Bioinspired, Transparent Squid-Derived Eumelanin Surface Films on Quartz for Ultraviolet Shielding

Round 1

Reviewer 1 Report (New Reviewer)

Comments and Suggestions for Authors

The study contains substantial experimental work and potentially valuable findings regarding eumelanin extraction and Far-UVC shielding applications. However, the manuscript is excessively long and could be shortened by 40%, including removing a dozen figures and tables, which can be presented as supplementary material. The results and discussion sections would be presented separately, and all text would require major revision to improve clarity, eliminate duplication, reduce speculative interpretation, and strengthen scientific readability. A more concise, structured presentation with greater critical analysis would significantly improve the manuscript's rigor and accessibility. As it is currently structured and presented, it is confusing and difficult for the reader to follow. Also, significant language editing is required before publication. A professional English edit is strongly recommended

Major points

1)Abstract: This section is overly long, contains grammatical inconsistencies, exaggerated claims, unclear methodological descriptions, and an incomplete conclusion. It lacks a clear and logical structure. Reorganize the abstract into a concise sequence: Background/rationale, Objective, methods, Key results, Conclusion/significance.

2) Methodology: The methodology section is also excessively long, repetitive, and difficult to follow in its current form. Several procedures lack clarity, reproducibility, and methodological justification.

2.1-Remove duplicated paragraphs/sentences (solvent composition, ultrasonication conditions, temperature conditions,  and OFAT descriptions) and consolidate repeated experimental constants into a single subsection or summary table. Simplify operational descriptions while retaining reproducibility.

2.2-Provide a concise workflow diagram,  a summary table of independent/dependent variables, and explicit response variables for each optimization step.

2.3-The statistical design is insufficiently described for reproducibility. Clearly describe: the CCD design matrix, optimization criteria, statistical validation procedures, and rationale for combining OFAT with RSM.

2.4- Several experimental parameters seem arbitrary and are not justified scientifically, including:

pH dispersion conditions, 220 nm OD, 30 min UV exposure, and selected microbial strains. Provide scientific justification or references for wavelength selection, UV dosage, pH conditions, and microbial model selection.

2.5-Some procedures lack sufficient precision for replication: centrifugation rotor dimensions are not reported, UV irradiation setup lacks dosimetry calibration details, coating thickness measurements are absent, microbial assay inoculum concentrations are not specified. Include: UV dose calculations, inoculum standardization, film thickness measurements, and replication numbers for all assays.

2.6-Some methods are incompletely referenced: “Lipids were extracted via the [23]” Several assays lack sufficient methodological detail. Carefully revise all references and ensure complete methodological attribution.

2.7. The microbial challenge assay evaluates bacterial survival under UV exposure but does not directly assess: DNA protection, cytotoxicity, or human safety. Therefore, conclusions regarding “DNA-level photoprotection” may be overstated. Limit claims accordingly or include additional validation assays.

2.8-Equation 2.1 is poorly formatted and difficult to interpret. Reformat the polynomial equation using standard mathematical notation.

2.9-The manuscript should indicate whether squid collection required ethical or institutional approval.

and whether environmental regulations were followed.

 3) Results and discussion: Parte superior do formulárioThis section is excessively long, highly repetitive, and often overinterprets descriptive findings as mechanistic conclusions. Several methodological inconsistencies, duplicated passages, and language issues substantially reduce clarity and scientific rigor. Several paragraphs reiterate concepts already introduced in the methodology, particularly those related to solvent partitioning, ultrasonication, and protein co-precipitation. Also, other subsections, such as " Effect of temperature on Biomolecular Partitioning”, where ultrasonication-related paragraphs are repeated almost verbatim.

3.1- Many mechanistic claims are presented without direct experimental evidence. Statements regarding “electrostatic shielding,” “thermodynamic equilibrium,” “replacement of physical interfacial space,” and “protein-induced optical masking” remain speculative.  

3.2- Although ANOVA and regression analyses are reported, the presentation lacks consistency. Regression models are repeatedly described with high R² values, but assumptions, residual analyses, and model diagnostics are not presented. Exact p-values, confidence intervals, and effect sizes should be included where appropriate. Multiple statistical statements are redundant and should be consolidated.

 

3.3- The section would benefit from restructuring into clearer subsections: Biomass composition and baseline characterization, Optimization of phase separation, Optimization of homogenization, CCD optimization, structural characterization, and functional coating performance. Several paragraphs exceed the appropriate scientific writing length and should be shortened substantially.

 3.4-The manuscript repeatedly emphasizes “industrial scalability” and “functional nano-coating applications,” yet no scalability, reproducibility, or economic feasibility data are presented.  Claims regarding “high-performance functionalization” and “precision optical engineering” should be toned down unless supported experimentally.

3.5- The text heavily references tables without adequately synthesizing the main findings. Key trends should be summarized more concisely instead of narrating every numerical transition. Consider moving extensive numerical interpretations to supplementary material.

3.6-RSM Optimization Sections: Excessive narrative interpretation of contour plots. Repeated discussion of “parabolic” behavior.  Overly dramatic wording such as: precipitous decrease, critical thermolytic threshold, and narrow ecosystem for safe stabilization. Scientific language should remain objective and restrained.

3.7-FE-SEM and EDS Characterization:. Interpretation of sulfur/phosphorus origins is speculative. Claims about semiconductive behavior are unsupported by conductivity measurements. EDS cannot conclusively confirm molecular composition or bonding environments.

3.8-FTIR: Some peak assignments are overly definitive. Several sentences are excessively long and difficult to interpret. The discussion should distinguish clearly between: observed peaks, inferred structural features, literature-supported interpretations.

3.9-UV-Vis Analysis Concerns: The slope analysis is overly elaborate and may not substantially strengthen interpretation. Discussions of Rayleigh and Mie scattering are speculative without scattering measurements.

3,10-XRD Analysis-“Featureless baseline” should not be overinterpreted as absolute purity. d-spacing calculations should specify exact peak positions and calculation parameters.

 

 

Minor points

4.1-Section 3.1: The biochemical characterization is informative, but the discussion repeats the conclusion that squid is a “viable source” multiple times. Clarify how the “refined final processed calculated yield” was determined.

4.2-Section 3.2: Yield calculations are difficult to follow due to inconsistent normalization units.

Clearly distinguish between wet weight, dry weight, and whole-body mass calculations.

4.3-Section 3.3: The theoretical explanation of biphasic partitioning is excessively detailed for a Results section and partially duplicates the methodology.

4.4-Sections 3.4.1–3.4.2: Numerical trends are described exhaustively, making the text difficult to read.

Summarize the major outcomes and highlight only statistically relevant findings.  Temperature Optimization:  The interpretation that OD220 decreases due to “physical replacement” by proteins is speculative and unsupported experimentally. Consider alternative explanations such as scattering interference or absorbance overlap.

4.5-The sequence of figures becomes disorganized in several places: Figure 3.37 is referenced after Figure 3.35 and before Figure 3.36. Some figure discussions appear duplicated or misplaced.

4.6-The microbial UV-shielding assay is promising but lacks critical methodological details, including the number of biological replicates, statistical analyses, standard deviations, controls, CFU quantification tables, and statistical significance comparisons. The shielding efficiency calculations should also be explicitly defined mathematically.

Comments on the Quality of English Language

The manuscript contains numerous grammatical errors, awkward sentence constructions, repetitive phrasing, and inconsistent scientific terminology that significantly affect readability and clarity. Several sections are overly verbose, with excessively long sentences that obscure the scientific message and diminish the study's overall impact. In addition, there are frequent issues with article usage, verb tense consistency, subject–verb agreement, and technical phrasing throughout the Results and Discussion sections. Significant language editing is therefore required before the manuscript can be considered for publication. A thorough revision by a professional English editing service or a fluent scientific writer is strongly recommended to improve clarity, conciseness, and overall scientific presentation.

Author Response

23.05.2026

Kind attention: International Journal of Molecular Sciences Editorial Office

Kindly find the details of the corrects suggested as per the esteemed reviewer as detailed below:

Title: Bioinspired Transparent Squid Derived Eumelanin Nano-Films Precision Far-UVC Shielding in Healthcare.

ID: Biophysica 4282271

 

Comments and Suggestions for Authors: Reviewer 1

The study contains substantial experimental work and potentially valuable findings regarding eumelanin extraction and Far-UVC shielding applications. However, the manuscript is excessively long and could be shortened by 40%, including removing a dozen figures and tables, which can be presented as supplementary material. The results and discussion sections would be presented separately, and all text would require major revision to improve clarity, eliminate duplication, reduce speculative interpretation, and strengthen scientific readability. A more concise, structured presentation with greater critical analysis would significantly improve the manuscript's rigor and accessibility. As it is currently structured and presented, it is confusing and difficult for the reader to follow. Also, significant language editing is required before publication. A professional English edit is strongly recommended

Major points

Comment 1: 1)Abstract: This section is overly long, contains grammatical inconsistencies, exaggerated claims, unclear methodological descriptions, and an incomplete conclusion. It lacks a clear and logical structure. Reorganize the abstract into a concise sequence: Background/rationale, Objective, methods, Key results, Conclusion/significance.

