Electrochemical Aptasensor Based on ZnO-Au Nanocomposites for the Determination of Ochratoxin A in Wine and Beer
Round 1
Reviewer 1 Report
This manuscript entitled “Electrochemical Aptasensor Based on ZnO-Au nanocomposites for the Determination of Ochratoxin A in Wine and Beer”, authors developed a ZnO-Au composites-based electrochemical assay by using methylene blue as the reporter to explore the interaction occurred on electrode interface. The aptasensor could provide versatile approaches for quantitative detection of various biomarkers. Overall, I think this work fits well in Process and recommend a revision.
Below are several points that the authors may consider to further improve the quality of this manuscript prior to publication.
1. In the experimental procedure, there were several irregularities in writing, such as “Zn(NO3)2·6H2O aqueous solution” and “586 μL of 10 mg·mL-1 HAuCl4·4H2O”.
2. During the EIS description, “initial potential” and “open potential” occurred simultaneously. It is understood that the phrase “open circuit potential” is commonly used for corrosion. Here it is best to replace “open circuit potential” with “initial potential”.
3. Electrochemical methods usually shown data instability, while in this article it shown good repeatability, did the ZnO-Au composites-based aptasensor construction could appropriate fix the aforementioned issues?
4. The text in Figures (Figure 2 and 3) needs to be enlarged to make it clear.
5. As shown in Figure 2A, the diffraction peak corresponding to the crystal plane (101) is so prominent. Does this crystal plane have an advantage on the load of gold nanoparticles?
6. It can be seen that the method, according to the manuscript (discussion & conclusion), has no major limitations (disadvantages). Please specify some at the end of the section Results and Discussion.
Comments for author File: 
Comments.pdf
Author Response
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Author Response File: Author Response.pdf
Reviewer 2 Report
Ref: processes-2265338
Title of the manuscript: “Electrochemical Aptasensor Based on ZnO-Au nanocomposites for the Determination of Ochratoxin A in Wine and Beer.”
In this paper, Zhang et al. reported an electrochemical aptasensor for Ochratoxin A (OTA) determination in real samples. The fabrication of the electrode involved ZnO-Au nanocomposite-modified GCE involving methylene blue (MB). In presence of OTA, the aptamer would attach itself to the OTA and further detaches itself from the electrode surface, thus preventing the MB from accessing the electrode surface, or the decrement in current is observed. While in absence of OTA, the MB would lead to a perfect MB/MBH2 electrochemical response with a higher peak current. I recommend the publication of the current manuscript after addressing the following points.
1. Why is the EIS data for ZnO/GCE> ZnO-Au/GCE> GCE. The reason for the support should be explained in the manuscript. Support the data with references.
2. Authors have reported in Figure 3, with an increase in OTA concentration, the current value of DPV decreases. The decrease in DPV can also be attributed to the incomplete attachment (lesser incubation time) or false response of the OTA binding. Hence, some strong proof of concept should be done using a traditional method and their relative comparison with respect to the electrochemical technique should be done (as a separate graph). Based on the relationship, the error percentage for each determination should be calculated in Figure 3.
3. The real sample analysis should be confirmed using 2 different traditional techniques.
4. A separate table should be tabulated for the literature review consisting of electrochemical and conventional techniques for OTA analysis.
Author Response
Please see the attached file.
Author Response File: Author Response.pdf
Reviewer 3 Report
In the paper ‘Electrochemical Aptasensor Based on ZnO-Au nanocomposites for the Determination of Ochratoxin A in Wine and Beer’, by Zhang S. and co-workers a novel aptasensor for Ochratoxin is investigated. The sensor is based on the immobilization of a c-DNA and the aptamer on a ZnOx-Au nanostructured support which is capable to bind methylene blue. In presence of Ochratoxin A, the aptamer shows a preferential affinity to the analyte leaving the surface of the electrode solely with a single stranded DNA.
After a careful reading of the paper, I would like to recommend the author to address the following points:
The authors state several times that their surface area is ‘larger’, but the paper is missing a proper quantification of this parameter. Could you please properly evaluate the surface area via Randles-Sevcik equation?
The concept presented in Figure 1 is not clear. If the aptamer is ‘leaving’ the surface due to the interaction with Ochratoxin why the surface is becoming more resistive? Is it related to the surface charges?
I do not understand at all the role of Nafion. This must be clarified in the text.
Figure S2 B is difficult to understand. I cannot clearly see the correspondence between the functionalization step and the impedance trace. Two traces are also the same color.
The caption of Figure S3 has no A and B labels
I do not understand if it is possible to re-use the sensor. It seems likely not since the aptamer is leaving the electrode, but I am wondering if the electrode surface can be re-loaded with new aptamer. Could you please comment about the reusability? The paragraph Specificity, repeatability and stability is not fully addressing this topic.
The following work should be cited: https://www.mdpi.com/2072-666X/13/6/834. It would be nice if you can compare this work with your results since their detection limit if 5 fg/ml.
Best Regards
Author Response
Please see the attached file.
Author Response File: Author Response.pdf
Round 2
Reviewer 2 Report
The manuscript can be considered for publication.
Author Response
Thanks the reviewer for the positive responses to our manuscript.
Reviewer 3 Report
Response 2 from the authors:
'Thanks the reviewer’s comments and suggestion. In Scheme 1, the aptamer captured OTA and detached from the electrode interface. Then, the double-strand DNA (dsDNA) was changed to single-strand DNA (ssDNA). In this case, the MB molecules would detached from the dsDNA and released to the solution, thus preventing MB/MBH2 from accessing the electrode surface for efficient electron transfer, and a weak peak current of MB was obtained. The surface didn’t become more resistive, but changing the distance between MB redox and the electrode. We have revised this part to make it more clear on page 5 in the revised manuscript.'
Do you observe a change in peak current dependency with the scan rate before and after the release of MB? This is also related to the Randles equation and it tells you if your electrode transfer is governed by diffusion or not.
Author Response
Thanks the reviewer’s comments. The peak current dependency with the scan rate before and after the release of MB were investigated. The effect of scan rates over the as-prepared electrode were performed at different sweep rates from 10 to 200 mV/s (Figure S4). The cathodic and anodic peaks over the as-prepared electrode before the release of MB increased with the increase of the sweep rates, indicating a typical surface-controlled process. After release of MB from the as-prepared electrode, the cathodic and anodic peak currents do not increase linearly with the increase of scan rates, but increased linearly with the increase of square root rates, suggesting the electrode reaction is not a surface controlled process but a diffusion-controlled one. We have revised this part to make it more clear in the revised manuscript.
