Iron atom–cluster interactions increase activity and improve durability in Fe–N–C fuel cells

Simultaneously increasing the activity and stability of the single-atom active sites of M–N–C catalysts is critical but remains a great challenge. Here, we report an Fe–N–C catalyst with nitrogen-coordinated iron clusters and closely surrounding Fe–N4 active sites for oxygen reduction reaction in acidic fuel cells. A strong electronic interaction is built between iron clusters and satellite Fe–N4 due to unblocked electron transfer pathways and very short interacting distances. The iron clusters optimize the adsorption strength of oxygen reduction intermediates on Fe–N4 and also shorten the bond amplitude of Fe–N4 with incoherent vibrations. As a result, both the activity and stability of Fe–N4 sites are increased by about 60% in terms of turnover frequency and demetalation resistance. This work shows the great potential of strong electronic interactions between multiphase metal species for improvements of single-atom catalysts.

N/C atoms" on Page 8, Line 12 is not convincing. Even if these clusters are really anchored by N/C atoms, how can the authors decide it is a N atom but not a C atom? Please see Adv. Mater. 2020, 32, 2004900 andSmall Methods 2021, 2001165. Will the theoretical results be different if the coordination atom changes? 6. Some missing data are suggested to be provided. 1) The ring and disk current curves recorded on RRDE.
2) The WT contour plots of Fe SA sample. 3) Fig. S18 only shows the LSV curves before and after 5k cycles, the chronoamperometric tests at 80 °C should also be given. 4) What is the spiking current shown in Fig. S17a?
Reviewer #3 (Remarks to the Author): Comments: This research has originality for synthesis of catalyst for efficient ORR system through the complex of Fe-N4 single atom site, Fe4-N6 nanocluster, Fe nanoparticle. Fe4-N= nanocluster can modulate the electronic structure of Fe-N4 site. Compared to Fe single atom catalysts, complex of Fe-N4 single atom site and Fe4-N6 nanocluster have high activity and durability. Since transition metal SACs can be replaced noble metal catalyst, attracting researchers in renewable energy society. In particular, Fe single atom catalyst has a problem of low durability in acid electrolytes, and this paper suggests a way to solve it. So, this paper is worth to be published in Nature Communications after revision of the manuscript following the comments below. 1) Through the Raman spectra data and difference of ORR limiting current density during the AST process, authors show that carbon corrosion does not occur. The pyrolysis temperature of M-N-C can affect the graphitic degree of carbon support and activity of the M-N4 site. Therefore, the authors need to show the relationship between the graphitic degree (Raman data) and activity (stability) difference at different temperature.
2) In DFT modeling, the authors suggested the complex of Fe-Nx site and Fe4-N6 site. In order to check the structure of the M-Nx site and the local coordination environment, fitting using EXAFS data is essential. However, the authors did not show the fitting data for the Fe-Nx site and Fe4-N6 site structure in EXAFS experimental data. The authors need to show experimental data for the Fe-Nx site and Fe4-N6 site structure presented in the modeling.
3) For site density and TOF measurements, the authors used nitrate stripping. Iron nanocluster and nanoparticles can be used for nitrate reduction (J. Water Process. Eng. 21,84-95,2018, Journal of Hazardous Materials,185,1513-1521, 2011. It is necessary to present reference or experimental results to ensure that the method using nitrate stripping is not affected by iron nanocluster and nanoparticles. 4) In Figure3a ORR performance data, FeSA/FeAC-2DNPC showed higher activity than FeSA/FeNP-2DNPC and FeSA-2DNPC. In a previous paper, the synergistic effect of clusters and single atoms was shown in acid electrolytes (Small Methods, 5, 2001165, 2021). As in the previous paper, it is necessary to explain whether the cluster acts as an active site and produces a synergistic effect. In addition, FeSA/FeNP-2DNPC has a lower Tafel slope value and a higher TOF value than that of FeSA-2DNPC. It is necessary to present experimental or DFT modeling results to explain why FeSA/FeNP-2DNPC ORR activity is lower than that of FeSA-2DNPC in half-cell test.

5)
In PEMFC full cell test, it should be clarified that the MEA is tested under atmospheric pressrure (1 bar) or applied pressure (ambient pressure+1 bar=2 bar). It is not clear with the information provided in the experimental part.

