Introduction

Nanozymes have the merits of low cost, high chemical stability, adjustable affinity for the substrate and good catalytic activity, therefore have been extensively investigated for substituting natural enzymes in diverse applications such as sensing and detection1,2, energy storage and battery engineering3, dye biodegradation4,5, point-of-care monitoring6, and cancer treatment7. Numerous studies on manganese oxide-based nanozymes have been reported both in vitro and in silico8,9,10,11. These works help frame the context of MnOx nanozyme research and support the scientific foundation of our study. Beyond single nanozyme systems, multinanozyme configurations have recently emerged to enhance sensitivity and selectivity in colorimetric sensing by integrating different nanozymes with complementary catalytic functions that act synergistically to overcome the limitations of individual systems. For instance, Hormozi Jangi et al. developed a hybrid system combining Au- and MnO₂-based nanozymes for glutathione detection, in which Au components enhanced selectivity while MnO₂ improved sensitivity. This synergistic design achieved superior analytical performance over single nanozyme counterparts12. Despite these valuable contributions, few studies have systematically explored the valence-dependent catalytic activity of different MnOx phases (MnO, Mn3O4, Mn2O3), especially in the context of their integration with low-cost, disposable, paper-based colorimetric platforms. Our study fills this gap by correlating manganese valence states with peroxidase-like activity and demonstrating the practical application of Mn3O4 nanozymes in a one-time-use bioassay with high selectivity and sensitivity toward H₂O₂. Peroxidase-mimicking properties of nanozymes have been widely studied for detecting reactive oxygen species (e.g., H2O2) through a colorimetric reaction, which has great potential for practical clinical diagnosis and food analysis13,14,15,16,17,18.

Ever since the first report on Fe3O4-based peroxidase nanozyme in 2007, enormous efforts have been paid to design various nanostructures, tailor their compositions, and make hybridizations19,20,21, however, only a few studies have been focused on identifying the active sites of nanozymes and investigating the key parameters that determine their catalytic activities. For instance, Sun et al. developed a fast co-precipitation method for the synthesis of ultrathin NiCo layered double hydroxide (LDH) at room temperature22. The obtained nanosheets possessed an average lateral size of 26 nm and an average thickness of 2 nm, so can be well dispersed in water. The elemental composition of NiCo LDH was tunable and the active sites were identified to be Co owing to the low redox potential of Co3+/Co2+ and the strong Lewis acidity of Co3+. Recently, the same group prepared ultrathin NiMn LDH. The nanosheets exhibited peroxidase mimicking property with the catalytic performance even superior to that of natural horseradish peroxidase due to the presence of Mn23.Typically, Mn shows multiple oxidation states in its compounds, which severely determines the final peroxidase-like properties; nevertheless, the in-depth research on the valence-dependent activities of Mn remain inadequate24,25,26,27.

Recent advancements in portable nanozyme-based sensing platforms have revolutionized analytical applications by combining low cost, ease of use, and real-time detection. Particularly, systems integrated with smartphone cameras and color-recognition software provide simple and cost-effective alternatives to traditional laboratory instruments, expanding the scope of on-site diagnostics and monitoring28,29,30. Alongside smartphone-assisted systems, paper-based sensing platforms have also gained attention due to their low-cost fabrication, flexibility, and disposability. When integrated with functional materials such as MOFs or nanozymes, these paper-based sensors exhibit enhanced sensitivity and selectivity for colorimetric and fluorescence-based assays. These attributes make them promising for environmental monitoring and point-of-care diagnostics31,32,33.Herein, we develop a systematic strategy to synthesize MnOx with controllable valance states through a two-step pyrolysis of Mn-based metal organic frameworks (MOFs). The peroxidase-like activity of the obtained MnOx was found to highly depend on the valance state of the Mn sites. The optimized MnOx was further utilized as nanozyme for single-use colorimetric bioassays for H2O2 detection, showing good selectivity, high sensitivity and a low detection limit.

Experimental section

Materials and reagents

Manganese acetate tetrahydrate (Mn(CH3COO)2•4H2O, AR grade, 99.9%), trimesic acid (BTC, AR grade, 99.9%), polyvinylpyrrolidonee (PVP, AR grade, 99.9%) were obtained from Shanghai Ling Feng Chemical Reagent Co., Ltd. 3,3’,5,5’-Tetramethylbenzidine (TMB), H2O2 (30%), dopamine (DA, AR grade) , ascorbic acid (AA, AR grade), sucrose (AR grade), and glucose (AR grade) were purchased from Shanghai Energy Chemical Co., Ltd.

