Surface Inactivation of Human Coronavirus by MACOMA™ UVA-TiO2 Coupled Photocatalytic Disinfection System
Round 1
Reviewer 1 Report
In this work, authors claimed the use of MACOMA UVA-TiO2 for Coronavirus inactivation purposes. In the abstract and introduction part, the authors introduced the current healthcare challenge because of the COVID-19 caused by SARS-CoV-2 coronavirus. However, in this work HCoV-OC43 was used. Although both in the beta coronaviruses family, are there any differences between this virus used and the one caused the COVID-19?
For the TiO2 used, is it anatase, rutile or brookite phase? How does it affect the disinfection? In this work, 80% TiO2 + 20% mineral binder suspension was used. How does the percent of the TiO2 and the particle size of the TiO2 will influence of the disinfection?
Author Response
We would like to thank the reviewers for their time and careful reading of the submitted manuscript. We have modified the revised manuscript accordingly and point-by-point responses are appended below.
1-In this work, authors claimed the use of MACOMA UVA-TiO2 for Coronavirus inactivation purposes. In the abstract and introduction part, the authors introduced the current healthcare challenge because of the COVID-19 caused by SARS-CoV-2 coronavirus. However, in this work HCoV-OC43 was used. Although both in the beta coronaviruses family, are there any differences between this virus used and the one caused the COVID-19?
Response:
Thank you for this comment, we would like to mention, which you have also pointed out, that the human coronavirus OC43 (HCoV-OC43) belongs to the same family of human coronaviruses and exhibits similar clinical presentation although not as severe as SARS-CoV-2. Additionally, HCoV-OC43 has been used as a surrogate of SARS-CoV-2 for evaluating the efficacies of viral inactivation systems. Therefore, we feel that it is justified to use HCoV-OC43 for evaluating the inactivation efficiencies of the system described in this manuscript.
2- For the TiO2 used, is it anatase, rutile or brookite phase? How does it affect the disinfection? In this work, 80% TiO2 + 20% mineral binder suspension was used. How does the percent of the TiO2 and the particle size of the TiO2 will influence of the disinfection?
Response:
TiO2 used in this study was in an anatase phase. The disinfection is through the photocatalytic activities of the TiO2 and we anticipate having an enhanced photocatalytic activity with higher percentages of the TiO2, the optimization to use 80% TiO2 was done to ensure that coating is stable and long-lasting. The particle size of TiO2 may also have altered disinfection but the optimization of the particle size is beyond the scope of this work.
Reviewer 2 Report
This paper entitled “Surface Inactivation of Human Coronavirus by MACOMA™ UVA-TiO2 coupled Photocatalytic Air Disinfection System.” systematically analysis thethe photocatalytic and vir- 11
ucidal activity of MACOMA™ TiO2 photocatalytic film activated by UVA-LED-12V-367nm 12
(MA- 717836-1) lamp against the HCoV-OC43. In the study, some interesting experimental results were obtained. I recommend that this paper can be accepted after carefully addressing the following concerns:
1. What is the innovation of this paper? TiO2 and UVA have been widely studied in the antibacterial and antiviral areas.
2. As “ Air Disinfection System” had been mentioned in the title, the related research is missing. Please add the related data.
3. The viral quantification is based on the standard curve in Fig.2A, however, the detailed information how this be achieved is missing, please include the information. In addition, the X-axis in Fig.2A start from 1 to 100,000,0, but the 100,000,0 come direct after 100,00, is the 100,000 missing? Please double check it.
4. In the paper, the TiO2 photocatalytic films deposited on 24mm x 24 mm glass coverslips are water-borne suspensions that consist of ~80% TiO2 + 20% mineral binder (inert, water-insoluble mineral carbonates, oxycarbonates, and hydrates). The following experiment used TiO2-coated coverslips and uncoated coverslips, are this uncoated coverslips bared coverslips or coverslips coated with 20% mineral binder? If uncoated coverslips are bared coverslips, the coverslips coated with 20% mineral binder should be included in the test.
Author Response
Comments and Suggestions for Authors
This paper entitled “Surface Inactivation of Human Coronavirus by MACOMA™ UVA-TiO2 coupled Photocatalytic Air Disinfection System.” systematically analysis the photocatalytic and virucidal activity of MACOMA™ TiO2 photocatalytic film activated by UVA-LED-12V-367nm (MA- 717836-1) lamp against the HCoV-OC43. In the study, some interesting experimental results were obtained. I recommend that this paper can be accepted after carefully addressing the following concerns:
We appreciate your time in reviewing our manuscript and the below comments were helpful in enhancing the overall quality of the manuscript.
- What is the innovation of this paper? TiO2and UVA have been widely studied in the antibacterial and antiviral areas.
Response:
We agree that TiO2 and UVA have been widely studied for their anti-microbial properties and the innovation of this study is the coating of the TiO2 with mineral binder and the combination of a low-power UVA light, which is harmless but still effective in disinfecting human coronavirus from the coated surfaces.
- As “ Air Disinfection System” had been mentioned in the title, the related research is missing. Please add the related data.
