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Abstract

Objectives: To fabricate iron-doped WS₂ (Fe-WS₂) nanozymes with prominent peroxidase-like (POD-like) activity and explore their application in biomolecule detection. Methods: Fe-WS₂ nanozymes were synthesized via a hydrothermal method, and their structural and chemical properties were characterized. The POD-like activity of the Fe-WS₂ nanozymes was evaluated at different pH, temperatures, and substrate concentrations. Kinetic parameters were determined using the Michaelis–Menten equation. The detection capabilities of the Fe-WS₂ nanozymes for H₂O₂ and glucose were assessed through colorimetric assays. Results: The synthesized Fe-WS₂ nanozymes exhibited a uniform size distribution with an average diameter of approximately 300 nm. The successful loading of iron ions onto the WS₂ nanosheets was confirmed by mapping and energy-dispersive X-ray spectroscopy analyses. The Fe-WS₂ nanozymes demonstrated enhanced POD-like activity compared to unloaded WS₂ nanozymes, as evidenced by the increased oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) in the presence of H₂O₂. The optimal conditions for POD-like activity were found to be at pH 4 and 55 °C. Kinetic analysis revealed that the Fe-WS₂ nanozymes had a high affinity for both H₂O₂ and TMB, with low Michaelis constants (K_m). The Fe-WS₂ nanozymes-based colorimetric assay exhibited a wide linear range (0.2 to 1 mM) for H₂O₂ detection, with a low detection limit of 7.27 × 10⁻⁷ M. For glucose detection, the assay showed good selectivity and sensitivity, attributed to the specificity of glucose oxidase in catalyzing glucose oxidation. Conclusions: This study successfully developed Fe-WS₂ nanozymes with remarkable POD-like activity, which were utilized to construct a sensitive and specific colorimetric biosensing system for the detection of H₂O₂ and glucose. The results indicate that the Fe-WS₂ nanozymes have great potential for applications in biomedical diagnostics due to their high catalytic efficiency, stability, and excellent biocompatibility. This work not only provides a novel inorganic nanozyme biosensor but also opens up new avenues for the application of other nanozyme biosensors in the biomedical field.

Keywords

peroxidase-like activity biomolecules detection bioassay nanotechnology

Introduction

Hydrogen peroxide (H₂O₂) and glucose serve as crucial biomarkers in human physiology, and their detection is essential for the diagnosis and treatment of numerous diseases.¹ Peroxidases have significant application value in H₂O₂ detection and glucose biosensing due to their high catalytic efficiency in decomposing H₂O₂.^2–4 Among natural enzymes, horseradish peroxidase stands as the most representative peroxidase, widely utilized for its abundant availability, low molecular weight, and ease of purification.^5,6 However, natural enzymes suffer from inherent limitations in practical applications, including poor stability, high cost, and difficult recovery, which restrict their large-scale utilization.⁷ To address these challenges, nanotechnology has given rise to a novel class of artificial enzymes, namely nanozymes.⁸ These nanomaterial-based enzyme mimics not only retain the catalytic activity and substrate specificity of natural enzymes but also demonstrate remarkable advantages, such as enhanced stability, low cost, and easy recyclability, thereby exhibiting tremendous potential in biomedical diagnostics and therapeutics.^9–11 Since the first report of Fe₃O₄ nanozymes in 2007, researchers have developed various nanozymes with peroxidase-, superoxide dismutase-, catalase-, and oxidase-like activities, which have been successfully applied in biosensing, environmental remediation, antibacterial, and antitumor therapies.^12–19

Among diverse nanozyme materials, two-dimensional transition metal dichalcogenides (2D TMDCs) have emerged as a current research focus due to their exceptional catalytic stability, facile synthesis methods, and low production costs.^20,21 Tungsten disulfide (WS₂) nanosheets, a typical 2D TMDC, owing to their unique photophysical and chemical properties, have been extensively used in biomedical applications.^22,23 However, unmodified WS₂ often demonstrates unsatisfactory performance, because of its rather low intrinsic catalytic activity and limited electron transfer efficiency. Fortunately, it has been recently reported that metal ion (including Ag, Ni, Pt, Mn, Pd, etc.) doping in the crystal lattice is a powerful modification approach to precisely regulate the electronic structure and catalytic performance of nanozymes.^24–28

