Portable DNA Probe Detector and a New Dry-QCM Approach for SARS-CoV-2 Detection
Round 1
Reviewer 1 Report
Comments and Suggestions for Authors
This work from Pojprapai et al introduces a new type of qcm based sensors applied for the detection of coronavirus. One of the originalities of this work is the possibility to work in the dry state for the detection. The positioning of this work is of interest and the potential for application is obvious. I therefore support publication of this work. After correction of the following issues
1- On the form of the manuscript.
a- The format for the formula of many chemical compounds is not correct (ex C2H5OH). Please format properly these formula.
b- Error bars are missing on figure 6.
c- The quality of figures 2, 4, 8 and 11 needs to be improved for readability.
2- On the positioning as compared with the state of the art.
a- The Sauerbrey model is only valid for films that are thin enough and rigid enough. Otherwise, I was models need to be applied that would take into account the dissipation of the film. Here they also need to add a sentence and a reference justifying why Sauerbrey model is applicable to their system.
b- The application is somewhat too focused on the application of this article. A few lines describing the general principle for the conception of a surface sensor are needed with appropriate references. Especially it is worth mentioning that different transduction modes are being developed by researchers. For instance consider the following references:
https://doi.org/10.1016/j.ccr.2022.214424 //
https://doi.org/10.1002/cnma.202300192 //
https://doi.org/10.1002/eom2.12094
3- On the scientific content of the manuscript.
a- The afm 3D images do not allow determining the quality of the surface coating (if it is indeed a continuous coating). Authors should provide 2D AFM images of the QCM sensors with the following purpose
- Images of different sizes and area on the crystal so that we can evaluate the surface coverage. This should be provided for the naked surface, the Sam-covered surface and the two hybridized surfaces
- Determination of the RMS from 3 different areas (with same image size and resolution) to have a statistically meaningful value for the naked surface, the Sam-covered surface and the two hybridized surfaces
b- Determination of the LOD and of the dynamic range of possible detection by the sensor is of a critical significance. Here, figure 5 does not allow determining the full linearity domain of the sensor. The authors need to perform additional experiments at larger and lower target concentrations in order to precisely determine when linearity is lost.
c- as the authors are rightly demonstrating that they can modulate the quantity of immobilized materials on top of their QCM sensor It would be interesting to establish the effect of this parameter on the detection performances.
d- The relative abundancy of each element can be determined from the xps measurements. From this data authors should be able to determine the relative proportion of SAMS versus immobilized and hybridized DNA on their surface. This would be a nice addition to the QCM analysis.
Author Response
Reviewer No.1
This work by Pojprapai et al. introduces a new type of QCM-based sensor that is applied to detect coronavirus. One of the originalities of this work is the possibility of working in the dry state for the detection. The positioning of this work is of interest and the potential for application is obvious. I therefore support the publication of this work. After the correction of the following issues
Ans: We appreciate the reviewer's comments and acknowledge their valuable input.
1- On the form of the manuscript.
a- The format for the formula of many chemical compounds is not correct (ex C2H5OH). Please format properly these formula.
Ans: The format of the formulae for all chemical compounds has been corrected as per the suggestions. The changes are throughout the manuscript and highlighted in yellow.
b- Error bars are missing in Figure 6.
Ans: New results and error bars are included. Respective changes are incorporated. To address this concern, we have added the explanation to the manuscript (highlighted in lines numbers 357 to 361, Figure number 8).
c- The quality of figures 2, 4, 8 and 11 needs to be improved for readability.
The quality of all figures is improved and amended.
2- On the positioning as compared with the state of the art.
a- The Sauerbrey model is only valid for films that are thin enough and rigid enough. Otherwise, I was models need to be applied that would take into account the dissipation of the film. Here they also need to add a sentence and a reference justifying why Sauerbrey model is applicable to their system.
