Here, we present atmospheric observations of gas-phase HClO3 and HClO4. Significant levels of HClO3 were observed during springtime at Greenland (Villum Research Station), Ny-Ålesund research station and over the central Arctic Ocean, on-board research vessel Polarstern during the Multidisciplinary drifting Observatory for the Study of the Arctic Climate (MOSAiC) campaign, with estimated concentrations up to 7 × 106 molecule cm−3. The increase in HClO3, concomitantly with that in HClO4, was linked to the increase in bromine levels. These observations indicated that bromine chemistry enhances the formation of OClO, which is subsequently oxidized into HClO3 and HClO4 by hydroxyl radicals. HClO3 and HClO4 are not photoactive and therefore their loss through heterogeneous uptake on aerosol and snow surfaces can function as a previously missing atmospheric sink for reactive chlorine, thereby reducing the chlorine-driven oxidation capacity in the Arctic boundary layer. Our study reveals additional chlorine species in the atmosphere, providing further insights into atmospheric chlorine cycling in the polar environment.Chlorine radicals are strong atmospheric oxidants known to play an important role in the depletion of surface ozone and the degradation of methane in the Arctic troposphere. Initial oxidation processes of chlorine produce chlorine oxides, and it has been speculated that the final oxidation steps lead to the formation of chloric (HClO 3 and HClO4 in the atmosphere and their widespread occurrence over the pan-Arctic during spring, providing further insights into atmospheric chlorine cycling in the polar environment.Observations are reported of HClO Chlorine atoms (Cl) are also a strong oxidant in the polar troposphere, where the levels of hydroxyl radicals, another major atmospheric oxidant, are relatively low4. It is also well established that the direct reaction with Cl provides a chemical sink of methane (CH4) in the atmosphere8. The presence of chlorine species such as molecular chlorine (Cl2) and bromine monochloride (BrCl) has been reported in the Arctic, attributed to direct emissions from snowpacks10 and heterogeneous reactions of chlorine species on snow grains and airborne aerosols11. Upon photolysis, these chlorine species release reactive Cl atoms (1−2), which rapidly react with O3 to form chlorine monoxide, ClO (3)3. ClO is subsequently oxidized by bromine monoxide (BrO) or ClO, producing chlorine dioxide (OClO); reacting with HO2 to form hypochlorous acid (HOCl); or reacting with nitric oxide (NO) and nitrogen dioxide (NO2) to produce Cl atoms and chlorine nitrate (ClONO2) (as shown in reactions 4−7)12. Cl atoms can also degrade other hydrocarbons (RH) to generate hydrochloric acid 13. HCl can be converted into chloride (Cl−) via hydrolysis on aerosol surfaces (9)14. Chloride can further undergo heterogeneous reactions with HOCl to produce Cl2 (10), which can, in turn, be photolyzed to recycle Cl atoms (1)15.Active chlorine cycling has been found in the Arctic boundary layer during the springtime following polar sunrise and is acknowledged to play a key role in the depletion of surface ozone (O3) and perchloric (HClO4) acids. The presence of atmospheric HClO4 was first proposed to be important in the polar stratosphere and is believed to be a missing atmospheric sink process of chlorine18. Recent studies have hypothesized the potential formation of HClO3 and HClO4 in the lower atmosphere through observations of significant chlorate (ClO3−) and perchlorate (ClO4−) levels in rainwater, snow, and Arctic ice core samples22. Therefore, the atmospheric occurrence of chlorine oxyacids could enhance the chlorine sink, thereby affecting the oxidation capacity of the atmosphere and potentially posing environmental threats once deposited to the Earth’s surface. However, to date, there exists no direct evidence of the presence of HClO3 and HClO4 in the atmosphere, thus, limiting our full understanding of the atmospheric chlorine cycle and its associated environmental impacts.Despite decades of research on chlorine cycling in the atmosphere, a largely unexplored aspect entails the formation of chlorine oxyacids, such as chloric  Polarstern during the Multidisciplinary drifting Observatory for the Study of the Arctic Climate (MOSAiC) expedition. The measurements show that both chlorine oxyacids are ubiquitous and widespread during spring in the Arctic region. We find that these atmospheric species are not photoactive and therefore represent a previously unconsidered atmospheric sink of reactive chlorine in the pan-Arctic boundary layer.Here, we present ambient observations of HClO3 and HClO4 measured at the Villum Research Station, Greenland, and during the MOSAiC campaign. Our observations in Greenland indicated a significant increase in the HClO3 signal measured with a nitrate-chemical ionization atmospheric pressure interface time-of-flight mass spectrometry , with an estimated concentration up to 1 × 106 molecules cm−3 in the spring of 2015. The HClO3 concentration began to increase when sunlight increased towards the end of February. HClO3 exhibited no diurnal pattern, but a unique feature is that a significant increase in HClO3 concentration was observed in coincidence with the depletion of O3, as shown in Fig. 3 peaked under relatively low O3 levels (<30 ppb). We also measured HClO3 with a nitrate CI-APi-TOF instrument during the MOSAiC expedition in 2019/2020 (Methods section). Similar to the observation in Greenland, the measurements onboard RV Polarstern in different seasons revealed a clear increment in HClO3 starting at the end of February, when solar radiation started to increase after the polar night. The estimated springtime concentration of HClO3 during the MOSAiC campaign ranged from approximately 1 × 105 to 7 × 106 molecules cm−3  to 8 × 104 molecules cm−3 and near detection limits (3 × 104) to 1 × 106 molecules cm−3, respectively, during springtime, which were typically lower than the HClO3 concentration. The lower concentration of HClO4 compared with that of HClO3 is consistent with the levels of ClO4− and ClO3− measured in Arctic ice cores, where the annual depositional flux of ClO4− was reported to be several times lower than that of ClO3− (refs. 22).As shown in Figs. 3 and HClO4 in the Arctic.The results obtained during our campaigns at the different locations and time periods over the Arctic demonstrate that these chlorine oxyacids are widespread Fig. , and tha14, the final oxidation steps leading to chlorine oxyacid formation are not well characterized. Here, we explore the potential formation mechanisms of the observed HClO3 and HClO4 during spring in the Arctic.Although the initial steps of atmospheric chlorine oxidation are well understood (1–10)3 and HClO4 coincides with the decrease in the O3 concentration. Springtime atmospheric surface ozone destruction is a well-known phenomenon in the Arctic and is typically linked to the shallow mixing layer and chemical reactions, including bromine and chlorine chemistry24. Our air mass backward trajectory analysis revealed that chlorine oxyacid-laden and O3-depleted air mass originated from the near ground surface, while the high O3 air mass originated from higher altitudes . However, the formation of HClO3 and HClO4 via heterogeneous reactions on the aerosol surface or direct emission from the surface of snowpacks is likely not the dominant pathway in the Arctic. This assumption is justified by the remarkably low vapor pressure 28 and high Henry’s law constant (KH) of HClO4 (9.9  ×  103 mol m−3 Pa−1)18. Although information for KH of HClO3 is not available, its value is very likely in between KH of HClO4 and KH of HOCl (6.5 mol m−3 Pa−1)29. These low vapor pressure and high KH suggest that the formed ClO3− and ClO4− on the aerosols or snow surface are unlikely being emitted directly as gas-phase HClO3 or HClO4 into the atmosphere. This is further supported by the detection of low HClO3 and HClO4 atmospheric concentrations in winter when the Arctic is covered by snow 30 may also not be important, as the results demonstrated no direct relationship between HClO3 (or HClO4) and our measured H2SO4 concentrations  during both the Greenland and MOSAiC campaigns 3. In addition to photolysis, the produced ClO can then react with BrO/ClO to produce OClO, or undergo loss through reactions with OH, HO2, NO and NO2, CH3OO, and CH3COOO2. Abundant BrO and ClO must have been present during the encountered ozone-depletion events, as have been previously demonstrated by many studies37, and significant levels of BrO have been observed in spring during the MOSAiC campaign38. By using the previously reported typical ranges of BrO, ClO, and HO2 levels during Arctic ozone-depletion events40, we estimate that the reaction rate of ClO + BrO is much higher than that of the ClO + ClO and ClO + HO2 channels , suggesting that the increase in BrO during ozone depletion events drives the OClO formation. The reaction of ClO + OH, ClO + CH3OO, and ClO + CH3COOO are insignificant; however, the presence of typical levels of NOx (NO and NO2) in the Arctic  can compete with BrO for ClO (section S1 in SI). Indeed, previous ground measurements have detected significant OClO, up to 24 ppt, in the Arctic springtime35. Further observational evidence for the key involvement of bromine chemistry in the chlorine oxyacids formations comes from our observations which demonstrated that the recorded bromide signal adhered to the increase in HClO3 and HClO4 combined with a drastic decline in the O3 concentration  reacting with NO to recycle ClO; and (iv) oxidation by O3 to produce ClO3 . These results suggest that a significant fraction of OClO can be directly oxidized by OH to convert into HClO3, and produce ClO to recycle OClO. This is consistent with previous experimental studies on the OH + OClO reaction, where HClO3 was suggested to be produced at low temperatures42. Despite the lower O3 concentration (a source of OH), previous studies have shown that HOx (OH and HO2) chemistry is active during springtime in the Arctic with a reported OH concentration of ≈105−106 molecules cm−3 (refs. 43). This can also be indicated by our observation of significant H2SO4 concentrations . Therefore, the HClO4 formation is likely regulated by the O3 levels as indicated by the higher mean HClO4 concentrations observed at relatively higher O3 levels during the depletion events  reacting with Cl to yield two ClO molecules; (ii) oxidation by OH to form HClO3 and HClO4 over the Arctic atmosphere are predominantly produced through homogeneous reactions of chlorine, involving photochemical processes of HOx and bromine chemistry.Based on these results, we conclude that the observed HClO3 and HClO4 in the troposphere through photodecomposition. Supplementary Fig. 3 and HClO4 within the 170–340 nm wavelength range (refer to the Methods section for details). At the relevant wavelengths under tropospheric conditions (>290 nm), the estimated cross-sections of HClO3 and HClO4 are very small, suggesting that these two chlorine species are not photolabile in the troposphere. Based on these cross-sections, the loss rate constants of these oxyacids against photolysis at noon in the Arctic springtime are calculated as 4.4 × 10−12 and 2.5 × 10−18 s−1 for HClO3 and HClO4, respectively  are also slow at low temperatures, with reported reaction rate coefficients of 1.00 × 10−31 and 5.8 × 10−13 cm3 molecule−1 s−1 at 253 K, respectively48. Assuming typical Cl (4 × 105 molecules cm−3)3 and OH (5  × 105 molecules cm−3)4 concentrations in the Arctic, the loss rates of kCl+HClO4[Cl] and kOH+HClO4[OH] are estimated to be 4.0 × 10−26 and 2.9 × 10−7 s−1, respectively.Another possible removal pathway of HClO2 cm−3) and humidity (which begins to increase in April) in the Arctic troposphere  of HClO3 and HClO4, previous studies have reported that the heterogeneous uptake coefficient of other chlorine acids, such as HCl, on aqueous aerosols reaches ≈0.2 at 273 K50. Both HClO3 and HClO4 are very strong acids with high electronegativity and can thus be easily ionized into ClO3−, ClO4−, and H3O+ in liquid water of the aerosol since HClO3 and HClO4 are highly soluble in water. The KH value of HClO4 in water was reported as 9.9 × 103 mol m−3 Pa−1 (ref. 18), while the KH value of HClO3 likely varies between the KH values of HClO4 and HOCl, with that of the latter compound reaching 6.5 mol m−3 Pa−1 (ref. 29). These higher KH values than that of HCl (KH = 0.2 mol m−3 Pa−1) may indicate that HClO3 and HClO4 could be efficiently accommodated on the surface of aerosol particles and that the fraction evaporating back into the gas phase could be small as well. By assuming that the heterogeneous uptake is accommodation limited and the γ values of HClO3 and HClO4 are similar to that of HCl (γ = 0.2)50, the estimated heterogeneous loss rate coefficients of HClO3 and HClO4 based on a typical aerosol surface area of 20 µm2 cm−3 during the MOSAiC campaign are 2.7 × 10−4 and 2.5 × 10−4 s−1, respectively (section S3 in SI). These rates are much (>3 orders of magnitude) higher than the rates of photodecomposition and radical attack (by OH and Cl) estimated above (<3 × 10−7 s−1). Therefore, the most relevant fate of HClO3 and HClO4 is their heterogeneous uptake by the surface of aerosol particles and subsequent deposition on the ground surface or undergo wet deposition. However, we cannot exclude the possibility of direct loss of these chlorine oxyacids to the snow surface , Cl− can be efficiently activated into reactive gas-phase chlorine again56. However, as HClO3 and HClO4 are not susceptible to photolysis and radical attack, and their conversion into ClO3− and ClO4− on aerosol surfaces or snowpacks is efficient in the Arctic boundary layer, the homogeneous formation of HClO3 and HClO4 could terminate chlorine recycling. Therefore, the formation of HClO3 and HClO4 is expected to affect the chlorine-mediated oxidation capacity in the Arctic troposphere. Furthermore, once HClO3 and HClO4 deposit on the ground surface , they may have environmental implications as their ions, ClO3− and ClO4−, can accumulate in the polar ice and marine sediment, and may present a toxicity risk to resident biota58.Atmospheric chlorine chemistry has been regarded as a “never-ending” reaction since there is no termination process in the cycle. Indeed, the formation of HCl can serve as a sink for chlorine compounds in the troposphere, where HCl is taken up by aerosols and converted into Cl3 and HClO4 in the atmosphere and their widespread occurrence over the pan-Arctic during spring. We propose a novel plausible mechanism for the formation and loss of chlorine oxyacids in the Arctic environment. The results provide evidence for chlorine oxyacids to be a previously unconsidered atmospheric sink for reactive chlorine, thereby providing further insights into chlorine chemistry in the Arctic region. We, therefore, conclude that the existence of HClO3 and HClO4 in the atmosphere should be considered when evaluating the environmental impacts of chlorine chemistry in the Arctic.In summary, our study revealed the observations of HClO44. The other field study was the MOSAiC expedition, which involved RV Polarstern drifting across the central Arctic from September 2019 to October 2020. The MOSAiC expedition track and detailed campaign information can be found in ref. 59.This study comprises data obtained during three field measurement campaigns over the Arctic within different time ranges. We conducted a measurement campaign at the Villum Research Station (Station Nord) in high Arctic Northern Greenland (81° 36’ N 16° 39’ W). This campaign started in mid-February 2015 and continued until the end of August 2015. The measurement station is located approximately 2 km from Station Nord, and the instrumentation was set up at the station location. We also conducted measurements at the atmospheric observatory, Gruvebadet, located 2 km southeast of Ny-Ålesund, Svalbard , from 28 March to 30 May 2017 (spring). Detailed information on the Greenland and Ny-Ålesund sampling site can be found in ref. 60 was employed in negative ion mode with nitrate (NO3−) ions as reagent ions to detect gas-phase HClO3 and HClO4. In Greenland, a straight, stainless steel inlet tube with a length of 1 m and an outer diameter of ¾ inch was applied at ~1.5 m above ground level (a.g.l.) to sample ambient air with a flow rate of 10 liters per minute (lpm). The inlet tube was heated to zero degrees Celsius. At Ny-Ålesund, the inlet tube length was 2 m (outer diameter of ¾ inch) and the sample was taken through the roof (height = 2 m a.g.l.), with a flow rate of 10 lpm. On RV Polarstern, the nitrate CI-APi-TOF instrument was set up in a Swiss Container on the bow deck of vessel61. The nitrate CI-APi-TOF inlet was connected to a new particle formation (NPF) inlet, through a core sampling flange system, accommodating a neutral air ion spectrometer utilized to create ~60 lpm inlet flow (height = about 15 m above sea level). The zero measurements were conducted occasionally with a high-efficiency particulate air (HEPA) filter for at least 40 min each measurement in both Greenland and MOSAiC, which cover different seasons during the measurement period.A state-of-the-art chemical ionization atmospheric pressure interface time-of-flight mass spectrometry (CI-APi-TOF) instrument3 was detected as ClO3− (82.954 m/z), and its isotope peak was clearly observed at 84.951 m/z, with a 35Cl:37Cl ratio of approximately 3:1  should be carefully identified, as it is difficult to distinguish it from the isotopic peak of deprotonated sulfuric acid, HSO4−  when the HSO4− signal is high. The m/z values of these two peaks are close to each other and may create interference with lower-mass resolution devices. Therefore, we considered the peak of 37ClO4− (100.946 m/z) to estimate the HClO4 signal in this study. We also detected the Br− signal from the peak at 78.919 m/z (together with NO3(HBr)− peak at 141.915 m/z; Supplementary Fig. 62. The raw data was pre-averaged over 10 min and processed with the MATLAB tofTools package according to the procedures described in Jokinen et al.60.HClO3− and ClO4− were the deprotonated products of HClO3 and HClO4, respectively. Quantum chemical calculations (section S4 in SI) indicated that the binding free energies of NO3− with HClO3 and HClO4 are 24.0 and 36.9 kcal mol−1, respectively , are similar to (the former is slightly lower than) that of 2SO4 (−1). However, similar to H2SO4 (−1), their binding free energies are higher than their deprotonation free energies . During the MOSAiC campaign, calibration was completed twice after the campaign, and the average of two-factor values was 6 × 109. This factor includes the losses at the NPF inlet. Based on these calibration factors, the detection limits of HClO3 and HClO4 were estimated as 3 × 103 and 7 × 103 molecules cm−3 , respectively, during the Greenland measurement campaign and 2 × 104 and 3 × 104 molecules cm−3 , respectively, during the MOSAiC measurement campaign. The detection limit for HClO3 measurements in Ny-Ålesund was calculated to be 1 × 103 molecules cm−3 .H3 and HClO4 concentrations in this study were computed with Eq. , was measured with a scanning mobility particle sizer (SMPS). The information on the SMPS setup in Greenland can be found in ref. 70. As for the MOSAiC campaign, the SMPS was measured from the United States Department of Energy Atmospheric Radiation Measurement (ARM) Aerosol Observation System container72. Water vapor was measured during the MOSAiC campaign via cavity ring-down spectroscopy (CRDS) with a commercial Picarro instrument (model G2401) also connected to the interstitial inlet of the Swiss container. Meteorological parameters (temperature and solar radiation) during the MOSAiC were obtained from the meteorological observatory Polarstern73. Details on the atmospheric and meteorological equipment during the MOSAiC campaign can be found in ref. 59 and ref. 74.O3 and HClO4, we applied the estimated absorption cross-sections of these two compounds (refer to section S5 in SI for the cross-sections computation) in an explicit Tropospheric Halogen Chemistry Model (THAMO): a one-dimensional atmospheric chemistry model75 that has been used in many previous studies  to simulate the halogen chemical processes (including the photolysis) in the boundary layer. The THAMO simulations were conducted for 24 h in an Arctic environment  during spring (1 May) to derive the photolysis rates of HClO3 and HClO4 in the Arctic boundary layer.To obtain the photolysis rates of HClOSupplementary InformationPeer Review File


Consequently, the open‐circuit voltage is extensively boosted from 1.02 to 1.14 V with the incorporation of Al2O3 filling the voids between grains, resulting in dense morphology of buried interface and reduced recombination centers. Finally, the impressive efficiencies of 23.1% (0.1 cm2) and 22.4% (1 cm2) are achieved with superior stability, which remain 96% (0.1 cm2) and 89% (1 cm2) of its initial performance after 1200 (0.1 cm2) and 2500 h (1 cm2) illumination, respectively. The dual modification provides a universal method to reduce interfacial defects, revealing a promising prospect in developing high‐performance PSCs and modules.Stability and scalability are essential and urgent requirements for the commercialization of perovskite solar cells (PSCs), which are retarded by the non‐ideal interface leading to non‐radiative recombination and degradation. Extensive efforts are devoted to reducing the defects at the perovskite surface. However, the effects of the buried interface on the degradation and non‐radiative recombination need to be further investigated. Herein, an omnibearing strategy to modify buried and top surfaces of perovskite film to reduce interfacial defects, by incorporating aluminum oxide (Al 2O3) nanoparticles are imbedded into the buried interface, which fills the voids and grain boundaries of perovskite, leading to compact morphology and reduced dangling bonds and defects. The suppressed trap‐assisted recombination, better energy alignment, and decreased J–V hysteresis in the modified device with Al2O3 nanoparticles and phenethylammonium bromide contribute to a significant increase in voltage and stability.The aluminum oxide (Al To date, the PCE of single‐junction PSCs has skyrocketed to 25.7% from the initial PCE of 3.8%. However, most of the PSCs with high PCE are small‐area cells and the PCE decreases remarkably when scaling up, so there is still much room to further improve the performance. On the other hand, to approach commercialization, considerable experimental efforts have been concentrated on improving the long‐term stability of PSCs. However, the long‐term stability of PSCs is still lagging behind commercial silicon solar cells. Massive defects could be formed at the surface and grain boundaries, resulting in serious non‐radiative recombination which significantly deteriorates the