Improving Surface Antimicrobial Performance by Coating Homogeneous PDA-Ag Micro–Nano Particles
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe article by Wang et. al on ‘Improving surface antimicrobial performance by coating homogeneous PDA-Ag micro-nano particles’ presents an interesting approach to coat titanium surfaces using silver-PDA conjugates. The silver nanoparticle has been synthesized using PDA as the reducing agent. The conjugates have been characterized using SEM, TEM, XPS etc. The conjugate finally tested for its antibacterial effect on gram-positive and gram-negative bacterial strains. The overall study is interesting, but the research work, experimental data especially the bacterial assay lack proper attention from the researchers. These experiments need significant revision and reconsideration from the authors before sending out for publication.
Verdict: Reject (and resubmit)
Comments:
1. Provide actual images of TC4 attached to conjugate and only TC4 on figure 1 to show actual surface loading.
2. Is there an effect of ultrasound to the stability or structural integrity of PDA particles. As the authors have used ultrasound to synthesize Ag nanoparticles, they should have checked its effect on PDA alone.
3. Is there any optimization in terms of concentration or time for Ag loading on PDA? Give details.
4. There are significant part missing in the experimental section. There is no mention of SEM, TEM, XPS, CLSM experiments. Please add all the relevant details.
5. Also, there is no reference for the CFU or biofilm assays. Please add relevant reference (ACS Appl. Bio Mater. 2019, 2, 4, 1772–1780). Also, there are missing references which are very relevant to the work (ACS Appl. Mater. Interfaces 2020, 12, 36, 40067–40077).
6. In Figure 3, what is the timepoint for reaction (24 or 48 hrs). Please mention. If the time points are same as of figure 2, why they have again shown in figure 3 c/d.
7. What is the average size of Ag NPs? Provide size distribution graph from TEM.
8. Change the inset color in figure 2, also change the scale bar color. Nothing is visible properly.
9. For Figure 4, does the EDS come from SEM. If so, do the author consider same area and scale when comparing composition of two samples.
10. In figure 7, what are the colony counts? Provide the cfu counts in graphical form with standard deviation.
11. For the biofilm assay, provide the images of CV-stained plates. Then provide the graphs with error bar and standard deviation.
12. The authors should do statistical analysis (determining the p-value) to mention significant changes.
13. Why untreated S. aureus shows very little CV staining? Is it consistent in triplicate experiments?
14. Figure 8 does not make any sense. The image quality is poor and certainly, the parameters used for image in panel b are different compared to others. The authors should always use same imaging parameters when comparing images.
15. Bacteria stain propidium iodide solution is a bacterial fluorescence staining dye and can be applied for microbial cell viability assay in different principles. It is an ethidium bromide analog that emits red fluorescence upon intercalation with double-stranded DNA. Though PI does not permeate viable cell membranes, it passes through injured cell membranes and stains the nuclei. PI is often used in combination with a fluorescein compound, such as CFDA, for simultaneous staining of viability and membrane injury (ref sigma-aldrich). The author should repeat and reevaluate the experiments.
16. The current findings of figure 8 actually means TC4@PDA-Ag lowers membrane injury or dead cell counts! Please review and check your experiments with available literature before sending for publications.
17. Line 120: for instead of from, line 122: aluminum?
Author Response
- Provide actual images of TC4 attached to conjugate and only TC4 on figure 1 to show actual surface loading.
Response: Thank you for the good suggestion. We have added actual images into Figure 1.
- Is there an effect of ultrasound to the stability or structural integrity of PDA particles. As the authors have used ultrasound to synthesize Ag nanoparticles, they should have checked its effect on PDA alone.
Response: Thank you for your comments. Ultrasonic treatment is required to disperse uniformly and then deposit PDA and PDA-Ag on TC4. Figures 3b, 3d, and 5d showed that ultrasound has almost no effect on the stability and structural integrity of PDA and PDA-Ag.
- Is there any optimization in terms of concentration or time for Ag loading on PDA? Give details.
Response: Thank you for your constructive suggestion. We added the optimization of loading silver concentration on PDA-Ag, and the results showed that when the silver nitrate concentration was 80mg/ml, the effect of PDA loading Ag was the best and tended to stabilize. The corresponding descriptions have been updated in section 2.2 and in the second paragraph of "Results and Discussion" of the latest version of the manuscript
- There are significant part missing in the experimental section. There is no mention of SEM, TEM, XPS, CLSM experiments. Please add all the relevant details.