Ans: The Sauerbrey equation is applicable to our system because the deposited film is sufficiently thin and rigid, ensuring that the frequency shift is predominantly mass-sensitive with negligible viscoelastic effects. According to Sauerbrey [40], the Sauerbrey model remains valid for films with a thickness in the nanometer range and a rigid structure, conditions that are met in our study. Kanazawa et al. [41] discussed the limitations of the Sauerbrey model and when viscoelastic effects must be considered, justifying why it remains valid for rigid films. Rodahl et al. [42] expanded on the conditions under which the Sauerbrey equation holds and when a viscoelastic model should be used. To address this concern, we have added further explanation to the manuscript (highlighted in lines 208 to 210), along with references [40–42].
b- The application is somewhat too focused on the application of this article. A few lines describing the general principle for the conception of a surface sensor are needed with appropriate references. Especially it is worth mentioning that different transduction modes are being developed by researchers. For instance consider the following references:
https://doi.org/10.1016/j.ccr.2022.214424, https://doi.org/10.1002/cnma.202300192, https://doi.org/10.1002/eom2.12094
Ans: We appreciate the reviewer’s suggestion to provide a broader context on the general principles of surface sensor conception and various transduction modes. To address this concern, we have added further information to the manuscript (highlighted in numbers 77 to 83) along with the references [20-22].
3- On the scientific content of the manuscript.
a- The AFM 3D images do not allow determining the quality of the surface coating (if it is indeed a continuous coating). Authors should provide 2D AFM images of the QCM sensors with the following purpose
- Images of different sizes and area on the crystal so that we can evaluate the surface coverage. This should be provided for the naked surface, the Sam-covered surface and the two hybridized surfaces
- Determination of the RMS from 3 different areas (with same image size and resolution) to have a statistically meaningful value for the naked surface, the Sam-covered surface and the two hybridized surfaces
Ans: We thank the reviewer for this valuable suggestion, which strengthens the characterisation of our sensor surface. To address this concern, we performed the experiment again for all the steps (fresh, P-DNA, P-DNA+T-DNA, P-DNA+covid-19 RNA). We took the data at three different areas covering multiple regions of the crystal and averaged the RMS values for four sets of samples. The image sizes and resolutions were kept consistent to ensure statistically meaningful comparisons. The amended text in the manuscript is “The initial root mean square (RMS) roughness of the fresh QC electrode was measured at 2.77 nm, which increased to 3.17 nm after P-DNA immobilisation, further increased to 3.60 nm followed by T-DNA hybridisation and increased to 4.17 nm after COVID-19 RNA hybridisation”. The fresh QC RMS value increased to 2.77 compared to the previously mentioned 1.7 because we used the cleaning procedure several times to regenerate the crustal, which might be the reason for the higher value. The new AFM images are added in Figure-9 in line 371; the RM values are changed in text in lines 378 to 381.
b- Determination of the LOD and of the dynamic range of possible detection by the sensor is of a critical significance. Here, figure 5 does not allow determining the full linearity domain of the sensor. The authors need to perform additional experiments at larger and lower target concentrations in order to precisely determine when linearity is lost.
Ans: We sincerely appreciate the reviewer’s insightful comment regarding the determination of the sensor's limit of detection (LOD). To address this concern, we conducted additional experiments covering both lower and higher target concentrations beyond the initially reported range in Figure 5. The new experimental data have been incorporated into an updated version of Figure 7, which now more clearly defines the sensor’s full linearity domain. The results and discussion section have also been revised to incorporate these new findings and comprehensively explain the sensor’s performance across its full detection range. To address this concern, we have updated the text in the manuscript (highlighted in line numbers 335 to 350, Figure number 7).
c- as the authors are rightly demonstrating that they can modulate the quantity of immobilized materials on top of their QCM sensor It would be interesting to establish the effect of this parameter on the detection performances.
Ans: In response, we have expanded our discussion to include an analysis of how varying immobilisation levels influence key performance metrics such as sensitivity, limit of detection, and signal stability. To address this concern, we have updated the text in the manuscript (highlighted in line numbers 343 to 349).
d- The relative abundancy of each element can be determined from the xps measurements. From this data authors should be able to determine the relative proportion of SAMS versus immobilized and hybridized DNA on their surface. This would be a nice addition to the QCM analysis.
