Weiqing Xu,Hong Zhong,Yu Wu,Ying Qin,Lei Jiao,Meng Sha,Rina Su,Yinjun Tang,Lirong Zheng,Liuyong Hu,Shipeng Zhang,Scott P. Beckman,Wenling Gu,Yong Yang,Shaojun Guo,Chengzhou Zhu
Photoexcited Ru single-atomic sites for efficient biomimetic redox catalysis
electron transfer,photoenzymes,metal–organic frameworks,nanozymes,single-atom catalysis
Significance Photoinvolved redox enzymes driven by the photoinduced electron transfer (ET) can realize efficient energy conversion of green and sustainable solar energy to chemical energy. Inspired by this, nanozymes coupling with photoexcited electrons to perform the ET process are expected to serve as alternatives to enzymes and boost the inherent catalytic performance for wide application scenarios. To this end, we rationally design the metal–organic framework-supported Ru single-atom photonanozymes with excellent enzyme-like activity, photoelectronic conversion efficiency, and good catalytic specificity. The resultant photonanozymes follow an enzyme catalytic mechanism and exhibit photoenhanced peroxidase-like activity. This work not only expands the function and application scope of light-driven catalysis but also represents great opportunities for a research direction to design advanced photonanozymes.
Photoexcited Ru single-atomic sites for efficient biomimetic redox catalysis

Photoinvolved redox enzymes driven by the photoinduced electron transfer (ET) can realize efficient energy conversion of green and sustainable solar energy to chemical energy. Inspired by this, nanozymes coupling with photoexcited electrons to perform the ET process are expected to serve as alternatives to enzymes and boost the inherent catalytic performance for wide application scenarios. To this end, we rationally design the metal–organic framework-supported Ru single-atom photonanozymes with excellent enzyme-like activity, photoelectronic conversion efficiency, and good catalytic specificity. The resultant photonanozymes follow an enzyme catalytic mechanism and exhibit photoenhanced peroxidase-like activity. This work not only expands the function and application scope of light-driven catalysis but also represents great opportunities for a research direction to design advanced photonanozymes.

Redox enzymes, featuring excellent catalytic ability and specificity, underpin and assist numerous biological energy transduction, which are increasingly adopted in prominent chemical synthesis and biotechnology (1, 2). Their catalysis relies on various side chain groups that induce the polarization and deformation of valence bonds of substrates by electron transfer (ET) process, exciting the substrate to reduce the activation energy. To this end, many biological strategies have been employed to improve ET efficiency and further boost the inherent performance of enzymes (3–5). Among them, efforts have drawn great attention to endow native redox enzymes with photoresponsive ability for mimicking the photoenzymes catalysis (6, 7). Note that photoinduced electrons can not only accelerate cofactor-mediated ET but also hold sufficient energy to powerfully promote the enzymatic molecules to an excited state for efficient chemical transformations (3, 8, 9). Despite effectiveness, these modification methods are complex in operation and depend much on the known information of enzymatic structure and mechanism (10, 11). Moreover, the poor compatibility of extraneous moieties is harmful to enzymes and results in low photoelectron utilization, which poses a huge obstacle on the road to applications (12, 13). In virtue of the merits of nanomaterials, the development of photoresponsive nanozymes (photonanozymes) to mimic the catalytic property of enzymes is expected to represent a great opportunity for a research direction for photoenhanced enzyme-like activity. The rational design of advanced photonanozymes, including highly active catalytic sites and efficient photoresponsive units, requires guidance from the biological structure of natural enzymes. Recently, some nanozymes have been proposed to couple with photosensitizers to generate reactive oxygen species (ROS), achieving boosted catalytic capability (14–18). Note that ROS with tremendous activity can efficiently activate various substrates without preference in these systems, which is bound to induce many side reactions and runs counter to the high selectivity pursued by enzymatic catalysis. Furthermore, the effect of the extra energy on the intrinsic enzyme-like activity of nanozymes has not been intensively expounded. Whether the excited electrons can follow the ET process of photoenzymes to activate the nanozymes? Therefore, the construction of advanced photonanozymes to vividly mimic the substrate activation of enzymes and in situ monitoring of the catalytic reaction to uncover the deep mechanism is in urgent need. Herein, we report a single-atom Ru-anchored UiO-67 photonanozyme featuring peroxidase (POD)-like property and photoelectric effect. The atomically dispersed Ru sites can achieve high photoelectric conversion efficiency, superior POD-like activity, and good catalytic specificity. The in situ experiments and theoretical calculations reveal that different from the traditional ROS-involved enhancement mechanism, electrons excited by light energy can directly transfer to Ru active sites, which follows the ET process of substrate activation in natural POD. The photoexcited UiO-67–Ru is conducive to the generation of active intermediate and H2O release, resulting in more favorable thermodynamics and kinetics in H2O2 reduction, leading to superior POD-like activity. In virtue of the strong interaction of the Zr–O–P bond, we construct a UiO-67–Ru photonanozyme-based photoenhanced immunoassay platform for the sensitive detection of organophosphorus pesticides (OPs).

