Oxygen-bridged W-Pd atomic pairs enable H₂O₂ activation for sensitive immunoassays

Abstract

Regulating the performance of peroxidase (POD)-like nanozymes is a prerequisite for achieving highly sensitive and accurate immunoassays. Inspired by natural enzyme catalysis, we design a highly active and selective nanozyme by loading atomically dispersed tungsten (W) sites on Pd metallene (W-O-Pdene) to construct an artificial three-dimensional (3D) catalytic center. The 3D asymmetric W-O-Pd atomic pairs can effectively stretch the O–O bonds in H₂O₂ and further promote the desorption of H₂O to enhance POD-like activity. Moreover, the W-O-Pd sites with unique spatial structures demonstrate satisfactory specificity for H₂O₂ activation, effectively preventing the interference of dissolved oxygen. Accordingly, the highly active and specific W-O-Pdene nanozymes are utilized for sensitive and accurate prostate-specific antigen (PSA) immunoassay with a low detection limit of 1.92 pg mL⁻¹, superior to commercial enzyme-linked immunosorbent assay.

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Introduction

Nanozymes with POD-like activity have become effective substitutes for natural POD and have been extensively investigated for colorimetric immunoassay.^1–6 Understanding and regulating the H₂O₂ activation of POD-like nanozymes plays a key role in improving the sensitivity and accuracy of immunoassay.^7–10 Although POD-like nanozymes have witnessed enormous progress in enhancing catalytic activity, limited studies have demonstrated improvement in catalytic specificity.^11,12 Although several efforts have been devoted to addressing this issue,^13–15 the rational design and controllable preparation of specific POD-like nanozymes are still impeded by an incomplete understanding of the reaction mechanism.^16,17 In particular, due to the structural complexity of nanozymes, the selective cleavage of the O–O bond in H₂O₂ activation is still ambiguous.^18–21

Horseradish peroxidases (HRP) are state-of-the-art biocatalysts and their catalytic pockets with unique electronic and geometrical structures significantly facilitate H₂O₂ activation.^22,23 In their three-dimensional catalytic pockets, the synergistic effects between catalytic sites and binding sites can accelerate the proton-coupled electron transfer of selective H₂O₂ activation.^24–26 The strong affinity between the H₂O₂ molecule and enzyme gives HRP excellent catalytic selectivity.²⁷ Hence, to significantly improve the catalytic activity and selectivity of nanozymes for H₂O₂ activation, rational design of the active sites of nanozymes requires vivid mimicking of the catalytic pocket of HRP. Not only the binding and catalysis sites but also the spatial configuration of the active sites should be designed.

Herein, W-O-Pdene nanozymes with artificial 3D catalytic centers (asymmetric W-O-Pd sites) are synthesized successfully as highly efficient POD mimics. Asymmetric W-O-Pd sites can provide the catalytic and binding sites for H₂O₂ activation, which not only improve the H₂O₂ adsorption but also reduce the energy barrier of H₂O desorption, thus efficiently achieving the improvement of H₂O₂ activation efficiency. Besides, owing to the unique spatial configuration, W-O-Pd sites can stretch the O–O bond of H₂O₂ and compress the O–O bond of O₂, resulting in selectively enhanced POD-like activity, effectively preventing the interference of dissolved oxygen. Accordingly, W-O-Pdene nanozymes display more than 6 times higher POD-like activity and better affinity for H₂O₂ than pure Pd metallene (Pdene) nanozymes. Finally, W-O-Pdene nanozymes are used in the immunoassay of the PSA, demonstrating an ultra-low limit (1.92 pg mL⁻¹) and practical feasibility in clinical diagnosis.

