Effect of N-(2-Aminoethyl)-3-Aminopropyltrimethoxysilane on the Adhesion of the Modified Silicone Tie-Coating to Epoxy Primer

Round 1

Reviewer 1 Report

Peer review on manuscript

“Effect of N-(2-Aminoethyl)-3-aminopropyltrimethoxysilane on the adhesion of the modified silicone tie-coating to epoxy primer.”

 

Introduction

 

The reviewed manuscript described a study reporting a simple modification method for silicone tie-paints securing adhesion between the epoxy anticorrosive primer and silicone fouling release coating and investigated the peel off and shear tests to evaluate the adhesion. It explains how understanding the influence of introducing N-(2-Aminoethyl)-3-aminopropyltrimethoxysilane (DAMO) is helpful in significantly improve the properties of the tie-coating.

 

Merits

Although the adhesion of coating layers for epoxy and silicone is well-researched in certain parts of its range, for example:

• (Holberg et al. 2020)
• (Svendsen et al. 2007)
• (Hu et al. 2020)

But the manuscript focused on some understudied approaches with a simple idea that is easy to implement. Therefore, DAMO’s effects on the improvement of the adhesion between the tie-coating and the epoxy primer presented have significant merit for understanding and possible future conversations of coating in the area.

 

Critique

 

The research is a well-written manuscript, and I am looking forward to seeing it published.

• Line 19 in the abstract section has a typo. DMAO>>DAMO

 

References

Holberg, Stefan, Ricardo Losada, Frances H. Blaikie, Helena H. W. B. Hansen, Sylvie Soreau, and Rob C. A. Onderwater. 2020. “Hydrophilic Silicone Coatings as Fouling Release: Simple Synthesis, Comparison to Commercial, Marine Coatings and Application on Fresh Water-Cooled Heat Exchangers.” Materials Today Communications 22:100750.

Hu, Peng, Qingyi Xie, Chunfeng Ma, and Guangzhao Zhang. 2020. “Silicone-Based Fouling-Release Coatings for Marine Antifouling.” Langmuir 36(9):2170–83.

Svendsen, Jacob R., Georgios M. Kontogeorgis, Søren Kiil, Claus E. Weinell, and Martin Grønlund. 2007. “Adhesion between Coating Layers Based on Epoxy and Silicone.” Journal of Colloid and Interface Science 316(2):678–86.

 

Author Response

Dear Reviewer:

Thank you for your comments concerning our manuscript entitled “Effect of N-(2-Aminoethyl)-3-aminopropyltrimethoxysilane on the adhesion of the modified silicone tie-coating to epoxy primer” (ID: coatings-1027135). These comments are all valuable and very helpful for revising and improving our paper, as well as the important guiding significance to our researches. We have studied comments carefully and have revised the full text. The language of this manuscript has been reviewed by a technical editor. Revised portion is marked using the “Track Changes” function in Microsoft Word. The responses to your comments are as follows.

1. Line 19 in the abstract section has a typo.                      Response:

Authors thanks for the comments of reviewer and revised the manuscript. Please find in revised manuscript.

Author Response File: Author Response.docx

Reviewer 2 Report

In this paper the authors report about the effect of DAMO mechanically mixed with modified silicone tie-coating based on PDMS as curing compound. They prepare the coating mixing various percentages of DAMO and studied the adhesion to epoxy primer of the silicone tie.-coating through the investigation of the interfacial morphology, tensile properties, chemical structure and interlaminar bonding strength and adhesion test.

The results reported in this paper are well written and reports interesting results that deserve publication and fits well with the scope of Coatings. The presented data has good quality and the data messages explained well and clear by the authors, supported with enough scientific data.
For all the above, I recommend to accept this article after minor revisions of the text. In particular:

1. the revision of the references to increase their uniformity;
2. pag 2 row 70 explain the acronym DAMO the first time in the text of the paper;
3. pag 6 row 186 Please check the sentence "One of the needed properties of the tie-coating was is excellent connection properties with..."
4. pag 8 row 231 Fig.8a should be replaced with Fig. 9a;
5. pag 9 row 242 Please check the sentence "All results show that the tie-coating was modified by DAMO would improve the interlaminar 242 bonding condition between the tie-coating and epoxy primer.".

