One-Step Electropolymerization of Azure A and Carbon Nanomaterials for DNA-Sensor Assembling and Doxorubicin Biosensing

Round 1

Reviewer 1 Report

In this study, new highly sensitive voltametric DNA-sensor has been developed for the detection of cytostatic drug doxorubicin based on the Azure A electropolymerized on various carbon nanomaterials. The results were investigated carefully in details. Some corrections need before publication:

1-     Why the R² of (PAA+fMWCNTs)/DNA+MB is low?

2-     Detail of morphology should be explained based on agglomeration procedure.

3-     English writing should be edited by native person

Author Response

• Why the R² of (PAA+fMWCNTs)/DNA+MB is low?

High dispersity and size distribution of the fMWCNTs applied as modifiers made worsen the  repeatability of the results obtained against those on the CB particles. This resulted in lower regression coefficient mentioned. Appropriate text was added to the manuscript (Section 3.4).

• Detail of morphology should be explained based on agglomeration procedure.

Changes in the morphology of the coatings (Fig. 4) have been described in terms of disaggregation caused by treatment protocols in the text as follows (Section 3.2):

“As was established by SEM experiments, dried AA+CB suspension forms on the GCE a dense and bundled coating containing some roundish aggregates with average diameter of 200-300 nm (Figure 4a). Their number sharply decayed after the electropolymerization step due to their disaggregation and conversion to fine-granular films. Smoothen and uniform surface was obtained due to filling the cavities and free space of the modification layer by the polymeric product (Figure 4 b).”

 And later on:

“Probably, such a treatment disaggregated the particles of the electrolysis products (Figure 4e). Removal of weakly bonded products and excessive monomer from the layer promote better reproducibility of the coating structure and redox signals related to its activity.”

• English writing should be edited by native person

We have reconsidered the English as requested.

Author Response File: Author Response.doc

Reviewer 2 Report

Journal: Journal of Carbon Research

Article title:  One-Step Electropolymerization of Azure A and Carbon Nanomaterials for DNA-Sensor Assembling and Doxorubicin Biosensing

In the current manuscript, the A. Profireva et al. developed voltammetric sensors for the detection of anticancer drugs like doxorubicin using a glassy carbon electrode modified with carbon nanoparticles and methylene blue. The interesting part of the research is that the authors discussed the utility of proposed electrodes to detect doxorubicin 0.03 pM. This is very important research in the area of sensors and its scope matches the MDPI Journal of Carbon research. But the article needs some major revisions to get published in MDPI biosensors. 

I recommend its publication with major revision.

1.                   In the abstract the authors need to explain how the modifier helps electron transfer reactions between a glassy carbon electrode and analyte.

2.                   At the introduction, the authors have to discuss the interferents for doxorubicin detection.

3.                   Why authors are using GCE why not CPE? 

4.                   In the introduction authors need to discuss the advantages of the electrochemical sensors for sensing doxorubicin over other classical analytical methods in a detailed manner.  

5.                   Literature review is poor, the authors need to discuss some of the recent literature related to sensors published in MDPI and other reputed publishers. It will be very much helpful for readers

a)       https://www.mdpi.com/2073-4360/13/12/1945

b)      https://pubs.rsc.org/en/content/articlelanding/2016/ay/c5ay03048j

c)       https://www.mdpi.com/1996-1944/15/18/6337

d)      https://www.mdpi.com/2227-9717/10/3/472

6.                   What is the area of the electrode after the modifications

7.                   Why the authors have not studied the impedance behavior of modified electrodes?

8.                   Why the LOD of the current electrodes is better than the previous electrodes?

9.                   The authors need to discuss the importance of polymerization on the carbon electrode surface please refer to the articles Pre-post redox electron transfer regioselectivity at the alanine modified nano graphene electrode interface, Chemical Physics Letters. Pre/post electron transfer regioselectivity at glycine modified graphene electrode interface for voltammetric sensing applications Journal of Electrochemical Science and Engineering.

10.               Conclusion is very short

I feel the theme of the article is interesting and attractive. The article becomes suitable for publishing in the MDPI Journal of Carbon research after modifying the manuscript by improving the literature review and solving the abovementioned concerns.  

