Parkinson's disease (PD) is characterized by degeneration of neurons, particularly dopaminergic neurons in the substantia nigra. PD brains show accumulation of α‐synuclein in Lewy bodies and accumulation of dysfunctional mitochondria. However, the mechanisms leading to mitochondrial pathology in sporadic PD are poorly understood. PINK1 is a key for mitophagy activation and recycling of unfit mitochondria. The activation of mitophagy depends on the accumulation of uncleaved PINK1 at the outer mitochondrial membrane and activation of a cascade of protein ubiquitination at the surface of the organelle. We have now found that SIAH3, a member of the SIAH proteins but lacking ubiquitin‐ligase activity, is increased in PD brains and cerebrospinal fluid and in neurons treated with α‐synuclein preformed fibrils (α‐SynPFF). We also observed that SIAH3 is aggregated together with PINK1 in the mitochondria of PD brains. SIAH3 directly interacts with PINK1, leading to their intra‐mitochondrial aggregation in cells and neurons and triggering a cascade of toxicity with PINK1 inactivation along with mitochondrial depolarization and neuronal death. We also found that SIAH1 interacts with PINK1 and promotes ubiquitination and proteasomal degradation of PINK1. Similar to the dimerization of SIAH1/SIAH2, SIAH3 interacts with SIAH1, promoting its translocation to mitochondria and preventing its ubiquitin‐ligase activity toward PINK1. Our results support the notion that the increase in SIAH3 and intra‐mitochondrial aggregation of SIAH3‐PINK1 may mediate α‐synuclein pathology by promoting proteotoxicity and preventing the elimination of dysfunctional mitochondria. We consider it possible that PINK1 activity is decreased in sporadic PD, which impedes proper mitochondrial renewal in the disease. Our study reports that SIAH3 is increased in PD brains. We also found that SIAH3, along with PINK1, is aggregated in the mitochondria of PD brains. The intra‐mitochondrial aggregation of SIAH3 and PINK1 inactivates PINK1, depolarizes the mitochondria, and leads to neuronal death. Pathogenic a‐synuclein pre‐formed fibrils (a‐SynPFF) increase SIAH3 levels and aggregation, suggesting that SIAH3 increase and its mitochondrial aggregation with PINK1 could help mediate a‐synuclein toxicity. HAhemagglutininPDParkinson's diseasePINK1PTEN‐induced putative kinase 1SIAHseven in absentia homologsiRNAshort interfering RNA1Parkinson's disease (PD) is a common neurodegenerative disorder. Its most striking neuropathological feature is the presence of inclusions called Lewy bodies and the degeneration of dopaminergic neurons in the substantia nigra pars compacta. Lewy bodies consist mainly of α‐synuclein aggregates and vesicular structures, including mitochondria , suggesting that the elevation of SIAH3 may contribute to mediate the pathogenicity of α‐synuclein. We also discovered that elevated SIAH3 in mitochondria co‐aggregates with PINK1 in PD brains and is present in Lewy bodies. In addition, we found that SIAH3 directly interacts with PINK1 and causes aggregation and inactivation of PINK1 in mitochondria, mitochondrial depolarization, and toxicity in cellular and neuronal models. In addition, we found that SIAH1 promotes proteasomal degradation of PINK1 and SIAH3 negatively regulates this process by promoting SIAH1 recruitment to mitochondria and its inactivation. SIAH3 may play a role in the pathogenesis of PD by promoting proteotoxicity and mitochondrial dysfunction. We raise the possibility that inactivation of PINK1 by SIAH3 may contribute to the mitochondrial dysfunction observed in sporadic PD.22.1A previous study identified SIAH3 as a mitochondrial protein that inhibits the translocation of Parkin to mitochondria, but the mechanism has remained unclear as SIAH3 lacks ubiquitin‐ligase activity  (characterized by an average size of 10–120 nm by electron microscopy) Figure , but notBecause PD is considered a proteinopathy  and especially the 60 amino acids (ΔN‐60) significantly reduced translocation of SIAH3 to mitochondria Figure . Using tWe next examined the aggregation of SIAH3 and PINK1 in the mitochondria of neurons. We observed that PINK1, together with full‐length SIAH3 but not SIAH3 ΔN‐30 and ΔN‐60, leads to mitochondrial clustering in neurons, as visualized by the mitochondrial marker GFP‐mito Figure . Concurr2.3Although the authors did not find an interaction between SIAH3 and PINK1 in the first screening study  Figure . This deFurther evidence for the role of SIAH3 in inhibiting SIAH1 activity is the fact that the expression of SIAH3 increases endogenous levels of SIAH1 and PINK1 Figure , whereas3PINK1 is important for maintaining mitochondrial homeostasis and mitophagy (Arena & Valente, PINK1 is a short‐lived protein that is degraded by the proteasome. This explains the low levels of PINK1 in the human brain and the paucity of sensitive antibodies for its detection (Gandhi et al., Ubiquitination and proteasomal degradation of PINK1 was shown to occur via the N‐end rule (Yamano & Youle, We now show that SIAH3 heterodimerizes with SIAH1, promoting its translocation to the mitochondria and inhibiting its ubiquitin‐ligase activity. This inactivation is consistent with our report showing that SIAH1 activity is inhibited once it translocates to the nucleus after binding to LRRK2 (Shani et al., Although the knockdown of SIAH3 was shown to increase the translocation of Parkin to mitochondria by depolarizing agents, the authors failed to identify an interaction between PINK1 and SIAH3 (Hasson et al., Our data suggest that the aggregation of SIAH3 and PINK1 in mitochondria is specific and not due to their mere overexpression. This is supported by SIAH3 constructs lacking a mitochondrial target sequence that do not translocate to mitochondria and do not promote aggregation of PINK1 in the organelle. In addition, under more physiological conditions, we observed aggregation of SIAH3 and PINK1 in mitochondria in neurons transduced with AAV particles under the synapsin promoter. Of note, the intra‐mitochondrial aggregates of SIAH3 and PINK1 are abundant in neurons but less voluminous than the aggregates observed in HEK293 cells, likely due to the lower ability of the synapsin promoter to induce protein expression compared with that of the CMV promoter. Moreover, under our experimental conditions, the expression of PINK1 alone in cells and neurons did not promote its substantial aggregation in mitochondria or spontaneous activation of mitophagy.The accumulation of dysfunctional mitochondria is recognized as a factor in the pathogenesis of PD (Schapira et al., Several studies have examined intra‐mitochondrial aggregates that occur under various circumstances, including hypoxia, aging, and the knockdown of mitochondrial proteases (Kaufman et al., It is long known that pathogenic α‐synuclein promotes mitochondrial dysfunction (Tanaka et al., Moreover, our findings suggest that the interaction of pathogenic α‐synuclein with mitochondria may represent an early event in the disease, as it is detected in the organelle of α‐SynPFF models but not in the mitochondria of PD. On the contrary, mitochondrial aggregation of SIAH3 is triggered by α‐SynPFF, and full pathology of PINK1 is observed in later stages of α‐synuclein pathology in PD brains, suggesting that co‐aggregation of SIAH3 and PINK1 in the mitochondria may represent a downstream process to pathogenic α‐synuclein. It is unclear how pathogenic α‐synuclein is removed from mitochondria during the disease. Further experiments with longer incubation times of α‐SynPFF in mouse brains and detailed analysis of mitochondria will help elucidate the dynamics of accumulation and aggregation of SIAH3 and PINK1 compared with α‐synuclein in the organelle in PD.Our study shows that SIAH3 is increased and aggregated in the mitochondria of PD along with PINK1. We also observed that aggregated PINK1 in mitochondria is less active and cannot properly clear depolarized mitochondria. We hypothesize that α‐synuclein may mediate mitochondrial damage by leading to the co‐aggregation of SIAH3 and PINK1 in the mitochondria during the course of the disease, making the organelle less available for mitophagy and contributing to its accumulation in sporadic PD. Moreover, the inactivation of SIAH1 upon translocation to the mitochondria promoted by SIAH3 may contribute to the proteotoxicity in the disease. SIAH3 now represents a novel target for improving mitochondrial function and proteostasis in PD.44.1g for 10 min, and the crude mitochondrial pellet was resuspended in 15% Percoll in cold homogenizing buffer, layered onto a discontinuous Percoll gradient of 23% over 40%, and then centrifuged for 20 min at 73,000× g at 4°C. The interface band between the 23% and 40% Percoll layers was collected and washed twice in cold homogenizing buffer. The mitochondrial pellet was suspended in homogenizing buffer.Mitochondria were isolated using a discontinuous Percoll gradient, as described (Haskin et al., A detailed description of the additional procedures utilized in the study is described in Fatimah Abd Elghani, Hazem Safory, Vered Shani, Sleman Bisharat, and Simone Engelender conceived or designed the work. Fatimah Abd Elghani, Hazem Safory, Haya Hamza, Mor Savyon, Malik Farhoud, Michal Toren‐Hershoviz, Zagorka Vitic, Kirsten Ebanks, Vered Shani, Sleman Bisharat, and Lihi Shaulov contributed data or analysis tools. Fatimah Abd Elghani, Hazem Safory, Kirsten Ebanks, Claude Brodski, Zhiyin Song, Rina Bandopadhyay, and Simone Engelender performed the analysis. Fatimah Abd Elghani, Hazem Safory, Claude Brodski, Zhiyin Song, Rina Bandopadhyay, and Simone Engelender wrote and edited the manuscript.None declared.Figure S1Click here for additional data file.Figure S2Click here for additional data file.Appendix S1Click here for additional data file.Appendix S1Click here for additional data file.Appendix S1Click here for additional data file.Appendix S1Click here for additional data file.


