Highly Sensitive Amperometric Sensor Based on Laccase-Mimicking Metal-Based Hybrid Nanozymes for Adrenaline Analysis in Pharmaceuticals

Round 1

Reviewer 1 Report

This paper reports the synthesis and application of nanolaccases (NLacs) in the development of amperometric sensors for the measurement of epinephrine. Compared to the natural laccase, the sensors reported in this paper showed a higher sensitivity, maybe a lower detection concentration of AD. Generally, it is an interest study. Some issues should be discussed with the authors.

1. Line 211, according to the Table 2, I do not think that the synthesized NLacs have a wide liner range for AD detection. Maybe, they have a lower detectable concentration of AD as compared with laccase/GE.
2. As shown in table 1, laccase has a higher specific activity than nAuCePt and nPtFe. Why do laccase/GE have a lower sensitivity than nAuCePt/GE and nPtFe/GE? Is the amount of laccase immobilized on the graphite electrode small or does this immobilization process cause the inactivation of laccase? Is there a better way for laccase immobilization?
3. In this study, the authors prepared a promising nAuCePt-based amperometric sensor. I have another question to discuss with the author, which is, how many times can this sensor be used?
4. Table 1 on page 5, “nPtFe” would this be “nFePt”, which should be the same as in the article.
5. As described by the authors in section of conclusion, the potential application of nAuCePt-based amperometric sensor is in medical diagnosis, I also think that it is an excellent application field. However, the related introduction lacks, thus I suggested that the authors added in the section of introduction.
6. Figures 1c and 1f are very unfriendly to the readers, I suggested that they should be modified.

Author Response

This paper reports the synthesis and application of nanolaccases (NLacs) in the development of amperometric sensors for the measurement of epinephrine. Compared to the natural laccase, the sensors reported in this paper showed a higher sensitivity, maybe a lower detection concentration of AD. Generally, it is an interest study. Some issues should be discussed with the authors.

1. Line 211, according to the Table 2, I do not think that the synthesized NLacs have a wide liner range for AD detection. Maybe, they have a lower detectable concentration of AD as compared with laccase/GE.

Thank you for your remark. Yes, we are agreeing with your comment. We have corrected this sentence.  

2. As shown in table 1, laccase has a higher specific activity than nAuCePt and nPtFe. Why do laccase/GE have a lower sensitivity than nAuCePt/GE and nPtFe/GE? Is the amount of laccase immobilized on the graphite electrode small or does this immobilization process cause the inactivation of laccase? Is there a better way for laccase immobilization?

From the obtained data, it is most likely that we did not achieve optimal conditions for laccase immobilization on GE. We have used the simplest method for such procedure, physical support of laccase by Nafion membrane. In future, we would test covalent methods for laccase immobilization on different electrodes to evaluate the best procedure. But we would like to emphasize that the aim of current paper was study of different NZs as laccase mimetics.

3. In this study, the authors prepared a promising nAuCePt-based amperometric sensor. I have another question to discuss with the author, which is, how many times can this sensor be used?

The constructed amperometric sensor based on nAuCePt was used at least 5 times.  

4. Table 1 on page 5, “nPtFe” would this be “nFePt”, which should be the same as in the article.

Thank you for your remark. In our paper, throughout the text manuscript, we have marked nanozymes as "nPtFe", and “nFePt” it is the same. That's why, we have corrected this inconsistency and have denoted throughout as “nPtFe”.

5. As described by the authors in section of conclusion, the potential application of nAuCePt-based amperometric sensor is in medical diagnosis, I also think that it is an excellent application field. However, the related introduction lacks, thus I suggested that the authors added in the section of introduction.

Thank you for your comment. In “Introduction” we have mentioned about the importance of adrenaline monitoring, in particular, for control of quality of water and for clinical diagnostics (line 56-66). However, in our work we investigated the specificity of our sensor only to some chemicals, which are used in pharmaceuticals. We didn’t study in our work the specificity of our sensor to structural analogues of AD, which are usually contained in biological liquids. That’s why, we didn’t mention in “Introduction” the potential application of nAuCePt-based amperometric sensor in medical diagnostics. We hope, that the study of possibility of nAuCePt-sensor application in medical diagnostics will be done in our future research.

