Tuning iron spin states in single-atom nanozymes enables efficient peroxidase mimicking

The large-scale application of nanozymes remains a significant challenge owing to their unsatisfactory catalytic performances. Featuring a unique electronic structure and coordination environment, single-atom nanozymes provide great opportunities to vividly mimic the specific metal catalytic center of natural enzymes and achieve superior enzyme-like activity. In this study, the spin state engineering of Fe single-atom nanozymes (FeNC) is employed to enhance their peroxidase-like activity. Pd nanoclusters (PdNC) are introduced into FeNC, whose electron-withdrawing properties rearrange the spin electron occupation in Fe(ii) of FeNC–PdNC from low spin to medium spin, facilitating the heterolysis of H2O2 and timely desorption of H2O. The spin-rearranged FeNC–PdNC exhibits greater H2O2 activation activity and rapid reaction kinetics compared to those of FeNC. As a proof of concept, FeNC–PdNC is used in the immunosorbent assay for the colorimetric detection of prostate-specific antigen and achieves an ultralow detection limit of 0.38 pg mL−1. Our spin-state engineering strategy provides a fundamental understanding of the catalytic mechanism of nanozymes and facilitates the design of advanced enzyme mimics.


Introduction
Peroxidases (POD), a family of enzymes that catalytically oxidize certain compounds in the presence of peroxides (H 2 O 2 in most cases), play a signicant role in biological systems, such as H 2 O 2 detoxication in the human body. 1,2 They can also be isolated from organisms and applied to various applications such as biosensing, therapy, and environmental protection. [3][4][5] However, their large-scale commercialization remains an ongoing issue because of the disadvantages of high cost, difficult recovery and variability. 6 Nanozymes, nanomaterials with enzyme-like characteristics, have been discovered as substitutes for natural enzymes with the merits of excellent stability, facile storage and easy production. [6][7][8][9][10] Nevertheless, one obstacle lying in nanozymes emanates from their low catalytic efficiency, which is mainly attributed to their insufficient intrinsic activity. 7 Overcoming these limitations to acquire highly active POD-like nanozymes is, therefore, a subject of interest and priority.
Compared with simulating functionally, mimicking natural enzymes from the perspective of biological structure may provide a more reasonable strategy to achieve high catalytic performance. [11][12][13][14] Single-atom nanozymes with well-dened electronic and geometric structures are appealing alternatives to natural enzymes as they can mimic the highly evolved catalytic center of natural enzymes at the atomic scale. 12,15,16 Given the similar heme structure of horseradish peroxidase (HRP), Fe singleatom nanozymes are promising to catalyze H 2 O 2 activation. 15,[17][18][19][20][21] More importantly, tuning the local environment of single atoms is expected to optimize their electronic structure for enhanced POD-like catalytic performance. For example, heteroatoms such as B, S and P have been introduced as "modulators" to decrease the energy barrier during the reduction of H 2 O 2 . 11,22,23 FeN 5 , in which the Fe single atom is coordinated by axial N atoms, emulates penta-coordinated heme systems and shows enhanced POD-like performance compared to its FeN 4 counterpart. 14 Furthermore, according to the frontier molecular orbital theory, the catalysis process depends on the electron donation and back-donation steps between the reactants/intermediates and metal centers. 24,25 During the reaction process involving the production of multistep intermediates, the number of unpaired electrons cannot always be conserved, resulting in the spin-related electron transfer and thus the d orbital occupation and spintronic conguration sensitive reaction kinetics and thermodynamics. 26 Inspired by this, tuning the spin state of metal atoms in single-atom nanozymes is expected to enhance their POD-like activity, realizing the efficient activation of H 2 O 2 and revealing the underlying relationships between spin states and performance.
