Electrochemical Biosensors Based on Conducting Polymers: A Review

Round 1

Reviewer 1 Report

The manuscript "Electrochemical biosensors based on conducting polymers: A review"  is well written and represents an overvieiw of the conducting polymers based biosensors, from their begining to the recent  trends in this field.

In order to improve the quality and the clarity of the manuscript I suggest the followings:

1. -correction/reformulation in/of the sentences from the below:

"Conducting polymers is a class of materials that..."

"...The potentiostatic techniques allow easy control of..."

"...when immobilization of oxidoreductases is preferentially done through..."

"Sometimes, the immobilization of the biomolecule can be facilitated by..."

"The presence of the conducting polymer aims at improving sensitivity and selectivity of the biosensor while..."

"...is mainly responsible from the long‐term efficiency..."

"The sensitivity of the biosensor 169 depends from..."

"However, these biosensors often suffer from their lack of selectivity since anything changing the..."

"Similarly, the detection of serotonin in banana was done by square wave voltammetry using conducting polypyrrole/Fe3O4 nanocomposites [87] when the detection of danazol was performed by differential pulse voltammetry using conducting electrodeposited polyaniline"

"In impedimetric immunosensors biosensors..."

"As a consequence many biosensors have been prepared until now that use conducting..."

"is applied for redox of..."

"This biosensor exhibited no interference with ascorbic acid and..."

"...as well as a satisfactory selectively since glucose"

"Figure 4. (a) Schematic of the biosensor elaboration"

"Figure 5. Schematic of the protocol for"

 

  2. In the sentence "The same group developed anotherimmunosensor for the
detection of carcinoma antigen‐125 (CA 125)" it is actually a different research team than that from reference [117].

 

  3. According to the manuscript, in the References list, the reference [125] should be replaced by [126] and [126] will become [125].

 

   4. A comparison between the analytical performances of different types of biosensors (enzymatic, immuno-, DNA and whole cells biosensors) in a table will provide conclusive evidences on their application.

 

Author Response

Reviewer 1 :

The manuscript "Electrochemical biosensors based on conducting polymers: A review"  is well written and represents an overview of the conducting polymers based biosensors, from their beginning to the recent trends in this field. à Thank you.

In order to improve the quality and the clarity of the manuscript I suggest the followings:

1. -correction/reformulation in/of the sentences from the below:

"Conducting polymers is a class of materials that..." done

"...The potentiostatic techniques allow easy control of..." done

"...when immobilization of oxidoreductases is preferentially done through..." done

"Sometimes, the immobilization of the biomolecule can be facilitated by..." done

"The presence of the conducting polymer aims at improving sensitivity and selectivity of the biosensor while..." done

"...is mainly responsible from the long‐term efficiency..." done

"The sensitivity of the biosensor 169 depends from..." done

"However, these biosensors often suffer from their lack of selectivity since anything changing the..." done

"Similarly, the detection of serotonin in banana was done by square wave voltammetry using conducting polypyrrole/Fe3O4 nanocomposites [87] when the detection of danazol was performed by differential pulse voltammetry using conducting electrodeposited polyaniline"à done

"In impedimetric immunosensors biosensors..." done

"As a consequence many biosensors have been prepared until now that use conducting..." done

"is applied for redox of..." done

"This biosensor exhibited no interference with ascorbic acid and..." done

"...as well as a satisfactory selectively since glucose" done

"Figure 4. (a) Schematic of the biosensor elaboration" done

"Figure 5. Schematic of the protocol for" done

2. In the sentence "The same group developed another immunosensor for the detection of carcinoma antigen‐125 (CA 125)" it is actually a different research team than that from reference [117]. This error has been corrected.
3. According to the manuscript, in the References list, the reference [125] should be replaced by [126] and [126] will become [125]. This error has been corrected.
4. A comparison between the analytical performances of different types of biosensors (enzymatic, immuno-, DNA and whole cells biosensors) in a table will provide conclusive evidences on their application. As suggested by the reviewer, additional Tables comparing the analytical performances of the different biosensors have been added (Tables 1, 2 and 3).

