Dual pH- and Temperature-Responsive Fluorescent Hybrid Materials Based on Carbon Dot-Grafted Triamino-Tetraphenylethylene/N-Isopropylacrylamide Copolymers

Round 1

Reviewer 1 Report

The manuscript entitled "Dual pH- and Temperature-Responsive Fluorescent Hybrid Materials Based on Carbon Dot-Grafted Triaminotetrastyrene/N-isopropylacrylamide Copolymers” describes the synthesis and carbon dots and their functionalization with Triaminotetrastyrene&N-isopropylacrylamide monomers to create hybrids with sensitivity to pH and temperature. The manuscript is well written, the abstract and introduction are good, and the results are clear and consistent. However, it has been very difficult to review the paper with the figures uploaded in different folders.

The main concern about this manuscript is that it is very similar to the next one already published by the authors:

Yang, S.; Liao, B.; Yi, S.; Liang, E.; He, B. Tetraphenylethylene and N-isopropylacrylamide block polymer-grafted carbon dots with their application in cellular imaging. Mater. Today Chem. 2022, 23.

Here they use a different ligand (with amino groups) but the reasons to change it are not described. The main differences between both studies, the comparison of the results and the progress (if any) that the new hybrids promote should be discussed to accept the manuscript in order to be accpeted for its publication.

In addition, some changes, addressed below, are needed to improve the readability of the manuscript. 

 Please, address the following suggestions:

Figures and schemes are missing in the main text. Please add the figures with the correct number and captions to the main manuscript.

Please, provide an SI document that contains all the SI figures with correct numbers and captions.

A scheme showing the chemical structure of the starting ligands and/or the final structure of the ligands attached to the CDs is recommended to follow the discussion of the results.

Scheme 1. Please, add solvent and temperature to the reaction scheme.

Title, Abstract and Page 2: Triaminotetrastyrene is not the correct name for the chemical compound shown in scheme 1 in the materials section. This name can be used as an acronym thereafter.

Page 4. FTIR characterization. Please add the label of the most important peaks to the spectra shown in figure S1. The bands at the region between 3200 and 3350 cm-1 assigned to the N-H stretching region of CDs-PNAT are already present in the spectrum of CD-DATC derivatives. Therefore, it can not be pointed out to confirm the functionalization with ATPE and NIPAM. Same with other bands.

Page 4. 1H-NMR characterization. All the peaks in the spectrum should be assigned. The image has very low resolution and the multiplet are not visible. Please, enhance the figure. Please, specify which spectrum is shown; derivative CDs-PNAT1, 2, 3 or 4? Have you seen differences in the intensity of the peaks with the quantity of ATPE? This will support the fluorescence result discussions.

Page 4. TEM characterization. Please, provide figures with similar scales so they can be comparable. Please, specify the solvent. Bigg aggregates are present in the image of CDs-PNATs, so the sentence “no significant agglomeration” can not be used. In addition, specify the derivative 1, 2, 3 or 4?

Page 4. Figure S3 shows the maximum at 400 nm under 340 nm excitation wavelength. Please, check this.

Page 5. 3.2.2. Please, specify the excitation wavelength.

Page 5. The fluorescence is not quenched with pH; it is the opposite: the increment in the pH increases the signal.

Page 6. Figure 9. The decrease in the diameter to 15 nm described in the text is not observed in the figure.

 

Author Response

Comments 1: The manuscript entitled "Dual pH- and Temperature-Responsive Fluorescent Hybrid Materials Based on Carbon Dot-Grafted Triaminotetrastyrene/N-isopropylacrylamide Copolymers” describes the synthesis and carbon dots and their functionalization with Triaminotetrastyrene&N-isopropylacrylamide monomers to create hybrids with sensitivity to pH and temperature. The manuscript is well written, the abstract and introduction are good, and the results are clear and consistent. However, it has been very difficult to review the paper with the figures uploaded in different folders.

The main concern about this manuscript is that it is very similar to the next one already published by the authors:

Yang, S.; Liao, B.; Yi, S.; Liang, E.; He, B. Tetraphenylethylene and N-isopropylacrylamide block polymer-grafted carbon dots with their application in cellular imaging. Mater. Today Chem. 2022, 23.

Here they use a different ligand (with amino groups) but the reasons to change it are not described. The main differences between both studies, the comparison of the results and the progress (if any) that the new hybrids promote should be discussed to accept the manuscript in order to be accpeted for its publication.

In addition, some changes, addressed below, are needed to improve the readability of the manuscript.

Response 1: Thanks for the reviewer’s professionalization and carefulness. We have made corresponding revisions in the revised draft to explain this. These sentences on page 2 line 14 to 17 “Building upon these foundational developments, we engineered a stimuli-responsive hybrid material through controlled copolymerization of Triamino-tetraphenylethylene (ATPE) and N-isopropylacrylamide (NIPAM) at optimized molar ratios, followed by covalent grafting onto CDs surfaces.” were revised into “Building upon previous developments where we grafted TPE-NIPAM copolymers onto CDs to prepare AIE nanoparticles [16], we encountered limitations in aqueous dispersion due to TPE's inherent hydrophobicity. To address this, we used triamino-tetraphenylethylene (ATPE), a hydrophilic and pH-responsive TPE derivative. Through controlled copolymerization of ATPE and NIPAM at optimized molar ratios followed by covalent grafting onto CDs surfaces, we successfully engineered a novel stimuli-responsive hybrid material system.” 

Comments 2:Figures and schemes are missing in the main text. Please add the figures with the correct number and captions to the main manuscript.

Please, provide an SI document that contains all the SI figures with correct numbers and captions.

Response 2: Thanks for the reviewer’s carefulness. Some figures and tables are used as “Supplementary Materials”. The correct number and captions were showed in the part of the “Supplementary Materials”. The Scheme and Figure captions including “Supplementary Information” (SI) were attached at the end of the article in the revised version of the carbon-3647589. The “Scheme and Figure captions” was added on page 10 in blue.

Comments 3:A scheme showing the chemical structure of the starting ligands and/or the final structure of the ligands attached to the CDs is recommended to follow the discussion of the results.

Scheme 1. Please, add solvent and temperature to the reaction scheme.