Iron is a typical active center element of many natural peroxidases with unique Fenton activity.²⁹ Additionally, Fe ions can minimize lattice distortion during doping and preserve the structure of the host semiconductor, due to their appropriate ionic radius. Motivated by the catalytic and biomedical advantages of iron, we have envisaged that the Fe-doped WS₂ nanosheets may exhibit efficient peroxidase-like (POD-like) nanozyme activity for biosensing. Therefore, we prepared iron-doped WS₂ (Fe-WS₂) nanozymes based on a hydrothermal method. The as-synthesized Fe-WS₂ can catalyze the decomposition of H₂O₂ into hydroxyl radicals (·OH), exhibiting excellent POD-like activity and high sensitivity. Furthermore, in vitro colorimetric biosensing of glucose and H₂O₂ by Fe-WS₂ as a catalytic material exhibited excellent specificity and ultra-sensitivity. Thus, a novel Fe-WS₂ nano-biosensor based on Fe-WS₂ nano-sensor was not only developed by such work, but also promoted further applications of inorganic nanozymes in biomedical diagnosis and therapy.

Experimental section

This research complies with all relevant safety protocols approved by Jinan University.

Materials

Commercial WS₂ (99.8%) was obtained from Alfa Aesar and used without further purification. FeCl₃·6H₂O, H₂SO₄ (95.0% to 98.0%, analytical reagent), potassium bromide, and ascorbic acid were obtained from Chron Chemical Co. Ltd (Chengdu, China). 3,3′-diaminobenzidine (DAB), 3,3′,5,5′-tetramethylbenzidine (TMB), 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), and o-phenylenediamine (OPD) were all from Aladdin Company (Shanghai, China). H₂O₂ (30%) was procured from Jinshan Chemical Reagent Co. Ltd (Chengdu, China). Glucose oxidase (GOx) was acquired from Shanghai Yuanye Bio-Technology Co. Ltd (China). Deionized (DI) water was used in the whole process.

Preparation of WS₂ nanosheets

The synthesis of WS₂ nanosheets was performed via ultrasound-assisted liquid exfoliation by using H₂SO₄ as an intercalation agent according to procedures previously reported with necessary optimizations.³⁰ First of all, the commercially available WS₂ bulks were ground with a ball mill for 6 h. Afterwards, the ground WS₂ (100 mg) powder was added to H₂SO₄ (100 mL) solvent and treated with an oil bath at 90 °C for 24 h. Subsequently, the H₂SO₄ remaining in the product was removed with DI water, and the product was sonicated for 8 h and then centrifuged at 6000 r/min for 10 min to acquire WS₂ nanosheets.

Preparation of Fe-WS₂ nanozymes

Fe-WS₂ was synthesized by the solvothermal method according to the previously reported method.³¹ In this protocol, ascorbic acid was used as an efficient reducing agent and stabilizer.^32,33 Briefly, WS₂ (15 mg), FeCl₃·6H₂O (15 mg), and ascorbic acid (15 mg) were solubilized in 30 mL of water under severe stirring with magnetic force, and finally, a mixed solution was formed. NaHCO₃ (10 mg) was inserted after 1 h and continued to stir magnetically for 20 min. Then the blended solution was placed into a 100 mL polystyrene-lined and highly pressure pot of stainless steel and heated in an oven at 150 °C for 6 h. It was washed three times with water for further use.

