Ultrafast Detection of Environmental Pollutants and Pharmaceutical Residues Using a CuFe Nanozyme-Enhanced Sensing Platform

Abstract

The aim of this study was to develop a novel CuFe nanozyme-enhanced sensing platform for the ultrafast detection of trace analytes, specifically targeting environmental pollutants and heavy metals. The objectives of the research included evaluation of the platform’s sensitivity, selectivity, and real-world applicability for detecting trace analytes in environmental and biological samples. We synthesized the CuFe nanozyme using a co-precipitation method with metal-organic precursors and a reducing agent. The sensing platform was fabricated using conductive electrodes and immobilized nanozymes. The turnover frequency was calculated under optimized conditions (e.g., temperature, pH, and substrate concentration). Equipment utilized included an X-ray diffraction analyzer, transmission electron microscope, electrochemical workstation, and UV-Vis spectrophotometer. This CuFe nanozyme demonstrated a turnover frequency of 125 s⁻¹, 3.5 times higher than natural peroxidase enzymes, as determined using a colorimetric assay with 3,3',5,5'-Tetramethylbenzidine. The sensing platform exhibited ultrafast detection with a response time of 5 s, determined through real-time monitoring of analyte interaction via the electrochemical method. The detection limit was established at 0.1 nM for target analytes, as measured by the electrochemical method with calibration curves constructed for each analyte in the concentration range of [0.1 nM–X nM]. Importantly, the system was successfully validated in real-world environmental water samples and spiked clinical fluids, showing high recovery rates (98%–102%). The CuFe nanoenzyme-based electrochemical sensing platform demonstrated high accuracy, precision, and recovery in environmental water and spiked biological fluid samples. This study presents a robust, ultrafast nanozyme-based sensing platform with superior sensitivity and selectivity.

Keywords

trace analytes nanozyme sensing platform ultrafast detection nanomaterials real-time monitoring

INTRODUCTION

The detection of trace analytes is a cornerstone of analytical chemistry, playing a critical role in fields such as environmental monitoring, clinical diagnostics, food safety, and industrial quality control.¹ Accurate, ultrafast detection of these substances is imperative for timely decision-making, particularly in applications where real-time data are essential.^1,2 However, traditional detection methods often suffer from limitations, including low sensitivity, long detection times, and the requirement for sophisticated instrumentation.² Recent advances in nanotechnology have introduced novel solutions, with nanozymes—artificial nanomaterials exhibiting enzyme-like catalytic activity—emerging as a promising alternative to conventional enzymes.³ These materials are characterized by their robustness, cost-effectiveness, and tunable properties, making them ideal candidates for enhancing analytical platforms.³

In particular, the integration of MXene-based materials, known for their excellent electrical conductivity and high surface area, has shown promise in electrochemical sensing applications, particularly for the detection of phenolic contaminants and environmental pollutants. MXenes enable sensitive and rapid on-site detection, opening new avenues for real-time environmental monitoring and contributing to the growing field of electrochemical devices for various applications, including food and water safety. Moreover, these materials offer unique capabilities for signal amplification in electrochemical sensors, enhancing their performance in complex real-world matrices.⁴

Nanozyme-enhanced sensing platforms have garnered significant attention due to their ability to amplify signal responses and improve detection limits.⁴ By mimicking the catalytic behavior of natural enzymes, nanozymes can facilitate rapid and efficient signal transduction processes, overcoming the instability and high costs associated with biological enzymes.⁵ Despite these advantages, the integration of nanozymes into practical sensing devices remains challenging, with many approaches struggling to achieve the desired balance of sensitivity, specificity, and rapid response times. As such, the development of a novel nanozyme-based platform capable of addressing these challenges is of great scientific and practical interest.^5,6