Streptococcus mutans biofilm formation.The purpose of this study was to compare the effect of hop extracts with diverse β‐acid concentrations on S. mutans isolates were treated with hop extract dilutions varying from 1:2 to 1:256. The minimum inhibitory, minimum bactericidal and minimum biofilm inhibitory concentrations were determined and the optical density was evaluated. Live/dead staining confirmed the bactericidal effects. Biofilm formation of several strains of S. mutans was significantly inhibited by hop extract dilutions of 1:2, 1:4, 1:8, 1:16 and 1:32. Strong negative correlations were observed between α‐ and β‐acid concentrations of the hop extracts and S. mutans total growth and biofilm formation.Ten different hop extracts, with α‐acid concentrations similar to those found in commercial beer products and β‐acid concentrations ranging from 2.6 to 8.1%, were added to distilled water to make standardized concentrations. S. mutans biofilm formation.The use of hop extracts prepared similarly to commercial beer decreased The inclusion of hops in the commercial beer products may provide beneficial health effects. Further studies are warranted to determine an effect in vivo on the development of dental caries. Humulus lupulus, commonly known as the hop plant, has been utilized for numerous purposes over the centuries, indicated for infection, cancer, atherosclerosis, diuresis, pain, inflammation and sleep disorders , UA130, OMZ‐175, LM7, A32‐2, NG8 and 10,449  in TSB was treated with 190 μl of 1:2–1:256 dilutions of each of the hop extracts in quadruplicate in TSB supplemented with 1% sucrose (TSBS) for 24 h in sterile 96‐well flat‐bottom microtitre plates . The optical density (OD) values of the total growth of the bacterial cultures were measured at 595 nanometres in a spectrophotometer . The MIC was defined as the lowest concentration of the hop extracts that yielded a significantly decreased change in OD compared to the 0 hop control. Bacterial cultures (10 μl) from the wells with hop extract dilutions of 1:2, 1:4, 1:8 and 1:16 were transferred onto blood agar plates  and incubated for 48 h to assess the MBC. The MBC was defined as the lowest concentration of the hop extracts that had no visible bacterial colonies on agar plates after 48 h of incubation.The minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of each hop extract for the The minimum biofilm inhibitory concentration (MBIC), defined as the lowest concentration of an agent that significantly inhibits the visible biofilm formation of a micro‐organism, was determined following the measurement of the total growth absorbance as optimized previously  for 10 min and examined under a fluorescent microscope  at 1000X.A pool of the hop extract was prepared by mixing equal volumes of each of the different hops. The pooled hop extract was diluted in TSBS and added (200 μl) to 24‐hour‐old established Each experiment was conducted in triplicate. The results are presented as mean and standard error of mean. SPSS Statistics version 16  was used for data analysis. Due to non‐normality, a rank transformation was used prior to one‐way ANOVA. The one‐way ANOVA and the post hoc Tukey multiple comparisons test were performed to compare the means of the hops extract treated and untreated groups. A significance level of α = 0.05 was adopted for statistical hypothesis testing. Spearman correlations were used to determine the statistical significance of correlations between the α‐ and β‐acid content with total growth and biofilm formation and with each other.S. mutans UA159 total growth and biofilm formation was noted with the addition of each hop extract compared to the controls , 1:4 (p < 0.01), 1:8 (p < 0.01), 1:16 (p < 0.01) and 1:32 (p = 0.05) dilutions. In addition, the MIC values for the pooled hops extract ranged from 1:8 to 1:64 with the exception of S. mutans NG‐8 that demonstrated an MIC with the pooled hops extract of <1:4. Two of the hop extracts (German Northern Brewer and Amarillo) displayed significant inhibition of biofilm formation at a 1:256 dilution  and biofilm formation , respectively, decreased in the presence of the hop mixtures. The 1:2, 1:4 and 1:8 dilutions of each hop extract demonstrated no growth in the MBC analysis while the 1:16 dilutions exhibited growth. Therefore, an MBC of 1:8 for all hops extracts was measured. The α‐ and β‐acid compositions of the 10 hop extracts were significantly correlated with total growth and biofilm formation (Table p < 0.0001).Strong negative correlations were demonstrated between the inhibition caused by the hop extracts and their α‐acid and β‐acid content as the S. mutans strains produced different levels of biofilm confirming our earlier results . This was also the case with MIC. In addition, live/dead staining of S. mutans UA159 demonstrated killing of the bacterium compared to the 0 hop control (Figure Each of the seven different s Figure . This inl Figure .S. mutans. Gram‐positive organisms, such as S. mutans, have lipophilic regions of the cell membrane which may serve as a site of antibacterial activity for the β‐acids of hop extracts (Gerhauser, S. mutans, and other Gram‐positive organisms, is thought to be associated with the high pKa of β‐acids, especially when compared to that of α‐acids (Bogdanova et al., These data suggest that both α‐ and β‐acids may inhibit total growth and biofilm formation of S. mutans UA159 compared to a control group in vitro. The strain of S. mutans used is the most commonly studied strain and has been evaluated by this lab in comparison to other strains with similar results using nicotine and fluoride, which suggest the effect of hop extracts may be effective against all strains of S. mutans (Huang et al., S. mutans. Our work also indicates the effectiveness of killing provided by the hop extracts. The exposure of the hop extracts to S. mutans, combined with the very high negative correlations of the different dilutions and biofilm growth, suggests α‐ and/or β‐acids were likely responsible for the strong inhibition of total growth and biofilm formation. However, since the α‐ and β‐acid compositions are correlated with each other and because previous reports indicate β‐acids are the major antimicrobial in hops, we speculate our results are due to the β‐acid in the 10 hop extracts and not the amount of α‐acids. While the individual hop dilutions varied in α‐ and β‐acid concentrations, each was prepared in a similar manner to how beer is commercially brewed today. Therefore, it is possible that hop extracts prepared by this method may have a role in preventing dental caries when these data are evaluated in humans.With an objective of assessing the benefit of diverse hop extracts, the current study examines the ability of the hop extract dilutions to inhibit total growth and biofilm growth of S. mutans in vitro and in vivo (Yaegaki et al., S. mutans growth while lactic acid production significantly decreased when exposed to the two most highly concentrated hop polyphenols, 0.1% and 0.5% (Yaegaki et al., Previous data have shown the benefit of specific hop products in vivo. For example, Yaegaki et al. evaluated if hop polyphenols, when harvested from raw hop plants and administered in salivary‐soluble table form in concentrations of 0.001%, 0.01%, 0.1% and 0.5%, could reduce the growth of Candida species (Leonida et al., More recently, Leonida et al studied the utility of encapsulating hop extracts in nanochitosan matrices as a means of improving the drug delivery system (Leonida et al., S. mutans biofilm formation was decreased. Moving forward, additional studies are warranted to determine the effect of hop extracts on other common species which cause dental caries. Furthermore, the effect of whole hop extracts on preformed biofilm should be evaluated to assess any potential benefit against pre‐existing plaque, both in vitro and in vivo. If successful, commercial beer may subsequently be assessed for its candidacy to inhibit biofilm formation in vivo.Using a more natural method of drug delivery, the use of whole hop extracts formulated in a similar technique as used in the beer brewing process may provide benefit against organisms causing caries. The value of these preparations was observed in vitro during our study as S. mutans at concentrations similar to what would be found in beer. Future in vivo investigations are required to confirm the clinical benefits of using hop extracts to prevent oral biofilm on teeth.The results of this study demonstrated the ability of hop extracts to inhibit growth, and initial and established biofilm formation of several different strains of The authors declare no conflict of interests.Appendix S1 Supporting informationClick here for additional data file.


Our preferred model assigns two low-frequencyvibrations of HO2NO2 as hindered rotors andcouples these to the external rotations. This model gives ΔrH°0(1a) = −93.9 ±1.0 kJ mol–1, which implies that ΔfH°0 HO2NO2 = −42.0 ± 1.0 kJ mol–1 (uncertaintiesare 2σ). A significant contributor to the uncertainty derivesfrom modeling the interaction between the internal and external rotors.Using this improved kinetics for reaction 1a/–1a, data at elevatedtemperatures, 353–423 K, which show no evidence of the expectedequilibration, have been reanalyzed, indicating that an additionalreaction is occurring that masks the equilibration. Based on a published ab initio study, this additional channel is assigned tothe bimolecular reaction HO2 + NO2 →H–NO2 + O2 (1b); H–NO2 is nitryl hydride and has not previously been directly observedin experiments. The output of the master equation analysis has beenparametrized and Troe expressions are provided for an improved descriptionof k1a and k–1a.A master equation (ME) analysis of available experimentaldatahas been carried out on the reaction HO For thereactionPeroxy radicals are ubiquitousin the oxidation of volatile organiccompounds (VOC). In combustion chemistry, they lead to branching,playing a central role in autoignition.K, andthe enthalpy change of reaction. In order to do this the rate coefficientsfor (k1(p), where it is defined mainlyby the determinations by Kurylo and Ouellette6 andthe more recent measurements by Christensen et al.7 and Bacak et al.,8 who usedmore precise experimental techniques. The IUPAC recommended high-and low-pressure limits are k1∞ = 4.0× 10–12 cm3 molecule–1 s–1 and k10 = 1.4 × 10–31 [N2] cm3 molecule–1 s–1; JPL recommends similar values. A problem with 3OH(used to generate HO2) and H2O form complexeswith HO2 and therefore need to be accounted for. The studyby Christensen et al.7 made measurementson the chaperone effect of CH3OH in order to assign k1 overthe ranges from 45 to 200 Torr and from 219 to 298 K. Using a highlysensitive chemical ionization mass spectrometer (CIMS), Bacak et al.8 studied the reaction with 1000 times lower radicalconcentrations, which in principle eliminates the interference ofcomplexes. This study was in agreement with the literature at 298K, but at low temperatures (223 and 200 K) their values for k1 are about a factor of 2 lower than the othermeasurements reported in the literature.The focus of this paper is to provide an explanation of the lackof equilibration observed in entsfor  and (−1a9 measured k–1a at lowtemperatures (<292 K) using FTIR detection of HO2NO2, pernitric acid, and more recently, Gierczak et al.10 measured k–1a between 331 and 350 K. Forward and reverse rate coefficients forpressure independent bimolecular reactions have been widely used todetermine enthalpies of reaction. Pressure-dependent reactions presenta greater challenge, and a master equation approach, as implementedin the present analysis, is essential.Master equation analysisprovides an accurate description of therate coefficients of a pressure dependent reaction, where the propertiesof the reactants and products are used in the calculation. The energyof the reaction is a fundamental parameter that links the forward and revek1a and k–1a, our previous experimental kineticdata4 are reanalyzed and show that thereis an additional loss channel for ab initio description of the 11In this paper, masterequation analysis using MESMER rigorouslyassesses the experimental rate data for 213 The application of the master equation to reactions in the gas phasehas been extensively discussed elsewhere.17 Here, the main points are summarized for a reversible reaction.The energy levels of HO2NO2 are partitionedinto a number of contiguous intervals or grains that are assignedvalues for the numbers of states they contain, average energies, and,where appropriate, average values of microcanonical rate coefficients.A single grain represents HO2 and pseudo first-order conditionsapply ([NO2] ≫ [HO2]). These grains formthe basis of the master equation representation of the system, anequation of motion of the grain probabilities, which is usually representedasp is a vector containingthe probability densities of the grains and the matrix M contains the transition rates between the grains either becauseof collisional activation/deactivation or because of reaction. Theevolution of p is limited by two constraints, mass (ordensity) conservation and detailed balance.We employa master equation analysis using the code MESMER.