Author Response

Dear Reviewer:

Thank you for your comments concerning our manuscript entitled “Effect of N-(2-Aminoethyl)-3-aminopropyltrimethoxysilane on the adhesion of the modified silicone tie-coating to epoxy primer” (ID: coatings-1027135). These comments are all valuable and very helpful for revising and improving our paper, as well as the important guiding significance to our researches. We have studied comments carefully and have revised the full text. The language of this manuscript has been reviewed by a technical editor. Revised portion is marked using the “Track Changes” function in Microsoft Word. The responses to your comments are as follows.

1. the revision of the references to increase their uniformity. Response:

Authors revised carefully the references. Please find in revised manuscript.

2. pag 2 row 70 explain the acronym DAMO the first time in the text of the paper.

Response:

Authors agreed with the comments of reviewer and revised the manuscript. Please find it in revised manuscript.

3. pag 6 row 186 Please check the sentence "One of the needed properties of the tie-coating was is excellent connection properties with...".

Response:

Authors revised the sentence as “Excellent connection property is very important for tie-coating”.

                                                                      

4. 4. pag 8 row 231 Fig.8a should be replaced with Fig. 9a. Response:

Authors revised it. Please find in revised manuscript.

5. pag 9 row 242 Please check the sentence "All results show that the tie-coating was modified by DAMO would improve the interlaminar 242 bonding condition between the tie-coating and epoxy primer."

Response:

Authors agreed with the comments of reviewer and revised the manuscript. The sentence was revised as “All the results show that DAMO modified tie-coating can the interlaminar bonding between the silicone coating and epoxy primer.”

Author Response File: Author Response.docx

Reviewer 3 Report

The authors provide an interesting study on how DAMO can impact the functional performance of tie-paints.  While the data presented does paint the picture that adding DAMO can improve the overall performance of tie-paints (albeit in a more complicated dose-dependent manner), there are major gaps in the story that need to be addressed before this paper can be published in Coatings. 

First and foremost, the Materials and Methods needs to be substantially updated to include a lot more information as to how the experiments were set up.  While this holds true for all section of the Materials and Methods, a list of some of the more presseing concerns are summarized below.  Without a clear understanding of how the experiments were carried out, it is impossible to determine whether the conclusions made by the author are scientifically valid.  

• The terms “filler” and “accessory ingredient” in numerous places, but they are never defined. This needs to be clarified in order to assess what chemical reactions are taking place, and the mechanisms for improved bonding.  For instance, how can the authors be certain that the chemical reaction is occurring directly between the epoxy primer and tie coating, and that there is no crosslinking between either of these and the “filler” or “accessory ingredient”? 
• Line 119: “the cast film samples were cut into 45 mm×4 mm dumbbell-shaped samples.” How were there samples cut?  More information must be provided so that the reader can assess whether cutting of the samples impacted the integrity of the sample.
• Line 139-142: What methods were taken to ensure that the same amount of tie-paint and epoxy primer were used for each experiment? Or were there different amount of each used in the various experiments?  Also, how were the samples bonded?  Were the two pieces held together with a constant force or weight?  How long were the samples allowed to cure, and was this constant for all samples?  How long were samples stored prior to measuring, and what were the storage conditions?  There is a lot of information missing in the methods that needs to be evaluated.  How the bonding was done would have a dramatic impact on the data obtained.  For instance, how the samples are applied, cured, stored and tested may have a significant impact on the fracture morphologies obtained using CLSM.

In the Results and Discussion section, there are several examples where a detailed discussion and analysis has not been conducted.  For instance, in section 3.1.1., the author just lists peaks and does not do any comparative analysis nor do they draw any conclusions.  There needs to be a detailed account of how (if) these peaks differ between samples, and what the difference mean.  For instance, what evidence is there that DAMO is doing anything?  The author claims that the peak at 3335 cm-1 is different in each because of the different DAMO applied, but are there any new bonds formed?

