ZIF-derived Co₃O₄/Fe₂O₃ dual-layer hollow nanoboxes as bimetallic nanozymes for detection of hydrogen peroxide and ascorbic acid

Abstract

Nanozymes, owing to their high stability and cost-effectiveness, present themselves as appealing alternatives to natural enzymes. The rational design of complex metal–organic framework (MOF) hybridization precursors offers significant opportunities for constructing diverse functional nanostructures. This paper introduces a novel strategy, assisted by MOF hybridization, for synthesizing Co₃O₄/Fe₂O₃ dual-layer hollow nanoboxes (DLHNBs) via anion exchange, coupled with low-temperature pyrolysis, employing ZIF-67 as the original template. This approach maximizes the advantages of hollow nanostructures, functioning as both nanoreactors and substrate channels, thereby mimicking the action of natural enzymes. The Co₃O₄/Fe₂O₃ DLHNBs exhibited lower steady-state kinetic parameters, indicating a superior affinity between Co₃O₄/Fe₂O₃ DLHNBs and H₂O₂. Furthermore, the sensing application of Co₃O₄/Fe₂O₃ DLHNBs nanozymes for the determination of H₂O₂ and ascorbic acid (AA) was explored by using a colorimetric method. The linear range for H₂O₂ detection was established at 100–1000 µM, with a limit of detection (LOD) of 2.13 µM. For AA, the linear range was determined to be 5–40 µM, with a calculated LOD of 0.15 µM. This work not only demonstrates the potential of peroxidase-like nanozymes for sensing applications but also provides a valuable reference for the design and synthesis of MOF-based nanozymes.

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1. Introduction

Ascorbic acid (AA), namely vitamin C, serves as a crucial redox cofactor in numerous biochemical pathways. Its distinctive role in human health encompasses the prevention and management of conditions such as the common cold, scurvy, and specific malignancies.^1,2 Furthermore, AA is extensively utilized as an antioxidant across diverse sectors, including pharmaceuticals, food preservation, and cosmetic formulations. The human liver lacks the ability to synthesize L-glutathione lactone oxidase, an essential enzyme involved in the conversion of blood glucose to AA. Consequently, it is vital for humans to obtain exogenous AA through their diet to sustain life.³ However, AA readily undergoes pH-dependent autoxidation producing hydrogen peroxide (H₂O₂). In the presence of catalytic metals, this oxidation is accelerated.⁴ As a potent oxidizing agent, H₂O₂ is extensively utilized in food production, industry, and medicine. Some diseases, such as aging and Alzheimer's disease,⁵ are associated with long-term exposure to high concentrations of H₂O₂. Therefore, there is an urgent need to develop a simple, rapid, sensitive, and cost-effective method for the detection of AA and H₂O₂ in food, drugs, the environment and living organisms.

To date, several methods have been developed for the detection of AA^6–10 and H₂O₂,^11–13 including chemiluminescence, fluorescence, electrochemistry, capillary electrophoresis, and high-performance liquid chromatography. While these techniques offer high accuracy and sensitivity, they often require sophisticated equipment and trained personnel. In contrast, colorimetric detection presents a simpler, faster, and more cost-effective alternative.^14,15 Natural enzymes can effectively catalyze substrates and can be applied in colorimetric detection. Nonetheless, they come with certain limitations, including stringent environmental requirements, limited stability, and high production costs. In contrast, nanozymes offer distinct advantages such as ease of synthesis, cost-effectiveness, recyclability, and robust resistance to harsh reaction conditions, positioning them as a promising alternative to natural enzymes.^16–21 Despite significant advancements in nanozymes’ research in recent years, their catalytic performance and specificity still fall short of those of natural enzymes. Therefore, the pursuit of nanozymes with excellent catalytic performances is a key focus in recent research endeavors.

Nanozymes are a class of nanomaterials with nanoscale sizes (1–100 nm), such as noble metals, metallic oxides, and carbon materials.²² Over the past decade, metal–organic frameworks (MOFs) have garnered significant interest as a new platform for nanozymes.^23,24 MOFs offer several advantages, including high stability, large specific surface areas, and unsaturated metal sites, and are extensively utilized across a range of applications, including bioanalysis and detection, environmental remediation,²⁵ gas separation,²⁶ energy storage and conversion,^27,28 catalysis,^29,30etc. MOFs and MOF-derived nanozymes demonstrate diverse catalytic properties, thereby aiding in the construction a variety of sensor systems for the detection of multiple sample substrates, such as diabetes-related glucose and α-glucosidase (α-Glu),³¹ antioxidants,³² nitrite,³³ and quercetin.³⁴ Therefore, nanoezyme sensors based on MOFs and their derivatives are becoming a research hotspot in fields such as analytical chemistry, biochemistry, biomedicine and environmental chemistry. It is anticipated that preparing multi-component and multi-catalytic center MOF-derived nanohybrids will lead to the development of highly active and specific nanozymes.

Herein, we introduce a new method of hybridization and derivatization of MOFs for the synthesis of Co₃O₄/Fe₂O₃ double-layer hollow nanoboxes (DLHNBs). Initially, ZIF-67 nanocubes (NCs) as the hard template are converted into ZIF-67@Co/Fe Prussian blue analog (PBA) core–shell nanocubes (CSNCs). Subsequently, the Co₃O₄/Fe₂O₃ DLHNBs were obtained by annealing the precursor of ZIF-67@Co/Fe CSNCs. Compared with Co₃O₄ single-layer hollow nanoboxes (SLHNBs) and Co₃O₄/Fe₂O₃ SLHNBs, Co₃O₄/Fe₂O₃ DLHNBs exhibit stronger peroxidase-like activity and show good selectivity and high sensitivity in the colorimetric detection of AA, thus holding great potential for practical applications.

