Elucidating Extracellular Vesicle Isolation Kinetics via an Integrated Off-Stoichiometry Thiol-Ene and Cyclic Olefin Copolymer Microfluidic Device
, Edgars Endzelins 3, Arturs Abols 2,3, Nadezda Romanchikova 3, Aija Line 3, Guido W. Jenster 4, Gatis Mozolevskis 1,2 and Roberts Rimsa 1,2,*Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsAuthors present a new microfluidic-mixer device production of which can be scaled up and optimized for capturing EVs by with magnetic particles. Their approach shows the possibility of measuring binding kinetics between EVs and nanobodies. Overall it can become a useful tool for semi-automated clinical diagnostics.
Comments, questions and suggestions:
1. Line 41: I recommend broadening the size range. A significant fraction of small EVs for instance is <100 nm. This is actually can be seen in Figure 3C.
2. I did not find where abbreviation MPs is defined. Is it Magnetic Particles? Should be specified in the beginning of the manuscript.
3. Line 145: “Zetzsizer Nano” typo (Zetasizer)
4. Under “EV concentration measurements” it would be useful to speify what parameters were used on Nanosight. Was the measurement done 5 times by manually updating sample with the syringe or at a certain flow rate by a syringe pump?
5. Authors should be consistent with the multiplication signs (e.g. 2.76·108 LNCaP cels vs p= 9*10-5 vs 2.76x108 LNCaP cells)
6. Fig 3a: Double check the calculations. It seems like that the ratio between number of EV/µg is low.
7. Fig 5 suggestion: It may be easier for the reader to have a figure showing integrated intensity of CD63 and CD9 vs mixing speed of both ON-chip and OFF-chip capture. Is there some relation that can be determined by regression of this data?
8. When mentioning the author the first letter of their name is not needed (e.g. K. Petkovica et al.)
9. Visually it seems that the relative intensity of the Standard Assay and Microfluidic assay WB are very close after 5-10 min. Can you explain what approach was used to analyze in ImageJ? I only found one sentence “To acquire numerical 241 band integrated density values, ImageJ software was used.” Did you use some chosen area of the WB results and were they the same for Standard and Microfluidics assay? Especially when looking at Figure 6B, 30 min data point, visually it seems that the microfluidic assay outperforms the standard assay and 50 min is nearly indistinguishable . My hypothesis is that the WB image analysis approach might not have been the best.
10. Related to question 8, if in fact the Standard assay outperforms Microfluidics can you provide some discussion on how Microfluidics can be improved to make it the same or better than the Standard Assay?
11. In supplementary information it would be nice to name figures (Figure S1, S2, etc.) especially since you used these names of figures in the manuscript.
12. Please label all protein ladders in the WB images provided in supplementary.
13. It may be better to move Table 1 to supplementary since it just specifies details of antibodies used for WB.
Comments for author File: 
Comments.pdf
Author Response
Thank you for comments, please see the attachment for changes and our comments.
Author Response File: Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsThis manuscript introduces an integrated off-stoichiometry thiol-ene and cyclic olefin copolymer microfluidic device,which incorporates functions for sample mixing and magnetic particle separation, and reveals the binding kinetics between extracellular vesicles (EVs) and anti-CD9 nanobodies.The manuscript provides detailed descriptions of the manufacturing method,workflow, preparation of magnetic EV-capture beads,EV capture efficiency, and other aspects, demonstrating the advantages and application potential of this device in EV isolation and detection.Despite its innovative nature, the manuscript still has issues and areas for improvement,requiring further revision and refinement:
1.The introduction section of the manuscript does not specify the type of antibody and the capture mechanism in detail.
2.The Transmission electron microscopy section in the Materials and Methods does not mention the purpose of this experiment, and the purpose of depositing AL2O3 in the Device fabrication section of the Materials and Methods is also not mentioned.It is recommended to complete these to make the logic clearer.
3.The milliliter unit is inconsistent in the manuscript,with both "ml" and "mL" being used.It is recommended to unify the unit for consistency.
4.There are expressions like "2.76·108 LNCaP cells","2.76×108 LNCaP cells" "3.92×109 LNCaP EV pts","6·109 LNCaP EV pts"in the manuscript.It is recommended to unify the multiplication symbols and notation for scientific notation.
5.The first word of the title of Figure 1 and Table 2 is not capitalized.
6.The annotation in Figure 2 is in lowercase "d",but it is referred to as uppercase "D" in the text.
7.The title of Figure 3 is in uppercase letters,but the letters in the figure are in lowercase.
8.The title format of the "Results and discussion" section, "Magnetic EV-capture beads featuring novel anti-CD9 nanobody",is inconsistent with the formats of the preceding and following titles in the manuscript.
Comments on the Quality of English LanguageThe English could be improved to more clearly express the research.
Author Response
Thank you for comments, please see the attachment for changes and our comments.
Author Response File: Author Response.pdf
