Electrochemical Strategies to Evaluate the Glycosylation Status of Biomolecules for Disease Diagnosis
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe authors review the electrochemical strategies for the detection of glycosylated biomolecules. This work is suitable for this journal. Comments:
The Abstract should be shortened.
The previous review papers on this topic should be commented in Introduction.
The references should be updated and recent works on electrochemical detection of glycosylation should be added. For example, biorecognition element-free electrochemical detection of recombinant glycoproteins, and their other works.
The general ways for the immobilization and recognition of biomolecules include covalent coupling reactions and non-covalent interactions of antigen–antibody, aptamer–target, glycan–lectin, avidin–biotin and boronic acid–diol. Besides, nitrilotriacetic acid-metal complexes for the immobilization and recognition of biomolecules should be added.
For boronic acid derivatives-based strategies, boronic acid-functionalized nanomaterials for the design of electrochemical biosensors, and biosensors with boronic acid-based materials as the recognition elements and signal labels should be added and discussed.
The subtitles should be revised with more detailed information, such as 3.4.
A scheme can be added to show the content of this review.
Author Response
Answer to Reviewer 1
The authors review the electrochemical strategies for the detection of glycosylated biomolecules. This work is suitable for this journal.
Response: Thank you very much for this nice comment and for taking the time to review our manuscript. Your invaluable comments and suggestions have been helpful in improving the clarity, quality, and overall impact of our work, and we sincerely appreciate your effort and consideration. All changes will be highlighted in yellow color in the manuscript.
Comment 1: The Abstract should be shortened.
Response 1: The abstract was shortened down to 200 words following your suggestion.
Comment 2: The previous review papers on this topic should be commented in introduction.
Response 2: Thank you for your comment. We added to the Introduction section a short paragraph of the three main reviews related to the topic. It is also shown below:
“For instance, Akiba et al. reported a review related to the topic in 2016 [16]. The authors reviewed the advancements in electrochemical biosensors designed to detect glycopro-teins, mainly related to cancer and diabetes diagnosis. This work summarizes fabrication strategies, recognition elements (antibodies, lectins, phenylboronic acids, molecularly im-printed polymers), and the role of nanomaterials like graphene, carbon nanotubes, and metal nanoparticles in enhancing sensitivity. The paper highlights challenges like selec-tivity and the need for label-free, single-step assays. Later Echevarri & Orozco reported a review focused on the development and application of electrochemical biosensors using glycans as biorecognition elements, covering from 2012 to 2022 [13]. Specifically, they aimed to the application of this glycan-based electrochemical biosensors on the detection of infectious diseases and cancer biomarkers. The paper also discusses structural glycobi-ology, biosensor classification, recent advances, and challenges such as glycan stability and reproducibility, highlighting opportunities for integrating nanomaterials and multi-plexed formats to improve clinical applicability and enable decentralized, rapid, and cost-effective disease diagnosis. Another recent and interesting review was reported by Hashkavayi et al. in 2025 and it is focused on the recent progress in glycan detection strategies on exomes, cancer cells and circulating cancer-derived glycoproteins, empha-sizing electrochemical biosensors [17]. This paper describes the characteristics of the most used affinity probes (lectins, aptamers, antibodies and boronic acid derivatives) and criti-cally discusses the advantages and disadvantages of each affinity probe. Ultimately, au-thors seek to guide innovations in glycobiosensors for cancer diagnostic toward reliable, portable and miniaturized tools with multiassay capabilities in combination with micro-fluidics”.
Comment 3: The references should be updated and recent works on electrochemical detection of glycosylation should be added. For example, biorecognition element-free electrochemical detection of recombinant glycoproteins, and their other works.
Response 3: Thank you for your suggestion. We checked again Web of knowledge database, and we didn’t find relevant articles about the evaluation of glycosylation status of glycoconjugates and cells. However, we found an interesting article, new reference [69], about one of the future research lines of this topic, i.e., the use of MOF as signal amplification strategy (Analytica Chimica Acta 1273 (2023) 341540, https://doi.org/10.1016/j.aca.2023.341540). We extended in section 2.3 the discussion about the use of MOF for improving the sensitivity for glycoprotein detection, and we added the new article. See below:
“Another interesting strategy is the use of metal organic frameworks (MOF) for signal amplification, such as that reported in [31]. In this work, ZIF-8 was preloaded with ferrocene (electroactive compound) yielding a LOD of 1 pg mL-1. MOFs have been previously used for signal amplification in the detection of other glycoproteins such as recombinant human erythropoietin with excellent LODs (pg mL-1 range) [69]”.