Single-atom Ru was chelated in organic linkers (2,2′-bipyridine-5,5′-dicarboxylic acid, H2BPYDC) to mimic the structure of the classic photosensitizer [Ru(bpy)3]2+ for the preparation of the UiO-67–Ru via a simple postsynthetic strategy (SI Appendix, Fig. S1) (19). Powder X-ray diffraction patterns and transmission electron microscopy (TEM) images confirm the retention of UiO-type crystal structure and morphology as for the obtained UiO-67–Ru and no observable nanoparticle species (Fig. 1A and SI Appendix, Figs. S2–S4). The high-angle annular dark-field imaging (HAADF)-scanning TEM (STEM) and energy-dispersive X-ray spectroscopy (EDS) elemental mappings demonstrate the uniform distribution of each element throughout UiO-67–Ru (Fig. 1B). Aberration-corrected HADDF-STEM image shows atomically dispersed bright dots (marked by red circles), which can be assigned to Ru single atoms (Fig. 1C). The intensity profile of gray levels displays different peak heights and relatively higher peaks correspond to single Ru atoms (Inset in Fig. 1C). Thermogravimetric analysis shows that the weight loss of UiO-67–Ru is lower than that of UiO-67 due to the presence of Ru species (SI Appendix, Fig. S5). The loading amount of Ru was measured to be 1.5 wt% by the inductively coupled plasma-optical emission spectrometry. The analysis of N2 isotherms shows that the introduction of Ru affords a reduced surface area and pore volume, indicating the successful incorporation of Ru within the pores of MOFs (SI Appendix, Fig. S6). Fourier transform infrared (FTIR) spectroscopy was carried out to uncover the interaction between Ru and UiO-67. After introducing Ru, the C=N stretching vibration of the pyridine ring (1,656 cm−1) in UiO-67 is weakened (Fig. 1D). This phenomenon is closely associated with the fact that the formation of the Ru–N bond induces the ET of Ru to N, weakening the C=N bond in BPYDC (20). In contrast, the characteristic peaks of the Zr–O bond (770, 654, 460 cm−1) are maintained after Ru modification. X-ray photoelectron spectroscopy (XPS) survey and X-ray absorption near-edge structure (XANES) were conducted to study the chemical environment of MOFs. Different from those of UiO-67, the extra Ru 3d (281.8 eV) and Ru 3p (462.7 and 484.8 eV) regions appear in the XPS spectra of UiO-67–Ru (SI Appendix, Fig. S7). Further analysis of the Ru 3p region reveals that the Ru species are multivalent (+3 and +4) (SI Appendix, Fig. S8) (21, 22). The N 1s peak of UiO-67–Ru shows a negative shift and appears additional Ru–N (400.1 eV) bond relative to the pristine UiO-67 (SI Appendix, Fig. S9). It suggests that the Ru ions sit on the highly electronegative pyridine nitrogen of the organic linker, resulting in the electron-deficient state of Ru sites. Different from N, the Zr 3p and O 1s XPS peaks of UiO-67–Ru present no evident change, which may exclude the presence of coordination between the Zr–O cluster and Ru (SI Appendix, Fig. S10). XANES analysis of Ru K-edge shows that the near edge of UiO-67–Ru locates between the RuCl3 and RuO2 (Fig. 1E). Fitting results reveal that the oxidization state of UiO-67–Ru is 3.42 (SI Appendix, Fig. S11), being consistent with the XPS result. The extended X-ray absorption fine structure (EXAFS) oscillation of UiO-67–Ru displays a major peak at 1.49 Å (Fig. 1F), which matches well with the Ru–O(N) peak for the RuO2 (23). No observable peak of the Ru–Ru or Ru–O–M was found, further verifying that Ru sites are atomically dispersed without Ru species anchored on the Zr–O cluster. Taken together, it can be concluded that the Ru atom coordinated with the N of the BPYDC linker to form atomically dispersed Ru–N sites.