Other issues in the Results and discussion section are as follows:

• Section 3.1.2: Based on what I see in Figure 4b, there is no clear evidence that the DAMO was more prevalent on the substrate side. What is the basis for this claim?  If there is any quantitative analysis used to make this claim, has statistical analyses been performed to verify that there are in fact significant differences between them?  In addition, from what I can see, this phenomenon seems to be depending on the amount of DAMO applied.  Does the author have any insights into why this may be the case?
• Section 3.2.: Has any statistical analyses been carried out to demonstrate that any difference are statistically significant? For instance, the authors claim that the fracture elongation of TD1.24 is higher than that of TD0, however, looking at the values and the error bars, I find it highly unlikely that there are any difference between these.  The author is making some sweeping conclusions in this section that are not true for all dependent variables examined. In addition, the author states that “This can be attributed to the crosslinking density changes with DAMO content increasing. The interaction force between the molecular chain was changed, which changes the deformation capacity of the samples. The crosslink density of TD0 is low due to insufficient TEOS content, a certain amount of DAMO was introduced led to an increased crosslinking density that improved the tensile strength.” (Lines 172-175).  The author does not provide any data supporting that cross-linking is even occurring and thus these statements are far overreaching.  It needs to be made very clear in the text that there are no data to support this theory that there are differences in cross-linking.
• Figure 9: These are nice pictures and help to support the argument, however, how representative are these pictures? These images are no doubt taken from areas where the adhesion/fracture took place.  However, this does not tell the whole story as there doesn’t appear to be any control of the area where bonding is actually occurring.  The authors describe the region of overlap between the two panels, but the area of adhesion seems to vary considerably (i.e. see Figure 7 inlays).
• Figure 11: Epoxy groups are highly reactive and can react with a variety of functional groups, not just amines. Thus, I find this figure an extremely oversimplified interpretation of what is happening.  Especially once you consider that there is also “filler” and “accessory ingredient” added.  Even in the figure, final molecule contains 3 hydroxyl groups that could potentially react with epoxy groups.

Some other general comments:

• The English needs to be improved. There are numerous grammatical errors throughout.  There are even some examples of sentence fragments being put together illogically (i.e. Lines 81-13).  There are also issues with spacing throughout (i.e. missing or extra spaces, particularly when looking at units). 
• Figure legends need to be improved. Legends should provide the reader with all of the necessary information needed to assess the data.  In most if not all figures, there isn’t even a description of what the samples are (i.e. TD1.24: what is this)?
• Only tested 1.24 and 2.45 for most analyses, but for APR sheer strength, there were many more DAMO amounts tested, some of which are also promising. Is there a reason why these other formulations were not tested?
• Figure 8: “The white part is tie-coating, the red part is epoxy primer”. I don’t see any red color here. 
• References: There are only 20 references for this paper, only two of which are after 2016.  Certainly, much progress has been made in the adhesive space pertaining to epoxy bonding that could provide some more insights and opportunities for discussion. 

Author Response

Dear Reviewer:

Thank you for your comments concerning our manuscript entitled “Effect of N-(2-Aminoethyl)-3-aminopropyltrimethoxysilane on the adhesion of the modified silicone tie-coating to epoxy primer” (ID: coatings-1027135). These comments are all valuable and very helpful for revising and improving our paper, as well as the important guiding significance to our researches. We have studied comments carefully and have revised the full text. The language of this manuscript has been reviewed by a technical editor. Revised portion is marked using the “Track Changes” function in Microsoft Word. The responses to your comments are as follows.

1. The terms “filler” and “accessory ingredient” in numerous places, but they are never defined. This needs to be clarified in order to assess what chemical reactions are taking place, and the mechanisms for improved bonding. For instance, how can the authors be certain that the chemical reaction is occurring directly between the epoxy primer and tie coating, and that there is no crosslinking between either of these and the “filler” or “accessory ingredient”?                                                         Response:

Author has redefined the terms in the manuscript. Please find in revised manuscript.

2.Line 119: “the cast film samples were cut into 45 mm×4 mm dumbbell-shaped samples.” How were there samples cut?  More information must be provided so that the reader can assess whether cutting of the samples impacted the integrity of the sample.         Response:

Authors agreed with the comments of reviewer and revised the manuscript. Please find in revised manuscript. Tool shown in Fig.1.

Fig.1 Dumbbell-shaped knife

 

3.Line 139-142: What methods were taken to ensure that the same amount of tie-paint and epoxy primer were used for each experiment? Or were there different amount of each used in the various experiments?

Response:

 In the experiment, the samples were prepared by air spraying method to ensure that the same coatings in the samples were made with the same spraying pressure, speed, and spraying times. After curing, the coating thickness was measured by a film thickness meter and the most qualified sample was selected. At the same time, the epoxy primer or tie-coating in the sample was made from the same batch of paint to avoid errors in multiple measurements.                                                                                      

 4.Also, how were the samples bonded?  Were the two pieces held together with a constant force or weight? 