Response:
Thank you for this comment. Since we have not used this system for disinfecting viruses in the air, we have changed Air Disinfection System to ‘Surface Inactivation of Human Coronavirus by MACOMA™ UVA-TiO2 coupled Photocatalytic Disinfection System’
The viral quantification is based on the standard curve in Fig.2A, however, the detailed information how this be achieved is missing, please include the information. In addition, the X-axis in Fig.2A start from 1 to 100,000,0, but the 100,000,0 come direct after 100,00, is the 100,000 missing? Please double check it.
Response:
As suggested, we have provided the following details on how this standard curve was generated and used for calculating the HCoV-OC43 virus genome in our samples.
Four ten-fold serial dilutions of HCoV-OC43 genomic RNA, obtained from the BEI Resources (Cat. # NR-52727 with 2.0x108genome equivalents/mL), were used for generating a standard curve of viral genome copies and corresponding Ct (cycle threshold value). The equation from this standard curve was used for quantifying the number of the HCoV-OC43 genome copies in the virus preparations.
Fig.2A. I changed the y-axis to a log10 scale with correct labeling. Thank you.
- In the paper, the TiO2photocatalytic films deposited on 24mm x 24 mm glass coverslips are water-borne suspensions that consist of ~80% TiO2 + 20% mineral binder (inert, water-insoluble mineral carbonates, oxycarbonates, and hydrates). The following experiment used TiO2-coated coverslips and uncoated coverslips, are this uncoated coverslip bared coverslips or coverslips coated with 20% mineral binder? If uncoated coverslips are bared coverslips, the coverslips coated with 20% mineral binder should be included in the test.
Response:
Thank you for pointing this out, we have corrected this in the text. The uncoated control coverslips used in the study were coated with the 20% mineral binder. The revised text reads as”
Glass coverslips (24mm x24 mm) with 20% mineral binder but without the TiO2, referred to as an ‘uncoated’ surface, are used as a control.
This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.
Round 1
Reviewer 1 Report
Recommendation: Reject
Comments: Authors have conducted the study of the inactivation of human coronavirus using macoma fn nano photocatalysts. The authors should have done a better literature investigation in the introduction part. This topic has been studied extensively since the break of the COVID-19, and the authors should really reflect those already published and distinguish the uniqueness of their work. There are some other questions that require the authors’ attention. I would not recommend this work to be published in Catalysts.
- For Figure 3A) FN NANO coated under UV rad and Figure 3B) FN NANO coated under UV rad, the uncertainties should be provided.
- Also in Figure 3, there is no significant difference in the coated and uncoated samples if taking the uncertainties into consideration. Statistically, it may be the same.
- Figure 4 does not have a scale.
- For Figure 4 B, C, D, and E, in the non-UV-exposure category, the total number of HCoV-OC43 virus-infected A549-hACE2 cells compared with that of A, are too few, and hard to tell the difference from the perspective of statistics. How reliable are these data and the conclusion?
Author Response
Comments: Authors have conducted the study of the inactivation of human coronavirus using macoma fn nano photocatalysts. The authors should have done a better literature investigation in the introduction part. This topic has been studied extensively since the break of the COVID-19, and the authors should really reflect those already published and distinguish the uniqueness of their work. There are some other questions that require the authors’ attention. I would not recommend this work to be published in Catalysts.
Response:
Thank you very much for the comments, we have revised the introduction by including additional studies on microbial inactivation. We have thoroughly studied the comments and revised our manuscripts to address specific comments. Detailed responses are appended here.
- For Figure 3A) FN NANO coated under UV rad and Figure 3B) FN NANO coated under UV rad, the uncertainties should be provided.
Response: As per suggestion, we have included the statistical analysis of viral inactivation due to the coating by determining the statistical significance between the residual viral copies as well as live virus on coated vs uncoated surfaces.
- Also, in Figure 3, there is no significant difference in the coated and uncoated samples if taking the uncertainties into consideration. Statistically, it may be the same.
Response: The data is presented on a log scale and there are at least 1-log lower viral copies on UV-Rad exposed FN-Nano coated surfaces as compared to the uncoated surfaces under similar exposure.
- Figure 4 does not have a scale.
Response: We have included a scale bar on the immunofluorescence panel.
- For Figure 4 B, C, D, and E, in the non-UV-exposure category, the total number of HCoV-OC43 virus-infected A549-hACE2 cells compared with that of A, are too few, and hard to tell the difference from the perspective of statistics. How reliable are these data and the conclusion?
Response: Panel is to show that nucleocapsid antibody specifically stains the virally infected cells and these infectious viruses was used for the assay.
We have counted the number of nucleocapsid stained cells per optical view and have included the average number from 3 such fields on each panel. These data clearly show that FN Nano coating reduces the number of live (infectious) virus, much quicker than without the coating under similar conditions (with UVA exposure).
Reviewer 2 Report
The research has a very good quality, above the average of reports on the subject.
Please review typing errors marked in the attached PDF file. Mainly the errors are about adding a space between numbers and measurement units, subscripts, among others. Also, please check if the abbreviation "min" or "mins" will be used for minutes.
Comments for author File: 
Comments.pdf
Author Response
The research has a very good quality, above the average of reports on the subject.