2. Experimental

2.1. Instruments and materials

Experimental instruments and materials are presented in the SI.

2.2. Preparation of Co₃O₄/Fe₂O₃ dual-layer hollow nanoboxes (DLHNBs)

The Co₃O₄/Fe₂O₃ dual-layer hollow nanoboxes (DLHNBs) can be synthesized in three steps. Synthesis of ZIF-67 nanocubes (NCs):³⁵ 1 mol of Co(NO₃)₂·6H₂O was dissolved in 10 mL of an aqueous solution containing 5 mg of cetyltrimethylammonium bromide (CTAB). This solution was then rapidly injected into 70 mL of an aqueous solution containing 0.05 mol of 2-methylimidazole (2-MIM) and stirred for 40 minutes at room temperature. The resulting product was collected via centrifugation and washed several times with ethanol.

Synthesis of ZIF-67@Co/Fe Prussian blue analog (PBA) core–shell nanocubes (CSNCs):³⁶ ZIF-67 NCs were dispersed in ethanol, while K₃[Fe(CN)₆] was dissolved in water to form a clear solution. The aqueous K₃[Fe(CN)₆] solution (0.015 M) was then rapidly injected into the ZIF-67 NCs dispersion (20 mL), with continuous stirring (800 rpm) at room temperature for at least 2 hours. The resulting product was collected by centrifugation and washed three times with both water and ethanol.

Lastly, ZIF-67@Co/Fe PBA CSNCs were heat-treated at 350 °C in air for 2 h to obtain Co₃O₄/Fe₂O₃ dual-layer hollow nanoboxes (DLHNBs). The synthesis steps of the control sample (Co₃O₄/Fe₂O₃ single-layer hollow nanoboxes and Co₃O₄ hollow nanoboxes) are described in the SI.

2.3. Catalytic reaction dynamics of Co₃O₄/Fe₂O₃ DLHNBs

The catalytic activities of Co₃O₄/Fe₂O₃ DLHNBs were evaluated through the oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) to ox-TMB in the presence of H₂O₂. The basic reaction solution was 1.86 mL of HAc-NaAc buffer (0.1 M, pH = 4), with the addition of 20 µL of TMB (0.5 mM) and 20 µL of H₂O₂ (1 mM). Subsequently, 100 µL of the catalyst dispersion (1 mg mL⁻¹) was introduced and mixed for 3 minutes at 37 °C. The reaction process was detected by the UV-Vis absorption spectrum recorded at 652 nm.

The steady-state kinetic parameters, i.e. the Michaelis constant (K_m) and the maximum reaction velocity (V_max), were determined by altering the concentration of TMB while keeping the concentration of H₂O₂ constant (1 mM), or by changing the H₂O₂ concentration while maintaining the concentration of TMB (0.5 mM). The variation of absorbance at 652 nm over time was recorded to monitor the color change of the reaction in time scan mode. By plotting the calculated initial rate versus substrate concentration, a Michaelis–Menten curve is obtained, which follows eqn (1):³⁷

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

where [S] is the the substrate concentration.

Additionally, the details of the catalytic reaction mechanism of Co₃O₄/Fe₂O₃ DLHNBs are described in the SI.

2.4. Detection of H₂O₂ and ascorbic acid based on nanozymes catalysis

Detection of H₂O₂: a total of 20 µL of TMB (0.5 mM) and 100 µL of dispersion liquid of Co₃O₄/Fe₂O₃ DLHNBs (0.05 mg mL⁻¹) were added to 1.86 mL of acetate buffer (pH 4.0) and mixed uniformly. Then, the various concentrations of H₂O₂ (ranging from 0 to 1 mM) were added to the above solution, and the reaction was carried out for 3 minutes at 37 °C in a water bath. The absorbance of solution at 652 nm was measured to ascertain the linear relationship.

Detection of AA: the mixed solution is the same as that for the test of H₂O₂. Then, 20 µL of 1 mM H₂O₂ was added to the mixture solution. Subsequently, 20 µL of AA with different concentrations (0–40 µM) was introduced to the solution, which was then incubated at 37 °C for 3 minutes. The absorbance of the solution was measured at 652 nm to establish a linear relationship between absorbance and concentration of AA.

To assess the practical applicability, we selected a commercial vitamin C injection for direct detection and commercial orange juice for spiked recovery experiments. The details of detection are described in the SI.

