Mapping Nanoparticles in Hydrogels: A Comparison of Preparation Methods for Electron Microscopy
Round 1
Reviewer 1 Report
The manuscript focuses on the morphology of polymers for sensing applications comparing hydrogels containing gold nanorods / gold nanoparticles and hydrogels not containing noble metal nanomaterials. The manuscript also deals with the determination of noble metal nanoparticle/nanorod distribution in several hydrogels based on polyacrylamide polymers with different scanning electron microscopy techniques.
The whole manuscript is well-written and references are used adequately. The study is interesting for a larger readership and therefore fits well within the scope of applied sciences.
In my opinion, the abstract is a bit long and thus the focus on the actual experiments and results is lost. I would recommend to shorten the abstract to below 250 words which is an arbitrary value but some shortening is important to draws more attention.
The introduction reviews the state of the art and leads to the motivation of the investigation.
The experimental details are explained thoroughly and indicate a reproducibility of the study.
I like how the authors critically discuss their results and clearly state, that a lot of effort and further investigations are necessary to completely interpret the morphology and nanoparticle distribution in the hydrogels.
My question is: Why do you not relating the morphology and particle distribution to the sensing applications of the hydrogels containing noble metal nanoparticles?
Furthermore, a thorough characterization of the hydrogels is missing.
From my perspective it is a bit odd to not characterize the hydrogels towards their mechanical and rheological behavior while focusing on studying the morphology.
Furthermore, in order to be able to better interpret the results, it would be quite helpful to have a comparison with other mapping techniques for the noble metal content in the polymers and especially on the surface.
Why are you not using other techniques such as XPS, TOF-SIMS or Scanning probe microscopy for the surface analysis and techniques such as EDX or PGAA for a general distribution analysis of your materials. Other mapping approaches like fluorescence microscopy, IR or Raman imaging might actually work as well.
Thus, I would like to ask you to provide a thorough characterization of your materials. Furthermore, you should answer, the question why you are not comparing your approach with a complementary technique and ideally try to compare your results.
However, I really liked your manuscript and would be happy to see it published with these slight improvements of the manuscript.
Author Response
The manuscript focuses on the morphology of polymers for sensing applications comparing hydrogels containing gold nanorods / gold nanoparticles and hydrogels not containing noble metal nanomaterials. The manuscript also deals with the determination of noble metal nanoparticle/nanorod distribution in several hydrogels based on polyacrylamide polymers with different scanning electron microscopy techniques.
The whole manuscript is well-written and references are used adequately. The study is interesting for a larger readership and therefore fits well within the scope of applied sciences.
In my opinion, the abstract is a bit long and thus the focus on the actual experiments and results is lost. I would recommend to shorten the abstract to below 250 words which is an arbitrary value but some shortening is important to draws more attention.
Thank you for the suggestion. The abstract is now shortened and reworked to clarify motivation and main findings. In addition the key message of introduction, section 3.1, section 3.2 and conclusion have been reworked to increase the readability.
The introduction reviews the state of the art and leads to the motivation of the investigation.
The experimental details are explained thoroughly and indicate a reproducibility of the study.
I like how the authors critically discuss their results and clearly state, that a lot of effort and further investigations are necessary to completely interpret the morphology and nanoparticle distribution in the hydrogels.
My question is: Why do you not relating the morphology and particle distribution to the sensing applications of the hydrogels containing noble metal nanoparticles?
We see the need for elucidating this and have now related earlier work on sensor GNP-hydrogels based on acrylamide or acrylic acid with the results in this manuscript in section 3.1 and 3.2.
Furthermore, a thorough characterization of the hydrogels is missing. From my perspective it is a bit odd to not characterize the hydrogels towards their mechanical and rheological behavior while focusing on studying the morphology.
We agree that it may look a bit odd to not perform a thorough characterization of the hydrogels towards mechanical and rheological properties. The swelling properties and molecular transport of sensor hydrogels have been discussed in earlier papers. To clarify the aim of the paper we have included a section about the earlier work on sensor hydrogel characterization in the introduction.
Furthermore, in order to be able to better interpret the results, it would be quite helpful to have a comparison with other mapping techniques for the noble metal content in the polymers and especially on the surface.
The interpretation of the results would be improved by having a comparison with other mapping techniques. The results obtained in section 3.1 and 3.2 are now compared with other results from similar characterization methods of noble metal nanoparticles in hydrogels
Why are you not using other techniques such as XPS, TOF-SIMS or Scanning probe microscopy for the surface analysis and techniques such as EDX or PGAA for a general distribution analysis of your materials.
