Synergistic Intra‐ and Inter‐Nanozyme Electron Transfer through Interfacial Assembly for Enhanced Multi‐Enzyme Activity

ABSTRACT

Prussian Blue composite nanozymes (PB C‐NZs) have been extensively employed in various biomedical applications. However, due to the complexity of its multi‐enzyme activities and structural components, designing material composition and processes, optimizing specific enzymatic properties of materials, and exploring the scientific mechanisms involved remain important challenges. Given the equal importance of electron transfer optimization and intrinsic redox properties, we propose an interfacial assembly strategy that leverages electron transfer and energy band structure, allowing for the synergistic interactions between internal and interfacial electrons of the nanocomposites (MoS₂/PB) and thereby enhancing the enzyme‐like activity. In the MoS₂/PB system, a distinct inter‐nanozyme electron transfer is operative, facilitating directional electron transport from MoS₂ to PB, thereby enhancing catalytic activity. Concurrently, band modulation effects induced by the interaction between MoS₂ and PB effectively enhance the reductase‐like catalytic activity. Notably, the expression of multiple enzyme activities can be enriched through band regulation. The comprehensive enzymatic activity tests demonstrated that MoS₂/PB exhibits enhanced multiple‐enzyme activities, including catalase, peroxidase, superoxide dismutase, glutathione peroxidase, S‐nitrosoglutathione reductase, and nitrite reductase. This study introduces a novel design concept for composite nanozymes based on electron transfer modulation, providing valuable insights and guidance for the development of high‐performance nanozymes.

A novel Prussian blue‐based composite nanoenzymes (PB C‐NZs)‐MoS₂/PB, was designed based on the electron transfer mechanism and transport pathway. The structure uses MoS₂ as the substrate, with PB grown on its surface through a surface assembly method. The relatively high energy band position of MoS₂ not only facilitates intrinsic electron transfer during the catalytic process but also promotes electron transfer from the interface to PB. The synergistic electron transfer within and between the nanoenzymes enhances the catalytic performance of MoS₂/PB, while the incorporation of MoS₂ enriches the multi‐enzyme activity expression of PB.

1. Introduction

Natural enzymes play a crucial role in biological systems and have been widely applied in the biomedical field. However, their poor chemical stability, limited application methods, and high production costs seriously hinder their practical implementation. Nanozymes, as emerging and efficient biocatalysts, exhibit the capability to regulate the levels of reactive oxygen species (ROS) in living organisms, thereby contributing to the homeostasis of the biological environments and demonstrating immense potential in biomedical applications.[1] Among them, Prussian Blue nanozymes (PBNZs), have attracted increasing attention in recent years and have been widely used in many diagnostic and therapeutic fields.[2, 3, 4] Their exceptional performance across diverse biological applications is mainly attributed to their unique structural units and excellent electron transfer efficiency.[5, 6, 7] Meanwhile, the development of PB C‐NZs, in which PB serves as the substrate, has advanced rapidly.[8] Existing studies have preliminarily explored the structure‐activity relationship of PB C‐NZs. The catalytic performance is enhanced by the affinity of the dual enzyme structure and the expression of multi‐enzyme activity of cascade nanozymes.[9, 10] The interfacial electric field and chemical bond “bridge” promote electron transfer in the catalytic process.[11, 12] In addition, the presence of dual active sites (Ce³⁺/Ce⁴⁺ and Mn³⁺/Mn²⁺) and charge‐mediated modulation of electron transfer and circulation in the composite structure can significantly optimize the catalytic activity of the composite structure.[13, 14] These indicate that electron transfer lies at the core of redox enzyme function, and highlight the potential of structural design to enhance electron transfer and thereby improve catalytic performance, providing a substantial research foundation for the rational construction of PB C‐NZs.[15, 16] Although the reported PB C‐NZs exhibit excellent catalytic performance by increasing catalytic sites and facilitating electron transfer through optimized pathways, further improvements remain possible when structural configuration and local electronic effects are considered. Meanwhile, many pathological conditions require the cooperative action of multiple enzyme‐like activities to achieve effective therapeutic effects.[17, 18, 19] Consequently, precise regulation of catalytic behavior and expression of multi‐enzyme activities via electronic effects remains a significant challenge.[20, 21] To address this issue, a precise design strategy is necessary to fully exploit the intra‐ and inter‐nanozyme electron transfer properties fully, thereby enhancing electron transfer efficiency and regulating the redox properties of PB C‐NZs. Notably, the adjustment of interfacial electronic effects and energy band positions can effectively regulate the redox properties. Nevertheless, the underlying mechanisms by which band structural differences mediate interfacial electron transfer and promote multi‐enzyme mimetic activity remain poorly understood. Therefore, elucidating the influence of energy band position on catalytic performance and enhancing the expression of multi‐enzyme activity by adjusting redox properties can not only enable the precise design of PB C‐NZs, but also be essential for the precise and efficient application of nanozymes in diverse biomedical fields.

