Electrochemiluminescence Sensor for Uric Acid Detection Using Natural Enzyme‐Confined Nanozyme Cascade

ABSTRACT

The detection of metabolites, such as uric acid (UA), is crucial for diagnosing and monitoring various metabolic disorders. Herein, an electrochemiluminescence (ECL) sensor for uric acid (UA) detection was constructed via integrating a cascade of bioenzyme (urate oxidase [UOx]) and confined nanozyme reactions. Gold nanoparticles (AuNPs) were confined within amino‐functionalized vertical mesoporous silica film (NH₂‐VMSF) via electrochemical deposition. The NH₂‐VMSF was synthesized on indium tin oxide (ITO) through electrochemical‐assisted self‐assembly (EASA) method. Amino groups on the NH₂‐VMSF surface served as anchoring sites for AuNPs, which were electrochemically reduced and confined within the nanochannels. The confined AuNPs exhibited strong pseudo‐peroxidase‐like activity, boosting ECL response of luminol‐H₂O₂. Compared to the NH₂‐VMSF/ITO electrode, the AuNPs‐deposited electrode produced a 3.4‐fold increase in ECL intensity. Urate oxidase catalyzed the oxidation of uric acid, producing H₂O₂, which acted as a co‐reactant for luminol, enabling indirect uric acid detection. The sensor demonstrated a detection range of 0.1–20 μM with a low limit of detection (LOD, 85 nM). The sensor was used for UA detection in complex biological samples (urine). The nanoconfined AuNPs provided a stable and effective environment for nanozyme‐catalyzed reactions, ensuring high sensitivity and long‐term stability.

A cascade electrochemiluminescence sensor integrating urate oxidase catalysis with nanochannel‐confined AuNPs nanozyme enables highly sensitive and selective detection of uric acid. The confined AuNPs exhibit strong peroxidase‐like activity, amplifying the luminol‐H₂O₂ signal by 3.3‐fold and achieving a low limit of detection.

1. Introduction

Uric acid (UA) represents the final catabolite of purine nucleotide metabolism in humans [1, 2]. Abnormal UA levels are associated with various health conditions: Elevated UA can form crystals due to its low solubility, potentially leading to gout, arthritis, kidney stones, and hyperuricemia; conversely, low UA levels may contribute to oxidative stress or conditions such as multiple sclerosis [3, 4]. Moreover, abnormal UA levels are linked to metabolic disorders like diabetes, obesity, hypercholesterolemia, and kidney dysfunction [5, 6]. Therefore, monitoring UA concentrations in bodily fluids serves as a critical diagnostic tool for purine metabolism‐related disorders, and the development of high‐sensitivity detection methods holds significant clinical value.

Currently, the most common approach for UA detection involves biosensors based on urate oxidase (UOx) by the enzyme's catalytic oxidation of UA to yield allantoin, CO₂, and H₂O₂. Several detection strategies, including colorimetric [7, 8], fluorescent [9, 10], electrochemical [11, 12], and electrochemiluminescence (ECL) strategies [13, 14], have been developed based on this enzymatic reaction. Among these, ECL, a process in which light is emitted from the excited states of electrochemically generated species, has attracted considerable interest for its rapid response, low background noise, controllable parameters, and broad dynamic range [15, 16, 17]. ECL occurs when electrochemically generated species undergo high‐energy electron transfer reactions to form excited states, which emit light upon returning to their ground states [18, 19, 20]. The luminol‐H₂O₂ system is a particularly popular ECL platform, owing to luminol's low oxidation potential, affordability, and stability [21, 22, 23, 24]. In this system, H₂O₂ serves as an efficient co‐reactant [25, 26]. The mechanism involves the initial deprotonation of luminol to form the luminol anion, followed by electrochemical oxidation to luminol radical. This radical then reacts with ROS generated from H₂O₂ to produce the excited‐state emitter, which emits light upon relaxation [27, 28]. Urate oxidase catalyzes the oxidation of uric acid, producing H₂O₂, which provides a suitable co‐reactant for constructing uric acid sensors using the luminol‐H₂O₂ system. However, the conversion of H₂O₂ to ROS is slow under neutral conditions, limiting both the reaction speed and the ECL intensity. Therefore, introducing a catalyst to promote H₂O₂ activation and ROS generation is critical for enhancing the ECL signal.

