Understanding the Dynamics of a Lipid Monolayer on a Water Surface under a Marangoni Flow

Round 1

Reviewer 1 Report

The manuscript reports results related to the tilting dynamics of phospholipid monolayer under the effect of tangential oscillations imposed to the surface layer by periodic Marangoni flow.
To this aim, the observed variations of the surface pressure during oscillations are nrelated to the molecule tilting utilising X-Ray reflectometry data obtained under static conditions (i.e., without Marangoni flow).

In spite interesting, in terms of the subject and of the results, in the present form, the manuscript contains several flaws. I recommend therefore the authors to do a deep revision of the paper, according to the following major remarks:

1. The English redaction is poor, so that the text in general, and some paragraphs in particular, are not clear and some concepts risk to be lost for the reader.
2. As for the next comments, many details about the origin of the studied phenomena and of the reported data and on their analysis are missing. It is therefore difficult to follow how the data from the different experiments and from the literature (ex., from reference 12) are logically connected and utilised to derive the reported results. I recommend adding more details on all these aspects.
3. The phenomenology and the explanation of the oscillatory Marangoni effect caused by the octanol should be reported in more details in the Introduction, in order to make also readers not fully aware of the
   phenomena to better understand your study.
4. Please, provide more details on the fitting procedure of X-ray reflectometry data (see lines 124-130).
5. The elastic energy (Fig. 4b) are estimated “…five times smaller than the van der Waals interactions between the hydrocarbon chains..”. Could you please comment the implication of this result?
6. The data in figure 8a are presumably obtained from the dynamic experiment. It should be useful to state it more clearly in the text. Moreover, it could also be useful to compare the Π₀-A data in the figure with those of the isotherms reported in reference 12.
7. The Marangoni flow caused by octanol produces a directional compression of the DSPC monolayer. I wonder if the resulting tilt of the molecules can really be compared with the static tilting data from X-ray reflectometry, which in fact correspond to a monolayer at equilibrium, where the phospholipid molecules interact isotropically. This is key point that deserves some discussion by the authors. In fact, the two situations, could in principle lead to different Π-A relations. In that respect, I am not sure that the cartoons referring to Π₀ in figure 9 really reflects the situation of a monolayer at equilibrium. I would expect instead molecules tilted off-plane by the angle measured by X-ray, but randomly oriented in-plane to the surface. The directional perturbation of the Marangoni flow would in this case induce a directional preference to the off-plane tilting of the molecules, which may result on a different molar area associated to the same change of surface pressure.
8. Please specify how the values of the molecular area of fig. 8a are obtained. Are they imposed by the amount of spread DSPC? Are they inferred from reference 12? Are they calculated from the tilt angle according to
   A=π*(lchain*sinβ)² ?  Using this latter equation to convert the tilting data from X-ray of fig. 6, how the resulting Π-A isotherm compares with the corresponding isotherm in reference 12 ? And how
   with the data of fig. 8b?

Other minor remarks:
- Page 2, Line 66: “The phospholipid, …..”
- Page 3, line 84-85: please specify more about the microscope (brand, company,…)
- Page 3, line 100: add reference 12 to “…in the literature…”

Author Response

We thank all the insightful comments. These comments have help make this manuscript stronger in terms of scientific rigor and clarity. We have addressed all the comments below and have also revised the manuscript accordingly. The corrected part is shown in red in the revised version. We appreciate the comments and hope that our responses are sufficient.

Comments:
The manuscript reports results related to the tilting dynamics of phospholipid monolayer under the effect of tangential oscillations imposed to the surface layer by periodic Marangoni flow.
To this aim, the observed variations of the surface pressure during oscillations are nrelated to the molecule tilting utilising X-Ray reflectometry data obtained under static conditions (i.e., without Marangoni flow).

Ans:
As you pointed out, X-Ray reflectometry data obtained under static conditions. To Fig. 6 was added to show that the X-ray data with Marangoni convection matches that under static conditions. 

Comments:
In spite interesting, in terms of the subject and of the results, in the present form, the manuscript contains several flaws. I recommend therefore the authors to do a deep revision of the paper, according to the following major remarks:
1. The English redaction is poor, so that the text in general, and some paragraphs in particular, are not clear and some concepts risk to be lost for the reader.
Ans:
We are very sorry for the lack of explanation. We added a lot of explanation as a whole which is shown in red.

Comments:
2. As for the next comments, many details about the origin of the studied phenomena and of the reported data and on their analysis are missing. It is therefore difficult to follow how the data from the different experiments and from the literature (ex., from reference 12) are logically connected and utilised to derive the reported results. I recommend adding more details on all these aspects.
3. The phenomenology and the explanation of the oscillatory Marangoni effect caused by the octanol should be reported in more details in the Introduction, in order to make also readers not fully aware of the phenomena to better understand your study.

Ans:
Thank you for your advice. The same point was made by other reviewers. So we added a detailed explanation about this phenomenon in the introduction.

Comments:
4. Please, provide more details on the fitting procedure of X-ray reflectometry data (see lines 124-130).

