Nitrogen-Doped Arginine Carbon Dots and Its Metal Nanoparticle Composites as Antibacterial Agent

Round 1

Reviewer 1 Report

Manuscript of Sahiner and co-workers describes the MW synthesis of N-doped carbon nanoparticles and the relative exhaustive characterization. These nanoparticles have been further combined with Ag and Cu, to obtain nanomaterials able to act as antibacterial agents and as inhibitors of AChE enzyme in the treatment of Alzheimer disease. In addition, studies on chelating properties towards Fe(II) ion have been described.

Manuscript is well written, and the topic of this paper is tuned to the target of the Journal. However, in my opinion, some points need to be addressed before the publication:

Major

• UV-Vis spectra: some clarifications require the “intense” bands at ca. 320 nm. The authors, in figure 5a, assign this band to the n-p* of the C=O groups of the NP. In my experience of no-N-doped CNPs, I have never seen this band. Is it possible that the band of p-p* of C=N bond is at 320 nm? The attribution of these transitions should be revised and properly commented in the main text.
• The emission spectra obtained by irradiation at different wavelengths show the same maximum? This point should be addressed in the main text
• Antibacterial and inhibition of AChE tests: control experiments by using simple Ag-nanoparticles and Cu-nanoparticles are mandatory to demonstrate the effect of the carbon nanoparticles in these studies.
• Literature must be improved: i) applications of carbon nanoparticles in catalysis missing; ii) the use of metal nanoparticles as antibacterial nanomaterials missing; iii) a reference of the correlation between Alzheimer disease and dyshomeostatis of metal ions is required

Minor:

• Abstract is too long (more than 300 words)
• Arginine chemical structure reported in Figure 1a must be redraw
• The authors reported a 77% of yield in the synthesis of the nanoparticles. This data cannot be obtained without a precise value of molecular mass of reagents and products. In this case, the molecular mass of carbon nanoparticles is not easy to calculate. Thus, I suggest to remove this data.
• TEM and DLS gave different results, in particular related to the lower dimensions of the nanoparticles (TEM : 2 nm, while DLS : 0.5 nm). These data should be properly addressed and compared
• Line 316: Figure 5 or Figure 6?

Author Response

Manuscript of Sahiner and co-workers describes the MW synthesis of N-doped carbon nanoparticles and the relative exhaustive characterization. These nanoparticles have been further combined with Ag and Cu, to obtain nanomaterials able to act as antibacterial agents and as inhibitors of AChE enzyme in the treatment of Alzheimer disease. In addition, studies on chelating properties towards Fe(II) ion have been described.

Manuscript is well written, and the topic of this paper is tuned to the target of the Journal. However, in my opinion, some points need to be addressed before the publication:

-We thank the reviewer for nice the nice comments about the manuscript and the following imrovements are done taking the reviewers’ suggestions point-by-point.

Major

• UV-Vis spectra: some clarifications require the “intense” bands at ca. 320 nm. The authors, in figure 5a, assign this band to the n-p* of the C=O groups of the NP. In my experience of no-N-doped CNPs, I have never seen this band. Is it possible that the band of p-p* of C=N bond is at 320 nm? The attribution of these transitions should be revised and properly commented in the main text.

-According to the literature, the peak at 320 nm can be attributed to n-π* transition of surface functional groups [8]. Also, ‘’C=O groups’’ was changed with ‘’surface functional group’’ in line 315.

• The emission spectra obtained by irradiation at different wavelengths show the same maximum? This point should be addressed in the main text.

-This point was mentioned in the revised text  on pp 16 as “The Arg CDs exhibit strong blue fluorescence at 425 nm maximum wavelength with the variation of the excitation between 310 and 350 nm and the highest PL intensity was obtained at 340 nm excitation wavelength.”

• Antibacterial and inhibition of AChE tests: control experiments by using simple Ag-nanoparticles and Cu-nanoparticles are mandatory to demonstrate the effect of the carbon nanoparticles in these studies.

-Antimicrobial activity of Ag NPs and Cu NPs were compared with the composite forms of metal NPs with Arg CDs. These required explanations were added in main text in line 339-341.

• Literature must be improved: i) applications of carbon nanoparticles in catalysis missing; ii) the use of metal nanoparticles as antibacterial nanomaterials missing; iii) a reference of the correlation between Alzheimer disease and dyshomeostatis of metal ions is required
  69. The use of carbon nanoparticles in catalysis applications were mentioned and referred in Line 56 and 69.
  70. The use of metal nanoparticles as antibacterial nanoparticles were also mentioned and referred in line 78-82, line 339-341, and line 417-419. These explanations were improved at this part.

• The ability of the compounds in metal chelation such as Fe⁺², Cu ⁺², Al ⁺³, Zn⁺² were also added in the treatment of Alzheimer Disease.

-The following reference was included in the revised manuscript:

41. 4 Cai, R., Wang, L.N., Fan, J.J., Geng, S.Q., and Liu, Y.M. (2019) New 4-N-phenylaminoquinoline derivatives as antioxidant, metal chelating and cholinesterase inhibitors for Alzheimer’s disease. Bioorg. Chem., 93 (July), 103328.
42. Wichur, T., Więckowska, A., Więckowski, K., Godyń, J., Jończyk, J., Valdivieso, Á.D.R., Panek, D., Pasieka, A., Sabaté, R., Knez, D., Gobec, S., and Malawska, B. (2020) 1-Benzylpyrrolidine-3-amine-based BuChE inhibitors with anti-aggregating, antioxidant and metal-chelating properties as multifunctional agents against Alzheimer’s disease. Eur. J. Med. Chem., 187, 111916.