Reviewer 2 Report

The manuscript is interesting,but has many grammatical errors in the current form that must be fixed and also requires additional work before the manuscript can be accepted for publication. The author is required to address the following:

1. Line 17, change “impedancemetry” to “impedometry”.
2. Line 18, change “involved” to “used”.
3. Line 48, the use of “whose” in referring to charge carriers is inappropriate, change to “their”.
4. Lines 77-81, the description of the common electropolymerisation methods should include potentiostatic, potentiodynamic and galvanostatic methods. You have mentioned the first two, but not the third in the obvious way it should be mentined. Your description of “chronopotentiometry where the current is kept constant with a variation of potential with time” come close to describing this approach not in the direct way it should be described. In galvanostatic polymerisation, a constant current is applied to initiate polymerisation and as more conducting film is grown the potential changes with time to reflect the conductivity.
5. Lines 81-83 should also include comments about galvanostatic polymerisation which considered to be better than the other two methods.
6. Line 97, the statement “a large amount of biomolecules must be directly attached” is incorrect. Several studies have shown that electroimmobilisation into conducting polymers permit the use of less biomolecules in biosensors.
7. Line 112, you need to define “GOX” as glucose oxidase (GOx).
8. Line 128, change “glucose oxidase (GOX)” to “GOx” if already defined on line 112.
9. Line 137, change “consists in the preparation” to “involves the preparation”.
10. Line 147, change “It can also be noticed” to “It is also important to note”.
11. Line 151, change “can be” to “is”.
12. Line 154, change “use original methods such as sinusoidal voltages” to “use sinusoidal voltages”. Describing this as original methods is confusing.
13. Line 164, change “aims at” to “is beneficial for”.
14. Line 168, change “from” to “for”.
15. Line 169, change “from” to “on”.
16. Line 234, change “when” to “and”.
17. Line 236, change “impedancemetry” to “impedometry”.
18. Lines 240, 242, 245, change “impedimetric) to “impedometric”.
19. Line 245, change “immunosensors biosensors” to “immunosensors”.
20. Line 261, as there is only one author for the manuscript, change “Our objective” to “My objective”.
21. Line 273, change “by” to “with”.
22. Line 276, insert “and” before “Os‐complex”.
23. Line 280, change “from” to “for”.
24. Line 308, Is “0.2 V vs. Ag” correct? Or should it be “0.2 V vs. Ag/AgCl”?
25. Lines 312-317, what is the importance of Figure 3? Discuss or delete.
26. Lines 353-354, change “as compared to most of the classical enzyme glucose sensors fabricated [114]” to “compared to most of the previously reported classical enzyme glucose sensors [114]”.
27. Line 369, change “impedimetric” to “impedometric”.
28. Line 380, change “impedimetric” to “impedometric”.
29. Line 387, change “so generate a production of H2O2” to “so generate H2O2”.
30. Line 396, delete “Indeed,”, it is unnecessary.
31. Lines 398 & 400, change “impedimetric” to “impedometric”.
32. Line 420, the discussion of MIP should start as a new paragraph.
33. Line 426, the term “last years” is unacceptable. It needs to be more specific, eg last five years or recent years.
34. Line 448, it is not correct to say “incorporated in DNA biosensors” better to say “used for fabrication of DNA biosensors”.
35. Line 459, change “it exists some works” to “some works exist”.
36. Lines 480 & 482, change “impedimetric” to “impedometric”.
37. Line 482, better to change “as done” to “as reported”.
38. Line 488, change “consisted in nanotubes” to “consisted of nanotubes”.
39. Lines 510-511, change “allowed to detect selectively” to “allowed selective detection of”.
40. Line 525, delete “over them”, it is not necessary.
41. Line 537, delete “be”, it is not necessary.
42. Line 546, change “allowed to decrease the cost” to “enabled a reduction in the cost”.
43. Line 547, change “to improve” to “improvement of”.
44. Line 554, change “with the glucose” to “to the glucose”.
45. Line 555, change “authors” to “researchers”.
46. Line 562, change “has been made to present” to “presents”.
47. Line 563, change “to outline” to “outlines”.
48. Line 566, change “impedimetric” to “impedometric”.
49. Line 568, change “mostly used” to “most commonly used”.
50. Line 569, change “even if adsorption or entrapment are also used” to “but adsorption and entrapment are also often used”.
51. Line 571, delete the term “handy” as it is not a scientific term; change “stable, handy sensing” to “and stable sensing”.
52. Authors should further improve manuscript by including a table each in sections 4.1 – 4.4 summarising the range and performances (including biomolecules/enzyme used, mode of detection, linear concentration range, sensitivity, detection limit, application to real samples, etc) of the biosensors that have been reported under each category.