Results and discussion

Fig. 1a exhibits a schematic diagram of the fabrication procedure of Pdene and W-O-Pdene nanozymes. The homogeneous reaction mixture, consisting of W(CO)₆, ascorbic acid, and Pd(acac)₂, was heated at 80 °C for one hour to obtain Pdene. In the process, the temperature-induced decomposition of W(CO)₆ led to the emission of carbon monoxide (CO), which acted as a ligand directing the atomic arrangement. The CO ligand exhibits a preference for binding to the low-index (111) facet of Pd, thereby facilitating anisotropic growth. W-O-Pdene with oxygen-bridged W-Pd atomic pairs is obtained after heating for 12 h. Transmission electron microscopy (TEM) and high-angle annular dark-field scanning TEM (HAADF-STEM) images provide evidence that the Pdene and W-O-Pdene exhibit a crosslinked curved 2D nanostructure (Fig. 1b and S1a, b,†). Moreover, the atomic force microscopy (AFM) images of Pdene and W-O-Pdene indicate an average thickness of only 1.2 nm and 3.4 nm, respectively (Fig. 1c and S2†). As described, W-O-Pdene possesses ultra-thin characteristics that can provide more active sites. The X-ray diffraction (XRD) pattern of W-O-Pdene indicates good crystallinity, which is consistent with the selected area electron diffraction (SAED) result (Fig. 1d and S3†). The diffraction peaks at 39.6, 45.8, and 67.3 are indexed to the 111, 200, and 220 planes of the fcc structure of W-O-Pdene. Aberration-corrected HAADF-STEM (AC-HAADF-STEM) characterization was performed. As illustrated in Fig. 1e and f, the heavy element W appears as dense bright contrast dots within the red circles. W-O-Pdene exhibits a lattice spacing of 0.235 nm, consistent with the high-resolution TEM images of Pdene and W-O-Pdene (Fig. S4†). Besides, the energy-dispersive X-ray spectroscopy elemental mapping demonstrates the homogenous distribution of Pd and W elements within the W-O-Pdene (Fig. 1g).

Fig. 1 (a) Scheme of the preparation of Pdene and W-O-Pdene. (b) HAADF-STEM image of W-O-Pdene. (c) AFM image of W-O-Pdene. (d) XRD spectra of W-O-Pdene and Pdene. (e) AC-HAADF-STEM image of W-O-Pdene. (f) 3d intensity profile of W-O-Pdene. (g) Elemental mapping of W-O-Pdene.

The chemical state of W-O-Pdene and Pdene was investigated by X-ray photoelectron spectroscopy (XPS). The location of the Pd⁰ peaks of W-O-Pdene in the Pd 3d spectra at 335.7 eV and 341.0 eV suggests a negative shift in comparison to Pdene (335.9 eV and 341.2 eV), indicating a clear electronic interaction (Fig. 2a). As displayed in Fig. 2b, two weak peaks at 37.7 eV and 35.5 eV can be attributed to W 4f_5/2 and W 4f_7/2 in W-O-Pdene. Notably, most of the W exists in the W⁶⁺ state, and no discernible peaks attributed to W⁰ are observed. X-ray absorption near-edge structure (XANES) and extended X-ray absorption fine structure (EXAFS) analyses were utilized to investigate the electronic structure and coordination environment in W-O-Pdene. The white line intensity of W-O-Pdene is between that of W-foil and WO₃, indicating that the valence state of W is between 0 and 6 (Fig. 2c). Meanwhile, the Fourier transform W L-edge EXAFS (FT-EXAFS) spectra of W-O-Pdene reveal a single peak centered at 1.2 Å, corresponding to the W–O scattering path (Fig. 2d). In particular, the absence of the W–W peak in the FT-EXAFS spectra confirms the atomic distribution of W. Fig. S5† presents the EXAFS fitting curves, and Table S1† provides the fitting results of W-O-Pdene. The coordination number of the W–O shell is about 3. Furthermore, wavelet transform (WT) analysis was employed (Fig. 2e). The maximum intensity of the W-foil is about 13.6 Å⁻¹, which corresponds to the W–W bond. The maximum intensity of W-O-Pdene is about 8.1 Å⁻¹, which is like that of WO₃ and corresponds to the W–O bond. Based on these results, the geometric configuration is optimized through first-principles calculations (Fig. S6†). In the most stable configuration of W-O-Pdene, W atoms are anchored on Pdene with three oxygen bridges. As displayed in Fig. 2f, the W-O-Pdene nanozyme possesses a spatially stable 3D structure. W-O-Pd asymmetric sites are successfully constructed as artificial 3D catalytic centers, and the W atom provides binding sites and catalytic sites for H₂O₂ activation. Like the HRP catalytic pocket, the artificial 3D catalytic centers possess unique spatial and electronic structures and can potentially enhance nanozyme activity and specificity.^28–31