Basic characterization

The morphology and energy-dispersive X-ray spectroscopy (EDS) elemental mapping of samples were visualized by transmission electron microscopy (TEM) (JEM-2100F, JEOL). The diameter of the samples was measured with ImageJ software. The hydrodynamic diameter, zeta potential, and stability analysis on samples were performed at room temperature (25 °C) by a dynamic light scattering (DLS) analyzer (Zetasizer ZS90, Malvern). Before measurement, the samples were dispersible in ultrapure water (100 μg/mL) by an ultrasonic dispersing technology using an ultrasonic cell crusher (300 W, 40 min). The crystalline structure was obtained using an X-ray powder diffraction (XRD) system with irradiation of Cu-Kα. Fourier transform-infrared (FT-IR) spectrometer (IR 200, Thermo Fisher Scientific, USA), ultraviolet (UV)-visible spectrophotometer (UV-6100, Mapada, China), and Raman spectrophotometer (Invia Reflex, Renishaw, UK) were used to determine the structure of the samples.

POD-like enzyme activity analysis

It was chosen to use TMB to evaluate the ability of Fe-WS₂ nanozyme to transform H₂O₂ into ·OH under diverse conditions in citric acid and Na₂HPO₄·12H₂O buffer solutions. The change in absorbance in the Fe-WS₂ nanozyme/H₂O₂/TMB system was examined by varying the pH, reaction temperature, TMB, H₂O₂, and material concentration. Measurements of UV absorbance of oxidized TMB (oxTMB) were carried out by adding H₂O₂ (30 mM), TMB (0.5 mM), and Fe-WS₂ nanozyme (50 µg/mL) to the reaction buffer and leaving them for 15 min at different reaction temperatures.

Kinetic assay

The steady-state dynamics behavior of Fe-WS₂ nanozyme was evaluated by changing the concentrations of TMB and fixing the H₂O₂ concentration, and conversely. The final concentration of Fe-WS₂ nanozyme was 50 µg·mL⁻¹, and the pH of the reaction system was adjusted to 4.0 with 0.1 M HAc-NaAc buffer solution. The reaction was monitored with the spectrophotometer at 652 nm. Which kinetic variables were obtained from the double inverse equation based on the Michaelis–Menten equation:

\frac{1}{ν} = \frac{K_{m}}{V_{max} [S]} + \frac{1}{V_{max}}

where K_m is the Michaelis constant, ν is the initial velocity, V_max is the maximal reaction velocity, and [S] is the concentration of substrate.

Glucose and H₂O₂ detection with Fe-WS₂ nanozyme

An appropriate amount of glucose was mixed with concentrations of GOx (1 mg/mL), TMB (0.5 mM), and Fe-WS₂ nanozyme (50 µg/mL) in the order from 1 to 10 mM. The reaction was carried out at 37 °C for 1 h under dark conditions. The absorbance of the mixed solutions at 652 nm was recorded by a spectrophotometer.

The detection was next performed using different concentrations of H₂O₂. And appropriate amounts of H₂O₂ were mixed with TMB (0.5 mM) and Fe-WS₂ nanozyme (10 µg/mL). The reaction was carried out at 37 °C for 5 min in the dark. As the reaction is relatively rapid, we used the nanomaterial concentration of 10 µg/mL for the H₂O₂ assay.

Meanwhile, in order to study Fe-WS₂ nanozyme as selective to H₂O₂, this experiment was set up to detect the material's resistance to H₂O₂ interference. Fe-WS₂ solution (50 μg/mL), TMB solution (0.5 mM) buffer (disodium hydrogen phosphate-citrate, pH = 4) was added to the solutions (1 mM) containing NaCl, MgCl₂, MnSO₄, CaCl₂, ZnCl₂, KCl, L-cysteine, amino acid, bovine serum albumin, glucose, and glutathione-S-transferase, respectively, and the reaction was carried out away from light to determine the absorption at 652 nm.

Results

The as-synthesized Fe-WS₂ nanozymes were prepared by a hydrothermal method (Figure 1). In this protocol, ascorbic acid was used as an efficient reducing and stabilizing agent, and FeCl₃·6H₂O was selected as the iron supply.^30–33

Figure 1.

