Polymer-Stabilized Silver (Gold)–Zinc Oxide Nanoheterodimer Structures as Antimicrobials

Round 1

Reviewer 1 Report

Nanostructures have unique property of enhanced stability and tunable surface chemistry and owing these characteristics, applications of these nanostructures have been recognized in healthcare and medicine. Many novel nanostructures are being developed to explore their antimicrobial potential and that has produced promising results to combat the rising issues related to infectious diseases. The present manuscript has given potential efforts to address the issue related to resistance development by pathogenic strains. The exploration of antimicrobial potential of Polymer-Stabilized Silver (Gold)-Zinc Oxide Nanoheterodimer structures in the present study is of great importance. However, in my opinion the study should be supported by some more experiments with significant data. Here are some suggestions to the manuscript:

1. The antimicrobial property of nanoheterodimer has been represented with MIC calculation, however, growth inhibition using plate method should be performed.

2. TEM micrographs should be replaced with another image having well-dispersion and density of nanoparticles. Also, histogram can be provided to reflect distribution of nanoparticles of varying size.

3. For XRD spectrum, miller indices are missing. Mention the hkl values in Figure S2 as well as in text section.

4. Similar set of experiments have been published in your previous manuscript entitled “Silver nanoparticles doped with silver cations and stabilized with maleic acid copolymers: specific structure and antimicrobial properties”. The present manuscript seems to be the repetition of same set of experiments and therefore limits the significance of the present study.

5. Please check the incomplete sentence, “Nanosilver is an inorganic nanoparticle with myriad potential applications including biomedical using’’       

6. Establish the significance of the current results in relation to prior research efforts, enhancing the article's overall impact.

7. Outline potential mechanistic approaches underlying the antimicrobial properties of the developed heteronanostructure.

8. The method section lacks the inclusion of statistical analysis.

9. The article should additionally focus on the assessment of toxicity and pharmacokinetics in relation to the newly formed nanoheterodimer structures.

10. Verify and rectify duplicated words in Table 1, address grammatical concerns in the instrumentation part (4th line), and correct a spelling error in the materials section (11th line).

Moderate editing of English language required

Author Response

Responses to Reviewer 1:

Authors are grateful to Reviewer for attentive reading of our paper and valuable remarks.

 

Comment: The antimicrobial property of nanoheterodimer has been represented with MIC calculation, however, growth inhibition using plate method should be performed.

Response: The microdilution method with MICs determination is the gold standard for determining the antimicrobial activity of studying substances. All other methods are secondary in relation to it, therefore, we suggested that "plate method" giving no additional data to MICs determination should not be performed.

Comment: TEM micrographs should be replaced with another image having well-dispersion and

density of nanoparticles. Also, histogram can be provided to reflect distribution of

nanoparticles of varying size.

Response: The TEM images in Fig. 2 have been changed.

Comment:  For XRD spectrum, miller indices are missing. Mention the hkl values in Figure S2 as well as in text section.

Response: Miller indexes have been in text.

Comment:  Similar set of experiments have been published in your previous manuscript entitled

“Silver nanoparticles doped with silver cations and stabilized with maleic acid copolymers:

specific structure and antimicrobial properties”. The present manuscript seems to be the

repetition of same set of experiments and therefore limits the significance of the present study.

 

Response: In this study, a new approach to the production of nanoheterodimers, contained nanometal and zinc oxide, is proposed. The proposed method of production of all such heterostructures is easy to implement - it is carried out in an alkaline environment in the absence of difficult-to-remove organic solvents and additionally introduced reagents. The use of polymers of various structures as stabilizing agents for nanostructures is a traditional approach. The physicochemical methods used to analyze the obtained structures represent the necessary evidence base. The study of antimicrobial properties of the obtained heterostructures was dictated by the presence of potentially active ingredients in their composition.

 

Comment:   Please check the incomplete sentence, “Nanosilver is an inorganic nanoparticle with

myriad potential applications including biomedical using’’

Response: This sentence have been excluded

Comment: Establish the significance of the current results in relation to prior research efforts,

enhancing the article's overall impact.

 

Response: In this study, water-soluble complex nanostructures of silver (gold) with zinc oxide stabilized by biocompatible non-toxic copolymers of maleic acid and also doped with zinc ions nanostructures were obtained using simple one-pot method. The proposed method of production of all such heterostructures is easy to implement - it is carried out in water medium, in the absence of difficult-to-remove organic solvents and additionally introduced reducing reagents. The obtained samples containing heterodimers can be stored in dry form. Our colloidal preparations contain a set of bioactive ingredients, and such composites have not been previously investigated as antimicrobials.

 

 

Comment: Outline potential mechanistic approaches underlying the antimicrobial properties of the developed heteronanostructure.

 

Response: Ions, released from the nanoheterodimer structures act on the thiol groups of the membrane proteins and enzymes [Hwang E.T., Lee J.H., Chae Y.J., Kim Y.S., Kim B.C., Sang B.I., Gu M.B. // Small. 2008. V. 6. P. 746–50], increasing membrane permeability and neutralize its charge [Ramalingam B., Parandhaman T., Das S.K. // ACS Appl. Mater. Interfaces. 2016. V. 8. № 7. P. 4963–4976] penetrate inside the microbial cell [Ivask A., Elbadawy A., Kaweeteerawat C., Boren D., Fischer H., Ji Z., Chang C.H., Liu R., Tolaymat T., Telesca D., Zink J.I., Cohen Y., Holden P.A., Godwin H.A. // ACS Nano. 2014. V. 8. № 1. P. 374–386; McQuillan J.S., Infante H.G., Stokes E., Shaw A.M. // Nanotoxicology. 2012. V. 6. № 8. P. 857–866], cause oxidative stress [Hwang E.T., Lee J.H., Chae Y.J., Kim Y.S., Kim B.C., Sang B.I., Gu M.B. // Small. 2008. V. 6. P. 746–50; Adeyemi O.S., Shittu E.O., Akpor O.B., Rotimi D., Batiha G.E. // EXCLI J. 2020. V. 19. P. 492–500; Xu H., Qu F., Xu H., Lai W., Andrew Wang Y., Aguilar Z.P., Wei H. // Biometals. 2012. V. 25. № 1. P. 45–53; Kim J.S., Kuk E., Yu K.N., Kim J.H., Park S.J., Lee H.J., Kim S.H., Park Y.K., Park Y.H., Hwang C.Y., Kim Y.K., Lee Y.S., Jeong D.H., Cho M.H. // Nanomedicine. 2007. V. 3. № 1. P. 95–101], as well as DNA damage [Vishnupriya S., Chaudhari K., Jagannathan R., Pradeep T. // Particle & Particle Systems Characterization. 2013. V. 30. № 12. P. 1056–1062], which leads to morphological changes in the microbial cell with membrane disorganization, lysis of the cell wall and microbial death [Akter S., Huq M.A. // Artif. Cells Nanomed. Biotechnol. 2020. V. 48 № 1. P. 672–682; Du J., Hu Z., Dong W., Wang Y., Wu S., Bai Y. // Microchemical J. 2019. V. 147. P. 333–338].

 

https://sciencejournals.ru/cgi/getPDF.pl?jid=prikbio&year=2021&vol=57&iss=6&file=PrikBio2106005Khina.pdf

 

Comment: The method section lacks the inclusion of statistical analysis.

 

Response: Statistical processing of data based on the results of 3 measurements was carried out.