In our previous work, we elucidated the electron transfer catalytic mechanism and catalytic pathway of PB.[22, 23] Accordingly, the design of related PB C‐NZs should consider the electronic structure and band positions to enable precise structural tailoring for applications in various biological environments. It is well established that electron transfer is closely related to the band gap and band position of a compound.[24] Furthermore, the band positions of individual components within the composite structure significantly affect the electron transfer pathway during catalysis, thereby influencing the expression of enzyme‐like activity.[25, 26, 27, 28] In short, selecting compounds with the exact electron transfer mechanism and appropriate band positions is essential for constructing PB C‐NZs. MoS₂ exhibits a metal valence state (Mo⁶⁺/Mo⁴⁺) similar to that of PB (Fe³⁺/Fe²⁺), and its relatively abundant redox electrons endow it with excellent electron transport properties.[29, 30] Notably, in the heterogeneous structure of MoS₂/Vo‐Fe₂O₃, interfacial electron transfer from Mo to Fe significantly enhances electron conductivity.[12] Similarly, in the structure of MIL‐101 (Fe)/MoS₂, the Mo⁶⁺/Mo⁴⁺ site can accelerate the cycle of Fe³⁺/Fe²⁺ in the catalytic process.[31] Therefore, the abundant redox electron pairs in MoS₂ and its ability to promote electron transfer through interfacial effects make it an excellent compound for constructing PB C‐NZs.

With these considerations in mind, we have developed a novel PB C‐NZs, MoS₂/PB, designed based on the electron transfer mechanism and transmission pathway. In this structure, MoS₂ is employed as the substrate, and PB is grown on its surface via a surface assembly approach. In addition to facilitating intrinsic electron transfer during catalysis, the relatively high energy band position of MoS₂ enables interfacial electron transfer toward PB, thereby enhancing the catalytic performance of the composite structure in specific reduction enzyme‐like reactions.[32] Simultaneously, the band overlap between MoS₂ and PB effectively reduces the band gap, further enhancing electron transfer ability and improving the expression of enzyme‐like activity.[33] The prepared MoS₂/PB thus reflects the intrinsic electron transfer behavior of PB.[14] More importantly, this composite achieves a certain degree of enhancement in reductase‐like activity through interfacial energy band differences, which, to our knowledge, is a design strategy that has not been reported before. Based on this construction mechanism, we have demonstrated the feasibility of the design concept for precisely enhancing the oxidase‐like activity of PB C‐NZs.[34] These perspectives on the design concept offer a broader framework for the rational design of PB C‐NZs with tunable electronic properties and advanced catalytic functions.

2. Results and Discussion

2.1. Preparation and Characterization of the PB Composite Nanozyme‐MoS₂/PB

A two‐step strategy was adopted to achieve the growth of PB on the surface of MoS₂, which served as the substrate, through a surface assembly approach, thereby constructing the MoS₂/PB composite nanozyme (Figure 1a). Briefly, MoS₂ with nanoflower morphology was first synthesized as a precursor using a hydrothermal method.[35] After purification, PBNZs were grown on their surface through coprecipitation. During the growth, PB modified the partial surface electronic structure of MoS₂. The successful preparation of the composite was initially confirmed through morphological characterization. As shown in Figure 1b and Figure S1a, both Transmission Electron Microscope (TEM) images and Scanning Electron Microscope (SEM) images reveal that the prepared MoS₂ exhibits a spherical nanoflower morphology. After surface assembly, distinct cubic structures approximately 30 nm in size were observed on the surface of the nanoparticles (Figure S1b,c). The original MoS₂ structure is covered on the surface to form a new nanoflower morphology (Figure 1c). The TEM image of PB obtained under the identical preparation conditions is shown in Figure S1d. To further validate the proposed surface assembly morphology of MoS₂/PB, High‐Angle Annular Dark Field Scanning Transmission Electron Microscopy (HAADF‐STEM) was employed to observe the contrast differences. The results clearly indicate that the central region of the composite exhibits a higher contrast, which is consistent with the difference in atomic numbers between Fe and Mo (Figure S2).

FIGURE 1.

(a) Schematic presentation of the generation of MoS₂/PB. (b) and (c) TEM images of MoS₂ and MoS₂/PB. (d) HAADF‐STEM‐EDS images of MoS₂/PB. (e) and (f) AC‐HRTEM images of MoS₂ and MoS₂/PB. (g) FT‐IR, (h) XRD, (i) UV–vis, and (j) Raman spectrum (pattern) of PB, MoS₂, and MoS₂/PB.

Additionally, the results of the Energy Dispersive Spectroscopy STEM (EDS‐STEM) further confirmed the successful synthesis of MoS₂/PB (Figure 1d). To investigate the microscopic structure of the nanomaterial, the Aberration Corrected High Resolution TEM (AC‐HRTEM) was selected. As shown in Figure 1e,f, the AC‐HRTEM image of MoS₂ displays lattice spacings attributed to MoS₂. In contrast, the image of the composite material MoS₂/PB reveals the lattice spacings corresponding to both MoS₂ and PB. Specifically, the lattice spacings of 0.252 nm and 0.628 nm correspond to the (002) and (102) planes of MoS₂, respectively, while the lattice spacing of 0.259 nm corresponds to the (400) plane of PB.[36, 37] Furthermore, the Fast Fourier Transform of AC‐HRTEM images demonstrates corresponding changes in crystal information (Figure S3). Elemental analysis provided further confirmation of the successful formation of the composite structure, as characteristic signals corresponding to Fe, Mo, and S were clearly detected (Figure S4). In summary, we successfully prepared the MoS₂/PB nanocomposite, and the optical image of its aqueous solution is presented in Figure S5.