Ans: 
The monolayer was divided into a hydrophobic and a hydrophilic layers. The model contains five adjustable parameters: two layer densities, two layer thick-nesses and a smearing parameter between the two layers. 

Comments:
5. The elastic energy (Fig. 4b) are estimated “…five times smaller than the van der Waals interactions between the hydrocarbon chains..”. Could you please comment the implication of this result?

Ans: 
Those values are five times smaller than the van der Waals interactions between the hydrocarbon chains of DSPC of 0.85 kJ/mol, indicating that DSPC molecules cannot be isolated from the monolayer film. 

Comments:
6. The data in figure 8a are presumably obtained from the dynamic experiment. It should be useful to state it more clearly in the text. Moreover, it could also be useful to compare the Π0-A data in the figure with those of the isotherms reported in reference 12.

Ans: 
We define the initial molecular area estimated from Fig. 2 at Π0 as A0 (the closed circles). While for the molecular area when the accepter surfactant is compressed at Π1 is defined as A1 (the open circles). 

Comments:
7. The Marangoni flow caused by octanol produces a directional compression of the DSPC monolayer. I wonder if the resulting tilt of the molecules can really be compared with the static tilting data from X-ray reflectometry, which in fact correspond to a monolayer at equilibrium, where the phospholipid molecules interact isotropically. This is key point that deserves some discussion by the authors. In fact, the two situations, could in principle lead to different Π-A relations. In that respect, I am not sure that the cartoons referring to Π0 in figure 9 really reflects the situation of a monolayer at equilibrium. I would expect instead molecules tilted off-plane by the angle measured by X-ray, but randomly oriented in-plane to the surface. The directional perturbation of the Marangoni flow would in this case induce a directional preference to the off-plane tilting of the molecules, which may result on a different molar area associated to the same change of surface pressure.

Ans: 
BAM images showed that DSPC had formed condensed domains. Since there is no space for taking random orientations, the phospholipid molecules tilt the same direction in the same domain.

Comments:
8. Please specify how the values of the molecular area of fig. 8a are obtained. Are they imposed by the amount of spread DSPC? Are they inferred from reference 12? Are they calculated from the tilt angle according to A=π*(lchain*sinβ)^2 ?  Using this latter equation to convert the tilting data from X-ray of fig. 6, how the resulting Π-A isotherm compares with the corresponding isotherm in reference 12 ? And how with the data of fig. 8b?

Ans:
As we mentioned above, the molecular area was derived from the Π-A isotherm at the corresponding surface pressure.They are much smaller than those calculated from the tilt angle according to A=π*(lchain*sinβ)^2. Therefore,this leads to a densely packed structure as shown in Fig. 10.  

Comments:
Other minor remarks:
- Page 2, Line 66: “The phospholipid, …..”
- Page 3, line 84-85: please specify more about the microscope (brand, company,…)
- Page 3, line 100: add reference 12 to “…in the literature…”

Ans:
We corrected them. Thank you.

Reviewer 2 Report

The authors have presented a manuscript that aims to investigate and understand the details of a lipid monolayer at the air/water interface under Marangoni flow.  In doing so they have carried out a series of experiments in a Langmuir trough studying the surface pressure oscillation, surface motion and use x-ray reflectivity on static systems to provide an envelope for aiding in understanding the observations.

The experiments appear to have produced high quality data that has been analysed and treated in an appropriate manner. There is a missing link in the manuscript though.  The title begins with "Understanding" and in lines 55 & 56 it is stated "... in order to understand the motion of fluid biological membranes."  Furthermore the introduction to the manuscript places the work in the context of fluid cell membranes with the aim of enhancing understanding of motion within membranes.

The paper does not really go beyond describing the phenomena observed and determining properties of the membrane.  The choice of DSPC lipid to study fluid phase monolayers does seem odd as it would be in the gel phase with experiments carried out at 23 ^oC.  The manuscript does not link the observations and interpretation to actually understanding the motion of biological membranes. Without this link the impact of and interest in the manuscript is likely to be rather low.

The impact of Marangoni flow on a monolayer film is not one that I was familiar with. The manuscript raised more questions that it answered for me. Why does the surface pressure oscillate ? What happens to the octane? I presume the the octane droplet is in place the entire experiment, where does the octane go ? I take it that it interacts with the membrane and then is squeezed out into solution allowing more octanol to then drive the next cycle. The manuscript doesn't really give any understanding but rather observations.

Author Response

We thank all the insightful comments. These comments have help make this manuscript stronger in terms of scientific rigor and clarity. We have addressed all the comments below and have also revised the manuscript accordingly. The corrected part is shown in red in the revised version. We appreciate the comments and hope that our responses are sufficient.

