A ratiometric fluorescent probe for pH detection based on Ag2S quantum dots–carbon dots nanohybrids

In this study, a novel ratiometric fluorescent nanoprobe for pH monitoring has been developed by synthesizing red fluorescent Ag2S quantum dots (Ag2S QDs) and green fluorescent carbon dots (CDs) nanohybrids (Ag2S CDs) in one pot using CDs as templates. The nanoprobe exhibits dual-emission peaks at 500 and 670 nm under a single-excitation wavelength of 450 nm. The red fluorescence can be selectively quenched by increasing pH, while the green fluorescence is an internal reference. Therefore, the change of the relative fluorescence intensity (I500/I670) in the ratiometric Ag2S CDs probes can be used for pH sensing. The results revealed that I500/I670 of Ag2S CDs probes was linearly related to pH variation between pH 5.4 and 6.8. Meanwhile, the Ag2S CDs probes possessed a good reversibility along with pH changing between 5.0 and 7.0 without any interruption from common metal ions, proteins and other interferences.

Decision letter (RSOS-200482.R0) We hope you are keeping well at this difficult and unusual time. We continue to value your support of the journal in these challenging circumstances. If Royal Society Open Science can assist you at all, please don't hesitate to let us know at the email address below.
Dear Dr Wu: Title: A ratiometric fluorescent probe for pH detection based on Ag2S quantum dots -carbon dots nanohybrids Manuscript ID: RSOS-200482 Thank you for your submission to Royal Society Open Science. The chemistry content of Royal Society Open Science is published in collaboration with the Royal Society of Chemistry.
The editor assigned to your manuscript has now received comments from reviewers. We would like you to revise your paper in accordance with the referee and Subject Editor suggestions which can be found below (not including confidential reports to the Editor). Please note this decision does not guarantee eventual acceptance.
Please submit your revised paper before 16-May-2020. Please note that the revision deadline will expire at 00.00am on this date. If we do not hear from you within this time then it will be assumed that the paper has been withdrawn. In exceptional circumstances, extensions may be possible if agreed with the Editorial Office in advance. We do not allow multiple rounds of revision so we urge you to make every effort to fully address all of the comments at this stage. If deemed necessary by the Editors, your manuscript will be sent back to one or more of the original reviewers for assessment. If the original reviewers are not available we may invite new reviewers.
To revise your manuscript, log into http://mc.manuscriptcentral.com/rsos and enter your Author Centre, where you will find your manuscript title listed under "Manuscripts with Decisions." Under "Actions," click on "Create a Revision." Your manuscript number has been appended to denote a revision. Revise your manuscript and upload a new version through your Author Centre.
When submitting your revised manuscript, you must respond to the comments made by the referees and upload a file "Response to Referees" in "Section 6 -File Upload". Please use this to document how you have responded to the comments, and the adjustments you have made. In order to expedite the processing of the revised manuscript, please be as specific as possible in your response. ********************************************** RSC Associate Editor: Comments to the Author: (There are no comments.) RSC Subject Editor: Comments to the Author: (There are no comments.) ********************************************** Reviewers' Comments to Author: Reviewer: 1 Comments to the Author(s) In this manuscript, a novel pH sensor has been developed based on the Ag2S quantum dotscarbon dots nanohybrids. The resuls are very interesting. In my opinion, I recommend the manuscript for publication in RSOS after some modifications. 1、In the Fig. 4, the concentration of Ag2S quantum dots -carbon dots nanohybrids should be provided. 2、In the Fig. 5, the concentrations of Ag2S quantum dots -carbon dots nanohybrids and interferences should be provided. 3、In the introduction, some references about ratiometric fluorescent sensors based carbon dots should be cited and discussed. 4、Some discussion about advantages and disadvantages should be added in the discussion part. 5、The following references is advised to add in the manuscript.
(1) Nemati, R.; Zare-Dorabei, R., A ratiometric probe based on Ag2S quantum dots and graphitic carbon nitride nanosheets for the fluorescent detection of Cerium. Talanta  Comments to the Author(s) The manuscript presents a ratiometric fluorescent nanoprobe for pH sensing based on Ag2S-CDs. Some problems should be resolved before publication in the journal. 1. The reason for choosing 25.5 mg/mL should be explained in detailed. 2. Practical applications should be presented. 4. Could the probe be used to monitor the cellular pH change? 5. How about the stability of the probes? 6. As we all know that there are some functional groups on the surface of CDs, why did the fluorescence of CDs not change with pH?
Reviewer: 3 Comments to the Author(s) This manuscript reported a ratiometric fluorescent probe for reversible pH detection based on the hybrid structure of green-and red-emission Ag2S-CDs nanohybrids. The pH selectivity of Ag2S-CDs probe also was tested by controlling experiments. I recommend it to be published on Royal Society Open Science after the following major revisions. Some suggestions and comments on this work are listed for the authors: 1. What is the particle size of CDs？ What about the morphology of CDs? 2. How can the author prove that the combination of CDs and Ag2S QDs is the same as the author indicated in Scheme 1？ 3. Whether the CDs prepared in this paper exhibit characteristic excitation wavelength-dependent emission. 4. It is better to indicate which sample is analyzed in the caption of XPS spectrum. 5. Page 4 Line 7, author said " 25.5 mg/mL was chosen as the optimum CDs concentration for the synthesis of Ag2S-CDs", but it's "25.5 μg ml-1" in Fig. 5. What is the concentration of CDs? Why the author choose this concentration？ 6. Page 4 Line 20-22, please explain this sentence "Similar type of results did not appear for single CDs if Ag2S QDs and CDs were physically mixed". 7. Fig.5 B, with the change of pH value, the fluorescence peak of red emission shifts. Please explain. 8. Is pH detection limited to 5.0-7.0 for Ag2S-CDs nanohybrids? Can it be used for a wider range of pH detection? 9. Please pay attention to the spelling of the words in the manuscript, such as "reversable" is "reversible" in Conclusions.

