Uric acid detection via dual-mode mechanism with copper-coordinated nitrogen-doped carbon dots as peroxidase mimics

Abstract

Monitoring disease-related biomarkers, such as uric acid in human body fluids, is essential for effective disease management and clinical diagnosis. In this study, copper-coordinated nitrogen-doped carbon dots (Cu@N-CDs) were synthesized via a simple hydrothermal method, achieving a remarkable photoluminescence quantum yield of 44.69%. The Cu@N-CDs emitted fluorescence at 460 nm upon excitation at 360 nm, making them highly suitable for sensitive biosensing applications. For uric acid detection, a Cu@N-CDs-based probe was developed and coupled with phenol (ph-OH) and 4-aminoantipyrine (AP-NH₂). In the presence of hydrogen peroxide (H₂O₂), generated through the enzymatic breakdown of uric acid by uricase, ph-OH and AP-NH₂ reacted to form a pink-colored compound. This compound quenched the fluorescence emission of Cu@N-CDs via an inner-filter effect, enabling fluorometric detection. Additionally, for colorimetric detection, the pink compound was quantified by measuring absorbance at 510 nm. The detection strategy utilized the peroxidase-mimetic activity of Cu@N-CDs, which was further enhanced by the presence of Cu. Under optimized conditions, the fluorometric method demonstrated a linear detection range of 0.01–700 μM, while the colorimetric method showed a range of 0.07–700 μM for uric acid. The developed approach proved highly effective in detecting uric acid in human blood serum and urine samples, yielding accurate results with acceptable recovery rates. This dual-mode detection method offers a reliable, sensitive, and cost-effective tool for monitoring uric acid levels, marking a significant advancement in clinical diagnostics and personalized healthcare.

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1. Introduction

Uric acid is a significant biomarker found in both urine and blood, playing a critical role in monitoring metabolic and renal health.¹ It is a by-product of purine metabolism, which occurs primarily in the liver, where purine bases are broken down to produce uric acid.² The levels of uric acid in the human body vary depending on factors such as sex, age, diet, and kidney function. Normal uric acid levels range from 0.088 to 0.455 mmol L⁻¹ in women and 0.149 to 0.416 mmol L⁻¹ in men.³ When uric acid levels exceed 0.420 mmol L⁻¹, a condition called hyperuricemia may develop, which is associated with various health complications.⁴ Low levels of uric acid in blood and urine may indicate underlying metabolic disorders or contribute to conditions such as hepatitis or nephritis.⁵ Conversely, elevated uric acid levels in blood or urine can result in the formation of uric acid crystals. These crystals often accumulate in joints, leading to a painful condition known as gout.^6,7 Chronic hyperuricemia is strongly associated with an elevated risk of cardiovascular diseases, including hypertension and atherosclerosis, as well as renal complications such as nephropathy and kidney stones. These health risks underscore the importance of regular monitoring of uric acid levels. Early detection facilitates timely interventions, enabling better management of metabolic, hepatic, and renal disorders, and reducing the likelihood of long-term complications. Monitoring also supports personalized treatment strategies, dietary adjustments, and lifestyle modifications to mitigate the adverse effects of abnormal uric acid levels. Uric acid has been previously quantified using various analytical techniques, including bi-enzyme colorimetry,⁸ chemiluminescence,⁹ UV/vis spectrometry,¹⁰ and High-Performance Liquid Chromatography (HPLC) with electrochemical detection.¹¹ These methods provide high sensitivity and selectivity but often involve lengthy procedures, high costs, and complex instrumentation. These limitations highlight the necessity for developing simpler, cost-effective, and efficient alternatives for uric acid detection. Fluorescence-based sensors provide several advantages, including simplicity, high sensitivity, rapid response times, and excellent selectivity. These attributes make fluorescence an accessible and effective technique for the detection and quantification of biomolecules. Additionally, the ability to operate under mild conditions and detect low concentrations further enhances the suitability of fluorescence-based sensors for various biological and clinical applications.^12–14

