Design of A Low-Cost and Disposable Paper-Based Immunosensor for the Rapid and Sensitive Detection of Aflatoxin B1
, Luiz H. C. Mattoso 1, Osvaldo N. Oliveira, Jr. 2 and Daniel S. Correa 1,*
Round 1
Reviewer 1 Report
In this manuscript, authors fabricated a paper-based electrochemical immunosensor for detection of a food carcinogenic mycotoxin - aflatoxin B1 (AFB1) within the range of 1 pg.mL-1 to 30 ng.mL-1 with LOD of 0.62 ng.mL-1. The immunosensor exhibited good repeatability, reproducibility, stability, and selectivity. From a technical point of view, (MWCNT)/chitosan is widely used in the preparation of electrochemical biosensors, and its preparation principle is similar to that of screen-printed electrodes.Possible innovations lie in the use of waterproof paper substrates and new conductive inks. And I recommend the manuscript to be published in Chemosensors journal after major revision.
Suggestions for revision are as follows:
- Antibody concentration has an important influence on sensor performance. Can the authors add an experiment to optimize antibody concentration? ( line 116-117)
- In addition, why does the author use 0.1M PBS solution instead of the commonly used 10mM PBS to rinse the electrode?
- Does the prepared sensor need to consider the influence of pH, temperature and other factors on the detection? The author needs to supplement relevant experiments.
- The antigen-antibody binding requires 10 minutes, plus the electrochemical impedance spectroscopy measurement time, is the whole determination process too long? Is there any way to shorten the measurement time? If the experimental results can be obtained faster, the commercial application value of this method will be greatly improved.
Author Response
Reviewer 1
In this manuscript, authors fabricated a paper-based electrochemical immunosensor for detection of a food carcinogenic mycotoxin - aflatoxin B1 (AFB1) within the range of 1 pg.mL-1 to 30 ng.mL-1 with LOD of 0.62 ng.mL-1. The immunosensor exhibited good repeatability, reproducibility, stability, and selectivity. From a technical point of view, (MWCNT)/chitosan is widely used in the preparation of electrochemical biosensors, and its preparation principle is similar to that of screen-printed electrodes..Possible innovations lie in the use of waterproof paper substrates and new conductive inks. And I recommend the manuscript to be published in Chemosensors journal after major revision.
A0: The authors thank the reviewer for the important suggestions and for recommending our manuscript for publication after revision.
Q.1 Antibody concentration has an important influence on sensor performance. Can the authors add an experiment to optimize antibody concentration? (line 116-117)
A1. Indeed, the sensor performance is highly influenced by antibody concentration. In our study, the optimized concentration of Anti-AFB1 was established according to previous studies reported in the literature, as shown below:
Xian Zhang, Chao-Rui Li, Wei-Cheng Wang, Jian Xue, Ya-Ling Huang, Xian-Xian Yang, Bin Tan, Xi-Peng Zhou, Chuang Shao, Shi-Jia Ding, Jing-Fu Qiu. A novel electrochemical immunosensor for highly sensitive detection of aflatoxin B1 in corn using single-walled carbon nanotubes/chitosan. Food Chemistry 2016,192 197–202.
Ma, H.; Sun, J.; Zhang, Y.; Xia, S. Disposable amperometric immunosensor for simple and sensitive determination of aflatoxin B1 in wheat. Biochem. Eng. J. 2016, 115, 38–46.
Ma, H.; Sun, J.; Zhang, Y.; Bian, C.; Xia, S.; Zhen, T. Label-free immunosensor based on one-step electrodeposition of chitosan-gold nanoparticles biocompatible film on Au microelectrode for determination of aflatoxin B1 in maize. Biosens. Bioelectron. 2016, 80.
Wang, H.; Zhang, Y.; Chu, Y.; Ma, H.; Li, Y.; Wu, D.; Du, B.; Wei, Q. Disposable competitive-type immunoassay for determination of aflatoxin B1 via detection of copper ions released from Cu-apatite. Talanta 2016, 147, 556–560.
Goud, K.Y.; Hayat, A.; Catanante, G.; Satyanarayana, S.M.; Gobi, K.V.; Marty, J.L. An electrochemical aptasensor based on functionalized graphene oxide assisted electrocatalytic signal amplification of methylene blue for aflatoxin B1 detection. Electrochim. Acta 2017, 244, 96–103.
Q2. In addition, why does the author use 0.1M PBS solution instead of the commonly used 10mM PBS to rinse the electrode?
A2. We thank the reviewer for noting this mistake. Actually, the PBS concentration used in our work was the standard 10mM PBS, which information was corrected in the revised manuscript.
Q3. Does the prepared sensor need to consider the influence of pH, temperature and other factors on the detection? The author needs to supplement relevant experiments.