Response: Thank you for the good suggestion. We have already added SEM, TEM, XPS, CLSM experimental details in section 2.4.
- Also, there is no reference for the CFU or biofilm assays. Please add relevant reference (ACS Appl. Bio Mater. 2019, 2, 4, 1772–1780). Also, there are missing references which are very relevant to the work (ACS Appl. Mater. Interfaces 2020, 12, 36, 40067–40077).
Response: Thank you for the good suggestion. We have already added the necessary references.
- In Figure 3, what is the time point for reaction (24 or 48 hrs). Please mention. If the time points are same as of figure 2, why they have again shown in figure 3 c/d.
Response: Thank you for your comments. It’s 24 hours. We apologize for the reading inconvenience caused by the image layout. In order to better express our meaning, we have split Figure 3 into Figures 4 and 5 in the updated manuscript.
- What is the average size of Ag NPs? Provide size distribution graph from TEM.
Response: Thank you for the good suggestion. We have already added the size distribution graph into TEM in Figure 4 of the updated manuscript.
- Change the inset color in figure 2, also change the scale bar color. Nothing is visible properly.
Response: Thank you for your comments. We have already changed the inset color and scale bar color of the Figure (Figure 3 in the updated manuscript).
- For Figure 4, does the EDS come from SEM. If so, do the author consider same area and scale when comparing composition of two samples.
Response: Thank you for your comments. The EDS come from SEM, and we added the SEM image into the Figure (Figure 6 in the updated manuscript).
- In Figure 7, what are the colony counts? Provide the cfu counts in graphical form with standard deviation.
Response: Thank you for your constructive suggestion. We added the colony counts information with standard deviation in the revised manuscript.
- For the biofilm assay, provide the images of CV-stained plates. Then provide the graphs with error bar and standard deviation.
Response: Thank you for your suggestion. We have already added CV-stained plates and revised the Figure in the revised manuscript (Figure 9 in the updated manuscript).
- The authors should do statistical analysis (determining the p-value) to mention significant changes.
Response: Thank you for your good suggestion. We did statistical analysis and mentioned significant changes in the revised manuscript.
- Why untreated aureus shows very little CV staining? Is it consistent in triplicate experiments?
Response: Thank you for your comments. We re-conducted the experiment and summarized the results of the three repeated experiments in Figure 9c of the revised manuscript.
- Figure 8 does not make any sense. The image quality is poor and certainly, the parameters used for image in panel b are different compared to others. The authors should always use same imaging parameters when comparing images.
Response: Thank you for your comments. Sorry for the reading inconvenience caused. We used the same face scanning method to obtain images. Firstly, we determined the top and bottom stop points for image acquisition and divided the depth of field into 5 sections, with one image collected in each section. Then, we merged them into one image. It may be due to the significant difference in depth of field between the original sample and the processed sample, causing the image to appear as a change in acquisition parameters.
- Bacteria stain propidium iodide solution is a bacterial fluorescence staining dye and can be applied for microbial cell viability assay in different principles. It is an ethidium bromide analog that emits red fluorescence upon intercalation with double-stranded DNA. Though PI does not permeate viable cell membranes, it passes through injured cell membranes and stains the nuclei. PI is often used in combination with a fluorescein compound, such as CFDA, for simultaneous staining of viability and membrane injury (ref sigma-aldrich). The author should repeat and reevaluate the experiments.
Response: Thank you for your good suggestion. The number of live bacteria attached to the surface was obtained by the plate counting method. We used propidium iodide for bacterial staining to directly and simply observe all bacteria attached to the sample surface under a fluorescence microscope. We understand that the combination of PI with fluorescein compound will be better for simultaneous staining of viability and membrane injury,and we will conduct this research in our future work.
- The current findings of figure 8 actually means TC4@PDA-Ag lowers membrane injury or dead cell counts! Please review and check your experiments with available literature before sending for publications.
Response: Thank you for your comments. We want to express the conclusion that PDA-Ag modified Ti C surfaces (TC4@PDA-Ag) possess bacterial adhesion resistance properties by this figure, which we have stated and appropriately cited in the updated manuscript.
- Line 120: for instead of from, line 122: aluminum?
Response: Sorry for the mistake. We have already revised them.