Carbon dots (CDs), a novel class of nanoscale fluorescent materials, have recently attracted significant attention owing to their numerous benefits, including straightforward synthesis, versatile surface modification capabilities, tunable structures, and excellent performance in applications such as catalysis and sensing.^15–18 They typically have particle sizes ranging from 1 to 10.5 nm.¹⁹ These nanoscale materials offer the ability to replicate the functions of natural enzymes, sharing comparable characteristics such as surface charge, shape, and size. Unlike natural enzymes, which are hindered by issues such as limited stability, high cost, and challenging storage requirements, CDs effectively address these limitations.²⁰ Doping with heteroatoms and surface functionalization are effective strategies for modifying the fluorescence properties of CDs. These modifications influence their electronic structures, particularly the energy band gap, enhancing their suitability for various analytical applications.^21–24 Metallic heteroatoms, particularly transition metals, possess additional electrons and unoccupied orbitals, facilitating the modification of electronic properties on the surface of CDs.^25–27 Moreover, metal ions can interact with CDs through complexation chemical reactions, further enhancing their functional properties.²⁸ Copper (Cu), a more cost-effective and accessible alternative to gold (Au), has been demonstrated to enhance the photochemical performance of CDs, including improved electron transfer and chemical reactivity capabilities.²⁹ Additionally, unsaturated copper species (Cu⁰ and Cu⁺) are anticipated to exhibit reductive properties, enabling them to scavenge hydroxyl radicals (˙OH) during Fenton reactions.³⁰

Building on these findings, a straightforward, reliable, and selective dual-mode sensor was developed for the detection of uric acid, incorporating both colorimetric and fluorometric capabilities. The probe utilized Cu@N-CDs, which exhibit peroxidase-like activity. In the presence of Cu@N-CDs, the enzymatic hydrolysis of uric acid by uricase produced H₂O₂, facilitating the coupling reaction between ph-OH and AP-NH₂. This reaction resulted in the formation of a pink-to-red chromogen with a strong absorbance peak at 510 nm. Concurrently, the fluorescence emission of Cu@N-CDs was quenched due to the presence of the colored chromogen, enabling dual-mode detection. The proposed sensor offers several advantages over conventional detection systems, including a broad dynamic linear range, high selectivity, ease of synthesis, and enhanced reliability. Notably, this dual-mode approach allows the detection of uric acid using two distinct techniques—colorimetry and fluorometry—by measuring signals at separate wavelengths. This capability provides greater analytical versatility and accuracy compared to traditional methods that typically rely on a single wavelength and detection mechanism. This innovative approach not only improves the sensitivity and reliability of uric acid detection but also demonstrates potential for broader applications in biomarker monitoring and clinical diagnostics.

2. Experimental

2.1. Materials and reagents

Uric acid (99.8%), uricase enzyme, ascorbic acid (AA, 98.7%), cysteine (Cys, 99.3%), glutathione (GSH, 98.4%), creatinine (CRE, 96.5%), glucose (Glu, 98.7%), dopamine (DA, 98.4%), quinine sulfate (98.8%), 3,3′,5,5′-tetramethylbenzidine (TMB, 98.7%), 4-aminoantipyrine (AP-NH₂), and phenol (ph-OH, AR) were sourced from Sigma Aldrich. Copper chloride dihydrate (CuCl₂·2H₂O), sodium dihydrogen phosphate, ethylenediamine, disodium hydrogen phosphate, sodium hydroxide, and hydrochloric acid were obtained from Merck. All chemicals and reagents used in this experiment were of analytical grade.

2.2. Instrumentation and quantum yield

All the relevant details regarding the instrumentation used and the calculation of the quantum yield are thoroughly described in the ESI.†

2.3. Preparation of Cu@N-CDs

A simple hydrothermal process was used to prepare Cu@N-CDs as follows: 1.08 g of CuCl₂·2H₂O was mixed with 1.28 mL of ethylenediamine and 30 mL of ultrapure water. The mixture was then sonicated for 5 minutes and autoclaved at 200 °C for ten hours. After cooling, the yellow-colored product was collected by centrifugation at 5000 rpm and filtered using a 0.22 μm filter paper. The resulting product was dialyzed in dialysis bags (MWCO = 3 kDa) against pure water for 24 hours. Finally, the filtrate was freeze-dried to obtain the CDs powder. For comparison, N-CDs and pristine CDs were prepared following the same procedure, using ethylenediamine and glucose as precursors, respectively.

