The Versatile Photo-Thermal Behaviour of a 2-Hydroxyazobenzene
Round 1
Reviewer 1 Report
The manuscript of P.L. Gentili and co-workers "The versatile photothermal behaviour..." describes photochromism of a chlorinated 2-hydroxyazobenzene in a protonated and deprotonated state in three different media.
While the manuscript is methodologically correct and presents consistent results, it is of very low originality. It is a recurrent, although careful characterization of a known compound. Potential applications are only signalized (conclusions: "All these results demonstrate that (E)-3,4,6-trichloro-2-(p-diazenil)-phenol is a versatile compound to be used as a molecular probe of different micro-environments.", and "Finally, the versatile photochromic properties of (E)-3,4,6-trichloro-2-(p-diazenil)-phenol and its photoacid behaviour make it a promising candidate for implementing photochromic oscillators, as mentioned in the introduction." or "It is worth pursuing the implementation of photochromic oscillators because
they promise to be a breakthrough in neuromorphic engineering").
Therefore, in my oppinion, the current form of the manuscript does not bring substantial novelty that would rationalize its publication in the proposed journal. I would love to see a real demonstration of an oscillatory system (like in the publication of Prof. Gentili from 2017 in Angewandte) involving the presented compound. Otherwise, I would recommend submission of this manuscript to much more specialized journal in the area of photochemistry, which would be more suitable place for demonstrating the obtained set of analytical data.
I would be also glad to see more broadly in the introduction, how the work of the Gentili group is located among other researchers working in the same area, as the significant amount of references are self-citations, which almost brings the impression that the authors are a single group working in this area (which is unlikely given the described importance of the field).
Author Response
Reviewer 1
Open Review
(x) I would not like to sign my review report
( ) I would like to sign my review report
English language and style
( ) English very difficult to understand/incomprehensible
( ) Extensive editing of English language and style required
( ) Moderate English changes required
( ) English language and style are fine/minor spell check required
(x) I don't feel qualified to judge about the English language and style
|
Yes |
Can be improved |
Must be improved |
Not applicable |
|
|
Does the introduction provide sufficient background and include all relevant references? |
( ) |
(x) |
( ) |
( ) |
|
Are all the cited references relevant to the research? |
( ) |
(x) |
( ) |
( ) |
|
Is the research design appropriate? |
(x) |
( ) |
( ) |
( ) |
|
Are the methods adequately described? |
(x) |
( ) |
( ) |
( ) |
|
Are the results clearly presented? |
(x) |
( ) |
( ) |
( ) |
|
Are the conclusions supported by the results? |
( ) |
( ) |
(x) |
( ) |
Comments and Suggestions for Authors
The manuscript of P.L. Gentili and co-workers "The versatile photothermal behaviour..." describes photochromism of a chlorinated 2-hydroxyazobenzene in a protonated and deprotonated state in three different media.
While the manuscript is methodologically correct and presents consistent results, it is of very low originality. It is a recurrent, although careful characterization of a known compound. Potential applications are only signalized (conclusions: "All these results demonstrate that (E)-3,4,6-trichloro-2-(p-diazenil)-phenol is a versatile compound to be used as a molecular probe of different micro-environments.", and "Finally, the versatile photochromic properties of (E)-3,4,6-trichloro-2-(p-diazenil)-phenol and its photoacid behaviour make it a promising candidate for implementing photochromic oscillators, as mentioned in the introduction." or "It is worth pursuing the implementation of photochromic oscillators because
they promise to be a breakthrough in neuromorphic engineering"). Therefore, in my opinion, the current form of the manuscript does not bring substantial novelty that would rationalize its publication in the proposed journal. I would love to see a real demonstration of an oscillatory system (like in the publication of Prof. Gentili from 2017 in Angewandte) involving the presented compound. Otherwise, I would recommend submission of this manuscript to much more specialized journal in the area of photochemistry, which would be more suitable place for demonstrating the obtained set of analytical data.
