Turn a Shrimp into a Firefly: Monitoring Tissue pH in Small Crustaceans Using an Injectable Hydrogel Sensor with Infrared Excitation and Visible Luminescence

Round 1

Reviewer 1 Report

The article in review is very well written and formally well supported. The structure of the document, the experimental procedure here described and the depth of the analysis is at a very high level.

My only  doubt would be if this the right journal to publish this particular work, as this paper presents, in depth, a experimental procedure related to the use of a new tool for study of in vivo animal physiology, with limited focus to the optical solution,  which is a more or less common technique in photonics.

A small detail requiring you attention regards  the presentation of results in equation 2 and figure 2 ( including legend and inserts) and the number of significant digits, which is not according to the standard presentation of results and uncertainties.

Author Response

The article in review is very well written and formally well supported. The structure of the document, the experimental procedure here described and the depth of the analysis is at a very high level.

Authors: Dear Reviewer 1, we would like to deeply thank you for critical reading of the manuscript and for high estimate of our work.

My only  doubt would be if this the right journal to publish this particular work, as this paper presents, in depth, a experimental procedure related to the use of a new tool for study of in vivo animal physiology, with limited focus to the optical solution,  which is a more or less common technique in photonics.

Authors: The scope of the journal and specifically of the special issue includes "Biophotonics, biomedical optics" and "Imaging systems", and the presented manuscript is directly related to these topics. Besides describing the new implantable optical sensors applicable to studies in animal physiology, the manuscript also presents the specific optical setup for their application. In our case the setup was critical for proper pH measurements due to variability of the ratio I595/I620 depending on I595/I571 and necessity to constantly keep all three spectrum peaks within the dynamical range of the spectrometer, which was achieved with the help of tightly selected filters LP570 and SP640 (otherwise, intensities at SNARF-1 wavelengths 595 and 620 nm would be negligible comparing to the peak of upconverting particles). However, to the best of our knowledge, such setups were not described previously for the sensors combining upconverting particles and fluorescent probes.

A small detail requiring you attention regards  the presentation of results in equation 2 and figure 2 ( including legend and inserts) and the number of significant digits, which is not according to the standard presentation of results and uncertainties.

Authors: We now unified the number of significant digits in both equations, as well as on Figure 2 and in its caption. We also exchanged the * sign with the more proper multiplication sign ×.

Thank you very much again!

With the best wishes,

Dr. Anton Gurkov and Prof. Maxim Timofeyev,

Irkutsk State University

Reviewer 2 Report

In this research paper, the authors reported an implantable hydrogel sensor to monitor the pH change in a shrimp. This work may provide inspirations for the pH monitoring in light-sensitive organisms. I recommend resubmission of this work after addressing the following issues.

1.      Consider changing “implantable sensor” to “injectable sensor”

2.      Please change the title. Something like “Monitoring tissue pH of amphipod using an injectable hydrogel sensor with infrared excitation and visible luminescence” make more sense. I suggest removing the words before colon since they do not accurately represent the sensing application reported in this paper. Is “Eulimnogammarus verrucosus” a type of shrimp? If yes, use shrimp in the title in place of amphipod.

3.      The sentence “The optical sensors are mostly based on some sensitive component dispersed within a biocompatible hydrogel and rely on external device detecting and analyzing the light obtained through the tissues” in line 36 to 38 needs more attention. Firstly, citation is needed to support this claim. Secondly, I don’t think the first half “The optical sensors are mostly based on some sensitive component dispersed within a biocompatible hydrogel” is correct. There are way more methods to build optical sensors besides hydrogels, which are only a specific group of materials.

4.      The authors mentioned “Fluorescent sensors” in line 41 to 42 but cited only one review paper in 2019. Please add more description about fluorescent sensors (with one to three sentences) and cite one recent research paper of hydrogel fluorescent sensor doi.org/10.1039/D2MH00537A.

5.      In the introduction part, please explain to the readers why the authors want to use hydrogels as the carrier.

6.      For Figure 1, please remove the brackets of (a), (b) and (c) and move it to the top left location to keep consistency. Similar suggestions for other figures as well.

7.      Legend of Figure 2a is missing.

Author Response

In this research paper, the authors reported an implantable hydrogel sensor to monitor the pH change in a shrimp. This work may provide inspirations for the pH monitoring in light-sensitive organisms. I recommend resubmission of this work after addressing the following issues.

Authors: Dear Reviewer 2, we would like to deeply thank you for critical reading of the manuscript and for the specific corrections provided.

