Graphene-Coated Iron Nitride Streptavidin Magnetic Beads: Preparation and Application in SARS-CoV-2 Enrichment
Round 1
Reviewer 1 Report
In the manuscript by Li J et al, entitled: “Graphene Coated Iron Nitride Streptavidin Magnetic Beads：Preparation and Application in SARS-CoV-2 Enrichment” the authors produced and characterized magnetic beads with increased water solubility and binding to streptavidin with the aim to provide magnetic beads for immuno-capture and enrichment of the SARS-CoV-2 that could be used for diagnostical purposes. The manuscript is mainly focused on the preparation and characterization of the magnetic beads. The preparation of the magnetic beads allows coupling with any biotinylated antibody. In a first step, the authors characterized the GO@FeN-MB using TEM, x-ray photoelectron spectroscopy analysis and Raman spectroscopy. In a second step, the saturation profile of the streptavidin-magnetic bead complex was determined. The authors optimized the preparation method of the so-called simulated virus (polystyrene beads with bound SARS-CoV-2 S spike protein) and claim that each polystyrene microsphere is coupled to about 30 spike proteins. Simulated virus and pseudoviruses were used to assess the binding properties of the prepared beads. As my expertise lies in virology, virus purification methods and electron microscopy, I cannot judge the Raman spectroscopy measurements. The manuscript has several major shortcomings which prevent it from being published without a major revision.
Major concerns:
- Authors claim that they developed a new type of beads suitable for immune-capture and enrichment methods of SARS-CoV-2 virus particles. However, no comparative experiments are shown using other types of magnetic beads functionalized with streptavidin which are commercially available. Overall, data is missing showing experiments comparing the new type of magnetic beads with other ‘’old’’ types of magnetic beads to validate the improvement.
- Authors claim the bead have a diameter of 100 nm but do not show data. It is not clear from the TEM data how 8-10 layers of graphene oxide were determined and there are not clearly annotated in the figure. Also, low magnification TEM of the beads should be shown together with a histogram of diameter size distribution. The authors claim that the oxidation process does not have a great impact on the morphology of the magnetic beads, however, neither TEM of beads prior to oxidation nor histogram showing size distribution is shown.
- It is not clearly explained what kind of SARS-CoV-2 pseudovirus was purchased and how this pseudovirus was characterized.
- The term “Simulated virus” is misleading. It would be more appropriate to use the term S spike coupled to polystyrene beads. Although the authors claim that the polystyrene beads contain on average 30 Spike, they provide no evidence to support the claim. The authors do not provide any information on the origin and purity of the spike protein.
- Importantly no data on recovery yield is reported. In such a study a given input of the pseudovirus should be incubated with the magnetic beads and eluted and the recovery yield of the pseudovirus should be determined. Authors should perform a pull-down of the pseudovirus followed by SDS-PAGE and Western Blot using the anti-S2 antibody where both input and eluate will be compared. Such experiments should be done also for another type of beads (commercially available) which should be used as a reference.
- Fluorescence microscopy images do not show negative control and scale bars not too small. Data in Fig.4 and 5: how many replicas were used for the experiment, what are the error bars?
Minor comments:
- The manuscript is not well written including the wrong syntax and there are several mistakes
- in the result part, the text is not ordered according to the order of the figures, instead, the text is jumping from figure 2c to 2b and 2a
- the word ‘’COVID-19’’ was used in the wrong context (conclusion). COVID-19 is a diseases caused by SARS-CoV-2 virus.
- figures: scale bars are too small or missing
- figure legends lack information
- figure 1, 4 and 5: images could be better arranged
- some references are missing
Author Response
our response to the reviewer’s comments is attached
Author Response File: 
Author Response.docx
Reviewer 2 Report
The direction of work is interesting and necessary. The method for obtaining a magnetic adsorbent is described well and in detail. The results obtained are well confirmed experimentally. The authors propose a new magnetic adsorbent based on iron nitride.
