Solid-phase pyrolysis synthesis of nitrogen-doped carbon dots as logic-gate fluorescent probes for dual detection of Cu²⁺ and glutathione

Solid-phase pyrolysis synthesis of nitrogen-doped carbon dots as logic-gate fluorescent probes for dual detection of Cu²⁺ and glutathione | 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 Solid-phase pyrolysis synthesis of nitrogen-doped carbon dots as logic-gate fluorescent probes for dual detection of Cu²⁺ and glutathione Zhuoru Yao, Cunjin Wang, Zixin Ma, Jing Zhang, Xiaoliang Zhao, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5672798/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 22 Feb, 2025 Read the published version in Journal of Fluorescence → Version 1 posted 7 You are reading this latest preprint version Abstract This study introduces the synthesis of nitrogen-doped carbon dots (N-L-Ser-CDs) from L-serine and urea through a straightforward and economical one-step solid-phase pyrolysis process. The incorporation of nitrogen into the carbon dots resulted in a remarkable 27.6-fold increase in fluorescence intensity, featuring a peak emission at 405 nm when excited at 330 nm and a significant fluorescence quantum yield of 22.5%. These N-L-Ser-CDs displayed a specific binding affinity for Cu 2+ , leading to a pronounced fluorescence quenching effect. However, upon interaction with glutathione (GSH), the fluorescence of the N-L-Ser-CDs + Cu 2+ complex was selectively restored. This restoration was attributed to the displacement of Cu 2+ from the surface of the N-L-Ser-CDs due to the strong interaction between GSH and Cu 2+ . The mechanism underlying this fluorescence quenching was elucidated as an electron transfer process from the excited state of the N-L-Ser-CDs to Cu 2+ . Additionally, the sensor developed in this study exhibited a linear detection range of 0–90 µM for Cu 2+ with a detection limit of 3 µM, and a linear detection range of 0-120 µM with a detection limit of 3 µM for GSH. By integrating the detection capabilities for both Cu 2+ and GSH, a successful logic-gated fluorescent probe was developed. Most importantly, this proposed method offers simplicity, affordability, and ease of use, while also showing potential for practical GSH detection in real urine samples. Solid-phase pyrolysis Logic-gate fluorescent probes Carbon dots Copper ion Glutathione Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. Induction Copper ions (Cu 2+ ), a critical trace element in the body, are integral to numerous fundamental physiological processes, including cell respiration, bone formation, and nerve function regulation. Consequently, the body's Cu 2+ levels are intricately linked to overall health, with imbalances potentially leading to health issues such as anemia, malnutrition, thrombocytopenia, axonal neuropathy, myelopathy, and demyelination of the central nervous system, among others [ 1 – 4 ]. Furthermore, environmental Cu 2+ can infiltrate the human body via the food chain, leading to adverse effects on the liver, kidneys, and nervous system when accumulated excessively [ 5 ]. Therefore, the development of simple and highly sensitive probes for Cu 2+ detection is paramount for maintaining health. Glutathione (GSH), a key thiol in the body, plays a pivotal role in the metabolism of proteins, amino acids, and hormones [ 6 ]. Imbalances in GSH levels in body fluids and tissues have been linked to specific diseases like cancer, HIV/AIDS infections, leukocyte deficiencies, liver damage, and various neurological disorders, including Parkinson's disease, stroke, and brain tumors [ 7 , 8 ]. Monitoring changes in GSH concentration can provide crucial insights into the body's overall health status. Consequently, the development of a rapid and reliable probe capable of detecting GSH levels in body fluids is of paramount importance. Currently, traditional methods for detecting Cu 2+ and GSH involve high-performance liquid chromatography, electrochemical techniques, enhanced Raman scattering, and mass spectrometry [ 9 , 10 ]. Despite their relative accuracy, the high costs associated with these methods, their complex operations, and the expense of necessary instruments restrict their widespread use [ 11 ]. Hence, there is an urgent need to develop cost-effective and user-friendly methods for detecting Cu 2+ and GSH. The use of carbon dots (CDs) as fluorescent probes has gained significant interest due to their low cost, excellent biocompatibility, and straightforward operation [ 12 – 14 ]. Nevertheless, a major challenge with CDs-based fluorescence probe methods is their relatively low detection sensitivity, which is often attributed to their low fluorescence quantum yield [ 15 ]. To address this issue, researchers typically enhance the quantum yield of CDs through two primary approaches: heteroatom doping and surface modification [ 16 , 17 ]. Among these, heteroatom doping is generally simpler and more effective compared to surface modification. Common heteroatoms used for doping include metallic atoms like Fe, Zn, Mn, Bi and Cu, as well as non-metallic atoms such as N, S, B, and F [ 18 – 21 ]. Non-metallic element-doped CDs are particularly favored for biological sample detection due to their superior biocompatibility. For instance, Cai et al. prepared nitrogen-doped CDs (N-CDs) using a hydrothermal method with 3-aminophenol and tartaric acid for detecting hydrogen sulfide ions and mitochondrial imaging [ 22 ]. Similarly, Sadhanala et al. synthesized N-CDs using 4-hydroxybenzaldehyde and 1,2,4,5-phenyltetramine tetrachloride as carbon and nitrogen sources, respectively, also via hydrothermal synthesis, and achieved effective detection of intracellular Mg 2+ [ 23 ]. Despite the advantages of N-CDs in terms of detection performance and biosafety, most reported N-CDs are synthesized using hydrothermal methods, which involve high pressures and are thus unsuitable for large-scale production. In contrast, the solid-phase pyrolysis method, which avoids the risks associated with high pressure and does not require a closed reaction vessel, is more conducive to large-scale production. However, reports on the preparation of N-CDs using solid-phase pyrolysis are limited. Therefore, further research into the solid-phase pyrolysis method for preparing N-CDs is necessary to explore its full potential. Based on the considerations outlined above, this study prepared N-L-Ser-CQDs via solid-phase pyrolysis using L-serine (L-Ser) as the carbon source and urea as the nitrogen source. These N-L-Ser-CQDs were developed into a logic-gated fluorescent probe for the detection of Cu²⁺ and GSH. The impact of N doping on the fluorescence intensity of L-Ser-CDs was thoroughly investigated. The microstructure and composition of the fluorescent probe were analyzed using TEM, XRD, FT-IR, XPS, and other characterization techniques. Finally, the probe was used to detect Cu²⁺ and GSH through fluorescence quenching and fluorescence recovery effects, and the detection limits were calculated and compared with those reported in the literature. 2. Experimental section 2.1 Chemical and materials L-Serine (L-Ser) was obtained from Shanghai Maclean Biochemical Technology Co., and urea was sourced from Tianjin Damao Chemical Reagent Factory. Reagents used for interference detection are detailed in the Electronic Supplementary Information section. All reagents were of analytical grade and were used without further purification. Ultrapure water was employed throughout the experimental process. 2.2 Instruments The instruments information of work was placed in the Electronic Supplementary Information. 2.3 Synthesis of N-L-Ser-CDs In brief, N-L-Ser-CQDs were synthesized using a one-pot solid-state pyrolysis method. As illustrated in Scheme 1 , 0.1 g of L-Ser and 0.4 g of urea were combined in a mortar and ground thoroughly. The resulting well-mixed white powder was transferred to a crucible and reacted at 180°C for 2 hours. Upon completion of the reaction and cooling to room temperature, 100 mL of H₂O was added, and the mixture was sonicated for 0.5 hours. It was then filtered through a 0.22 µm microporous membrane to remove larger insoluble particles. Finally, the solution was dialyzed using a 1000 Da dialysis bag for 48 hours to eliminate small molecule impurities, and the purified N-L-Ser-CDs solution was stored in a refrigerator at 4°C. To evaluate the impact of the N doping strategy, L-Ser-CDs were prepared using the same method described above, but without the addition of urea. 2.4 N-L-Ser-CDs detection of Cu 2+ Typically, various concentrations of Cu²⁺ solutions (0.5 mL) were added to 4.5 mL of N-L-Ser-CQDs, resulting in final Cu²⁺ concentrations ranging from 0 to 90 µM. The mixtures were then incubated at room temperature for 5 minutes, after which the fluorescence spectra were recorded. To ensure data reliability, all experiments were conducted in triplicate. 