2.2. Structural Components and Crystal Structure

To further verify the successful synthesis of MoS₂/PB, Fourier Transform Infrared (FTIR) spectroscopy was performed on the composite material as well as on each component. The FTIR spectra of MoS₂, PB, and MoS₂/PB exhibit significant differences (Figure 1g). Before assembly, MoS₂ shows characteristic absorption bands at 1105, 1277, 1419, and 1636 cm⁻¹. The bands at 1105, 1277, and 1636 cm⁻¹ are assigned to the stretching vibration of the C─O group, O─C─O group, and C═O group of polyvinylpyrrolidone (PVP), respectively. Additionally, the band at 1419 cm⁻¹ corresponds to the stretching vibration of the S−Mo−S bond.[38] After surface assembly of PB, the out‐of‐plane and in‐plane bending vibrations of the Fe─C bond are observed at 500 and 601 cm⁻¹. Moreover, a strong C≡N stretching vibration of Fe─C≡N─Fe is observed at 2083 cm⁻¹, which is characteristic of the PB structure. The peak representing the S─Mo─S bond at 1419 cm⁻¹ exhibits a red shift, which can mainly be attributed to the introduction of CN⁻ during the formation of PB.[39] Moreover, the change in Zeta potential before and after assembly further confirms the growth of PB on the MoS₂ surface (Figure S6). To further investigate the crystal composition of MoS₂/PB, X‐ray diffraction (XRD) analysis was performed. As shown in Figure 1h, the characteristic diffraction peaks of MoS₂/PB are located at 11.73°, 33.20°, and 59.93°, corresponding to the (002), (100), and (008) lattice planes of MoS₂.[40] Additionally, the characteristic diffraction peaks at 17.19°, 24.65°, 35.27°, and 39.55° are observed, which are attributed to the (200), (220), (400), and (420) lattice planes of PB.[41] The intensity of the characteristic peak corresponding to MoS₂ is reduced, likely due to the growth of PB on its surface, which partially covers the crystal information of MoS₂. To determine the crystalline phase of MoS₂, Ultraviolet‐Visible (UV–vis) and Raman spectroscopy were performed. The UV–vis spectrum shows three characteristic peaks at 431, 609, and 668 nm. Given that the 1T‐MoS₂ lacks UV–vis absorption features, MoS₂ is preliminarily confirmed to have a 2H crystalline phase (Figure S7).[42] The MoS₂/PB curve shows a characteristic peak of PB at 700 nm after surface assembly (Figure 1i). Additionally, the Raman spectrum of MoS₂/PB reveals three characteristic peaks, one of which, 2153.4 cm⁻¹ corresponds to C≡N (Figure 1j and Figure S8a). A broader characteristic peak in the range of 1200–1800 cm⁻¹ is attributed to PVP outside MoS₂ (Figure S8b,c). Importantly, two characteristic Raman peaks at 378.7 cm⁻¹ (E¹ _2g) and 403.9 cm⁻¹ (A_1g) are observed in both MoS₂ and MoS₂/PB, further confirming the existence of the 2H‐MoS₂ phase (Figure S8d).[42] These results confirm that MoS₂/PB with high phase purity and high crystallinity has been successfully prepared.

Although the information regarding the preparation of MoS₂/PB is clear, its assembly process requires further investigation. The preparation strategy of MoS₂/PB leverages the fact that the surface electronic structure of MoS₂ changes under acidic conditions, influencing the charge dynamics.[43] While the MoS₂ is exposed to acidic conditions, a positive charge is generated on its surface. This results in the electrostatic adsorption of CN⁻, which subsequently reacts with the Fe²⁺ and Fe³⁺, facilitating the PB assembly on the MoS₂ surface. To validate this hypothesis, MoS₂ was analyzed under acidic conditions. After adding K₄[Fe(CN)₆] and removing the added molecules, the results show that as acidity increases, the Zeta potential of MoS₂ shifts in the positive direction. Notably, this potential can be restored to its original value when the solution returns to neutral (Figure S9a,b). After the addition of K₄[Fe(CN)₆], the potential change trend remains consistent, and the 2H crystal phase of MoS₂ is preserved (Figure S9c,d). Furthermore, Raman spectroscopy and TEM images further confirm the crystal stability of MoS₂ under these preparation conditions (Figures S10,S11). These findings provide comprehensive evidence supporting the composition, crystal structure, and formation process of MoS₂/PB.

2.3. Examinations of Microstructure and Electronic Structure

The enzyme‐like catalytic mechanism of PB is predominantly governed by the electron transfer processes, so the electronic structure and valence state characteristics of PB C‐NZs are crucial for elucidating their catalytic behavior. To further probe the structural modifications induced by PB surface assembly and to examine the surface chemical composition of the MoS₂/PB composite, X‐ray photoelectron spectroscopy (XPS) was employed. Additionally, the result can also be used to further analyze whether there is a 1T crystal phase in the MoS₂ structure. The XPS spectrum of MoS₂, MoS₂‐CN⁻, and MoS₂/PB, shown in Figure 2, provides insights into the local electronic structure and valence state information. Six main peaks are observed at binding energies of 163, 231, 283, 399, 531, and 708–728 eV, corresponding to S 2p, Mo 3d, C 1s, N 1s, O 1s, and Fe 2p, respectively (Figure S12a).[44, 45]

FIGURE 2.