The general distribution analysis with XPS, TOF-SIMS, EDAX or PGAA would help to confirm the results obtained. From earlier LSPR sensor measurements we observed a stable LSPR signal for different hydrogel swelling equilibriums indicating that GNP are contained in the gel. From these results we have assumed that the GNP are also contained in the hydrogel during the sample preparations for fixation, dehydration, and drying. Thus, the general distribution analytical techniques were omitted due to this assumption. This argumentation is included in the manuscript in the experimental section 2.5 elucidation the limitations in the experiments.
Concerning the use of scanning probe microscopy, it was challenging to obtain even surfaces of the hydrogel from cross-sectional sample preparations by using ultramicrotomy due to hydrogel rehydration. Due to the complication in performing cross-sectional sample preparations, we did not utilize scanning probe microscopy techniques. This argumentation is also included in section 2.5.
Other mapping approaches like fluorescence microscopy, IR or Raman imaging might actually work as well.
Thank you. This was a very interesting suggestion. When planning the experiments, we did not consider optical methods as feasible due to the limited resolution often offered by these techniques to identify individual GNRs orientation and positions. However, with super-resolution optical microscopy or with multivariate analysis used with Raman imaging, or also with TOF-SIMS, a characterization of the GNR-hydrogel can be obtained without performing sample preparations that are influencing the hydrogel structure. We will certainly consider using these techniques in future experiments.
Thus, I would like to ask you to provide a thorough characterization of your materials. Furthermore, you should answer, the question why you are not comparing your approach with a complementary technique and ideally try to compare your results.
Thank you for those suggestions. The swelling properties and molecular transport of sensor hydrogels have been discussed in earlier papers. To clarify the aim of the paper we have included a paragraph about the earlier work on sensor hydrogel characterization in the introduction. Also, the results obtained in section 3.1 and 3.2 are now compared with other results from similar characterization methods of noble metal nanoparticles in hydrogels
However, I really liked your manuscript and would be happy to see it published with these slight improvements of the manuscript.
Thank you for your comments and it was nice to hear that you found our manuscript interesting. The comments you suggested we think elucidated the aim of the manuscript as well as the limitations of the experiments conducted. Now the results in this paper are also more related to earlier results on LSPR sensor hydrogels with GNPs as well as compared with other similar characterization methods.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Development of innovative materials is essential for new technologies and this simply can happen by innovative methods combining hydrogels with nanoparticles (metals, metal oxides and non‐metals). This combination is an effective tactic for providing superior and miscellaneous functionality to composite materials. Gold as nanoparticle and Poly acrylic acid and polyacrylamide copolymer were employed for fabrication of gold-hydrogel composite using photopolymerization technique and SEM was used for morphology analysis.
I believe the focus of this study is analysing the morphology of hydrogel (mainly the surface) and studying the distribution of nanoparticles was excluded. There is no reliable evidence for this claim. Also the study lacks proper control, while two different methods of sample preparation (CPD and HMDS) were compared (I do not believe that gold particles on carbon film plays a role as control). Moreover, the result of study are not able to support some claims. For example, in lines 174-176 it was stated that: “The pore sizes of the HMDS dried hydrogels are comparable to the SEM images of CPD hydrogels. This shows that using HMDS for drying hydrogels for SEM imaging may serve as a suitable alternative to the CPD technique. The embedding of GNRs in the hydrogel have minor influence on the polymer network structure.” It is not clear how the pore size or shape were measured and why this comparison could lead to the conclusion? Presumably, the authors considered CPD as a gold standard for preparation of samples for SEM? Also where is the evidence for negligible effect of embedding nanoparticles on the network structure?
The following are some of my comments:
The title needs to be modified to reflect the study. Distribution and applications are excluded in this research.
The abstract should be summarized to reflect the key finding of the study.
Why the SEM was performed on the surfaces of two halve of the samples only and samples’ cross section didn’t used.
While elemental distribution can be performed using EDAX mapping, why this technique wasn’t considered to support some part of the results (claims)?
Some uncertainty and ambiguity also exist in the body of manuscript including (not limited to):
Line 202: “Some collapsing characteristics can be observed for the hydrogel without GNRs.”
Line 209: The GNR-hydrogel exhibits rather a highly entropic polymer network in comparison to the AAM-hydrogel with GNRs.
