Advanced Ga2O3/Lignin and ZrO2/Lignin Hybrid Microplatforms for Glucose Oxidase Immobilization: Evaluation of Biosensing Properties by Catalytic Glucose Oxidation
, Adam Piasecki 3 and Teofil Jesionowski 1,*
Round 1
Reviewer 1 Report
The present article by Jedrzak and co-workers describes the synthesis and development of a hybrid microplatform for glucose sensing. The study combines the synthesis of oxides-lignin hybrids and the subsequent immobilization of glucose oxidase enzyme units for selective detection of glucose.
The general scope of the manuscript seems to fit better in a more specific journal devoted to sensor applications. After carefully reading the manuscript I can not consider its acceptance with this present form.
Please find below the comments/suggestions/corrections that would be necessary prior to further evaluation:
1) Introduction part: It would be necessary to expand the introduction and describe into further detail, the current state of the art and the comparison with other systems available in the literature (i.e. other metal oxides, other type of hybrid systems containing carbon-based systems, etc).
2) It would be beneficial for the manuscript to include some sort of description of the synthesis methodology as part of the 2.1 section of results. Include supporting schemes. Include the synthesis schemes. Describe how the samples are formed. Include additional images, provide statistics about the particle size distributions.
3) Line 95. Figure 1 is confusing. Have the authors assigned the TEM images correctly? The images corresponding to the hybrids appear much “cleaner” than the plain oxides. For the sake of clarity, I would also recommend renaming the different images with A,B,C,D letters avoiding A` types.
4) Line 104. It would be interesting to include EDX-mapping analysis to evaluate the distribution of lignin within the oxides.
5) XRD analysis should be provided with the identification of the oxide phases generated.
6) Line 175. How do the authors determine the bonding between the enzyme and the lignin? It seems very speculative. The authors should consider the use of FT-IR for comparative purposes.
7) Line 175. How do the authors quantify the amount of enzyme? Please expand the explanation with further details.
8) Line 205. Have the authors performed a control experiment with CPE+Lignin-GOx+Ferrocene (in the absence of the oxides)? Please complete this control experiment for comparison and to evaluate the critical effect of adding Ga and Zr oxides.
9) Additional discussion of the results should be included in the revised manuscript.
10) The authors should also comment and include additional results on the reusability of these sensor platforms.
Author Response
Answers to Reviewer 1
1) Introduction part: It would be necessary to expand the introduction and describe into further detail, the current state of the art and the comparison with other systems available in the literature (i.e. other metal oxides, other type of hybrid systems containing carbon-based systems, etc).
Thank you for the comment. The Introduction was enriched about description and the comparisons with other similar available hybrid system in literature.
2) It would be beneficial for the manuscript to include some sort of description of the synthesis methodology as part of the 2.1 section of results. Include supporting schemes. Include the synthesis schemes. Describe how the samples are formed. Include additional images, provide statistics about the particle size distributions.
Thank you for that comment. The description of methodology, synthesis details and additional schemes were added to the manuscript (new synthesis schemes in the Chapter 3.2. - 3.5.). Moreover, the extra SEM images and DLS (including PdI) measurements of the components and the final materials were added to the manuscript.
3) Line 95. Figure 1 is confusing. Have the authors assigned the TEM images correctly? The images corresponding to the hybrids appear much “cleaner” than the plain oxides. For the sake of clarity, I would also recommend renaming the different images with A,B,C,D letters avoiding A` types.
Thank you that you focused on the aspects. The images were verified and the renaming of them were changed, as you recommend.
4) Line 104. It would be interesting to include EDX-mapping analysis to evaluate the distribution of lignin within the oxides.
Thank you for the valuable comment. The distribution of lignin on the TEM and SEM images were confirmed. In the estimated time, there is no access to the equipment to carried out the EDX-mapping.
5) XRD analysis should be provided with the identification of the oxide phases generated.
Thank you for that comment. We used the commercially available β-Ga2O3 and β-ZrO2 oxides, so we are sure of the identification of them by manufacturer's data.
6) Line 175. How do the authors determine the bonding between the enzyme and the lignin? It seems very speculative. The authors should consider the use of FT-IR for comparative purposes.
Thank you for the question. We assume that there are creating hydrogen bonds, van der Waals interactions and covalent bonding (amide) too. The lignin are very various moieties-rich molecules (including carbonyl, carboxylic, hydroxyl). A detailed description about the bonding between the enzyme and the lignin has been attached to the manuscript. The mechanism of that was presented in the literature and also mentioned in the manuscript. (i.e. Colloids Surf., B. 162, 2018, 90-97).
7) Line 175. How do the authors quantify the amount of enzyme? Please expand the explanation with further details.
