Electrochemical Immuno- and Aptasensors for Mycotoxin Determination
Round 1
Reviewer 1 Report
The authors reviewed recent technologies in immuno- and aptasensors for mycotoxin detection. The references are new and the manuscript provided the summary and discussion from the state of the recent technologies.
However, some part of the manuscript dealt with too much basic information. For example, in the part of “3.1. Immunosensors – General Principles and Ab Immobilization”, the principles of immobilization provided too many details so that the readers may fall into the other points. Could the authors reduce the length of the manuscript?
Overall, the manuscript provides useful information but is publishable in Chemosensors after the revision.
Author Response
In accordance with the opinion of esteemed Reviewer 1, the length of part 3.1 was reduced about twice in part related to immobilization principles.
Reviewer 2 Report
The manuscript reports a selection of methods to determine the presence and concentration of mycotoxins in vegetable or food samples. The author reports both classical and biosensor-based methods based on either antibodies (or fragments) or DNA aptamers.
The topic is very relevant and this work provide a useful contribute to the field. The structure of the manuscript is generally clear and some topics are explained in detail. However, Some parts of the manuscript are not detailed enough or need a revision to properly address the topic. Overall, I support the publication of a revised version of this work that addresses the following points:
- The manuscript only describes label free methods of the electro-chemical type. For a more consistent overview, the author should briefly mention the development on optical methods for food analysis (see for example 10.1515/nanoph-2016-0158 and 10.1016/j.snb.2018.03.004 0925-4005).
- The introduction to aptamers in Section 4.1 is misleading and incomplete. The description based only on G4 quadruplex (Figure 12) is not appropriate in this context, and unclear anyway. This part needs a thorough revision.
- In general, Section 4 (Aptasensors) is rather incomplete and does not properly sample the fields. It contains a single subsection: 4.1. Please, revise.
- The part at lines 526-544 at page 22, is not related to aptasensors, although it falls under Section 4.1. Please, correct.
- In all the tables, the LOD values are reported in many different units of measure. Please, try to use as few unit of measure as possible, in order to facilitate the comparison.
- In figure 3B, it look that some of the antibodies are binding the antigen by the Fc side. Please, correct.
- The manuscript needs to be revised for proper use of English language at least in some paragraph (see for instance page 6, lines 222-230).
Author Response
1. The manuscript only describes label free methods of the electro-chemical type. For a more consistent overview, the author should briefly mention the development on optical methods for food analysis (see for example 10.1515/nanoph-2016-0158 and 10.1016/j.snb.2018.03.004 0925-4005).
The methods mentioned by Reviewer 2 has been added to the Conclusion briefly mentioned in Conclusions as follows:
It should be also mentioned, that other than electrochemical transducers are successfully used for mycotoxin determination. They offer various opportunities both in the measurement mode and reliability of the response [179, 180] especially in analysis of food and biological tissues. Although they are considered as a tool for quantification of aptamer – mycotoxin interaction [15] and seem less appropriate for design of portable (bio)sensors, it can be expected that the gap between the electrochemical and optical techniques disappears in the nearest future. Most progress can be related to the use of quantum dots and SERS techniques together with new types of nanomaterials like reduced graphene oxide and metal-organo frameworks.
A brief description of optical sensors has been also added to the Section 3 in accordance with the Reviewer 3 comment.
2. The introduction to aptamers in Section 4.1 is misleading and incomplete. The description based only on G4 quadruplex (Figure 12) is not appropriate in this context, and unclear anyway. This part needs a thorough revision.
Other mechanisms of the signal generation have been presented in Section 4.2. The following text was introduced at the beginning of the Section 4.
Aptamers are synthetic DNA/RNA sequences obtained by combinatorial chemistry from the random library of oligonucleotides by affinity chromatography [120, 121]. Aptamers usually contain up to 60 nucleotides and can recognize a wide variety of targets, from small ions to cells and Ab molecules. strand. Contrary to DNA probes applied in DNA sensors intended for hybridization event detection, aptamer do not have direct analogs in nature. They are selected from random oligonucleotide libraries that can contain up to 1015 individual sequences. This is made by special protocols, among them Systematic Evolution of Ligands by Exponential Enrichment (SELEX) is mostly known [15]. Until 2007, aptamers mostly consisted of RNA, recently DNA aptamers have become dominating in analytical applications. They are stable to ward nuclease digestion, show excellent batch-to-batch reproducibility of main properties, including analyte binding, and rather low cost in mass production.