Please review typing errors marked in the attached PDF file. Mainly the errors are about adding a space between numbers and measurement units, subscripts, among others. Also, please check if the abbreviation "min" or "mins" will be used for minutes.
Response:
Thank you very much for reading this manuscript, we have addressed all the comments in this revised manuscript.
Reviewer 3 Report
- This paper reported a FN NANO and MACOMA UV-A LED-12V-367nm (MA-FN 717836-1) based disinfection system which provides a rapid and complete surface inactivation of human coronavirus. But in part of virus inactivation assay, the copies of viral genomic RNA from UV-treated, uncoated coverslips declined 99.8181% within 60 mins, and 99.9739% within 120 min. The result was approximately the same as the copies of viral genomic RNA from UV-treated, FN NANO coated coverslips declined 99.9927% within 60 mins, and 100% within 120 min. The infectivity assay was the same. Can we think that the FN NANO has almost no effect in this disinfection system, mainly due to the effect of UVA? In order to save costs, should we consider using only UVA for disinfection?
- UVA has a strong penetrating power, can penetrate most transparent glass and plastic, can penetrate the surface of the skin, penetrate into the tissues below the dermis, destroy the internal structure of the skin, and make the skin aging. This reason leads to limitations in application, not as the author said that provides a rapid, and complete surface inactivation of human coronavirus in a human-safe manner.
Comments for author File: Comments.pdf
Author Response
Comments and Suggestions for Authors
- This paper reported a FN NANO and MACOMA UV-A LED-12V-367nm (MA-FN 717836-1) based disinfection system which provides a rapid and complete surface inactivation of human coronavirus. But in part of virus inactivation assay, the copies of viral genomic RNA from UV-treated, uncoated coverslips declined 99.8181% within 60 mins, and 99.9739% within 120 min. The result was approximately the same as the copies of viral genomic RNA from UV-treated, FN NANO coated coverslips declined 99.9927% within 60 mins, and 100% within 120 min. The infectivity assay was the same. Can we think that the FN NANO has almost no effect in this disinfection system, mainly due to the effect of UVA? In order to save costs, should we consider using only UVA for disinfection?
Response: According to the observed results, the presence of UVA radiation led to an effective 2 log10 and 3 log10 reduction in the amount of infectious virus on uncoated coverslips within 60 and 120 min, respectively. The presence of TiO2 (in the form of FN NANO coating) is seen to further aid/accelerate this deactivation to 4 log10 and complete reduction, respectively, within the mentioned time window. Since, TiO2 absorbs UVA radiation, thereby making it extremely effective against skin damage, utilizing UVA radiation alone for surface disinfection is not a recommended option, in my humble opinion.
- UVA has a strong penetrating power, can penetrate most transparent glass and plastic, can penetrate the surface of the skin, penetrate into the tissues below the dermis, destroy the internal structure of the skin, and make the skin aging. This reason leads to limitations in application, not as the author said that provides a rapid, and complete surface inactivation of human coronavirus in a human-safe manner.
Response: We agree with the reviewer that UVA rays, with strong penetration effect, are extremely unsafe for humans. However, presence of non-toxic TiO2 nanoparticles, that act as a UV filter, provides the invaluable protection to human skin from the harmful UVA rays. Therefore, surface decontamination using UVA radiation coupled with FN NANO coating can be considered as a better deactivation solution as compared to just the UVA system.
Round 2
Reviewer 1 Report
1) Only A549 was used for this study, which is too few. At least another cell line should be used for this in vitro inactivation study.
2) The localization result is not trustworthy. The number of cells in the immunofluorescence images is much fewer than that of a typical cell transfection. In contrast, a typical localization should show a much larger number of cells to make it meaningful statistically. A good example is shown in the reference: Oncotarget 2015, 6, 30, 30263-30276.
3) Additionally, with such few number of cells, with a random selection of view regions, you will get a huge statistical difference, and hard to tell if it is because of the inactivation or just from the manual selection. Such data will not support the conclusions which the authors are trying to claim.
4) Some minor details: Scale bars should be provided for each of the figures, not only the first one.
Reviewer 3 Report
This paper analysis the human coronavirus inactivation ability of FN NANO photocatalytic film under the UV-A LED activation. Both RT-PCR and the cell infection assay were used in this paper to calculate the inactivation efficiency of this system. I don’t think the novelty of this work can meet the standard of catalysts.
- For the figure1, the standard curve A and B have only three points, which is a little bit less for standard curve. In the figure1C the scale bar is missing for all microscope pics, the same as figure 4 B, C, D, and E.
- For the figure3, the uncoated glass treated with UV-A can reach more than 90% reduction of the virus. After 120 min treatment, 99.9739% (RNA copies) and 99.9746% reduction without FN NANO photocatalytic is quite close to 100%. I would believe that only UV-A can inactive the virus on the glass, there is no need to use FN NANO photocatalytic film. On the other side, the UV-A shouldn’t be used in this test, since it’s effect on the virus inactivation.
- UV activated TiO2 based bacteria and virus inactivation have been widely studied. I didn’t see the innovation point of this work.