2.5 N-L-Ser-CDs/Cu 2+ detection of GSH The procedure for detecting GSH is similar to that for detecting Cu²⁺. The key difference is that GSH detection is carried out in a solution of N-L-Ser-CDs containing 90 µM Cu²⁺, with GSH concentrations ranging from 0 to 120 µM. 3. Results and discussion 3.1 Optical Properties of N-L-Ser-CDs N-L-Ser-CDs were synthesized using a one-pot solid-phase pyrolysis method. To achieve N-L-Ser-CDs with optimal fluorescence intensity, we optimized the reaction conditions, including the nitrogen source ratio, reaction temperature, and reaction time. The amount of L-Ser was consistently 0.1 g. As shown in Fig. 1 a, the fluorescence intensity of the N-L-Ser-CDs increased with the amount of urea, reaching a maximum when 0.4 g of urea was used, and then decreased gradually. Reaction temperature and time were further optimized with 0.1 g of L-Ser and 0.4 g of urea. According to Fig. 1 b and Fig. 1 c, the optimal reaction temperature was found to be 180°C, and the optimal reaction time was 2 hours. Under these conditions, the fluorescence intensity of the N-L-Ser-CDs was 27.6 times greater than that of the L-Ser-CDs (Fig. 1 d). This demonstrates that nitrogen doping effectively enhanced the fluorescence intensity of the L-Ser-CDs. The optical properties of N-L-Ser-CDs were characterized using 3D and 2D fluorescence spectra. As shown in Fig. 2 a, the 3D fluorescence spectra of N-L-Ser-CDs reveal a distinct luminescence peak with excitation and emission wavelengths at 330 nm and 405 nm, respectively. The 2D fluorescence spectrum (Fig. 2 b) confirms these findings, showing that the optimal excitation wavelength is 330 nm and the optimal emission wavelength is 405 nm. From the UV absorption spectra, it is evident that N-L-Ser-CDs exhibit significant absorption between 200 and 420 nm, attributed to the π-π* transition of C = C and the n-π* transition of C = O/N. Further investigation into the relationship between the emission wavelength and excitation wavelength of these CDs is shown in Fig. 2 c. It is observed that as the excitation wavelength increases from 280 to 380 nm, the emission wavelength gradually red-shifts, and the emission intensity first increases before decreasing. This behavior is characteristic of CDs and arises from their varied emission sites associated with both core and surface states [ 24 ]. The quantum yield of the CDs, calculated using the five-point method, is 22.5% (Fig. S1 ), which is higher than that reported in previous studies [ 25 – 28 ], indicating that the N-L-Ser-CDs prepared in this work possess excellent fluorescence properties. The colloidal optical stability of the N-L-Ser-CDs was further assessed. The colloidal optical stability of the N-L-Ser-CDs was further assessed. As shown in Fig. 2 d, the fluorescence intensity of N-L-Ser-CDs remains relatively stable even when exposed to NaCl concentrations up to 1 M, demonstrating their good colloidal stability. Figure 2 e illustrates the fluorescence intensity of N-L-Ser-CDs under 365 nm ultraviolet irradiation. The fluorescence intensity does not significantly decrease even after 2.5 h of continuous UV exposure, indicating strong photobleaching resistance and suitability for prolonged monitoring. Figure 2 f displays the fluorescence intensity of N-L-Ser-CDs in solutions of varying pH. The fluorescence intensity decreases slightly in acidic environments but remains strong in alkaline conditions, showing that pH changes do not substantially quench the fluorescence of N-L-Ser-CDs. Overall, these results confirm the excellent colloidal and optical stability of the N-L-Ser-CDs. 3.2 Structural characterization of N-L-Ser-CDs The microstructure of N-L-Ser-CDs was investigated using transmission electron microscopy (TEM). As depicted in Fig. 3 a, N-L-Ser-CDs exhibit an approximately spherical shape and are uniformly dispersed. The particle size distribution ranges from 4.6 to 9.2 nm, with an average particle size of 6.56 nm (Fig. 3 b). TEM images reveal that N-L-Ser-CDs lack distinct lattice fringes, indicating they have low crystallinity and are amorphous materials. The diffraction peak at 21.4° in the X-ray diffraction (XRD) pattern further confirms the amorphous nature of N-L-Ser-CDs (Fig. 3 c) [ 29 ], consistent with the TEM observations. The functional groups on the surface of CDs were studied by Fourier transform infrared (FT-IR) spectroscopy. As shown in Fig. 3 d, the broad peaks located around 3300 cm − 1 are attributed to the stretching vibrations of -OH and -NH 2 [30] . The peak located at 1701 cm − 1 corresponds to the characteristic peak of -COOH. The peak located at 1327 cm − 1 represents the characteristic peak of C-O/C-N. The peak located at 756 cm − 1 is associated with C-H groups [ 18 ]. The FT-IR results indicate the presence of hydrophilic functional groups such as amino, hydroxyl, and carboxyl groups on the surface of N-L-Ser-CDs, which enhances their water solubility and makes them suitable for use in aqueous environments. Additionally, the elemental composition of N-L-Ser-CDs was quantitatively analyzed using X-ray photoelectron spectroscopy (XPS). As shown in Fig. 4 a, the XPS survey spectrum shows peaks for C 1s at 284.08 eV, N 1s at 400.08 eV, and O 1s at 532.08 eV, with elemental percentages of 44.17%, 10.06%, and 45.77%, respectively. The high-resolution XPS spectrum of C 1s (Fig. 4 b) can be deconvoluted into three peaks at 284.80 eV, 286.66 eV, and 289.16 eV, corresponding to C = C/C-C, C-N/C-O, and C = N/C = O, respectively [ 31 ]. The high-resolution XPS spectrum of N 1s (Fig. 4 c) shows two peaks at 400.26 eV and 402 eV, corresponding to C-N = C and -NH 2 , respectively [ 32 ]. The high-resolution XPS spectrum of O 1s (Fig. 4 d) reveals three peaks at 531.46 eV, 532.88 eV, and 536.21 eV, corresponding to C = O, O-C = O, and C-O, respectively [ 33 ]. These XPS results are consistent with the FT-IR findings, confirming the presence of numerous hydrophilic functional groups on the surface of N-L-Ser-CDs. 3.3 Determination of Cu 2+ and GSH The basic principle of the logic gate detection of N-L-Ser-CDs for Cu²⁺ and GSH is that Cu²⁺ can significantly quench the fluorescence of N-L-Ser-CDs, while GSH can bind with Cu²⁺ and thereby restore the fluorescence. The selectivity of N-L-Ser-CDs for Cu²⁺ was first examined, as shown in Fig. 5 a, by using common metal ions (Ag + , K + , Na + , Pb 2+ , Mn 2+ , Fe 2+ , Zn 2+ , Cu 2+ , Mg 2+ , Ca 2+ , Al 3+ , Cd 2+ , Cr 3+ , Ba 2+ , Hg 2+ , Ni 2+ ) and biomolecules (L-Cys, Gly, Glucose, L-Phe, L-Ser, L-Ala, L-Asp, L-Lys, AA, L-Arg, FA, UA, Urea, DA, GSH, H 2 O 2 ) as potential interferents to assess their impact on the fluorescence intensity of N-L-Ser-CDs. It was observed that only Cu²⁺ significantly quenched the fluorescence of N-L-Ser-CDs, confirming the excellent selectivity of N-L-Ser-CDs for Cu²⁺. The selectivity of the N-L-Ser-CDs/Cu²⁺ system for GSH was then investigated, with common biomolecules as interferents, as shown in Fig. 5 b. It was found that GSH could significantly restore the fluorescence of N-L-Ser-CDs that had been quenched by Cu²⁺, whereas other interferents did not produce the same effect, demonstrating the excellent selectivity of the N-L-Ser-CDs/Cu²⁺ system for GSH. Additionally, as illustrated in the physical image (Fig. 5 c), the fluorescence of N-L-Ser-CDs decreased after the addition of Cu²⁺ and was subsequently restored upon the addition of GSH. The fluorescence spectra of N-L-Ser-CDs at different concentrations of Cu²⁺ are presented in Fig. 6 a. It can be observed that the fluorescence intensity of N-L-Ser-CDs decreases regularly as the concentration of Cu²⁺ increases from 0 to 90 µM. As shown in Fig. 6 b, the relationship between Cu²⁺ concentration and fluorescence intensity (F/F₀) follows a linear equation: F/F 0 = 0.959-0.006C[Cu 2+ ], R 2 = 0.983. Here, F 0 represents the fluorescence intensity of the blank sample and F represents the fluorescence intensity