PONE-D-25-09375Chemometrics-aided Surface-enhanced Raman spectrometric detection and quantification of GH and TE hormones in bloodPLOS ONE

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Reviewer #1: Partly

Reviewer #2: Partly

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Reviewer #1: No

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: No

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5. Review Comments to the Author

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Reviewer #1: This manuscript presents a quantitative detection of hormones using SERS with a portable Raman spectrometer. The SERS spectra of whole blood from rats injected with hormones are distinguished from those of control samples (without hormone injection) through chemometric analysis. Furthermore, the authors examine temporal variations at different time points following hormone injection. Notably, hormone concentrations are predicted via SERS using an artificial neural network (ANN) model, and the temporal trends appear to be roughly consistent with those obtained through ELISA. The rapid detection of hormones is of significant importance, as it pertains to health monitoring, doping control, and related applications. However, there may be statistical issues. Therefore, I can recommend publication after resolving the following concerns.

Fig. 1: Are these spectra averaged? If so, the number of spectra used to obtain the average should be specified. Additionally, are these spectra normalized? If so, this should be explicitly stated.

Lines 189–192: Does the observed difference exceed the spectral deviation? A statistical analysis should be conducted to confirm this.

Fig. 4: The 3D plots provide limited clarity in distinguishing the temporal variations among (a)–(d). To enhance interpretability, corresponding 2D plots should also be provided.

[Minor Comments]

Section 2.2: The fabrication method or source of AgNPs should be specified.

Lines 88–89: "30" and "2" -> "Thirty" and "Two."

Lines 123, 125: The meaning of the asterisks should be clarified. Are they needed?

Lines 143–145: This paragraph can be merged with the subsequent one, as they discuss a similar topic.

Lines 162–164: These sentences can be integrated into the following paragraph due to thematic continuity.

Lines 199–201: These sentences can be incorporated into the next paragraph, as they address a related topic.

Lines 242–244: This paragraph can be merged with the preceding one due to their shared subject matter.

Lines 245–246: This paragraph can be combined with the following one for coherence.

Line 271: A new paragraph can begin here, and the sentences up to line 275 can be merged with the subsequent paragraph, as they introduce and expand upon a related topic.

Lines 296–302: This paragraph can be combined with the following one for improved cohesion.

Reviewer #2: 1, The abstract needs to be re-phrased. For example, sentences like, "Current methods of detecting them suffer from being costly, time-consuming, non-portable and others." Then, "Secondly, use of artificial neural network (ANN) models in predicting levels of these hormones in blood". Do these sentences look complete? Again, "This work explored, first, Surface-Enhanced Raman Spectroscopy (SERS)....." does not sound professional. I would appreciate it if the authors could re-phrase and reorganize these few lines. Also, I expect the authors must adopt a more professional approach in writing the abstract.

2, In the Introduction section, "The primary methods currently used to measure these hormone levels in blood are currently based on immunoassays and mass spectrometry as described in (5)". Can the authors kindly elaborate here? Include a few more references and talk about why SERS is advantageous as compared to other techniques/approaches.

3, In Fig. 1, rats injected both GH only....rats injected both TE only... Remove both.

4, For Figs. 1, 3 and 5, the Raman spectrum for different conditions cannot be easily distinguished. Although for Fig.1 it's not that bad, however, for Figs.3 and 5, the individual spectrum/peaks are not clearly visible.

It is advisable to vertically shift the individual spectrum for better clarity.

5, "The SERS spectra of blood from non-injected rats and the injected, as shown in Fig 1, are nearly identical, with a few minor band intensity variations suggesting subtle but distinct biochemical changes....". It is good to explain/justify why there is not much change/shift observed between the non-injected and injected spectra. What do the authors mean by subtle but distinct biochemical changes?

6, The authors must try to enhance the overall readability of the paper to ensure it is accessible to a broad audience. Also, please check the reference formatting.

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Reviewer #1: No

Reviewer #2: No

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Chemometrics-aided Surface-enhanced Raman spectrometric detection and quantification of GH and TE hormones in blood

PONE-D-25-09375R1

Dear Dr. Ondieki,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

Tanveer A. Tabish

Academic Editor

PLOS ONE

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