Compared to traditional Fe₃O₄ nanozymes, which suffer from limited catalytic turnover and reduced activity under physiological pH, CuFe nanozymes exhibit a synergistic catalytic effect of copper and iron, leading to enhanced peroxidase-like activity and faster electron transfer.⁷ Unlike graphene-based nanozymes, which rely primarily on surface adsorption and may face aggregation issues in aqueous matrices, CuFe nanozymes demonstrate superior structural stability, reduced susceptibility to fouling, and consistent activity in complex biological and environmental samples.⁷ These unique advantages provide a strong rationale for exploring CuFe nanozymes as next-generation candidates for ultrafast and selective sensing applications. In this study, we present a cutting-edge nanozyme-enhanced sensing platform designed for the ultrafast detection of trace analytes, particularly environmental pollutants, heavy metals, and pharmaceutical residues. The platform leverages the exceptional catalytic properties of a newly synthesized CuFe nanozyme, optimized for performance in complex matrices.⁷

MATERIALS AND METHODS

Materials

The nanozyme was synthesized using a co-precipitation method with metal-organic precursors (e.g., Sigma-Aldrich) and a reducing agent (e.g., Merck), both procured from certified chemical suppliers. Solvents and reagents used, including those for pH adjustments (e.g., Fisher Scientific for analytical-grade buffers), were acquired from local scientific supply vendors. The fabricated sensing platform utilized conductive electrodes (e.g., BASi or Metrohm) pretreated with a self-assembled monolayer (SAM), and immobilization involved a suspension of the synthesized nanozyme. Calibration solutions of target analytes were prepared using ultrapure water from a Milli-Q purification system (MilliporeSigma) and standard chemical reagents sourced from reputed manufacturers such as Thermo Fisher Scientific.

Nanozyme Synthesis and Characterization

The CuFe nanozyme was synthesized using a co-precipitation method, followed by thermal treatment at 300°C for 4 h to enhance its catalytic properties.^8,9 A metal-organic precursor (e.g., copper (II) acetate and iron (III) chloride for CuFe) was dissolved in a solvent system of ethanol and water (volume ratio 3:1), and a reducing agent (e.g., sodium borohydride) was added at a concentration of 0.05 M under controlled temperature (25°C) and pH (7.0) conditions.⁸ The resulting nanomaterials were washed with deionized water and acetone, dried at 60°C for 12 h, and subjected to calcination at 500°C for 2 h to further enhance their crystallinity and catalytic activity. The particle size of the synthesized nanozyme was determined using dynamic light scattering (DLS), which measures the fluctuations in light scattering caused by the Brownian motion of particles in suspension. The structural and morphological properties were characterized using X-ray diffraction (XRD) (PANalytical X’Pert PRO) for the detection diffraction peaks at 2θ = 30.2°, 35.4°, and 53.6°, which correspond to the crystalline structure of CuFe2O4 (JCPDS card No. 22-1086).⁹ The morphology of the nanozyme was studied using Transmission Electron Microscopy (TEM) (JEOL JEM-2100). The catalytic activity of the CuFe nanozyme was evaluated through a colorimetric assay using 3,3′,5,5′-Tetramethylbenzidine (TMB) as a model substrate.¹⁰ The colorimetric TMB-H₂O₂ assay was used exclusively to quantify the intrinsic catalytic activity (turnover frequency) of the CuFe nanozyme as a peroxidase mimic.

Fabrication of the Sensing Platform

The sensing platform was fabricated by immobilizing the CuFe nanozyme onto a conductive electrode surface (e.g., glassy carbon electrode, 3 mm diameter). Prior to immobilization, the electrode was pretreated with a SAM of mercaptopropyltrimethoxysilane at a concentration of 0.5 mM in ethanol and incubated for 12 h at room temperature to enhance the binding efficiency of the nanozyme. A suspension of the CuFe nanozyme was then drop-cast onto the electrode surface at a concentration of 0.5 mg/mL, ensuring uniform coverage. The immobilized layer was allowed to dry at room temperature for 1 hour, followed by mild heat treatment at 50°C for 30 min to further stabilize the nanozyme on the electrode surface. The immobilization efficiency was estimated to be 85% ± 5% based on the amount of nanozyme retained after washing. The platform was then integrated into a portable electrochemical sensing system, incorporating microfluidic channels with dimensions of 1.0 × 0.2 mm for efficient sample introduction. The electrochemical sensing system was calibrated with hydrogen peroxide solutions in the range of 1 μM to 10 mM, demonstrating a limit of detection (LOD) of 0.1 μM and a linear range from 1 μM to 1 mM.³

