However, the slow and complex oxygen evolution reaction at the anode limits the efficiency. Co3O4 with added iron is a capable catalyst for this reaction, but the role of iron is presently unclear. To investigate this topic, we compare epitaxial Co3O4(111), CoFe2O4(111), and Fe3O4(111) thin film model electrocatalysts, combining quasi in-situ preparation and characterization in ultra-high vacuum with electrochemistry experiments. The well-defined composition and structure of the thin epitaxial films permits the obtention of quantitatively comparable results. CoFe2O4(111) is found to be up to about four times more active than Co3O4(111) and about nine times more than Fe3O4(111), with the activity depending acutely on the Co/Fe concentration ratio. Under reaction conditions, all three oxides are covered by oxyhydroxide. For CoFe2O4(111), the oxyhydroxide’s Fe/Co concentration ratio is stabilized by partial iron dissolution.Water electrolysis to produce ‘green H Cobalt-based oxidic anodes with added iron are good electrocatalysts for alkaline oxygen evolution reaction, but the role of iron is still unclear. Here the authors investigate oxygen evolution reaction activity of three well-defined epitaxial thin-film electrodes to address this issue. H2 produced this way is called green hydrogen when the energy stems from renewable sources2. The limiting factor in this process is the sluggish kinetics of the oxygen evolution reaction (OER) and its high thermodynamic potential.Recent years have seen a push towards renewable energy sources in the transition away from our dependence on fossil fuels. However, due to natural fluctuations of sources such as solar, wind, and tidal energy, an efficient energy storage path is needed for situations where the production level does not match the consumption. One potential route for energy storage is the electrocatalytic production of H3. On the other hand, a number of oxides of less expensive, abundant metals have shown comparable reactivity in alkaline media, particularly oxide spinels containing Co, Ni, and Fe4. According to current knowledge, the real catalysts are not the oxides themselves, but surface oxyhydroxide layers which form under OER conditions8. Thus, the oxides are just pre-catalysts. Pure iron oxide was not predicted to yield a good catalyst9, but it was discovered that Fe-impurities in the electrolyte could enhance the activity of other oxides and oxyhydroxides11. This also holds for Co and Ni-based catalysts13, and it has been proposed that the active sites in iron-containing mixed oxyhydroxide catalysts are iron-based16. A recent study by Haase et al. reveals the presence of oxyl species on cobalt oxyhydroxide during OER, which may also play a relevant role for the reaction17.Catalysts based on noble metals such as Ru and Ir exhibit excellent OER performance in acidic media, but their high cost conflicts with a large-scale useUnderstanding catalytic processes on nanoparticulate ‘real’ catalysts is demanding. For catalysis, the surface structure is relevant, but this can be very complex in such systems, and usually only incomplete knowledge is available. Moreover, the catalyst’s composition and structure are often inhomogeneous, which additionally enhances the complexity. Epitaxial thin film model catalysts are not afflicted by these issues: they have a well-defined surface structure and composition, and all parts of the surface can be equally well reached by the electrolyte. Furthermore, electrical conductivity, and its variation over the surface area can be a problem for the study of nanoparticulate ‘real’ catalysts, whereas homogeneous thin films with a thickness of just a few nanometres suffer much less from this issue even if the film material is nominally insulating. Consequently, results for epitaxial films can be more easily interpreted, and data for different films can be compared quantitatively if the experimental conditions are identical.3O4(111) and CoOOH(001)20, CoOx nano-islands on Au(111)22, and Fe3O4(001) and (110) single crystal surfaces24. Building on the work of Bergmann et al. on polycrystalline Co3O4 thin film catalysts25, Reikowski et al. elegantly showed that a thin layer of CoOOH is formed on the surface of Co3O4(111) in a rapid and reversible process that begins before the onset of OER20. Chung et al. recently reported that Fe ions in iron oxyhydroxides are “dynamically stable”, i.e., the ions are continuously dissolved from and redeposited onto the surface26. Müllner et al. used single crystal Fe3O4 samples to show that the (001) and (110) surfaces are stable during OER, and that the (110) surface is more reactive23. Further, an operando study by Grumelli et al. demonstrated that the (√2×√2)R45° surface reconstruction of Fe3O4(001) is maintained deep into the OER regime and has a large effect on the OER kinetics24. Han et al. prepared CoxFe3-xO4 films with different preferential orientations via electrodeposition on Cu single crystal substrates27 and found that the OER activity depends on the surface orientation, with (110) > (111) > (001). The same orientation-reactivity trend was found by Poulain et al. for NiO thin films6.Only very few experimental OER studies have been performed on well-ordered surfaces within the Co-Fe-O system. These include Co28. We found the best OER reactivity and stability for mixed Co1+δFe2−δO4(111) layers (|δ| <0.2) with a low Fe concentration, while pure cobalt oxide and iron oxide layers lag behind. However, Co3O4(111) freshly introduced into the electrolyte had an initial similar activity as the best Co1+δFe2−δO4(111) film, pointing towards a high activity of cobalt hydroxide which is unstable under OER conditions. Furthermore, for Co1+δFe2−δO4(111), we observed a reduction of the Fe concentration by Fe dissolution into the electrolyte during OER, accompanied by an increase of the OER activity. Our results reveal that the cobalt/iron ratio is an important parameter for the OER reactivity of the films, while the surface morphology plays only a minor role.The central topic of the present study is the investigation of the effect of the Co/Fe concentration ratio on the OER activity for films of the same surface orientation3O4(111). We assign this to remnants of the (oxy)hydroxide layers formed during OER25. They might be disordered and thus be partially responsible for the diffuse LEED background. The XPS data discussed later clearly reveal the presence of such layers.Well-defined epitaxial thin films were prepared via oxidative physical vapour deposition as discussed in the experimental section. The samples were characterized in situ with X-ray photoelectron spectroscopy (XPS), low-energy electron diffraction (LEED), and scanning tunnelling microscopy (STM) before and after the electrochemistry experiments. After more than 2 h under OER conditions, the oxide films still exhibited a visible LEED pattern, see Fig. 3O4(111), Fe3O4(111) and Co1+δFe2−δO4(111) films. The step height of the terraces on all three films is ~4.8 Å, which is the distance between equivalent layers along [111]. The Fe3O4(111) film has terraces larger than 100 nm across, while the Co1+δFe2−δO4(111) terraces are smaller, approximately 20–60 nm wide. Co3O4(111) exhibits an island-like structure, with terrace widths in the range of 10–30 nm. RMS roughness values are listed in Table 29 from several 500 × 500 nm STM images) is the scaling or roughness factor listed in Table 2) by the scaling factor we can get an estimate of the electrochemically active surface area (ECSA) and find that the ECSA is not much different between the different as-prepared films.The STM images in Fig. 3O411, Fe3O4s30, we divided the double-layer capacitance measured with potentiostatic electrochemical impedance spectroscopy (PEIS)  surface are ~3 times larger than those determined for metallic Co30, which may be due to different surface structures and oxidation states.In order to determine the specific capacitance, C1+δFe2−δO4(111) surface structure has not yet been reported. Quantitative XPS analysis finds an enhanced iron concentration in the surface-sensitive spectra for as-prepared Co1+δFe2−δO4(111)  and Co1+δFe2−δO4(111). Thus, the combined information from STM, LEED, and XPS infers that tetrahedrally terminated Fe3O4(111) terminates most of the Co1+δFe2−δO4(111) surface  and Fe3O4(111) are both terminated by tetrahedral layers33, and Co1+δFe2−δO4(111) might share this termination, being a mixture of both.An investigation of the Co3O4(111) and Co1+δFe2−δO4(111) STM images recorded before and after electrochemistry  surface is more granular after electrochemistry: the density of islands has approximately doubled, and the RMS roughness is ~40% larger (Table 3O4(111) on the terraces, accompanied by a notable surface area increase and a Cs decrease. This may be related to the oxidation of the film (see discussion below) or structural rearrangements during the formation of the oxyhydroxide layer.Comparison of the Fe3O411 and Cos and metal 2p XPS spectra are compiled in Fig. s spectra of all three films (panels a-c) show the main oxide peak at ~530 eV and after electrochemistry an additional small peak at about 1.5 eV higher binding energy, which we assign to surface hydroxide. Oxyhydroxide O1s peaks overlap with the oxide bulk peak and are therefore not clearly detectable34. Yang et al. identified the cobalt hydroxide O 1s bulk peak at 531.2 eV, somewhere between the surface hydroxide and the bulk oxide peak35, which makes it hard to clearly identify it. Since the layer may also contain oxyhydroxide, we use the notation ‘(oxy)hydroxide’ when we refer to the full layer. Assuming a flat and homogeneous layer, we find a surface (oxy)hydroxide layer thickness in the range of 3 Å for all three oxides. The whole surface layer may not be much thicker, as concluded from the intensity of the LEED spots after OER  it was checked whether the hydroxide stems from the electrochemical experiments by recording an O 1s spectrum from a sample area not exposed to the electrolyte  and Co1+δFe2−δO4(111) samples.O 1Contaminants were absent according to the XPS spectra, except for a minute amount of adventitious carbon (<1 monolayer). Substrate material (Pt or Au) could not be detected  film, Fig. 2+ intensity after OER. This will (in part) be due to oxyhydroxide formed during OER, but in view of the limited thickness of the layer, one may expect that also part of the oxide becomes oxidized to Fe2O3. Such an oxidation process has also been observed for Fe3O4(100) by Müllner et al.23. and for Fe3O4 nanocubes by Hsu et al.36. The Fe3O4(111) LEED pattern of the oxide after OER shows spots at similar positions as before OER, see Fig. 2O3), which is cubic and can be formed by dissolution of tetrahedrally-coordinated iron out of the Fe3O4 lattice leading to a Fe oxidation state of 3+ without changing the lattice structure. As shown in Table 3O4(111) film has decreased by ~25 %, which we attribute to the oxidation process.The Fe 2ure Fe3O411 film, 3O4(111), the Co 2p spectrum (panel d) is very similar to spectra