English

• The widespread use of horseradish peroxidase (HRP) is plagued by issues such as poor stability, high costs, and complex purification processes, prompting the need for artificial enzymes [1-3]. Following the groundbreaking work in 2007, extensive explorations into nano-peroxidase alternatives have emerged, leading to research on nanomaterials displaying enzyme-like activities, coined nanozymes [4-7]. Nanozymes possess enzyme-like activities while offering benefits like simplified preparation, improved stability, customizable surfaces, and adaptable properties [8-14]. However, despite the increasing numbers, particularly in peroxidase-like nanozymes, improving their activity remains pivotal [15,16]. Furthermore, most peroxidase-like nanozymes lack specificity, potentially inducing competitive actions such as oxidase-like activities and affecting result accuracy [17,18]. Thus, achieving the full potential of peroxidase-like nanozymes is still hindered, necessitating further advancements.

  Platinum group metal-based nanomaterials have emerged as versatile nanozymes, boasting unique physicochemical characteristics, excellent enzyme-like activity, remarkable stability, and a vast surface area, which make them conducive for biomedical applications [19-23]. Notably, the exploration into osmium (Os) nanozymes, a subset within the platinum group metals, has witnessed a surge in recent years, leading to profound insights into their peroxidase-like activity and potential applications [24-28]. These studies have illuminated the inherent peroxidase-like activity of Os nanozymes, highlighting their surface modification, exceptional peroxidase-like activity, and minimal oxidase-like activity under acidic conditions, thereby positioning them as candidates for peroxidase-based applications. Nevertheless, owing to its precious metal nature, its cost remains relatively high, necessitating a critical evaluation of potential enhancements in activity or cost-saving measures before practical and large-scale application.

  Extensive research has revealed that bimetallic or polymetallic nanomaterials display enhanced activity compared to their monometallic counterparts [29-34]. This heightened performance stems from the distinct physicochemical properties inherent in each metal, their inter-metal interactions, and their synergistic effects when used in conjunction with supports. Inspiringly, strategies aimed at improving enzyme-like activity and reducing costs associated with noble metal-based nanozymes involve tailoring compositions through the integration of base metals, presenting a promising solution [35-38]. For example, Xi et al. devised Ni-Pt nanoparticles exhibiting peroxidase-like behavior, proving effective in detecting carcinoembryonic antigens [36]. Their findings underscore that the exceptional peroxidase-like efficacy of Ni-Pt nanoparticles arises from a unique surface structure that mitigates the adsorption of critical intermediates during catalysis. Additionally, Tang et al. introduced high-indexed intermetallic Pt₃Sn, demonstrating remarkable peroxidase-like activity and specificity in immunoassay applications [37]. Hence, the incorporation of bimetallic nanozymes could hold the promise of yielding benefits, including heightened activity, cost-effectiveness, or engendering suitable applications.

  Despite notable progress in synthesizing Os nanozymes and exploring their enzyme-like activities, the development of Os-based alloy or bimetallic nanozymes remains limited. Therefore, our approach focuses on engineering a nickel (Ni)-supported Os bimetallic nanozyme to enhance peroxidase-like activity while ensuring exceptional specificity. In this work, we synthesized Ni₂/Os nanoclusters by utilizing K₂OsCl₆ and NiCl₂ as the metal precursors, employing NaBH₄ as the reducing agent, and capitalizing on hyaluronate (HA) as both a stabilizer and a ligand. This approach resulted in the creation of HA-Ni₂/Os, revealing a substantial augmentation in peroxidase-like activity, surpassing the performance of non-Ni-supported Os nanozyme (Scheme 1). In-depth analyses unraveled the properties of the HA-Ni₂/Os, shedding light on their exceptional peroxidase-like activity. Furthermore, harnessing the remarkable peroxidase-like activity of HA-Ni₂/Os, coupled with the presence of carboxyl groups within the HA structure that facilitates effective crosslinking, empowered their integration into an immunoassay for squamous carcinoma antigen (SCCA) detection (Scheme 1). These outcomes establish this Ni-supported Os bimetallic nanozyme, characterized by enhanced peroxidase-like activity and cost efficiency, as a promising candidate for a wide array of peroxidase-like applications and endeavors.

  Scheme 1

  

  Scheme 1.  Schematic representation of HA-Ni₂/Os with specifically improved peroxidase-like activity and HA-Ni₂/Os-based SCCA immunoassay.