With the advent of nanotechnology, various nanomaterials have been explored to improve the performance of ECL sensors [29, 30, 31]. Nanozymes with peroxidase (POD)‐like activity have attracted increasing attention for amplifying luminol‐H₂O₂ ECL reactions. Among different nanozymes, gold nanoparticles (AuNPs) are particularly suitable for constructing ECL sensing interfaces. AuNPs have high surface area, excellent biocompatibility, and good electron‐transfer capability [32, 33, 34]. Moreover, AuNPs exhibit POD‐like activity and can promote H₂O₂ activation to generate ROS, thereby facilitating luminol oxidation and enhancing ECL emission. Additionally, AuNPs possess excellent electrical conductivity and can facilitate interfacial electron transfer. Unlike many nanozymes that require separate synthesis and immobilization (e.g., metal oxide nanozymes Fe₃O₄, CeO₂, MnO₂), AuNPs can be conveniently introduced onto conductive substrates by mild electrochemical deposition, avoiding complicated synthetic procedures. Notably, the catalytic performance of AuNPs is strongly associated with their particle size, dispersion state, accessible surface area, and interactions with the supporting interface [35, 36, 37, 38]. Therefore, constructing a confined and stable environment for AuNPs is important for maintaining their catalytic activity and sensing reproducibility.

Confining AuNPs within mesoporous silica film represents an effective strategy [39, 40]. Vertically ordered mesoporous silica film (VMSF) is a suitable electrode‐modification layer because of its high density, uniform, ultrasmall nanochannels, good mechanical stability, and anti‐fouling ability [41, 42, 43, 44, 45]. These attributes help reduce contamination from complex biological samples, making VMSF an ideal material for constructing biosensors [46, 47, 48]. Moreover, the highly ordered nanochannels can provide a confined microenvironment for guest nanomaterials, which helps improve their dispersion and suppress aggregation or detachment during measurements [49, 50]. In particular, amino‐functionalized VMSF (NH₂‐VMSF) can provide abundant amino groups on the nanochannel walls. These amino groups are expected to interact with Au species via Au–N coordination, which helps stabilize AuNPs inside the nanochannels and suppresses aggregation and detachment.

Herein, an ECL sensor for highly sensitive detection of UA was developed by confining AuNPs within VMSF nanochannels and coupling the resulting catalytic interface with urate oxidase (UOx). The sensor amplifies the luminol‐H₂O₂ ECL signal generated from UOx‐catalyzed UA oxidation, enabling sensitive UA detection. The NH₂‐VMSF modified ITO electrode (NH₂‐VMSF/ITO) was easily fabricated to provide ordered nanochannels and amino‐rich confined spaces. AuNPs were subsequently introduced into the NH₂‐VMSF nanochannels by electrochemical deposition. AuNPs were selected because of their reported POD‐like catalytic activity, excellent conductivity, chemical stability, and compatibility with electrochemical fabrication. The amino groups in NH₂‐VMSF can interact with Au species through Au–N interactions, contributing to the high stability of AuNPs. Owing to the POD‐like activity of the confined AuNPs, the AuNPs@NH₂‐VMSF/ITO electrode achieved 3.4 times higher ECL signal than that of the NH₂‐VMSF/ITO electrode. For UA detection, UOx specifically catalyzes UA oxidation to produce H₂O₂, which then serves as the co‐reactant in the luminol ECL system. The confined AuNP nanozymes further promote H₂O₂ activation and amplify the luminol ECL emission. Therefore, the proposed sensing strategy integrates the molecular specificity of UOx, the catalytic amplification ability of AuNP nanozymes, and the stability and anti‐fouling advantages of NH₂‐VMSF nanochannels, enabling sensitive detection of UA.