In this study, we develop spin-dependent Fe single-atom nanozymes by employing Pd nanoclusters (Pd NC ) as "modulators" (named FeNC-Pd NC ) for the rst time to achieve enhanced POD-like activity. The synergistic effect between Pd NC and Fe single atoms results in the outstanding POD-like activity of FeNC-Pd NC in comparison to FeNC. Studies show that Pd NC promotes the formation of a greater amount of medium spin (MS) Fe(II) atoms, which are more active than low spin (LS) Fe(II) owing to the weakened binding strength towards oxygen-containing intermediates and accelerated H 2 O 2 activation. The spin-dependent H 2 O 2 reduction opens an avenue for the rational design of highly active POD mimicking.

Results and discussion
As displayed in Fig. 1a, FeNC-Pd NC was prepared via the connement effect of metal-organic frameworks (MOFs). Briey, FeNC was prepared by pyrolysis of Fe-doped zeolitic imidazolate frameworks (ZIF-8) (Fig. S1 †). Then, Pd 2+ ions were incorporated into FeNC through a micropore adsorption strategy and in situ reduced to form Pd NC . 27 The rich metal nodes of ZIF-8 and the pores aer annealing act as two "claws", anchoring Fe single atoms and Pd NC . 28 To explore the role of Pd NC , NC-Pd NC without Fe single atoms was also prepared using ZIF-8 as the precursor. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images of FeNC-Pd NC display its dodecahedral shape inherited from ZIF-8 ( Fig. 1b and S2 †). The high-angle annular dark-eld scanning TEM (HAADF-STEM) image conrms the highly dense and uniformly distributed Pd NC conned in the carbon with an average size of 2.13 nm (Fig. 1c). The Brunauer-Emmett-Teller (BET) area of FeNC-Pd NC , FeNC and NC-Pd NC is 547.0, 554.9 and 550.5 m 2 g −1 , respectively ( Fig. S3 and Table S1 †), which guarantees the accessibility of active sites. 29 The aberrationcorrected HAADF-STEM (AC-HAADF-STEM) image shown in Fig. 1d reveals that numerous Fe single atoms surround Pd NC . The enlarged AC-HAADF-STEM and corresponding energy dispersive spectroscopy (EDS) mapping conrm the existence of C, N, O, Fe, and Pd elements and the uniformly distributed Pd species in Pd NC (Fig. S4 †). X-ray diffraction (XRD) patterns exhibit Pd(111) characteristic peaks in both FeNC-Pd NC and NC-Pd NC , conrming the successful introduction of Pd NC into FeNC-Pd NC (Fig. S5 †). Inductively coupled plasma optical emission spectrometry (ICP-OES) analysis shows that the contents of Pd and Fe in FeNC-Pd NC were ∼5.6 and ∼0.36 wt%, respectively. Raman spectra (Fig. S6 †) indicate that both FeNC-Pd NC and FeNC have rich disorientated graphene structures with a high ratio of D and G band intensities (I D /I G ). 30 X-ray photoelectron spectroscopy (XPS) analysis was performed to understand the chemical states of various elements in the nanozymes. The C 1s spectra shown in Fig. S7a † exhibit four peaks corresponding to sp 2 -C, sp 3 -C, C-N x, and C]O/C-O. 31 The N 1s spectra can be deconvoluted into four peaks at 398.5, 399.5, 400.4, and 401.1 eV, which are attributed to pyridinic N, FeN, pyrrolic N, and graphitic N, respectively ( Fig. S7b †). 32 The high ratios of FeN content in FeNC-Pd NC (23.1%) and FeNC (21.6%) indicate the presence of numerous Fe single-atom species (Table S2 †). In particular, the binding energy of Fe 2p in FeNC-Pd NC is higher than that in FeNC, whereas Pd shows a decreased binding energy compared to NC-Pd NC ( Fig. 1e and S8 †). This suggests that electron transfer occurs from Fe single atoms to Pd NC , owing to the difference in the electronegativities of Pd and Fe 2+ . 33 Moreover, the decreased electron density on the Fe center in FeNC-Pd NC may signicantly regulate its catalytic behavior.