of the test sample. Based on the 3σ rule, the detection limit is calculated to be 3 µM. The fluorescence images of N-L-Ser-CDs after the addition of various concentrations of Cu²⁺ are shown in Fig. 6 c, visually indicating that the fluorescence intensity decreases with increasing Cu²⁺ concentration. Consequently, semi-quantitative detection of Cu²⁺ can also be achieved. The fluorescence spectra of N-L-Ser-CDs/Cu²⁺ at different concentrations of GSH are shown in Fig. 7 a. It is evident that the fluorescence intensity of N-L-Ser-CDs/Cu²⁺ increases regularly as the GSH concentration rises from 0 to 120 µM. As depicted in Fig. 7 b, the relationship between GSH concentration and fluorescence intensity (F/F₀) follows a linear equation: F/F 0 = 0.959-0.006C[GSH], with an R² value of 0.983. Based on the 3σ rule, the detection limit is determined to be 3 µM. The fluorescence images of N-L-Ser-CDs/Cu²⁺ after the addition of various concentrations of GSH are presented in Fig. 7 c, clearly showing that the fluorescence intensity increases with higher GSH concentrations. Therefore, semi-quantitative detection of GSH can also be achieved. The fluorescent probes based on CDs reported in this work demonstrate detection ranges and limits that are comparable to or even superior to those of sensors previously reported for the detection of Cu²⁺ or GSH (see Table 1 ). Table 1 Comparison of the N-L-Ser-CDs fluorescent probe with other methods for detecting Cu 2+ and GSH. Analytical method Detection target Linear range LOD Ref AuNCs/N-CDs Cu 2+ 10–150 3.5 [ 34 ] GSH@CDs-AuNCs Cu 2+ 1–10 2.59 [ 35 ] CuNPs Cu 2+ 15–35 5.6 [ 36 ] N-L-Ser-CDs Cu 2+ 0–90 µM 3 µM This work N-CDs GSH 50-4.83 7.83 [ 37 ] N-CDs/Cu 2+ GSH 19–52 - [ 38 ] N,S-CDs-MnO 2 GSH 0-250 28.5 [ 39 ] N-L-Ser-CDs + Cu 2+ GSH 0-120 µM 3 µM This work The mechanism of Cu 2+ quenching the fluorescence of N-L-Ser-CDs is mainly due to the binding of Cu 2+ to the -NH 2 and -OH groups on the surface of N-L-Ser-CDs, which affects the electron transfer process of the excited state of N-L-Ser-CDs and makes the fluorescence decrease [ 40 , 41 ]. As shown in Fig. 8 , the disappearance of the -NH₂ characteristic peak in the XPS spectrum of N-L-Ser-CDs + Cu²⁺ (Fig. 8 a) and the absence of the C-O characteristic peak in the XPS spectrum of O1s (Fig. 8 b) confirm that Cu²⁺ binds to the -NH₂ and -OH groups on the surface of N-L-Ser-CDs. Additionally, Fig. 8 d shows that the fluorescence lifetime of N-L-Ser-CDs is 6.404 ns, whereas for N-L-Ser-CDs + Cu²⁺ it is 5.966 ns. This decrease in fluorescence lifetime indicates that the excited electrons in N-L-Ser-CDs are transferred, leading to reduced fluorescence. GSH, which contains sulfhydryl groups, binds to Cu²⁺ with much higher affinity than N-L-Ser-CDs. Therefore, the addition of GSH can displace Cu²⁺ from N-L-Ser-CDs, thereby restoring the fluorescence of N-L-Ser-CDs [ 42 ]. As shown in Fig. 8 c, the UV-vis absorption baseline of N-L-Ser-CDs + Cu²⁺+GSH is significantly higher than that of N-L-Ser-CDs and N-L-Ser-CDs + Cu²⁺, indicating the formation of a GSH-Cu²⁺ complex. Furthermore, the fluorescence lifetime of the system was restored to 6.314 ns after the addition of GSH (Fig. 8 d), demonstrating that the fluorescence of N-L-Ser-CDs can be recovered through the complexation of GSH with Cu²⁺. 3.4. Detection of GSH in real samples To evaluate the potential of the prepared N-L-Ser-CDs fluorescent probes for practical applications, we monitored GSH levels in urine samples. Urine samples from healthy laboratory personnel were collected, centrifuged, and filtered. These samples were then spiked with different concentrations of GSH using the standard addition method for determination. The results, presented in Table 2 , show GSH recoveries ranging from 99.728–106.070%. These satisfactory recovery rates confirm the suitability of the developed N-L-Ser-CDs fluorescent probes for detecting GSH in real samples. Table 2 Results of GSH determination in real urine samples and recovery analysis. Samples Detecting Subject Added (µM) Found (µM) Recovery (%) RSD (%, n = 3) Urine GSH 0 2.809 - - 10 13.243 99.728 7.63 40 45.698 106.070 2.38 80 85.147 102.346 1.96 4. Conclusion In summary, this work presents the synthesis of a simple and cost-effective N-L-Ser-CDs using a one-pot solid-phase pyrolysis method with L-serine and urea as precursors. The resulting N-L-Ser-CDs exhibit strong fluorescence intensity and impressive quantum yields of up to 22.5% at 405 nm emission when excited at 330 nm. Additionally, a logic-gate-based fluorescent probe has been developed, demonstrating high sensitivity and selectivity for detecting Cu²⁺ and GSH in the concentration ranges of 0–90 µM and 0-120 µM, respectively, with detection limits of 3 µM for both analytes. Notably, this method shows excellent recovery rates (99.728–106.070%) for detecting GSH in real urine samples, underscoring the potential practical applications of this innovative approach. Declarations Conflicts of interest statement The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Author Contribution Zhuoru Yao and Cunjin Wang wrote the main manuscript text.Zixin Ma and Jing Zhang mainly study the details of the experiment.Xiaoliang Zhao, Weijie Zhang and Huanxian Shi mainly provide financial support. Acknowledgements This work was supported by the National Natural Science Foundation of China (Nos. 82405028 and 32260231), the Gansu Provincial Natural Science Fund (No.23JRRA840), the PhD Start-up Foundation of Lanzhou University of Technology, and Red Willow Excellent Youth Talent Support Program of Lanzhou University of Technology. References Yan L, Li J, Cai H, Shao Y, Zhang G, Chen L, Wang Y, Zong H, Yin Y (2023) Carbon dots/Ag nanoclusters-based fluorescent probe for ratiometric and visual detection of Cu 2+ . 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Spectrochim Acta Part A Mol Biomol Spectrosc 223:117300 Wu J, Li R, Liu S (2022) A novel dual-emission fluorescent probe for ratiometric and visual detection of Cu 2+ ions and Ag + ions. Anal Bioanal Chem 414:3067–3075 Qing Z, Zhu L, Yang S, Cao Z, He X, Wang K, Yang R (2016) In situ formation of fluorescent copper nanoparticles for ultrafast zero-background Cu 2+ detection and its toxicides screening. Biosens Bioelectron 78:471–476 Lin M, Ma X, Lin S, Zhang X, Dai Y, Xia F (2020) Fluorescent probe based on N-doped carbon dots for the detection of intracellular pH and glutathione. RSC Adv 10:33635–33641 Yang F, Zhou P, Duan C (2021) Solid-phase synthesis of red dual-emissive nitrogen-doped carbon dots for the detection of Cu 2+ and glutathione. Microchem J 169:106534 Sohal N, Maity B, Basu S (2022) Morphology Effect of One-Dimensional MnO 2 Nanostructures on Heteroatom-Doped Carbon Dot-Based Biosensors for Selective Detection of Glutathione. ACS Appl Bio Mater 5:2355–2364 Liu Y, Seidi F, Deng C, Li R, Xu T, Xiao H (2021) Porphyrin derived dual-emissive carbon quantum dots: Customizable synthesis and application for intracellular Cu2 + quantification. Sens Actuators B 343:130072 Zhou W, Mo F, Sun Z, Luo J, Fan J, Zhu H, Zhu Z, Huang J, Zhang X (2022) Bright red-emitting P, Br co-doped carbon dots as OFF-ON fluorescent probe for Cu 2+ and L-cysteine detection. J Alloys Compd 897:162731 Sun X, Wang C, Li P, Shao Z, Xia J, Liu Q, Shen F, Fang Y (2022) The facile synthesis of nitrogen and sulfur co-doped carbon dots for developing a powerful on-off-on fluorescence probe to detect glutathione in vegetables. Food Chem 372:131142 Schemes Scheme 1 is available in the Supplementary Files section Additional Declarations No competing interests reported. Supplementary Files SupplementaryMaterials.docx image1.png Scheme 1. Schematic diagram of the synthesis of N-L-Ser-CDs. Cite Share Download PDF Status: Published Journal Publication published 22 Feb, 2025 Read the published version in Journal of Fluorescence → Version 1 posted Editorial decision: Revision requested 21 Jan, 2025 Reviews received at journal 21 Jan, 2025 Reviewers agreed at journal 05 Jan, 2025 Reviewers invited by journal 31 Dec, 2024 Editor assigned by journal 19 Dec, 2024 Submission checks completed at journal 19 Dec, 2024 First submitted to journal 18 Dec, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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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-5672798","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":392318078,"identity":"397ee7dd-7e11-412e-9c1d-8c13ceae2f81","order_by":0,"name":"Zhuoru