Nanosensors for Exhaled Breath Condensate: Explored Models, Analytes, and Prospects

Response to Reviewers’ Comments

Summary

I would like to thank the reviewers for taking the time to provide their comments and recommendations to improve the quality of this manuscript. Please find the detailed responses (point-by-point; in blue) below and the corresponding revisions/corrections in track changes in the re-submitted files.

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Comments 1: The manuscript entitled "Nanosensors for Exhaled Breath Condensate: Explored Models, Analytes,and Prospects" discusses about application of nanoparticles in fibrication of sensors for detection of biomarkers in exhaled breath condensate. This review can be considered for publication after major revision and addressing the following comments point by point:

1-Please reconstruct the sentences such as 

.....as a biological sample for biobanking and a diagnostic gateway for lung diseases, but also for lung microbiota dysbiosis....

Thank you. I hope the statement reads better now as revised in lines 8,9 as per your recommendation.

.....include gases (H2O2 and VOCs), cations (polyamines), fatty acids [1], carcinogenic antigens, cytokines, and aldehydes with almost half of the studies tracing drug and antibiotics....

thank you for highlighting this unclear statement. It was revised as depicted in lines 14,15.

2- Please Remove citation from abstract 

The first citation is removed from the abstract, thanks for pointing out this mistake.

3-Althogh the subject is about nanosensors but it seems nonsignificant all over the text. It should be highlighted. So it can be highly suggested to add more contents about sensors and biosensors to the text specially introduction 

The introduction focuses on the description of EBC and modes of collection ending with the use of nanomaterial to reveal EBC signature. I  added general information about nanosensors with more focus on nanosensors design and properties throughout the entire document. [Page 2; Lines 63- 82]

4- The classifications of biosensors such as optical, electrochemical, magnetic,... are not mentioned in the text. 

The nanosensors were categorized based on their core elements, such as silver, gold, cupper.. Such properties are tabulated in either section (a) or (b) of corresponding sensor type and read-out method. Yet, I added general description of such classification in the introduction as per your recommendation. p.2; lines 75,76

5- The manuscript is poorly graphically produced. For each section (nanoparticles) add figures from literature with complying copyright.

Definitely, schematic drawings can help visualize the design of nanosensors with their functionalized part and detection mode. Yet, due to the time restriction in resubmitting the revised version of the manuscript, I am afraid securing copyrights and preparing for compiled figure is out of reach.

6-Why was HPLC method mentioned in table 1.2 while title is about nanosensors ?!

HPLC is the read-out method of the collected metabolites by the magnetic NPs. The nanosensor detected and extracted the metabolites from EBC.

7- According to section 2, it seems this review is systematic. Why is flowchart of data collection missed?

The exclusion and inclusion criteria to select the studies are mentioned. A flowchart would help if this review were to run a meta-analysis with data quantification. The review just describes existing selected methods while evaluating the quality of the findings.

8- It is highly suggested to prepare a schematic figure includes subject 

True, an illustration of the subjects or biomarkers can help the reader assimilate the content. If deemed necessary, I can ask the editor for an additional revision period to illustrate this figure. However, I added a graphical abstract in figure 1 as depicted in p.2.

9- The discussion is poorly written. Please add more critical topics

Thank you for pointing this out. Few adjustments are added, with the hope it reads better now.

10- The conclusion is poorly written. Please

complete it.

Thank you for raising your concern. A short paragraph was shifted from the discussion to the conclusion section to present our final comments and future perspectives. Hope it reads better now.

I would like to thank the reviewers for taking the time to provide their comments and recommendations to improve the quality of this manuscript. Please find the detailed responses (point-by-point) below and the corresponding revisions/corrections in track changes in the re-submitted files.

Comments 2: This paper presents a review of the use of nanosensors to analyze EBCs as a non-invasive technique for detecting different diseases. It explains the different types of nanomaterials used in these sensors and the detection mechanisms to identify various biomarkers. In this way, it aims to detect oxidative stress, tissue damage, and respiratory diseases.

As the author herself points out, this kind of review is not easy to carry out because the literature includes a wide variety of studies without a shared methodology, standardized sample collection, homogeneous populations, or consistent types of sensors and materials. For this reason, it is more difficult to draw clear conclusions.

I think it is a good paper with a very detailed and thorough literature review, but I found some areas that could be improved. Here are some suggestions:

1. It would be helpful to include the fabrication techniques used in the studies described, as well as other fabrication details like nanoparticle size, layer thickness, or type of functionalization—if this information is available in the original sources. This would improve the comparison between studies.

True, in the nanosensor part of the tables, major properties, if applicable, are inserted. The nanosensor column is shifted to the second table (b) for better visibility.

1. It would also be useful to clarify whether the samples analyzed in each study are in vivo or if the studies use gas mixtures with the biomarkers being studied (as could be the case with VOCs).

Across all studies, air was collected either using a mouthpiece, Rtube device, or in a bag and various biomarkers were examined. Table section (a) depicts such details.

1. In the studies that focus on VOCs, it would be helpful to specify which volatile compounds were identified as biomarkers, when this information is available. Even though many articles use a "smell fingerprint" to analyze samples, specific VOCs are sometimes identified through other analytical techniques.

