Preparation of Magnetic Molecularly Imprinted Polymer for Methylene Blue Capture

Round 1

Reviewer 1 Report (Previous Reviewer 2)

In this work, the authors prepared a magnetic molecularly imprinted polymer for the selective removal of methylene blue using magnetic ferric oxide as carrier and nylon-6 as functional monomer. The results show that the material has a good selective adsorption effect on methylene blue. However, some details require more work in whole the manuscript. In general, I think this paper should be published by Magnetochemistry after minor revision.

（1） The adsorption kinetic results should be presented in the manuscript, which have great significance for the comprehensive investigation of adsorbent performance.

（2） It is suggested that the relevant description of methylene blue in the introduction be placed in the first part of the preface to make the introduction smoother.

 Minor editing of English language required

Author Response

The authors gratefully thank the Referee for the constructive comments and recommendations, which help to improve the readability and quality of the paper. All the comments are addressed accordingly and have been incorporated into the revised manuscript. Detailed responses to the comments and recommendations are as follows. Please note that all the comments are bold-faced, and the authors' reply follows immediately below the comments.

The adsorption kinetic results should be presented in the manuscript, which have great significance for the comprehensive investigation of adsorbent performance.

Thank you for your valuable comments. We have inserted some data about adsorption kinetic in the Subsection 3.2. . Despite the fact that adsorption kinetics is a very important factor affecting assay performance, the adsorption for all MMIP samples reached adsorption equilibrium for 1-3 min. Therefore, it is quite difficult to analyze the rate of the adsorption quantitatively and calculate the rate constant. 

It is suggested that the relevant description of methylene blue in the introduction be placed in the first part of the preface to make the introduction smoother.

Thank you for your valuable comments. We have revised the text according to reviewer’s suggestion.

Reviewer 2 Report (Previous Reviewer 3)

In this work, the nylon-based magnetic molecularly imprinted polymer (MMIP) for the capture of MB dye was successfully prepared. As for the questions raised before, the authors have modified or supplemented the relevant statements and data presentation. The properties of MMIP were comprehensively characterized by various methods of TEM, AFM, DLS and FTIR. The contents of material stability, selectivity and practical application effect were also added. Meanwhile, the logical hierarchy and result description of this revised manuscript are clearer and more concise. For these reasons, I consider the article acceptable. Before full acceptance, authors are expected to make minor changes to the keywords and formatting of the subheadings (i.e., 2.9, 3.4, and 3.5).

Author Response

The authors gratefully thank the Referee for the constructive comments and recommendations, which help to improve the readability and quality of the paper. All the comments are addressed accordingly and have been incorporated into the revised manuscript. Detailed responses to the comments and recommendations are as follows. Please note that all the comments are bold-faced, and the authors' reply follows immediately below the comments.

Before full acceptance, authors are expected to make minor changes to the keywords and formatting of the subheadings (i.e., 2.9, 3.4, and 3.5).

Thank you for your valuable comments. We have revised formatting. However, we don’t clearly understand what we have to do with keywords. We have revised some keywords, according to previously published works on this topic.

Reviewer 3 Report (New Reviewer)

The manuscript describes the preparation of a series of magnetic molecularly imprinted polymers (MMIPs) with methylene blue (MB) as the template by using the off-the-shelf polymer technology. The nylon-6 polyamide polymer and magnetic nanoparticles were used as a matrix and magnetic core, respectively. The resulting MMIPs showed good selectivity toward MB in comparison with its analogues and high template binding capacities. They could be easily separated from the analyte solutions under the magnetic field and could be reused at least 4 times. Moreover, their application as solid-phase extraction sorbents was also demonstrated by analyzing MB from the lake water. I recommend the manuscript to be published in the journal after the authors address the following issues:

1)      There are too many figures in the manuscript. Figures 1 and 13 can be moved to the Supporting Information. Figures 2 and 3 are basically the same and can be combined into one figure.

2)      Please also present the AFM images of MMIP_2 and MMIP_5 in the Figure 6. The Scatchard plot of MNIP_0.5 should also be presented in Figure 11.

