Protein-assisted scalable mechanochemical exfoliation of few-layer biocompatible graphene nanosheets

A facile method to produce few-layer graphene (FLG) nanosheets is developed using protein-assisted mechanical exfoliation. The predominant shear forces that are generated in a planetary ball mill facilitate the exfoliation of graphene layers from graphite flakes. The process employs a commonly known protein, bovine serum albumin (BSA), which not only acts as an effective exfoliation agent but also provides stability by preventing restacking of the graphene layers. The latter is demonstrated by the excellent long-term dispersibility of exfoliated graphene in an aqueous BSA solution, which exemplifies a common biological medium. The development of such potentially scalable and toxin-free methods is critical for producing cost-effective biocompatible graphene, enabling numerous possible biomedical and biological applications. A methodical study was performed to identify the effect of time and varying concentrations of BSA towards graphene exfoliation. The fabricated product has been characterized using Raman spectroscopy, powder X-ray diffraction, transmission electron microscopy and scanning electron microscopy. The BSA-FLG dispersion was then placed in media containing Astrocyte cells to check for cytotoxicity. It was found that lower concentrations of BSA-FLG dispersion had only minute cytotoxic effects on the Astrocyte cells.


Recommendation?
Major revision is needed (please make suggestions in comments)

Comments to the Author(s)
The paper investigates exfoliation of graphite by ball milling in the presence of BSA as "green" stabilizer. The resultant material is characterised by XRD, TEM, SEM/EDX and Raman spectroscopy as function of milling time and BSA concentration. Qualitatively, it was found that relatively short milling times can be used for the exfoliation which avoids contamination of the graphene with the milling media. The results are interesting and the paper is well written. The introduction is relatively lengthy, but provides a good overview of the literature. Acceptance after the following revisions is suggested: 1) The main issue is that the observations are all rather qualitative in nature. The authors are encouraged to make a bit more quantitative analysis. i) The dispersed concentration could be estimated based on UVVis spectroscopy. While the protein will also give signal (see for example SI of Nature Commun. 2018, 9 (1), 1577), the extinction at long wavelength can be used to analyse the dispersed concentration as function of time ii) The Raman spectra should be investigated in more detail. For example, the authors are encouraged to analyse D/G ratio and G band width which allows to draw (semi-quantitative) conclusions on edge and basal plane defectiveness (2D Mater. 2017, 4 (2), 025039). In addition the 2D band shape could be analysed to get a more quantitative picture on degree of exfoliation (at least as comparison); example procedures are described in various articles(Carbon 2015, 81 (0), 284-294, Nanoscale 2016, 8 (7), 4311-4323., Carbon 2013, 53, 357-365. Carbon 2020, 161, 181-189.). While all these methods have some restrictions, they would allow for a more solid sample comparison. 2) The plot of Raman D/G ratio as function of time in Figure 7a shows a significant amount of scatter. Is this related to spot to spot variations in each sample from the three measurements that were averaged? The authors are encouraged to average over more spots and include the standard deviation in the figure. For Figure 7b, they have chosen to show the data at 45h which follows a decreasing D/G ratio in the order no BSA, 1:10 BSA and 1:2 BSA. However, the plot above shows this is more or less a coincidence which varies for the different times. As such, it is not representative and it cannot be concluded that the D/G ratio is lower in the presence of BSA (as currently stated) 3) The sample preparation for each characterization should be included in the experimental section.