 

Comment: The article should additionally focus on the assessment of toxicity and pharmacokinetics in relation to the newly formed nanoheterodimer structures.

 

Response: The study of toxicity and pharmacokinetics is a separate large study and it will be reflected in subsequent publications.

 

 

 

Comment: Verify and rectify duplicated words in Table 1, address grammatical concerns in the

instrumentation part (4th line), and correct a spelling error in the materials section (11th line).

 

Response: The text has been corrected.

 

Please see the attachment.

 

Author Response File: Author Response.pdf

Reviewer 2 Report

The article "Polymer-Stabilized Silver (Gold)-Zinc Oxide Nanoheterodimer Structures as Antimicrobials" presents interesting results on new structures based on metallic nanoparticles. Unfortunately, the article suffers from a large number of shortcomings and errors that need to be clarified:

- supplementary data: Fig 1. The data were poorly prepared: the baseline was not subtracted; the position of the bands was not marked, and the bands were not assigned to the characteristic bands, especially those written about in the text; not very clear markings; the small font making it challenging to read,

- Abstract and text: values without units.

- Introduction: line 51: abrupt transition from Ag to Au. In addition, the properties and influence of Ag are described quite accurately, and Au is described superficially in the context of the most detailed description of ZnO. There is a considerable disproportion of data and literature references between these fragments. The negative aspects of Ag and Au are not mentioned. 

lines 112-120: very cursory introduction to polymers. No literature references and no comparisons with other similar solutions. No information on why these particular copolymers were chosen. What advantages and disadvantages are related to nanostructures?

It is worth sorting out the data and completing the introduction significantly!

- Methodology: line 132: abbreviations not explained. Why does information about synthesis appear in this part when it should be mentioned only about materials?

line 136: What was the purity of the reagents?

Why is the methodology for XPS very detailed and the other techniques very cursory and abbreviated without any measurement details?

- Synthesis: line 233: why is there different metal content? On what basis were such concentrations selected? Why composite with Au0 was based on only one copolymer and not similar to the Ag0 with three considered?

line 235: The procedure is worth describing in a little more detail and not just providing a literature reference.

- Results: 

Is ZnO in the form of nanoparticles crystallised in the form of structures? Here there are analytical errors because, in EDS, it is written about Zn, in XRD about crystalline phases; in TEM, there is no evidence of crystalline forms of ZnO and the textual notation and determinations of the matter was treated even differently!

line 305: Average of what? Crystallinity, Ag nanoparticles, Au? Why is XRD data not shown for all samples (all polymers and all combinations shown in Table 2)?

line 306: How was the % content of the different phases of the composite determined since no Rietveld analysis was done and not even mentioned?

Scheme 1: How is it known that Ag0 is surrounded by the polymer chain and not hooked up to C=O? Why is Au0 not mentioned? Why are there bacterial cells in this scheme? Not described R fragments:

line 260: unexplained abbreviation

lines 269+ fig.1, no reference to the text and what the authors describe in the text and on the spectrum, i.e. no indication confirming the presence of ZnO or Zn2+, no description of the y-axis, not very clear markings, I would have used a legend to present the data instead of a caption under the figure. Why are the results not shown for all the samples in Table 2?

lines 279-288: Where do such conclusions come from when no result confirms the described validity of the results? Why was IR not done for all samples presented in Table 2?

Fig. 2 shows practically nothing: no description of what can be seen in the picture, no histogram of size distribution with statistical analysis, no TEM-XRD confirming the presence of Ag0, Au0 and ZnO, no TEM-EDS confirming what the authors write about in the text. Why are the results not shown for all samples, as in Table 2?

Fig. 3 illegible picture. The caption shows the Zn 3p and Au 4f range while there are two diagrams, and I assume one for samples with Ag. How do these results relate to each other? Why aren't all the lines visible from the fit described? Why do the same intensities characterise the doublet for Au?

The XPS analysis needs to be more accurate and chaotic, e.g. lines 335-337. Where does the positive and negative shift come from when it is not shown anywhere? How do the XPS results relate to Table 2 when only one sample is shown? The lack of O 1s, Ag 2p, and C 1s lines could be more coherent, not relating to all the samples tested. What was obtained? What are the differences between samples? Where do these differences come from?

Table 2: No pure polymers as references

Differences should be described, and symbols explained

- conclusions: lines 425-438: not needed

line 450: what about Au?

lines 450-457: in my opinion, no fragment is needed

It should be improved.

Author Response

Response to Reviewer 2:

Authors are grateful to Reviewer for attentive reading of our paper and valuable remarks.

 

Comment, Supplementary data, Fig 1: The data were poorly prepared: the baseline was not subtracted; the position of the bands was not marked, and the bands were not assigned to the characteristic bands, especially those written about in the text; not very clear markings; the small font making it challenging to read

 

Response: Data on the FTIR spectroscopy were placed in the Supplemental data section because this method provides incomplete information on the composition of heterodimers, the receipt and use of which is the purpose of the work. The FTIR spectroscopy does not reflect the presence of metal nanoparticles in the samples and can only qualitatively confirm the introduction of zinc oxide into the samples. The method is also sensitive to the content of this component in the system. If 3-5% of this component is present in the samples, it is the content on detection limit

of the device, so the region of 400-800 cm^–1 is presented in more detail. The copolymers used in the work have been known for a long time, so we focused on the above mentioned component of heterodimers. The baseline is automatically taken into account when registering spectra. The spectra of all polymer-stabilized heterodimers were additionally placed in the Supplemental data section. The quality of the spectra was adjusted.

 

 

Comment, Abstract and text: values without units.

Response:  The units of measurement are specified: Minimum inhibitory concentrations (MIC) of Ag/ZnO samples based on all copolymers were in the ranges of 1.4-1.7 mg/mL – for C. albicans, 2.9-6.8 mg/mL – for E. coli and 23-27 mg/mL for S. aureus; MIC values of Au/ZnO samples were 472 mg/mL for S. aureus, and 945 mg/mL for C. albicans and E. coli.

 The value of Fractional Inhibitory Concentration Index – has no units of measurement.

Comment: Introduction, line 51: abrupt transition from Ag to Au. In addition, the properties and influence of Ag are described quite accurately, and Au is described superficially in the context of the most detailed description of ZnO. There is a considerable disproportion of data and literature references between these fragments. The negative aspects of Ag and Au are not mentioned. 

lines 112-120: very cursory introduction to polymers. No literature references and no comparisons with other similar solutions. No information on why these particular copolymers were chosen. What advantages and disadvantages are related to nanostructures?

It is worth sorting out the data and completing the introduction significantly!

 

Response:  The texts below have been added to the manuscript.

 

There are few data on the use of nanogold as a bactericide, and it has been shown that the antimicrobial activity of nanosilver exceeds that of nanogold [23]. Although silver nanoparticles have been shown to be more toxic than nanogold, nanosilver has a relatively low toxicity to human cells in comparison with lower forms of life [24-26].

 

In particular, organic polymers can act as stabilizers of bioactive nanoparticles, preventing their aggregation by reducing their surface energy [51]. Water-soluble polymers can promote the binding of metal nanoparticles to the pathogen cell due to high-active potential interactions between repeating polymer units and multivalent surface properties of bacteria [65]. In comparison with a number of natural and synthetic polymers, maleic acid (anhydride) copolymers have a complex of positive properties. 

Comment, Methodology: line 132: abbreviations not explained.

Response:  We have added the explanations.