High‐resolution XPS spectra of MoS₂, MoS₂‐CN⁻, and MoS₂/PB. (a), (d), and (g), Mo 3d. (b), (e), and (h), S 2p. (c), (f), and (i), Fe 2p.

The XPS spectrum of the Mo element in the MoS₂ structure reveals seven distinct spectral bands after curve fitting, among which the peaks at 225 and 227 eV with lower binding energy are attributed to S²⁻ 2s (Figure 2a).[45] The valence states of the Mo element in the MoS₂ spectrum include Mo⁴⁺ 3d_5/2, Mo⁴⁺ 3d_3/2, and Mo⁶⁺ 3d_3/2, reflecting the sulfidation state of Mo species in the synthesized MoS₂ structure. Specifically, the peaks centered at 227.8 and 229.2 eV are attributed to the Mo⁴⁺ 3d_5/2, while the peaks centered at 230.9 and 232.3 eV correspond to Mo⁴⁺ 3d_3/2. The peak centered at 234.4 eV is assigned to the Mo⁶⁺ 3d_3/2, consistent with previously reported MoS₂ spectra.[42, 46] Therefore, the Mo in the structure exists in two sulfidation states, Mo⁴⁺ and Mo⁶⁺, whose coexistence will form relatively independent electron cycling states contributing to enzyme‐mimicking activity.[47] Furthermore, the XPS spectrum of the S element in MoS₂ reveals four distinct spectral bands, corresponding to 1T‐MoS₂ S²⁻ 2p_3/2, 1T‐MoS₂ S²⁻ 2p_1/2, 2H‐MoS₂ S²⁻ 2p_3/2, and 2H‐MoS₂ S²⁻ 2p_1/2, which reflect the valence states of sulfur species and the crystal phase information of MoS₂ (Figure 2b). So, the Mo 3d and S 2s spectra indicate the coexistence of both the 2H phase and a 1T phase of MoS₂.[48] The peaks centered at 160.7 and 162.1 eV are attributed to 1T‐MoS₂ S²⁻ 2p_3/2 and 1T‐MoS₂ S²⁻ 2p_1/2, respectively, while the peaks centered at 161.3 and 163.3 eV correspond to 2H‐MoS₂ S²⁻ 2p_3/2 and 2H‐MoS₂ S²⁻ 2p_1/2, respectively. In contrast, the XPS spectrum does not display any identifiable peaks of Fe, confirming the absence of Fe‐related spectral information in the MoS₂ structure (Figure 2c).

To further verify the stable adsorption of CN⁻ on the MoS₂ surface, XPS analysis of MoS₂‐CN⁻ was performed, and the overall spectrum is presented in Figure S12b. Notably, in the MoS₂‐CN⁻, the partial binding energies of both Mo and S orbitals are shifted compared to MoS₂. (Figure 2d,e). The specific values ​​are shown in Tables S1,S2. This should be caused by the surface adsorption of CN⁻ and Fe²⁺. Among the N 1s spectra, four bands are revealed, with four peaks centered at 394.8, 397.2, 399.4, and 400.1 eV, corresponding to the bonding states of C─N, C≡N, Mo─N, and PVP─N.[44] The presence of a C≡N peak at 397.2 eV provides strong evidence that CN⁻ can be adsorbed on the MoS₂ surface, facilitating subsequent assembly processes (Figure S13a,b). Additionally, the N spectrum of MoS₂/PB reveals the characteristic peak of C≡N at 397.8 eV, which is absent in the spectrum of MoS₂ (Figure S13c). This finding aligns with the previous analysis of the assembly process, further confirming the incorporation of PB into the composite structure. Simultaneously, the overall spectrum does not show a prominent peak for the Fe 2p orbital. However, upon further analysis, peaks are observed at 708.1 and 721.2 eV (Figure 2f), corresponding to Fe²⁺ 2p_3/2 and Fe²⁺ 2p_1/2, respectively. This phenomenon is likely due to the interaction of CN⁻ with the MoS₂ surface or the adsorption of free Fe²⁺ onto unsaturated sulfur sites.