Reviewer 2 Report
Comments and Suggestions for AuthorsI have reviewed the manuscript entitled “Improving surface antimicrobial performance by coating homogeneous PDA-Ag micro-nano particles”. The novelty is unclear, and I have the following comments for the authors to address.
1- Please state the novelty of your work, I feel confused to find what is new in this work!
2- Abstract, please provide numerical values for the findings. Just like what you did for the activity. Also, please provide a short scientific explanation.
3- Why did you put abbreviations in the abstract if you didn’t use them again in the abstract? Such as sem?
4- Please treat the abstract as a separate part from the other manuscript sections in terms of putting abbreviations.
5- The English is good. However, I detected some mistakes. Please revise the paper carefully.
6- “to reduce silver ions to silver nanoparticles” How the ions are reduced to NPs?? Please check and clarify.
7- You repeated the word nanoparticles on many occasions. Please use NPs.
Also, please define the abbreviation at its first appearance. Do not repeat, e.g. PDA.
8- Section 2-2, is this method yours, or have you adopted it from another work? If it is yours, please clarify why you selected these operation conditions. If it is not yours, please add a reference(s).
9- Please avoid long sentences as in 161-164 and 206-210. Please apply through the whole manuscript.
10- In Fig 2 a and b, the distribution curve is not clear, please try to make it obvious.
11- Fig 3, why they are not all TEM? Also, SEM and TEM in the same figure for different materials do not make sense. Please either make them all TEM or separate.
12- Title of figure 8 is wrong.
Comments on the Quality of English LanguageNeeds another round of revision.
Author Response
1. Please state the novelty of your work, I feel confused to find what is new in this work!
Response: Thank you for your comments. We have clarified in the last paragraph of the “Introduction”.
2. Abstract, please provide numerical values for the findings. Just like what you did for the activity. Also, please provide a short scientific explanation.
Response: Thank you for your good suggestion. We have already revised them in the updated manuscript.
3. Why did you put abbreviations in the abstract if you didn’t use them again in the abstract? Such as sem?
Response: Thank you for your comments. We removed the unnecessary abbreviations.
4. Please treat the abstract as a separate part from the other manuscript sections in terms of putting abbreviations.
Response: Thank you for your comments. We removed the unnecessary abbreviations.
5. The English is good. However, I detected some mistakes. Please revise the paper carefully.
Response: Thank you for your comments. We have checked and revised the entire manuscript.
6. “to reduce silver ions to silver nanoparticles” How the ions are reduced to NPs?? Please check and clarify.
Response: Thank you for your comments. We mentioned and explained it in the third paragraph of the “Introduction” and the second paragraph of the “Results and discussion”.
7. You repeated the word nanoparticles on many occasions. Please use NPs.
Also, please define the abbreviation at its first appearance. Do not repeat, e.g. PDA.
Response: Thank you for your comments. We have already revised them in the updated manuscript.
8. Section 2-2, is this method yours, or have you adopted it from another work? If it is yours, please clarify why you selected these operation conditions. If it is not yours, please add a reference(s).
Response: Thank you for your good suggestion. We referred to previous research and added some of our own optimization steps. References and necessary explaination were added in the updated manuscript.
9. Please avoid long sentences as in 161-164 and 206-210. Please apply through the whole manuscript.
Response: Thank you for your good suggestion. We have already revised the sentences in the revised manuscript.
10. In Fig 2 a and b, the distribution curve is not clear, please try to make it obvious.
Response: Thank you for your suggestion. We have redrawn the distribution curve (Figure 3 in the updated manuscript).
11. Fig 3, why they are not all TEM? Also, SEM and TEM in the same figure for different materials do not make sense. Please either make them all TEM or separate.
Response: Thank you for your constructive suggestion. We apologize for the reading inconvenience caused by the image layout. In order to better express our meaning, we have separated Figure 3 ( Figures 4 and 5 in the updated manuscript).
12. Title of figure 8 is wrong.
Response: Thank you for your comments. We have already revised the figure title.
Reviewer 3 Report
Comments and Suggestions for Authors- 2. Materials and methods. Include City for these companies: Aladdin Reagent Co., Sinopharm Chemical Reagent Co., Biochemical Technology Co., Beijing Wokai Biotechnology Co., Qingdao high tech Industrial Park Haibo Biotechnology Co. Because nobody knows these companies
- Lines 84 and 85 why selected these conditions: The deionized water (90 mL) and ethanol (40 mL) were mixed at room temperature, and was gently stirred for 30 min after adding ammonia solution
- Line 94, finally dried in vacuum at 60 ℃ for subsequent use, include information time, dried overnight, 5h, etc.