Response:

Regarding the binding method of the samples, no external force was used for fixation. First, the epoxy primer was sprayed on the treated substrate, after the surface is dry, the prepared tie-paint was sprayed on the epoxy primer surface. When preparing samples, ensure that other conditions other than the sample research variables are the same. The samples were flat placed in a high-low temperature damp heat test box at 25 degrees Celsius and humidity of 60% for curing, and take it out for testing after 7 days.

5.How long were the samples allowed to cure, and was this constant for all samples? 

Response:

The samples were allowed to cure for 7 days in a high-low temperature damp heat test box at 25 degrees Celsius and humidity of 60%, except for the research variables, other constants were the same.

 

6.How long were samples stored prior to measuring, and what were the storage conditions?  

Response:

The samples were flat placed in a high-low temperature damp heat test box at 25 degrees Celsius and humidity of 60% for curing, and take it out for testing after 7 days.

                                                                  

7. 7. In the Results and Discussion section, there are several examples where a detailed discussion and analysis has not been conducted. For instance, in section 3.1.1., the author just lists peaks and does not do any comparative analysis nor do they draw any conclusions. There needs to be a detailed account of how (if) these peaks differ between samples, and what the difference mean.                                                         Response:

It can be seen from the Fig.3 that there peaks between samples mentioned in the manuscript (except at 3335cm^-1) have no obvious difference.

 

8. 8. For instance, what evidence is there that DAMO is doing anything?

Response:

It can be known from chemical knowledge that the alkoxy group in DAMO will undergo cross-linking reaction with PDMS and TEOS under the action of the catalyst to complete the connection between DAMO and PDMS in the tie-paint, and realize the introduction and fixation of amino groups. Please find in revised manuscript.

 

9. The author claims that the peak at 3335 cm-1 is different in each because of the different DAMO applied, but are there any new bonds formed?

Response:

Due to the DAMO content was less and the limitations of ATR-FTIR testing, it is difficult to in-depth investigate on amino groups, so it is not certain whether a new bond is formed.

 

10. Section 3.1.2: Based on what I see in Figure 4b, there is no clear evidence that the DAMO was more prevalent on the substrate side. What is the basis for this claim?

Response:

This claim was obtained by XPS quantitative analysis. Please find in revised manuscript.

 

11. If there is any quantitative analysis used to make this claim, has statistical analyses been performed to verify that there are in fact significant differences between them?

Response:

This claim has been verified by repeated experiments, and the results were statistical analysed tend to be consistent.

 

12. In addition, from what I can see, this phenomenon seems to be depending on the amount of DAMO applied. Does the author have any insights into why this may be the case?

Response:

The results from XPS show that even if the DAMO content is less or more in the coating, the DAMO was more prevalent on the substrate side, so this conclusion was inferred.

 

13. Section 3.2.: Has any statistical analyses been carried out to demonstrate that any difference are statistically significant? For instance, the authors claim that the fracture elongation of TD1.24 is higher than that of TD0, however, looking at the values and the error bars, I find it highly unlikely that there are any difference between these. The author is making some sweeping conclusions in this section that are not true for all dependent variables examined.

Response:

The result was obtained from the same sample through 3 repeated experiments, and meets the statistical requirements. After inspection, the result was a writing error, which has been revised in the revised manuscript, please find in revised manuscript.

 

14. In addition, the author states that “This can be attributed to the crosslinking density changes with DAMO content increasing. The interaction force between the molecular chain was changed, which changes the deformation capacity of the samples. The crosslink density of TD0 is low due to insufficient TEOS content, a certain amount of DAMO was introduced led to an increased crosslinking density that improved the tensile strength.” (Lines 172-175). The author does not provide any data supporting that cross-linking is even occurring and thus these statements are far overreaching. It needs to be made very clear in the text that there are no data to support this theory that there are differences in cross-linking.

Response:

Authors agreed with the comments of reviewer and revised the manuscript. Please find in revised manuscript.

 

15. Figure 9: These are nice pictures and help to support the argument, however, how representative are these pictures? These images are no doubt taken from areas where the adhesion/fracture took place. However, this does not tell the whole story as there doesn’t appear to be any control of the area where bonding is actually occurring. The authors describe the region of overlap between the two panels, but the area of adhesion seems to vary considerably (i.e. see Figure 7 inlays).