The electronic structure of the MoS₂/PB surface plays a crucial role in the catalytic process. The XPS spectrum of the Mo element reveals seven distinct spectral bands after curve fitting, with the peak at the lower binding energy attributed to S²⁻ 2s. Notably, the valence states of the Mo in the MoS₂/PB structure remain consistent, exhibiting two valence states, Mo⁴⁺ 3d_5/2, Mo⁴⁺ 3d_3/2, and Mo⁶⁺ 3d_3/2 (Figure 2g). Specifically, the peaks centered at 228.8 and 230.1 eV correspond to Mo⁴⁺ 3d_5/2, while the peaks at 231.9 and 233.2 eV correspond to Mo⁴⁺ 3d_3/2. The peak centered at 236.0 eV is attributed to the Mo⁶⁺ 3d_3/2. Similarly, four distinct spectral bands are observed in the XPS spectrum of the S element of MoS₂/PB (Figure 2h). The peaks at 161.2 and 162.9 eV are assigned to 1T‐MoS₂ S²⁻ 2p_3/2 and 1T‐MoS₂ S²⁻ 2p_1/2, respectively, while the peaks centered at 161.9 and 164.1 eV are attributed to the 2H‐MoS₂ S²⁻ 2p_3/2 and 2H‐MoS₂ S²⁻ 2p_1/2, respectively.[45] It is noteworthy that, in the MoS₂/PB structure, the binding energies of both Mo and S orbitals exhibit a noticeable shift toward higher binding energy compared to those in MoS₂ (Table S1,S2). The results indicate the presence of electron‐withdrawing groups in the surrounding environment, which can be attributed to the assembly of PB on the MoS₂ surface.[49]

As expected, the Fe spectrum of MoS₂/PB exhibits significant features, with seven distinct spectral bands identified after curve fitting (Figure 2i). Among these, the peaks at 716.9 and 727.6 eV are satellite peaks. The remaining five peaks correspond to the characteristic Fe 2p signals, reflecting the valence states of the iron element in PB within the MoS₂/PB composite. Specifically, the three peaks centered at 708.5, 709.1, and 721.3 eV are attributed to Fe²⁺ 2p_3/2, Fe²⁺ 2p_3/2, and Fe²⁺ 2p_1/2, respectively. Meanwhile, the two peaks centered at 712.5 and 723.8 eV are attributed to the Fe³⁺ 2p_3/2 and Fe³⁺ 2p_1/2, respectively. Notably, the proportion of Fe²⁺ in the surface structure is relatively high, consistent with most current reports [23]. Furthermore, the valence distribution of the Fe in PB synthesized using the same method is also in agreement (Figure S14). Interestingly, the binding energy positions of the Fe element's different valence states exhibit a shift toward lower binding energies, suggesting the presence of electron‐donating groups in the surrounding environment (Table S3).[49] This should be caused by the internal MoS₂. The offset in binding energy of the two further illustrates the success of the assembly.

What is particularly noteworthy is the significant increase in the content of Mo⁶⁺ within the MoS₂ structure after treatment or assembly. Specifically, the proportion of Mo⁶⁺ in MoS₂ is 6% of the Mo content, whereas in MoS₂/PB, it rises to 16% (Table S4). Analysis of MoS₂ at different stages suggests that this phenomenon is likely due to partial oxidation of the MoS₂ surface under acidic conditions, resulting in an increased Mo⁶⁺ content (Figure S15).[50] Importantly, a higher Mo^{6 +} content is associated with the increased availability of electron transfer–active sites, which may facilitate charge transport and thereby contribute to the enhanced activity observed in certain enzyme‐mimicking reactions.

2.4. Electron Transport and Catalytic Mechanisms

Electron transfer capability is a critical determinant of the catalytic performance of PB‐based nanozymes. Moreover, elucidation of the electron transfer pathways is essential for gaining insight into their enzyme‐mimicking catalytic behavior. Therefore, UV–visible Diffuse Reflectance Spectroscopy (DRS) was employed to calculate the band gap (Eg) value (Figure S16). Subsequently, the valence band (VB) and conduction band (CB) positions were determined using UV Photoelectron Spectroscopy (UPS), enabling a detailed analysis of the difficulty and pathways of the electron transfer.

The Eg values of PB, MoS_2, and MoS₂/PB were estimated to be 2.13, 2.07, and 1.97 eV, respectively, demonstrating that the combination of PB and MoS₂ slightly reduces Eg (Figure 3a–c). This reduction in Eg facilitates easier electron movement within the composite structure.[51] Furthermore, the UPS results reveal that the VB binding energies of PB, MoS₂, and MoS₂/PB are 3.05, 1.87, and 2.58 eV, respectively (Figure 3d–f), allowing for the determination of the corresponding energy band positions (Figure S17). Specifically, the valence band potential (E_VB) and conduction band potential (E_CB) of PB are approximately 3.05 and 0.92 eV, respectively, while those of MoS₂ are approximately 1.87 and −0.20 eV, respectively. For MoS₂/PB, the E_VB and E_CB are approximately 2.58 and 0.61 eV, respectively. Notably, the E_CB of MoS₂/PB shifts in the positive direction compared to PB, a change that is anticipated to enhance the catalytic effect of specific reduction enzyme‐like reactions. In summary, the composite structure of MoS₂/PB not only promotes electron transfer to enhance enzyme‐like activity but also has the potential to improve the reductive enzyme‐like activity of PB.[52] As shown in Figure 3g, the positions of VB and CB indicate that PB and MoS₂ can promote electron transfer because of their overlapping energy band potentials. Notably, the CB potential of MoS₂ is more negative, enabling electrons from MoS₂ to easily migrate to the surface of PB, resulting in interfacial electron transfer.[53] More importantly, since the mechanism of the related enzymatic reaction is redox‐based, MoS₂/PB can acquire electrons from the reaction, thereby supporting a catalytic cycle. Consequently, the electron transfer pathway of MoS₂/PB is illustrated in Figure 3h. Within the composite structure, two relatively independent intra‐electron cycles are established—one associated with PB and the other with MoS₂.[54] Additionally, some electrons migrate from MoS₂ to the PB surface, generating interfacial electron transfer, further enhancing the catalytic performance.