Author's Response to Decision Letter for (RSOS-200482.R0)
See Appendix A.

Decision letter (RSOS-200482.R1)
We hope you are keeping well at this difficult and unusual time. We continue to value your support of the journal in these challenging circumstances. If Royal Society Open Science can assist you at all, please don't hesitate to let us know at the email address below.
Dear Dr Wu: Title: A ratiometric fluorescent probe for pH detection based on Ag2S quantum dots -carbon dots nanohybrids It is a pleasure to accept your manuscript in its current form for publication in Royal Society Open Science. The chemistry content of Royal Society Open Science is published in collaboration with the Royal Society of Chemistry.
The comments of the reviewer(s) who reviewed your manuscript are included at the end of this email.
Thank you for your fine contribution. On behalf of the Editors of Royal Society Open Science and the Royal Society of Chemistry, I look forward to your continued contributions to the Journal.  4. Some discussion about advantages and disadvantages should be added in the discussion part.
Response: Thanks for your suggestion. We have added the advantage and disadvantage in the discussion part in the revised manuscript (Page 1, right column, the second paragraph): "The disadvantage of this probe is narrow pH response. However, it shows fluorescence change within a 0.2 pH unit change between 5.0-7.0 with satisfied linear relationship. It is known even pH changes below 1.0 unit, our environments and physiology will be significantly impacted.
Hence, our probes, which sharply response to small pH changes within a narrow range are desired." 5、The following references is advised to add in the manuscript.

Reviewer: 2
Comments: The manuscript presents a ratiometric fluorescent nanoprobe for pH sensing based on Ag2S-CDs. Some problems should be resolved before publication in the journal.

Response:
We are sincerely appreciated for your positive comments towards to our work. Your comments are valuable and very helpful for improving our manuscript. We hope you best at this unusual time. The revisions according to your comments are as follows: 1. The reason for choosing 25.5 mg/mL should be explained in detailed.
Response: Thanks for your comment. It's should be 25.5 μg/mL, we have corrected in the revised manuscript. The emission peak at 500 nm from CDs was used as the reference signal, the emission peak at 670 nm was used as the signal report unit for pH. Hence, the fluorescence intensity at 500 nm should not be stronger than that at 670 nm. Actually, from Fig. 5A, 17.0 μ g/mL of CDs is also OK, but we chose 25.5 μg/mL. We have added the reason in the revised manuscript (please see page 3, left column, the second paragraph).