Response to comments Reviewer #1 (Remarks to the Author):
In the manuscript by Wan et al., the authors report the construction of Fe-N-C electrocatalyst with single-atom/cluster hybrid active sites for high-performance fuel cells. The iron single atoms and clusters are stabilized by N/C on carbon support and closely adjacent, thus creating a strong interaction. It is interesting that this strong interaction simultaneously increases the activity and stability of the single-atom active sites. In recent years, stability challenge has become a central issue for the development of M-N-C fuel cell catalysts, while the mitigation strategies are still limited. This work is the first to show that the synergy between singleatom/cluster could improve the fuel cell stability. The authors also performed molecular dynamics simulations to investigate the mechanism for the stability enhancement, and proposed a pinning effect of iron clusters, which can shorten the amplitude of Fe-N bonds of the adjacent Fe-N4 active sites and thus improve their stability.
The presented data are well organized and the editing quality of the manuscript is acceptable.
Due to the promising results and the apparent scientific quality, I would recommend the manuscript for publication in Nature Communications after solving the following issues.

R:
We are grateful to the reviewer for the support and valuable comments/suggestions. Comment 1: It is often very challenging to well define the structure of metal clusters. In DFT calculations, why do the authors select an Fe4-N6 structure for Fe clusters? Please justify.

R:
We understand the reviewer's concern about the theoretic model. Our model is based on the electron microscopic characterization and literature. A survey of previous literature shows the following two types of practices when modeling the hybrid active site: Type Ⅰ (No. 1&2): the iron cluster is supported on pure carbon adjacent to an Fe-Nx site.
However, we believe that Fe cluster is more likely to coordinate with N because N is ubiquitous and more electronegative than C.
Type Ⅱ (No. 3&4): the iron cluster is connected to an Fe-Nx site via Fe-Fe bonding. This type is also less likely because the HADDF-STEM characterization shows that there is a gap between the Fe single atom and the cluster. Therefore, it is reasonable for us to setup a model of N-coordinated iron cluster adjacent to an Fe-Nx site. Here, Fe4-N6 is a simplified model for iron clusters as it is difficult to define the exact structure of metal clusters. In revision, we also try a larger cluster model consisting of 13 Fe atoms. The DFT calculation result shows that the regulation effect of Fe13-N6 is similar to that of Fe4-N6 ( Supplementary Fig. 35

R:
We are sorry for the insufficient experimental details.
The yield of CQD was about 25 wt% from the raw ZIF-8 precursor. The yield of the catalyst was about 20 wt% based on CQD.
The concentration of the CQD colloidal solution was determined by a dry weighing method.
A 100-µl aliquot of CQD colloidal solution was pipetted onto microscope slides and dried on a hot plate, and then the remaining solid was weighed.
The ink was subjected to sonication for 10 min and stirring for 10 h to make a uniform suspension.
The carbon paper (GDS2240 with a microporous layer) was obtained from Ballard.
We have added the above information in the Methods section.

Comment 4:
It would be better if the fuel cell stability of the catalyst is compared with the literature.

R:
We appreciate the reviewer's valuable suggestion. The fuel cell stability of M-N-C catalysts measured under the 1 bar H2-air is compared in Table R1.
The manuscript has been accordingly revised: "This stability performance is very promising compared with reported M-N-C catalysts (Supplementary Table 7)." Page 16.

R:
We are sorry for the typos. We checked the manuscript thoroughly and corrected the errors in the revised manuscript.

Reviewer #2 (Remarks to the Author):
In this manuscript, Wan and coworkers reported a system involving Fe-N-C sites and Ncoordinated Fe clusters for its application in acidic fuel cells. The physicochemical properties of catalysts were well investigated using HAADF-STEM, XPS, and XAS. DFT and MD simulations provided convincing support to the proposed reaction mechanism. Due to the strong electronic interplay within the short interaction distance, the binding strength of intermediates on SAs was optimized by surrounding clusters, which boosted the ORR reaction activity, as manifested by the half-wave potential of 0.81 V and the TOF of 2.82 s −1 . Moreover, the bond amplitude of Fe-N4 was shortened by the incoherent vibration, thus leading to better demetallation resistance and stability in acidic conditions. Overall, it is an interesting work and offers some possible explanations about the existing problems of SA/cluster ORR catalysts, especially on the stability enhancement. But at the same time, there are some key opinions and analyses presented in this manuscript that I could not fully agree with. Considering the impact of Nat. Commun., I suggest a major revision and another run of review. The following concerns should be well addressed, and I hope it can achieve a better quality before being considered.