performance of PSCs. Therefore, it is important to passivate the defects at the surface and grain boundaries of perovskite film to realize the high efficiency and outstanding stability of PSCs.Over the past decades, photovoltaic (PV) technologies as power supply systems have received much attention due to the increased need for energy‐harvesting devices. Among the various techniques, perovskite solar cells (PSCs) are the most popular topic,1 whic butylammonium, and oleylammonium salts. Specifically, You and co‐workers reported highly efficient PSCs with a certified PCE of 23.32% and an open‐circuit voltage (VOC) as high as 1.18 V through forming a 2D PEA2PbI4 perovskite layer above 3D perovskite films using phenethylammonium iodide (PEAI). The defects and non‐radiative recombination were indispensably suppressed. It is well known that the 2D perovskite layer is prepared via a post‐treatment protocol, in which isopropanol (IPA) is applied to dissolve the organic ammonium salts. Nevertheless, IPA is detrimental to the 3D perovskite by dissolving formamidinium iodide (FAI). Recently, Mohite's group fabricated phase‐pure 2D halide perovskite with different thicknesses on 3D perovskite by choosing a suitable solvent acetonitrile (MeCN) to dissolve the 2D perovskite precursor, whereas the solubility of 3D perovskite is neglected in MeCN. This method is conducive to protecting the underlying 3D perovskite. A high PCE of 24.5% and remarkable stability of T99 > 2000 h were achieved.So far, a plethora of strategies have been proposed to passivate the defects of perovskite films. The generally used approach for passivating the perovskite top surface is to construct a 2D/3D interface using a solution‐based deposition of organic ammonium salts such as phenylethylammonium,10 butx/perovskite interface was modified by implementing organic cations with plentiful —NH2 groups, for example, guanidinium (GA) cations, leading to a notable increase of 65 mV in VOC and a PCE to 22.9%. Vaynzof and co‐workers adopted dual interfacial modification to simultaneously improve the VOC and fill factor (FF) on the basis of depositing large organic cation PEAI on the top and the buried perovskite film. This protocol is beneficial to grow high‐quality perovskite film, as well as the passivation of the top surface of perovskite film. As a result, maximum VOC and FF values of 1.184 V and 85% were reached, respectively. Huang et al. have revealed that extensive voids near the buried interface were generated during the deposition process of perovskite, due to the evaporation of residual dimethyl sulfoxide (DMSO) solvent, restricting the PCE and stability of PSCs under illumination. Therefore, modifying the buried and top interfaces of perovskite film is imperative for obtaining top‐performing PSCs. Nevertheless, most approaches focused on the top surface of perovskite film. There are scarce works concentrated on solving the bottleneck issues of the buried interface of perovskite film, predominately because the buried interface is not exposed, unlike the top surface of perovskite. It is difficult to straightforwardly mitigate the imperfections of the buried interface, leading to a huge room for improvement in the PV performance. Apart from defect passivation and non‐radiation recombination suppression, the buried interface is also vital for the crystal growth process. Among the several works devoted to modifying the buried contact layer, dielectric layer passivation has been applied for high‐performance PSCs, like PMMA and polystyrene (PS) interfacial layer, but the effect on the underlying dielectric layer is still not clear yet. Researchers have also investigated dielectric inorganics (Al2O3 and ZrO2) to passivate and self‐encapsulate the perovskite film. However, it is not widely used due to the sensitivity of electric tunneling to the thickness of the dielectric layer. Furthermore, the synchronous regulation of the buried and top interfaces has rarely been investigated by a combination of the dielectric layer and the organic passivation layer. Hence, rational modification for buried and top interfaces of perovskite film is desperately necessary to reduce undesired charge carrier recombination for high‐performance PSCs.In recent years, unremitting efforts have also been made to reduce the buried interfacial defects and improve the carriers' transport beneath the perovskite. For example, the NiO However, it is still challenging to deposit well‐ordered and compact SAMs layer due to the chemical properties of the substrate surface. The presence of imperfections at the substrate reduces the potential binding sites and the absorbability of SAMs on the substrate, and pinholes might be generated in the SAMs which undoubtedly connect the perovskite and bottom electrode, leading to an increment of recombination centers. Therefore, in order to reduce the defect densities and non‐radiative recombination at the interfaces of perovskite film, we propose an effective strategy using aluminum oxide (Al2O3) nanoparticles as a dielectric layer and alkylammonium salt phenethylammonium bromide (PEABr) as a passivation layer. Al2O3 nanoparticles were introduced into the heterointerface between HTL and perovskite, hampering the direct contact between perovskite and substrate, which result in a 90 mV increase in VOC based on [2‐ethyl] phosphonic acid (MeO‐2PACz) HTL and MA‐free perovskite. On the other hand, the effect of PEABr has been studied systematically, which was employed to modify both the buried and top interfaces of perovskite film. It was found that the VOC just increased by 20 mV when only PEABr was applied without Al2O3 nanoparticles. When Al2O3 nanoparticles and PEABr were simultaneously inserted into the buried perovskite film, and the perovskite top surface was concurrently passivated using PEABr, it is intriguing that 120 mV raise in VOC was observed. The significant increase in VOC is mainly attributed to the reduced non‐radiative recombination at the buried interface because the Al2O3 nanoparticles could fill the void between grains and enlarge the grain size, hampering the accumulation of carriers and improving the collection of carriers. The dual modification proposed in this work endows a remarkable increase in PCE from 19.8% to 23.1% based on the precursor composition of Cs0.15FA0.85Pb(I0.95Br0.05)3. Excitingly, the long‐term and humidity stabilities were also extensively ameliorated. The large‐area device with an area of 1 cm2 delivered a PCE as high as 22.4%, showing superior long‐term stability with the remaining 89% PCE of its initial value after continually illuminating for 2500 h. Owing to the scalable property of SAMs, we also fabricated the module based on a 5 cm × 5 cm substrate showing a PCE of 18.7%. The improved PV performance could be mainly attributed to the high‐quality perovskite film, better energy level alignment, faster charge extraction, and reduction of non‐radiative recombination at both the buried and top interfaces. Particularly, the smooth and compact morphology of the buried interface makes a great contribution to improving the PCE and stability. These results prove that this work paves the way for effectively passivating the defects at the buried and top interfaces of perovskite film and reveals the relationship between the morphology of the buried interface and the device performance.Here, self‐assembled monolayers (SAMs) were selected as HTL because of their outstanding properties such as easy preparation, minimal required concentration, and scalable fabrication.22 How22.1 Accordingly, dual surface modification was adopted to improve the device's PV performance, and four different kinds of inverted structures were designed, as shown in Figure61BM/C60/BCP/Ag prepared by a simple one‐step protocol where ethyl acetate (EA) is used as antisolvent. For the purpose of decreasing the defect densities of both interfaces, that is, buried and top surfaces of the perovskite layer, Al2O3 nanoparticles and PEABr were employed as the interface modification layers. For simplicity, surface treatment using Al2O3 nanoparticles on MeO‐2PACz is referred to as ST‐Al2O3 in the following context  of 24.3 mA cm−2, and an FF of 80%, leading to a PCE of 19.8%. After the modification using Al2O3 nanoparticles, the PCE increases to 21.8%, mainly attributed to a significant increase in VOC, which is enhanced to 1.11 V. The significantly improved VOC may attribute to the suppressed trap‐assisted recombination because the incorporation of Al2O3 fills the grain boundaries and regulates the nucleate and growth process, reducing the contact area of perovskite/HTL and the dangling bonds at the interface, thereby decreasing the interfacial defects, similar to a recent report. As for the device of D‐PEABr, the VOC shows a slight increase to 1.04 V, while the FF is clearly improved with respect to the control device. Surprisingly, the VOC could be further improved to 1.14 V based on the dual modification, which induces an extraordinary improvement in PCE of as high as 23.1% for the best‐performing ST‐Al2O3&D‐PEABr device. This efficiency is also exciting compared to other MA‐free perovskite devices with inverted structure  measurement was performed to study the surface chemical properties. The XPS survey spectra of MeO‐2PACz, MeO‐2PACz/Al2O3, and MeO‐2PACz/Al2O3/PEABr films are shown in Figure 2O3 and MeO‐2PACz/Al2O3/PEABr films, indicating the presence of Al2O3 nanoparticles on the MeO‐2PACz surface, as evidenced by the XPS spectra of O 1s core level  origination from MeO‐2PACz and the surface of In2O3 which is important to the absorption of MeO‐2PACz on the substrate. Additionally, we find the contribution of C—O—C belonging to the methoxy groups of MeO‐2PACz in accordance with the previous study. The peak at the highest binding energy may originate from other hydroxides and adventitious contaminants. We speculate that P—O species could not be resolved here because of limited sensitivity and possible overlap with the contaminant peaks. It should be mentioned that the O 1s peak shapes of MeO‐2PACz/Al2O3 and MeO‐2PACz/Al2O3/PEABr films are various from MeO‐2PACz film, and the peak of —OH deviate to higher binding energies compared with MeO‐2PACz film, which may be related to the introduction of Al2O3. Since the peak of the Al—O bond in Al2O3 is located at 531.5 eV, which is very close to the peak of —OH (531.3 eV). Additionally, the EDX element mapping and line scan also illustrate that the Al2O3 nanoparticles can be spin‐coated onto the surface of the MeO‐2PACz film , and C—O/C=O (≈286 to 289 eV), where C—C and C—N bonds belong to the FA+ and PEA+ of perovskite film, and the N+=CH—N and C—O/C=O are assigned to FA+ and oxygen/moisture, respectively. A slight shift of Pb 4f is detected, which is attributed to the interaction between the Pb2+ and —NH2 group of PEABr, indicating the effective passivation of the perovskite surface, and Br/Pb atomic ratio of ST‐Al2O3&D‐PEABr film increases compared to that of ST‐Al2O3&B‐PEABr film, suggesting that more Br− anions are accumulated at the perovskite surface. Besides, the ultraviolet photoelectron spectroscopy (UPS) measurement was conducted to explore the impact of surface treatments on the energy level alignment between individual layers. Figure 61BM, C60, BCP, and Ag are obtained from references. It is found that the synergistic effect of surface modifications using Al2O3 nanoparticles and PEABr at the buried interface results in a better energy