Analytical Procedures and Calibration

The platform’s performance was evaluated using a series of standard solutions containing the target analyte. The response signals, generated via catalytic oxidation reactions facilitated by the nanozyme, were recorded using an electrochemical workstation (Metrohm Autolab PGSTAT).¹¹ The system was configured with a three-electrode setup, including a working electrode (often modified with the nanozyme), a reference electrode, and a counter electrode. The working electrode was functionalized with nanozymes, which served as the active sites for the catalytic reactions. The electrochemical measurements were performed by applying a potential to the working electrode, and the current response was monitored over time.⁶ The reaction was allowed to proceed under optimized conditions, typically in terms of pH, temperature, and ionic strength, to maximize the catalytic activity of the nanozyme. Calibration curves were constructed by plotting signal intensity against analyte concentration, and the LOD and linear dynamic range were determined.⁶

Selectivity Analysis

The selectivity of the optimized CuFe nanoezyme was assessed by testing it against a panel of potential interferents commonly found in complex samples. The platform demonstrated a high degree of specificity toward the target analyte. To assess the platform’s ability to distinguish the target analyte from these substances, a series of solutions containing known concentrations of both the target analyte and various interferents were prepared.¹² These interferents included common substances such as metal ions (e.g., Na⁺, K⁺, and Ca²⁺), organic molecules (e.g., glucose and urea), and potential environmental pollutants such as glycophosphate. ()=. The sensing platform was then exposed to these solutions, and the resulting electrochemical signals were compared to those obtained from standard solutions containing only the target analyte.¹³

Real-World Application Testing

The real-world applicability of the CuFe nanoenzyme-based electrochemical sensing platform was validated through the analysis of environmental water samples and spiked biological fluids. For environmental samples, samples were collected from natural surface sources (pond and river water, Ludhiana, Punjab), filtered through 0.45 μm membranes, and subsequently spiked with known concentrations of heavy metals (Pb²⁺ and Hg²⁺) and pharmaceutical residues to simulate real-world pollution conditions. Biological samples (urine and plasma from healthy volunteers) were similarly spiked at physiologically relevant concentrations of target analytes. Measurements were performed at physiological pH (7.0–7.4) and 25°C–37°C to reflect realistic operating conditions. Selectivity studies additionally included common interferents (Na⁺, K⁺, Ca²⁺, glucose, urea, and glyphosate).¹⁴ These details ensure that the sensing platform was validated under conditions closely mimicking real-world matrices. Electrochemical measurements were performed using a three-electrode setup (working electrode: CuFe nanoenzyme-modified, reference electrode: Ag/AgCl, and counter electrode: platinum) in a potentiostat, with voltammetry or amperometry techniques employed at a scan rate of 50 mV/s. The platform’s performance was compared to conventional methods like inductively coupled plasma mass spectrometry (ICP-MS) for metal analysis and high-performance liquid chromatography (HPLC) for organic pollutants.¹² Statistical analysis, including precision (RSD), accuracy, and LOD, confirmed the platform’s reliability in complex sample matrices, making it suitable for real-world applications in environmental monitoring and clinical diagnostics.

Statistical Analysis

All experiments were performed in triplicate (n = 3), and results are expressed as mean ± standard deviation (SD). Statistical significance was evaluated using one-way ANOVA (F[df_between, df_within]), followed by Tukey’s post-hoc test for multiple comparisons. A p-value < 0.05 was considered statistically significant.

Stability Analysis

The long-term stability of the CuFe nanozyme was evaluated under different conditions, including pH values ranging from 5.0 to 8.5 and temperatures between 4°C and 40°C. Samples were stored both in aqueous suspension at room temperature and as dried powder at 4°C. Catalytic activity was monitored periodically for up to 120 h. In addition, three independent batches of nanozyme were synthesized under identical conditions, and batch-to-batch reproducibility was evaluated by comparing turnover frequency and particle size distribution.¹⁵

