Development of Electrochemical Biosensor Platforms for Determination of Environmental Viral Structures
Round 1
Reviewer 1 Report
1. The tittle need to be reviewed to avoid tautology (i.e. “Development of Electrochemical-Based Biosensor Platforms for Determination of Environmental Viral Structures) (line 2-3).
2. All species names used in the manuscript should be italicize.
3. How did confirm the electro activity of the species?
4. Why did you choose CuO for the modification of the PGEs?
5. There are some grammatical errors that need to be corrected in the manuscript (Avoid using preposition at the beginning of the paragraph). Please correct them all.
6. Add more discussion to your Electrochemical Analysis and SEM-EDX results.
Author Response
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Reviewer 2 Report
The abstract is not informative. It does not explain the methods and work in general. The introduction section is too long and it includes general information on viral infections which is not appropriate for a bioanalytical paper. The methods based on cyclic voltammetry are not the best for this kind of studies. It would be much better to use impedance spectroscopy which provides more quantitative results. Overall, the approach is very standard and the results are rather expected and not impressive.
Author Response
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Reviewer 3 Report
In this manuscript, the authors developed an electrochemical sensor for the detection of SARS-CoV-2 N protein based on the modification of pencil graphite electrodes (PGE) with hydroxyapatite (HAP) and copper oxide were functionalized with N protein antibody.
The manuscript is well-organized and fairly satisfied. But there are some issues to be addressed for the publication:
1. In the introduction of pencil graphite (Line 112-114). The authors should add references to explain why using the pencil graphite as an electrode with advantages as superior sensitivity, modifiable electroactive surface area, etc.
2. In line 124-125, the authors should add the reason for utilizing the square wave voltammetry (SWV) as an electrochemical technique for antigen detection (e.g., SWV's principle).
3. Line 219-221, Please add nucleocapsid protein, represents for NP.
4. Line 278, Please change redoks to redox.
5. Please enhance the resolution in CV results of Figure 3.
6. In the section 3.1. Characterization of the prepared electrodes. The authors should add the XRD or XPS results to confirm the structure of unmodified PGE, Copper oxide modified PGE, Hydroxyapatite and copper oxide modified PGE.
7. Please enhance the quality of all images in the manuscript.
Author Response
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Reviewer 4 Report
Comments to the author:
Paper by Ekici et al. presents a thorough and insightful description and explanation of all the factors related to viral pathogen electrochemical detection. This is a very interesting manuscript addressing all the parameters that researchers have to target when designing their platform to successfully detect and quantify viral pathogens. The article is well documented and presents well to the reader, introducing the global current context of pandemics and the main types of viruses of concent. However some more explanation on certain chosen parameters are needed to be expanded for a less experienced experimentalist audience.
The authors should modify and update the manuscript based on the following comments:
1.- the authors describe that the use of CaP, due to its ability to exchange ions, increases the surface area, which increases the conductivity. This is completely not supported by any evidence whatsoever in the manuscript and it is mixing different concepts without any second thoughts.
The authors should perform and include new experiments where they use inner-sphere redox probes (such as hexamine ruthenium chloride), and perform scan rate studies to calculate the electroactive area of this electrodes (use the well known and used Randles Sevcik equations).
In additions, they claim this also increases conductivity which is totally unsupported again, the authors should analyse and report the conductivity of their electrodes with a multi-meter and then include EIS analysis about the different resistances and conductivities of their system.
Both these parameters are the most basic studies when performing electrochemistry and the authors not only have failed to address, but have mixed electrochemical active area and conductivity, which makes it even more confusing and wrong for the less experienced readers.
2.- the authors rightfully present the benefits of amperometric sensors for POC applications, when compared to GC/HPLC-MS devices, however they do not mention any of the drawbacks of electrochemical and screen-printed based biosensors such as manufacture, stability, commercialisation etc. Also, if the authors present this work as a POC application, please explain how could that be, describing the current situation of portable hardware equipment for portable electrochemistry, its pros and cons; if not, how can the reader understand their on-site applications.
3.- The authors only introduce which counter and reference electrodes are being used in line 231 in the main body of the manuscript. Could the authors please describe within the experimental section which exact electrodes are being used? (they mention Ag/AgCl but there are many different types of these for example, which might not be the exact same one as the ones used within the literature manuscripts they have based their method on, which might cause a drift in voltages).
4.- Figure 2 includes SEMs of the bare and modified electrodes. In particular Figure 2a, d and g include some text to show the scale. However all these micrographs included in Figure 2 show extremely low quality legends and the scale and dimensions of the objects included herein are totally illegible. Could the authors please modify these figures to improve the manuscript’s readability?
5.- Several typos are within the manuscript, please improve the overall writing and English of the manuscript. (line 267 and 278 “figure.”, line 278 and 283 “redoks”.
6.- The authors mention from line 278 that when PGEs are modified, they exhibit increased redox peak currents and decreased peak-to-peak separation. However they totally fail to explain what these mean to the reader. Please do tell and explain the reader why are you doing this, what does it mean and why is it important to do. Also this is not totally true, as the use of 0.1M CuSo4 actually exhibited a smaller current than 0.05 or 0.2M. Could the authors please explain that. Also have these results been performed in triplicate and if so could the authors please include their respective deviations?
7.- Figure 3, 4 and 5 are of extremely low quality and not good for publication. The different CVs are their use of colour is misleading and not consistent. Also the some of the legend texts are in a foreign language (F3). Please change all the axis./
8.-From line 319 the authors describe the 10minute modification as the one with the best analytical sensitivity and linearity. However they do totally fail to describe the other two methods as non-linear, and they also offer higher delta currents. The selection of 10minute as optimal is merely arbitral here and experiment should be repeated.
9.- Figure 6 includes what the author describe as a very precise measurement, however it is clear upon inspection of this figure that most of the points within the calibration plot overlap between themselves, which goes against the good performance of the sensor.
10.- Could the authors present the selectivity of their proposed electrochemical sensor when other similar proteins are present within the sample?
Could the authors explore their proposed biosensor performance in more clinical samples? (serum, saliva, plasma, blood?)
11.- Finally, I would suggest to move most of the text from the conclusion section to the end of the main body and only include the actual real conclusions onto this section.
Perhaps a "future trends" paragraph would bring further insights into the aspects that are hindering the commercialisation and real world application of these kind of electrochemical sensors towards clinical setups.
Author Response
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Author Response File: Author Response.pdf
Round 2
Reviewer 2 Report
The revision was insufficient. The authors didn't follow the reviewer's suggestions.
Author Response
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Reviewer 4 Report
The authors should address this reviewer's requests #1 (electrochemical active area calculations with outer sphere redox probes}) where SEM is not enough and comment 6# where the authors do not even attempt to explain their own manuscript to the reader; if they want to have this reviewer to suggest the editors to accept it.
Authors miss to explain their own paper to those less experienced researchers, that is the only basic thing that one should attempt doing.
Author Response
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