To further elucidate the changes in the local chemical conguration of Fe species aer the introduction of Pd NC , the Fe K-edge X-ray absorption ne structure (XAFS) was obtained. As depicted in Fig. 1f, the Fe K-edge extended-XAFS (EXAFS) Fourier-transformed magnitudes of FeNC-Pd NC and FeNC show major peaks at ∼1.4Å, which is consistent with that of ironporphyrin (Fe Pc) and attributed to the backscattering between Fe and N atoms. 11,34 The FeN coordination numbers in FeNC-Pd NC and FeNC obtained via the quantitative EXAFS tting analysis were 4.1 and 4.2, respectively (Fig. S9 and  Table S3 †). X-ray absorption near edges structure (XANES) spectra were obtained to identify the valence state of Fe species. As displayed in Fig. 1g, the absorption edges of Fe K-edge XANES curves of FeNC-Pd NC and FeNC are between Fe Pc and Fe 2 O 3 . In addition, the valence state of FeNC-Pd NC is more positive than that of FeNC, which indicates the decreased electron density and is consistent with the XPS results. 35,36 First derivative XANES spectra were obtained to reveal the oxidation state of Fe (Fig. S10 †). Overall, the characteristic Fe 2+ peak (∼7122.6 eV), similar to that of Fe Pc, appears for both FeNC-Pd NC and FeNC, while the peak of FeNC-Pd NC shows a slight positive shi compared to that of FeNC, indicating its relatively positive charge upon the integration of Pd NC .
The charge redistribution primarily dominates the electronic conguration of Fe(II), i.e., the spin state, and thus regulates the catalytic activity. N coordinated Fe(II) exhibits multiple states, such as LS (d xy 2 d xz 2 d yz 2), MS (d xy 2 d xz 2 d yz 1 d z 2 1) and high spin (HS; d xy 2 d xz 1 d yz 1 d z 2 1 d x 2 −y 2 1) owing to the variation of the coordination environment (Fig. S11 †). 37 In the case of LS Fe(II), the Fe atom splits into fully occupied low-level orbitals whereas in MS Fe(II), one electron from the d yz orbital will hop to the d z 2 orbital, resulting in unpaired electrons. To verify the spin redistribution experimentally, the zero-eld cooling temperature-dependent (ZFC-T) magnetic susceptibility (c m ) study was carried out. As shown in Fig. 2a, the total effective magnetic moment (m eff ) was obtained according to the equa- of single spin electrons (n). 38 The n of Fe(II) increases from ∼0.52 in FeNC to ∼2.18 in FeNC-Pd NC , conrming the increase in spin state. For quantication, Mössbauer spectroscopy analysis, which is a powerful tool for identifying the spin polarization conguration, was conducted. Three different doublets (D1-D3) are tted for FeNC-Pd NC and FeNC, corresponding to LS Fe(II), MS Fe(II), and HS Fe(II), respectively. 39 FeNC-Pd NC possesses a higher content of MS Fe(II) (33.4%) than FeNC (17.1%) ( Fig. 2b and Table S4 †). This could be attributed to the strong interactions between the Fe single atoms and Pd NC in FeNC-Pd NC that effectively reshaped the electronic structure of Fe, achieving the Fe 3d electron-spin conguration transition from LS to MS. Based on this, the electronic spin information of Fe single atoms was further investigated through density functional theory (DFT) calculations. FeN 4 embedded in graphene was used to simulate FeNC, while the FeNC coupling with Pd (111) facets was used to simulate FeNC-Pd NC . As shown in Fig. 2c, the projected density of states (DOS) diagrams reveal that the distance between the dband center of spin up and spin down (j3 d-up -3 d-down j) increases with the introduction of Pd NC , indicating a higher spin state for the Fe center. The integral projected density of states analysis of Fe 3d orbital splitting reveals that the increasing spin state mainly originated from the increased Fe d z 2 contributions (Fig. 2d), which is also visualized by the spin density diagram (Fig. S12 †). Compared to FeNC, the highest occupied molecular orbital (HOMO) of Fe(II) in FeNC-Pd NC shows one d z 2 orbital perpendicular to the plane within the introduction of Pd NC compared to FeNC (Fig. 2d below).