Authors’ reply: This article gives concrete experimental and computational proofs that the (E)-3,4,6-trichloro-2-(p-diazenil)-phenol (t-DZH) and its conjugated phenoxide base (t-DZ) are valuable probes of micro-environments. Their performances as probes ground on their photochromic properties. Such properties have been investigated in three different media: (1) pure acetonitrile, (2) in water and acetonitrile mixed in a 1/1 volume ratio, and (3) in an aqueous micellar solution of 3-(N,N-Dimethylmyristylammonio)propanesulfonate (SB3-14). The analyses of the experimental data have been supported by quantum-mechanical DFT calculations and the Maximum Entropy Method. Cutting-edge algorithms, such as “Fuzzy Entropy” and “Colourability” have been applied to characterize the performances of t-DZH and t-DZ as probes of micro-environments.
Furthermore, the photochromic t-DZH and t-DZ are appropriate for implementing neural surrogates in the phasic excitable regime. To highlight this result, a new figure (Figure S17) and new sentences have been added. In paragraph 2.4 of the revised version of the manuscript, we explain that “phasic excitable neurons respond to external stimuli in an analog manner: the extent of DpH (shown in Figure 8) is proportional to the irradiation intensity when the concentration of t-DZH has been fixed. As soon as the irradiation is discontinued, the compound recovers the original state spontaneously and reversibly, as any phasic excitable neuron does. The reversibility is shown in Figure S17”.
The new figure S17 is shown in the Supplementary materials and n the pdf.
Figure S17. Three consecutive DpH cycles generated by the irradiation of t-DZH in H2O/CH3CN=1/1 (A) and SB3-14 (B).
Furthermore, a new sentence has been added to the conclusions of the revised version of the manuscript. The new sentence in the conclusions is reported below.
“Finally, the versatile photochromic properties of (E)-3,4,6-trichloro-2-(p-diazenil)-phenol and its photoacid behaviour make it appropriate for implementing neural surrogates in phasic excitable regime (as shown in Figure 8 and S17) and a promising candidate for implementing photochromic oscillators, as mentioned in the introduction.”
Finally, further work is underway to test the possibility of using t-DZH in the implementation of brand-new photochemical oscillators. In case of success, the results will be presented in the next articles.
Therefore, we are convinced that this work deserves to be published in the special issue “Cutting-edge Physical Chemistry Research in Europe” of the journal Molecules.
I would be also glad to see more broadly in the introduction, how the work of the Gentili group is located among other researchers working in the same area, as the significant amount of references are self-citations, which almost brings the impression that the authors are a single group working in this area (which is unlikely given the described importance of the field).
Authors’ reply: Just a few groups are working on the implementation of neural surrogates in wetware. They are mentioned in references 12, 13, 14, and 15. Reference 15 is a book and contains various contributions in the field of reservoir computing in wetware. We added one more citation: a review paper published rightly in January 2023 by Prof. Adamatzky and his collaborators. It is reference 16 of the revised version of the manuscript. As far as we know, no groups are working on the implementation of photochemical oscillators based on photochromic compounds. A pioneer in this field is Dr Jean-Claude Micheau (CNRS UMR 5623, IMRCP, University Toulouse III, Paul Sabatier, F-31062 Toulouse Cedex, France). He is collaborating with us, and he is cited in references 19 and 20 of the revised version of our manuscript. Finally, we are tracing a new path in the field of neuromorphic engineering by proposing the optical communication between neural surrogates implemented in wetware. These are the reasons why there are self-citations in this part of the introduction.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
t-DZH and its derivatives with the excellent photochromic properties have been previously examined for their potential application in light-dependent pH tailors and oscillators. Pier and etc provides theoretical understanding about the molecular orbitals of t-DZH that contribute to the photochemical properties. Secondly, the authors examined the photochromic properties of its deprotonated form as t-DZ with the very distinguishable photoisomerization properties from t-DZH. The paper is suitable to be published in Molecules after addressing the following minor points.
1. What is the kinetic rate of photoinduced Trans to Cis isomerization of t-DZH and t-DZ?
2. The authors noticed the isosbestic point during the photolysis of t-DZH in SB3-14 (Figure 4) and in CH3CN (Figure S11). Whether Cis t-DZH has distinguished absorption wavelength compared to trans t-DZH in these two solvents should be clarified.
3. Why the thermal transition of cis t-DZ to Trans t-DZ is significantly slower than the transition rate from cis t-DZH to trans t-DZH?
