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题目：Depletable peroxidase-like activity of Fe₃O₄ nanozymes accompanied with separate migration of electrons and iron ions

期刊：《Nature Communication》

原文链接：https://doi.org/10.1038/s41467-022-33098-y

汇报人：张秋平+2020级硕士

As pioneering Fe₃O₄ nanozymes, their explicit peroxidase (POD)-like catalytic mechanism remains elusive. Although many studies have proposed surface Fe2+-induced Fenton-like reactions accounting for their POD-like activity, few have focused on the internal atomic changes and their contribution to the catalytic reaction. Here we report that Fe²⁺ within Fe₃O₄ can transfer electrons to the surface via the Fe²⁺-O-Fe³⁺ chain, regenerating the surface Fe²⁺ and enabling a sustained POD-like catalytic reaction. This process usually occurs with the outward migration of excess oxidized Fe³⁺ from the lattice, which is a rate-limiting step. After prolonged catalysis, Fe₃O₄ nanozymes suffer the phase transformation to γ-Fe₂O₃ with depletable POD-like activity. This self-depleting characteristic of nanozymes with internal atoms involved in electron transfer and ion migration is well validated on lithium iron phosphate nanoparticles. We reveal a neglected issue concerning the necessity of considering both surface and internal atoms when designing, modulating, and applying nanozymes.

Since the first discovery of Fe₃O₄ nanoparticles (NPs) with intrinsic peroxidase (POD)-like activity in 2007, nanomaterial-based artificial enzymes (nanozymes) and their extensive applications have rapidly attracted attention over the past decade. Recently, research efforts on nanozymes have gradually shifted from application-oriented to mechanism-oriented. For example, single-atom nanozymes centered on different metal species have been synthesized with well-defined structures and coordination environments, which facilitate the identification of catalytic centers and unravel the catalytic mechanisms at the atomic level. Besides, the high substrate selectivity of nanozymes has been achieved by the bionic principle of natural substrate channeling and stepwise screening or by molecular blotting techniques. Given the intricate structure-activity relationships and restricted characterization techniques, however, it is still challenging to understand the explicit mechanism of most nanozymes.

a Illustration of the synthesis process of IONPs. b The specific activity (anano) of these three IONPs with TMB as colorimetric substrates. c Diagram of the cyclic catalysis assay. d Kinetic study of anano values of Fe₃O₄ NPs with the days of cyclic catalytic reaction. Error bars represent standard deviation from three independent measurements. e Comparison of Fe L2,3 spectra of Fe₃O₄ NPs before and after 5 days of cyclic POD-like reactions. f The fitted Fe2p XPS spectra of Fe₃O₄ NPs recycled after catalysis on days 0, 1, 3, and 5. g The Fe L-edge NEXAFS spectra of Fe₃O₄ NPs and recycled Fe₃O₄ NPs after 5 days of catalysis in comparison with the reference spectra of FeSO₄ and Fe₂O₃. h Raman spectra of Fe₃O₄ NPs recycled after catalysis on days 0, 1, 3, and 5. i TEM, HRTEM images, and SAED pattern of Fe₃O₄ NPs and recycled Fe₃O₄ NPs after 5 days of catalysis. Images were collected at least three times for each type of NPs.

Supplementary Fig. 11 The inside Fe ²⁺ transfer electron to particle surface to maintain the POD-like catalytic capacity of Fe₃O₄ nanozymes.

Not only the surface Fe²⁺ but also the interior Fe²⁺ of the Fe₃O₄ nanozymes were gradually oxidized by prolonging the reaction time. Simultaneously, the catalytic activity of the recovered NPs gradually decreases with the increase of their oxidation state. Therefore, we suggest that the involvement of Fe²⁺ inside the particles is responsible for the prolonged catalytic capacity of Fe₃O₄ nanozymes. Specifically, as shown in Supplementary Fig. 11, when the surface Fe²⁺ is oxidized to Fe³⁺ by the Fenton-like reaction, the adjacent Fe²⁺ inside the particle will continuously transfer its electron outward via the Fe²⁺-O-Fe³⁺ chain in the lattice to maintain the catalytic activity of the surface Fe atoms. However, this replenishment of electrons is not infinite. When all the interior Fe²⁺ are oxidized to Fe³⁺, the Fe₃O₄ phase is transformed to γ-Fe₂O₃ without electrons being transferred to the surface, resulting in the reduction of catalytic activity or even inactivation.