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  Fig. 1A delineated the procedure for modifying Ni₂/Os bimetallic nanozymes with HA as a ligand. The synthesis of Ni₂/Os occurred through a one-pot method, involving the reduction of K₂OsCl₆ and NiCl₂ in HA using NaBH₄. Observable to the naked eye, the mixture underwent a noticeable color change from light green to brown post-reaction, suggestive of an Os transformation. In the pre-exploratory phase, we have respectively synthesized several polysaccharides (HA, carboxymethyl cellulose, fucoidan, and chondroitin sulfate) modified Ni₂/Os nanozymes and compared their peroxidase-like activities, as shown in Fig. S1 (Supporting information), with HA-Os demonstrating the most favorable catalytic performance, highlighting the advantages of HA as a ligand. We further compared HA-Ni₂/Os with several reported Os nanozymes and found that HA-Ni₂/Os exhibited the best peroxidase-like activity among them (Table S1 in Supporting information). Several characterizations were conducted to validate the preparation, morphology, and structure of HA-Ni₂/Os. The TEM image depicted in Fig. S2A (Supporting information) exhibited the well-dispersed morphology of HA-Ni₂/Os. Moreover, the hydrated particle size of HA-Ni₂/Os was calculated to be approximately 33.45 nm, consistent with the size observed in the inset TEM image (Fig. 1B), indicating the encapsulation of Ni₂/Os within HA. The zeta potential of HA-Ni₂/Os was also measured to be −37.1 mV (n = 3). Additionally, the HRTEM image of HA-Ni₂/Os in Fig. S2B (Supporting information) revealed crystal lattice spacings, corresponding to the lattice constants of the (002) plane of Os and the (111) plane of Ni, respectively. Upon further magnification, the average particle size of Ni₂/Os without HA was calculated to be approximately 1.62 nm at the nanocluster level (Fig. 1C). As illustrated in Fig. S3 (Supporting information), high-angle annular dark-field scanning TEM (HADDF-STEM) images and corresponding energy dispersive spectroscopy (EDS) mapping showed that Os and Ni elements were well distributed in HA-Ni₂/Os. Moreover, comprehensive EDS analysis showed the presence of C, N, O, Ni, and Os in HA-Ni₂/Os, indicating the smooth binding of HA to Ni₂/Os (Fig. S4 in Supporting information). Table S2 (Supporting information) provided the elemental composition in HA-Ni₂/Os, confirming a Ni/Os ratio of approximately 2:1. The XPS patterns illustrated the distribution of C, O, N, Ni, and Os in HA-Ni₂/Os (Fig. S5 in Supporting information). The corresponding fitted peaks for C and O were provided in Figs. S6A and B (Supporting information). The 2p orbital pattern of Ni indicated that both Ni⁰ and Ni²⁺ were present. The binding energies of Ni 2p_1/2 and Ni 2p_3/2 were 870 eV and 852.5 eV, respectively (Fig. 1D). Ni²⁺ accounted for 42% and Ni⁰ for 58% of the total. The 4f orbital profile of Os showed the presence of both Os⁰ and Os⁴⁺. The binding energies of Os⁰ were 53.8 eV and 50.7 eV, and the binding energies of Os⁴⁺ were 54.4 eV and 52.8 eV (Fig. 1E). The ratio of Os⁴⁺ was 46% and the ratio of Os⁰ was 54%. The peak area ratio of Ni⁰:Os⁰ was approximately 2:1, indicating the synthesis of HA-Ni₂/Os. These findings collectively contribute to a comprehensive understanding of the morphology, composition, and structural attributes of HA-Ni₂/Os.

  Figure 1

  

  Figure 1.  (A) Schematic representation of the synthesis process. (B) The hydrodynamic size distribution of HA-Ni₂/Os. Inset: TEM image of single HA-Ni₂/Os. (C) Gaussian fitting of the size distribution of Ni₂/Os in HA-Ni₂/Os (100 random). XPS spectra of (D) Ni 2p and (E) Os 4f in HA-Ni₂/Os.