Comment 4: The general ways for the immobilization and recognition of biomolecules include covalent coupling reactions and non-covalent interactions of antigen–antibody, aptamer–target, glycan–lectin, avidin–biotin and boronic acid–diol. Besides, nitrilotriacetic acid-metal complexes for the immobilization and recognition of biomolecules should be added.
Response 4: Thank you for your comment, but we partially agree with it. The general ways for the immobilization and recognition of biomolecules is a very important topic, but it has been previously discussed and reviewed in a lot of reviews ([1] https://doi.org/10.1016/j.aca.2023.342044; [2] DOI: https://doi.org/10.1039/D0AN01410A; [3] https://doi.org/10.3390/s16122045; [4] https://doi.org/10.1016/j.aca.2024.343277; [5] https://doi.org/10.3390/molecules27238533; [6] https://doi.org/10.1016/j.colsurfb.2021.112148) and book chapters (Advances in Clinical Chemistry, Volume 93, 2019 Elsevier Inc. ISSN 0065-2423 https://doi.org/10.1016/bs.acc.2019.07.001). We do not intend to divert the focus of this review article, addressing topics that have been widely discussed and commented on previously. We aim to review articles related to the evaluation of glycosylation status or level of biological compounds by electrochemical methods, highlighting the singular and specific strategies developed to achieve this goal. The ways and mechanism of immobilization of biorecognizing elements and the interaction between them and the target analyte is very general and common to any sensor (optical, electrochemical, piezoelectric…).
In any case, agreeing with you that it is a very relevant topic, we have decided to add a paragraph indicating a series of review articles and book chapters related to the topic. Below is said paragraph:
“On the other hand, there are multiple reviews focused on the biorecognition events used for the detection of glycoproteins, but none summarizes the use of this recognition event to evaluate the glycosylation status/level. Therefore, this review does not describe the general ways for the immobilization of biorecognition elements (covalent and non-covalent) and for the recognition of biomolecules (biorecognition element/analyte interaction). Interested readers are invited to check the following reviews and book chapters [13, 16-21]”.
Comment 5: For boronic acid derivatives-based strategies, boronic acid-functionalized nanomaterials for the design of electrochemical biosensors, and biosensors with boronic acid-based materials as the recognition elements and signal labels should be added and discussed.
Response 5: Thank you for the comment. We consider that the explanation of general strategies to develop boronic acid-based sensors is out of the scope of this review. As it was aforementioned, boronic acid-based sensor topic has been exhaustively reviewed in the literature, there is even a specific review about this topic (Boronate affinity material-based sensors for recognition and detection of glycoprotein, Analyst, 2020,145, 7511-7527, https://doi.org/10.1039/D0AN01410A), which is cited in our review (ref. 72). In this review, we aim to survey electrochemical strategies applied to the assessment of glycosylation level or status of a specific glycoprotein or cell, trying to fulfill a gap found in literature. We briefly commented on chemistry interaction between boronic acid derivatives and diols group from glycans (section 2.3), but we are interested in the singular and specific strategies developed to measure on the same sensor/approach two signals: (i) the specific glycoform/glycan and (ii) the total glycoprotein/cell content. To our best knowledge, this topic was not reviewed until now.
Anyway, as stated in response 4, we added a short paragraph suggesting relevant references about this topic.
“On the other hand, there are multiple reviews focused on the biorecognition events used for the detection of glycoproteins, but none summarizes the use of this recognition event to evaluate the glycosylation status/level. Therefore, this review does not describe the general ways for the immobilization of biorecognition elements (covalent and non-covalent) and for the recognition of biomolecules (biorecognition element/analyte interaction). Interested readers are invited to check the following reviews and book chapters [13, 16-21]”.
Comment 6: The subtitles should be revised with more detailed information, such as 3.4.
Response 6: Thank you for the comment. The information of subtitles 2.3 and 3.4 was extended to make it easier for readers to know the content. Now they are as follows:
2.3. General discussion about the strategies for the evaluation of glycosylation degree of glycoconjugates.
3.4. General discussion about the assessment of glycosylation in biological structures.
Comment 7: A scheme can be added to show the content of this review.
Response 7: We agree with your comment. A scheme showing the review content was added to the introduction along with a short description.
Reviewer 2 Report
Comments and Suggestions for AuthorsIn this article, the authors reviewed recent developments in electrochemical biosensors for evaluating aberrant glycosylation at the glycoform level—rather than mere glycoprotein concentration—highlighting sensing strategies, materials, and platforms for point-of-care diagnosis of cancer, metabolic, immune, and neurodegenerative diseases, along with current challenges and future directions. Overall, the work could be suitable for publication in Chemosensors. However, several points require attention:
- In Section 2.1.1 (Aptamer-based strategies), the authors should briefly explain the role and working principle of aptamers in electrochemical detection.