reported by others37 and is not affected very much by the OER process, which means that the oxide is mostly stable under the chosen OER conditions. The features in the difference spectrum at around 780 eV are consistent with the formation of CoOOH at the surface38. Likewise, the small shift of the main lines to slightly higher binding energy would be compatible with the formation of CoOOH and Co(OH)235.For Co1+δFe2−δO4(111) film, the Co 2p spectra recorded before and after electrochemistry (panel e) are also very similar, with very small CoOOH-related features in the region around 780 eV. Within the composition range |δ| <=0.2, the spectra do not vary much and therefore only data for a selected film  is different from the corresponding Co3O4(111) spectrum in panel d, which results from the fact that the cobalt ions in Co1+δFe2−δO4(111) are in a 2+ oxidation state, and therefore, the Co 2p spectrum is similar to that of CoO37.For the Cop and Fe 2p spectra of the Co1+δFe2−δO4(111) films reveals a decrease of the Fe concentration during electrochemistry in all experiments. Table A quantitative evaluation of the Co 21+δFe2−δO4(111) surface compositions were observed when the samples were placed in the electrolyte without applied potential, which shows that the iron loss only occurs under OER conditions  is reduced after OER, revealing that part of the surface iron is oxidized. Oxyhydroxide formation and oxidation of the iron oxide at the surface may be responsible for this. The Co2+ ions are apparently not further oxidized under the given OER conditions, similar to the observation for Co3O4(111).The Fe2), see Fig. Cyclic voltammetry (CV) was applied to identify the influence of Fe on the surface redox electrochemistry. Before a 2-h OER run, linear sweep voltammograms (LSVs) were recorded from the open-circuit voltage (OCV) up to OER conditions  film, a redox feature is visible at ~1.46 V. This feature has typically been assigned to a Co3+/Co4+ redox transition40. The assignment is likely a strong simplification as it neglects metal-ligand charge reorganization leading to electron–hole formation at surface oxygen ions, similar to IrOx41. There is no indication of a peak previously reported between 1.0 and 1.3 V for other Co-based electrocatalysts42. We note that a peak at this potential has been observed more frequently for layered CoOOH-like structures than for Co3O4-like oxides25.For the Co3O4(111) film exhibits broad oxidation features centred at approximately 0.73 V  CV data published by Grumelli et al.24. However, Calvillo et al. observed peaks at ~1.25 V during the first two CVs for Fe3O4/Pd(001) films43.The first anodic linear sweep of the Fe3O4(111) CVs  films had no distinguishable redox features. This may be because the Fe in Co1+δFe2−δO4 is already in a 3+ oxidation state44, which is probably also the case for most of the surface iron atoms . Additionally, the presence of Fe has been shown to shift the Co oxidation peaks to higher potentials28. Therefore, the peak that we observe at 1.46 V for Co3O4(111) may be obscured by the onset of OER if it does exist for Co1+δFe2−δO4(111).The initial anodic scan of the Co2 in the preceding CV scan  activity after OER is at a significantly higher potential than before OER. Co3O4(111) has a similar onset potential to Fe3O4(111) in the cathodic scan after OER, but is much better in the subsequent anodic scan, while the Co1+δFe2−δO4(111) film has a lower onset potential after OER.After the initial CVs, the samples were exposed to OER conditions for 2 h using the potential where the current density reached 1 mA/cmRHE are compiled in Fig. RHE). Following this, the potential was brought back to that for j = 1 mA/cm2 and held for at least 2 h. Then, a further CV scan was performed . Figure VRHE = 1.63 V during the first CV and the CV after 2 h at OER conditions. Thus, Fig. 1+δFe2−δO4(111) (δ = 0.2) was the most active catalyst at this point. We also found a clear trend in the dependence of the activity on the Fe concentration in Co1+δFe2−δO4(111) in the given concentration range: films with a lower Fe concentration have the higher activity, see panel b. The dependence is significant—the activity varies by several times with a change in Fe concentration of only a few percent.OER activity data for a potential of 1.63 V7, which is soluble in basic media, and dissolves slowly during OER, with the rate of dissolution depending on the potential applied45. At high anodic potentials it is rapidly oxidized to FeO42−, which is highly reactive but also highly soluble45. An ICP-MS (inductively coupled plasma mass-spectrometry) characterization of the iron content in the electrolyte revealed a slight increase after electrochemistry, which corresponds to the dissolution of just 0.4 nm of Fe3O4. This is a negligible part of the Fe3O4(111) film, which had a thickness between 10 and 15 nm. The data in Fig. 3O4(111) is the lowest of the three catalysts. After a brief improvement at the beginning, it degrades until it finally reaches about 50% of the starting activity. This degradation may in part be due to the oxidation towards Fe2O3, which is an electrical insulator, but the fact that the activity is the smallest at all abscissa points in Fig. 3O4(111) simply has a low OER activity, as also proposed by other authors9.Under OER conditions, the iron oxide film is covered by an FeOOH layeroperando Raman spectra were recorded. The spectra support the XPS result that part of the film transforms to γ-Fe2O3 during OER  layer grown in ultra-high vacuum (UHV) is very thin, thinner than on electrochemically grown Co3O4(111) layers. It may be assumed that structural imperfections in the latter give rise to this difference.To obtain insights into the state of the thin films during OER, 3O4(111) has a detrimental effect on the activity: before electrochemistry, the Co3O4(111) activity is higher than that of the other oxides, but the activity decreases once OER conditions have been reached and it keeps decreasing with increasing OER time. This is also visible in Supplementary Fig. 3O4(111) OER current density decreases continuously after the start of the experiment. During the cathodic scan after the EC experiments, the activity is as small as that of Fe3O4(111) while the Co3O4(111) OER activity improves again when the potential is decreased and the film exposed to reducing conditions, see the last data point in Fig. 47 is higher than that of the thermodynamically more stable oxyhydroxide layer which forms under OER reaction conditions.Figure 1+δFe2−δO4(111) due to oxide’s high surface iron concentration, which may be the reason for the similar activities of Fe3O4(111) and Co1+δFe2−δO4(111) in the initial phase of the experiment, Fig. 3O4(111) and Co1+δFe2−δO4(111) CA curves in the first ~50 s, see Supplementary Fig. 3O4(111) and Co1+δFe2−δO4(111) CA curves deviate more and more with increasing time. Burke et al. found that OOH mixed oxyhydroxide electrodes dissolve in a KOH electrolyte during OER when the Fe concentration is larger than 54%, but they are stable at lower Fe concentrations28. We find a reduction of the surface Fe concentration to similar values, see Table 1+δFe2−δO4(111) after OER. An ICP-MS characterization of the electrolyte after OER showed the same Fe loss as for the Fe3O4(111) film , but no increase of the Co concentration in the electrolyte, indicating that Co is not lost from the film. We hypothesize that iron dissolves into the electrolyte from the oxyhydroxide layer without re-deposition. To test this hypothesis, a Co1+δFe2−δO4(111) film was prepared, but in the final preparation step, pure Fe was deposited instead of a Fe/Co mixture. After oxidizing the film, the excess Fe was still not well-mixed within the film, and it remained as an Fe3O4(111)-like layer on the surface. A long-term OER CA scan , where the current mostly decreases as a function of time, see Supplementary Fig. An oxyhydroxide layer with an initially high iron concentration may form on Co3O4(111), Co1+δFe2−δO4(111) (|δ| <=0.2), and Fe3O4(111). Epitaxial, well-characterized and defect-poor UHV-grown cobalt ferrite layers are used here for the first time for OER studies and permit to quantitatively assess the effect of the iron concentration on the reactivity, which may be a topic for future studies.Summing up, we have performed a comparative study of the OER activity of epitaxial crystalline thin oxide film model catalysts: Co3O4(111) and Co1+δFe2−δO4(111) were largely stable under the chosen OER conditions, but Fe3O4(111) was found to partially transform to the cubic γ-Fe2O3 phase. Co1+δFe2−δO4(111), the most active catalyst, was found to lose iron during OER via dissolution, eventually converging towards a stable low Fe concentration, while cobalt dissolution was not detected. The Co3O4(111) surface appears to be somewhat granular after 2 h of OER, while the surface island structures of Co1+δFe2−δO4(111) and Fe3O4(111) remained nearly unchanged.Co3O4(111) has an initial high OER activity which decreases when the OER reaction is running, which we attribute to a higher activity of the surface layer formed after introduction into the electrolyte comprising a higher density of reduced Co2+ sites and hydroxyls. In contrast, the Co1+δFe2−δO4(111) activity increases during exposure to OER conditions and decreases upon exposure to reducing conditions. We attribute this finding to the compositional adaptations leading to elevated Fe surface concentration passivating the surface.CoWe feel that the most important aspect of the thin-epitaxial-film approach is a significant reduction of uncertainties inherent to less well-defined pre-catalyst systems, which permits quantitative comparison of the specific activity for different oxides. Given the homogeneity of the surfaces, we can use laterally averaging spectroscopy methods without averaging over differing areas. These data will constitute a feedstock for theoretical modelling which is likely needed to further unravel mechanistic details of the reaction process but also require more advanced surface sensitive chemical spectroscopy. Upcoming studies will also involve the effect of the facet terminating the surface and thereby, provide further needed insights to yield in a comprehensive picture of the oxygen evolution surface of Co-based electrocatalysts for alkaline water splitting.