3. Results and discussion

3.1. Synthesis and characterization of Co₃O₄/Fe₂O₃ DLHNBs

The concise preparation process of nanozymes is illustrated in Scheme 1. Cubic ZIF-67 nanoparticles were selected as precursors due to their distinctive reactivity, enabling three pathways to produce cobalt-based metal oxides with varying chemical compositions and unique shell structures. The Co₃O₄/Fe₂O₃ dual-layer hollow nanoboxes (DLHNBs) were synthesized through a two-step method (Route III). Initially, homogeneous cobalt-based zeolite imidazolium skeleton nanocubes were fabricated using a modified surfactant method. This was followed by the preparation of ZIF-67@Co/Fe PBA core–shell nanocubes (CSNCs) via an anion-exchange reaction with K₃[Fe(CN)₆]₃, employing ZIF-67 NCs as a sacrificial template. Ultimately, Co₃O₄/Fe₂O₃ DLHNBs were formed by thermally calcining ZIF-67@Co/Fe PBA CSNCs under air. The ZIF-67 cubes and Co/Fe PBA outer shells were transformed into Co₃O₄ inner hollow cubes and Co₃O₄/Fe₂O₃ shells, respectively. For comparison, Co₃O₄ SLHNBs were synthesized through direct thermal conversion of ZIF-67 NCs (Route I). Additionally, interior ZIF-67 was entirely etched away after the anion-exchange reaction, resulting in the formation of Co/Fe PBA hollow nanocubes. Then, Co₃O₄/Fe₂O₃ single-layer hollow nanoboxes (SLHNBs) were subsequently formed by calcining the Co/Fe PBA hollow nanocubes in air (Route II).

Scheme 1 Schematic illustration of routes towards MOF-derived Co₃O₄/Fe₂O₃ DLHNBs, Co₃O₄/Fe₂O₃ SLHNBs and Co₃O₄ SLHNBs.

Images obtained from transmission electron microscopy (TEM) and field emission scanning electron microscopy (FESEM) confirmed the morphology and structure of the products at each step. The initial precursor of ZIF-67 NCs shows uniform size distribution, smooth surface, and solid nanocubes (Fig. 1a and Fig. S1). Then, the anion exchange process involves the reaction of ZIF-67 NCs with [Fe(CN)₆]³⁻ ions. Upon the introduction of K₃[Fe(CN)₆] solution into the ZIF-67 suspension for approximately 1 hour, a notable color change in the reaction solution from purple to brick-red was observed. The [Fe(CN)₆]³⁻ ions rapidly reacted with the Co²⁺ ions to form ultra-small Co/Fe PBA subunits. The ZIF-67@Co/Fe PBA CSNCs retained the cubic shape and the size of the original ZIF-67 (Fig. S2), although their surfaces were covered by the Co/Fe PBA subunits. TEM images further elucidated the structure of the products (Fig. 1b and Fig. S2), revealing a clear gap between the outer shell and the inner solid core. This gap is attributed to the Co/Fe PBA shell serving as an isolation belt that restricts further anion-exchange reaction between the outer [Fe(CN)₆]³⁻ ions and the inner Co²⁺ ions.

Fig. 1 TEM images of (a) ZIF-67 NCs, (b) ZIF-67@Co/Fe PBA CSNCs, and (c) Co₃O₄/Fe₂O₃ DLHNBs, respectively. (d) SEM image, (e) selected-area diffraction pattern, (f)–(i) HRTEM images and (j)–(o) EDS mapping images of Co₃O₄/Fe₂O₃ DLHNBs.

When ZIF-67@Co/Fe PBA CSNCs were calcined at 350 °C under an air atmosphere, the final products of Co₃O₄/Fe₂O₃ DLHNBs were obtained. In Fig. 1c, Co₃O₄/Fe₂O₃ presents a special double-shell hollow nanoboxes’ structure. Furthermore, Co₃O₄/Fe₂O₃ maintained the cubic skeleton, but the six faces of nanocubes obviously collapsed (Fig. 1d). Fig. 1e displays a selected area electron diffraction (SAED) pattern, and the brightest internal diffraction ring is allocated to the (220) and (111) lattice planes of Co₃O₄ and the (311) lattice plane of Fe₂O₃, respectively.^38,39 The high-resolution TEM (HRTEM) images in Fig. 1f–i reveal that the planar spacings of the (220) and (111) faces of Co₃O₄ are 0.28 nm and 0.47 nm,^13,38 respectively, while the planar spacing of the (311) face of Fe₂O₃ measures 0.25 nm.³⁹ Elemental mapping images (Fig. 1j–o) indicate that cobalt (Co) and oxygen (O) are uniformly distributed throughout the nanostructures, whereas iron (Fe) atoms predominantly reside in the outer shell. This distribution is attributed to the transformation during annealing of ZIF-67@Co/Fe PBA CSNCs under air, where the inner ZIF-67 cube and the outer Co/Fe PBA shell are transformed into interior Co₃O₄ and exterior Co₃O₄ and Fe₂O₃, respectively.

For comparative analysis, Co₃O₄ SLHNBs and Co₃O₄/Fe₂O₃ SLHNBs were prepared, respectively. In Route I, solid ZIF-67 was pyrolysed to obtain Co₃O₄ SLHNBs. In Fig. 2a and b, the Co₃O₄ SLHNBs presented the hollow nanobox structure. The SEM image shows the smooth surface of Co₃O₄ SLHNBs, which maintained the original cubic structure. In Route II, Co₃O₄/Fe₂O₃ SLHNBs were synthesized by a two-step process. First, ZIF-67 was rapidly etched following the addition of acetic acid, which resulted in the outward diffusion of Co²⁺ ions. Subsequently, Co²⁺ ions and [Fe(CN)₆]³⁻ ions primarily reacted on the surface of the ZIF-67 skeleton, causing the generation of the Co/Fe PBA hollow nanocubes (Fig. S3). TEM and SEM images indicate that the diameter of the Co/Fe PBA cubes is comparable to that of the original ZIF-67; however, they exhibit a distinct hollow structure (Fig. S3). Secondly, the Co/Fe PBA nanocubes were calcined under air to obtain Co₃O₄/Fe₂O₃ SLHNBs. The TEM and FESEM images (Fig. 2d–f) demonstrate that Co₃O₄/Fe₂O₃ SLHNBs retained the nanocube morphology, which was inherited from the hollow structure of Co/Fe PBA nanocubes.