FIGURE 3.

Bandgap of PB (a), MoS₂ (b), and MoS₂/PB (c). UPS spectra of PB (d), MoS₂ (e), and MoS₂/PB (f). (g) Schematic diagram of electron transfer. (h) Schematic diagram of the electron transport pathway.

2.5. Density Functional Theory (DFT) on the Interfacial Electron Transfer of MoS₂/PB

For composite nanozymes that operate via electron transfer mechanisms, interfacial electronic effects play a crucial role in governing the expression of enzyme‐like activities. Given that MoS₂ is confirmed to consist of both 1T and 2H crystalline phases, the coexistence of these phases is expected to modulate interfacial charge transfer behavior, thereby influencing the catalytic performance of the composite nanozyme. Therefore, in order to verify the interfacial electron transfer effect between MoS₂ and PB, DFT calculations were performed on the two interfacial electronic environments of 1T‐MoS₂/PB and 2H‐MoS₂/PB, and the optimized crystal structure is shown in Figure S18. The results of differential charge density calculations show that obvious charge transfer can be observed at the interface for both 1T‐MoS₂ and 2H‐MoS₂ crystals, indicating that the MoS₂/PB interface can effectively transfer electrons from MoS₂ to PB (Figure 4a).[55, 56] The energy band arrangement provides another theoretical support for this transfer. It is worth noting that after the interfacial electron transfer occurs, the valence state of Fe ions near the interface also changes. According to our previous research, it can be concluded that this interfacial effect is consistent with the electron transfer mechanism of PBNZs; that is, this interfacial effect can be used to increase the electron transfer path. This phenomenon is expected to improve the expression of enzyme‐like activity. Simultaneously, this valence state conversion of the adjacent interface may also bring about a longer‐lasting and stable interfacial electron transfer efficiency. It can be seen from the charge density diagram mapped to the z‐axis that, compared with 2H‐MoS₂, 1T‐MoS₂ shows a more favorable electron transfer from MoS₂ to PB (Figure 4b,c). The Bader charge meter shows that 1T‐MoS₂ also transfers 0.24 e to PB, while 2H‐MoS₂ transfers 0.12 e to PB. This difference can also be verified in the simulation results of the work function (Figure S19).[34] Meanwhile, theoretical analysis of the density of states further confirms the above results. Simply put, in both the 1T‐MoS₂/PB and 2H‐MoS₂/PB structures, PB occupies the portion of the unoccupied states closest to the Fermi level, making it easier for PB to gain electrons to fill these unoccupied states. For MoS₂, the occupied states in 2H‐MoS₂ are shifted to the left compared to 1T‐MoS₂, so electrons from MOS₂ in the 1T system are more easily transferred to the PB structure (Figure 4d,e).[57] Further calculations of the PDOS for both structures show that the unoccupied states of PB are mainly contributed by Fe, while the occupied states of MoS₂ are mainly composed of Mo. Therefore, during electron migration, the electrons mainly migrate from the occupied states of Mo to the unoccupied states of Fe in PB (Figure S20). This difference between different crystal phases will also provide new ideas for the design of subsequent composite nanozymes. In addition, electron energy loss spectroscopy (EELS) reveals a significant red shift in the Fe L₃ edge and a corresponding blue shift in the Mo M₃ edge in MoS₂/PB (Figure 4f,g, and Figure S21). These spectral shifts provide strong evidence for the interfacial electron transfer from Fe to Mo.[58, 59, 60] Furthermore, surface photovoltage (SPV) and transient photovoltage (TPV) measurements demonstrate that the interfacial architecture of MoS₂/PB generates a driving force for electron migration (Figure S22).[61, 62, 63] In short, whether it is 1T‐MoS₂ or 2H‐MoS₂, its electron migration to PB will drive a more efficient reduction enzyme‐like properties.[64] This is also in line with the design concept of improving the catalytic performance of reducing enzymes and enriching the expression of multi‐enzyme activities through intra‐ and inter‐nanozyme electron transfer (Figure S23).

FIGURE 4.

(a) Differential charge density (DCD) structure diagram of 1T‐MoS₂/PB and 2H‐MoS₂/PB. The yellow and cyan areas represent the gain and loss of electrons, respectively. (b) and (c) Differential charge Z‐direction plane average charge density of 1T‐MoS₂/PB and 2H‐MoS₂/PB. (d) and (e) Total density of states (TDOS) of 1T‐MoS₂/PB and 2H‐MoS₂/PB. (f) EELS spectra of the Fe L‐edge. (g) EELS spectra of the Mo M‐edge.