Ans: We appreciate the reviewer’s suggestion to use XPS to quantify the relative proportions of SAMs, immobilised, and hybridised DNA. However, due to the inherent limitations of XPS in detecting thin DNA layers—such as weak phosphorus signals and surface charging effects, this approach may not provide reliable quantification. Instead, we rely on QCM, which offers a direct and real-time mass measurement of DNA immobilisation and hybridisation. Additionally, AFM imaging already supports our surface characterisation. Based on these factors, we believe that XPS is not the most suitable technique for quantifying DNA coverage in this study. Also, we did not perform a high-resolution scan of all the elements during the experiment. Our aim was to show qualitative (especially for S2p of Au-S bonding), and we did not expect quantitative analysis.
Instead, we added more analysis on the Au4f peak, why it is negatively shifted, how the oxide or thiolate layer was removed and seen in XPS spectra, how the Ar plasma cleaning helped us to provide the active Au surface (+ve charge), and how it became advantageous for SAM formation via Au-S (-ve charged) bond and correspondingly a -ve energy shift instead of +ve energy shift. It provided the most important information through this analysis. To address this concern, we have added the text in the manuscript (in line numbers 395 to 403).
Reviewer 2 Report
Comments and Suggestions for Authors
The scope of the manuscript is significant, particularly considering the emergence of the recent global pandemic. This study presents initial findings on the rapid and direct detection of SARS-CoV-2 using the Quartz Crystal Microbalance (QCM) method. A novel dry-QCM approach was developed to simplify the experimental workflow. Key findings include an estimated probe density of 0.51×10120.51 \times 10^{12} probes/cm² (below the critical limit), a hybridization efficiency of 58.9%, a detection limit of approximately 0.74 pM, and high selectivity for SARS-CoV-2 target DNA.
While the manuscript's scientific approach is not highly original, it is of considerable interest due to the development of the dry-QCM method. Unfortunately, this method is poorly described. The manuscript should be revised to include a detailed explanation of the wet and dry-QCM approaches, with emphasis on the techniques used to wash away nonspecific molecules adsorption.
Several major revisions are necessary before publication:
- Lines 80-85: The first part of the introduction is highly detailed and appears overly weighted compared to the discussion on QCM. A more in-depth comparison between the references mentioned in lines 80-85 and the improvements introduced by this work is required.
- Introduction: The state of the art for the dry-QCM approach should be included.
- Throughout the manuscript: Review and correct the chemical formulas (e.g., "H2SO4" should be formatted as H2SO4H_2SO_4). Additionally, using "Figure" instead of "Fig" throughout the text would enhance consistency and readability.
- Line 164: The experiments conducted at liquid-surface and air-surface interfaces need further elaboration. Supporting data for these experiments are unavailable, which prevents adequate evaluation.
- Line 172: The process of drying the sensors and the measures taken to prevent nonspecific adsorption of species or buffer salts during drying should be clarified.
- Line 267: In Fig. 6, amend the text to reflect "23.3°" correctly.
- Line 376: Add a description of how the shelf life was evaluated in the experimental section and discuss it in the results section.
Addressing the above points will significantly improve the clarity and completeness of the manuscript. Once these essential revisions are implemented, a second round of review will likely be necessary.
Author Response
------------------------Reviewer No.2-------------------------------
The scope of the manuscript is significant, particularly considering the emergence of the recent global pandemic. This study presents initial findings on the rapid and direct detection of SARS-CoV-2 using the Quartz Crystal Microbalance (QCM) method. A novel dry-QCM approach was developed to simplify the experimental workflow. Key findings include an estimated probe density of 0.51×10120.51 \times 10^{12} probes/cm² (below the critical limit), a hybridization efficiency of 58.9%, a detection limit of approximately 0.74 pM, and high selectivity for SARS-CoV-2 target DNA.