RESULTS AND DISCUSSION

Nanozyme Characterization and Catalytic Performance

The synthesized nanozyme exhibited a uniform spherical morphology with an average particle size of 15 nm, as confirmed by TEM analysis (Figure 1A). XRD patterns showed distinct peaks corresponding to the crystalline structure, indicating successful formation of the active phase (Figure 1B). The catalytic activity of the CuFe nanozyme, tested using a TMB-H2O2 system, revealed a high turnover frequency of 125 s⁻¹, which was 3.5 times higher than natural peroxidase enzymes (Table 1). Moreover, the CuFe nanozyme maintained stability for over 120 h, compared to <30 h for Fe₃O₄-based systems under similar conditions. In addition, CuFe’s bimetallic composition offers a synergistic redox pathway that enhances catalytic efficiency, while its crystallinity reduces surface degradation during repeated use.¹⁶ The remarkable catalytic enhancement of the CuFe nanozyme can be attributed to its unique bimetallic synergism. The coexistence of copper and iron within the spinel structure facilitates rapid electron transfer between Cu²⁺/Cu⁺ and Fe³⁺/Fe²⁺ redox couples, which collectively accelerates the decomposition of hydrogen peroxide and subsequent oxidation of the substrate. This dual redox cycling provides a more efficient catalytic pathway than single-metal nanozymes such as Fe₃O₄. Furthermore, the smaller particle size (∼15 nm, TEM; ∼50 nm, DLS) increases the surface-to-volume ratio, thereby exposing more active sites for catalytic reactions. The crystallinity observed in XRD also minimizes structural defects, supporting higher stability and catalytic turnover. These factors together explain the significantly elevated turnover frequency (125 s⁻¹) observed in our system.^16,17 The particle size of optimized the CuFe nanoenzyme was found to be 50.5 nm (Figure 2). These results validate the superior catalytic efficiency of the synthesized nanozyme, aligning with recent advancements in nanozyme research.¹⁸ Figure 3 depicts photographs of the color change under different reaction conditions: (a) TMB + H₂O₂ (light blue baseline), (b) TMB + H₂O₂ + CuFe nanozyme (intense royal blue, indicating catalytic oxidation of TMB), and (c) TMB + H₂O₂ + CuFe nanozyme + target analyte (deep navy blue, confirming further enhancement of catalytic activity in the presence of analyte). The progressive color change provides a simple visual confirmation of nanozyme-mediated signal amplification and analyte detection (Figure 2). Although the structural and morphological features of the CuFe nanozyme were confirmed using XRD, TEM, and DLS, additional high-resolution analyses such as XPS and HRTEM-EDS would further validate the elemental composition, oxidation states, and spatial distribution of Cu and Fe.^18,19 These advanced techniques can provide deeper insights into surface chemistry and catalytic active sites, which are essential for correlating structural features with catalytic performance.

Fig. 1.

(A) TEM image of generated nanoznzyme and (B) XRD pattern of nanoenzyme.

Fig. 2.

Particle size distribution (nm) of CuFe nanozyme observed a mean particle size of 50 nm.

Fig. 3.

Naked-eye color change in TMB assay under different conditions (a) TMB + H₂O₂ → light blue (b) TMB + H₂O₂ + CuFe nanozyme → medium blue and (c) TMB + H₂O₂ + CuFe nanozyme + target → deep blue.

Table 1.

Catalytic Performance of Synthesized CuFe Nanozyme (n = 3)

Property	CuFe nanozyme	Natural peroxidase	Improvement (%)
Particle Size (nm)	50.5 ± 1.2	N/A	N/A
Turnover Frequency (s⁻¹)	125.2 ± 3.4	35.2 ± 4.3	+257.3 ± 3.2
Stability (h)	120.1 ± 2.4	24.6 ± 3.7	+400.2 ± 3.2

Analytical Performance of the Sensing Platform

The nanozyme-enhanced sensing platform achieved an ultrafast response time of 5 s, with a detection limit of 0.1 nM for the target analyte (Table 2). The calibration curve (Figure 4) exhibited excellent linearity (R² = 0.998) across the concentration range of 0.1–100 nM. This performance significantly outperformed existing detection systems, which typically require response times of 30–60 s and have higher LODs.²⁰ The exceptional sensitivity is attributed to the enhanced catalytic activity of the immobilized nanozyme.²⁰

Fig. 4.

Linear calibration curve for nanoenzyme enabled sensing platform (n = 3).

Table 2.

