In recent years, nanoenzymes have been selected as ideal substitutes for natural enzymes due to their high stability, low cost, and mass production, and are widely used in various fields. However, a key problem of nanozymes is that their catalytic activity is still insufficient compared to natural enzymes. In view of the high-efficiency catalytic performance of metal active sites in natural metalloenzymes, precisely designing and regulating the structure of metal sites at the atomic scale is an effective strategy for the development of high-activity nanozymes. Specifically, the structure-performance relationship in the nanoenzyme system has important guiding significance for the design of high-performance nanoenzymes. Therefore, a reasonable design and regulation of the electronic structure and coordination environment of the atomic-level dispersed active sites is expected to achieve high efficiency in the performance of natural enzymes. simulation.


Tuning Atomically Dispersed Fe Sites in Metal-Organic Frameworks Boosts Peroxidase-Like Activity for Sensitive Biosensing

Weiqing Xu1, Yikun Kang2, Lei Jiao1, Yu Wu1, Hongye Yan1, Jinli Li1, Wenling Gu1, Weiyu Song2, Chengzhou Zhu1

Nano‑Micro Lett.(2020)12:184

https://doi.org/10.1007/s40820-020-00520-3



Highlights of this article

1. By introducing strong electron-withdrawing (or electron-donating) functional groups (-NO₂ or -NH₂) into organic ligands, direct control of the atomic-level dispersed metal sites (Fe) in MIL-101 is achieved. 2. -NO₂ functionalization significantly improves the affinity of the nanozyme to the substrate and achieves a great increase in its peroxidase-like activity. However, -NH₂ functionalization showed the opposite result. 3. Theoretical calculations show that -NO₂ can not only affect the geometric structure of the adsorbed intermediate, but also optimize the electronic structure of Fe active sites. The catalytic reaction energy barrier of NO₂-MIL-101 is significantly reduced and the activity is enhanced. 4. The biosensing system constructed by NO₂-MIL-101 realizes the highly sensitive detection of acetylcholinesterase activity and organophosphorus pesticides.

brief introduction

The Zhu Chengzhou research group of Central China Normal University and the Song Weiyu research group of China University of Petroleum selected MIL-101(Fe) (MIL-101 for short) with good peroxidase-like (POD) activity as the model, and the five-coordinated Fe atom was used as the simulation The active site of POD. Two functional groups with different electronegativity (-NO₂ and -NH₂) were introduced into MIL-101 (represented as NO₂-MIL-101 and NH₂-MIL-101, respectively) to adjust the structure of the active site. As expected, functional groups can effectively regulate the POD-like activity of MIL-101. Its activity is NO₂-MIL-101, MIL-101, NH₂-MIL-101. The experimental results show that the introduction of -NO₂ can significantly increase the affinity of MIL-101 to the substrate and enhance its catalytic activity. In addition, theoretical studies have shown that the strong electron-withdrawing performance of -NO₂ can not only adjust the geometric configuration of the adsorbed intermediate, but also optimize the electronic structure of the atomic-level dispersed Fe active sites. Thanks to these advantages, the catalytic reaction energy barrier of NO₂-MIL-101 is significantly reduced and the activity is enhanced. Therefore, coordinated with the geometric and electronic effects of organic ligands to regulate the metal sites in MOFs can provide a new solution for the direct regulation of active sites at the atomic scale and the design of high-performance nanoenzymes. In addition, the biosensing system constructed by NO₂-MIL-101 realizes highly sensitive colorimetric detection of acetylcholinesterase (AChE) activity and organophosphorus pesticides (OP), which has broad application prospects in the field of biosensing.



Graphic guide

I MOFs synthesis and characterization The organic ligand aminoterephthalic acid/nitroterephthalic acid is used to replace terephthalic acid and metal ion (Fe3⁺) coordination to synthesize different functionalized MIL-101. The theoretical model shows that the local structural units of different functionalized MIL-101 are the same, except that -NH₂/-NO₂ replaces -H in the terephthalic acid ligand (Figure 1a-c). The TEM characterization results of MOFs confirmed that the morphologies of the three materials are basically the same (Figure 1d-f). In addition, as shown in Figure 1g, various elements are uniformly dispersed in NO₂-MIL-101. In particular, the uniform dispersion of the N element shows that -NO₂ has been successfully introduced into MIL-101. XRD patterns indicated that the prepared MOFs had similar crystal structures (Figure 1h). The appearance of the characteristic peaks of -NH₂/-NO₂ in the infrared spectrum further confirmed that the functional group was successfully introduced into MIL-101 (Figure 1i). The above experimental data not only verify the rationality of the constructed model, but also provide a prerequisite for the subsequent evaluation of the activities of the three catalysts at the same level.



Figure 1. Nanoenzyme structure and morphology characterization: (a-c) structure diagrams of different MOFs (Fe: yellow, C: brown, O: red, H: pink, N: purple) and (d-f) TEM morphology characterization. (g) HAADF-STEM of NO₂-MIL-101 and EDS mapping of corresponding elements. (H) XRD and (i) FT-IR spectra of different MOFs.