Author Response

The manuscript is interesting, but has many grammatical errors in the current form that must be fixed and also requires additional work before the manuscript can be accepted for publication. The author is required to address the following:

1. Line 17, change “impedancemetry” to “impedometry”. done
2. Line 18, change “involved” to “used”. done
3. Line 48, the use of “whose” in referring to charge carriers is inappropriate, change to “their”. done
4. Lines 77-81, the description of the common electropolymerisation methods should include potentiostatic, potentiodynamic and galvanostatic methods. You have mentioned the first two, but not the third in the obvious way it should be mentioned. Your description of “chronopotentiometry where the current is kept constant with a variation of potential with time” come close to describing this approach not in the direct way it should be described. In galvanostatic polymerisation, a constant current is applied to initiate polymerisation and as more conducting film is grown the potential changes with time to reflect the conductivity. done
5. Lines 81-83 should also include comments about galvanostatic polymerisation which considered to be better than the other two methods. done
6. Line 97, the statement “a large amount of biomolecules must be directly attached” is incorrect. Several studies have shown that electroimmobilisation into conducting polymers permit the use of less biomolecules in biosensors. done
7. Line 112, you need to define “GOX” as glucose oxidase (GOx). done
8. Line 128, change “glucose oxidase (GOX)” to “GOx” if already defined on line 112. done
9. Line 137, change “consists in the preparation” to “involves the preparation”. done
10. Line 147, change “It can also be noticed” to “It is also important to note”. done
11. Line 151, change “can be” to “is”. done
12. Line 154, change “use original methods such as sinusoidal voltages” to “use sinusoidal voltages”. Describing this as original methods is confusing. done
13. Line 164, change “aims at” to “is beneficial for”. done
14. Line 168, change “from” to “for”. done
15. Line 169, change “from” to “on”. done
16. Line 234, change “when” to “and”. done
17. Line 236, change “impedancemetry” to “impedometry”. done
18. Lines 240, 242, 245, change “impedimetric) to “impedometric”. done
19. Line 245, change “immunosensors biosensors” to “immunosensors”. done
20. Line 261, as there is only one author for the manuscript, change “Our objective” to “My objective”. done
21. Line 273, change “by” to “with”. done
22. Line 276, insert “and” before “Os‐complex”. done
23. Line 280, change “from” to “for”. done
24. Line 308, Is “0.2 V vs. Ag” correct? Or should it be “0.2 V vs. Ag/AgCl”? done
25. Lines 312-317, what is the importance of Figure 3? Discuss or delete. I think that the presence of Figure 3 is important to give an example of results obtained with a voltammetric biosensor. This Figure is discussed from line 304 to line 312.
26. Lines 353-354, change “as compared to most of the classical enzyme glucose sensors fabricated [114]” to “compared to most of the previously reported classical enzyme glucose sensors [114]”. done
27. Line 369, change “impedimetric” to “impedometric”. done
28. Line 380, change “impedimetric” to “impedometric”. done
29. Line 387, change “so generate a production of H2O2” to “so generate H2O2”. done
30. Line 396, delete “Indeed,”, it is unnecessary. done
31. Lines 398 & 400, change “impedimetric” to “impedometric”. done
32. Line 420, the discussion of MIP should start as a new paragraph. done
33. Line 426, the term “last years” is unacceptable. It needs to be more specific, eg last five years or recent years. done
34. Line 448, it is not correct to say “incorporated in DNA biosensors” better to say “used for fabrication of DNA biosensors”. done
35. Line 459, change “it exists some works” to “some works exist”. done
36. Lines 480 & 482, change “impedimetric” to “impedometric”. done
37. Line 482, better to change “as done” to “as reported”. done
38. Line 488, change “consisted in nanotubes” to “consisted of nanotubes”. done
39. Lines 510-511, change “allowed to detect selectively” to “allowed selective detection of”. done
40. Line 525, delete “over them”, it is not necessary. done
41. Line 537, delete “be”, it is not necessary. done
42. Line 546, change “allowed to decrease the cost” to “enabled a reduction in the cost”. done
43. Line 547, change “to improve” to “improvement of”. done
44. Line 554, change “with the glucose” to “to the glucose”. done
45. Line 555, change “authors” to “researchers”. done
46. Line 562, change “has been made to present” to “presents”. done
47. Line 563, change “to outline” to “outlines”. done
48. Line 566, change “impedimetric” to “impedometric”. done
49. Line 568, change “mostly used” to “most commonly used”. done
50. Line 569, change “even if adsorption or entrapment are also used” to “but adsorption and entrapment are also often used”. done
51. Line 571, delete the term “handy” as it is not a scientific term; change “stable, handy sensing” to “and stable sensing”. done
52. Authors should further improve manuscript by including a table each in sections 4.1 – 4.4 summarising the range and performances (including biomolecules/enzyme used, mode of detection, linear concentration range, sensitivity, detection limit, application to real samples, etc) of the biosensors that have been reported under each category.

