Testing of Diamond Electrodes as Biosensor for Antibody-Based Detection of Immunoglobulin Protein with Electrochemical Impedance Spectroscopy

Round 1

Reviewer 1 Report

This manuscript reports an experimental study of the electrochemical biosensing of immunoglobuline G (IgG) on boron-doped diamond (BDD) electrodes by using electrochemical impedance spectroscopy (EIS). The goal of this study was to perform an evaluation of this methodology for virus detection.

The manuscript is well written (i.e., English is satisfactory and manuscript has logical build-up); however, it suffers from several major deficiencies, primarily the absence of substantial EIS data that could support positive statements on described methodology. A biosensor using EIS and antibody-modified BDD electrodes was not realized here, and reported data and manuscript figures do not provide significant progress to the state of the art in my view.

Hence, I do not recommend publication of the manuscript in C – Journal of Carbon Research.

1. Manuscript title is too generic. Which “proteins”? And why did the authors not include the term “virus/viruses” in the title? Basically, the development of a fast a reliable virus detection was pursued! Moreover, presented data do not form enough base for a well-supported “evaluation” (1^st noun in title) either.

2. The presented work is incomplete. In Results and Discussion paragraphs, the authors admit that no biosensing capability was achieved with as-modified BDD electrodes. The plasma-treated BDD electrodes without fluorescence marker showed virtually similar fluorescence signal as with marker. Furthermore, the recorded EIS data do not show a clear increase in charge transfer resistance (i.e., no impedance rise in Fig. 6, nor increase of the semicircle in the Nyquist plot in Fig. 7).

At this stage, it is unclear whether the electrode functionalization was inadequate/instable or (any of) the other experimental conditions (e.g., frequency range in EIS, ratio of electrolyte volume/electrode surface area, EC cell design) are still to be optimized. Overall, the study does not provide sufficient relevant new data or concepts to warrant its publication.

3. Essential literature on the topic is missing. Several recent studies did report on the successful use of BDD electrodes and EIS for virus detection, but were not cited, e.g.:

- https://doi.org/10.1016/j.bios.2022.114222 (SARS-CoV-2)

- https://doi.org/10.1016/j.diamond.2021.108542 (SARS-CoV-2)

- https://doi.org/10.1016/j.snb.2018.10.005 (influenza virus)

- https://doi.org/10.1073/pnas.1603609113 (influenza virus)

Note that different electrode functionalization approaches were successfully introduced in above-listed studies, i.e. the electroreduction of diazonium salts and H-termination followed by OH-functionalization.

4. It is unclear what is meant by “a principle evaluation” (abstract, line 20) and “to evaluate … in principle” (p.3, line 113).

5. The authors do not explain the rationale behind their choice to use modified Fc-part in the recombinant IgG and 1,4-conjugate addition reaction (see Fig. 1).

6. Relevant info regarding the growth of BDD is missing in Section 2.3.1, such as the choice of filament material, substrate temperature, and system pressure.

7. Figures 2 and 3 (schematic of EC cell and optical images, resp.) provide little novel information as the experimental setups are pretty straightforward in our community on electrochemical biosensing. They could be part of Supplementary Information.

Text improvements:

p. 2, line 46: replace reference error by “[2]”.

p. 2, line 90: remove 2^nd “is”.

p. 3, line 147: write “3-aminopropyl-trimethyloxysilane”

p. 6, line 216: what is meant by “10 … 12”?

p. 10, line 325: write “[6]”.

Author Response

Response to Reviewer 1 Comments

27.11.2022

1. Manuscript title is too generic. Which “proteins”? And why did the authors not include the term “virus/viruses” in the title? Basically, the development of a fast a reliable virus detection was pursued! Moreover, presented data do not form enough base for a well-supported “evaluation” (1^stnoun in title) either.

“proteins” changed to “immunoglobulin protein”.

We avoid “virus” in the title because we have not analysed viruses or virus particles. The detection of viruses is the future target of the work.

“evaluation” replaced by “testing”.

2. The presented work is incomplete. In Results and Discussion paragraphs, the authors admit that no biosensing capability was achieved with as-modified BDD electrodes. The plasma-treated BDD electrodes without fluorescence marker showed virtually similar fluorescence signal as with marker. Furthermore, the recorded EIS data do not show a clear increase in charge transfer resistance (i.e., no impedance rise in Fig. 6, nor increase of the semicircle in the Nyquist plot in Fig. 7).

At this stage, it is unclear whether the electrode functionalization was inadequate/instable or (any of) the other experimental conditions (e.g., frequency range in EIS, ratio of electrolyte volume/electrode surface area, EC cell design) are still to be optimized. Overall, the study does not provide sufficient relevant new data or concepts to warrant its publication.