Synthesis of Mn-BTC and MnOx

In a typical reaction to synthesize Mn-BTC, 0.22 g of trimesic acid was dissolved in 20 mL of ethanol, while 0.18 g of manganese acetate tetrahydrate and 2.50 g of polyvinylpyrrolidone were dissolved in a mixture of 10 mL of ethanol and 10 mL of deionized (DI) water. The two solutions were subsequently mixed and stirred for 30 min, then aged at 200 °C for 3 h. The resulting white precipitates (Mn-BTC) were collected, washed, and dried in an oven at 60 °C.

The MnOx/C nanocomposites were obtained by thermal treatment of the above obtained Mn-BTC precursors in a N2 atmosphere at 650 °C for 2 h with a ramping rate of 5 °C min−1. Then, the MnOx powder was obtained by heating the composites in air at 200, 300 and 400 °C for 2 h by setting the ramping rate at 1 °C min−1. To evaluate the potential interference from common coexisting substances in food systems, several representative molecules were tested, including ascorbic acid (AA), dopamine (DA), glucose, sucrose, and potassium chloride (KCl), each at a final concentration of 0.1 mM in the reaction system.

Characterization

UV-Vis spectroscopy (UV-1600, Mapada), X-ray diffraction (XRD, Rigaku-Ultima III, λ = 1.5418 Å), field emission scanning electron microscopy (FESEM, JSM-7800F, JEOL) equipped with an energy dispersive spectroscopy (EDS) detector, transmission electron microscopy (TEM, JEM-1400F, JEOL), and X-ray photoelectron spectroscopy (XPS, PHI Quantera spectrometer) were used to characterize the absorption properties, crystal structures, morphologies, and bonding states of the MnOx/C nanocomposites.

To evaluate the peroxidase-like activity of the nanozymes, the MnOx/C nanocomposites were first dispersed in deionized water by ultrasonication to obtain a homogeneous suspension at a concentration of 1 mg·mL⁻1. In a typical reaction system, 10 μL of the Mn3O4 nanozyme suspension was added into 1.0 mL of phosphate buffer solution (PBS, 10 mM, pH = 5.35), which contained 0.2 mL of TMB (1 × 10⁻3 M) and an appropriate concentration of H₂O₂. This system was used to assess the catalytic efficiency by monitoring the colorimetric reaction. The apparent kinetic parameters of the nanozyme can be calculated according to the Michaelis–Menten equation:

$$v = {{v_{{{\text{max}}}} \times \left[ {\text{S}} \right]} \mathord{\left/ {\vphantom {{v_{{{\text{max}}}} \times \left[ {\text{S}} \right]} {\left( {{\text{K}}_{{\text{m}}} + \left[ {\text{S}} \right]} \right)}}} \right. \kern-0pt} {\left( {{\text{K}}_{{\text{m}}} + \left[ {\text{S}} \right]} \right)}}$$
(1)

where the Michaelis–Menten constant Km describes the affinity between nanozyme and substrate; v and vmax are the initial reaction velocity and the maximal velocity, respectively; and [S] represents the concentration of substrate.

Evaluation of nanozyme activity

The oxidase-like activity of the Mn3O4 nanozyme was further evaluated based on the colorimetric reaction system described above. The catalytic performance was investigated under various experimental parameters to determine the optimal operating conditions, including TMB concentration, H₂O₂ dosage, pH (2.5–10), and temperature (25–60 °C). UV–Vis spectroscopy at 652 nm was employed to monitor the oxidation of TMB, and the catalytic efficiency was quantitatively analyzed. The apparent kinetic parameters were subsequently calculated using the Michaelis–Menten equation and Lineweaver–Burk plots, confirming typical enzyme-like kinetics. The results demonstrated strong substrate affinity and high catalytic activity, consistent with previously reported temperature-resilient Mn–MOF nanozymes34,35.

Sensing assay and design

Based on the catalytic oxidation reaction, a paper-based colorimetric platform was developed for portable sensing. A defined volume of Mn3O4 nanozyme suspension was drop-cast onto cellulose pads and dried at room temperature. After adding a TMB/H₂O₂ mixture, the resulting color change was recorded using a smartphone camera. The color intensity was quantitatively analyzed via RGB extraction using ImageJ software, enabling low-cost and on-site detection applications. The proposed paper-based platform is designed for single use and therefore not suitable for reproducibility testing36.

