A Simple Yet Effective Preanalytical Strategy Enabling the Application of Aptamer-Conjugated Gold Nanoparticles for the Colorimetric Detection of Antibiotic Residues in Raw Milk

Round 1

Reviewer 1 Report

In this paper, the authors investigated an aptamer-conjugated gold nanoparticles (AuNPs) for colorimetric detection of antibiotics in raw milk. The proposed idea is not conceptually novel because the similar methods using aptamer-conjugated AuNPs have been widely used for the colorimetric detection of antibiotics (Ref 1-3). However, this work is meaningful in that it provides the important results for the detection of antibiotics in the real, raw milk samples. Moreover, the work was systematically investigated and executed, and the manuscript was well written and organized. Therefore, I think that the manuscript is worthy of publication in Sensors after accommodating the following comments.

Ref 1) https://www.sciencedirect.com/science/article/pii/S0003269711002326?via%3Dihub

Ref 2) https://pubs.rsc.org/en/content/articlelanding/2014/ay/c3ay41816b

Ref 3) https://www.mdpi.com/2079-4991/11/4/840

• It would be better to substitute “ultrasensitive” into “sensitive” in the whole manuscript. In addition, it would be better to change the title in order to focus on the pretreatment methods for practical application.
• The authors need to discuss why NAS kanamycin has the optimal molar ratios of 1:60, which is different from others (1:20). In addition, it would be better to discuss why A520/A720 is better than A520/A620.
• The sentence in line 302 is not appropriately placed. Instead of mentioning their recent work, it would be better to suggest/discuss the appropriate reasons.
• In Figure 6, the authors should explain how the protein removal efficiency (98.13 or 96.98%) was calculated.

Author Response

Dear reviewer 1: We greatly appreciate the effort the referees have put into reviewing our work and the opportunity that we have been given to further revise the manuscript. We have replied to the criticisms and have incorporated into the manuscript important changes that we believe have clarified and improved it significatively.

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

title is too long, please revise

abstract is premetive and must be revised to reflect the significance of research.

language must be revised

more details on the preparation of the aptamer-conjugated gold nanoparticles should be added.

please include the TEM images of gold nanoparticles before and after sensing.

the significance of colorimetric sensors muust be discussed in the introduction section;

Author Response

Dear reviewer 2: We greatly appreciate the effort the referees have put into reviewing our work and the opportunity that we have been given to further revise the manuscript. We have replied to the criticisms and have incorporated into the manuscript important changes that we believe have clarified and improved it significatively.

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

The authors reported the preparation of a nanoaptasensor based on gold nanoparticles and its use in a simple methodology for the detection of antibiotics in milk. Specifically, the analytical methodology was tested for the detection of different common antibiotics including kanamycin, oxytetracycline, sulfadimethoxine, and ampicillin. The influence of different components of milk on the activity of the sensors was also considered.

The manuscript demonstrated the potentialities of the new methodology based on the Au-sensor for practical applications in the cattle sector. The general aim of the manuscript might be of interest, and the language is quite fluent (but improvable).

I’d recommend considering accepting the paper after addressing all these points:

• Please revise the overall style of language. I’m sure that some corrections and the reformulation of some sentences will make the manuscript more pleasantly readable.
• An important issue to address is to apply the same punctuation to all the data reported in the text. Sometimes “.” is used to separate thousands, sometimes to separate decimals (as it should be in English), such as in line 28, 39, 201, ….
• Lines 60-79: I suggest expanding a little the discussion related to Au nanoparticles and their utilization for example in catalysis. For example, add some references such as DOI: 10.1016/j.aca.2019.12.026 or 10.1021/acssuschemeng.9b00198
• Considering the possibility of using the sensors in the cattle sector, isn’t it a problem to use an expensive material such Au nanoparticles, is it? Please add some explanations in the text justifying the use of a precious metal.
• Table 3: did the authors performed only one analysis of the components of raw milk and whey? No deviations are reported.
• Figures 2, 3, 5, 8, and others are not complete. It is difficult to understand the figures without continuously check the text and the captions of the figures. Please add some details. Importantly, it is not clear if the sensor is detecting the all amount of the antibiotics put in water/milk.
• Table 5: please emphasize the novelty of the work respect to the literature.
• Table 5: Is the range the same for the different antibiotics? Otherwise, is should be reported. I also suggest adding the range in M (adding one line for each antibiotic).
• Is it possible to recover the Au NPs after utilization? Is it possible to sequentially regenerate them?
• I cannot find a clear discussion the results of the different characterization techniques (described in 2.3). For example, where are the TEM images? All these analyses should be reported before and after utilization of the Au NPs in the analytical methodology. Please also provide XRD.

Author Response

Dear reviewer 3: We greatly appreciate the effort the referees have put into reviewing our work and the opportunity that we have been given to further revise the manuscript. We have replied to the criticisms and have incorporated into the manuscript important changes that we believe have clarified and improved it significatively.

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

my concerns were addressed by authors.

Author Response

We truly appreciate the effort the referees have put into reviewing our work. Please see the attachement.

Reviewer 3 Report

The authors addressed almost all the issues reported. It should be highlighted that the authors did a quite relevant job, and the quality of the manuscript has increased substantially.

Still two points haven't been fully completed:

- Lines 60-79: I suggest expanding a little the discussion related to Au nanoparticles and their utilization for example in catalysis. For example, add some references such as DOI:10.1016/j.aca.2019.12.026 or 10.1021/acssuschemeng.9b00198. None of the suggested references was added.

- I cannot find a clear discussion the results of the different characterization techniques (described in 2.3). For example, where are the TEM images? All these analyses should be reported before and after utilization of the Au NPs in the analytical methodology. Please also provide XRD. The authors clearly justified the absence of XRD, however, their reply should be included in some way in the text (or at least some of the important references the authors reported in the responses must be included as reference literature with additional characterization techniques/data)

After completing addressing these points, I recommend accepting the manuscript for publication.

Author Response

We truly appreciate the effort the referees have put into reviewing our work. Please see the attachment.

Author Response File: Author Response.pdf

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.