Fluorimetric Detection of Single Pathogenic Bacterium in Milk and Sewage Water Using pH-Sensitive Fluorescent Carbon Dots and MALDI-TOF MS

Round 1

Reviewer 1 Report

Nice work.

The figures illustrate the written text, from my point of view, they are allways to little in the presentation.

You are summarizing in the discussion: ‚… CDs solution … was … fluorescently optimized. ‘. I agree with your statement of extensive characterization of the CDs, but I do not see the optimization process you did.

Author Response

Reviewer 1: Comments and Suggestions for Authors;

Nice work.

The authors are thankful for the appreciation and careful review of the manuscript. We have revised the manuscript in response to the valuable comments and useful suggestions. Further, the language of manuscript was improved for grammatical errors, typos, punctuation, and sentence structure and presentation as can be seen from the corrections sprinkled throughout the manuscript to meet the standard of journal “Microorganisms”. All the changes are highlighted yellow in the revised manuscript.

Comment: 1

The figures illustrate the written text, from my point of view, they are allways to little in the presentation.

Response

Thank you for the constructive comment. In the original manuscript draft we have revised the font size of the text illustration in some figures (Scheme 1, figure 3a, Scheme 2 and figure 4) where necessary, to be clearly visible to readers. Moreover, we have revised and explained all the captions of figures to provide enough explanations to the results presented. We have changed the captions of Scheme 1 (at line number 88-90), Scheme 2 (at line number 250-253), and Figure 4 (at line number 273-278) of the revised manuscript.  

Comment: 2

You are summarizing in the discussion: ‚… CDs solution … was … fluorescently optimized. ‘. I agree with your statement of extensive characterization of the CDs, but I do not see the optimization process you did.

Response:

Thank you for the valuable comment. The explanations about the CDs optimization process is presented in supplementary materials and clearly highlighted the statement in results section at line 168-169 (“The fluorescent CDs solution was synthesized by successive carbonization of sucrose and optimized fluorescently under variable pH values during the synthesis (Figure S1).”). In order to make readers more clear, we added one sentence at line 280-282 in discussion section of the revised manuscript as follows: “The fluorescent CDs solution synthesized by successive carbonization of sucrose was characterized and optimized fluorescently by comparing the fluorescent intensity at variable pH values of CDs solutions (Figure S1, detailed in the supplementary materials).”

Reviewer 2 Report

The paper describes the use of fluorescent carbon dots for its use in the detection of e. coli bacteria in two different real mediums such as milk and sewage. The authors study the influence of the pH in the luminescence and also the changes in the intensity depending of the excitation wavelength. The methodology followed and the results are convincing. However, although the results are clear, the working mechanism need a more deeply knowledge. For instance, why one of the emissions is more sensitive to the pH/concentration than the other or the explanation of the inverse behaviour (Ex 350 nm, increase and Ex 410 nm decrease). Probably, all this is related with the different size distribution and the origin of the emission sites in the carbon dots. All the above, will help to design adequately this type of sensor materials.

Therefore, I recommend the manuscript for publication in this journal after a proper discussion on the fundamental working mechanisms.

Author Response

Reviewer 2: Comments and Suggestions for Authors;

We are thankful for your time and useful comments. We have revised the manuscript according the valuable comments and useful suggestions. The responses to the raised comments are presented in the following context. Furthermore, we proofread the manuscript for grammatical errors, typos, punctuation, and sentence structure and presentation as can be seen from the corrections sprinkled throughout the manuscript to meet the standard of journal “Microorganisms”. All the changes are highlighted yellow in the revised manuscript.

Comment

The paper describes the use of fluorescent carbon dots for its use in the detection of e. coli bacteria in two different real mediums such as milk and sewage. The authors study the influence of the pH in the luminescence and also the changes in the intensity depending of the excitation wavelength. The methodology followed and the results are convincing. However, although the results are clear, the working mechanism need a more deeply knowledge. For instance, why one of the emissions is more sensitive to the pH/concentration than the other or the explanation of the inverse behaviour (Ex 350 nm, increase and Ex 410 nm decrease). Probably, all this is related with the different size distribution and the origin of the emission sites in the carbon dots. All the above, will help to design adequately this type of sensor materials.

Therefore, I recommend the manuscript for publication in this journal after a proper discussion on the fundamental working mechanisms.

Response:

Thank you for the valuable comment and useful suggestion.

According to the comments, we added more discussion about the inverse fluorescent behavior of CDs on 350 and 410 nm excitation wavelength at line number 326-333 as follows:

“When excited at an excitation wavelength of 350 nm, the fluorescence intensity of the mixture increased along with the increasing bacterial cell density. However, the fluorescence intensity of the mixture decreased along with the increasing bacterial cell density at excitation wavelength of 410 nm (Figure 3a). As discussed earlier, the key factors possibly contributing to the inverse fluorescence behavior of CDs, are the different emission sites on the surface of CDs as well as their different sizes [38]. Another possible reason is the gradual ionization of –COOH groups on the surface of CDs under various pH which result in the dispersion of CDs and enhance fluorescence activity [39].”

In order to explain the behavior deeply, we also revised some sentences at line number 298-306 as follows:

“The fluorescent behavior of the CDs as demonstrated in Figure 2a further revealed that when excited at a wavelength of 350 nm, the fluorescence intensity of synthesized CDs was decreased with the increasing pH value; however, the fluorescence intensity was increased with the increasing pH value at an excitation wavelength of 410 nm. These variations in fluorescence performance of CDs could be attributed to the presence of different functional groups, such as –OH and –COOH, on their surface [39]. With the increasing pH, the –COOH groups are ionized gradually, which results in a variation of surface charge of CDs. At the same time, the electrostatic interaction between the –COOH group results in the dispersion of CDs that result in an enhanced fluorescence activity, thus confirming the pH-dependent fluorescence performance of CDs [39].”

Furthermore, we discussed the fundamental working mechanism at line number 334-337 of the revised manuscript as follows:

“In general, the exclusive fluorescent properties of CDs put forward their potential application by measuring the pH sensitivity, and the variations in pH of the bacterial culture that could be used as an indicator for the detection of bacterial cells. Relying on these principles, bacterial cell density can be fluorometrically detected by applying the ratiometric CDs-based pH probe.