Λ is a diagonal matrixcontaining the eigenvalues of M, U is amatrix of the corresponding eigenvectors, and p0 is a vector containing the initial grain densities. The number ofeigenvalues is equal to the total number of grains; all the eigenvaluesare negative. They can be divided into two types: internal energyrelaxation eigenvalues (IEREs), which relate to the collisional relaxationof the internal energy of the system and chemically significant eigenvalues(CSEs); the magnitude of the reciprocal CSEs relate to the time scalesof the chemical reactions and the number of CSEs is related to thenumber of chemical species included in the master equation. Generally,and certainly in the reactions analyzed here, the CSEs are significantlysmaller in magnitude than the IEREs—energy relaxation occurson time scales that are shorter than those of the chemical reaction.Under these circumstances, the rate coefficients for the chemicalreactions can be determined from an analysis of the chemically significanteigenvalues and eigenvectors. In the present system, there are twoCSEs, one of which, λ1, relates to equilibrium andis zero. The nonzero λ2 = −(k1a[NO2] + k–1a), and Kc = k1a[NO2]/ k–1a.The solution to Supporting Information. The association reaction is barrierless and the limiting high-pressurerate coefficient for association, k1a∞, is parametrized as A(T/298 K)n, and the microcanonical rateconstants for dissociation of HO2NO2*, k(E), are determined by inverse Laplacetransformation.18 The rates of reactionfrom HO2 + NO2 into a particular grain of HO2NO2 are determined by detailed balance at a specific,fixed [NO2].19 An exponentialdown model, coupled with detailed balance, is used for the probabilitiesof collisional energy transfer between the grains, based on the parametrization⟨ΔE⟩down = ⟨ΔE⟩down,298(T/298 K)m.4 All of theavailable experimental rate data were fitted to the master equationmodel, using the minimization of χ2ki,exp is the ith experimentalrate coefficient, ki,model is the modelresult under the same condition of T and p, σi is the standard deviation of the experimental rate coefficient,and N is the total number of experimental measurements.The analysis was conducted, as were the experiments, under pseudo-first-orderconditions ([NO2] ≫ [HO2]), and [NO2] was given a fixed value of 1015 molecules cm–3). The variable parameters in the fitting processwere ΔrH°0, A, n, ⟨ΔE⟩down,298 and m. The best available experimental data wereused in a global fit for k1a[NO2] ≫ k–1a or the reverse), although experiments underequilibrating conditions are considered in MESMER includes a facility that allows pressure- and temperature-dependentforward and reverse rate coefficients to be fitted to a reaction model.The molecular constants for the reactants and product are fed intothe model and are listed in the 2NO2 were treated as hinderedrotors, HO2—NO2 and HO–ONO2, where the hindering potential was calculated using M062X/6-31+G**.A 1-D hindered rotor (HR) model was also used in our analysis of CH3 + CH3,20 and the methodused is discussed in detail in our analysis of H + C2H4.21 However, for the present reaction,coupling between the internal and external rotors in HO2NO2 is significant. We are unable to model this interactionwith a full quantum mechanical model. To overcome this problem, theMESMER analysis was carried out in four ways:(i)The two 1-D HRs were treated quantummechanically and uncoupled from the external rotors.(ii)The internal and external rotorswere both treated classically but coupled. This method accounts forthe Coriolis interaction, the change in external moments of inertiawith internal rotation, and the kinematic coupling between the internalrotors.(iii)2–NO2 rotor was treated classically and coupledto the externalrotors while the HO–ONO2 rotor was treated quantummechanically but uncoupled. The HO2–NO2 rotor has the greater effect on the external moments of inertia;it also has a smaller energy level spacing than the HO–ONO2 rotor.The HO(iv)Bothinternal rotors were treatedclassically but uncoupled.The vibrational and rotationalconstants for each species wereobtained from experimental data. However, the two lowest frequencyvibrations of HO22 and is describedin the MESMER manual.13 The input parametersand methods for these calculations are given in the Supporting Information.The classical coupled rotors option in MESMER is basedon the methoddiscussed by Gang et al.33a23 Kurylo et al.24 Christensen et al.7 and Bacak et al.8 for the forward reaction  and needs to be properly accounted for to determine k1a accurately. Bacak et al. argued that becausetheir experiments used 1000 times lower HO2 radical concentrations,the HO2 self-reaction, and hence the uncertainty in itsrate constant at low T, did not impact on their results.The data were reanalyzed using (i) just the data of Bacak at al.,together with the dissociation data, and (ii) all the data at 298K and above, plus the low T data of Bacak et al.,and the dissociation data. The results are shown in the Supporting Information and demonstrate that thelow T data of Bacak et al. are inconsistent withthe Mesmer model and higher T association and thedissociation rate data. Both JPL25 andthe IUPAC26 do not include the low temperaturedata from Bacak et al. in their evaluations as they noted that thesedata fall about a factor of 2 below the fit based on the other data.Further MESMER analysis was then performed without the 223 and 200K data from Bacak et al.8 The removal ofthese 9 points improved the χ2 from 446 to 132, emphasizingthat these points are incompatible with rest of the data.Bacak et al. pointed out that in the other studies,the HOk–1a, are plotted in Thereverse, dissociation data, 3b4 we presented pulsed photolysis results for RO2 + NO2 ⇋ RO2NO2  under equilibrating conditions , allowingthe direct determination of the equilibrium constant and of reactionenthalpies. HO2 was monitored by LIF detection of the OHproduct, following pulsed photolysis. It was noted that attempts tofit HO2 + NO2 data under similar conditions(353–423 K) failed to return a consistent value for k–1a:2 or HO2NO2. The possibility thatHONO is a product from HO2 + NO2 had previouslybeen explored and ruled out by Dransfield et al.27 and Tyndall et al.28 Tyndallet al. placed an upper limit of 5 × 10–16 cm3 molecule–1s–1 on thesecond-order rate coefficient for the contribution of this reactionto 11 explored theoretically the bimolecular channels of k1b = 1.3 × 10–15 cm3 molecule–1 s–1. Nitryl hydride has not beenobserved experimentally, but it has been explored theoretically ina number of previous papers. Asatryan et al.29 calculated a ∼ 200 kJ mol–1 barrier forHNO2 to isomerize to HONO, where HONO is more stable. Accordingto the Active Thermochemical Tables (ATcT)30 HONO is 36 kJ mol–1 more stable than nitryl hydride,based largely on the calculations of Klippenstein et al.31 This conclusion is supported by the calculationsof Asatryan et al.29 and Zhang et al.11 Therefore, production of nitryl hydride from The traces did not exhibit distinctivebiexponential equilibrating behavior, see k1a and k–1a are fixed to the values from our MESMER analysis,see k1a and k–1a rate coefficients at the temperaturesand pressures of our high temperature experiments In the absence ofNO2, HO2 is lost via self-reaction sufficientlyslowly ([HO2] ∼ 1013 molecules cm–3) that it is reasonably approximated as a first orderloss:To explore the possibility of 2 is also lost via diffusion,which is evident fromthe differences observed in experiments carried out at 100 and 400Torr. Therefore, HO2 diffusional loss was assigned a differentrate coefficient at each experimental pressure (see Supporting Information):HO4 it produces OH and interferes with the detectionmethod used for HO2:OH,PNA was determined inour previous paper as 0.15 ± 0.03;4 the photolysis produces a ∼ 10% increase, X, in the baseline given byAn additional complication is the interferencefrom the photolysisof pernitric acid, the addition product of k1b was assigned an Arrhenius form, k1b = A1b × exp, where A1b was fixed to 1 × 10–10 cm–3 molecule–1 s–1; the experiments were not carried out overa large enough temperature range to treat both A1b and Ea,1b as variable parameters. The value of Ea,1b given in A1b; i.e., thebarrier for k1b itselfis well-defined over the temperature range investigated. The reactionscheme and analytical solution were given in our previous publication.4 It is given in an appropriately modified formin the Supporting Information, where ithas been extended to include loss of HO2 by 2–NO2, As k4 multiplied by 0.64, the square root of the reciprocal ratio of thereagent masses. The traces were analyzed globally32 in order to test the mechanism and improve the parameterrecovery, and the results for the variable parameters are given in T = 423 K). Alsoincluded in this example is a trace calculated under identical conditionswith the HNO2 channel, Diffusional loss of peroxynitric acid2 which was used for the weightingof each trace in the global analysis. In this global analysis, k1a and k–1a are fixed to our MESMER model—see 2] is the experimentalamount added for each trace. From 2/point inthe global analysis is 1.12, which indicates that the overall fitis not much worse than the individual fits. This indicates that themodel adequately describes all the data. The rate coefficient for Ea,1b. From k1a and k–1a determined in Supporting Information.Before carrying out theglobal analysis, each trace was fittedto the model to obtain the best fit on an individual basis. Each fityielded a value for χ44a2H4 used electronic structure calculationsof the properties of the tight transition state, floating its energyin the global fit to experimental data.21 The calculated and fitted transition state energies agreed well.Such an approach is not feasible for HO2 + NO2 because there is no barrier to reaction and a variational transitionstate method is required to calculate the microcanonical rate constantsfor association and dissociation. The computational costs of suchan approach in global fitting to experimental data is prohibitive.Instead, we use the inverse Laplace transform (ILT) method, basedon the high pressure limit for association, k∞, to determine the microcanonical rate coefficientswhich implicitly accounts for rotational effects.18 The convolution used in the ILT method is taken over allstates, including rotation. The parameters for k∞, A, and n  – where p = N2 - have been parametrized using a Troe formalism34 and the parameters are given in 14 – 1025 molecules cm–3 and thefit reproduces the Master Equation results within 7%. Also includedin 35 and JPL,36 where it is noted that each uses a differentTroe formalism. The Troe parametrization used is given in the Supporting Information.The Master Equation results for 26 and JPL,25 and theresults at 1 bar N2 are given in Assessment of the literature data have been carriedout by IUPACk1a = 7.5 × 10–13 cm3 molecule–1 s–1 at 298 K when there is generalagreement from experiments that k1a is ∼1.4 × 10–12 cm3 molecule–1 s–1.8 The origin of this discrepancy is unclear. Note that none of theanalyses includes the data of Bacak et al. in their recommendation.From this figure, it can be seen that the presentresults are ingood agreement with JPL, especially close to 298 K. However, the IUPACevaluation is not in agreement and is typically a factor of 2 belowthe other evaluations. The obvious problem with the IUPAC evaluationis that it recommends k1a and k–1a were simultaneously fitted in order to yield the most robust descriptionof the reaction. From 8 The IUPAC recommendationof k–1a was based on a Troe-typefit to the literature data for k–1a while the JPL recommendation assigned the equilibrium constant, Kc,1a with k1a to best match the k–1a data, i.e., k–1a = k1a × Kc,1a. k–1a versus temperature from