3)      The binding kinetics should be studied and presented in the manuscript.

4)      During the preparation of MMIPs, the template was removed by washing the polymers with an aqueous solution containing 5% glacial acetic acid and 5% sodium dodecyl sulfate (SDS) at 60 °C for 1 h. However, the MMIPs were washed with an aqueous solution containing 1% glacial acetic acid and 1% SDS at 60 °C for 1 h for the reusability test. Why change the washing solutions in the latter case?

5)      Since the specificity coefficients (Cs) of the MMIPs are different at different analyte concentrations. Please provide the MB concentration for the mentioned Cs values in line 306 in page 9.

6)      The reusability efficiency of MMIP_0.5 is much lower than MMIP_2 and MMIP_5 (Figure 12), which might be attributed to the much lower content of the magnetic nanoparticles in MMIP_0.5, which led to the lower recycled amount of MMIP_0.5 during the reusability test. This point should be made clear in the manuscript.

Author Response

The authors gratefully thank the Referee for the constructive comments and recommendations, which help to improve the readability and quality of the paper. All the comments are addressed accordingly and have been incorporated into the revised manuscript. Detailed responses to the comments and recommendations are as follows. Please note that all the comments are bold-faced, and the authors' reply follows immediately below the comments.

There are too many figures in the manuscript. Figures 1 and 13 can be moved to the Supporting Information. Figures 2 and 3 are basically the same and can be combined into one figure.

Thank you for your valuable comments. However, such Figures were inserted due to the problems with understanding of work design and some experimental procedures according to referees’ reviews.

Please also present the AFM images of MMIP_2 and MMIP_5 in the Figure 6. The Scatchard plot of MNIP_0.5 should also be presented in Figure 11.

Thank you for your valuable comments. We have inserted AFM images of MMIP_2 in the Figure 6 and Scatchard plot of MNIP_0.5 in Figure 11. Unfortunately, AFM data for MMIP_5 could not be received.

The binding kinetics should be studied and presented in the manuscript.

Thank you for your valuable comments. We have inserted some data about adsorption kinetics in the Subsection 3.2. Despite the fact that adsorption kinetics is a very important factor affecting assay performance, the adsorption for all MMIP samples reached adsorption equilibrium for 1-3 min. Therefore, it is quite difficult to analyze the rate of the adsorption quantitatively and calculate the rate constant. These results indicate that MMIPs exhibit a good imprinting effect. 

During the preparation of MMIPs, the template was removed by washing the polymers with an aqueous solution containing 5% glacial acetic acid and 5% sodium dodecyl sulfate (SDS) at 60 °C for 1 h. However, the MMIPs were washed with an aqueous solution containing 1% glacial acetic acid and 1% SDS at 60 °C for 1 h for the reusability test. Why change the washing solutions in the latter case?

Thank you for your question. There is no difference between 1% or 5% solutions for such experiment. A 1% solution is enough for MB clearance. However, for the MMIP synthesis, we decided to use 5% for precise cleaning.

Since the specificity coefficients (Cs) of the MMIPs are different at different analyte concentrations. Please provide the MB concentration for the mentioned Cs values in line 306 in page 9.

Thank you for your valuable comments. We have inserted in the text concentration for Cs values.

The reusability efficiency of MMIP_0.5 is much lower than MMIP_2 and MMIP_5 (Figure 12), which might be attributed to the much lower content of the magnetic nanoparticles in MMIP_0.5, which led to the lower recycled amount of MMIP_0.5 during the reusability test. This point should be made clear in the manuscript.

Thank you for your valuable comments. We have revised the text according to reviewer’s suggestion.

Reviewer 4 Report (New Reviewer)

Title: Preparation of Magnetic Molecularly Imprinted Polymer for  Methylene blue. What for? Waste wather management? You should provide more meaningful title.

