Detection of Ovarian Cancer Biomarker Lysophosphatidic Acid Using a Label-Free Electrochemical Biosensor

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In the paper entitled "Detection of Ovarian Cancer Biomarker Lysophosphatidic Acid using a Label-free Electrochemical Biosensor", Ivanova and co-workers described the fabrication and electrochemical evaluation of novel phospholipid sensor based on recognition element (peptide) indirectly embedded on the surface of the screen-printed electrode by thienyl linker. The results of the studies seem very interesting and worth publishing in Elecrochem. However, some minor issues should be addressed:

1.    Section 3.2. – The E_1/2 peak currents observed on the CV voltammogram vary between the used analyte concentration by 100 mV. Please explain this phenomenon.
2.    Despite the fact depicted in the Conclusion section that the stability tests weren’t performed, it is important to say something about the potential reusability of the sensor. Have the Authors assessed this issue in repeatable cycles of scanning and washing? What medium could be used in sensor regeneration?
3.    Sensor selectivity against LPA – is it reliable despite the presence of the other phospholipids in serum?

Author Response

We would like to express our gratitude to the reviewer 1 for their insightful comments and constructive feedback on our manuscript. Below is a point-by-point response addressing each of the reviewers' comments and suggestions. We have carefully considered each remark and incorporated relevant revisions to enhance the clarity, and coherence of our study. The revised parts in the manuscript are highlighted in yellow to facilitate easy identification of the changes made. We believe that these revisions have significantly strengthened the quality and comprehensiveness of our work.

 

1. Section 3.2. – The E_1/2peak currents observed on the CV voltammogram vary between the used analyte concentration by 100 mV. Please explain this phenomenon.

Respond: Thank you for the comment. To address the comment, we added the following sentences to section 3.2.

Lines 346-351: In addition to the change in the current intensity, we also observed variations in peak potentials (Fig. 7A), which became more pronounced when the biosensor was exposed to 10 µM LPA. This could be due to the displacement of actin by LPA, altering the electrochemical behavior of the SPE. Such changes affect the electron transfer kinetics and consequently the redox reaction of [Fe(CN)₆]^3-/4- occurring at the electrode interface, leading to shifts in the peak potentials [36].

 

2. Despite the fact depicted in the Conclusion section that the stability tests weren’t performed, it is important to say something about the potential reusability of the sensor. Have the Authors assessed this issue in repeatable cycles of scanning and washing? What medium could be used in sensor regeneration?

Respond: Thank you for the comment. We added the following sentence in the conclusion to highlight the stability of SPE modification. However, our attempt for regenerate the surface of SPE after final modification wasn’t successful.   

Line 452-455: While stability testing to determine the shelf-life was not conducted in this proof-of-concept work, the stability of the modified SPE in electrochemical environment was assessed by conducting at least 7 CV cycles after each modification step.

 

3. Sensor selectivity against LPA – is it reliable despite the presence of the other phospholipids in serum?

Respond: The selectivity of the biosensor towards LPA is based on the specific interaction between LPA and the gelsolin protein used as the biorecognition element. Previous studies have demonstrated the selectivity of the gelsolin-actin system for this purpose. We conducted control experiments using goat serum samples to assess this selectivity, which showed that in the absence of LPA, the current does not change. However, further investigations using human serum are necessary to evaluate the effects of other phospholipids on the performance of our biosensor. As mentioned in the manuscript, this will be the focus of future work and was beyond the scope of our current study. Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This study presents the development of an innovative label-free electrochemical biosensor that employs a gelsolin-actin biorecognition element for the detection of lysophosphatidic acid (LPA), a potential early biomarker for ovarian cancer. The demonstrated high sensitivity and specificity for LPA underscore its potential as a reliable tool for early stage diagnosis of ovarian cancer. However, the study also raises several concerns that demand further discussion:

 

1.     The article should provide reference levels of LPA in healthy individuals, which is crucial for interpreting the clinical significance of the measured LPA levels. 