Typically, Fe-based catalysts strongly bind to oxygen, circumscribing proton-electron transfer and catalytic activity. During the H 2 O 2 reduction procedure, the 2p orbital of O in H 2 O 2 /intermediates and 3d orbital of Fe sites hybridize, making the 3d electronic state act as spin-dependent gates to regulate electron transfer and orbital interaction in the catalytic reaction. 24 To conrm this phenomenon, the H 2 O 2 reduction process in both LS and MS Fe(II) was theoretically predicted. Due to the scaling relationships of different intermediates, O* is chosen as the model to study its interaction with Fe sites. 40 Besides, the interaction between Fe-d xy /d x 2 −y 2 orbitals and adsorbed O* is negligible because they cannot mix based on symmetry conservation. 41 As shown in Fig. 2e, the interaction between the Fe center and adsorbed O* changes along with the change in the spin state of Fe(II). In particular, a half-lled d z 2 orbital in MS Fe(II) can accept fewer valence electrons from *O with the generation of one s* orbital, favorable for the desorption of H 2 O 2 /intermediates to trigger a catalytic cycle (Fig. S13 †). In this case, the local microenvironment disturbance is exerted to modulate the electronic hopping and occupation of the 3d orbital, achieving the spin reconguration and regulating the enzyme-like activity.
The POD-like catalytic activity of FeNC-Pd NC was quantitively assessed using a chromogenic biochemical reaction of 3,3 ′ ,5,5 ′tetramethylbenzidine (TMB), which can be oxidized by H 2 O 2 to ox-TMB in blue color (at 652 nm). 42,43 As expected, FeNC-Pd NC exhibits two times the catalytic activity of FeNC (Fig. 3a). Besides, the specic activity (SA) value of FeNC-Pd NC was 95.68 U mg −1 , which is more than three-fold higher than that of FeNC (30.27 U mg −1 ) (Fig. 3b). Meanwhile, the injection of SCN − prominently impairs the POD-like performance of FeNC-Pd NC , conrming the main role of Fe single atoms in the POD-like property (Fig. S14 †). 13 Thus, the above results illustrate that Fe single atoms play a critical role in POD-like activity and the existence of Pd NC can signicantly boost their activity, despite the sluggish kinetics of H 2 O 2 reduction at Pd NC . Generally, the unsatisfactory specicity of nanomaterials has been a major constraint in the development of nanozymes. 44 Therefore, the oxidase (OXD)-like activity, the major interfering reaction to POD-like activity was also evaluated (Fig. 3c and S15 †). Unlike the POD-like activity of FeNC-Pd NC , the OXD-like activity shows negligible enhancement aer the integration of Pd NC . Moreover, the POD-like activity of FeNC-Pd NC is much higher than that of the addition of FeNC and NC-Pd NC activities, which demonstrates the synergistic effect between Pd NC and Fe single atoms. The Michaelis-Menten equation was applied to assess the POD-like activity of nanozymes (Fig. 3d, S16 and S17 †). The Michaelis-Menten constant (K m ), maximum velocity (V max ), and K cat (K cat = V max /[S]) are listed in Table S5 †. FeNC-Pd NC exhibits higher K cat (5.10 s −1 ) than FeNC (2.56 s −1 ) towards H 2 O 2 , indicating its higher POD-like activity. More importantly, FeNC-Pd NC also has competitive catalytic activity compared with other reported nanozymes. Both FeNC-Pd NC and FeNC exhibit high pH-dependent properties similar to natural HRP and higher temperature tolerance than HRP (Fig. S18 †). The asprepared nanozymes maintain their POD-like activity aer longterm storage and the treatment of strong acidic and alkaline solutions compared to HRP, suggesting that they can perfectly surmount the disadvantage of the inferior stability of natural enzymes ( Fig. S19 and S20 †). Therefore, FeNC-Pd NC is a promising alternative to natural enzymes.