Lines 43-45: „MMIPs show excellent selective binding to target compounds and can be 44 quickly separated and easily removed from samples using an external magnet, avoiding the time-consuming and tedious steps (centrifugation or filtration) [1,2,18,6,9– 46 12,15–17].” Magnetic separation is easy and simple in laboratory conditions, but is it applicable in industrial conditions for wastewater treatment?

Lines 81-82: “MB has antiseptic, photo-inactivates multidrug-resistant bacteria, and cancer treatment [40,43].” Rephrase this sentence.  For example: Due to its photochemical properties methylene blue inactivates multidrug-resistant bacteria, and kill cancer cells.

Lines 95-110: Remove the entire paragraph. This paragraph can be used for the results, discussion or conclusion sections. In the introduction, the results are not presented or discussed.

Line 125: “The resulting MNPs have an average size of 11.0 ± 2.0 nm using TEM.” This is your result. You should move it to result section and provide corresponding figure of TEM.

Result and discussion section:

The results and discussion section is written as follows: The obtained result is announced and then the reason for such a result is discussed. For example:

Result: Fe3O4@mSiO2@DMIP and Fe3O4@mSiO2@NIP were regularly spherical with a rough surface and a diameter ranging from 120 nm to 300 nm and 100 nm to 270 nm, respectively (Fig. 2).

Discussion: The broad size distribution of the Fe3O4@mSiO2@DMIP and Fe3O4@mSiO2@NIP particles may be attributed to the uneven stirring speed and reaction temperature during polymerisation.

Lines 202-206: This paragraph is more suited to the introduction than to the results and discussion.

Line 207-208: “For MMIP and MNIP synthesis, 208 MNP water suspension and 2,2,2-trifluoroethanol (TFE) solution of nylon-6 were used.” This sentence belongs to the material and methods section.

Line 223-224: “To synthesize MMIPs, we dissolved nylon-6 in TFE and mixed it with MB and MNPs  aqueous solution under vigorous stirring (Figure 3).“ This is not a result. This sentence belongs to the material and methods section.

Line 223-231: “To synthesize MMIPs, we dissolved nylon-6 in TFE and mixed it with MB and MNPs 224 aqueous solution under vigorous stirring (Figure 3). For the MNIPs, we haven’t used tem225 plate molecule MB for the synthesis. The reaction mixture was stirred (1400 rpm) at 25 °C 226 for 24 h. Afterward, MMIPs and MNIPs were magnetically separated and washed with 227 water. To extract the template molecules from the polymer structure, MMIPs were incu228 bated with the aqueous solution containing 5 % glacial acetic acid and 5 % sodium dodecyl 229 sulfate (SDS) under stirring (1400 rpm) at 60 °C for 1 h. For comparison, MNIPs have also 230 been incubated with acid and SDS solution. MMIPs and MNIPs were magnetically sepa231 rated and washed with water.” This whole paragraph is for the material and methods section. Explanation: The material and methods section exists to describe how the experiment was done with the help of which apparatus and chemicals and under what conditions, so that if someone wants to repeat your experiment, they could do it based on your description in the material and methods.

237-238: “The obtained MMIPs and MNIPs were characterized by DLS, transmission electron mi238 croscopy (TEM), and atomic force microscopy (AFM).”-delete this sentence.

Lines 347-351: “ To aliquots of MMIPs and MNIPs sus348 pensions, various amounts of MB, TB, or PH dyes were added and incubated for 12 h at 349 25 °C under stirring (1400 rpm). Afterward, the mixtures were magnetically separated and 350 analyzed by UV-vis at 660 nm (MB), 620 nm (TB), and 390 nm (PH). The amount of dye  was calculated using a serial dilution of a dye standard solution.” Move the entire paragraph to the material and methods section.

Conclusion

The conclusion is written as an abstract, which is wrong and distracts the readers. The conclusion should only contain the conclusions you reached based on your results. For example, by examining the effectiveness of three MMIP samples of different concentrations in removing MB, sample XY proved to be the best. It was also found that the XZ sample performed best for reuse. Based on the established efficiency, stability and physico-chemical properties, sample XY is suitable for further testing for potential application in wastewater treatment.