 

Comment: Why does information about synthesis appear in this part when it should be mentioned only about materials?

 

Response:  We have removed the information about synthesis.

 

 

Comment, line 136: What was the purity of the reagents?

 

Response:  The sentence “All reagents were of the analytical grade and were used without purification.” has been added.

 

Comment: Why is the methodology for XPS very detailed and the other techniques very cursory and abbreviated without any measurement details?

 

Response:  The fact is that XPS is a surface sensitive method with sampling depth less 10 nm, so measurements are very sensitive to experimental conditions such as sample preparations, vacuum level and surface charging during spectra recording. Thus, the data obtained by XPS are more difficult to interpret than those obtained by bulk sensitive methods what requires a more detailed description of Experimental.

 

Comment, Synthesis, line 233: why is there different metal content? On what basis were such concentrations selected? Why composite with Au0 was based on only one copolymer and not similar to the Ag0 with three considered?

 

Response: For composites VM (EM, SM)/Ag⁰/ZnO the molar ratio of units of maleic acid residues of copolymer to silver cations and zinc cations 1/0.3/0.15 was used.  A large load on precursor cations led to the production of less stable colloids, and a smaller one was not advisable due to the continued use of composites as bactericides. The different metal content was dictated by the different in the molecular weights of the alternating units of copolymers, on the basis of which the ratios were calculated. For VM and SM the molecular weights of units are close - 227 and 220, respectively, and for EM – 144. Based on the literature data on the lower biological activity of nanogold in comparison with that of nanosilver, we chose one composite with nanogold for comparison. The obtained data on the bactericidal properties of composites confirmed this.

 

 

 

Comment, line 235: The procedure is worth describing in a little more detail and not just providing a literature reference.

 

Response: We have described this procedure in more detail.

 

 

Comment, Results: 

Is ZnO in the form of nanoparticles crystallised in the form of structures? Here there are analytical errors because, in EDS, it is written about Zn, in XRD about crystalline phases; in TEM, there is no evidence of crystalline forms of ZnO and the textual notation and determinations of the matter was treated even differently!

Response: TEM and XRD are methods based on different physical principles. TEM is a qualitative characteristic of the samples for the presence of crystal structures. TEM cannot detect crystallinity directly and quantitatively. This task can be done by XRD. EDS can detect the spectral bands for Zn but cannot detect crystal phases. Thus, all these methods provide pieces of information to obtain detailed picture.

 

Comment, line 305: Average of what?

Response: Size of nanoparticles. The corresponding sentence has been changed.

 

Comment: Crystallinity, Ag nanoparticles, Au? Why is XRD data not shown for all samples (all polymers and all combinations shown in Table 2)?

 

Response: The main purpose of the work is to synthesize and study the properties of heterodimeric structures and samples containing silver or gold nanoparticles stabilized by copolymers were obtained and characterized by us earlier, and in this work were used as a controls. Polymer Zinc salt does not have crystallinity.

 

Comment, line 306: How was the % content of the different phases of the composite determined since no Rietveld analysis was done and not even mentioned?

 

Response: The content of crystal phases of the heterodimers was calculated using DIFFRAC TOPAZ software (Coelho, A. TOPIC 5.0, Bruker AXS GmbH, Karlsruhe (Germany), 2012; Bruker AXS uses Rietveld refinement of collected X-ray data). This sentence was in the first version of manuscript.

 

 

Comment, Scheme 1: How is it known that Ag0 is surrounded by the polymer chain and not hooked up to C=O? Why is Au0 not mentioned? Why are there bacterial cells in this scheme? Not described R fragments.

 

Response: This is a simplified scheme, so only the most active silver nanoparticle was included. Also, in order to simplify the scheme, the arrangement of the active components of the system is given, which is dictated by the fact that the polar zinc oxide molecule, unlike the nonpolar silver nanoparticle, tends to the more polar structural elements of the polymer chain. R fragments are specified. Bacterial cells are pictured because the samples obtained were used as potential bactericides. The R fragments were described in the Scheme.

 

Comment: lines 269+ fig.1, no reference to the text and what the authors describe in the text and on the spectrum, i.e. no indication confirming the presence of ZnO or Zn2+, no description of the y-axis, not very clear markings, I would have used a legend to present the data instead of a caption under the figure. Why are the results not shown or all the samples in Table 2?

 

Response: The references have been added. The text has been corrected. We have moved the mention of Figure 1. The UV spectroscopy method is not a method for identifying zinc cations, therefore we did not give the spectra of samples containing these cations. The y-axis has been descibed (absorbance). The appearance of zinc oxide in a system containing nanosilver or nanogold is accompanied by the appearance of the wide absorption band in the 365-375 nm what was previously noted [59,61,62].

 

 

Comment, lines 279-288: Where do such conclusions come from when no result confirms the described validity of the results? Why was IR not done for all samples presented in Table 2?

 

Response: Data on the FTIR spectroscopy were placed in the Supplemental data section because this method provides incomplete information on the composition of heterodimers, the receipt and use of which is the purpose of the work. The FTIR spectroscopy cannot detect Au⁰ and Ag⁰ states in the samples and can only qualitatively confirm the introduction of zinc oxide into the samples. The method is also sensitive to the content of this component in the system. If 3-5% of this component is present in the samples, it is the content on the edge of the sensitivity of the device, so the region of 400-800 cm^–1 is presented in more detail. The copolymers used in the work have been known for a long time, so we focused on the above component of heterodimers. The baseline is automatically taken into account when registering spectra. The spectra of all polymer stabilized heterodimers were additionally placed in the Supplemental data section.

 

Comment: Fig. 2 shows practically nothing: no description of what can be seen in the picture, no histogram of size distribution with statistical analysis, no TEM-XRD confirming the presence of Ag0, Au0 and ZnO, no TEM-EDS confirming what the authors write about in the text. Why are the results not shown for all samples, as in Table 2?

Response: We have changed Fig. 2. Samples of polymer-stabilized silver and gold nanoparticles that do not contain zinc oxide were used as a comparison in the context of this work. Their properties were described earlier [66,67]. The methods are nevertheless informative in relation to metal cations. Polymer zinc salts do not have crystallinity.

 

Comment: Fig. 3 illegible picture. The caption shows the Zn 3p and Au 4f range while there are two diagrams, and I assume one for samples with Ag. How do these results relate to each other? Why aren't all the lines visible from the fit described? Why do the same intensities characterise the doublet for Au? The XPS analysis needs to be more accurate and chaotic, e.g. lines 335-337. Where does the positive and negative shift come from when it is not shown anywhere? How do the XPS results relate to Table 2 when only one sample is shown? The lack of O 1s, Ag 2p, and C 1s lines could be more coherent, not relating to all the samples tested. What was obtained? What are the differences between samples? Where do these differences come from?