Author Response
Reviewer 2
Open Review
(x) I would not like to sign my review report
( ) I would like to sign my review report
English language and style
( ) English very difficult to understand/incomprehensible
( ) Extensive editing of English language and style required
( ) Moderate English changes required
(x) English language and style are fine/minor spell check required
( ) I don't feel qualified to judge about the English language and style
|
Yes |
Can be improved |
Must be improved |
Not applicable |
|
|
Does the introduction provide sufficient background and include all relevant references? |
(x) |
( ) |
( ) |
( ) |
|
Are all the cited references relevant to the research? |
(x) |
( ) |
( ) |
( ) |
|
Is the research design appropriate? |
(x) |
( ) |
( ) |
( ) |
|
Are the methods adequately described? |
(x) |
( ) |
( ) |
( ) |
|
Are the results clearly presented? |
(x) |
( ) |
( ) |
( ) |
|
Are the conclusions supported by the results? |
(x) |
( ) |
( ) |
( ) |
Comments and Suggestions for Authors
t-DZH and its derivatives with the excellent photochromic properties have been previously examined for their potential application in light-dependent pH tailors and oscillators. Pier and etc provides theoretical understanding about the molecular orbitals of t-DZH that contribute to the photochemical properties. Secondly, the authors examined the photochromic properties of its deprotonated form as t-DZ with the very distinguishable photoisomerization properties from t-DZH. The paper is suitable to be published in Molecules after addressing the following minor points.
Authors’ reply: We thank reviewer 2 for his/her appreciation of our work.
- What is the kinetic rate of photoinduced Trans to Cis isomerization of t-DZH and t-DZ?
Authors’ reply: The kinetic constants of the photo-induced trans-to-cis isomerization have been calculated as it has been asked by reviewer 2. The results are now reported in Table S8 of the revised version of the Supplementary Materials. We report Table S8 and its caption below.
Table S8. Kinetic constant values k(s-1) for the photochemical conversion of trans to cis determined by fitting the DA changes vs. time through the following function: .
|
|
SB3-14 |
H2O/CH3CN=1/1 |
CH3CN |
|
t-DZH |
0.018 |
0.012 |
0.045 |
|
t-DZ |
0.0077 |
0.010 |
0.010 |
- The authors noticed the isosbestic point during the photolysis of t-DZH in SB3-14 (Figure 4) and in CH3CN (Figure S11). Whether Cis t-DZH has distinguished absorption wavelength compared to trans t-DZH in these two solvents should be clarified.
Authors’ reply: The spectral position of the isosbestic point for the t-DZH and c-DZH depends on the solvent. It is at 304 nm in SB3-14, 300 nm in H2O/CH3CN=1/1, and 297.5 nm in CH3CN. This information has been added to the revised version of our paper as suggested by reviewer 2. It is on page 7, line 183.
- Why the thermal transition of cis t-DZ to Trans t-DZ is significantly slower than the transition rate from cis t-DZH to trans t-DZH?
Authors’ reply: The thermal reaction from c-DZ to t-DZ is significantly slower than that from c-DZH to t-DZH because the hydrogen atom of the hydroxyl group (which is present only in c-DZH and not c-DZ) establishes a hydrogen bond with the azo group. Such a hydrogen bond catalyzes the cis-to-trans conversion through the formation of the tautomeric phenylhydrazone with a partial breaking of the N=N bond, as we explain at the end of paragraph 2.3 and anticipate in the introduction of the revised version of our manuscript.
“The kinetics and mechanism of the thermal back reaction from cis to trans of hydroxyazobenzenes has been the subject of many theoretical and experimental investigations [29]. The presence of a hydrogen bond between the hydroxyl and azo groups favors the formation of a hydrazone-like intermediate with a partial breaking of the N=N bond. Hence, the rotation around the N-N bond can occur, facilitating the recovery of the more stable trans isomer.”
Author Response File: Author Response.pdf
Reviewer 3 Report
The authors perform an investigation of photochromism in a 2-hydroxyazobenzene (DZH) and its conjugate phenoxyde base DZ. Three different solvents are considered, and the spectral features of DZH and DZ are reported and analyzed also with the aid of quantum chemistry computations. The trans-cis photoisomerization and the thermal cis-trans are also investigated.