While the manuscript's scientific approach is not highly original, it is of considerable interest due to the development of the dry-QCM method. Unfortunately, this method is poorly described. The manuscript should be revised to include a detailed explanation of the wet and dry-QCM approaches, with emphasis on the techniques used to wash away nonspecific molecules adsorption.
Ans: We appreciate the reviewer’s suggestions and concerns about the manuscript. We have revised it thoroughly and added more optimization data (e.g., probe concentration, multiple probe incubation, and extended range of LoD test). We believe these additions help the manuscript describe the key findings in the dry-QCM approach.
Several major revisions are necessary before publication:
Lines 80-85: The first part of the introduction is highly detailed and appears overly weighted compared to the discussion on QCM. A more in-depth comparison between the references mentioned in lines 80-85 and the improvements introduced by this work is required.
Ans: We appreciate the reviewer’s insights in the manuscript. The first part has been streamlined to provide a more balanced discussion, and we have included a more in-depth comparison between the references cited in lines 80-85. More in-depth analyses of QCM and findings have been added to the manuscript. To address this concern, we added the following sentence to the manuscript (highlighted in lines 84 to 114), and references are changed accordingly [23-38].
Introduction: The state of the art for the dry-QCM approach should be included.
Ans: We acknowledge the need to discuss the dry-QCM approach in greater detail. We have incorporated a section in the introduction outlining dry-QCM techniques' importance. To address this concern, we added the following sentence to the manuscript in lines 119 to 125).
Throughout the manuscript: Review and correct the chemical formulas (e.g., "H2SO4" should be formatted as H2SO4H_2SO_4). Additionally, using "Figure" instead of "Fig" throughout the text would enhance consistency and readability.
Ans: The chemical formulae are formatted throughout the manuscript according to the reviewer's suggestions. Additionally, we have amended Figure instead of Figure throughout the text to enhance consistency and readability.
Line 164: The experiments conducted at liquid-surface and air-surface interfaces need further elaboration. Supporting data for these experiments are unavailable, which prevents adequate evaluation.
Ans: We appreciate the reviewer’s comment regarding the experiments conducted at liquid-surface and air-surface interfaces. This study primarily performed QCM measurements in dry (air-phase) and wet (liquid-phase) conditions to evaluate surface functionalisation and biomolecular interactions. However, the signal stability is very poor when the sensor is in contact with liquid. The particular results are attached in the Appendix as supporting data. The amended text is reflected in line numbers 225 to 229.
Line 172: The process of drying the sensors and the measures taken to prevent nonspecific adsorption of species or buffer salts during drying should be clarified.
To address this concern, the methodology is added to the manuscript in lines numbers 174 to 196 and Figure 1. Specially designed glassware was used to hold the QC and liquid; the slow rocking ensures that the excess unbound DNA is removed.
Line 267: In Fig. 6, amend the text to reflect "23.3°" correctly.
Ans: The authors performed the contact angle measurement again, and new images and values were incorporated into the manuscript. The new results with corresponding changes are in 357 to 361 and figure 8.
Line 376: Add a description of how the shelf life was evaluated in the experimental section and discuss it in the results section.
Ans: A detailed description and experimental findings of the shelf-life evaluation are added to the manuscript. To address this concern, a separate section (section number 3.8), experimental procedure (in line numbers 194 to 197), results and discussion (line numbers 488 to 495), and corresponding figure (figure number 14) are added to the manuscript. Thanks to the reviewer for the valuable suggestion.
Addressing the above points will significantly improve the clarity and completeness of the manuscript. Once these essential revisions are implemented, a second round of review will likely be necessary.
Ans: All the above points have significantly improved the clarity and completeness of the manuscript. The revisions have been implemented as per the suggestions of the eminent reviewer.
Round 2
Reviewer 1 Report
Comments and Suggestions for Authors
The authors have brought satisfactory improvements to their manuscript
Reviewer 2 Report
Comments and Suggestions for Authors
Authors carried out a profound revision of the manuscript. They have adequately addressed all my comments raised in the previous round.
In my opinion the manuscript could be accepted for publication.
