A novel microfluidic compact disc to investigate electrochemical property changes between artificial and real salivary samples mixed with mouthwashes using electrical impedance analysis

Aung Thiha; Fatimah Ibrahim; Karunan Joseph; Bojan Petrović; Sanja Kojić; Nuraina Anisa Dahlan; Nurul Fauzani Jamaluddin; Saima Qureshi; Goran M. Stojanović

doi:10.1371/journal.pone.0280381

Peer Review History

Original SubmissionSeptember 1, 2022
24 Oct 2022 Decision Letter - Ajaya Bhattarai, Editor PONE-D-22-24397A Novel Microfluidic Compact Disc to Investigate Electrochemical Property Changes Between Artificial and Real Salivary Samples Mixed with Mouthwashes Using Electrical Impedance AnalysisPLOS ONE Dear Dr. Ibrahim, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. ============================== ACADEMIC EDITOR: Due to a minor revision provided by one reviewer and a rejection by another, the academic editor has suggested a major revision.============================== Please submit your revised manuscript by 8 December 2022. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols. We look forward to receiving your revised manuscript. Kind regards, Ajaya Bhattarai Academic Editor PLOS ONE Journal Requirements: When submitting your revision, we need you to address these additional requirements. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf. 2. Please provide additional details regarding participant consent. In the ethics statement in the Methods and online submission information, please ensure that you have specified what type you obtained (for instance, written or verbal, and if verbal, how it was documented and witnessed). If your study included minors, state whether you obtained consent from parents or guardians. If the need for consent was waived by the ethics committee, please include this information. Once you have amended this/these statement(s) in the Methods section of the manuscript, please add the same text to the “Ethics Statement” field of the submission form (via “Edit Submission”). For additional information about PLOS ONE ethical requirements for human subjects research, please refer to http://journals.plos.org/plosone/s/submission-guidelines#loc-human-subjects-research. 3. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager. Please see the following video for instructions on linking an ORCID iD to your Editorial Manager account: https://www.youtube.com/watch?v=_xcclfuvtxQ. Additional Editor Comments: One reviewer suggested minor revision and one rejected the manuscript. Therefore, the academic editor suggests major revision. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: No Reviewer #2: Partly ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: N/A Reviewer #2: N/A ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: The manuscript contains a number of preliminary n=1 type experiments and there is no clear results. If the three samples of artificial or real saliva are replicates then the data is highly variable. If they are three different samples then it is not explained how they are different, There is no statistical analysis of the samples so it is unclear what are the significant results. The labelling on the graphs is very poor. The RGB/ spectral analysis of listerine when diluted with saliva also makes no sense While the application of microfluidics and impedance to the measurement of saliva is of interest this paper does not in anyway progress the field. Reviewer #2: The manuscript submitted by Fatimah Ibrahim, Bojan Petrović et al. describes a microfluidic compact disc consisting of six unique chambers that run simultaneously from sample loading, holding, mixing and analysis used for the detection of the electrochemical property of different types of saliva and mouthwashes. The manuscript is quite interesting but there are several issues that needs to be addressed by the authors before being accepted: 1. In the abstract please look at the phrase „On the other hand, electrochemical impedance property of artificial saliva, a commonly used drug for the treatment of xerostomia or dry mouth syndrome was also studied.” The drug is missing from this phrase 2. The microfluidic compact disc contained Al based electrodes. There are no issues related to the strong adsorption of the proteins from real saliva at the surface of those electrodes? 3. Are other similar approaches in the literature? Maybe it will be interesting to compare the findings from this manuscript withe the published results. 4. How the dick could be integrated into a point-of-care system? What parameters would be monitored by the end users of such a system? 5. The quality of figure 8 must be improved since the mentioned green color could not be observed. ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: No ******** [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0280381.r001
Revision 1
13 Dec 2022 Author Response Reviewer 1: "The manuscript contains a number of preliminary n=1 type experiments and there is no clear results. If the three samples of artificial or real saliva are replicates then the data is highly variable. If they are three different samples then it is not explained how they are different, There is no statistical analysis of the samples so it is unclear what are the significant results. The labelling on the graphs is very poor. The RGB/ spectral analysis of listerine when diluted with saliva also makes no sense While the application of microfluidics and impedance to the measurement of saliva is of interest this paper does not in anyway progress the field." Response: " Thank you for the comment and suggestions. We have revised the manuscript performing additional experiments using real saliva samples from 3 different healthy volunteers instead of one reported in previous version. Methodology section was updated and error bars were included in Fig 4, Fig 5, Fig 6 and Fig 7. In short, three real saliva samples from 3 healthy subjects were obtained and used in the experiments. The microfluidic CD has six chambers and mouthwashes were mixed with artificial and real saliva in each single run (disc spin). Electrical impedance analysis was recorded for 3 times in each run. The entire experiment was repeated for 3 runs, resulting in n =27 for real saliva samples and n = 9 for artificial saliva (supplementary data is attached). The results indicate this novel microfluidics platform can reliably detect saliva conductivity enabling future clinical applications using rapid and low cost fabrication technique. We have performed statistical analysis to access the effect of mixing on electrical parameters on both real and artificial saliva samples. RGB spectral analysis was demonstrated to show homogenous mixing in microfluidics CD (not the color analysis itself) as laminar flow in microchannels could result in non-homogenous mixture of fluids. Our microfluidics have incorporated specially designed channel to promote mixing of fluids. We have added clarity in the manuscript for contribution of this research (marked in red font color). This paper demonstrate a new low cost fabrication technique for Compact Disc microfluidics using simple