I recommend you to use grammar checker "Grammarly"( Its free of charge).

Author Response

The authors gratefully thank the Referee for the constructive comments and recommendations, which help to improve the readability and quality of the paper. All the comments are addressed accordingly and have been incorporated into the revised manuscript. Detailed responses to the comments and recommendations are as follows. Please note that all the comments are bold-faced, and the authors' reply follows immediately below the comments.

Title: Preparation of Magnetic Molecularly Imprinted Polymer for Methylene blue. What for? Waste wather management? You should provide more meaningful title.

Thank you for your valuable comments. However, as it has been mentioned, methylene blue was used as a model molecule for molecularly imprinting design presentation. In this way, we have only minimally revised a title of the work.

Lines 43-45: „MMIPs show excellent selective binding to target compounds and can be 44 quickly separated and easily removed from samples using an external magnet, avoiding the time-consuming and tedious steps (centrifugation or filtration) [1,2,18,6,9– 46 12,15–17].” Magnetic separation is easy and simple in laboratory conditions, but is it applicable in industrial conditions for wastewater treatment?

Thank you for your question. Yes, magnetic separation may be used for industrial wastewater treatment. Of course, it depends on water amount.^1–4

(1)       He, C.; Fang, K.; Gong, G.; Liu, J.; Song, X.; Liang, R.; He, Q.; Yuan, Q.; Wang, K. Advanced Organic Recovery from Municipal Wastewater with an Enhanced Magnetic Separation (EMS) System: Pilot-Scale Verification. Water Res. 2022, 217, 118449.

(2)       Tseng, J. Y.; Chang, C. C.; Tu, C. W.; Yuan, M. H.; Chang, C. Y.; Chang, C. F.; Chen, Y. H.; Shie, J. L.; Ji, D. R.; Liu, B. L.; Franzreb, M. Application of High-Gradient Magnetic Separation for the Recovery of Super-Paramagnetic Polymer Adsorbent Used in Adsorption and Desorption Processes. Processes 2023, 11 (3), 965. https://doi.org/10.3390/pr11030965.

(3)       Prabhu, S. M.; Rane, N. R.; Li, X.; Otari, S. V.; Girawale, S. D.; Palake, A. R.; Kodam, K. M.; Park, Y.-K.; Ha, Y.-H.; Yadav, K. K.; Khan, M. A.; Jeon, B.-H. Magnetic Nanostructured Adsorbents for Water Treatment: Structure-Property Relationships, Chemistry of Interactions, and Lab-to-Industry Integration. Chem. Eng. J. 2023, 468, 143474.

(4)       Macaskie, L. E.; Redwood, M. D. ( 12 ) Patent Application Publication ( 10 ) Pub . No .: US 2008 / 0225123 A1 Patent Application Publication, 2008.

Lines 81-82: “MB has antiseptic, photo-inactivates multidrug-resistant bacteria, and cancer treatment [40,43].” Rephrase this sentence.  For example: Due to its photochemical properties methylene blue inactivates multidrug-resistant bacteria, and kill cancer cells.

Thank you for your valuable comments. We have revised the text according to reviewer’s suggestion.

Lines 95-110: Remove the entire paragraph. This paragraph can be used for the results, discussion or conclusion sections. In the introduction, the results are not presented or discussed.

Thank you for your valuable comments. We have revised the text according to reviewer’s suggestion.

Line 125: “The resulting MNPs have an average size of 11.0 ± 2.0 nm using TEM.” This is your result. You should move it to result section and provide corresponding figure of TEM.

The magnetic nanoparticles (MNPs) were synthesized according to the previously published method. In this way, we are not provided TEM data.⁵

(5)       Bulgakova, A.; Chubarov, A.; Dmitrienko, E. Magnetic Nylon 6 Nanocomposites for the Microextraction of Nucleic Acids from Biological Samples. Magnetochemistry 2022, 8 (8), 85.