IUPAC, JPL, and Leeds, where itcan be seen that there is fair agreement. In general, the Leeds assessmentis closer in agreement with IUPAC, but all are within 50% of eachother at all temperatures. This agreement between the assessmentsis also the case for k–1a∞.In our MESMER analysis, both 4b–1 for each of thefour models. However, it is noted that the four models predict significantlydifferent thermodynamic parameters. The 0 K reaction enthalpies derivedirectly from the MESMER model and are influenced by the densitiesof states at all energies. The fully classical models show a largeincrease in the magnitude of the reaction enthalpy, but the differencebetween the coupled and uncoupled models is relatively small (0.6kJ mol–1). This suggests that these models significantlyoverestimate the magnitude of the reaction enthalpy because of theuse of the classical description of the internal rotors. Model iiireduces this change in the reaction enthalpy, because the HO–ONO2 rotor is treated quantum mechanically. The effect on theexternal coupling will be underestimated, but probably only slightlybecause the changes in the external moments of inertia resulting fromthis internal rotation are small. The energy level spacing in theHO2–NO2 internal rotation is significantlysmaller than that of HO–ONO2, and so the classicaldescription introduces a smaller error into the densities of states;by contrast, its impact on the external rotors is greater, so thatinclusion of this coupling is important. Model iii therefore providesthe best compromise. The use of a classical model for one of the internalrotors and the neglect of coupling for the other both introduce significanterrors, so the uncertainty should be increased. A final value of −93.9± 1.0 kJ mol–1 is proposed. The resulting enthalpyof formation for HO2NO2, ΔfH°0, is 41.9 ± 1.0 kJ mol–1 based on the enthalpies of formation of HO2 and NO2 of 15.1 kJ mol–1 and 36.9 kJmol–1 at 0 K, taken from ATcT.The enthalpy and entropyof rH°298(1a) and ΔrS°298(1a) from Sander et al.,30 Gierczak etal.,10 and Zabel.9 Sander and Petersen23 analyzed the equilibriumdata using a second-law approach, where entropy is not fixed but determinedfrom the temperature dependence of the equilibrium constant. Thissecond-law approach is known to produce large errors as the equilibriumdata are determined over a limited temperature range, and the valuescan be skewed as the entropy and enthalpy changes are highly correlated.Gierczak et al.10 and Zabel.9 used a third-law method, calculating the entropyusing a vibration only model . rS°298| >170J mol–1 K–1, where slight differencesarise from the vibrational frequencies used, either experimental orcalculated, or a combination of the two. Consequently, these two studiesyield similar enthalpies of reaction; see 2/degrees of freedomequal to 1.59. In our recent paper on the reaction between OH andisoprene under equilibrium conditions,37 it was demonstrated that a vibration only model returned an incorrectvalue for the enthalpy of the reaction, which was better assignedwhen the low frequency vibrations were described as hindered rotors.39) are the benchmark for thermodynamic data. The major contributorto the provenance of the ATcT values for HO2NO2 is the high level electronic structure calculation of Szakacs etal.40  agree with the vibration onlycalculations of Gierczak et al.10 and Zabel.9 Since these studies used an RRHO model, thisresult suggests that the effect of the anharmonicity on ΔrS°298 is small. Calculationsfor other molecules.43 also show that the effects of anharmonicity on theentropy are small at the temperatures of this study (up to 358 K).The present study shows that the influence on the entropy and enthalpyof internal rotation and its coupling with external rotation is substantial.The ATcT value for ΔrS°298(1a) = 163.9 J mol–1 K–1 includes anharmonicity and the coupling between the internal andexternal rotors. The Active Thermochemical Tables  for Δ4cIn the 44 and it was concluded that k1c < 5 × 10–16 molecules cm–3 s–1; the rate coefficient for the bimolecularproduct channel at 295 K from –15 moleculescm–3 s–1, which is too fast tobe consistent with the results of Tyndall et al. The theoretical studyon 11 is more recent and provides explanation of ourresults as it predicts that the fastest bimolecular channel is k1b. Nitryl hydride , wherethe literature data were fitted simultaneously to provide the bestoverall parametrization of this reaction. By treating two low frequenciesin HO2NO2 as hindered rotors (HR), this leadsto a better fit, and four HR rotors models were considered, uncoupledand coupled with the external rotors.The kinetics of both the forward and reverse reactions1a/−1a),,−1a), HO8The master equation analysis provides a good fit toall the literature data except for the low temperature measurementsof Bacak et al.2 + NO2 kinetic decay traces over the temperature range353–423 K were reanalyzed where k1a and k1-a were fixedto the values from our MESMER analysis. This analysis revealed thatthe approach to equilibrium is obscured by an additional bimolecularchannel, which leads to loss from the equilibrating system. Basedon the literature, this channel is assigned to Previously determined high temperature HO2–NO2 rotor was treated classically and coupled to the external rotorswhile the HO–ONO2 rotor was treated quantum mechanicallybut uncoupled appears to provide the best available description ofthe system, based on a comparison of the four models used. The useof a classical basis for the HO2–NO2 rotorundoubtedly introduces error beyond the statistical error derivedfrom the fit to the experimental data. All the fits using the hinderedrotor models are excellent, and the use of a quantum mechanical coupledmodel would undoubtedly provide a determination of the reaction enthalpyof very high accuracy.The thermochemistryof


However, the side-on configuration of O2 on the catalysts surface will lead to the formation of H2O, which seriously limits the selectivity and activity of 1O2 production. Herein, we show a robust N-doped CuO (N–CuO) with Pauling-type (end-on) adsorption of O2 at the N–Cu–O3 sites for the selective generation of 1O2 under direct-current electric field. We propose that Pauling-type configuration of O2 not only lowers the overall activation energy barrier, but also alters the reaction pathway to form 1O2 instead of H2O, which is the key feature determining selectivity for the dissociation of Cu–O bonds rather than the O–O bonds. The proposed N dopant strategy is applicable to a series of transition metal oxides, providing a universal electrocatalysts design scheme for existing high-performance electrocatalytic 1O2 production.Due to environmentally friendly operation and on-site productivity, electrocatalytic singlet oxygen ( 2 on the catalysts conventionally leads to reduction of O2 to water. Here, the authors propose a nitrogen doping strategy with Pauling-type adsorption of O2 for selective electrocatalytic singlet oxygen production.Side-on configuration of O Advanced oxidation processes (AOPs) are state-of-the-art water treatment technologies, which can achieve deep oxidation of refractory organic pollutants because of the production of reactive oxygen species (ROS) such as hydroxyl radicals (•OH), hydrogen peroxide (H2O2), singlet oxygen (1O2), superoxide radicals (O2•−) and so on3. As one of the reactive ROS, 1O2 has become a favorable candidate for the treatment of refractory organic pollutants in the environmental field because of its mild oxidation ability (2.2 V vs NHE) with aromatic organic compounds, higher tolerance to water matrixes, inhibited generation of halide disinfection byproducts and its relatively long lifetime (over an hour in gas phase and 10−6–10−3 s in solution)7. The mild oxidation ability of 1O2 can selectively oxidize unsaturated organics through electrophilic addition and electron abstraction reaction8. Hence, 1O2 is an effective ROS to remove aromatic organic pollutants from wastewater. Greater recognition of the conspicuous significance of 1O2 has inspired research for more effective 1O2 production. At present, the common approaches of 1O2 production in the practical treatment of wastewater by AOPs mainly include the activation of H2O2 and peroxymonosulfate10. However, despite the substantial development of 1O2 production, the dependence of external chemicals, the introduction of potential emerging pollutants and complicated operation have thus far limited their practical applications. Therefore, the direct chemical synthesis of 1O2 from the ground state oxygen molecules via photocatalysis and electrocatalysis (EC) could be a potential alternative strategy for 1O2 production, due to the environmentally friendly operation and on-site productivity12. In particular, electrocatalytic 1O2 production, a highly automated, straightforward and propitious technology, can offer a more accessible 1O2 production pathway and is more promising for industrial scale-up, has obtained extensive research interest. However, various ROS will be involved in EC process due to the low selectivity of O2 activation, which seriously limits the application of 1O2 production15.Global environmental deterioration has been recognized as one of the most important challenges in the 21st century, especially refractory organic contaminants in environmental water adversely affect the ecological system and potentially threaten human health2 can be adsorbed on the metal oxide or sulfide electrocatalysts surface in “side-on” adsorption configuration (including Griffiths-type and Yeager-type) in the electrocatalytic O2 activation process  configuration can minimize O–O bond breaking, leading to the cleavage of M–O bond rather than O–O bond, which will ultimately prevent the cleavage of O–O bonds and facilitate the formation of –O–O– intermediates (the most important intermediates of 1O2)20. Furthermore, the reduction or disproportionate reaction of –O–O– intermediates will generally form the 1O2  due to the in-situ formation of active Co–OOH species and the suitable regulation of free energy for adsorption of the OER intermediates22. Enlightened by the above analysis, controllable synthesis of local coordination environment of metal center with non-metal heteroatom-doping is highly desirable to improve the 1O2 generation in EC process.Generally, Oess Fig. 16. The “2O (Eqs. )17–19, w1O2 (Eq. )21. In ttes (Eq. )21. Bene1O2 production by using oxygen gas under a direct current (DC) electric field, in which the surface local coordination environment of Cu–O4 is regulated to N-Cu-O3 moiety. Results elucidats that the selective generation of 1O2 is achieved in EC process by tailoring the electronic structure and coordination environment of N-doped CuO, which is in sharp contrast to the •OH intermediates generated in the formation of H2O in undoped CuO electrocatalytic system. Combining experimental and theoretical investigations, it is found that the adsorption of O2 on N–Cu–O active sites is “end-on” type, which promotes the formation of •OOH, leading to an efficient pathway for 1O2 selective generation. The concept of using non-metal heteroatom doping metal oxides to simultaneously boost 1O2 selective production shall provide a design guide to develop more advanced electrocatalytic systems for extensive applications.Herein, we develop an N-doped CuO electrocatalyst for direct electrocatalytic 2H6N2·HCl) oxidation etching strategy  by using multiple approaches, and the performance of N–CuO  of SOSG is observed in the EC process, indicating its excellent 1O2 generation performance. While for CuO, no obvious change of fluorescence intensity at 529 nm of SOSG is observed, which is very close to the blank experiment , indicating that the N dopant on CuO surface is responsible for the enhanced 1O2 generation activity and selectivity , as they would form the corresponding endoperoxide (DMA-O2)21. The fluorescence intensity of DMA in the presence of N–CuO decreases rapidly, while the CuO system decreases slowly, indicating the ROS generated in the N–CuO system have higher reaction rate with DMA and might be the 1O2 . Instead, •OH is negligible produced within the N–CuO system, completely demonstrating the superior selectivity of N–CuO catalysts in the 1O2 production  process25.Furthermore, electron paramagnetic resonance (EPR) measurements were employed to gain more substantive evidence for the ROS species in different systems. Figure 1O2 is an important ROS for electron-rich organic pollutants degradation, the degradation