Specific and Simultaneous Detection of E. coli O157:H7 and Shiga-like Toxins Using a Label-Free Photonic Immunosensor

Round 1

Reviewer 1 Report

The manuscript attempts to describe a label-free photonic inmunosensor to detect E.coli O157:H7 and Shiga-like toxins. However, significant shortcomings justify the rejection of the manuscript. Line numbers are not present, so I will provide general comments.

1) The abstract should briefly report the detailed methods and main quantitative/qualitative results (at least the LOD/LOQ achieved).

2) The introduction provides a good background. However, food safety legal requirements are not reported. Also, a brief introduction to the work carried out and methods is needed in the final paragraph. The scientific soundness of the text must be improved (e.g., "molecular proliferation"?). As it appears, the sensor will serve more for food safety than for disease diagnosis.

3) The Materials and Methods sections must be rearranged as it is very difficult to understand what was used in the experiments and what and how the experiments were performed. Please start describing/listing with reagents followed by equipment used. A sample and assay description should follow this. include some schematic to facilitate the readers. Again, use appropriate scientific writing style.

4) The results section must be improved. Tables 1 and 2 can be reported as graphs or included as they are in the supplementary information attachment. Improve the scientific writing style in reporting the results and compare those with analogous studies and legal requirements (if any). Ensure this section presents the results following the methods described. 

Author Response

Please, see attachment

Author Response File: Author Response.pdf

Reviewer 2 Report

In the manuscript entitled as " Specific and simultaneous detection of E.coli O157:H7 and Shiga-like toxins using a label-free photonic inmunosensor", the innovative technique for the simultaneous detection of E. coli O157:H7 and its Shiga-like toxins in food samples by utilizing a photonic label-free biosensor coupled with a microfluidic system. A comprehensive validation was carried out using this detection approach against several replicates of minced beef samples inoculated with varying concentrations of the target bacteria and their respective verotoxins. This practical method of structural design showcasing the sensitive and rapid detection of E. coli O157:H7 in the field of novel quality control and food hygiene methodology. Therefore, I would like to support the publication of this manuscript with a few comments listed below.

1. The layout of the pictures in this article is blur and confusing (e.g., different sizes of pictures, sloppy picture cropping), and the descriptions of the pictures are ambiguous, which is not conducive to the readers' understanding of the content of the article.

2. This article does not describe the preparation process of photonic biosensor in detail, including material selection, surface functionalization steps, integration with microfluidic system, etc. It is suggested to add these details.

3. There is no picture for the experimental process in this article, which is suggested to be added.

4. This article does not describe the statistical analysis method in detail, including the software used, the type of statistical test and the significance level.

5. There is no stability and reusability test for the photonic biosensor in this article, do the authors have further testing plan?

In conclusion, it is suggested to major revise the manuscript before the publication.

Author Response

Pleas, see attachment

Author Response File: Author Response.pdf

Reviewer 3 Report

The manuscript by Blanco et al presents an innovative method for the simultaneous detection of E. coli O157:H7 and its Shiga-like toxins in food samples through a photonic label-free biosensor coupled with a microfluidic system. The results demonstrate the biosensor's efficacy in sensitive and rapid detection, demonstrating its potential applications in food safety and hygiene. Considering the novelty, the use of ring resonator transduction for this purpose is innovative and provides a rapid, sensitive alternative to traditional detection methods. The results are mostly sound, methodology seems to be robust. However, I have the following suggestions for the authors to consider as improvements - 

1. Additional testing across a wider range of food matrices is highly encouraged to establish the biosensor's applicability in diverse food safety contexts.

2. Assessment of the biosensor's stability over time and potential for reusability is also an important missing aspect, which limits its practicality and cost-effectiveness for routine use in food safety testing.

3. A more detailed comparison with existing detection methods, including PCR and ELISA, in terms of cost, time efficiency, and ease of use, would provide a clearer understanding of the biosensor's advantages and limitations.

4. Development of automated systems for sample preparation and analysis using the photonic biosensor could enhance its efficiency and user-friendliness.

5 (Optional) Implementing field tests in real-world food production and processing environments could validate the biosensor's performance under practical conditions and facilitate its adoption in the industry.

Overall, I suggest the above revision before the manuscript can be re-considered for publication.

Author Response

Please, see the atttachment

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Dear Authors,

Thank you for addressing my points. Unfortunately, the manuscript still lacks scientific soundness and can not be accepted until improvements are made.

Please refer to the following additional notes:

1) The introduction should provide a background and aim of the study, whereas the results section should present the results and then compare them with previous results. As such, I recommend you include the 3.2 paragraph in the introduction, avoiding making claims about how good/excellent the developed sensor is (especially if you have not yet reported the results or provided evidence), as this is not scientifically acceptable.

