A Novel Synthesis of a Magnetic Porous Imprinted Polymer by Polyol Method Coupled with Electrochemical Biomimetic Sensor for the Detection of Folate in Food Samples
Round 1
Reviewer 1 Report
In recent years, molecularly imprinted polymers (MIPs) are treated as artificial antibodies and have received great attention in molecular recognition and biosensors. In this manuscript, the authors synthesized magnetic molecularly imprinted polymers and used them to detect folate in different food samples. The works look good and the paper is well-organized. However, some issues are still founded, which should be addressed before publication.
1. The imprinted polymers are the most important work in the manuscript and should be included in the title.
2. For the Thermogravimetric analysis, the degradation of both materials should start around 100%.
3. Advanced MIP-based electrochemical sensor works are recommended to be highlighted, like Biosensors and Bioelectronics (2021) 191, 113434.
4. The sensing ability should be compared with other reported works, especially MIPs works.
5. Error bars should be given in Figure 10. Also, why does the signal decrease after adding the interference samples? Usually, the interference may cause more background noise here.
6. English still needs to be polished.
Author Response
Please see the attachment, thanks in advance!
Author Response File: 
Author Response.pdf
Reviewer 2 Report
The work aims at presnting an electrochemical sensor for detection of Folates, yet it fails to cover the basic optimization parameters that has to be considered for an electrochemical volatmmetric sensor, where, no information was given regarding the optimization of the sensor composition, mechanism of interaction with the target template, Effect of pH, scan rate, incubation time, accumulation time and potential, No information regarding electrochemical characterization using CV or EIS, ......plotting a calibration curve using SWV is not efficient enough to consider this work as an electrochemical sensor.
The role of using magnetic MIps is not clear at all.
The optimization of the ratio between template and monomer was not given as and was not tested neither in computational calculations or pracrically.
Figure one has to be in English
A comparison should be given with previous work as this is not the first sensor for folate.
for interference, what about other vitamin B members??
Author Response
Please see file attachment, thanks in advance!
Author Response File: Author Response.pdf
Reviewer 3 Report
The authors prepared a new molecularly imprinted sensor for the quantification of folate in food samples. A series of experiments were carried out to characterize the sensor and to demonstrate its sensing performances. The sensor was also successfully employed to quantify folate in real-world samples. This manuscript is well designed and carried out. Publication is recommended. Several minor issues are raised below.
1. The linear dynamic range in the abstract is inconsistent with that in Fig. 8.
2. How long is the readsorption time of MMIPs?
3. Dynamic light scattering for Fe3O4, MMIPs and MNIPs should be supplied.
4. Section 3.1: Please provide graphs for nitrogen adsorption and pore size distribution.
5. Section 3.9: The concentration of interfering substances should be more than 10 times that of folate.
6. The advantages of this detection method compared to other existing techniques should be more thoroughly described.
7. A recent, relevant review could be cited.
Sci. Bull. 2022, 67, 853
Author Response
Please see the attachment file, thanks in advance!
Author Response File: Author Response.pdf
Reviewer 4 Report
In this research article, the authors describe the development of an electrochemical biomimetic sensor for folate detection in foods. The authors put a lot of effort in the synthesis and characterization of the magnetic porous polymer, treating only superficially the sensing part. In summary, the authors synthesized magnetic molecularly imprinted polymers (MMIPs), which they subsequently used to modify a CPE, however the modification steps and conditions were not provided. MMIPs are widely used in analytical and electroanalytical chemistry, but the authors managed to develop a new, specific synthesis route, which allows the detection of folate, a very important vitamin for humans. The new material was thoroughly characterized, and its efficiency was proven. For the sensor development however, insufficient data is presented.
The reviewer rejects the article in its current form and would like to suggest the authors to write 2 separate articles, one focusing on the synthesis of the magnetic porous polymer and its characterization, with only a short description of its application for folate detection and direct it towards a journal focusing on nanomaterials. For the second article (starting with chapter 3.6) the authors need to put more effort in the presentation and characterization of the newly developed biomimetic sensor and resubmit it to Chemosensors.
Some specific comments on the manuscript:
· Large parts of the introduction lack proper citation: e.g. lines 55-60, 62-67, 82-85
· Lines 142-146 – they are repetition of 136 -141, delete them
· All terms in equation 1 should be defined
· Line 207, BET method was not defined
· Notation of units in text and table 1 differ, check and use same notation
· Line 211: delete word “analysis”
· A more detailed analysis on the SEM images is required. There are 2 images with different sized NPs, the bigger ones, presumably Fe3O4@SiO2, the presence of silica should be addressed.
· Figure 2 – there are 3 SEM images and 4 legends – please check accuracy of text and image
· Line 239: “An increase in the average particle size of the materials was observed after polymerization” – please provide numerical values
· Figure 5 -legend: highlight (b), (c) and (d) in figure. (a) is missing.