Author Response

In the current manuscript, the A. Profireva et al. developed voltammetric sensors for the detection of anticancer drugs like doxorubicin using a glassy carbon electrode modified with carbon nanoparticles and methylene blue. The interesting part of the research is that the authors discussed the utility of proposed electrodes to detect doxorubicin 0.03 pM. This is very important research in the area of sensors and its scope matches the MDPI Journal of Carbon research. But the article needs some major revisions to get published in MDPI biosensors. 

I recommend its publication with major revision.

1. In the abstract the authors need to explain how the modifier helps electron transfer reactions between a glassy carbon electrode and analyte.

Mechanism of signal generation has been explained in the Abstract as follows:

Carbon materials promote electorpolymerization of the Azure A dye applied as a matrix for DNA molecules saturated with Methylene blue (MB) molecules, Interaction with intercalator (doxorubicin) liberates the MB molecules and changes is redox activity

2. At the introduction, the authors have to discuss the interferents for doxorubicin detection.

The following text was added to the Introduction:

Doxorubicin determination is based on the intercalation phenomena so that all the other anthracycline antitumor drugs exert similar effect on the response. Meanwhile, previously it was shown that sulfonamide drugs do not interfere with the anthracycline influence [14]. The effect of stabilizers applied in appropriate medicines (mannite, lactose etc.), serum proteins, urine components and plasma electrolytes can be suppressed by dilution of the samples. The effect of such a protocol should be assessed on the stage of sample treatment optimization.

3. Why authors are using GCE why not CPE? 

GCE shows higher reproducibility of the surface characteristics and better mechanical durability against carbon paste electrodes (CPE). In this work, the formation of layer formed by carbon nanomaterials calls for more durable platform allowing better description of the effects observed on the stages of CB/MWCNTs/Azure A deposition and following electropolymerization. The following text was added:

GCE was used as a transducer due to its higher mechanical durability and more reproducible surface characteristics against other common electrodes, e.g., carbon paste and pencil graphite electrodes [33].

4. In the introduction authors need to discuss the advantages of the electrochemical sensors for sensing doxorubicin over other classical analytical methods in a detailed manner.

Conventional methods of doxorubicin determination have been described in Introduction. The following text was added to stress the advantages of electrochemical sensors:

Electrochemical methods of doxorubicin determination offer many advantages over conventional instrumentation techniques, i.e., simpler design, cost- effective measurement protocol, intuitively understandable mechanism of signal generation, simple robust design of appropriate sensors compatible with point-of-care diagnostics devices and application outside biochemical laboratory. 

5. Literature review is poor, the authors need to discuss some of the recent literature related to sensors published in MDPI and other reputed publishers. It will be very much helpful for readers
6. a)       https://www.mdpi.com/2073-4360/13/12/1945
7. b)      https://pubs.rsc.org/en/content/articlelanding/2016/ay/c5ay03048j
8. c)       https://www.mdpi.com/1996-1944/15/18/6337
9. d)      https://www.mdpi.com/2227-9717/10/3/472

We thank esteemed Reviewer for suggestion though we cannot agree with the statement about poor literautre review. About all of the materials used in electrochemical DNA sensors have been mentioned. We have also added references provided in the above list except that marked as (a) „Albendazole Release from Silica-Chitosan Nanospheres. In Vitro Study on Cervix Cancer Cell Lines” because there is no relation to the topic of investigation. The following text was added:

Carbon nanomaterials are well compatible with other additives accumulating analytes or providing selectivity of their determination by electrochemical methods [23, 24]. … GCE was used as a transducer due to its higher mechanical durability and more reproducible surface characteristics against other common electrodes, e.g., carbon paste and pencil graphite electrodes [33].

[23] Srivastava, J.; Singh, M. A biopolymeric nano-receptor for sensitive and selective recognition of albendazole. Anal. Methods 2016, 8, 1026-1033. DOI: 10.1039/c5ay03048j

[24]     Revanappa, SK.; Soni, I.; Siddalinganahalli, M.; Jayaprakash, G.K.; Flores-Moreno, R.; Nanjegowda, C.B. A Fukui analysis of an arginine-modified carbon surface for the electrochemical sensing of dopamine. Materials 2022, 15, 6337. https://doi.org/10.3390/ma15186337