Title, Abstract and Page 2: Triaminotetrastyrene is not the correct name for the chemical compound shown in scheme 1 in the materials section. This name can be used as an acronym thereafter.

Response 3: Thanks for the reviewer’s professionalization. We have accepted the suggestions. The relevant revisions are shown in the revised Title, Abstract, main text and Scheme 1( in blue). 

Comments 4:Page 4. FTIR characterization. Please add the label of the most important peaks to the spectra shown in figure S1. The bands at the region between 3200 and 3350 cm-1 assigned to the N-H stretching region of CDs-PNAT are already present in the spectrum of CD-DATC derivatives. Therefore, it can not be pointed out to confirm the functionalization with ATPE and NIPAM. Same with other bands.

Response 4: We appreciate the reviewer's professional comments. The key characteristic peaks have been clearly indicated in the revised Figure S1. During our experimental process, the CD-DATC sample might have been contaminated, as it does not show the characteristic double peaks at 3200-3350 cm^-1. We have now provided the revised FTIR spectrum of CD-DATC. The double peaks at 3200-3350 cm^-1 represent typical N-H stretching vibrations, which are exclusively present in CDs-PANT. This observation confirms the successful grafting of the polymer onto the CDs.

Comments 5:Page 4. 1H-NMR characterization. All the peaks in the spectrum should be assigned. The image has very low resolution and the multiplet are not visible. Please, enhance the figure. Please, specify which spectrum is shown; derivative CDs-PNAT1, 2, 3 or 4? Have you seen differences in the intensity of the peaks with the quantity of ATPE? This will support the fluorescence result discussions.

Response 5: We sincerely appreciate the reviewers for their invaluable suggestions. In the previously submitted version, we labeled the chemical structures corresponding to several characteristic absorption peaks in Figure S2. The resolution of the figure had been improved. In response to the comments, we have provided a clearer ¹H-NMR spectrum of CD-PNAT4 and carried out a detailed peak assignment analysis for all peaks. For the ¹H-NMR measurements of other samples, taking into account both the experimental cost and efficiency, we chose the representative CD-PNAT4 sample for testing. This selection was founded on the following experimental findings:  

• with the increase in the feeding amount of ATPE, the color of the samples deepened gradually, and their dispersibility in a weakly acidic aqueous solution (pH = 6.8) decreased remarkably.
• The variations in these physical properties exhibited a distinct correlation with the ATPE content. Thus, we contend that testing a single typical sample (CD-PNAT4) suffices to comprehensively reflect the relevant regularities.

It is worth noting that we chemically grafted a polymer onto the surface of the carbon dots. Compared with small molecule compounds, polymers are mixtures, and the assignment of their ¹H-NMR peaks is to some extent different from that of pure small molecule compounds. Therefore, in the aspect of material chemical structure characterization, we mainly focus on the changes of key chemical functional groups and the successful verification of grafting reactions. By selecting representative sample (CD-PNAT4) and combining the results of FTIR, we believe that the current structural characterization can fully reflect the chemical properties of the materials. We earnestly hope that the reviewers will find our explanation acceptable. The sentences “The amide protons (–CONH–) manifested as a multiplet spanning δ = 4.08–4.17 ppm. Diagnostic aromatic proton resonances for benzoxazine-bound ArH appeared as three distinct peaks at δ = 5.91, 5.98, and 6.21 ppm, while the primary amine (–NH₂) resonance emerged at δ = 5.34 ppm” were revised into “The amide protons (–CONH–) manifested as a multiplet spanning δ = 4.08–4.17 ppm. Diagnostic aromatic proton resonances for benzoxazine-bound ArH appeared as three distinct peaks at δ = 5.61, 6.25, and 6.75 ppm, while the primary amine (–NH₂) resonance emerged at δ = 5.80–5.98 ppm.”   

Comments 6:Page 4. TEM characterization. Please, provide figures with similar scales so they can be comparable. Please, specify the solvent. Bigg aggregates are present in the image of CDs-PNATs, so the sentence “no significant agglomeration” can not be used. In addition, specify the derivative 1, 2, 3 or 4?

Response 6: Thanks for the reviewer’s professionalization again. These suggestions are fantastic for the improvement of our paper. We were shameful for our poor and ambiguous description in the “no significant agglomeration”. The sentence “In contrast, CDs-PNATs showed a size range of approximately 20–30 nm, with relatively homogeneous dispersion and no significant agglomeration” on page 5 line 5 was revised into “In contrast, CDs-PNATs showed a size range of approximately 20–30 nm, with relatively homogeneous dispersion”. The solvent used for the characterization of TEM was THF. Regarding the issue of the TEM morphologies ratio, the current ratio value we had chosen should be reasonable. If a uniform scale is applied, it will affect both the image quality and the length of the scale. Thank you for reviewer’ understanding.

Comments 7:Page 4. Figure S3 shows the maximum at 400 nm under 340 nm excitation wavelength. Please, check this.

Page 5. 3.2.2. Please, specify the excitation wavelength.

Page 5. The fluorescence is not quenched with pH; it is the opposite: the increment in the pH increases the signal.

Page 6. Figure 9. The decrease in the diameter to 15 nm described in the text is not observed in the figure.

Response 7: We sincerely appreciate the reviewer's valuable comments. Regarding the revisions: for Figure S3, we have corrected the maximum wavelength to 425 nm (marked in blue in the revised manuscript on page 5 in 3.2.1). In 3.2.2, the sentence “On basis of the above characterization of fluorescence performances, in the subsequent study of fluorescence response performance, we uniformly adopted 360 nm as the excitation wavelength.” was added on page 7 in 3.2.2. The sentence “...attributable to reduced phenylamino density enabling...” on page 7 in 3.2.2 was revised into “ ...attributable to elevated phenylamino density enabling...”. The sentence “...displayed higher transition...” on page 7 in 3.2.2 was revised into “ ...displayed lower transition...”. The sentence “...exhibited linear fluorescence quenching...” on page 8 2^nd paragraph was revised into “ ..exhibited linear fluorescence enhancement...” (highlighted in blue). For Figure 9, we acknowledge that the original presentation showing 15 nm nanoparticle size at 35 °C could be misleading. We have carefully revised this figure to ensure accurate representation of the data.