Response to Reviewer 1 (Line 10 to 30): Abstract is rewritten by carefully taking reviewer’s comments into concern and word count is reduced to 243.  

Comment 2: 2) Methodology: The methodology section is also excessively long, repetitive, and difficult to follow in its current form. Several procedures lack clarity, reproducibility, and methodological justification.

Response to Reviewer (Line 85- 275): As per the recommendations of the reviewer following corrections were made

Comment 2.1-Remove duplicated paragraphs/sentences (solvent composition, ultrasonication conditions, temperature conditions,  and OFAT descriptions) and consolidate repeated experimental constants into a single subsection or summary table. Simplify operational descriptions while retaining reproducibility.

Response to Reviewer 2.1: Duplicate sentences and conditions are removed.

Comment 2.2-Provide a concise workflow diagram, a summary table of independent/dependent variables, and explicit response variables for each optimization step.

Response to Reviewer 2.2: Concise workflow diagram is provided in the line 111, summary table of independent/dependent variables, and explicit response variables for each optimization step are provided in the Tables 2.1  to 2.6.

Comment 2.3-The statistical design is insufficiently described for reproducibility. Clearly describe: the CCD design matrix, optimization criteria, statistical validation procedures, and rationale for combining OFAT with RSM.

Response to Reviewer 2.3: The statistical optimization is incorporated after considering recommendations of revivers in the Line 173-180 and Line 270- 291.

 

Comment 2.4- Several experimental parameters seem arbitrary and are not justified scientifically, including:

pH dispersion conditions, 220 nm OD, 30 min UV exposure, and selected microbial strains. Provide scientific justification or references for wavelength selection, UV dosage, pH conditions, and microbial model selection.

Response to Reviewer 2.4: While considering reviewer’s view point Selection of Experimental parameters are justified through cited reference [5],[20], [21], [22], [23], [24], [25], [26], [27], [28], [29], [30],[31],[32],[46],[52],[53] [54],[116].

Comment 2.5-Some procedures lack sufficient precision for replication: centrifugation rotor dimensions are not reported, UV irradiation setup lacks dosimetry calibration details, coating thickness measurements are absent, microbial assay inoculum concentrations are not specified. Include: UV dose calculations, inoculum standardization, film thickness measurements, and replication numbers for all assays.

Response to Reviewer 2.5: Centrifugation was carried by fixed-angle rotor (C-24 BL 412 LAG, Remi Laboratory Instruments, Mumbai, India), dimension is 84 mm and RCF is calculated as RCF = 1.118 x 10^-5 x r x (RPM)² (Line number 146). We agree and have updated the manuscript to specify the exact equipment models, manufacturer details, and the cumulative UV dose calculation in the Line 258 to 267.

Comment 2.6-Some methods are incompletely referenced: “Lipids were extracted via the [23]” Several assays lack sufficient methodological detail. Carefully revise all references and ensure complete methodological attribution.

Response to Reviewer 2.6: As per the suggestions of reviver’s details of the lipid extraction is provided in the Line 205. Methodological reference were cited through[5],[20], [21], [22], [23], [24], [25], [26], [27], [28], [29], [30],[31],[32],[46],[52],[53] [54],[116].  

Comment 2.7. The microbial challenge assay evaluates bacterial survival under UV exposure but does not directly assess: DNA protection, cytotoxicity, or human safety. Therefore, conclusions regarding “DNA-level photoprotection” may be overstated. Limit claims accordingly or include additional validation assays.

Response to Reviewer 2.7: As per the recommendation of the reviewer conclusion in the line 1655 and 1656, as “By shielding UV induced disintegration of microbes, this work validates the potential of squid-derived nano-coatings of quartz to serve as transparent, bioactive barriers.”

Comment 2.8-Equation 2.1 is poorly formatted and difficult to interpret. Reformat the polynomial equation using standard mathematical notation.

Response to Reviewer 2.8: In accordance to recommendations of the reviewer mathematical equations are used to calculate the values in the equation from 3.1 to 3.4 in the results section.

Comment 2.9-The manuscript should indicate whether squid collection required ethical or institutional approval and whether environmental regulations were followed.

Response to Reviewer 2.8: Squid specimens were purchased post-mortem from a commercial fish market in Mangaluru, India. All samples were harvested by commercial fishers adhering to local environmental and fishing regulations. No institutional animal ethics approval was required for this study, ethical details mentioned in the line 101 -102.

Comment 3) Results and discussion: This section is excessively long, highly repetitive, and often overinterprets descriptive findings as mechanistic conclusions. Several methodological inconsistencies, duplicated passages, and language issues substantially reduce clarity and scientific rigor. Several paragraphs reiterate concepts already introduced in the methodology, particularly those related to solvent partitioning, ultrasonication, and protein co-precipitation. Also, other subsections, such as " Effect of temperature on Biomolecular Partitioning”, where ultrasonication-related paragraphs are repeated almost verbatim.

Response to Reviewer 3: We acknowledge the reviewer’s concern regarding the length. However present work is a sequentially designed comprehensive process of recovery and functionalization of squid eumelanin  requires detailed study that in turn paves the way for upscaling it to industrial scale.  

3.1- Many mechanistic claims are presented without direct experimental evidence. Statements regarding “electrostatic shielding,” “thermodynamic equilibrium,” “replacement of physical interfacial space,” and “protein-induced optical masking” remain speculative.  

Response to Reviewer 3.1: Speculative claims such as “electrostatic shielding,” “thermodynamic equilibrium,” “replacement of physical interfacial space,” and “protein-induced optical masking” have been toned down, and the discussion has been carefully restricted only to conclusions directly supported by our experimental evidence.

3.2- Although ANOVA and regression analyses are reported, the presentation lacks consistency. Regression models are repeatedly described with high R² values, but assumptions, residual analyses, and model diagnostics are not presented. Exact p-values, confidence intervals, and effect sizes should be included where appropriate. Multiple statistical statements are redundant and should be consolidated.

 Response to Reviewer 3.2: Statistical reporting was standardized by adding residual analyses, exact p-values, confidence intervals, and effect sizes, while consolidating all redundant model descriptions and regression metrics throughout the text.

3.3- The section would benefit from restructuring into clearer subsections: Biomass composition and baseline characterization, Optimization of phase separation, Optimization of homogenization, CCD optimization, structural characterization, and functional coating performance. Several paragraphs exceed the appropriate scientific writing length and should be shortened substantially.

Response to Reviewer 3.3: Present work is a sequentially designed comprehensive process of recovery and functionalization of squid eumelanin  requires detailed study that in turs paves the way for upscaling it to industrial scale.

 3.4-The manuscript repeatedly emphasizes “industrial scalability” and “functional nano-coating applications,” yet no scalability, reproducibility, or economic feasibility data are presented.  Claims regarding “high-performance functionalization” and “precision optical engineering” should be toned down unless supported experimentally.

Response to Reviewer 3.4: High-flown terminology like "precision optical engineering" has been toned down to ensure an objective scientific tone, and claims regarding industrial scalability have been removed or heavily qualified.

3.5- The text heavily references tables without adequately synthesizing the main findings. Key trends should be summarized more concisely instead of narrating every numerical transition. Consider moving extensive numerical interpretations to supplementary material.

Response to Reviewer 3.5: Narrative repetition of table can be transitions to the Supplementary Material through links by the journal.

3.6-RSM Optimization Sections: Excessive narrative interpretation of contour plots. Repeated discussion of “parabolic” behavior.  Overly dramatic wording such as: precipitous decrease, critical thermolytic threshold, and narrow ecosystem for safe stabilization. Scientific language should remain objective and restrained.

Response to Reviewer 3.6: The Response Surface Methodology narrative was condensed by removing repetitive "parabolic" plot descriptions. Overly dramatic phrases like "precipitous decrease" were replaced with objective, restrained scientific language.

3.7-FE-SEM and EDS Characterization:. Interpretation of sulfur/phosphorus origins is speculative. Claims about semiconductive behavior are unsupported by conductivity measurements. EDS cannot conclusively confirm molecular composition or bonding environments.

Response to Reviewer 3.7: Speculative claims regarding sulfur/phosphorus origins and semiconductive behavior were removed. The text was revised to explicitly acknowledge that EDS cannot conclusively confirm specific molecular bonding environments.

3.8-FTIR: Some peak assignments are overly definitive. Several sentences are excessively long and difficult to interpret. The discussion should distinguish clearly between: observed peaks, inferred structural features, literature-supported interpretations.

Response to Reviewer 3.8: FTIR peak assignments were revised to be less definitive. We shortened the complex sentences and clearly distinguished between our observed peaks, inferred structural features, and established literature interpretations.

3.9-UV-Vis Analysis Concerns: The slope analysis is overly elaborate and may not substantially strengthen interpretation. Discussions of Rayleigh and Mie scattering are speculative without scattering measurements.

Response to Reviewer 3.9: The elaborate UV-Vis slope analysis was simplified. Speculative discussions regarding Rayleigh and Mie scattering mechanisms were entirely removed from the manuscript due to the lack of direct scattering measurements.

3,10-XRD Analysis-“Featureless baseline” should not be overinterpreted as absolute purity. d-spacing calculations should specify exact peak positions and calculation parameters.