2.6. Simulate Multi‐Enzyme Activity

After determining the surface electronic structure and energy band information of MoS₂/PB, its enzymatic activity was evaluated as a novel PB C‐NZs. PB C‐NZs are primarily known for their excellent oxidation/reduction properties, which enable them to regulate biological signaling molecules within the microenvironment of organisms for therapeutic purposes.[65] In this study, a simulation‐based method was employed to assess the activities of six related enzymes: catalase (CAT), peroxidase (POD), superoxide dismutase (SOD), glutathione peroxidase (GSH‐Px), S‐nitrosoglutathione reductase (GSNOR), and nitrite reductase (NiRs). Notably, the enzymatic activities of GSNOR and NiRs have not been previously reported for PB C‐NZs. The detection principles and methods were consistent with those described previously.[66, 67, 68]

To more explicitly demonstrate that the enhanced enzymatic activity of MoS₂/PB is due to electron transfer, 2‐hydroxybenzoic acid was chosen to investigate whether it is affected by the Fenton reaction. The results are shown in Figure S24 and reveal that neither MoS₂/PB nor MoS₂ and PB alone exhibit any Fenton reaction, and free Fe²⁺ does not interfere with the reaction. Therefore, the enhanced electron transfer efficiency, resulting from the unique electron transfer mechanism in the structure, will be the fundamental reason for the excellent enzyme‐like activity of MoS₂/PB. To evaluate the enzymatic activity of MoS₂/PB, the previously reported method was first employed to assess CAT activity. As shown in Figure 5a, MoS₂/PB demonstrated superior CAT‐like activity compared to both PB alone and single‐component MoS₂. The catalysis performance of MoS₂/PB at varying concentrations toward hydrogen peroxide revealed that MoS₂/PB maintained its excellent CAT‐like activity (Figure S25). Next, the POD‐like activity of MoS₂/PB was examined. The results showed that MoS₂/PB also exhibited highly efficient enzyme‐like activity (Figure 5b and Figure S26). The enhanced POD‐like activity of MoS₂/PB was attributed primarily to the improved electron transfer ability of PB and the composite structure. Furthermore, the SOD activity, which is crucial for scavenging ROS and providing anti‐inflammatory effects, was also tested. As anticipated, MoS₂/PB displayed significant SOD‐like activity (Figure 5c). Even with increasing concentration or prolonged reaction times, the composite maintained stable SOD‐like activity (Figure S27). In contrast, single‐component MoS₂ exhibited negligible SOD‐like activity. This observation underscores the role of the MoS₂/PB composite structure in enhancing enzyme‐mimicking activity through intra‐ and inter‐nanozyme electron transfer. Furthermore, the synergistic effect of inter‐nanozyme can be further observed through POD‐like kinetics (Figure S28).

FIGURE 5.

Multi‐enzyme‐like activity results of PB, MoS_2, and MoS₂/PB. (a) CAT, n = 3. (b) POD. (c) SOD, n = 3. (d) NiRs, n = 3. (e) GSNOR. (f) GSH‐Px, n = 3.

The three enzyme‐mimicking activities are primarily attributed to PB nanozymes, whereas MoS₂ in the composite mainly serves to enhance electron transfer efficiency and introduce synergistic effects. Therefore, to comprehensively elucidate the enzyme‐like catalytic properties of MoS₂/PB composite, special attention should be paid to the catalytic activities that are weak or absent in PB nanozymes. Through extensive experimentation, we identified that the MoS₂/PB composite exhibits remarkable catalytic activities analogous to glutathione peroxidase (GSH‐Px), S‐nitrosoglutathione reductase (GSNOR), and nitrite reductase (NiRs). Notably, these enzyme‐like activities are significantly enhanced in the MoS₂/PB composite compared to the negligible activity observed in PB nanozymes.

The NiRs activity can be evaluated by detecting the nitrite concentrations. As shown in Figure S29a,b, the selected detection reagent does not interfere with the experimental results. The catalytic activity is quantified by measuring absorbance at 385 nm, where the lower absorbance corresponds to higher catalytic efficiency. Figure 5d demonstrates that while PB exhibits the ability to promote nitrite decomposition, MoS₂ shows a pronounced catalytic effect. Notably, the MoS₂/PB exhibits excellent NiRs‐like activity, which increases with increasing concentration (Figure S29c).

Based on the previous valence state and energy band analyses, we hypothesize that the MoS₂/PB composite structure will be more beneficial to the reduction enzyme‐like reaction. To verify this hypothesis, we selected two reductive enzyme‐like, GSNOR and GSH‐Px. The results of GSNOR are shown in Figure 5e. The principle of this assay is based on detecting the amount of NO released, where a higher absorbance at a specific wavelength indicates greater NO release. The results demonstrate that, compared to PB or MoS₂, MoS₂/PB exhibits the most vigorous GSNOR‐like activity. Similarly, its catalytic ability increases with concentration. Furthermore, MoS₂/PB retains excellent enzymatic activity over a specific period (Figure S30). The GSH‐Px activity is determined by measuring the content of GSH, with a lower absorbance indicating lower content. The results show that MoS₂/PB demonstrates superior GSH‐Px‐like catalytic activity compared to both PB and MoS₂ (Figure 5f and Figure S31). The GSH‐Px‐like activity of PB alone is negligible.