2. Materials and Methods

2.1. Chemical and Materials

Tetraethoxysilane (TEOS), glutaraldehyde (GA), 3‐aminopropyltriethoxysilane (APTES), cetyltrimethylammonium bromide (CTAB), 3,3′,5,5′‐tetramethylbenzidine (TMB), K₃[Fe (CN)₆], 1,4‐benzoquinone (BQ), tert‐butanol (TBA), NaH₂PO₄, Na₂HPO₄, glucose (Glu), luminol, L‐cysteine (L‐cys), UA, dopamine (DA), ascorbic acid (AA), urate oxidase (UOx), H₂O₂, and NaNO₃ were sourced from Aladdin Biochemical Technology Co. Ltd. (Shanghai, China). All chemicals were analytical grade, and solution was prepared using ultrapure water (18.2 MΩ·cm).

2.2. Measurements and Instrumentations

Transmission electron microscopy (TEM) characterizations were performed with 100 kV accelerating voltage (HT7700, Japan). Scanning electron microscopy (SEM) images were recorded at 5 kV accelerating voltage (SU8010, Hitachi, Japan). For SEM sample preparation, a glass cutter was used to create a fresh cross section of the electrode surface, which was then mounted on the sample holder, gold‐coated, and analyzed. Cyclic voltammetry (CV) experiments were performed on an Autolab electrochemical workstation (PGSTAT302N, Metrohm, Switzerland). ECL experiments were performed with an MPI‐E II analyzer (Ruimai Analytical Instrument Co. Ltd., Xi'an). All electrochemical and ECL experiments were performed in a three‐electrode cell, consisting of a bare or modified ITO working electrode, a platinum sheet (1 cm × 1 cm) counter electrode, and an Ag/AgCl (saturated KCl) reference. X‐ray photoelectron spectroscopy (XPS) data were acquired on a PHI 5300 spectrometer (PE Ltd., United States) equipped with a Mg Kα X‐ray source (250 W, 14 kV). Ultraviolet–visible (UV–vis) absorption spectra were recorded on a UV‐2450 spectrophotometer (Shimadzu Corporation, Japan).

2.3. Fabrication of NH₂‐VMSF/ITO and AuNPs@NH₂‐VMSF/ITO Electrodes

NH₂‐VMSF modified ITO electrodes were prepared via EASA method [51, 52]. Cleaned ITO substrates (sheet resistance < 17 Ω/sq., ITO thickness: 100 ± 20 nm) were immersed in a precursor solution of TEOS, APTES, and CTAB in ethanol‐NaNO₃. A constant current of −350 μA was applied for 10 s, after which the electrodes were rinsed, dried, and annealed at 120 °C overnight. The resulting electrode containing surfactant micelles (SM@NH₂‐VMSF/ITO) was treated with 0.1 mol/L HCl‐ethanol for 5 min to remove the template, yielding NH₂‐VMSF/ITO. AuNPs were then electrodeposited onto the electrode by applying a constant potential of −0.5 V in 0.5% HAuCl₄ for 2–5 s. The final AuNPs@NH₂‐VMSF/ITO electrodes were rinsed and stored at 4 °C.

2.4. ECL Detection of UA Using AuNPs@NH₂‐VMSF/ITO Electrode

The analytical performance of the AuNPs@NH₂‐VMSF/ITO electrode for UA was determined by measuring its ECL response during continuous potential scanning (0–0.8 V, 100 mV/s) in a solution of 0.01 M PBS (pH 8.0) containing 10 μg/mL UOx, 100 μM luminol, and varying concentrations of UA. For real sample analysis, human urine (from a healthy male) was diluted 50‐fold with PBS (0.01 M, pH 8.0) before detection.

3. Results and Discussion

3.1. Catalytic Cascade Strategy Involving Bioenzyme and Nanochannel‐Confined Nanozyme for UA Detection

Schematic illustration of the ECL sensor construction for UA detection was presented in Figure 1. The sensor relied on the combination between VMSF‐confined AuNPs and enzymatic catalysis. NH₂‐VMSF was first grown on ITO electrodes by EASA method. This nanofilm featured uniform nanochannels that acted as nanoreactors, where AuNPs were electrochemically deposited and stably anchored via Au–N bonds, achieving high dispersion and preventing aggregation. UOx was then introduced to specifically oxidize UA, yielding H₂O₂ in situ. The generated H₂O₂ diffused into the nanochannels and was subsequently converted to reactive oxygen species (ROS) by the peroxidase‐like activity of the confined AuNPs, triggering high ECL response. The sensor offered three key advantages. First, the uniform nanochannels of VMSF ensured high structural integrity and resistance to interference, minimizing contamination from complex biological samples. Second, AuNPs exhibited multifunctionality, serving as peroxidase mimics, enhancing conductivity, and boosting ECL signal. Third, the combination between UOx and AuNPs optimized both enzyme selectivity and nanocatalyst efficiency. Moreover, this design with nanochannel‐confined nanocatalyst and natural enzyme cascade can be explore to the detection of other H₂O₂‐dependent analytes (e.g., glucose and cholesterol) by changing the bioenzyme.