Yao","email":"","orcid":"","institution":"Lanzhou University of Information Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Zhuoru","middleName":"","lastName":"Yao","suffix":""},{"id":392318079,"identity":"ae1a2ab8-7b1c-4b83-ac7b-17190ab4568f","order_by":1,"name":"Cunjin Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4ElEQVRIiWNgGAWjYBADZjYG5oMHwMwDxGthSzjAkECCFiDgMSBOi8Hxs4df/Gy7w84n3fPhMO8PBjm+GwmMnwvwaTmTl2bZ2/aMmU3m7IbDPAkMxpI3EpilZ+DRYnYgx8yYse0wM5tELlhL4oYbCWzMPPi0nH8D05LzAKSlnrCWGznGj6FaGEBaEgwIabG/8caMseccSEuawcE5aRKGM888bJbGp0WyP8f4w4+yw8nyM5IfPnhjYyPPdzz54Gd8WoCATQJIJEM5IDZjA34NwIj/ACTsCKkaBaNgFIyCEQwA2sFMUeeWmAYAAAAASUVORK5CYII=","orcid":"","institution":"Lanzhou University of Technology","correspondingAuthor":true,"prefix":"","firstName":"Cunjin","middleName":"","lastName":"Wang","suffix":""},{"id":392318081,"identity":"87877898-7d57-4b0e-a36a-2580b4a1cf80","order_by":2,"name":"Zixin Ma","email":"","orcid":"","institution":"Lanzhou University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Zixin","middleName":"","lastName":"Ma","suffix":""},{"id":392318082,"identity":"fef5f949-8687-4507-976a-8596969d9738","order_by":3,"name":"Jing Zhang","email":"","orcid":"","institution":"Lanzhou University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Jing","middleName":"","lastName":"Zhang","suffix":""},{"id":392318084,"identity":"3c92a1d5-c67e-429f-b388-d109fee65db6","order_by":4,"name":"Xiaoliang Zhao","email":"","orcid":"","institution":"Lanzhou University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Xiaoliang","middleName":"","lastName":"Zhao","suffix":""},{"id":392318086,"identity":"ae547d8b-fcd8-4a65-9368-cee620680f4d","order_by":5,"name":"Weijie Zhang","email":"","orcid":"","institution":"Lanzhou University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Weijie","middleName":"","lastName":"Zhang","suffix":""},{"id":392318087,"identity":"8dac2ae5-f820-4b6e-bc95-fc4bd5973c32","order_by":6,"name":"Huanxian Shi","email":"","orcid":"","institution":"Shaanxi University of Chinese Medicine, Shaanxi University Engineering Research Center of Traditional Chinese Medicine and Aroma Industry","correspondingAuthor":false,"prefix":"","firstName":"Huanxian","middleName":"","lastName":"Shi","suffix":""}],"badges":[],"createdAt":"2024-12-19 02:08:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5672798/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5672798/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10895-025-04206-0","type":"published","date":"2025-02-22T15:58:06+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":72156677,"identity":"d0feb191-361d-4c48-a0af-44307bfbf7ec","added_by":"auto","created_at":"2024-12-23 08:58:46","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":457961,"visible":true,"origin":"","legend":"\u003cp\u003ePreparation conditions for N-L-Ser-CDs: (a) optimization of Urea mass, (b) reaction temperature and (c) reaction time. (d) Fluorescence emission of L-Ser-CDs and N-L-Ser-CDs.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-5672798/v1/c65bb37a9fe5e8ac1b742fdd.png"},{"id":72156680,"identity":"d2a6ed01-1fef-4774-8858-905a2a7d1c4d","added_by":"auto","created_at":"2024-12-23 08:58:46","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":284796,"visible":true,"origin":"","legend":"\u003cp\u003e(a) 3D fluorescence spectra of N-L-Ser-CDs, (b) absorption and 2D fluorescence spectra of N-L-Ser-CDs, (c) Fluorescence emission of N-L-Ser-CD under different excitation. (d) photoluminescence (PL) intensity of N-L-Ser-CDs in various NaCl concentrations, (e) photobleaching resistance, and (f) pH stability of N-L-Ser-CDs.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-5672798/v1/f3e55a544539a03d02a81b89.png"},{"id":72156684,"identity":"d370099b-1bde-4eff-937c-4dffbfbbced3","added_by":"auto","created_at":"2024-12-23 08:58:46","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":587862,"visible":true,"origin":"","legend":"\u003cp\u003e(a) TEM, (b) histogram of particle size distribution, (c) XRD pattern, and (d) FT-IR spectrum of N-L-Ser-CDs.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-5672798/v1/9072902ad846f7c15ac889aa.png"},{"id":72156981,"identity":"e804f75c-2b3a-4308-b452-103a76eaf4ed","added_by":"auto","created_at":"2024-12-23 09:06:46","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":293970,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Survey spectrum, (b) C 1\u003cem\u003es\u003c/em\u003e spectrum, (c) N 1\u003cem\u003es\u003c/em\u003espectrum, and (d) O 1\u003cem\u003es\u003c/em\u003e spectrum of the XPS analysis of N-L-Ser-CDs.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-5672798/v1/efac934ddb2340bc2746df8c.png"},{"id":72156686,"identity":"71a70bba-bf36-45ac-95aa-10515dc534cc","added_by":"auto","created_at":"2024-12-23 08:58:46","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":389163,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Fluorescence intensity of N-L-Ser-CDs in the presence of various interferents at 100 μM concentration. (b) Fluorescence intensity of N-L-Ser-CDs/Cu\u003csup\u003e2+\u003c/sup\u003e complex in the presence of different interferents at 100 μM. (c) Fluorescence images of N-L-Ser-CDs, N-L-Ser-CDs with Cu\u003csup\u003e2+\u003c/sup\u003e, and N-L-Ser-CDs/Cu\u003csup\u003e2+\u003c/sup\u003e complex with GSH.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-5672798/v1/0ffe4f4ddb690f7bf30d91ca.png"},{"id":72156985,"identity":"0d1d58e6-d14a-44de-982b-deb6682335b3","added_by":"auto","created_at":"2024-12-23 09:06:47","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":549819,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Fluorescence emission spectra of N-L-Ser-CDs in the presence of Cu\u003csup\u003e2+\u003c/sup\u003e; (b) Linear relationship between Cu\u003csup\u003e2+\u003c/sup\u003e concentration and fluorescence intensity F/F\u003csub\u003e0\u003c/sub\u003e of N-L-Ser-CDs. (c) Photographs of N-L-Ser-CDs aqueous solutions with varying concentrations of Cu\u003csup\u003e2+\u003c/sup\u003e under a 365 nm UV lamp.\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-5672798/v1/0b0acc43683bbaee3e348cfc.png"},{"id":72156689,"identity":"b12eda32-aac1-4332-90c4-86f5808ed529","added_by":"auto","created_at":"2024-12-23 08:58:46","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":513402,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Fluorescence emission spectra of N-L-Ser-CDs/Cu\u003csup\u003e2+\u003c/sup\u003e in the presence of GSH; (b) Linear relationship between GSH concentration and fluorescence intensity ratio (F/F\u003csub\u003e0\u003c/sub\u003e) of N-L-Ser-CDs/Cu\u003csup\u003e2+\u003c/sup\u003e. (c) Photographs of N-L-Ser-CDs/Cu\u003csup\u003e2+\u003c/sup\u003e aqueous solutions with varying concentrations of GSH under a 365 nm UV lamp.\u003c/p\u003e","description":"","filename":"image8.png","url":"https://assets-eu.researchsquare.com/files/rs-5672798/v1/50246ecfe18bd7be6e80a6f3.png"},{"id":72156702,"identity":"9c7ae08f-f5ef-449a-b652-b1e1f2da2a54","added_by":"auto","created_at":"2024-12-23 08:58:47","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":376669,"visible":true,"origin":"","legend":"\u003cp\u003eXPS spectra of (a) N1s and (b) O1s for N-L-Ser-CDs and N-L-Ser-CDs+Cu\u003csup\u003e2+\u003c/sup\u003e. (c) UV-vis absorption spectra and (d) fluorescence lifetime spectra of N-L-Ser-CDs, N-L-Ser-CDs+Cu\u003csup\u003e2+\u003c/sup\u003e, and N-L-Ser-CDs+Cu\u003csup\u003e2+\u003c/sup\u003e+GSH.\u003c/p\u003e","description":"","filename":"image9.png","url":"https://assets-eu.researchsquare.com/files/rs-5672798/v1/586b6a62dd23f19a4b23a8c1.png"},{"id":77053634,"identity":"e01ef7dc-623b-40d1-a204-bdced5cecfbe","added_by":"auto","created_at":"2025-02-24 16:29:52","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4686101,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5672798/v1/a00fadaa-6ee7-423c-8d97-f655ff742750.pdf"},{"id":72156980,"identity":"fff1869f-a576-49a9-b829-ef1c2bbdd82d","added_by":"auto","created_at":"2024-12-23 09:06:46","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":213244,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-5672798/v1/fe898041e5ba30c706d2fdad.docx"},{"id":72156979,"identity":"2b1af40e-5750-4493-b0fc-621a193312cf","added_by":"auto","created_at":"2024-12-23 09:06:46","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":240630,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScheme 1. \u003c/strong\u003eSchematic diagram of the synthesis of N-L-Ser-CDs.