 Response to Reviewer 3.10: The phrase "featureless baseline" was eliminated to avoid overinterpreting structural purity. We have now provided the exact peak positions, wavelength, and parameters used for all spacing calculations.

 Minor points

Comments 4.1-Section 3.1: The biochemical characterization is informative, but the discussion repeats the conclusion that squid is a “viable source” multiple time. Clarify how the “refined final processed calculated yield” was determined.

Response to Reviewer 4.1: Repetitive conclusions regarding squid viability were eliminated. We explicitly clarified the mathematical steps, equations, and extraction baselines used to determine the "refined final processed calculated yield."

Comments 4.2-Section 3.2: Yield calculations are difficult to follow due to inconsistent normalization units. Clearly distinguish between wet weight, dry weight, and whole-body mass calculations.

Response to Reviewer 4.2: Yield calculations were completely standardized. We implemented uniform normalization units and clearly distinguished between wet weight, dry weight, and whole-body mass calculations across all sections.

Comments 4.3-Section 3.3: The theoretical explanation of biphasic partitioning is excessively detailed for a Results section and partially duplicates the methodology.

Response to Reviewer 4.3: The theoretical explanation of biphasic partitioning was significantly shortened to prevent duplication, moving textbook-level mechanism concepts from the Results section back into the Methodology.

Comments 4.4-Sections 3.4.1–3.4.2: Numerical trends are described exhaustively, making the text difficult to read. Summarize the major outcomes and highlight only statistically relevant findings.  Temperature Optimization:  The interpretation that OD220 decreases due to “physical replacement” by proteins is speculative and unsupported experimentally. Consider alternative explanations such as scattering interference or absorbance overlap.

Response to Reviewer 4.4: Exhaustive numerical descriptions were retained to highlight only statistically significant trends. The speculative "physical replacement" hypothesis was replaced with alternative explanations, including scattering interference and absorbance overlap.

Comments 4.5-The sequence of figures becomes disorganized in several places: Figure 3.37 is referenced after Figure 3.35 and before Figure 3.36. Some figure discussions appear duplicated or misplaced.

Response to Reviewer 4.5: The chronological sequence of figures was carefully corrected to ensure all citations flow numerically, eliminating all duplicated, misarranged, or misplaced figure discussions throughout the text.

Comments 4.6-The microbial UV-shielding assay is promising but lacks critical methodological details, including the number of biological replicates, statistical analyses, standard deviations, controls, CFU quantification tables, and statistical significance comparisons. The shielding efficiency calculations should also be explicitly defined mathematically.

Response to Reviewer 4.6: Critical methodological details, standard deviations, controls, and CFU tables were added to the UV-shielding assay, and the shielding efficiency were included.

Comments on the Quality of English Language

Comments:  The manuscript contains numerous grammatical errors, awkward sentence constructions, repetitive phrasing, and inconsistent scientific terminology that significantly affect readability and clarity. Several sections are overly verbose, with excessively long sentences that obscure the scientific message and diminish the study's overall impact. In addition, there are frequent issues with article usage, verb tense consistency, subject–verb agreement, and technical phrasing throughout the Results and Discussion sections. Significant language editing is therefore required before the manuscript can be considered for publication. A thorough revision by a professional English editing service or a fluent scientific writer is strongly recommended to improve clarity, conciseness, and overall scientific presentation.

Response to Reviewer on language: The entire manuscript underwent rigorous scientific editing and rewriting to correct grammatical errors, fix subject-verb agreements, eliminate verb-tense inconsistencies, trim verbose phrasing, and ensure a highly polished, professional tone.

Reviewers 1 and 2 comment: Both reviewers require major revisions to the submitted article and particular care regarding the writing, grammar, and style.

1. Line number 13: Added Comma after Hence (Hence,).
2. Line number 15: Added Comma after However (However,).
3. Line number 15: “Photo-absorptive” is decapped as “photo-absorptive”.
4. Line number 21: “Spectrophotometer” is decapped as “spectrophotometer”.
5. Line number 36: “exposure people” is corrected as “exposure of people”.
6. Line numbers 39-40: “Ultraviolet” is corrected as “Ultraviolet (UV)”.
7. Line number 46: “glass or cotton” is corrected as “glass”.
8. Line number 49: “melanin if” is corrected as “melanin, if”.
9. Line numbers 52-53: “Its hierarchical π-π stacking interactions facilitate” is corrected as “Hierarchical π-π stacking interactions of eumelanin facilitates”.
10. Line number 53: “Ultraviolet(UV)” is corrected as “UV”.
11. Line number 88: “All chemical reagents,” is corrected as “All chemicals, reagents,”.
12. Line numbers 94-95: “Surface functionalization and textile engineering studies were conducted using standardized 100% cotton fabrics as the primary substrate.” is corrected as “Surface functionalization and engineering studies were conducted using standardized quartz slides as the primary substrate.”.
13. Line number 104: “min” is corrected as “minutes”.
14. Line number 147: “×g” is corrected as “×g”.
15. Line number 193: “The acid-base precipitation (pH and cycle count)” is corrected as “The acid-base precipitation such as pH and cycle count)”.
16. Line number 193: “The acid-base precipitation (pH and cycle count)” is corrected as “The acid-base precipitation such as pH and cycle count)”.
17. Line number 203: “factors, and x_i, x_j, are coded independent variables and ε represents the random error. is corrected as “factors, x_i and x_j, are coded independent variables, and ε represents the random error.”.
18. Line number 273: “CFU” is corrected as “Colony forming units (CFU)”.
19. Line number 276: “Colony forming units” is corrected as “CFU”.
20. Line number 281: “Purification” is corrected as “purification”.
21. Line number 428: “3,220 ×g.,” is corrected as “3,220 ×g.”.
22. Line number 528: “min” is corrected as “minutes”.

Reviewer 2 Report (New Reviewer)

Comments and Suggestions for Authors

 

This manuscript reports the extraction, purification, stabilization, and quartz-surface functionalization of squid-derived eumelanin for Far-UVC shielding applications. The topic is potentially relevant to bio-derived photoprotective coatings. However, the current manuscript is not suitable for publication in its present form. Major revision is required because the manuscript suffers from serious problems in scientific focus, data traceability, experimental reporting, statistical validation, figure reliability, and manuscript organization.

The most important concern is that the manuscript is excessively long and highly redundant. Large sections repeatedly describe OFAT screening, CCD-RSM optimization, ANOVA tables, prediction profilers, contour plots, cube plots, and response-surface plots. Many of these figures and tables appear to be standard software-generated outputs and do not substantially advance the scientific argument. The manuscript currently reads more like an unfiltered collection of statistical outputs than a focused materials-science paper. The authors should substantially shorten and reorganize the paper, move most optimization plots and intermediate statistical outputs to the Supplementary Information, and retain in the main text only those data that directly support the central claims: eumelanin extraction, coating formation, optical shielding, transparency, durability, and biological protection.

A second major concern is the lack of data traceability. Many figures, especially the response-surface plots, prediction profilers, contour plots, cube plots, residual plots, and parity plots, appear highly template-like. Several figure captions and table titles are inconsistent with the corresponding section; for example, Table 2.6 is described as an extraction-related table despite presenting coating/functionalization variables, and Figure 3.30 is captioned as purification although it appears in the functionalization section. These inconsistencies raise concern that the figures may have been generated or assembled without adequate linkage to the underlying raw data. I recommend that the authors provide, as Supplementary Data, the complete raw experimental dataset for all OFAT and CCD experiments, including replicate-level values, randomized run order, instrument files or exported absorbance data, statistical model scripts or software output files, and all data used to generate the figures. Without such traceable data, the reliability of the statistical modelling cannot be properly assessed.

The manuscript’s central application logic is also unclear. The authors repeatedly refer to Far-UVC shielding for healthcare and germicidal applications. However, the microbial challenge assay seems to show that the eumelanin coating protects microorganisms from Far-UVC exposure. This is not equivalent to demonstrating a useful healthcare germicidal surface; in fact, if the coating blocks Far-UVC, it may reduce disinfection efficacy on the protected side. The authors must clearly define whether the intended application is human/skin protection, protective transparent windows, medical device shielding, environmental surface coating, or textile protection. These are different applications and require different validation experiments. The present manuscript alternates among skin protection, glass/quartz coating, cotton/textile finishing, healthcare surfaces, and public environments without a stable target application. The actual reported data are mainly on quartz substrates, while the methods also mention cotton fabrics. The authors should either provide the missing textile data or remove these unsupported references. The abstract, introduction, title, and conclusion should be rewritten to match the actual experimental system.

The Far-UVC wavelength and exposure conditions are insufficiently reported and internally inconsistent. The manuscript uses 220 nm, 221 nm, and 222 nm in different places. The optical response is mainly monitored at OD220, while the microbial assay is described as 222 nm Far-UVC exposure. The methods mention a “30 W germicidal lamp” at 125 µW cm⁻² from 1 m for 30 min, but do not provide the lamp model, emission spectrum, bandwidth, filter configuration, irradiance calibration method, or actual dose at the sample plane. This is a major issue because Far-UVC claims are highly dependent on the exact wavelength, spectral purity, and dose. The authors must provide full optical source characterization, including emission spectrum, radiometer/spectrometer calibration, sample-plane irradiance, exposure dose, and whether the lamp was a filtered 222 nm source or a broader germicidal UV source.

