Revealing the Effects of Three Different Antimicrobial Agents on E. coli Biofilms by Using Soft-Probe Scanning Electrochemical Microscopy
Round 1
Reviewer 1 Report
1. State clearly the novelty in the introduction
2. How to conduct the sterilization of the antimicrobial agent for the experiments?
3. What is the difference of terms used, SECM vs SEM?
4. The EDX measurement shoud be conducted.
5. The nanoparticles formed should be characterized at least using XRD and TEM.
Author Response
Please see the attachment.
Author Response File: 
Author Response.docx
Reviewer 2 Report
An interesting method for analysis of biofilms under treatment
I have couple of questions about the methodology.
1) With respect to the flashlight experiments. Is there anything you can add to the introduction about the effect of the presence of light of different energies on biofilms?
2) Please include a schematic or photo for the SECM assembly used in the experiment.
3) You mention the limited penetration of silver nanoparticles into biofilms as a reason for its limited effectiveness. Can you include proposals for how to determine this for future experiments. Think of SEM elemental analysis or dark field microscopy.
4) It is very hard to make out the text on the microscope images (figures 2, 5, and 7. Please change from a white text to one that is clearer to read.
The English shows moderate issues with some mistakes in word choice and order. I would suggest a proof read before resubmission.
Author Response
Please see the attachment.
Author Response File: Author Response.docx
Reviewer 3 Report
In this work the Authors proposed the use of soft-probe scanning electrochemical microscopy and confocal laser scanning microscopy to study the effects of different antimicrobial agents and treatments on E. coli biofilm. The topic has certainly a great scientific interest and the methodology are showed in an accurate way.
However, few but important points along the manuscript need major revision:
- The LSPR (Localized Surface Plasmon Resonance) peak of metal nanoparticles undergoes a wavelength red shift when the size of the NPs increases, and a wavelength blue shift when the size of the NPs decreases. In this case the findings show an opposite trends, which is impossible. Specifically, the AgNPs-PVP in EG show smaller size than the other ones, so the wavelength should move towards lower values of wavelengths. Please check these results.
- The absorbance decreases of the AgNP spectra after 24 hours occurs for all the spectrum wavelengths, so it means that there isn’t a decrement in the peak absorption but just in the “zero adjustment” of the instrument.
- The Authors should comment the comparison of their results with the literature. Are there any other published works which proved the bioactivity of the same antimicrobial agents/treatments or which used the same techniques (SECM and/or confocal microscopy) but with different antimicrobials?
Just as a minor request, some comments and curiosities are reported below:
- Why three different AgNPs are tested? Could the PVP or the EG have some influence in the antibacterial activity?
- It seems that the current in the SECM curves decreased more with the use of flashlight rather then using sodium azide and AgNPs. Is this difference significative? Some comments about this point could be interesting.
In conclusion, major revisions are necessary for paper publication.
Comments for author File: 
Comments.pdf
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
Reviewer 4 Report
The present work concerns the use of scanning electrochemical microscopy (SECM) for studying the effects of antimicrobial agents on biofilm. In particular, the SECM of E. coli biofilms treated with sodium azide, silver nanoparticles and flashlight is displayed with the purpose of explaining the mode of action for each treatment. To understand the inhibitory mechanism of the chosen antimicrobial agents, live/dead staining confocal laser scanning microscopy (CLSM) is also employed as well as Cristal Violet (CV) staining is performed to evaluate the effect on the intact biomass of biofilm. Since the demand of new tool for understanding the mechanism of bacterial infections the is still growing up, the present study could be suitable for the publication in the Topical Collection “Feature Papers for Applied Nano”. However, minor revision is required to improve the current version of the manuscript.
Comments for author File: Comments.pdf
Author Response
Please see the attachment.
Author Response File: Author Response.docx
Round 2
Reviewer 3 Report
The manuscript is suitable for the publication.