level alignment, reducing the energy loss. Meanwhile, the energy level offset between perovskite and PCBM decreases after the PEABr treatment, which may be attributed to the formation of dipole moments between Br anions and PEA cations. Compared to PEAI passivation, a larger energy shift could be expected for PEABr because of the higher electronegativity of Br− than I−, which leads to a more appropriate energy level alignment for transporting electrons, similar to previous reports. The UPS results account for a significant improvement in VOC, which is in good agreement with the PV performance discussed before.To study the top surface chemistry of perovskite film with and without passivation of PEABr, we fabricated ST‐Alectively.34 A sα) of 1°. For measuring the perovskite film of the buried interface, the perovskite film was peeled off from the device encapsulated by UV‐curing glue and glass. It can be seen that an extra diffraction peak of perovskite film at the buried side appears with respect to the top perovskite film. The new peak could be assigned to the (003) plane of PbI2, and the area ratio of perovskite (100) to PbI2 (003) is calculated, as shown in Figure 2O3&D‐PEABr film suggests a superior crystal quality of perovskite film, probably owing to decreased defect states and improved morphology at the buried interface of perovskite film. According to previous reports, the 2D perovskite phase might be formed on the 3D perovskite surface after the passivation of organic ammonium salts on the top surface of the perovskite film. However, we do not observe the peaks of the 2D phase for D‐PEABr and ST‐Al2O3&D‐PEABr films. When decreasing the α to 0.5° and 0.3° , as shown in Figure α. For control, ST‐Al2O3, and D‐PEABr films, the position of the (100) plane shifts to lower angles as increasing the α, corresponding to the expansion of lattice spacing. However, the (100) lattice spacing of ST‐Al2O3&D‐PEABr film hardly changes, suggesting that the residual stress is released which is conducive to improving the performance of the device, in consistence with XPS and UPS results.Grazing incidence X‐ray diffraction (GIXRD) was performed to investigate the crystallinity and residual strain of perovskite films. Figure  of PbI2,25 and2O3, D‐PEABr, and ST‐Al2O3&D‐PEABr perovskite films are shown in Figures 2O3 nanoparticles can be observed in ST‐Al2O3 and ST‐Al2O3&D‐PEABr films. The grain size distribution suggests that the mean grain sizes of ST‐Al2O3, D‐PEABr, and ST‐Al2O3&D‐PEABr films increase slightly compared with the control film, probably attributing to the enhanced contact angle  and MeO‐2PACz/PEABr (3.98 nm) films. The increased surface roughness is attributed to the coverage of Al2O3 nanoparticles with a size of around 15–50 nm on the MeO‐2PACz substrate. The increased roughness will enlarge the crystal grains and reduce the voids in‐between perovskite grains, which decreases the surface recombination centers, thereby suppressing the non‐radiative recombination at the interface. On the other hand, the hole carries can transport through the space between Al2O3 nanoparticle which is infilled by perovskite. Both positive effects are beneficial to improve the photoelectric performance of devices. For ST‐Al2O3&D‐PEABr perovskite film, the RMS value decreases to 7.84 nm, compared to the ST‐Al2O3&B‐PEABr perovskite film (8.57 nm)  and the atomic force microscope (AFM). The top‐view SEM, cross‐section SEM, and the crystal size distributions of control, ST‐Al  and time‐resolved PL (TRPL) spectroscopies for the control, ST‐Al2O3, D‐PEABr, and ST‐Al2O3&D‐PEABr perovskite films. The PL spectra of perovskite film treated with PEABr show a slight blue shift, which could be ascribed to the introduction of the Br element, slightly increasing the bandgap  and slow (τ2) decay lifetime related to trap‐assisted recombination at the surface and bulk, respectively. The average carrier lifetime (τave) can be determined to be 217.08, 643.93, 362.12, and 666.55 ns for the control, ST‐Al2O3, D‐PEABr, and ST‐Al2O3&D‐PEABr perovskite films, respectively. The improved PL intensity and longer carrier lifetimes of treated perovskite films than the control can be ascribed to the larger grain size and improved film quality, demonstrating reduced defect densities and suppressed trap‐assisted recombination. To shed light on this, the space‐charge‐limited current (SCLC) measurement of hole‐only devices was conducted to evaluate the trap‐state density (nt) of perovskite with or without surface modifications, as shown in Figure nt is estimated from the formula shown in Supporting Information. The VTFL is 0.712, 0.457, 0.627, and 0.341 V for the control, ST‐Al2O3, D‐PEABr, and ST‐Al2O3&D‐PEABr, respectively, corresponding to the nt of 1.25 × 1016, 8 × 1015, 1.10 × 1016, and 5.97 × 1015 cm−3. The reduction of nt can be explained by the improved crystallization and passivated defects by Al2O3 nanoparticles and PEABr, resulting in a substantial increase in VOC and FF for the modified PSCs which is consistent with the above discussions. As normally acknowledge, the VOC loss originating from non‐radiative recombination decreases with increasing the electroluminescence (EL) efficiency (EQEEL). Figure EL as a function of applied voltage curves of ST‐Al2O3, D‐PEABr, and ST‐Al2O3&D‐PEABr PSCs. It is noted that the control device could not operate as light‐emitting diodes (LEDs), so the EQEEL of the control is not presented here. Clearly, the ST‐Al2O3&D‐PEABr device exhibits the highest EQEEL with respect to other devices, indicative of the minimal VOC loss, further illustrating the positive impact of Al2O3 nanoparticles and PEABr on suppressing the non‐radiative recombination via modulating the morphology and passivating defects at both buried and top interfaces of perovskite film.To further reveal the effect of surface treatments using Alap Figure5a.[41Rrec). Figure Rrec of devices with modification is dramatically increased, indicating the significantly retarded charge recombination. To provide a deep insight into the carrier dynamic of PSCs, we performed the Mott–Schottky measurement to extract the built‐in potential (Vbi). The capacitance–voltage (C–V) curves are provided in Figure Vbi can be deduced from the crossing with the x‐axis. Clearly, the Vbi remarkably increases  compared to the control (1.01 V), suggesting that the dual modification method is conducive to separate, transport, and extract the photogenerated charge carriers. Figure VOC as a function of light intensity, and the ideal factor (nID) can be evaluated from the equation described in supporting information. The nID values greater than and closer to 1 represent the appearance of defect‐assisted recombination and reduced defect‐assisted recombination in the device, respectively. It is found that the nID values show a clear decrease after modifications of Al2O3 nanoparticles and PEABr, proving that the modified devices have fewer defects, leading to lower non‐radiative recombination and improved VOC.In addition, electrochemical impedance spectroscopy (EIS) was conducted to collect the recombination resistance Rrec. Figu2O3 nanoparticles and PEABr in different compositions, Cs0.15FA0.85PbI3 and Cs0.05FA0.80MA0.15PbI2.5Br0.5 perovskite systems were prepared, as shown in Figure 0.15FA0.85PbI3 system, the VOC is extensively improved to 1.13 V for ST‐Al2O3&D‐PEABr device, compared with that of control with the VOC of 0.93 V, and the VOC could be increased to 1.16 (ST‐Al2O3&D‐PEABr) from 0.98 V (control) for Cs0.05FA0.80MA0.15PbI2.5Br0.5 perovskite system. These observations manifest that the dual modification strategy could be easily adopted in other perovskite systems to repress the notorious charge recombination and improve performance. In addition, we fabricated the 1 cm2 perovskite device based on the structure of ST‐Al2O3&D‐PEABr and Cs0.15FA0.85Pb(I0.95Br0.05)3 composition to validate the potential of these modifications in the scalable fabrication of PSCs, and the J–V curves are shown in FigureVOC = 1.17 V, JSC = 23.63 mA cm−2, FF = 80.93%) is achieved. Furthermore, the large‐area module with the structure of ST‐Al2O3&D‐PEABr based on 5 cm × 5 cm substrate was fabricated through the spin coating and laser scribing methods, and the structure of the module is shown in Figure J–V curves of the module , and the optical image of the module and laser patterning of P1, P2, and P3 lines are shown in Figure 2O3 nanoparticles and PEABr at both buried and top interfaces of perovskite films can not only be adequate in different perovskite composition systems but also be expanded to large‐area PSCs and modules, indicating the outstanding compatibility in realizing high‐performance PSCs.To verify the universality of the Ale Figure , the mod2.52 flowed box at the temperature of 25 °C. As shown in Figure 2O3 and D‐PEABr can maintain for 600 and 400 h before the PCE decreased to 80% of the original value, respectively. It is worth mentioning that the ST‐Al2O3&D‐PEABr device exhibits 96% of the initial PCE after operating for 1200 h, indicative of much better operational stability than that of control. Moreover, we also measured the humidity stability by aging perovskite films at a high humidity condition of 98 ± 1%, as displayed in Figure 2O3 at the buried interface hardly changes within 5 days, demonstrating that the introduction of Al2O3 nanoparticles could dramatically hamper the penetration of moisture and other external chemicals through the perovskite undersurface. The improved humidity stability may be attributed to the improved perovskite quality since the embedding of Al2O3 nanoparticles into the perovskite film induces the formation of compact morphology at the buried interface. Most astonishingly, the device with an area of 1 cm2 displays remarkable operational stability, as shown in Figure 2‐filled glovebox for more than 1150 h , and the best PCE is as high as 23.1% with VOC of 1.14 V, JSC of 24.9 mA cm−2, and FF of 82%. Meanwhile, the ST‐Al2O3&D‐PEABr device with an area of 0.2025 cm2 (without mask) has excellent long‐term operational stability, maintaining 96% of its original PCE after operating for 1200 h. Most interestingly, the ST‐Al2O3&D‐PEABr perovskite film exhibits outstanding moisture stability. Moreover, this modification strategy could be easily expanded into other perovskite systems, large‐area solar cells, and modules. Particularly, the large‐area solar cell shows a PCE of 22.4% and excellent long‐term stability with 89% PCE of the initial value after MPP tracking for 2500 h. The improved performance could be mainly attributed to the suppression of non‐radiative recombination because of the morphology changes at the buried interface and reduced imperfections. Moreover, the suppressed ion migration owing to decreased defects and alleviated J–V hysteresis also account for improved operational stability. This work presents an important insight for modifying the buried interface using a dielectric layer of Al2O3 nanoparticles and passivating the perovskite surface using PEABr, which plays a significant role in manipulating the energy level alignment, perovskite film morphology, film quality, and suppression of trap‐assisted recombination.In summary, we propose an omnibearing strategy for the inverted PSCs to reduce the defect densities at the buried interface of perovskite film, as well as the top interface employing AlThe authors declare no conflict of interest.Supporting InformationClick here for additional data file.