 

Response: Figure 3 shows the spectra of two samples Ag0/ZnO (1) and Au0/ZnO (2), which is noted in the caption. The spectrum of the Ag0/ZnO sample (1) is a Zn 3p spectrum consisting of two peaks belonging to the Zn 3p_1/2 and Zn 3p_3/2 sublevels with a branching ratio of 0.5 (the value of 0.5 is constant for the 2p levels, for the 4f levels it is equal to 4/3) and a satellite peak, which was subtracted for clarity. The spectrum of the Au0/ZnO sample (2) is more complex because it includes the spectra of Zn 3p and Au 4f core-levels, which partially overlap, as seen in the Figure 3 and a satellite peak, which was subtracted for clarity Characteristics (such as the value spin-orbit splitting and Gaussian widths) obtained by describing the Zn 3p spectrum of the Ag0/ZnO sample, which is noted in the text. But, since this region of the spectrum cannot be described by one spin-orbit doublet, another one was introduced with the same characteristics, but with different binding energies. As is known, the binding energy of a photoelectron peak is a characteristic of the chemical state of an atom. Thus, we can state that there are two chemical states of zinc atoms. As for the low-energy region characteristic of the Au 4f levels, it is also described by two spin-orbit doublets, whose Au 4f_7/2 photoelectron peaks are shifted relative to the standard value for pure gold (84.0 eV)  by -0.1 and +0.8 eV, which made it possible to assert the presence of Au-Zn-O and Au-O-Zn groups, respectively. Since the performed analysis made it possible to adequately interpret the obtained Zn 3p and Au 4f spectra, there was no need for a similar analysis of the O 1s and C 1s spectra. The comment of the reviewer to analyze Ag 2p line looks rather strange since binding energy of Ag 2p_3/2 level is more 3 keV while kinetic energy of photons emitted by X-ray source is near 1.5 keV.

 

 

 

Comment, Table 2: No pure polymers as references. Differences should be described, and symbols explained

 

Response: Copolymers of maleic acid are used only as carriers of various bioactive and bactericidal agents and do not have the properties of the latter. [Patel H., Raval D.A., Madamwar D., Patel S.R. Polymeric prodrug: Synthesis, release study and antimicrobial property of poly(styrene-co-maleic anhydride)-bound acriflavine. Angew. Macrom. Chem. 1998;263:25–30;  Jeong J.H., Byoun Y.S., Ko S.B., Lee Y.S. Chemical modification of poly(styrene-alt-maleic anhydride) with antimicrobial 4-aminobenzoic acid and 4-hydroxybenzoic acid. J. Ind. Eng. Chem. 2001;7:310–315; Jeong J.H., Byoun Y.S., Lee Y.S. Poly(styrene-alt-maleic anhydride)-4-aminophenol conjugate: Synthesis and antibacterial activity. React. Funcct. Polym. 2002;50:257–263. doi: 10.1016/S1381-5148(01)00120-1; Akshatha Nagaraja, Manohara Dhulappa Jalageri, Yashoda Malgar Puttaiahgowdaa,Kakarla Raghava Reddy, Anjanapura V. Raghu. A review on various maleic anhydride antimicrobial polymers. J. Microbiol. Methods 163 (2019) 105650 https://doi.org/10.1016/j.mimet.2019.105650]. The symbols were explained.

 

Comment, conclusions: lines 425-438: not needed

line 450: what about Au?

lines 450-457: in my opinion, no fragment is needed

 

Response: We have removed the phrase:” With approximately equal molar mass of maleic acid copolymers, ethylene residues in polymeric chain create fewer steric difficulties compared to styrene or N-vinylpyrrolidone residues”. We have mentioned on gold containing composites.

 

Comments on the Quality of English Language

Response:  The text has been corrected.

 

Please see the attachment

Author Response File: Author Response.pdf

Reviewer 3 Report

The manuscript presents a simple one-pot method for preparing silver (gold) and zinc oxide colloidal nanohybrid structures doped with zinc ions. It's an interesting job. At present, there are still some problems in the manuscript which need the author to consider seriously. The modification suggestions are as follows:

1、Transmission electron microscopy (TEM) is a conventional method for the characterization of nanomaterials. ​It is suggested that the authors complement the characterization of the diffraction pattern of the prepared nanomaterial. Is the fabricated nanomaterial a single crystal diffraction pattern, a polycrystalline diffraction pattern, or an amorphous diffraction pattern? It is hoped that these characterization can be supplemented to give the reader a clearer understanding of the prepared nanomaterial.

Author Response

Responses to Reviewer 3:

Authors are grateful to Reviewer for attentive reading of our paper and valuable remarks.

 

Comments: Transmission electron microscopy (TEM) is a conventional method for the characterization of nanomaterials. ​It is suggested that the authors complement the characterization of the diffraction pattern of the prepared nanomaterial. Is the fabricated nanomaterial a single crystal diffraction pattern, a polycrystalline diffraction pattern, or an amorphous diffraction pattern? It is hoped that these characterizations can be supplemented to give the reader a clearer understanding of the prepared nanomaterial.

Response:  The TEM images in Fig. 2 have been changed. Detailed information about the composition of the crystalline phases of the composites, determined by XRD, has been given.

Please see the attachment

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Authors have revised the manuscript and have addressed the comments. However, it is strongly recommended to include some more set of experiments. The manuscript should be therefore submitted for major corrections. Here are some additional comments to the manuscript:

1. For MIC calculation, authors have taken positive control without the composites. However, positive control should be a marketed drug that gives positive result against the microbe under consideration and negative control is kept without tested compound and only inoculated with microbial suspension. Author needs to rectify the error.

2. SAED pattern has been represented in Figure 2, however, it is missing in the result and discussion part.

3. Different scales have been used for recording TEM micrographs. Author should mention the reason.

4. Different size ranges have been mentioned for Ag⁰/ZnO and Au⁰/ZnO composites in XRD analysis, however, in case of TEM single average size range has been mentioned. Elaborate the findings.

Minor editing of English language required

Author Response

 

The responses to Reviewer 1.

 

Authors are grateful to Reviewer for attentive reading of our paper and valuable remarks.

 

Comment:

For MIC calculation, authors have taken positive control without the composites. However, positive control should be a marketed drug that gives positive result against the microbe under consideration and negative control is kept without tested compound and only inoculated with microbial suspension. Author needs to rectify the error.

 

Response:

When determining the sensitivity of microorganisms to antimicrobial substances, two controls can be used - positive and negative. The purpose of a positive control (the growth of a microorganism in a well with a nutrient broth without an antibiotic) is to prove the viability of the culture and the possibility of its growth under the applied incubation conditions. The purpose of the negative control (lack of growth of the microorganism in the well with a nutrient broth and an antibiotic, but without a test strain) is to prove the absence of contamination of the test system and the tested antimicrobial substances. This approach is classical for such experiments. This information is completely enough for assessing the validity of the experiment. Making corrections is not needed.

 

Comment:

SAED pattern has been represented in Figure 2, however, it is missing in the result and discussion part.

 

Response:

The TEM gives a qualitative assessment of the composition of the crystal phase, so we added the phrase: “In addition to the TEM, the XRD gives a more detailed qualitative and quantitative composition of the crystal phases of heterodimers. SAED patterns demonstrate concentric rings with intermittent dots, indicating that these nanoparticles are crystalline in nature”. Table 1 contains XRD characteristics of the composites.

 

Comment:

Different scales have been used for recording TEM micrographs. Author should mention the reason.

 

Response:

The scales have been corrected.

 

Comment:

Different size ranges have been mentioned for Ag0/ZnO and Au0/ZnO composites in XRD analysis, however, in case of TEM single average size range has been mentioned. Elaborate the findings.

 

Response:

The phrase: “In addition to the TEM, the XRD gives a more detailed qualitative and quantitative composition of the crystal phases of heterodimers” has been added. Table 1 contains XRD characteristics of the composites.

 

Comment:

Comments on the Quality of English Language Minor editing of English language required.

 

Response:

 

It has been improved.

