Specific Detection of PE-Included Vesicles Using Cyclic Voltammetry
Round 1
Reviewer 1 Report
The authors demonstrate that cyclic voltammetry (CV) measurements can be used for the detection of lipid vesicles containing phosphatidylethanolamine (PE). Experiments were carried out with vesicles consisting of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE). The measurements are based on a detection of the interaction of DPPE-containing vesicles with a gold electrode which has surface-bound cynnamycin, a PE-binding cyclic peptide antibiotic.
Although the concept of the work is interesting and straightforward (illustrated in Fig. 1) and experimental data are shown that under certain conditions, CV measurements detect DPPE-containing vesicles (Fig. 2 and Fig. 3), it is not clear whether these are preliminary findings or whether the CV measurements show a DPPE or cynnamycin concentration-dependency that could be used for a quantitative analysis. The last part of the manuscript is rather unclear and should be re-written (from section 3.3. on). It is important that the interested reader clearly understand the details of the experiments. The statement in the conclusion "However, the vesicles made with 90% DPPC/10% DPPE on the monolayer prepared respectively with 99% MUD/1% MHA to 90% MUD/10% MHA showed the steady decrease in the CV response." This sentence is also part of the abstract and probably the key message of the paper. Does the sentence mean that there is a linear dependence of CV response on MHA (i.e. bound cynnamycin) content? If so, where are the data to show that this is the case?
The conditions of the experiments shown in Figure 2 are not clear. MUD/MHA. Is this 90% MUD/10% MHA, like in Figure 3? If so, the dashed CV curve shown in Figure 3 is probably the same as the dashed CV curve shown in Figure 2. If this is correct, it should be mentioned in the legend of Figure 3.
Author Response
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Author Response File: 
Author Response.doc
Reviewer 2 Report
In this work the authors aim to study the specific binding between cinnamicyn and artificial vesicles incorporating PE in their lipid composition using the cyclic voltammetric technique. The experimental design of the work is correct, but the description of the CV experiments and some other parts of the manuscript must be improved before its publication.
Abstract and conclusions
Abstract and conclusions are identical. Authors must revise and change the conclusions. The conclusions should explain better the results of the work with respect its objectives.
Material and methods:
The authors must clarify if the phospholipid composition of the vesicle bilayer is expressed as percentage in weigh or is a molar ratio.
The authors must explain better the process performed for the vesicle adsorption on the gold/monolayer/cinnamicyn surface because it is not clear if the vesicle solution was injected on the electrode surface directly before it was introduced in the electrochemical cell. The time employed in the adsorption process is not explained either.
To perform the CV experiments the redox prove used is K3Fe(CN)6, but it is not clear what is the composition and pH of the electrolyte solution employed in the electrochemical cell. To compare with other systems the authors should indicate the reaction area of the working electrode.
Results and Discussion:
In the first paragraph in section 3.1, when the authors discus the blocking effect of the monolayer on the faradaic response of the redox prove in figure 2, they say “The exclusion possibility is described further later”. It is not clear where that discussion is made in the manuscript. On the other hand, the effect of a change in a biomimetic monolayer composition affects slightly the capacitive current of the voltametric response (see for example J. Hoyo et al Eur. Phys. J. E (2016) 39: 39) when lipid structure is similar, and it cannot be appreciated in the faradaic response of the redox prove.
On the first paragraph in section 3.2, authors should say: “……………. electronic transfer” and not " .... electrolyte transfer".
In the section 3.3, the authors calculate the charge transferred from the voltmmetric curve. The authors should indicate if the values in Table 1, correspond to cathodic charge, anodic charge, or total charge.
In the second paragraph, in section 3.3, authors should say: “ ....charge transfer was blocked by the monolayer of the pure MUD or 90% MUD/10% MHA..." and not “ ....charges were blocked by the monolayer of the pure MUD or 90% MUD/10% MHA...".
Figures:
In all the CV figures, the authors have changed the more recommended convention to represent the potential axe, as a conventional mathematical axe, that is, with the negative values of the potential at the left side respect to the zero value.
Author Response
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Author Response File: Author Response.doc
Reviewer 3 Report
The authors investigated the specificity of interaction between cinnamycin and PE. However, it is not clear what the experiments and results are adding more to the research field. The impact of the research study is unclear.
The manuscript needs a full English and style revision.
The introduction should give more information on the specificity of binding and its importance. The specificity of interaction is not clear in the introduction. The peptide-lipid interaction is defined as hydrophilic (headgroup) and hydrophobic (tail) interaction. Since all the lipids are amphiphilic, what does it make specific?
All the figures are missing a clear and descriptive caption.
Most of the control data described in the results and discussion session are not shown. This also makes it difficult to evaluate the relevance of the shown data.
The line format in figure 3 and 4 should be clearer.
Author Response
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Author Response File: Author Response.doc
Round 2
Reviewer 3 Report
The reviewers' comments are poorly addressed.
The manuscript still needs an English and style revision.
The introduction should still give more information on the specificity of binding and its importance.
All figures are still missing a clear and descriptive caption.
Most importantly, it is not clear what the experiments and results are adding to the research field. The impact of the research study is unclear.
Author Response
Please see the attachment.
Author Response File: Author Response.doc