The results and discussion section is written as follows: The obtained result is announced and then the reason for such a result is discussed. For example:

Result: Fe3O4@mSiO2@DMIP and Fe3O4@mSiO2@NIP were regularly spherical with a rough surface and a diameter ranging from 120 nm to 300 nm and 100 nm to 270 nm, respectively (Fig. 2).

Discussion: The broad size distribution of the Fe3O4@mSiO2@DMIP and Fe3O4@mSiO2@NIP particles may be attributed to the uneven stirring speed and reaction temperature during polymerisation.

Lines 202-206: This paragraph is more suited to the introduction than to the results and discussion.

Line 207-208: “For MMIP and MNIP synthesis, 208 MNP water suspension and 2,2,2-trifluoroethanol (TFE) solution of nylon-6 were used.” This sentence belongs to the material and methods section.

Line 223-224: “To synthesize MMIPs, we dissolved nylon-6 in TFE and mixed it with MB and MNPs  aqueous solution under vigorous stirring (Figure 3).“ This is not a result. This sentence belongs to the material and methods section.

Line 223-231: “To synthesize MMIPs, we dissolved nylon-6 in TFE and mixed it with MB and MNPs 224 aqueous solution under vigorous stirring (Figure 3). For the MNIPs, we haven’t used tem225 plate molecule MB for the synthesis. The reaction mixture was stirred (1400 rpm) at 25 °C 226 for 24 h. Afterward, MMIPs and MNIPs were magnetically separated and washed with 227 water. To extract the template molecules from the polymer structure, MMIPs were incu228 bated with the aqueous solution containing 5 % glacial acetic acid and 5 % sodium dodecyl 229 sulfate (SDS) under stirring (1400 rpm) at 60 °C for 1 h. For comparison, MNIPs have also 230 been incubated with acid and SDS solution. MMIPs and MNIPs were magnetically sepa231 rated and washed with water.” This whole paragraph is for the material and methods section. Explanation: The material and methods section exists to describe how the experiment was done with the help of which apparatus and chemicals and under what conditions, so that if someone wants to repeat your experiment, they could do it based on your description in the material and methods.

237-238: “The obtained MMIPs and MNIPs were characterized by DLS, transmission electron mi238 croscopy (TEM), and atomic force microscopy (AFM).”-delete this sentence.

Lines 347-351: “ To aliquots of MMIPs and MNIPs sus348 pensions, various amounts of MB, TB, or PH dyes were added and incubated for 12 h at 349 25 °C under stirring (1400 rpm). Afterward, the mixtures were magnetically separated and 350 analyzed by UV-vis at 660 nm (MB), 620 nm (TB), and 390 nm (PH). The amount of dye  was calculated using a serial dilution of a dye standard solution.” Move the entire paragraph to the material and methods section.

Conclusion

The conclusion is written as an abstract, which is wrong and distracts the readers. The conclusion should only contain the conclusions you reached based on your results. For example, by examining the effectiveness of three MMIP samples of different concentrations in removing MB, sample XY proved to be the best. It was also found that the XZ sample performed best for reuse. Based on the established efficiency, stability and physico-chemical properties, sample XY is suitable for further testing for potential application in wastewater treatment.

Thank you for your valuable comments. We have revised the text according to reviewer’s suggestion. However, we have used non-standard MMIP synthesis. In this way, due to the previous round of reviewing, we have inserted some information about synthesis design.

Round 2

Reviewer 3 Report (New Reviewer)

The authors have adequately responsed my comments and revised their manuscript. I recommend the manuscript to be published in its present form.

Reviewer 4 Report (New Reviewer)

Thank you for your answers.

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.

Round 1

Reviewer 1 Report

This work investigates the preparation and characterisation of MMIP which were then tested for three dyes retention.

