Development of Terahertz Time-Domain Spectroscopy for Properties Analysis of Highly Diluted Antibodies

Round 1

Reviewer 1 Report

I would like to thank all the Authors for their efforts.

I ensure the authors that I do not have any potential conflict of interest with regards to this paper.

I also would like to clarify that I have got no intention to accuse the authors for any type of plagiarism.

Over all it is a self-repetitive work even if the authors have tried to show a novelty by changing a minute amount of mixture. I do not find the research design  appropriate. I do not believe if the research is conducted correctly. I reject the article due to serious flaws. Please find my comments below.

With my best regards,

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• The authors prefer to use IR spectrometer to extract the distances by using the given analytical formula in equation 1. (It is not explained where the 5000 comes from). Even if one can easily extract the cuvette wall thicknesses and the distance in between them from the time domain signal with a standard THz transmission scheme, since the cuvette spacing is larger than 50um.

• It is not clear if the authors have used THz TDS or the IR-spectroscopy to extract the real part of the index of refraction.

• The authors use the given formula in equation 2, to extract the imaginary part of the dielectric function which is irrelevant if they have used a THz TDS. This is a methodological error.

Since, the major advantages of THz-TDS is that the transient THz electric field is measured, and therefore, the constituent elements of the pulse which are the amplitude and phase that have connection with the absorption coefficient and the complex refractive index of the sample can be determined. In a THz TDS measurement, the time dependent pulsed electric field transmitted through the sample and through the free space (reference signal) is acquired separately. Then the temporal profiles are converted into the frequency domain by applying a standard Fast Fourier Transform (FFT) technique, and the material transfer function is derived. transfer function correlates with the signal amplitude and phase changes due to the absorption and refraction processes in each sample. The frequency-dependent complex index of refraction  is directly correlated with the complex dielectric function.  Moreover, starting from the complex dielectric function one can extract the frequency dependent real conductivity. Even if the extraction methodology is well established in the literature, it is not always so trivial, that one needs to itteratively minimize the periodic Fabry–Perot oscillations produced inside the target to achieve reliable results.

• Later on the authors express the model including the Debye relaxation, bending and stretching modes and then fit the parameters with the imaginary part of the dielectric function. And extract the physical parameters as given in table 1. The authors keep the dielectric relaxation time constant referring to the literature data” t=7.315 ps at 30°C [55]).”

I did not find the given value in the cited work. More over the given reference is focused on ice investigation and not liquid water. Electro optical parameters of water show high diversities in between the two phases. 

• The proposed work addresses “Application of Terahertz Time-Domain Spectroscopy…” yet neither time domain nor frequency domain spectra is shown.
  
  
• Same for the IR-spectroscopy data, there is no IR spectra shown. More over there is no strong correlation and/or clear explanation given regarding to the importance of the use of IR-spectroscopy as a supporting tool.

• The dilutions (“HD Abs to IFN-γ” and “Saturation control”) are sampled as water-ethanol mixture while the “intact lactose” is prepared with water only. The THz field strongly interacts with polar molecules and polar liquids (ethanol, 2-propanol, water vs…) tend to absorb the THz intensity. Yet, as well studied in the literature there is a discernible difference in between ethanol and water in terms of absorbance and dielectric properties. And the difference is far larger (the given difference may be due to the water vs methanol mix effect) when compared with the un-discernible inclusion of lactose as justified by the authors.

Such that “The solutions of the saturation control and  intact lactose showed a statistically significant difference only with regard to the value of ω1.”

“we take into account only water bands and completely exclude from the consideration the  spectral contribution of lactose itself. This is absolutely justified, since the coefficient of water absorption in the studied region is 1.5–3 orders of magnitude greater than the absorption of lactose. If we take into account that the lactose content in the solution is 10%, then the contribution of lactose  to the absorption does not exceed 0.3%.”

“Corresponding spectra of solutions of the remaining studied samples (HD Abs to IFN-γ and Saturation control) are very similar”

• There are some misleading phrases throughout the text. For instance “The spectra of aqueous solutions in the terahertz frequency domain generally do not contain clearly defined spectral bands; therefore, to obtain interpretable information, the spectra recorded need to undergo mathematical processing.” This is not correct, THz-TDS gives the electro-optical properties of the studied system without any sort of mathematical processing. The atmospheric absorption lines not being observed in the liquid form has a strong physical reason.

