCF-10A normal cells were chosen as models. Initially, MTT assays were conducted to assess the biocompatibility and safety of O-QD@Ag. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, when the concentrations of O-QD and O-QD@Ag ranged from 10 \u0026micro;g/mL to 100 \u0026micro;g/mL, the cell viability remained above 80%, indicating good biocompatibility. Based on the fluorescence intensity data, a concentration of 100 \u0026micro;g/mL was selected for subsequent experiments. Next, the ability of O-QD@Ag to detect O₂⁻ at the cellular level was assessed as a fluorescence probe. MDA-MB-436 cells were treated with O-QD@Ag, followed by the addition of different concentrations of O₂⁻ (0 mM, 0.5 mM, 1 mM, 1.5 mM, 2 mM), with the O₂⁻-free group serving as a control. The results showed that the fluorescence intensity of O-QD@Ag gradually recovered with increasing O₂⁻ concentrations, and this recovery was concentration-dependent (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea), which was consistent with the quantitative analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003eTo further investigate the application of this probe for detecting endogenous O₂⁻, lipopolysaccharide (LPS) was used to induce oxidative stress in the cells, thereby increasing the intracellular ROS levels\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. MDA-MB-436 cells were pretreated with LPS for 60 min to elevate the O₂⁻ content, followed by 30 min of incubation with the probe and imaging, which showed fluorescence signal recovery. To confirm that this signal change was indeed caused by O₂⁻, Tiron, a specific scavenger of O₂⁻\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e, was used to incubate the LPS-pretreated cells for 30 min, after which the probe was added for incubation and imaging (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The results demonstrated that Tiron could reverse the fluorescence signal changes induced by LPS. In conclusion, the cell imaging experiments confirmed that the O-QD@Ag probe is capable of effectively detecting endogenous O₂⁻ in cells.\u003c/p\u003e\u003c/div\u003e"},{"header":"4 Conclusion","content":"\u003cp\u003eIn summary, we successfully developed an O-QD@Ag fluorescent probe with yellow-green fluorescence properties. The probe exhibits a remarkable fluorescence recovery pattern in response to varying concentrations of superoxide anion (O₂⁻) solutions, with a strong linear correlation between the normalized fluorescence intensity and O₂⁻ concentration (R\u0026sup2; = 0.994). The effective detection range of the probe is 0.2-2 \u0026micro;M. Therefore, due to the high specificity between O-QD@Ag and superoxide anions, this probe allows for dynamic monitoring of intracellular O₂⁻ levels. We believe that this probe holds significant potential for further applications in the detection and prognosis of pathological processes in chronic diseases such as cancer, cardiovascular disorders, and neurodegenerative diseases.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflicts of interest\u003c/h2\u003e \u003cp\u003e\u0026ldquo;The authors have no relevant financial or non-financial interests to disclose.\u0026rdquo;\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eEthics approval\u003c/h2\u003e \u003cp\u003eThis is a critical study, so no ethical approval was involved in this study.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was financially supported by the Chongqing Bureau of Science and Technology (CSTB2024NSCQ-KJFZMSX0046). Funding results of the Action plan for high-quality development of graduate students of Chongqing University of Technology(gzlcx20243224).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eJiao Zhang: Data curation, Writing \u0026ndash; original draft. Bin Liao：Investigation, Resources, Formal analysis. YuXin Pei: Investigation, Formal analysis. ZhiYan Liu: Investigation, Formal analysis. YuXin Huang: Investigation, Resources. YuanQiang Wang: Investigation, Resources. Qinghua Yu: Conceptualization, Funding acquisition, validation, Writing \u0026ndash; review \u0026amp; editing, Supervision. Wanyi Chen: Conceptualization, Writing \u0026ndash; review \u0026amp; editing, Supervision, Project administration.