Fig. 2 TEM and SEM images of (a)–(c) Co₃O₄ SLHNBs and (d)–(f) Co₃O₄/Fe₂O₃ SLHNBs.

The different materials were characterized structurally and compositionally using X-ray diffraction (XRD). As shown in Fig. 3a, the XRD patterns confirmed the formation of phase-pure ZIF-67.³⁵ Following the reaction with K₃[Fe(CN)₆], in addition to the diffraction peaks of ZIF-67, two new peaks at 25.0° and 35.5° were observed, corresponding to the (002) and (400) planes of Co/Fe PBA, which confirm the formation of the composite ZIF-67@Co/Fe PBA. Additionally, after etching the ZIF-67 NCs and reacting with K₃[Fe(CN)₆], XRD analysis confirmed the crystal structure of Co/Fe PBA (Fig. 3a), with peaks’ compliance with Co/Fe PBA (JCPDS No. 75-0039), which is the precursor of Co₃O₄/Fe₂O₃ SLHNBs. Subsequently, during air oxidation, the ZIF-67 NCs, the Co/Fe PBA, and the ZIF-67@Co/Fe PBA were entirely converted into Co₃O₄ SLHNBs, Co₃O₄/Fe₂O₃ SLHNBs, and Co₃O₄/Fe₂O₃ DLHNBs, respectively (Fig. 3b). After air oxidation of ZIF-67 NCs, the diffraction peaks are assigned to Co₃O₄ (PDF#42-1467)(Fig. 3b). After air oxidation of Co/Fe PBA and ZIF-67@Co/Fe PBA, the diffraction peaks show Co₃O₄ (PDF#42-1467) and Fe₂O₃ (PDF#33-0664),^38,39 which convincingly verify the evolution of the material composition (Fig. 3b).

Fig. 3 (a) XRD patterns of ZIF-67 NCs, Co/Fe PBA SLHNBs and ZIF-67@Co/Fe PBA CSNCs. (b) XRD patterns of Co₃O₄ SLHNBs, Co₃O₄/Fe₂O₃ SLHNBs and Co₃O₄/Fe₂O₃ DLHNBs. High resolution XPS spectra of (c) Co 2p and (d) Fe 2p.

The elemental states and chemical structures of ZIF-67@Co/Fe PBA CSNCs and Co₃O₄/Fe₂O₃ DLHNBs were analyzed using X-ray photoelectron spectroscopy (XPS) (Fig. 3c and d). In the high-resolution spectra of Co₃O₄/Fe₂O₃ DLHNBs, two broad peaks at 795.5 eV and 780.3 eV correspond to Co 2p_1/2 and Co 2p_3/2, respectively (Fig. 3c). The observed energy difference of approximately 15 eV between these spin–orbit lines is consistent with standard Co₃O₄ spectra.^13,38 Following air oxidation, the Co 2p_3/2 and Co 2p_1/2 orbitals of the Co₃O₄/Fe₂O₃ DLHNBs shifted toward lower binding energies. The Co 2p_3/2 peaks were fitted into 779.8 eV and 781.8 eV, attributed to Co³⁺ and Co²⁺, respectively. Similarly, the Co 2p_1/2 peaks were deconvoluted into two peaks assigned to Co³⁺ (794.8 eV) and Co²⁺ (796.2 eV).^40,41 After air oxidation, the Fe 2p_3/2 and Fe 2p_1/2 orbitals in the Co₃O₄/Fe₂O₃ DLHNBs shifted toward higher binding energies, which indicated that there is an electron transfer between Co and Fe. The oxidation state of iron increases, while that of cobalt decreases (Fig. 3d). The Fe 2p_3/2 peaks are fitted into two peaks at 709.8 eV and 712.7 eV, corresponding to Fe³⁺ and Fe²⁺, respectively. Additionally, the Fe 2p_1/2 peaks are fitted into two peaks attributed to Fe³⁺ (724.7 eV) and Fe²⁺ (726.2 eV).⁴² The composition and valence state of the surface elements have been confirmed by XPS.

Fourier-transform infrared (FTIR) spectroscopy was employed to confirm the formation of ZIF-67 NCs, ZIF-67@Co/Fe PBA CSNCs, and Co/Fe PBA SLHNBs. As shown in Fig. S4a, the peaks observed in the 600–1500 cm⁻¹ range and at 1578 cm⁻¹ for ZIF-67 NCs and ZIF-67@Co/Fe PBA CSNCs are indicative of the imidazole ring and the C=N bond, respectively. In addition, the peaks at 3132 and 2925 cm⁻¹ correspond to the aliphatic chain and the C–H stretching vibration, respectively. For Co/Fe PBA, the bands at 2078, 460, and 594 cm⁻¹ are attributed to the C≡N group, Co–CN bending vibrations, and Fe-CN bending vibrations, respectively. Following pyrolysis in air, the characteristic absorption peaks of ZIF-67 NCs, ZIF-67@Co/Fe PBA CSNCs, and Co/Fe PBA SLHNBs vanished, while two new peaks emerged at 571 and 664 cm⁻¹ (Fig. S4b), which correspond to the stretching vibrations of M–O (where M represents Co or Fe). In addition, compared with Co₃O₄ SLHNBs, Co₃O₄/Fe₂O₃ SLHNBs and Co₃O₄/Fe₂O₃ DLHNBs showed the obvious differences around 2000 cm⁻¹ and 1300 cm⁻¹ (Fig. S4b), indicating the existence of water molecules in them. These data are consistent with the XRD and XPS results.