2.4. Detection of uric acid

The detection involved a hydrolysis step using the uricase enzyme to generate H₂O₂, followed by determination using Cu@N-CDs. The hydrolysis process was conducted as follows: 350 μL of various concentrations of uric acid were mixed with 50 μL of 2.5 mg per mL uricase enzyme and 100 μL of phosphate buffer (pH 8.5), then incubated at 37 °C for 20 minutes. For the detection step, 300 μL of 15.0 mM ph-OH, 300 μL of 15.0 mM AP-NH₂, 600 μL of phosphate buffer (pH 3.5), and 300 μL of 5.0 mg per mL Cu@N-CDs were added to the hydrolyzed solution (uric acid/uricase). The mixture was incubated at 55 °C for 25 minutes. Fluorescence emissions were recorded at 460 nm with an excitation wavelength of 360 nm, and colorimetric determination was performed at the maximum wavelength of 510 nm.

2.5. Steady-state kinetic assays

To study the kinetics of Cu@N-CDs, the material was tested with varying concentrations of H₂O₂ and TMB, and the time-dependent absorbance changes were recorded. H₂O₂ concentrations were varied in the range of 0.1–2.2 mM, while the TMB concentration was kept constant at 12 mM. Conversely, the H₂O₂ concentration was fixed at 0.8 mM, and the TMB concentration was varied from 2 to 26 mM. In all experiments, the absorbance of the blue-colored reaction product was measured at 654 nm. The kinetic parameters K_m (Michaelis constant) and V_m (maximum reaction velocity) were determined using the Michaelis–Menten equation (eqn (1)) and the Lineweaver–Burk equation (eqn (2)). In these equations, V represents the initial reaction velocity, and [S] is the substrate concentration.

V = V_max × [S]/(K_m + [S]) (1)

1/V = (K_m/V_max)(1/[S]) + (1/V_max) (2)

2.6. Preparation of samples

Blood plasma was collected from healthy, non-smoking volunteers at Assiut University Hospital with informed consent. For serum preparation, 0.5 mL plasma was mixed with 0.75 mL acetonitrile and centrifuged at 4000 rpm for 20 minutes. The supernatant was stored at 4 °C for analysis. Urine samples (1.0 mL) were similarly centrifuged and filtered using medium-grade paper to remove particulates.

2.7. Ethical approval

Ethical approval was obtained from the Institutional Ethics Committee, University of Assiut. Written informed consent was secured from all participants before sample collection. All procedures complied with applicable regulations for the use of human specimens in biomedical research. Participant privacy and safety were maintained during collection, handling, analysis, and disposal of samples.