 

2.     The article described the use of gelsolin bound to Ni-NTA surfaces for the biosensor preparation. However, details on the specific mechanism enabling this interaction are not shown. It is necessary to include relative information in the section 'Materials and Methods'.

 

3.     The authors claim that DTT_COOH offers better surface coverage and a more uniform self-assembled monolayer due to the presence of an additional thiol group is compelling and aligns well with the known chemistry of thiol-gold interactions. Are there additional quantitative data or imaging results supporting this conclusion, such as the surface coverage density?

 

4.     The authors state that "the SPE surface exhibited lesser hydrophilicity when MUA was employed as the linker for immobilizing gelsolin-actin." Please provide a more detailed explanation of why the use of MUA as a linker results in decreased surface hydrophilicity after immobilizing gelsolin-actin.

 

5.     The authors describe assessing the antifouling properties of the developed biosensor by comparing changes in current after modification with DTT_COOH (as shown in Figure S5) and following exposure of the biosensor in goat serum (illustrated in Figure 8A). It appears there is a repetition in these results. The manuscript claims that "by using a new linker, DTT_COOH, we successfully engineered a SAM on the gold surface less prone to fouling compared to MUA, a linear thiol linker." While this statement highlights a potentially significant advancement in the design of biosensors, the paper currently lacks sufficient evidence to support this conclusion. 

 

6.     The authors note, "This observation confirmed that the replacement of actin by LPA, which lacks redox activity, could result in decreased electroactivity and a subsequent reduction in current intensity." However, the statement does not fully address how the decreased redox activity attributed to actin removal surpasses the simultaneous increased redox activity of the [Fe(CN)₆]³⁻/⁴⁻. Please provide more evidence to support this explanation. In addition, the interplay of these counteractive effects suggests that the role of [Fe(CN)₆]³⁻/⁴⁻ might compromise the sensitivity enhancements. Have alternative configurations without [Fe(CN)₆]³⁻/⁴⁻ been considered or tested? 

 

8.     The manuscript presents contradictory outcomes in buffer solution and goat serum, raising fundamental questions about the effects of LPA on the current changes observed. While the authors hypothesize that the presence of oxidative species in goat serum inhibits the involvement of actin in the electron transfer process, this explanation requires further substantiation. For instance, if oxidative species in goat serum can modify actin, it is also possible that these species could directly influence the electrochemical behavior of the electrode itself. 

 

By addressing these concerns, the study could provide a more reliable validation of the sensor's performance and its potential for the early detection of ovarian cancer.

Author Response

[Electrochem] Manuscript ID: electrochem-2942603  

We would like to express our gratitude to the Reviewer 2 for their insightful comments and constructive feedback on our manuscript. Below is a point-by-point response addressing each of the reviewers' comments and suggestions. We have carefully considered each remark and incorporated relevant revisions to enhance the clarity, and coherence of our study. The revised parts in the manuscript are highlighted in yellow to facilitate easy identification of the changes made. We believe that these revisions have significantly strengthened the quality and comprehensiveness of our work.

 

1. The article should provide reference levels of LPA in healthy individuals, which is crucial for interpreting the clinical significance of the measured LPA levels. 

Respond: Thank you for the suggestion, to address this comment we added the following sentences to the manuscript:

Lines 391-395:  A study with 100 healthy volunteers found that LPA levels range from 0.14–1.64 μM [38]. Another study with 27 healthy volunteers and 51 patients with benign ovarian tumor, found that their respective median LPA levels are 1.86 and 6.82 μM [3]. Separate research indicates that in cases of OC, LPA levels are significantly higher that these of healthy individuals, ranging from 5.4 to 200 μM. Given these findings, the obtained LOD and LOQ fall within the range of LPA levels observed in healthy individuals and early stages of OC, confirming the potential of the developed biosensor for OC screening [37].

2. The article described the use of gelsolin bound to Ni-NTA surfaces for the biosensor preparation. However, details on the specific mechanism enabling this interaction are not shown. It is necessary to include relative information in the section 'Materials and Methods'.