Decision letter (RSOS-200911.R0)
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Once again, thank you for submitting your manuscript to Royal Society Open Science and I look forward to receiving your revision. If you have any questions at all, please do not hesitate to get in touch. ********************************************** RSC Associate Editor: 1 Comments to the Author: (There are no comments.) RSC Associate Editor: 2 Comments to the Author: (There are no comments.) ********************************************** Reviewers' Comments to Author: Reviewer: 1 Comments to the Author(s) The authors present a method to produce few layer graphene using ball milling in presence of bovine serum albumin (BSA). This method for graphene exfoliation from graphite have already been presented in multiple reports using single proteins or full serum, and there are significant contributions to the field that were not cited in this manuscript ( The fact that BSA help stabilizing the flakes produced by the milling exfoliation is very well known therefore the reviewer feels it is difficult to find elements of novelty or insight in this manuscript. The authors should highlight more the specific rationale of the study, the increment over the published works and better link the results obtained to the application. The material could have been characterized more in terms of yield, lateral size, overtime stability etc., trying to correlate these features to the milling conditions (absence/presence of BSA, BSA/graphite ratio, milling time, etc).
Also, the material is called biocompatible but the biological investigation is quite poor. In Fig. 8 only one optical microscope image is shown for each condition. The use of PI to detect cell necrosis is mentioned in materials but the results are not shown. The choice of astrocytes over other cell lines is not explained. The author could improve this part by adding a quantification of the cell viability with some assay. Another important aspect is the influence of BSA (that is probably highly aggregated or unfolded after the long treatment) and the influence of the contaminants on the viability. The author should use appropriate controls to investigate these aspects. Comments to the Author(s) The paper investigates exfoliation of graphite by ball milling in the presence of BSA as "green" stabilizer. The resultant material is characterised by XRD, TEM, SEM/EDX and Raman spectroscopy as function of milling time and BSA concentration. Qualitatively, it was found that relatively short milling times can be used for the exfoliation which avoids contamination of the graphene with the milling media. The results are interesting and the paper is well written. The introduction is relatively lengthy, but provides a good overview of the literature. Acceptance after the following revisions is suggested: 1) The main issue is that the observations are all rather qualitative in nature. The authors are encouraged to make a bit more quantitative analysis. i) The dispersed concentration could be estimated based on UVVis spectroscopy. While the protein will also give signal (see for example SI of Nature Commun. 2018, 9 (1), 1577), the extinction at long wavelength can be used to analyse the dispersed concentration as function of time ii) The Raman spectra should be investigated in more detail. For example, the authors are encouraged to analyse D/G ratio and G band width which allows to draw (semi-quantitative) conclusions on edge and basal plane defectiveness (2D Mater. 2017, 4 (2), 025039). In addition the 2D band shape could be analysed to get a more quantitative picture on degree of exfoliation (at least as comparison); example procedures are described in various articles(Carbon 2015, 81 (0), 284-294, Nanoscale 2016, 8 (7), <a href="tel:4311-4323">4311-4323</a>., Carbon 2013, 53, 357-365. Carbon 2020, 161, 181-189.). While all these methods have some restrictions, they would allow for a more solid sample comparison.
2) The plot of Raman D/G ratio as function of time in Figure 7a shows a significant amount of scatter. Is this related to spot to spot variations in each sample from the three measurements that were averaged? The authors are encouraged to average over more spots and include the standard deviation in the figure. For Figure 7b, they have chosen to show the data at 45h which follows a decreasing D/G ratio in the order no BSA, 1:10 BSA and 1:2 BSA. However, the plot above shows this is more or less a coincidence which varies for the different times. As such, it is not representative and it cannot be concluded that the D/G ratio is lower in the presence of BSA (as currently stated) 3) The sample preparation for each characterization should be included in the experimental section.

Author's Response to Decision Letter for (RSOS-200911.R0)
See Appendix A.

Comments to the Author(s)
The authors improved the manuscript adding some rationale for the investigation and the analysis of lateral size and defect density.

Comments to the Author(s)
The authors have addressed all comments made by the referees and revised their manuscript appropriately. Unfortunately, the additional experiments both reviewers asked for could not be performed which would have improved the quality of the manuscript signficantly. Nonetheless, it contain a sufficient amount of novelty and publication is recommended.

Decision letter (RSOS-200911.R1)
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Reviewer: 1
Comments to the Author(s) The authors improved the manuscript adding some rationale for the investigation and the analysis of lateral size and defect density.

