Label-free sensitive detection of influenza virus using PZT discs with a synthetic sialylglycopolymer receptor layer

We describe rapid, label-free detection of Influenza A viruses using the first radial mode of oscillations of lead zirconate titanate (PZT) piezoelectric discs with a 2 mm radius and 100 µm thickness fabricated from a piezoelectric membrane. The discs are modified with a synthetic sialylglycopolymer receptor layer, and the coated discs are inserted in a flowing virus suspension. Label-free detection of the virus is achieved by monitoring the disc radial mode resonance frequency shift. Piezo transducers with sialylglycopolymer sensor layers exhibited a long lifetime, a high sensitivity and the possibility of regeneration. We demonstrate positive, label-free detection of Influenza A viruses at concentrations below 105 virus particles per millilitre. We show that label-free, selective, sensitive detection of influenza viruses by home appliances is possible in principle.


Recommendation?
Major revision is needed (please make suggestions in comments)

Comments to the Author(s)
The authors applied interesting approaches to detect viruses and their experimental data suggest a potential of resonator systems as sensors for viruses. However, to confirm their claims they need to provide more experimental evidences. Please find the questions and comments as below to improve the study and manuscript. _ There are multiple grammatical errors, so please carefully check English throughout the manuscript.
_ In the introduction the authors emphasize the need to detect virus particles in the air, but they performed detection of viruses in solution. Please rewrite the introduction section to minimize this mismatch.
_ Potential readers would not be convinced with the authors' claim that the detection method is very specific to influenza virus. It is because there is no experimental evidence for this claim. To prove their statement authors need to perform detection experiments with other enveloped viruses (for example, HIV-1, MLV, VSV, and etc.).
_ Sialic moiety can also react to other enveloped viruses. So the authors need more discussion for potential interactions between this moiety and other enveloped viruses.
_ In addition, the authors claim that the method is specific to influenza A viruses. They need to justify why other influenza virus strains will not interact with the sialic moiety.
_ Please provide more explanations on how current work is different from the authors' previous work (the 12th reference).
_ They also need to show the effect of flow rate on virus detection performance. In other word, they need to justify why the specific flow rate was chosen during detection experiment.
_ Getting physical images with AFM is not sufficient to confirm the existence of virus particles. Do you have other specific methods to prove that you actually obtained resonance signals from virus particles? One suggestion is to saturate virus particles with sialic molecules and then apply the sample onto your system. The samples should not generate resonance signals that were obtained with viruses not pre-treated with the moiety.
_ If there are molecules with sialic moiety not in excess compared with the number of virus particles, the authors should not obtain the resonance signals linearly increasing with the number of virus particles applied to the system. Please address this point by providing new experimental data or logical statement. _ Please discuss why certain times are needed to reach state-state level of frequency in terms of physical theory about resonance.

Review form: Reviewer 2
Is the manuscript scientifically sound in its present form? Yes

Are the interpretations and conclusions justified by the results? Yes
Is the language acceptable? Yes

Recommendation?
Major revision is needed (please make suggestions in comments)