The metallic copper nanocrystals formed from CuCl precursor present preponderant Cu(100) and Cu(111) facets, likely to favour the adsorption of key *C1 and *C2 intermediates. Strikingly, the production rate of propylene drops substantially when carbon monoxide is used as the reactant. From the electrochemical reduction of isotope-labelled carbon dioxide mixed with carbon monoxide, we infer that the key step for propylene formation is probably the coupling between adsorbed/molecular carbon dioxide or carboxyl with the *C2 intermediates that are involved in the ethylene pathway.The conversion of carbon dioxide to value-added products using renewable electricity would potentially help to address current climate concerns. The electrochemical reduction of carbon dioxide to propylene, a critical feedstock, requires multiple C–C coupling steps with the transfer of 18 electrons per propylene molecule, and hence is kinetically sluggish. Here we present the electrosynthesis of propylene from carbon dioxide on copper nanocrystals with a peak geometric current density of −5.5 mA cm The electrochemical reduction of carbon dioxide to propylene requires multiple C–C coupling steps and the transfer of 18 electrons, posing kinetic and thermodynamic barriers. Now the electrosynthesis of propylene from carbon dioxide using copper nanocrystals has been demonstrated, with analysis revealing that the key step for its formation is probably the coupling between carbon dioxide or carboxyl with intermediates involved in the ethylene pathway. With copper-based catalysts, electrochemical CO2 reduction has shown an appreciable activity for the production of multiple C1 (refs. 6) and C2 chemicals9. Although C3+ terminal oxygenates such as n-propanol and n-butanol could be produced from CO2 reduction12, C3+ hydrocarbons, such as propylene (CH2=CH–CH3), have rarely been observed as products. Propylene, a critical chemical feedstock, has reached an annual global capacity of 130 Mt in 2019, requiring an input of energy equivalent to the one from about 190 million barrels of crude oil and entailing around 80 Mt of CO2 emission . The electrosynthesis of propylene from CO2, yielding a negative carbon footprint, is an attractive strategy for producing this indispensable feedstock for the polymer industry and is yet to be achieved.The electrochemical conversion of carbon dioxide  of 0.9% at −1.8 V versus RHE14. Recently, Pablo-García et al. proposed that the production of propylene could be traced to the allyl alkoxy (CH2=CHCH2O) intermediate, easy desorption of which in an alkaline microenvironment results in the unfavourable production of propylene15. This conclusion helps to explain why propylene is rarely being produced/detected in CO2 reduction, which contrasts with the production of ethylene18. In-depth understanding of the reaction pathway towards propylene formation is warranted for designing catalysts for this reaction.Lee et al. observed propylene formation from electrochemical CO2 reduction with appreciable selectivity and production rate. By carrying out well-designed control experiments, including the reductions of CO, CO2/CO, CO2/He and 13CO2/CO mixtures, we propose that propylene generation shares a highly protonated *C2 intermediate with ethylene generation, and *CO is unlikely to be the *C1 intermediate that couples with *C2 species for propylene formation. This contrasts with the n-propanol pathway where *CO is proposed to be the key *C1 precursor participating in the *C1–*C2 coupling.Here we synthesize copper nanocrystals (CuNCs), the surfaces of which predominantly consist of Cu(100) and Cu(111) facets, enabling the electrosynthesis of propylene from CO19, was electrochemically pre-reduced to form copper NCs. X-ray diffraction and high-resolution X-ray photoelectron spectroscopy (XPS) confirm the presence of CuCl after electrochemical roughening  and Cu(111) facets conjoined are also discernible.CuNCs were then used as electrocatalysts and characterized after 10 min of CO nm Fig. , consist nm Fig. , represe nm Fig. 22, respe2 with high reaction rates at moderate potentials in aqueous 1 M KOH with additional 0.2 M CsI (refs. 24)  adsorption to reveal the difference in their catalytic activity. Two cathodic peaks at approximately −0.02 and −0.06 V versus RHE, which could be respectively assigned to the UPD of lead on Cu(111) and Cu(100) facets, are observed  and Cu(100) were calculated to be 1.3 for CuNCs and 2.7 for polycrystalline copper, respectively (Supplementary Table − adsorption peaks on Cu(100) and Cu(111) are present at ~0.36 and ~0.46 V versus RHE29, respectively  and hydroxide (OHjspecific and turnover frequency (TOF) 31, while coexistence of Cu(111) could provide a conjoined interface for stabilizing key intermediates for multi-carbon products22.We then normalized the partial current of propylene against the electrochemical surface area of two catalysts . Akhade et al. reported that a small quantity of KI improves current density at more negative potentials on a copper electrode by enhancing the reaction energetics of *CO coupling due to the presence of I− ions in the electrochemical double layer39. This is consistent with the increased CO2 reduction current observed on our CuNCs at potentials less than −0.6 V  and Cu(111) facets are still present at ~0.37 and ~0.48 V versus RHE, respectively. Further analysis of their charges evidenced a ratio of 1.05 between the active area of Cu(111) and Cu(100)  of propylene and the j of a specific C2 product . We also introduced CO into the feed gas to form mixtures of CO/CO2 to tune the production rate of the products of interest /*CO leads to the formation of n-propanol. This is consistent with the finding of Pablo-García et al., showing that the lowest activation barrier for C3 backbone formation is the coupling of CH2CH + CHO or CH3CH + CHO(H), where CHO(H) is formed from *CO hydrogenation15. The difference in the structure of *C1 intermediate between the propylene pathway and the n-propanol pathway leads to the formation of n-propanol being less affected by the change of feed gas  compared to the standard mass spectra of two chemicals  to gain more insights into the *Cals Fig. .Fig. 4GC12CO2 reduction show the same ionized molecules and fragments as compared to their respective standard gas  and 3  for ethylene and propylene, respectively  and 12C2H4 (from the 12CO–12CO pathway), with a low percentage of 12C2H4. The MS signals of propylene also show a wide range of m/z ratio from 40 to 45, with the main fragment peaks locating at 44 and 45  and Cu(111) facets, providing active centres for binding the key *C2 molecules. The intermediate species involved in this process are more numerous than the ones revealed by our study. Nevertheless, the mechanism uncovered in our study offers opportunities for designing advanced catalysts for the efficient production of this crucial chemical feedstock. Although still far from large-scale practical implementation, this study opens a pathway to the electrosynthesis of propylene via electrochemical CO2 reduction.Transfer of 18 electrons is required for the production of one propylene molecule from three COAll chemicals are used as received without further purification. Deionized water  was used for preparing solutions and washing samples.1. Here, a 920 nm-thick Cu film (99.995%) was deposited via sputtering  onto a GDL . The Cu/GDL substrate was then electrochemically roughened in 0.1 M KCl electrolyte by repeating five cycles of triangular potential scans. Each triangular potential scan includes three steps: (1) a chronoamperometric step at an applied potential of 0.24 V versus RHE for 10 s; (2) a linear sweep voltammetric step from 0.24 to 1.74 V versus RHE at a scan rate of 500 mV s−1; (3) a chronoamperometric step at an applied potential of 1.74 V versus RHE for 5 s. The as-prepared GDL-supported CuCl films were then rinsed thoroughly with deionized water and dried using compressed air. The CuCl films were pre-reduced in a custom-designed flow cell at a constant current density of −30 mA cm−2 for ~80 s. The aqueous electrolyte was prepared by dissolving 56.11 g of KOH (Reactolab) and 51.96 g of CsI  into 1 litre of deionized water.The synthesis procedure for the CuCl layer was modified from a previous studyλ = 1.5409 Å) with a grazing incident beam. XPS was performed using a PHI VersaProbe II scanning XPS microprobe . Analysis was carried out using a monochromatic Al Kα X-ray source of 24.8 W power with a beam size of 200 µm.The surface morphologies of copper-based samples were acquired using a field emission scanning electron microscope (Zeiss Merlin). Image acquisition was carried out via an in-lens detector under an accelerating voltage of 15 kV. High-resolution transmission electron micrographs of the catalysts were obtained using a transmission electron microscope  equipped with a high-angle angular dark field detector. X-ray diffraction was measured on a Bragg–Brentano instrument  was used . The flow rate of the cathodic chamber was also confirmed at the GC outlet by a soap-bubbled volumetric meter. The gases employed in this study include 12CO2 , 13CO2 , 12CO , helium  and their mixtures. The ratio between 12CO2/12CO, 13CO2/12CO or 12CO2/He was tuned by adjusting the corresponding flow rate of each gas, with the total flow rate being 10 cm3 min−1.The electrolysis was performed using a Gamry (Interface 1000) potentiostat and each reduction reaction lasted for ∼3,000 s. 2. A gas diffusion layer with sputtered 200-nm-thick platinum (99.995%) was used as the counter electrode and a Ag/AgCl electrode  was used as the reference electrode. The electrochemical potential of the Ag/AgCl was calibrated daily against an RHE  immersed in 0.1 M HClO4 solution, prepared by diluting 0.862 ml of HClO4  into 100 ml deionized water. All of the potentials measured in this work were referenced to the RHE using the following conversion:For each measurement, fresh working electrodes and electrolytes were used. The geometric surface area of the working electrode was 0.33 cm−1 and holding at 180 °C for 2.5 min; (3) increasing to 260 °C with a ramp rate of 40 °C min−1 and holding at 260 °C for 3.5 min. The total run time was 14.5 min. The pulse discharge detector signals were calibrated using standard gas mixtures (Carbagas) with all relevant gases, including H2, CO, CH4, C2H4, C2H6 and C3H6. The liquid products that dissolved in the electrolyte were collected after electrolysis. The electrolyte was mixed with D2O  and an internal standard consisting of 25 mM phenol  and 5 mM dimethyl sulfoxide . Then the prepared solution was analysed on a 1H NMR spectrometer  and water suppression technique was applied , CH3COONa , C2H5OH , C3H7OH  and C3H6O  in 1 M KOH were used for calibration.During the electrolysis three gas aliquots were automatically injected into an online GC . The first aliquot was sampled at ∼600 s after the start of reaction to ensure adequate equilibrium of gas phase. The gas products were separated by a micropacked shincarbon column (Restek) and quantified by a pulse discharge detector (Vici). The oven was programmed as follows: (1) holding at 60 °C for 3.5 min; (2) increasing to 180 °C with a ramp rate of 40 °C minThe partial current density of each product is calculated by multiplying the FE by the total geometric current density. The average of multiple measurements was used in this work.−1 cm−2, is calculated based on the specific surface areas of catalysts determined by lead UPD measurements:The TOF, in units of nmol sThe TOF value given in this work corresponds to the average of three independent measurements.13CO2  was also used as the reactant gas. Pure 13CO2 and mixed 13CO2–12CO feeding experiments were carried out using the same protocol as mentioned above. Gas products were collected into a withdrawal valve after the system reached equilibrium and were injected by microsyringe into a GC–MS instrument (Agilent 7890B/5977A). A GC equipped with a capillary column  was used for chemical separation with helium  as the carrier gas. The GC was interfaced with an MS , which was operated with a filament current of 34.6 μA and electron energy of 70 eV in electron ionization mode. The data acquisition and processing were performed with GC/MSC MassHunter Acquisition software. The signals were referenced to National Institutes of Standard (NIST) library.Isotopically labelled − adsorption on copper active sites was performed by CV using N2-saturated 1 M KOH solution with 0.2 M CsI additive as the electrolyte. All the measurements were carried out using the same flow cell as used to perform CO2 electroreduction; electrolyte and helium was continuously purged into the corresponding chambers with flow rates of 0.25 cm3 min−1 and 10 cm3 min−1, respectively. Both copper-based electrodes were pre-reduced at −30 mA cm−2 for ∼80 s and then the potential was kept at 0 V versus RHE for 3 min immediately before measuring OH− adsorption. The CV curves were recorded within a potential range from 0 to 0.6 V versus RHE at a scan rate of 100 or 50 mV s−1. The charge of OH− adsorption on the respective facet of Cu(100) and Cu(111) was calculated by integrating the corresponding peak. Reference values of 8.22 and 2.16 μC cm−2 were used for calculating the surface areas of Cu(100) and Cu(111), respectively2.Electrochemical OH2-saturated 0.1 M HClO4 solution with 10 mM Pb(OAc)2 into the flow reactor. The catalysts were first preconditioned at −30 mA cm−2 for ∼80 s and the potential was kept at the initial potential of the CV scans for an additional 3 min, followed by immediately recording the CV curves from −0.12 to 0.31 V versus RHE for CuNCs and from −0.16 to 0.19 V versus RHE for polycrystalline copper at a scan rate of 10 mV s−1. The facet areas of Cu(100) and Cu(111) were calculated by normalizing the deposition charge to the reference values: 262 μC cm−2 for (100) and 285 μC cm−2 for (111), respectively2. As a control experiment, the lead UPD of the CuNC and polycrystalline catalysts that were prepared onto the non-porous glass was also conducted in a three-electrode set-up.Lead UPD measurement was carried out by flowing a N2 reduction. Each reaction was performed at −250 mA cm−2 (corresponding to ∼0.60 V versus RHE) for ∼3,000 s and the other parameters were the same as the ones used for CO2 reduction. Aqueous 1 M KOH solutions containing 0.2 M CsI additive, with different concentrations of allyl alcohol, that is, 1, 5, 10 and 50 mM, were respectively pumped into the reactor at a flow rate of 0.25 cm3 min−1. Helium was fed into the cathodic gas camber at a flow rate of 10 cm3 min−1. The gas products in helium matrix were automatically sampled into an online GC and the collected electrolytes after electrolysis were analysed by 1H NMR. The feed rate of allyl alcohol (vf) and the production rate of propylene (vp) were first calculated from the following equations:cAA is the concentration of allyl alcohol;velectrolyte is the flow rate of electrolyte (0.25 cm3 min−1);The electrochemical reduction of allyl alcohol was carried out in the same flow cell used for COSWE is the geometric surface area of working electrode (0.33 cm2 in our flow cell);npropylene is the number of moles of propylene detected by GC; andt2 is the time required to fill up the sample loop (20 μl)vp as a function of vf at different concentrations of allyl alcohol, a linear correlation between these two parameters was obtained with a slope of 0.026  is calculated to be  is calculated as (Supplementary Table vp-total represents the total production rate of propyleneThe percentage of propylene being produced from the reduction of allyl alcohol (Any methods, additional references, Nature Portfolio reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at 10.1038/s41557-023-01163-8.Supplementary InformationSupplementary Figs. 1–25, Tables 1–12, and refs. 1 and 2.