Schematic illustration of the Fe-WS₂-based biosensing system for H₂O₂ and glucose detection.

Concretely speaking, benefitting from the POD-like catalytic performance of Fe-WS₂, versatile enzyme cascade-based colorimetric bioassays for the detection of H₂O₂ and glucose have been devised using Fe-WS₂-triggered reaction of TMB as an amplifier. In this study, GOx catalyzes the oxidation of glucose in the presence of O₂, yielding gluconic acid and H₂O₂ as byproducts. Subsequently, the as-synthesized Fe-WS₂ nanozyme decomposes the generated H₂O₂ via POD-like activity, producing •OH that oxidizes colorless TMB into a blue-colored product (oxTMB). Notably, the POD-like activity of Fe-WS₂ is H₂O₂-concentration-dependent, resulting in a progressive intensification of the colorimetric signal with elevated H₂O₂ and glucose concentrations.

The morphological and structural characterizations of both WS₂ nanosheets and Fe-WS₂ were systematically investigated. TEM images revealed that the lamellar morphology of both WS₂ nanosheets and Fe-WS₂, with differences in their physical dimensions (Figure 2(a) and (b)). Quantitative analysis from TEM images demonstrated that Fe doping induced a size increase, from an average lateral dimension of 122.47 ± 3.95 nm for WS₂ to 211.72 ± 45.35 nm for Fe-WS₂ (Supplemental Figure S1). This dimensional enhancement was further corroborated by DLS measurements, which showed a consistent shift in hydrodynamic diameter distribution (Figure 2(e) and (f)), suggesting successful Fe integration and potential surface modification. Furthermore, EDS elemental mapping analysis confirmed the homogeneous distribution of Fe throughout the WS₂ nanosheets, with quantitative analysis revealing an Fe loading of 1.35% (Figures 2(c) and (d)). The zeta potential measurements revealed significant alterations upon Fe incorporation (Figure 2(g)). The shift from −37.45 mV for WS₂ nanosheets to −14.2 mV for Fe-WS₂ indicates modified surface properties. Besides, comprehensive structural characterization was performed using FT-IR, XRD, and Raman spectroscopy (Figure 2(h) and (i) and Supplemental Figure S2). FT-IR spectra confirmed the preservation of characteristic WS₂ vibrational modes while revealing new Fe–S bonding signatures (674 cm⁻¹) (Supplemental Figure S2). XRD patterns maintained all major WS₂ diffraction peaks with inconspicuous peak shifting, indicating that Fe doping occurred without disrupting the host crystal structure (Figure 2(h)). Raman spectroscopy showed the distinctive E_2g and A_1g modes of WS₂ at ∼350 cm⁻¹ and ∼416 cm⁻¹, respectively, with the weakened peaks of Fe-WS₂ attributable to Fe doping (Figure 2(i)). As evidenced by spectroscopic characterizations above, this controlled doping level was achieved while maintaining the structural integrity of the WS₂ nanosheets.

Figure 2.

Structural characterization analysis of WS₂ nanosheets and Fe-WS₂. TEM images of WS₂ nanosheets (a) and Fe-WS₂ nanozymes (b). (c) The elemental mapping of Fe-WS₂ nanozyme. (d) EDS analysis of synthesized Fe-WS₂. Hydrodynamic diameter distributions of WS₂ nanosheets (e) and Fe-WS₂ (f) (n = 3). (g) The zeta potentials of WS₂ nanosheets and Fe-WS₂. XRD patterns (h) and Raman spectra (i) of WS₂ nanosheets and Fe-WS₂.