 

6. Figures 1c and 1f are very unfriendly to the readers, I suggested that they should be modified.

We have significantly improved Figures 1c and f.

 

 

Author Response File: Author Response.pdf

Reviewer 2 Report

The paper focuses on laccase-like nanozymes  based on nFePtPd, nCoPtPd or nAuPtPd, the assessment of  their catalytic activity and  their application for the development of amperometric sensors of adrenaline. The paper is well-structured and the conclusions are supported by the data. The paper falls within the scope of the journal and might be of interest for its readers. My suggestions/comments are shown below:

1. Fig 1 c and f, merely appear as screenshots, but the presentation should be improved.
2. In Figure 3,4,5,6 the scale, the labels and the numbers are very small in size and very difficult to read.
3. Table 2 is very informative.  The authors should expand their discussion on this table, highlighting advantages and disadvantages of their systems compared to those described in literature in terms of  sensitivity, apparent Michaelis-Menten constant, linear range and limit of detection.
4. Uncertainty values should be determined for all data reported (currently some, but not all uncertainty values are shown).
5. The authors stated “The proposed amperometric sensors are extremely sensitive, easy-to-use and cost-effective.” The authors should compare their sensors with similar systems regarding their cost-effectiveness and usability.

Author Response

The paper focuses on laccase-like nanozymes  based on nFePtPd, nCoPtPd or nAuPtPd, the assessment of  their catalytic activity and  their application for the development of amperometric sensors of adrenaline. The paper is well-structured and the conclusions are supported by the data. The paper falls within the scope of the journal and might be of interest for its readers. My suggestions/comments are shown below:

1. Fig 1 c and f, merely appear as screenshots, but the presentation should be improved.

We have improved Fig. 1c and Fig. 1f.

 

2. In Figure 3,4,5,6 the scale, the labels and the numbers are very small in size and very difficult to read.

It has corrected.

 

3. Table 2 is very informative.  The authors should expand their discussion on this table, highlighting advantages and disadvantages of their systems compared to those described in literature in terms of  sensitivity, apparent Michaelis-Menten constant, linear range and limit of detection.

 

We have discussed in text manuscript (in part 3. “Results and Dicsussion”) more widely the advantages and disadvantages, sensitivity, linear range and limit of detection and etc of previously published sensors presented in Table 2.

“The nAuCePt/GE was shown to possess the highest sensitivity to AD in comparison with the reported (bio)sensors, this value showing 384-fold increase as compared to that for laccase/GE (Table 2)”.

We have added some sentences concerning the Table 2 in the text of manuscript:

“As it was demonstrated in Table 2, for AD detection not only laccase preparations, but various nanomaterials may be used [17-28] Some of the reported sensors exhibit exelent analytical properties, having low limits of detection (2 nM [27] or 9 nM [26]) and wide linear ranges (for example, from 0.7 µM to 1200 µM [22]). But the main disadvantages of these sensors are insufficient sensitivity [20] and high working potentials (+400 mV) [27]”.

4. Uncertainty values should be determined for all data reported (currently some, but not all uncertainty values are shown).

We have included uncertainty values in Table 3. “Results of adrenaline estimation in pharmaceutical”.

5. The authors stated “The proposed amperometric sensors are extremely sensitive, easy-to-use and cost-effective.” The authors should compare their sensors with similar systems regarding their cost-effectiveness and usability.

We have changed the indicated phrase by the following words: “labour-effective”, because, on this stage of research, it is not easy to calculate real cost-effectiveness of using NZs instead of natural enzymes. In respect of labour-effectiviness, it is obvious that immobilization of NZs is more simple procedure, compared to enzyme laccase. NZs, due to their chemical composition, can be more easily immobilized of electrode surface, than natural enzymes, for which the immobilization procedure is still problematic in biosensorics.

 

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

The authors have made a good effort in revising their manuscript.