Inclusion of Biological Targets in the Analysis of Electrical Characteristics of Non-Thermal Plasma Discharge

Round 1

Reviewer 1 Report

It is noteworthy that the authors are telling the truth (other authors hesitate to say this) that no cause-and-effect relationships has been established between specific NTP effector(s) and observed cellular outcomes (lines 39-40). The authors also correctly say that the influence of the electrical properties of the cells was ignored. Hence the interesting hypothesis of the study: these properties have a significant effect on NTP generated RONS concentrations in their surrounding media. For an electrophysicist, this position seems very plausible. But it's experimental verification is necessary, which is what the authors of this publication have done. I must say right away that the authors' conclusion was confirmed by them experimentally: "Biological cells are dynamic targets that are electrically active and influence the properties of NTP" (line 88). The experiments take into account various factors that can cause disturbances in the charging and discharging of targets. The interpretation of the experimental data is correct.

There is one note to the work:

Lines 119-120. «The voltage was set to 8.5 kV and the frequency 120 was set to 250 Hz for all NTP exposures». It would be good if the authors explained why these voltage parameters were chosen. Will the facts found by the authors be local, inherent only in these voltage parameters, or are they still natural? And they will manifest themselves at other voltage parameters. In fact, answering this question, the authors will set new tasks for those who will do research in the direction they started. Their wording can be added to the conclusions of the article.

Author Response

Please see the attached letter.

Author Response File: Author Response.pdf

Reviewer 2 Report

REVIEW

Manuscript ID: plasma-2543086
Title: Inclusion of biological targets in the analysis of electrical
characteristics of non-thermal plasma discharge

The problem considered in the article is important. It should be noted that when the plasma is exposed to samples placed in it, there is an influence of objects on the plasma parameters. This is not only obvious geometrical changes in the discharge structure and the influence of samples on the electrodynamics of the discharge, but also the arrival of various chemical compounds emitted by the samples during their treatment. In this case, the component composition of the plasma and all its parameters change. This is one of the directions of plasma chemistry research. Such influence has a fundamental character and is now intensively studied when analyzing the processes of surface modification in plasma, in particular, plasma processing of various polymers. In other words, the plasma and the processed sample form a self-consistent system with mutual influence. Such self-consistency of the processes is now beyond doubt.

In the case of plasmas for impact on biological objects, the main attention was paid to the influence of active plasma particles on them. The peer-reviewed work investigates the little-studied phenomenon of the influence of biological samples on nonequilibrium plasma. The research is carried out using a barrier discharge in the design of which a great influence of treated biological samples on the discharge parameters is implied.

The topic of the article is important and interesting, and deserves to be presented to the scientific community, especially to specialists in plasma medicine. However, when reading the article, a number of questions and comments arose, which should be taken into account in the version of the article revised for publication.

Comments:

1.       The plasma gas should be specified in the section 2.4,. The source of hydrogen-containing compounds in the discharge is not clear. This should be explained in the paper.

2.       When describing the plasma source, it is necessary to specify the parameters of the voltage pulse: rise time and fall time.

3.       The authors used a system without gas flow (the flow rate of plasma-forming gas is not specified). It is known that such systems are extremely inconvenient for studying the mechanisms of processes in plasma, since the plasma composition changes in time, and all discharge parameters change accordingly.

4.       4. The authors attribute all changes in the plasma to electrodynamic factors (influence of dielectric properties of samples) and demonstrate it by their experiments. But this is one of the reasons, as noted above. When using different samples differently can change the gas environment due to the arrival of impurities from the processed samples. This factor is not analyzed by the authors, but it can be decisive. It is known that small gas additives can strongly change the kinetics of processes in the plasma. In other words, the paper does not consider possible changes in the gas medium (only RONS were measured), which may lead to changes in the Lissajous figures, by which the equivalent capacitance was determined. A detailed analysis of the composition of the gas medium, at least of its main components, for example, is desirable. This comment can be considered as a recommendation for the future, but should be discussed in the article and if the research is continued, to give special attention to this issue.

 The article can be published after taking into account the comments.

Comments for author File: Comments.docx

Author Response

Please see the attached letter.

Author Response File: Author Response.pdf

Reviewer 3 Report

The work presented by the Authors is original and interesting. The influence of biological targets on the electrical characteristics of plasma system is a novel topic very much of interest for the scientific community. The work deserves publication after some revisions necessary to clarify and integrate few points. The following comments should be of use to the Authors for the revision:

Figure 2: The positioning of the HV probe is unclear. It appears to be in contact with the dielectric of the HV electrode or the plasma. Is that the case?

Lines 171 and 172: The supplement of 900mL to a 100µL sample looks extreme. Are the units corrects? Please verify

Material and methods: What was the density of the cell suspension prepared before the plasma exposure?

Material and methods, line 116: It is not clear the composition of the samples composed of lysed cells. Where they suspended in DMEM10? Was the medium freeze and then thawed? Please clarify this point

Material and methods: The Authors should provide additional detail on the glass coverslip and if the same model was used for all of the experiments. If available the capacitance of the system with the glass coveslip but without the medium could be an interesting piece of information to add.

Introduction: The topic of the impact of the target electrical characteristic in plasma medicine has been recently addressed, among others, in a work from the group of Robert and Pouvesle (ref below). Not only the biological substrate but also the supporting vessel (microplate, glass cover, etc…) affect the plasma characteristics and must be taken into account. I invite the Authors to take into account this piece of literature and integrate the introduction and discussion as they deem appropriate.
               - “Mimicking of human body electrical characteristic for easier translation of plasma biomedical studies to clinical applications” IEEE TRPMS (2029) DOI:10.1109/TRPMS.2019.2936667

Figure 4 E and F: It is not clear why the energy per cycle for live cells in 4E is much lower than the energy per cycle on live cell at the beginning of NTP treatment as reported in 4F. Please clarify this point in the text concerning the two figures.

Discussion, lines 322-330: The first paragraph seems to imply that only in the case of FE-DBD the biological substrate is part of the circuit and its electrical properties can affect the operation. It has been demonstrated by several works that substrates (biological and non biological ones) can greatly influence plasma jet operation. Please rephrase so to avoid any ambiguity.

Figure 8: The variable resistance in the gap is not labelled.

Author Response

Please see the attached letter.

Author Response File: Author Response.pdf