The Road to Unconventional Detections: Paper-Based Microfluidic Chips

Round 1 Reviewer 1 Report

The authors have reviewed the latest development in microfludic paper based devices. The manuscript is well written but there are some minor issues which need to be addressed

1. Figures are not enough so more figures can be added

2. Application part is too small. There are many applications which needs to be added.

3. Schematic of nucleic acid isolation using paper based device need to be added for more clear picture to the readers.

4. Various types of inks used in designing the paper based electrodes that need to be addressed.

5. Various types of lateral flow based assay is missing.

6. Schematic of cell analysis also need to be added

7. Multiplexed analysis with the paper based devices need to be elaborated.

8. Smartphone app linkage with the paper based devices is also missing.

Author Response

Dear Reviewer:

We are grateful to you for reading and evaluating our work. Your constructive, critical, and scientific ideas have helped us improve the quality of the manuscript. The whole manuscript has been properly revised in response to your recommendations. We have marked the main changes in YELLOW in the revised manuscript, and all the answers have been listed one by one in response to the raised issues.

We hope that the revised version is now suitable to be published.

Response to the reviewers' comments： 

1. Figures are not enough so more figures can be added

Response:  We agree with the reviewer's suggestion and we have added three new figures. These figures include a summary figure as the graphical abstract (line#23) and two explanatory figures about nucleic acid preparation (Fig. 6, line#386) and cell analysis (Fig. 7, line#432) for enhancing readers' understanding.

2. Application part is too small. There are many applications which needs to be added.

Response：Each subsection has been updated with new data (lines#342-344, 354-360, 374-379, 422-426,436-439, 444，and 447-450) highlighting several key applications of paper-based microfluidic chips. Moreover, to make the application section more understandable we have also added two new figures (Fig. 6, line#386 and Fig. 7, line#432).

3. Schematic of nucleic acid isolation using paper based device need to be added for more clear picture to the readers.

Response：An explanatory figure (Fig. 6, line#386) for DNA extraction has been added as suggested by the reviewer.

4. Various types of inks used in designing the paper based electrodes that need to be addressed.

Response：We agree with the reviewer that various types of ink should be mentioned in the text. Therefore, we have added some common and recently developed inks used in electrode preparation (line#285-288).

5. Various types of lateral flow based assay is missing.

Response：Many thanks for highlighting this significant point. Since lateral flow analysis is related to chromogenic methods, we have added its details to the same section (line#247-253).

6. Schematic of cell analysis also need to be added

Response：We have added a schematic diagram (Fig. 7, line#432) showing the method of cell analysis.

7. Multiplexed analysis with the paper based devices need to be elaborated.

Response：Multiplexed analysis has been explained in detail in a new paragraph. (line#354-360).

8. Smartphone app linkage with the paper based devices is also missing.

Response：We are very thankful to the reviewer for mentioning this essential point. We agree with the reviewer that smart apps linked to paper chip devices must be part of this review. We have added a new subsection 4.4 (lines#324-339) that discusses the combination of paper-based chips with electronic devices including smart phones. Moreover, we have also discussed this point in section 6 (lines#475-479).

Author Response File: Author Response.docx

Round 1 Reviewer 2 Report

The authors of the manuscript titled "The Road to Unconventional Detections: Paper-Based Microfluidic Chips" wrote a review paper which is based on the capabilities and advantages of paper-based microfluidic chips for biomedical applications. It mainly focuses on efficiency, accuracy, integration, and innovation. In addition, it discusses how paper-based microfluidic chips are made, examined, and utilized to aid in biological research and functional integration. The topic is within the scope of the journal. However, the manuscript needs major and sincere revision before being accepted.

1)    How is continuous progress observed in the transition from single-channel detection to multi-channel detection? Authors should include a detailed description of this somewhere in their manuscript.

2)    The overall manuscript requires an English language check. It is suggested to be consistent about the tense used.

3)    Line 100, it's soaked the filter paper in an alkyl ketene dimer (AKD) solution. The authors should describe how it soaked the filter paper in AKD.

4)     Figure 5, Some texts are blurry. Authors need to improve the image quality.

5)    A graphical representation of the total process is another effective method to make the task more understandable and appealing. It's important to remember that the document is meant for readers and is a way for scientists to share their findings with the public. It shouldn't be self-explanatory.