The manuscript is well written and deserves to be published after the authors have addressed the following remarks.
1) According to the authors, the observed red shift (with respect to azobenzene?) of the n-pi* band of t-DZH is expected. However, they should be more explicit on this point. In fact, on the one hand, the formation of an intramolecular hydrogen bond should bind the n orbital, shifting the n->pi band to the blue. On the other hand, there are two n orbitals... So, it is not that clear to me why the red shift is expected.
2) In order to have a rough idea about the strength of the intramolecular H bond in t-DZH, the author could perform a DFT calculation to obtain the energy of the t-DZH conformer obtained rotating the H atom around the C-O axis by 180 degrees (i.e., with the H atom facing Cl instead of N). That could be reported in the supporting information.
3) It is well known that phenol is much stronger acid in the excited S1 state than in the ground state. Now, the pH jump noted by the authors is rather due to photoisomerization (ground state c-DZH being stronger acid than t-DZH). Nevertheless, could the excited species have a role in the pH jump? Maybe the authors could briefly comment on this.
Author Response
Reviewer 3
Open Review
(x) I would not like to sign my review report
( ) I would like to sign my review report
English language and style
( ) English very difficult to understand/incomprehensible
( ) Extensive editing of English language and style required
( ) Moderate English changes required
( ) English language and style are fine/minor spell check required
(x) I don't feel qualified to judge about the English language and style
|
Yes |
Can be improved |
Must be improved |
Not applicable |
|
|
Does the introduction provide sufficient background and include all relevant references? |
(x) |
( ) |
( ) |
( ) |
|
Are all the cited references relevant to the research? |
(x) |
( ) |
( ) |
( ) |
|
Is the research design appropriate? |
(x) |
( ) |
( ) |
( ) |
|
Are the methods adequately described? |
(x) |
( ) |
( ) |
( ) |
|
Are the results clearly presented? |
(x) |
( ) |
( ) |
( ) |
|
Are the conclusions supported by the results? |
(x) |
( ) |
( ) |
( ) |
Comments and Suggestions for Authors
The authors perform an investigation of photochromism in a 2-hydroxyazobenzene (DZH) and its conjugate phenoxyde base DZ. Three different solvents are considered, and the spectral features of DZH and DZ are reported and analyzed also with the aid of quantum chemistry computations. The trans-cis photoisomerization and the thermal cis-trans are also investigated.
The manuscript is well written and deserves to be published after the authors have addressed the following remarks.
Authors’ reply: We thank reviewer 3 for his/her appreciation of our work.
1) According to the authors, the observed red shift (with respect to azobenzene?) of the n-pi* band of t-DZH is expected. However, they should be more explicit on this point. In fact, on the one hand, the formation of an intramolecular hydrogen bond should bind the n orbital, shifting the n->pi band to the blue. On the other hand, there are two n orbitals... So, it is not that clear to me why the red shift is expected.
Authors’ reply: As usual in azobenzenes, the transition n-pi* is towards the red if compared with the pi-pi* transitions. The original statement has been changed to make it clearer. Now, it is:
“The transition involving the lone pair of the nitrogen atom and the antibonding orbital of the azo group is shifted towards the red if compared with the transitions. Moreover, it is forbidden and dark according to the quantomechanical calculations (see Table 1). It is responsible for the long weak tail (having ) that extends up to 600 nm.”
2) In order to have a rough idea about the strength of the intramolecular H bond in t-DZH, the author could perform a DFT calculation to obtain the energy of the t-DZH conformer obtained rotating the H atom around the C-O axis by 180 degrees (i.e., with the H atom facing Cl instead of N). That could be reported in the supporting information.
Authors’ reply: We thank reviewer 3 for his/her suggestion. We performed a DFT calculation to estimate the strength of the intramolecular hydrogen bond established between the hydroxylic and azo groups. The result is reported in the main text of the revised paper. On page 11, lines 325-327, we say:
“The intramolecular hydrogen bond that establishes between the hydroxylic and the azo groups is of the order of 4.73 Kcal/mol according to the DFT simulations (see Figure S15).”