Thank you for the question. The quantify the amount of the enzyme was determined by the Bradford measurement. The assay is a spectrophotometric measurement (at wavelength λ = 595 nm) of a Coomassie Brillant Blue G-250 combination with amino acid residues containing a positive charge. These include mainly arginine, and to a slightly lesser extent: histidine, lysine, proline, tryptophan and tyrosine. The used dye links with the mentioned amino acids causing the colour changes from brown to blue. The intensity of the colour depends on the concentration of amino acids contained in the analyzed solution. The Bradford method belongs to relatively universal and precision analyzes.
8) Line 205. Have the authors performed a control experiment with CPE+Lignin-GOx+Ferrocene (in the absence of the oxides)? Please complete this control experiment for comparison and to evaluate the critical effect of adding Ga and Zr oxides.
Thank you for the comment. Verification of the lignin, as a component of immobilization, was impossible by Bradford method, due to the fact that the kraft lignin is well-soluble in water. We strongly believe that we are not able to construct biosensor material using only lignin as a matrix for enzyme immobilization. There could be some drawback such as electrode fouling. We assume that solid oxides are necessary to maintain a proper stability of enzyme at the surface of the electrode. This fact was presented in the previous works (i.e. Sens. Actuators B, 2018, 256, 176-185). The presence of these hybrid materials make a possible to immobilize the enzyme, and there is also a synergistic effect of combination inorganic (metal oxides) and organic (lignin) parts to make them more efficient carriers for that.
9) Additional discussion of the results should be included in the revised manuscript.
Thank you. Additional discussion of the work and more details, additional findings and new synthesis schemes was included in the manuscript.
10) The authors should also comment and include additional results on the reusability of these sensor platforms.
Thank you that you focused on the crucial biosensor's feature. Herein, we presented a novel materials, their physicochemical and morphological properties. Moreover, we proposed a potential applications for the enzyme immobilization and biosensing performance by carrying out the fundamental research. We are still working to improve our biosensors' systems, so the work is in progress. So, the manuscript presents the novel platforms with catalytic possible to biosensor application, however there is still basic findings in application.
Reviewer 2 Report
The proposed manuscript reports Ga2O3 or ZrO2/lignin hybrid materials that immobilize glucose oxidase (GOx) for the detection of glucose. This hybrid matrix showed not only a good platform for enzyme immobilization but also induced electrocatalytic properties by mixing ferrocene and carbon paste. The proposed manuscript is well written. Taking into account the quality and scope of the journal, I would recommend the acceptance of this manuscript to publish in ‘Catalysts’.
Herewith I have added a few comments that will help authors to improve the current manuscript:
The authors should add a schematic diagram that depicts the overall idea of the proposed manuscript. The authors should add a discussion about why Ga2O3 or ZrO2/lignin hybrid materials used in this study? Which hybrid materials showed better enzyme immobilization and catalytic activity? The authors should compare the activity of free GOx and immobilized GOx. It will help to understand the inhibitory effect of the proposed hybrid nanomaterials on the immobilized enzymes. The authors should check the stability and reusability of immobilized GOx. The applicability of the proposed systems should be validated with spiked glucose in biological fluids. The authors should check selectivity with other sugars. The universality of the proposed system should be discussed. It will expand the scope of the manuscript. The authors should add error bars in the figure (5, 7B, and 8B). It will highlight the repeatability of the proposed system. The authors should compare this study with other reports of GOx immobilization with the hybrid matrix. The authors should discuss other GOx immobilization methods by referring these research articles (such as Sensors and Actuators B: Chemical, 2017, 249, 321-330; Sensors and Actuators B: Chemical, 2019, 283, 749-754; Applied Surface Science, 2016, 364, 752-757).Author Response
Answers to Reviewer 2
The proposed manuscript reports Ga2O3 or ZrO2/lignin hybrid materials that immobilize glucose oxidase (GOx) for the detection of glucose. This hybrid matrix showed not only a good platform for enzyme immobilization but also induced electrocatalytic properties by mixing ferrocene and carbon paste. The proposed manuscript is well written. Taking into account the quality and scope of the journal, I would recommend the acceptance of this manuscript to publish in ‘Catalysts’.
Herewith I have added a few comments that will help authors to improve the current manuscript:
The authors should add a schematic diagram that depicts the overall idea of the proposed manuscript. The authors should add a discussion about why Ga2O3 or ZrO2/lignin hybrid materials used in this study? Which hybrid materials showed better enzyme immobilization and catalytic activity? The authors should compare the activity of free GOx and immobilized GOx (lit). It will help to understand the inhibitory effect of the proposed hybrid nanomaterials on the immobilized enzymes. The authors should check the stability and reusability of immobilized GOx. The applicability of the proposed systems should be validated with spiked glucose in biological fluids. The authors should check selectivity with other sugars. The universality of the proposed system should be discussed. It will expand the scope of the manuscript. The authors should add error bars in the figure (5, 7B, and 8B). It will highlight the repeatability of the proposed system. The authors should compare this study with other reports of GOx immobilization with the hybrid matrix. The authors should discuss other GOx immobilization methods by referring these research articles (such as Sensors and Actuators B: Chemical, 2017, 249, 321-330; Sensors and Actuators B: Chemical, 2019, 283, 749-754; Applied Surface Science, 2016, 364, 752-757).
