N-Doped Carbon Nanoparticles as Antibacterial Agents on Escherichia coli: The Role of the Size and Chemical Composition of Nanoparticles
, María Dolores Merchán 1,2,*, Pilar Pérez 3 and María Mercedes Velázquez 1,2Round 1
Reviewer 1 Report
Major revision.
Comments
The authors reported the N-doped carbon nanoparticles as antibacterial agents: the role of the chemical composition on the antibacterial activity. The author studied only bacterial application and missing the novelty. I suggest following comments to improve the quality of manuscript. Paper is very short and need more supporting data.
1. Abstract is not clear which contain only literature review. I suggest highlighting the aim of this work, method used and important results.
2. Authors should highlight the importance and novelty in the introduction as well as cite more relevant references such as
https://www.mdpi.com/2076-3921/11/6/1064
https://www.nature.com/articles/s41598-023-28356-y
10.1021/acs.langmuir.0c02293
https://pubs.acs.org/doi/10.1021/acsomega.2c00848
https://apb.tbzmed.ac.ir/Article/APB_258_20140507125858
The author should compare the results from the above references and explain the novelty.
3. Author mentioned in the abstract and in the discussion they used TEM/ SEM to measure particle size. However, I can’t find the SEM/TEM images in the manuscript. I strongly suggest adding morphological analysis.
4. Author mentioned in the discussion session that the reduction rate of antibacterial capacity increases with nanoparticle concentration until…. However, the reason for such behavior is not explained well. The author needs to present some supporting evidence.
5. Author mentioned at the end of the discussion section It is interesting to note, that the bactericidal character of nanoparticles comes from the chemical interaction between the N- functional groups attached to the wall of the bacteria,
Add the reaction mechanism here.
6. There are many grammatical errors, recheck the manuscript once again for all typo errors.
There are many grammatical errors, recheck the manuscript once again for all typo errors.
Author Response
Author’s comments in italics
Reviewer 1
The authors reported the N-doped carbon nanoparticles as antibacterial agents: the role of the chemical composition on the antibacterial activity. The author studied only bacterial application and missing the novelty. I suggest following comments to improve the quality of manuscript. Paper is very short and need more supporting data.
- Abstract is not clear which contain only literature review. I suggest highlighting the aim of this work, method used and important results.
We agree with the reviewer, in the new version of the manuscript, the abstract has been written in order to introduce the changes suggested.
- Authors should highlight the importance and novelty in the introduction as well as cite more relevant references such as
https://www.mdpi.com/2076-3921/11/6/1064
https://www.nature.com/articles/s41598-023-28356-y
10.1021/acs.langmuir.0c02293
https://pubs.acs.org/doi/10.1021/acsomega.2c00848
https://apb.tbzmed.ac.ir/Article/APB_258_20140507125858
The author should compare the results from the above references and explain the novelty.
We thank the reviewer for the valuable comment. For this reason, the abstract and the introduction have been modified for adding the novelty of the work including the references suggested by the reviewer. See the new version of the manuscript
- Author mentioned in the abstract and in the discussion they used TEM/ SEM to measure particle size. However, I can’t find the SEM/TEM images in the manuscript. I strongly suggest adding morphological analysis.
We agree with the reviewer and a new figure is included in the corrected version of the manuscript. Also a new paragraph is inserted analyzing the morphology of the nanoparticles.
“As can be observed in Figure 1, CNPs synthesized from different carbonaceous materials present different sizes and morphologies. Nanoparticles synthesized from carbon nanofibers (CNPNF, Figure 1 a) are mainly constituted by small carbon dots of size close to 10 nm in diameter. These results are in agreement with previously published results in which a frequency analysis of the images was performed, showing single-mode distribution peaks with maximum frequencies centered at 10 nm in diameter [65]. For the rest of the materials, the TEM images show large and aggregated carbon nanoparticles formed during the solvent evaporation and due to the hydrodynamic processes [69, 70] (Figures 1 b), 1c) and 1d)). To avoid this aggregation we used Nanoparticle Tracking Analysis, NTA to obtain the diameter of the free nanoparticles [65]. The values obtained for the diameter of the four types of nanoparticles are collected in Table 1”.
- Author mentioned in the discussion session that the reduction rate of antibacterial capacity increases with nanoparticle concentration until…. However, the reason for such behavior is not explained well. The author needs to present some supporting evidence.
We appreciate the reviewer's comment and for this reason the Monod model has been explained in more detail in the discussion section. In addition, the values obtained in this work have been compared with those obtained by other authors.
- Author mentioned at the end of the discussion section It is interesting to note, that the bactericidal character of nanoparticles comes from the chemical interaction between the N- functional groups attached to the wall of the bacteria. Add the reaction mechanism here.