图4. （a）化学共沉淀法和（b）高温热分解法制备的Fe₃O₄纳米酶的UV-Vis吸收光谱随空气氧化时间的变化。（c）不同方法制备的Fe₃O₄NPs的anano值随空气氧化时间的变化。

Variation of UV-Vis-NIR absorption of a cc-Fe3O4 NPs and b TD-Fe3O4 NPs with aeration oxidation time. Insets are photos of the suspensions corresponding to oxidation times at 0, 0.5, 1, 3, 5, 8, 10, and 12 h. All spectra and photos were obtained at the same Fe concentration. c Changes in anano of the oxidized cc-Fe3O4 NPs and TD-Fe3O4 NPs during the aeration oxidation. Error bars represent standard deviation from three independent measurements.

Analogous to aerated oxidation, the rapid electron and ion migration are also considered to facilitate the POD-like catalysis of Fe₃O₄ NPs, with the only difference being that the electron receptor changed from O₂ in aerated oxidation reaction to H₂O₂ in POD-like reaction. To prove this, the POD-like activity of cc-Fe₃O₄ NPs and TD-Fe₃O₄ NPs as well as their variation with aerated oxidation time were investigated. As seen in Supplementary Fig. 13, the POD-like activity of cc-Fe₃O₄ NPs was higher (2.8 folds) than that of TD-Fe₃O₄ NPs, despite TD-Fe₃O₄ NPs having a smaller hydrodynamic diameter and negative surface potential contributing to a strong affinity with TMB. Aeration oxidation kinetic studies show that the POD-like activity of both Fe₃O₄ NPs decreased with oxidation time (Fig. 2c), along with slight fluctuations in hydrodynamic size and surface potential (Supplementary Fig. 14). However, the decline rate of cc-Fe₃O₄ NPs was faster than TD-Fe₃O₄ NPs, particularly in the initial oxidation stage. This phenomenon is consistent with the changes in NIR spectra shown in Fig. 2a, b. These results further confirm that the more lattice defects of Fe₃O₄ NPs, the easier the migration of excess Fe ions, and thus the higher the POD-like activity. It also means that Fe₃O₄ NPs with more defect sites are easier to be depleted when involved in a POD-like reaction due to their excellent catalytic capability.

The catalytic POD-like reaction of Fe₃O₄ NPs occurs along with internal electron transfer and excess Fe ions migration. After prolonged catalysis, Fe₃O₄ NPs suffer the phase transformation to γ-Fe₂O₃ NPs with depletable POD-like activity.

The rapid electron hopping between Fe²⁺ and Fe³⁺ on the B-sites, creating an intermediate valence state of Fe^2.5+, contributes to the conductivity of magnetite at room temperature, exhibiting a half-metallic nature. This electron-hopping process has been reported to be limited to available Fe²⁺–Fe³⁺ pairs and thus highly depends on the degree of non-stoichiometry of magnetite. Oxidizing Fe₃O₄ to non-stoichiometry magnetite (Fe_3-δO₄) or to γ-Fe₂O₃, the Fe²⁺ in B-sites can be replaced by Fe³⁺ and vacancies, which can be written as (Fe³⁺)A[Fe_2-6δ^2.5+]B[Fe₅δ³⁺□δ]BO4 (□ indicates vacancy; δ indicates vacancy parameter, 0 < δ ≤ 1/3). Thus, the number of available Fe²⁺–Fe³⁺ pairs decreases while isolated Fe³⁺ increases. Besides, the formation of cation vacancies due to the surface migration of excess Fe³⁺ can also disrupt the fast electron-hopping between Fe ions in B-sites. According to the local charge compensation model, each vacancy is electrically equivalent to an extra −5/² charge at one B-site, which has to be neutralized by the excess positive charge at the adjacent B-sites. Thus, each vacancy traps 5 Fe³⁺ and no longer involves in the conduction process. In general, this disturbed electron-hopping process caused by the reduction of Fe²⁺–Fe³⁺ pairs and the formation of cation vacancies is thought to impair the electron transfer to the surface when Fe₃O₄ nanozymes participate in the sustained POD-like reaction, leading to their depletable catalytic activity.