As suggested by the reviewer, additional Tables summarizing the analytical performances of the different biosensors have been added: Table 1 comparing the different glucose enzymatic biosensors, Table 2 comparing the different immunosensors, and Table 3 comparing the different DNA biosensors. No Table related to whole cell biosensors has been added since we believe that these sensors are not comparable since there are too many differences between each other (mode of detection, nature of the biological cell, nature of the sensitive layer,…).

Reviewer 3 Report

This review covers broad diapason of conducting polymer application areas therefore it will receive very broad interest from scientific auditorium and it is recommended for publication after some minor revisions and improvements:

Some conducting polymers such as polypyrrole are especially suitable for the development of molecularly imprinted polymer based sensor, therefore, chapter which is addressing development and application of molecularly imprinted conducting polymers should be extended by disscusing some additional important publications in this area (Characterization of Caffeine-Imprinted Polypyrrole by a Quartz Crystal Microbalance and Electrochemical Impedance Spectroscopy. Sensors and Actuators B-Chemical 2015, 212, 63-71. which should be overviewed and discussed in the chapter which is related to development analytical application of molecularly imprinted polymers.

Author Response

This review covers broad diapason of conducting polymer application areas therefore it will receive very broad interest from scientific auditorium and it is recommended for publication after some minor revisions and improvements: Thank you.

Some conducting polymers such as polypyrrole are especially suitable for the development of molecularly imprinted polymer based sensor, therefore, chapter which is addressing development and application of molecularly imprinted conducting polymers should be extended by discussing some additional important publications in this area (Characterization of Caffeine-Imprinted Polypyrrole by a Quartz Crystal Microbalance and Electrochemical Impedance Spectroscopy. Sensors and Actuators B-Chemical 2015, 212, 63-71. which should be overviewed and discussed in the chapter which is related to development analytical application of molecularly imprinted polymers.

As suggested by the reviewer, a section dedicated to molecularly imprinted polymers has been added to the manuscript. In particular, this reference has been added as well as several works dedicated to molecularly imprinted polypyrrole.

Reviewer 4 Report

Review manuscript ‘Electrochemical biosensors based on conducting polymers: A review’ is very beneficial (preparation, methods, useful overviews) and reviews some important researches in the area of application of conducting polymers. This review has every opportunity to have an interest of a very large scientific auditorium.