Respond: We appreciate the reviewer’s suggestion, and to address it with add the following to the manuscript:

Lines 162–166: We use a poly histidine-tagged gelsolin that binds effectively to Ni-NTA via the imidazole rings of histidine residues. This binding is possible due to the additional free coordination sites on the Ni²⁺ in Ni-NTA, which are occupied by the imidazole ring of histidine acting as ligand.

 

3. The authors claim that DTT_COOHoffers better surface coverage and a more uniform self-assembled monolayer due to the presence of an additional thiol group is compelling and aligns well with the known chemistry of thiol-gold interactions. Are there additional quantitative data or imaging results supporting this conclusion, such as the surface coverage density?

Respond: We appreciate the reviewer’s comments, this statement is based on our previous study using QCM. We added the following sentence to clarify:

Lines 256-259: Our previous study, employing a quartz crystal microbalance, demonstrated that DTT_COOH provides superior surface coverage and anti-fouling properties compared to MUA, possibly due to the additional thiol group [22].

4. The authors state that "the SPE surface exhibited lesser hydrophilicity when MUA was employed as the linker for immobilizing gelsolin-actin." Please provide a more detailed explanation of why the use of MUA as a linker result in decreased surface hydrophilicity after immobilizing gelsolin-actin.

 Respond: We appreciate the reviewer’s comments, we added the following sentences to explain

 Lines 315- 318: The higher contact angle observed when using MUA compared to DTT_COOH may indicate lesser immobilization of gelsolin-actin that led to less hydrophilicity. This observation provides further confirmation of the superior effectiveness of DTT_COOH in developing the biosensor compared to MUA.  

5.The authors describe assessing the antifouling properties of the developed biosensor by comparing changes in current after modification with DTT_COOH (as shown in Figure S5) and following exposure of the biosensor in goat serum (illustrated in Figure 8A). It appears there is a repetition in these results. 

Respond: Thank you for informing us for this repetition, appreciated it. Figure S5 was removed from SI.

6.The manuscript claims that "by using a new linker, DTT_COOH, we successfully engineered a SAM on the gold surface less prone to fouling compared to MUA, a linear thiol linker." While this statement highlights a potentially significant advancement in the design of biosensors, the paper currently lacks sufficient evidence to support this conclusion. 

Respond: We acknowledge your concern about the statement regarding the use of DTT_COOH as a linker. We want to clarify that our study represents a proof-of-concept, and while our initial results are promising, further validation is needed. We plan to conduct more extensive studies to fully evaluate the performance of DTT_COOH in preventing fouling and enhancing biosensor functionality in human serum.

7. The authors note, "This observation confirmed that the replacement of actin by LPA, which lacks redox activity, could result in decreased electroactivity and a subsequent reduction in current intensity." However, the statement does not fully address how the decreased redox activity attributed to actin removal surpasses the simultaneous increased redox activity of the [Fe(CN)₆]³⁻/⁴⁻. Please provide more evidence to support this explanation. In addition, the interplay of these counteractive effects suggests that the role of [Fe(CN)₆]³⁻/⁴⁻might compromise the sensitivity enhancements. Have alternative configurations without [Fe(CN)₆]³⁻/⁴⁻ been considered or tested? 

Respond: Thank you for your insightful comments and valuable suggestions. We performed scan rate experiments (Figure 5) and we discussed the results that confirmed the involvement of actin in electron transfer. We are agreed that using another redox probe can provide valuable information, however since our previous work (Bioelectrochemistry 2023, 153, 108466. https://doi.org/10.1016/j.bioelechem.2023.108466) also confirmed the involvement of actin in electron transfer, we didn’t test another alternative redox probe.

8. The manuscript presents contradictory outcomes in buffer solution and goat serum, raising fundamental questions about the effects of LPA on the current changes observed. While the authors hypothesize that the presence of oxidative species in goat serum inhibits the involvement of actin in the electron transfer process, this explanation requires further substantiation. For instance, if oxidative species in goat serum can modify actin, it is also possible that these species could directly influence the electrochemical behavior of the electrode itself. 