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  Typically, the oxidative transformation of TMB to generate blue compounds in the presence of H₂O₂ catalyzed by HA-Ni₂/Os confirmed the inherent peroxidase-like activity, a validation supported by the observed absorption peak at 652 nm (Fig. 2A). This study was primarily dedicated to exploring the distinct benefits linked to employing Ni/Os bimetallic nanozymes with peroxidase-like activity. The substantial impact of Ni incorporation into Os nanozymes, compared to diverse transition metals, has been discerned as pivotal in significantly influencing peroxidase-like activity, as highlighted (Fig. S7 in Supporting information). Furthermore, we examined the influence of Ni doping on Os by synthesizing nanozymes with different Ni/Os ratios and observing consequent alterations in their peroxidase-like activity. Fig. S8 (Supporting information) presented the XPS results for various HA-Ni_n/Os corresponding to distinct Ni/Os ratios, including HA-Os, HA-Ni/Os, and HA-Ni₃/Os. Upon comparing the peroxidase-like activity among different HA-Ni_n/Os, a notable revelation emerged: the absorbance at 652 nm for HA-Ni₂/Os was more than twice as high as that observed for non-Ni-supported Os (Fig. 2B). Furthermore, our investigation extended to the analysis of products within the H₂O₂ + HA-Ni_n/Os system using electron spin resonance (ESR) spectroscopy, employing dimethylpyridine N-oxide (DMPO) as a spin trap (Fig. 2C). Notably, in the ESR spectra of the DMPO + H₂O₂ + HA-Ni₂/Os group, the DMPO/•OH adducts exhibited the most prominent peaks. These observations suggest that the ·OH generated during the HA-Ni₂/Os catalyzed process facilitates the effortless oxidation of TMB.

  Figure 2

  

  Figure 2.  (A) UV–vis spectrum: TMB + H₂O₂, TMB + HA-Ni₂/Os. and TMB + H₂O₂ + HA-Ni₂/Os. Inset: Schematic representation and corresponding photographs. (B) Histogram: the absorbance at 652 nm of TMB + H₂O₂ for different HA-Ni_n/Os. (C) ESR spectrum of H₂O₂ with different nanozymes employing DMPO as a spin trap. Steady-state kinetic parameters of different HA-Ni_n/Os toward (D) H₂O₂ and (E) TMB. (F) Peroxidase (POD), superoxide dismutase (SOD), catalase (CAT), and oxidase-like activities of different HA-Ni_n/Os. The error bar represents the standard deviation from the repeated experiments after three times.

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  Fig. S9 (Supporting information) illustrated the steady-state kinetic parameters of various HA-Ni_n/Os towards H₂O₂ and TMB, fitted by the Michaelis-Menten equation. K_M values signify the affinity between the catalyst and the substrate, with lower K_M values indicating stronger substrate affinity. Notably, HA-Ni₂/Os exhibited superior affinities for H₂O₂ (K_M = 69.4 mmol/L) and TMB (K_M = 0.15 mmol/L) compared to others (Figs. 2D and E). Consequently, HA-Ni₂/Os could generate elevated levels of peroxidase-like activity compared to the other HA-Ni_n/Os due to higher substrate affinity. Furthermore, the specific activity (SA) serves as a metric for the nanozyme's enzyme-like activity and can be quantified [39]. Herein, a single unit of activity (U) is defined as the amount of Os in HA-Ni_n/Os capable of catalyzing the formation of 1 µmol product per minute at 37 ℃ and pH 4. SA is determined as the amount of U per gram of Os in HA-Ni_n/Os. This quantification enabled an evaluation of various HA-Ni_n/Os and provided a standardized measure for comparative assay. As calculated in Fig. S10 (Supporting information), the specific activity of HA-Ni₂/Os (1224 U/mg) surpassed that of HA-Os (500 U/mg) by over two-fold, consistent with the findings presented in Fig. 2B. This substantial difference underlined the enhanced catalytic efficiency of HA-Ni₂/Os compared to HA-Os. Additionally, Table S3 (Supporting information) offered a summary of the kinetic parameters and specific activity units of HA-Ni_n/Os, conclusively affirming the superior peroxidase-like activity of HA-Ni₂/Os. Additionally, a comprehensive assessment encompassing the superoxide dismutase (SOD)-like activity, catalase (CAT)-like activity, and oxidase-like activity of HA-Ni_n/Os was also conducted (Fig. 2F). Intriguingly, results demonstrated that the introduction of Ni did not exert any discernible influence on other activities, apart from its substantial effect on enhancing the peroxidase-like activity. This phenomenon in augmenting only the peroxidase-like activity suggests a controlled catalytic modification induced by the presence of Ni within the Os bimetallic nanozyme, emphasizing its unique role in improving the peroxidase-like activity.