6)    Line 278, It's suggested that authors write in detail about the CuCo-CeO2 nano-spheres. 

7)    Line 288-289, What are the specific wavelengths and emit photons at longer wavelengths to obtain analytical results?

8)    Line 292-294, what the authors mean by ROR1 ? Explain in detail.

9)    Line 338, Can authors elucidate more about polymerase chain reaction (PCR)?

10) Line 383-384, “It can specifically capture Salmonella typhimurium and conduct rapid and sensitive colorimetric detection by urease activity inhibition method.” Authors should provide more the detail of urease activity inhibition method. It will help the newbie readers to understand the topic in detail.

Author Response

Dear Reviewer:

We are grateful to you for reading and evaluating our work. Your constructive, critical, and scientific ideas have helped us improve the quality of the manuscript. The whole manuscript has been properly revised in response to your recommendations. We have marked the main changes in YELLOW in the revised manuscript, and all the answers have been listed one by one in response to the raised issues.

We hope that the revised version is now suitable to be published.

Response to the reviewers' comments：

• How is continuous progress observed in the transition from single-channel detection to multi-channel detection? Authors should include a detailed description of this somewhere in their manuscript.

Response：Many thanks for highlighting this significant point. We have added a new paragraph and discussed the main method of multiplexing, the reasons behind its development, and its significance for paper chips (line#354-360).

• The overall manuscript requires an English language check. It is suggested to be consistent about the tense used.

Response：The current manuscript has been revised with the assistance of a native English speaker experienced in this field.

• Line 100, it's soaked the filter paper in an alkyl ketene dimer (AKD) solution. The authors should describe how it soaked the filter paper in AKD.

Response： The specific method for soaking the filter paper in AKD has been described (lines#108-110).

• Figure 5, Some texts are blurry. Authors need to improve the image quality.

Response：We have optimized the image quality, and the text is now clearer (Fig.5, line#281).

• A graphical representation of the total process is another effective method to make the task more understandable and appealing. It's important to remember that the document is meant for readers and is a way for scientists to share their findings with the public. It shouldn't be self-explanatory.

Response：We are very thankful to the reviewer for mentioning this essential point. We agree with the reviewer that a graphical abstract will enhance its understandability. Therefore, a graphic abstract has been added (line#23).

• Line 278, It's suggested that authors write in detail about the CuCo-CeO2 nano-spheres.

Response： Thanks for your expert opinion. We have added a supplement about the details of CuCo-CeO₂ nano-spheres (lines#294-299).

• Line 288-289, What are the specific wavelengths and emit photons at longer wavelengths o obtain analytical results?

Response：We have added a clearer description and explanation of how fluorophores work (lines#306-310).

• Line 292-294, what the authors mean by ROR1 ? Explain in detail.

Response：A supplement to the description of ROR1 (lines#422-426) has been added.

• Line 338, Can authors elucidate more about polymerase chain reaction (PCR)?

Response：A more detailed description of PCR has been added that explains the role of PCR in the gene extraction function of the paper chips (line#374-379).

• Line 383-384, “It can specifically capture Salmonella typhimurium and conduct rapid and sensitive colorimetric detection by urease activity inhibition method.” Authors should provide more the detail of urease activity inhibition method. It will help the newbie readers to understand the topic in detail.

Response：A more detailed description of the urease activity inhibition method has been added. (lines#267-274).

Author Response File: Author Response.docx

Round 1 Reviewer 3 Report

The proposed review describes the use of paper-based microfluidic devices for unconventional detection of analytes. In general, the work is well written and contains an adequate number of references. The schematic organization of the topics is appropriate, and the reading of the manuscript is fluid and pleasant.

That said, I must add that there is an extensive literature reviewing paper-based microfluidic devices in depth (10.1002/elps.201900258, 10.1007/s10337-013-2413-y, 10.1016/j.trac.2018.08.018, 10.1021/acs.chemrev.0c01335). Many other authors have reviewed the same topic from different approaches, generating many reports, both general (on paper microchips in general) and limited to specific techniques of both manufacturing, detection and use.

Having clarified this, the work deals in an extensive, clear, and well-documented manner with the different manufacturing techniques (even leaving aside some, https://doi.org/10.1039/C4AN00230J). Instead, the detection techniques are initially explained superficially and, in some cases, inadequately. The advantages and disadvantages of the detection methods are not clearly detailed and the information that many of the colorimetric, electrochemical, and fluorescent methods have already been brought to portability is omitted. In some sense, the review places more emphasis on molecular biology applications, which is plausible, but is not reflected in the title of the manuscript.