R:
We are grateful to the reviewer for the support and valuable comments/suggestions. We have accordingly revised the manuscript: "However, so far, the ORR activity of M-N-C in acidic media is still significantly lower than that of Pt-based catalysts due to insufficient accessible active sites and less competitive TOF." Page 3.
2) We are sorry for the inaccurate statement. We agree that the metal nanoparticles/clusters in the mentioned references might be connected to carbon support via Fe-C bonding. However, the dissolution of these clusters in acid indicates weak support-cluster interactions. We have accordingly modified this statement: "It suggests that these metal NPs/ACs are weakly anchored (or bonded) on the carbon support, which may result in a very limited regulation effect on the electronic configuration of the SA sites." Page 4.

Comment 2:
In the acidic ORR performance tests ( Fig. 3a), the FeSA, FeSA/cluster, and FeSA/particle display very similar Eonset and E1/2 values. The enhanced TOF value of FeSA/cluster actually stems from the lower Fe site density as determined by NO stripping.
Speaking from the aspects of mass activity and atomic utilization efficiency, which are more important indices in practical fuel cells, the FeSA/cluster even exhibits much worse performance.
How do the authors evaluate these disadvantages?

R:
The main advantage of Fe-N-C catalysts over Pt/C is negligible cost. For PGM catalysts, Pt mass activity and atomic utilization efficiency are important indices. But for PGM-free catalysts, it is more meaningful to evaluate the catalyst in terms of the area current density and volumetric current density of the fuel cell cathode. Therefore, US DOE targets are 0.44 A mgPt −1 @0.9ViR-free for PGM catalysts, while 300 A cm −3 @0.8ViR-free and 0.044 A cm −2 @0.9ViRfree for PGM-free catalysts. In this context, FeSA/FeAC-2DNPC is obviously the best among three catalysts. Besides, the core value of this report is a new mechanism for the activity/stability enhancement of Fe-N-C catalysts by Fe clusters, whose cost is negligible.
Comment 3: Carbon corrosion and demetallation are two factors that can affect each other.
According to the literature report, Angew. Chem. Int. Ed., 2015, 54, 12753-12757, carbon oxidation occurs at a high potential (> 0.9 V) with the destruction of active Fe-N-C sites which was believed to be the main reason for the activity degradation of catalysts in acidic medium.
Since the authors observed negligible ID/IG ratio change in Raman spectra, the stable carbon structure should be responsible for the robust Fe-N-C sites and the enhanced stability. The sentence "the impact of carbon corrosion can be ruled out" on Page 13, Line 16 is not proper then. Except for the demetallation of SA, nanoparticles and clusters also suffer from the risk of leaching. In ACS Energy Lett., 2019, 4, 1619 and ACS Catal. 2021, 11, 484, the low or nonactive Fe clusters derived from Fe single atoms during PEMFC operation were confirmed to be easy to leach from the carbon matrix. What about the possibility of the aggregation of SA into clusters in this work? Is the bond in the Fe cluster stable enough? Will the formation and leaching of clusters affect the density of SA and weaken the synergies between SA and cluster?

R:
We appreciate the reviewer's insight into the degradation mechanisms. Specifically: Carbon corrosion: We fully agree that carbon oxidation is an indispensable cause of activity degradation. We apologize for inaccurate words that misled reviewers. Actually, the Raman spectra were not collected before and after the AST test. Instead, we compared the Raman spectra of the three fresh catalysts to check whether they differ in the degree of graphitization, as this is also a factor affecting catalyst stability. The results show that the three catalysts had a similar degree of graphitization. We have modified our statement: "Therefore, the difference in the stability of the three catalysts is independent of the graphitic degree of the carbon supports." Page 13.  Fig. 28) shows the well-retained single atoms and clusters, indicating the leaching rate was quite slow. We also noticed that the TOF of FeSA/FeAC-2DNPC decreased slightly from 2.60 to 2.39 s −1 , but still much higher than 1.79 s −1 of the isolated Fe-N4 site in FeSA-2DNPC, suggesting the interaction between SA and cluster still existed.
Effects of cluster formation and leaching: As discussed above, the Fe ions leached from the Fe-N4 site and then possibly formed FexO clusters. In this case, the density of SA surely decreased as measured by our nitrite stripping experiment (18.7% reduction after 5k potential cycles), while the FexO clusters (if any) were inactive and did not have a synergy with the Fe-N4 sites. The pristine N/C-coordinated clusters also leached, which may weaken the synergies between SA and cluster to some extent, as manifested by the reduced TOF value.