The interest to aptamers as biorecognition elements in food analysis, including mycotoxin detection, is related to their high sensitivity (dissociation constants in nanomolar range). Besides, they are easily modified to involve labels and functional groups required for aptamer immobilization and detection. In some cases, aptamer molecule can contain a long linker consisted of homonucleic sequence (oligoT as example) or lineal hydrocarbon radical (C12-C16) with terminal amino group. They allow partially suppress possible steric limitations of the reaction with the analyte molecule that take place near the electrode surface.
3. In general, Section 4 (Aptasensors) is rather incomplete and does not properly sample the fields. It contains a single subsection: 4.1. Please, revise.
Section 4.2 “4.2. Aptasensors – assembling and signal transduction” was introduced
4. The part at lines 526-544 at page 22, is not related to aptasensors, although it falls under Section 4.1. Please, correct.
Indeed, the description corresponded to the ref. [160] with pinhole aptamer hybridized with enzyme bearing auxiliary DNA sequence. The paragraph was re-written to avoid confusing
Ultra-low levels of mycotoxins to be detected dictate the choice of enzyme label. Appropriate reaction scheme [160] is similar to electrochemical ELISA technique but mostly uses alkaline phosphatase which has sensitive detection schemes based on α-naphtol oxidation (Fig. 11).
5. In all the tables, the LOD values are reported in many different units of measure. Please, try to use as few unit of measure as possible, in order to facilitate the comparison.
Most of the units used are derivatized from molar concentration (pM, fM, nM) and correspond to those mentioned in original sources.
6. In figure 3B, it looks that some of the antibodies are binding the antigen by the Fc side. Please, correct.
The Figure was corrected as required.
7. The manuscript needs to be revised for proper use of English language at least in some paragraph (see for instance page 6, lines 222-230).
English language was revised as requested.
Reviewer 3 Report
The manuscript is interesting and reviews electrochemical immune and aptasensors for mycotoxins detection which is a quite important topic. It is well organized,easy to read, and provides useful information for the readers.
I have two main concerns:
1) the title of the manuscript " Immuno- and Aptasensors for Mycotoxin Determination" is a bit misleading since it reviews specifically electrochemical sensors. Therefore I suggest to change the title to " Electrochemical Immuno- and Aptasensors for Mycotoxin Determination". In addition it should be clearly stated in the abstract.
2) Although other methods for mycotoxin determination (chromatography, ELISA) are included and explained in brief, different types of sensors, i.e., optical, QCM etc., are not mentioned at all. I consider that a short part of the text in 3.1 section, prior to the specifics for antibody immobilization, should be devoted to immuno- and aptasensors based on different transducers (optical, QCM) than electrochemical, since considerable research has been published recently (such as the reports: Liu et al., Sensors 2018, 18(6), 1769; Karczmarczyk et al., Talanta 2017 166, 193; Pagkali et al., J. Hazard. Mat. 2017, 323, 75; Nabok et al., Sensors and Actuators, B: Chemical 2017, 247, 975; Al-Rubaye et al., Sensors and Actuators, B: Chemical 2018, 263, 248; Al-Rubaye et al., Toxins 2018, 10 ,291; Tsounidi et al., Sensors and Actuators, B: Chemical 2019, 282, 104; Pagkali et al., J. Hazard. Mat. 2018, 359, 445-453, just to mention few of them.
Based on the above, I would like the authors to respond positively to the comments and I suggest acceptance of the manuscript after minor revision.
Author Response
1) the title of the manuscript " Immuno- and Aptasensors for Mycotoxin Determination" is a bit misleading since it reviews specifically electrochemical sensors. Therefore I suggest to change the title to " Electrochemical Immuno- and Aptasensors for Mycotoxin Determination". In addition it should be clearly stated in the abstract.
We agree with this comment, though electrochemical aptasensors have been mentioned at the end of Introduction section as main topic of the review. The following title is suggested:
Electrochemical Immuno- and Aptasensors for Mycotoxin Determination
Also, second sentence of the Abstract was modified as follows (“electrochemical” term added):
In this review, electrochemical biosensors based on immunological reactions and aptamers are considered in the determination of mycotoxins as one of most common contaminants able to negatively affect human health.