 

 

 

 

Author Response File: Author Response.pdf

Reviewer 2 Report

I want to thank the authors for the answers. However, most of them are entirely unsatisfactory for me, and the article still enables corrections. See below:

- Supplementary data: Fig 1. The data needs to be prepared much better: the baseline is still not subtracted, and the answer that the software made a baseline correction is naive because you can correct it yourself. The position of the bands still needs to be marked, and others, especially visible in spectra 2 and 3, are a noise but not a real band. The bands were still not assigned to the characteristic bands, especially those written about in the text; not very clear markings; the small font makes it challenging to read,

- Introduction: There is still an abrupt transition from Ag to Au. In addition, the properties and influence of Ag are described quite accurately, and Au, despite some extra sentences, is still described superficially in the context of the most detailed description of ZnO. There is still a considerable disproportion of data and literature references between these fragments. The negative aspects of Ag and Au are not mentioned. 

There is still a cursory introduction to polymers, primarily that the paper is based on the polymers. No literature references and no comparisons with other similar solutions. No detailed information on why these particular copolymers were chosen. What advantages and disadvantages are related to nanostructures, and the sentence “In comparison with a number of natural and synthetic polymers, maleic acid (anhydride) copolymers have a complex of positive properties” is not enough?

!It is worth sorting out the data and completing the introduction significantly!

- Methodology: 

I am not satisfied with the answer because XRD or FTIR are also tricky techniques and enable more detailed information about the way of the measurements. Hence, “Why is the methodology for XPS very detailed and the other techniques cursory and abbreviated without any measurement details?” remains.

- Results: 

The answer needs to be more satisfactory. I know the fundamentals of each method, but I need answers to my questions. Therefore, I leave my question unresolved: “Is ZnO in the form of nanoparticles crystallised in the form of structures? Here, there are analytical errors because, in EDS, it is written about Zn, in XRD about crystalline phases; in TEM, there is no evidence of crystalline forms of ZnO and the textual notation and determinations of the matter were treated even differently!” Additionally, the histogram of the particle distribution was not fitted and discussed., Similar SEAD data were fitted and not explained. 

My question remains: “Crystallinity, Ag nanoparticles, Au? Why is XRD data not shown for all samples (all polymers and all combinations shown in Table 2)?”

My question remains, “How was the % content of the different phases of the composite determined since no Rietveld analysis was done and not even mentioned?”, because you mentioned only the software but did not describe the method.

My doubts and questions remain: “Scheme 1: How is it known that Ag0 is surrounded by the polymer chain and not hooked up to C=O? Why is Au0 not mentioned? Why are there bacterial cells in this scheme? Not described R fragments (there are three):

There is still no indication confirming the presence of ZnO or Zn2+. I would have used a legend instead of a caption under the figure to present the data. Why are the results not shown for all the samples in Table 2?

My question to Fig. 3 remains: “Why do the same intensities characterise the doublet for Au?”. Still, “The XPS analysis needs to be more accurate and chaotic. Where does the positive and negative shift come from when it is not shown anywhere? How do the XPS results relate to Table 2 when only one sample is shown?” I'm afraid I have to disagree with the authors because the situation observed on the Zn, Ag, or Au core lines should also be visible on O 1s. In comparison, other information should be visible when interpreting C 1s lines. Therefore, I suggest supplying such an information.

Equations should be prepared in the equation editor with an explanation of the parameters. 

- Conclusions: What about Au?: This information is marginally introduced, but the paper summarized the data. It is strange. The last fragment, in my opinion, is unnecessary.

It can be corrected.

Author Response

 

The responses to Reviewer 2

 

Authors are grateful to Reviewer for attentive reading of our paper and valuable remarks.

 

Comment:

Supplementary data: Fig 1. The data needs to be prepared much better: the baseline is still not subtracted, and the answer that the software made a baseline correction is naive because you can correct it yourself. The position of the bands still needs to be marked, and others, especially visible in spectra 2 and 3, are a noise but not a real band. The bands were still not assigned to the characteristic bands, especially those written about in the text; not very clear markings; the small font makes it challenging to read.

 

Response:

When processing FTIR data, we were guided by the ways of submitting such data in this journal, for example:

 

Appl. Sci. 2023, 13(17), 9650; https://doi.org/10.3390/app13179650

 

Appl. Sci. 2023, 13(17), 9705; https://doi.org/10.3390/app13179705

 

Appl. Sci. 2023, 13(7), 4517; https://doi.org/10.3390/app13074517

 

Appl. Sci. 2023, 13(6), 3924; https://doi.org/10.3390/app13063924

 

 

 

 

 

 

 

 

 

 Appl. Sci. 2023, 13(16), 9098; https://doi.org/10.3390/app13169098

 

Appl. Sci. 2023, 13(8), 5162; https://doi.org/10.3390/app13085162

 

Data on the FTIR method were placed in the Supplemental Materials because this method provides incomplete information on the composition of heterodimers, the receipt and use of which is the purpose of the work. The FTIR method does not reflect the presence of metal nanoparticles in the samples and can only qualitatively confirm the introduction of zinc oxide into the samples. The method is also sensitive to the content of this component in the system. If 3-5% of this component is present in the samples, it is the content on the edge of the sensitivity of the device. The nature of the spectra corresponds to the composition of the composite and the amount of zinc oxide in the heterodimers. The quality of the spectra was adjusted.

 

Comment:

Introduction: There is still an abrupt transition from Ag to Au. In addition, the properties and influence of Ag are described quite accurately, and Au, despite some extra sentences, is still described superficially in the context of the most detailed description of ZnO. There is still a considerable disproportion of data and literature references between these fragments. The negative aspects of Ag and Au are not mentioned.

 

Response:

We have completed the introduction.

“Unlike nanosilver, gold nanoparticles were much less often used as bactericides. For example, nanogold had almost negligible effect on E. coli, Staphylococcus aureus, Bacillus subtilis and Agrobacterium tumefaciens [Nyberg, L.; Turco, R. F.; Nies, L. Assessing the impact of nanomaterials on anaerobic microbial communities. Environ. Sci. Technol. 2008, 42, 1938-1943. https://doi.org/10.1021/es072018g; Wang, C.; Wang, L.; Wang, Y.; Liang, Y.; Zhang, J. Toxicity effects of four typical nanomaterials on the growth of Escherichia coli, Bacillus subtilis and Agrobacterium tumefaciens. Environ. Earth Sci. 2012, 65, 1643-1649. https://doi.org/10.1007/s12665-011-1139-0].

Greater bactericidal activity was observed for silver nanoparticles, than for nanogold [Ahmad, T.; Wani, I. A.; Manzoor, N.; Ahmed, J.; Asiri, A. M. Biosynthesis, structural characterization and antimicrobial activity of gold and silver nanoparticles. Colloids Surfaces B-Biointerfaces, 2013, 107, 227-234. DOI: 10.1016/j.colsurfb.2013.02.004; Hernandez-Sierra, J. F.; Ruiz, F.; Cruz Pena, D. C.; Martinez-Guttierrez, F.; Martinez, A. E.; De Jesus Pozos Guillen, A.; Tapia-Perez, H.; Martinez Castanon, G. The antimicrobial sensitivity of Streptococcus mutans to nanoparticles of silver, zinc oxide, and gold. Nanomed.:Nanotechnol., Biol. Med. 2008, 4, 237-240. DOI: 10.1016/j.nano.2008.04.005; Amin, R. M.; Mohamed, M. B.; Ramadan, M. A.; Verwanger, T.; Krammer, B. Rapid and sensitive microplate assay for screening the effect of silver and gold nanoparticles on bacteria. Nanomedicine, 2009, 4, 637-643. DOI: 10.2217/nnm.09.50; Zhou, Y.; Kong, Y.; Kundu, S.; Cirillo, J.; Liang, H. Antibacterial activities of gold and silver nanoparticles against Escherichia coli and bacillus Calmette-Guerin. J. Nanobiotechnol. 2012, 10, 19. https://doi.org/10.1186/1477-3155-10-19].