Lastly, the N₂ adsorption–desorption isotherms and pore sizes of Co₃O₄/Fe₂O₃ DLNBs, Co₃O₄/Fe₂O₃ SLHNBs, and Co₃O₄ SLHNBs were analyzed separately (Fig. S5). The N₂ adsorption–desorption isotherms for all catalyst materials exhibited IV isotherm characteristics. The pore size distribution from 2 to 15 nm confirms that three materials display the characteristics of mesoporous materials. The Brunauer–Emmett–Teller (BET) specific surface areas of the Co₃O₄/Fe₂O₃ DLHNBs, Co₃O₄/Fe₂O₃ SLHNBs, and Co₃O₄ SLHNBs, obtained after pyrolysis, were 39.07, 27.57, and 22.93 m² g⁻¹, respectively. Notably, the Co₃O₄/Fe₂O₃ DLHNBs exhibited the largest specific surface area, suggesting that these materials possess a greater number of active sites. The mesoporous structure facilitates improved electron and substance transfer, contributing to the excellent catalytic performance of the materials.

3.2. Catalytic activity and mechanism of Co₃O₄/Fe₂O₃ DLHNBs

When 3,3′,5,5′-tetramethylbenzidine (TMB) is oxidized, its oxidation product, ox-TMB, appears blue to the naked eye and exhibits a maximum absorption peak at 652 nm.^43,44 This characteristic makes TMB an effective substrate for studying nanoenzymatic activity. To investigate the peroxidase-mimicking properties of various materials, this experiment employed TMB as the substrate for the chromogenic reaction. Materials with peroxidase-like activity can catalyze the oxidation of the TMB by H₂O₂, turning it from colorless to blue (ox-TMB). The enzyme catalyzes the oxidation of TMB hydrogen peroxide. The absorbance measured at 652 nm is directly proportional to the peroxidase-mimicking activity of the materials; thus, a higher absorbance indicates greater catalytic activity.

ZIF-67 NCs, ZIF-67@Co/Fe PBA CSNCs, and Co₃O₄/Fe₂O₃ DLHNBs were added in equal concentrations and volumes to the TMB and H₂O₂ systems. After a reaction time of 3 minutes, the UV-vis absorption spectra obtained are presented in Fig. 4A. For Co₃O₄/Fe₂O₃ DLHNBs, the characteristic absorption peak of ox-TMB was significantly higher in the presence of TMB and H₂O₂ compared to that of ZIF-67 NCs and ZIF-67@Co/Fe PBA CSNCs, indicating enhanced peroxidase-mimicking activity of Co₃O₄/Fe₂O₃ DLHNBs. Notably, Co₃O₄/Fe₂O₃ DLHNBs were also able to catalyze TMB oxidation in the absence of H₂O₂. This observation suggests that Co₃O₄/Fe₂O₃ DLHNBs exhibit both oxidase and peroxidase activities. The exceptional peroxidase catalytic performance of Co₃O₄/Fe₂O₃ DLHNBs may stem from the synergistic effects of these two properties. Furthermore, Co₃O₄ SLHNBs, Co₃O₄/Fe₂O₃ SLHNBs, and Co₃O₄/Fe₂O₃ DLHNBs were introduced into the TMB/H₂O₂ system under the same reaction conditions. The UV-vis absorption spectra are presented in Fig. 4B. The results indicate that when Co₃O₄ SLHNBs, Co₃O₄/Fe₂O₃ SLHNBs, and Co₃O₄/Fe₂O₃ DLHNBs served as catalysts, pronounced absorption peaks at 652 nm were observed, suggesting that these three materials display comparable peroxidase-like activities. Notably, Co₃O₄/Fe₂O₃ DLHNBs demonstrated a higher catalytic oxidation rate of TMB. This enhanced activity can be attributed to the advantages of the dual-shell structure of Co₃O₄/Fe₂O₃ DLHNBs and the synergistic interactions among the complex chemical components.

Fig. 4 UV-visible absorption spectra of (A) different systems in the buffer solution (pH = 4), (B) H₂O₂ and TMB with different catalysts. (C) Effects of radical trapping agents on A652 of the TMB/H₂O₂/Co₃O₄/Fe₂O₃ DLHNBs system. (D) Fluorescence intensity of the H₂O₂ + Co₃O₄/Fe₂O₃ DLHNB system generated ˙OH captured by PTA changes with concentration. (E) EPR spectra of DMPO spin-trapping for the ˙OH.