We appreciate the reviewer's comment. Our results show the existence of two mechanism actuating simultaneously, but it is difficult to separate these mechanisms to study each of them independently. However, our results show that the bactericidal character of these nanomateriales can be related to the N-groups, therefore, according to the information reported in literature, (Materials Chemistry and Physics 295 (2023) 127135 or RSC Adv. 10 (2020) 41202–41208), these N-groups are able to transfer electrons to the bacteria producing Reactive Oxygen Species. To confirm the groups and reactions involved in this interaction it is necessary to design other type of experiments focused in the study of interactions between these nanomaterials and molecules with functional groups that mimic those existing in the walls of the bacteria. However, these studies exceed the objective of this work and will be carried out in the future.
- There are many grammatical errors, recheck the manuscript once again for all typo errors.
In response to the reviewer's comment, the text has been revised to eliminate grammatical errors.
Reviewer 2 Report
The manuscript titled “N-doped carbon nanoparticles as antibacterial agents: the role of the chemical composition on the antibacterial activity” presents an important subject of study, however the work is quite different from its title. This is mainly because it only focuses on a class of bacteria, which is Escherichia coli, which does not mean that it is general for everything. Neither is the methodology to quantify the concentration of bacteria the most appropriate and must be modified to have a greater impact. Nor is the methodology to quantify the concentration of bacteria the most appropriate and must be modified to have a greater impact. The role of the chemical composition in the antibacterial activity should also be evaluated by physiological responses and thereby support the results of the model. The introduction should be directly focused on the topic with the objectives and clear novelty.
There are no further comments
Author Response
Author’s comments in italics
Reviewer 2
Comments and Suggestions for Authors
The manuscript titled “N-doped carbon nanoparticles as antibacterial agents: the role of the chemical composition on the antibacterial activity” presents an important subject of study, however the work is quite different from its title. This is mainly because it only focuses on a class of bacteria, which is Escherichia coli, which does not mean that it is general for everything. Neither is the methodology to quantify the concentration of bacteria the most appropriate and must be modified to have a greater impact. Nor is the methodology to quantify the concentration of bacteria the most appropriate and must be modified to have a greater impact. The role of the chemical composition in the antibacterial activity should also be evaluated by physiological responses and thereby support the results of the model. The introduction should be directly focused on the topic with the objectives and clear novelty.
We agree with the reviewer and the title and the manuscript have deeply been modified to include the above suggestions.
Reviewer 3 Report
The following suggestions can be addressed:
1. The second and third paragraphs are too small and incomplete. These two paragraphs must be improved by addressing recent works like [https://doi.org/10.1016/j.molliq.2020.112586; https://doi.org/10.1002/slct.201804021; https://doi.org/10.1002/slct.202003033] and are merged to highlight the rational progress to the recent status.
2. Picture of the plates and graph should be separated for better presentation. The inset representation procedure is not correct. So, the result section should be improved according to modified figure numbers.
3. Figure 2 and Figure 3 must have statistical information for the calculation of significant change.
Author Response
Reviewer 3
Comments and Suggestions for Authors
The following suggestions can be addressed:
- The second and third paragraphs are too small and incomplete. These two paragraphs must be improved by addressing recent works like:
https://doi.org/10.1016/j.molliq.2020.112586
https://doi.org/10.1002/slct.201804021
https://doi.org/10.1002/slct.202003033
and are merged to highlight the rational progress to the recent status.
We thank the reviewer for the valuable comment. For this reason, the abstract and the introduction have been modified for adding the novelty of the work including the references suggested by the reviewer. See the new version of the manuscript
- Picture of the plates and graph should be separated for better presentation. The inset representation procedure is not correct. So, the result section should be improved according to modified figure numbers.
We thank the comment of the referee and following its suggestion figure 1 has been divided in two new figures.
- Figure 2 and Figure 3 must have statistical information for the calculation of significant change.
Following the referee's suggestion, we have indicated in the text the procedure for calculating the error bars in the figures that appear.
Statistical analysis. The assays were performed in triplicates and repeated in two series. Experimental data are presented as mean ± standard deviation.
In addition, we have reviewed them and found errors in figures 2 and 3, which are detailed below.
In Figures 2 and 3 of the original manuscript, the error bars corresponded to the relative errors, not to the absolute error calculated from the standard deviation, accordingly, in the new version of the manuscript the absolute error was represented.
To calculate the errors of K, we have compared the relative errors of the experimental data with the error obtained from the fit procedure for each set of data. Since the later was always higher than the error of the experimental results, we have considered the biggest one, from fits, as the error of K values
Reviewer 4 Report
The manuscript by López-Díaz et al. entitled
“N-doped carbon nanoparticles as antibacterial agents: the role of the chemical composition on the antibacterial activity”
is in a scope of a journal Coating and may be recommended for publishing after considering few suggestions.