On the other hand, detailing the detection methods repeats the need for trained personnel to carry out the measurements and results interpretation, which is true. But it is omitted to issue an opinion about this same point in advanced techniques of molecular biology.

It is noteworthy that many the references in this manuscript are very ingenious and well-crafted works, the authors have carried out an adequate bibliographic search.

Due to the above, I advise rejecting this work since the developed topic has already been extensively treated, its approach is not particularly innovative, and the different techniques are not equally reviewed according to the aim exposed in the title, the abstract and the introduction..

Author Response

Dear Reviewer:

We are grateful to you for reading and evaluating our work. Your constructive, critical, and scientific ideas have helped us improve the quality of the manuscript. The whole manuscript has been properly revised in response to your recommendations. We have marked the main changes in YELLOW in the revised manuscript, and all the answers have been listed one by one in response to the raised issues.

We hope that the revised version is now suitable to be published.

Response to the reviewers' comments：

1. The proposed review describes the use of paper-based microfluidic devices for unconventional detection of analytes. In general, the work is well written and contains an adequate number of references. The schematic organization of the topics is appropriate, and the reading of the manuscript is fluid and pleasant.

Response: We are thankful to the reviewer for these kind comments.

2. That said, I must add that there is an extensive literature reviewing paper-based microfluidic devices in depth (1002/elps.201900258, 10.1007/s10337-013-2413-y, 10.1016/j.trac.2018.08.018, 10.1021/acs.chemrev.0c01335). Many other authors have reviewed the same topic from different approaches, generating many reports, both general (on paper microchips in general) and limited to specific techniques of both manufacturing, detection and use.

Response:

Thanks for reviewing our manuscript thoroughly and for providing a list of relevant publications that discuss paper-based microfluidic devices. Having read these papers, we also consider these papers to be worth reading. The manuscripts are unique in the sense that they have targeted specific areas. For instance, [10.1002/elps.201900258] focuses on biomarker detection, [10.1007/s10337-013-2413-y] discusses paper-based chromatographic filtration and mixing device, [10.1016/j.trac.2018.08.018] highlights the analysis and detection methods of paper-based microfluidic chips, and [10.1021/acs.chemrev.0c01335] summarizes the paper-based chips preparation methods, especially the fluid control method without explaining the detail of the application of paper-based chips.

On the other hand, our review article summarizes a large amount of recent literature, keeping pace with the development of paper chip technology. The manuscript describes the direction of paper chips in the field of biomedicine. The review examines key developments in paper-based microfluidic chips from three perspectives: channel preparation methods, analytical techniques, and their current applications. Moreover, we have discussed some new opportunities for innovations that could move us beyond the current state of the art. These opportunities could enable these chips to be used for societal purposes.

During revision, we strictly followed your instructions and emphasized novelty and innovation. We have changed the title, added a new paragraph at the end of the introduction (lines#36-40), revised each section (preparation methods, analysis techniques, and application parts) extensively and added new tables for comparing the advantages and disadvantages of different preparation and analysis methods (Table 2, line#79, Table 3, lines#240).

The reviewer may kindly approve that this article has enough innovation and academic value to be suitable for publication.

3. Having clarified this, the work deals in an extensive, clear, and well-documented manner with the different manufacturing techniques (even leaving aside some, https://doi.org/10.1039/C4AN00230J). Instead, the detection techniques are initially explained superficially and, in some cases, inadequately. The advantages and disadvantages of the detection methods are not clearly detailed and the information that many of the colorimetric, electrochemical, and fluorescent methods have already been brought to portability is omitted.

Response:

We are sorry for our inadequate explanation and thankful to reviewer for highlighting this issue. In each subsection of the analysis part, the latest literature has been discussed and cited. A new table (Table. 3, line#240) has been added to compare different analytical techniques used on paper-based chips. In the colorimetric method section (4.1), we have added two supplements that describe lateral flow analysis in detail (lines #247-253) and explain the role of nano-probes in the urease activity inhibition method (lines #267-274). The electrochemical method (4.2) and fluorescence method (4.3) subsections have also been expanded to include information about inks (lines#285-288), CuCo-CeO2-nanospheres (lines#295-299) and fluorophores (lines#306-310).