Catalytic mechanism and proposed pathway

The peroxidase-mimicking mechanism of Mn3O4 nanozyme was investigated to elucidate its redox behavior and reactive species generation during the colorimetric oxidation process. The catalytic activity originates from the intrinsic multivalent states of manganese ions (Mn2⁺/Mn3⁺), which enable rapid electron transfer and continuous redox cycling analogous to natural peroxidases. Upon interaction with hydrogen peroxide (H₂O₂), Mn2⁺ species are oxidized to Mn3⁺, generating highly reactive hydroxyl radicals (•OH) through a Fenton-like reaction. These radicals subsequently oxidize chromogenic substrates such as TMB, producing the characteristic blue oxidized product (oxTMB) that confirms the peroxidase-like pathway.

The catalytic mechanism can be described by the following reactions:

$${\text{Mn}}^{{2 + }} {\text{ + H}}_{{2}} {\text{O}}_{{2}} \to {\text{Mn}}^{{3 + }} { + } \bullet {\text{OH + OH}}^{ - }$$
(2)

This redox cycle ensures sustained radical generation and continuous catalytic turnover. The coexistence of Mn ions with different oxidation states and abundant surface oxygen vacancies facilitates electron delocalization and enhances H₂O₂ adsorption and activation. Moreover, the structural stability and high surface-to-volume ratio of Mn3O4 contribute to efficient ROS production under mild conditions37,38.

Results and discussion

Despite the extensive research on Mn-based nanostructures for catalytic and sensing applications, most prior studies have concentrated on a single oxidation state and have not systematically elucidated the relationship between Mn valence and catalytic performance. In addition, the integration of Mn-based nanozymes into low-cost, portable, and disposable sensing platforms has received relatively little attention. To overcome these limitations, we developed a controllable two-step pyrolysis strategy that enables precise tuning of Mn valence states and evaluation of their influence on peroxidase-like activity. This approach focuses on the valence-dependent catalytic behavior of Mn-based nanozymes.

The two-step calcination process resulted in a hybrid structure composed of MnOx nanocrystals homogeneously embedded in carbon frameworks (MnOx/C) due to the decomposition of organic ligands and the formation of Mn–O bonds (Fig. 1).

Fig. 1
figure 1

The controllable preparation of MnOx compounds.

Full size image

The derivatives obtained at a calcination temperature of 200, 300, and 400 °C are respectively presented in Fig. 2a–h, which inherited the spherical morphology of the Mn-BTC precursor. The diameter distribution statistics in Fig. 2i reveal that the diameter of Mn-BTC, MnO, Mn3O4, and Mn2O3 nanoparticles are 1.9 μm, 1.05 μm, 0.95 μm, and 0.65 μm, respectively, which demonstrates an inverse correlation between the diameter of the derivatives and the calcination temperature. The corresponding XRD patterns in Fig. 2j indicate the successful conversion of the Mn-BTC precursors into MnO (JCPDS 07–0230), Mn3O4 (JCPDS 24–0734), and Mn2O3 (JCPDS 41–1442) when the calcination temperature was set at 200, 300, 400 °C, respectively. Moreover, the EDS mappings further confirm the homogenous elemental distribution of MnOx and carbon frameworks in the nanocomposites (Fig. S1). TEM image in Fig. S2 suggests that the hierarchical morphology of MnOx maintains. The valence state distribution of Mn in the derivatives can be determined from the high-resolution XPS spectra of Mn 2p (Fig. 2k). Deconvolution of the Mn 2p₃/₂ peak reveals contributions from Mn2⁺ (~ 640.9 eV), Mn3⁺ (~ 642.1 eV), and Mn4⁺ (~ 643.8 eV). These three oxidation states coexist in the material, with Mn2⁺ dominating at lower temperatures. As the calcination temperature increases, the content of Mn2⁺ decreases while that of Mn3⁺ gradually increases, which agrees with the phase evolution observed in the XRD results. These findings further confirm the progressive oxidation of Mn during thermal treatment and the tunability of Mn valence through calcination. The oxidation state of Mn plays a crucial role in determining the peroxidase-like catalytic activity of MnOx materials. Previous reports have demonstrated that Mn3⁺, owing to its moderate redox potential and strong Lewis acidity, can effectively decompose H₂O₂ into hydroxyl radicals (•OH), which in turn oxidize TMB to its colored product39. As depicted in Fig. 2l, the content of Mn2+ decreases with increasing calcination temperature, while the content of Mn3+ gradually increases. These results demonstrate that the valence states of Mn in MnOx can be regulated by controlling the calcination temperature in the two-step synthesis process, which is crucial for further investigations into the relationship between Mn valence states and the peroxidase-like catalytic activity of MnOx.

Fig. 2
figure 2

(a–d) Low-magnification and (e–h) high-magnification SEM images, (i) Diameter distribution and (j) XRD patterns of Mn-BTC, MnO, Mn3O4 and Mn2O3. (k) Mn 2p spectra of MnO, Mn3O4, and Mn2O3. (l) Percentage of Mn2+, Mn3+ and Mn4+ in MnO, Mn3O4, and Mn2O3.