3. Results and discussions

3.1. Characterization

The morphological characteristics of Cu@N-CDs were analyzed using Transmission Electron Microscopy (TEM), revealing a spherical shape with an average size of 3.2 nm (Fig. S1A and B†). The HRTEM inset in Fig. S1A† shows lattice fringes of 0.23 nm, corresponding to the [100] facet of sp²-graphitic carbon.³¹ The FT-IR spectra of N-CQDs and Cu@N-CDs (Fig. S1C†) display characteristic bands at: 3290 cm⁻¹: ν(OH), 3180 cm⁻¹: ν(NH), 2860 cm⁻¹: ν(CH₂),1695 cm⁻¹: ν(C=O), 1620 cm⁻¹: δ(OH), and 560 cm⁻¹: δ(NH).^32,33 Additionally, a new band at 690 cm⁻¹, attributed to ν(Fe–O), confirms the successful incorporation of Fe during Cu@N-CD fabrication. The Raman spectra (Fig. S1D†) exhibit two prominent bands: 1390 cm⁻¹ (D-band): associated with disordered carbon, 1595 cm⁻¹ (G-band): related to graphitic carbon. The D/G intensity ratios were 0.90 and 1.25 for N-CDs and Cu@N-CDs, respectively, indicating a higher degree of disordered carbon in Cu@N-CDs.³⁴ Fig. 1A presents the EDX pattern of Cu@N-CDs, showing identifiable peaks for Cu, C, N, and O. The PXRD patterns of N-CDs and Cu@N-CDs are displayed in Fig. 1B. The N-CDs exhibit a broad peak at approximately 22.3°, corresponding to graphitic carbon,^35,36 while additional diffraction peaks are observed in Cu@N-CDs, attributed to Cu doping. Fig. S2† presents the XPS analysis of the as-prepared Cu@N-CDs. Fig. S2A† displays the full XPS survey spectrum of Cu@N-CDs, showing distinct and well-separated peaks for C 1s (287.8 eV), N 1s (398.4 eV), O 1s (533.3 eV), and Cu 2p (947.6 eV). Fig. S2B† highlights the deconvoluted spectrum of C 1s, with three peaks at 284.5 eV, 286.4 eV, and 287.6 eV, attributed to C=C, C–O/C–N, and C=O, respectively. Fig. S2C† illustrates the deconvoluted spectrum of N 1s, showing peaks at 398.8 eV, 399.2 eV, and 399.9 eV, corresponding to pyridinic N, pyrrolic N, and graphitic N, respectively. Fig. S2D† explores the deconvoluted spectrum of O 1s, with sharp peaks at 531.4 eV and 533.8 eV, associated with C=O and C–O, respectively. Fig. S2E† demonstrates the deconvoluted spectrum of Cu 2p, revealing five distinct peaks. Peaks at 932.6 eV and 934.8 eV correspond to Cu 2p_3/2 of Cu(I) and Cu(II), while peaks at 951.8 eV and 953.7 eV correspond to Cu 2p_1/2 of Cu(I) and Cu(II). A weak, broad peak at 943.4 eV is identified as the Cu 2p_3/2 satellite peak.³⁷ Fig. 2 presents the absorption and fluorescence characterization of the as-prepared Cu@N-CDs. Fig. 2A shows the absorption spectrum with distinct absorption bands at 220 nm and 335 nm, attributed to π–π* and n–π* transitions, respectively. Additionally, the figure includes the excitation and emission spectra of Cu@N-CDs, observed at 360 nm and 460 nm, respectively. Fig. 2B demonstrates the dependency of emission wavelength and intensity on the excitation wavelength within the range of 310–380 nm. The results indicate that emission intensities increase and emission wavelengths exhibit a red shift with increasing excitation wavelength. This behavior suggests variations in the distribution of surface functional groups on the surface of Cu@N-CDs.^38,39 The stability of Cu@N-CDs probe was evaluated under various conditions, including different ionic strengths, different ion solutions (100 mM), pH values, irradiation times, and temperature values (Fig. S3†). The results demonstrate the probe's robustness across these conditions. Additionally, the quantum yield of the as-prepared CDs, N-CDsm and Cu@N-CDs were determined to be 33.78%, 37.65%, and 44.69%, respectively, calculated using quinine sulfate as the standard (refer to ESI† for details). The high quantum yield (44.69%) of the Cu@N-CDs is primarily attributed to the synergistic effects of nitrogen doping and copper coordination. Nitrogen atoms introduce surface functional groups such as pyridinic, pyrrolic, and graphitic nitrogen, which passivate surface defects and enhance electron delocalization, promoting radiative recombination. Simultaneously, copper ions (Cu⁺/Cu²⁺) coordinate with these nitrogen sites, modulating the electronic band structure and suppressing non-radiative pathways by minimizing trap states.⁴⁰ This coordination also enhances charge carrier separation and stabilizes excited states. Additionally, the well-defined graphitic core structure and uniform nanoscale size (∼3.2 nm) contribute to efficient fluorescence emission. These combined structural and electronic features result in the remarkable photoluminescence efficiency observed in the Cu@N-CDs.

Fig. 1 EXD (A) and PXRD (B) patterns of Cu@N-CDs.

Fig. 2 (A) UV/vis and excitation/fluorescence spectra while (B) emission-dependent excitation of Cu@N-CDs.