  To decipher the underlying mechanism behind the peroxidase-like activity of HA-Ni₂/Os, further investigation employed focused density-functional theory (DFT) calculations centered on the H₂O₂ cleavage and TMB oxidation, exploring the surfaces of Ni, Os, and Ni/Os (HA-Ni, HA-Os, and HA-Ni₂/Os). For this purpose, we constructed computational models of the Ni (111) slab, Os (002) slab, and NiOs (200) slab which emulate the catalytic surfaces of Ni, Os, and Ni/Os, respectively. Previous DFT studies have proposed some potential mechanisms for peroxidase-like metal nanozymes, yet there is no mention to our knowledge, of the specific interaction or mechanism involving Ni/Os-based peroxidase-like process [36,40-42]. Accordingly, the H₂O₂ cleavage had three reaction steps on the metal surfaces (Fig. 3A): Firstly, the molecule of H₂O₂ adsorbed at the metal surface (Ⅰa). Then, the cleavage of H₂O₂ to form two OH* adsorbates (Ⅱa). Finally, the rearrangement of the two OH* adsorbates to form H₂O* and O* adsorbates (Ⅲa). These three steps were written as follows: * + H₂O₂ → H₂O₂* (Ⅰa), H₂O₂* + * → 2OH* (Ⅱa), 2OH* → H₂O* + O* (Ⅲa). The adsorption of the H₂O₂ molecule on the Ni, Os, and Ni/Os surfaces had a negative energy of −0.1079 eV, −0.3259 eV, and −0.4644 eV, respectively (Fig. 3B). Comparison of Ni and Os, H₂O₂ adsorbed more easily on the Ni/Os surface. The subsequent cleavage of the H₂O₂* → 2OH* on the Ni, Os, and Ni/Os surfaces had an energy change of −3.6652 eV, −2.6286 eV, and −3.6615 eV, respectively. Although step (Ⅱa) was more likely to occur on the Ni surface than Os and Ni/Os, Ni/Os had the strongest ability to generate OH* in a combined comparison due to the better ability to adsorb H₂O₂ on step (Ⅰa) than others. Thus, the strong peroxidase-like activity of NiOs could be attributed to the strongest affinity for H₂O₂. Fig. 3C also depicted the sequential stages of TMB oxidation on Ni, Os, and Ni/Os surfaces. Upon comparison, the rate-determining step was identified as C₁₆H₁₉N₂* → C₁₆H₁₈N₂* + (H⁺ + e⁻), revealing energy values of 0.3889 eV for Ni, 0.1467 eV for Os, and 0.0599 eV for Ni/Os (Fig. 3D). Significantly, Ni/Os surface displayed the lowest free energy barriers at this critical step, substantiating its superior capability in promoting TMB oxidation, thus indicating the probable occurrence of reaction. Following DFT calculations, it was inferred that the Ni/Os surface exhibited accelerated •OH generation through easier H₂O₂ adsorption, while demonstrating heightened susceptibility to TMB oxidation.

  Figure 3

  

  Figure 3.  (A) Proposed H₂O₂ cleavage pathway on the Ni/Os surface. (B) Free energy diagrams for H₂O₂ cleavage on Ni, Os, and Ni/Os surfaces. (C) Proposed TMB oxidation pathway on the Ni/Os surface. (D) Free energy diagrams for TMB oxidation on Ni, Os, and Ni/Os surfaces.