−11 mbar was used for sample preparation and surface characterization with XPS, LEED, and STM at room temperature. For some measurements the surface sensitivity of XPS was enhanced by measuring at non-normal detection angles. Unless stated otherwise, measurements were made at normal emission geometry (0°) using Mg Kα radiation (1253.6 eV). Further details of the UHV chamber may be found in the supporting information.An UHV chamber with a base pressure of 4 × 101+δFe2−δO4(111) on Pt(111) and Co3O4(111) on Au(111). A well-established preparation procedure was employed for Fe3O4(111) films on Pt(111)48. All samples were prepared and characterized quasi in situ in UHV before and after OER without exposure to air at any point to avoid surface contaminations. ‘Quasi in-situ’ means here that all experimental steps were done in the same system, but in different interconnected chambers and environments , without intermediate exposure to air during the sample transfer.We have developed recipes for the production of well-ordered epitaxial thin films of Co+-sputtering and annealing in UHV until a sharp LEED pattern with a low background intensity was observed and no traces of carbon were detected with XPS. Fe and Co were deposited using e-beam assisted evaporators  with the deposition rates being calibrated with a quartz microbalance. Deposition rates of 1.2 Å/min for Fe and 0.6 Å/min for Co were employed, except for the growth of Co3O4(111), where a Co deposition rate of 1.3 Å/min was used.The Pt(111) and Au(111) crystal substrates  were prepared through cycles of Ar3O4(111) film was grown on a Pt(111) substrate by molecular beam epitaxy (MBE) using a slightly modified version of the procedure described by Sala et al.48. First, 4.1 Å of Fe was deposited at room temperature. The sample was then heated in 1 × 10−6 mbar O2 from room temperature up to 1000 K, where it was held for 2 min. The O2 pressure was not reduced until the sample had cooled down to 500 K after annealing. This led to the formation of an FeO(111) layer covering the whole substrate. Then, in at least 3 cycles, ~20 Å of Fe were deposited at room temperature followed by annealing in 5 × 10−6 mbar of O2 for 20 min. This led to films approximately 10–15 nm thick. The oxidation temperature was limited to 850 K to avoid de-wetting of the film. This limited temperature resulted in broader LEED spots and smaller terraces, but partial exposure of the Pt substrate could have led to misleading electrochemical data and therefore had to be avoided.The Fe3O4(111) on Ir(100) films has been published32, but in view of the high OER activity of Ir we decided to avoid this metal as underlayer and developed a new procedure to prepare Co3O4(111) on Au(111). First, a buffer layer of 2.4 nm of Co was deposited at ~250 K. Following this, 1.6 nm of Co was deposited in 5 × 10−6 mbar O2 at ~350 K. While maintaining this O2 pressure, the sample was heated to 650 K and kept at this temperature while 1.9 nm of Co was deposited. After this, the sample was annealed at 650 K for 10 min. The valve allowing O2 into the chamber was closed when the annealing was stopped. The thickness of these films was approximately 10–12 nm.A recipe for the preparation of Co1+δFe2−δO4(111) films were prepared on Pt(111) by following a procedure similar to that used for growing Fe3O4(111) films on Pt(111)49. First, an FeO layer was prepared as detailed above. Following this, Fe and Co were simultaneously deposited at room temperature for ~18 min, followed by annealing in 1 × 10−5 mbar O2 for at least 30 min at 850 K. This deposition and oxidation cycle was repeated at least three times. Annealing temperatures were limited to 850 K, as for Fe3O4(111). The stoichiometry of the layers was judged from Fe 2p and Co 2p XPS spectra. If required, it was adjusted by the addition of iron or cobalt combined with annealing and oxidation. The small differences in the final concentrations of iron and cobalt within these films, denoted Co1+δFe2−δO4(111), did not lead to large differences in the LEED and STM data. These films were 15–20 nm thick. Some STM images and LEED patterns of as-prepared Co3O4(111), Co1+δFe2−δO4(111), and Fe3O4(111) are shown in Supplementary Fig. Co50 the Fe3O4(111) surface is terminated with a layer of iron atoms under UHV conditions. In the bulk, these iron ions would be in tetrahedral positions. At the surface, the Co–O coordination in Co3O4(111) is unsaturated (lower than 4-fold), but the layer is still commonly called ‘tetrahedral’. Based on an IV-LEED study of Co3O4(111)/Ir(100)33, Co3O4(111) is terminated with a ‘tetrahedral’ Co layer.According to published studiesWe investigated the electrochemical (EC) performance of the thin films in an electrochemical cell attached to the UHV chamber, allowing transfer in situ without exposure to air. Details of the electrochemistry setup can be found in Supplementary Fig. 2. The following CV cycle was between 1 VRHE and the potential corresponding to 1 mA/cm2. Then a PEIS spectrum was recorded with the potential at this value. The measured resistance was used for iR correction. Following this, chronoamperometry (CA) data were recorded with the sample at the same potential for approximately 2 h to observe time-dependent reactivity changes. Then, another CV was measured: the first LSV was cathodic, starting from the potential applied during OER down to 1 VRHE, followed by an anodic scan to the potential where the current density reached 1 mA/cm2. After this, a PEIS spectrum was measured at OER conditions (1 mA/cm2). Finally, the seal between the sample and the cell was removed while this potential was still being applied. This was to ensure that the sample did not return to OCV in the electrolyte, as this might induce additional changes at the surface, which might affect the post-reaction surface characterization results. All given potentials and all potential scales are iR-corrected and referenced to the reversible hydrogen electrode (RHE).The following protocol was used for the electrochemical measurements. First, the electrolyte was introduced with the sample at OCV. Then, at OCV + 0.04 V, PEIS was measured, followed by cyclic voltammograms (CVs). The first anodic sweep was from the previous potential, slightly above OCV, to the potential where the current density reached 1 mA/cmIn all the electrochemical experiments, we used 0.1 M KOH and calibrated the Ag/AgCl reference electrode against a commercial RHE in the same electrolyte to compensate for any deviations from the nominal pH. For the reference electrode calibration, we measured the open circuit potential between the Ag/AgCl reference electrode and the RHE in the electrolyte after equilibration for at least 15 min. [We note that a potentially wrong calibration of the reference electrode might cause stronger deviations in the current compared to the variations in the OH- concentrations due to errors in the weighting of the chemicals.] We did not specify the pH of the electrolyte in the manuscript to avoid any misleading or wrong statements.The ohmic resistance of the electrochemical setup was determined from a Nyquist plot obtained via potentiostatic impedance spectroscopy as the high-frequency resistance HFR (HFR = Re(Z) at Im(Z) = 0 Ω).Operando Raman measurements were conducted using a Renishaw (InVia Reflex) confocal Raman microscope. The electrochemical measurements were performed in a 0.1 M KOH electrolyte in a home-built spectro-electrochemical cell made of Teflon and controlled by a Biologic SP240 potentiostat. The cell was equipped with a reference electrode  and a counter electrode (Pt foil). The substrate crystal with the thin film was fixed in place using Kapton tape. The sample was transferred from the UHV chamber to the cell through air. Further details of the operando Raman measurements may be found in the SI.Supplementary information


In vitro, Mn3O4 nanozymes are confirmed to reprogram both the imbalance of metabolism in chondrocytes and the uncontrolled inflammatory response stimulated by hydrogen peroxide. In vivo, a cross‐linked chondroitin sulfate (CS) hydrogel is designed as a substrate for Mn3O4 nanozymes to treat osteoarthritis in mouse models. As a result, even in the early stage of OA (4 weeks), the therapeutic effect of the Mn3O4@CS hydrogel is observed in both cartilage metabolism and inflammation. Moreover, the Mn3O4@CS hydrogel maintained its therapeutic effects for at least 7 days, thus revealing a broad scope for future clinical applications. In conclusion, these results suggest that the Mn3O4@CS hydrogel is a potentially effective therapeutic treatment for osteoarthritis, and a novel therapeutic strategy for osteoarthritis based on nanozymes is proposed.Osteoarthritis, a chronic degenerative cartilage disease, is the leading cause of movement disorders among humans. Although the specific pathogenesis and associated mechanisms remain unclear, oxidative stress‐induced metabolic imbalance in chondrocytes plays a crucial role in the occurrence and development of osteoarthritis. In this study, a trimanganese tetroxide (Mn Trimanganese tetroxide nanozyme with superoxide dismutase (SOD)‐like and catalase (CAT)‐like activities reduces oxidative stress‐induced damage in cartilage. Cross‐linked chondroitin sulfate carried with trimanganese tetroxide nanozyme performed well in osteoarthritis mice models. It presents a novel therapeutic strategy for osteoarthritis based on nanozymes. A recent study reported that nearly 25% of adults are expected to suffer from OA by 2030 owing to the rapid growth of the aging population. Although OA has a high prevalence and disability, effective treatments for this widespread disease are lacking. Surgery is the only option for patients with late‐stage disease. For early‐stage patients, there is still no therapy to reverse or delay the progression of OA. Cartilage degradation, osteophyte formation, and chronic inflammatory reactions are the three typical pathological processes of OA. Although the pathogenic mechanism of OA remains unclear, oxidative stress‐induced inflammation has been shown to be a critical part of its occurrence and progression. It is widely believed that the cartilage is formed by chondrocytes and extracellular matrix (ECM). In the cartilage, chondrocytes generate constituents of the ECM, which, in turn, provides a survival environment for the chondrocytes; this positive feedback supports the metabolic balance in the cartilage. However, this positive feedback is disrupted by inflammation induced by oxidative stress because of an imbalance in the biomechanics of joints in the early stage of OA. Next, the expression of collagen and aggrecan, which are key constituents of ECM in the cartilage, is inhibited. Moreover, inflammatory cytokines and matrix hydrolases, such as interleukin‐1 (IL‐1), tumor necrosis factor α (TNF‐α), and matrix metalloproteinase (MMP13), which activate the inflammatory cascade to further exacerbate the progression of OA, are produced and released. Consequently, the initial handling of oxidative stress is the fundamental treatment to reverse the progression of OA.Osteoarthritis (OA), a chronic degenerative cartilage disease characterized by joint pain and dysfunction, is the leading cause of movement disorders in humans.1 To d In chondrocytes, ROS levels are regulated by superoxide dismutase (SOD) and catalase (CAT), which convert ROS to molecular oxygen (O2) and water (H2O). This means that SOD and CAT form the first line to protect chondrocytes against oxidative damage; thus, we developed a new therapeutic strategy based on SOD and CAT activity. However, there are few SOD and CAT bioenzymes in the knee joint cartilage due to the uneven location of bioenzymes in the body, which limits the application of this therapy in