3.2. Quenching mechanism and peroxidase-mimetic activity of Cu@N-CDs

In the presence of H₂O₂, AP-NH₂ undergoes oxidative coupling with ph-OH to form a red-colored product exhibiting a maximum absorbance at 510 nm. The UV-vis absorption spectra for various systems, including Cu@N-CDs, Cu@N-CDs/H₂O₂, Cu@N-CDs/ph-OH, Cu@N-CDs/AP-NH₂, and Cu@N-CDs/H₂O₂/AP-NH₂/ph-OH, are presented in Fig. S4A.† Notably, a distinct absorption band at 510 nm was observed exclusively in the Cu@N-CDs/H₂O₂/AP-NH₂/ph-OH system, indicating the successful formation of the colored product. In contrast, systems lacking Cu@N-CDs exhibited no significant UV-vis absorption at 510 nm (Fig. S4B†), underscoring the critical role of Cu@N-CDs in catalyzing the reaction. To further investigate the individual contributions of Cu@N-CDs, AP-NH₂, ph-OH, and H₂O₂, a systematic survey of various combinations was conducted (Fig. S4C†). The results confirmed that the characteristic absorption band at 510 nm emerged only in the presence of Cu@N-CDs, H₂O₂, AP-NH₂, and ph-OH. It was clearly observed that only weak absorption bands appeared when CDs or N-CDs were used as substitutes for Cu@N-CDs. In contrast, a prominent absorption band was recorded exclusively with Cu@N-CDs, highlighting the critical role of copper doping in enhancing the catalytic efficiency of the platform (Fig. S4D†). It is important to note that doping CDs with copper (Cu) and nitrogen (N) significantly modulates their surface states and intrinsic electronic structures, leading to enhanced fluorescence emission properties (Fig. S4E†). This modification not only improves the optical characteristics of the CDs but also broadens their potential applications in catalytic and sensing systems. The quenching behavior of the red-colored product on the fluorescence emission of Cu@N-CDs was analyzed, as shown in Fig. S5A.† It was observed that only the system containing H₂O₂, AP-NH₂, and ph-OH (resulting in the red-colored product, quinoneimine) effectively quenched the fluorescence emission of Cu@N-CDs. In contrast, other systems exhibited negligible quenching effects, underscoring the specific interaction between the red-colored product and Cu@N-CDs. Furthermore, the absorption spectrum of the red-colored product was found to overlap with the emission spectrum of Cu@N-CDs, indicating the presence of an inner-filter effect between the two systems, Fig. S5B.†⁴¹ To further validate this interaction, fluorescence lifetimes of Cu@N-CDs were measured both before and after the addition of the red-colored product. The results revealed that the fluorescence lifetimes were 4.67 ns for Cu@N-CDs and 4.65 ns for the Cu@N-CDs/quinoneimine, demonstrating minimal variation (Fig. S5C†). This suggests that static quenching is the primary mechanism underlying the observed fluorescence suppression.^42,43

3.3. Optimization of reaction variables

To improve detection sensitivity, various factors influencing the system's sensing performance were systematically evaluated (Fig. S6†). Fig. S6A† illustrates the effect of pH on the detection of uric acid. The results revealed that the catalytic performance of Cu@N-CDs was enhanced under acidic conditions, which promote the Fenton-like reaction facilitated by the incorporation of copper atoms into the CDs' framework.^44,45 Fig. S6B† illustrates the influence of hydrolysis temperature within the range of 40–65 °C on the fluorescence response. The optimum fluorescence response was observed at 55 °C, identified as the optimal temperature for uric acid hydrolysis. Beyond this temperature, a noticeable decline in fluorescence readings was recorded, likely due to the decomposition of H₂O₂ and the resultant red-colored product, which diminishes the system's overall stability and sensitivity. Fig. S6C† examines the effect of Cu@N-CDs concentration on the catalytic performance within the range of 3.0–6.5 mg mL⁻¹. The results indicated that the optimum fluorescence response was achieved at a Cu@N-CDs concentration of 5.0 mg mL⁻¹. Beyond this concentration, no significant change in fluorescence response was observed, suggesting a saturation point in the catalytic activity. Conversely, at concentrations below 5.0 mg mL⁻¹, the fluorescence response was notably reduced, likely due to insufficient Cu@N-CDs to effectively drive the catalytic reaction. Additionally, the effect of hydrolysis time on the catalytic performance was investigated over a range of 10–40 minutes (Fig. S6D†). The results showed that the optimum hydrolysis time was 25 minutes, which was subsequently adopted as the optimal value for further analyses. Finally, the effect of ph-OH and AP-NH₂ concentrations on the fluorescence response was evaluated (Fig. S6E and F†). The results indicated that the optimal concentrations for both compounds were 15.0 mM, yielding the highest fluorescence responses.