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  In clinical diagnostics, tumor markers like squamous cell carcinoma antigen (SCCA) are identified or found to have abnormal expression in patients' serum [43,44]. The presence of SCCA closely correlates with the advancement of lung cancer, presenting substantial promise in early detection initiatives [45,46]. Hence, the creation of methods tailored for SCCA holds importance. This study further employed the HA-Ni₂/Os-linked immunoassay to evaluate its efficacy in detecting SCCA (Scheme 1). Initially, mouse anti-SCCA polyclonal antibodies recognizing SCCA were pre-coated onto the plates. Subsequently, the carboxyl groups within the HA-Ni₂/Os structure were activated and cross-linked with the amino groups in rabbit anti-SCCA polyclonal antibodies using a traditional crosslinking method (EDC/NHS). This crosslinking, in combination with the TMB-H₂O₂ system, enabled the detection of SCCA. The optimization of the TMB-H₂O₂ system catalyzed by HA-Ni₂/Os revealed that the most effective conditions encompassed a pH of 4, temperature of 37 ℃, reaction duration of 20 min, with a final concentration of 0.2 mmol/L for TMB and 5 mmol/L for H₂O₂ (Fig. S11 in Supporting information). The immunoassay utilizing HA-Ni₂/Os exhibited a linear detection range spanning 2–36 pg/mL for SCCA, boasting a limit of detection (LOD) of 1.55 pg/mL (n = 3) as the constructed calibration curve (Fig. 4A). Meanwhile, we analyzed SCCA by the traditional HRP immunoassay, which had a linear range of 2–32 pg/mL and constructed a standard curve equation of y = 0.0253x - 0.0190, which led to the calculation of the limit of detection (LOD) of 1.38 pg/mL (Fig. S12 in Supporting information). This indicated that our constructed assay could replace the traditional HRP assay. Fig. 4B also demonstrated the specificity of the HA-Ni₂/Os-based immunoassay for SCCA detection by replacing SCCA with interfering substances, including carcinoembryonic antigen, β-amyloid 1–42, prostate-specific antigen, leukocyte-derived chemokine 2, and the proto-oncogene human epidermal growth factor receptor 2, set at concentrations of 16 pg/mL. The concentrations of SCCA detected in human serum samples using the HA-Ni₂/Os-based immunoassay exhibited consistency with the outcomes obtained from the HRP-based ELISA (Fig. 4C). A strong correlation (R² = 0.992) and a close slope (0.999) were observed between the data of each method, confirming the suitability of HA-Ni₂/Os-based immunoassay in real samples.

  Figure 4

  

  Figure 4.  (A) Linear calibration plot of Δ_{A450 nm} with various SCCA concentrations. (B) Selectivity of HA-Ni₂/Os-based SCCA immunoassay. Inset: Corresponding photographs. (C) Correlation analysis between the HA-Ni₂/Os-based and HRP-based SCCA immunoassay in separately quantifying SCCA from clinical serum samples. (D) Catalytic stability of HA-Ni₂/Os after incubation at room temperature for one month. The error bar represents the standard deviation from the repeated experiments after three times.

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  Compared to recent SCCA detection methods, this assay showcases a detection range and limit that are either lower or comparable to those of existing techniques (Table S4 in Supporting information). While certain methods, like electrochemical immunoassays, provide heightened sensitivity, their practicality is hampered by operational complexity and the necessity for specialized operators [47]. Surface-enhanced Raman scattering testing necessitates expensive and sophisticated instruments [48]. Herein, the immunoassay developed in this study offers visual simplicity and straightforward operation. Notably, the HA-Ni₂/Os also exhibit remarkable catalytic stability, retaining over 90% of their peroxidase-like activity even after being stored at room temperature for a month, a feature unparalleled by natural peroxidase (Fig. 4D) [49]. Additionally, we compared the stability of HA-Ni₂/Os-linked with rabbit anti-SCCA polyclonal antibody (HA-Ni₂/Os-conjugate) and HRP-linked with rabbit anti-SCCA polyclonal antibody (HRP-conjugate) after incubation at room temperature for one week (Fig. S13 in Supporting information). Impressively, the HA-Ni₂/Os-conjugate demonstrated notably superior stability compared to the HRP-conjugate. These results underscore the robustness and reliability of HA-Ni₂/Os as a nanozyme, positioning it as a preferred choice for immunoassays. Overall, this immunoassay exhibits substantial practical value in real-world applications. Finally, we also summarised the cost of the immunoassay method for SCCA based on HA-Ni₂/Os from nanozyme synthesis, HA-Ni₂/Os-conjugate preparation to SCCA assay, as shown in Table S5 (Supporting information), the cost of spiking each well plate each time was about ¥ 0.23.