· Keep same notations, sometimes it is MIP, sometimes MMIP
· HPLC measurements can also be conducted as a validation for the obtained SWV results
On the sensor description, characterization, and its analytical performances – mandatory aspects to be considered for article acceptance:
· Sensor configuration is not presented. How were the CPE modified with the MMIPs?
· Spectra (CV, SWV) in the absence of folate are not presented.
· How was the optimal pH value of 4 established?
· The biomimetic sensors principle of detection was not highlighted.
· Again, notations in text and figure differ: MIP/CPE or MMIPs/CPE
· Ionic strength of acetate buffer is not given.
· By calculating the capacitance in a non-faradaic region form the CVs in Fig. S4, the authors can comment on the electrode surface modification in both MMIP and MNIP scenarios, using the formula C = current density / scan rate for a fixed non faradaic potential.
· Electrochemical impedance spectroscopy provides additional information on the polymer porosity and electron transfer at the interface – only then the authors can talk about a complete electrochemical profile of the modified electrode / sensor
· Line 337 – what do the authors mean by „polishing the electrode surface”. The MMIP modification itself can be polished and reused? How thick is the MMIP layer? How many times can it be polished? How do the authors assure repeatability in these conditions?
· Line 350 – correct pH value
· Electrode stability was not discussed. A number of 50 CV cycles should be executed in the presence and absence of folate and the results must be discussed.
· How was the interference study conducted in different food samples? Are the results the same in any of the samples? That is unlikely in the reviewers opinion.
· Food sample preparation was not described. Were the samples adjusted to a pH of 4?
· Why did the authors choose antibiotics as interferents in food samples? Their chemical structure and/or oxidation potential do not justify them as interferents.
· Why was it necessary to use the spiking method? For e.g. broccoli naturally contains folate – could it not be detected without spiking the sample? – This can be validated using HPLC.
· Comparison with other folate sensors form literature is missing. A table comparing the performances should be added.
Author Response
Please see the attachment, thanks in advance!
Author Response File: Author Response.pdf
Round 2
Reviewer 2 Report
Unfourtantly, the autrhors ignored completly the fisrst major comment regarding the optimization of the sensor composition, mechanism of interaction with the target template, Effect of pH, scan rate, incubation time, accumulation time and potential, No information regarding electrochemical characterization using CV or EIS, ....
Inorder to consider that a new sensor is presented, all these parameters which influence the response has to be tested, once more plotting a calibration curve using SWV is not efficient enough to consider this work as an electrochemical sensor.
Author Response
Dear Rewiever
Please see the attachment
Best regards
Pilar
Author Response File: Author Response.pdf
Reviewer 4 Report
In the revised version of the article there is significant improvement, but not yet ready for publishing. The authors describe the development of an electrochemical biomimetic sensor for folate detection in foods. In summary, the authors synthesized magnetic molecularly imprinted polymers (MMIPs), which they subsequently used to modify a CPE. MMIPs are widely used in analytical and electroanalytical chemistry, but the authors managed to develop a new, specific synthesis route, which allows the detection of folate, a very important vitamin for humans. The new material was thoroughly characterized, and its efficiency was proven. For the sensor development and performances however more data is required.
Specific comments on the revised manuscript:
· Line 47 “(Robinson, 2000)” – correct to reference number
· Large parts of the introduction lack proper citation: e.g. lines 55-60, 62-67, 82-85
- The authors say that it was corrected, but no insertion of references was noted. More specifically “The natural forms of folate 53 include the following: single carbon derivatives of tetrahydrofolate (H4 folate) and dif-54 ferent substituents to N5 or N10. The major sources of folate in food and metabolic reac-55 tions are H4 folate, 5-methyltetrahydrofolate (5-CH3-H4folate), 5-formyltetrahydrofolate 56 (5-CHO-H4folate), 10-formyltetrahydrofolate (10-CHO-H4folate) and oxidized 57 10-formyl-folic acid (10-CHO-PteGlu), and their interconversion products including 58 5,10-methylentetrahydrofolate (5,10-CH2-H4folate) and 5,10-methenyltetrahydrofolate 59 (5,10-CH+-H4folate). The multiplicity and complexity nature of folate forms and the 60 ability to convert and reconvert them into other forms makes the analysis of this group of 61 vitamins extremely challenging. A wide range of methods have been employed for the 62 analysis of folates. Apart from the general extraction procedures which include extrac-63 tion in buffer solution, heating, and other clean-up procedures (such as solid-phase ex-64 traction), all the methods applied for the analysis of folates require the conversion of 65 polyglutamate folate - the naturally occuring folate in food, into monoglutamate folate 66 through the application of pteroyl polyglutamate hydrolases (PPH).” – There are at least 2 statements here that need citations!
· Lines 133, 136 – L-1 – use superscript, carefully check whole manuscript for such errors
· Chapter 2.5 – please also add approximately how many times the surface can be regenerated. Could this approach be an advantage of the developed sensor towards single use electrodes – from an economical perspective? Please also comment on this.
· A more detailed analysis on the SEM images is required. There are 2 images with different sized NPs, the bigger ones, presumably Fe3O4@SiO2, the presence of silica should be addressed.