Moreover, for highlighting importance of the smart apps linked to paper chip devices, we have added a new subsection 4.4 (lines#324-339) that discusses the combination of paper-based chips with electronic devices including smart phones. We have also discussed this point in section 6 (lines#475-479).

4. In some sense, the review places more emphasis on molecular biology applications, which is plausible, but is not reflected in the title of the manuscript.

Response:

We are very thankful to the reviewer for mentioning this essential point. We agree with the reviewer that the title of the manuscript does not fully reflect the manuscript’s content. Therefore, we have changed the title now it reads as “Paper-based microfluidic chips: The legend of Biomedical Engineering”.

5. On the other hand, detailing the detection methods repeats the need for trained personnel to carry out the measurements and results interpretation, which is true. But it is omitted to issue an opinion about this same point in advanced techniques of molecular biology.

Response:

We agree that detailed analysis methods often rely on professional experimental equipment and professionals. Precise analysis results conflict with the advantages of low-cost and easy portability of paper chips and the development direction of out-of-laboratory testing. Researchers are also working to solve this problem. The use of paper chips in combination with electronic devices such as smartphones and blood glucose meters is a step forward in this direction. We have added a new subsection 4.4 (lines#324-339) for discussing this point in detail.

Author Response File: Author Response.docx

Round 2 Reviewer 2 Report

Accept in present form.

Author Response

Dear Reviewer:

Thanks for accepting our manuscript and for your constructive, critical, and scientific ideas that have helped us improve the quality of the manuscript. English has been revised and improved with the assistance of experts.

We hope that the revised version is now publishable.

Author Response File: Author Response.docx

Round 2 Reviewer 3 Report

The work of the authors on the manuscript has been very useful for this review. The modification of the title gives more clarity to the final objective of this revision, adjusting the contents in a more adequate way. From my point of view, the manuscript is ready to be accepted after some minor revisions that I detail below:

- Line 73 mentions "hydrophobic channels" in paper-based chips. In general, the structure of paper-based microfluidic devices seeks to generate hydrophilic channels flanked by hydrophobic barriers. Please clarify that line so as not to generate confusion in the reader.

-In section 4.3 electrochemiluminescence is presented as a detection method, in my opinion single electrode electrochimiluminescence (https://doi.org/10.1039/D2SD00041E) should be considered by the authors together with those already mentioned in the manuscript.

I thank the authors for their dedication and effort to improve the review and I recommend its publication after these minor modifications.

Author Response

Dear Reviewer:

We are grateful to you for your constructive, critical, and scientific ideas that have helped us improve the quality of the manuscript. We have revised the manuscript according to your recommendations and marked the main changes in YELLOW. Answers have been listed one by one in response to the raised issues.

We hope that the revised version is now publishable.

Response to the reviewers' comments：         

1. The work of the authors on the manuscript has been very useful for this review. The modification of the title gives more clarity to the final objective of this revision, adjusting the contents in a more adequate way. From my point of view, the manuscript is ready to be accepted after some minor revisions that I detail below:

Response: We are thankful to the reviewer for these kind comments, and have addressed the minor issues in the revised version.

2. Line 73 mentions "hydrophobic channels" in paper-based chips. In general, the structure of paper-based microfluidic devices seeks to generate hydrophilic channels flanked by hydrophobic barriers. Please clarify that line so as not to generate confusion in the reader.

Response: Many thanks for highlighting this significant point. We have revised the description of the channel structure of paper-based microfluidic devices accordingly (lines#72-73).

3. In section 4.3 electrochemiluminescence is presented as a detection method, in my opinion single electrode electrochimiluminescence (https://doi.org/10.1039/D2SD00041E) should be considered by the authors together with those already mentioned in the manuscript.

Response: Many thanks for highlighting this point. We agree with the reviewer that single electrode electrochimiluminescence must be part of this review. We have described it in the revised version and included some valuable research about it (lines#318-321).

4. I thank the authors for their dedication and effort to improve the review and I recommend its publication after these minor modifications.

Response: We are very grateful to you for these encouraging remarks and for putting your time and energy into helping us improve the quality of the manuscript. We hope our recent revision has made the manuscript suitable for publication.

Thanks again.

Author Response File: Author Response.docx