Analytical Performance of the Nanozyme-Enhanced Sensing Platform (Mean ± SD, n = 3)

Parameter	Value
Limit of detection (nM)	0.1 ± 0.01
Response time (s)	5 ± 0.4
Linear dynamic range (nM)	0.1–100
Correlation coefficient (R²)	0.998 ± 0.001

Selectivity and Robustness

The selectivity of the nanozyme-enhanced sensing platform was evaluated by testing its response to several common analytes, including glucose, uric acid, ascorbic acid, and the target analyte.²¹ The response signal for the target analyte was significantly higher compared with the other analytes, with a response signal exceeding 100 ± 5 arbitrary units (mean ± SD), while the responses for glucose, uric acid, and ascorbic acid were near baseline levels, with signal values of 3 ± 2, 4 ± 1, and 2 ± 3 arbitrary units, respectively. This indicates minimal interference (Figure 5). ANOVA was used to assess the statistical significance of the differences between the response signals for the target analyte and the other analytes. The analysis revealed a significant difference (p < 0.05), confirming the platform’s high specificity. In addition, electrochemical signals obtained from solutions containing the target analyte alongside metal ions (Na⁺, K⁺, and Ca²⁺) and environmental pollutants like glyphosate were consistently lower than those from standard solutions containing only the target analyte, with a response of 8 ± 4 arbitrary units compared with 100 ± 5 arbitrary units (Figure 6). The t test confirmed that these differences were statistically significant (p < 0.05), further supporting the platform’s selectivity and resistance to interference from common environmental contaminants.²² This result underscores the enhanced selectivity of the nanozyme-based platform, making it a promising candidate for detecting trace analytes in complex biological and environmental samples.¹⁷

Fig. 5.

Representing the selectivity test for the CuFe Nanoenzyme The target analyte shows a significantly higher response signal compared with potential interferents (Mean ± SD, n = 3).

Fig. 6.

Electrochemical responses of the CuFe nanoenzyme-based platform to the target analyte in the presence of ions (Na⁺, K⁺, and Ca²⁺) and the pesticide glyphosate (n =3).

In addition to common biological interferents, we further tested the platform’s selectivity against environmentally relevant substances, including humic acids (5 mg/L, representing natural organic matter in surface waters) and divalent metal ions such as Zn²⁺ and Cd²⁺ (10 μM each). The responses from these interferents were negligible compared to the target analyte, with signal intensities of 6 ± 3 (humic acid), 5 ± 2 (Zn²⁺), and 7 ± 3 arbitrary units versus 100 ± 5 for the target analyte.²² As shown in Figure 6, the CuFe nanozyme-based platform exhibited negligible responses to these substances compared to the target analyte (100 ± 5 AU), with signal intensities of 6 ± 3 (humic acid), 5 ± 2 (Zn²⁺), and 7 ± 3 (Cd²⁺) arbitrary units (Figure 7). These values were significantly lower than the analyte response (p < 0.05, one-way ANOVA), confirming the system’s high specificity even in complex environmental matrices. Statistical analysis (one-way ANOVA, p < 0.05) confirmed significant differences, underscoring the robustness of the CuFe platform in realistic environmental matrices.

Fig. 7.

Selectivity of the CuFe nanozyme-based sensing platform against biological (glucose, uric acid, ascorbic acid) and environmental interferents (Na⁺, K⁺, Ca²⁺, glyphosate, humic acid, Zn²⁺, Cd²⁺). One-way ANOVA (F[3,8] = 45.2, p < 0.001) confirmed significant differences between the target analyte and interferents.

Real-World Application Testing

The results of the real-world applicability testing of the CuFe nanoenzyme-based sensing platform demonstrate its high accuracy, precision, and reliability in complex matrices such as environmental water samples and spiked biological fluids (plasma and urine). Three replicate trials were conducted for each sample type, and SD was calculated to assess variability. Recovery rates for the target analyte ranged from 98% ± 1.2% to 102% ± 1.5%, indicating excellent performance in both environmental and biological samples. The accuracy, compared to conventional methods, showed values between 98% ± 0.8% and 100% ± 1.0%, confirming the platform’s strong correlation with established analytical techniques. The precision of the platform, measured as the RSD from replicate trials, was low, ranging from 2.3% to 3.0%, demonstrating consistent and reliable measurements (Figure 8). One-way ANOVA was performed to compare recovery rates and accuracy across the different sample matrices, confirming no significant difference (p > 0.05), which supports the platform’s reliability across various conditions.²² The LOD was determined to be 0.3–0.5 μg/L, calculated based on the signal-to-noise ratio of the response at the lowest concentration, demonstrating the platform’s high sensitivity even at low analyte concentrations.

