Enhanced Catalytic Surfaces for Catechol Sensing: Combining Grafted Aryldiazonium Derivative with Cross-Linking Dopamine or Coupling Tyrosinase Immobilizations

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors

from my point of view an interesting study. I have only two comments:

- the application of enzymes like tyrosinase is well established, can you outline the controls for this enzyme in detail, and also comment on your enzyme as fungi have multiple tyrosinase enzymes and thus the commercial source might be a mix and this might effect reproducibility.

- molecular oxygen is not a cofactor of tyrosinase it is a co-substrate; please change

Author Response

Dear Reviewer 1,

We sincerely appreciate the time and effort you have dedicated to reviewing our manuscript, as well as your comments and suggestions.

Regarding your first point about the controls for tyrosinase, we would like to clarify that the enzyme used in our study was purchased from Sigma-Aldrich and is extracted from Agaricus bisporus, as specified by the manufacturer. The company maintains rigorous quality control measures to ensure that its products are highly reproducible. Additionally, the activation process applied to commercial screen-printed electrodes further enhances this reproducibility. As a result, we expect a high degree of reproducibility in our results.

With respect to your second point, we appreciate your correction regarding the role of molecular oxygen in the enzymatic reaction. We have revised the manuscript to clearly indicate that molecular oxygen functions as a co-substrate, not as a cofactor, in the catalytic mechanism of tyrosinase. The revised text now states: “Briefly, in the presence of molecular oxygen as co-substrate, TYR initially hydroxylates monophenolic substrates to produce o-diphenols…”

Once again, we sincerely thank you for your thorough review and constructive feedback. Your expertise has been invaluable in enhancing the clarity and scientific rigor of our manuscript.

Reviewer 2 Report

Comments and Suggestions for Authors

In this paper the authors describe the modifying of different surfaces of screen-printed  electrodes by the electrochemical reduction of 4-nitrobenzenediazonium salts followed by the subsequent modification either by cross-linking with dopamine or subsequent diazotation + coupling with tyrosinase enzyme. Thereafter the application of these electrodes for catechol is presented. The article is very lengthy, and the information is not clearly presented. The authors should restructure the manuscript for better clarity. Additionally, certain aspects remain unclear and need to be addressed before the article can be considered for publication.

The authors mention the fabrication of a SPCE-GRAFT-GLU-DA biosensor (or DA-biosensor) that exhibits catalytic activity toward the ferri/ferrocyanide redox couple. However, the modified electrodes do not meet the criteria for a biosensor. What serves as the recognition layer in this case? Did the authors investigate potential interferences? This electrode cannot be classified as a biosensor, as many substances could produce a similar response. It merely demonstrates a catalytic effect on different redox processes.

It is not clear why the authors have chosen all these SPE substrates: carbon, carbon modified with MWCNTs, and gold. The rationale behind these choices, as well as the results related to each substrate, are not well explained and are difficult to follow. I suggest that the authors present the results for each substrate in separate subchapters and conclude by highlighting the most promising one

It is not clear what for the CA abbreviation is used – see page 4 line 140, page 15 line 482, and page 17 line 535

 

Author Response

Dear Reviewer 2,

We sincerely appreciate the time and effort you have dedicated to reviewing our manuscript, as well as your comments and suggestions you provided. Your feedback has been invaluable in enhancing the clarity, structure, and overall quality of our work.

We have revised the whole manuscript based on your comments on condensing some sections. Despite this, we have found that attempting to merge or summarize sections makes the manuscript more difficult to follow, and valuable information is even lost at some point. Therefore, we have maintained the original structure of the manuscript with some minor modifications that we hope will make it easier for the reader to follow the text.

In response to your inquiry regarding the classification of the SPCE-GRAFT-GLU-DA sensor, we have reviewed the terminology used. We have decided to refer to this surface as the "DA-sensor" rather than a biosensor, given that it does not include a bioreceptor. In contrast, the SPCE-GRAFT-Diazo-TYR, which includes an immobilized enzyme, is now designated as the "TYR-Biosensor."

Additionally, we recognize the importance of performing interference studies and evaluating matrix effects in real samples. As noted in the manuscript, the results presented are preliminary. We acknowledge that further studies are necessary to fully characterize the sensors response, and we have clarified in the text that these additional studies will be addressed in future research.