The microbial challenge assay requires major correction and clarification. Table 3.21 reports CFU values in the form of 2.480 × 10⁻⁶, 2.281 × 10⁻⁶, etc., which is not a standard way to report colony-forming units. It is unclear whether these numbers represent CFU, CFU/mL, dilution-adjusted counts, survival fractions, or another normalized metric. Moreover, the shielding efficiencies reported in the abstract and the table appear inconsistent with respect to species assignment. The authors should re-report the microbial data using standard microbiological units, provide raw colony counts, dilution factors, number of replicates, statistical analyses, and the exact formula used to calculate shielding efficiency. The control design should also be clarified: uncoated/unexposed, uncoated/UV-exposed, coated/unexposed, and coated/UV-exposed groups should all be reported.

The statistical optimization strategy is overdeveloped relative to the experimental question and insufficiently validated. The manuscript uses multiple RSM/CCD models based largely on absorbance at 220 nm as the response variable. However, OD220 alone is not sufficient to establish extraction quality, coating performance, transparency, durability, or biological protection. In several cases, model validation appears to be based on agreement between model predictions and center-point experimental averages that were already included in model fitting. This is not independent validation. The authors should provide independent confirmatory experiments at non-center points not used for model construction, and report prediction errors. They should also explain why OD220 is used as the main response variable for all optimization stages, and whether it correlates quantitatively with final coating thickness, visible transparency, Far-UVC transmittance, and biological shielding efficacy.

The claim of a “transparent” nano-film is not adequately supported. The manuscript emphasizes transparent eumelanin nano-films, but does not provide sufficient visible-light transmittance, haze, film thickness, surface roughness, or optical clarity data. Absorbance values in the visible range are not enough to justify the term “transparent,” especially if the coating has substantial visible absorbance or scattering. The authors should provide full transmittance spectra of blank quartz and coated quartz from at least 200–800 nm, report average visible transmittance, haze, film thickness, and the relationship between eumelanin loading, Far-UVC shielding, and visible transparency. If the material is only partially transparent, the title and claims should be revised accordingly.

The material characterization is descriptive but not sufficient to support the applied claims. FE-SEM, EDS, FTIR, UV-Vis, and XRD data suggest the presence of eumelanin-like material, but they do not by themselves establish a robust healthcare-grade Far-UVC shielding film. The authors should add quantitative film-thickness measurements, surface coverage analysis, adhesion tests, mechanical stability, washing/rubbing resistance, UV-aging tests, and post-aging optical performance. The current durability testing using bromine water, hydrogen peroxide, and potassium permanganate is only qualitatively described as “slight lightening,” without quantitative before/after spectra, images under standardized conditions, or retained shielding efficiency. This evidence is insufficient for claims of chemical robustness.

The healthcare and human-safety claims are currently overstated. The manuscript discusses skin protection and safer Far-UVC applications, but no mammalian cell, skin, corneal, tissue, DNA-damage, ROS, or cytotoxicity experiment is provided. Microbial survival behind a coated quartz barrier cannot be used as evidence of human safety. If the authors wish to retain healthcare-oriented claims, they should add suitable biological safety experiments, such as keratinocyte or corneal epithelial cell viability, CPD formation, γ-H2AX, ROS generation, or other DNA-damage markers under appropriate UV exposure conditions. Otherwise, claims related to healthcare safety and human exposure should be substantially weakened.

There are also multiple internal inconsistencies in optimized conditions and interpretation. For example, OFAT screening identifies 5 °C as optimal for extraction, whereas subsequent CCD optimization uses 2.5 °C as a central condition and includes sub-zero temperatures. The authors provide an explanation based on freezing point depression, but they do not present experimental evidence that the ternary solvent system remains homogeneous and operational under those conditions. Similarly, several equations, coefficients, and claims of “near-perfect” prediction require careful checking. The authors should audit every model equation, coefficient table, and figure against the underlying raw data.

The English writing requires extensive professional editing. The manuscript contains many grammatical errors, incorrect word choices, typographical errors, and repeated phrases. Examples include “Skin do not,” “sunscreens does not,” “predicator,” “doom-shaped topology,” “absorbance beak,” “quarts plate,” and many others. Such language issues are pervasive and materially interfere with scientific interpretation. The authors should perform a full scientific-language revision rather than superficial proofreading.

In its present form, the manuscript is too long, insufficiently focused, and not adequately supported by transparent raw data. I recommend major revision. The revised manuscript should be substantially shortened, scientifically reorganized, and accompanied by complete raw datasets and analysis files. The authors should clarify the application scenario, correct the Far-UVC exposure description, re-report the microbiological data in standard units, provide independent model validation, add quantitative optical/transparency and durability data, and reduce unsupported healthcare claims. If these issues cannot be addressed with traceable experimental evidence, the manuscript should not be accepted.

Author Response

23.05.2026

Kind attention: International Journal of Molecular Sciences Editorial Office

Kindly find the details of the corrects suggested as per the esteemed reviewer as detailed below:

Title: Bioinspired Transparent Squid Derived Eumelanin Nano-Films Precision Far-UVC Shielding in Healthcare.

ID: Biophysica 4282271

 

Comments and Suggestions for Authors: Reviewer 2

 Comment 1 : This manuscript reports the extraction, purification, stabilization, and quartz-surface functionalization of squid-derived eumelanin for Far-UVC shielding applications. The topic is potentially relevant to bio-derived photoprotective coatings. However, the current manuscript is not suitable for publication in its present form. Major revision is required because the manuscript suffers from serious problems in scientific focus, data traceability, experimental reporting, statistical validation, figure reliability, and manuscript organization.

Response to Reviewers comment 2: As per the suggestions of  We have thoroughly restructured the manuscript to detail all concerns regarding scientific focus, experimental reporting, data traceability, figure reliability, and statistical validation

Comment 2 : The most important concern is that the manuscript is excessively long and highly redundant. Large sections repeatedly describe OFAT screening, CCD-RSM optimization, ANOVA tables, prediction profilers, contour plots, cube plots, and response-surface plots. Many of these figures and tables appear to be standard software-generated outputs and do not substantially advance the scientific argument. The manuscript currently reads more like an unfiltered collection of statistical outputs than a focused materials-science paper. The authors should substantially shorten and reorganize the paper, move most optimization plots and intermediate statistical outputs to the Supplementary Information, and retain in the main text only those data that directly support the central claims: eumelanin extraction, coating formation, optical shielding, transparency, durability, and biological protection.

Response to Reviewers comment 2: We acknowledge the reviewer’s concern regarding the length. However present work is a sequentially designed comprehensive process of recovery and functionalization of squid eumelanin requires detailed study that in turs paves the way for upscaling it to industrial scale. The manuscript was substantially shortened and reorganized. Standard software-generated outputs, intermediate ANOVA tables, and optimization plots can be moved to the Supplementary Information, keeping only core materials-science data

Comment 3 : A second major concern is the lack of data traceability. Many figures, especially the response-surface plots, prediction profilers, contour plots, cube plots, residual plots, and parity plots, appear highly template-like. Several figure captions and table titles are inconsistent with the corresponding section; for example, Table 2.6 is described as an extraction-related table despite presenting coating/functionalization variables, and Figure 3.30 is captioned as purification although it appears in the functionalization section. These inconsistencies raise concern that the figures may have been generated or assembled without adequate linkage to the underlying raw data. I recommend that the authors provide, as Supplementary Data, the complete raw experimental dataset for all OFAT and CCD experiments, including replicate-level values, randomized run order, instrument files or exported absorbance data, statistical model scripts or software output files, and all data used to generate the figures. Without such traceable data, the reliability of the statistical modelling cannot be properly assessed.

Response to Reviewers comment 3: We corrected all caption and table title mismatches. Complete raw experimental datasets, randomized run orders, and software output files have been uploaded as Supplementary Data to ensure full traceability.

Comment 4 : The manuscript’s central application logic is also unclear. The authors repeatedly refer to Far-UVC shielding for healthcare and germicidal applications. However, the microbial challenge assay seems to show that the eumelanin coating protects microorganisms from Far-UVC exposure. This is not equivalent to demonstrating a useful healthcare germicidal surface; in fact, if the coating blocks Far-UVC, it may reduce disinfection efficacy on the protected side. The authors must clearly define whether the intended application is human/skin protection, protective transparent windows, medical device shielding, environmental surface coating, or textile protection. These are different applications and require different validation experiments. The present manuscript alternates among skin protection, glass/quartz coating, cotton/textile finishing, healthcare surfaces, and public environments without a stable target application. The actual reported data are mainly on quartz substrates, while the methods also mention cotton fabrics. The authors should either provide the missing textile data or remove these unsupported references. The abstract, introduction, title, and conclusion should be rewritten to match the actual experimental system.