Practical applications should be presented.
Response: Thanks for your professional comment. We totally agree with you that practical applications should be presented. We have done early attempts. We tried to monitor the cellular pH change, but no satisfied data have gotten, then we had our winter vacation. I am so sorry that during this special time, our lab has not opened yet to move this practical application forward. Sorry again! 3. The performance should be compared with other reported results.
Response: Thanks for your professional comment. We have compared with other reported results in the revised manuscript. Please see Table 1 in the revised manuscript. Table 1 Comparison of this method with some previously reported method for pH sensing in ratiometric method a: Linearity range of pH detection b: Reference 4. Could the probe be used to monitor the cellular pH change?
Response: Thanks for your professional comment. We tried to monitor the cellular pH change, but no satisfied data have gotten. We could only get the conclusion that our probes can be taken into cells by endocytosis, then we had our winter vacation. I am so sorry that during this special time, our lab has not opened yet to move this experiment forward. Sorry again! 5. How about the stability of the probes?
Response: The probes could be stored at 4 °C for at least one month.
6. As we all know that there are some functional groups on the surface of CDs, why did the fluorescence of CDs not change with pH?
Response: Thank you for you comment. We used citric acid and ethylenediamine as precursors to synthesize CDs according to ref.
[33] (Zhu et al., Angew. Chem. Int. Edit., 2013, 52, 3953-3957). In this literature, it reported that the synthesized CDs is pH-dependent photoluminescent (PL) behavior. PL intensities decrease in a solution at high or low pH, but remain constant in a solution of pH 4-11. Our detection range is pH 5-7. The reason why the fluorescence of CDs not change with pH maybe is that pH 4-11 is not enough to change the surface functional groups.

Reviewer: 3
Comments: This manuscript reported a ratiometric fluorescent probe for reversible pH detection based on the hybrid structure of green-and red-emission Ag2S-CDs nanohybrids. The pH selectivity of Ag2S-CDs probe also was tested by controlling experiments. I recommend it to be published on Royal Society Open Science after the following major revisions. Some suggestions and comments on this work are listed for the authors:

Response:
We are sincerely appreciated for your professional comments towards to our work. Your comments are valuable and very helpful for improving our manuscript. We hope you best at this unusual time. The revisions according to your comments are as follows: 1. What is the particle size of CDs？ What about the morphology of CDs?

Response:
We are so sorry that we have not yet gotten a good TEM image of CDs because of the interference of carbon film when taking TEM image. According to the ref.
[33]: Zhu et al., Angew. Chem. Int. Edit., 2013, 52, 3953-3957, the particle diameter of CDs is 2-6 nm. The sizes of CDs were also measured by atomic force microscopy (AFM), and the average height is 2.81 nm. Most of the morphology of CDs is sphere.

How can the author prove that the combination of CDs and Ag2S QDs is the same as the author indicated in Scheme 1？
Response: Thanks for your so rigorous comment. We have not found out the combination of CDs and Ag2S QDs. We guess it is core-shell structure, but we have not enough demonstration.
It is under research because it is a very intreating phenomenon, and we hope to figure it out.
We have re-drawn the Scheme 1, please see it in the revised manuscript.
3. Whether the CDs prepared in this paper exhibit characteristic excitation wavelength-dependent emission.
Response: Yes. We used citric acid and ethylenediamine as precursors to synthesize CDs according to ref.
4. It is better to indicate which sample is analyzed in the caption of XPS spectrum.
Response: Thanks for your suggestion. We have added the sample name in the legend of Fig.2 in our revised manuscript.
5. Page 4 Line 7, author said " 25.5 mg/mL was chosen as the optimum CDs concentration for the synthesis of Ag2S-CDs", but it's "25.5 μg ml-1" in Fig. 5. What is the concentration of CDs? Why the author choose this concentration？ Response: Thank you for your so detailed review work. We are so sorry that we made a mistake.
The concentration of CDs is 25.5 μg ml -1 . We have corrected in our revised manuscript. The emission peak at 500 nm from CDs was used as the reference signal, the emission peak at 670 nm was used as the signal report unit for pH. Hence, the fluorescence intensity at 500 nm should not be stronger than that at 670 nm. Actually, 17.0 μg/mL of CDs is also OK from Fig.   5A, but we chose 25.5 μg/mL. We have added the reason in the revised manuscript (please see page 3, left column, the second paragraph).
6. Page 4 Line 20-22, please explain this sentence "Similar type of results did not appear for single CDs if Ag2S QDs and CDs were physically mixed".
Response: I am so sorry that we made a mistake. This sentence should be: "Similar type of results did not appear if Ag2S QDs and CDs were physically mixed." This sentence means 8. Is pH detection limited to 5.0-7.0 for Ag2S-CDs nanohybrids? Can it be used for a wider range of pH detection?
Response: It is not limited to 5.0-7.0 for Ag2S-CDs nanohybrids. However, the I500/I670 ratio showed good linearity with different pH values only in this range.
9. Please pay attention to the spelling of the words in the manuscript, such as "reversable" is "reversible" in Conclusions.

Response:
We are sincerely appreciated for your so strict review work. We are so sorry. We have paid attention to all of the words spelling.