[33] David, I.G.; Buleandra, M.; Popa, D.E.; Cheregi, M.C.; David, V.; Iorgulescu, E.E.; Tartareanu, G.O. Recent developments in voltammetric analysis of pharmaceuticals using disposable pencil graphite electrodes. Processes 2022, 10, 472. https://doi.org/10.3390/pr10030472

6. What is the area of the electrode after the modifications.

Simultaneous deposition of electroactive poly(Azure A) film did not allow assessing the electroactive area based on the common approach (ferricyanide ions voltammetry and Randles-Sevcik equation) because the ferri/ferrocyanide peaks were in the potential range of the redox activity of the polymer. In the absence of the dye, the electroactive are of GCE covered with CB in chitosan increased by 1.7 times and that of fMWCNTs by 2.0 times against bare GCE.  We could also refer to the Figure 3, where the cyclic voltammograms of the polymerized dye are compared for various carbon nanomaterials tested. One could see the difference in the CB and fMWCNTs coatings is not as high. The following text was added:

Section 2.3: Based on the ferricyanide cyclic voltammogram and Randles-Sevcik equation, the ratio of effective and geometric surface area was equal to 1.9 for CB and 2.0 for fMWCNTs (both dispersed in chitosan).

7. Why the authors have not studied the impedance behavior of modified electrodes?

Electrochemical impedance spectroscopy is quite sensitive to the full coverage of the surface with modifying layers. Here, the use carbon nanomaterials and especially fMWCNTs resulted in formation of rather porous spongy coatings with numerous defects of the film. This is not so important for direct current voltammetry that gives an average current but crucial for charge transfer resistance assessment. Besides, as was mentioned above, the redox activity of ferri/ferrocyanide redox probe overlapped with that of polymeric dye. For this reason, we decided not to use EIS for surface layer assembling control. The following text was added:

Assembling of the surface layer is commonly monitored by EIS. However, in this case, the use of carbon nanomaterials forming rather porous spongy layers with numerous defects prevented full coverage of the surface with appropriate components. Besides, the redox active area of ferri/ferrocyanide redox probe and that of polymeric dyes overlapped. This made the use of the impedimetric method not informative. For this reason, the morphology of the layers was investigated using SEM

8. Why the LOD of the current electrodes is better than the previous electrodes?

The following text was added to the comments to Table 2 (Section 3.4):

One could see, joint deposition of carbon nanomaterials, DNA and polymeric dye offers synergistic effect due to dense contacts of all the reactants in the composite film, better electric wiring of the redox active species responsible for the analytical signal. The MB displaced by doxorubicin in the DNA helix releases from the inner space of the layer, and its effect on the electron exchange chain becomes higher than for conventional modifiers of the electrode. Similar sensitivity toward doxorubicin was also reported for other DNA sensors with carbon nanomaterials mentioned.

9. The authors need to discuss the importance of polymerization on the carbon electrode surface please refer to the articles Pre-post redox electron transfer regioselectivity at the alanine modified nano graphene electrode interface, Chemical Physics Letters. Pre/post electron transfer regioselectivity at glycine modified graphene electrode interface for voltammetric sensing applications Journal of Electrochemical Science and Engineering.

We have mentioned both the articles [53, 54] in the Discussion and conclusion section.

10. Conclusion is very short

The conclusion contained all the explanations of the mechanism of the signal generation and importance of the surface layer components. I was also extended by addition of the following sentences:

Application of electrochemical impedance spectroscopy was found ineffective due to overlapping of the redox activity potentials of the polymer and redox probe (ferri/ferrocyanide ion pair) and not full coverage of the underlying electrode surface with the polymer.

Synergistic effect of the surface layer components resulted in remarkable sensitivity of the response toward doxorubicin as model intercalator. Positive effect of carbon nanomaterials has been previously proved for other redox active modifiers, e.g., for the detection of electron transfer of glycine adsorbed on the graphene nanosheets [53]. Alanine was found to be active in the redox reactions on the graphene interface [54]. In both cases, the mechanism of the transfer of the amino acids on the carbon nanomaterials and regioselectivity of redox reactions were confirmed by DFT calculations.

Author Response File: Author Response.doc

Round 2

Reviewer 1 Report

The correction are acceptable.

Reviewer 2 Report

The authors made sufficient changes and the article can be accepted in its present form.