xurographic tools and integration of microfluidics with conductive electrodes for electrical impedance analysis. In addition, we demonstrate our microfluidic platform by performing fluidic mixing and impedance analysis on 6 analytes simultaneously. Our research demonstrated rapid, multiplex, low cost salivary analysis tool and open the way for further applications. First, by applying this system, it would be possible to conduct clinical studies with minimal amounts of salivary analytes in a faster and more efficient way. Then, at the level of individual point of care applications, with this study we have shown that the analysis of the electrical properties of saliva is feasible and simple, and each of the analyzed parameters is clinically relevant, both for healthy patients and for patients with oral diseases and disorders in the composition of saliva. In the conducted study, the saliva of healthy volunteers, without oral and general diseases was used, so the obtained values were in the physiological range, as expected. Future clinical studies will, we believe, confirm the effectiveness of this system in evaluating the electrical properties of saliva in patients with various disorders. However, even in healthy patients at low risk for developing oral diseases, it is possible to assess which type of the most commonly used mouthwash solutions lead to the most favorable changes in the composition of saliva, with the least chance of adverse effects. Finally, in patients with reduced salivary secretion, it is possible to evaluate the effect of the artificial saliva that is used, as well as the combination of artificial saliva and antiseptic solutions that will reduce the symptoms, complaints and reduce the risk of oral diseases occurrence in patients with xerostomia." Reviewer 2: "The manuscript submitted by Fatimah Ibrahim, Bojan Petrović et al. describes a microfluidic compact disc consisting of six unique chambers that run simultaneously from sample loading, holding, mixing and analysis used for the detection of the electrochemical property of different types of saliva and mouthwashes. The manuscript is quite interesting but there are several issues that needs to be addressed by the authors before being accepted:" Response: "Thank you for the suggestions. We have improved our manuscript following comments and suggestion from the reviewer. " 1. In the abstract, please look at the phrase “On the other hand, electrochemical impedance property of artificial saliva, a commonly used drug for the treatment of xerostomia or dry mouth syndrome was also studied”. The drug is missing from this phrase. Response: We have revised the statement. “On the other hand, the electrochemical impedance property of artificial saliva, a commonly used moisturizing agent and lubricant for the treatment of xerostomia or dry mouth syndrome was also studied.” " 2. The microfluidic compact disc contained Al based electrodes. There are no issues related to the strong adsorption of the proteins from real saliva at the surface of those electrodes? Response: In our study, we haven’t observed any adsorption of proteins. We rinsed the CD with DI water between each run with real saliva. No major changes were observed during repeated recordings. 3. Are other similar approaches in the literature? Maybe it will be interesting to compare the findings from this manuscript with the published results. Response: To the best of our knowledge, we haven’t found similar approach using microfluidics CD and impedance analysis of saliva in the literature. 4. 1. How the disc could be integrated into a point-of-care system? What parameters would be monitored by the end users of such system? Response: We have revised and included the suggestions in the manuscript. The possibilities of integrating this system into various clinical applications are numerous. First, by applying this system, it would be possible to conduct clinical studies with minimal amounts of salivary analytes in a faster and more efficient way. Then, at the level of individual point of care applications, with this study we have shown that the analysis of the electrical properties of saliva is feasible and simple, and each of the analyzed parameters is clinically relevant, both for healthy patients and for patients with oral diseases and disorders in the composition of saliva. In the conducted study, the saliva of healthy volunteers, without oral and general diseases was used, so the obtained values were in the physiological range, as expected. Future clinical studies will, we believe, confirm the effectiveness of this system in evaluating the electrical properties of saliva in patients with various disorders. However, even in healthy patients at low risk for developing oral diseases, it is possible to assess which type of the most commonly used mouthwash solutions lead to the most favorable changes in the composition of saliva, with the least chance of adverse effects. Finally, in patients with reduced salivary secretion, it is possible to evaluate the effect of the artificial saliva that is used, as well as the combination of artificial saliva and antiseptic solutions that will reduce the symptoms, complaints and reduce the risk of oral diseases occurrence in patients with xerostomia. 5. The quality of figure 8 must be improved since the mentioned green color could not be observed. Response: Thank you. It was a mistake to have written ‘as shown in Figure 8’. Color is not clearly observable with naked eye. However, RGB analysis showed the difference in RGB values. https://doi.org/10.1371/journal.pone.0280381.r002
28 Dec 2022 Decision Letter - Ajaya Bhattarai, Editor A Novel Microfluidic Compact Disc to Investigate Electrochemical Property Changes Between Artificial and Real Salivary Samples Mixed with Mouthwashes Using Electrical Impedance Analysis PONE-D-22-24397R1 Dear Dr. Ibrahim, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Ajaya Bhattarai Academic Editor PLOS ONE Additional Editor Comments (optional): The revised manuscript looks good. Reviewers' comments: <quillbot-extension-portal></quillbot-extension-portal> https://doi.org/10.1371/journal.pone.0280381.r003
Formally Accepted
6 Feb 2023 Acceptance Letter - Ajaya Bhattarai, Editor PONE-D-22-24397R1 A Novel Microfluidic Compact Disc to Investigate Electrochemical Property Changes Between Artificial and Real Salivary Samples Mixed with Mouthwashes Using Electrical Impedance Analysis Dear Dr. Ibrahim: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Ajaya Bhattarai Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0280381.r004

Open letter on the publication of peer review reports

PLOS recognizes the benefits of transparency in the peer review process. Therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. Reviewers remain anonymous, unless they choose to reveal their names.

We encourage other journals to join us in this initiative. We hope that our action inspires the community, including researchers, research funders, and research institutions, to recognize the benefits of published peer review reports for all parts of the research system.

Learn more at ASAPbio .