After the successful fabrication of the Fe-doped tungsten disulfide nanozyme, the POD-like activity of Fe-WS₂ was systematically evaluated using TMB as the chromogenic substrate. Upon oxidation, TMB undergoes a characteristic colorimetric transition, producing a blue-colored oxidized product (oxTMB) with a distinct absorption maximum at 652 nm (Figure 3(a)). Negligible oxTMB absorption enhancement in the H₂O₂- or Fe-WS₂-treated group could be observed, while the Fe-WS₂ + H₂O₂ + TMB reaction system exhibited the strongest absorbance at 652 nm after just 5 min of incubation at room temperature. In addition, control experiments with Fe ions alone and WS₂ without doping have now been included in Supplemental Figure S3. A negligible absorption change with the WS₂ alone-treated group could be observed, while an enhanced absorption can be found in the group of Fe³⁺ treatment. Besides, after Fe was doped with WS₂, the oxTMB of the Fe-WS₂-treated group showed the highest absorption peak, confirming the synergistic role of Fe-doped WS₂. The underlying mechanism of Fe-doping-facilitated POD-like activity illustrates that the electron cloud is affected by Fe embedded in the crystal lattice, resulting in more active electron-deficient or electron-rich centers for the catalytic process. To further confirm the POD-like activity of Fe-WS₂ nanozyme, comparative chromatography experiments with other typically peroxidase substrates (e.g. ABTS, OPD, and DAB) were also carried out (Figure 4(c)), where similar experimental phenomena were observed, demonstrating efficient catalytic activity of Fe-WS₂.

Figure 3.

(a) Schematic illustration of the catalytic oxidation of different peroxide substrates between Fe-WS₂ nanozyme and H₂O₂. (b) Absorption spectra of oxTMB in various reaction systems. (c) UV–Vis absorption spectra of the oxides of TMB, OPD, ABTS, and DAB in the presence of the Fe-WS₂ nanozyme and H₂O₂.

Figure 4.

The relative POD-like catalytic activity of WS₂ and Fe-WS₂ nanozyme depends on (a) concentration, (b) H₂O₂, (c) TMB, (d) pH, and (e) temperature. The maximum value in each curve is set to 100%.

Similar to natural enzymes, the catalytic activity of nanozymes is highly dependent on both substrate concentration and reaction conditions.⁸ To systematically evaluate the POD-like activity of WS₂ nanozymes before and after Fe loading, we conducted parallel experiments under identical reaction conditions. As demonstrated in Supplemental Figure S3, Fe-WS₂ exhibited significantly enhanced enzymatic activity compared to its unmodified counterpart (WS₂ and free Fe³⁺), confirming the crucial role of iron incorporation in boosting catalytic performance. To gain deeper insights into the catalytic behavior of the Fe-WS₂ nanozyme, we conducted comprehensive investigations on several key parameters affecting its POD-like activity, including Fe-WS₂ concentration, H₂O₂ concentration, TMB concentration, reaction temperature, and pH value. As illustrated in Figure 4 and Supplemental Figures S4 to S8, the enzymatic activity showed a positive correlation with increasing concentrations of the nanozyme material, H₂O₂ substrate, and TMB chromogenic substrate, as evidenced by the progressively intensified UV–visible (UV–vis) absorption corresponding to the oxidation product oxTMB. Besides, this concentration-dependent activity enhancement eventually reached a plateau at higher concentrations. Through careful optimization of environmental factors, we identified that the Fe-WS₂ nanozyme achieved maximum relative activity at pH 4.0 and 55 °C, with activity declining outside these optimal conditions. The experimental results show that the nanozyme not only possesses excellent enzyme-like activity, but also provides some experimental basis for its use in biological assays.