2) Although other methods for mycotoxin determination (chromatography, ELISA) are included and explained in brief, different types of sensors, i.e., optical, QCM etc., are not mentioned at all. I consider that a short part of the text in 3.1 section, prior to the specifics for antibody immobilization, should be devoted to immuno- and aptasensors based on different transducers (optical, QCM) than electrochemical, since considerable research has been published recently (such as the reports: Liu et al., Sensors 2018, 18(6), 1769; Karczmarczyk et al., Talanta 2017 166, 193; Pagkali et al., J. Hazard. Mat. 2017, 323, 75; Nabok et al., Sensors and Actuators, B: Chemical 2017, 247, 975; Al-Rubaye et al., Sensors and Actuators, B: Chemical 2018, 263, 248; Al-Rubaye et al., Toxins 2018, 10 ,291; Tsounidi et al., Sensors and Actuators, B: Chemical 2019, 282, 104; Pagkali et al., J. Hazard. Mat. 2018, 359, 445-453, just to mention few of them
We appreciate the recommendation to make reader familiar with all of the sensors developed for mycotoxin determination. However, this is hardly possible within the review format and requires much more space. Besides, Section 3 mentioned is devoted to immunosensors and the articles recommended cover both aptasensing and immunosensing. For this reason we suggest to put very brief description of alternative sensors prior to Section 3. as the Item 2.3 (Other sensors for mycotoxin determination)
The following text was added:
Although the topic of the present review is focused on electrochemical biosensors, other principles of mycotoxin determination should be mentioned though some of them are rather far from portable design of electrochemical devices. Appropriate devices cover last five years.
Among optic sensors, monolithically integrated optoelectronic biosensor has been proposed for OTA determination in beer [55]. Broad-band Mach-Zehnder interferometer was produced on silicon chip (37 mm2) with one arm coated with immobilized Ab. Competitive immunoassay made it possible to detect down to 2.0 ng/mL with recoveries ranging from 90.6 to 116%. Similar design was used in polarization reflection ellipsometry based on silicon wafers consisted of Si3N4 – SiO2 sandwich [56]. Immobilization of monoclonal Ab against AFB1 allowed detecting 0.01 ng/ml of the analyte by shift of refractive index. Total internal reflectance ellipsometry has been applied for detection of OTA bonded to specific aptamers immobilized on gold coated glass slices [57]. Similar technique was exploited here for detection of AFB1 and AFM1 with specific aptamers immobilized on the surface of golden films [58]. Method proposed was limited in detection of mycotoxins with 0.01 ng/mL level and was successfully applied for investigation of binding kinetics and calculation of binding constants.
A miniaturized optical immunosensor based on White Light Reflectance Spectroscopy (WLRS) has been described for sensitive detection of AFM1 in milk [59]. Silicon chip with a SiO2 layer on top was covered with AFM1-BSA conjugate. Indirect competitive immunoassay allowed detecting 6 pg/mL of the analyte within 25 min.
Among mass sensitive sensors, quartz crystal microbalance with dissipation monitoring (QCM-D) can be mentioned [60]. In sandwich immunoassay, 0.2-40 ng/mL of OTA were measured using Au nanoparticles as labels of secondary Abs for signal amplification.
[55] Pagkali, V.; Petrou, P. S.; Salapatas, A.; Makaronab, E.; Peters, J.; Haasnoot, W.; Jobst, G.; Economou, A.; Misiakos, K.; Raptis, I.; Kakabakos, S. E. Detection of ochratoxin A in beer samples with a label-free monolithically integrated optoelectronic biosensor. J. Hazard Mat. 2017, 323, 75—83. DOI: 10.1016/j.jhazmat.2016.03.019
[56] Nabok, A.; Al-Jawdah, A. M.; Tsargorodska, A. Development of planar waveguide-based immunosensor for detection of low molecular weight molecules such as mycotoxins. Sens. Actuators B. 2017, 247, 975-980. DOI: 10.1016/j.snb.2017.01.197
[57] Al-Rubaye, A.; Nabok, A.; Catanante, G.; Marty, J.-L.; Takacs, E.; Szekacs, A. Detection of ochratoxin A in aptamer assay using total internal reflection ellipsometry. Sens. Actuators B 2018, 263, 248-251. DOI: 10.1016/j.snb.2018.01.220
[58] Al-Rubaye, A. G.; Nabok, A.; Catanante, G.; Marty, J.-L.; Takács, E.; Székács, A.Label-free optical detection of mycotoxins using specific aptamers immobilized on gold nanostructures. Toxins 2018, 10, 291 DOI: doi:10.3390/toxins10070291
[59] Tsounidi, D.; Koukouvinos, G.; Petrou, P.; Misiakos, K.; Zisis, G.; Goustouridis, D.; Raptis, I.; Kakabakos, S. E. Rapid and sensitive label-free determination of aflatoxin M1 levels in milk through a White Light Reflectance Spectroscopy immunosensor. Sens. Actuators B. 2019, 282, 104-111.