3.4. Dual-mode detection of uric acid

The colorimetric and fluorometric determination of uric acid using the uricase enzyme was enhanced by the peroxidase-like activity of fluorescent Cu@N-CDs (Scheme 1). In the colorimetric method, the red-colored product, exhibiting an absorption peak at 510 nm, was formed through the coupling oxidation reaction between AP-NH₂ and ph-OH in the presence of H₂O₂. The degradation of uric acid by the uricase enzyme generated H₂O₂, thereby increasing the red product's formation. This red-colored product quenched the fluorescence emission of Cu@N-CDs, enabling the fluorometric quantification of uric acid.

Scheme 1 Dual-mode detection of uric acid via Cu@N-CDs, AP-NH₂, uricase, and ph-OH.

The colorimetric and fluorometric data for the quantification of uric acid are presented in Fig. 3. Fig. 3A shows the colorimetric absorption spectra, while Fig. 3B illustrates the corresponding calibration plot for uric acid determination, facilitated by enzymatic hydrolysis in the presence of AP-NH₂ and ph-OH. The results indicate that increasing uric acid concentrations led to a rise in absorbance values at 510 nm within the range of 0.07–700 μM. The linear regression equation describing the relationship between absorbance and uric acid concentration is given as: A = 0.123 + 0.0014[uric acid] with a determination coefficient (r²) of 0.9907. The detection limit was calculated to be 0.02 μM. Fig. 3C illustrates the fluorescence spectra of Cu@N-CDs after the successive addition of uric acid concentrations (0.01–700 μM), facilitated by enzymatic hydrolysis in the presence of AP-NH₂ and ph-OH. The fluorescence emission of Cu@N-CDs was progressively quenched with increasing uric acid concentrations. Fig. 3D presents the calibration plot of fluorescence responses (F⁰/F) versus uric acid concentration, with a regression equation of F⁰/F = 1.05 + 0.001[uric acid] and a determination coefficient (R²) of 0.9940. The detection limit was calculated to be 0.001 μM. Table 1 summarizes the analytical performance of the proposed system for uric acid determination in comparison with other systems. The proposed system demonstrates notable advantages, including a wide linear range and a low detection limit, making it superior to existing methods. Furthermore, the reproducibility of the proposed methods was evaluated, as shown in Table 2. The results revealed that both the colorimetric and fluorometric methods exhibit a high degree of reproducibility, as evidenced by the low relative standard deviation (RSD) percentages.

Fig. 3 (A) Absorption spectrum of AP-NH₂/ph-OH/uricase with Cu@N-CDs in the presence of varying concentrations of uric acid (0.07–700 μM); (B) calibration plot showing the relationship between absorbance values and uric acid concentration; (C) fluorescence spectra of AP-NH₂/ph-OH/uricase with Cu@N-CDs in the presence of varying concentrations of uric acid (0.01–700 μM); (D) calibration plot illustrating the relationship between fluorescence responses (F⁰/F) and uric acid concentration.