Full size image

The peroxidase-like properties of MnOx were probed using TMB as the allochronic substrate in the presence of H2O2. As shown in Fig. 3a, a strong absorption peak at 652 nm is observed upon adding MnOx (herein we use Mn3O4 as an example) in the mixture of H2O2 and TMB; on the contrary, no absorption peaks were found in the absence of Mn3O4 or H2O2 in the system. In addition, Mn3O4 has a higher catalytic activity by exhibiting a stronger absorption peak at 652 nm, as compared to MnO and Mn2O3 (Fig. 3b).

Fig. 3
figure 3

(a) UV–Vis absorption spectra of TMB + H2O2, TMB + Mn3O4, and TMB + H2O2 + Mn3O4 solutions. (b) Catalytic performance comparison with MnO, Mn3O4 and Mn2O3 as POD-like nanozyme. (c) Fluorescence emission spectra revealing the generation of •OH through the catalytic reaction using TA as a fluorescent probe. (d) The calalytic mechanism of Mn3O4. POD-like activity of Mn3O4 nanozyme against (e) pH value, (f) reaction temperature, and (g) storage time.

Full size image

Fluorescence spectroscopy was employed to elucidate the catalytic mechanism by examining the generation of hydroxyl radicals (•OH) with the assistance of terephthalic acid (TA) as the capture agent40,41,42. TA can act as a capture molecule and react with ·OH to generate fluorescent 2-hydroxyterephthalic acid (TAOH)43. As shown in Fig. 3c, a strong fluorescence peak is observed at 430 nm upon the addition of H2O2 into TA, suggesting the generation of •OH by the decomposition of H2O2. To better visualize the catalytic mechanism, a schematic illustration is shown in Fig. 3d. Mn3O4 catalyzes the decomposition of H₂O₂ into hydroxyl radicals (•OH), which subsequently oxidize the colorless TMB into a blue-colored product (oxTMB). The color change correlates with H₂O₂ concentration and is used for quantitative analysis. This mechanism underpins both the solution-based and paper-based bioassays developed in our work. The fluorescence resonance peak becomes stronger after adding Mn3O4 into the system, which indicates that the catalyst promoted the generation of •OH. Therefore, it can be concluded that the colorless TMB is oxidized by •OH to produce blue color. The concentration of H2O2 can be indirectly evaluated based on the color change. The catalytic performance of Mn3O4 was further examined in PBS buffer with varied pH values from 2.5 to 10 at an increasing reaction temperature from 25 to 60 °C. Mn3O4 exhibits the highest peroxidase-like activity at a pH of 5.35 (Fig. 3e). The activity initially increases with the rising reaction temperature and peaks at 35 °C, then gradually decreases when the reaction temperature is further increased, with a ~ 60% retention at 60 °C (Fig. 3f). Besides, Mn3O4 shows excellent long-term catalytic stability without negligible degradation after being stored for 10 days (~ 96% retention of the activity as shown in Fig. 3g), which is superior to that of natural enzyme. All of the above results demonstrate that Mn3O4 can function as peroxidase-mimicking nanozyme with high activity and chemical stability.

To strengthen the catalytic assessment, a comparative analysis was performed with reference to recent studies on Mn-based oxidase-like nanozymes. The catalytic behavior of Mn3O4 was consistent with temperature-resilient Mn–MOF systems34,35, in which robust frameworks and redox-active Mn centers sustain high catalytic efficiency under diverse environmental conditions. These findings demonstrate the excellent oxidase-like activity and environmental adaptability of Mn3O4, underscoring its potential for reliable and versatile sensing applications.

Subsequently, the steady-state kinetic of Mn3O4 was evaluated using the Michaelis–Menten model under the optimized testing conditions (Fig. 4). The Michaelis–Menten curves were experimentally obtained by changing the contents of H2O2 and TMB; the corresponding Lineweaver Burk diagrams were calculated to determine the kinetic parameters. The Km and vmax values for the substrates of H2O2 and TMB were respectively calculated to be 0.043 mM, 19.22 × 10−8, and 0.054 mM, 8.15 × 10−8.

Fig. 4
figure 4

(a) The dependence of the reaction velocity on the H2O2 concentration (the concentration of TMB was fixed at 166 μM). (b) The corresponding Lineweaver–Burk plot. (c) The dependence of the reaction velocity on the TMB concentration (the concentration of H2O2 was fixed at 314 μM). (d) The corresponding Lineweaver–Burk plot. The test conditions: 10 μL of 1 mg mL−1 Mn3O4 nanozymes in 1 mL of 10 mM PBS (pH = 5.35) at 35 °C. The error bars were obtained based on three sets of measurement.