Comment 4:
The fabrications of FeSA, FeSA/cluster, and FeSA/particle catalysts were realized by varying the ratio of TPI precursors. By this approach, without any spatial confinement, the distribution and population of each Fe species are quite hard to control. For example, in Fig.   1d, the distance between SA and the adjacent cluster was measured at around 0.5 nm. However, there is a large amount of isolated SAs that distribute far away from the cluster and they are not taken into account. It is strongly suggested to reconsider the future of the proposed strategy of using Fe clusters as "boosters" for enhancing the intrinsic ORR activity/stability of M-N-C.
The difficulty in the accurate synthesis of these structures would definitely cause reproducibility issues and increase the production cost. Besides, is it possible to precisely calculate the ratio of atomic Fe species and Fe clusters in the synthesized samples? We had noticed there were many isolated SAs that distribute far away from the cluster, which should be responsible for the fast drop of fuel cell performance of FeSA/FeNP-2DNPC during the initial 32 h (Fig. 4a).
As for the ratio of atomic Fe species and Fe clusters, it is hard to calculate via XPS or XAS because the Fe clusters are oxidized. A feasible method is to count a large number of single atoms and clusters in HAADF-STEM images. Based on three different areas of FeSA/FeAC-2DNPC ( Figure R1), a total of 908 iron single atoms and 90 clusters have been counted.
Therefore, the ratio of atomic Fe species and Fe clusters in the catalyst is about 10:1. Some seemingly large clusters are composed of several small adjacent clusters while some bright spots may be due to the image overlap of single Fe atoms. We have added a description in the Catalyst synthesis and characterization section: "From HAADF-STEM images, we estimate that the average diameter of Fe ACs is 0.7 nm and the ratio of SA to AC is about 10:1 ( Supplementary Fig. 9). We note that there is a fraction of SAs far away from the ACs, which should behave like regular single-atom active sites." Page 8.

R:
We thank the reviewer for the comments on the computational model. Figure R1, the average diameter of Fe clusters is around 0.7 nm. It is a common practice in the literature to use a simplified model of four iron atoms to represent an iron cluster. We also tried a larger model consisting of 13 Fe atoms (Fe-N4-OH/Fe13-N6) in DFT calculations. As shown in Figure R2, the boosting effect of Fe13 cluster is similar to that of the Fe4 cluster and the limiting energy barrier is still much smaller than that of Fe-N4 (0.38 vs. 0.53 eV). 2) We thank the reviewer for pointing out the inaccuracy in the fitting of XPS spectra. We checked our XPS raw data and found that we had mixed up the Fe 2p data for FeSA-2DNPC

1) As shown in
and FeSA/FeNP-2DNPC during the data fitting. We are very sorry for this mistake. The raw Fe 2p data are shown in Figure R3a. Although the signal-to-noise ratio is less satisfactory due to low iron contents, Fe 0 (~706.7 eV) can be seen in FeSA/FeNP-2DNPC, while is not discernible in FeSA-2DNPC and FeSA/FeAC-2DNPC. According to the reviewer's suggestion, we refit the  Since N is more electronegative than C, the Fe atom is more likely to coordinate with N, which can be corroborated by the fact that Fe-N4 sites are prevalent in Fe-N-C system while Fe-C4 sites have been rarely reported. Therefore, it is reasonable for the Fe cluster to coordinate with N. We also calculated the carbon coordinated clusters (Fe-N4/Fe4-C6), as presented in the recommended literature. As shown in Figure R4, the regulation effect of the Fe4-C6 cluster on the Fe-N4 site is rather weak and limiting energy barrier is the same as the bare Fe-N4 site.
The manuscript has been revised accordingly: "Two variants of the cluster are further investigated using models of Fe13-N6 and Fe4-C6. The calculations show that the Ncoordinated iron cluster has a much more significant boosting effect on the adjacent Fe-N4 site than the C-coordinated iron cluster, while the number of Fe atoms in the cluster plays a less significant role (Supplementary Figs. 35 and 36)." Page 19. the LSV curves before and after 5k cycles, the chronoamperometric tests at 80 °C should also be given. 4) What is the spiking current shown in Fig. S17a?