Fig. 2 XPS spectra of W-O-Pdene and Pdene for Pd 3d (a) and W 4f (b). (c) W L-edge XANES spectra of WO₃, W-O-Pdene, and W foil. (d) FT-EXAFS spectra of WO₃, W-O-Pdene, and W foil. (e) WT images of WO₃, W-O-Pdene, and W-foil. (f) W-O-Pdene and the HRP structural model diagram.

The typically chromogenic reaction (TMB/H₂O₂) was used to verify the POD-like activity of W-O-Pdene and Pdene. As displayed in Fig. 3a, the W-O-Pdene can rapidly oxidize TMB and produce an obvious absorbance change at 652 nm with the assistance of H₂O₂, and the absorbance of the W-O-Pdene system is higher than that of the Pdene system. Without nanozymes, the TMB/H₂O₂ system cannot trigger the absorbance changes. As shown in Fig. S7,† the Michaelis–Menten equation verified the kinetic analysis of the POD-like activity of different nanozymes at different concentrations of H₂O₂ and TMB, respectively. Table S2† displays the kinetic constants (V_max and K_m). On the one hand, W-O-Pdene nanozymes possess higher V_max for both H₂O₂ and TMB than Pdene nanozymes, showing their higher catalytic activity; on the other hand, W-O-Pdene nanozymes have lower K_m value than Pdene, which indicates that W-O-Pdene has a better affinity to H₂O₂. The specific activity (SA) is defined as activity units per milligram of nanozymes which can quantitatively evaluate the POD-like activity.³² As shown in Fig. 3b, the SA of W-O-Pdene (256 U mg⁻¹) increases more than 6 times compared with Pdene (40 U mg⁻¹) and is higher than that of HRP (208 U mg⁻¹), which means that W-O-Pdene has better H₂O₂ catalytic activity than Pdene and HRP. As shown in Table S3,† compared to noble metal nanoparticle nanozymes, W-O-Pdene nanozymes have the best POD-like activity. The activation energy (E_a) of POD-like activity was calculated based on the Arrhenius equation to reveal the catalytic mechanism.^33–35 As displayed in Fig. 3c, W-O-Pdene nanozymes (0.68 KJ mol⁻¹) have a lower E_a value for POD-like activity than that of Pdene nanozymes (1.67 KJ mol⁻¹), indicating that W-O-Pdene nanozymes can better express POD-like activity. Like natural HRP, W-O-Pdene nanozymes display pH-dependent characteristics (Fig. S8a†). Compared with natural HRP, W-O-Pdene nanozymes possess high-temperature tolerance (Fig. S8b†). Meanwhile, W-O-Pdene has better recyclability than HRP; after five cycles the POD-like activity remained unchanged (Fig. S8c†). XRD (Fig. S9a†) and TEM (Fig. S9b†) of W-O-Pdene after the reaction have not changed, indicating that W-O-Pdene has excellent stability.

Fig. 3 (a) Absorbance spectra of the H₂O₂-TMB system catalyzed by W-O-Pdene and Pdene. (b) The specific activity of W-O-Pdene, Pdene, and HRP. (c) E_a of W-O-Pdene and Pdene in H₂O₂ activation. (d) POD-like and OXD-like activity of W-O-Pdene and Pdene. (e) EPR spectra of W-O-Pdene and Pdene. (f) In situ ATR-FTIR spectra of the H₂O₂ catalytic reaction on W-O-Pdene. (g) Schematic of W-O-Pdene for H₂O₂ and O₂ activation.