- The manuscript would benefit from a clearer discussion of signal readout mechanisms and data interpretation strategies for the different electrochemical techniques described.
- The authors state that “the second most employed strategy is the use of boronic acid derivatives as capture probes for HbA1c”. Please explain why this approach is widely used and support the statement with appropriate references.
- The statement “the limit of detection was high (below mg mL⁻¹)” is unclear and potentially contradictory; this sentence should be revised for clarity and technical accuracy.
- In Section 2.1.3 (Other strategies), the authors should first briefly introduce and define these alternative approaches before presenting specific examples.
- The quality and resolution of several figures should be improved.
- The statement that “lectins emerge as ideal biorecognition probes for identifying and capturing specific monosaccharides and glycans on the surface of cancer cells” should be further justified, with an explanation of the underlying biochemical specificity.
- While the advantages of electrochemical biosensors are discussed, the manuscript should also address their limitations and drawbacks. Relevant literature may be cited, for example: Electrochemical biosensors: The beacon for food safety and quality, Food Chemistry, 2025, 143284.
- Finally, the authors may consider briefly discussing the potential feasibility of wearable or flexible electrochemical platforms for glycosylation analysis.
Author Response
Answer to Reviewer 2
In this article, the authors reviewed recent developments in electrochemical biosensors for evaluating aberrant glycosylation at the glycoform level—rather than mere glycoprotein concentration—highlighting sensing strategies, materials, and platforms for point-of-care diagnosis of cancer, metabolic, immune, and neurodegenerative diseases, along with current challenges and future directions. Overall, the work could be suitable for publication in Chemosensors. However, several points require attention:
Response: Thank you very much for trusting in this work and for taking the time to review our manuscript. We sincerely appreciate your effort and consideration. All changes will be highlighted in yellow color in the manuscript.
Comment 1: In Section 2.1.1 (Aptamer-based strategies), the authors should briefly explain the role and working principle of aptamers in electrochemical detection.
Response 1: We are sorry, because maybe our explanations were not clear. We tried to explain the role of aptamer in the process of electrochemical detection in each article. Following your recommendation, we expanded the explanation of each article (highlighted in yellow color). We show you below one example (new words are in bold):
“The assay consisted of adding the hemolyzed blood sample on the aptasensor, so the target analytes (Hb or HbA1c) are captured by their corresponding aptamer. Then [Fe(CN)6]4−/3− solution was added acting as redox probe and square wave voltammetry (SWV) was carried out, monitoring the oxidation/reduction reaction of the redox probe. The signal from redox probe was registered before (no analyte) and after sample addition (with analyte), so the signal was expressed as the difference between both peak intensities. A decrease of peak current happens on the different electrodes when the target proteins are bound to their specific aptamer, due to the blockage of electrode surface by the bulky proteins, preventing the access of the redox probe to the electrode”.
Moreover, regarding the interaction chemistry between the aptamer and the target glycoprotein, there are a lot of reviews and books that describe this interaction and, in our opinion, this fact is well-known by potential readers (analytical chemists). Therefore, delving into this topic would greatly expand the discussion of the work, blurring the true novelty of our review work, which is none other than describing the electrochemical strategies to evaluate the glycosylation status or level of biological compounds.
Anyway, we added to section 2.1.1 a short paragraph with relevant reviews, for interested readers. See below:
“Aptamer is a very selective ligand, and it consists of a short DNA or RNA strands, which is generated using the systematic evolution of ligands by exponential enrichment (SELEX) technology. The chemistry of the aptamer-glycoprotein interaction is well known, and these recent reviews can be found for more details [17, 19]”.
Comment 2: The manuscript would benefit from a clearer discussion of signal readout mechanisms and data interpretation strategies for the different electrochemical techniques described.
Response 2: Thank you for the comment. We explained further the readout mechanism and data interpretation of the discussed articles throughout the review (highlighted in yellow color). Below we show you one of the examples from section 2.1.1. Aptamer-based strategies (new words are in bold).
“According to the authors, when the aptamer captures the protein, the distance from the ferrocene to the electrode surface is increased, so oxidation process of ferrocene is hindered (see Figure 2). This means that the current signal obtained by DPV decreases with the concentration of the analyte (inversely proportional)”
Comment 3: The authors state that “the second most employed strategy is the use of boronic acid derivatives as capture probes for HbA1c”. Please explain why this approach is widely used and support the statement with appropriate references.