\u003c/p\u003e\u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eThe raw data supporting the conclusions of this article will be made available by the authors on request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSies H, Jones DP (2020) Nat Rev Mol Cell Biol 21:363\u0026ndash;383\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSun M, Yu H, Zhu H, Ma F, Zhang S, Huang D, Wang S (2014) Anal Chem 86:671\u0026ndash;677\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXiao H, Zhang W, Li P, Zhang W, Wang X, Tang B (2020) \u003cem\u003eAngewandte Chemie (International ed. in English)\u003c/em\u003e, 59, 4216\u0026ndash;4230\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou J, Jangili P, Son S, Ji MS, Won M, Kim JS (2020) Adv Mater (Deerfield Beach Fla) 32:e2001945\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGaladari S, Rahman A, Pallichankandy S, Thayyullathil F (2017) Free Radic Biol Med 104:144\u0026ndash;164\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi X, Zhang B, Yan C, Li J, Wang S, Wei X, Jiang X, Zhou P, Fang J (2019) Nat Commun 10:2745\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVictor VM, Rocha M, Sol\u0026aacute; E, Ba\u0026ntilde;uls C, Garcia-Malpartida K (2009) Hern\u0026aacute;ndez-Mijares. 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225:121996\u003c/span\u003e\u003c/li\u003e \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":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-fluorescence","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jofl","sideBox":"Learn more about [Journal of Fluorescence](https://www.springer.com/journal/10895)","snPcode":"10895","submissionUrl":"https://submission.nature.com/new-submission/10895/3","title":"Journal of Fluorescence","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Carbon dots, Fluorescence sensor, Superoxide Anions, Nanoprobe","lastPublishedDoi":"10.21203/rs.3.rs-6467425/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6467425/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSuperoxide anion (O₂⁻), as a crucial member of reactive oxygen species (ROS), plays a pivotal role in cellular function regulation within biological systems and has been extensively implicated in the pathogenesis and progression of numerous diseases. In this paper, we successfully prepare a switching fluorescent probe (O-QD@Ag) for visual monitoring of superoxide anion (O₂⁻) by coating silver nanolayer on the surface of carbon quantum dots(CQDs). The experimental results show that the fluorescence signal of the composite is quenched in the presence of silver nanolayers. However, in the presence of O₂⁻, the fluorescence signal recovers due to the oxidation etching process of silver nanolayer, and there is a good linear relationship between the fluorescence intensity of the probe and the O₂⁻ concentration. In addition, O-QD@Ag composite exhibits excellent biocompatibility and low toxicity, which is characterized by low toxicity in normal MCF-10A and MDA-MB-436 cancer cells, and is suitable for selective detection of intracellular O₂⁻. To sum up, the probe not only provides an effective means to monitor the pathological process of chronic diseases such as cancer, cardiovascular diseases and neurodegenerative diseases but also shows the potential value of application in disease prognosis evaluation.\u003c/p\u003e","manuscriptTitle":"Switching-Type Fluorescence Probe Based on Carbon Dot Nanocomposites for the Visual Detection of Superoxide Anions","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-08 12:19:18","doi":"10.21203/rs.3.rs-6467425/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-05-22T16:31:45+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-14T17:56:14+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"58637048505710912553374291252909636841","date":"2025-05-05T20:41:58+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-05-05T16:12:17+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-04-24T14:34:31+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-04-24T14:32:27+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Fluorescence","date":"2025-04-17T03:02:44+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":"ff4ed421-f81d-4ade-9c82-228086014261","owner":[],"postedDate":"May 8th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-07-14T16:04:43+00:00","versionOfRecord":{"articleIdentity":"rs-6467425","link":"https://doi.org/10.1007/s10895-025-04414-8","journal":{"identity":"journal-of-fluorescence","isVorOnly":false,"title":"Journal of Fluorescence"},"publishedOn":"2025-07-11 15:57:17","publishedOnDateReadable":"July 11th, 2025"},"versionCreatedAt":"2025-05-08 12:19:18","video":"","vorDoi":"10.1007/s10895-025-04414-8","vorDoiUrl":"https://doi.org/10.1007/s10895-025-04414-8","workflowStages":[]},"version":"v1","identity":"rs-6467425","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6467425","identity":"rs-6467425","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}