BK virus (BKV) is a very common pathogen infecting up to 90% of the general population . While uN = 397) for the presence of serum IgG antibodies against the structural BKV protein VP1, using an ELISA assay   IgG concentration of 23 13–38] µg/ml Below is the link to the electronic supplementary material.


Mammalian target of rapamycin complex 1 (mTORC1) is highly activated in diabetes, and the decrease of low‐density lipoprotein receptor‐associated protein 1 (LRP1) in brain microvascular endothelial cells (BMECs) is a key factor leading to amyloid‐β (Aβ) deposition in the brain and diabetic cognitive impairment, but the relationship between them is still unknown.fl/+) mice to investigate the role of mTORC1 in regulating LRP1‐mediated Aβ efflux and diabetic cognitive impairment at the tissue level.In vitro, BMECs were cultured with high glucose, and the activation of mTORC1 and sterol‐regulatory element‐binding protein 1 (SREBP1) was observed. mTORC1 was inhibited by rapamycin and small interfering RNA (siRNA) in BMECs. Betulin and siRNA inhibited SREBP1, observed the mechanism of mTORC1‐mediated effects on Aβ efflux in BMECs through LRP1 under high‐glucose conditions. Constructed cerebrovascular endothelial cell‐specific Raptor‐knockout (Raptorfl/+ diabetic mice had significantly inhibited activation of mTORC1 and SREBP1, increased LRP1 expression, increased Aβ efflux, and improved cognitive impairment.mTORC1 activation was observed in HBMECs cultured in high glucose, and this change was confirmed in diabetic mice. Inhibiting mTORC1 corrected the reduction in Aβ efflux under high‐glucose stimulation. In addition, high glucose activated the expression of SREBP1, and inhibiting of mTORC1 reduced the activation and expression of SREBP1. After inhibiting the activity of SREBP1, the presentation of LRP1 was improved, and the decrease of Aβ efflux mediated by high glucose was corrected. RaptorInhibiting mTORC1 in the brain microvascular endothelium ameliorates diabetic Aβ brain deposition and cognitive impairment via the SREBP1/LRP1 signaling pathway, suggesting that mTORC1 may be a potential target for the treatment of diabetic cognitive impairment. fl/+ mice are used to exclude the effects of other brain cells and tissues and to avoid the manipulation of mTORC2 signaling. The results first confirm that Mammalian target of rapamycin complex 1 in the brain microvascular endothelial ameliorates diabetic Aβ brain deposition and cognitive impairment via SREBP1/LRP1 signal pathway.Raptor Epidemiological studies have shown that diabetes is associated with dementiaThere are characteristic pathological changes associated with AD in the brain tissue of patients with diabetic cognitive impairment, such as amyloid plaques (mainly amyloid‐β), neuronal fibers, and entanglement.As a multisubunit complex of mammalian target of rapamycin (mTOR), mammalian target of rapamycin complex 1 (mTORC1) is composed of regulatory associated protein of mTOR (Raptor), proline‐rich AKT substrate 40 kDa (PRAS40), and mammalian lethal with sec‐13 (mLST8). Raptor is an mTOR‐binding protein that regulates mTORC1 activity by binding to the downstream mTORC1 substrates phosphoprotein 70 ribosomal protein S6 kinase (P70S6K) and 4 E binding protein 1 (4EBP1) and promotes translation and protein synthesis.22.1tm1.1Dmsa/J mice with SP‐A Cre transgenic mice that express Cre recombinase in cerebrovascular endothelial cells to generate cerebrovascular endothelial cell‐specific knockout mice (Raptorfl/fl). Due to the Raptorfl/fl genotype, Cre + mice did not develop after birth, and so they could not eat and died. Therefore, we used Raptorfl/+; Cre+ (Raptorfl/+) male mice as the experimental group and wild‐type male mice as the control group. The experimental mice were housed in a specific pathogen‐free environment with free access to food and light and dark cycles for 12 h. After 1 week of adaptation, wild‐type mice and Raptorfl/+ mice were randomly divided into the control and diabetic groups. Diabetic mice were intraperitoneally injected with 60 mg/kg/d STZ for five consecutive days, and the control group were injected with the same dose of sodium citrate buffer. After 1 week, mice with blood glucose ≥16.7 mmol/L were considered as having type 1 diabetes and were prepared for subsequent experiments. There were four groups of animals, including the control group, Raptorfl/+ group, diabetes group, and Raptorfl/+ diabetes group, with 15 mice in each group. Finally, excluding accidental death and modeling failure, the number of mice in each group was 8, 12, 9, and 8, respectively. This study conformed to the principles of animal protection and institutional ethics in China. All animal protocols were approved by the Animal Care and Use Committee, Tongji Medical College, Huazhong University of Science and Technology. The IACUC approval number was [2019] 2570.We crossed Raptor2.2Details can be found in Supplementary Material 2.32 and 95% air) to the recommendations. Cells were cultured in starvation solution for 24 h before various treatments. HBMECs were pretreated with rapamycin (20 nM) and Betulin (50 mM) for 1 h before high‐glucose treatment.Human brain microvascular endothelial cells (HBMECs) were purchased from ScienCell Research Laboratories (ScienCell 1000) and cultured in 1640 medium supplemented with 10% fetal bovine serum according at 37°C in a humidified atmosphere (5% CO2.44/cm2 on top of semipermeable Transwell polycarbonate tissue culture inserts (pore size 0.4 μm and bottom diameter 6.5 mm) (Corning Costar Corp.). The fluid was changed every 2 days. The transendothelial resistance was monitored by an EVOM epithelial voltmeter with an STX2 electrode (World Precision Instruments). When the resistance value peaked, the cells formed a continuous monolayer and were used as a blood–brain barrier model for subsequent functional experiments  to eliminate exploratory reflexes as follows: 20 s of noise stimulation, and at the end of the noise during the last 2 s and electric shock (0.5 mA) was administered at intervals of 30 s after repeated noise stimulation and electric shock, for a total of five cycles. After 24 h, the mice were placed in a Skinner box, and the percentage of time the mice spent freezing (freezing time) was recorded within 5 min.2.5.2of to determine the cognitive index was RI = tC/(tA + tC) × 100%.The new object recognition experiment included an adaptation period, familiarity period, and test period, for a total of 3 days. The mice were placed in a test room 3 days in advance to eliminate the stress response. On Day 1, the mice were placed in the test box without any objects allowed to move freely for 5 min. On Day 2, after 30 min of adaptation, the mice were placed in a test box with two identical objects (A and B) in the two corners, and then the mice were placed back in the box with the two objects in the middle of the test box. Then the mouse's nose or mouth was ≤2 cm away from the object indicated observation, and the exploration of the two objects within 5 min was recorded by the Ethovision XT animal trajectory tracking system . On Day 3, object B was replaced with object C, which had a different shape, and the remaining steps were the same as those performed on Day 2. The exploration time of objects A and C was recorded as tA and tC, respectively. The test chamber was wiped with 75% alcohol before each mouse and object were placed to eliminate odor effects. The formula 2.6g for 5 min. The obtained tissue was added to 18% dextran and centrifuged at 4°C and 2500 g for 20 min to obtain the initial sample of brain microvasculature. Then, the sample was resuspended in PBS and filtered through a 40 μm cell filter. The brain microvasculature at the top of the filter was used for the experiment.After the behavioral experiment, three mice in each group were anesthetized. After successful anesthesia, the mice were perfused with 4°C saline until the liver, lung, and tongue were white and then were perfused with 4% paraformaldehyde in the original position until the mice were stiff. After the brain tissue was removed, it was immersed in 4% paraformaldehyde and fixed overnight at 4°C. Six mice in each group were immediately frozen in liquid nitrogen and stored at −80°C for Western blot, qRT‐PCR, and ELISA analysis. The experimental method to prepare brain microvessels was described previouely.2.7We performed immunofluorescence staining on brain tissue sections and HBMECs. The tissue sections were dewaxed with xylene and then rehydrated in ethanol. High glucose‐treated HBMECs were fixed on cell slides with 4% paraformaldehyde. The fixed sections and cell slides were transferred to blocking solution (goat serum) for 1 h. The blocked HBMECs were incubated overnight at 4°C with primary antibodies against SREBP1  and p‐mTOR . The tissue sections were incubated overnight at 4°C with primary antibodies against SREBP1 , LRP1 , Raptor  and CD31 . Immunoreactions were observed using CY3‐conjugated affinity‐pure goat anti‐rabbit IgG  and FITC‐conjugated affinity‐pure goat anti‐mouse IgG . Nuclei were stained with Hoechst (Invitrogen). Antifluorescent quenchers were added, and the slides were observed under a microscope (Olympus CX31 microscope).2.8Tissue sections were dewaxed with xylene and gradiently hydrated in ethanol for antigen repair. Endogenous peroxidase was blocked with 3% hydrogen peroxide at room temperature for 20 min, and the sections were then incubated in 10% goat serum for 30 min. The washed sections were incubated with primary antibodies against Aβ  at 4°C overnight. On the next day, the sections were reacted with HRP‐labeled goat anti‐Ig G polymer for 30 min, and DAB was used to observe the color reaction. Target detection was performed under a microscope (Olympus CX31 microscope).2.9g for 10 min. The supernatant protein concentration of the supernatant was determined by a BCA kit  to ensure the same quality of each sample. After being leveled, the supernatant was fully mixed with loading buffer and boiled for 10 min. Equal amounts of denatured supernatant were transferred to a nitrocellulose membrane after SDS‐PAGE, blocked with 5% skimmed milk powder, and incubated with the corresponding primary antibody and enzyme‐labeled secondary antibody. Protein expression was observed with an enhanced chemiluminescence detection reagent. The results were recorded by a ChemiDoc imaging system (Bio‐Rad) and analyzed quantitatively by ImageJ software.The lysis buffer  was added to the prepared tissue and cell samples and was lysed at 4°C for 30 min and nuclear and cytoplasmic protein extraction kits (Beyotime Biotechnology) were used to separate cytoplasmic and nuclear proteins. The lysate was collected and centrifuged at 13,902 2.10Total RNA was extracted from prepared cerebral microvessels using TRIzol reagent. cDNA was reverse transcribed using PrimeScript RT Master Mix. A SYBR Premix Ex Taq kit and an iCycler Real‐Time PCR detection system (Bio‐Rad) were used for quantitative PCR. RNA expression levels were analyzed by normalizing to β‐actin, and the value was determined by the 2‐ΔΔCt method. The primer sequences can be found in Table 2.11As described previously,Total TRAMA‐Aβ40/42 and total inulin were the sums of the apical and basolateral chambers. TRAMA‐Aβ40/42: Abs/Em = 544/572 mm; FITC‐inulin: Abs/Em = 490/520 mm.2.12Experimental procedures were performed based on the product instructions. Briefly, the standard sample was diluted to 50 μL, and the concentrations were 120, 80, 40, 20, and 10 pg/mL. The blank and sample wells were set up, and the sample was added to make a final 5× dilution of the sample. The enzyme was added, and the plate was incubated and washed, after which the color liquid was added and incubated at 37°C in the dark for 10 min, and the reaction was terminated. The absorbance  of each well was measured at a wavelength of 450 nm wavelength after zeroing with the blank well.2.13t‐test, and data between multiple groups were analyzed by one‐way analysis of variance (ANOVA) and Tukey's post hoc test. Two‐way ANOVA and Bonferroni post hoc correction were used for multiple comparisons. The relevant experimental results were verified at least three times and are displayed as the mean ± SEM. If p < 0.05, the difference was considered significant. In addition, the graphical abstract was created with FigDraw.Statistical analyses were performed using GraphPad Prism 7.0 software. The Shapiro–Wilk test was used to verify the normality of the distribution of the data. Significant differences between the two groups were analyzed by unpaired 33.1It is known that the activation of mTORC1 in the hippocampus occurs in the early stage of AD and is consistent with the degree of cognitive impairment in AD patients.3.2The role of cerebral vascular endothelial LRP1 in mediating Aβ deposition has been widely recognized. We observed the activation of mTORC1 in the diabetic cerebrovascular endothelium. To explore whether mTORC1 regulates LRP1‐mediated Aβ efflux, some experiments were performed. First, we used rapamycin to inhibit mTORC1 in HBMECs and observed that the efflux of Aβ40 and Aβ42 damaged by high glucose was improved by rapamycin Figure , and tra3.3Studies have shown that in diabetes, mTORC1 can be activated to induce the transcriptional expression and functional activation of Sterol‐regulatory element binding protein 1 (SREBP1).3.4To determine whether SREBP1 is involved in high glucose‐induced LRP1‐mediated Aβ efflux disorder in the cerebral vascular endothelium, we targeted SREBP1 with inhibitors, silenced genes and measured the mRNA and protein expression levels of SREBP1 and LRP1 in HBMECs stimulated with high glucose by real‐time quantitative PCR and Western blotting. The results showed that the expression of LRP1 decreased under high‐glucose stimulation. When SREBP1 was inhibited, the downregulation of LRP1 expression caused by high glucose was improved Figure . Consist3.5fl/+ mice, Raptor expression in brain tissue was ablated, as shown by double immunofluorescence staining of CD31 positive regions in brain tissue with the monoclonal antibody against Raptor .None.SupinfoS1Click here for additional data file.


