Fabrication of Zinc Protoporphyrin-Modified Gold Electrode for Sensitive and Fast Detection of Vascular Endothelial Growth Factor

Round 1

Reviewer 1 Report

The authors provide a scientifically sound report on fabrication and thorough characteristics of highly modified gold electrodes. The results are presented clearly and they are pretty convincing. All in all - I find this submission highly valuable and worth publication.

I have only two major remarks, which I would like to be adressed by the Authors:

1. I would like to read more and see some data regarding the stability of such eleborated electrodes? Did the Authors tested it?
2. The selectivity tests seem to be a bit sketchy. Could the Authors provide the results of selective detection of VEGF from more complex matrixes? 

Besides, there are some minor/editing lapses:

1. line 25,26 (and throughout the manucsript) - what is the unit of the electrode's sensitivity? It needs some explanation and/or correction.
2. lines 35-37 - the Authors describe the structure of ZnPP - the picture would be really helpfull.
3. line 51 - how does the BSA act as a non-specific linkage preservetion agent? Some explanation and a reference is missing
4. line 65,71 and through the text - some consistency in the way of expressing the concentration would help the readability
5. line 65 - what was the method of preparation such diluted solution? Was it really just 156 nano grams/mL?
6. line 69 - cysteine is an amino acid, not a nucleic acid
7. line 87, 88 - what was the solvent? NMP? 
8. line 105 - delete one space in the K3Fe(CN)6 formula 
9. line 117 - delete the word "peaks" in "...587 nm peaks
10. Figure 4 - some data is missing on (b) I=f(t) diagram - there should be two lines for the Au/ZnPP: with and without the light treatment. Also - there is no description for the blue line.
11. Figure 5 - it would be sufficient to wirte contact angles directly on the photos, this huge diagram seems an exagerration for that data presentation.
12. Figure 6 - the description of the diagram (a) is insufficient - which particular coloured lines describe which electrode and reaction time?
13. line 259 some words are missing: excellent specitificty IN RESPECT TO?   

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

The study proposal is interesting and important in the development of sensors for the detection of vascular endothelial growth factor. However, some adjustments and additions must be made to the manuscript.

1) Introduction 

In the introduction, is need to add information about avastin. The authors should describe the use of avastin, its importance and mention some studies (if  possible) its use as a material in the construction of chemical sensors.

2) Introduction 

It is missing to describe the importance of studying "vascular endothelial growth factor". And indicate works that study the development of sensors for this affinity.

3) 2.3. Fabrication of VEGF biosensors with and without light treatment - pg. 2

According to the authors, the sensor was constructed with "20 μL of 31.25 μg / mL avastin and 0.136 mg / mL BSA were sequentially reacted with 88 Au / ZnPP for 1.5 and 1 h to immobilize the Au / ZnPP electrode as Au / ZnPP / avastin......"

Have these parameters and concentrations of the modifiers been studied previously? If so, the authors should mention that the parameters were previously optimized. 

4) 2.4. Detection of VEGF and selectivity tests - pg.3

K3Fe(CN)6/K4Fe(CN)6  rather than K3Fe(CN)6/K4Fe(CN)6•3H2O

As it is a solution, there is no need to indicate H2O.

5) 3.5. VEGF detection and specificity test

A description of the sensor response mechanism is missing.

Author Response

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Author Response File: Author Response.pdf

Reviewer 3 Report

In this manuscript, the authors developed a zinc protoporphyrin-modified gold electrode for sensitive and fast detection of VEFG. I recommend its publication in chemsensors after the following major issues are addressed:

1. The figure 1 is very poor, the color, configuration and layout should be improved.
2. There is no visualized experiments data about the zinc protoporphyrin-modified gold electrode, such as SEM, EDS, and AFM.
3. In figure 7, what is the principle of the drop in current? There is no experiments date about the resistance.
4. In figure 4b, in order to understand better about Au/ZnPP electrodes with and without light treatment, the professional photoelectrochemical measurements should be performed and some pertinent literature (Anal. Chem., 2018, 90, 9591; Biosens. Bioelectron., 2016, 75, 359) should be cited.
5. The principle of the detection of VEGF is not clear. Please draw a SCHEME for the detection of VEGF and describe it clearly.
6. Please check your manuscript thoroughly and carefully to refine the language. The description was too verbose.
7. To give readers a more comprehensive research background electrochemical biosensing, some recent progress deserves citation and discussion (Anal. Chem., 2019, 91, 3604; Anal. Chem., 2017, 89, 12293) in the introduction.

Author Response

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Author Response File: Author Response.pdf

Round 2

Reviewer 3 Report

The revised manuscript has addressed the issues proposed by the reviewer. Now it can be accepted in this current version.