Furthermore, as suggested by reviewer 3, we report the image of the two conformers used to estimate the strength of the intramolecular hydrogen bond in Figure S15 of the Supplementary Materials. Finally, the atomic coordinates of the two conformers have been added.
3) It is well known that phenol is much stronger acid in the excited S1 state than in the ground state. Now, the pH jump noted by the authors is rather due to photoisomerization (ground state c-DZH being stronger acid than t-DZH). Nevertheless, could the excited species have a role in the pH jump? Maybe the authors could briefly comment on this.
Authors’ reply: The DpHs reported in Figure 8 have been recorded under stationary irradiation. Therefore, they are generated only by forming c-DZH at the ground state. As reviewer 3 noticed, phenol groups become stronger acid in their excited S1 states. However, time-resolved techniques and pulsed excitation are required to detect the DpHs generated by the excited S1 states of phenols. Moreover, it is worth noticing that it is tough to achieve significant DpH using excited-state photoacids due to their fast relaxation from the high-acidity excited state to the low-acidity ground state.
Author Response File: Author Response.pdf
Reviewer 4 Report
With reference to the manuscript "The versatile photo-thermal behavior of a 2-Hydroxyazobenzene" by: Pier Luigi Gentili, Antonio Capaccioni, Raimondo Germani, and Simona Fantacci (Molecules 2157817) I would like to emphasize once again that in my opinion it represents a high scientific level . I believe that the investigated effects of photo- and thermoisomerization of (E)-3,4,6-trichloro-2-(p-diazenil)-phenol (t-DZH) and its deprotonated counterpart dissolved in various wet media are very interesting from the point of view of basic research. In this particular case, we are dealing with a molecular system that simultaneously reacts to UV light and changes in the environment related to specific intermolecular interactions. These changes can be observed directly with the unaided eye or with the help of more accurate instruments that allow you to determine RGB chromatic coordinates. Although the authors quite strongly indicate the potential application of their molecular system in the technique of neural networks, it seems that this type of application is quite problematic, mainly due to the very long cis-to-trans isothermal relaxation time. A more realistic application of this type of systems can be expected in advanced detection and determination of the concentration of various chemical compounds (including pollutants) in water environments.
Some reservations may be raised about the length of the text of the manuscript, especially since the authors added an extensive supplement to it. The preparation of test materials, the measurement methods used, as well as the test results are presented in detail. In addition, the analysis of the spectroscopic test results was supported by DFT calculations. This approach is particularly welcome by potential readers who are directly involved in similar research and can directly verify the data presented or perform analogous research on similar systems for comparison purposes. A rather casual suggestion to the authors is that it might be more beneficial for a wider range of interested readers to limit the information provided in the supplement.
The scientific language of the manuscript is correct, no terminology errors were noted.
Submitted for publication in Molecules, this manuscript is an interesting and valuable contribution to the research on photo- and thermoisomerization in molecular systems and the search for the possibility of using such systems in practice.