 Respond: Thank you for your insightful comments and valuable suggestions. You rightly point out the possibility that oxidative species present in goat serum could influence both the behavior of actin and the electrochemical properties of the electrode itself. We added the following sentence to address your comment.

Lines 413-417:  There is also a possibility that redox active species present in goat serum could influence the electrochemical properties of the electrode. Comparing the current of the developed biosensor after exposing to the buffer solution and goat serum in absence of LPA (blank solutions) suggested that components of goat serum may directly block the electrode, possibly due to hydrophobic components, despite the "water barrier" introduced during the modification process.

 

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

A new molecule, 3-dithio-threitol propanoic acid (DTT_COOH), is synthesized and used as a linker in SAMs based electrochemical sensor for the detection of LPA by means of gelsolin-actin biorecognition system. It is suggested that the linker forms  a "water barrier"  due to a hydration network within the SAM that has antifouling properties. Fouling of the electrode surfaces  is a common troublesome phenomenon when using electrochemical sensors in physiological media.

Point to be considered:

1.       Impedance measurements are missing in the films characterization. What is the difference in resistance and capacitance of the modified electrode in the case the two modifiers DDT_COOH and MUA  (measurement should be done before the NHS/EDC reaction)?

2.       Fig 4 should show CV’ s  and DPV’s for  0 mM [Fe(CN)₆]^3−/4− at two types of modified surfaces  DDT_COOH and MUA to allow comparison of the reversibility of the electrode process of this marker after each step of surface modification with special focus on the values of background currents.

3.       What is the surface coverage for DDTCOOH and MUA on the electrode keeping in mind the area of each of these molecules.

4.       Is there any oxidation of gold and reduction of Au oxide seen in 0.1M H2SO4? How do these Au/AuO voltammetric peaks look like in both cases? Especially the modification keeping water in the layer is interesting.

5.       The authors write: Despite the presence of hydrophilic groups of carboxyl and hydroxyl in DTTCOOH, the orientation of the molecules may expose hydrophobic chains to the surface, which could contribute to the hydrophobicity. This should made more clear by an appropriate drawing of the layer organization because scheme 2 does not explain this difference in hydrophobicity.

6.       Fig. 7 page Another explanation of decreasing current with increasing LPA concentration would be simply decrease of reversibility of the marker system because of blocking by LPA of the defects in the film. Background current without marker in the solution should be also compared in the presence and absence of increasing concentration of LPA. Impedance measurements would be useful again  to evaluate changes in the resistance of the electrode.

7.       The Authors write: We hypothesized that the presence of goat serum altered the behavior of the SAM on the electrode surface, consequently reducing or inhibiting the involvement of actin in the electron transfer process. There can be another hypothesis to check: Components of goat  serum block the electrode despite the “water barrier” because the current is much smaller than in buffer solution without any addition of LPA. Compare Figures 7 and 8 without addition of LPA. This also points to the need of resistance measurements describing the properties of the layers at all stages. Oxidative species present in the goat serum may not play any role but hydrophobic components do because they additionally block the film improving its barrier properties towards [Fe(CN)₆]^3−/4− which is reflected in the increased deviation from reversibility and in consequence,  lower currents.

8.       What are the interferents in the case of this LPA sensor?

Author Response

[Electrochem] Manuscript ID: electrochem-2942603  

We would like to express our gratitude to the Reviewer 3 for their insightful comments and constructive feedback on our manuscript. Below is a point-by-point response addressing each of the reviewers' comments and suggestions. We have carefully considered each remark and incorporated relevant revisions to enhance the clarity, and coherence of our study. The revised parts in the manuscript are highlighted in yellow to facilitate easy identification of the changes made. We believe that these revisions have significantly strengthened the quality and comprehensiveness of our work.

 

1. Impedance measurements are missing in the films characterization. What is the difference in resistance and capacitance of the modified electrode in the case the two modifiers DDT_COOHand MUA (measurement should be done before the NHS/EDC reaction)

Respond: Thank you for your feedback. We appreciate your suggestion for applying impedance measurements in characterizing the films. While our focus is on cyclic voltammetry and contact angle measurements, we understand and acknowledge the value of impedance analysis in surface characterization. However, it should be noted that this work represents a proof-of-concept rather than an exhaustive exploration of surface chemistry. We'll consider it for future studies to evaluate differences in resistance and capacitance between DTT_COOH and MUA-modified electrodes.