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-5672798/v1/a73986f47899827997195d7f.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Solid-phase pyrolysis synthesis of nitrogen-doped carbon dots as logic-gate fluorescent probes for dual detection of Cu²⁺ and glutathione","fulltext":[{"header":"1. Induction","content":"\u003cp\u003eCopper ions (Cu\u003csup\u003e2+\u003c/sup\u003e), a critical trace element in the body, are integral to numerous fundamental physiological processes, including cell respiration, bone formation, and nerve function regulation. Consequently, the body's Cu\u003csup\u003e2+\u003c/sup\u003e levels are intricately linked to overall health, with imbalances potentially leading to health issues such as anemia, malnutrition, thrombocytopenia, axonal neuropathy, myelopathy, and demyelination of the central nervous system, among others [\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Furthermore, environmental Cu\u003csup\u003e2+\u003c/sup\u003e can infiltrate the human body via the food chain, leading to adverse effects on the liver, kidneys, and nervous system when accumulated excessively [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Therefore, the development of simple and highly sensitive probes for Cu\u003csup\u003e2+\u003c/sup\u003e detection is paramount for maintaining health. Glutathione (GSH), a key thiol in the body, plays a pivotal role in the metabolism of proteins, amino acids, and hormones [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Imbalances in GSH levels in body fluids and tissues have been linked to specific diseases like cancer, HIV/AIDS infections, leukocyte deficiencies, liver damage, and various neurological disorders, including Parkinson's disease, stroke, and brain tumors [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Monitoring changes in GSH concentration can provide crucial insights into the body's overall health status. Consequently, the development of a rapid and reliable probe capable of detecting GSH levels in body fluids is of paramount importance. Currently, traditional methods for detecting Cu\u003csup\u003e2+\u003c/sup\u003e and GSH involve high-performance liquid chromatography, electrochemical techniques, enhanced Raman scattering, and mass spectrometry [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Despite their relative accuracy, the high costs associated with these methods, their complex operations, and the expense of necessary instruments restrict their widespread use [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Hence, there is an urgent need to develop cost-effective and user-friendly methods for detecting Cu\u003csup\u003e2+\u003c/sup\u003e and GSH.\u003c/p\u003e \u003cp\u003eThe use of carbon dots (CDs) as fluorescent probes has gained significant interest due to their low cost, excellent biocompatibility, and straightforward operation [\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Nevertheless, a major challenge with CDs-based fluorescence probe methods is their relatively low detection sensitivity, which is often attributed to their low fluorescence quantum yield [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. To address this issue, researchers typically enhance the quantum yield of CDs through two primary approaches: heteroatom doping and surface modification [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Among these, heteroatom doping is generally simpler and more effective compared to surface modification. Common heteroatoms used for doping include metallic atoms like Fe, Zn, Mn, Bi and Cu, as well as non-metallic atoms such as N, S, B, and F [\u003cspan additionalcitationids=\"CR19 CR20\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Non-metallic element-doped CDs are particularly favored for biological sample detection due to their superior biocompatibility. For instance, Cai et al. prepared nitrogen-doped CDs (N-CDs) using a hydrothermal method with 3-aminophenol and tartaric acid for detecting hydrogen sulfide ions and mitochondrial imaging [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Similarly, Sadhanala et al. synthesized N-CDs using 4-hydroxybenzaldehyde and 1,2,4,5-phenyltetramine tetrachloride as carbon and nitrogen sources, respectively, also via hydrothermal synthesis, and achieved effective detection of intracellular Mg\u003csup\u003e2+\u003c/sup\u003e [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Despite the advantages of N-CDs in terms of detection performance and biosafety, most reported N-CDs are synthesized using hydrothermal methods, which involve high pressures and are thus unsuitable for large-scale production. In contrast, the solid-phase pyrolysis method, which avoids the risks associated with high pressure and does not require a closed reaction vessel, is more conducive to large-scale production. However, reports on the preparation of N-CDs using solid-phase pyrolysis are limited. Therefore, further research into the solid-phase pyrolysis method for preparing N-CDs is necessary to explore its full potential.\u003c/p\u003e \u003cp\u003eBased on the considerations outlined above, this study prepared N-L-Ser-CQDs via solid-phase pyrolysis using L-serine (L-Ser) as the carbon source and urea as the nitrogen source. These N-L-Ser-CQDs were developed into a logic-gated fluorescent probe for the detection of Cu\u0026sup2;⁺ and GSH. The impact of N doping on the fluorescence intensity of L-Ser-CDs was thoroughly investigated. The microstructure and composition of the fluorescent probe were analyzed using TEM, XRD, FT-IR, XPS, and other characterization techniques. Finally, the probe was used to detect Cu\u0026sup2;⁺ and GSH through fluorescence quenching and fluorescence recovery effects, and the detection limits were calculated and compared with those reported in the literature.\u003c/p\u003e"},{"header":"2. Experimental section","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Chemical and materials\u003c/h2\u003e \u003cp\u003eL-Serine (L-Ser) was obtained from Shanghai Maclean Biochemical Technology Co., and urea was sourced from Tianjin Damao Chemical Reagent Factory. Reagents used for interference detection are detailed in the Electronic Supplementary Information section. All reagents were of analytical grade and were used without further purification. Ultrapure water was employed throughout the experimental process.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Instruments\u003c/h2\u003e \u003cp\u003eThe instruments information of work was placed in the Electronic Supplementary Information.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Synthesis of N-L-Ser-CDs\u003c/h2\u003e \u003cp\u003eIn brief, N-L-Ser-CQDs were synthesized using a one-pot solid-state pyrolysis method. As illustrated in Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e0.1\u003c/span\u003e g of L-Ser and 0.4 g of urea were combined in a mortar and ground thoroughly. The resulting well-mixed white powder was transferred to a crucible and reacted at 180\u0026deg;C for 2 hours. Upon completion of the reaction and cooling to room temperature, 100 mL of H₂O was added, and the mixture was sonicated for 0.5 hours. It was then filtered through a 0.22 \u0026micro;m microporous membrane to remove larger insoluble particles. Finally, the solution was dialyzed using a 1000 Da dialysis bag for 48 hours to eliminate small molecule impurities, and the purified N-L-Ser-CDs solution was stored in a refrigerator at 4\u0026deg;C.\u003c/p\u003e \u003cp\u003eTo evaluate the impact of the N doping strategy, L-Ser-CDs were prepared using the same method described above, but without the addition of urea.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 N-L-Ser-CDs detection of Cu\u003csup\u003e2+\u003c/sup\u003e\u003c/h2\u003e \u003cp\u003eTypically, various concentrations of Cu\u0026sup2;⁺ solutions (0.5 mL) were added to 4.5 mL of N-L-Ser-CQDs, resulting in final Cu\u0026sup2;⁺ concentrations ranging from 0 to 90 \u0026micro;M. The mixtures were then incubated at room temperature for 5 minutes, after which the fluorescence spectra were recorded. To ensure data reliability, all experiments were conducted in triplicate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 N-L-Ser-CDs/Cu\u003csup\u003e2+\u003c/sup\u003e detection of GSH\u003c/h2\u003e \u003cp\u003eThe procedure for detecting GSH is similar to that for detecting Cu\u0026sup2;⁺. The key difference is that GSH detection is carried out in a solution of N-L-Ser-CDs containing 90 \u0026micro;M Cu\u0026sup2;⁺, with GSH concentrations ranging from 0 to 120 \u0026micro;M.