- Manuscript has some typos, revise and correct them
- Figure 1 was no possible to se the information of top arrows, improve it
- Line 121 define CFU, maybe colony formation units (CFU)
- Figure 5 needs to improve quality
- Conclusion needs to improve
- manuscript has some interesting results but need to improve all figures discussion
- manuscript has only one reference from 2024, include more references from 2024
- Manuscript has some characterization about SEM and antimicrobial properties, but needs to include one more characterization, maybe RMN, FT-IR, etc.
Author Response
- Materials and methods. Include City for these companies: Aladdin Reagent Co., Sinopharm Chemical Reagent Co., Biochemical Technology Co., Beijing Wokai Biotechnology Co., Qingdao high tech Industrial Park Haibo Biotechnology Co. Because nobody knows these companies.
Response: Thank you for your good suggestion. We added the information in the section 2.1.
- Lines 84 and 85 why selected these conditions: The deionized water (90 mL) and ethanol (40 mL) were mixed at room temperature, and was gently stirred for 30 min after adding ammonia solution. Line 94, finally dried in vacuum at 60 ℃for subsequent use, include information time, dried overnight, 5h, etc.
Response: Thank you for your good suggestion. The drying time is 12 h and we added it into the updated manuscript. In addition, we referred to previous research and added some of our own optimization steps. References and necessary explaination were added in the updated manuscript.
- Manuscript has some typos, revise and correct them
Response: Thank you for your comments. We have checked and revised the entire manuscript.
- Figure 1 was no possible to see the information of top arrows, improve it.
Response: Thank you for the good suggestion. We have already improved the Figure quality. (Figure 1 in the updated manuscript).
- Line 121 define CFU, maybe colony formation units (CFU)
Response: Thank you for the good suggestion. We have already defined it in the updated manuscript.
6. Figure 5 needs to improve quality
Response: Thank you for the good suggestion. We have already improved the Figure quality. (Figure 7 in the updated manuscript).
7. Conclusion needs to improve.
Response: Thank you for the good suggestion. We have already revised the “Conclusion”.
8. Manuscript has some interesting results but need to improve all figures discussion
Response: Thank you for the good suggestion. We have already revised the figures discussion.
9. Manuscript has only one reference from 2024, include more references from 2024
Response: Thank you for the good suggestion. We have added some articles related to the paper published in 2024.
10. Manuscript has some characterization about SEM and antimicrobial properties, but needs to include one more characterization, maybe RMN, FT-IR, etc.
Response: Thank you for the good suggestion. We speculate that you would like us to conduct RMN or FT-IR testing in order to have a clearer understanding of the content and functional groups of the main elements on the surface, which we have used EDS and XPS to analyze the content, valence states, and possible functional groups of the main elements on the sample surface. We also believe that more characterization can better display the surface information and physicochemical properties of the sample. We will pay more attention to related testing in future research.
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsI am not convinced with PI staining of the bacterial biofilm. Figure 10 is very confusing and the authors should provide some other proof to corelate with the result. SEM images of the biofilms after treatment and comparing those with an untreated biofilm would give a better perspective.
Author Response
I am not convinced with PI staining of the bacterial biofilm. Figure 10 is very confusing and the authors should provide some other proof to corelate with the result. SEM images of the biofilms after treatment and comparing those with an untreated biofilm would give a better perspective.
Response: Thank you for your comments. We are sorry for the reading inconvenience caused to you. (1)We used PI staining to observe and analyze the adhesion of bacteria on the surface of the sample, rather than bacterial biofilm. (2)Figure 10 is to observe the adhesion of bacteria on the surface of the sample over a large range. In order to better characterize the adhesion information of bacteria on the surface of the sample, we conducted SEM analysis according to the suggestions of the reviewer. The results were shown in Figure 11, and the sample surface possessed significant resistance to E. coli and S. aureus attachment.
Reviewer 2 Report
Comments and Suggestions for AuthorsPlease add the novelty of the work in the abstract too.
Author Response
1. Please add the novelty of the work in the abstract too.
Response: Thank you for your good suggestion. We have already added the novelty of the work in the abstract.