1. Consider changing “implantable sensor” to “injectable sensor”

Authors: Our original intention in using “implantable sensor” was more general meaning of the term and the possible perception of “injectable sensor” by the readers as amorphous (non-resilient) hydrogel, which is not the case for the developed filamentous sensor. However, we agree that “injectable sensor” can be better perceived as something easily implantable with a needle without any surgery (as indeed was performed in our work). So, we exchanged “implantable sensor” with “injectable sensor” in the title and in relevant places over the manuscript as suggested.

2. Please change the title. Something like “Monitoring tissue pH of amphipod using an injectable hydrogel sensor with infrared excitation and visible luminescence” make more sense. I suggest removing the words before colon since they do not accurately represent the sensing application reported in this paper. Is “Eulimnogammarus verrucosus” a type of shrimp? If yes, use shrimp in the title in place of amphipod.

Authors: We indeed found the wording “Monitoring tissue pH … using an injectable hydrogel sensor” better than the original one and would like to thank you for the suggestion. However, we cannot agree with removing the part before the colon. This is a catchy phrase, which is intended to make the article easily recognizable by the reader and also quickly describes the suggested procedure since these animals indeed look similar to fireflies with the injected sensor! (Please check the graphical abstract besides the Figure 4). The species Eulimnogammarus verrucosus belongs to the order Amphipoda, which is evolutionary and anatomically close to the order Decapoda, some representatives of which are called shrimps. However, amphipods are sometimes also informally called freshwater shrimps due to their visual and anatomical similarity to these decapods (this issue is mentioned in the 5th paragraph of Introduction). The technique presented in the manuscript is not restricted to amphipods by any parameter and can be readily applied to other crustaceans with similar size, anatomy and optical properties of the exoskeleton such as true shrimps (infraorder Caridea within order Decapoda) and prawns (suborder Dendrobranchiata within order Decapoda), as well as representatives of completely different taxons such as young mantis shrimp (order Stomatopoda). That's why we insist on writing “small crustaceans” instead of just amphipods and on using informal term “shrimp” (as the most understandable to a broader audience) in the title.

So, we changed the title as follows: «Turn a shrimp into a firefly: Monitoring tissue pH in small crustaceans using an injectable hydrogel sensor with infrared excitation and visible luminescence».

3. The sentence “The optical sensors are mostly based on some sensitive component dispersed within a biocompatible hydrogel and rely on external device detecting and analyzing the light obtained through the tissues” in line 36 to 38 needs more attention. Firstly, citation is needed to support this claim. Secondly, I don’t think the first half “The optical sensors are mostly based on some sensitive component dispersed within a biocompatible hydrogel” is correct. There are way more methods to build optical sensors besides hydrogels, which are only a specific group of materials.

Authors: We completely agree that there is a plenty of structural bases and principles to build some optical sensors, but here we talk specifically about implantable sensors, which implies the necessity to simultaneously (i) deal with the foreign body response in order to prolong the functional lifetime of the sensor and (ii) make the carrier permeable to the molecule of interest. For hydrophilic molecules of interest (which is usually the case for animal tissues), such carriers are mostly hydrogels (DOI: 10.1002/adfm.202007226; DOI: 10.1038/s41578-022-00483-4; DOI: 10.1186/s12951-023-01873-8; DOI: 10.1021/acs.nanolett.1c00887; DOI: 10.1016/j.mvr.2019.02.002). The additional references along with the relevant explanations are now added.

4. The authors mentioned “Fluorescent sensors” in line 41 to 42 but cited only one review paper in 2019. Please add more description about fluorescent sensors (with one to three sentences) and cite one recent research paper of hydrogel fluorescent sensor doi.org/10.1039/D2MH00537A.

Authors: Again, here we aimed to talk specifically about implantable fluorescent sensors, which was probably not clear from the original sentence. We completely updated the reference list specifically for this topic of continuous long-term monitoring (since fluorescent sensors in general or fluorescent labels for bioimaging are enormous topics that are not directly relevant to the manuscript), expanded the description and included some recent publications. The suggested article DOI: 10.1039/D2MH00537A is indeed a very interesting research, and the proposed mechanism potentially can be utilized in some implantable sensors in the future, but right now we do not see its relevance to our manuscript.

5. In the introduction part, please explain to the readers why the authors want to use hydrogels as the carrier.

Authors: The explanation has now been added to the first paragraph of Introduction along with the references.

6. For Figure 1, please remove the brackets of (a), (b) and (c) and move it to the top left location to keep consistency. Similar suggestions for other figures as well.

Authors: The journal guidelines do not mention any rules regarding the panel naming in figures, so we checked the recently published papers in the special issue and found that 10 of them used brackets, while only 5 did not. Thus, we kept the brackets around the letters as in the majority of the papers, but indeed moved some of them to keep consistency within each figure (updated Figs 1 and 3) as suggested by the Reviewer.