We are well aware that we present only limited novelty. However, telling about experimental results that did not prove the initial theses are also relevant for the scientific community, in our opinion. It gives relevant information for other groups to develop different approaches and stimulates feedback for us about alternatives.

3. Essential literature on the topic is missing. Several recent studies did report on the successful use of BDD electrodes and EIS for virus detection, but were not cited, e.g.:

- https://doi.org/10.1016/j.bios.2022.114222  (SARS-CoV-2)

- https://doi.org/10.1016/j.diamond.2021.108542  (SARS-CoV-2)

- https://doi.org/10.1016/j.snb.2018.10.005  (influenza virus)

- https://doi.org/10.1073/pnas.1603609113  (influenza virus)

Note that different electrode functionalization approaches were successfully introduced in above-listed studies, i.e. the electroreduction of diazonium salts and H-termination followed by OH-functionalization.

All four papers and two additional ones (https://doi.org/10.3390/ijms23126768, https://doi.org/10.1149/1945-7111/ac5d91) were added to the introduction.

4. It is unclear what is meant by “a principle evaluation” (abstract, line 20) and “to evaluate … in principle” (p.3, line 113).

Replaced by “initial tests/testing”.

5. The authors do not explain the rationale behind their choice to use modified Fc-part in the recombinant IgG and 1,4-conjugate addition reaction (see Fig. 1).

We thank Reviewer 1 for that comment: This strategy was envisaged to give well defined attachment of the antibodies on the electrode surface through the cysteine engineered Fc part of the antibody. Alternative lysine reactive bioconjugation is known to lead to also to attachement via lysine residues in the fab part potentially blinding the antibodies from recognition of their antigens and furthermore, conjugation via cysteine residue after partial reducing of disulfide bridges between the light and heavy chains of the antibody are known to negatively impact the overall stability of antibodies. We added this explanation to our manuscript.

6. Relevant info regarding the growth of BDD is missing in Section 2.3.1, such as the choice of filament material, substrate temperature, and system pressure.

The three parameters were added.

7. Figures 2 and 3 (schematic of EC cell and optical images, resp.) provide little novel information as the experimental setups are pretty straightforward in our community on electrochemical biosensing. They could be part of Supplementary Information.

As this does not influence the content itself, we would like to put this decision to the editor.

Text improvements:

p.2, line 46: replace reference error by “[2]”.

This error does not appear in our submitted manuscript. I kindly ask the editorial office to check.

p.2, line 90: remove 2^nd“is”.

Done.

p.3, line 147: write “3-aminopropyl-trimethyloxysilane”

Done.

p.6, line 216: what is meant by “10 … 12”?

Changed to only one value “12 mL”.

p.10, line 325: write “[6]”.

Done.

Please see separate word file

Author Response File: Author Response.pdf

Reviewer 2 Report

The authors have set out to investigate the possibility of using boron-doped diamond electrodes as biosensors for protein detection in viruses with EIS. The reviewer notes the following comments regarding the manuscript:

This needs to be correctly added. E.g. line 45 p2 - [Error! Reference source not found.]

Abbreviation should be noted at the first presence of the term… e.g. line 84 p2 - ……an Fc-engineered antibody is used, which carries a free cysteine in the fragment crystallisable (Fc) part…….

It would be better to include more references in discussing the related problem

More experiments are needed for the study to understand if the EIS biosensing system is possible for virus detection using BDD electrodes as the authors intend to.

Control studies need to be conducted with other electrodes to be able to compare the obtained results.

Author Response

Response to Reviewer 2 Comments

15.11.2022

This needs to be correctly added. E.g. line 45 p2 - [Error! Reference source not found.]

This error does not appear in our submitted manuscript. We kindly ask the editorial office to check.

Abbreviation should be noted at the first presence of the term… e.g. line 84 p2 - ……an Fc-engineered antibody is used, which carries a free cysteine in the fragment crystallisable (Fc) part…….

“Fc-engineered” was deleted to solve this issue.

It would be better to include more references in discussing the related problem

Six additional references were added in the introduction.

More experiments are needed for the study to understand if the EIS biosensing system is possible for virus detection using BDD electrodes as the authors intend to.

Control studies need to be conducted with other electrodes to be able to compare the obtained results.

That is indeed our intention as stated in the outlook.

Please see separate word file

Author Response File: Author Response.docx

Reviewer 3 Report

Reviewer report on Manuscript Draft ‘Evaluation of diamond electrodes as biosensor for antibody-based detection of proteins with electrochemical impedance spectroscopy’.