Gold NPs exert their antibacterial activities mainly by two ways: one is to collapse membrane potential, inhibiting ATPase activities to decrease the ATP level; the other is to inhibit the subunit of ribosome from binding tRNA. Gold NPs enhance chemotaxis in the early-phase reaction. The action of gold NPs did not include reactive oxygen species (ROS)-related mechanism, the cause for cellular death induced by most bactericidal antibiotics and nanomaterials [Cui, Y.; Zhao, Y.; Tian, Y.; Zhang, W.; Lü, X.; Jiang, X. The molecular mechanism of action of bactericidal gold nanoparticles on Escherichia coli. Biomaterials, 2012, 33, 2327-2333. doi: 10.1016/j.biomaterials.2011.11.057] 

The disruption of the cell membrane of the pseudomonas aurigenosa anduncontrolled leakage of cell DNA was reported after interacting with diaminopyrimidineethiolfunctionalised, cationic 3 nm AuNPs [Zhao, Y.; Tian, Y.; Cui, Y.; Liu, W.; Ma, W.; Jiang, X. Small Molecule-Capped Gold Nanoparticles as Potent Antibacterial Agents That Target Gram-Negative Bacteria. J. Am. Chem. Soc. 2010, 132, 12349-12356. https://doi.org/10.1021/ja1028843]

As for the toxicity of nanosilver and gold, it was carried out a comparative assessment of adverse bioactivity of virtually equidimensional gold and silver nanoparticles administered to rats at equal mass doses either intratracheally or intraperitoneally. It has demonstrated that NS is more toxic than NG, notwithstanding the latter’s higher accumulation in the RES-abundant organs [Katsnelson, B. A.; Privalova, L. I.; Gurvich, V. B.; Makeyev, O. H.; Shur, V. Y.; Beikin, Y. B.; Sutunkova, M. P.; Kireyeva, E. P.; Minigalieva, I. A.; Loginova, N. V.; et al. Comparative in Vivo Assessment of Some Adverse Bioeffects of Equidimensional Gold and Silver Nanoparticles and the Attenuation of Nanosilver’s Effects with a Complex of Innocuous Bioprotectors. Int. J. Mol. Sci. 2013, 14, 2449-2483. https://doi.org/10.3390/ijms14022449].

In addition, it has been shown that gold has low toxicity to biological systems, then silver whether bacteria, animal, or human [Jin, Y.; Zhao, X. Cytotoxicity of photoactive nanoparticles. In Safety of Nanoparticles: From Manufacturing to Medical Applications. Editor Webster, T.; Springer Science + Business Media, LLC, New York, 2008, pp.19-31].

 

Comment:

There is still a cursory introduction to polymers, primarily that the paper is based on the polymers. No literature references and no comparisons with other similar solutions. No detailed information on why these particular copolymers were chosen. What advantages and disadvantages are related to nanostructures, and the sentence “In comparison with a number of natural and synthetic polymers, maleic acid (anhydride) copolymers have a complex of positive properties” is not enough? !It is worth sorting out the data and completing the introduction significantly!

 

Response:

We have completed the introduction:

Previously, polyvinylpyrrolidone was used to obtain heterostructures, while a 2-step process was used [Attia, Y. A.; Mohamed, Y. M. A.; Awad, M. M.; Alexeree, S. Ag doped ZnO nanorods catalyzed photo-triggered synthesis of some novel (1H-tetrazol-5-yl)-coumarin hybrids. J. Organomet. Chem. 2020, 919, 121320. https://doi.org/10.1016/j.jorganchem.2020.121320]. This polymer has been used to help deposit Zn oxide on AuNPs. Growing oxide shells on seed nanoparticles requires the control of several processes: (a) the nucleation and growth of the shell material; (b) the “wetting” of the shell material on the seeds; and (c) the aggregation of the nanoparticles [Sun, H.; He, J.; Wang, J.; Zhang, S. Y.; Liu, C.; Sritharan, T.; Mhaisalkar, S.; Han, M. Y.; Wang, D.; Chen, H. Investigating the Multiple Roles of Polyvinylpyrrolidone for a General Methodology of Oxide Encapsulation. J. Am. Chem. Soc. 2013, 135, 9099-9110. https://doi.org/10.1021/ja4035335].

Synthesis of ZnO-Ag heterostructure was carried out by a precipitation method with cellulose nanofibers as a stabilizer (with the formation of Ag nanoparticles on ZnO particles) [Azizi, S.; Ahmad, M.B.H.; Hussein, M.Z.; Ibrahim, N.A. Synthesis, Antibacterial and Thermal Studies of Cellulose Nanocrystal Stabilized ZnO-Ag Heterostructure Nanoparticles. Molecules 2013, 18, 6269–6280. https://doi.org/10.3390/molecules18066269].

Using dextran as both reductant and stabilizing agent, ZnO-Au NPs composites were synthesized and possessed a petal-like morphology [ Xiang, S.; Mtng, Q.; Zhang, K.; Gu, Y.; Liu, W.; Yang, B. Facile Synthesis of ZnO-Au Nanopetals and Their Application for Biomolecule Determinations. Chem. Res. Chinese Universities, 2019, 35, 924-928. doi:10.1007/s40242-019-9063-z]

 

Comment:

Methodology: I am not satisfied with the answer because XRD or FTIR are also tricky techniques and enable more detailed information about the way of the measurements. Hence, “Why is the methodology for XPS very detailed and the other techniques cursory and abbreviated without any measurement details?” remains.

 

Response:

XRD or FTIR are tricky techniques of course.  In the XRD method, the Miller indices are indicated. The text below has been added. The peaks corresponding to Ag crystal phase are at ~38.1, 44.2, 64.4, 78.3° and to Au crystal phase are at ~38.2, 54.5, 64.7 and 78.7° and ZnO (zincite) are at ~31.8, 34.4, 36.1, 47.5, 56.7, 63.0, 66.5, 68.0, 69.1, 72.6, and 79.0°. By intensity, the diffraction peaks are located in the following sequence of Miller indexes: Ag(Au) - 111, 002, 022, 311, and ZnO - 010, 002, 011, 011, 012, 020, 004 (Figure S2). The FTIR method is very useful and informative, especially with regard to organic structures, polymers. But in the context of this work, it can only be an auxiliary tool, since the work is devoted to the production and use of inorganic heterostructures.

 

 

Comment:

Results: The answer needs to be more satisfactory. I know the fundamentals of each method, but I need answers to my questions. Therefore, I leave my question unresolved: “Is ZnO in the form of nanoparticles crystallised in the form of structures? Here, there are analytical errors because, in EDS, it is written about Zn, in XRD about crystalline phases; in TEM, there is no evidence of crystalline forms of ZnO and the textual notation and determinations of the matter were treated even differently!” Additionally, the histogram of the particle distribution was not fitted and discussed., Similar SEAD data were fitted and not explained. My question remains: “Crystallinity, Ag nanoparticles, Au? Why is XRD data not shown for all samples (all polymers and all combinations shown in Table 2)?”