To investigate the catalytic mechanism of Co₃O₄/Fe₂O₃ DLHNBs, we conducted free radical scavenging experiments to identify the active species involved. p-Benzoquinone (pBQ) and L-histidine (L-His) were employed to capture superoxide anions (˙O₂⁻) and singlet oxygen (¹O₂), respectively, and isopropyl alcohol (IPA) was used to capture hydroxyl radicals (˙OH).^45,46Fig. 5C indicates that the participation of pBQ and L-His had minimal impact on catalytic performance, suggesting that ˙O₂⁻ and ¹O₂ may not be generated during the reaction process. In contrast, the addition of IPA significantly reduced the relative activity of the system, indicating that ˙OH was produced, playing a crucial role in the follow-up catalytic reaction. Additionally, terephthalic acid (PTA), a well-established probe, efficiently captures ˙OH and forms 2-hydroxyterephthalic acid, which exhibits a strong fluorescence signal at 428 nm. The intensity of this fluorescence emission peak increased with the concentration of Co₃O₄/Fe₂O₃ DLHNBs (Fig. 5E). Electron spin resonance (ESR) spectroscopy is a reliable method for characterizing free radicals. Thus, we utilized ESR to confirm the production of ˙OH during the catalytic process of Co₃O₄/Fe₂O₃ DLHNBs. 4,4-Dimethyl-2-pyridyl oxide (DMPO) was chosen as the trapping agent to capture ˙OH, forming DMPO/ ˙OH spin adducts.^45,46 As shown in Fig. 5D, the reaction system containing both Co₃O₄/Fe₂O₃ DLHNBs and H₂O₂ exhibited a characteristic four-line spectrum of DMPO/ ˙OH with relative intensities of 1 : 2 : 2 : 1, further confirming the generation of ˙OH radicals. Based on the above analysis, we propose a catalytic mechanism for the generation of hydroxyl radicals (˙OH) and the oxidation of TMB by Co₃O₄/Fe₂O₃ DLHNBs. First, H₂O₂ is adsorbed onto the surface of Co₃O₄/Fe₂O₃ DLHNBs. Subsequently, the metal ions (M²⁺, where M = Co or Fe) within the catalyst reduce H₂O₂ to ˙OH through a Fenton-like reaction (eqn (2)). Eqn (3) acts as a side reaction, facilitating the re-formation of the metal ions in the +2 valence state. Finally, the colorless TMB molecules are oxidized by ˙OH to yield blue ox-TMB (eqn (4)).

≡M²⁺ + H₂O₂ → ≡M³⁺ + ˙OH + OH⁻ (2)

≡M³⁺ + H₂O₂ → ≡M²⁺ + ˙OOH + H⁺ (3)

˙OH + TMB (colorless) + H⁺ → ox-TMB (blue) + H₂O (4)

Fig. 5 (a) and (c) Michaelis–Menten curves for Co₃O₄ SLHNBs, Co₃O₄/Fe₂O₃ SLHNBs and Co₃O₄/Fe₂O₃ DLHNBs with different concentrations of TMB and H₂O₂, respectively; (b) and (d) the corresponding double-reciprocal plots.

Given the close relationship between simulated peroxidase activity and pH, we first investigated the effects of varying pH levels on the peroxidase activity of Co₃O₄/Fe₂O₃ DLHNBs. As illustrated in Fig. S6a, Co₃O₄/Fe₂O₃ DLHNBs exhibited relatively high catalytic activity in a weakly acidic environment. In contrast, their simulated peroxidase activity was significantly lower under neutral and alkaline conditions. Notably, the highest catalytic activity was observed at a buffer pH of 4 (Fig. S6b). Consequently, a HAc-NaAc buffer solution with a pH of 4 was selected for subsequent experiments.

The simulated peroxidase activity exhibits properties similar to those of natural enzymes, and its catalytic performance is also influenced by temperature. Thus, we investigated the effect of temperature on the catalytic activity of Co₃O₄/Fe₂O₃ DLHNBs within the range of 20–60 °C. As shown in Fig. S6c, the catalytic activity of Co₃O₄/Fe₂O₃ DLHNBs initially increased with temperature, reaching a maximum at 50 °C, after which it gradually declined. This indicates that the optimal reaction temperature for Co₃O₄/Fe₂O₃ DLHNBs is 50 °C (Fig. S6d). The decrease in catalytic activity at elevated temperatures may be attributed to the instability of TMB oxidation intermediates under high-temperature conditions. To facilitate experimental operations and enable comparisons of peroxidase catalytic activity at the human normal body temperature, a reaction temperature of 37 °C was selected for subsequent studies.

We also investigated the effect of catalyst concentration on the catalytic activity of Co₃O₄/Fe₂O₃ DLHNBs. As depicted in Fig. S6e, at the optimal pH of 4 and a temperature of 37 °C, the catalytic activity of Co₃O₄/Fe₂O₃ DLHNBs was low at lower catalyst concentrations. However, as the catalyst concentration increased, the catalytic activity correspondingly improved. Notably, when the catalyst concentration exceeded 50 µg mL⁻¹, the catalytic activity stabilized and showed minimal change (Fig. S6f). In practical applications, the reproducibility and stability of the catalyst are crucial. To assess these factors, we prepared six batches of Co₃O₄/Fe₂O₃ DLHNBs using the same method. The results indicated that the nanoenzyme activities of the different batches were comparable, confirming the good reproducibility of the catalyst (Fig. S7a). Furthermore, the catalyst maintained high peroxidase-like activity even after 120 days of storage at room temperature, demonstrating the excellent storage stability of Co₃O₄/Fe₂O₃ DLHNBs (Fig. S7b). In addition, the stability of the Co₃O₄/Fe₂O₃ nanozymes after the catalytic reaction has been evaluated by TEM and XRD. The TEM image and XRD pattern are shown in Fig. S8a and b, respectively. The morphology and phase composition of the Co₃O₄/Fe₂O₃ nanozymes have no obvious change, which indicated that the Co₃O₄/Fe₂O₃ nanozymes have good stability.