Also, please try to avoid repetitions throughout the manuscript.

2) Methods should be discussed in the methods section. Avoid repeating the method description in the result section; briefly mention the methods if needed. Please check how iELISA works, as you state that an indirect ELISA requires only one antibody. 

3) Results section needs more work. Results should be reported scientifically and objectively:

a) The significance of the p-value is discussed before the results are reported. Above all, results supported by statistical analysis (e.g., p values) should be reported scientifically. The same applies to PPV and NPV values. Please refer to the book statistics for guidance.

b) A sensorgram should be included. 

c) avoid judging the results (e.g., "positive finding").

d) "Promising results in this context suggest that the biosensor can detect very low concentrations of E. coli O157:H7 and its verotoxins in meat samples, highlighting its potential for sensitive and reliable detection in real-world applications." -> Again, you need to provide objective evidence for this statement.

f) Provide a comparison table comparing your results with previous work and then comment on this, describing the pros, cons, and limitations of your sensor.

The scientific soundness is missing, and the paper needs further work.

Author Response

Response to Reviewer 1 Round 2

Comments:

 Point 1: Dear Authors,Thank you for addressing my points. Unfortunately, the manuscript still lacks scientific soundness and can not be accepted until improvements are made.Please refer to the following additional notes:1) The introduction should provide a background and aim of the study, whereas the results section should present the results and then compare them with previous results. As such, I recommend you include the 3.2 paragraph in the introduction, avoiding making claims about how good/excellent the developed sensor is (especially if you have not yet reported the results or provided evidence), as this is not scientifically acceptable.Also, please try to avoid repetitions throughout the manuscript.

Response 1: Thanks for your contribution. In response to your appreciation, We have included, as you indicated, paragraph 3.2 in the Introduction. At the same time, we have tried to avoid repetitions throughout the new version of the manuscript and avoiding statements about how good the developed sensor is, with the aim of improving scientific writing.

Point 2: Methods should be discussed in the methods section. Avoid repeating the method description in the result section; briefly mention the methods if needed. Please check how iELISA works, as you state that an indirect ELISA requires only one antibody.

Response 2: In response to the request, part of the introduction has been transfer from the section Results 3.1 iELISAs and sensitivity studies to the section Introduction and Methods 2.2. As a result, the results of this method are now strictly described in section 3.1 of the manuscript.

Point 3: Results section needs more work. Results should be reported scientifically and objectively. The significance of the p-value is discussed before the results are reported. Above all, results supported by statistical analysis (e.g., p values) should be reported scientifically. The same applies to PPV and NPV values. Please refer to the book statistics for guidance.

Response3: Thanks again for your appreciation. In response to your request, The significance of the p value is now discussed in the manuscript in the results section after reporting the results. Similarly, in order to include the PPV and NPV values description, the text in section 3.2 has been thoroughly restructured.

Point 4: A sensorgram should be included.

 Response 4: As per the reviewer's request, we have made changes to Figure 3. Figure 3  already includes a  sensorgram, which previously represented a schematic sensor detection sensogram. Additionally, we have updated the title of the figure to better reflect its content.

Point 5: Avoid judging the results (e.g., "positive finding").

Response 5: Thanks again for your appreciation. We have modified the value judgments that appeared in the Results and Conclusions section.

Point 6: "Promising results in this context suggest that the biosensor can detect very low concentrations of E. coli O157:H7 and its verotoxins in meat samples, highlighting its potential for sensitive and reliable detection in real-world applications." -> Again, you need to provide objective evidence for this statement.

Response 6: We appreciate the reviewer's input. As per your feedback, we have included additional information regarding the detection and quantification limits achieved by the sensor currently under development. Furthermore, we have conducted a comparison of these limits with those of other biosensors that have already been developed..

Point 7: Provide a comparison table comparing your results with previous work and then comment on this, describing the pros, cons, and limitations of your sensor.

Response 7: Thank you for your valuable contribution. In response to your suggestion, we have incorporated Tables 3 and 4 into the manuscript's results section. This addition enhances the discussion of our findings, emphasizing the advantages of our immunosensor detection methods compared to others and showcasing relevant studies in the field.

Reviewer 2 Report

The revised version is recommended to be accepted. 

Author Response

Thank you for your contribution. We have made modifications to the Introduction and Results sections in accordance with your criteria and those of reviewer 1. Additionally, we have improved the Methods section by adding descriptions and citations. Furthermore, details have been added regarding the text, figures, and an additional table to enhance the research design in the Results section. As a result, the Conclusions section is now supported by the Results.