performance of CuO and N–CuO was investigated with sulfamethoxazole (SMX) as a target pollutant11. As shown in Fig. k value) of the N–CuO system based on the degradation rate is 0.12 min−1, and almost 6.0 times higher than that of CuO system. Notably, the optimized N–CuO catalyst with a TOF of 0.41 min−1 for SMX degradation exhibits the better catalytic oxidation performance among reported catalysts  and sulfadiazine (SDZ) was investigated. Results demonstrate that the degradation efficiencies of SFX and SDZ are 91.1 and 95.7% after 30 min, and the k values are 0.08 min−1 and 0.10 min−1, showing the efficient removal ability of N–CuO system for electron-rich pollutants  with similar N dopant methods. Results validate that these catalysts have different degrees of enhancement of 1O2 generation, elucidating that N dopant strategy is universal and feasible to promote the formation of singlet oxygen in EC system with N dopant strategy. Furthermore, the facet effect of N–CuO was investigated, and results show that N-doping effect accounts for the higher catalytic performance of N–CuO than that of facet effect  of N species doped in CuO  image indicate the (002) and (222) planes of N–CuO and the corresponding fast Fourier transform (FFT) image shows the high orientation along the [128. Moreover, the HAADF-TEM reveals a regular periodic arrangement of Cu atoms without obvious vacancy defects in N–CuO, further confirming the structural stability and robustly excluding the possibility of copper substitutions by nitrogen of N–CuO sample. The corresponding elemental mapping and energy dispersive X-ray spectrum (EDX) elemental mapping of HAADF-TEM verify that Cu, O, and N elements are uniformly distributed on the surface of N–CuO  in area-scan mode, indicating the presence of N that changed the excitation of the Cu 2p1/2 or Cu 2p3/2 electrons from the inner shell into the first available unoccupied, which might be a result of Cu–N interaction on the surface  are slightly larger than those of pristine CuO , further suggesting that N dopant leads to lattice distortion. Similarly, the Raman peaks of N–CuO appear blue shift compared with CuO, which is due to the increase in grain size along with the N doping  spectrometry of the N–CuO was employed. Peaks at 78.9–79.04, 79.9, and 80.9 Da are ascribed to CuNH2+, CuNH3+, and CuNH4+ respectively, suggesting that N element is bonded to Cu in the form of enhanced chemical bond, rather than a simple physical or chemical adsorption process , NO2 (404.3 eV), and NO3− (407.3 eV) and yet is closer to that of NH3 (398.7 eV), suggesting that the valence state of the N species may be between −2 and −3  analysis of Cu 2 −3 Fig. . That ca2O  k2-weighted EXAFS spectra  and extended X-ray absorption fine structure (EXAFS) spectra were collected for Cu elements. The normalized XANES spectra show that the white line intensity of N–CuO is higher than Cu foil and Cu2O Fig. , suggesttra Fig.  demonstrces Fig. . Evident37. The coordination numbers for O and N atoms are calculated as 3.0 ± 0.3 and 1.0 ± 0.1 at distances of 1.93 ± 0.01 and 2.03 ± 0.02 Å, respectively (Supplementary Table 4), which is different from the Cu (N–Cu–O3) in N–CuO with three O atoms and one N atom , the potential-dependent in situ Raman spectroelectrochemistry, in situ Fourier transform infrared (FTIR), EPR spectra and DFT calculations. Firstly, the CuO and N–CuO electrodes were investigated to get further insight into the enhanced electrocatalytic activity after N doping. Results show that N doping results in different electrochemical behavior for the molecular oxygen activation process . The Raman spectrum of the initial sample shows only there are peaks at 295.2, 342.7, and 633.5 cm–1 at open circuit potential (OCP), which are attributable to Ag, Bg, and Bg modes of CuO crystals39. The Raman peaks of CuO appear blue shift with the potential decrease to −1.0 V, while no new peaks appear even under more negative potentials. In sharp contrast, as the potential decreases to −0.4 V, the N–CuO sample shows a Raman peak around 872 cm−1, which is possibly associated with O–O stretching mode of superoxide species on the surface of N–CuO (CuII–O2•)41. Further decreasing the potential, the Raman peak becomes slightly stronger. To exclude the •OOH species adsorbed or desorbed on N–CuO surfaces originating from breakage of water molecules, the control in situ Raman experiment saturated with N2 was employed  and the •OOH desorption process. When the potential decreases from open circuit potential (OCP) to –0.8 V, the intensity of peak located at around 1135–1160 cm−1 appears and increases, which can be assigned to the stretching mode of –O–O– adsorbed on N–CuO (correspond to the end-on Cu–O2• on the surface of N–CuO)43. That is the accumulation of the intermediate species (CuII–O2•). While with the further decrease of potential, the intensity of peak barely changes, corresponds to the •OOH desorption process42. In comparation, no new peaks appear around 1135–1160 cm−1 for CuO even at more negative potentials, verifying that there are different O2 adsorption configurations on CuO and N–CuO surfaces  signal is detected for N–CuO system, while there is no DMPO-OOH signal in CuO system, confirming that CuII–O2• detected in in situ Raman and FTIR spectra may undergo the desorption process. Furthermore, the amount of •OOH produced in this period are then quantified using nitroblue tetrazolium chloride (NBT)44. As shown in Supplementary Fig. −1 •OOH are accumulated in N–CuO system, giving solid evidence for the desorption step of CuII–O2• and then the formation of free •OOH. In view of the •OOH is the major intermediate in 2e− ORR reaction, we then monitored linear sweep voltammetry (LSV) and ROS generation with the addition of H2O2 in EC system, confirming that N–CuO could suppress the generation of •OH in N–CuO system and the main source of 1O2 comes from •OOH , tryptophan , and catalase (the scavenger for H2O2) on the EPR signal were investigated. As it is seen in Fig. 1O2 generation46. To further confirm the role of •OOH for the 1O2 generation, the quenching process of •OOH in the electrocatalysis system was investigated  is able to remove the electron of •OOH, it is determined to be used to investigate the role of •OOH for 1O2 generation in the N–CuO system48. The 1O2 generation had an obvious decrease in the N–CuO system after adding of 5 mmol L−1 K2Cr2O7  around the N coordination  than the Cu–O4 (CuO) sites on account of the change of Cu coordination environment, which helps to lower the *OOH intermediates formation energy in N–CuO; hence, these charge-deficiency Cu atoms (N–Cu–O3) in the N–CuO could serve as preferential active sites, which could more effectively absorb the O2 via Pauling-type adsorption compared to the Yeager-type adsorption on dual Cu sites with the uniform distribution of localized charge of CuO, hence promoting the formation of *OOH  to 1O2 generation is downhill in the free energy, which is much more favorable thermodynamics for 1O2 generation  is thertep Fig. .2 would be more favorable for 1O2 production because of lower energy barriers (0.16 eV), in comparation with the Yeager-type adsorption mode of CuO (0.32 eV)  Fig. . In brie1O2 generation selectivity and activity of metal oxides is demonstrated. The combined characterization results reveal that N species appear in the form of O substitutional N in N–CuO, and the coordination environment of Cu atoms is coordinated with one N and three O, which is identified unambiguously to be the active sites for the O2 Pauling-type adsorption and then 1O2 generation. Furthermore, DFT calculations and spectroscopy characterizations suggest that N dopant results in charge deletion on the Cu atoms surrounding with N atoms, favouring the breakage of the Cu–O bonds rather than O–O bonds, which promotes the formation of *OOH, an important precursor of 1O2. This approach is general and applicable to a variety of electrocatalysts, including CuO, CeO2, NiO, and so on. As an outcome, a 6.0 times faster SMX degradation kinetics in N–CuO than CuO system is obtained. This work represents the collaborative effect between atomic coordination environment and the adsorption configuration of the reactants, which opens up a strategy for designing various nitrogen-doped metal oxides for the electrocatalytic 1O2 generation reactions and is promising for the application of environmental remediation.In summary, the selectivity of the catalyst is related to the adsorbed intermediates and the reaction pathways, then an effective N dopant strategy for improving 2O was synthesized by adapted from known literature procedures52. In a typical procedure for Cu2O, 10.0 mL of 0.1 mol L−1 EDTA solution was added to 10.0 mL of 0.1 mol L−1 CuSO4 solution. The blue color of the CuSO4 solution became deep. Then, 10.0 mL of 0.1 mol L−1 glucose solution was added to this solution, and the solution was then stirred well for 15 min at room temperature. Finally, 10.0 mL of 1.0 mol L−1 NaOH was added to it, and the solution was heated in an oil bath at 80.0 °C for 30 min. The color of the resulting solution changed from blue to green to red. The red particles were collected by centrifugation and washed several times with deionized water and absolute ethanol, and dried well at 60.0 °C overnight for further characterization.Cu2O cubes (40.0 mg) were dispersed into 30.0 mL deionized water containing 4.0 mL NaOH (1.0 mol L−1). Then, acetamidine hydrochloride aqueous solution (1.0 mol L−1) was added to the above mixture under stirring. After 90 min, the precipitate was collected by centrifugation, washed with water and ethanol several times. The preparation of the working electrode was similar with above method, except immersing the carbon felt  in the above mixture.The as-prepared Cus at 284.8 eV. Electron paramagnetic resonance  tests were used to characterize ROS in heterogeneous EC process. Total organic carbon (TOC) was analyzed on a Shimadzu VCSH TOC analyzer (Japan).The morphology and structure of the samples were examined by field emission scanning electron microscope , transmission electron microscope  and HAADF-STEM energy-dispersive X-ray spectrometry  detectors. The EELS spectra under area-scan mode were recorded with an FEI Titan Themis scanning transmission electron microscope operating at 300 kV quipped with a Gatan image filter (GIF) spectrometer. The crystal phases of the samples were analyzed by X-ray diffraction (XRD) by using Rigaku Corporation (Japan) with Cu Kα radiation (λ = 1.54178 Å), operated at an accelerated voltage of 40 kV and an emission current of 40 mA. Raman spectrum was collected on a Renishaw Invia Reflex Raman microscope equipped with a 532 nm excitation laser under the power of 20 mW. Time-of-flight secondary ion mass spectrometry (TOF-SIMS) measurements were carried out using TOF.SIMS.5-NSC instrument  allowed the surface chemistry characterization of investigated samples. Surface chemical compositions of catalysts were determined by X-ray photoelectron microscopy . The binding energies were corrected to C1Raman spectroscopy was recorded on the same Renishaw InVia reflex Raman microscope under an excitation of 532 nm laser under controlled potentials by an electrochemical workstation . The electrolytic cell was homemade by Teflon. The working electrode was set to keep the plane of the sample perpendicular to the incident laser. Pt wire as the counter electrode was rolled to a circle around the working electrode. Saturated calomel electrode (SCE) was used as the reference electrode.53. The prepared CF loaded with catalysts was served as cathode equipped with anode using the platinum electrode (2 cm × 1 cm), with a spacing of 1.0 cm between them. Then 65 mL simulated wastewater containing 50.0 mg L−1 pollutant  and 50.0 mmol L−1 Na2SO4 solution was electrolyzed under aeration of O2 with a flow rate of 100 mL min−1. In addition, the pH value of simulated wastewater was adopted to 3 by 0.5 mol L−1 H2SO4 and 0.5 mol L−1 NaOH solution.The evaluation of pollutants degradation performance was performed in a single-cell for electrolyzation in two-electrode system with the cell voltage of 3.0 V2O2, O2•− and 1O2, respectively), which were added before the EC reaction. The radical species were further detected by EPR technology, where 5, 5-dimethyl-1-pyrroline (DMPO) and 2,2,6,6-tetramethyl-4-piperidinol (TEMP) were used as a spin-trapping reagent. The detailed parameters are as follows: 100.0 μL of aqueous suspension of samples were mixed with 1.0 mL of TEMP (50.0 