Some minor addition, improvements are recommended in order to improve this Review:

• Part, which describes applicability of conducting polymers in amperometric sensors could be extended taking more significant attention to possible involvement of conducting polymers in direct charge transfer from redox enzymes to electrode, e.g. PPy can be involved into direct electron transfer from heme-dependet PQQ dehydrogenase, electrochemically generated polyphenanthroline and some Carbazole derivatives can transfer charge from glucose oxidase, Some recent investigations on interaction of PPy with active site of glucose oxidase (FAD) can be also overviewed and discussed etc.
• Part, which describes development of immunosensors could be extended/improved by taking considerable attention to molecularly imprinted polymers (MIPs) based on conducting polymer – polypyrrole. MIPs-based affinity sensors are very suitable for the determination of various compounds. Conducting polymer – polypyrrole -  is very suitable for the development of MIPs towards various compounds due to easy over-oxidation of formed polypyrrole layer and in such way the formation of carboxylic, hydroxylic and other oxidized groups, which can be involved into molecular recognition of imprinted analyte. Some additional references in this area could be added in order to support great applicability of this conducting polymer in MIPs based electrochemical sensors.

The review is structured and clear, responds interest of electrochemical biosensors. The manuscript can be published, although after some minor additions (recommendation) it could be improved greatly.

Author Response

Review manuscript ‘Electrochemical biosensors based on conducting polymers: A review’ is very beneficial (preparation, methods, useful overviews) and reviews some important researches in the area of application of conducting polymers. This review has every opportunity to have an interest of a very large scientific auditorium. Thank you.

Some minor addition, improvements are recommended in order to improve this Review:

• Part, which describes applicability of conducting polymers in amperometric sensors could be extended taking more significant attention to possible involvement of conducting polymers in direct charge transfer from redox enzymes to electrode, e.g. PPy can be involved into direct electron transfer from heme-dependet PQQ dehydrogenase, electrochemically generated polyphenanthroline and some Carbazole derivatives can transfer charge from glucose oxidase. Some recent investigations on interaction of PPy with active site of glucose oxidase (FAD) can be also overviewed and discussed etc.

As suggested by the reviewer, a paragraph dedicated to biosensors for which a direct charge transfer takes place from redox enzymes to electrode has been added to the manuscript.

Additional paragraph: “An ultimate goal of the biosensors is to eliminate the usage of the mediator toward lower fabrication cost and complexity while increasing the durability of the biosensor. Therefore, the third-generation biosensors based on the direct electron transfer from immobilized enzyme to the working electrode are a more progressive type of sensor. Such direct electron transfer have been evidenced from redox enzymes to electrode in conducting polymer-based biosensors by Ramanivicius et al. who reported for the first time that direct electron-transfer processes between a polypyrrole entrapped quinohemoprotein alcohol dehydrogenase from Gluconobacter sp. 33 and a platinum electrode take place via the conducting-polymer network [115]. The cooperative action of the enzyme-integrated prosthetic groups is assumed to allow this electron-transfer pathway from the enzyme’s active site to the conducting-polymer backbone. This electron-transfer pathway leads to a significantly increased linear detection range of an ethanol sensor. Since this work, dehydrogenase based bioelectrocatalysis has been increasingly exploited in order to develop electrochemical biosensors with improved performances since dehydrogeases are able to directly exchange electrons with an appropriately designed electrode surface, without the need for an added redox mediator, allowing bioelectrocatalysis based on a direct electron transfer process [116]. A direct electron transfer van also occur from immobilized glucose oxidase via grafted and electropolymerized 1,10-phenanthroline [117]. Such polymer-modified biosensor showed superior electron transfer to/from flavine adenine dinucleotide cofactor of GOx as well as an excellent selectivity towards glucose and a good operational-stability. Similarly, a biosensor based on electrodeposited polycarbazole was fabricated and exhibited good electrocatalytic activity toward enzymatic glucose sensors with a high sensitivity, a wide linear range of detection up to 5 mM due to direct electrode transfer from the enzyme to electrode and direct glucose oxidation on the electrode [118].”