OA. Moreover, owing to the rapid consumption of biological enzymes, frequent supplementation of biological enzymes is needed in the joints. Furthermore, supplemental biological enzymes can only be supplied through intraarticular injections, owing to the special structure of the knee joint. However, frequent intra‐joint injections of bioenzymes result in a huge physiological and psychological burden on patients. Additionally, application and storage are difficult because of the poor stability of bioenzymes, which further limits their use in OA. Thus, anti‐ROS therapy based on bioenzymes for OA treatment is almost impossible.Reactive oxygen species (ROS) produced in mammalian cells cause oxidative stress in OA chondrocytes.12 In  Nanozyme, a type of nanomaterial that possesses intrinsic enzyme‐like activity, catalyzes enzyme substrates efficiently under mild conditions and exhibits catalytic efficiency and enzymatic reaction kinetics similar to those of natural enzymes. Compared with bioenzymes, nanozymes still maintain 85% catalytic activity even in strong acids/bases (pH ≈2–10) or over a wide temperature range (≈4–90 °C), which suggests broad clinical application prospects. As described above, stable nanozymes may solve the problem of biological enzymes being easily inactivated and rapidly consumed. This indicates that application in OA may avoid frequent intra‐joint injections to remarkably reduce the physiological and psychological burden on patients. Recently, an increasing number of studies have reported that nanozymes are used in oncology therapy, Crohn's disease, hepatitis, brain injury, and others. Most recently, researchers have found that well‐designed nanozymes could act as SOD or CAT to regulate ROS levels, which enables an appropriate cue for ROS‐related OA therapy. Most nanozyme‐related studies focus on cancer therapy because of the ease of operation and availability of animal models. The application of nanozymes for OA therapy is in urgent demand, but a great challenge exists in interdisciplinary research between materials science and biomedical engineering. In recent years, Mn3O4 nanozymes have attracted considerable attention because of their highly enhanced SOD‐mimicking performance. As one of the essential trace elements in the human body, Mn has great biocompatibility, and its metabolite can also be used in other physical activities. In other words, the use of Mn3O4 nanozymes in treatment is safe with few side effects. Moreover, owing to the special structure of the knee joint, the use of nanozymes is challenging. The cartilage comprises a sparse population of chondrocytes within a dense ECM without blood or lymphatic vessels. When oxidative stress is experienced in the joint, the cartilage is vulnerable to oxidation and is a difficult target for drug delivery. Thus, in the current study, we designed a Mn3O4 nanozyme with a small particle size (6 nm) to rapidly reach the chondrocytes. Furthermore, compared with other nanozymes such as Fe3O4 and Fe2O3, the Mn3O4 nanozyme in this study had higher SOD activity. Compared with CeO2 and V2O5, the catabolite of Mn3O4 nanozymes can be used for other physiological activities of the body. Thus, the degradation and metabolism of Mn3O4 nanozymes have great advantages for OA treatment.Fortunately, recent studies have shown that some nanomaterials that can reduce oxidation by denaturation in the cellular microenvironment act as bioenzymes to eliminate excessive intracellular ROS.15 Nan Thus, a better method to deliver Mn3O4 nanozymes is required to avoid elimination. To avoid frequent intraarticular injections and maintain lubricity in the joint, a crosslinked chondroitin sulfate (CS) hydrogel was used to carry Mn3O4 nanozymes through intraarticular injections. In this study, we proposed a strategy for OA therapy using Mn3O4 nanozymes. To mimic the OA environment, a human chondrocyte cell line (SW1353) was stimulated with hydrogen peroxide to simulate a high‐oxidative‐stress microenvironment. The SOD‐mimicking performance of the Mn3O4 nanozymes was assessed using this system in vitro. Moreover, crosslinked CS hydrogel was accepted in the current study as a carrier for Mn3O4 nanozymes and used in OA mouse models to assess the therapeutic effect of this treatment strategy. Collectively, this study presents the effects of Mn3O4 nanozymes in protecting chondrocytes against oxidative stress and proposes associated treatment strategies.In joints, synovial vasculature and lymphatics are abundant and rapidly clear small molecules and large macromolecules.9 Thus22.13O4 nanozymes were synthesized using a chemical reaction method at 100 °C, and the synthesis process of Mn3O4 is shown in Figure3O4 nanoparticles was characterized using high‐resolution transmission electron microscopy (HRTEM). As shown in Figure 3O4 nanoparticles at low magnification , is shown in Figure 3O4. X‐ray photoelectron spectroscopy (XPS) was used to characterize the valence states of the synthesized nanoparticles. As shown in Figure 2p clearly shows peaks of Mn2+ and Mn3+, which confirms the composition of Mn3O4. The SOD and CAT activities of Mn3O4 nanozymes were also assayed in this study. The peaks in the Fourier transform infrared (FTIR) spectrum shown in Figure −1 indicate the existence of COO‐. The peaks located at 2921.8 and 2851.8 cm−1 indicate N–H bonds, and the broad peak located at 3363.4 cm−1 indicates C–H bonds. The FTIR results indicated some residual oleic acid and olamine molecules only on the surface of the Mn3O4 nanozymes without any other foreign substances. To further confirm the effect of residual oleic acid and olamine molecules on cells, a live/dead assay in SW1353 cells treated with Mn3O4 nanoparticles was performed. As shown in Figure 3O4 nanoparticles produced in this study had great biocompatibility. To assess the SOD‐like activity of the Mn3O4 nanozymes, the SOD enzyme was used as a standard for further quantification in this study, and the associated results are displayed in Figure 3O4 nanozymes had high SOD‐like activity. After the average calculation, the specific values of the SOD enzyme‐like activities were 120.00 U mg−1. Moreover, according to the kinetic curve of the enzymatic reaction  of Mn3O4 nanozymes at different time points were dose‐dependent. The SOD catalytic initial velocity of Mn3O4 nanozyme was detected according to the kinetics tests method under different concentrations of O2−. The reaction curve was shown in Figure Vmax was 0.0486 × 107 mol s−1 and the Km was 0.017 mmol L−1. As described above, the validity of nanozymes was still high, even in strong acid/base or wide temperature ranges, which suggests broad clinical application prospects. Thus, the SOD activity of Mn3O4 nanozymes in a strong acid/base (pH = 4/10) and over a wide temperature range (4 and 80 °C) was tested in this study. As indicated in Figure 3O4 nanozymes was hardly affected. At the reaction temperatures of 4 and 80 °C, the activity of Mn3O4 nanozymes was still hardly affected, while the SOD enzyme showed nearly no enzyme activity  exceeded the upper limit of kit detection. Moreover, the activity at several concentrations (1–32 ng µL−1) of Mn3O4 nanozymes at different time points was dose‐dependent, according to the kinetic curve of the enzymatic reaction  and under a wide temperature range (4 and 80 °C), the CAT activity of the Mn3O4 nanozymes was still maintained; meanwhile, the CAT enzyme was nearly inactivated, and its activity was the same as the SOD activity  of Mn2+ and Mn3+ is smaller than Mn4+ at acidic conditions and shows similar stability at alkalinity conditions, therefore, the Mn3O4 nanozyme shows stable activity under different PH. It is also an interesting study for the application of extreme conditions in the future. However, in this study, the application of Mn3O4 nanozyme was under physiological conditions. Collectively, Mn3O4 nanoparticles presented SOD and CAT activities in a normal reaction environment.Mnn Figure  analysiss Figure , 128 ng 2.23O4 nanozymes on chondrocytes, an inflammatory microenvironment induced by high levels of oxidative stress was created by adding H2O2, and then treated with Mn3O4 nanozymes. Cells cultured in a tissue culture plate (TCP) without any treatment were used as negative controls. Collagen II, the main component of the cartilage matrix, is produced by normal chondrocytes to maintain cartilage matrix metabolism and protects the cartilage against degeneration in the development of OA. As shown in Figure3O4 nanozyme group, which suggests that Mn3O4 nanozymes do not inherently affect the expression of collagen II. However, comparing the TCP group, H2O2‐treated group, and H2O2 and Mn3O4 nanozyme group, the expression of collagen II was significantly downregulated by a factor of two after stimulation with H2O2 but was rescued by Mn3O4 nanozyme treatment. The results shown in Figure 3O4 nanozymes rescued the expression of collagen II in chondrocytes under oxidative stress. Aggrecan, a proteoglycan originally isolated from cartilage, is another component of the cartilage matrix produced by normal chondrocytes. The normal supply of aggrecan maintains the balance of cartilage tissue metabolism and reduces the inflammation caused by joint instability. Figure 3O4 nanozymes did not inherently affect the expression of aggrecan in the TCP and Mn3O4 nanozyme groups. A nearly threefold decrease in aggrecan expression was observed after H2O2 treatment and was rescued by Mn3O4 nanozyme treatment. Decreased aggrecan levels in the H2O2‐treated group revealed that H2O2‐induced high ROS levels obstructed the normal expression of aggrecan in chondrocytes, leading to cartilage metabolic disorders. However, the addition of Mn3O4 nanozymes restored the function of chondrocytes, which was confirmed by comparing the H2O2‐treated group with the H2O2‐and Mn3O4 nanozyme‐treated groups. In summary, these results revealed that the function of chondrocytes was destroyed by H2O2 and restored by Mn3O4 nanozymes.To assess the therapeutic effect of Mn In chondrocytes, iNOS is activated and released by oxidative stress‐induced inflammation, whereas iNOS contributes to oxidative stress by producing more oxidative agents. As shown in Figure 3O4 nanozyme groups, indicating that the addition of Mn3O4 nanozymes did not interfere with the normal function of chondrocytes. A nearly threefold increase in the expression of iNOS was found between the TCP and H2O2‐treated groups, which indicates that high iNOS levels are induced by oxidative stress. Next, the increased iNOS levels were repressed by the addition of Mn3O4 nanozymes. In addition to iNOS, cyclooxygenase 2 (COX2), which is involved in the antioxidant defense system under oxidative stress, is activated in chondrocytes after H2O2 stimulation. After activation by oxidative stress, the iNOS‐induced COX2‐mediated pathway further disturbs the balance between oxidative stimulators and inhibitors, leading to an inflammatory reaction. The results, as shown in Figure 2O2 treatment successfully activated the expression of COX2, which was in