FIGURE 1.

Schematic illustration of the ECL sensor construction for UA detection based on natural enzyme‐confined nanozyme cascade.

3.2. Characterization of NH₂‐VMSF/ITO and AuNPs@NH₂‐VMSF/ITO Electrodes

The morphology of NH₂‐VMSF/ITO was characterized using TEM. Figure 2A,B showed the top and cross‐sectional views of NH₂‐VMSF/ITO, revealing uniform nanochannels with an average nanochannel size of about 2–3 nm and a film thickness of approximately 95 nm.

FIGURE 2.

(A) TEM top view of NH₂‐VMSF. (B) TEM cross‐sectional view of NH₂‐VMSF.

To confirm that AuNPs were confined within the nanochannels rather than on the surface, the morphologies of NH₂‐VMSF/ITO and AuNPs@NH₂‐VMSF/ITO were compared using SEM. The images (Figure 3A,B) revealed that the surface morphology remains unchanged after AuNPs deposition, supporting the confinement of AuNPs in nanochannels. Subsequent removal of the NH₂‐VMSF film by NaOH treatment exposed the underlying structure, where SEM imaging (Figure 3C) clearly showed aggregated nanoparticles. EDS mapping analysis (Figure 3D) confirmed these aggregates to be gold, providing direct evidence that the AuNPs were originally confined within and protected by the nanochannels.

FIGURE 3.

(A,B) SEM image of NH₂‐VMSF/ITO (A) or AuNPs@NH₂‐VMSF/ITO (B). (C) SEM image of AuNPs@NH₂‐VMSF/ITO after removal of NH₂‐VMSF by immersion the electrode in 0.5 M NaOH. (D) EDS Au mapping of the AuNPs@NH₂‐VMSF/ITO.

XPS was used to further verify the presence of AuNPs and analyze the surface composition of the electrodes. Figure 4A,B showed the XPS spectra of NH₂‐VMSF/ITO and AuNPs@NH₂‐VMSF/ITO, where the latter showed a distinct Au 4f peak at around 85 eV, confirming the presence of the gold element. The high‐resolution Au spectrum (Figure 4D) revealed two peaks at Au 4f_7/2 and Au 4f_5/2, verifying the presence of gold on the electrode surface.

FIGURE 4.

(A) XPS spectra of NH₂‐VMSF/ITO. (B,C) High‐resolution Au_4f XPS spectra of AuNPs@NH₂‐VMSF/ITO. (D) CV curves of NH₂‐VMSF/ITO and AuNPs@NH₂‐VMSF/ITO in 0.5 M H₂SO₄. (E) CV and (F) EIS of NH₂‐VMSF/ITO and AuNPs@NH₂‐VMSF/ITO in 0.1 M KCl containing 2.5 mM Fe (CN)₆ ^3−/4−.

The electrochemical properties of the modified electrodes were investigated by CV in 0.5 M H₂SO₄. As shown in Figure 4D, the CV curve of AuNPs@NH₂‐VMSF/ITO exhibited a distinct oxidation peak at approximately 1.0 V, corresponding to the formation of gold oxide, and a reduction peak at around 0.9 V, attributed to the reduction of gold oxide. The appearance of these characteristic peaks confirmed the successful electrodeposition of AuNPs onto the NH₂‐VMSF/ITO electrode. To further evaluate the interfacial properties, electrochemical impedance spectroscopy (EIS) and CV were performed using Fe (CN)₆ ^3−/4− as a redox probe. Compared to the unmodified NH₂‐VMSF/ITO, the AuNPs@NH₂‐VMSF/ITO electrode exhibited a significantly higher peak current, a reduced peak‐to‐peak separation, and a lower charge transfer resistance (Figure 4E,F). These results demonstrated that the confined AuNPs effectively facilitate electron transfer at the electrode interface.