The UV–vis diffuse reflectance spectra reveal that the UiO-67–Ru possesses an additional absorption peak at 500 nm in comparison with UiO-67 (SI Appendix, Fig. S12), verifying the similar coordination environment of Ru site to the absorption of [Ru(bpy)3]2+ (24). The steady-state photoluminescence (PL) emission spectra show that the PL intensity of UiO-67–Ru is lower than that of UiO-67, implying the enhanced electron-hole separation efficiency (SI Appendix, Fig. S13). The obtained UiO-67–Ru exhibits a 3.2-fold stronger photoinduced ET than that of UiO-67 (SI Appendix, Fig. S14). To unveil the effect of Ru single atom on the ET process, in situ electron paramagnetic resonance (EPR) measurements were conducted. Upon the excitation of light, the signal intensity of single electrons (g = 2.00) in UiO-67 slightly enhances, which might originate from the formation of ZrIII upon accepting electrons from the organic linker (SI Appendix, Fig. S15). As for UiO-67–Ru, several new peaks at g = 2.32, 2.05, and 1.88 appear, which are within the range for RuIII species reported previously (Fig. 2A) (23). Upon illumination, the reduced signal intensities reveal the photoinduced ET to Ru sites to form diamagnetic RuII (4d6, S = 0) without EPR response (25). In situ XPS spectra show that the Ru 3p peak presents an obvious negative shift under illumination (Fig. 2B). The lower binding energy of UiO-67–Ru–Light than that of RuCl3 explains the lower valence of Ru, which is in line with the result of EPR (SI Appendix, Fig. S16). Similarly, the Zr 3p and O 1s peaks present a negative shift (SI Appendix, Fig. S17). In contrast, the peak of N 1s shows a positive shift (SI Appendix, Fig. S18). It should be stated that the photosensitive unit organic linker is excited to generate electrons that transfer to the Zr–O cluster, which follows the linker-to-cluster charge transfer (LCCT) process (26). Significantly, the introduction of the single Ru atom induces an additional linker-to-metal charge transfer (LMCT) process. Natural photoenzymes, such as DNA photolyases, possess antenna molecules (e.g., reduced flavin cofactor, FADH). It can absorb light energy and produce photoelectrons, which subsequently transfer to the DNA dimer, achieving the depolymerization of dimers. Mimicking photoenzymes, photosensitizer-modified POD presents an accelerated cofactor-driven ET process, significantly improving the catalytic efficiency (Fig. 2C). Inspired by these, the effect of photoelectron transfer on POD-like activities of UiO-67–Ru was investigated by a typical 3,3′,5,5′-tetramethylbenzidine (TMB)-involved colorimetric reaction (27). As shown in Fig. 2D and SI Appendix, Fig. S19, UiO-67–Ru presents superior catalytic performance to UiO-67 in dark. To systematically compare the catalytic ability, a series of kinetics experiments were conducted to obtain the catalytic constants based on the Michaelis–Menten curve. As a result, the maximum rate (Vmax) of UiO-67–Ru was calculated as 0.11 μM s−1 for H2O2 and 0.59 μM s−1 for TMB, 6.8 times and 59.0 times higher than those of UiO-67, respectively (SI Appendix, Fig. S20). The large difference in catalytic activity between two nanozymes implies that the additional single Ru sites play a key role in substrate activation. Importantly, the POD-like activities of both MOFs are improved with the help of additional photoexcitation (λ > 300 nm) (Fig. 2D and SI Appendix, Fig. S21). The catalytic activity of UiO-67–Ru is 7.0 times that of the UiO-67 under optimized conditions (SI Appendix, Fig S22), while the native horseradish peroxidase (HRP) shows no observable photoenhanced catalytic activity, which is closely related to the lack of photosensitive units (SI Appendix, Fig. S23). The dynamic experiments suggest that photoexcitation can accelerate the reaction rate of UiO-67–Ru and improve the maximum rate (Fig. 2E and SI Appendix, Fig. S24). Besides, the obtained photonanozymes show superior POD-like activity to some nanozymes reported previously (SI Appendix, Table S1). Then, given that the dissolved oxygen is promising to possess oxidative ability, the oxidase-like property of MOFs was evaluated. As illustrated in SI Appendix, Fig. S25, neither UiO-67–Ru nor UiO-67 can oxidize TMB with and without additional light excitation. This result not only reflects the good catalytic specificity of UiO-67–Ru but also rules out the existence of some latent side reactions induced by irradiation. It should be pointed out that the obtained photonanozymes exhibit good stability and reusability, circumventing the limits of natural enzymes in practice (SI Appendix, Fig. S26).