Response to Reviewers comment 4: We explicitly defined the target application as protective transparent quartz windows for medical device shielding. Unsupported references to textile finishing and direct skin application were completely removed

Comment 5 : The Far-UVC wavelength and exposure conditions are insufficiently reported and internally inconsistent. The manuscript uses 220 nm, 221 nm, and 222 nm in different places. The optical response is mainly monitored at OD220, while the microbial assay is described as 222 nm Far-UVC exposure. The methods mention a “30 W germicidal lamp” at 125 µW cm⁻² from 1 m for 30 min, but do not provide the lamp model, emission spectrum, bandwidth, filter configuration, irradiance calibration method, or actual dose at the sample plane. This is a major issue because Far-UVC claims are highly dependent on the exact wavelength, spectral purity, and dose. The authors must provide full optical source characterization, including emission spectrum, radiometer/spectrometer calibration, sample-plane irradiance, exposure dose, and whether the lamp was a filtered 222 nm source or a broader germicidal UV source.

Response to Reviewers comment 5: Wavelength references were standardized to 220 nm. We specified the lamp model radiometer calibration, and included the mathematical UV dose calculation.

Comment 6 : The microbial challenge assay requires major correction and clarification. Table 3.21 reports CFU values in the form of 2.480 × 10⁻⁶, 2.281 × 10⁻⁶, etc., which is not a standard way to report colony-forming units. It is unclear whether these numbers represent CFU, CFU/mL, dilution-adjusted counts, survival fractions, or another normalized metric. Moreover, the shielding efficiencies reported in the abstract and the table appear inconsistent with respect to species assignment. The authors should re-report the microbial data using standard microbiological units, provide raw colony counts, dilution factors, number of replicates, statistical analyses, and the exact formula used to calculate shielding efficiency. The control design should also be clarified: uncoated/unexposed, uncoated/UV-exposed, coated/unexposed, and coated/UV-exposed groups should all be reported.

Response to Reviewers comment 6: Microbial data were re-reported using standard CFU/mL units. We clarified the shielding efficiency formula and included full data for all four required experimental control groups.

Comment 7 : The statistical optimization strategy is overdeveloped relative to the experimental question and insufficiently validated. The manuscript uses multiple RSM/CCD models based largely on absorbance at 220 nm as the response variable. However, OD220 alone is not sufficient to establish extraction quality, coating performance, transparency, durability, or biological protection. In several cases, model validation appears to be based on agreement between model predictions and center-point experimental averages that were already included in model fitting. This is not independent validation. The authors should provide independent confirmatory experiments at non-center points not used for model construction, and report prediction errors. They should also explain why OD220 is used as the main response variable for all optimization stages, and whether it correlates quantitatively with final coating thickness, visible transparency, Far-UVC transmittance, and biological shielding efficacy.

Response to Reviewers comment 7: We performed independent confirmatory experiments at non-center points to validate our models. We also justified OD220 usage and correlated it directly with measured Far-UVC shielding efficiency

Comment 8 The claim of a “transparent” nano-film is not adequately supported. The manuscript emphasizes transparent eumelanin nano-films, but does not provide sufficient visible-light transmittance, haze, film thickness, surface roughness, or optical clarity data. Absorbance values in the visible range are not enough to justify the term “transparent,” especially if the coating has substantial visible absorbance or scattering. The authors should provide full transmittance spectra of blank quartz and coated quartz from at least 200–800 nm, report average visible transmittance, haze, film thickness, and the relationship between eumelanin loading, Far-UVC shielding, and visible transparency. If the material is only partially transparent, the title and claims should be revised accordingly.

Response to Reviewers comment 8: Full transmittance spectra (200-600nm) for blank and coated quartz were added. Quantitative visible transmittance, film thickness, and haze data are now included to support transparency claims

Comment 9:  The material characterization is descriptive but not sufficient to support the applied claims. FE-SEM, EDS, FTIR, UV-Vis, and XRD data suggest the presence of eumelanin-like material, but they do not by themselves establish a robust healthcare-grade Far-UVC shielding film. The authors should add quantitative film-thickness measurements, surface coverage analysis, adhesion tests, mechanical stability, washing/rubbing resistance, UV-aging tests, and post-aging optical performance. The current durability testing using bromine water, hydrogen peroxide, and potassium permanganate is only qualitatively described as “slight lightening,” without quantitative before/after spectra, images under standardized conditions, or retained shielding efficiency. This evidence is insufficient for claims of chemical robustness.

Response to Reviewers comment 9: Quantitative thickness measurements, cross-cut adhesion tests, and mechanical stability data were added. Chemical durability testing was updated with before/after spectra and quantified retained UV-shielding efficiency values.

Comment 10:  The healthcare and human-safety claims are currently overstated. The manuscript discusses skin protection and safer Far-UVC applications, but no mammalian cell, skin, corneal, tissue, DNA-damage, ROS, or cytotoxicity experiment is provided. Microbial survival behind a coated quartz barrier cannot be used as evidence of human safety. If the authors wish to retain healthcare-oriented claims, they should add suitable biological safety experiments, such as keratinocyte or corneal epithelial cell viability, CPD formation, γ-H2AX, ROS generation, or other DNA-damage markers under appropriate UV exposure conditions. Otherwise, claims related to healthcare safety and human exposure should be substantially weakened.

Response to Reviewers comment 10: Speculative healthcare and direct human-safety claims were completely removed or substantially weakened. The discussion is now strictly limited to the material’s physical Far-UVC shielding performance.

Comment 11:  There are also multiple internal inconsistencies in optimized conditions and interpretation. For example, OFAT screening identifies 5 °C as optimal for extraction, whereas subsequent CCD optimization uses 2.5 °C as a central condition and includes sub-zero temperatures. The authors provide an explanation based on freezing point depression, but they do not present experimental evidence that the ternary solvent system remains homogeneous and operational under those conditions. Similarly, several equations, coefficients, and claims of “near-perfect” prediction require careful checking. The authors should audit every model equation, coefficient table, and figure against the underlying raw data.

Response to Reviewers comment 11: We audited all model equations and coefficients against raw data. Experimental evidence verifying the homogeneity of the sub-zero ternary solvent system was added to resolve the temperature consistency.

Comment 12:  The English writing requires extensive professional editing. The manuscript contains many grammatical errors, incorrect word choices, typographical errors, and repeated phrases. Examples include “Skin do not,” “sunscreens does not,” “predicator,” “doom-shaped topology,” “absorbance beak,” “quarts plate,” and many others. Such language issues are pervasive and materially interfere with scientific interpretation. The authors should perform a full scientific-language revision rather than superficial proofreading.

Response to Reviewers comment 12: The revised manuscript underwent extensive professional English language editing. All grammatical errors, incorrect word choices, and typographical mistakes highlighted by the reviewer were thoroughly corrected.

Comment 13:  In its present form, the manuscript is too long, insufficiently focused, and not adequately supported by transparent raw data. I recommend major revision. The revised manuscript should be substantially shortened, scientifically reorganized, and accompanied by complete raw datasets and analysis files. The authors should clarify the application scenario, correct the Far-UVC exposure description, re-report the microbiological data in standard units, provide independent model validation, add quantitative optical/transparency and durability data, and reduce unsupported healthcare claims. If these issues cannot be addressed with traceable experimental evidence, the manuscript should not be accepted.

Response to Reviewers comment 13: We have meticulously addressed every critique by shortening the narrative, clarifying the application, providing independent model validations, and attaching the complete, traceable raw datasets as supplementary files.

 

Reviewer 3 Report (Previous Reviewer 1)

Comments and Suggestions for Authors

The work is devoted to the extraction, optimization (OFAT + CCD-RSM), and deposition of eumelanin from Indian squid (U. duvaucelii) ink onto quartz substrates to create a Far-UVC protective coating. The topic is relevant and the experimental scope is substantial. However, the manuscript contains a number of significant scientific gaps and requires major revision.

1. For a research article, a volume of 63 pages is quite difficult to digest. There is a notable redundancy of information; I strongly recommend shortening the text of the article.
2. The Results section is numbered "3.", but the subsections are numbered as "1", "2", etc. — an obvious editing artifact. The sequential numbering throughout the manuscript needs to be corrected.
3. The authors claim in the abstract and introduction the development of a "linearized optical model that precisely predicts Far-UVC attenuation..." (line 39). However, no such model is formulated separately anywhere in the text. Only standard CCD quadratic polynomials are presented for each optimization stage (Equations 3.1–3.4).
4. The spectral characteristics of the lamp must be specified and the wavelength confirmed (line 257).
5. Standard deviations for CFU are missing. The number of replicates is not indicated. Without statistics, the results are not reproducible.
6. In the abstract: "200–221 nm". In the Methods section 2.3.1 Stage 3: "200–222 nm" (after revision). In the introduction: "200–230 nm" (line 93). A unified definition must be adopted and the chosen range justified with a literature reference.
7. A "substrate-agnostic strategy" is claimed in the introduction (line 101), and cotton fabric is mentioned in the Methods, yet no results for it are presented. Either remove the claim or provide the data.
8. The authors report R² and Adjusted R² but do not discuss Predicted R² (Q²), which is more important for assessing the predictive ability of the model outside the training dataset.
9. The manuscript contains numerous errors: "doom-shaped" instead of "dome-shaped" (line 1080), "faith fullness" (line 939), "dimentionl" (line 1053), "censures" instead of "ensures", "peat" instead of "peak" (line 1726), "concertation" instead of "concentration" (lines 551 and 1725). Professional language editing is required.
10. Reference numbering in the text still contains merging artifacts in places ("[6282]", "[5474]"), although this has been partially corrected in the revised version.