Response to Reviewers
We are thankful to the reviewers for their time and consideration while making their thoughtful comments. We have done our utmost to work on the reviewers' comments and build our manuscript according to their expectations.
The reviewers' comments have been copied verbatim and an answer to their comments are provided directly below. The citations for the above papers have been added. Although, the comparison between our work and the above reports 1-4 presents common ground, our onestep method on fabricating bio-compatible few layer graphene (BSA-FLG), using a planetary mill, in the presence of BSA has not been replicated. In addition, most of the reports mentioned in this comment have performed exfoliation using ultrasonic exfoliation, whose pitfalls have been explained in the introduction of our paper. We have also included it here for the reviewer's convenience -"However, sonication works through the principle of cavitation assisting exfoliation leading to excessive local heat generation which causes the material being sonicated to be subjected to enormous pressure and sharp temperature changes 5-7 making it highly restrictive for industrial applications." 2. The fact that BSA help stabilizing the flakes produced by the milling exfoliation is very well known therefore the reviewer feels it is difficult to find elements of novelty or insight in this manuscript. The authors should highlight more the specific rationale of the study, the increment over the published works and better link the results obtained to the application After reviewing the literature extensively, we have not found papers that apply planetary ball milling to produce BSA-FLG. Also, we focused on doing a systematic study of different parameters, i.e., BSA quantity and milling time to indicate the quality of BSA-FLG formed through various forms of characterization. Moreover, we explained the pitfalls and advantages of extreme parameter values during the fabrication process.
We have modified our introduction to highlight the rationale of our research-"This approach may be applied in areas that require a cheap one-step fabrication method for FLG while recognizing the impact of milling speed and BSA-graphite concentrations." 3. The material could have been characterized more in terms of yield, lateral size, overtime stability etc., trying to correlate these features to the milling conditions (absence/presence of BSA, BSA/graphite ratio, milling time, etc).
In this manuscript, we focused on characterization of manufactured graphene in order to show the quality of graphene. We have shown Raman spectroscopy, TEM, and biocompatibility of graphene. We appreciate the comment and we agree with the reviewer on the future steps of this work in terms of yield and lateral size correlation to the milling.
Furthermore, we added the following analysis -"The nanographite size ( _ ) has usually been characterized using / . The qualitative control of structural transformation in various graphitic materials can be calibrated by Tuinstra and Koenig's (T-K) empirical relation 8 . The amount of initial defects is given by −1 and referred to as the defect density . _ is proportional to the quantity of disorder in nano crystallites. Furthermore, the defect distance in graphene containing 0-dimensional point defects can also be obtained through the correlations given below 9,10 .  4. Also, the material is called biocompatible but the biological investigation is quite poor. In Fig. 8 only one optical microscope image is shown for each condition.
The work done on integrating BSA-FLG with Astrocytes was intended to be preliminary in nature in this specific manuscript, therefore limited resources were directed towards those experiments here. However, we have performed further investigations on biocompatibility of graphene in below papers: 5. The use of PI to detect cell necrosis is mentioned in materials but the results are not shown.
We have modified the results section to explain the effect of PI -"Propidium Iodide (PI) dye, which detects cell viability, was used to identify cell death. It does not penetrate live cells containing intact membranes, however it does enter damaged/dead cells and intercalates with the RNA and DNA bases, thereby binding to them." "PI dye was used to identify the dead cells and they have been marked in red [ Figure 8 a-d]" 6. The choice of astrocytes over other cell lines is not explained. The author could improve this part by adding a quantification of the cell viability with some assay. Another important aspect is the influence of BSA (that is probably highly aggregated or unfolded after the long treatment) and the influence of the contaminants on the viability. The author should use appropriate controls to investigate these aspects.
We thank the reviewer for this careful comment. We did not have a particular reason for choosing Astrocytes. Our group has been working with Astrocytes and we have encapsulated them in graphene fibrous structures as 3D cell culture The problem with references has been corrected. 9. Starting graphite spectrum in Fig 7b show high presence of defects. The reference for the commercial starting material is missing.