In addition to POD-like activity, nanozymes usually have oxidase (OXD)-like activity which can catalyze O₂ and oxidize chromogenic substrates, seriously affecting the accuracy of immunoassay. Herein, the OXD-like and POD-like activities of W-O-Pdene nanozymes and Pdene nanozymes are investigated (Fig. 3d). Impressively, compared with Pdene, W-O-Pdene nanozymes possess lower OXD-like and higher POD-like activity, demonstrating that W-O-Pdene nanozymes can enhance POD-like activity and decrease OXD-like activity. As shown in Fig. S10,† the Michaelis–Menten equation verifies the kinetic analysis of the OXD-like activity of the W-O-Pdene nanozymes and Pdene nanozymes at different concentrations of TMB. W-O-Pdene nanozymes have lower V_max and higher K_m than that of Pdene nanozymes, indicating that Pdene nanozymes have better O₂ catalytic activity (Table S4†). Electrochemical tests were performed to further verify the specificity of the nanozymes, using nanozyme-modified glassy carbon electrodes to evaluate the catalytic activity of H₂O₂ and O₂. As displayed in Fig. S11a,† W-O-Pdene nanozymes demonstrate a larger H₂O₂ reduction current than that of Pdene nanozymes, indicating that W-O-Pdene nanozymes have outstanding H₂O₂ reduction activity. In contrast, Fig. S11b† indicates that W-O-Pdene nanozymes have excellent specificity for H₂O₂ activation. These results convincingly demonstrate that W-O-Pdene nanozymes possess excellent specificity for H₂O₂ activation. Next, the active intermediates of the POD-like reaction system were confirmed by using isopropanol (IPA) as the ·OH scavenger. As shown in Fig. S12a,† when IPA is added to the POD-like catalytic system, a significant decrease in the absorbance value emerges, indicating that the produced ·OH is one of the active intermediates. Moreover, terephthalic acid (TA) can be used as a typical fluorescent probe to further detect the resulting ·OH. As shown in Fig. S12b and c,† the Fenton reaction system can react with TA and generate an obvious fluorescence signal, indicating that TA can be used as an ·OH fluorescent probe. Similarly, both Pdene and W-O-Pdene reaction systems can also react with TA to generate significant fluorescence signals, which can determine the existence of ·OH intermediates. To obtain deeper insights into the catalytic process, electron paramagnetic resonance (EPR) tests were conducted. 5,5-dimethyl-1-pyrroline N-oxide (DMPO) is used as the spin trap of ·OH. As displayed in Fig. 3e, the higher EPR signal of the W-O-Pdene catalytic system is observed, indicating that more ·OH is produced than that of the Pdene. To examine the other reactive oxygen species, nitro blue tetrazolium chloride (NBT) and Dimethyl sulfoxide (DMSO) were used to detect ·O₂⁻and absorbed oxygen (*O) respectively. NBT can react with ·O₂⁻ to produce blue absorption at about 560 nm. Fig. S13a† indicates that there is no ·O₂⁻ in the catalytic system. DMSO can scavenge *O and ·OH, and tert-butanol (TBA) can only scavenge ·OH. As displayed in Fig. S13b,† ·OH is the main reactive intermediate in the Pdene nanozyme and W-O-Pdene nanozyme catalytic systems. Then, in situ ATR-FTIR spectroscopy was employed to monitor the process of H₂O₂ activation. After the addition of H₂O₂, two signals at 1580 cm⁻¹ and 1339 cm⁻¹ can be detected respectively (Fig. 3f), which can be assigned to the H–O–H bending mode and O–H of absorbed H₂O_2.^36,37 Besides, peaks at 1278 cm⁻¹ can be assigned to the stretching vibration of O–O in H₂O₂, which gradually decreased because of the partial dissociation of H₂O₂.³⁸ These results indicate that the O–O bond in H₂O₂ can be stretched in the catalytic process (Fig. 3g).