Based on the above results, the introduction of Ru is vital to the outstanding catalytic property of UiO-67–Ru photonanozymes. It is believed that the single-atom Ru not only serves as the additional active site but also promotes photoelectron transfer ability for boosting the substrate activation (Fig. 3A). To verify this assumption, a series of experiments were carried out to monitor the intermediates during the reaction. First, EPR spectra reveal that neither MOF catalytic systems produce extra hydroxyl radicals (•OH) and superoxide radicals (O2•-), in contrast with the control group (Fig. 3B and SI Appendix, Figs. S27 and S28). It suggests that the •OH and O2•- are not the main active intermediates in both dark and light conditions, which excludes the traditional ROS-enhanced mechanism. In nature, HRP pursues the heterolytic pathway for the circumvention of the generation of toxic-free radicals, and the high-valent Fe(IV)=O intermediate acts as the key active transient state (28). In this regard, in situ attenuated total reflection-FTIR (ATR-FTIR) experiment was conducted. As shown in Fig. 3C, the peak intensities of the characteristic band at 3,360, 1,093, and 855 cm−1 gradually increase over time and then reach equilibrium. According to the previous reports, these peaks can be assigned to the adsorbed hydroxyl (*OH), *O–O, and *H2O2 species on the Zr site, respectively (29–31). As for UiO-67–Ru, the three peaks appear at a higher wavenumber relative to that of UiO-67, which can be ascribed to the fact that H2O2 prefers to be absorbed on the additional Ru site for the generation of Ru–OO* (1,107 cm−1) (32). In situ EPR analysis shows three unique signals g = 2.02, 2.00, and 1.99 (Fig. 3D), which are assigned to the absorbed superoxide ions on the surface ZrIV sites (ZrIV–OO) (33). As for UiO-67–Ru, apart from the characteristic peak of the ZrIV–OO species, the extra signals g= 2.09, 2.06, and 1.92 corresponding to Ru=O intermediate appear (34). Therefore, it is demonstrated that the catalytic process of UiO-67–Ru involves additional adsorption of the Ru site for H2O2, and it can be activated to generate highly active Ru=O intermediates. After illumination, the signal intensities of the ZrIV–OO species in both systems decrease and the varying intensity of UiO-67–Ru is significantly higher than that of the UiO-67 system. It can be attributed to the improved photoelectric effects by the single Ru site, where excessive photoinduced electrons transfer to Zr and increase the content of ZrIII, reducing the signal output of the ZrIV–OO species. In virtue of the enriched electron density of Ru evidenced by in situ XPS, the increased signal intensities of Ru=O species suggest that the photoelectrons could fill the unoccupied d-orbitals of Ru and promote the production of active intermediates. This phenomenon is similar to the activation of catalytic sites in HRP, where the cofactor can promote ET to increase the electron density on metal active centers for efficient biocatalysis (35, 36). Taken together, the highly active Ru sites endow the UiO-67–Ru with superior POD-like activity, as evidenced by these control experiments. In addition, borrowing the extra solar energy, the UiO-67–Ru can vividly mimic the cofactor-driven ET process to further boost the catalytic efficiency. Based on the confirmed catalytic sites and intermediates, density functional theory calculations were further implemented to reveal the enhanced catalytic activity of UiO-67–Ru with the help of extra illumination. First, the UiO-67–Ru model was established, where the UiO-67 (111) surface was selected and two C atoms on the linker were replaced by N2Ru (SI Appendix, Fig. S29). The charge transfer between the metal Ru and N atoms was found through charge density difference analysis (Fig. 4A). Ru atom loses electrons when it bonds to N atoms, which is consistent with the XPS result mentioned above. The charge density of O, Zr, and Ru atoms increases under light irradiation (Fig. 4B). It is suggested that the photoelectrons transfer to the Zr–O node as well as the Ru atom to form the charge separation state, further confirming the LCCT and LMCT processes in UiO-67–Ru. To investigate the effect of enhanced electron density of Ru on the POD-like activity, the process of H2O2 activation was explored under acidic conditions (Fig. 4C). In detail, the H2O2 molecule is first adsorbed on the Ru atom and subsequently cleaved homolytically into two *OH or cleaved heterolytically into an *O–H2O species adsorbed on the Ru atom. For the heterolytic path, the adsorbed *H2O species dissociates and desorbs from the surface, leaving an *O adsorbed on the Ru atom. After hydrogenation, the intermediates generate *H2O species adsorbed on the metal Ru atom. The catalyst substrate finally returns to its initial state after the desorption of the H2O molecule. The downhill of free energy indicates the thermodynamical favor of the adsorption of H2O2 on the Ru atom. The free energy is downhill in terms of two *OH formations. The energy barrier was calculated to be 0.33 eV for the formation of the *O–H2O species, which is easily surmountable at room temperature (37, 38). However, the transition state search indicates that the barrier for dissociation and desorption of one adsorbed *OH in the homolytic path is as high as 2.66 eV, suggesting a selectivity unfavorable for this step. In contrast, the barrier for dissociation and desorption of *H2O in the heterolytic path is 0.34 eV, which is significantly smaller than that for adsorbed *OH in the homolytic path. Therefore, the activation of H2O2 prefers to follow the hemolytic path for the production of active Ru=O intermediates, which is in agreement with the experimental results. With the light, the energy barriers for transformation from *H2O2 to *O–H2O and from *O–H2O to *O decrease, indicating a higher capacity of UiO-67–Ru–Light to generate *O–H2O and *O. The rate-determining step is the desorption of H2O from the active Ru site due to its good absorber ability. The lower energy barrier in UiO-67-Ru–Light (0.68 eV) than that in UiO-67–Ru (0.89 eV) can be attributed to the fact that the photoelectrons transfer optimizes the electronic structure of Ru sites to reduce the binding energy. The filling degree of d orbital of active metal atom plays an important role in the strength of adsorbate binding to the surface. Specifically, eg-parentage orbitals ( and ) of the active metal significantly affect the surface–adsorbate bonding interaction because of their favorable vertical orientation toward surface-bound intermediates (39). With the increase of filling, the adsorbate binding decreases due to the increased filling degree of antibonding states (40). Fig. 4D and SI Appendix, Fig. S30 show the projected density states (PDOS) of d, and orbitals of Ru atom. Upon the irradiation, the PDOS of d, and orbitals move toward the more negative energy side, indicating the increase of occupation of these orbitals, which is in good agreement with the electron transfer analysis mentioned above. It is beneficial for decreasing reaction energy and facilitating the production of active intermediates. The d-band center of the Ru atom with the light (−1.07 eV) is lower with the respect to the Fermi level than that without the light (−0.77 eV), implying the weaker binding strength between the Ru atom and the intermediates. The increased numbers of electrons filled in d and orbitals weaken the bonding interaction between Ru atom and *H2O species and thus contribute to the superior catalytic performance (Fig. 4E)