Thank you for the comments and crucial tips for our research. We enriched the manuscript based on your information. The schematic diagrams, detailed descriptions, corrected figures, additional literatures' information and extra comparison to other materials were added to the manuscript. We also presented the immobilization efficiency based on these materials and other ones. In the work, we presented a new carriers (Ga2O3/Lig-GOx; ZrO2/Lig-GOx), and their physicochemical and morphological properties too. The glucose oxidase as a selected enzyme is very selective to glucose determination, well described in the literature, in the manuscript included. The attractive alternatives of the materials shedding a new light in a potential application for the enzyme immobilization and biosensing by carrying out the basic experiments.
Reviewer 3 Report
The manuscript “Advanced Ga2O3/lignin and ZrO2/lignin hybrid microplatforms for glucose oxidase immobilization: Evaluation of biosensing properties by catalytic glucose oxidation” is of interest and contains interesting science but some control experiments are missing.
The authors modify both Ga2O3 and ZrO2 with APTES. They should perform CHN analysis to see how much N is in the sample and how many amino groups they might have. The assumption is that these will react with the lignin after oxidation of the lignin. Both for the lignin after oxidation and for the modified oxides a titration would reveal the number of acid and amino groups respectively.
The authors are not really clear on how they follow the immobilisation. Is it enzyme in solution that is probed by Bradford assay to see how much is left? The authors should also check activity before and after immobilisation. And the authors should verify that the immobilised enzyme is actually active.
The enzyme contains a co-factor. This might wash out in the immobilisation process. It might also be that only the co-factor binds well to the carrier. The authors need to perform blanks on the CV experiments to rule out that what they observe is not an artefact but the activity of an enzyme immobilised in its active form. Also the authors need to prove that the enzyme does not leach and it is leached enzyme that gives the effect observed.
Then the authors need to perform stability tests. Now only CV shows activity. But for how long? What is the storage stability?
Once these points have been answered the manuscript can be resubmitted.
But for how long? What is the storage stability?
Once these points have been answered the manuscript can be resubmitted.
Author Response
Answers to Reviewer 3
The manuscript “Advanced Ga2O3/lignin and ZrO2/lignin hybrid microplatforms for glucose oxidase immobilization: Evaluation of biosensing properties by catalytic glucose oxidation” is of interest and contains interesting science but some control experiments are missing.
The authors modify both Ga2O3 and ZrO2 with APTES. They should perform CHN analysis to see how much N is in the sample and how many amino groups they might have. The assumption is that these will react with the lignin after oxidation of the lignin. Both for the lignin after oxidation and for the modified oxides a titration would reveal the number of acid and amino groups respectively.
Thank you for the comment. The CHN analysis was performed and the results are summarized in the Table 2 in the manuscript. The increase of the nitrogen content in the samples after APTES-modification were confirmed.
The authors are not really clear on how they follow the immobilisation. Is it enzyme in solution that is probed by Bradford assay to see how much is left? The authors should also check activity before and after immobilisation. And the authors should verify that the immobilised enzyme is actually active.
Thank you for the question. The quantify the amount of the enzyme was determined by the Bradford measurement. The assay is a spectrophotometric measurement (at wavelength λ = 595 nm) of a Coomassie Brillant Blue G-250 combination with amino acid residues containing a positive charge. These include mainly arginine, and to a slightly lesser extent: histidine, lysine, proline, tryptophan and tyrosine. The used dye links with the mentioned amino acids causing the colour changes from brown to blue. The intensity of the colour depends on the concentration of amino acids contained in the analyzed solution. The Bradford method belongs to relatively universal and precision analyzes. The efficiency of glucose oxidase immobilization was added in the manuscript. The results came from electrochemical assays confirm that the enzyme is active by characteristic curve, described as a Michaelis–Menten kinetics presented in the manuscript indeed.
The enzyme contains a co-factor. This might wash out in the immobilisation process. It might also be that only the co-factor binds well to the carrier. The authors need to perform blanks on the CV experiments to rule out that what they observe is not an artefact but the activity of an enzyme immobilised in its active form. Also the authors need to prove that the enzyme does not leach and it is leached enzyme that gives the effect observed.