line with the expression of iNOS. Treatment with Mn3O4 nanozymes protected chondrocytes against oxidative stress‐induced inflammation. To assess the anti‐ROS effect of the Mn3O4 nanozymes, a DCFH‐DA fluorescent probe was used to test the level of cellular ROS. As shown in Figure 2O2 treatment significantly increased the level of ROS, and Mn3O4 nanozymes inhibited this increase. Moreover, a superoxide assay further confirmed that H2O2 treatment successfully destroyed the mitochondrial respiratory chain, and Mn3O4 nanozymes effectively reduced superoxide accumulation  and 3‐amino‐1,2,4‐triazole  were used to inhibit the expression of intracellular SOD and CAT biological enzymes. As shown in Figure 3O4 nanozymes increased intracellular SOD and CAT activities, respectively. In addition, after DDC+3‐AT treatment, ROS levels were increased by H2O2 but suppressed by Mn3O4 nanozymes  mediates the expression of inflammatory cytokines, suppresses the synthesis of collagen and aggrecan, and induces chondrocyte cell death.24 In s Figure . Collect3O4 nanozymes in OA treatment, the expression of matrix proteins and associated inflammatory factors was quantified in both gene transcription and protein translation. Because collagen II and aggrecan are the main components of the cartilage matrix, their production determines the level of cartilage degeneration to some extent. Figure2O2 treatment, and this repression was reversed by Mn3O4 nanozyme treatment. The mRNA level of aggrecan was decreased to 10% after H2O2 treatment but was rescued by Mn3O4 nanozyme treatment , also called aggrecanase 5, which maintains cartilage matrix balance by degrading aggrecan, shows abnormally high expression during the progression of OA. As indicated in Figure 2O2 remarkably activated the transcript level of ADAMTS‐5, which further reduced the content of aggrecan through proteolysis. Mn3O4 nanozymes inhibited the increased mRNA levels of ADAMTS‐5 stimulated by H2O2, which demonstrates that Mn3O4 nanozymes maintain the balance of cartilage tissue by slowing the hydrolysis rate of aggrecan. Matrix metalloproteinase 13 (MMP13), also called collagenase 3, plays a crucial role in cartilage destruction by hydrolyzing collagen. In the cartilage matrix, MMP13 and ADAMTS‐5 participate in the balance between hydrolysis and the production of matrix proteins, which form the matrix microenvironment. A significant result was that the mRNA level of MMP13 increased after stimulation with H2O2, but this increase was repressed by Mn3O4 nanozyme treatment  mouse model was established, and Mn3O4@CS hydrogel treatment was performed. As shown in Figure3O4@CS hydrogel treatment group, the degree of damage was significantly improved, while the improvement in the CS hydrogel treatment group was fairly limited, which confirmed the therapeutic effect of the Mn3O4@CS hydrogel treatment. After protein fixation with paraformaldehyde, 5‐µm‐thick paraffin tissue slices were prepared, and HE staining was performed to assay cartilage disruption and associated inflammatory changes. As shown in Figure 3O4@CS hydrogel treatment markedly reduced cartilage disruption and inflammatory cell recruitment caused by DMM surgery, whereas the therapeutic effects for the CS hydrogel treatment group were not evident. To further assess the degree of cartilage damage, safranin O/fast green staining was performed. In this staining, owing to the difference between bone and cartilage tissues, the cartilage tissue was dyed red by safranin‐O, while the bone tissue was dyed green by fast green. As shown in Figure 3O4@CS hydrogel injection. To further quantify the degree of cartilage damage, the OARSI and histological scores of both the tibial plateau and femoral condyle in all groups were determined. As shown in Figure 3O4@CS treatment. Similar results were observed for cartilage PG depletion and cartilage surface erosion score  assay and superoxide anion assays were performed as previously reported. As shown in Figure 3O4@CS hydrogel treatment. The inhibition ability of superoxide anions was also elevated after Mn3O4@CS hydrogel treatment of the knee joint. Collectively, as a well‐accepted mouse model, the DMM mouse model, which displayed high similarity with the normal progression of human OA, provides an excellent model to assess the effect of Mn3O4 in vivo. As described above, the inflammation and imbalance in cartilage metabolism caused by DMM surgery were successfully rescued by Mn3O4@CS hydrogel treatment.To further quantify the differences in expression in these groups, total mRNA was extracted from each joint, followed by qPCR. An inspection of 33O4 nanozymes on chondrocytes during the progression of cartilage degeneration  acetate tetrahydrate, 1‐octadecene, oleylamine, oleic acid, sodium hydroxide, chondroitin 4‐sulfate sodium salt (CS), and 1,4‐butanediol diglycidyl ether were purchased from Shanghai Macklin Biochemical Co. Ltd. (Shanghai). Acetone, n‐hexane, and hydrochloric acid were purchased from Sinopharm Chemical Reagent Co. Ltd. All the chemicals were used without further purification.3O4 nanozymes were synthesized by the thermal decomposition method, as described in a previous report. Briefly, 0.49 g manganese (II) acetate tetrahydrate was added to a mixed solution of 1.14 g oleic acid, 6.05 g oleylamine, and 30 mL 1‐octadecene. The mixture was stirred for 4 h at room temperature, and then slowly heated to 100 °C. Then, 0.38 mL of 0.05 M manganese (II) acetate tetrahydrate aqueous solution was added to the mixed solution before the heating temperature reached 100 °C. The reaction was allowed to proceed for 10 min and then cooled to room temperature. Mn3O4 nanozymes were obtained by adding a mixture of acetone and n‐Hex.Mns report.31 Bri3O4@CS hydrogel was obtained by adding 0.1–0.4 g of crosslinked CS and 10–160 µg of Mn3O4 nanozymes to 1 mL of normal saline.CS hydrogel was obtained by a crosslinking reaction between 1,4‐butanediol diglycidyl ether and CS. The reaction method was as follows: 0.5 g of CS was added and adequately dissolved in a solution of 4 mL of 15 wt% aqueous NaOH and 9 mL of acetone. Then, 0.3 mL of 1,4‐butanediol diglycidyl ether was added to the mixed solution. The mixture was stirred vigorously and heated to 100 °C for 5 h. After the reaction, hydrochloric acid was used to normalize the pH of the solution to neutral, and crosslinked CS was obtained. The obtained powder was washed thrice with acetone and dried in an oven. The CS hydrogel was obtained by adding 0.4 g of crosslinked CS to 1 mL of normal saline. Similarly, the Mn3O4 nanozymes were characterized using TEM and HRTEM , respectively. Scanning electron microscopy  was used to characterize the morphology of the obtained hydrogels. XRD patterns  were used to characterize the crystal structures of the obtained nanozymes and hydrogels. The element composition and atomic valence state of the obtained Mn3O4 nanozymes were analyzed by XPS. FTIR spectra of the obtained nanomaterials and hydrogels were obtained using an IR spectrophotometer . Elemental distribution and quantification of the hydrogel were performed using an X‐ray electron probe microanalyzer (EPMA).The morphology and crystal structure of the synthesized Mn3O4  and SOD enzyme   were tested in this study. Briefly, WST‐8/enzyme working solution(160 µL), reaction‐starting liquid (20 µL), and sample or control blank (20 µL) were incubated at 37, 4, or 80 °C for 30 min, and the absorbency was assayed by a microplate reader at 450 nm. ΔA450 = A450control1− A450sample. Inhibition percentage = (A450control 1− A450sample)/ (A450control 1− A450control 2) × 100%. In this study, the SOD enzyme (#S0086) was used to test the reliability of the experiment and quantify the activity of Mn3O4 nanozymes. Moreover, based on these kits, acetic acid and sodium hydroxide were used to change the pH of the reaction solution (a pH of 4 or 10). Similar to the SOD activity, the CAT activity of Mn3O4 nanozymes was assayed by a catalase assay kit . According to the protocol, 250 mM hydrogen peroxide solution was prepared and calibrated by NanoDrop at 240 nm. Next, 250 mM hydrogen peroxide solution (10 µL), catalase detection buffer  and catalase standard or sample (5 µL) were mixed and incubated at 25 °C. After 2 min of incubation, 10 µL of the reaction solution was mixed with 200 µL of color‐developing working fluid and assayed by a microplate reader at 520 nm.SOD activity was assayed by a Total Superoxide Dismutase Assay Kit with WST‐8 . As per the instructions from the reagent vendor, several concentrations of Mn2O2 (40 nM) and Mn3O4 nanozymes (8 µg ml−1) to detect the anti‐inflammatory and matrix‐protective effects of Mn3O4 nanozymes in vitro. SW1353 cells were divided into four groups and cultured and stimulated in 1) phosphate‐buffered saline (PBS), 2) H2O2, 3) H2O2 + Mn3O4 nanozymes, or 4) Mn3O4 nanozymes for 24 h. The SW1353 cells were then extracted for protein and mRNA quantification.SW1353 cells were cultured in Leibovitz's L‐15 medium with 10% fetal bovine serum (FBS). Before stimulation, SW1353 cells were seeded in 12‐well plates and treated with HThe animals used in this study were wild‐type male mice , which were provided by Beijing Vital River Laboratory Animal Technology  and maintained under pathogen‐free conditions at the Shandong University Qilu Hospital Animal Center. All experimental procedures were approved by the Animal Research Ethics Committee of the Qilu Hospital of Shandong University (#DWLL‐2021‐053).3O4 in vivo, as previously reported. In this study, the mice were divided into four groups (n = 6): sham operation, DMM, Mn3O4@CS treatment, and CS treatment groups. The mice in the sham operation group underwent the same operation, but without ligation. In the DMM group, the mice underwent DMM surgery without any injections. In the Mn3O4@CS treatment group, the mice underwent DMM operation and were treated with Mn3O4@CS hydrogel  articular injection once a week. In the CS treatment group, the mice underwent the same operation and were injected with CS hydrogel into the articulation once a week. After 4 weeks, the animals were sacrificed. Knee joint tissues from each mouse were collected and labeled for further evaluation.The DMM mouse model was established to confirm the therapeutic effect of MnTo analyze articular erosion, the knee joint was fixed for at least 48 h and scanned using a micro‐CT imaging system .p < 0.05 was considered statistically significant. The significance level was defined as ns (no significance), *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.The number of replicates and animals is indicated in the figure legends or the Experimental Section. The results are shown as average values ± SD of the mean (S.E.M.). For normally distributed data sets with equal variances, a one‐way analysis of variance (ANOVA) followed by a Tukey post‐hoc test was carried out across groups. Statistical analysis was carried out using GraphPad Prism 8 Software. The authors declare no conflict of interest.Supporting InformationClick here for additional data file.