I agree, being able to specify typical VOCs from a breath sample is vital for diagnostic/therapeutic uses of the nanosensors; yet, most of the read-out methods did not reply on GC-MS/SPME analysis after EBC collection. I couldn’t find enough sources. Moreover, having tens if not hundreds of various VOCs/ breath would make it even harder to enlist them.

1. Some inconsistencies were found in the use of units and notation throughout the paper, which should be revised. Here are some examples:
• In Table 1.1, in the first row with “Commercial RTube technique”, the column "Outcomes" mentions “20 L of formic acid”, which seems to be an error—it might refer to 20 µL.
• In Table 4.1, the population of newborns or premature babies is associated with vancomycin detection levels of 0.56 g/mL. However, in Table 4.2 and on page 17, the levels are shown in µg/mL.

Thank you for pointing out this important notice. The units and notations were revised as recommended.

1. To improve the comparison between studies, it is recommended to include the same kind of information in Tables 1.1, 2.1, 3.1, 4.1, and 5.1. For example, population data (number of samples, age ranges), diseases studied, and references for each study.

Due to space limit and for a better vision of the data, the age and sample size were removed to focus mainly on the method of EBC extraction and the nanosensor outcomes. The condenser type is shifted to table .b.

1. It's recommended to include references in every table, even if the main researcher’s name is already mentioned (as in Tables 1.2 to 5.2). This would make reading and consulting the paper easier.

Thank you for your suggestion. References are added to all tables.

1. The relationship between the tables ending in ‘.1’ and ‘.2’ is not clearly explained in the text. I assume that the ‘.1’ tables group several studies, which are then detailed in the ‘.2’ tables, but it would be good to clarify this.

A brief explanation is added in p.3.; lines 152-154: “Each table is divided into two sections: (a) Parameters Used in the EBC Methodology and Nanosensor-Based Analysis, and (b) Performance of the Categorized Nanosensor System.”

1. Several cases were found where the reference in the text doesn’t seem to match the citation. I suggest doing a careful review of the references to make sure they are correct. Here are some examples:
• Citation [1] on page 12 (line 168) and in Table 5.2 appears to be wrong. The text refers to “Hasanzadeh et al.”, but the reference doesn’t match.
• Citation [2] on page 3 (line 109) also seems incorrect—it’s about safety in biological sample preservation and transport. Also, citation [2] is repeated in the “Linear range” column in Tables 1.2, 3.2, and 4.2, which might be an error.
• In section 4, citations like [71] in line 60, [75] in line 61, [71] again in line 87, or [63] in line 88 seem to be incorrectly linked with the text.

The citations were all checked and properly matched in the text and table.

1. Please make sure that all acronyms are defined the first time they appear in the text and are also included in the list of abbreviations. For example, I think LR and DEF are not explained. Also, avoid repeating acronyms already defined, like EBC, which appears both in the abstract and again in line 27 (first line of the introduction).

LR abbreviation is enlisted in line 596

DEF abbreviation added in line 590

EBC and other acronyms are checked throughout the document.

Formatting comments:

1. There is a page numbering error starting from page 17, and the line numbers stop and restart several times. This makes it hard to refer to specific parts of the text. It’s recommended to fix this.

I fixed the issue by setting the page and line numbers to ‘continue from the previous section’ and applying continuous line numbering across the entire document. Just one page shows the line numbering to the left (p17, lines 323 to 361). Thank you for spotting this problem.

1. Please revise the formatting of chemical formulas, ions, and scientific notation to make sure subscript and superscript are used correctly (e.g., H₂O₂, Fe₃O₄, Tb³⁺, CO₂, N₂, NO, NH₃).

Thank you for raising this important point; indeed, such corrections are a must.

1. Please review the formatting of the tables (e.g., vertical borders and font style/size). Tables 2.1 and 2.2 don’t match the style of the others. Also, check column alignment, as in Table 5.1 where “Condenser” and “Type” are not correctly placed under their headers.

Thank you for pointing out the formatting inconsistencies in the tables. I have reviewed and updated all tables to ensure a consistent font size, style, and border format throughout.

1. In Table 2.2, bullet points are used even when there's only one item in a cell. It’s better to remove them in those cases for a cleaner look.

Sure, your suggestion is taken into consideration. The appearance of cells looks better without the bullet points. This was applied to all tables.

1. Please correct unit typos, such as writing ‘ml’ instead of ‘mL’, or using ◦C instead of °C throughout the document.

Based on the SI unit, milliliter is abbreviated as mL (Milliliter (mL) Unit Definition | Math Converse). I prefer to keep it as such. Regarding the degree Celsius, the typos are put in place.

4. Response to Comments on the Quality of English Language

The manuscript was checked by an external reader, with English language edits tracked and highlighted in red where applicable.

5. Additional clarifications based to the comments received by email:

1.       Subtables are not recommended; if necessary, subtables could be
numbered by Latin letters with parentheses, e.g., (a), (b), (c), etc.,
or (A), (B), (C), etc., which should be put before the table.

Additionally, use the format of Table 1a,b in the main text.

 As suggested, tables numbering is adjusted.

2.       Please insert all authors in the references part, not only one author.
e.g., Journal article: Fisher, J.A.; Krapf, C.B.E.; Lang, S.C.; Nichols, G.J.;
Payenberg, T.H.D. Sedimentology and architecture of the Douglas Creek
terminal splay, Lake Eyre, central Australia. Sedimentology 2008, 55, 1915–1930.