mmol L−1) solution. The •OH trapping-EPR test was also performed as described above, except the use of DMPO (50.0 mmol L−1) as the spin-trapping agent. As for O2•−, 1 mL methanol and water  containing DMPO (50.0 mmol L−1) were mixed with reaction liquid.Radical quenching tests were conducted to identify the dominant radicals in the EC system with catalase, superoxide dismutase (SOD) and tryptophan (Trp)  under ambient conditions using a transmission mode. The storage ring conditions of BSRF was operated at the energy of 2.5 GeV and a maximum current of 250 mA with double-crystal Si (111) as monochromator for energy selection.The Cu 54 to perform all spin-polarization DFT calculations within the generalized gradient approximation (GGA) using the Perdew–Burke–Ernzerhof (PBE) formulation55. We chose the projected augmented wave (PAW) potentials to describe the ionic cores and take valence electrons into account using a plane wave basis set with a kinetic energy cutoff of 400 eV57. Partial occupancies of the Kohn-Sham orbitals were allowed using the Gaussian smearing method and a width of 0.05 eV. The electronic energy was considered self-consistent when the energy change was smaller than 10−4 eV. A geometry optimization was considered convergent when the energy change was smaller than 0.05 eV Å−1. Finally, the adsorption energies (Eads) were calculated as:Ead/sub, Ead, and Esub are the total energies of the optimized adsorbate/substrate system, the adsorbate in the gas phase, and the clean substrate, respectively. The Brillouin zone integral uses the surfaces structures of 2 × 2 × 1 monkhorst pack K point sampling for structures and we fixed the bottom layers. The free energy (ΔG) for elemental reaction step were calculated as:E is the difference between the total energy, ΔEZPE and ΔS are the differences in the zero-point energy and the change of entropy, T is the temperature (T = 300 K in this work), respectively.We employed the first-principlesSupporting Information


Aspergillus brasiliensis (AbVS). These enzymatic reactions resulted in the formation of several new terpene analogs that were isolated and structurally characterised by NMR spectroscopy. Several DMAPP analogs showed a changed reactivity giving access to compounds with unusual skeletons. Their formation is mechanistically rationalised and the absolute configurations of all obtained compounds were determined through a stereoselective deuteration strategy, revealing absolute configurations that are analogous to that of the natural enzyme product variediene.Five analogs of dimethylallyl diphosphate (DMAPP) with additional or shifted Me groups were converted with isopentenyl diphosphate (IPP) and the fungal variediene synthase from  Five DMAPP analogs with additional carbon atoms or with changed methylation patterns were converted with IPP and the bifunctional variediene synthase from Aspergillus brasiliensis AbVS to yield diterpene analogs with unusual skeletons. The DMAPP analogs opened new reaction pathways through the stabilisation of cationic charges at alternative carbon atoms that are naturally not observed. These transformations are achieved by the Mgrmediate.2, 3 TTaxus brevifoliaCatenulispora acidiphilaAspergillus brasiliensis (AbVS),Emericella variecolor (EvVS),It is well known that prenyltransferases can use analogs of the terpene monomers dimethylallyl diphosphate (DMAPP) and isopentenyl diphosphate (IPP).1, Scheme A, followed by Wagner‐Meerwein rearrangement and ring opening to B with a specific stereochemical course that places Me16 in the pro‐S and Me17 in the pro‐R position. A reverse 14,11 ring closure with simultaneous 2,10 cyclisation leads to C that is the precursor of 1 by deprotonation.AbVS is a bifunctional enzyme with a prenyltransferase (PT) and a terpene cyclase (TC) domain that naturally converts DMAPP and IPP through geranylgeranyl diphosphate (GGPP) into variediene (E)‐3‐methylpent‐2‐enyl diphosphate (2), (Z)‐3‐methylpent‐2‐enyl diphosphate (3), 2,3‐dimethylbut‐2‐enyl diphosphate (4), (E)‐2‐methylbut‐2‐enyl diphosphate (5) and (Z)‐2‐methylbut‐2‐enyl diphosphate (6) were used  is an analog of variediene methylated at C16. Its formation can be explained by the same sequence of steps as for the biosynthesis of 1, suggesting that the additional carbon introduced from 2 does not cause a change in the reactivity of the intermediates towards 8. In contrast, for compound 10 the cyclisation cascade is disturbed at intermediate A2, the analog of A in Scheme B analog, but instead reacts through 1,2‐hydride shift and deprotonation to 10. The altered reactivity in A2 is likely a consequence of a steric repulsion in the enzyme's active site that forces the sec‐Bu group in A2 with rotation around the C14−C15 bond into a different conformation as in A1 (and as for the iPr group in A). While in A1 (and A) the empty p‐orbital at C15 may be parallel to the σ‐orbital of the C13−C14 bond, the conformational change in A2 may lead to a parallel orientation of the empty p‐orbital at C15 and the σ‐orbital for the C14−H bond, with the consequence of a downstream 1,2‐hydride shift instead of a Wagner‐Meerwein rearrangement. A compound with the same skeleton as 10, but without the additional Me group shown in blue, has first been obtained by total synthesis11) from the brown alga Dilophus spiralis.10 was designated homodolabellatriene.For enzymatic conversions with AbVS, the DMAPP analogs . The substrate analog 4 also opens a new reaction path through 1,11–10,15 cyclisation to A3, followed by deprotonation to isohomodolabellatriene I (13), or a 1,2‐methyl shift to B3 and loss of a proton to isohomodolabellatriene II  by Stl‐SS from Emericella variecolor a similar cyclisation reaction is observed,16) obtained from farnesyl diphosphate (FPP) to formation of the six‐membered ring compound 18 from 10‐methyl‐FPP (17) was recently described for dauc‐8‐en‐11‐ol synthase (DcS) from Streptomyces venezuelae  through 1,11‐10,15‐cyclisation  from Streptomyces coelicolor A3(2)21 by GC/MS ‐ and (S)‐IPPE)‐ and (Z)‐IPP.13C‐substitutions were introduced. All combinations of the DMAPP analogs 2–6 with the four labelled substrates (R)‐ and (S)‐IPP and (E)‐ and (Z)‐IPP were converted with AbVS, with addition of FPPS and GGPP synthase (GGPPS) from Streptomyces cyaneofuscatus8, 10, 13, 14 and 20 as shown in Schemes 1 and the intermediates along its cyclisation cascade . Herein, we show for the first time that both domains of a fungal bifunctional terpene synthase can collaborate in the enzymatic conversion of analogs of the terpene monomer DMAPP that is elongated by the PT domain with IPP to obtain the corresponding GGPP analogs. Further conversion by the TC domain leads to analogs of diterpene hydrocarbons. Depending on the methylation pattern of the DMAPP analogs, in several cases new reaction paths that are not observed with the natural substrate GGPP were opened, because charges in the cationic intermediates can be stabilised at alternative carbon atoms. This approach gives enzymatic access to compounds that are naturally not obtained. While the cyclisation mechanism can change according to the reactivity implemented in the DMAPP analogs, the absolute configurations of the obtained compounds that are determined during the first cyclisation step, with all subsequent transformations being diastereoselective, are analogous to that of the natural enzyme product 1. Besides the varidiene synthase[The bifunctional diterpene synthase AbVS naturally converts DMAPP and IPP into the diterpene hydrocarbon variediene  should be addressed to the authors.Supporting InformationClick here for additional data file.


The modified CaCO3 filler was mixed with PBAT and melt extruded to prepare biodegradable PBAT/CaCO3 composites. The characterization results show that gas—solid fluidization method combines the traditional wet modification method’s excellent modification effect with the scalability of the traditional dry modification method. The effects of modification methods and amount of CaCO3 filling on the crystallinity, mechanical, and rheological properties of PBAT/CaCO3 composites were compared. The results demonstrated that PBAT/CaCO3 composites containing 30% gas—solid fluidization modified CaCO3 could still maintain excellent overall performance. As a result, this work provides a simple, efficient, and scalable method for chemically modifying inorganic fillers and preparing biodegradable composites.Modifying biodegradable poly  (PBAT) plastic with inorganic fillers is critical for improving its overall performance, lowering the costs, and expanding its application scope. The chemical modification method for the inorganic filler determines the application performance of PBAT composites. In this work, gas—solid fluidization method was developed as a simple, efficient, and scalable strategy for chemically modifying CaCO The widespread application of traditional petroleum-based plastics has resulted in serious environmental pollution issues. As a result, countries worldwide have enacted laws and regulations to encourage the use of biodegradable materials ; however3 being one of the most commonly used fillers in the polymer industry 2 solution in situ and discovered that the corresponding water contact angle could reach 127.5°. In contrast, the water contact angle for dry-method-modified CaCO3 was only 110.3° . Titanate was supplied by Shanghai Macklin Biochemical Technology Co. (China). And liquid paraffin was provided by Sinopharm Chemical Reagent Co. .PBAT (TH801T) pellets were provided by Xinjiang Lanshan Tunhe Polyester Co. (China). The PBAT samples had a melt flow index of 3.0–5.0 g/10 min (2.16 kg at 190°C), density of 1.20–1.28 g/cm3 powder was dried at 80°C for 12 h before surface modification treatment to remove surface moisture. CaCO3 was gas—solid fluidized and modified by adding it into a glass fluidization reactor; 2.5% titanate coupling agent was added in the atomizing cup and mixed with liquid paraffin in a 1:1 mass ratio. The fluidization reactor was then placed in a vertical tube furnace. Next, the titanate coupling agent and liquid paraffin were atomized by an air compressor and reacted with CaCO3 at 60°C for 30 min , and then 2.5 wt.% titanate coupling agent and liquid paraffin were added into it and stirred at 60°C for 30 min. This sample was then filtered and dried at 80°C for 12 h.The pristine CaCOr 30 min ; for the3 in various mass ratios  for tensile performance tests.PBAT and CaCOs ratios  were mixs ratios ; the mel3 particles was studied using Fourier transform infrared (FT-IR) spectroscopy (3 particles are O–C–O in-plane bending vibration (728 cm−1), O–C–O out-of-plane bending vibration (882 cm−1), C–O asymmetric stretching vibration , and C–O symmetric stretching vibration . As compared to unmodified CaCO3, modified CaCO3  has characteristic absorption peaks of–CH3  and–CH2–  stretching vibration. Meanwhile, compared with dry- and wet-modified CaCO3, the gas-solid fluidization modified product has stronger absorption peaks for–CH3 and–CH2– stretching vibration, verifying its excellent functionalization effect. These findings indicate that the long-chain alkyl groups of the titanate coupling agent have been encapsulated on the surface of CaCO3 particles. In addition, the modified CaCO3 particles exhibit a distinct absorption peak for C–Ti–O–CaCO3 at 1,024 cm−1, indicating that the surface groups of CaCO3 is converted from–OH to C–Ti–O–CaCO3 and the coupling agent is tightly adsorbed on the CaCO3 particle surface.The surface chemical composition of the modified CaCOtroscopy ; the res3 particles before and after modification. The modified CaCO3 showed a progressive mass loss in the temperature range of 180°C–630°C due to the decomposition of titanate coupling and liquid paraffin attached to the CaCO3 particle surface , dry-modified CaCO3 (2.85%), and wet-modified CaCO3 (4.83%). According to these findings, the gas–solid fluidization modification allows more titanate coupling agents to coat the surface of CaCO3.Thermal stability is an important parameter that could quantitatively reflect the amount of modification of coupling agent molecules ; therefo surface . Followi3 surface properties are crucial in determining its interfacial compatibility with polymer matrix  is estimated from ΔHm/ΔHm0 ωPBAT × 100%, where ΔHm is the fusion heat of the sample, ΔHm0 is the melting enthalpy that equal 114J/g for PBAT, and ωPBAT is the mass content of PBAT in the composite.The degree of crystallinity  were increased by 3%, 14%, and 17%, respectively, compared with the unmodified CaCO3; and the corresponding elongation at break increased by 30%, 42%, and 44%, respectively. The relatively higher tensile strength and elongation at break of PBAT composites filled with gas–solid fluidization modified CaCO3 were attributed to the uniform distribution of the modifier molecules on the surface of CaCO3 during the gas—solid fluidized modification method, which resulted in better dispersion of the modified CaCO3 in the PBAT matrix and promoted the cross-linking of PBAT with the modified CaCO3, significantly improving the strength and toughness of the composite. As a result, the overall mechanical properties of the composites, such as strength and toughness, were significantly improved.The tensile strength and elongation at break of PBAT composites filled with 30 wt.