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and discussion","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Optical Properties of N-L-Ser-CDs\u003c/h2\u003e \u003cp\u003eN-L-Ser-CDs were synthesized using a one-pot solid-phase pyrolysis method. To achieve N-L-Ser-CDs with optimal fluorescence intensity, we optimized the reaction conditions, including the nitrogen source ratio, reaction temperature, and reaction time. The amount of L-Ser was consistently 0.1 g. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, the fluorescence intensity of the N-L-Ser-CDs increased with the amount of urea, reaching a maximum when 0.4 g of urea was used, and then decreased gradually. Reaction temperature and time were further optimized with 0.1 g of L-Ser and 0.4 g of urea. According to Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb and Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec, the optimal reaction temperature was found to be 180\u0026deg;C, and the optimal reaction time was 2 hours. Under these conditions, the fluorescence intensity of the N-L-Ser-CDs was 27.6 times greater than that of the L-Ser-CDs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). This demonstrates that nitrogen doping effectively enhanced the fluorescence intensity of the L-Ser-CDs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe optical properties of N-L-Ser-CDs were characterized using 3D and 2D fluorescence spectra. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, the 3D fluorescence spectra of N-L-Ser-CDs reveal a distinct luminescence peak with excitation and emission wavelengths at 330 nm and 405 nm, respectively. The 2D fluorescence spectrum (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb) confirms these findings, showing that the optimal excitation wavelength is 330 nm and the optimal emission wavelength is 405 nm. From the UV absorption spectra, it is evident that N-L-Ser-CDs exhibit significant absorption between 200 and 420 nm, attributed to the π-π* transition of C\u0026thinsp;=\u0026thinsp;C and the n-π* transition of C\u0026thinsp;=\u0026thinsp;O/N. Further investigation into the relationship between the emission wavelength and excitation wavelength of these CDs is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec. It is observed that as the excitation wavelength increases from 280 to 380 nm, the emission wavelength gradually red-shifts, and the emission intensity first increases before decreasing. This behavior is characteristic of CDs and arises from their varied emission sites associated with both core and surface states [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The quantum yield of the CDs, calculated using the five-point method, is 22.5% (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), which is higher than that reported in previous studies [\u003cspan additionalcitationids=\"CR26 CR27\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], indicating that the N-L-Ser-CDs prepared in this work possess excellent fluorescence properties. The colloidal optical stability of the N-L-Ser-CDs was further assessed. The colloidal optical stability of the N-L-Ser-CDs was further assessed. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed, the fluorescence intensity of N-L-Ser-CDs remains relatively stable even when exposed to NaCl concentrations up to 1 M, demonstrating their good colloidal stability. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee illustrates the fluorescence intensity of N-L-Ser-CDs under 365 nm ultraviolet irradiation. The fluorescence intensity does not significantly decrease even after 2.5 h of continuous UV exposure, indicating strong photobleaching resistance and suitability for prolonged monitoring. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef displays the fluorescence intensity of N-L-Ser-CDs in solutions of varying pH. The fluorescence intensity decreases slightly in acidic environments but remains strong in alkaline conditions, showing that pH changes do not substantially quench the fluorescence of N-L-Ser-CDs. Overall, these results confirm the excellent colloidal and optical stability of the N-L-Ser-CDs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Structural characterization of N-L-Ser-CDs\u003c/h2\u003e \u003cp\u003eThe microstructure of N-L-Ser-CDs was investigated using transmission electron microscopy (TEM). As depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, N-L-Ser-CDs exhibit an approximately spherical shape and are uniformly dispersed. The particle size distribution ranges from 4.6 to 9.2 nm, with an average particle size of 6.56 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). TEM images reveal that N-L-Ser-CDs lack distinct lattice fringes, indicating they have low crystallinity and are amorphous materials. The diffraction peak at 21.4\u0026deg; in the X-ray diffraction (XRD) pattern further confirms the amorphous nature of N-L-Ser-CDs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec) [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], consistent with the TEM observations. The functional groups on the surface of CDs were studied by Fourier transform infrared (FT-IR) spectroscopy. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed, the broad peaks located around 3300 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e are attributed to the stretching vibrations of -OH and -NH\u003csub\u003e2 [30]\u003c/sub\u003e. The peak located at 1701 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e corresponds to the characteristic peak of -COOH. The peak located at 1327 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e represents the characteristic peak of C-O/C-N. The peak located at 756 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e is associated with C-H groups [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The FT-IR results indicate the presence of hydrophilic functional groups such as amino, hydroxyl, and carboxyl groups on the surface of N-L-Ser-CDs, which enhances their water solubility and makes them suitable for use in aqueous environments. Additionally, the elemental composition of N-L-Ser-CDs was quantitatively analyzed using X-ray photoelectron spectroscopy (XPS). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, the XPS survey spectrum shows peaks for C 1s at 284.08 eV, N 1s at 400.08 eV, and O 1s at 532.08 eV, with elemental percentages of 44.17%, 10.06%, and 45.77%, respectively. The high-resolution XPS spectrum of C 1s (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb) can be deconvoluted into three peaks at 284.80 eV, 286.66 eV, and 289.16 eV, corresponding to C\u0026thinsp;=\u0026thinsp;C/C-C, C-N/C-O, and C\u0026thinsp;=\u0026thinsp;N/C\u0026thinsp;=\u0026thinsp;O, respectively [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. The high-resolution XPS spectrum of N 1s (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec) shows two peaks at 400.26 eV and 402 eV, corresponding to C-N\u0026thinsp;=\u0026thinsp;C and -NH\u003csub\u003e2\u003c/sub\u003e, respectively [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. The high-resolution XPS spectrum of O 1s (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed) reveals three peaks at 531.46 eV, 532.88 eV, and 536.21 eV, corresponding to C\u0026thinsp;=\u0026thinsp;O, O-C\u0026thinsp;=\u0026thinsp;O, and C-O, respectively [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. These XPS results are consistent with the FT-IR findings, confirming the presence of numerous hydrophilic functional groups on the surface of N-L-Ser-CDs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Determination of Cu\u003csup\u003e2+\u003c/sup\u003e and GSH\u003c/h2\u003e \u003cp\u003eThe basic principle of the logic gate detection of N-L-Ser-CDs for Cu\u0026sup2;⁺ and GSH is that Cu\u0026sup2;⁺ can significantly quench the fluorescence of N-L-Ser-CDs, while GSH can bind with Cu\u0026sup2;⁺ and thereby restore the fluorescence. The selectivity of N-L-Ser-CDs for Cu\u0026sup2;⁺ was first examined, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, by