2. Fig 4 should show CV’ s and DPV’s for 0 mM [Fe(CN)₆]^3−/4−at two types of modified surfaces  DDT_COOH and MUA to allow comparison of the reversibility of the electrode process of this marker after each step of surface modification with special focus on the values of background currents.

Respond: Thank you for your feedback. We updated Figure 4 to include DPV of both MUA and DTT_COOH. We  also add the CV of SPE modification with MUA and DTT_COOH  to the SI.

 

3. What is the surface coverage for DDT_COOH and MUA on the electrode keeping in mind the area of each of these molecules.

Respond: Thank you for your insightful comment. We added the following sentences to address your comment.

Lines 271-276: Furthermore, using Randles-Sevcik equation (assuming all parameters in the  equation except current and surface area remain the same after modification)  and the average oxidation peaks from Fig. 4A and 4C, we were able to estimate the surface coverage of DTT_COOH and MUA to be 18.06% and 8.16%, respectively. As such, DTTCOOH has a better estimated surface coverage by more than 10%, supporting the previous statement.

 

4. Is there any oxidation of gold and reduction of Au oxide seen in 0.1M H2SO4? How do these Au/AuO voltammetric peaks look like in both cases? Especially the modification keeping water in the layer is interesting.

Respond: Thank you for your insightful comment. While we didn't specifically test for the oxidation of gold and reduction of Au oxide in 0.1M H₂SO₄ in this study, we appreciate the suggestion for further investigation, which could indeed provide valuable insights into the electrochemical behavior of the electrodes. This investigation is out of scope of this study, and we will consider this suggestion for future studies to enhance our understanding of the effects of surface modification on the electrochemical properties of the electrodes.

5. The authors write: Despite the presence of hydrophilic groups of carboxyl and hydroxyl in DTTCOOH, the orientation of the molecules may expose hydrophobic chains to the surface, which could contribute to the hydrophobicity. This should made more clear by an appropriate drawing of the layer organization because scheme 2 does not explain this difference in hydrophobicity.

Respond: Thank you for your comment. We acknowledge that our initial conclusion regarding the hydrophobicity of the DTTCOOH-modified surface may have been strong. Upon re-evaluation, we observed only a slight increase in the contact angle, indicating a minimal change in hydrophobicity. We have revised the manuscript accordingly to reflect this more nuanced interpretation.

Lines 307-311: Figure 6 illustrates the contact angles of the SPEs following each modification step. Introduction of DTT_COOH to the surface resulted in a slightly increased contact angle compared to the bare gold surface, which may be due to hydrophobic nature of the molecule. In contrast, the SPE modified with MUA exhibited less hydrophobicity, indicating linear immobilization of MUA and exposure of carboxylic acid groups on the surface (Figure 6F).

 

6. 7 page Another explanation of decreasing current with increasing LPA concentration would be simply decrease of reversibility of the marker system because of blocking by LPA of the defects in the film. Background current without marker in the solution should be also compared in the presence and absence of increasing concentration of LPA. Impedance measurements would be useful again to evaluate changes in the resistance of the electrode.

Respond: We apricate the reviewer’s suggestion. We added the following sentences to address the comment.

Lines 339-342: It is also probable that LPA molecules, upon binding to the biorecognition surface, may create a barrier for electron transfer. This interference could disrupt the reversibility of redox reaction or the efficient flow of electrons transfer, thereby contributing to the observed decrease in current intensity.