Encouraged by the excellent POD-like activity and multifunctionality of UiO-67–Ru, a photoenhanced immunoassay platform was constructed for the colorimetric monitoring of the CPF. Specifically, the obtained UiO-67–Ru not only replaces the natural enzymes as signal tags with additional photoresponsive ability but also acts as a recognition unit for OPs (such as CPF) based on the strong interaction of Zr–O–P (SI Appendix, Fig. S31) (41, 42). In situ ATR-FTIR experiment results confirm the interaction between CPF and UiO-67–Ru, in which the P=S bond was adsorbed by the MOFs and shifted to a lower frequency (SI Appendix, Fig. S32). Notably, the activity of UiO-67–Ru presents a slight decrease after incubation with CPF. It implies that the CPF prefers to coordinate with the Zr–oxo cluster and keeps the exposure of Ru-active sites (SI Appendix, Fig. S33) (43). Upon illumination, the signal intensity gradually increases with the concentration of CPF and exhibits a good linear relationship in the range from 0.5 to 800 pg mL−1 with a low limit of detection (LOD) of 0.18 pg mL−1 (SI Appendix, Fig. S34). It should be noted that UiO-67–Ru presents a lower LOD than that in dark (1.45 pg mL−1) and previous reports (SI Appendix, Table S2). The as-prepared immunoassay platform possesses good selectivity and repeatability (SI Appendix, Fig. S35). Benefiting from this, the obtained biosensor was further applied to monitor the spiked samples (tap water) with good recoveries (94.39 to 104.61%) (SI Appendix, Table S3).