 

Response:

The EDS method was presented as a link – we removed this mention. TEM visualizes the shape and size of particles in solution, and histograms indicate the distribution of crystalline particles in the sample.

We have added next clarifications:

 “TEM micrographs of colloidal solutions of prepared Au⁰/ZnO and Ag⁰/ZnO composites show relatively monodispersed mostly 6-20 nm nanoparticles with a shape close to spherical.”

“In addition to the TEM, which gives a qualitative assessment of the composition of the crystal phase, the XRD method gives a more detailed qualitative and quantitative composition of the crystal phases of heterodimers. SAED patterns demonstrate concentric rings with intermittent dots, indicating that these nanoparticles are crystalline in nature”.

In addition to heterodimers, which are the main subject of consideration in this work, the remaining compounds are known and are given as a comparison in the study of antibacterial properties. In our opinion, the description of known compounds will complicate the perception of the essence of this study.

 

Comment:

My question remains, “How was the % content of the different phases of the composite determined since no Rietveld analysis was done and not even mentioned?”, because you mentioned only the software but did not describe the method.

 

Response:

In the experimental part, it is noted on the basis of which program (DIFFRAC TOPAZ software (Coelho, A. TOPIC 5.0, Bruker AXS GmbH, Karlsruhe (Germany), 2012;) the % content of the different phases of the composite was calculated. Rietveld analysis is a standard method for calculating composite phases, included in textbooks, and in our opinion, there is no need to provide details of the calculation method, as well as to describe the basics of other methods used in this work.

 

Comment:

Scheme 1: My doubts and questions remain: “Scheme 1: How is it known that Ag0 is surrounded by the polymer chain and not hooked up to C=O? Why is Au0 not mentioned? Why are there bacterial cells in this scheme? Not described R fragments (there are three): There is still no indication confirming the presence of ZnO or Zn2+. I would have used a legend instead of a caption under the figure to present the data.

 

Response:

The caption has been changed. The detailed description of legend is in the main text.

 

A silver and gold hydrosol is a typical lyophobic colloid, and the adsorption layer favors the formation of a solvate layer with the result that the particle surface becomes lyophilic and the lyophobic sol becomes much less sensitive to coagulation by electrolytes [L A Dykman, V A Bogatyrev, "Gold nanoparticles: preparation, functionalisation and applications in biochemistry and immunochemistry", RUSS CHEM REV, 2007, 76 (2), 181–194, DOI: https://doi.org/10.1070/RC2007v076n02ABEH003673]. Therefore, such an arrangement of metal nanoparticles in the scheme, in our opinion, is quite acceptable. In this scheme, we additionally mentioned Au⁰. Bacterial cells are present in this scheme, because in this work the antibacterial properties of the obtained heterodimers were investigated.

Linear alternating copolymers of maleic acid were used as polymers of stabilizers of heterodimers, those in polymer molecules alternated dimeric units consisting of residues of maleic acid and a comonomer, which is indicated by the symbol “R”. And these comonomers are the residues of N-vinyl-2-pyrrolidone, ethylene or styrene. Samples containing zinc ions were prepared with the addition of a zinc salt to the copolymer or heterodimer contained copolymer.

 

Comment:

Why are the results not shown for all the samples in Table 2?

 

Response:

From the many samples presented in Table 2, heterodimers were selected that differed in elemental composition (Ag or Au) with approximately the same phase composition.

 

 

Comment:

My question to Fig. 3 remains: “Why do the same intensities characterise the doublet for Au?”. Still, “The XPS analysis needs to be more accurate and chaotic. Where does the positive and negative shift come from when it is not shown anywhere? How do the XPS results relate to Table 2 when only one sample is shown?” I'm afraid I have to disagree with the authors because the situation observed on the Zn, Ag, or Au core lines should also be visible on O 1s. In comparison, other information should be visible when interpreting C 1s lines. Therefore, I suggest supplying such an information. Equations should be prepared in the equation editor with an explanation of the parameters.

 

Response:

 

In the previous response we wrote that branching ratio (i.e. Au 4f_7/2 / Au 4f_5/2) is 4/3. It means the intensities of the peaks are different. The relative intensities of both doublets are presented in Table S1 (Supplementary Materials).

 

In the previous response we also wrote “As for the low-energy region characteristic of the Au 4f levels, it is also described by two spin-orbit doublets, whose Au 4f_7/2 photoelectron peaks are shifted relative to the standard value for pure gold (84.0 eV) by -0.1 and +0.8 eV, which made it possible to assert the presence of Au-Zn-O and Au-O-Zn groups, respectively.”

 

Negative shift by -0.1 eV means 84.0 - 0.1 = 83.9 eV

Positive shift by +0.8 eV means 84.0 + 0.8 = 84.8 eV

 

These values are presented in Table S1 (Supplementary Materials) as well.

 

These terms are traditional in XPS.

 

The O 1s, C 1s and N 1s have been added Supplementary Materials.

 

 

 

 

Comment:

Conclusions: What about Au?: This information is marginally introduced, but the paper summarized the data. It is strange. The last fragment, in my opinion, is unnecessary.

 

Response:

Gold-containing heterodimers turned out to be significantly less active in comparison with silver-containing composites, which was discussed in the section "Results", so in “Conclusions” section only mentions this fact. We have deleted the last fragment.

 

Author Response File: Author Response.pdf

Round 3

Reviewer 1 Report

Authors have substantially restructured the manuscript entitled “Polymer-Stabilized Silver (Gold)-Zinc Oxide Nanoheterodimer Structures as Antimicrobials” “Applied Sciences” Manuscript ID: applsci-2551035 following the corrections as suggested. I, therefore recommend this article to further process for publication in the present form.

Minor editing of English language required

Author Response

The text has been corrected.

Reviewer 2 Report

Thank you for the answer. Some data are satisfactorily corrected, but others still need to be corrected.

- The infrared spectra should be corrected for the baseline in the supplement. The measurement apparatus does not do this, while in post-process analysis, it is worth comparing the spectra against each other and looking for differences between them

- line 49: add gold nanoparticles (Au NPs). 

- Names of bacteria, either binary (Escherichia coli) or abbreviated (E. coli etc.).

- I wouldn't say I like too much the statement nanosilver and nanogold; better to use Ag NPs and Au NPs.

-lines 58-59 in one line because they treat the same thing

-the titles of subsection 2.3 on synthesis are worth unifying, especially since 2.3.2 does not match 2.3.2.2.

-Additionally, the histogram of the particle distribution needed to be fitted and discussed. SEAD data should be fitted to estimate the phase. It should be discussed more precisely, especially in the context of metallic or oxidized phases., What do the "size" data in Table 1 mean, and where do they come from?

-Why is XRD data not shown for all samples (all polymers and all combinations shown in Table 2)?" I'm afraid I have to disagree with you, and Ritvield is valuable for estimating the parameters of your systems. So please supply the data. Please mark the different phases on the diffractograms. XRD provides information about the size of the crystallites. Then where do the values from line 364 come from? Please complete the formula.

- lines 371-373: where do the data come from?

- line 380: don't understand why? "...satellite peak at 86.39 eV (subtracted for clarity)..."

Why are the XPS lines of Zn 3p and Zn 2p not shown for other silver compounds?

