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Peer-Review Record

Synthesis and Characterization of Bio-Active GFP-P4VP Core–Shell Nanoparticles

Catalysts 2020, 10(6), 627; https://doi.org/10.3390/catal10060627
Reviewer 1: Anonymous
Reviewer 2: Anonymous
Reviewer 3: Anonymous
Catalysts 2020, 10(6), 627; https://doi.org/10.3390/catal10060627
Received: 4 May 2020 / Revised: 27 May 2020 / Accepted: 3 June 2020 / Published: 5 June 2020

Round 1

Reviewer 1 Report

This paper describes the preparation of nanoparticles conjugated with the green fluorescence properties. The works is fully out of the scope of this journal, it is nothing related to catalysis. I´m surprised how this articles went even to referees. 

The work is well developed though although it has a notably lack of contribution to the field of protein immobilization and biomaterials. 

I suggest the author to complement these results with some application and then submit the manuscript to another journal more interdisciplinary or with an scope in biomaterials.  

Author Response

Response: Thank you very much for your comments. This is an invited article for the special issue "Bio-Inspired Materials: Learning from Nature to Improve Catalysts". Although this article is not dedicated to catalysts, the protein stabilization method can be easily extended to enzyme catalysts because enzymes are a classification of proteins. Enzyme immobilization is a direction that our group is now engaged in.

Reviewer 2 Report

This manuscript describes the synthesis and characterization of bio-active GFP-P4VP core-shell nanoparticles. The authors were able to successfully prepare CSNPs by simple methods. They show the presence of the protein on the surface using Gold conjugation methods. 

The general experiments, analysis, and results are good. This paper merits publication in Catalysts. 

Corrections:

1. Abstract:  Line 16-17 (from top of the journal) "... maintain bioactivity in a variety of chemical environments".  There are no experiments reported that describes the bioactivity in various chemical environments.  This should be removed or need to include additional experiments to support this claim.

2. Figure 3 (page 5). It was difficult to understand the presence of circular region with high density gold on the circumference inside the particles (see pictures c - h). Maybe, this is cleared in the movie (which is not included in the manuscript - see next comment).  If the above circular region are actually present in these particles, an explanation should be given because this could affect some of the measurements/interprtaions.  

2. Line 151 (page 6). Don't see the relevance of the broken sentence "A movie after construction".  If there are movies taken to show the spherical shape, please describe it clearly.

3. Figure 5. The smaller peak at around 425 nM is characteristic of P4VP. It was not clear why the G2 partcle has much more P4VP than G3. Their preparation methods and other experiments show that the size of the P4VP core increases from G1 to G3.  It will be helpful to understand and explain this anomaly. 

4. Lines 165-167 (page 7). Figure 5 should be changed to Figure 4d.

5. Reference 29 is not complete.

6. References 12, 37, 46, etc. are missing last page.

 

 

Author Response

This manuscript describes the synthesis and characterization of bio-active GFP-P4VP core-shell nanoparticles. The authors were able to successfully prepare CSNPs by simple methods. They show the presence of the protein on the surface using Gold conjugation methods. 

The general experiments, analysis, and results are good. This paper merits publication in Catalysts. 

Response: Thank you very much for the positive comments from the reviewer.

  1. Abstract:  Line 16-17 (from top of the journal) "... maintain bioactivity in a variety of chemical environments".  There are no experiments reported that describes the bioactivity in various chemical environments.  This should be removed or need to include additional experiments to support this claim.

Response: The statement regarding bioactive stability in a variety of chemical environments has been removed from this manuscript. We plan to include such results in future work.

  1. Line 151 (page 6). Don't see the relevance of the broken sentence "A movie after construction".  If there are movies taken to show the spherical shape, please describe it clearly.

Response: Thank you for pointing this out, this line has been removed from the manuscript.

  1. Figure 5. The smaller peak at around 425 nM is characteristic of P4VP. It was not clear why the G2 partcle has much more P4VP than G3. Their preparation methods and other experiments show that the size of the P4VP core increases from G1 to G3.  It will be helpful to understand and explain this anomaly.

Response: Based on the particle size and the fluorescence intensity of the GFP, we assume the total P4VP and GFP surface coverage in the sample is consistent with the other samples. Therefore at this time we cannot properly describe the cause of this phenomenon, but plan on investigating in future work.

  1. Lines 165-167 (page 7). Figure 5 should be changed to Figure 4d.

Response: This has been fixed, thank you.

  1. Reference 29 is not complete.

Response: Reference 29 has been properly completed.

  1. References 12, 37, 46, etc. are missing last page.

Response: These references have been updated to include their ending page numbers, thank you.

Reviewer 3 Report

This paper describes the formation and some characteristic of GFP-polymer core-shell nanoparticles (CSNPs). Using Poly(4-vinylpyrine) as the polymer core, the formation of CSNPs is proved by fluorescence, TEM, and DLS methods. GFP is well distributed on the surface of the polymer. The size of NP can be tuned by varying the ratio between GFP and polymer. It is also shown by fluorescence that GFP is still active.

My main concern is related to the stabilization effect claimed by the authors which is not supported by any experiments. Florescence of GFP in the CSNP as a function of time should be provided before publication.

Minor point: In Figure 2, the red curve is not visible.

Author Response

This paper describes the formation and some characteristic of GFP-polymer core-shell nanoparticles (CSNPs). Using Poly(4-vinylpyrine) as the polymer core, the formation of CSNPs is proved by fluorescence, TEM, and DLS methods. GFP is well distributed on the surface of the polymer. The size of NP can be tuned by varying the ratio between GFP and polymer. It is also shown by fluorescence that GFP is still active.

Response: Thank you very much for the positive comments from the reviewer.

  1. My main concern is related to the stabilization effect claimed by the authors which is not supported by any experiments. Florescence of GFP in the CSNP as a function of time should be provided before publication.

Response: Thank you very much for your comment. However, due to the outbreak of the pandemic, we are unable to complete the suggested experiment now, and we are not sure when the laboratory can be opened for research. However, we have determined that this method can be used to stabilize many other proteins, such as BSA, lipase, CAZyme, and have already filed a patent for this method. We believe that this method is effective for stabilizing GFP.

  1. Minor point: In Figure 2, the red curve is not visible.

Response: The figure has been updated so that the red curve is more visible. Thank you

Round 2

Reviewer 1 Report

I am still convince that this work meets neither the scope of the journal nor the topic of the special issue . I think that to publish this story within this special issue this immobilization chemistry should be proven with an enzyme. Even for an immobilization protocol for biocatalytic approach many things need to be studied to validate it. For example, the protein leaching was not studied and this becomes very important when we want to re-use the immobilized enzyme. How this process is going affect the enzyme activity and stability?

Therefore, my opinion keeps being the same that this article is too preliminary to be published in a journal that falls in the catalysis field. 

Author Response

Response: Thank you very much for your comments. As I mentioned, this is an invited article for the special issue "Bio-Inspired Materials: Learning from Nature to Improve Catalysts". I believe this article is within the scope of this special issue focusing on Bio-Inspired Materials. In addition, I agree that this article is not dedicated to catalysts, however, the protein stabilization method can be easily extended to enzyme catalysts because enzymes are a classification of proteins. Enzyme immobilization is a direction that our group is now engaged in.

Reviewer 3 Report

the reviewer notes that the authors were not able to provide any further experiments because of pandemic situation.

Nevertheless, this manuscript can be published as is.

Author Response

Response: Thank you very much for the understanding and positive comments.

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