To understand the potential catalytic intermediates, electron paramagnetic resonance (EPR) spectroscopy was performed by using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as an cOH trapping agent. No signal of cOH was detected in HRP/H 2 O 2 and all nanozymes/H 2 O 2 systems except for the Fe 2+ /H 2 O 2 system (Fig. 3e). The existence of cOH radical scavenger t-butyl alcohol has little effect on the activity of FeNC-Pd NC and FeNC, indicating that cOH is not a critical active intermediate in the catalytic reaction (Fig. S21 †). Inspired by the catalytic mechanism of natural HRP, the Fe(IV)]O intermediate is considered as the active intermediate. 45,46 Then a low-temperature (77 K) EPR experiment was performed. As expected, rhombic signals at g = 2 were detected aer the reaction of FeNC-Pd NC and FeNC with excess H 2 O 2 , suggesting the formation of compound oxoferryl Fe(IV)]O and the porphyrin cation radical (named as the oxo-iron porphyrin p-cation radical [Fe(IV)]O Por +c ]) during the reaction process (Fig. 3f). 47 Notably, the intensity of FeNC-Pd NC is higher than that of FeNC. Considering the heme-like structure of FeNC similar to natural HRP, the essential active intermediate Fe(IV)]O might have formed via the bound reactive oxygen species (ROS) pathway while Pd NC is expected to enhance the production of Fe(IV)]O species.
DFT calculations were performed to theoretically understand the effect of the spin state on the POD-like activity. As shown in Fig. 4a and S22, † the H 2 O 2 heterolytic dissociation route is energetically more favorable for FeNC and FeNC-Pd NC . Moreover, the H 2 O* desorption on FeNC is the rate-determining step (RDS) with an energy barrier of 0.28 eV due to the strong binding between Fe single atoms and oxygen-containing species. Encouragingly, this step is exothermic for FeNC-Pd NC , indicating that Pd NC integration facilitates the timely H 2 O desorption. Fe(IV)]O, a critical intermediate for TMB oxidation similar to that observed for natural HRP, is produced by the dehydration of the adsorbed H 2 O 2 as follows: H 2 O 2 / O* + H 2 O. 48 As shown in Fig. 4b, the H 2 O 2 dehydration process is not thermodynamically favorable for FeNC with an energy barrier of 0.75 eV. Aer the integration of Pd NC , this energy barrier is decreased to 0.36 eV on FeNC-Pd NC , promoting the formation of Fe(IV)]O intermediate sites. As mentioned previously, the spin state of Fe(II) uctuates the interaction between Fe sites and *O, which further improves its oxidation capacity towards TMB. Next, the effect of Pd NC integration on TMB oxidation was evaluated. The oxidation of TMB proceeded through the N-H bond cleavage of TMB and transformation H to Fe(IV)]O along with transfer of an electron from the substrate to the nanozymes. As a result, the dissociation energy of TMB decreases from 0.55 eV in FeNC to 0.24 eV in FeNC-Pd NC , thus rendering it more favorable to oxidize TMB (Fig. 4c).
The orbital interactions between H 2 O* and Fe sites were explored. The COHP and DOS analysis reveals that the energy level of the bonding molecular orbital of FeNC-Pd NC shis to lower energy, whereas that of the antibonding molecular orbital shis to higher energy, which weakens the Fe-O bond strength in the H 2 O* adsorption conguration (Fig. 4d and S23 †). Furthermore, an axial s* antibonding molecular orbital is observed for H 2 O* on FeNC-Pd NC owing to the electrons in the higher level d z 2 orbital, while the antibonding contribution is mainly from p* antibonding orbitals for FeNC. This unique s* antibonding orbital contributes to the timely H 2 O desorption from FeNC-Pd NC . These DFT results conrm that FeNC-Pd NC with a higher ratio of MS Fe(II) exhibits highly efficient POD-like activity.