Manuscript reports on the antibacterial activity of N-doped carbon nanoparticles against E.coli bacteria. The preparation of the mentioned photoluminescent carbon nanoparticles and their chemical and structural characterization by XPS, FTIR, TEM and Raman spectroscopy have been reported previously (Ref. 47).
The presented research is devoted to analysis using a Ligand-Substrate model based on Monod´s equation, which allows to interpret the dependence of affinity of E.coli bacteria with the nanomaterial structure, particularly the nanoparticle size and content of nitrogen groups. Results indicated increasing affinity constant with increasing content of nitrogen forms and decreasing nanoparticle size, i.e. increasing surface area of nanoparticles, suggesting the importance of adsorption in the antibacterial process and bacteria damage.
There are quite many reports demonstrating the adsorption processes of bacteria(s) to nanoparticles followed by bacteria disruption evidenced by SEM/TEM, kill-time kinetics, and determination of MIC and MBC dependence for different nanocomposites and against various bacteria(s) as indirect evidence of influence of structural parameters of nanocomposites on antibacterial activity. However, the approach using Monod equation is quite unique.
Introduction section contains compiled results from the literature, i.e. 47 reasonably recent references, on carbon nanomaterials' bactericidal properties and mainly on the bactericidal mechanism. However, this section does not contain any report on composites, i.e. metal/metal oxides and graphene/carbon nanomaterial. Also, the efficiency of the antibacterial mechanism for different systems can be discussed, i.e. expressed by measurable parameters in IC50, MIC, and MBC.
Section 2.3. (XPS) is not necessary. This research has been carried out in Ref. 47. The results are cited in Table 1, where an error in citation occurs, i.e. instead of Ref. 39 Ref. 47 should be cited. Also, providing the atomic percent or weight percent of nitrogen forms may be useful.
The experimental section contains only the antibacterial test. More parameters may be evaluated from the dependence of the number of colonies versus concentration for different nanoparticles, e.g. parameter IC50.
The Monod model may be discussed in detail and previous applications of this model can be mentioned in the section Introduction.
More detailed characterization of carbon nanoparticles using XRD, for evaluating the crystallite size (Scherrer’s equation) and determining if carbon or graphene (Bragg’s equation for evaluating an interlayer distance) dots are present, may be additionally performed. It may be also helpful to provide a more detailed interpretation of UV-vis spectra published in Ref. 47. Carbon nanoparticle size mentioned in Table 1 (citation from Ref. 47) results from agglomeration since the nanoparticles of size smaller than about a few nm can pass through 2 kDalton membrane.
Parameters of graphene structures such as height and diameter may be determined from XRD (002) and (100) reflexes (see Stobinski et al. Journal of Electron Spectroscopy and Related Phenomena 195 (2014) 145-154).
More detailed interpretation of Raman spectra from Ref. 47 may be also performed, i.e. determination of cluster size (see Ferrari and Robertson, Phys. Rev. B 61(20) 14095-14107).
Abstract is suggested to be revised since it should also contain briefly stated results.
The manuscript is proposed to be reconsidered after major revision.
Author Response
Reviewer 4
Comments and Suggestions for Authors
Manuscript reports on the antibacterial activity of N-doped carbon nanoparticles against E.coli bacteria. The preparation of the mentioned photoluminescent carbon nanoparticles and their chemical and structural characterization by XPS, FTIR, TEM and Raman spectroscopy have been reported previously (Ref. 47).
The presented research is devoted to analysis using a Ligand-Substrate model based on Monod´s equation, which allows to interpret the dependence of affinity of E.coli bacteria with the nanomaterial structure, particularly the nanoparticle size and content of nitrogen groups. Results indicated increasing affinity constant with increasing content of nitrogen forms and decreasing nanoparticle size, i.e. increasing surface area of nanoparticles, suggesting the importance of adsorption in the antibacterial process and bacteria damage.
There are quite many reports demonstrating the adsorption processes of bacteria(s) to nanoparticles followed by bacteria disruption evidenced by SEM/TEM, kill-time kinetics, and determination of MIC and MBC dependence for different nanocomposites and against various bacteria(s) as indirect evidence of influence of structural parameters of nanocomposites on antibacterial activity. However, the approach using Monod equation is quite unique.
Introduction section contains compiled results from the literature, i.e. 47 reasonably recent references, on carbon nanomaterials' bactericidal properties and mainly on the bactericidal mechanism. However, this section does not contain any report on composites, i.e. metal/metal oxides and graphene/carbon nanomaterial. Also, the efficiency of the antibacterial mechanism for different systems can be discussed, i.e. expressed by measurable parameters in IC50, MIC, and MBC.