Response:

We have prepared three batches of samples in total, each batch of samples prepared under the same conditions. The same method was adopted for each batch of samples, and six fractures are obtained for each sample for observation. The results are analyzed and counted, and representative pictures are selected.

 

16. Figure 11: Epoxy groups are highly reactive and can react with a variety of functional groups, not just amines. Thus, I find this figure an extremely oversimplified interpretation of what is happening. Especially once you consider that there is also “filler” and “accessory ingredient” added. Even in the figure, final molecule contains 3 hydroxyl groups that could potentially react with epoxy groups.

Response:

The filler titanium dioxide, silicon dioxide and auxiliary component leveling agent, TEOS, etc. in the experiment do not have the ability to react with epoxy groups. The relevant literature shows that the reaction between epoxy group and hydroxyl groups can be completed at a certain temperature and the presence of a catalyst[1]. In this reaction, with regard to the reaction mechanism of the amino group and the epoxy group, only the reaction mentioned in the manuscript has been agreed by everyone at present,, but there are different views on the reaction mechanism when hydroxyl groups exist[2].

[1]Zhang, Z. M.; Guo, X. Y.; Tong, Z. Synthesis and Characterization of Bisphenol A Glycidyl Dimethacrylate. Chemical Times,2006,6,7-9.

[2]Zhao, J. Research on Main Performance Characterization Technology of Bisphenol A Epoxy Resin System. 2016.

 

17. The English needs to be improved. There are numerous grammatical errors throughout. There are even some examples of sentence fragments being put together illogically (i.e. Lines 81-13). There are also issues with spacing throughout (i.e. missing or extra spaces, particularly when looking at units).

Response:

Authors agreed with the comments of reviewer and revised the manuscript. Please find in revised manuscript.

18. Figure legends need to be improved. Legends should provide the reader with all of the necessary information needed to assess the data. In most if not all figures, there isn’t even a description of what the samples are (i.e. TD1.24: what is this)?

Response:

The naming of the sample has been explained in 2.2. The sample was named TDx, x represents the tie-coating with x mass% of DAMO in the tie-coating. For example, TD1.24 indicates that the DAMO content in the tie-coating is 1.24wt%.

19. Only tested 1.24 and 2.45 for most analyses, but for APR sheer strength, there were many more DAMO amounts tested, some of which are also promising. Is there a reason why these other formulations were not tested?

Response:

The core of the research in this manuscript is the adhesion of the tie-coating on the epoxy primer. Four methods (interlaminar adhesion, shear strength, CLAM and SEM) were used to investigate the adhesion. The fracture morphology of the samples showed similar phenomena within a certain DAMO content range according to SEM and CLSM. So three typical samples were selected, and each sample represents the characteristics of sample near this DAMO content.

 

20. Figure 8: “The white part is tie-coating, the red part is epoxy primer”. I don’t see any red color here.

Response:

Authors agreed with the comments of reviewer and revised the manuscript. Please find in revised manuscript. The red part is not obvious due to poor light when taking pictures.

 

21. References: There are only 20 references for this paper, only two of which are after 2016. Certainly, much progress has been made in the adhesive space pertaining to epoxy bonding that could provide some more insights and opportunities for discussion.

Response:

Authors revised carefully the references. Please find in revised manuscript.

 

Author Response File: Author Response.docx

Round 2

Reviewer 3 Report

I thank the Authors for their revisions.  There are still a few issues that need to be addressed prior to publication (see below).  For the most part, I think that the authors just need to incorporate their responses into the actual manuscript so that the reader has all of the information necessary.  The authors' explanations were done well in general, but will not provide any clarity to the reader of the manuscript.

1) The English still needs to be improved, with a particular emphasis on the edited text in red. 

2) Responses to issues with the materials and methods section should be incorporated into the actual manuscript, rather than just explained in the Response to Reviewers.  While this has clarified many issues for me, the reader will still have no clue.

3) Regarding section 3.1.1, the authors suggest in the paper and in their Response to Reviewers that there was a crosslinking between DMAO and PDMS/TEOS as this reaction is well known. While I recognize that this chemical reaction is well documented, my point is still that the authors have done nothing to show that there is any cross-linking occurring in there studies, and in fact, the FTIR data seem to suggest that no reaction took place.  The peaks at 3335 cm-1 just confirm that DAMO has been mixed into the tie-coating, but does not confirm that any crosslinking reaction took place.  Since there is no direct evidence that cross-linking reaction occurred, I caution the authors against suggesting that the data result due to cross-linking.  This is pure speculation, and though I am okay publishing as such, it needs to be pointed out to the reader that there is no direct evidence of cross-linking. 