A3. We thank the reviewer for the observations. As the electrode surface was modified by using chitosan, a biopolymer soluble in an acid medium (pH < 6.5), and anti-AFB1, which is sensitive to acid and basic mediums (pH < 7; pH >8), we performed all the experiments at pH 7.4 to avoid the solubilization of chitosan and the denaturation of the antibody. Furthermore, the temperature during the experiments was kept at 25 °C to preserve the anti-AFB1 activity.
Q4. The antigen-antibody binding requires 10 minutes, plus the electrochemical impedance spectroscopy measurement time, is the whole determination process too long? Is there any way to shorten the measurement time? If the experimental results can be obtained faster, the commercial application value of this method will be greatly improved.
A4. In our study, the antibody-antigen binding was set for 10 min. aiming to ensure completion of interaction, once the minimum time required for binding in conditions of analysis similar to our work is 6 min (please see refs. displayed below). In addition, the measurement time for AFB1 detection took nearly 2 min. Therefore, the total time for analysis by using the sensors developed in this study was nearly 12 min, which is significantly lower than the time required for conventional analytical methods as enzyme immunoabsorbent (ELISA) and chromatographic assays.
Lili Yu, Yang Zhang, Chenyi Hu, Hui Wu, Yayun Yang, Chusen Huang, Nengqin Jia. Highly sensitive electrochemical impedance spectroscopy immunosensor for the detection of AFB1 in olive oil, Food Chemistry 2015, 176, 22–26.
Arati Sharma, A. Kumar, Raju Khan. Electrochemical immunosensor based on poly(3,4-ethylenedioxythiophene) modified with gold nanoparticle to detect aflatoxin B1, Materials Science and Engineering C 2017, 76, 802–809.
Reviewer 2 Report
Good paper.To be improved final discussion where not all the described results find evidence.Bibliography needs updating.Waterptoff paper needs some further information to be possibly otherwhere applied.How detection limit was determined?Do the authors foresee any limit of food to which the method could not be possibly adopted(considering.the teduced cases of food here considered)
Author Response
Reviewer 2
Q1. Good paper to be improved final discussion where not all the described results find evidence bibliography needs updating. Waterptoff paper needs some further information to be possibly otherwhere applied.
A1: The authors thank the reviewer for the valuable suggestions and comments. As requested, changes were made in the revised manuscript.
Q2. How detection limit was determined?
A2. The detection limit (D.L.) was calculated from the ratio between the standard deviation (σ) value corresponding to the response of three measurements by using an AFB1 solution at a fixed concentration (lowest concentration detected), and the slope (S) of the calibration curve (D.L. = 3.3 σ/S). This information is highlighted in section 3.2 of the revised manuscript.
Q3. Do the authors foresee any limit of food to which the method could not be possibly adopted (considering the reduced cases of food here considered)?
A3. We developed a sensing platform which D.L. is below the maximum allowed level of ABF1 in food established by the European Union and the U.S. Food and Drug Administration (FDA) legislation, which are 2 ng mL-1 and 20 ng mL-1, respectively. Although a reduced number of real samples were analyzed in this study, we believe that the use of the developed immunosensor can be extended to other real foods samples that are favorable to the appearance of mycotoxins, but this can be accomplished in future investigation.
Reviewer 3 Report
The design of paper-based electrode with cut-printing technique for fabrication of disposable sensor is feasible. Also the strategy of immune approaching is reasonable for sensing response. These two aspects built the substrate of this manuscript.
But, there is a significant shortage in experiments.
The EIS investigation is unfaithful. It might be out the experience of the authors.
Firstly, this is not a complete EIS curve, at least it did not give the part of Warberg's impedance. And, thereafter, the authors did not take the work of equivalent circuit fitting. On my opinion, the acquired data by authors are totally not the Rct, or else its decline along with the target concentration is unaccountable.
Author Response
Reviewer: 3-
Q1. The design of paper-based electrode with cut-printing technique for fabrication of disposable sensor is feasible. Also the strategy of immune approaching is reasonable for sensing response. These two aspects built the substrate of this manuscript.
A1. The authors thank the reviewer for the positive evaluation of our manuscript.
But, there is a significant shortage in experiments. The EIS investigation is unfaithful. It might be out the experience of the authors. Firstly, this is not a complete EIS curve, at least it did not give the part of Warberg's impedance. And, thereafter, the authors did not take the work of equivalent circuit fitting. On my opinion, the acquired data by authors are totally not the Rct, or else its decline along with the target concentration is unaccountable.
A1. In this study, the Rct values were calculated from Nyquist plots shown in Figure 5 and the data were analyzed and fitted by using a Randle’s equivalent circuit (added as inset of figure 5 in the revised manuscript) as illustrated in Figure below, where “Rs” represents the electrolyte resistance, “Rct” corresponds to the charge (electron) transfer resistance, “Cdl” represents the interface capacitance, and Zw is the Warburg impedance. The semicircular region located at high frequencies is associated to an interfacial charge-transfer process and its diameter corresponds to the charge transfer resistance (Rct), whereas diffusional processes can be ascribed to the linear region at low frequencies. Such piece of information was included in the revised manuscript.