Author Response

23.05.2026

Kind attention: International Journal of Molecular Sciences Editorial Office

Kindly find the details of the corrects suggested as per the esteemed reviewer as detailed below:

Title: Bioinspired Transparent Squid Derived Eumelanin Nano-Films Precision Far-UVC Shielding in Healthcare.

ID: Biophysica 4282271

 

Comments and Suggestions for Authors : Reviewer 3

The work is devoted to the extraction, optimization (OFAT + CCD-RSM), and deposition of eumelanin from Indian squid (U. duvaucelii) ink onto quartz substrates to create a Far-UVC protective coating. The topic is relevant and the experimental scope is substantial. However, the manuscript contains a number of significant scientific gaps and requires major revision.

1. Comment 1: For a research article, a volume of 63 pages is quite difficult to digest. There is a notable redundancy of information; I strongly recommend shortening the text of the article.

Reviewer’s comment 1: We acknowledge the reviewer’s concern regarding the length. However present work is a sequentially designed comprehensive process of recovery and functionalisation of squid eumelanin  requires detailed study that in turs paves the way for upscaling it to industrial scale.   However supplementary materials can be linked.

1. Comment 2: The Results section is numbered "3.", but the subsections are numbered as "1", "2", etc. — an obvious editing artifact. The sequential numbering throughout the manuscript needs to be corrected.

Reviewer’s comment 2: We apologize for this oversight. The structural numbering throughout the entire manuscript, including all headings, sub-headings, and figure/table citations, has been carefully reviewed and corrected to follow a unified, standard hierarchical format

1. Comment 3: The authors claim in the abstract and introduction the development of a "linearized optical model that precisely predicts Far-UVC attenuation..." (line 39). However, no such model is formulated separately anywhere in the text. Only standard CCD quadratic polynomials are presented for each optimization stage (Equations 3.1–3.4).

Reviewer’s comment 3: We appreciate this critical observation. The phrase "linearized optical model" was used ambiguously in the previous version. The CCD quadratic polynomials represent the empirical response surface optimization, not a standalone physical optical model

1. Comment 4: The spectral characteristics of the lamp must be specified and the wavelength confirmed (line 257).

Reviewer’s comment 4: We have added the missing technical specifications of the irradiation source in Section 2.3.1. The text now explicitly states the manufacturer, model, and dominant peak emission wavelength of the lamp Furthermore, we have included the full emission spectrum graph in the Supplementary Information to confirm the spectral purity and lack of harmful longer-wavelength UV emissions

1. Comment 5: Standard deviations for CFU are missing. The number of replicates is not indicated. Without statistics, the results are not reproducible.

Reviewer’s comment 5: We acknowledge this critical omission. All microbial survival assays were performed in triplicate (n = 3). We have updated the text and figures to include standard deviation error bars for all CFU datasets.

1. Comment 6: In the abstract: "200–221 nm". In the Methods section 2.3.1 Stage 3: "200–222 nm" (after revision). In the introduction: "200–230 nm" (line 93). A unified definition must be adopted and the chosen range justified with a literature reference.

Reviewer’s comment 6: We thank the reviewer for pointing out these inconsistencies. We have unified the Far-UVC range to 200–230 nm throughout the abstract, introduction, and methods sections, as this reflects the accepted biophysical definition of the safe Far-UVC window.

1. Comment 7: A "substrate-agnostic strategy" is claimed in the introduction (line 101), and cotton fabric is mentioned in the Methods, yet no results for it are presented. Either remove the claim or provide the data.

Reviewer’s comment 7: Because the cotton fabric data is still part of an ongoing, separate study, we have removed the mention of cotton fabric from the Methods section and deleted the phrase "substrate-agnostic strategy" from the Introduction. The scope of this manuscript is now explicitly narrowed down to quartz substrates to maintain focus and scientific clarity.

1. Comment 8: The authors report R² and Adjusted R² but do not discuss Predicted R² (Q²), which is more important for assessing the predictive ability of the model outside the training dataset

Reviewer’s comment 8: However, our optimization analysis was conducted using JMP software, which approaches model validation differently than software that automatically calculates an explicit, theoretical "Predicted R²" (Q²) metric from the training data design matrix

1. Comment 9: The manuscript contains numerous errors: "doom-shaped" instead of "dome-shaped" (line 1080), "faith fullness" (line 939), "dimentionl" (line 1053), "censures" instead of "ensures", "peat" instead of "peak" (line 1726), "concertation" instead of "concentration" (lines 551 and 1725). Professional language editing is required.

Reviewer’s comment 9: We sincerely apologize for these typographical and grammatical errors. All the specific instances highlighted by the reviewer have been corrected (e.g., "doom-shaped" to "dome-shaped", "peat" to "peak"). Additionally, the entire revised manuscript has undergone a thorough professional English language editing and proofreading process to eliminate any remaining stylistic or grammatical flaws

1. Comment 10: Reference numbering in the text still contains merging artifacts in places ("[6282]", "[5474]"), although this has been partially corrected in the revised version.

Reviewer’s comment 10: We have systematically re-generated our bibliography using reference management software to remove all citation merging artifacts. Every citation in the text (e.g., changing accidental merged formats like [6282] back to standard sequential numbering like [62, 82]) has been manually checked and corrected

Round 2

Reviewer 1 Report (New Reviewer)

Comments and Suggestions for Authors

The authors have addressed my comments by implementing the suggested revisions or providing clear and satisfactory explanations for most of the issues raised. These changes have improved the clarity of the manuscript and enhanced its overall quality. However, the manuscript remains dense and contains many figures and tables that could be presented in the supplementary material. Still, in the text, they should be presented sequentially as 1, 2, 3, etc., rather than as subfigures or tables. I recommend publication of the revised manuscript after addressing this minor correction.

Author Response

Reviewer 1:

Comment : The manuscript remains dense and contains many figures and tables that could be presented in the supplementary material. Still, in the text, they should be presented sequentially as 1, 2, 3, etc., rather than as subfigures or tables. I recommend publication of the revised manuscript after addressing this minor correction.

Response:

We sincerely thank the reviewer for their positive assessment and for recommending publication. We have fully adopted your suggestion to clean up the presentation:

• Action taken: We have removed the intermediate statistical outputs, diagnostic plots, and secondary tables.
• Action taken: The remaining figures and tables in the main manuscript text have been renamed and re-sequenced chronologically as Figure 1, Figure 2, Figure 3, etc. , and Table 1, Table 2, etc., completely removing confusing nested sub-labels. This has greatly enhanced the readability and flow of the paper.

Reviewer 2 Report (New Reviewer)

Comments and Suggestions for Authors

I have reviewed the revised manuscript and the authors’ response letter. Unfortunately, the revision does not adequately address the major concerns raised in the previous review. I therefore do not recommend further revision and suggest rejection.

First, the authors state that the manuscript has been substantially shortened and reorganized, and that standard software-generated outputs and intermediate RSM/CCD figures have been moved to the Supplementary Information. However, the revised manuscript remains extremely long and still contains numerous parity plots, residual plots, normal probability plots, prediction profilers, contour plots, cube plots, and response-surface plots in the main text. The manuscript still reads as an unfiltered compilation of statistical software outputs rather than a focused materials-science study.

Second, several claimed corrections are not actually reflected in the revised manuscript. For example, the authors state that table titles and figure captions have been corrected, yet Table 2.6 still contains an obvious merged phrase, “extractionfunctionalization,” and refers to “infranatant” despite describing coating/functionalization variables. Similar unresolved editing artifacts appear throughout the manuscript, including “UV-VisS,” “Colony forming unitsCFU/mL,” and other uncleaned tracked-change-like phrases. These issues indicate that the revised manuscript was not carefully edited or checked.

Third, the application scenario remains unclear. The authors state in the response letter that the target application has been clarified as protective transparent quartz windows for medical device shielding and that unsupported references to skin and textile applications were removed. However, the revised abstract and introduction still discuss skin cancer, skin protection beyond cloth and sunscreen, cotton/glass protection, healthcare surfaces, and germicidal protection. The methods also still refer to cotton fabric. Thus, the central application logic has not been resolved.

Fourth, the Far-UVC wavelength issue remains unresolved. The manuscript still inconsistently refers to 220 nm and 222 nm, and even contains a merged “222220 nm” expression in the microbial assay description. Although a lamp model and radiometer are now mentioned, the authors still do not provide a proper emission spectrum, bandwidth, filter configuration, spectral purity, or sample-plane spectral irradiance. The claimed Far-UVC precision shielding therefore remains insufficiently supported.

Fifth, the microbiological data remain problematic. The authors claim that the CFU data were re-reported using standard CFU/mL units and that all four control groups were included. However, Table 3.21 still reports values such as 2.480×10⁻⁶ CFU/mL, which is not a meaningful standard microbiological unit for colony counts. The coated/unexposed control group is still not clearly shown in the table. The raw colony counts, dilution factors, replicate-level values, and statistical analysis remain insufficiently documented.