Author Response File: Author Response.pdf

Reviewer 2 Report

In general, the paper is well prepared and presents new and interesting information; however, I cannot recommend this manuscript because all figures and tables are missing from both the main text and the supplementary files, making it impossible to adequately evaluate the manuscript.

Detailed comments will be possible after including figures.

Author Response

Reviewer #2:

Comments 1: In general, the paper is well prepared and presents new and interesting information; however, I cannot recommend this manuscript because all figures and tables are missing from both the main text and the supplementary files, making it impossible to adequately evaluate the manuscript.

        Detailed comments will be possible after including figures.

Response 1: First of all, we sincerely appreciate your recognition of our work. We are very sorry that we were not ready for the submission of the manuscript prepared on our part. We have already uploaded the figures and tables into the submission system; nonetheless, further verification may be required from the journal's editorial office. Should it be necessary, we kindly request the editorial office to assist in resubmitting the revised version for your review.

Author Response File: Author Response.pdf

Reviewer 3 Report

In the manuscript titled “Dual pH- and Temperature-Responsive Fluorescent Hybrid Materials Based on Carbon Dot-Grafted Triaminotetrastyrene/N-isopropylacrylamide Copolymers”, the authors report a series of pH- and temperature-responsive copolymers synthesized via reversible addition-fragmentation chain-transfer (RAFT) polymerization. The copolymers undergo transition between emissive state (fluorescence due to aggregation-induced emission effect) and non-emissive state in response to temperature or pH-change. The current study closely resembles the group’s previous work (Reference #16), with the primary distinction being the incorporation of amine groups in the aggregating molecule thus making the copolymer pH-responsive. The reviewer does not recommend publication of the manuscript. Few suggestions for the authors:

1. The copolymers have carbon dots (CDs) as end group, which make the copolymers fluorescent in non-aggregated state. Does CDs influence the polymer properties in other ways? In other words, what is the specific role or necessity of CDs in the copolymer?
2. Pg 4, Sec 3.2.1, 1^st paragraph: The authors have written, “This divergence correlates with the structural characteristics of the hybrids: the ATPE moieties within CDs-PNATs undergo AIE under acidic conditions.” Just saying that the ATPE moieties within CDs-PNATs undergo AIE under acidic conditions contradicts (to some extent) the following statements made by the authors in the Introduction: “Under acidic conditions (pH < pKa), protonation of ATPE stabilizes materials dispersion through electrostatic repulsion, effectively suppressing the AIE effect. Conversely, at elevated pH (pH > pKa), deprotonation induces ATPE aggregation via hydrophobic interactions, leading to progressively enhanced AIE-driven fluorescence at 500 nm.”

While it is true that CD-PNAT3 and CD-PNAT4 exhibit AIE at pH 4.8 (certainly acidic), the explanation could be more accurate. I think a brief explanation on the effect of pH (pH>pKa or pH<pKa) and the resulting balance between the hydrophilicity of the protonated amines and the hydrophobicity of the phenyl groups would be helpful to the readers.

3. Pg 5, Sec 3.2.2, 3^rd paragraph: The authors have written, “CDs-PNAT2 exhibited analogous behavior with a lower transition pH (at 4.98), attributable to reduced phenylamino density enabling earlier deprotonation-induced aggregation.” This sentence might be confusing to some of the readers because CD-PNAT2 has higher phenylamino content compared to CDs-PNAT1 as per Table S1. Please clarify.
4. In the same paragraph as in #2 the authors have written, “In contrast, CDs-PNAT3 and CDs-PNAT4 displayed higher transition pH values (at 4.22 and 4.11 respectively), consistent with their elevated phenyl-amino content requiring stronger acidity for complete protonation.” The transition pH values for CDs-PNAT1 and CDs-PNAT2 as reported by the authors are 5.23 and 4.98 respectively, which means that CDs-PNAT3 and CDs-PNAT4 displayed lower transition pH values. Please correct this error.
5. Figures, tables, and schemes are missing from the main manuscript draft. I believe there is a separate file that the authors need to upload.
6. For all figures with the fluorescence emission data (main manuscript or SI) please mention the concentration of the polymer solution taken.
7. Figure S4: Why do CDs-PNAT1 exhibit much stronger fluorescence intensity compared to CDs-PNAT2, CDs-PNAT3, and CDs-PNAT4. Wouldn't one expect polymers containing a higher proportion of aggregating molecules i.e., phenylamino groups to exhibit stronger fluorescence?

Same comment for Figure S5.

8. Table S1: It will be helpful to the readers if instead of the weight ratios, the authors express in terms of the equivalents of chain transfer agent and monomers taken. Also, please determine the degree of polymerization of the two monomers in the copolymer series.

Please see the comments above.

Author Response

Reviewer #3:

Comments 1:In the manuscript titled “Dual pH- and Temperature-Responsive Fluorescent Hybrid Materials Based on Carbon Dot-Grafted Triaminotetrastyrene/N-isopropylacrylamide Copolymers”, the authors report a series of pH- and temperature-responsive copolymers synthesized via reversible addition-fragmentation chain-transfer (RAFT) polymerization. The copolymers undergo transition between emissive state (fluorescence due to aggregation-induced emission effect) and non-emissive state in response to temperature or pH-change. The current study closely resembles the group’s previous work (Reference #16), with the primary distinction being the incorporation of amine groups in the aggregating molecule thus making the copolymer pH-responsive. The reviewer does not recommend publication of the manuscript. Few suggestions for the authors:

1.The copolymers have carbon dots (CDs) as end group, which make the copolymers fluorescent in non-aggregated state. Does CDs influence the polymer properties in other ways? In other words, what is the specific role or necessity of CDs in the copolymer?