7. The Authors write: We hypothesized that the presence of goat serum altered the behavior of the SAM on the electrode surface, consequently reducing or inhibiting the involvement of actin in the electron transfer process. There can be another hypothesis to check: Components of goat serum block the electrode despite the “water barrier” because the current is much smaller than in buffer solution without any addition of LPA. Compare Figures 7 and 8 without addition of LPA. This also points to the need of resistance measurements describing the properties of the layers at all stages. Oxidative species present in the goat serum may not play any role but hydrophobic components do because they additionally block the film improving its barrier properties towards [Fe(CN)₆]^3−/4− which is reflected in the increased deviation from reversibility and in consequence, lower currents.

Respond: Thank you for this insight. We agree with the hypothesis that components of goat serum could block the electrode surface and inhibit electron transfer is a valid alternative explanation for the observed changes in current.  We added the following sentences based on your suggestion.

Lines 413-417: There is also a possibility that redox active species present in goat serum could influence the electrochemical properties of the electrode. Comparing the current of the developed biosensor after exposing to the buffer solution and goat serum in absence of LPA (blank solutions) suggested that components of goat serum may directly block the electrode, possibly due to hydrophobic components, despite the "water barrier" introduced during the modification process.

 

8. What are the interferents in the case of this LPA sensor?

Respond: Common interferents in biological samples could be other phospholipids, proteins, lipids, and ions, which may affect the electrochemical response of the LPA sensor. Our results indicating that biological components of goat serum don’t affect the performance of our biosensor. 

 

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

I appreciate the authors' efforts to revise and improve the manuscript. Unfortunately, I have not observed significant enhancements in the revisions, and the revision does not adequately address my concerns. Below are my detailed comments on the authors' responses:

 

Point 2: I suggest revising this wording, as the term "possible" may be perceived as too speculative for an academic paper. If the described binding mechanism is well-supported by evidence, a more assertive term would enhance the clarity and authority of the statement.

 

Point 3: The reference provided by the authors does not appear to support their claim, as it states, "While CAG is useful for confirming functionalization, it does not indicate the SAM’s density." Could the authors provide additional support for their conclusions regarding the density of the SAM?

 

Point 4: The authors’ response has not adequately addressed my concern regarding the decrease in hydrophilicity upon immobilization of GA on the MUA-based surface. This is particularly perplexing given the previous claim that 'The gelsolin-actin complex on the surface significantly increased its hydrophilicity.' Further data to explain this apparent contradiction would be helpful.

 

Point 5: It seems that Figure S5 duplicates the content of Figure 8A. Instead of eliminating Figure S5, I recommend providing the appropriate figure that accurately represents the changes.

 

Point 6: Though the initial results are promising, they do not provide solid support for the conclusion as stated. Further validation is needed before such a definitive statement can be made.

 

Point 7: The authors' response has not sufficiently addressed how the decrease in redox activity due to actin removal surpasses the simultaneous increase in redox activity of [Fe(CN)₆]³⁻/⁴⁻. Additional evidence regarding how these contrasting effects are reconciled would be helpful.

 

Point 8: The response does not adequately address how LPA and goat serum contribute to the observed changes in current. There is no evidence provided that components of goat serum, which may directly block the electrode, would necessarily inhibit the electron transfer of actin. A more detailed examination of these effects is needed to substantiate the conclusions drawn.

 

 

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

There are still corrections to be made:

1. The Authors should use the  common methods for evaluating the coverage of electrode by adsorbed species. The approach provided below based on current will not give a correct answer since several factors may lead to a decrease of current measured. This is why I asked for gold oxidation peak.

Lines 271-276: Furthermore, using Randles-Sevcik equation (assuming all parameters in the equation except current and surface area remain the same after modification) and the average oxidation peaks from Fig. 4A and 4C, we were able to estimate the surface coverage of DTT_COOH and MUA to be 18.06% and 8.16%, respectively. As such, DTTCOOH has a better estimated surface coverage by more than 10%, supporting the previous statement.

2. Still the Authors should show in form of data table that the mentioned interferences do not affect their results (as it can be expected)

 

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 3

Reviewer 3 Report

Comments and Suggestions for Authors

nThe Authors did not include EIS measurements that I suggested to do and would enhance the quality of the paper. Perhaps it is time to learn impedance methods especially when dealing with film covered electrodes. Otherwise, the paper is ready for accepting.