The applicability of the assay was demonstrated.Lytic polysaccharide monooxygenases (LPMOs) are industrially relevant enzymes that utilize a copper co-factor and an oxygen species to break down recalcitrant polysaccharides. These enzymes are secreted by microorganisms and are used in lignocellulosic refineries. As such, they are interesting from both the ecological/biological and industrial perspectives. Here we describe the development of a new fluorescence-based kinetic LPMO activity assay. The assay is based on the enzymatic production of fluorescein from its reduced counterpart. The assay can detect as little as 1 nM LPMO with optimized assay conditions. Furthermore, the reduced fluorescein substrate can also be used to identify peroxidase activity as seen by the formation of fluorescein by horseradish peroxidase. The assay was shown to work well at relatively low H This futile cycle has been used as a proxy for LPMO activity using ascorbate or Amplex Red as the probe  into the chromophore coerulignone (Lytic polysaccharide monooxygenases (LPMOs) are important industrial enzymes for the production of lignocellulosic ethanol . LPMOs ahe probe . While Lnsume it , and thiulignone . Anotherulignone . This asulignone . BranderThermoascus aurantiacus (TaAA9A) (We have investigated fluorescein (Fl) as an alternative to Ph as an LPMO activity probe. Fl shares the xanthene structure with Amplex Red and the triphenol methane structure with Ph. Due to these similarities, we hypothesized that reduced fluorescein (rFl) could be used as an LPMO substrate. Fl is one of the original synthetic dyes, first synthesized by Baeyer in 1871 and is one of the most fluorescent organic molecules . To test(TaAA9A) . This is(TaAA9A) , 2021. F2.Thermoascus aurantiacus AA9 (TaAA9A) UniProtKB: G3XAP7, Thermothielavioides terrestris AA9 (TtAA9E) UniProtKB: D0VWZ9, Neurospora crassa AA9 (NcAA9E) UniProtKB: Q7RWN7, and Neurospora crassa AA9 (NcAA9A) NCBI: XP_959499 were kindly donated by Novozymes A/S. Lentinus similis AA9 (LsAA9A) UniProtKB: A0A0S2GKZ1, Pseudomonas fluorescens CopC UniProtKB: A0A0D0TME7, Lactobacillus plantarum AA10 (LpAA10A) UniProtKB: A0A165NIT9, Streptomyces coelicolor AA10 (Sc10AA10B) UniProtKB: A3KIM2, Thermobifida fusca AA10 (TfAA10A) UniProtKB: Q47QG3, Bacillus subtilis laccase (CotA) UniProtKB: H8WGE7 were expressed as described previously (Synechocystis flavodoxin (IsiB) UniProtKB: P27319 and Arabidopsis thaliana cytochrome P450 reductase (ATR) UniProtKB: F4JQY4 were kindly provided by Dr. Silas Busck Mellor (University of Copenhagen). All enzymes were purified to homogeneity before use. Alcohol dehydrogenase, ascorbate oxidase, superoxide dismutase, galactose isomerase, galactose oxidase, laccase from Trametes versicolor and horseradish peroxidase (HRP) were purchased from Merck . CBHII and β-glucosidase were purchased from Megazyme . The protein concentration of LPMOs were determined by acid hydrolysis followed by separation and quantification of individual amino acids with ion-exchange-HPLC  unless otherwise stated. eviously . Synechonge-HPLC . Flavodo2.1.A stock solution of rFl was prepared by reducing fluorescein with zinc powder. For this, 1 g fluorescein, 6 g zinc powder and 2.1 g NaOH were suspended in 500 ml water and boiled for 12 h as follows. The ingredients were mixed in a glass bottle with a magnetic stir bar with the lid off. A small glass funnel was placed on top of the bottle, and the normal lid was loosely placed over it. The bottle was then placed on a stir plate, the heating plate was set to 120°C, and the mixture was left to slow-boil overnight. Glacial acetic acid (3.6 mL) was then added, and the solution boiled for a further 2 h. The rFl solution was allowed to cool to room temperature, and the zinc oxide was removed by decanting the rFl solution into a new flask. Some fresh zinc powder was added, and the solution was stored at 4°C for over a month without loss of activity. The rFl stock concentration was determined to be 5 mM by first oxidizing a 0.125% solution to completion with HRP and comparing the emission of fluorescence at 528 nm to a standard curve for Fl .2.2.2O2. In one instance, ascorbate was added instead of DHA. The reactions were prepared in 100 mM citrate–phosphate buffer, pH 7.25, and H2O2 was always added last. Samples were prepared in triplicates in black 200 μL microtiter plates . The plate reader reports relative fluorescence units (RFU) and in assay conditions the conversion factor is ε = 181,100 RFU/μM. This number together with the enzyme concentration C is used to calculate turn over frequencies of the enzymatic oxidation of rFl: TOF = rate / (ε x C).The enzymatic production of fluorescein was determined by fluorescence reading using a Biotek Synergy H1 plate reader  with excitation at 488 nm and emission at 528 nm. Assay conditions as follows: 0–1 μM Cu(II)-LPMO, 0–250 μM dehydroascorbic acid (DHA), 6.35 μM rFl and 0–1,000 μM H2.3.2O2, 6.35 μM rFl. These are the conditions used to determine the assay limit of detection and the protocol goes as follows (96 samples). Step1: Prepare a black microtiter plate with 50 μL protein containing samples. Step 2: Prepare an assay mix of 18 mL 100 mM citrate–phosphate buffer, pH 7.25, 4.8 μL 50 mM DHA, 30 μL 5 mM rFl and 2.4 μL 0.1 M H2O2. Step 3: Add 150 μL of the assay mix to each protein sample. Step 4: Place the microtiter plate in a spectrophotometer and read the fluorescence intensity  for 30 min. Prior to each reading, shake the plate using a double-orbital movement for 5 s.During the course of this study, the assay conditions for rFl oxidation were optimized in a detailed assay protocol to give final concentrations of 10 μM DHA, 10 μM H3.2O2 at an equimolar concentration with DHA was included due to the finding that the LsAA9A activity was strictly dependent on H2O2 (2O2 in a citrate–phosphate buffer at pH 7.24. This resulted in a clear time-and enzyme-concentration-dependent increase in Fl fluorescence (2 was used instead of the enzyme showed that “free” Cu(II) did not cause any increase in fluorescence, and that Cu(II) without DHA or H2O2 did not lead to the development of fluorescence  that was expected to be a positive control . Clearlying LPMO . Laccaseing LPMO . HRP exhn of DHA .TaAA9A was diluted in LB media or milli Q water. LB media decreased the rFl oxidation rate but the activity was clearly above the assay background with enzyme concentrations as low as 75 nM Cu(II)-TaAA9A -)-TaAA9A .3.2.TaAA9A in presence of 0–250 μM DHA or 0–250 μM ascorbate and 100 μM H2O2. Addition of DHA enhances the enzyme activity by a factor of 100, and the full effect is realized at 100 μM DHA with only a small diminishing effect of adding more. Addition of ascorbate also enhances the enzyme activity to a similar level, but the full effect is reached at 3–7.5 μM and adding more gives a diminishing effect  from samples with or without enzyme as the threshold for assay sensitivity. These limits are visualized in the dose response plot and show that the limit of detection (LOD) in the rFl assay is 0.5 nM Cu-TaAA9A. However, the blank is slightly below the linear dose response curve and will overestimate the quantification limit. The assay cannot distinguish between 0.5 nM and 1.0 nM Cu-TaAA9A and more reasonable assessment of sensitivity is that limit of quantification (LOQ) of the rFl assay is 1 nM Cu-TaAA9A. We repeated the screening of LPMOs in the lower concentration range, and found a relatively similar assay response as in the concentrated samples (The low concentration optima for both ascorbate and Hactivity . We chosrelation . Breslma samples .4.4.1.2O2 has been demonstrated and used to develop an enzyme activity assay. The product is the stable and fluorescent Fl compound and the reaction can be monitored continuously by fluorescence spectrophotometry. The rFl compound was produced in the laboratory by boiling Fl with solid zinc followed by neutralization and filtration. A commercial preparation of rFl was tested and gave similar reaction rates, but the larger background level complicated analysis.An LPMO catalyzed reaction between rFl and HTaAA9A. The assay has a linear dose response in the concentration range of 1–1,000 nM. Breslmayr et al. has reported an LPMO sensing DMP peroxidase assay that can be measured continuously by absorption spectrophotometry and is linear in the concentration range of 12.5–1,000 nM NcAA9C (TaAA9A in LB medium.The rFl assay was found to be a sensitive probe for LPMO activity with a sensitivity of 1 nM of M NcAA9C . The lowM NcAA9C . A third4.2.2O2 to oxidize its substrates, but surprisingly, H2O2 was not required for the oxidation of rFl. This suggests that the rFl assay can differentiate between the enzyme classes. However, for the most accurate results, it is recommended to use the assay when LPMOs are the primary contributors to oxidase activity. The mechanism by which the two enzyme classes differ is beyond the scope of this study and will require further research.HRP showed assay activity in the same range as LPMOs and it is possible that the rFl assay can also be used to determine peroxidase activity. Almost by definition, HRP uses H4.3.2O2 (The LPMO dependent oxidation of rFL was enhanced by 100 μM DHA which is similar to the effect reported on rPh oxidation . The rPh2O2  and to a2O2 . It is p2O2 .4.4.2O2 until a plateau was observed at 6–250 μM. The onset of the plateau is at a concentration similar to the 6.35 μM rFl used in the assay, and this indicates that the oxidation of rFl can be coupled to consumption of H2O2. The stoichiometry between rFl and H2O2 together with the enabling role of reductants suggests a reaction mechanism similar to the peroxidase driven mechanism first suggested by 2O2 and can probably be attributed to the stability of the used model LPMO, TaAA9A. This enzyme has previously been shown to tolerate 200 μM H2O2 (LsAA9A has been shown to tolerate 80 μM H2O2 when in reaction with soluble cellulose-oligosaccharides (2O2 in reaction with chitin (2O2. The DMP assay use 100 μM H2O2 and we suggest that the lower level of H2O2 in the rFl assay makes it preferable for assaying LPMOs.The rFl oxidation was found to increase with H μM H2O2  when in charides  and CBP2h chitin . These t5.2O2. This LPMO activity assay may be useful for screening purposes when the H2O2 sensitivity of an LPMO is not yet established, and for detailed characterization of individual LPMOs.A protocol has been developed for a fast and sensitive LPMO activity assay based on enzymatic oxidation of rFl into Fl. The reactivity of different LPMOs toward rFl varies. The assay has a sensitivity range of 1 nM –1,000 nM LPMO and uses a relatively low concentration of just 10 μM HThe original contributions presented in the study are included in the article/SB conceptualized the rFl to Fl oxidation and its use as a LPMO-specific substrate. JI performed the experiments. KJ oversaw the study. All authors contributed to the article and approved the submitted version.This study was supported by the Novo Nordisk Foundation, grant nos. NNF17SA0027704 and NNF20OC0059697 to KJ.