Table 1 Comparison between analytical parameters of the proposed dual-mode system and other colorimetric and fluorometric systems for detection of uric acid

System + uricase enzyme	Substrate	Linear range (μM)	LOD (μM)	Reaction time (minutes)	Reference
Colorimetric
Fe3O4@MnO2 NS	TMB	200–650	21	1	46
MoS2/PDA/CoCu	TMB	0.5–200	0.13	40	47
Polypyrrole	TMB	200–1000	65	70	48
Cu@hydroxyapatite	TMB	1–270	0.24	100 (s)	49
Au@cellulose membrane	TMB	7–1400	7	18	50
N-CDs	OPD	0.5–150	0.06	30	51
Cu@N-CDs	AP-NH2/ph-OH	0.07–700	0.02	25	This work
Fluorometric
Papain	OPD	10–1000	4.6	15	52
Fe/N-CDs	OPD	0.5–100	0.14	30	53
BSA@Au NCs	TMB	0.50–50	0.39	60	54
Cu@N-CDs	AP-NH2/ph-OH	0.01–700	0.001	25	This work

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Table 2 Reproducibility of the colorimetric and fluorometric methods (n = 5)

Concentration (μM)	Colorimetric			Fluorometric
	Found (μM)	Recovery %	RSD %	Found (μM)	Recovery %	RSD %
5.0	4.97	99.4	2.43	5.17	103.4	2.67
100.0	99.21	99.21	1.87	102.73	102.7	3.25
500.0	505.41	101.2	2.98	507.27	101.5	3.53

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3.5. Steady-state kinetics

The steady-state kinetics of the fabricated Cu@N-CDs were illustrated in Fig. S7A and B† by recording the absorbance at 654 nm. Additionally, the Lineweaver–Burk plots were constructed and presented in Fig. S7C and D.† Table 3 provides a summary of the kinetic parameters of Cu@N-CDs, comparing them with horseradish peroxidase (HRP) and other peroxidase mimics. The data clearly show that Cu@N-CDs exhibit a high V_max and a low K_m, indicating their superior catalytic efficiency compared to HRP and other nanozymes. These findings highlight the potential of Cu@N-CDs for the effective determination of H₂O₂ and uric acid.

Table 3 Comparison of K_m and V_max between Cu@N-CDs and other nanozymes

Nanozyme	Substrate	Km (mm)	Vmax (×10^−7 M s^−1)	Reference
Horseradish peroxidase	H2O2	3.70	8.71	55
	TMB	0.43	10.0
Hemin-graphene	H2O2	2.74	3.53	56
	TMB	4.84	4.69
Fe-MIM/ZIF-8	H2O2	0.02	8.05	57
	TMB	2.51	20.07
Vanadium penta-oxide	H2O2	0.29	20.06	58
	TMB	0.69	27.15
MIL-53(Fe)	H2O2	0.04	1.86	59
	TMB	1.08	8.78
Fe-MIL-88-NH2	H2O2	0.21	7.04	60
	TMB	0.28	10.47
MoS2@MgFe2O4	H2O2	0.24	3.78	61
	TMB	0.81	14.13
2D Fe-CTF	H2O2	0.17	61.5	62
	TMB	0.04	66.2
Au–Ag/L-Cys-rGO	H2O2	22.8	1.06	63
	TMB	0.27	0.82
Cu@N-CDs	H2O2	0.12	57.34	This work
	TMB	0.08	52.87

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3.6. Stability, reproducibility, and reversibility

The stability of the synthesized Cu@N-CDs was evaluated over a period of 56 days. During this time, the relative activity (RA %) was monitored and found to decrease slightly by the end of the period. This decline suggests good long-term stability of the nanozyme. The observed reduction in RA % may be attributed to changes in the particle size of the catalyst over time. Furthermore, the reproducibility of the proposed sensor was assessed by preparing eight different batches under identical fabrication conditions. The relative standard deviation (RSD %) across the batches was found to be no more than 3.43%, demonstrating the sensor's reliability for detecting uric acid. Static quenching typically involves the formation of a non-fluorescent complex, which is generally not reversible, especially under the experimental conditions used in the study.