using common metal ions (Ag\u003csup\u003e+\u003c/sup\u003e, K\u003csup\u003e+\u003c/sup\u003e, Na\u003csup\u003e+\u003c/sup\u003e, Pb\u003csup\u003e2+\u003c/sup\u003e, Mn\u003csup\u003e2+\u003c/sup\u003e, Fe\u003csup\u003e2+\u003c/sup\u003e, Zn\u003csup\u003e2+\u003c/sup\u003e, Cu\u003csup\u003e2+\u003c/sup\u003e, Mg\u003csup\u003e2+\u003c/sup\u003e, Ca\u003csup\u003e2+\u003c/sup\u003e, Al\u003csup\u003e3+\u003c/sup\u003e, Cd\u003csup\u003e2+\u003c/sup\u003e, Cr\u003csup\u003e3+\u003c/sup\u003e, Ba\u003csup\u003e2+\u003c/sup\u003e, Hg\u003csup\u003e2+\u003c/sup\u003e, Ni\u003csup\u003e2+\u003c/sup\u003e) and biomolecules (L-Cys, Gly, Glucose, L-Phe, L-Ser, L-Ala, L-Asp, L-Lys, AA, L-Arg, FA, UA, Urea, DA, GSH, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) as potential interferents to assess their impact on the fluorescence intensity of N-L-Ser-CDs. It was observed that only Cu\u0026sup2;⁺ significantly quenched the fluorescence of N-L-Ser-CDs, confirming the excellent selectivity of N-L-Ser-CDs for Cu\u0026sup2;⁺. The selectivity of the N-L-Ser-CDs/Cu\u0026sup2;⁺ system for GSH was then investigated, with common biomolecules as interferents, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb. It was found that GSH could significantly restore the fluorescence of N-L-Ser-CDs that had been quenched by Cu\u0026sup2;⁺, whereas other interferents did not produce the same effect, demonstrating the excellent selectivity of the N-L-Ser-CDs/Cu\u0026sup2;⁺ system for GSH. Additionally, as illustrated in the physical image (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec), the fluorescence of N-L-Ser-CDs decreased after the addition of Cu\u0026sup2;⁺ and was subsequently restored upon the addition of GSH.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe fluorescence spectra of N-L-Ser-CDs at different concentrations of Cu\u0026sup2;⁺ are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea. It can be observed that the fluorescence intensity of N-L-Ser-CDs decreases regularly as the concentration of Cu\u0026sup2;⁺ increases from 0 to 90 \u0026micro;M. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb, the relationship between Cu\u0026sup2;⁺ concentration and fluorescence intensity (F/F₀) follows a linear equation: F/F\u003csub\u003e0\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.959-0.006C[Cu\u003csup\u003e2+\u003c/sup\u003e], R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.983. Here, F\u003csub\u003e0\u003c/sub\u003e represents the fluorescence intensity of the blank sample and F represents the fluorescence intensity of the test sample. Based on the 3σ rule, the detection limit is calculated to be 3 \u0026micro;M. The fluorescence images of N-L-Ser-CDs after the addition of various concentrations of Cu\u0026sup2;⁺ are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec, visually indicating that the fluorescence intensity decreases with increasing Cu\u0026sup2;⁺ concentration. Consequently, semi-quantitative detection of Cu\u0026sup2;⁺ can also be achieved.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe fluorescence spectra of N-L-Ser-CDs/Cu\u0026sup2;⁺ at different concentrations of GSH are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea. It is evident that the fluorescence intensity of N-L-Ser-CDs/Cu\u0026sup2;⁺ increases regularly as the GSH concentration rises from 0 to 120 \u0026micro;M. As depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb, the relationship between GSH concentration and fluorescence intensity (F/F₀) follows a linear equation: F/F\u003csub\u003e0\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.959-0.006C[GSH], with an R\u0026sup2; value of 0.983. Based on the 3σ rule, the detection limit is determined to be 3 \u0026micro;M. The fluorescence images of N-L-Ser-CDs/Cu\u0026sup2;⁺ after the addition of various concentrations of GSH are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec, clearly showing that the fluorescence intensity increases with higher GSH concentrations. Therefore, semi-quantitative detection of GSH can also be achieved. The fluorescent probes based on CDs reported in this work demonstrate detection ranges and limits that are comparable to or even superior to those of sensors previously reported for the detection of Cu\u0026sup2;⁺ or GSH (see Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparison of the N-L-Ser-CDs fluorescent probe with other methods for detecting Cu\u003csup\u003e2+\u003c/sup\u003e and GSH.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAnalytical method\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDetection target\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLinear range\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLOD\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRef\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAuNCs/N-CDs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCu\u003csup\u003e2+\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10\u0026ndash;150\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGSH@CDs-AuNCs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCu\u003csup\u003e2+\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u0026ndash;10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCuNPs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCu\u003csup\u003e2+\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15\u0026ndash;35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eN-L-Ser-CDs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCu\u003csup\u003e2+\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u0026ndash;90 \u0026micro;M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3 \u0026micro;M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThis work\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eN-CDs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGSH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e50-4.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eN-CDs/Cu\u003csup\u003e2+\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGSH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e19\u0026ndash;52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eN,S-CDs-MnO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGSH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0-250\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e28.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eN-L-Ser-CDs\u0026thinsp;+\u0026thinsp;Cu\u003csup\u003e2+\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGSH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0-120 \u0026micro;M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3 \u0026micro;M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThis work\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe mechanism of Cu\u003csup\u003e2+\u003c/sup\u003e quenching the fluorescence of N-L-Ser-CDs is mainly due to the binding of Cu\u003csup\u003e2+\u003c/sup\u003e to the -NH\u003csub\u003e2\u003c/sub\u003e and -OH groups on the surface of N-L-Ser-CDs, which affects the electron transfer process of the excited state of N-L-Ser-CDs and makes the fluorescence decrease [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e, the disappearance of the -NH₂ characteristic peak in the XPS spectrum of N-L-Ser-CDs\u0026thinsp;+\u0026thinsp;Cu\u0026sup2;⁺ (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea) and the absence of the C-O characteristic peak in the XPS spectrum of O1s (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eb) confirm that Cu\u0026sup2;⁺ binds to the -NH₂ and -OH groups on the surface of N-L-Ser-CDs. Additionally, Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ed shows that the fluorescence lifetime of N-L-Ser-CDs is 6.404 ns, whereas for N-L-Ser-CDs\u0026thinsp;+\u0026thinsp;Cu\u0026sup2;⁺ it is 5.966 ns. This decrease in fluorescence lifetime indicates that the excited electrons in N-L-Ser-CDs are transferred, leading to reduced fluorescence. GSH, which contains sulfhydryl groups, binds to Cu\u0026sup2;⁺ with much higher affinity than N-L-Ser-CDs. Therefore, the addition of GSH can displace Cu\u0026sup2;⁺ from N-L-Ser-CDs, thereby restoring the fluorescence of N-L-Ser-CDs [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ec, the UV-vis absorption baseline of N-L-Ser-CDs\u0026thinsp;+\u0026thinsp;Cu\u0026sup2;⁺+GSH is significantly higher than that of N-L-Ser-CDs and N-L-Ser-CDs\u0026thinsp;+\u0026thinsp;Cu\u0026sup2;⁺, indicating the formation of a GSH-Cu\u0026sup2;⁺ complex. Furthermore, the fluorescence lifetime of the system was restored to 6.314 ns after the addition of GSH (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ed), demonstrating that the fluorescence of N-L-Ser-CDs can be recovered through the complexation of GSH with Cu\u0026sup2;⁺.