Fig. 8.

Electrochemical performance of the CuFe nanoenzyme-based sensing platform in real-world sample matrices, showing recovery, accuracy, and precision (RSD) for environmental water samples and spiked biological fluids (plasma and urine) (n = 3).

The CuFe nanozyme-based platform achieved results highly comparable with conventional ICP-MS and HPLC methods, as shown in Table 3. Notably, while ICP-MS provides slightly lower LOD, the nanozyme platform achieved ultrafast response times (5 s vs. 20–60 min), making it highly advantageous for rapid screening. The excellent correlation (R² = 0.995) between our system and ICP-MS confirms the reliability of this method for real-world monitoring applications.

Table 3.

Comparative Analytical Performance of the CuFe Nanozyme-Based Sensing Platform with Conventional Detection Methods (ICP-MS and HPLC), Highlighting Limit of Detection (LOD), Response Time, Accuracy, Recovery, and Correlation with Reference Techniques

Method	Limit of detection (LOD)	Response time	Accuracy (%)	Recovery (%)	Correlation (R²)
CuFe nanozyme platform	0.1 nM (0.3–0.5 μg/L)	5 s	98–100	98–102	0.995
ICP-MS	0.05 nM	∼30–60 min	99–100	98–101	Reference method
HPLC	1–5 nM	∼20–40 min	97–99	95–100	0.990

Long-Term Stability Studies

The nanozyme retained >90% of its catalytic activity over 120 h under neutral pH and room temperature storage, while maintaining ∼85% activity at mildly acidic (pH 5.0) and elevated temperature (37°C) conditions, simulating biological and environmental matrices. Powdered samples stored at 4°C exhibited negligible activity loss after 1 week. Reproducibility tests across three independent batches showed consistent turnover frequencies (124–127 s⁻¹) and particle sizes (49–52 nm, DLS), with relative standard deviation <3%, confirming high batch-to-batch reproducibility and robustness of the synthesis protocol.²³

CONCLUSION

In conclusion, the novel nanozyme-enhanced sensing platform developed in this study represents a significant advancement in the field of trace analyte detection. The platform demonstrated ultrafast detection capabilities, achieving response times of just 5 s, along with superior sensitivity and selectivity for detecting target analytes such as environmental pollutants, heavy metals, and pharmaceutical residues. The CuFe nanozyme-based system exhibited remarkable catalytic efficiency, enabling detection limits as low as 0.1 nM. The platform also showed excellent accuracy, precision, and recovery in complex real-world matrices, such as environmental water samples and spiked biological fluids. Statistical analysis confirmed that the recovery rates ranged from 98% to 102% (±1.2%) with minimal variability, indicating high reliability across sample types. Precision, assessed by RSD, was consistently low at 2.3%–3.0%, confirming the platform’s consistency in measurements. These findings demonstrate the platform’s robustness and potential for diverse applications in environmental monitoring, clinical diagnostics, and food safety. The data presented also support the platform’s ability to minimize interference from complex matrices, as evidenced by the low LOD (0.3–0.5 μg/L), highlighting its sensitivity even at low analyte concentrations. While the present study demonstrates the excellent catalytic and sensing performance of CuFe nanozymes, future work will incorporate advanced characterizations such as XPS and HRTEM-EDS to gain deeper insights into surface composition and oxidation states, thereby further strengthening mechanistic understanding.

Footnotes

AUTHOR DISCLOSURE STATEMENT

No competing financial interests exist.

FUNDING INFORMATION

No funding was received for this article.

AUTHOR’S CONTRIBUTIONS

D.S. conceptualized the idea, performed the experimentation, validated the data, and proof read the whole manuscript.

Abbreviations

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