  In summary, this study has successfully introduced a bimetallic nanozyme, HA-Ni₂/Os, developed by incorporating Ni and Os with HA. Experiments have confirmed their formation and peroxidase-like behavior. The role of Ni in augmenting the peroxidase-like activity of Os nanozyme has been demonstrated, showcasing an over two-fold increase in activity compared to non-Ni-supported Os nanozyme. DFT results also indicate HA-Ni₂/Os in enhancing H₂O₂ adsorption and TMB oxidation, boosting their peroxidase-like activity. Importantly, the support of Ni does not significantly alter the other enzyme-like activities of Os nanozymes, thereby enabling Ni to selectively enhance their peroxidase-like activity. Utilizing the enhanced peroxidase-like activity of HA-Ni₂/Os and HA's carboxyl groups, a sensitive and selective nanozyme-linked immunoassay achieved an LOD of 1.55 pg/mL for SCCA detection. Moreover, HA-Ni₂/Os presented excellent stability unparalleled by natural enzymes. This research underscores Ni's pivotal role in improving the peroxidase-like performance of Os-based bimetallic nanozymes, promising advancements in analytics and diagnostics.

  Ehical statement

  Clinical human samples were obtained from the Second Affiliated Hospital of Fujian Medical University. This study was approved by the Ethics Committee of the Second Affiliated Hospital of Fujian Medical University (Ethics Number: 2021–421 and 2023–20).

  Declaration of competing interest

  The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

  CRediT authorship contribution statement

  Shaobin He: Conceptualization, Investigation, Methodology, Project administration, Writing – original draft. Xiaoyun Guo: Investigation, Methodology, Writing – original draft. Qionghua Zheng: Investigation, Validation. Huanran Shen: Investigation. Yuan Xu: Investigation. Fenglin Lin: Investigation. Jincheng Chen: Conceptualization, Funding acquisition, Project administration, Supervision, Writing – review & editing. Haohua Deng: Methodology. Yiming Zeng: Conceptualization, Supervision, Writing – review & editing. Wei Chen: Conceptualization, Funding acquisition, Project administration, Supervision, Writing – review & editing.

  Acknowledgments

  The authors gratefully acknowledge financial support from the Natural Science Foundation of Fujian Province (No. 2022J01271), the Joint Funds for the Innovation of Science and Technology, Fujian Province (No. 2023Y9226), the Introduced High-Level Talent Team Project of Quanzhou City (No. 2023CT008), and the Doctoral Research Foundation Project of the Second Affiliated Hospital of Fujian Medical University (No. BS202201).

  Supplementary materials

  Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.cclet.2024.110096.

  
  
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  Scheme 1  Schematic representation of HA-Ni₂/Os with specifically improved peroxidase-like activity and HA-Ni₂/Os-based SCCA immunoassay.

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  Figure 1  (A) Schematic representation of the synthesis process. (B) The hydrodynamic size distribution of HA-Ni₂/Os. Inset: TEM image of single HA-Ni₂/Os. (C) Gaussian fitting of the size distribution of Ni₂/Os in HA-Ni₂/Os (100 random). XPS spectra of (D) Ni 2p and (E) Os 4f in HA-Ni₂/Os.

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  Figure 2  (A) UV–vis spectrum: TMB + H₂O₂, TMB + HA-Ni₂/Os. and TMB + H₂O₂ + HA-Ni₂/Os. Inset: Schematic representation and corresponding photographs. (B) Histogram: the absorbance at 652 nm of TMB + H₂O₂ for different HA-Ni_n/Os. (C) ESR spectrum of H₂O₂ with different nanozymes employing DMPO as a spin trap. Steady-state kinetic parameters of different HA-Ni_n/Os toward (D) H₂O₂ and (E) TMB. (F) Peroxidase (POD), superoxide dismutase (SOD), catalase (CAT), and oxidase-like activities of different HA-Ni_n/Os. The error bar represents the standard deviation from the repeated experiments after three times.

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  Figure 3  (A) Proposed H₂O₂ cleavage pathway on the Ni/Os surface. (B) Free energy diagrams for H₂O₂ cleavage on Ni, Os, and Ni/Os surfaces. (C) Proposed TMB oxidation pathway on the Ni/Os surface. (D) Free energy diagrams for TMB oxidation on Ni, Os, and Ni/Os surfaces.

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  Figure 4  (A) Linear calibration plot of Δ_{A450 nm} with various SCCA concentrations. (B) Selectivity of HA-Ni₂/Os-based SCCA immunoassay. Inset: Corresponding photographs. (C) Correlation analysis between the HA-Ni₂/Os-based and HRP-based SCCA immunoassay in separately quantifying SCCA from clinical serum samples. (D) Catalytic stability of HA-Ni₂/Os after incubation at room temperature for one month. The error bar represents the standard deviation from the repeated experiments after three times.

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