We appreciate the reviewer's comments, a paragraph has been added in the text comparing the IC50 values obtained in this work with those obtained for other carbon and carbon-metal oxide nanoparticles.
“In the Monod’s model, 1/K represents the concentration at which 50% of the bacteria have been inhibited. Our results show 1/K values ranging between 23.3 and 34.5 g/ml. This value can be compared with the same parameter obtained by other authors, who obtain IC50 between 550 and 700 g/ml for carbon dots doped with N [42], or IC50 of 440 µg/ml for Carbon Quantum Dots obtained by hidrothermal synthesis [44]. Hybrid GO-CuO nanoparticles [52] were tested as an antibacterial agent against E. coli, and the IC50 was much higher, between 2-3 mg/mL. Lower IC50 values, 2-3 µg/ml have been found for E. coli antibacterial activity for GO and Ag hybrid materials [47, 48]”.
Section 2.3. (XPS) is not necessary. This research has been carried out in Ref. 47. The results are cited in Table 1, where an error in citation occurs, i.e. instead of Ref. 39 Ref. 47 should be cited. Also, providing the atomic percent or weight percent of nitrogen forms may be useful.
At the reviewer's suggestion, the description of XPS measurements has been eliminated from the experimental procedure
The experimental section contains only the antibacterial test. More parameters may be evaluated from the dependence of the number of colonies versus concentration for different nanoparticles, e.g. parameter IC50.
We agree with the reviewer's comment and we have therefore obtained the IC50 value and the maximum efficiency and compared them with those values obtained by other authors with carbonaceous and non-carbonaceous nanomaterials.
The Monod model may be discussed in detail and previous applications of this model can be mentioned in the section Introduction.
We agree with the reviewer's comment, however, since the monod model is a model proposed in the 1950s we did not feel it appropriate to put it in the introduction. A more detailed description of the model and its application has been included in the discussion section.
More detailed characterization of carbon nanoparticles using XRD, for evaluating the crystallite size (Scherrer’s equation) and determining if carbon or graphene (Bragg’s equation for evaluating an interlayer distance) dots are present, may be additionally performed. It may be also helpful to provide a more detailed interpretation of UV-vis spectra published in Ref. 47. Carbon nanoparticle size mentioned in Table 1 (citation from Ref. 47) results from agglomeration since the nanoparticles of size smaller than about a few nm can pass through 2 kDalton membrane.
We greatly appreciate the reviewer's comment. Due to their size and the presence of functional groups, these materials are so poorly crystalline that only small bands of carbonates used to neutralize the acid in the synthesis step were observed. Since there were no defined bands of graphene compounds, it was very difficult to study the size of the microcrystal.
The size of the nanoparticle was obtained using transmission electron microscopy (TEM) for CNPNF. In the other three materials the drying process necessary to prepare the sample produces agglomeration of material which makes it difficult to obtain its size, for this reason, it have used NTA since it is methods to obtain the nanoparticle diameter in solution avoiding the aggregation processes induced by de-wetting.
Parameters of graphene structures such as height and diameter may be determined from XRD (002) and (100) reflexes (see Stobinski et al. Journal of Electron Spectroscopy and Related Phenomena 195 (2014) 145-154).
More detailed interpretation of Raman spectra from Ref. 47 may be also performed, i.e. determination of cluster size (see Ferrari and Robertson, Phys. Rev. B 61(20) 14095-14107).
We appreciate the reviewer's suggestions; however, we believe that microcrystal size and nanoparticle size is being confused.
The publications that reviewer suggests relate the characteristics of the Raman spectra of carbonaceous materials (ID/IG ratio) with the crystalline structure of the material, but never with the size of the nanoparticle. Moreover, in cases where the materials have many defects, the relationship applied in the publications suggested by the reviewer are no longer valid as we have shown in recently published work in our group.
- Phys. Chem. C 2015, 119, 10123-10129
- Phys. Chem. C 2017, 121, 20489-20497.
Microcrystal size and nanoparticle size could only coincide in the case the nanoparticles were monocrystalline, which does not seem to be the case of these nanoparticles due to the high number of functional groups that they possess, determined by XPS.
Abstract is suggested to be revised since it should also contain briefly stated results.
According to the reviewer's comment, the introduction has been thoroughly revised.
Round 2
Reviewer 1 Report
The Author responded to all my comments and revised the manuscript. I agree to publish this paper now.
Minor correction which can be done during proof reading
Reviewer 4 Report
The manuscript has improved significantly.
I have found only one misprint on p. 8.
Instead of "hidrothermal" "hydrothermal" should be used.