Author Response
Reviewer 4
Open Review
( ) I would not like to sign my review report
(x) I would like to sign my review report
English language and style
( ) English very difficult to understand/incomprehensible
( ) Extensive editing of English language and style required
( ) Moderate English changes required
( ) English language and style are fine/minor spell check required
(x) I don't feel qualified to judge about the English language and style
|
Yes |
Can be improved |
Must be improved |
Not applicable |
|
|
Does the introduction provide sufficient background and include all relevant references? |
(x) |
( ) |
( ) |
( ) |
|
Are all the cited references relevant to the research? |
(x) |
( ) |
( ) |
( ) |
|
Is the research design appropriate? |
(x) |
( ) |
( ) |
( ) |
|
Are the methods adequately described? |
(x) |
( ) |
( ) |
( ) |
|
Are the results clearly presented? |
(x) |
( ) |
( ) |
( ) |
|
Are the conclusions supported by the results? |
(x) |
( ) |
( ) |
( ) |
Comments and Suggestions for Authors
The manuscript “The versatile photo-thermal behaviour of a 2-Hydroxyazobenzene” by Pier Luigi Gentili, Antonio Capaccioni, Raimondo Germani and Simona Fantacci presents the results of experimental research on the photochromic properties of (E)-3,4,6-trichloro-2-(p-diazenil)-phenol (t-DZH) and its conjugated phenoxide base (t-DZ) dissolved in three different media. Spectrophotometric and photochemical studies in the UV-Vis range were supported by quantum-mechanical DFT calculations using Gaussian09 software using B3LYP as the exchange–correlation functional and 6-311++g** as the basis set. The spectrophotometric thermal isomerization of the cis-to-trans isomers was analyzed through the Maximum Entropy Method using the MemExp Software. For solutions of the cis and trans forms, the chromaticity and RGB coordinates from the transmittance spectra were calculated using the CIE standardized sensitivity of human eye. These parameters are important when t-DZH and t-DZ are used as potential molecular probes of micro-environments, as well as photochemical oscillators for use in pacemaker neurons communicating through optical signals in wetware. The results of the study are described in detail and fully in the manuscript (some of the results are included in the Supplement). A detailed description of the preparation and synthesis of materials is presented, and the methods used are discussed in detail. The presented list of literature on the subject is extensive enough. The manuscript is well structured, stylistically correct, and generally needs no linguistic correction. The presented particular scientific problem is original and may be interesting from the point of view of potential applications of the presented molecular system, and may also be an inspiration for research on other similar systems. In my opinion, the submitted manuscript may be considered for publication in Molecules (MDPI) as presented.
Authors’ reply: We thank reviewer 4 for his/her appreciation of our work.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
The authors have sufficiently addressed my previous comment "I would love to see a real demonstration of an oscillatory system ... involving the presented compound." by adding the new experiment depicted in Figure S17. The concerns regarding high rate of self-citations vs. other groups were also convincingly addressed.
The purpose of the demonstrated research has been more clearly spelled out "phasic excitable neurons respond to external stimuli in an analog manner: the extent of DpH (shown in Figure 8) is proportional to the irradiation intensity when the concentration of t-DZH has been fixed. As soon as the irradiation is discontinued, the compound recovers the original state spontaneously and reversibly, as any phasic excitable neuron does. The reversibility is shown in Figure S17”."
And the authors also underlined more clearly, where novelty points are localized in their manuscript (e.g. ". Cutting-edge algorithms, such as “Fuzzy Entropy” and “Colourability” have been applied to characterize the performances of t-DZH and t-DZ as probes of micro-environments.")
Taking all the arguments into account, I recommend publication of the new version of manuscript with minor revision - the authors should read the manuscript again (or ask a friend from outside their group) in order to make more understandable to the readers (general audience, not necesarilly fully versed in the issues of neuronal surrogates in wetware and photochromic oscillators). Please, highlight better the novelty aspects of the manuscript, the same way as you did in the response to my comments. As I mentioned before, the novelty presented in the first version of the manuscript is (at least from the point of view of the photochromic researchers' community) not demonstrated clearly (as said, the compound is known, photochromism is standard). There has to be explicit, brief and clear explanation to the fact why this particular compound has been chosen and why the obtained results actually matter (more than photoswitching of any other photochromic compound). The updated version of the manuscript already contains the answer (the DpH pattern of this particular compound follows the pattern of neuronal behavior). Please, add a brief discussion why the t-DZ/t-DZH is the optimal choice here.
Author Response
Open Review
(x) I would not like to sign my review report
( ) I would like to sign my review report
English language and style
( ) English very difficult to understand/incomprehensible
( ) Extensive editing of English language and style required
( ) Moderate English changes required
( ) English language and style are fine/minor spell check required
(x) I don't feel qualified to judge about the English language and style
|
Yes |
Can be improved |
Must be improved |
Not applicable |
|
|
Does the introduction provide sufficient background and include all relevant references? |
(x) |
( ) |
( ) |
( ) |
|
Are all the cited references relevant to the research? |
(x) |
( ) |
( ) |
( ) |
|
Is the research design appropriate? |
(x) |
( ) |
( ) |
( ) |
|
Are the methods adequately described? |
(x) |
( ) |
( ) |
( ) |
|
Are the results clearly presented? |
(x) |
( ) |
( ) |
( ) |
|
Are the conclusions supported by the results? |
(x) |
( ) |
( ) |
( ) |
Comments and Suggestions for Authors
The authors have sufficiently addressed my previous comment "I would love to see a real demonstration of an oscillatory system ... involving the presented compound." by adding the new experiment depicted in Figure S17. The concerns regarding high rate of self-citations vs. other groups were also convincingly addressed.