To elucidate the catalytic mechanism of Fe-WS₂ nanozymes and quantitatively assess the POD-like activity, we conducted a comprehensive steady-state kinetic analysis. The kinetic studies were performed using a well-established methodology where we systematically varied the concentration of one substrate (either H₂O₂ or TMB) while maintaining the other substrate at a constant concentration. This experimental approach allowed us to obtain classical Michaelis–Menten saturation curves across an optimized range of substrate concentrations, as shown in Figure 5(a) and (c). From these kinetic analyses, we derived two fundamental enzymatic parameters: the maximum reaction velocity (V_max) and the Michaelis constant (K_m). The K_m value serves as a crucial indicator of enzyme-substrate affinity, with lower values signifying stronger binding interactions between the nanozyme and its substrate. Our detailed kinetic measurements revealed that the Fe-WS₂ nanozymes exhibited significantly higher affinity (lower K_m) for H₂O₂ compared to TMB, suggesting that the catalytic oxidation process is likely initiated through preferential activation of H₂O₂ molecules at the active sites (Figure 5(b) and (d) and Supplemental Table S1). The calculated V_max values further confirmed the robust catalytic efficiency of the Fe-WS₂ nanozymes, demonstrating their capacity to achieve high catalytic efficacy under optimal conditions. The superior catalytic performance, combined with the inherent stability of the nanomaterial, establishes Fe-WS₂ as a promising candidate for developing reliable and efficient molecular detection platforms. This advancement provides a solid foundation for designing novel nanozyme-based biosensing systems with potential applications in clinical diagnostics, environmental monitoring, and biomedical research.

Figure 5.

The steady-state kinetic assay of Fe-WS₂ nanozyme. Reaction rates were correlated with different concentrations of H₂O₂ ((a) 30 mM H₂O₂) or TMB ((c) 0.5 mM TMB). Double inverse plots of velocity versus different concentrations of (b) H₂O₂ and (d) TMB.

The above experiments demonstrated the excellent POD-like activity of Fe-WS₂ nanozymes, and we constructed a colorimetric biosensing system for H₂O₂ based on Fe-WS₂ nanozymes, as shown in Figure 6(a). The absorbance intensity increased dramatically with increasing H₂O₂ concentration (Figure 6(b) and (c)), and simultaneously, the Fe-WS₂ nanozymes-H₂O₂–TMB system was able to detect 0.2 to 1 mM H₂O₂ with excellent linearity (Figure 6(d)), and the limit of detection (LoD) (signal-to-noise ratio = 3) was calculated to be 7.27 × 10⁻⁷ M, which superior to other nanomaterials (Supplemental Table S2). Furthermore, the selectivity of the Fe-WS₂ nanozyme for H₂O₂ was assessed by performing control experiments using other proteins, biomolecules, and metal ions (Figure 6(e)). The high selectivity of the biosensor for H₂O₂ was demonstrated by the fact that only H₂O₂ generated significant UV–Vis absorption at the characteristic peak of 652 nm compared to other proteins, biomolecules, and metal ions. This result indicates that the developed colorimetric method has favorable selectivity for the detection of glucose due to the high specificity of GOx in catalyzing the oxidation of glucose.

Figure 6.

Schematic diagram of the response mechanism of the H₂O₂ bioassay. (a) Reaction mechanism for the detection of H₂O₂. UV–Vis absorbance in Fe-WS₂/TMB systems containing different H₂O₂ concentrations (b, c) (n = 3). (d) The corresponding calibration curves (n = 3). (e) Selectivity of Fe-WS₂ nanozyme for the detection of H₂O₂ (n = 3).

Glucose is the main source of energy supply in living organisms, and changes in blood levels, either low or high, can lead to disease. Therefore, it is important to know how to effectively monitor the level of glucose in the blood, and the results obtained from the test allow for effective management and control of the patient. We assayed glucose by glucose oxidation catalyzed with GOx and TMB oxygenation catalyzed by Fe-WS₂ nanozymes (Figure 7(c)). It also should be noted that this sensing system exhibits an excellent selectivity toward glucose among different saccharides, profiting from the high specificity of GOx (Supplemental Figures S9 and S10). As shown in Supplemental Figure S9, single GOx or glucose cannot evoke the chromogenic reaction of TMB due to the lack of H₂O₂. Only when GOx and glucose are present conjointly will a significant colorimetric signal be generated. Firstly, GOx catalyzed the glucose oxidation to generate H₂O₂ in the presence of O₂; subsequently, Fe-WS₂ nanozymes catalyze the oxidation of H₂O₂ generated in situ. Therefore, glucose can be assayed by measuring the absorbance of TMB at 652 nm. As shown in Figure 7(a) and (b), it demonstrated promising results by detecting glucose at different concentrations. This result indicates that the developed colorimetric method has promising selectivity for the detection of glucose due to the specificity with which GOx catalyzes the oxidation of glucose. Therefore, compared with other nanomaterial-based bioassays that have been reported, the Fe-WS₂ nanozyme-based catalytic system possesses great potential for detection applications with higher selectivity and sensitivity in glucose biosensing. Therefore, compared with other reported nanomaterial-based bioassays, the Fe-WS₂ nanozymes-based catalytic system possesses significant potential for detection applications with higher selectivity and sensitivity in glucose biosensing.