Response 1: Thanks for the reviewer’s professionalization and carefulness. In our study, we have designed a core-shell structured AIE nanoparticle, in which carbon dots (CDs) serve as the core and the polymer functions as the shell. The rationale behind designing such a core-shell structured nanoparticle is two-fold. Firstly, CDs, as a type of fluorescent entity, can guarantee that the nanoparticles maintain their fluorescence properties even in the absence of aggregation-induced emission (AIE) phenomena. This is particularly relevant when the nanoparticles are dispersed in an organic solvent such as THF. Secondly, CDs, in their role as the core, can effectively inhibit the excessive aggregation of the polymer shell. This helps to prevent the unnecessary aggregation of single-chain polymers, which could otherwise affect the performance and properties of the nanoparticles. We tended to focus more on explaining the role of polymers but overlook the background introduction of CDs, which made the section of “introduction” difficult to understand. According to the reviewer's suggestion, these sentences on page 2 line 14 to 17 “Building upon these foundational developments, we engineered a stimuli-responsive hybrid material through controlled copolymerization of Triamino-tetraphenylethylene (ATPE) and N-isopropylacrylamide (NIPAM) at optimized molar ratios, followed by covalent grafting onto CDs surfaces.” were revised into “Building upon previous developments where we grafted TPE-NIPAM copolymers onto CDs to prepare AIE nanoparticles [16], we encountered limitations in aqueous dispersion due to TPE's inherent hydrophobicity. To address this, we used triamino-tetraphenylethylene (ATPE), a hydrophilic and pH-responsive TPE derivative. Through controlled copolymerization of ATPE and NIPAM at optimized molar ratios followed by covalent grafting onto CDs surfaces, we successfully engineered a novel stimuli-responsive hybrid material system”.

  Comments 2:2. Pg 4, Sec 3.2.1, 1st paragraph: The authors have written, “This divergence correlates with the structural characteristics of the hybrids: the ATPE moieties within CDs-PNATs undergo AIE under acidic conditions.” Just saying that the ATPE moieties within CDs-PNATs undergo AIE under acidic conditions contradicts (to some extent) the following statements made by the authors in the Introduction: “Under acidic conditions (pH < pKa), protonation of ATPE stabilizes materials dispersion through electrostatic repulsion, effectively suppressing the AIE effect. Conversely, at elevated pH (pH > pKa), deprotonation induces ATPE aggregation via hydrophobic interactions, leading to progressively enhanced AIE-driven fluorescence at 500 nm.”

While it is true that CD-PNAT3 and CD-PNAT4 exhibit AIE at pH 4.8 (certainly acidic), the explanation could be more accurate. I think a brief explanation on the effect of pH (pH>pKa or pH<pKa) and the resulting balance between the hydrophilicity of the protonated amines and the hydrophobicity of the phenyl groups would be helpful to the readers.

Response 2: We sincerely appreciate the reviewer's valuable suggestions. Regarding this particular explanation, we fully accept the reviewer's comments and have accordingly revised the relevant section in the manuscript. The sentence on page 5 in 3.2.1 “This divergence correlates with the structural characteristics of the hybrids: the ATPE moieties within CDs-PNATs undergo AIE under acidic conditions [29,30]” was revised into “The observed divergence arises from pH-dependent states of ATPE units in CDs-PNATs. At pH < pKa, protonated ATPE maintains dispersion through electrostatic repulsion, suppressing AIE. At pH > pKa, deprotonation induces ATPE aggregation via hydrophobic interactions, enhancing AIE fluorescence at 500 nm.”, which was highlighted in blue for easy reference. The relevant references [29,30] had been deleted. So the original citation numbers have been rearranged.

Comments 3:1.Pg 5, Sec 3.2.2, 3rd paragraph: The authors have written, “CDs-PNAT2 exhibited analogous behavior with a lower transition pH (at 4.98), attributable to reduced phenylamino density enabling earlier deprotonation-induced aggregation.” This sentence might be confusing to some of the readers because CD-PNAT2 has higher phenylamino content compared to CDs-PNAT1 as per Table S1. Please clarify.

Response 3: We were shameful for our poor and ambiguous description. It was our fault, we have corrected it. The sentence “...attributable to reduced phenylamino density enabling...” on page 7 in 3.2.2 was revised into “ ...attributable to elevated phenylamino density enabling...”. The sentence “...displayed higher transition...” on page 7 in 3.2.2 was revised into “...displayed lower transition...”. The sentence “...exhibited linear fluorescence quenching...” on page 8 2^nd paragraph was revised into “ ...exhibited linear fluorescence enhancement...” (highlighted in blue).  

Comments 4:2. In the same paragraph as in #2 the authors have written, “In contrast, CDs-PNAT3 and CDs-PNAT4 displayed higher transition pH values (at 4.22 and 4.11 respectively), consistent with their elevated phenyl-amino content requiring stronger acidity for complete protonation.” The transition pH values for CDs-PNAT1 and CDs-PNAT2 as reported by the authors are 5.23 and 4.98 respectively, which means that CDs-PNAT3 and CDs-PNAT4 displayed lower transition pH values. Please correct this error.

Response 4: It was our fault, we have corrected it. The sentence “...attributable to reduced phenylamino density enabling...” on page 7 in 3.2.2 was revised into “ ...attributable to elevated phenylamino density enabling...”. The sentence “...displayed higher transition...” on page 7 in 3.2.2 was revised into “...displayed lower transition...”. The sentence “...exhibited linear fluorescence quenching...” on page 8 2^nd paragraph was revised into “ ...exhibited linear fluorescence enhancement...” (highlighted in blue).  

Comments 5: 3. Figures, tables, and schemes are missing from the main manuscript draft. I believe there is a separate file that the authors need to upload.

Response 5: We are very sorry that we were not ready for the submission of the manuscript prepared on our part. We have already uploaded the figures and tables into the submission system; nonetheless, further verification may be required from the journal's editorial office. Should it be necessary, we kindly request the editorial office to assist in resubmitting the revised version for your review.

 Comments 6: 4.For all figures with the fluorescence emission data (main manuscript or SI) please mention the concentration of the polymer solution taken.

Response 6: Thanks for the reviewer’s professionalization. We marked the concentration of the solution on the relevant figure captions. The changes are respectively shown in Figure 2, 4, 8, 9 and Figure S3, S4, S5, S6.

Comments 7: 5. Figure S4: Why do CDs-PNAT1 exhibit much stronger fluorescence intensity compared to CDs-PNAT2, CDs-PNAT3, and CDs-PNAT4. Wouldn't one expect polymers containing a higher proportion of aggregating molecules i.e., phenylamino groups to exhibit stronger fluorescence?