Comments:
The authors have presented a manuscript that aims to investigate and understand the details of a lipid monolayer at the air/water interface under Marangoni flow.  In doing so they have carried out a series of experiments in a Langmuir trough studying the surface pressure oscillation, surface motion and use x-ray reflectivity on static systems to provide an envelope for aiding in understanding the observations.
The experiments appear to have produced high quality data that has been analysed and treated in an appropriate manner. There is a missing link in the manuscript though.  The title begins with "Understanding" and in lines 55 & 56 it is stated "... in order to understand the motion of fluid biological membranes."  Furthermore the introduction to the manuscript places the work in the context of fluid cell membranes with the aim of enhancing understanding of motion within membranes.
The paper does not really go beyond describing the phenomena observed and determining properties of the membrane.  The choice of DSPC lipid to study fluid phase monolayers does seem odd as it would be in the gel phase with experiments carried out at 23 oC.  The manuscript does not link the observations and interpretation to actually understanding the motion of biological membranes. Without this link the impact of and interest in the manuscript is likely to be rather low.

Ans:
Thank you for your very reasonable point. Certainly, there was a big gap between the introduction and the following parts. In accordance with your suggestions, we have made corrections so that the content of this study will lead to an understanding of cell membranes.

Comments:
The impact of Marangoni flow on a monolayer film is not one that I was familiar with. The manuscript raised more questions that it answered for me. Why does the surface pressure oscillate ? What happens to the octane? I presume the the octane droplet is in place the entire experiment, where does the octane go ? I take it that it interacts with the membrane and then is squeezed out into solution allowing more octanol to then drive the next cycle. The manuscript doesn't really give any understanding but rather observations.

Ans:
We are very sorry for the lack of explanation. We added a lot of explanation as a whole which is shown in red.
Microscopically, 1-octanol dissolved from the droplet adsorbs at the air/water interface. The local surface pressure around the capillary gradually increases in accordance with the adsorption amount and beyond the threshold value, an abrupt burst of Marangoni flow is generated at the water surface. It pushes the lipid monolayer outward subsequently dissolves in the water. This process occurs faster for the more soluble surfactant, leading to a decreasing oscillation period. In this system, the lipid monolayer is rapidly compressed by the mechanical energy of Marangoni convection generated by the donor surfactant, and then slowly returns to its original state, repeating elastic motion periodically.

 

Reviewer 3 Report

This is a follow-up work to the authors previous research on the oscillation of surface tension under Marangoni convection. In the present work, the authors investigated the elastic motion of lipids with more detail, including calculating the elastic energy and tilt angle of lipids.

• The manuscript is well written and results are presented clearly.
• What’s new about the present work compared to the work that has been published on JPCL? The novelty should be emphasized in the abstract and conclusions.
• Did the authors investigate conditions with higher surface pressure? According to Figure 2(a), would k decrease with increasing surface pressure above 40 mN/m? Or are the values irrelevant since the oscillation of surface tension does not occur at such surface pressure?
• In Figure 4(a), what do the arrows indicate?
• Caption of Figure 8 is confusing. “Closed and open circles represent data for the initial Π0 and the final Π1” – are those referring to initial and final states since Π0 is the x-axis?

Author Response

We thank all the insightful comments. These comments have help make this manuscript stronger in terms of scientific rigor and clarity. We have addressed all the comments below and have also revised the manuscript accordingly. The corrected part is shown in red in the revised version. We appreciate the comments and hope that our responses are sufficient.

Comments:
This is a follow-up work to the authors previous research on the oscillation of surface tension under Marangoni convection. In the present work, the authors investigated the elastic motion of lipids with more detail, including calculating the elastic energy and tilt angle of lipids.
The manuscript is well written and results are presented clearly.
What’s new about the present work compared to the work that has been published on JPCL? The novelty should be emphasized in the abstract and conclusions.

Ans:
Previous studies have found that changes in surface tension are consistent with changes in lipid surface pressure investigated using in-situ X-ray reflectivity. Further analysis of the structure of lipids in the current study shows that lipid molecules are in rotational rather than translational motion 

Comments:
Did the authors investigate conditions with higher surface pressure? According to Figure 2(a), would k decrease with increasing surface pressure above 40 mN/m? Or are the values irrelevant since the oscillation of surface tension does not occur at such surface pressure?

Ans: 
We didn't try to investigate conditions above 40 mN/m. Considering with the cross section of the DSPC molecule is 0.4 nm^2, the monolayer of DSPC collapses above 40 mN/m. Therefore, the lipid monolayer may not behave as an elastic film.

Comments:
In Figure 4(a), what do the arrows indicate?

Ans:
See the scale on the left for the pink simbols and the scale on the right for the blue symbols.

Comments:
Caption of Figure 8 is confusing. “Closed and open circles represent data for the initial Π0 and the final Π1” ? are those referring to initial and final states since Π0 is the x-axis?

Ans:
Sorry for confusing. We corrected it.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The new version of the manuscript has been significantly improved, acccordingly to the comments and suggestions raised by the referees, that have been taken into seriuos consideration.  After some revision of the English redaction and with the addition of important informations and comments, the presented experiments and results are now much more clear, enhancing the comprehension and scientific content of the work.

I recommend therefore the publication of the manuscript.

Reviewer 3 Report

Comments have been addressed by the authors.