3.3. Kinetics of the Co₃O₄/Fe₂O₃ DLHNB catalysis reaction

The catalytic kinetic process of Co₃O₄/Fe₂O₃ DLHNBs was further investigated based on the principles of enzyme catalysis kinetics. Given that peroxidase operates as a two-substrate reaction, we fixed the concentration of one substrate (either TMB or H₂O₂) while varying the concentration of the other substrate to obtain the steady-state kinetic parameters of the peroxidase-mimicking activity of Co₃O₄/Fe₂O₃ DLHNBs. In Fig. 5(a) and (c), with the increasing concentration of the substrate, the initial reaction rate was increased. However, at high concentrations of TMB and H₂O₂, the reaction exhibited inhibition and approached saturation, consistent with the typical Michaelis–Menten curve.^47,48 The Michaelis–Menten curve was fitted using a Lineweaver–Burk double reciprocal linear approach, revealing a strong linear relationship as illustrated in Fig. 5(b) and (d). From the slope and intercept of the Lineweaver–Burk plot, we calculated the catalytic kinetic parameters of Co₃O₄/Fe₂O₃ DLHNBs. The Michaelis constant (K_m) indicates the affinity between the enzyme and the substrate; a smaller K_m value signifies a stronger affinity.⁴⁹ As summarized in Table S1, the K_m value of Co₃O₄/Fe₂O₃ DLHNBs when using H₂O₂ as the substrate was found to be 1.8, 2.25, 4.46, 168.8, and 185.5 times lower than those of Co₃O₄/Fe₂O₃ SLHNBs, Co₃O₄ SLHNBs, HRP, Co₃O₄ NPs, and Fe₃O₄, respectively. This demonstrates that Co₃O₄/Fe₂O₃ DLHNBs exhibit superior affinity for H₂O₂. Furthermore, Co₃O₄/Fe₂O₃ DLHNBs displayed lower K_m values and higher V_max compared to natural peroxidases and other artificial peroxidases.

3.4. Detection of H₂O₂ and AA using bimetallic nanozymes

Given the high peroxidase-like activity of Co₃O₄/Fe₂O₃ DLHNBs, we developed a colorimetric sensing platform for the highly sensitive detection of H₂O₂. The sensing principle is based on a Fenton-like reaction mediated by Co₃O₄/Fe₂O₃ DLHNBs, which generates hydroxyl radicals (˙OH) (eqn (3)). The produced ˙OH then catalyzes the oxidation of TMB, resulting in a colorimetric reaction (eqn (4)). The absorbance of the blue oxTMB product is directly proportional to the concentration of H₂O₂, allowing for the establishment of a colorimetric method for H₂O₂ detection. As illustrated in Fig. S9a, under optimal conditions, the absorbance at 652 nm (A652) increases with rising concentrations of H₂O₂. The corresponding linear fitting curve, shown in Fig. S9b, indicates a linear range from 0.1 to 1 mM, with a limit of detection (LOD) as low as 2.13 µM, which is better than other methods reported by literature studies and listed in Table S2. The catalytic selectivity of the Co₃O₄/Fe₂O₃ DLHNB catalyst was studied. We assessed the influence of potential interfering ions, including Na⁺, K⁺, Ba²⁺, Ca²⁺, Cu²⁺, Mn²⁺, Zn²⁺, and Ni²⁺, on the absorbance of the solution (Fig. S9c). The concentration of these ions was set at 10 mM, which is ten times the concentration of H₂O₂ (1 mM). Under these conditions, no significant changes were observed in the absorbance or color of the solution (Fig. S9d). These results indicate that the colorimetric method utilizing Co₃O₄/Fe₂O₃ DLHNBs demonstrates high selectivity for detecting H₂O₂, effectively minimizing interference from other ions.

The ability of certain antioxidants or reducing agents to inhibit the TMB color response can be leveraged as a sensing strategy. As the hydroxyl group on the dienol in ascorbic acid (AA) has strong reducing properties, which can reduce blue ox-TMB to its colorless form,^50,51 we selected AA as a representative reducing agent and established a colorimetric method using Co₃O₄/Fe₂O₃ DLHNBs (Fig. 6a). AA is a vital vitamin essential for human health, playing a significant role in various metabolic processes. However, both excessive and insufficient levels of AA can contribute to numerous diseases, including Alzheimer's disease, cancer, urinary stones, and diabetes. Therefore, developing a simple and effective method for determining AA is of great significance.⁵² As illustrated in Fig. 6b, the oxTMB produced by the TMB/H₂O₂/Co₃O₄/Fe₂O₃ DLHNB system gradually became colorless upon the addition of 5–40 µM AA, resulting in a decrease of UV-vis absorbance with increasing AA concentration. The standard curve for AA was fitted, yielding the linear equation y = −0.02997x + 1.92974. A strong linear relationship was observed between absorbance and AA concentration at 652 nm, with an R² value of 0.9999 (Fig. 6c). The limit of detection (LOD) for AA was approximately 0.15 µM, which outperforms other catalysts, as shown in Table 1.