Same comment for Figure S5.

Response 7: Thank you for the reviewers’ attention. Regarding the phenomenon that the apparent fluorescence intensity of CDs-PANT1 in Figure S4 is higher than that of CDs-PNAT2, CDs-PNAT3 and CDs-PNAT4, it should be particularly noted that this is not due to the difference in the intrinsic luminescence intensity of the samples, but rather is caused by different test parameter settings. Specifically, CDs-PANT1, lacking the AIE effect, has a relatively weak fluorescence intensity. Therefore, a larger slit width was adopted during the test (excitation slit 10 nm, emission slit 5 nm). Other samples exhibited significant aggregation-induced emission (AIE) effects, with relatively strong fluorescence intensities. If the same slit widths were used, signal saturation might occur in the region with the strongest AIE effect. To avoid this issue, we selected smaller slit widths (excitation slit 2 nm, emission slit 5 nm) for testing. This differential setting resulted in the apparent differences in fluorescence intensity shown in the figures, which actually reflected our optimization of the testing process according to the characteristics of different samples. The issue in Figure S5 is also attributed to the same parameter setting.

Comments 8: 1.Table S1: It will be helpful to the readers if instead of the weight ratios, the authors express in terms of the equivalents of chain transfer agent and monomers taken. Also, please determine the degree of polymerization of the two monomers in the copolymer series.

Response 8: We sincerely appreciate the valuable suggestions from the reviewers. The reason for using the weight ratio instead of the equivalent ratio for the reactant feeding ratio is elaborated as follows. The chain transfer agent (DATC) is grafted onto the surface of CDs to form the CD-DATC complex. As nanoparticles, the molecular weight of CDs is extremely challenging to measure accurately. Consequently, it is not feasible to adopt the conventional equivalent ratio measurement method. Instead, we have to choose the weight ratio.

  Regarding the determination of the polymerization degree of the two monomers within the polymer series, in theory, it can be calculated by the ratio of the peak area of the monomer's characteristic peak to that of the characteristic peak of the chain transfer agent (-CH₃) on the surface of carbon dots in the ¹H NMR spectrum. Nonetheless, our earlier investigations (as detailed in our previously published work, see Re. RSC Advances, 4, 57683, 2014) have revealed that the polymerization degree measured by this approach exhibits a relatively large error, rendering it of limited practical utility. Hence, in the present study, no quantitative characterization of the polymerization degree of this series of polymers was conducted.

Reviewer 4 Report

Reviewer comments

       The manuscript presents an innovative study on dual pH- and temperature responsive fluorescent hybrid materials based on carbon dot-grafted copolymers of triaminotetrastyrene and N-isopropylacrylamide. The topic aligns well with the journal's scope and demonstrates potential for smart sensing applications. However, there are several critical areas that require further clarification. Key concerns include the need for deeper mechanistic insights, additional characterization to confirm polymer grafting and structural integrity, and improved quantitative analysis of fluorescence response and reversibility. Furthermore, selectivity, reproducibility, and stability under practical conditions are not sufficiently addressed. I believe the manuscript has merit, but major revisions are necessary before it can be considered for publication. I encourage the authors to carefully address the detailed comments and questions to enhance the scientific rigor and overall impact of the work.

1. What is the exact role of carbon dots (CDs) in the hybrid system are they acting purely as a fluorescence donor, or do they also influence polymer behavior or aggregation?
2. The authors mention pKa-dependent fluorescence switching but do not provide experimental pKa values for the ATPE moieties. Can the authors estimate or determine the pKa to support the fluorescence transitions observed?
3. The fluorescence intensity changes are attributed to AIE effects. Were fluorescence lifetime measurements conducted to confirm the non-radiative decay suppression mechanism?
4. How do the authors ensure that the observed fluorescence changes are not due to pH- or temperature-dependent degradation or detachment of the polymer chains from the CDs?
5. Can the authors clarify whether the sensor operates effectively in complex media (e.g., biological fluids, ionic strength variation)? What is the selectivity and sensitivity under real conditions?
6. What is the response time and recovery time of the sensor under both pH and temperature switching? Are these parameters suitable for real-time sensing?
7. Regarding reversibility: how many cycles can the material endure without significant signal degradation? Only 4 cycles are shown what happens after 10 or 20?
8. What are the long-term photostability and environmental stability (e.g., under light, air, moisture) of the CDs-PNAT films for practical sensing deployment?
9. Why were specific molar ratios of ATPE to NIPAM chosen for the copolymer synthesis?
10. What is the exact chemical structure of Acr-ATPE?
11. How do the authors distinguish between CD emission and ATPE-based AIE emission in the overlapping spectra?
12. What is the long-term stability (weeks/months) of these hybrid materials under ambient conditions?
13. Have the authors confirmed covalent bonding (vs. physical adsorption) of polymers on the CDs? XPS or detailed FTIR peak shifts are needed.
14. How scalable is the synthesis process for practical device or commercial applications?

Author Response

Reviewer #4:

Comments 1: 1.What is the exact role of carbon dots (CDs) in the hybrid system are they acting purely as a fluorescence donor, or do they also influence polymer behavior or aggregation?

Response 1: We sincerely appreciate the suggestions put forward by the reviewers. The AIE nanoparticles we designed feature a core-shell structure, with carbon dots (CDs) serving as the core and polymers as the shell. The merits of this design are elaborated as follows. First, CDs, functioning as the fluorescent core, are capable of preserving the fluorescence characteristics of the nanoparticles even under non-AIE conditions (for example, in a THF dispersion system). Second, the CDs core structure can efficiently modulate the aggregation behavior of the polymer shell. It can not only induce the AIE effect but also prevent the excessive aggregation of single-chain polymers.

Comments 2: 2. The authors mention pKa-dependent fluorescence switching but do not provide experimental pKa values for the ATPE moieties. Can the authors estimate or determine the pKa to support the fluorescence transitions observed? 