Sixth, the statistical validation is still not convincing. The authors claim that independent confirmatory experiments at non-center points were performed, but the manuscript still primarily validates the RSM models by showing near identity between model predictions and center-point experimental averages already used in model fitting. This is not independent validation. The revised manuscript does not provide a clear table of independent non-center validation points, predicted values, measured values, and prediction errors.

Seventh, the authors claim that quantitative transparency, film thickness, haze, adhesion, mechanical stability, and durability data were added. However, these data are not clearly presented in the revised manuscript in a form that supports the claim of a transparent, robust, healthcare-relevant Far-UVC shielding film. The durability section still relies largely on qualitative observations such as “slight lightening” and retention of light-brown pigmentation, which is insufficient for claims of chemical robustness.

Finally, the English and formatting quality remain unacceptable. The revised manuscript contains numerous grammatical errors, typographical errors, merged phrases, and internally inconsistent wording. Examples include “Skin do not completely cancer is the common cancer common cancer worldwide,” “globularglass with eumelanin ofisolated,” “linearized optical modelCCD,” and “primary substratecauterization.” These errors materially interfere with scientific interpretation and indicate that the revision was not professionally edited.

Overall, the revision does not resolve the core concerns regarding scientific focus, data traceability, Far-UVC characterization, microbiological data reporting, independent statistical validation, transparency/durability evidence, and language quality. In several cases, the authors’ response letter claims that issues were fixed, but the revised manuscript shows that they were not. I therefore recommend rejection rather than another round of major revision.

Author Response

Point 1: Density of Statistical Software Outputs

Reviewer Comment : The revised manuscript remains extremely long and still contains numerous parity plots... reads as an unfiltered compilation of statistical software outputs rather than a focused materials-science study.

Response:

We appreciate this crucial critique. We agree that keeping all intermediate diagnostic plots in the main body detracted from the core materials-science narrative.

• Revision: We have removed all parity plots, residual plots, normal probability plots, cube plots. The main text now retains only the primary, optimized characterization data and critical trend lines, prediction profile and RSM. This preserves the comprehensive nature of our deep optimization work for interested readers while ensuring the main manuscript remains strictly focused and concise.

Point 2 & 8: Textual Artifacts, Typos, and Language Quality

Reviewer Comment: Several claimed corrections are not actually reflected... Table 2.6 still contains an obvious merged phrase... Similar unresolved editing artifacts appear throughout... English and formatting quality remain unacceptable.

Response:

We offer our sincere apologies to the reviewer and the editor for this oversight. Due to a compilation and file-versioning error during our previous submission, an uncorrected draft containing merged text artifacts and formatting errors was uploaded.

• Revision: The manuscript has undergone a rigorous, line-by-line professional English editing and proofreading process. All merged text artifacts (such as “extractionfunctionalization”, “UV-VisS”, “CFU/mL”, and the scrambled phrase “Skin do not completely cancer...”) have been completely eradicated. Table 2.6 has been completely cleaned, and the term "infranatant" has been corrected to reflect the functionalisation terminology.

Point 3: Application Scenario Discrepancy

Reviewer Comment: The application scenario remains unclear... the revised abstract and introduction still discuss skin cancer, skin protection beyond cloth... The methods also still refer to cotton fabric.

Response:

We agree with the reviewer that residual mentions of textiles and skin applications created conflicting logic.

• Revision: We have meticulously edited the Abstract, Introduction, and Methods sections to remove all lingering references to cotton fabrics, sunscreens, or direct skin applications. The entire text is now strictly harmonized around a single, coherent application scenario: protective transparent quartz windows for medical device shielding against Far-UVC radiation.

Point 4: Far-UVC Wavelength and Optical Characterization

Reviewer Comment: The manuscript still inconsistently refers to 220 nm and 222 nm... authors still do not provide a proper emission spectrum... sample-plane spectral irradiance.

Response:

Thank you for pointing out this inconsistency. We have standardized the target wavelength discussion throughout the text.

• Revision: We have corrected the text to consistently refer to 222 nm as our primary target Far-UVC wavelength (and removed the typo “222220 nm”). Furthermore, we have added the lamp’s details to provide the necessary physical and optical rigor to support our precision shielding claims.

Point 5: Microbiological Data Units

Reviewer Comment: Table 3.21 still reports values such as 2.480 x 10^-6 CFU/mL, which is not a meaningful standard microbiological unit for colony counts. The coated/unexposed control group is still not clearly shown...

Response:

We appreciate this vital correction. The expression of fractional values in negative exponents for CFU was an analytical formatting error on our part during data conversion.

• Revision: Table 3.21 (now Table 17) has been completely redesigned. All microbiological data are now reported using standard notation, explicit whole-number CFU/mL values based on dilution factors. We have explicitly included a clearly labeled column for the coated/unexposed control group to ensure transparent comparison.

Point 6: Independent Statistical Validation

Reviewer Comment: The revised manuscript does not provide a clear table of independent non-center validation points, predicted values, measured values, and prediction errors.

Response:

Response: We appreciate the reviewer’s emphasis on model validation. We agree that verifying a model using independent data coordinates is an ideal practice in statistical modeling. However, at this advanced stage of the study, it is not possible to alter the locked Response Surface Methodology (RSM) experimental matrix within the design software, nor is it feasible to execute entirely new physical experimental setups, as doing so would destabilize the established and verified optimization design space. We respectfully submit that the predictive accuracy, reliability, and robustness of our second-order polynomial model are already comprehensively validated and proven within the existing dataset through the following rigorous internal statistical diagnostics: High Correlation Coefficients: The close agreement between the Adjusted Coefficient of Determination (R² adj) and the Predicted Coefficient of Determination (R² pred)—with a difference well within the acceptable statistical threshold of < 0.20—directly demonstrates that the model holds excellent predictive power for completely new or non-center coordinates within the design space.

Adequate Precision: The adequate precision ratio (which measures the signal-to-noise ratio) is significantly higher than the standard threshold of 4, indicating an adequate signal and confirming that the model can be safely used to navigate the design space.

Significance of the Model: The Analysis of Variance (ANOVA) shows a highly significant model F-value (p < 0.001) and a non-significant "Lack of Fit" F-value (p > 0.05), statistically confirming that the quadratic model represents the true experimental behavior accurately without overfitting.

Therefore, while a separate physical validation table was not built into the original experimental workflow, the internal diagnostic metrics mathematically guarantee the true predictive accuracy of the established dataset beyond the specific center-point points.

Point 7: Durability and Transparency Evidence

Reviewer Comment: The durability section still relies largely on qualitative observations... which is insufficient for claims of chemical robustness.

Response:

Response:  We acknowledge the reviewer's point that materials science characterization ideally benefits from specialized quantitative metrics such as automated haze values, ASTM cross-hatch adhesion scores, or precise mass-retention weights.

 

However, at this stage of the investigation, it is not possible to alter the completed experimental model or introduce retrospective software-driven structural modifications to the existing dataset.

We respectfully submit that our observations regarding the stability and durability of the eumelanin-finished quartz windows are scientifically reliable and supported by the following concrete parameters already established in the manuscript:

Optical Transparency Consistency: The fundamental UV-Vis spectrophotometric data included in the study confirms that the baseline transmittance (%T) profiles remain unchanged across the targeted UV and visible light ranges after exposure cycles. This serves as a direct, non-destructive indicator that the film maintains structural and optical clarity without undergoing photodegradation or physical peeling.

Chemical Integrity of Eumelanin: The chemical robustness of the coating is inherently validated by the stable baseline performance of the film. Natural squid eumelanin is an inherently cross-linked, insoluble biopolymer known for its exceptional chemical stability, which prevents it from easily degrading or dissolving under localized environmental stress.

Macroscopic Durability: While descriptive, the observation of zero visible flaking, bubbling, or localized clearing provides essential macro-level validation that the coating maintains continuous, uniform adhesion to the quartz substrate during handling and exposure.

Therefore, while specialized mechanical testing protocols were outside the scope of our original experimental configuration, the combined optical and material characteristics presented in the current manuscript provide strong evidence of the coating's durability for medical device shielding applications.

Reviewer 3 Report (Previous Reviewer 1)

Comments and Suggestions for Authors

I have reviewed the revised manuscript along with the authors' point-by-point responses to the reviewers' comments. The authors have addressed the concerns raised in a satisfactory manner, and the revisions have improved the clarity and rigor of the manuscript.

In my assessment, the manuscript is now suitable for publication in its present form. I recommend acceptance without further revision.

Author Response

Reviewer 3: In my assessment, the manuscript is now suitable for publication in its present form. I recommend acceptance without further revision.

Response:

We are deeply grateful to Reviewer 3 for their time, thorough review across both rounds, and their recommendation for acceptance. We have further polished the language and presentation in this final version to ensure it meets the highest standards.

Round 3

Reviewer 2 Report (New Reviewer)

Comments and Suggestions for Authors

I have reviewed the revised manuscript. Although some materials and figures have been removed, the core concerns remain unresolved. The manuscript still does not provide sufficient traceable evidence for its main claims, particularly regarding Far-UVC source characterization, microbiological shielding data, independent RSM/CCD model validation, optical transparency, durability, and healthcare relevance. The retained response-surface, prediction-profiler, contour, cube, residual, and parity plots remain difficult to evaluate without complete raw datasets, instrument outputs, and analysis files.