Fig. 6 (a) Scheme of the detection mechanism of the sensing system. (b) UV-vis absorption spectra of the TMB + Co₃O₄/Fe₂O₃ DLHNB system with varying concentrations of DA and (c) fitted linear curve for AA. (d) and (e) The absorbance (652 nm) values caused by different species (ascorbic acid, glutamic acid, dopamine, histidine, lysine, urea, phenylalanine, glutathione, and cysteine).

Table 1 Comparison of different AA detection methods using different nanomaterials

Nanozymes	Linear range (µM)	LOD (µM)	Ref.
Pal@Co3O4	1–60	0.7	54
PCN-224-Fe/Co	1–200	0.2	55
Co3O4 hollow nanocages	0.1–2	0.15	56
Fe3O4/CoFe-LDH	0.5–10	0.2	57
Fe–N–C	0–40	—	58
ZnO–Co3O4 NFs/Ti3C2Tx	2.5–100	0.58	59
Mn@Co3O4	1–8	0.4	60
Co3O4/Fe2O3 DLHNBs	5–40	0.15	This work

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Several potential interfering substances, including glucose (Glc), dopamine (DA), histidine (His), lysine (Lys), uric acid (UA), phenylalanine (Phe), glutathione (GSH), and cysteine (Cys), were tested to evaluate the selectivity of the sensing platform for AA (Fig. 6d). The concentration of these interfering agents was set at 50 µM, matching the concentration of AA. As shown in Fig. 6e, AA produced the most significant change in absorbance among all tested substances. Notably, the absorbance of GSH and Cys also exhibited marked changes. If N-ethylmaleimide (NEM) is used as a masking agent, it will effectively mitigate the influence from GSH and Cys without affecting AA detection.⁵³ Therefore, the AA sensing platform we developed demonstrates a high level of selectivity for detecting AA.

To evaluate the practicability of the proposed sensing platform, AA in vitamin C injection was quantified and tested by using a standard addition method. In Tables S3, the relative standard deviation (RSD) of the method was less than 2.7%, with recoveries ranging from 97.3% to 104.2%. We validated the colorimetric method using high-performance liquid chromatography (HPLC), and the results indicated no significant differences between the analytical outcomes of the HPLC method and the colorimetric method. The colorimetric method based on Co₃O₄/Fe₂O₃ DLHNBs is more convenient and rapid, and does not require expensive equipment.

4. Conclusions

In conclusion, we successfully synthesized unique Co₃O₄/Fe₂O₃ DLHNBs through successive anion exchange and annealing strategies. These DLHNBs exhibited excellent peroxidase-like activity, which can catalyze the decomposition of H₂O₂ to produce hydroxyl radicals (˙OH), effectively oxidizing TMB to blue ox-TMB. The structural and compositional advantages of the Co₃O₄/Fe₂O₃ DLHNBs significantly enhance their peroxidase-like activities compared to both the Co₃O₄/Fe₂O₃ SLHNBs and the Co₃O₄ SLHNBs. Leveraging the properties of Co₃O₄/Fe₂O₃ DLHNBs, we developed a fast, cost-effective, and efficient colorimetric sensing platform for the detection of H₂O₂ and AA, achieving LODs as low as 2.13 µM and 0.15 µM, respectively. This work presents an effective strategy for utilizing metal–organic framework (MOF) materials to enhance the activity of nanozymes, paving the way for the design and synthesis of bimetallic nanozymes for applications in pharmaceutical analysis, clinical diagnostics, environmental monitoring and the food industry.

Author contributions

Rongguan Lv: conceptualization, resources, formal analysis, visualization, writing – review & editing. Mengting Shao: conceptualization, investigation, validation, data curation. Yuan Xiao: writing – review & editing. Shufei Liu: software, formal analysis, investigation, validation. Jingjing Jiang: conceptualization, resources. Haiyan Yu: formal analysis, validation. Fang Guo: supervision, validation. Yongjun He: conceptualization, resources. Ming Chen: conceptualization, resources, data curation, writing – review & editing, supervision.

Conflicts of 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.

Data availability

The data underlying this article will be shared by the corresponding author upon reasonable request.

The data supporting this article have been included as part of the supplementary information (SI). The supplementary information includes chemicals and materials, apparatus, synthetic procedure of Co-Fe PBA, Co₃O₄/Fe₂O₃ and Co₃O₄ SLHNBs; SEM, TEM images and FTIR spectra of ZIF-67 NCs, ZIF-67@Co-Fe PBA CSNCs, Co-Fe PBA SLHNBs; FTIR spectra, N₂ adsorption-desorption isotherms and pore-size distribution of three catalysts; the effect of pH, temperature, and catalyst concentration on the activities of Co₃O₄/Fe₂O₃ DLHNBs; the stability of Co₃O₄/Fe₂O₃ DLHNBs; detection of H₂O₂; comparison of K_m of different nanozymes (Table S1); comparison of linear range and LOD for H₂O₂ sensing with other mimetic peroxidase materials (Table S2). See DOI: https://doi.org/10.1039/d5nj03834k.

Acknowledgements

The funding support from the Priority Academic Program Development of Jiangsu Higher Education Institutions is acknowledged.

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Footnote

† These authors contributed equally to this work.

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