Figure 7.

The response mechanism and detection capability of the glucose bioassay. (a) UV absorbance in Fe-WS₂ nanozyme/TMB systems containing different glucose concentrations. (b) Glucose concentration-dependent UV absorbance calibration curves and images. (c) Response mechanism of the glucose assay bioassay.

Further, we conducted a CCK-8 assay to explicitly highlight the biocompatibility of Fe-WS₂ nanozymes. As shown in Supplemental Figure S11, Fe-WS₂ nanozymes exhibit a high tolerance dose in the mouse fibroblast cell line L929, and the relative cell viability remains above 80% even at concentrations of 100 μg/mL, respectively. These findings demonstrate its promising potential for in vivo biosensing applications.

Discussion

Although the Fe-WS₂ nanozymes demonstrate high selectivity in the detection of H₂O₂ and glucose, their performance in complex biological samples (e.g. serum and urine) may be affected by interfering substances. Additionally, long-term stability remains a challenge, as Fe-WS₂ nanozymes may undergo aggregation or catalytic deactivation during prolonged storage and usage, potentially compromising their enzymatic activity. Besides, the pH and temperature optima (pH 4, 55 °C) are far from physiological conditions (pH 7.4, 37 °C).

To address these limitations and further advance the practical application of Fe-WS₂ nanozymes, the following research avenues are proposed:

Clarify the structure. It is critically important to investigate the atomic-level coordination of Fe sites through further characterization (e.g. density functional theory analysis) to elucidate the structure–activity relationship between the nanomaterial and its performance.

Surface functionalization and modification. Enhancing selectivity and stability through surface engineering strategies, such as polymer coating or biomolecule conjugation (e.g. peptides, antibodies, or DNA aptamers), to mitigate interference from complex matrices and improve biocompatibility.

Multimodal sensing platforms. Although suboptimal under physiological conditions, such properties can be advantageous for biosensing applications.^34–36 The thermostability at 55 °C suggests robust performance under storage, potentially extending shelf-life and reliability. Secondly, the acidic pH optimum could enable targeted sensing in specific pathological microenvironments (e.g. inflammatory sites or tumor microenvironments where pH is often lower). Integrating Fe-WS₂ nanozymes with thermal, electrochemical, and magnetic sensing modalities to develop multifunctional biosensors with improved sensitivity, selectivity, and cross-validation capabilities.

Clinical translation and validation. While this study has validated the Fe-WS₂ nanozyme efficacy in biomolecule detection, its real-world application in disease diagnostics requires further systematic investigation.

These future studies will be crucial in bridging the gap between laboratory research and clinical implementation, ultimately facilitating the development of reliable nanozyme-based diagnostic tools.