Response 3: We are sorry for the misunderstanding. We meant that “the second most employed strategy is the use of boronic acid derivatives as capture probes for HbA1c” in the context of the evaluation of glycosylation status (glycoform / total glycoprotein ratio), not in a general context. We simply make this assertion based on the number of related articles we found in the bibliography. For that reason, we change the sentence as follows:
“In the context of evaluation of glycosylation status, the second most employed strategy is the use of boronic acid derivatives as capture probe for HbA1c”.
Moreover, we explained in section 2.3. the properties of boronic acid derivatives, see below:
“Boronic acid derivatives can specifically react with cis-diols, forming five or six-membered cyclic esters. This binding is reversible and pH dependent [70]. They were used as capture [28, 30] and detection probe [31] for the selective quantification of HbA1c.”
Comment 4: The statement “the limit of detection was high (below mg mL⁻¹)” is unclear and potentially contradictory; this sentence should be revised for clarity and technical accuracy.
Response 4: We agree with you. We changed the sentence as follows: “the limit of detection was in the mg mL-1 range, but it…”
Comment 5: In Section 2.1.3 (Other strategies), the authors should first briefly introduce and define these alternative approaches before presenting specific examples.
Response 5: Thanks for your comment. This section is actually about a glycoprotein biomarker other than hemoglobin (Hb) for diabetes management (gHSA) and the different electrochemical sensors used to measure it. Therefore, we have modified subheading 2.1.3, which is now "2.1.3 Other strategies for diabetes management." We have also added an introductory paragraph to explain the meaning of "Other strategies". The sentence is as follows:
“As has been observed in the previous subsections, the main glycoprotein biomarker for the diagnosis and prognosis of diabetes is HbA1c. However, there is a potential biomarker that is gaining recognition as a useful alternative or complementary test in specific clinical situations: glycated human serum albumin (gHSA).
Comment 6: The quality and resolution of several figures should be improved.
Response 6: Thank you for your comment, new figures have been added to manuscript at 300dpi resolution, however limitations derived from original images resolution cannot be solve on our part.
Comment 7: The statement that “lectins emerge as ideal biorecognition probes for identifying and capturing specific monosaccharides and glycans on the surface of cancer cells” should be further justified, with an explanation of the underlying biochemical specificity.
Response 7: Thank you for this insightful comment. We added to section 3.1 two interesting references about the biochemical specificity and the importance of lectins in oncology field (new references 77 and 78). In addition, a proper paragraph was introduced in this section, describing the origin of specificity of lectins towards glycans.
“In this sense, it seems that lectins emerge as ideal biorecognizing probe for identifying and capturing specific monosaccharides and glycans on the surface of cancer cells [82]. Lec-tins can distinguish fine structural differences in glycans (branching patterns, linkages (α/β), terminal epitopes, and monosaccharide composition). The lectin–glycan binding depends on highly defined physicochemical and geometric features within lectin binding sites, including pocket depth, charge, hydrophobicity, and aromatic stacking. These features create highly selective interactions with particular glycan motifs (e.g., sialylated, high mannose, branched N glycans) [83].”
Comment 8: While the advantages of electrochemical biosensors are discussed, the manuscript should also address their limitations and drawbacks. Relevant literature may be cited, for example: Electrochemical biosensors: The beacon for food safety and quality, Food Chemistry, 2025, 143284.
Response 8: We partially agree with you. Throughout the review, drawbacks and limitations of each article were commented. Even in the conclusion section, it is pointed out the main concern about the use of these sensors in clinical practice: lack of demonstration of their utility in the analysis of real-world samples, including clinical validations to define their sensitivity and specificity for disease diagnosis.
However, the general drawbacks of electrochemical biosensors were not commented on in the introduction section, so we add a short paragraph including the suggested reference:
“However, there are still several concerns for the transference to the industry related to long-term stability and the user-friendliness [14, 15]”.
Comment 9: Finally, the authors may consider briefly discussing the potential feasibility of wearable or flexible electrochemical platforms for glycosylation analysis.
Response 9: Thank you for your suggestion. In our research group, we have given this topic considerable thought. In our opinion, the development of wearables for the continuous monitoring of glycoproteins or their glycosylation levels for the diseases discussed in our review does not seem very relevant, as these are analytes whose concentration varies very slowly over time. However, the possibility of developing a sensor that allows for continuous detection of the appearance of circulating tumor cells is a highly relevant application with enormous potential. Therefore, we have added a brief comment to the conclusions, as shown below.
“In fact, microfluidics combined with electrochemical sensors offers the possibility of developing wearable devices that, who knows, could continuously monitor the release of CTCs in the bloodstream in the near future.”
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsAccept in present form
Reviewer 2 Report
Comments and Suggestions for AuthorsAccept it.