Response 2: We sincerely appreciate the valuable suggestions from the reviewers. Regarding the determination of the pKa values of CDs-PANT nanoparticles, an important phenomenon was observed through experiments: the pKa value of the nanoparticles showed a negative correlation with the APTE content (an increase in APTE content leaded to a decrease in the pKa value). The reason why the pKa values were not systematically determined in this study is mainly based on the following considerations:

• The core objective of the research is to clarify the pH-induced AIE phenomenon and its mechanism;
• The focus is on the critical pH conditions for AIE occurrence and their regulatory patterns;
• The quantitative relationship between the pKa value and the critical pH for AIE belongs to a deeper level of mechanism issue, which is beyond the main scope of the current research. This discovery provides important clues for further in-depth research. We will further explore the correlation mechanism between pKa values and AIE performance in our future work.   

    According to the reviewers’ suggestion, the sentence “We will further explore the correlation mechanism between pKa values and AIE performance in our future work.” was added into the part of “Conclusions” on page 11.

Comments 3: 3. The fluorescence intensity changes are attributed to AIE effects. Were fluorescence lifetime measurements conducted to confirm the non-radiative decay suppression mechanism?

Response 3: We would like to express our gratitude to the reviewers for their attention and suggestions. Regarding the issue of fluorescence lifetime testing, we have not yet conducted relevant experimental research. However, the results of dynamic light scattering (DLS) tests show that when the fluorescence signal increased, the hydrodynamic diameter of the nanoparticles showed an increasing trend, which confirmed the occurrence of aggregation. Conversely, when the fluorescence signal decreased, the hydrodynamic diameter significantly reduced, indicating that the system undergoes a depolymerization process. Based on the correspondence between the above DLS experimental data and the changes in fluorescence intensity, we speculated that this fluorescence behavior could be closely related to AIE effect.

Comments 4: 4. How do the authors ensure that the observed fluorescence changes are not due to pH- or temperature-dependent degradation or detachment of the polymer chains from the CDs?  

Response 4: We would like to express our gratitude to the reviewers for their attention and suggestions. Regarding the aggregation behavior of CDs-PANT, we further verified the following phenomena through dynamic light scattering (DLS) experiments. When the pH value increased or the temperature raised to a specific threshold, the hydrodynamic diameter of CDs-PANT significantly increased, indicating a clear aggregation phenomenon in the system; conversely, when the pH value decreased or the temperature drops, its hydrodynamic diameter significantly decreased, suggesting a depolymerization process in the system. Notably, due to the strong covalent bond connection between the polymer and the surface of CDs, these covalent bonds can remain stable under mild conditions (such as non-extreme acidic or alkaline environments or excessively high temperatures), effectively preventing the risk of polymer detachment from the surface of CDs. This characteristic ensured the structural stability of the material under conventional usage conditions, providing a reliable guarantee for practical applications.

Comments 5: 5. Can the authors clarify whether the sensor operates effectively in complex media (e.g., biological fluids, ionic strength variation)? What is the selectivity and sensitivity under real conditions? 

Response 5: Thank you for the valuable suggestions provided by the reviewer. The issues indeed have significant research value and application significance. It should be noted that the current focus of this study is mainly on the design and preparation of materials, and a systematic exploration of its practical application in the field of sensors has not yet been fully conducted. The valuable suggestions have pointed out an important research direction for us. If this material is applied to sensor development, we will fully consider and adopt all the suggestions you have made, including (specifically list 1-2 key suggestions). These suggestions have important guiding significance for improving the sensing performance of the material, and we will give them priority consideration in our subsequent research.

Comments 6: 6. What is the response time and recovery time of the sensor under both pH and temperature switching? Are these parameters suitable for real-time sensing?

Response 6: Thank you for the valuable suggestions provided by the reviewer. Regarding the response time of the pH/temperature-induced AIE effect, our experimental observations indicated that the system exhibited an immediate response to pH/temperature stimuli, and no significant delay was detected under the current test conditions. However, it should be noted that the technical limitations of the existing test system (such as insufficient time resolution of fluorescence signal acquisition and relatively slow solution mixing/temperature control response speed) could have an impact on the precise capture of rapid kinetic processes. Based on this, we plan to introduce ultrafast spectroscopic techniques such as time-correlated single photon counting (TCSPC) in subsequent studies to further improve the time resolution and obtain more accurate response kinetic parameters. This improvement will provide important support for in-depth exploration of the molecular mechanism of the AIE effect.

Comments 7: 7. Regarding reversibility: how many cycles can the material endure without significant signal degradation? Only 4 cycles are shown what happens after 10 or 20?

Response 7: We are grateful to the reviewer for the valuable suggestions. Regarding the phenomenon observed in the cycling experiments, we have indeed conducted multiple repeated experiments for verification. The experimental data showed that when the cycling times exceeded 4, the fluorescence signal tended to stabilize and no longer changes significantly. After analysis, we believed that this could be due to the gradual deposition of inorganic salt NH4Cl on the membrane surface during the cycling process, which affected the aggregation-disaggregation dynamic equilibrium of APTE molecules. This deposit may restrict the conformational changes of APTE molecules through physical hindrance, thereby leading to the plateau of the fluorescence response. We will further verify this hypothesis in subsequent studies.

Comments 8: 8. What are the long-term photostability and environmental stability (e.g., under light, air, moisture) of the CDs-PNAT films for practical sensing deployment? 

Response 8: Thank you for the valuable suggestions provided by the reviewer. Regarding this issue, we have not conducted in-depth research on it yet, but we have included it in our subsequent research plan. Considering the significance and academic value of this problem, we will systematically explore this research direction in our future work in order to achieve more comprehensive research results.

Comments 9: 9. Why were specific molar ratios of ATPE to NIPAM chosen for the copolymer synthesis? 

Response 9: During the experiment, we found that the molar ratio of APTE had a significant regulatory effect on the AIE effect. Specifically, when the molar ratio of APTE was relatively low, the system hardly showed a distinct AIE effect; while when the molar ratio of APTE was too high, it could lead to an overly strong AIE effect, thereby affecting the overall performance of the material. Based on this discovery, we systematically studied the influence of different molar ratios of APTE on the material performance and ultimately determined an optimized molar ratio range. Notably, when the AIE effect occurred, its intense fluorescence signal completely masked the intrinsic fluorescence of CDs, a phenomenon clearly demonstrated in Figure 4c and 4d.