Conclusions

In conclusion, iron-loaded WS₂ nanozymes with outstanding POD-like activity were fabricated successfully by the hydrothermal method, which can be used for ultrasensitive biosensing. Due to the successful loading of Fe ions on WS₂ nanosheets, Fe-WS₂ nanozyme enabled enhanced catalytic activity of its inherent POD-like enzymes and excellent stability. We have the advantages of easy preparation, high stability, low cost, and favorable biocompatibility of Fe-WS₂ nanozymes. Most significantly, the fabricated Fe-WS₂ nanozyme catalytic system exhibits satisfactory sensitivity and specificity in the ultrasensitive bioassay of H₂O₂ and glucose. Thus, our work not only provides a novel inorganic nanozymes biosensor that can be used in various fields, but also provides some experimental basis for the application of other inorganic nanozymes biosensors in biomedical diagnostic fields.

Supplemental Material

sj-docx-1-sci-10.1177_00368504251362318 - Supplemental material for Iron-doped tungsten disulfide nanozyme with peroxidase-like activity for hydrogen peroxide (H₂O₂) and glucose detection

Supplemental material, sj-docx-1-sci-10.1177_00368504251362318 for Iron-doped tungsten disulfide nanozyme with peroxidase-like activity for hydrogen peroxide (H₂O₂) and glucose detection by Jing Zhou, Guobo Du, Yi Li, Shiyao Liu, Xiaoyu Chen, Yue Peng and Xiaoming Wang in Science Progress

Footnotes

Acknowledgments

We gratefully acknowledge the Institute of Nanomedicine Innovation Research and Transformation at Affiliated Hospital of North Sichuan Medical College for providing experimental support and technical assistance.

ORCID iD

Jing Zhou

Ethical statement

Ethical approval was not required for the use of the cell line in this study, as it is a commercially available, well-established cell line (L929, CL-0137 from Wuhan Pricella Biotechnology Co. Ltd).

Author contributions

YL, SL, and XC performed the experiments. JZ contributed to the scheme and figures. GD and YL discussed the data. JZ prepared the original draft. GD and XW contributed to the revision of the original draft. SL and YP conceived the idea and designed the experimental plan. GD acquired the funding.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was financially supported by the Research project of Affiliated Hospital of North Sichuan Medical College (2022JC012), Young Elite Scientists Sponsorship Program by CAST (YESS) (2022-2024QNRC002), Central Nervous System Drug Key Laboratory of Sichuan Province (230011-01SZ), Key Research and Development Program of Chengdu (2022-YF05-02071-SN), Research and development project of Affiliated Hospital of North Sichuan Medical College (2023-2ZD004), the Fundamental Research Funds for the Central Universities, Southwest Minzu University (ZYN2024006), and Scientific Research Development Plan of The Affiliated Hospital of North Sichuan Medical University in 2024 (2024PTZK003).

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Data availability statement

The data that support the findings of this study are available within the paper and its supplementary information files. Data are from three independent experiments and presented as mean ± SD (n = 3).

Supplemental material

Supplemental material for this article is available online.

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Supplementary Material

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Iron-doped tungsten disulfide nanozyme with peroxidase-like activity for hydrogen peroxide (H 2 O 2 ) and glucose detection

Abstract

Keywords

Introduction

Experimental section

Materials

Preparation of WS2 nanosheets

Preparation of Fe-WS2 nanozymes

Basic characterization

POD-like enzyme activity analysis

Kinetic assay

Glucose and H2O2 detection with Fe-WS2 nanozyme

Results

Discussion

Conclusions

Supplemental Material

sj-docx-1-sci-10.1177_00368504251362318 - Supplemental material for Iron-doped tungsten disulfide nanozyme with peroxidase-like activity for hydrogen peroxide (H2O2) and glucose detection

Footnotes

Acknowledgments

ORCID iD

Ethical statement

Author contributions

Funding

Declaration of conflicting interests

Data availability statement

Supplemental material

References

Supplementary Material

Preparation of WS₂ nanosheets

Preparation of Fe-WS₂ nanozymes

Glucose and H₂O₂ detection with Fe-WS₂ nanozyme

sj-docx-1-sci-10.1177_00368504251362318 - Supplemental material for Iron-doped tungsten disulfide nanozyme with peroxidase-like activity for hydrogen peroxide (H₂O₂) and glucose detection