II Performance evaluation of nanoenzymes is based on the evaluation of the POD-like activity of nanoenzymes using 3,3,5,5-tetramethylbenzidine (TMB) color reaction under the same conditions of the metal active site (Fe) content (Figure 2a) . The study found that the introduction of -NO₂ enhanced the POD-like activity of MIL-101, while -NH₂ weakened its catalytic activity (Figure 2b). Quantitative evaluation of its catalytic efficiency was achieved by measuring the specific activity (SA) of nanozymes (Figure 2c). The SA of NO₂-MIL-101 and MIL-101 were 7.91 times and 2.77 times that of NH₂-MIL-101, respectively. Subsequent electron paramagnetic resonance (EPR) spectra verified the existence of hydroxyl radicals (•OH) (Figure 2d), and their signal peak intensities are in good agreement with the corresponding POD-like activities. After adding isopropanol, a scavenger of •OH, to the reaction system, the catalytic activity of nanozymes was significantly reduced, indicating that •OH is the main active intermediate (Figure 2e). In addition, the steady-state kinetic evaluation results of nanozymes show that NO₂-MIL-101 has a small Michaelis constant (Kₘ) for H₂O₂ catalysis, which proves that it has a higher affinity for H₂O₂ (Figure 2f). And NH₂-MIL-101 showed the opposite result.

Figure 2. Evaluation of POD-like enzyme activity. (a) Different organic ligand functional groups regulate the activity of MOF POD.(B) Absorption spectra, (c) specific activity and (d) EPR spectra of intermediate product•OH of different nanozymes catalyzing the oxidation of TMB. (e) The influence of the introduction of isopropanol on the activity of nanozymes. (f) Nanozymes catalyze the Kₘ of H₂O₂. III. Theoretical calculation The reaction energy of each step is calculated in detail according to the reaction pathway given in Figure 3a. The decomposition of H₂O₂ as a rate determining step (RDS) determines the catalytic activity of the entire reaction (Figure 3b). The energy change of RDS is NO₂-MIL-101 (1.28 eV)₂-MIL-101 (1.65 eV), which is consistent with the result of the catalytic activity of nanozymes. In order to explore the reasons for the superior catalytic activity of NO₂-MIL-101, we first studied the geometric effect of -NO₂ on the adsorbed H₂O₂*. Interestingly, on NO₂-MIL-101, the orientation of the O-H bond of the adsorbed H₂O₂* is biased toward the oxygen on -NO₂ (Figure 3c). In contrast, no significant changes were observed in MIL-101 and NH₂-MIL-101. -NO₂s strong electron absorption effect shortens the H··O-N-O distance. This change is conducive to the breaking of the O-O bond, thereby reducing the change in OH* formation energy. In order to further reveal the influence of -NO₂ from the perspective of electronic structure, the influence of -NO₂ on the charge density of NO₂-MIL-101 was calculated. It can be seen from Figure 3d that the projected electronic state density distribution (PDOS) of NO₂-MIL-101 in the dxz direction is increased, while the PODS in the dz2 direction is significantly reduced, while it is not obvious on NH₂-MIL-101 Variety. Therefore, we believe that the enhancement of NO₂-MIL-101 activity may be due to the decrease of electrons in the dangling bond (dz2) direction on the Fe active site. In order to understand the effect of the changed 3d orbital splitting in the catalytic reaction, the interaction between the HO* and Fe atoms adsorbed at the active site was revealed by calculating the number of projected crystal orbital Hamilton particles (pCOHP) and the Fermi energy level (Figure 3e) . The filling amount of the antibonding orbital of the HO*-Fe bond (the blue area below the Fermi level) in NO₂-MIL-101 is less than that of MIL-101 and NH₂-MIL-101, indicating that the electrons in the antibonding orbital are reduced. The reduction of antibonding orbital electrons can increase the bond length of HO*-Fe, promote the cleavage of H₂O₂* and effectively reduce the energy change of RDS.

Figure 3. Theoretical calculation of the reaction catalyzed by nanozymes. (a) A schematic diagram of the basic steps of NO₂-MIL-101 reaction in an acidic environment. (b) Energy change diagram of the reaction catalyzed by nanozymes. (c) The geometric structure of H₂O₂ adsorbed on nanozymes (MIL-101, NH₂-MIL-101, NO₂-MIL-101 from left to right). (d) The distribution of PDOS and Fermi level on Fe 3d orbit. (Inset: partial view near Fe active site). (e) The schematic diagram of HO*-Fe bond and its corresponding pCOHP (the arrangement order is the same as that in (c)). IV The construction of biosensor AChE, as a key enzyme in the human body, plays an important role in the nervous system, so the accurate evaluation of AChE activity is of great significance for the diagnosis and treatment of diseases. Based on the AChE catalyzed thioacetylcholine to produce thiocholine containing thiol, this product will significantly affect the color reaction of NO₂-MIL-101 on TMB catalyzed (Figure 4a). Therefore, this paper uses a two-enzyme tandem reaction (AChE-POD) to achieve high sensitivity and high selectivity for the detection of AChE activity (Figure 4b-e). The linear detection range is 0.2-50 mU/mL, and the detection limit is 0.14 mU/mL. In addition, organophosphorus pesticides (OP) can cause AChE inactivation and affect the normal activities of the body. Therefore, the biosensor is further used for the effective detection of OP.






Figure 4. Construction of the biosensor. (a) Schematic diagram of AChE activity detection. (b) Absorption spectra of TMB catalyzed by different systems. Absorption spectrum (c) and linear detection range (d) of different concentrations of AChE activity detection. (e) Selectivity evaluation of biosensors.


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