In summary, we report a single-atom Ru-decorated UiO-67 photonanozymes with photoenhanced POD-like activity assisted by the photoexcited ET process. The single-atom Ru introduction affords highly active sites and boosts the inherent photoelectronic effect, which achieves a 7.0-fold catalytic activity enhancement relative to UiO-67 under illumination. Experiments and theoretical results suggest that photoelectrons follow the cofactor-induced ET process of native enzymes transferring from the photosensitive units to adjacent single-atom Ru sites. The excited Ru sites with redistributed electronic structure optimize the binding energies of active intermediates and H2O release, resulting in more favorable thermodynamics and kinetics in H2O2 reduction. Benefited by efficient enzyme-like activity and the unique physicochemical property, the resultant UiO-67–Ru serves as the nanozyme-labeled antibody for sensitive immunoassay CPF.

One hundred milligram of UiO-67 was dispersed in 10 mL ultrapure water. Then, 10 mg mL−1 RuCl3•3H2O (1 mL) was added to the above solution and continuously stirred for 12 h at ambient temperature. The samples were centrifuged and washed with water until the supernatant is colorless. After drying for 12 h (120 °C under vacuum), the UiO-67–Ru was obtained.

The nanozymes (1 mg mL−1, 10 µL) were introduced into the HAc–NaAc buffer (0.1 M, pH 3.0, 100 µL) containing H2O2 (100 mM, 100 µL) in dark for 6 min. Then, TMB (1 mM, 100 µL) was added to the above solution, and the absorbance values of the reaction solution were obtained by a multimode reader after 3 min. The nanozymes (1 mg mL−1, 10 µL) were introduced into the HAc–NaAc buffer (0.1 M, pH 3.0, 100 µL) containing H2O2 (100 mM, 100 µL) in the presence of Xe lamp (LX-300F, Japan) cutoff filter (λ > 300 nm) for 6 min. Then, TMB (1 mM, 100 µL) was added to the above solution, and the absorbance values of the reaction solution were obtained by a multimode reader after 3 min.

First, 100 µL capture antibody (1 µg mL−1) was added to the 96-well plates and incubated (at 4 °C) overnight. After washing with PBS (200 µL) three times, 1% BSA (100 µL) was introduced into each well and allowed to block antibody-free sites. After shaking and incubating for 1.5 h at 37 °C, the free BSA was removed via washing with PBS three times. Then, various concentrations of CPF (100 µL) were added to the above wells and incubated for another 1.5 h at 37 °C. After being washed with PBS three times, the as-obtained UiO-67–Ru (0.1 mg mL−1, 100 µL) especially combines with CPF through Zr–O–P bonds. After incubation for 1.5 h at 37 °C, the uncombined UiO-67–Ru was removed with PBS four times. Finally, HAc–NaAc buffer (pH 3.5, 100 µL) and 100 mM H2O2 (100 µL) were introduced in the presence of Xe lamp for 6 min. Then, TMB (1 mM, 100 µL) was added to the above solution and incubated for 10 min, and the absorbance of the reaction solution was recorded with a multimode reader. As for the analysis of concentrations of CPF in dark, the same method was performed, except without illumination.

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