Thank you for the comment. We followed by the literature recipes and our research's team experiences. Moreover, the CV analysis presented the peaks and specific curves came from enzyme, related to oxidation of glucose to glucolactone came from glucose oxidase, so the enzyme is presented and is active in the examined carriers. (i.e. Sens. Actuators B, 2018, 256, 176-185)
Then the authors need to perform stability tests. Now only CV shows activity. But for how long? What is the storage stability? Once these points have been answered the manuscript can be resubmitted. But for how long? What is the storage stability? Once these points have been answered the manuscript can be resubmitted.
Thank you that you focused on the crucial biosensor's feature. Herein, we presented a novel micromaterials, their synthesis, physicochemical and morphological properties too. Moreover, we proposed a potential applications for the enzyme immobilization and biosensing by carrying out only the basic research. We are still working to improve our biosensors' systems, so the work in progress. So, the manuscript presents the novel microplatforms with catalytic possible to biosensor application, however there is still basic findings in application. The verification of stability and optimization study are in progress.
Round 2
Reviewer 1 Report
I believe the authors have improved some of the issues, especially those addressed by the other referees.
Regarding my own recommendations, I do not feel the authors have provided sufficient justification to avoid performing some of the requested analyses (i.e. SEM and/or TEM EDX analysis, XRD or reproducibility/reusability tests).
Therefore, I would encourage the authors to perform those analyses and request additional time to the editorial team if needed.
I feel the work looks much better now but it still requires some additional work on the characterization of the materials.
Author Response
Answers to review
I believe the authors have improved some of the issues, especially those addressed by the other referees. Regarding my own recommendations, I do not feel the authors have provided sufficient justification to avoid performing some of the requested analyses (i.e. SEM and/or TEM EDX analysis, XRD or reproducibility/reusability tests).
Therefore, I would encourage the authors to perform those analyses and request additional time to the editorial team if needed. I feel the work looks much better now but it still requires some additional work on the characterization of the materials.
Thank you for the valuable comment. The SEM, TEM images in the manuscript were included. In addition, the Energy Dispersive Spectroscopy (EDS) for an elements distribution analysis and mapping were carried out. The obtained results were collected, summarized and described in Table 1 and Figs. 3-6 and in the manuscript (chapter 2.1 Surface morphology) included. Moreover, thank you that you mentioned on the biosensor’s features like reproducibility/reusability test. Each of the electrochemical systems were tested three times. Herein, we focused on novel microstructured materials, their synthesis, physicochemical and morphological properties and characterization. We just proposed a potential application for the enzyme immobilization and biosensing by carrying out only the basic research. We are still working to improve our biosensors systems, so the work in progress and it takes longer period of time. So, the manuscript presents the novel microplatforms with catalytic possible to biosensor application, however there is still basic findings in application. The verification of stability and optimization study are in progress. The crucial analysis of the microstructured materials like TEM, SEM, EDS, zeta potential, DLS, PdI, EA (elemental analysis,) FTIR, TGA, Bradford method, AFM and elect
Reviewer 2 Report
The experimental data of the revised manuscript is well designed and conducted. The revised manuscript is well written, and the data is well presented. The authors have correctly answered all comments made by the reviewers. Taking into account the quality and scope of the journal, I would recommend the acceptance of this manuscript in the present form to publish in ‘Catalysts’.
Author Response
Thank you
Reviewer 3 Report
The revised manuscript can now be accepted
Author Response
Thank you
Round 3
Reviewer 1 Report
The authors included additional SEM-EDX analysis but omitted again XRD analyses. I can not understand why the authors are so reluctant to perform/include such an accessible routine analysis to confirm/discard the potential presence of addional phases, especially in the composites.
Overall, the paper has been nicely improved from the first version and I would only recommend minimal modifications:
1) Substitute SEM-EDX images of the plain oxides by XRD spectra comparing all samples. Place SEM-EDX of the plain oxides as Supporting Information.
2) Check scale bars in the SEM-EDX images of the composites to make them readable.
Author Response
Answer to Reviewer
The authors included additional SEM-EDX analysis but omitted again XRD analyses. I can not understand why the authors are so reluctant to perform/include such an accessible routine analysis to confirm/discard the potential presence of additional phases, especially in the composites.
Overall, the paper has been nicely improved from the first version and I would only recommend minimal modifications:
1) Substitute SEM-EDX images of the plain oxides by XRD spectra comparing all samples. Place SEM-EDX of the plain oxides as Supporting Information.
2) Check scale bars in the SEM-EDX images of the composites to make them readable.
Thank you for the comments. After your valuable suggestions, we enriched the manuscript with XRD assays and the obtained results are included in the chapter 2.1 Surface morphology. We decided to present the EDS-SEM and XRD data, findings in the work. In addition, the scale bars in the SEM-EDX images were corrected and they are readable.
We decided to include all data in main manuscript due to open online submission system.
Our manuscript was improved due to your recommended assays, and now we hope that the current form of the work will be fully accepted.