The difference lies in the fact that the catalytic reactions of GSNOR and GSH‐Px are mainly rely on their reduction enzyme‐like properties, distinct from the redox enzyme‐like discussed earlier. MoS₂ exhibits vigorous catalytic activity, likely due to the presence of Mo⁴⁺ in its structure, which can be converted into Mo⁶⁺, thereby providing more electrons. The enhanced catalytic effect of MoS₂/PB can be attributed to the improvement in electron transfer efficiency due to multiple electron transfer pathways and the enhancement of electron transfer from MoS₂ to PB at the interface, as well as the inter‐nanozyme electron transfer, which further improves the reduction enzyme‐like performance. These results highlight the structural advantages of the rationally designed MoS₂/PB composite. Collectively, the enhanced catalytic performance and the multiple enzyme‐like activities confirm that our preliminary design strategy for PB‐based composite nanozymes is reasonable and effective. Notably, the multi‐enzyme‐like activities of MoS₂/PB are primarily reflected in its ability to regulate the homeostasis of nitric oxide (NO) and glutathione (GSH). Accumulating evidence has demonstrated that these two signaling molecules play crucial roles in mediating cellular signaling pathways and maintaining redox homeostasis, thereby contributing to therapeutic outcomes in a variety of disease models.[69, 70, 71] These observations strongly highlight the considerable biomedical potential of PB‐based composite nanozymes (PB C‐NZs) constructed under the proposed design strategy.

3. Conclusion

In summary, we have rationally designed and constructed a novel PB C‐NZs, MoS₂/PB, via surface assembly, guided by electron‐transfer mechanisms. The formation process was systematically investigated, and comprehensive structural and compositional characterizations were conducted. Band gap measurements and energy band position analyses confirmed that the MoS₂/PB structure facilitates efficient electron transfer. DFT calculations further supported the electron transfer at the MoS₂/PB interface. Notably, 1T‐phase MoS₂ was identified as more favorable for interfacial electron transport, offering valuable insights for the future design of composite nanozymes. Interfacial electron transfer from MoS₂ to PB significantly enhances reductive enzyme‐like catalytic activity, consistent with the observed structural features. The MoS₂/PB composite exhibits markedly improved enzyme‐mimicking performance, particularly in reductive enzyme‐like activities such as GSNOR and GSH‐Px. Importantly, this enhancement is achieved without altering the intrinsic catalytic mechanism, owing to synergistic intra‐ and inter‐nanozyme electron transfer. In conclusion, the MoS₂/PB integrates multiple catalytic activities, demonstrating strong potential for biomedical applications. Furthermore, the design strategy and mechanistic insights presented in this work provide a novel approach for developing high‐performance composite nanozymes.

4. Experimental Section

4.1. Preparation of MoS₂ Nanoflower

The MoS₂ nanoflowers were prepared by the hydrothermal method. Specifically, the raw materials were ammonium heptamolybdate as the molybdenum source, thiourea as the sulfur source, and polyvinyl pyrrolidone as the dispersant. The process was to add 60 mg of ammonium heptamolybdate, 76 mg of thiourea, and 600 mg of polyvinyl pyrrolidone to 17 mL of deionized water under constant stirring and continue stirring for 30 min after being fully dissolved. Then, it was transferred to a hydrothermal reactor of appropriate volume, heated at 220 °C for 12 h, and then slowly cooled. The resulting mixture was repeatedly centrifuged with pure water and washed several times to finally obtain MoS₂ nanoflowers. The above solution was freeze‐dried to obtain a powder sample, which was then dissolved using ultrapure water as a solvent, and the concentration was calibrated with mass fraction.

4.2. Preparation of PB

The PB were prepared using a co‐precipitation method with a dual iron source strategy. Specifically, 55 mL of ultrapure water was added to the reaction vessel, and the pH was adjusted to 3 using HCl (6M) as the base solution. Subsequently, 10 mL each of two pre‐prepared solutions, K₄[Fe(CN)₆] and citric acid/FeCl₃ solution, were added simultaneously. After the addition was complete, the mixture was stirred at room temperature, and the reaction was terminated after 2 h. Then, the reactants were centrifuged and washed several times to remove unreacted molecules, finally yielding PBNZs.

4.3. Preparation of MoS₂/PB

The co‐precipitation method was selected to prepare PB and assemble it on the MoS₂ surface. Specifically, the above‐mentioned fixed concentration MoS₂ solution (20 mL) was dispersed in ultrapure water (35 mL). After being fully mixed, a certain amount of HCl was added to adjust the pH to 3. After continuous stirring for 30 min, 10 mL of the previously prepared K₄[Fe(CN)₆], citric acid solution, and FeCl₃ solution were added in sequence. After being added in sequence, the mixture was stirred at room temperature, and the reaction was terminated after 2 h. Then, the reactants were centrifuged and washed several times to remove unreacted molecules, and MoS₂/PB was finally obtained.

4.4. Statistical Analysis

Quantitative date were presented as the mean ± standard deviation (mean ± SD), based on a sample size of n = 3.

The other detailed experimental processes were available in the Supporting Information.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Supporting File: advs74093‐sup‐0001‐SuppMat.doc.

Data Availability Statement

The data that support the findings of this study are available from the cor‐responding author upon reasonable request.