The authors claim that iron nitride is the best magnetic carrier, with better magnetic properties than magnetite. There is no data on the method of obtaining iron nitride and its magnetic properties. There is no evidence that this adsorbent is better than magnetite. Iron nitrides are highly susceptible to corrosion in aqueous solutions. The material can greatly reduce the magnetic properties in aqueous solution.
In the process of obtaining iron nitride coated with graphene, all iron nitride could turn into iron carbide, with a significant decrease in magnetic properties. It is known that iron carbide is a harder magnetic material than iron nitride. It has a large residual magnetization after magnetization, and the particles stick together into one lump.
It is necessary to make a description of the properties of the magnetic adsorbent.
Author Response
our response to the reviewer’s comments is attached
Author Response File: Author Response.docx
Reviewer 3 Report
Jianxing Li et al. synthesized streptavidin-functionalized immunomagnetic core-shell (FeN/graphene) bead. Considering the urgency of COVID-19 issue, this is a timely prepared report. However, the study is somewhat unclear and characterization is not fully conducted. Also, there are numerous grammatical errors and typos throughout the manuscript. Hence, I recommend authors to improve the manuscript by conducting experiments and correct errors in the manuscript.
In Fig 1, TEM of GO@FeN-MB is provided. Provide low magnification data so that distribution and uniformity can be observed.
Provide VSM, DLS (or PSA) data for the IMB synthesized in this study.
Authors claimed that streptavidin is covalently bound on the surface of the magnetic bead. Provide FT-IR for streptavidin, magnetic bead w/o streptavidin, and magnetic bead with streptavidin.
In Fig 2c, x-axis range is different. Use the same scale. Although authors deconvoluted the peaks, convoluted fit is not shown. Considering deconvoluted C-C peak fit is larger than the raw data (see 280-282 eV region), obviously, the overall fit is not matching well with the measured raw data. Also, difference of 289 eV peaks is not so obvious between two graphs.
Fig 4 and 5 showed different fluorescent colors but any explanation and discussion are provided. Provide UV-vis spectra for absorption behaviors.
Authors are claiming enhanced water solubility by the oxidation of G@FeN-MBs. However, any quantification is not conducted.
As shown in Fig 4a, intensity of fluorescent color is not uniform in the samples. In particular, bottom image has dark color at the center of the sample. Also, the upper image has no fluorescent at all for the sample on the very left. Any explanation? It seems that streptavidin is not uniformly reacted.
In Fig 4 and 5, it is hard to see scale bars.
Author Response
our response to the reviewer’s comments is attached
Author Response File: Author Response.docx
Round 2
Reviewer 1 Report
The authors have addressed most of the raised concerns. However, there are still some changes that would improve the manuscript:
1) Figure 1d and Figure 2b (after oxidation) are identical, which is inappropriate. Figure 1 and 2 should be rearranged: Figure 1 should contain TEM before and after oxidation showing low mag overview micrographs as well as high magnification images with the carbon layers (before and after oxidation).
2) Figure 2 should contain the histogram of diameter size distribution and photos of the Eppendorf tubes. Why are the samples in the tubes shown twice (Figure 2c)?
3) The abstract should be improved for clarity. It would be beneficial to provide an explanation of the differences between carbon-coated and conventional beads and the reason why carbon bears were tested here.
4) Authors could suggest how to improve the dispersity of the beads
5) remove the term ‚brand new‘ from the abstract
6) Line 231: remove ‚very‘
7) 312 and 313: remove ‚real‘ (not need to say that)
8) 312: there should be a space: 100 nm
9) Conclusion: Again authors could state here the benefit of the carbon-coated beads and compare them with other beads. Conclusions should be written with capitol C.
Author Response
Response to the reviewer 1’s comments has been upload as a Word file.
Reviewer 3 Report
Authors answered most previous concerns and the manuscript is greatly improved. While it will be better if authors can provide better fitting for deconvoluted XPS results, main claims are mostly supported by the experimental results. Hence, I can recommend for publication of this manuscript in Magnetochemistry.
Author Response
Thank you for your attentive review and valuable suggestions for improving our manuscript.