3.7. Anti-interference studies

The ability of an analytical method to selectively detect the target analyte is critical for its practical applications. To evaluate selectivity and anti-interference properties, various potential interfering species were tested, including 500 μM ions (Na⁺, K⁺, Ca²⁺, Mg²⁺, HCO₃⁻) and 20 μM biomolecules (glucose, cysteine, epinephrine, dopamine, glutathione, and ascorbic acid) (Fig. 4). The results indicate that the colorimetric and fluorometric methods demonstrate good anti-interference capabilities for uric acid detection. This selectivity is likely due to the specific hydrolysis of uric acid catalyzed by the uricase enzyme. However, certain compounds, such as ascorbic acid, cysteine, dopamine, epinephrine, and glutathione, were found to interfere with the reaction. These interferences are attributed to the compounds' ability to reduce hydroxyl radicals (OH˙), which are crucial for catalyzing the oxidative coupling mechanism.^64,65 Since the concentrations of ascorbic acid, cysteine, dopamine, epinephrine, and glutathione in urine and serum are significantly lower than that of uric acid, their interference can be minimized by diluting the biological fluids.^66,67 Although Fe³⁺ can quench fluorescence in controlled environments, their low free-ion concentration, protein-bound state, and dilution during sample preparation make their quenching effect on Cu@N-CDs negligible in practical biological applications.

Fig. 4 Anti-interference studies were conducted on the Cu@N-CDs/AP-NH₂/ph-OH/uricase system for the determination of uric acid using the colorimetric method ((A) with a uric acid concentration of 30 μM) and the fluorometric method ((B) with a uric acid concentration of 120 μM). The concentrations of interfering ions and biomolecules were set at 500 μM and 20 μM, respectively.

3.8. Real samples analysis

Serum and urine samples were analyzed using the Cu@N-CDs/AP-NH₂/ph-OH/uricase system (Table 4). The results from serum samples were compared with those obtained from a biochemistry analyzer. The recoveries were found to range from 98.9% to 103.7%, with RSDs between 2.67% and 4.21%. These results confirm the reliability and accuracy of the system for detecting uric acid in biological fluids.

Table 4 Detection of uric acid in human serum and urine samples using fluorometric method-based Cu@N-CDs/AP-NH₂/ph-OH/uricase system (n = 3)

Samples	Biochemistry analyzer	Proposed	Added	Found	Recovery %	RSD %
Serum 1	465 μM	474 μM	1.0 μM	470.7 μM	99.1	2.78
			5.0 μM	488.9 μM	102.1	3.23
			10.0 μM	488.1 μM	100.8	3.76
Serum 2	486 μM	468 μM	1.0 μM	463.8 μM	98.9	2.89
			5.0 μM	477.3 μM	100.9	3.65
			10.0 μM	485.4 μM	101.5	4.21
Urine 1	2.34 mM	—	1.0 mM	3.43 mM	102.7	2.67
			2.0 mM	4.43 mM	102.1	3.52
			3.0 mM	5.32 mM	99.6	3.20
Urine 2	2.23 mM	—	1.0 mM	3.35 mM	103.7	3.76
			2.0 mM	4.34 mM	102.6	3.29
			3.0 mM	5.28 mM	100.9	4.08

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4. Conclusions

This study successfully developed a dual-mode biosensor for uric acid detection based on copper- and nitrogen-doped carbon dots (Cu@N-CDs) synthesized via a hydrothermal method. The Cu@N-CDs exhibited a high photoluminescence quantum yield of 34.87%, enabling their application in both fluorometric and colorimetric detection modes. The probe, in combination with phenol (ph-OH), 4-aminoantipyrine (AP-NH₂), and hydrogen peroxide (H₂O₂) enzymatically generated by uricase, enabled sensitive detection of uric acid. Detection was achieved through fluorescence quenching of Cu@N-CDs at 460 nm and absorbance measurements of the pink-colored product at 510 nm. The fluorometric method demonstrated a detection range of 0.01–700 μM, while the colorimetric method exhibited a range of 0.07–700 μM. This dual-mode approach proved highly effective for analyzing uric acid in serum and urine samples, delivering high accuracy and recovery. It offers a cost-effective and reliable tool for clinical diagnostics and personalized healthcare applications.

Data availability

Data will be available upon request from the corresponding authors.

Conflicts of interest

The authors state that there are no conflicts of interest, financial or personal, that could have affected the integrity of this work.

Acknowledgements

The authors would like to extend their sincere appreciation to the Researchers Supporting Project, King Saud University, Riyadh, Saudi Arabia for funding this work through the project number (RSP2025R457).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d5ra01797a

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