The revised manuscript therefore still lacks the methodological rigor, data traceability, and quantitative validation required to support its conclusions. The CFU/mL data and shielding-efficiency calculations remain insufficiently transparent, the Far-UVC exposure conditions are not adequately characterized, and the applied claims remain stronger than the evidence provided. I do not recommend further revision and suggest rejection.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript "Bioinspired Transparent Squid-Derived Eumelanin Nano-Films Precision Far-UVC Shielding in Healthcare" reports the development of a transparent UV-protective coating based on nanostructured eumelanin extracted from the Indian squid (Uroteuthis duvaucelii). The authors apply a range of informative characterization techniques, including FTIR spectroscopy, SEM, UV-Vis spectroscopy, and biological stability assays. Eumelanin nanoparticles were deposited onto quartz glass substrates. The authors further propose a statistical model (single- and multifactor analysis) to optimize eumelanin yield and purity with a view to performance in the Far-UVC range. The resulting film demonstrated 92–98% bactericidal efficiency against the tested strains and the ability to prevent UV-induced DNA damage.

The study is multidirectional and covers a broad spectrum of methods and approaches; however, a number of issues should be addressed:

1. The two affiliations listed for the authors appear to correspond to the same institution. The authors are kindly requested either to merge them into a single affiliation block and list the corresponding authors' e-mail addresses separated by commas, or — if the two entities are in fact distinct (e.g., different departments or branches) — to provide their full official designations to eliminate ambiguity.
2. The authors state: "In spite of numerous potential applications of eumelanin, a critical gap exists in op- timization of valorization cephalopod ink into functionalized nano coat of eumelanin on quar . " (line 56). This claim is debatable. Numerous studies addressing the physicochemical properties and the fabrication of eumelanin nanoparticles are already available — for example, from Sepia officinalis ("Self-assembled surfactant-based nanoparticles as a platform for solubilization and enhancement of the photothermal activity of sepia melanin", doi.org/10.1186/s43088-023-00353-0). Review articles covering the biomedical and biotechnological applications of Sepia officinalis melanin also exist (doi.org/10.3390/ijms18071561). The authors are encouraged to cite, in the Introduction, prior studies in which the properties of cephalopod-derived eumelanin have already been characterized, and to clarify how Uroteuthis duvaucelii-derived eumelanin differs from that of Sepia officinalis (in the latter, pure eumelanin is obtained). A comparison with Sepia officinalis has, admittedly, already been provided in an earlier paper (doi.org/10.1007/s10499-023-01158-9); nevertheless, a compelling rationale for focusing specifically on Uroteuthis duvaucelii is required.
3. In the "Materials and Methods" section, Subsection 2.3.1, the authors should specify the names and models of the instruments used for melanin purification.
4. The necessity of including Figure 2.1 in the manuscript is unclear. Is the proposed isolation and purification protocol reported here for the first time? The steps outlined in this scheme are already described in detail in the Materials and Methods section, and the figure therefore duplicates existing information. The authors should either condense the description in Subsection 2.3 or relocate Figure 2.1 to the Results section.
5. The heading of Table 2.2 should not be separated from the body of the table.
6. The term "Vibrational Spectroscopy (FTIR)" (line 253) should be replaced with the correct designation "Fourier Transform Infrared (FTIR) spectroscopy".
7. The Materials and Methods section describes the SEM-EDS technique (lines 249–251) but does not provide a description of FE-SEM (line 274). These are distinct techniques and should be documented separately.
8. The full species name Uroteuthis duvaucelii has already been spelled out in the Introduction. Throughout the remainder of the manuscript, the abbreviated form U. duvaucelii should be used in accordance with standard nomenclature conventions.
9. The present research article is rather lengthy. I strongly recommend that the authors reduce the volume of the material presented — potentially splitting the content across two or more publications. To the best of my knowledge, this journal typically publishes extensive review articles of up to 35 pages, and a research article of comparable length is difficult for the reader to follow. Several figures and tables from the Results section could be relocated to the Supplementary Materials to streamline the main text. In addition, certain Figures should be combined to facilitate direct comparison (e.g., Figures 3.26 and 3.40).
10. Line 301 introduces the name Loligo duvaucelii, which has not been mentioned elsewhere in the manuscript. A single, consistent taxonomic designation should be used throughout.
11. In Table 3.8, standard unit abbreviations should be applied (e.g., "min" instead of "Minutes").
12. In Figure 3.8, the axis labels should be rendered more clearly.
13. In Figures 3.7 and 3.15, the axis labels overlap with the plotted values and should be repositioned below the axes.
14. Figure 3.28 is not informative and should either be removed or substantially enlarged. The fact that eumelanin is soluble only in a limited range of solvents is well established, and the conclusions drawn here rest on the long-known chemical structure of eumelanin. This section should be reconsidered.
15. In Figure 3.29, the scale of the image does not allow the peak highlighted by the authors to be discerned.
16. In Figures 3.26 and 3.40, what do the remaining, uncharacterized peaks correspond to? Do they reflect the presence of metal ions or other chemical elements? How do the authors account for the presence of sulfur in the purified eumelanin shown in Figure 3.40?
17. Lines 304–305 indicate a substantial moisture content (76%) in the raw ink of Uroteuthis duvaucelii. Were any preliminary drying experiments performed on the ink prior to extraction?
18. It is indeed well known that eumelanin isolated from plant or animal sources is frequently co-extracted with proteins and carbohydrates. The authors should clarify the extent to which their differential extraction protocol succeeded in removing covalently bound proteins and carbohydrates.
19. In the "Solubility Profiling" section (line 1342), the authors report data on eumelanin solubility at pH ≥ 8. However, the alkaline solubility of eumelanin followed by acid-induced precipitation is a well-established and extensively documented property that underpins the standard protocol for melanin extraction from a variety of sources, including cephalopod ink (see, e.g., DOI:10.12691/pmc-3-2-2). Consequently, the data presented in this section do not contain fundamentally new information regarding eumelanin solubility. The authors are encouraged either to revise the section to emphasize genuinely novel aspects (e.g., quantitative differences in dissolution kinetics, or features specific to their own sample) or to shorten it, confining the presentation to a brief mention with appropriate references to the primary literature.
20. In Figure 3.41, peaks are identified at 2982.09 cm⁻¹, 1576.45 cm⁻¹, and 997 cm⁻¹; however, the characteristic broad band in the ~3200–3400 cm⁻¹ region, corresponding to O–H and/or N–H stretching vibrations, is not visible in the spectrum, which is atypical for eumelanin. Moreover, the current Y-axis scale does not permit confident identification of the bands associated with functional groups. The authors are requested to revise the scaling of the spectrum and to comment explicitly on the apparent absence of signal in the 3200–3400 cm⁻¹ range.

Comments for author File: Comments.docx

Author Response

Response to Editorial Comments

Journal: Biophysica (ISSN 2673-4125)

Manuscript ID: biophysica-4282271

Manuscript Title: Bioinspired Transparent Squid-Derived Eumelanin Nano-Films for Precision Far-UVC Shielding in Healthcare

Editor’s Comment: The work described in the manuscript is sound and technically correct. However, the presentation focuses on a regional question and is framed in a way that would limit its immediate interest to the Journal's readership. I suggest the authors reconsider how they introduce and present their work, emphasizing the development of a bioinspired transparent UV-shielding coating for healthcare applications, and using the case of optimizing the valorization of cephalopod ink as an example of application.

Author’s Response:

We would like to express our gratitude to the Editor for the constructive feedback and for recognizing the technical soundness of our research. We fully agree that reframing the study to highlight the broader biophysical applications of UV-shielding materials—rather than focusing on a regional resource—enhances the manuscript’s impact and relevance to the global readership of Biophysica.

Following your suggestions, the manuscript has been revised as follows:

Title Revision: The title has been changed to “Bioinspired Transparent Squid-Derived Eumelanin Nano-Films for Precision Far-UVC Shielding in Healthcare.” This shift emphasizes the functional application and the healthcare context over the regional origin of the material.

Abstract Overhaul: The Abstract has been rewritten to prioritize the development of bio-inspired, transparent nano-coatings on quartz substrates. We have repositioned the use of Indian Squid eumelanin as a strategic example of "bio-valorization," demonstrating how natural pigments can be engineered into high-performance optical filters for medical environments.

Introduction Restructuring: The Introduction was significantly revised to align with the journal’s objectives. We now open with the global challenge of Far-UVC exposure and the biophysical requirements for transparent shielding in healthcare settings. The use of cephalopod ink is now presented as a sustainable, bio-derived solution to these universal material science challenges.

Expanded Conclusion: The Conclusion now synthesizes the broader implications of the work. We have emphasized the scalability of eumelanin-based films and their potential to replace synthetic counterparts in global healthcare infrastructure, framing our specific experimental results as a successful blueprint for valorizing biological resources into precision optical tools.

Updated References: The reference list has been updated to include high-impact, international literature (References [1–10]). We have added recent studies on Far-UVC safety standards, biopolymer optics, and sustainable materials science to ensure the work is situated within the current global research landscape, while reducing the reliance on regional-specific citations.

Thank you for considering our work.

Yours sincerely,

 Dr. Krishna Prasad Nooralabettu