To reveal the internal mechanism of artificial 3D catalytic centers to enhance the activity and specificity of nanozymes, density functional theory (DFT) calculations were performed. At first, the impacts of active sites are evaluated by calculating the O–O bond distance of H₂O₂ and O₂ adsorbed on Pdene and W-O-Pdene (Fig. 4a). The dual active sites (W-O-Pd) of W-O-Pdene nanozymes can alter the adsorption conformation of H₂O₂ and thus achieve the extension of the O–O bond, which facilitates the activation of H₂O₂. Impressively, when O₂ is adsorbed on the W-O-Pd site, the O–O bond of O₂ is compressed and thus inhibits the O₂ activation. Moreover, the analysis of charge density difference (Fig. 4b) reveals the significant electronic interaction in W-O-Pdene. Then, the projected density of states (PDOS) of the Pd d-band is employed further to indicate the electronic interaction effects (Fig. 4c). The d-band center of W-O-Pdene (−1.768 eV) exhibits a downshift relative to that of Pdene (−1.465 eV), indicating that the electronic interaction of W-O-Pd can optimize the electronic structure of W-O-Pdene nanozymes and accelerate the desorption process of intermediates. Furthermore, Fig. 4d displays the schematic representation of the fundamental reaction step and energy change diagram of H₂O₂ activation on nanozymes. H₂O₂ is first absorbed on the W-O-Pd active sites. Then H₂O₂ is cleaved into two absorbed hydroxyl groups (OH*). Next, the two absorbed OH* are protonated to constitute H₂O* and desorb subsequently. W-O-Pdene nanozymes have a better capacity for capturing H₂O₂ because the adsorption energy of W-O-Pdene (−1.53 eV) is lower than that of Pdene (−1.03 eV). The step of desorption of the H₂O can be regarded as the rate-determining step (RDS). The RDS energy barrier of W-O-Pdene (0.82 eV) is lower than that of Pdene (1.19 eV), explaining the superiority of the POD-like activity of W-O-Pdene nanozymes. This result is consistent with the PDOS analysis. Besides, the weakened H₂O* binding on W-O-Pdene can be further evidenced by crystal orbital Hamilton population (COHP) analysis (Fig. 4e); pCOHP analysis reveals that the -ICOHP value at the Fermi level is lower for W-O-Pdene (2.434 eV) than that of Pdene (2.916 eV), which means that the H₂O* is easier to desorb on W-O-Pdene. Accordingly, W-O-Pdene nanozymes have high activity and specificity for the catalysis of H₂O₂, which is due to the synergistic effect of Pd sites and axial W atoms in the artificial 3D catalytic centers. On the one hand, the axial W atoms play an electronic role in the regulation of the Pd sites, resulting in a better desorption to enhance POD. On the other hand, the axial W atoms play an important role in binding for catalysis of H₂O₂, superior spatial configuration improving the specificity (Fig. S14†).

Fig. 4 (a) Distance of O–O in H₂O₂ and O₂ on Pdene and W-O-Pdene, respectively. (b) The charge density difference of W-O-Pdene (yellow: charge accumulation; cyan: charge depletion). (c) PDOS of the Pd d-band for W-O-Pdene and Pdene. (d) Schematic representation of the fundamental reaction step. Energy change diagram of H₂O₂ activation on W-O-Pdene and Pdene. (e) COHP of H₂O* on Pdene and W-O-Pdene.