Comments 10: 10.What is the exact chemical structure of Acr-ATPE?

Response 10: The exact chemical structure of Acr-ATPE was shown in Scheme 1. To facilitate a deeper understanding of the chemical structure of this material among readers, we have revised Scheme 1 to explicitly illustrate the structure of CDs-PNAT within the Scheme 1.

Comments 11: 11.How do the authors distinguish between CD emission and ATPE-based AIE emission in the overlapping spectra?

Response 11: In the fluorescence property analysis, we observed the following phenomenon. Before the AIE phenomenon occurred, the characteristic fluorescence peak of CDs at 410 nm could be clearly identified; when the AIE effect began to show but was still weak (as shown in Figure 4a and 4b), the short-wave emission peak of CDs near 410 nm and the long-wave emission peak of ATPE could still be clearly distinguished in the system; however, when the AIE effect significantly intensified (as shown in Figure 4c and 4d), due to the sharp increase in the fluorescence intensity of ATPE, its emission signal completely masked the characteristic fluorescence peak of CDs, making it difficult to distinguish the fluorescence spectra of the two. This phenomenon intuitively revealed the significant impact of the AIE effect on fluorescence detection.

Comments 12: 12.What is the long-term stability (weeks/months) of these hybrid materials under ambient conditions?

Response 12: According to our experimental observations, this hybrid material exhibited outstanding stability at room temperature. When it was stored away from light, its performance remained stable for more than one year without significant attenuation.

Comments 13: Have the authors confirmed covalent bonding (vs. physical adsorption) of polymers on the CDs? XPS or detailed FTIR peak shifts are needed. 

Response 13: We are grateful to the reviewers for their valuable suggestions. Regarding the characterization of polymer-grafted CDs, we have provided the following experimental evidence to support it.
  Firstly, the TEM test results showed that the size of the grafted nanoparticles was significantly larger than that of the original CDs. This phenomenon is difficult to explain by simple physical adsorption (which usually only involves the coating of 1-2 polymer chains). In addition, our previous research had confirmed that as the molecular weight of the grafted polymer increased, the size of the nanoparticles observed by TEM also increased accordingly.
  Secondly, in the RAFT polymerization experiment, we ensured the smooth progress of the living polymerization process by strictly controlling the ratio of the initiator to the chain transfer agent ([I]/[CTA] ≪ 1), thereby effectively avoiding the formation of free polymers.
  It should be pointed out that, due to the thickness of the polymer shell layer in the core-shell structure being much greater than the size of the carbon dot core (about 2 nm), XPS and FTIR were indeed difficult to directly prove the existence of the grafted structure. These characterization methods were more suitable for analyzing the surface chemical composition rather than the spatial configuration features.

Comments 14: 14.How scalable is the synthesis process for practical device or commercial applications? 

Response 14: Thank you for the reviewer’s attention. Regarding the preparation of large-scale sample, we had successfully achieved stable synthesis of gram-scale (up to 10 grams) samples in our laboratory. From the perspective of the technical route, this synthesis method had good scalability potential. By further optimizing the reactor design and process parameters, it is expected to achieve commercial-scale production. However, during the industrialization process, it may still be necessary to solve engineering problems such as thermodynamic control of the reaction and purification efficiency of the product to ensure the stability and economy of the process.

Round 2

Reviewer 2 Report

1. What is the grafting density of the copolymer brushes?

2. What type of copolymer brushes was fabricated—statistical or block? The reactivity ratios of the monomers should be considered.

3. A table showing the molar concentrations of NIPAM and Acr-ATPE should be included in the main text.

4. A table presenting the LCST values of the various samples at different pH levels should be added to the main text.

The paper is very interesting and highly relevent to topic, the experimental methodology appears sound, and the manuscript is generally well-organized. However, several important details are either missing or insufficiently discussed, which limits the reproducibility and full understanding of the results. 

The authors should report the grafting density and molecular weight of the copolymer brushes formed on the substrate. these parameters play a key role in determining the conformation, swelling behavior, and responsive properties of the polymer brushes. If direct measurement is not feasible, please estimate or discuss the expected range and its influence on brush behavior.

It is not clear whether the synthesized copolymers are statistical, gradient, or block in nature. Given that the architecture of the copolymer can drastically affect phase transition behavior and inter-chain interactions, this should be explicitly clarified. In addition, a discussion of the reactivity ratios of NIPAM and Acr-ATPE is warranted to help readers assess the likely copolymer composition profile and monomer distribution.

A table summarizing the initial molar feed ratios or concentrations of NIPAM and Acr-ATPE used in the synthesis should be included in the main text. This is essential for reproducibility and for evaluating the effect of composition on the observed thermoresponsive behavior.

The LCST values reported in the manuscript are a key outcome, particularly in relation to environmental pH. To make the data more accessible and comparable, the authors should include a comprehensive table summarizing LCST values of all investigated samples across the pH range studied. This will also facilitate a better understanding of the role of the acidic/basic functionality of Acr-ATPE in modulating phase transitions.

An appropriate discussion on the molecular mechanisms underlying the LCST shifts should be included. I recommend incorporating insights from the following highly relevant study, which will significantly strengthen this section: https://doi.org/10.3390/polym17111580

Author Response

Comments 1: 1. What is the grafting density of the copolymer brushes?

The authors should report the grafting density and molecular weight of the copolymer brushes formed on the substrate. these parameters play a key role in determining the conformation, swelling behavior, and responsive properties of the polymer brushes. If direct measurement is not feasible, please estimate or discuss the expected range and its influence on brush behavior.