Regarding the selection of substrates (SPCE, MWCNT-SPCE, AuBT-SPE), the objective our study was to develop an electrografting procedure of organic monolayers that can be applied to different materials. Carbon SPEs were selected due to their high affinity with organic molecules and significantly lower cost compared to other materials used as working electrodes. Specifically, SPCE was selected as the optimal substrate because of its performance and cost-effectiveness, while MWCNT-modified SPCEs, despite their excellent catalytic properties, demonstrated less satisfactory performance in this study. Gold SPEs were incorporated for SERS characterization, which confirmed the covalent bonding of the aryl radicals. We have organized the results of electrografting for each substrate into separate subchapters and included a concluding discussion to highlight the most promising material.

Furthermore, we have corrected the typographical errors of the abbreviations. The abbreviation “CC” is now used on pages 4 and 15 to denote catechol, and the meaning of “CA” as chronoamperometry has been clearly specified in the table footnotes, thereby eliminating any ambiguity.

It is important to mention that the work presented in this manuscript is based on a patent. Our approach was designed to demonstrate the versatility of the electrografting process for a wide range of materials, and to highlight the significance of the reactivity of arenediazonium ions for the covalent immobilization of biomolecules and bioreceptors. This methodology facilitates the development of more robust catalytic surfaces, with potential for industrial applications, particularly due to the low cost of SPCE electrodes.

Once again, we would like to express our sincere gratitude for your thorough review and constructive feedback. Your expertise has played a pivotal role in enhancing both the clarity and scientific rigor of our manuscript.

Reviewer 3 Report

Comments and Suggestions for Authors

Title: Getting enhanced catalytic surfaces for catechol sensing! Combination of grafted aryldiazonium derivative and either cross-linking dopamine or coupling tyrosinase immobilizations

General Comments:

The manuscript presents a study on the development of catalytic surfaces for catechol sensing using grafted aryldiazonium derivatives with dopamine or tyrosinase immobilizations. The topic is relevant and fits well within the scope of Applied Sciences. The novelty lies in the comparison of two immobilization strategies and their efficiency in voltammetric catechol quantification. However, several issues related to English grammar, typographical errors, clarity, and scientific presentation need attention before the manuscript can be considered for publication.

Specific Comments:

1. Title: The exclamation mark in the title seems informal. Suggestion: "Enhanced Catalytic Surfaces for Catechol Sensing: Combination of Grafted Aryldiazonium Derivatives with Cross-linking Dopamine or Coupling Tyrosinase Immobilizations."

1. Line 9: "The research work presented here" is redundant. Suggestion: "This study describes the development of catalytic surfaces..."
2. Line 12: "resulting in the formation of a covalently bonded aromatic layer on the electrode surface" — this phrase is repetitive in the text. Condense for clarity.
3. Line 20: "...signal on this catalytic DA-biosensor about 14 times higher..." — rephrase. Suggestion: "...a signal approximately 14 times higher..."
4. Line 77: "based on a granted Patent" — should be "based on a granted patent."
5. Line 67: "that plays an important role in the nervous system (neurotransmitter)" — rephrase. Suggestion: "which functions as a neurotransmitter in the nervous system."
6. Figures 2 and 3: Ensure that figure legends are self-explanatory. Consider adding a brief note on experimental conditions.
7. Line 213: The sentence "which facilitates the reduction of the nitrophenyl group" is redundant. Suggestion: Remove or rephrase to avoid redundancy.
8. Table 1: Consider adding a brief discussion highlighting the advantages of the proposed method compared to others in the table.
9. The comparison between dopamine and tyrosinase immobilization is a strong point. However, further discussion on the practical implications of these findings (e.g., cost, scalability) would strengthen the paper.
10. Line 576: The statement about the enzyme biosensor being 10 times more sensitive could benefit from a brief discussion on why this difference is significant in real-world applications.
11. Section 2.4.1 (Electrografting): Include specifics on electrode surface preparation to improve reproducibility.
12. Line 155: "...for 5 cycles at a scan rate of 100 mVs-1" — mention why 5 cycles were chosen.
13. The conclusion is comprehensive but reiterates points excessively. Condense to highlight key findings and the potential for future research.

Overall recommendations:

1. Revise for grammar, punctuation, and typographical consistency throughout the manuscript.
2. Improve figure legends for better standalone clarity.
3. Condense repetitive sections to improve readability.
4. Emphasize the scientific novelty and practical implications of the findings.