• The research design is not appropriate. One should extract the complex dielectric function of each matrix element separately and/or just measure the complex dielectric function of  the host media(pure water, pure ethanol, water-ethanol) and then by using a mean field theory one may extract the inclusion lactose from the mixed sample (lactose-ethanol-water). Later, one may fit the final data to extract other parameters.

• There are unnecessarily many self-citations (I would not say inappropriate) in the references. More over the basics of the methodologies are already previously published by the authors (for example “REF 22.Terahertz Spectroscopy Applied for Investigation of Water Structure”).

Author Response

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Author Response File: Author Response.docx

Reviewer 2 Report

Gramatical errors across the publication. Secondly, an explanation is required on what procedure for measuring spectra excludes unwanted water artifact. Finally, how does this detection compare against other techniques is not clear. A more detailed analysis is required including error bars. The approach requires tracking a third decimal change in the spectroscopy, it seems to imply there might be a high error in detection. It would be good to show repeatability and a paragraph describing why this method should be used against conventional UV -VIS or mid IR spectroscopy would be better. 

 

Author Response

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Author Response File: Author Response.docx

Reviewer 3 Report

In this paper, the authors present a new method (THz-TDS) for the analysis of highly diluted antibodies. It's more a methodology paper than a real application method for pharmaceutical analysis. 

The authors need to improve the english writing to make it easier for the reader to understand this paper. Be also more concise to improve the clarity of  your study.

The figure 1 is not really pertinent and a better choice of the illustrations as NIR spectrum, for example, will have been more suitable.

The authors need to be more precise when talking about NIR spectroscopy. Almost all the time they are using, IR ou FT-IR spectroscopy, while conventionnaly it will imply MIR spectroscopy. They thus need to correct it to be more precise.

In the title, the author are talking about "pharmaceutical analysis" but to be honest we really need to search for the pharmaceutical analyses. They only used what we may say "pharmaceutical samples" to develop their approach and no real application of it is presented. Especially since for a pharmaceutical application of a method it is mandatory to validate it according to the current standards. It could thus be confusing for the reader.

Author Response

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Author Response File: Author Response.docx

Reviewer 4 Report

The paper by Penkov et al. addresses the application of the terahertz time-domain spectroscopy to analyze the aqueous solutions of lactose saturated with highly diluted antibodies. Predominantly, the experiments were planned and performed properly but there are few drawbacks that need to be addressed before publication.

• The authors stated that the final solution contained a mixture of 12, 30, and 50 centesimal dilutions of antibodies to IFN-γ. Which centesimal dilutions were taken into account in a model?
• How about water evaporation of the samples during measurements?
• In my opinion, the authors should present the results of the change in the absorption coefficient of the samples at any stage of the dilution level.
• Moreover, the structure of water is very sensitive to various perturbations, such as variations in temperature or additives. These variations can be reflected by the change of the NIR spectra of water in aqueous solutions. Therefore, multivariate statistical analysis (e.g. partial least squares-discriminant analysis or support vector machine) should be employed to extract the spectral variation.

To sum up, although, this work describes a potentially suitable tool I feel that it lacks proof of concept. Therefore, I recommend a major revision along the lines of suggestions given above.

Author Response

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Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

I would like to thank all the Authors for their efforts.

Authors are representing a different modality of using THz-TDS technique for sensing minute amounts of Antibodies in an aqueous solution. The authors try to show evidence from the statistical differences extracted from the fitting parameters. Even if the idea is interesting, the authors do not show any spectroscopic evidence of sensing minute changes neither in the time domain nor in the frequency domain regarding the claimed sensing in between the water and the antibody solution.

I do not think if the research design is appropriate. I reject the article due to this reason.

With my best regards,

Reviewer 2 Report

Accept in current form

Reviewer 3 Report

The authors has taken into account all the comments. They tried their best to answer them.

Reviewer 4 Report

In my opinion, the manuscript is ready to publish.