Line 239: “these small-sized contrast features are difficult to quantize as a distribution of GNRs due to the low contrast intensity”
And finally the study needs limitation section. In this section limitation for sample preparation, SEM imaging and etc. should be included. Using ImagJ software would be helpful as extra analysis to support the study including pore size measurement, distribution and orientation analyses and maybe to pinpoint the gold nanoparticle (based on contrast differences as explained).
More importantly, what is the key message of this study to take home? The significance of the study needs to be clearly explained.
Author Response
Development of innovative materials is essential for new technologies and this simply can happen by innovative methods combining hydrogels with nanoparticles (metals, metal oxides and non‐metals). This combination is an effective tactic for providing superior and miscellaneous functionality to composite materials. Gold as nanoparticle and Poly acrylic acid and polyacrylamide copolymer were employed for fabrication of gold-hydrogel composite using photopolymerization technique and SEM was used for morphology analysis.
I believe the focus of this study is analysing the morphology of hydrogel (mainly the surface) and studying the distribution of nanoparticles was excluded. There is no reliable evidence for this claim.
This is true. The focus on studying distribution in the manuscript is now redirected towards the aim of verifying LSPR coupling observed in our earlier studies by trying to characterize GNR positions.
Also the study lacks proper control, while two different methods of sample preparation (CPD and HMDS) were compared (I do not believe that gold particles on carbon film plays a role as control).
We agree that the particles on carbon film does not play as a role as a control. This was maybe miscommunicated in the manuscript. The role of this sample preparation is now clarified as a sample preparations for comparison for how GNRs may appear in an SEM image.
Moreover, the result of study are not able to support some claims. For example, in lines 174-176 it was stated that: “The pore sizes of the HMDS dried hydrogels are comparable to the SEM images of CPD hydrogels. This shows that using HMDS for drying hydrogels for SEM imaging may serve as a suitable alternative to the CPD technique. The embedding of GNRs in the hydrogel have minor influence on the polymer network structure.” It is not clear how the pore size or shape were measured and why this comparison could lead to the conclusion? Presumably, the authors considered CPD as a gold standard for preparation of samples for SEM? Also where is the evidence for negligible effect of embedding nanoparticles on the network structure?
These statements come with no evidence and we see the need for additional measurements as support. We have followed your suggestions of using ImageJ for pore size analysis and have now included mean and standard deviation of the Feret diameters of these pores to support the statements. Also, the CPD may not be considered as gold standard for preparation of samples for SEM, but we have observed limited sources in literature of using HMDS for drying hydrogels as an alternative to CPD and high/low temperature drying. We chose to focus on comparing HMDS drying with CPD since it may relate to comparisons for biological samples dried with HMDS and CPD. The limitations of comparing only CPD and HMDS for drying have now been included in the limitation section 2.5.
The following are some of my comments:
The title needs to be modified to reflect the study. Distribution and applications are excluded in this research.
Thank you for the suggestion. The title is now modified to reflect the study where distribution and applications are excluded.
The abstract should be summarized to reflect the key finding of the study.
We agree that the abstract was too long and not properly reflecting the study. The abstract has now been reworked.
Why the SEM was performed on the surfaces of two halve of the samples only and samples’ cross section didn’t used.
The rehydration of the hydrogel after drying complicated the cross-sectional sample preparation (hydrogel interpenetrated with epoxy polymers and cured) when performing ultramicrotomy in water. The water reduced the stability of the hydrogel network and resulted in dissociation. This argumentation is included in the limitation section 2.5.
While elemental distribution can be performed using EDAX mapping, why this technique wasn’t considered to support some part of the results (claims)?
We see that EDAX could be performed to support some parts of the results with claims. From earlier LSPR sensor measurements we observed a stable LSPR signal for different hydrogel swelling equilibriums indicating that GNP are contained in the gel. From these results we have assumed that the GNP are also contained in the hydrogel during the sample preparations for fixation, dehydration, and drying. Thus, the general distribution analysis with .e.g. EDAX were omitted due to this assumption. This argumentation is included in the manuscript in the limitation section 2.5.
Some uncertainty and ambiguity also exist in the body of manuscript including (not limited to):
Line 202: “Some collapsing characteristics can be observed for the hydrogel without GNRs.”
Line 209: The GNR-hydrogel exhibits rather a highly entropic polymer network in comparison to the AAM-hydrogel with GNRs.
Line 239: “these small-sized contrast features are difficult to quantize as a distribution of GNRs due to the low contrast intensity”
Thank you for elucidation the ambiguity of typical paragraphs in the manuscript. This have now been reworked and the Feret diameters of the pore sizes found by using ImageJ have been used for this.