The main therapeutic task is carried out by the 2D NiCoOx nanosheets, which have four multienzyme‐mimicking activities: peroxidase‐ (POD), oxidase‐ (OXD), glutathione peroxidase‐ (GPx), and catalase‐ (CAT) mimicking activities. These activities induce the reversal of the hypoxic microenvironment, endogenous glutathione depletion, and a continuous ROS output. The ROS‐induced pyroptosis process is carried out via the ROS‐NLRP3‐GSDMD pathway, and the exogenous US activation boosts the multienzyme‐mimicking activities and favors the incremental ROS generation, thus inducing mitochondrial dysfunction. The anti‐cancer experimental results support the dominance of NiCoOx nanosheet‐induced pyroptosis. This work expands on the biomedical applications of engineering 2D materials for US‐augmented catalytic breast cancer nanotherapy and deepens the understanding of the multienzyme activities of nanomaterials.Overcoming apoptosis resistance is necessary to ensure an effective cancer treatment; however, it is currently very difficult to achieve. A desirable alternative for cancer treatment is the targeted activation of pyroptosis, a unique type of programmed cell death. However, the pyroptosis inducers that are efficient for cancer therapy are limited. This work reports the engineering of 2D NiCoO x nanosheets as proptosis inducers have been developed to increase the rate of deleterious reactive oxygen species generation for the ultrasound‐augmented catalytic nanotherapy of breast cancer.The 2D multienzyme‐mimicking NiCoO These nanoenzymes also offer exceptional advantages for medicinal applications, particularly for cancer treatment. Representatively, nanozymes with peroxidase (POD)‐mimicking and oxidase (OXD)‐like catalytic capabilities can catalyze the disintegration of hydrogen peroxide (H2O2) or oxygen (O2) and generate highly toxic ROS that can induce the apoptosis or necrosis of cancer cells. Thus, the application potential of nanoenzymes for ROS‐mediated tumor‐nanocatalytic treatment is promising. Unfortunately, the highly complex tumor microenvironment (TME) influences the effect of the nanoenzyme‐enabled catalytic procedures, such as hypoxia, mildly acidic environment, and overexpression of H2O2 and glutathione (GSH). To overcome these limitations, H2O2‐responsive nanoenzymes with dual‐ or multienzyme‐mimicking activities have been explored with broad interest. Besides the above two types of enzyme activities, the glutathione peroxidase (GPx)‐ and catalase (CAT)‐like activities also play important roles. In recent years, nanoenzymes that catalyze reactions of multivalent metal ions  mimic the function of natural enzymes and play a significant role in regulating the TME, thus enhancing their therapeutic effect. CoFe2O4 nanoflowers can act as POD‐ and CAT‐mimicking nanoenzymes to catalyze the decomposition of H2O2 into hydroxyl radicals (•OH) and O2 for sonodynamic therapy (SDT) and chemodynamic therapy (CDT). Zhang and co‐workers prepared MnFe2O4 nanoenzymes with GPx‐ and CAT‐like activities that can overcome tumor hypoxia and consume GSH by continuously catalyzing H2O2 to generate O2, to achieve a better therapeutic effect. However, nanozymes that concurrently have POD‐, OXD‐, CAT‐, and GPx‐mimicking activities under weak acidic conditions have rarely been explored.According to the laws of enzyme kinetics under physiological conditions, nanoenzymes are nanomaterials that can catalyze specific chemical reactions and that have enzyme‐mimicking properties.1 Nano ferroptosis, pyroptosis, and so on. Pyroptosis is a form of necrosis that regulates cell death and is performed by the gasdermin (GSDM) family. Although GSDMs are expressed in an inactive form, some proteinases activate them via proteolysis. Cleaved gasdermin D (GSDMD) releases the N‐terminal fragment (N‐GSDMD), which forms membrane pores, and then releases pro‐inflammatory molecules into the extracellular environment, thus triggering inflammation and an immunological reaction. Ultrasound (US), which is a non‐invasive sound wave that can penetrate deep tissues, possesses acoustic cavitation effects. Therefore, the mass diffusion of the nanoenzyme can be improved through US, which significantly improves the enzyme‐like activity and corresponding reaction rates. Therefore, appropriately integrating the US‐enhanced enzyme‐like activities into one nanoenzyme can effectively inhibit tumor growth and recurrence. Overall, pyroptotic cell death is caused by the activation of multiple cellular protein complexes called inflammasomes; for example, the nucleotide binding oligomerization domain‐like receptor protein 3 (NLRP3) inflammasome acts as a regulatory factor of pyroptosis. The activated NLRP3 inflammasomes then transform protoproteinase‐1 into caspase‐1. In contrast with apoptosis, pyroptosis and ferroptosis are immunogenic programmed cell death (PCD) processes, constituting powerful anti‐cancer strategies because they can initiate antitumor immune responses by delivering adequate cytokines and damage‐associated molecular patterns (DAMPs). US‐induced pyroptosis can maximize the significant control of tumor growth, thus generating a strong and lasting antitumor response.At present, nanoenzyme‐induced cell death has been applied to multiple processes, including apoptosis,12 fer In particular, 2D nanomaterials have become a promising nanoplatform for various biomedical applications due to their specific physical and chemical properties and high drug loading capacity. By taking advantage of the interactions between the TME and 2D nanomaterials, an efficient TME‐targeted tumor treatment can be actualized by developing TME‐responsive 2D nanomaterials, which would enable the application of 2D nanomaterials to catalytic medicine and to efficient tumor treatments. To demonstrate our points, herein, morphology‐controlled 2D NiCoOx nanozymes were successfully constructed using the hard template method. The 2D NiCoOx nanosheets act as multienzyme‐mimicking pyroptosis inducers for US‐augmented catalytic breast cancer nanotherapy without complicated side effects 2·6H2O with Ni(NO3)2·6H2O, the addition of hydrophilic polyvinylpyrrolidone (PVP) and urea, and the lyophilization and calcination of the Ni‐Co precursor. The morphology of the 2D NiCoOx nanosheets according to the transmission electron microscopy (TEM) observations is presented in Figure x nanosheets have an ultrathin sheet‐shaped structure. The TEM image of the 2D NiCoOx nanosheets shows that they exhibit high dispersibility, with an average particle size of 178 nm, according to the statistical analysis  images  analysis was performed to determine the components, purity, and crystal structure of the fabricated 2D NiCoOx nanosheets , (400), and (440) planes, respectively, and were consistent with the standard values corresponding to NiCo2O4 (JCPDS 20–0781). The band gap value of the 2D NiCoOx sheets recorded by the derivative Tauc's plot was around 1.43 eV /Ni3+ (855.5 eV). The high‐resolution Co 2p spectrum can also be fitted to two satellites and two spin‐orbit peaks with binding energies, presented in the valence state of Co2+ (779.3 eV)/Co3+ (781.2 eV), respectively  upon adding H2O2  with a maximum absorption peak at 652 nm. The absorbance at 652 nm in the NiCoOx + H2O2 group was significantly higher than in the NiCoOx or H2O2 alone groups, validating the dramatic oxidation of TMB  was used to detect the superoxide anion radical (·O2−) and singlet oxygen (1O2) after US irradiation . The characteristic peak of DPBF at 416 nm at different NiCoOx concentrations decreased gradually  measurement  spectra showed that BSA was successfully grafted onto the surface of the 2D NiCoOx nanosheets  using confocal laser scanning microscopy (CLSM). As shown in Figurex nanosheets entered the 4T1 cells for each of the different culture durations , which can be demonstrated by the red fluorescence emitted by RB and the corresponding linear‐scan spectrogram. The intensity of the red fluorescence gradually increased as the incubation duration increased.Based on its intrinsic non‐toxicity, desirable biodegradability, and water‐solubility, bovine serum albumin (BSA) is the main available albumin extensively used in nanobiomedicine.30 The2O2, and NiCoOx, only produce weak fluorescence intensities. Notably, the obvious green fluorescence observed in 4T1 cells indicated that the 2D NiCoOx nanosheets induce the production of small amounts of ROS under US irradiation, while the further combination with H2O2 under acidic conditions significantly increases the intracellular ROS levels. Therefore, the chemodynamic effect generated by the 2D NiCoOx nanosheets and US under acidic conditions (pH 6.5) can markedly improve the generation efficiency of •OH  Figure  and neutH Figure .x nanosheets, the viability of 4T1 cells decreased. Next, we investigated the effect of different concentrations of H2O2 and of the US time on the cytotoxicity. Tumor cells featured higher endogenous H2O2 levels than normal cells. To further model the TME and more comprehensively understand the cytotoxic properties of the 2D NiCoOx nanosheets, 4T1 cancer cells were treated using different NiCoOx concentrations  under neutral (pH 7.4) and acidic (pH 6.5) environmental conditions in the presence of H2O2 (50 or 100 µm) for 24 h  and 31.9% , respectively . As shown in Figure h Figure . In an ay Figure . For they Figure . As the y Figure .x nanosheets on tumor cells were further confirmed using calcein‐AM and propidium iodide (PI) staining experiments , and the combination of NiCoOx + H2O2 and US stimulation effectively amplified the multienzyme‐mimicking activities of the 2D NiCoOx nanozymes, leading to apoptosis progress revulsion  , whereas the fluorescence intensity observed in the NiCoOx + H2O2 and NiCoOx + H2O2 groups was lower than that found in the control group, and that observed in the NiCoOx + US + H2O2 group was almost non‐existent, indicating that the 2D NiCoOx nanosheets have an excellent capability of scavenging the intracellular GSH through their US‐augmented enzymatic activity , which is representative of mitochondrial membrane disruption. Consequently, the 2D NiCoOx nanosheets were significantly cytotoxic in acidic medium, whereas they did not lead to significant cell death in neutral medium , their pyroptosis capability could be further improved  and interleukin‐1β (IL‐1β) in the 4T1 cells apparently increased; however, no significant release of LDH or IL‐1β was observed in the control group, indicating that the NiCoOx + US treatment highly induces pyroptosis  and a C‐terminal repressor structure (C‐GSDMD), which can be proteolytically activated by certain proteases.34 Thee Figure . To furtd Figure . The exps Figure . All of 2.5x multienzyme‐mimicking nanosystems, transcriptome analysis was performed to compare the changes in the messenger RNA (mRNA) expression in 4T1 cancer cells treated with 2D NiCoOx in the presence of H2O2. In total, 1786 genes were differentially expressed from those expressed in the US‐treated NiCoOx group, including 1104 up‐regulated mRNAs and 682 down‐regulated mRNAs , tumor necrosis factor (TNF)‐related genes , the antiviral protein genes , interferon‐related genes , and chemokine genes , the obvious appearance of these up‐regulated gene changes confirmed that the 2D NiCoOx nanosheets can activate a pyroptosis‐binding inflammatory reaction. These findings demonstrate that the US‐induced 2D NiCoOx nanosheets can alleviate tumor hypoxia and further induce pyroptosis.After determining the gene expression differences, the specific regulatory genes corresponding to the main influencing pathways were presented in a