3.3. Amplification of ECL by Confinement of AuNPs

The signal amplification effect of AuNPs confined within NH₂‐VMSF on the luminol‐H₂O₂ ECL system was evaluated by comparing the ECL signal of different electrodes. As shown in Figure 5A, luminol only generated a weak ECL signal on NH₂‐VMSF/ITO electrode in the presence of H₂O₂. After the deposition of AuNPs, the ECL signal increased significantly, approximately 3.4 times higher than that of the NH₂‐VMSF/ITO electrode. ECL experiments were also performed to compare confined and non‐confined AuNPs systems. As shown in Figure S1 in the supporting information (SI), the ECL responses of bare ITO, AuNPs/ITO, NH₂‐VMSF/ITO, and AuNPs@NH₂‐VMSF/ITO were compared. For bare ITO, the ECL signal exhibited a signal decay of approximately 45% during repeated scanning. After AuNPs were directly deposited on bare ITO without nanochannel confinement, the ECL signal decreased sharply with a decay of ~88%. This poor stability might be attributed to possible detachment or rearrangement of AuNPs during repeated measurements. In contrast, NH₂‐VMSF/ITO showed a stable ECL response with only 6.2% signal decay, indicating the good stability of the VMSF interface. More importantly, AuNPs@NH₂‐VMSF/ITO showed both enhanced ECL intensity and excellent stability, with a low relative standard deviation (RSD) of 1.4%. These results demonstrated that NH₂‐VMSF nanochannels effectively confined and stabilized AuNPs, suppressing aggregation and detachment, and allowed the catalytic activity of AuNPs to be retained during repeated ECL measurements. As shown in Figure S2 (SI), the ECL signal first increased and then decreased with increasing deposition time, reaching the maximum at 2 s. The increase at short deposition times was attributed to the increased number of catalytic Au sites. However, excessive deposition might partially block the nanochannels and hinder the diffusion of H₂O₂, luminol, or reaction intermediates, resulting in decreased ECL intensity. Thus, 2 s was selected for electrodeposition of AuNPs.

FIGURE 5.

(A) Comparison of ECL intensities obtained with different electrodes. (B) UV–vis absorption spectra and digital photoes of the solution (inset) demonstrating the catalytic oxidation of TMB by H₂O₂ in the absence (blank, ①) and presence of NH₂‐VMSF/ITO (②) or AuNPs@NH₂‐VMSF/ITO (③). (C) Evaluation of the role of reactive oxygen species in the ECL mechanism using BQ and TBA as scavengers. (D) Assessment of the ECL responses of AuNPs@NH₂‐VMSF/ITO toward various substances.

The peroxidase‐like activity of AuNPs was assessed using the TMB‐H₂O₂ system. Figure 5B demonstrates that, in the presence of AuNPs, the TMB‐H₂O₂ solution turned a deeper blue, accompanied by a substantial increase in absorbance at 652 nm, a signature of oxidized TMB (oxTMB). This confirmed the intrinsic POD‐like activity of the confined AuNPs, which efficiently catalyzed the decomposition of H₂O₂ and the subsequent oxidation of the substrate, thereby enabling signal amplification for ECL.

To explore the mechanism of this amplification, radical scavenging experiments were conducted. As shown in Figure 5C, the ECL signal was significantly quenched upon the addition of tert‐butanol (TBA) and benzoquinone (BQ), scavengers for •OH [53] and •O₂ ⁻ [54, 55, 56], respectively. This suggested that both •OH and •O₂ ⁻ were key intermediates in the ECL process. The formation of these reactive oxygen species was directly linked to the catalytic activity of AuNPs towards H₂O₂, and their subsequent interaction with luminol was responsible for the enhanced ECL emission.