And finally the study needs limitation section. In this section limitation for sample preparation, SEM imaging and etc. should be included.
Thank you for this suggestion. We found it fitting to add a limitation section (section 2.5) in the experimental section to clarify the restrains for the experimental methods used.
Using ImagJ software would be helpful as extra analysis to support the study including pore size measurement, distribution and orientation analyses and maybe to pinpoint the gold nanoparticle (based on contrast differences as explained).
This suggestion was very helpful. ImageJ has been used to support and clarify the discussion of the results in the manuscript. Paragraphs were also added in section 3.1. and 3.2 concerning the feasibility of using threshold filters to pinpoint the gold nanoparticles in the SEM images.
More importantly, what is the key message of this study to take home? The significance of the study needs to be clearly explained.
We agree that the key message was not clearly explained in parts of the manuscript. Abstract, introduction, section 3.1, section 3.2 and conclusion have been reworked to elucidate the significance of the study., as well as to increase the readability.
Author Response File: Author Response.pdf
Reviewer 3 Report
The paper by Muri et al. report on plasmonic nanoparticles in hydrogels for biosensing applications. The degree of innovation is good, as well as the potential impact. I recommend publication pending some amendments.
1) The potential use of noble-metal nanoparticles as nanofillers for composites for thermoplasmonic applications has not been clarified to the readers, in spite of the recent results reported by several groups [1-3].
2) Figure 1 is useless
3) The interesting paper on hydrogels by Alzari and coworkers should be cited [4], as well as their work on nanocomposites [5].
[1] Overcoming temperature polarization in membrane distillation by thermoplasmonic effects activated by Ag nanofillers in polymeric membranes, Desalination (2018) doi:10.1016/j.desal.2018.03.006.
[2] Photothermal membrane distillation for seawater desalination, Adv. Mater. 29 (2017) 1603504.
[3] When plasmonics meets membrane technology, J. Phys.: Condens. Matter 28 (2016) 363003.
[4] Stimuli responsive hydrogels prepared by frontal polymerization, Biomacromolecules 10 (2009) 2672.
[5] Tailoring the physical properties of nanocomposite films by the insertion of graphene and other nanoparticles, Compos. Part B: Eng. 60 (2014) 29.
[6] Noble Metals on the Nanoscale: Optical and Photothermal Properties and Some Applications in Imaging, Sensing, Biology, and Medicine, Acc. Chem. Res., 2008, 41 (12), pp 1578–1586.
Author Response
The paper by Muri et al. report on plasmonic nanoparticles in hydrogels for biosensing applications. The degree of innovation is good, as well as the potential impact. I recommend publication pending some amendments.
1) The potential use of noble-metal nanoparticles as nanofillers for composites for thermoplasmonic applications has not been clarified to the readers, in spite of the recent results reported by several groups [1-3].
Thank you for suggesting very interesting references. A paragraph about thermoplasmonics is now included in the introduction and clarified for the readers.
2) Figure 1 is useless
This is true. Thank you for confirming this. We were not sure whether this figure was informative or not.
3) The interesting paper on hydrogels by Alzari and coworkers should be cited [4], as well as their work on nanocomposites [5].
We see that these references are important for the manuscript and they have now been included in the introduction.
[1] Overcoming temperature polarization in membrane distillation by thermoplasmonic effects activated by Ag nanofillers in polymeric membranes, Desalination (2018) doi:10.1016/j.desal.2018.03.006.
[2] Photothermal membrane distillation for seawater desalination, Adv. Mater. 29 (2017) 1603504.
[3] When plasmonics meets membrane technology, J. Phys.: Condens. Matter 28 (2016) 363003.
[4] Stimuli responsive hydrogels prepared by frontal polymerization, Biomacromolecules 10 (2009) 2672.
[5] Tailoring the physical properties of nanocomposite films by the insertion of graphene and other nanoparticles, Compos. Part B: Eng. 60 (2014) 29.
[6] Noble Metals on the Nanoscale: Optical and Photothermal Properties and Some Applications in Imaging, Sensing, Biology, and Medicine, Acc. Chem. Res., 2008, 41 (12), pp 1578–1586.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
The authors improved their manuscript by editing abstract, introduction and stated the outline of their investigation more precisely.
Furthermore, the authors defended why they are not using complementary techniques for the characterization of the GNP-hydrogels and added references concerning the material characterization.
I would have preferred a thorough material characterization within this manuscript but I can understand the authors and would recommend publication of the manuscript.
Reviewer 2 Report
The reviewer is happy with those responses.