Minor:

• Abstract is too long (more than 300 words)

-Done As suggested and it is now <300 words.

• Arginine chemical structure reported in Figure 1a must be redraw

-Chemical structure of arginine was redrawn in Figure 1a.

• The authors reported a 77% of yield in the synthesis of the nanoparticles. This data cannot be obtained without a precise value of molecular mass of reagents and products. In this case, the molecular mass of carbon nanoparticles is not easy to calculate. Thus, I suggest to remove this data.

-This value was deleted in the main text as suggested.

• TEM and DLS gave different results, in particular related to the lower dimensions of the nanoparticles (TEM : 2 nm, while DLS : 0.5 nm). These data should be properly addressed and compared.

-Size range of Arg CDs was changed to 1 -10 nm for TEM images. As shown in Figure 1c, this distribution of Arg CDs in aqueous solution is in agreement with the TEM images as the particle sizes are in 0.5-10 nm range with an average size of 2.8±0.85 nm. This explanation included in the revised manuscript on pp 6 as “Small size of Arg CDs up to 1 nm was not very clearly visualized by TEM images because of the lesser number of particles at these sizes.

• Line 316: Figure 5 or Figure 6?

-we are sorry, this as Figure 6 and changed accordingly.  

Author Response File: Author Response.docx

Reviewer 2 Report

The authors describe preparation of arginine carbon dots and their combination with silver and copper nanoparticles. The nanoparticles were characterized by TEM, DLS, zeta potential, FT-IR, X-ray, fluorescence and UV-vis spectroscopy. Antimicrobial behaviour on S. aureus and E. coli was determined.  Moreover, the utility for Alzheimer disease treatment was suggested.

Some suggestions to improve the manuscript are below.

In the text after full names Staphylococcus aureus and Escherichia coli shortcuts should be used and italics checked. Full description of suppliers should be given. Tris is not explained. What is prm (142), Cds (147)? Is the concentration 200 mg/mL (177) correct? There are Tris –HCI, MgCI.6H₂0 in part 2.6.. Figure 1c, is it intensity? How is it that ones Arg CDs are 2-10 nm (233) and later 2-20 nm (278)? From TEM it is not clear. What about DLS and zeta potential of Arg-Ag CDs and Arg-Cu CDs? Figure 6 b and c, does it mean that inhibition is 100 % for 0 concentrations of dots and Gentamicin? The part about chelating of Fe(II) is not clear, what concentration is given in Fig.8? with could be (359), ACheE (363), natue (282), cabalities (383)?     

Author Response

The authors describe preparation of arginine carbon dots and their combination with silver and copper nanoparticles. The nanoparticles were characterized by TEM, DLS, zeta potential, FT-IR, X-ray, fluorescence and UV-vis spectroscopy. Antimicrobial behaviour on S. aureus and E. coli was determined.  Moreover, the utility for Alzheimer disease treatment was suggested.

Some suggestions to improve the manuscript are below.

-We thank the reviewer for nice the nice comments about the manuscript and the following improvements are done taking the reviewers’ suggestions point-by-point.

In the text after full names Staphylococcus aureus and Escherichia coli shortcuts should be used and italics checked.

-Done as suggested.

Full description of suppliers should be given. Tris is not explained. There are Tris –HCI, MgCI.6H₂0 in part 2.6..

-The full description of chemicals and supplier is given in the revised manuscript as suggested.

What is prm (142), Cds (147)?

-We apologize for the mistake it as corrected as “Prm” and “Cds” were changed as “rpm” and “CDs”, respectively.

Is the concentration 200 mg/mL (177) correct?

-This concentration is correct.

Figure 1c, is it intensity?

-Y axis is intensity and (nm) is removed.

 How is it that ones Arg CDs are 2-10 nm (233) and later 2-20 nm (278)? From TEM it is not clear.

-The size range of Arg CDs were corrected as 1-10 nm on pp 6 and 8 on the revised manuscript.

What about DLS and zeta potential of Arg-Ag CDs and Arg-Cu CDs?

-DLS and zeta potential measurements of Arg-Ag CDs and Arg-Cu CDs were not added to the manuscript because they are almost the same.

Figure 6 b and c, does it mean that inhibition is 100 % for 0 concentrations of dots and Gentamicin?

-As seen in the Figure 6b and c, 100% inhibition for 0 concentrations of dots and Gentamicin were represented the control group which does not contain any sample.

The part about chelating of Fe(II) is not clear, what concentration is given in Fig.8? with could be (359), ACheE (363),

-It was corrected as “Arg based CDs particle suspension at 2000 µg/mL stock solution was prepared and diluted to 666, 333, 166.5, 83.3 µg/mL with DI. From these solutions, 140 µL was put into the well plate. Next, 20 µL of 1 mM Fe(II) solution was added to each one these wells and the absorbance values were measured at 562 nm. Following the addition of 40 µL ferrozine of 2.mM, the total solution volumes were completed to 200 µL and the final concentration of CD materials were 1400, 466, 233,116.5, 58.3, 29.1 µg/mL.”

The wrong word was corrected as AChE at 363 line.

natue (282), cabalities (383)?     

-We apologize for the mistakes; these were corrected as “nature” and “abilities”.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

manuscript is now suitable for the publication

Reviewer 2 Report

Thanks for the improvements.