-The authors partially addressed this comment. Some comments like “Upon ploymerization an increase of the particle size could be observed, however structural uniformity ….. and distribution …. did/did not change….”
· Line 239: “An increase in the average particle size of the materials was observed after polymerization” – please provide numerical values
- The authors say it is visible in the figure. It is visible, but not clear! The authors should state “After silica polymerization, the nanoparticle diameter increased to 200 something nm”
· Figure 3(c) shows a big clump. Does this correspond to aggregation of the MMIPs? If yes, this could be a problem in the CPE/MMIP electrode surface homogeneity. Please comment on this.
· How was the optimal pH value of 4 established?
- The reply provided by the authors to the reviewer should also be added to the manuscript, chapter 2.4
· Chapter 3.8 – Regarding repeatability, was the electrode surface polished in between the n = 5 measurements? This is an important parameter, please specify.
· What about reproducibility? Were there several electrodes fabricated and tested or the surface regeneration was enough?
· Electrode stability was not discussed. A number of 50 CV cycles should be executed in the presence and absence of folate and the results must be discussed.
- The authors confuse repeatability with stability. Stability means that the electrode needs to be tested only in buffer solution, no analyte, for a very high number of scans. The reviewer suggested CVs because it is easier compared to do 50 SWV (at least). In this type of measurement it is important that the shape of the CV doesn’t change much from the first to the last scan, otherwise, the electrode is not stable. This can be added to supplementary data, but it must be specified.
· How was the interference study conducted in different food samples? Are the results the same in any of the samples? That is unlikely in the reviewers opinion.
- The reviewer thinks the authors misunderstood the question. The sentence „Figure 10 shows the results obtained from the interference analysis conducted using some potential interfering compounds in the presence of the analyte in different food samples” is confusing. The authors should choose 1 food sample, e.g. broccolli and do the interferent study in that specific food sample. Otherwise how did the authors determine the interferences in different food samples and are representing only one graphic?
· Food sample preparation was not described. Were the samples adjusted to a pH of 4?
- The authors only responded to the 2nd question. Please add it also to the manuscript, it is important information. Regarding the first question/statement: „Food sample preparation was not described” – please at least mention in the supplementary files if the fruits and vegetables were just liquefied with a blender or they were also boiled and prepared in some other manner. This is important to mention, because the reader needs to understand if the developed electrode could be used in an orange juice (home made or commercial) and it would highlight the electrodes potential to be commercialized.
· Comparison with other folate sensors form literature is missing. A table comparing the performances should be added.
- The authors misunderstood the reviewers request. Table S1 highlights the detection of different analytes in different samples. The reviewer wants a comparison between the performances of CPE/MMIP (LoD = 1 * 10-7 M) and other sensor/biosensor architectures for folate detection with their LoDs. Some articles about electrochemical folate detection in blood but also food samples: https://0-doi-org.brum.beds.ac.uk/10.1016/j.bios.2016.05.095, https://0-doi-org.brum.beds.ac.uk/10.1007/s11694-022-01421-2
Author Response
Dear Rewiever
Please see the attachment
Best regards
Pilar
Author Response File: Author Response.pdf
Round 3
Reviewer 2 Report
The authors added effect of accumulation time and pH while other basic variables for a sensor optimization are still ignored.
Author Response
Ms. Ref. No.: chemosensors-1910240
Title: A novel synthesis of magnetic porous polymer by polyol method coupled with electrochemical biomimetic sensor for detection of folate in food samples
Comments:
Firstly, we thank you for all the excellent comments that improve our article. The Responses are highlighted in yellow color.
Response: Thanks for the comment. The introduction has been improved and some new refrences were also added. Moreover, the manuscript is also correct for minor spell checks in English. Please also see the comments raised by reviewer 4.
Author Response File: Author Response.pdf
Reviewer 4 Report
In the revised version of the article there is significant improvement, the authors provided all the requested data and they tackled all enquiries. However, the reviewer insists that the authors add the valuable graphs and explanations provided in the cover letter to the manuscript and supplementary material.
Specific comments on the revised manuscript:
· What about reproducibility? Were there several electrodes fabricated and tested or the surface regeneration was enough?
è Thank you for the explanation and please add it to the manuscript, with proper citation of previous work, especially since the authors mention reproducibility in line 413!
· Electrode stability was not discussed. A number of 50 CV cycles should be executed in the presence and absence of folate and the results must be discussed.
è The authors must add text and figure to the manuscript. It could go under chapter 3.8 by changing the title to „Characteristics of MMIPs/CPE” or similar and address repeatability, stability and previously explained reproducibility. It is enough to add to figure 9 the stability using only CVs, the SWV graph can be added to supplementary materials.
· How was the interference study conducted in different food samples? Are the results the same in any of the samples? That is unlikely in the reviewers opinion.
è The explanation provided to the reviewer is nice and clear, but please add it to the manuscript, there could be other readers who will get confused if the original version is kept.
Author Response
"Please see the attachment."
Author Response File: Author Response.pdf