% modified CaCO3 contents and modification methods. According to the cross-sectional comparison in 3 was uniformly dispersed on the cross-sectional surface of the blends, and the surface was relatively flat, but when the filler content was increased to 40 wt.%, the tendency to form agglomerates was more obvious, and the interfacial compatibility was poor, resulting in poor performance of the composite. As a result, the optimal amount of CaCO3 filler preferred to be added is 30 wt.%. According to the longitudinal comparison in 3 is due to poor dispersion uniformity and poor titanate modification effect by dry modification, which further leads to poor particle–matrix interfacial compatibility. In contrast, PBAT composites filled with gas—solid fluidization modified CaCO3 resulted in improved CaCO3 particle agglomeration, with large crystal size and low degree of agglomeration. Furthermore, the CaCO3 filler was well embedded in the PBAT matrix, and the two were more tightly bonded, resulting in improved composite performance.Morphological analysis of the blends is frequently used to help us understand their compatibility and dispersion. 3. To generate biodegradable PBAT/CaCO3 composites, the modified CaCO3 filler was melt blended with PBAT and injection molded. The effects of various modification methods and filling amounts on the crystalline, mechanical, and rheological properties of PBAT/CaCO3 composites were investigated for comparison with dry and wet methods. The results showed that PBAT composites filled with gas—solid fluidization modified CaCO3 (at 30 wt.%) improved crystallinity (33.39%), and had excellent mechanical and rheological properties with uniform dispersion and interfacial compatibility. Therefore, the strategy proposed in this work provides an efficient modification method for preparing PBAT/CaCO3 composites that are low in cost and high in performance and can be applied more effectively in the field of ground film and other plastics.A gas—solid fluidization method was proposed in this work to modify CaCO


However, the poor cell affinity of ZrO2 for bone regeneration and tissue binding, as well as its shrinkage due to crystal phase transformation during heat treatment, limits its clinical use and processing plasticity. This study aims to investigate an appropriate ZrO2–SiO2 composite recipe for ceramic 3D printing processes that can strike a balance between the mechanical properties and cell affinity needed in clinical applications. Specimens with different ZrO2–SiO2 composite recipes were fabricated by a selective laser gelling method and sintered at temperatures ranging from 900 to 1500 °C. The S5Z5 composite, which consists of 50 wt% ZrO2, 35 wt% SiO2 and 15 wt% SiO2 sol, showed an appropriate compressive strength and bending strength of 82.56 MPa and 55.98 MPa, respectively, at a sintering temperature of 1300 °C. The shrinkage rate of the S5Z5 composite was approximately 5% when the sintering temperature was increased from 900 to 1500 °C. All composites exhibited no cytotoxicity after 144 h of MG63 cell incubation, and the S5Z5 composite exhibited the most obvious cell affinity among the composite recipes. From these results, compared with other composites, the S5Z5 composite was shown to possess mechanical properties and a cell affinity more comparable to those of natural human bone.Zirconia (ZrO Natural bone is often used for implantation since it provides an ideal environment for cell growth, thereby shortening the bone regeneration period. However, patients may experience adverse immunological reactions, which increased the risk of contracting transmissible diseases via implants. In addition, the quantity of available natural bone implants is insufficient to meet the demands of clinical surgery3. Accordingly, tissue engineering (TE), including the engineering of cells, growth factors, and scaffolds, has been explored to overcome these aforementioned difficulties. Scaffolds are of special importance since they provide an environment for cellular adhesion, proliferation, and differentiation5. The materials of scaffolds must possess appropriate mechanical properties to ensure structural stability during bone reconstruction7. Hence, the selection of scaffold materials and the approaches for fabricating scaffolds remain crucial research topics8.The clinical demand for the orthopedic treatment of large bone defects has been increasing over the years because of increased rates of osteoporosis and critical accidents. Currently, bone regeneration therapy employs autografts or allografts to enhance bone healing in large bone defects9. Metals can offer sufficient support to maintain the structure of a scaffold, but their surfaces need to be modified to enhance their bioactivity11. Polymers have good flexibility similar to that of natural bone, but their rapid degradation and material weakness limit their applicability14. Ceramics possess good mechanical properties and cell affinity, which make them suitable for cell growth17. In particular, ZrO2 has long been used in clinical applications because of its mechanical properties and resistance to fracture19. However, ZrO2 is not bioactive and cannot chemically or biologically bond to bones20. The low cell affinity together with the high thermoshrinkage of ZrO2 may limit its application in scaffold manufacturing and medical utilization21. On the other hand, SiO2 has excellent cell and tissue affinities via the interaction of its silanol group with the calcium and phosphate ions in biological fluids23. Studies have demonstrated that ZrO2 can react with SiO2 in the liquid phase to form ZrO2–SiO2 compounds24. These ZrO2–SiO2 compounds have shown good biological properties suitable for medical applications since they have the ability to release silicate ions that facilitate the growth and differentiation of osteoblasts25. However, the influence of the ZrO2–SiO2 composition on the mechanical properties of these compounds was not investigated in these previously reported studies.Metals, polymers, ceramics, and composite materials are common scaffold materials used in clinical applications28. However, it has been very difficult to manufacture this desired structure until the advent of 3D printers. 3D printing technology can facilitate the fabrication of complex structures based on a layer-by-layer principle31. Hung et al. used a fused deposition manufacturing (FDM) process and employed polyurethane/hyaluronan/TGFβ3 to form soft scaffolds that promoted the self-aggregation of mesenchymal stem cells (MSCs) and induced the chondrogenic differentiation of MSCs to produce a matrix for cartilage repair32. Wang et al. used sodium alginate/polyvinyl formal composite as a raw material to fabricate porous scaffolds for bone remolding33. Weinand et al. used hydrogels as a binder to fabricate porous β-TCP scaffolds with a 3D printing method, which led to better regeneration of bone tissue with MSCs34. Lee et al. used binder jetting to build polycaprolactone/chitosan bone scaffolds with an interconnected structure that facilitated the spreading and proliferation of MSCs after an apatite-coating treatment35. A selective laser gelling (SLG) process for the manufacturing of 3D parts called ceramic laser gelling (CLG) was developed36 and applied to fabricate a CaCO3–SiO2 interporous bioceramic scaffold37. Near-zero volume shrinkage of the specimens after sintering was realized since inorganic materials (SiO2 sol in this case) were used as the binder. In another study, when an inorganic binder was used in the SLG process, the volume shrinkage was found to be lower than that observed when an organic binder was used in other 3D printing processes38.Ideal bone scaffolds should have highly interconnected porous structures that can induce the formation of bone from the surrounding tissue or act as a template for growing cells for bone tissue regeneration3–SiO2 composites in a previous study was 47 MPa, but this is much lower than that of human bone, which is 100–230 MPa39. Hence, the objective of this study was to determine an appropriate bioceramic material and sintering temperature that could improve the mechanical properties of and impart satisfactory biological properties to specimens prepared by the SLG process. ZrO2–SiO2 compounds have been suggested to have these desired properties and therefore were selected for this study.The compressive strength of CaCO2–SiO2 composite recipes were designed, and specimens manufactured by the SLG process were sintered at various temperatures40. Their mechanical properties, microstructures, and cell affinities were investigated. X-ray diffraction (XRD) analysis was conducted for a detailed interpretation of the experimental results. Finally, the most appropriate recipe was applied to fabricate a biomimetic bone scaffold, and its feasibility in clinical applications was assessed.To achieve the required properties, various ZrO2–SiO2 recipes and the heat treatment temperatures, the compressive and bending strengths of the samples prepared by three different recipes at different temperatures of heat treatment were determined, and the results are shown in Fig. 2, which is between 800 and 1200 °C, depended on the manufacturing process. The gradual recrystallization of gelled SiO2 increased with increasing temperature, which could strengthen the binding force between particles and lead to better mechanical properties. When the sintering temperature was raised from 1300 to 1500 °C, the mechanical properties of S3Z7 were enhanced, but those of S5Z5 and S7Z3 deteriorated. Figure 2 with increasing temperature was verified by observing the obvious melting of gelled SiO2 on the specimen. From the high-magnification SEM microphotographs, the grain size of ZrO2 increased with increasing temperature. Oh proposed that the recrystallization of gelled SiO2 would further improve the mechanical properties of ZrO2–SiO2 compounds41. In addition, SEM and EDX elemental analyses of S5Z5 at a heat treatment temperature of 1300 °C were conducted, as shown in Fig. 2 particles in recrystallized SiO2, and the weight ratios of O, Si and Zr were 31.6%, 14.7% and 53.7%, respectively.To understand the relationship between the mechanical properties induced by various ZrOThe low-magnification 500×) SEM microphotographs of S3Z7 and S7Z3 sintered at 900 °C were compared to that of S5Z5, as shown in Fig. 00× SEM m43. As a result, the density was reduced from 2.5 to 2.3 g/cm3, and expansion of SiO2 occurred accordingly44. The transformation of quartz to cristobalite occurred over a temperature interval from approximately 1300 °C up to the melting point of SiO2 at 1710 °C, and the transformation from quartz to an intermediate amorphous state reached a maximum at 1677 °C. From XRD analysis, the phase transformation from quartz into cristobalite was observed in the specimens prepared by three recipes, especially in S5Z5 and S7Z3, over a temperature range of 1100 to 1500 °C during heat treatment.The XRD results of the specimens prepared by three recipes at sintering temperatures of 1100 °C, 1300 °C, and 1500 °C are shown in Fig. Based on an analysis of the resulting mechanical properties, S7Z3 was eliminated as a potential candidate material since it showed insufficient compressive and bending strengths. The S3Z7 and S5Z5 specimens were heat treated at 1300 °C and 1500 °C, respectively, and were kept for further consideration based on the shrinkage of the fabricated specimens.2 powder took place at all temperatures, as depicted in Fig. 2 content that was treated at a higher temperature. Since the size of the SiO2 powder particles was far larger than that of the ZrO2 powder particles, the spaces between the SiO2 powder particles would be filled by the