3.4. Feasibility of Uric Acid Detection

ECL experiments were performed using externally added H₂O₂ to directly compare the responses of NH₂‐VMSF/ITO and AuNPs@NH₂‐VMSF/ITO under identical conditions. As shown in Figure S3 (SI), the NH₂‐VMSF/ITO electrode produced an ECL intensity of 4598 a.u. in 0.01 M PBS containing 100 μM luminol and 10 μM H₂O₂, whereas the AuNPs@NH₂‐VMSF/ITO electrode generated a much stronger ECL intensity of 25,204 a.u. The ECL signal was enhanced by approximately 5.4‐fold after the introduction of AuNPs. Since the only difference between the two electrodes was the presence of AuNPs confined within the NH₂‐VMSF nanochannels, this result directly demonstrates that AuNPs played a critical catalytic role in amplifying the luminol‐H₂O₂ ECL reaction. Considering the strong correlation between the luminol ECL signal intensity and H₂O₂ concentration, this system showed promise for detecting substrates associated with H₂O₂ generation. In this study, UA was chosen as the model analyte to investigate its potential for generating H₂O₂ under the catalysis of uricase and triggering an ECL response. As illustrated in Figure 5D, when only UA or uricase was present, the AuNPs@NH₂‐VMSF/ITO electrode produced negligible ECL signals. However, when both UA and uricase were introduced, the ECL intensity increased significantly. This enhancement was attributed to the specific oxidation of UA by UOx, resulting in the production of H₂O₂, which was then decomposed by the confined AuNPs to generate ROS. These ROS interacted with luminol, resulting in emission of light. These findings confirmed that the AuNPs@NH₂‐VMSF/ITO electrode facilitates UA detection in the presence of UOx.

3.5. ECL Detection of Uric Acid

Based on the specific catalytic reaction of UOx and the signal amplification strategy of AuNPs nanozymes, an ECL sensor for UA detection was developed. The working principle can be summarized as follows: in the presence of dissolved oxygen, UOx specifically catalyzed the oxidation of UA to generate H₂O₂, which was then catalyzed by AuNPs confined within NH₂‐VMSF nanochannels to produce ROS. These ROS reacted with luminol to form excited intermediates, which released strong ECL signals upon returning to their ground state. This design integrated enzyme substrate specificity with nanomaterial signal amplification, offering a sensitive method for UA detection.

As shown in Figure 6A, as the concentration of UA increased, the ECL intensity of luminol also increased, indicating that the amount of H₂O₂ generated was positively correlated with UA concentration. Furthermore, the confined AuNPs effectively amplified this signal. The calibration curve in Figure 6B showed a good linear relationship between ECL signal intensity and UA concentration within the 0.1–20 μM range. The linear regression equation was I _ECL = −72.8 + 1048 C_UA, with a correlation coefficient R ² = 0.990. The limit of detection (LOD) was 85 nM (S/N = 3). Table S1 in supporting information (SI) compared the UA detection performance using different sensors. The fabricated ECL sensor exhibited a low LOD. Thus, the sensor has advantages including simple fabrication, easy operation, and high sensitivity.

FIGURE 6.

(A) ECL responses of AuNPs@NH₂‐VMSF/ITO to various UA concentrations (in 100 μM luminol + 10 μg/mL UOx). (B) Corresponding calibration curve for UA detection. (C) Operational stability of the sensor under continuous UA measurement. (D) Selectivity of the sensor for UA detection.

3.6. Anti‐Interference, Reproducibility, and Stability of the Constructed Sensor

The performance of the ECL sensor for practical application was assessed in terms of its stability, reproducibility, and selectivity. The ECL signal from the AuNPs@NH₂‐VMSF/ITO electrode remained stable during continuous measurements of UA, with RSD of only 1.5% (Figure 6C). Furthermore, the fabrication reproducibility was confirmed by testing five electrodes prepared independently, and ECL responses to 1 μM UA showed good consistency, with RSD of 2.7%, indicating good reproducibility. The high stability and reproducibility of the electrodes are attributed to two factors. On the one hand, the spatial confinement of AuNPs by the NH₂‐VMSF nanochannels prevented nanoparticle aggregation and detachment. On the other hand, the covalent Au‐N bonds between AuNPs and amino groups of the NH₂‐VMSF walls enhanced the structural stability of the composite, ensuring consistent electrode performance.