- lines 397-402: the authors write about CLS with no reference data for Au0 and Au+; if there is, it must be written explicitly. As a result, this hinders reception, and I recommend correcting the text.

- line 405-406: it should be written instead of "two main symmetric Ag 3d5/2 and Ag 3d3/2 peaks" to "doublet".

- line 409: different values than in line 406. why aren't the Ag 3dd spectra for the other systems shown?

- line 417: why were no results commented for the other lines? Where does nitrogen come from?

- line 420: TEM results do not confirm "...core-shell structure...", nor "...zinc oxide nanoparticles are the outer layer...". similarly, lines derived from Ag-O were obtained. Then how can Ag be Ag0

- lines 464-471: the formulas should be written with an equation editor, and the symbols in the formulas should be labelled. Another time, I ask the same thing!

English could be proofread,

Author Response

Authors are grateful to Reviewer for attentive reading of our manuscript and valuable comments.

 

Comment: The infrared spectra should be corrected for the baseline in the supplement. The measurement apparatus does not do this, while in post-process analysis, it is worth comparing the spectra against each other and looking for differences between them

Response: The spectra have been corrected.

 

Comment, line 49: add gold nanoparticles (Au NPs). 

Response: We have added (AuNPs).

 

Comment: Names of bacteria, either binary (Escherichia coli) or abbreviated (E. coli etc.).

Response: Names of bacteria have been corrected.

 

Comment: I wouldn't say I like too much the statement nanosilver and nanogold; better to use Ag NPs and Au NPs.

Response:  Nanosilver and nanogold have been changed with Ag NPs and Au NPs.

 

Comment: lines 58-59 in one line because they treat the same thing

Response: The text has been corrected.

 

Comment: The titles of subsection 2.3 on synthesis are worth unifying, especially since 2.3.2 does not match 2.3.2.2.

Response: The titles have been unified.

 

Comment: Additionally, the histogram of the particle distribution needed to be fitted and discussed. SEAD data should be fitted to estimate the phase. It should be discussed more precisely, especially in the context of metallic or oxidized phases. What do the "size" data in Table 1 mean, and where do they come from?

Response: As for the histograms, the next text: “TEM micrographs of colloidal solutions of all prepared Au⁰/ZnO and Ag⁰/ZnO composites show relatively monodispersed nanoparticles, predominantly  6-20 nm in size with a shape close to spherical (Figure 2). The size and shape of the particles shown in the micrographs practically did not depend on the nature of the polymers used as stabilizers of nanoparticles” has been added. The content of crystal phases of the heterodimers was calculated using DIFFRAC TOPAZ software (Coelho, A. TOPIC 5.0) using Powder X-ray diffractometry phase analysis of the samples. We have added explanations in Table 1. The number and size refer to the crystalline phases found in the samples.

 

Comment: Why is XRD data not shown for all samples (all polymers and all combinations shown in Table 2)?" I'm afraid I have to disagree with you, and Ritvield is valuable for estimating the parameters of your systems. So please supply the data. Please mark the different phases on the diffractograms. XRD provides information about the size of the crystallites. Then where do the values from line 364 come from? Please complete the formula.

Response:  The work is devoted to the synthesis of heterodimers and their use as potential bactericides. The remaining compounds are given for comparison with the properties of heterodimers when studying their antibacterial properties. In our opinion, the description of known compounds make it difficult to understand the essence of this study. The stabilizer polymers and their salts we use are amorphous [Jing Huang, S. Richard Turner, Recent advances in alternating copolymers: The synthesis, modification, and applications of precision polymers, Polymer, Volume 116, 5 May 2017, Pages 572-586, https://doi.org/10.1016/j.polymer.2017.01.020]. Amorphous polymers can be defined as polymers that do not exhibit any crystalline structures in X-ray or electron scattering experiments when bombarded with X-rays, and does not form a diffraction patterns.

 

The content of crystal phases of the heterodimers was calculated using DIFFRAC TOPAZ software (Coelho, A. TOPIC 5.0, Bruker AXS GmbH, Karlsruhe (Germany), 2012; Bruker AXS uses Rietveld refinement of collected X-ray data). The characteristic mean crystallite size of  nanoparticles was calculated from the broadening of the peaks using the Scherrer formula [Patterson, A.L. The Scherrer Formula for X-Ray Particle Size Determination. Phys. Rev. 1939, 56, 978. 826 https://doi.org/10.1103/PhysRev.56.978. ]

Scherrer formula:

D_ℎkl = Kl/β_hkl cosθ,

 where K – the particle shape factor, 0.9, λ – X-ray wavelength, β_hkl – the half-width of (hkl) reflection, θ = 2θ/2 – Bragg angle corresponding to (hkl) reflection.

We labeled the different phases in the diffraction patterns.

Comment,  lines 371-373: where do the data come from?

Response: This data is from Table 1. We have indicated this.

 

Comment:  line 380: don't understand why? "...satellite peak at 86.39 eV (subtracted for clarity)..."

Response: The satellite peak, like the background of inelastically scattered electrons, does not carry additional information about the chemical state of zinc atoms; therefore, in order to simplify the figure, it was subtracted from the integral spectrum.

 

 

Comment: Why are the XPS lines of Zn 3p and Zn 2p not shown for other silver compounds?

Response: From the samples studied in this work, heterodimers were selected that differed in elemental composition (Ag or Au) and had approximately the same phase composition.

 

Comment, lines 397-402: the authors write about CLS with no reference data for Au0 and Au+; if there is, it must be written explicitly. As a result, this hinders reception, and I recommend correcting the text.

Response: The text has been corrected.

 

Comment:  line 405-406: it should be written instead of "two main symmetric Ag 3d5/2 and Ag 3d3/2 peaks" to "doublet".

Response: “two main symmetric Ag 3d5/2 and Ag 3d3/2 peaks” on our opinion means that it is a pure doublets, while asymmetric peaks can contain a different hidden chemical state with low intensity. Symmetric is a keyword in this case.

 

Comment:  line 409: different values than in line 406. why aren't the Ag 3dd spectra for the other systems shown?

Response: These values are different because they are related to different samples: CLS by 0.13 (binding energy = 367.4 eV) is for our sample (Ag⁰/ZnO structure stabilized with VM) while 367.6 eV is for Ag-doped ZnO from reference [95].

 

Comment:  line 417: why were no results commented for the other lines? Where does nitrogen come from?

Response: The C 1s and O 1s spectra were added at the request of the reviewer. Since they do not directly characterize the charge states of Au and Ag, they were not considered in the previous version of the manuscript. A brief commentary on these spectra is given in the Supporting materials.

The presence of nitrogen in the VM/Ag⁰/ZnO and VM/Au⁰/ZnO samples is due to the presence of N-vinylpyrrolidone residues in the structure of the copolymer VM - Poly(N-vinyl-pyrrolidone-alt-maleic anhydride), which was used as stabilizer of heterodimers.

 

Comment, line 420: TEM results do not confirm "...core-shell structure...", nor "...zinc oxide nanoparticles are the outer layer...". similarly, lines derived from Ag-O were obtained. Then how can Ag be Ag0.

Response: We removed mention of the core-shell structure, since this assumption was based only on literature data.

 

Comment, lines 464-471: the formulas should be written with an equation editor, and the symbols in the formulas should be labelled. Another time, I ask the same thing!

Response: The formulas were rewritten using the equation editor.

 

Comments on the Quality of English Language. English could be proofread.

Response: The text has been corrected.