a The crystal structure of LiFePO4 and FePO4 viewed along the a, b, c-axis. The olivine structure is maintained during Li-ions insertion and extraction. b The SEM image of as-synthesized LiFePO4 NPs. The image was collected at least three times. Inset is a photo of LiFePO4 NPs aqueous solution. c The POD-like activity of LiFePO4 NPs (6.25 μg Fe/mL) with TMB (1.7 mM) as colorimetric substrates under the presence of H2O2 (0.8 M) in 0.2 M acetate buffer (pH = 3.6). d ESR spectra of spin adducts DMPO/·OH produced by LiFePO4 NPs (10 μg/mL) in the presence or absence of H2O2 (0.165 M) in 0.2 M acetate buffer (pH = 3.6). e Comparison of the anano of as-synthesized LiFePO4 NPs and cc-Fe3O4 NPs. Error bars represent standard deviation from three independent measurements. f Diagram of the POD-like catalytic reaction process of LiFePO4 NPs and Fe3O4 NPs.

Rod-like LiFePO₄ NPs with an average length of 321.9 nm and width of 172.2 nm (Fig. 4b) were successfully synthesized using the solvothermal method59 and characterized via various methodologies. As expected, the POD-like activity of LiFePO₄ NPs was demonstrated with different chromogenic substrates including TMB, ABTS, and OPD (Fig. 4c). Also, they follow pH, temperature as well as NPs concentration dependence, and the Michaelis–Menten kinetics. The optimal pH is about 4.0. The ·OH was shown to be generated in a time- and concentration-dependent manner (Fig. 4d), which is similar to Fe₃O₄ NPs. We then compared the POD-like activity of LiFePO₄ NPs and cc-Fe₃O₄ NPs using two oppositely charged substrates (TMB and ABTS) at pH 3.6. The results consistently show that LiFePO₄ NPs had higher catalytic ability than cc-Fe₃O₄ NPs, and the anano of LiFePO₄ NPs was approximately four times than that of cc-Fe₃O₄ NPs, despite their larger particle size (Fig. 4e). These results imply that LiFePO₄ NPs may share a similar POD-like catalytic mechanism with Fe₃O₄ NPs, differing in that the rapid Li+ migration in the lattice of LiFePO₄ NPs confers them a superior POD-like catalytic activity (Fig. 4f).

In summary, the catalytic mechanism of the POD-like activity of Fe₃O₄ nanozymes is elucidated by characterizing the chemical composition and catalytic activity of the Fe₃O₄ NPs recycled from the long-term POD-like catalysis. These studies indicate that not only the surface Fe²⁺, but also the internal Fe²⁺ contribute to the POD-like activity of Fe₃O₄ nanozymes. The Fe²⁺ inside the particle can transfer electrons to the surface, regenerating the surface Fe²⁺ that is constantly involved in the sustained catalytic reaction. This process is usually accompanied by the outward migration of excess oxidized Fe³⁺ from the interior of the crystal, which is considered as a rate-limiting step. Analogous to the low-temperature oxidation of magnetite, Fe₃O₄ NPs participated in the POD-like reaction are eventually oxidized to γ-Fe₂O₃ NPs with a reduced POD-like capacity. Furthermore, this mechanism is well-validated on LiFePO₄ NPs. This work reveals the depletable property of Fe₃O₄ nanozymes differing from natural enzymes and highlights the potential contribution of internal metal atoms in nanozymes-catalyzed reactions. Meanwhile, these findings provide a theoretical basis for the mechanistic study and rational design of other related nanozymes.