Design and Evaluation of a Competitive Phosphorescent Immunosensor for Aflatoxin M1 Quantification in Milk Samples Using Mn:ZnS Quantum Dots as Antibody Tags

Round 1

Reviewer 1 Report

This paper describes the development of a competitive immunosensor for aflatoxin M1 quantification based on metal-doped quantum dots as tags. This is a very interesting work in which the authors compare different assay schemes in terms of analytical performance for AFM1 quantification. As a result, the selected immunosensor provides a detection limit for AFM1 quantification of 0.002 ug L-1, which greatly satisfies the maximum tolerable limit of 0.05 ug L-1 of AFM1 in milk. These results are very promising, since they demonstrate the good performance of phosphorescent biosensors as a simple and fast alternative to conventional chromatographic analytical methods. The paper is well written, making a good use of the English language. The Introduction provides a good background to the reader and the basic references to understand the context of the work.  The manuscript includes high quality figures properly illustrating the key concepts of the work. The results are concise and potentially applicable in practical scenarios. Finally, the Conclusions are well supported by the obtained data. All in all, this is a very good work and an excellent match for MDPI Chemosensors. Therefore, I recommend its publication in its present form.

Author Response

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Reviewer 2 Report

the manuscript appear to intend to present the advantages of “novel” Mn-doped ZnS QDs in comparison with other metal QDs based on less toxic metals. Nevertheless, this QDs are well known (e.g. DOI: 10.1016/j.bios.2013.04.024), the production protocol was already published, the characterization and comparison with other alternative QDs is missing. Also, it is not obvious why the direct link of the QDs to model analyte (aflataoxin) leads too poor results while Ab-QDs performs well.

The title is not informative

Abstract must be rewritten to describe the developed system and its performances.

There are numerous pages and fig.1 that are just a generic description of any immunoassay and could be inserted in any manuscript without any modification (but do not provide any interest).

Author Response

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Reviewer 3 Report

This paper reported the fabrication of immunosensor for aflatoxin M1 quantification. The work idea is interesting, but results presentation and discussion should be revised. In my humble opinion, this manuscript can be published after major revision.

Specific comments:

• Lines 152-153: “Mn-doped ZnS QDs having their surface modified with Dihydrolipoic acid (DHLA), for water stabilization”. What did authors mean writing “for water stabilization”?
• Lines 155-157: “A DLS spectrum of the product from the synthesis is provided in Fig. S1 in the ESM, showing that aqueous-stabilized nanoparticles were synthesized with a hydrodynamic radius of around 11 nm.” Please, change this information, because it can be seen in Fig. S1 that diameter of Qdots were 11 nm. Please, add information about DLS instrument, which was used for measurements, to the paper. Moreover, particles size distribution by number (%) were added as Fig. S1 and Fig. S2. According to my information, in such situation DLS software calculate the possible “real” size of particles, but not the hydrodynamic diameter or radius. For such statement, which was mentioned before, it is necessary to add addition graph, which will represent particle hydrodynamic size distribution by intensity (%). In my humble opinion, it is more informative to show distribution by intensity (%) in scientific papers. Please, change “print screen” pictures, which were taken from DLS software program (Figure S1 and S2), to “drawn” graphs.

• Figure 2 and Figure 3: The numbers on Y axis should be added with dots, but not with commas (Also, please do it for Figure 5B). The mistake in title of X axis (“wavelenght" should be changed to “wavelength”) should be corrected. The same mistake is in Figure 5A.
• Lines 262-263: “sensitivity achieved was not enough (observed detection limits (DLs) were too high for the desired application)”. The value of detection limit should be provided. At the moment it is not clear how detection limit was calculated.
• The part “3.1. Synthesis of the antibody-QDs bioconjugates” should be provided in the beginning of Results.
• In results parts authors mentioned that “maximum phosphorescence signals were obtained for a 1:30 molar ratio.” It is not clear how optimal ratio was obtained, Please, provide results which will proof this statement.
• In my opinion statement “the inhibition curve” (Lines 322-323) is not correct to use.
• The legend in Figure 5A should be fixed. Please, fix “ng ml^-1” writing. It is not necessary to write AMF1 in the legend.
• Inset in graph 5B should be added in better resolution. In my opinion, the units on X axis should be changed.

Author Response

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Round 2

Reviewer 2 Report

The interest and novel aspects of the manuscript are limited.

Author Response

Dear reviewer, under the light of your comment, we have included a new paragraph at the end of the conclusions section of the revised manuscript trying to further highlight the novelty, potential impact and interest of the here-developed phosphorescent competitive immunosensor:

“In brief, we have demonstrated the feasibility of the use of Mn:ZnS QDs as highly valuable phosphorescent labels in the development of a quantitative immunoassay for sensitive AFM1 detection in milk samples (a demand of high interest in food quality control). In addition, it is important to point out that it was not necessary to perform any complex sample pre-treatment and an aqueous sample dilution is only required for the success of the quantification.

It is envisaged that the competitive phosphorescent immunoassays format here developed have wide potential applicability in different areas such as bioscience, food analysis, clinical, etc. , just by selecting an adequate selective receptor specific of the desired analyte, making it possible to expand the applicability to other relevant problems. Although in the present study AFM1 was used as a target analyte, the approach here developed (based on the labelling of a secondary antibody with the phosphorescent QDs) can be directly translated for detection or quantification of any other environmental, clinical or food relevant small molecules by just selecting an appropriate capture primary antibody"

Reviewer 3 Report

I am grateful for providing answers.

In my opinion, paper can be accepted for publication.

Author Response

Dear reviewer, thank you very much for your positive comments about our revised manuscript.