7. Legend of Figure 2a is missing.

Authors: We checked the legend, but the caption to Fig 2a was present in the file. We also double checked the presence of other figure legends.

Thank you very much again!

With the best wishes,

Dr. Anton Gurkov and Prof. Maxim Timofeyev,

Irkutsk State University

Round 2

Reviewer 2 Report

Please continue to revise and resubmit this work after addressing the following issues.

1.     Original comment #2: “Turn a shrimp into a firefly:” is redundant and inaccurate, though catchy. This may be good for a “News and Views” (such as https://www.nature.com/articles/s41570-022-00428-z ) and Review type paper, but not for a research article.

2.     In line 37, “usually” in “which is usually some hydrogel” is not fair. Hydrogel is just a specific type and please change the wording.

3.     Original comment #4: For references to mention (not to discuss in details), it is fine to be not directly related. Recent papers of this type should be beneficial for broadening the readership of this paper to people who are generally interested in fluorescent sensors. The authors should cite this paper rather than get limited to “Implantable optical sensors”.

4.     Original comment #6: Brackets are fine, but do move “(a) (b) (c)” to the top left location! (I would be really surprised to see that the authors didn’t even know this general rule, if they disagree…)

5.     Figure 2a: what do lines with different colors represent? e.g., what does cyan line mean? How about red? I cannot get this information from the figure per se. That is why I mean “legend is missing”

Author Response

Reviewer 2

Please continue to revise and resubmit this work after addressing the following issues.

1. Original comment #2: “Turn a shrimp into a firefly:” is redundant and inaccurate, though catchy. This may be good for a “News and Views” (such as https://www.nature.com/articles/s41570-022-00428-z ) and Review type paper, but not for a research article.

Authors: We do not agree. As we already stated, to our opinion, this phrase nicely describes the resulting look of the studied animals during implementing the suggested measurement procedure.

2. In line 37, “usually” in “which is usually some hydrogel” is not fair. Hydrogel is just a specific type and please change the wording.

Authors: We provide two references to the mentioned phrase. The first one "Dealing with the Foreign-Body Response to Implanted Biomaterials: Strategies and Applications of New Materials" is a recognized recent review in the topic of implantable devices. According to the review, the majority of carriers reducing the immune response to implantable devices (both conventionally used such as alginate, PEG, gelatin, polyacrylamide, PHEMA and especially more modern modifications such as zwitterionic materials and modified alginates) are hydrogels. The title of another review speaks for itself: "Hydrogel interfaces for merging humans and machines". So, we do not see what exactly is wrong with the phrase that we use here.

3. Original comment #4: For references to mention (not to discuss in details), it is fine to be not directly related. Recent papers of this type should be beneficial for broadening the readership of this paper to people who are generally interested in fluorescent sensors. The authors should cite this paper rather than get limited to “Implantable optical sensors”.

Authors: So, the Reviewer, in fact, agrees with us that the suggested paper doi.org/10.1039/D2MH00537A is not directly related to our manuscript. Quick search for "fluorescent sensors" in Google Scholar since 2021 (please check https://scholar.google.com/scholar?as_vis=1&q=%22fluorescent+sensors%22+&hl=ru&as_sdt=0,5&as_ylo=2021) gave us almost 10 000 publications. Why should we cite one specific article among thousands of similarly low relevance? The topic of our manuscript is specifically implantable optical sensors, and the Reviewer did not provide any clear reason why exactly we should "broaden" the scope of this part of Introduction.

4. Original comment #6: Brackets are fine, but do move “(a) (b) (c)” to the top left location! (I would be really surprised to see that the authors didn’t even know this general rule, if they disagree…)

Authors: As far as we can see, other authors and the editors of Photonics also do not know this so-called "general rule" since the recent examples with practically any panel labeling can be easily found even within the same special issue:

- https://www.mdpi.com/2304-6732/10/6/670

- https://www.mdpi.com/2304-6732/10/5/582

- https://www.mdpi.com/2304-6732/10/2/135

- https://www.mdpi.com/2304-6732/10/4/449

- https://www.mdpi.com/2304-6732/10/4/443

5. Figure 2a: what do lines with different colors represent? e.g., what does cyan line mean? How about red? I cannot get this information from the figure per se. That is why I mean “legend is missing”

Authors: Thank you for this suggestion; here different colors indicate individual spectra, but it could indeed be unclear. In the updated manuscript we have added the clarification to the figure caption.

With the best wishes,

Dr. Anton Gurkov and Prof. Maxim Timofeyev,

Irkutsk State University