The Figure shows the Randle’s equivalent circuit model used to fit and analyze the impedance data.
Wu Z, Wang B, Cheng Z, Yang X, Dong S, Wang E. A facile approach to immobilize protein for biosensor: Self-assembled supported bilayer lipid membranes on glassy carbon electrode. Biosens Bioelectron 2001, 16, 47–52.
Reviewer 4 Report
- How many replications were included for each AfB1 concentration point?
- Were the two maize flour samples used to have two extraction solutions and each solution spiked with AFB1?
Figure 1. The illustration is good but the figure does not provide additional information than what is described in the text. Consider removing this figure.
line 65: consider replacing "describe" with "developed"
line 93: replace "Next, they" with "The electrodes"
line 107: delete "obtained"
line 108: replace "submitted" with "placed"
line 153: consider changing "non infected" with " AFB1-free"
line 206, 207: do you mean "anti-AFB1"?
line 224: delete "with"
line 250: delete " It is worth mentioning that"
line 269-270: This statement is not correct. The maize flour samples did not contain aflatoxin. The samples were used to obtain and extract solution that was spiked with AFB1.
line 278: consider replacing "real" with "using extraction solutions from"; consider replacing "had remarkable" with "showed high"
line 279 consider replacing "common interferent" with "another mycotoxin"
Author Response
Reviewer: 4
The authors thank the reviewer for the evaluation and important suggestions to improve the manuscript.
Q1. How many replications were included for each AfB1 concentration point?
A1. All measurements were performed in triplicate. This information has been inserted in the revised manuscript.
Q2. Were the two maize flour samples used to have two extraction solutions and each solution spiked with AFB1?
A2. Yes, the reviewer is correct. Two extraction solutions were prepared from two non-infected maize flours samples purchased from a local market, and solutions spiked with AFB1 at concentrations ranging from 1 to 30 ng mL-1 were prepared from these extracts. The procedure full procedure is described in item 2.7 of the revised manuscript.
Q3. Figure 1. The illustration is good but the figure does not provide additional information than what is described in the text. Consider removing this figure.
A3. The purpose of Figure 1 is to provide t the readers a concise and visual summary of the procedure for preparing the sensing platforms.
Q4. line 65: consider replacing "describe" with "developed"
A4. We thank the reviewer for the suggestion. Change was made in the revised manuscript.
Q5. line 93: replace "Next, they" with "The electrodes"
A5. Changes made accordingly.
Q6. line 107: delete "obtained"
A6. Changes made accordingly
Q7. line 108: replace "submitted" with "placed"
A7. Changes made accordingly
Q8. line 153: consider changing "non infected" with " AFB1-free"
A8. We thank the reviewer for the observation. Corrections were performed in the revised manuscript.
Q9. line 206, 207: do you mean "anti-AFB1"?
A9. We thank the reviewer for the observation. Corrections were performed in the revised manuscript.
Q10. line 224: delete "with"
A10. Changes made accordingly.
Q11. line 250: delete " It is worth mentioning that"
A11. Changes made accordingly.
Q12. line 269-270: This statement is not correct. The maize flour samples did not contain aflatoxin. The samples were used to obtain and extract solution that was spiked with AFB1.
A12. We thank the reviewer for the observation. Changes were made to the revised manuscript
Q13. line 278: consider replacing "real" with "using extraction solutions from"; consider replacing "had remarkable" with "showed high"
A13. We thank the reviewer for the observation. The suggestions were implemented in the revised manuscript.
Q14. line 279 consider replacing "common interferent" with "another mycotoxin"
A14. We thank the reviewer for the observation. The suggested alteration was implemented in the revised manuscript.
Round 2
Reviewer 1 Report
I agree to accept.
Author Response
The authors thank the reviewer for to accept our work.
Reviewer 3 Report
It looks like that the authors did not understand my question in first round reviewing.
They discussed a simplest R(Q(RW)) mode to fit the EIS data, but the real case is not this mode. There clearly presents two semi-circles on EIS curve, thus they must select another fittest equivalent circuit.
Author Response
Response sheet:
Reviewer: 3-
Q1. It looks like that the authors did not understand my question in first round reviewing. They discussed a simplest R(Q(RW)) mode to fit the EIS data, but the real case is not this mode. There clearly presents two semi-circles on EIS curve, thus they must select another fittest equivalent circuit.
A1. The authors thank the reviewer for clarifying his/her question. As requested, we tested several other and more complex equivalent circuits and could find one in which the results showed better values for x2 (10-4) and a lower error associated with the parameters involved in the equivalent circuit. In terms of Rct values, the change was minimal, so we did not change the fit values employed for the calculation of the detection limit. The new equivalent circuit was included in the revised manuscript, as well as proper changes were made in the revised manuscript.