The focus of the work is on the synthesis and characterization of MMIP materials, the novelty of their procedure must be stressed in the introductory part. Additional characterisation must be included FTIR and XRD, UV/VIS DRS. However, adsorbed amounts of dye are far less than in the literature, ranging up to several hundred mg/g. If the authors have decided on this type of application, they should provide an overview, of their results compared to those already published. Also, some examples of interactions in the system, after performing FTIR analysis. It should be borne in mind that MB is easily subject to discolouration, so its presence must be monitored in the UV area, as well [10.3390/catal12050519}. The details in the experimental section should be supplemented with that information, at which wavelength it was recorded, as well as excitation and emission for fluorescence spectroscopy. Give a proposition on how to regenerate magnetic material after dye adsorption. 

Reviewer 2 Report

In this work, nylons-6 and Fe₃O₄ were used to prepare molecularly imprinted materials for the selective separation of methylene blue (MB). The synthesis method was simple, and the prepared material had good adsorption selectivity for MB. However, the clarity and the research integrality require more work in whole the manuscript. In general, I think this paper should not be published by Magnetochemistry.

1.     The writing logic of the introduction should be adjusted, is it to use molecular imprinting for the separation of MB or to select MB as the target for the purpose of using molecular imprinting?

2.     The sequence of section 2 should be adjusted, such as materials, synthesis, characteristic and so on.

3.     As a complete molecular imprinting research work, it is necessary to add some content, such as infrared analysis characterization could be used to characterize whether the material is successfully synthesized, adsorption kinetics and adsorption isotherm analysis, adsorption mechanism analysis, etc.

4.     The first time the abbreviation appears, the full name should be written. Such as SDS in line 103. The writing logic of the whole manuscript needs to be reorganized, and the language should be polished.

Reviewer 3 Report

This manuscript developed a convenient approach for the preparation of MMIP based on nylon-6 and magnetic nanoparticles. The MMIPs exhibited good adsorption ability and selectivity for MB. However, in view of insufficient performance evaluation of MMIPs, as well as inadequate innovation and non-standard writing, I don't think this paper is suitable to be published in Magnetochemistry. Here are some of my comments.

1. The abstract mentioned "The magnetic properties of the MMIP allow magnetic separation, imaging, hyperthermia, and selective template release. ", but MMIPs have nothing to do with it. Keywords are also very unreasonable.

2. What are the improvements of such MMIPs compared with other reported MIPs using methylene blue as the target molecules? They need to be discussed in the introduction and results.

3. In the synthesis of MMIPs, MB and nylon were used as a template molecule and a matrix, respectively. What are the components of functional monomers, cross-linker and initiators?

4. The MB-absorbed MMIPs were incubated with the aqueous solution containing 5% glacial acetic acid and 5% SDS under stirring (1300 rpm) at 90 °C for 1 h. Will this elution method lead to the reduction of the reproducibility of MMIPs, and how about the stability of such materials?

5. The characterizations of materials were insufficient, which can be supplemented by FT-IR, XRD, BET, etc.

6. Why does increasing the amount of magnetic core lead to different changes in the sizes of MMIPs and MNIPs? And the characterization description of DLS (Table 1) was interspersed again in that of SEM, which seems to be illogical and confused.

7. What are the analytical instruments and detection conditions for MB?

8. Figures 6, 7 and 8 are interrelated, but the scheme of dyes binding to the MMIPs and MNIPs in Figure 6 was not specifically described.

9. How about the detection effect of MMIPs on MB in actual samples?

10. The descriptions of synthesized materials in the manuscript include MB-MMIP, MMIP and MMIPs, which is very puzzled.

11. There are many expressions or grammatical errors in the manuscript, such as

—Molecularly imprinted materials are prepared using a polymer and a template molecule.

—The resulting polymer is called molecularly imprinted polymers (MIPs) or in the presence of the magnetic core in the structure magnetic MIPs (MMIPs).

—Nylon provides high physical and chemical stability, and low toxicity, which allows using this biocompatible polymer as medical plastic for various tools manufacturing.

—At the same time, with an increase in the MNP content, the size of the MMIP decreases, and the size of the MNIP increases.

—However, the Qc for TB dye is much lower, which may be explained by the great structural similarity of PH to MB.

The authors need to carefully check and revise the whole manuscript.

Comments for author File: Comments.docx