As the specific marker for prostate cancer, PSA has been widely used for the early diagnosis of prostate cancer during routine check-ups.^39–42 To investigate the potential application of W-O-Pdene for immunoassays, a colorimetric nanozyme-linked immunosorbent assay (NLISA) of PSA was performed.^43–46 As displayed in Fig. 5a, NLISA is implemented through the typical sandwich immunoassay; anti-PSA coating monoclonal antibody (Ab₁) can capture the target PSA, subsequently, PSA is captured by anti-PSA labeling monoclonal antibody (Ab₂) to form the sandwich immunocomplexes. As displayed in Fig. 5b and c, with the increase of PSA concentration, the associated absorbance signals are consequently increased, demonstrating the feasibility of NILISA for the detection of PSA. The interference of dissolved oxygen with the background signal was investigated. As displayed in Fig. 5d, HRP has low background signals because of the superior specificity. Compared with Pdene nanozymes, W-O-Pdene nanozymes can decrease the background signal of immunoassay. Fig. 5e displays the limit of detection (LOD) of NLISA and ELISA. Interestingly, the LOD of W-O-Pdene-involved NLISA is determined to be 1.92 pg mL⁻¹, and the LOD of HRP-involved ELISA and Pdene-involved NLISA is calculated to be 5.88 and 38.02 pg mL⁻¹, respectively. The enhancement in the detection sensitivity for W-O-Pdene NLISA can be ascribed to the high catalytic efficiency and specificity of the W-O-Pdene. Furthermore, the specificity of PSA detection is demonstrated by the negligible responses observed for other biomarkers such as C-reactive protein (CRP), carcinoembryonic antigen (CEA), glucose oxidase (GOX), and bovine serum albumin (BSA) at a concentration of 2 ng mL⁻¹ (Fig. 5f). Due to the superior sensing performance of NLISA, it is hopeful to be used in clinical diagnosis. Therefore, to verify the practicability in clinical diagnosis, NLISA was employed to detect human serum samples. As displayed in Fig. 5g and Table S5,† NLISA gets a linear fit greater than 99% with the chemiluminescence (CL) method in the hospital, verifying the practicability of NLISA in practical applications.

Fig. 5 (a) Schematic illustration of the colorimetric immunoassay of PSA. (b) Calibration curves of proposed W-O-Pdene-involved immunoassay and HRP-involved immunoassay. Linear ranges of the detection results from NLISA (C). (d) The background signals are caused by OXD-like activity. (e) LOD of different nanozyme-involved immunoassays. (f) Selectivity of W-O-Pdene-involved immunoassay. (g) The correlation curve of the NLISA method and the CL method of the real sample detection of PSA.

Conclusions

Inspired by the natural HRP catalytic pocket, W-O-Pdene nanozymes with artificial 3D catalytic centers are prepared successfully and demonstrate specific H₂O₂ activation efficiency. The catalytic activity and selectivity of W-O-Pdene nanozymes are systematically investigated based on experimental and theoretical analysis. 3D asymmetric W-O-Pd atomic pairs can selectively regulate the O–O bonding in H₂O₂ and O₂, and thus achieve the selective H₂O₂ activation. Besides, the asymmetric W-O-Pd atomic pairs can regulate the d-band center to accelerate the desorption of H₂O, which improves the catalytic activity of W-O-Pdene nanozymes. As a typical application, a colorimetric immunoassay for PSA is established, demonstrating better sensitivity than an enzyme-linked immunosorbent assay. This work offers new insight into fabricating unique POD-like nanozymes with high activity and selectivity.

Data availability

The data supporting this article have been included as part of the ESI.†

Author contributions

Conceptualization: L. J.; formal analysis: C. C., D. Y., X. J., R. L., L. H., and X. L.; funding acquisition: X. L., L. J., and Y. Z.; methodology: C. C. and L. J.; writing – original draft: C. C.; writing – review & editing: L. J., Y. Z., C. Z., and X. L. All authors discussed the results and assisted during manuscript preparation.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

The authors gratefully acknowledge the financial support of the National Natural Science Foundation of China (22174110, 22127803, and 22304096), the project ZR2023QB006 supported by Shandong Provincial Natural Science Foundation, supported by the China Postdoctoral Science Foundation under Grant Number 2023M741853, the Qingdao Postdoctoral Science Foundation (QDBSH20230202031), and the Plan for Youth Innovation Team of Colleges in Shandong Province.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4sc04711g

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