The purpose of the demonstrated research has been more clearly spelled out "phasic excitable neurons respond to external stimuli in an analog manner: the extent of DpH (shown in Figure 8) is proportional to the irradiation intensity when the concentration of t-DZH has been fixed. As soon as the irradiation is discontinued, the compound recovers the original state spontaneously and reversibly, as any phasic excitable neuron does. The reversibility is shown in Figure S17”."
And the authors also underlined more clearly, where novelty points are localized in their manuscript (e.g. ". Cutting-edge algorithms, such as “Fuzzy Entropy” and “Colourability” have been applied to characterize the performances of t-DZH and t-DZ as probes of micro-environments.")
Taking all the arguments into account, I recommend publication of the new version of manuscript with minor revision - the authors should read the manuscript again (or ask a friend from outside their group) in order to make more understandable to the readers (general audience, not necesarilly fully versed in the issues of neuronal surrogates in wetware and photochromic oscillators). Please, highlight better the novelty aspects of the manuscript, the same way as you did in the response to my comments. As I mentioned before, the novelty presented in the first version of the manuscript is (at least from the point of view of the photochromic researchers' community) not demonstrated clearly (as said, the compound is known, photochromism is standard). There has to be explicit, brief and clear explanation to the fact why this particular compound has been chosen and why the obtained results actually matter (more than photoswitching of any other photochromic compound). The updated version of the manuscript already contains the answer (the DpH pattern of this particular compound follows the pattern of neuronal behavior). Please, add a brief discussion why the t-DZ/t-DZH is the optimal choice here.
Authors’ reply: We thank reviewer 1 for his/her suggestion. In the new version of our manuscript, we have stressed the novelty aspects of the manuscript as we did in response to the previous reviewer’s comments. At the end of the introduction, on page 3 of the revised version of our manuscript, we have added the following statements:
“In this work, (E)-3,4,6-trichloro-2-(p-diazenil)-phenol and its conjugated base are also evaluated as probes of different micro-environments. Their performances as probes are assessed through cutting-edge algorithms, such as “Fuzzy Entropy” and “Colourability”.
Moreover, as suggested by reviewer 1, we have added new sentences in paragraph 2.4 wherein we explain why we have chosen (E)-3,4,6-trichloro-2-(p-diazenil)-phenol. The new sentences are also reported below.
“The photo-acid behaviour makes t-DZH capable of communicating with other compounds chemically through the release of a proton. This property extends the possibility of coupling a pair of photochromic compounds that can interplay through light and protons. They might allow the implementation of the first photochemical oscillator as a dynamical model of a pacemaker neuron. Anyway, a solution of the simple t-DZH can be exploited to mimic the dynamics of neurons in the phasic excitable regime, which respond to external stimuli in an analog manner [24,39]. The extents of DpH (shown in Figure 8) are proportional to the irradiation intensity when the concentration of t-DZH has been fixed. As soon as the irradiation is discontinued, the compound recovers the original state spontaneously and reversibly, as any phasic excitable neuron does. The reversibility is shown in Figure S17.”
Finally, the reasons why the results matter are highlighted in the revised version of the Conclusions when we say:
“…The experimental results and their interpretation through cutting-edge algorithms, such as Fuzzy Entropy and Colourability, demonstrate that (E)-3,4,6-trichloro-2-(p-diazenil)-phenol is a versatile compound to be used as a molecular probe of different micro-environments…
…the versatile photochromic properties of (E)-3,4,6-trichloro-2-(p-diazenil)-phenol and its photoacid behaviour make it appropriate for implementing neural surrogates in the phasic excitable regime and a promising candidate for implementing photochromic oscillators, as mentioned in the introduction. t-DZH can interplay with other photochromic compounds not only through light but also through photo-induced proton exchange….”
Author Response File: Author Response.pdf