Comments for author File: Comments.pdf

Comments on the Quality of English Language

The manuscript requires moderate revisions to improve the quality of English language. Specific examples, such as redundant phrases in the abstract (Line 9) and awkward sentence structures (Line 67), have been highlighted in the review. Additionally, punctuation and capitalization errors (Line 77) need correction for consistency.

Author Response

Dear Reviewer 3,

We would like to express our sincere gratitude for the time you dedicated in reviewing our manuscript and the constructive suggestions you provided. Your feedback has been essential in improving both the clarity and scientific rigor of our work.

In response to your comments, we have made the following revisions throughout the manuscript. The title has been revised to remove the exclamation mark, now reading: “Getting enhanced catalytic surfaces for catechol sensing: Combination of grafted aryldiazonium derivative and either cross-linking dopamine or coupling tyrosinase immobilizations”. Redundant phrases have been eliminated, and specific sentences have been rephrased for conciseness.

Regarding the figures, the legends for Figures 2 and 3 have been updated to include experimental conditions such as pH, concentrations, and scan rate. This addition ensures that the figures are self-explanatory, while other instrumental conditions like the initial and end potentials are clearly visible in the figures. For Table 1, we have added a brief discussion that not only highlights the analytical performance of our proposed method compared to others but also addresses practical aspects such as fabrication cost.

We would like to emphasize that the selection of five cycles during the electrografting step is supported by the literature; specifically, based on the findings reported in the article “Simple diazonium chemistry to develop specific gene sensing platforms. Revenga-Parra, M. et al. Analytica Chimica Acta 813 (2014) 41–47; http://dx.doi.org/10.1016/j.aca.2014.01.026”. Moreover, the conclusion has been revised to focus on the key results and potential for future research without unnecessary repetition.

Once again, we extend our sincere gratitude for your detailed review and the constructive recommendations. Your expertise has significantly contributed to refining our manuscript, and we are confident that these revisions have significantly enhanced the overall quality and clarity of our work.

Reviewer 4 Report

Comments and Suggestions for Authors

This paper studies an electrochemical sensing platform for catechol detection. The shielded carbon electrode (SPCE) was electrochemically grafted by electrochemical reduction of 4-nitrobenzene diazonium salt (4-NBD+), and further surface modified by dopamine (DA) or tyrosinase (TYR) to improve the catalytic performance. The paper proposes two strategies:
1. DA-sensor (SPCE-GRAFT-GLU-DA): dopamine is cross-linked with glutaraldehyde to stably fix it on the electrode surface, thereby providing higher catalytic activity and achieving sensitive detection of catechol.
2. TYR-sensor (SPCE-GRAFT-Diazo-TYR): An in situ diazotization reaction generates surface-modified diazo groups, which are coupled with the phenolic group of tyrosinase to improve the catalytic efficiency.
These two sensors show excellent sensitivity and stability in the electrochemical detection of catechol, and the tyrosinase-modified sensor is more sensitive than the dopamine-modified sensor.
Compared with existing enzyme sensors and nanomaterial-modified electrodes, this method is shown to be competitive in terms of cost, manufacturing simplicity, and stability.

However, this study still has some minor limitations:

1. Lack of actual sample verification and further exploration of its applicability in complex matrices is needed.

2. Failure to adopt a more sensitive electrochemical detection method may limit its application in trace detection.

Overall, this paper provides valuable contributions to the application of electrochemical grafting technology in the field of biosensors and points out the direction of improvement for subsequent research.

Author Response

Dear Reviewer 4,

We sincerely appreciate the time and effort you have dedicated to reviewing our manuscript. Your comments and constructive feedback have been invaluable in improving the quality of our work. We are pleased that you recognize the potential of our electrochemical (bio)sensors and acknowledge the advantages they offer in terms of cost, manufacturing simplicity, and stability.

We fully agree with your observation regarding the need for real sample verification and further exploration of the sensors’ applicability in complex matrices. Indeed, investigating potential interferences and matrix effects would be a crucial step toward assessing the practical implementation of our (bio)sensors. While these aspects were not the primary focus of our current study, they will undoubtedly be considered in future research to further validate the performance of our sensing platforms.

Regarding your recommendation to employ more sensitive electrochemical techniques, such as differential pulse voltammetry (DPV), for trace detection, we acknowledge that this approach could enhance detection limits and broaden the applicability of our sensors. However, as our study is based on a patented methodology, our primary objective was to develop and characterize the sensor surfaces rather than optimize detection techniques.

Once again, we appreciate your feedback, which has helped us enhance the quality of our manuscript.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The suggested changes have been made