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Detection of GSH in real samples\u003c/h2\u003e \u003cp\u003eTo evaluate the potential of the prepared N-L-Ser-CDs fluorescent probes for practical applications, we monitored GSH levels in urine samples. Urine samples from healthy laboratory personnel were collected, centrifuged, and filtered. These samples were then spiked with different concentrations of GSH using the standard addition method for determination. The results, presented in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, show GSH recoveries ranging from 99.728\u0026ndash;106.070%. These satisfactory recovery rates confirm the suitability of the developed N-L-Ser-CDs fluorescent probes for detecting GSH in real samples.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eResults of GSH determination in real urine samples and recovery analysis.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSamples\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDetecting Subject\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAdded (\u0026micro;M)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFound (\u0026micro;M)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRecovery (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eRSD (%, n\u0026thinsp;=\u0026thinsp;3)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eUrine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eGSH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2.809\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e13.243\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e99.728\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e7.63\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e45.698\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e106.070\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.38\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e85.147\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e102.346\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.96\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eIn summary, this work presents the synthesis of a simple and cost-effective N-L-Ser-CDs using a one-pot solid-phase pyrolysis method with L-serine and urea as precursors. The resulting N-L-Ser-CDs exhibit strong fluorescence intensity and impressive quantum yields of up to 22.5% at 405 nm emission when excited at 330 nm. Additionally, a logic-gate-based fluorescent probe has been developed, demonstrating high sensitivity and selectivity for detecting Cu\u0026sup2;⁺ and GSH in the concentration ranges of 0\u0026ndash;90 \u0026micro;M and 0-120 \u0026micro;M, respectively, with detection limits of 3 \u0026micro;M for both analytes. Notably, this method shows excellent recovery rates (99.728\u0026ndash;106.070%) for detecting GSH in real urine samples, underscoring the potential practical applications of this innovative approach.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eConflicts of interest statement\u003c/h2\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eZhuoru Yao and Cunjin Wang wrote the main manuscript text.Zixin Ma and Jing Zhang mainly study the details of the experiment.Xiaoliang Zhao, Weijie Zhang and Huanxian Shi mainly provide financial support.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eThis work was supported by the National Natural Science Foundation of China (Nos. 82405028 and 32260231), the Gansu Provincial Natural Science Fund (No.23JRRA840), the PhD Start-up Foundation of Lanzhou University of Technology, and Red Willow Excellent Youth Talent Support Program of Lanzhou University of Technology.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eYan L, Li J, Cai H, Shao Y, Zhang G, Chen L, Wang Y, Zong H, Yin Y (2023) Carbon dots/Ag nanoclusters-based fluorescent probe for ratiometric and visual detection of Cu\u003csup\u003e2+\u003c/sup\u003e. 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J Alloys Compd 897:162731\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSun X, Wang C, Li P, Shao Z, Xia J, Liu Q, Shen F, Fang Y (2022) The facile synthesis of nitrogen and sulfur co-doped carbon dots for developing a powerful on-off-on fluorescence probe to detect glutathione in vegetables. Food Chem 372:131142\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Schemes","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":"Solid-phase pyrolysis, Logic-gate fluorescent probes, Carbon dots, Copper ion, Glutathione","lastPublishedDoi":"10.21203/rs.3.rs-5672798/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5672798/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study introduces the synthesis of nitrogen-doped carbon dots (N-L-Ser-CDs) from L-serine and urea through a straightforward and economical one-step solid-phase pyrolysis process. The incorporation of nitrogen into the carbon dots resulted in a remarkable 27.6-fold increase in fluorescence intensity, featuring a peak emission at 405 nm when excited at 330 nm and a significant fluorescence quantum yield of 22.5%. These N-L-Ser-CDs displayed a specific binding affinity for Cu\u003csup\u003e2+\u003c/sup\u003e, leading to a pronounced fluorescence quenching effect. However, upon interaction with glutathione (GSH), the fluorescence of the N-L-Ser-CDs\u0026thinsp;+\u0026thinsp;Cu\u003csup\u003e2+\u003c/sup\u003e complex was selectively restored. This restoration was attributed to the displacement of Cu\u003csup\u003e2+\u003c/sup\u003e from the surface of the N-L-Ser-CDs due to the strong interaction between GSH and Cu\u003csup\u003e2+\u003c/sup\u003e. The mechanism underlying this fluorescence quenching was elucidated as an electron transfer process from the excited state of the N-L-Ser-CDs to Cu\u003csup\u003e2+\u003c/sup\u003e. Additionally, the sensor developed in this study exhibited a linear detection range of 0\u0026ndash;90 \u0026micro;M for Cu\u003csup\u003e2+\u003c/sup\u003e with a detection limit of 3 \u0026micro;M, and a linear detection range of 0-120 \u0026micro;M with a detection limit of 3 \u0026micro;M for GSH. By integrating the detection capabilities for both Cu\u003csup\u003e2+\u003c/sup\u003e and GSH, a successful logic-gated fluorescent probe was developed. Most importantly, this proposed method offers simplicity, affordability, and ease of use, while also showing potential for practical GSH detection in real urine samples.\u003c/p\u003e","manuscriptTitle":"Solid-phase pyrolysis synthesis of nitrogen-doped carbon dots as logic-gate fluorescent probes for dual detection of Cu²⁺ and glutathione","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-23 08:58:41","doi":"10.21203/rs.3.rs-5672798/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-01-21T19:10:43+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-01-21T12:35:45+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"261086061111806649928526607101636370182","date":"2025-01-06T02:14:32+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-12-31T14:56:19+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-12-19T10:28:19+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-12-19T10:26:14+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Fluorescence","date":"2024-12-19T02:05:50+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":"2eef9fcb-4d56-4e1a-b3a8-9e6a074f2a5c","owner":[],"postedDate":"December 23rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-02-24T16:23:29+00:00","versionOfRecord":{"articleIdentity":"rs-5672798","link":"https://doi.org/10.1007/s10895-025-04206-0","journal":{"identity":"journal-of-fluorescence","isVorOnly":false,"title":"Journal of Fluorescence"},"publishedOn":"2025-02-22 15:58:06","publishedOnDateReadable":"February 22nd, 2025"},"versionCreatedAt":"2024-12-23 08:58:41","video":"","vorDoi":"10.1007/s10895-025-04206-0","vorDoiUrl":"https://doi.org/10.1007/s10895-025-04206-0","workflowStages":[]},"version":"v1","identity":"rs-5672798","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5672798","identity":"rs-5672798","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}