Response 1: Thanks for the reviewer’s professionalization. The determination of the grafting density of polymers on the surface of CDs has indeed been a persistent challenge in the research of this field. In our previously earlier  published papers, we made attempts to measure the molecular weight of carbon-dot-polymer nanoparticles using Gel Permeation Chromatography (GPC) and estimate the molecular weight of the grafted polymers via Nuclear Magnetic Resonance (NMR), aiming to calculate the grafting density. However, the results of two independent experiments exhibited substantial discrepancies, with the grafting density values being 16 and 4 per CD respectively (as detailed in our previously published work, see Res. Carbon, 73, 155, 2014 and RSC Advances, 4, 57683, 2014, respectively). This deviation primarily stemmed from the significant errors between the molecular weight of the nanoparticles measured by GPC and the molecular weight of the grafted polymers estimated by NMR, as compared to their true values. At present, it remains extremely challenging to accurately measure the true molecular weights of both directly. Consequently, in our subsequent research, we refrained from further quantitative discussions regarding the grafting density of this system. In our same research, we have made every effort to maintain a consistent grafting density through the meticulous control of the stoichiometric ratio, which aims to mitigate any direct influence on the properties that could arise from variations in the grafting density.

Comments 2: 2.What type of copolymer brushes was fabricated—statistical or block? The reactivity ratios of the monomers should be considered.

It is not clear whether the synthesized copolymers are statistical, gradient, or block in nature. Given that the architecture of the copolymer can drastically affect phase transition behavior and inter-chain interactions, this should be explicitly clarified. In addition, a discussion of the reactivity ratios of NIPAM and Acr-ATPE is warranted to help readers assess the likely copolymer composition profile and monomer distribution.

Response 2: We sincerely appreciate the reviewer for the insightful and professional suggestions. As shown in Scheme 1, both monomers, NIPAM and Acr-ATPE, are involved in the polymerization reaction, resulting in the formation of graft polymer brushes with a random copolymer structure. During the design of experimental, we considered that the Acr-ATPE monomer might exhibit slightly lower reactivity than the NIPAM monomer due to its greater steric hindrance. However, constrained by current research conditions, we were unable to quantitatively characterize the reactivity ratios of the two monomers. This issue holds significant research value, and we will conduct an in-depth investigation through systematic kinetic studies in follow-up work.  

Comments 3: 3.A table showing the molar concentrations of NIPAM and Acr-ATPE should be included in the main text.

A table summarizing the initial molar feed ratios or concentrations of NIPAM and Acr-ATPE used in the synthesis should be included in the main text. This is essential for reproducibility and for evaluating the effect of composition on the observed thermoresponsive behavior.

Response 3: Thanks for the reviewer’s valuable suggestions. As shown in Table S1, we have listed the carbon dots used in the synthesis of different CD-PANT polymers along with the mass ratios of the two monomers. Due to the difficulty in accurately determining the molecular weight of carbon dots, we could only provide the mass ratios rather than molar ratios of the components.  

Comments 4: A table presenting the LCST values of the various samples at different pH levels should be added to the main text.

The LCST values reported in the manuscript are a key outcome, particularly in relation to environmental pH. To make the data more accessible and comparable, the authors should include a comprehensive table summarizing LCST values of all investigated samples across the pH range studied. This will also facilitate a better understanding of the role of the acidic/basic functionality of Acr-ATPE in modulating phase transitions.

An appropriate discussion on the molecular mechanisms underlying the LCST shifts should be included. I recommend incorporating insights from the following highly relevant study, which will significantly strengthen this section: https://doi.org/10.3390/polym17111580

Response 4: We thank the reviewer for the valuable suggestions. In our preliminary studies, we primarily focused on the fluorescent properties of the materials in response to temperature and pH. However, we did not thoroughly investigate the influence of pH on the LCST and therefore did not conduct LCST tests under different pH conditions. Based on the literature guidance provided by the reviewer, we have supplemented the analysis of how pH affects the LCST in the discussion section. The sentence “Notably, the differential fluorescence transition temperatures observed across CD-PANT systems could stem from both varying APTE content and structure-specific pH modulation of LCST behavior in the polymers [29]” was marked in blue in the revised manuscript on page 9 to 10 in section 3.2.3. We cited this paper “https://doi.org/10.3390/polym17111580” to better assist in the explanation of our thesis. After we cited this new reference, the original references sequence changed from 29-33 to 30-34. So the original citation numbers have been rearranged. In our future studies, we will follow the reviewers’ suggestions and systematically investigate the regulatory mechanism of pH on the LCST.

 The other revisions.

Revised: 1. There was an error in the caption of Table S1, and we have made the correction in the file of “revised Supplementary Materials carbon II-3647589”.

2. In the last revision, on basis of the reviewers’suggestions we revised two Supplementary Materials, Figure S1 and S2, but did not upload them to the submission system. We have completed the upload this time. 

Author Response File: Author Response.pdf

Reviewer 3 Report

In the revised draft of the manuscript titled “Dual pH- and Temperature-Responsive Fluorescent Hybrid Materials Based on Carbon Dot-Grafted Triaminotetrastyrene/N-isopropylacrylamide Copolymers”, the authors have addressed the comments thoughtfully and thoroughly. They have carefully noted all the errors and made the necessary corrections in the revised draft. For the comments where no changes were made, they have provided clear and reasonable justifications for their decisions. The reviewer recommends publication of the manuscript in its present form.

Please see above.

Author Response

Thanks again for the professional opinions and suggestions of the reviewer. We will also continue to improve the relevant research work under the guidance of the reviewer.

Reviewer 4 Report

The authors have thoroughly revised the manuscript in response to the reviewers' comments. All previously raised concerns regarding the experimental methodology, data interpretation, language clarity, and overall presentation have been adequately addressed. The revised version reflects a significant improvement in both scientific quality and readability. The authors have provided clear justifications and incorporated necessary changes throughout the manuscript. Based on the comprehensive revisions and satisfactory responses to all queries, I find the current version acceptable for publication in its present form.

The authors have thoroughly revised the manuscript in response to the reviewers' comments. All previously raised concerns regarding the experimental methodology, data interpretation, language clarity, and overall presentation have been adequately addressed. The revised version reflects a significant improvement in both scientific quality and readability. The authors have provided clear justifications and incorporated necessary changes throughout the manuscript. Based on the comprehensive revisions and satisfactory responses to all queries, I find the current version acceptable for publication in its present form.

Author Response

Thanks again for the professional opinions and suggestions of the reviewer. We will also continue to improve the relevant research work under the guidance of the reviewer.

Round 3

Reviewer 2 Report

The authors answered to all my comments and the paper can be accepted in its present form.

The authors answered to all my comments and the paper can be accepted in its present form.