Comments to the Author(s)
The paper titled "Label-free sensitive detection of influenza virus using PZT discs with a synthetic sialylglycopolymer receptor layer", designed and measured a biosensor for the rapid and label-free detection of Influenza A viruses. It shows the principle of label-free, selective, sensitive detection of Influenza viruses for home appliances. After a deep consideration, I think this paper can be accepted after a major revision with the following comments: 1) In the introduction, there are some other methods to detect the viruses, except the methods mentioned in this paper, such as the terahertz TPS, which is given in the following references: The editors assigned to your paper ("Label-free sensitive detection of influenza virus using PZT discs with a synthetic sialylglycopolymer receptor layer") have now received comments from reviewers. We would like you to revise your paper in accordance with the referee and Associate Editor suggestions which can be found below (not including confidential reports to the Editor). Please note this decision does not guarantee eventual acceptance.
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Once again, thank you for submitting your manuscript to Royal Society Open Science and I look forward to receiving your revision. If you have any questions at all, please do not hesitate to get in touch. Comments to the Author(s) The authors applied interesting approaches to detect viruses and their experimental data suggest a potential of resonator systems as sensors for viruses. However, to confirm their claims they need to provide more experimental evidences. Please find the questions and comments as below to improve the study and manuscript. _ There are multiple grammatical errors, so please carefully check English throughout the manuscript. _ In the introduction the authors emphasize the need to detect virus particles in the air, but they performed detection of viruses in solution. Please rewrite the introduction section to minimize this mismatch. _ Potential readers would not be convinced with the authors' claim that the detection method is very specific to influenza virus. It is because there is no experimental evidence for this claim. To prove their statement authors need to perform detection experiments with other enveloped viruses (for example, HIV-1, MLV, VSV, and etc.).
_ Sialic moiety can also react to other enveloped viruses. So the authors need more discussion for potential interactions between this moiety and other enveloped viruses.
_ In addition, the authors claim that the method is specific to influenza A viruses. They need to justify why other influenza virus strains will not interact with the sialic moiety.
_ Please provide more explanations on how current work is different from the authors' previous work (the 12th reference).
_ They also need to show the effect of flow rate on virus detection performance. In other word, they need to justify why the specific flow rate was chosen during detection experiment.
_ Getting physical images with AFM is not sufficient to confirm the existence of virus particles. Do you have other specific methods to prove that you actually obtained resonance signals from virus particles? One suggestion is to saturate virus particles with sialic molecules and then apply the sample onto your system. The samples should not generate resonance signals that were obtained with viruses not pre-treated with the moiety.
_ If there are molecules with sialic moiety not in excess compared with the number of virus particles, the authors should not obtain the resonance signals linearly increasing with the number of virus particles applied to the system. Please address this point by providing new experimental data or logical statement. _ Please discuss why certain times are needed to reach state-state level of frequency in terms of physical theory about resonance.

Reviewer: 2
Comments to the Author(s) The paper titled "Label-free sensitive detection of influenza virus using PZT discs with a synthetic sialylglycopolymer receptor layer", designed and measured a biosensor for the rapid and label-free detection of Influenza A viruses. It shows the principle of label-free, selective, sensitive detection of Influenza viruses for home appliances. After a deep consideration, I think this paper can be accepted after a major revision with the following comments: 1) In the introduction, there are some other methods to detect the viruses, except the methods mentioned in this paper, such as the terahertz TPS, which is given in the following references:  , 2017, vol.8, no.8. [2] D Cheng, X He, X Huang, B Zhang, G Liu, G Shu, "Terahertz biosensing metamaterial absorber for virus detection based on spoof surface plasmon polaritons" International Journal of RF and Microwave Computer-Aided Engineering, 2018 (7) 2) Much more description and explaining about fig.3 and 4 should be added in the text. 3) In fig.3(b), I am not very clear how much is the viurs concentration? And can you plot the curves of concentration versus frequency shift? In addition, once the viurs concentration changes, will the amplitude in Fig. 3
Decision letter (RSOS-190255.R1) 19-Aug-2019 Dear Dr Erofeev, I am pleased to inform you that your manuscript entitled "Label-free sensitive detection of influenza virus using PZT discs with a synthetic sialylglycopolymer receptor layer" is now accepted for publication in Royal Society Open Science.
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Response to Referees
Reviewer: 1 Remark: The authors applied interesting approaches to detect viruses and their experimental data suggest a potential of resonator systems as sensors for viruses. However, to confirm their claims they need to provide more experimental evidences. Please find the questions and comments as below to improve the study and manuscript. There are multiple grammatical errors, so please carefully check English throughout the manuscript.

Answer:
The grammatical errors have been corrected by our colleague whose native language is English.

Remark:
In the introduction the authors emphasize the need to detect virus particles in the air, but they performed detection of viruses in solution. Please rewrite the introduction section to minimize this mismatch.

Answer:
We rewrote the introduction and mentioned that the problem of effectively sampling airborne virus particles has already been solved. A combination of such a sampler and a sensor working in solution could be very effective for virus detection.

Remark:
Potential readers would not be convinced with the authors' claim that the detection method is very specific to influenza virus. It is because there is no experimental evidence for this claim. To prove their statement authors need to perform detection experiments with other enveloped viruses (for example, HIV-1, MLV, VSV, and etc.).

Answer:
The development of highly specific receptor layer was not a goal of our study. Sialylglycoconjugates are widely used for influenza virus detection. We used a well-known receptor molecule, which can reliaby bind the chosen target avian Influenza A virus, with an innovative polymer matrix to develop a new type of micromechanical sensor based on the first radial mode resonant frequency shift. Determining the specificity of the sialyl-based receptor is a separate biochemical investigation.

Remark:
Sialic moiety can also react to other enveloped viruses. So the authors need more discussion for potential interactions between this moiety and other enveloped viruses.