R:
1) The ring and disk current curves recorded on RRDE for Figure 3a,b are provided, as shown in Figure R5. 2) The WT contour plots of FeSA-2DNPC and FeSA/FeNP-2DNPC are provided, as shown in

Reviewer #3 (Remarks to the Author):
This research has originality for synthesis of catalyst for efficient ORR system through the complex of Fe-N4 single atom site, Fe4-N6 nanocluster, Fe nanoparticle. Fe4-N6 nanocluster can modulate the electronic structure of Fe-N4 site. Compared to Fe single atom catalysts, complex of Fe-N4 single atom site and Fe4-N6 nanocluster have high activity and durability. Since transition metal SACs can replace noble metal catalyst, attracting researchers in renewable energy society. In particular, Fe single atom catalyst has a problem of low durability in acid electrolytes, and this paper suggests a way to solve it. So, this paper is worth to be published in Nature Communications after revision of the manuscript following the comments below.

R:
We are grateful to the reviewer's support and valuable comments/suggestions.

Comment 1:
Through the Raman spectra data and difference of ORR limiting current density during the AST process, authors show that carbon corrosion does not occur. The pyrolysis temperature of M-N-C can affect the graphitic degree of carbon support and activity of the M-N4 site. Therefore, the authors need to show the relationship between the graphitic degree (Raman data) and activity (stability) difference at different temperature.

R:
We appreciate the suggestion. We adjusted the temperature of the second pyrolysis (T2 /°C) from 800 to 1000 °C. The samples were denoted as x%-T2, where x% means the mass ratio of TPI relative to CQD in the precursor. The Raman spectra of these samples are shown in Figure   R8. The ORR activity of the samples was evaluated by rotating ring disk electrode (RRDE) in O2-saturated 0.5 M H2SO4 solution at room temperature. The stability was evaluated by 10,000 potential cycling from 0.6 to 1.0 V vs. RHE in O2-purged 0.5 M H2SO4 at a scan rate of 50 mV s −1 and a rotation rate of 300 rpm at room temperature. The results are shown in Figure R9.
Based on these data, we correlate the graphitic degree (ID/IG), activity (E1/2), and stability (∆E1/2 after 10,000 cycles) with T2 ( Figure R10). We can draw the following conclusions: 1) Increasing T2 can increase the graphitic degree of the catalyst, while the Fe content does not influence the graphitic degree of the catalyst.
2) Increasing T2 can increase the catalyst activity, possibly due to the thermally driven evolution of ideal local structures of Fe-N4 sites, with one exception (30%TPI-1000). We speculate that the high Fe content plus high pyrolysis temperature led to Fe agglomeration into inactive Fe nanoparticles and thus decreased the activity.
3) Increasing T2 can improve the catalyst stability, which could be partially attributed to the enhanced graphitic degree. We note that when T2 was increased from 900 to 1000 °C, the stability of the 15%-T2 series showed a greater improvement relative to the other two groups, suggesting the emergence of a new stability enhancement mechanism.
The manuscript has been accordingly revised: "The pyrolysis temperature was optimized to 1000 °C to achieve the best activity and stability  The high temperature is crucial for the formation of optimal active sites and highly graphitic carbon support." Page 7.
"The approximately equal ID/IG ratios indicate a similar degree of graphitization of the three catalysts, which is controlled by the pyrolysis temperature regardless of the iron content ( Supplementary Fig. 7a)." Page 13.     Figure R11 and Table R2.   N is coordination number, R is the distance between absorber and backscatter atoms, σ 2 is Debye-Waller factor to account for both thermal and structural disorders, ΔE0 is inner potential correction; R factor indicates the goodness of the fit. Error bounds (accuracies) that characterize the structural parameters obtained by EXAFS spectroscopy were estimated as N ± 20%; R ± 1%; σ 2 ± 20%; ΔE0 ± 20%. S0 2 was fixed to 0.9 as determined from Fe foil fitting. Fitting range: 2.5 ≤ k (/Å) ≤ 10.8 and 1 ≤ R (Å) ≤ 3. iron) had also been tested and they concluded that the method should work provided the catalyst is not highly active for hydrogen evolution, which would mask the stripping charge. Therefore, the stripping method is generally applicable for the vast majority of Fe-N-C catalysts.
We have added a brief note in the Methods section to clarify this issue: "We note that nitrite ions may be reduced by the trace amount of metallic iron (if any) in the catalysts. However, the major products are ammonium and nitrogen gas, which should not interfere with adsorption and subsequent stripping of nitrite on Fe-Nx sites." Page 27.
Comment 4: In Figure 3a ORR performance data, FeSA/FeAC-2DNPC showed higher activity than FeSA/FeNP-2DNPC and FeSA-2DNPC. In a previous paper, the synergistic effect of clusters and single atoms was shown in acid electrolytes (Small Methods, 5, 2001165, 2021). As in the previous paper, it is necessary to explain whether the cluster acts as an active site and produces a synergistic effect. In addition, FeSA/FeNP-2DNPC has a lower Tafel slope value and a higher TOF value than that of FeSA-2DNPC. It is necessary to present experimental or DFT modeling results to explain why FeSA/FeNP-2DNPC ORR activity is lower than that of FeSA-2DNPC in half-cell test.