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Nine women  completed a CPET, passively rested for 5 min, and then completed a verification phase at 90% of peak power output to determine the highest 2 from the CPET (2CPET) and verification phase (2verification) on 2 separate days. Analyses included a two-way repeated measures ANOVA, intraclass correlation coefficients , standard errors of the measurement (SEM), minimal differences (MD), and coefficients of variation (CoV). There was no test (test 1 versus test 2) × method (CPET vs. verification phase) interaction (p = 0.896) and no main effect for method (p = 0.459). However, test 1 (39.2 mL·kg−1·min−1) was significantly higher than test 2 (38.3 mL·kg−1·min−1) (p = 0.043). The 2CPET  and 2verification  demonstrated “excellent” reliability. Two subjects demonstrated a test 1 2CPET that exceeded the test 2 2CPET, and one subject demonstrated a test 1 2verification that exceeded the test 2 2verification by more than the respective CPET and verification phase MD. One subject demonstrated a 2CPET that exceeded the 2verification, and one subject demonstrated a 2verification that exceeded the 2CPET by more than the MD. These results demonstrate the importance of examining the individual responses in the measurement of the 2max and suggest that the MD may be a useful threshold to quantify real individual changes in 2.This study aimed to investigate the test–retest reliability, mean, and individual responses in the measurement of maximal oxygen consumption ( A 95% cAdditionally, the minimal difference to be considered real (MD) was calculated using the equation: 2). An a priori alpha level was set at 0.05, and all of the data were analyzed using IBM SPSS Statistical Software Version 28 .Based on previous recommendations , a CoV o2CPET, test 2 2CPET, test 1 2verification, and test 2 2verification) are listed in 2 demonstrated no significant test x method interaction  and no main effect for the method , but there was a significant main effect for the test . Followup comparisons indicated that collapsed across the method , test 1 (39.2 ± 7.2 mL·kg−1·min−1) was significantly higher than test 2 (38.3 ± 7.7 mL·kg−1·min−1). The mean ± SD TLim for test 1 and test 2 for the CPET and verification phase, as well as the peak power output (PPO) from the CPET, the verification phase power output, and the maximal HR, RER, and RPE for the CPET and verification phase are listed in Lim for the CPET was longer than the verification phase (p < 0.001), and the peak power output (p < 0.001) and RER (p < 0.001) during the CPET were greater than the verification phase. There were no interactions or main effects for the test or method for the maximal HR or RPE (p = 0.062–0.512).The individual responses and the mean ± SD for each variable , but there was no difference between the test 1 2verification and the test 2 . The ICC values of the 2CPET (R = 0.984) and the 2verification (R = 0.964) indicated both methods demonstrated ‘excellent’ test–retest reliabilities [2CPET (1.98%) and 2verification (3.30%) were both below the 10% threshold used to be considered reliable [−1·min−1; 0.12 L·min−1) for the 2CPET test–retest. In addition, one subject exceeded the MD  for the 2verification test–retest. Lastly, one subject demonstrated a 2CPET that was greater than the 2verification by a value that exceeded the MD (2.14 mL·kg−1·min−1), and one subject demonstrated a 2verification that was greater than the 2CPET by a value that was greater than the MD , this mean difference reflected a real difference, based on the MD, for only two of the nine subjects. The use of several indices of reliability allows for the determination of the absolute and relative reliability, which enables an individual to compare across studies [2CPET and 2verification in this study were consistent with previous work examining the CPET and verification phase protocols  [2CPET and 2verification can be reliably determined in younger, healthy, and well-motivated subjects.The purpose of this study was to use a test–retest approach to examine the reliability, mean, and individual differences between the revalent . It is orevalent . Howeverrevalent . In addiesponses ,27,28,29 and CoV . Althoug studies ,40. The .1–3.8%) ,27,28,292max has examined the mean responses of the 2 determined from the CPET compared to the 2 determined from the verification phase. Other works have demonstrated no mean difference in the 2max between the CPET and verification phase, yet still recommend its use in all populations [2 (2max). In the current study, there was no main effect for method . This lack of difference between the CPET and the verification phase is consistent with previous studies [2max was significantly greater than the verification phase. However, in contrast to other works [2 from test 1 in this study was significantly greater than test 2, collapsed across the CPET and verification phase. It is important to note that the mean difference between test 1 (39.2 ± 7.2 mL·kg−1·min−1) and test 2 (38.3 ± 7.7 mL·kg−1·min−1) was 0.9 ± 1.2 mL·kg−1·min−1 (~2.2%), and there were no differences in the time to exhaustion, power output, or maximal HR between test 1 and test 2. On an individual basis, only two of the nine subjects for the CPET and one of the nine subjects for the verification phase test–retest exceeded the MD for the test–retest responses. These findings demonstrate a potential pitfall of using the mean responses alone to evaluate the changes in the 2max across time or as the result of a training or dietary intervention. That is, evaluation of the mean response alone would suggest a significant change across time; however, this reflected a real difference for only three out eighteen total test–retest responses. Thus, these findings illustrate that using the mean responses may not be sufficient to fully examine the proper methodology for the measurement of the 2max and highlight the potential usefulness in examining changes across time and the need for the examination of individual responses.Previous work investigating the need for a verification phase in the determination of the ulations ,25. Howeulations ,24, the  studies ,7,8,11 b studies ,28,29,41er works ,27,28,292 obtained from the CPET is truly the maximal 2 that an individual is capable of producing. However, in past works, the threshold that has been used to determine whether there were individual differences was set at 2–3% between measures [2 in addition to the biological variability associated with the measurement. The use of the minimal difference (MD) to be considered real provides a threshold with increased statistical backing to determine whether the differences between the CPET and the verification phase are real differences or are just due to the error of the measure [2 from the CPET and verification phase has previously been quantified in men and women during treadmill running [−1·min−1) may allow future researchers to examine the individual responses in the 2max to potential changes in interventional studies. Using this approach, one individual demonstrated a 2verification that exceeded the 2CPET by more than the MD, while one individual demonstrated a 2CPET that was greater than the 2verification by more than the MD. These data suggest that a verification phase is not necessary in the measurement of the 2max in healthy well-motivated women on a cycle ergometer. In addition, the MD may provide a valuable tool to examine individual differences in the 2max across time or as the result of an intervention.The utility of the verification phase is to determine on an individual basis whether the measures ,7,12,14. measure . The MD  running ,28 and i running , but it 2max for those subjects who exceeded the MD for the test–retest responses may be a result of the increased variation in the 2 due to biological factors and may highlight ‘responders’ vs. ‘non-responders’ [2 values can vary even when there are minor variations (<3 h) in the time of day that the testing is repeated. Furthermore, Lebrun et al. [2max can vary across the menstrual cycle phases. The individual menstrual cycle phase was not tracked in the current study; however, all test–retest responses were recorded within 48–72 h. Although it is possible that menstrual cycle phase transitions introduced sufficient variability to alter the 2max, based on the timing of the testing protocol and the lack of effect for these same subjects on the verification phase 2 responses, this seems unlikely to be the primary driver of the variations in the 2max. Future researchers should examine factors that may impact maximal day-to-day performance to determine the possible magnitude of these effects. In addition, future studies should quantify the MD using a larger sample size. The variation in the ponders’  due to fponders’  or the mponders’ , in addiponders’ . Howeverponders’  demonstrn et al.  demonstr2CPET and 2verification, and both measures demonstrated ‘excellent’ test–retest reliabilities. Therefore, these data support the claims made in previous work [2max. In addition, this study has added to the quantification of the MD that has previously been determined for men in running and cycling and in women in running but had not been derived for women in cycling. There were two individuals who demonstrated differences in the 2CPET test–retest and one individual who demonstrated differences in the 2verification test–retest. Thus, the few individual differences in combination with the lack of mean difference between the CPET and verification phase responses, suggested that the verification phase may not be necessary in healthy motivated women. The MD allows for the examination of individual responses with a threshold that is based on the standard error of the measure and presents an improvement on the 2–3% threshold, which has previously been used. Thus, the MD may prove useful for other researchers to examine the individual 2max responses to potential training or nutritional intervention studies.The results of the current study suggested that its use in healthy well-motivated women who are accustomed to maximal exhaustive exercise may not be necessary. The performance of additional maximal tests increases the demand on subjects to push themselves to their limit more than may be necessary and increases the burden on researchers to perform these additional tests. Day et al. , and morous work ,30, whic