 

[115] Ramanavicius, A.; Habermüller, K.; Csöregi, E.; Laurinavicius, V.; Schuhmann, W. Polypyrrole-Entrapped Quinohemoprotein Alcohol Dehydrogenase. Evidence for Direct Electron Transfer via Conducting-Polymer Chains. Anal. Chem. 1999, 71, 3581-3586

[116] Bollela, P.; Gorton, L.; Antiochia, R. Direct Electron Transfer of Dehydrogenases for Development of 3rd Generation Biosensors and Enzymatic Fuel Cells. Sensors 2018, 18, 1319.

[117] Oztekin, Y.; Ramanaviciene, A.; Yazicigil, Z.; Solak, A.O.; Ramanavicius, A. Direct electron transfer from glucose oxidase immobilized on polyphenanthroline-modified glassy carbon electrode. Biosens. Bioelectron. 2011, 26, 2541-2546.

[118] Bagdziunas, G.; Palinauskas, D. Poly(9H-carbazole) as a Organic Semiconductor for Enzymatic and Non-Enzymatic Glucose Sensors. Biosensors 2020, 10, 104.

• Part, which describes development of immunosensors could be extended/improved by taking considerable attention to molecularly imprinted polymers (MIPs) based on conducting polymer – polypyrrole. MIPs-based affinity sensors are very suitable for the determination of various compounds. Conducting polymer – polypyrrole -  is very suitable for the development of MIPs towards various compounds due to easy over-oxidation of formed polypyrrole layer and in such way the formation of carboxylic, hydroxylic and other oxidized groups, which can be involved into molecular recognition of imprinted analyte. Some additional references in this area could be added in order to support great applicability of this conducting polymer in MIPs based electrochemical sensors.

As suggested by the reviewer, a section dedicated to molecularly imprinted polymers has been added to the manuscript. In particular, this reference has been added as well as several works dedicated to molecularly imprinted polypyrrole.

Round 2

Reviewer 2 Report

The authors have addressed most of the issues I raised previously, but the following issues must also be addressed before acceptance for publication:

1. Line 80, delete “the current” from the sentence “….where the current a constant current …”
2. Line 84, change “one” to “approach”.
3. Line 290, change “doesn’t exhibited” to “does not exhibit”.
4. Line 355, change “toward” to “to”.
5. Line 370, change “van” to “can”.
6. Line 376, change “direct electrode” to “direct electron”.
7. Line 378, change “Table 1. Comparison of glucose amperometric biosensor performances.” to “Table 1. Comparison of conducting polymer-based glucose amperometric biosensor performances”
8. Line 444, change “Table 2. Comparison of immunosensor performances” to “Table 2. Comparison of conducting polymer-based immunosensor performances”
9. Line 526, change “Table 3. Comparison of DNA biosensor performances.” to “Table 3. Comparison of conducting polymer-based DNA biosensor performances.”
10. It is good that the author has included 3 new Tables, but no references were made to the Tables. Key features of each Table should be discussed. At least, there should be a statement that “Table 1 summarises the performances ……”, “Table 2 summarises the performances ……”, “Table 3 summarises the performances ……”

Author Response

The authors have addressed most of the issues I raised previously, but the following issues must also be addressed before acceptance for publication:

1. Line 80, delete “the current” from the sentence “….where the current a constant current …”  done
2. Line 84, change “one” to “approach”.  done
3. Line 290, change “doesn’t exhibited” to “does not exhibit”.  done
4. Line 355, change “toward” to “to”.  done
5. Line 370, change “van” to “can”.  done
6. Line 376, change “direct electrode” to “direct electron”.  done
7. Line 378, change “Table 1. Comparison of glucose amperometric biosensor performances.” to “Table 1. Comparison of conducting polymer-based glucose amperometric biosensor performances”  done
8. Line 444, change “Table 2. Comparison of immunosensor performances” to “Table 2. Comparison of conducting polymer-based immunosensor performances”  done
9. Line 526, change “Table 3. Comparison of DNA biosensor performances.” to “Table 3. Comparison of conducting polymer-based DNA biosensor performances.”  done
10. It is good that the author has included 3 new Tables, but no references were made to the Tables. Key features of each Table should be discussed. At least, there should be a statement that “Table 1 summarises the performances ……”, “Table 2 summarises the performances ……”, “Table 3 summarises the performances ……”  done