R:
We investigated the ORR activity of the cluster by DFT calculations. The Fe4 surface in Fe-N4-OH/Fe4-N6 is chosen as the active site. As shown in Figure R12, a large energy uphill (1.37 eV) for OH* desorption is observed, implying the formation of a permanent OH ligand. After the attachment of the OH ligand, however, the situation becomes even worse ( Figure R13). The ultralarge ∆G (3.64 eV) for the formation of OH* (O* + H+ + e − → OH*) indicates that the reaction is impossible to proceed. Therefore, the iron cluster, at least in the considered model, does not act as an active site. Instead, it acts as a booster to enhance the intrinsic ORR activity/stability of the Fe-N4 site.
The manuscript has been accordingly revised: "The Fe4 in Fe-N4-OH/Fe4-N6 is predicted with inferior activity (Supplementary Figs. 33 and 34), indicating the cluster mainly acts as an activity booster." Page 19.  On the activity difference between FeSA/FeNP-2DNPC and FeSA-2DNPC: Actually, FeSA/FeNP-2DNPC has a slightly lower Tafel slope value and a slightly higher TOF value (57.2 mV dec −1 , 2.01 s −1 ) than those of FeSA-2DNPC (57.8 mV dec −1 , 1.79 s −1 ). The apparent ORR activity (expressed by E1/2) of FeSA/FeNP-2DNPC (0.786 V) is slightly lower than that of FeSA-2DNPC (0.795 V). As you see, the electrochemical properties of these two catalysts are rather similar. This is because the iron nanoparticles in FeSA/FeNP-2DNPC are encapsulated by thick layers of graphitic carbon (typically > 2 nm, as shown in Supplementary   Fig. 13). The possible synergistic effect between single-atom Fe-N4 and the Fe NPs should be rather weak. Therefore, the TOF of the active sites in FeSA/FeNP-2DNPC is only marginally enhanced. In our study, we conclude that the closely adjacent iron clusters serve as a more powerful promoter of the activity of Fe-N4 compared with the encapsulated iron NPs. On the other hand, the formation of iron NPs consumes considerable iron sources and leads to the decreased density of Fe-N4 sites, which is confirmed by the nitrite stripping experiments.
FeSA/FeNP-2DNPC has an SD of 30.2 μmol g −1 , which is much smaller than that of FeSA-2DNPC (41.4 μmol g −1 ). The slightly high TOF but much lower SD make the apparent activity of FeSA/FeNP-2DNPC lower than that of FeSA-2DNPC in half-cell test.

Comment 5:
In PEMFC full cell test, it should be clarified that the MEA is tested under atmospheric pressure (1 bar) or applied pressure (ambient pressure+1 bar=2 bar). It is not clear with the information provided in the experimental part.

R:
We are sorry for the insufficient experimental information. The pressure conditions for each fuel cell data were specified in the figure captions. For example, 1 bar H2-O2 means that the absolute pressure for H2 and O2 is both 1 bar, which is achieved by applying 0.5 bar backpressure. For clarity, we have added this information to the Methods section.