chord diagram after the KEGG pathway analysis. Notably, the most significantly regulated genes belonged to the NOD‐like receptor signaling pathway Figure . Notably2.6x nanosheets, we further evaluated their antitumor therapeutic effect in vivo. Female BALB/c mice aged 3–4 weeks were subcutaneously injected with 4T1 cells on the right side to establish a breast cancer model, and the tumor‐bearing mice were irregularly divided into four groups (n = 5 per group): 1) control group; 2) US group; 3) NiCoOx group; 4) NiCoOx + US group (intravenous injection (i.v.)). The detailed treatment protocol of the tumor‐bearing mice is shown in Figurex nanosheets, the majority of Ni was distributed in the spleen and liver tissues of the 4T1 tumor‐bearing mice, in addition to their efficient accumulation at tumor sites  1, 3, 7, and 11 days after tail vein injection of a NiCoOx aqueous solution for 15 min and 4 h. The body weight and tumor volume of the mice were monitored every 2 days during the whole treatment period. As expected, no apparent body weight changes were observed for any of the groups, which show the high therapeutic biosafety of the treatment , which is attributed to the catalytic therapeutic effect of the 2D NiCoOx nanozymes  rate to quantitatively evaluate the desirable antitumor therapeutic efficiency of the nanoenzymes enhanced by US. Compared with that of the NiCoOx group (the relative TGI rate was 0.14%) at the end of day 14, the TGI rate of the US‐induced NiCoOx group was 71.8%, surpassing that of the US group (−0.04%)  Figure . Moreovex + US group show serious apoptosis  staining of the tumor slices after treatment further suggest that the tumors in the NiCoO3x nanosheets, including ROS‐associated POD‐ and OXD‐mimicking activities, hypoxia reversal involving a CAT‐like activity, and a GSH‐consumption capability involving GPx‐like activity. US stimulation improved the multienzyme‐like activities and enhanced ROS production, which caused mitochondrial dysfunction. The pyroptosis process can be specifically induced by the ROS generated by the multienzyme‐mimicking with US activation, which was demonstrated by caspase‐1 activation and the release of N‐GSDMD and LDH/IL‐1β. The subsequent transcriptome sequencing analysis revealed that pyroptosis was caused by the ROS‐NLRP3‐GSDMD pathway. Importantly, the US‐augmented catalytic tumor nanotherapy produced remarkable therapeutic results, as evidenced by the systematic in vitro and in vivo evaluations (4T1 breast tumor model). Collectively, our results show that the engineered 2D nanosheets/nanoenzymes with US‐augmented multienzyme‐mimicking activities constitute a novel strategy to induce pyroptosis, which enables a highly efficient catalytic tumor nanotherapy.In summary, we developed specific US‐augmented multienzyme‐mimicking strategies for an efficient tumor nanotherapy by engineering distinct 2D NiCoO nanosheets/nanoenzymes to improve the rate of harmful ROS generation for catalytic tumor nanotherapy and cell pyroptosis. A variety of multienzyme‐mimicking activities were observed in the designed/fabricated 2D NiCoOThe authors declare no conflict of interest.Supporting InformationClick here for additional data file.


High concentrations of unconjugated bilirubin (UCB) have toxic effects. The aim of our study was to find a way to elevate UCB tolerance or inhibit its toxicity in neurocytes. It has been reported that cystatin C (CST3) concentrations have a significant positive correlation with total bilirubin (TB) levels and a negative correlation with albumin levels. In addition, CST3 can directly bind UCB, decrease human umbilical vein endothelial cells’ permeability, improve blood–brain barrier integrity after ischemic brain injury in mice, and induce autophagy. We hypothesized that CST3 could increase the solubility of UCB, decrease permeability of neurocytes, induce autophagy of neurocytes, and alleviate bilirubin-induced damage. To verify our hypothesis, we measured TB and conjugated bilirubin levels, and the permeability and autophagy of neurocytes treated with UCB and CST3. Our findings suggest that CST3 can protect against UCB-induced damage in neurocytes and that autophagy played an important role in this process. Total bSustained higher UCB levels can damage various organs . StudiesCystatin C (CST3) is a low molecular weight ≈13.3 kDa) protein produced by nucleated cells , and is .3 kDa pr22.1ad libitum access to standard laboratory diet and water. They were euthanized after 3 days and the tissues were fixed in 4% formalin or 2.5% glutaraldehyde.A total of 15 male C57BL/6 mice (6–8 weeks) were used in this study. Ten mice injected intravenously with 150 μg/g of UCB . They then were evenly divided into two groups and injected intravenously with 5 μg/g of CST3  or PBS . The animals were maintained under controlled conditions with a temperature of 24 ± 2°C, humidity of 55–65%, a 12 h light/dark cycle, and 2.2The mice need to find and stand on a platform submerged under 5 mm of water in a circular pool by observing four visual cues in different locations around the pool. On the training days, the mice were given a maximum of 60 s to find the platform, and recording was stopped when the mice remained on the platform for 5 s. Mice that did not find the platform within 60 s were guided to the platform and allowed to remain on it for 10 s. The training phase lasted for 4 days, during which the mice were placed in the pool four times every day. On the fifth day, the test was performed by placing the mice in the pool from which the platform had been removed and allowing them to explore for 60 s. The time and distance they swam in the platform quadrant or in the other three quadrants were measured.2.3For Masson’s staining, the brain samples were treated with hematoxylin for 5 min, with deionized water for 30 s three times, and with Masson’s dye  for 7 min. Then, the brain sections were treated with 2% glacial acetic acid solution and 10% molybdic acid for 5 min. After aniline blue staining for 5 min and gradient alcohol washing, neutral gum was used to seal the slide, and the section was observed under a microscope.2.4TB and CB were measured using TB assay kit  and a direct bilirubin assay kit , respectively. UCB content was defined by TB minus CB levels (µmol/L).2.5HT22 cells were cultured in RPMI-1640 medium . The medium was changed every 2–3 days. CST3 proteins were obtained from ProSpec and blocking peptides for CST3 were purchased from Abgent. The proteins were dissolved in double distilled water. UCB of 100 µg/mL was added to the cells to establish high UCB cell model.2.6Total RNA was extracted from HT22 cells using TRIzol reagent . We synthesized cDNA using the FastQuant RT Kit  following the manufacturer’s instructions. qPCR was performed on an IQ5 thermal cycler  using the following cycling conditions: predenaturation at 95°C for 15 min, 40 cycles of denaturation at 95°C for 10 s, annealing at 60°C for 15 s, extension at 72°C for 20 s, and 71 cycles of melt curve analysis at 60°C for 10 s. The following primers were used in reactions:CST3 primer sequences: F CAACAAAGCCAGCAACGACA, R TCTTGGTACACGTGGTTCGG and β-actin primer sequences: F AGAGGGAAATCGTGCGTGAC, R CAATAGTGATGACCTGGCCGT.2.7Proteins were extracted from the brain samples using a Proteins Extraction Kit  and quantified with BCA-Reagents . Proteins were separated by SDS–PAGE at 160 V on a 12% gel  for 1 h and then transferred to a 0.22 μm nitrocellulose filter membrane at 200 mA for 3 h. The membranes were incubated with autophagy related primary antibodies diluted as follows: anti-CST3 antibody , anti-mTOR antibody , anti-mTOR (phospho S2448) antibody , anti-LC3 antibody , anti-Beclin-1 antibody  diluted 1:1,000, and anti-GAPDH antibody  diluted 1:2,000. Membranes were then incubated with secondary antibodies diluted at 1:5,000. The membranes were washed and bands were visualized with enhanced chemiluminescence immunoblotting detection reagents . For autophagic protein, bafilomycin A1 was used at a final concentration of 200 nM and added during the last 3 h. Images were gotten by Image Lab Software , and semiquantitative analysis was performed by normalizing the protein bands to the internal control GAPDH.2.8We performed MTT  assays to examine the viability of HT22 cells treated with CST3 (100 pg/μL), UCB (170 µM), or bafilomycin A1 (200 nM and added during the last 3 h of treatment). Cells treated with solvent were used as the negative control. HT22 cells were plated at a density of 2,000 cells/well and allowed to adhere for 6 h, followed by treatment with CST3 protein and UCB. Wells containing 100 μL medium alone (without cells) were used as blank controls. MTT assays were performed after treatment for 48 h. The blank control served as baseline. Each experiment was repeated three times, and the results are presented as a percentage of viable cells calculated by the following equation:  × 100 = percentage of viable cells.2.9The activity of P450 was determined using a P450 activity ELISA kit  in accordance with the manufacturer’s instructions.2.105) were seeded onto polycarbonate cell culture inserts of a 2-well Transwell system (Costar) until they formed a complete monolayer. Then FITC–BSA was added to the upper chamber and the fluorescence was evaluated in the lower chamber after adding FITC–BSA for 24 h .HT22 cells (2 × 102.115) were seeded onto polycarbonate cell culture inserts of a 24-well Transwell system (Costar) until they formed a complete monolayer. Then 100 µg/mL UCB was added to the upper chamber and concentration of UCB in lower chamber was evaluated after 6 h.HT22 cells . After the normality test and variance homogeneity test of measured data, comparisons between different groups were performed using Student’s Ethical approval: The research related to animals’ use has been complied with all the relevant national regulations and institutional policies for the care and use of animals. All animal experiments were approved by the Animal Experiments and Experimental Animal Welfare Committee of Beijing friendship hospital, Capital Medical University (21-2036).33.1To investigate the role of CST3 on UCB toxicity, we established CST3 overexpression and knockdown HT22 cells , which w3.2To investigate the mechanism of the protective effect of CST3 on hyperbilirubinemia, we measured the solubility of UCB, and found that CST3 could increase the solubility of UCB in a cell-free system , but it 3.3To further explore the mechanism of the protective effect of CST3 on hyperbilirubinemia, we studied the autophagy pathway and found that CST3 could enhance autophagy flux in HT22 cells  and the 3.4We found enhanced autophagy in the neurocytes of CST3-treated high concentration bilirubin (HBR) mice by transmission electron microscopy .4We found that CST3 could reduce UCB toxicity by increasing the solubility of UCB, decreasing the permeability of HT22 cells, and promoting autophagy of HT22 cells. However, the effects of increasing solubility and decreasing permeability are intriguing. It might be because noncovalent binding between UCB and CST3 ,12, whicEffects of exogenous CST3 to enhance viability and autophagy of neurocytes suggest that CST3 might serve as a potential drug for the treatment of hyperbilirubinemia. In addition, CST3 is an endogenous protein and should have few side effects. Excessive CST3 is also excreted through urine.In conclusion, we demonstrated that CST3 could alleviate UCB-induced damage to neurocytes by increasing UCB solubility, decreasing the permeability of neurocytes, and promoting autophagy, and that autophagy played the most important role in this process.