Subsequently, we performed a proof-of-principle demonstration. FeNC-Pd NC was applied as a label in the colorimetric nanozyme-linked immunosorbent assay (NLISA), and prostate-specic antigen (PSA) was chosen as the model analyte (Fig. 5a). [49][50][51] Typically, the surface of FeNC-Pd NC is positively charged, which can conjugate with the labeling antibody of PSA (Ab 2 ) as a signal tag through electrostatic interaction (Fig. S24 †). The red uorescence emission in the confocal laser scanning microscope demonstrates the successful immobilization of Ab 2 (labeled by rhodamine B) on FeNC-Pd NC (Fig. S25 †). The absorption spectra and corresponding calibration curves were obtained, where FeNC-Pd NC NLISA exhibits a wide detection range of 1-2000 pg mL −1 with a lower variable coefficient of 0.18-5.18% (Fig. 5b and S26 †). The limit of detection (LOD) dened by the 3SD method is 0.38 pg mL −1 . In contrast, the LOD of the traditional HRP-based enzyme-linked immunosorbent assay (ELISA) is 5.38 pg mL −1 with a detection range of 10-1000 pg mL −1 (Fig. S27 †). The sensitivity of FeNC-Pd NC towards PSA is also higher than those in previous reports, suggesting that FeNC-Pd NC has potential immunoassay applications (Table S6 †).
In terms of specicity, an obvious characteristic intensity was observed for PSA, while there are negligible changes in the signal intensity for other biomarkers and metal ions (BSA, CEA, ESR1, HER2, CAT, GO x , Zn 2+ , K + , and Na + ) compared to the blank samples (Fig. 5c). In the presence of all of the above interferents, the FeNC-Pd NC NLISA maintains its high-intensity signal for PSA, indicating its good selectivity and antiinterference ability. Furthermore, FeNC-Pd NC NLISA exhibits outstanding repeatability and reproducibility with low calculated relative standard deviations (RSD; 4.23% and 1.92%, respectively) (Fig. S28 †). Stability tests were conducted for ten days, where the RSD was 3.29% (Fig. S29 †). By taking advantage of its high activity, selectivity, and stability, FeNC-Pd NC NLISA was used to detect PSA in real serum samples. As expected, the results of FeNC-Pd NC NLISA are consistent with those of chemiluminescence analysis, indicating its remarkable feasibility and accuracy for practical application in the proposed assay (Fig. 5d). Thus, FeNC-Pd NC NLISA has potential applications in clinical diagnosis and can be extended for the detection of other biomarkers.

Conclusions
Spin-dependent Fe single-atom nanozymes have been developed to achieve enhanced POD-like activity. Experimental and theoretical calculations reveal that the electron-withdrawing effect of Pd NC induces the LS to MS transition of the Fe single atom with the increased electron occupation in the d z 2 orbital. The charge transfer and orbital interactions between the Fe sites and intermediates display spin-dependent features, where the higher electron occupation in the d z 2 is benecial for the generation of the essential Fe(IV)]O intermediates and promotes the timely desorption of H 2 O*. Thanks to the superior catalytic activity, FeNC-Pd NC can be used to construct NLISA for sensitive detection of PSA, exhibiting satisfactory selectivity and sensitivity. This spin-state engineering strategy for the design of nanozymes not only provides a fundamental understanding of their catalytic mechanism but also promotes the design and development of enzyme-like catalysts for the future.

Data availability
The data supporting the ndings of this study are available within the article and in the ESI. †

Author contributions
C. Z. and Y. Y. conceptualized and supervised this study. X. W. designed the experiments and wrote the paper. S. S. and W. S. performed the theoretical calculations. Y. W., W. X., Y. C., Z. W.,

Conflicts of interest
There are no conicts of interest to declare.