4) I am happy to hear that the quantitative analysis was been verified via repeated experiments, however, the statistical analysis used (as well as the software) needs to be identified in the materials and methods.  The confidence level and the number of replicates also needs to be identified. 

5) Again, with regards to my comment #12, it is okay to me that this conclusion was inferred, but this needs to be specifically described in the manuscript so that the reader knows that this is speculation. 

6) Regarding comment #15, the authors response needs to be incoropated into the materials and methods so that the reader understands the experimental design and how representative the results actually are. 

7) Regarding comment #18, the Figure legends have not been improved, nor has the manuscript been improved to identify what TD1.24 is.  Again, I would have guessed as much, but in a scientific manuscript, this should not be left to chance and should be very carefully described to avoid all confusion. 

 

 

Author Response

Dear Reviewer:

Thank you for your comments concerning our manuscript entitled “Effect of N-(2-Aminoethyl)-3-aminopropyltrimethoxysilane on the adhesion of the modified silicone tie-coating to epoxy primer” (ID: coatings-1027135). These comments are all valuable and very helpful for revising and improving our paper, as well as the important guiding significance to our researches. We have studied comments carefully and have revised the full text. The language of this manuscript has been reviewed by a technical editor. Revised portion is marked using the “Track Changes” function in Microsoft Word. The responses to your comments are as follows.

1.The English still needs to be improved, with a particular emphasis on the edited text in red.                                                                   Response:

Authors agreed with the comments of reviewer and revised the manuscript by review the related literature and consult others. Please find in revised manuscript.

2. Responses to issues with the materials and methods section should be incorporated into the actual manuscript, rather than just explained in the Response to Reviewers. While this has clarified many issues for me, the reader will still have no clue.

Response:

Authors agreed with the comments of reviewer and revised the manuscript. Please find in revised manuscript. (Lins112-185)

3. Regarding section 3.1.1, the authors suggest in the paper and in their Response to Reviewers that there was a crosslinking between DMAO and PDMS/TEOS as this reaction is well known. While I recognize that this chemical reaction is well documented, my point is still that the authors have done nothing to show that there is any cross-linking occurring in there studies, and in fact, the FTIR data seem to suggest that no reaction took place. The peaks at 3335 cm-1 just confirm that DAMO has been mixed into the tie-coating, but does not confirm that any crosslinking reaction took place. Since there is no direct evidence that cross-linking reaction occurred, I caution the authors against suggesting that the data result due to cross-linking.  This is pure speculation, and though I am okay publishing as such, it needs to be pointed out to the reader that there is no direct evidence of cross-linking.

Response:

Authors agreed with the comments of reviewer, I am deeply sorry that my less rigorous to bring you unnecessary troubleand. I have revised the manuscript, please find in revised manuscript. (Lins196-197)

4. I am happy to hear that the quantitative analysis was been verified via repeated experiments, however, the statistical analysis used (as well as the software) needs to be identified in the materials and methods.  The confidence level and the number of replicates also needs to be identified. 

Response:

Each coating was tested three samples, and the result was directly given by the ESCALAB XI+ X-ray photoelectron spectrometer (XPS) in Excel format, and only a simple analysis was required. The confidence level of the data was judged by calculating the variance of the data, Excel can do it.(Lins 118-119)

5. Again, with regards to my comment #12, it is okay to me that this conclusion was inferred, but this needs to be specifically described in the manuscript so that the reader knows that this is speculation.

Response:

Authors agreed with the comments of reviewer and revised the manuscript. Please find in revised manuscript. (Lins205-207)

 

6. Regarding comment #15, the authors response needs to be incoropated into the materials and methods so that the reader understands the experimental design and how representative the results actually are.

Response:

Authors agreed with the comments of reviewer and revised the manuscript. Please find in revised manuscript. (Lins133-135)

                                                                  

7. 7. Regarding comment #18, the Figure legends have not been improved, nor has the manuscript been improved to identify what TD1.24 is. Again, I would have guessed as much, but in a scientific manuscript, this should not be left to chance and should be very carefully described to avoid all confusion.                               Response:

Authors agreed with the comments of reviewer and revised the manuscript. Please find in revised manuscript.( Lins108-109 and Figure.3)

Author Response File: Author Response.docx