Prochlorococcus is an abundant photosynthetic bacterium in the open ocean, where nitrogen (N) often limits phytoplankton growth. In the low-light-adapted LLI clade of Prochlorococcus, nearly all cells can assimilate nitrite (NO2−), with a subset capable of assimilating nitrate (NO3−). LLI cells are maximally abundant near the primary NO2− maximum layer, an oceanographic feature that may, in part, be due to incomplete assimilatory NO3− reduction and subsequent NO2− release by phytoplankton. We hypothesized that some Prochlorococcus exhibit incomplete assimilatory NO3− reduction and examined NO2− accumulation in cultures of three Prochlorococcus strains  and two Synechococcus strains (WH8102 and WH7803). Only MIT0917 and SB accumulated external NO2− during growth on NO3−. Approximately 20–30% of the NO3− transported into the cell by MIT0917 was released as NO2−, with the rest assimilated into biomass. We further observed that co-cultures using NO3− as the sole N source could be established for MIT0917 and Prochlorococcus strain MIT1214 that can assimilate NO2− but not NO3−. In these co-cultures, the NO2− released by MIT0917 is efficiently consumed by its partner strain, MIT1214. Our findings highlight the potential for emergent metabolic partnerships that are mediated by the production and consumption of N cycle intermediates within Prochlorococcus populations.Prochlorococcus, the numerically dominant photosynthetic cell in the subtropical open ocean. In laboratory cultures, some Prochlorococcus cells release extracellular NO2− during growth on NO3−. In the wild, Prochlorococcus populations are composed of multiple functional types, including those that cannot use NO3− but can still assimilate NO2−. We show that metabolic dependencies arise when Prochlorococcus strains with complementary NO2− production and consumption phenotypes are grown together on NO3−. These findings demonstrate the potential for emergent metabolic partnerships, possibly modulating ocean nutrient gradients, that are mediated by cross-feeding of N cycle intermediates.Earth’s biogeochemical cycles are substantially driven by microorganisms and their interactions. Given that N often limits marine photosynthesis, we investigated the potential for N cross-feeding within populations of  Prochlorococcus and its close relative, Synechococcus, are globally abundant non-diazotrophic cyanobacteria that are jointly responsible for approximately 25% of marine net primary production , which is a preferred N source for cyanobacteria likely because it requires less reducing power to assimilate compared to more oxidized N sources  at all examined light intensities during growth on NO3−  are consistent with rates measured for marine diatoms when normalized to either C quotas (as a proxy for biomass) or cell volumes. In batch culture experiments, Thalassiosira pseudonana was observed to produce NO2− during growth on NO3− at a maximum rate of 2.5 × 10−14 µmol s−1 cell−1 (2.2 × 10−6 nmol NO2− cell−1 d−1)  . Note th 46 µm−3 , while L.14 µm−3 . Thus, T2− release by MIT0917 were with respect to the N requirements of low-light-adapted Prochlorococcus. To evaluate this question, we assumed a cellular N quota of 4.3 fg N cell−1 for LLI Prochlorococcus . While a broad range of NO2− excretion to NO3− uptake ratios have been observed previously  often reach maximum abundance within the subsurface chlorophyll maximum layer  to estimate the fraction of cells in LLI populations that possessed each version of the nitrate assimilation gene cluster .In both the subtropical North Pacific and North Atlantic oceans, LLI um layer  and in tum layer . Our datentified ? To addrentified  derived 2− but not NO3−) dominated the LLI Prochlorococcus populations throughout the year and generally exceeded 60% of total LLI genomes  generally dominated the NO3− assimilating functional types in LLI Prochlorococcus populations at the North Atlantic’s BATS station (2−) in the North Pacific compared to the North Atlantic suggest that the configuration of the NO3− assimilation gene cassette encoding a type II NirA provides a selective advantage to cells in the stable, well-stratified, and generally N-limited waters of the North Pacific Subtropical Gyre.At both sites, we observed that MIT1214-like cells  with either Prochlorococcus MIT0915 or Prochlorococcus MIT0917 (each of which can use both NO3− and NO2−) in medium containing NO3− as the sole N source.Given that LLI features  coexist features , we next−2 s−1 of blue light produced nitrite at rates from 4.6 × 10−8 to 5.4 × 10−8 nmol NO2− cell−1 d−1  for growth on NO2−  would be consistent with its significantly lower growth rate when co-cultured with MIT0915.One possible explanation for the MIT1214 strain’s continued slow growth in the presence of MIT0915 may be the release of N-containing organic compounds by its partner strain. abolites  that couabolites . Alternansporter —in contr culture . Supply Prochlorococcus and the N cycle by demonstrating that some Prochlorococcus divert a sizable fraction of transported NO3− to extracellular pools of NO2− when grown under N-replete batch culture conditions. An intriguing feature of this phenomenon is the degree of phenotypic variability  exhibited incomplete assimilatory NO3− reduction. It is possible that the divergent version of the NirA NO2− reductase possessed by MIT0917—and similar Prochlorococcus in the field—has distinct biochemical features. One hypothesis is that this divergent NirA has a higher substrate affinity, perhaps providing these cells with an advantage under chronically N-limited conditions, such as at HOT in the North Pacific Subtropical Gyre (2− reduction step of the NO3− assimilation pathway that results in cellular accumulation of NO2− because NO3− reduction outpaces the kcat of this divergent NirA. This NO2− could then diffuse out of the cell as nitrous acid (HNO2) which would make up a small fraction of the intracellular NO2− pool at cellular pH . At an NO3− concentration that was representative of these intermittent pulses of NO3− in the wild, MIT0917 released NO2− at a cell-specific rate of 6.1 × 10−8 (±0.28 × 10−8) nmol NO2− cell−1 d−1 . Additional study is warranted to examine the conditions under which Prochlorococcus populations are either net producers or net consumers of NO2− and evaluate how microbial populations and communities modulate the availability of various N sources that ultimately impact production and remineralization processes. At the population level, the dynamic assembly of distinct functional types of Prochlorococcus could emerge through interactions that are mediated, in part, by cross-feeding of NO2−. We posit that trait variability and the selection of complementary functions might facilitate robustness or resiliency in microbial populations. Prochlorococcus, as a key primary producer in the tropical and subtropical oceans, offers an extremely valuable lens through which to constrain the rules under which emergent features arise.As we have demonstrated, different functional types of inimized . ConsequProchlorococcus strains MIT0915 (LLI clade), MIT0917 (LLI clade), and MIT1214 (LLI clade), the high-light-adapted Prochlorococcus strain SB (HLII clade), as well as Synechococcus strains WH8102 (clade III) and WH7803 (clade V) (The cultures used in this study included the low-light-adapted clade V) .3− assimilating strains  were grown in triplicate as pure cultures using Pro99 medium  with the 800 µM ammonium chloride (NH4Cl) omitted and replaced by 800 µM sodium nitrate (NaNO3). The cultures were grown in 35 mL of medium in borosilicate glass culture tubes at a temperature of 24°C and under continuous illumination intensities of 6, 36, 52, and 85 µmol photons m−2 s−1. Given that the strains represent multiple clades of Prochlorococcus and Synechococcus that span a wide range of depths and light wavelengths in the euphotic zone, we used white light provided by fluorescent lamps. The LLI strains (MIT0915 and MIT0917) had poor and inconsistent growth at the highest light intensity, so these strains were only examined at the three lower light intensities . Cultures were monitored daily by removing 0.5 mL of culture in order to determine cell abundances with flow cytometry and NO2− concentrations with the Griess colorimetric method . Net cell-specific NO2− production rates were calculated as the change in NO2− concentration relative to the logarithmic mean of cell concentrations—to account for exponential growth—for successive time points during the log-linear portion of the growth curve.The NO3− as the sole N source at 24°C. In order to approximate the light intensities and wavelengths that LLI Prochlorococcus typically experience in the wild (−2 s−1 of continuous blue light (in lieu of white light). As controls, all strains were grown as pure cultures, in duplicate, under the same temperature and light conditions. The MIT1214-MIT0915 and MIT1214-MIT0917 co-cultures, as well as the MIT0915 and MIT0917 pure cultures, used Pro99 medium  with 800 µM sodium nitrate (NaNO3) as the sole N source. Pure cultures of MIT1214 were grown in Pro99 medium using 100 µM sodium nitrite (NaNO2) as the sole N source to serve as a control for growth on NO2−. Given that some strains of Prochlorococcus might be inhibited by NO2− (2 was used (instead of 800 µM) to minimize potential growth inhibition due to NO2− toxicity. MIT1214 was also grown in Pro99 medium using 800 µM NO3− as the sole N source to serve as control for the absence of growth on NO3−. The co-cultures and pure cultures were sampled daily over two sequential transfers to monitor cell abundances with flow cytometry, NO2− concentrations using the Griess method, and to preserve cells on filters for strain-specific cell abundance measurements . Given that the cell size and fluorescence properties of MIT0915, MIT0917, and MIT1214 overlap when examined using flow cytometry , we used quantitative PCR to assess the cell abundance of each strain in co-culture. Sample processing, standards, reaction conditions, and amplification efficiencies for the qPCR assays are detailed in the Supplementary Materials and Methods.MIT1214 was co-cultured, in triplicate, with either MIT0915 or MIT0917 in 35 mL of medium in borosilicate glass culture tubes using NOthe wild , 43, whi by NO2− , a lower