To evaluate its selectivity, the sensor was tested against various potential interferents, such as ascorbic acid (AA), dopamine (DA), glucose (Glu), and L‐cysteine (L‐cys), under the same experimental conditions. As seen in Figure 6D, when the sensor was exposed to these interferents in the UA detection system, the ECL signals remained nearly identical to those of the blank control containing only UA. Moreover, the signal from mixed samples containing all interferents showed no significant change, indicating that the sensor demonstrated good selectivity for UA. This selectivity was primarily due to the enzyme specificity of UOx. Specifically, the generation of H₂O₂ was strictly dependent on the enzymatic reaction between UOx and UA, preventing interference from other substances that do not participate in this reaction.

3.7. Real Sample Analysis

To validate its reliability for real‐world applications, the ECL sensor was employed to quantify UA in complex biological matrices. A human urine sample was diluted and different concentrations of UA standard were added to simulate pathological conditions. Subsequent analysis using the standard addition method resulted in satisfactory recoveries (93.0%–107%) and low RSD (1.3%–3.4%, n = 3), confirming detection accuracy in complex sample matrices (Table 1). These promising results indicate the sensor's potential utility for clinical UA monitoring.

TABLE 1.

Uric acid detection in urine samples using standard addition method.

Sample	Added concentration (μM)	Found concentration (μM)	RSD (%)	Recovery (%)
Urine	0.00	6.13	2.3	‐—
	1.00	7.06	1.3	93.0
	5.00	11.2	2.8	101
	10.00	16.1	3.4	99.7

4. Conclusions

In this study, a uric acid sensor platform was developed that utilizes nanochannel‐confined AuNPs to enhance the luminol‐H₂O₂ ECL signal, coupled with UOx to achieve high‐sensitivity and high‐selectivity detection of uric acid. The NH₂‐VMSF structure, with its uniform nanochannels (approximately 2–3 nm) and excellent film‐forming properties, provided a stable confinement space for AuNPs, effectively preventing aggregation and detachment, thus ensuring the long‐term stability and catalytic activity of the composite. The AuNPs confined within the VMSF nanochannels served two key roles. Firstly, they improved the electron transfer properties, boosting electrochemical kinetics at the electrode interface. Secondly, they acted as nanozymes to catalyze the decomposition of H₂O₂ into ROS, which significantly amplified ECL signal in the luminol‐H₂O₂ system and enhanced luminescent efficiency by lowering luminol's oxidation potential. This study presents a strategy for constructing AuNPs nanozyme, enabling the detection of a wide range of biomarkers through H₂O₂‐mediated ECL signal amplification. The resulting sensor, owing to the antifouling properties of VMSF and the specificity of urate oxidase, exhibits strong anti‐interference capability and high accuracy for uric acid detection in complex biological samples. Despite these advantages, several limitations should be noted. The analytical performance depends on the activity of UOx, which may be influenced by storage conditions, pH, and temperature. In addition, systematic optimization of nanochannel structural parameters, such as nanochannel size and film thickness, may further improve the sensing performance and will be investigated in future work.

Author Contributions

Lanlan Wu: data curation, investigation, writing – original draft. Xiaochun Deng: data curation, writing – original draft, investigation. Jiyang Liu: conceptualization, supervision, funding acquisition, writing – review and editing.

Funding

This work was supported by the National Natural Science Foundation of China, 10.13039/501100001809, 22374131.

Supporting information

Figure S1: ECL intensity–time curves recorded at (A) bare ITO and AuNPs/ITO; (B) NH₂‐VMSF/ITO and AuNPs@NH₂‐VMSF/ITO electrodes. The measurements were performed in 0.01 M PBS (pH = 8) containing 100 μM luminol, 10 μg/mL urate oxidase (UOx), and 10 μM uric acid (UA).

Figure S2: (A) ECL responses of the AuNPs@NH2‑VMSF/ITO electrode acquired at various deposition time in the presence of luminol (100 μM), urate oxidase (10 μg/mL), and UA (10 μM).

Figure S3: ECL intensity–potential curves of NH2‑VMSF/ITO and AuNPs@NH2‑VMSF/ITO electrodes in 0.01 M PBS (pH = 8) containing 100 μM luminol and 10 μM H2O2.

Table S1: Performance for UA detection using different sensors.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.