In this research authors performed a principle evaluation of Boron-doped diamond (BDD)-based electrochemical impedance spectroscopy (EIS) for biosensing. As an easily available standard biomaterial, human immunoglobulin G (IgG) was used as analyte. Niobium plates were coated via hot-filament activated chemical vapor deposition with polycrystalline diamond, doped with boron for electrical conductivity. An anti-human IgG antibody was immobilised on the BDD electrodes as biosensing component. During EIS measurements, both impedance over frequency curves and Nyquist plot showed no clear sign of a change of the charge transfer resistance.

This manuscript is in the scope of journal, it is rather well written and interestingly addressed. Manuscript contributes to the field of bioanalytical chemistry and bioelectrochemistry. Therefore, the manuscript can be published after some minor improvements:

 Introduction is short not very informative and overviews just very limited number of references, therefore, some recent references on the development of electrochemical impedance spectroscopy (EIS) based immunosensors from MDPI (Electrochemical determination of interaction between SARS-CoV-2 spike protein and specific antibodies. International Journal of Molecular Sciences 2022, 23, 6768.) and some other journals (Towards an electrochemical immunosensor for the detection of antibodies against SARS-CoV-2 spike protein.   Journal of The Electrochemical Society 2022, 169, 037523.) could be overviewed and discussed.

Some corrections in expressions are required, e.g. in line 167 authors are stating that ‘2.3.3. Immobilisation of biomarker antibody on electrodes’, however it is not very correct immobilization of antibodies against biomarkers should be immobilized.

Figures 4 and 5. could be supported by error bars if experiment was performed several times.

Chapter ‘3.2. Electrochemical impedance spectroscopy results’ should be advanced and improved, e.g.: Figure 7 represent EIS data in Nyquist plot, impedance imaginary part over real part, these data should be assessed using equivalent circuit suitable for the assessment of EIS data of EIS-based immunosensors (Electrochemical impedance spectroscopy of polypyrrole based electrochemical immunosensor. Bioelectrochemistry 2010, 79, 11–16.), these EIS data should be assessed using the most suitable equivalent circuit, the most important characteristics of equivalent circuits should be calculated and discussed.

Discussion part is not very sound and should be improved.

Conclusions should be derived and presented.

Author Response

Response to Reviewer 3 Comments

15.11.2022

Introduction is short not very informative and overviews just very limited number of references, therefore, some recent references on the development of electrochemical impedance spectroscopy (EIS) based immunosensors from MDPI (Electrochemical determination of interaction between SARS-CoV-2 spike protein and specific antibodies. International Journal of Molecular Sciences 2022, 23, 6768.) and some other journals (Towards an electrochemical immunosensor for the detection of antibodies against SARS-CoV-2 spike protein. Journal of The Electrochemical Society 2022, 169, 037523.) could be overviewed and discussed.

These two and four additional references (https://doi.org/10.1073/pnas.1603609113, https://doi.org/10.1016/j.snb.2018.10.005, https://doi.org/10.1016/j.diamond.2021.108542, https://doi.org/10.1016/j.bios.2022.114222) were added to the introduction.  

Some corrections in expressions are required, e.g. in line 167 authors are stating that ‘2.3.3. Immobilisation of biomarker antibody on electrodes’, however it is not very correct immobilization of antibodies against biomarkers should be immobilized.

New headline: 2.3.3 Immobilisation of antibody on electrodes

Figures 4 and 5. could be supported by error bars if experiment was performed several times.

We checked this in advance of the first submission, but unfortunately the fluorescence data sources to not allow the creation of error bars in these value representations.

Chapter ‘3.2. Electrochemical impedance spectroscopy results’ should be advanced and improved, e.g.: Figure 7 represent EIS data in Nyquist plot, impedance imaginary part over real part, these data should be assessed using equivalent circuit suitable for the assessment of EIS data of EIS-based immunosensors (Electrochemical impedance spectroscopy of polypyrrole based electrochemical immunosensor. Bioelectrochemistry 2010, 79, 11–16.), these EIS data should be assessed using the most suitable equivalent circuit, the most important characteristics of equivalent circuits should be calculated and discussed.

(Martin)

Discussion part is not very sound and should be improved.

Thank you for the hint. However, we do not understand in which way it could be improved. We discuss possible reasons for the unsuccessful results and give an outlook about future work.

Conclusions should be derived and presented.

The conclusions are included in chapter Discussion, see answer to comment above.

Please see separate word file

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

The authors have improved their manuscript text significantly. In most cases, they considered my suggestions to improve.

Personally, I still judge the presented work as premature because no biosensing capability was achieved. Simply said (and I repeat): it remains unclear whether the electrode functionalization was inadequate or instable, or the experimental conditions are still to be optimized.

Overall, the study thus provides low novelty/usability of data and theses.

Reviewer 2 Report

The authors have tried to answer most of reviewer's comments in improving the manuscript.

Reviewer 3 Report

Corrections were performed, the manuscript has been improved and now it is suitable for publishing.