ZrO2 powder particles and their agglomerates. This action, together with the recrystallization of the SiO2 gel, caused the densification of the specimen structure and a reduction in the specimen porosity, as shown in Fig. 2 content. Therefore, S7Z3 showed the largest volume increase among the three composites. The increase in volume could compensate for the reduction in porosity discussed previously, and it led to small variations in the porosities of S5Z5 and S7Z3 over the temperature range of 1300–1500 °C. The greater size of ZrO2 at higher temperatures, as discussed before, also contributed to the increase in the volume. On the other hand, the volume was reduced by the agglomeration of the ZrO2 powder particles. This action dominated the volume change of S3Z7 for all investigated temperatures. S5Z5 and S7Z3 showed an affected volume change over the temperature range of 900–1100 °C. However, starting at 1300 °C, the volume increased because the reasons discussed above became more significant, and hence, the behavior was exhibited as shown in Fig. Figure 2–SiO2 composites, the samples after heat treatment at 1300 °C were used for further cell affinity and proliferation tests. Figure 2) and Z10 (pure ZrO2) were used as the positive and negative controls, respectively, to determine if the cell affinity would be affected by the specific compositions of the ZrO2–SiO2 recipes. The OD values were detected for all samples after 24 h of cell culturing, indicating the initial adherence of the cells to the samples. Significantly, cell growth was observed after a long culturing time of 144 h for all the samples, and hence, the proliferation of the cells on the ZrO2–SiO2 samples could be considered evident and satisfactory. It was also noted from the figure that S5Z5 showed the best cell proliferation.After optimizing the thermoshrinkage and mechanical strength of the ZrO45. The number of MG63 cells increased, and the actin of MG63 connected with the surrounding cells when the culturing time was extended to 144 h.A fluorescent stain was used to assess the state of cell adhesion by checking if the cells maintained their original shape, and the results are shown in Fig. The S5Z5 composite was employed to manufacture a biomimetic bone scaffold because its properties of thermoshrinkage, compressive strength, and cell affinity were better than those of S3Z7 and S7Z3 when the green parts of the fabricated ceramic composite specimens were heat-treated at a temperature of 1300 °C. The digital files of the outer shape and inner structure for the scaffold were transformed and merged by software for the fabrication process, as shown in Fig. 2–SiO2 bone graft manufactured by the SLG process could result in improved mechanical properties and impart satisfactory biological properties for clinical use. As a result of analyzing the compressive and bending strengths at different sintering temperatures, it was observed that the compressive and bending strengths of S7Z3 were lower than those of S5Z5 and S3Z7 in the temperature range of 900–1300 °C during heat treatment. Compared to the other composites, S7Z3 exhibited the worst mechanical properties and more microcracks on the surface. Additionally, the microstructure of agglomerated ZrO2 in S7Z3 was not as dense as that in S5Z5 and S3Z7. In Fig. 2 is more obvious than agglomerated ZrO2 in S7S3 compared to S5Z5 and S3Z7. This phenomenon was due to the difference in the particle sizes and weight ratios of ZrO2 and SiO2. The average particle sizes of ZrO2 and SiO2 were 0.1 μm and 25 μm, respectively, and the specific surface areas of S3Z7 and S5Z5 were higher than that of S7Z3. Therefore, the microstructure of agglomerated ZrO2 in S7Z3 was not as dense as that in S5Z5 and S3Z7 after sintering. The reduction in the mechanical strength due to the formation of micropores within the structure affected the mechanical support provided by ZrO2. The existence of Zr, Si, and O ions may indicate the formation of a ZrO2–SiO2 compound from the solid reaction of ZrO2 and SiO2 (powder and/or sol)24. This solid reaction could enhance the bonding strength between the two different materials46. Similar ZrO2–SiO2 compounds were found for all three composites at various heat treatment temperatures. The formation of ZrO2–SiO2 compounds was found to be a temperature-dependent reaction, which could facilitate an improvement in the mechanical properties of the composites with increasing temperature. The maximum compressive and bending strengths of pure SiO2 were 33.6 and 26.2 MPa after heat treatment at 1300 °C. In comparison, the maximum compressive and bending strengths of the three composites, especially S5Z5, were higher than those of pure SiO2. From the SEM analysis, it was observed that there was no obvious damage on the surface of S5Z5. The microstructure of S3Z7 was dense with many micropores on the surface. The reason for this could be the higher specific surface area of the ZrO2 powder compared to that of the SiO2 powder. The effect of the reduction in the mechanical strength of the structure due to the formation of micropores was larger than the effect of the improvement in the mechanical properties of the structure owing to ZrO2. As a result, the compressive and bending strengths of S3Z7 were lower than those of S5Z5. For S7Z3, its microstructure was not as dense as that of S3Z7. Furthermore, there were microcracks on its surface. The formation of microcracks played a major role in the reduction of the compressive and bending strengths. As shown in Fig. 2 because of its phase transformation at a high temperature. A similar transformation of amorphous SiO2 to cristobalite, which influenced mechanical properties, has also been reported by He et al.47.This study aimed to investigate whether an appropriate ZrOAccording to the results, large volume shrinkage and small porosity were observed for S3Z7, which was not considered an appropriate composite for the 3D printing of bone scaffolds for medical applications. The S7Z3 composite was excluded due to its unsatisfactory mechanical properties, as described in “2 , which has long been used in clinical applications 48. Only for ZrO2 was the reduction of cell proliferation reduced greatly. Various ZrO2–SiO2 compounds for the manufacturing of bone scaffolds by the SLG method were investigated in this study. S5Z5 shows more suitable mechanical properties and cell affinity for manufacturing artificial bone scaffolds for medical implantation, and its introduction into the bone-repair market can be expected in the near future.Based on the results of the MTT assay and fluorescence staining, it was observed that none of the specimens exhibited cytotoxicity. A lower cytotoxicity of a material could provide better environments for cellular adhesion, spread, and proliferation. From Fig. 2 laser and scanner, (2) scrapper, (3) slurry feeder, (4) platform, (5) servo motor, and (6) control unit. In the 3D printing process, the platform moves to its starting position first by the action of the servo motor. This is followed by slurry injection through the slurry feeder. Next, the scraper paves the slurry to ensure a uniform layer of a specific thickness. Then, the platform moves downward along the layer thickness, and at the same time, the slurry feeder and scraper return to their original positions. The whole procedure is repeated until the green part of the ceramic composite specimen is finished. A schematic of the building process for the green part of a ceramic composite specimen on the platform is shown in Fig. The 3D printer used in this study was self-designed and assembled, as shown in Fig. 2 powder, SiO2 powder, and SiO2 sol were used as raw materials for the SLG process. The ZrO2 and SiO2 powders acted as fillers to reinforce the structures of the fabricated specimens, while the SiO2 sol was used as a binder in the construction of the bioceramic green parts of the fabricated specimens. The powder sizes of ZrO2 and SiO2 were 0.1 μm and 25 μm, respectively. The SiO2 sol consisted of 60 wt% water and 40 wt% SiO2 nanoparticles that were 40 nm in size. The ZrO2 and SiO2 were mixed in various proportions so that the appropriate material composition resulting in the best mechanical properties and cell affinity could be obtained. Three recipes were tested, as shown in Table 2 and ZrO2 in the compounds. The ceramic slurries containing the ZrO2 powder, SiO2 powder, and SiO2 sol were further blended by ball milling with a rotational speed of 100 rpm for 1 h to ensure that they were mixed homogeneously.ZrO2 sol. Coagulation of SiO2 leading to gelling takes place because of the change in the pH or moisture evaporation as a result of the dissolution of the –OH terminus of the silicic acid (Si(OH)4) of SiO2 sol, which generates a condensation reaction49. Hence, the region not irradiated by a CO2 laser can be readily removed by deionized water, and complex structures, such as undercuts, overhangs, or inner channel structures, can be fabricated by the SLG process29.The principle of part fabrication in this study is based on the irreversible sol–gel reaction of the SiO2–SiO2 compounds were fabricated by using the following process parameters: a laser power of 7 W, laser scanning speed of 100 mm/s, laser scanning hatch of 0.1 mm, and layer thickness of 0.1 mm. The compressive and bending strengths of the specimens were tested in accordance with the JIS R1608 and JIS R1601 standards, respectively. The specimens used for the compressive strength test were shaped as disks that were 6 mm in diameter and 12.5 mm in height, and the specimens used for the bending strength test were shaped as cuboids that were 30 mm in length, 4 mm in width, and 3 mm in height. The dimensions of the specimens used for evaluating the porosity and volume shrinkage were 10 mm × 10 mm × 4 mm. To determine the appropriate sintering temperature, the testing specimens were sintered at 900 °C, 1100 °C, 1300 °C and 1500 °C for 2 h with a heating rate of 5 °C/min.The specimens used for evaluating the compressive strength, bending strength, volume shrinkage, and porosity of the ZrO50. To evaluate the volume shrinkage, a digital microscope  was used to measure the dimensions of the fabricated specimen, and then the volume changes as a percentage before and after heat treatment were calculated. Three specimens were tested for each condition, and crystalline phase analysis of the specimens was conducted by X-ray diffraction (XRD) analysis  with CuKα radiation. Each test was performed for 2θ values between 20° and 60° at a step size of 5°/min. The topographies, elemental compositions, and crystalline structures of the specimens were observed using scanning electron microscopy (SEM) and energy-dispersive X-ray spectrometry (EDX) .A universal test machine  was used to test the average compressive and bending strengths of the fabricated specimens according to JIS R1608 and JIS R1601. The maximum values of the compressive and bending strengths were determined by the first inflection points of the compression and bending curves, respectively. Archimedes’ principle was used to evaluate the porosity of the specimens, and the numerical value of the porosity was calculated as follows:2–SiO2 composites was tested by inoculating MG63 cells (104 cells/well) directly onto the surface of the cuboid specimens (4.5 mm × 4.5 mm × 1 mm) in a 96-well tissue-culture plate. The cell-culture temperature was kept at 37 °C with a CO2 concentration of 5%. MG63 proliferation was determined using the MTT  assay with an ELISA reader  to measure the optical density (OD 570 nm). Cell adhesion and spreading were observed using stereomicroscopy  and imaged  after 24 h and 144 h of culturing, respectively. The ceramic specimens were washed with PBS and fixed with 2.5% paraformaldehyde at 25 °C. After the cells were fixed, the nuclei and actin of the MG63 cells were stained at 37 °C with DAPI (FluoroPure™ Grade) and phalloidin (Fluor 488) for 5 and 10 min, respectively. Five specimens were used for the biological tests.The cell affinity of the ZrOTo manufacture a biomimetic bone scaffold, a digital file of the scaffold’s outer shape was obtained by scanning a human femur bone model with a 3D scanner . A digital file of the scaffold’s inner structure was obtained by scanning a cancellous bone model using μCT . These two digital files were transformed into STL files by the Mimics software and merged by the Magics software. The manufacturing parameters were input into the developed machine for the fabrication process, as shown in Fig.