[60] Karczmarczyk, A.; Haupt, K.; Feller, K.-H. Development of a QCM-D biosensor for Ochratoxin A detection in red wine. Talanta 2017, 166, 193-197. DOI: 10.1016/j.talanta.2017.01.054
Reviewer 4 Report
Evtugyn et al. made a detailed review of the immunosensors and aptasensors to mycotoxin detection. The review is comprehensive, and the audiences are wide. However, the manuscript needs improvement. I would like to recommend its publication after revision.
In the chemical reaction (1) between line 172 and line 173, authors are suggested to use Kon and Koff. If not, authors should explain the meaning of K1 and K-1
In line 265, “affine immobilization” indicating “affinity immobilization”? meanwhile, protein A/G should be included after “avidin-biotin binding”
In line 292 covalent immobilization, authors are recommended to include the references of NHS conjugation chemistry on gold surface for label-free propose. Like 10.1002/adma.201700057 or 10.1016/j.bios.2014.11.025.
In figure 6, authors are recommended to separate the APTES polycondensation and thiolated chemical for SAM on both gold and silver by Fig. 6A and Fig. 6B, respectively. In line 310, put the Fig. 6B in the sentence. Need a silver surface modification reference. May refer. 10.1021/ac504110t and 10.1021/ac402126k. Fig. 6b may use Au/Ag electrode.
In figure 8, The conjugation between biotin and antibodies is hard by simply mixing. Authors may consider using biotin-NHS instead of biotin. Figure name may use biotinylation instead of biotinylating.
In table 1, the production protocol is hard to follow. reference [90] “K = (8.5-27) 108 M-1” missed some words?
Table 2 is hard to follow
There are two “table 2”s
In the second table 2, reference [133], “LOD” is missing in the sensitivity
In line 502, table 3, I would like to use “dynamic range” instead of “conc. range”
The paragraph style needs improvement. Like line 126 and line 142, the font size of 2.2. is smaller than the one of 2.2.1
Author Response
1. In the chemical reaction (1) between line 172 and line 173, authors are suggested to use Kon and Koff. If not, authors should explain the meaning of K1 and K-1
The following equation was modified as requested (see file eq.doc attached)
2. In line 265, “affine immobilization” indicating “affinity immobilization”? meanwhile, protein A/G should be included after “avidin-biotin binding”
Changes suggested were made, ‘affine‘ words were substituted with ‘affinity”
3. In line 292 covalent immobilization, authors are recommended to include the references of NHS conjugation chemistry on gold surface for label-free propose. Like 10.1002/adma.201700057 or 10.1016/j.bios.2014.11.025.
In figure 6, authors are recommended to separate the APTES polycondensation and thiolated chemical for SAM on both gold and silver by Fig. 6A and Fig. 6B, respectively. In line 310, put the Fig. 6B in the sentence. Need a silver surface modification reference. May refer. 10.1021/ac504110t and 10.1021/ac402126k. Fig. 6b may use Au/Ag electrode.
In figure 8, The conjugation between biotin and antibodies is hard by simply mixing. Authors may consider using biotin-NHS instead of biotin. Figure name may use biotinylation instead of biotinylating.
Section describing immobilization was significantly reduced following the Reviewer 1 comment. For this reason, detailed discussion of the above problems was found impossible, Fig.8 was deleted.
4. In table 1, the production protocol is hard to follow. reference [90] “K = (8.5-27) 108 M-1” missed some words?
The following description of the protocol was inserted:
scFv formed in recombinant E. coli after cloning the scFv-coding gene from hybridoma 2C12. The scFv formed inclusion bodies in the cytoplasm of E. coli required in vitro refolding.
5. Table 2 is hard to follow
We agree with this comment and we would prefer another format of the Table but should follow the template provided
6. There are two “table 2”s
We thank reviewer for this correction, Tables were re-numbered
7. In the second table 2, reference [133], “LOD” is missing in the sensitivity
Misprint was corrected
8. In line 502, table 3, I would like to use “dynamic range” instead of “conc. range”
We have added suggestion to Table 2 and 4 describing immune- and aptasensors
9. The paragraph style needs improvement. Like line 126 and line 142, the font size of 2.2. is smaller than the one of 2.2.1
Paragraph style was checked and corrected
Author Response File: 
Author Response.pdf