Full size image

These results confirm that the Mn3O4 nanozyme follows typical Michaelis–Menten kinetics similar to natural peroxidases. The relatively low Km values indicate a strong substrate-binding affinity, superior to most reported metal-oxide nanozymes. Moreover, the observed kinetics agree with recent findings on Mn- and Ag-based oxidase- or peroxidase-like nanozymes, where multivalent metal centers and surface oxygen vacancies jointly promote substrate activation and electron transfer during catalysis44,45,46.

Food safety is of significance to human health. Although H2O2 is a widely adopted agent for food preservation, its content should be strictly controlled to avoid any harm to the consumers47,48,49,50,51. In this work, Mn3O4 were utilized for colorimetric detection of H2O2 in the mixture of TMB and H2O2. As exemplified by Mn3O4 shown in Fig. 5a, the intensity of the absorption peak increases as the concentration of H2O2 rises. Figure 5b displays the corresponding absorbance as a function of the concentration of H2O2, which has a good linearity (R2 = 0.9992) in the range of 0.01–0.20 mM. The limit of detection (LOD) can be calculated to be 0.25 μM based on the formula:

$${\text{LOD}} = {{{3}\sigma } \mathord{\left/ {\vphantom {{{3}\sigma } {{\text{slope}}}}} \right. \kern-0pt} {{\text{slope}}}}$$
(3)
Fig. 5
figure 5

(a) UV–Vis absorbance as a function of H2O2 concentration. (b) The absorbance as a function of the H2O2 concentration, showing good linearity. (c) The detection of H2O2 by the paper-based colorimetric bioassay. (d) The selective detection of H2O2 using Mn3O4 nanozymes.

Full size image

where σ represents the standard deviation of background signal, the slope is the linear calibration in Fig. 5b. Benefiting from the excellent peroxidase-like activity of Mn3O4, a colorimetric bioassay was fabricated by coating nanozyme particles on cellulose paper, as a proof-of-concept disposable biosensor for the detection of H2O2. The color change of the bioassay induced by the mixture of TMB and H2O2 was recorded by photographs; the relative intensity of green color in the broadband color (G/R + G + B) was analyzed as a function of the H2O2 content (Fig. 5c). A low LOD of 6.77 μM were obtained in the first linear range (0.16–1.26 mM) for the paper bioassay, which is lower than the allowance level of US FDA regulations (15 μM). Furthermore, the detection of H2O2 can be interfered by other components such as ascorbic acid (AA), dopamine (DA), sucrose, glucose and K+ in food, it is critical for the biosensor to have a high selectivity. In our experiment, even in the presence of these substances, the content of H2O2 can be selectively determined (Fig. 5d), indicating the paper bioassay is applicable for fast and low-cost food analysis.

Although this study demonstrates the promising oxidase-like activity and sensing potential of the Mn3O4 nanozyme, several limitations remain. The catalytic evaluation and sensing performance were primarily conducted for H₂O₂ detection under controlled laboratory conditions; therefore, further validation involving diverse analytes and complex real samples is necessary to assess its practical applicability. In future studies, efforts should focus on optimizing the nanozyme synthesis and surface modification to improve selectivity and sensitivity, integrating it into portable or microfluidic sensing systems, and expanding its applications in biomedical diagnostics, food safety, and environmental monitoring.

Conclusion

In summary, we have developed a valence-controlled synthesis method for MnOx nanozymes and systematically investigated their peroxidase-like catalytic activities. Among them, Mn3O4 exhibited the best catalytic performance due to its optimal Mn2⁺/Mn3⁺ ratio, promoting efficient •OH generation from H₂O₂. Spectroscopic and kinetic analyses confirmed the mechanism. Based on this, we constructed a disposable paper-based colorimetric bioassay using Mn3O4, which showed high sensitivity, good selectivity, and a low detection limit for H₂O₂. Importantly, this sensing mechanism is highly compatible with portable platforms. The Mn3O4 nanozyme can be stably coated on low-cost cellulose paper and integrated into smartphone-readable colorimetric strips. Due to its visual signal output, low power requirement, and simple fabrication, the platform is suitable for on-site, user-friendly detection without complex instruments. Therefore, our work not only deepens the mechanistic understanding of MnOx nanozymes, but also demonstrates a practical pathway to translate nanozyme catalysis into portable, low-cost diagnostic tools for food safety, clinical analysis, and environmental monitoring.