Functionalized Surfaces as a Tool for Virus Sensing: A Demonstration of Human mastadenovirus Detection in Environmental Waters

Round 1

Reviewer 1 Report

The presented manuscript shows the development of a method for improved detection of Human mastadenovirus in water samples by pre-concentration with immuno-modified magnetic particles.

The scientific work is properly presented and discussed, improved sensitivity comparing to the standard sample analysis (PCR without pre-concentration) is clearly shown.

There are only 5 minor issues needed to be corrected before the final publication.

1. Formatting of the numbers and units: 'degree Celsius' should always be formatted as, e.g., 80 °C (see here: https://www.e-education.psu.edu/styleforstudents/c2_p10.html)
2. Inspect the text carefully for the formatting of the uppercase symbols, especially in the numbers and symbols. For instance, here - page 3, line 112: went from 10-1 to 10-13 (should be '10^-1 to 10^-13'), line 120: 36.15 hab/km2 (should be '36.15 hab/km²')
3. Formating exponential numbers in the following format '5.83E+06' should be unified with the format shown in section 2.
4. Authors use two ways of expressing the concentration of genomic copies of the virus - GC/mL or GC/5 µL - this should be unified.
5. The statistical difference is correctly calculated; however, the graphical presentation should be improved - Figures 4, 5 and 7. The statistical difference should be indicated inside the bar graphs using lines and symbols (ns, *, **, ***, etc.).

Author Response

Dear Reviewer,

Thanks for your valuable comments, we have addressed them in the manuscript:

1. The units have been reviewed and formatted accordingly.
2. We have also reviewed and formatted the uppercase symbols throughout the text.
3. The exponential numbers format has been unified.
4. The concentration has been unified to GC/5µL.
5. We have tried to add the error bars, but we believe that the graph then contains too much information and less clear. We would prefer to keep the error bar information in the table.

Reviewer 2 Report

Gularte et al. provide a method taking advantage of immunomagnetic separation as a concentration tool and qPCR for Human mastadenovirus detection in environmental waters. The work is executed at a very high professional level in terms of the experimental design and data presentation. Different concentration methods were compared. SEM and HIM were used to assess the surface of the magnetic beads. The performance of monoclonal Ab and polyclonal Ab was also assessed. The reported method can become useful in water quality control. I'm also supporting the idea of using a microfluidic device to improve the performance of IMS next. I recommend accepting this paper for publication in Chemosensors after revisions according to the following comments.

1. The authors claim that " qPCR has some disadvantages; this method alone cannot discriminate between infectious and noninfectious viral particles". How is the use of IMS able to address this problem? Are the antibodies used in this study able to discriminate between the infectious virus and noninfectious virus? 
2. The IMS method concentrated 1 mL of the water sample to 200 uL(5 times). The ultracentrifugation method concentrates 36 mL of sample to 2mL (18 times). The ultracentrifugation method is able to process a bigger volume of samples and has a higher concentration factor. Why IMS provides better detection sensitivity? What's the maximal sample volume that can be processed by IMS? It will be better to provide more discussion on this.
3. Polyclonal antibodies showed way better detection sensitivity in figure 4. But in water sample analysis, the polyclonal antibodies did not show this advantage. Can you provide some explanations?
4. In row 91, [a], [b-d] need to be clarified.
5. In row 140, I guess "108" should be 10⁸
6. The consistency of formatting needs to be improved, Such as 10-6, 4.00E+02, 10^-13.
7. In row 350 and 351, the unit used is GC/5ul. But the unit used in Figure 5 is GC/5mL. They should be consistent. Also, GC/ml is preferred.

Author Response

Dear Reviewer,

Thanks for your valuable comments, we have addressed them in the manuscript:

1. qPCR cannot discriminate between infectious and noninfectious viral particles because this technique can also detect genomic fragments of the target virus. Since IMS concentrates viral genomes packed in capsid proteins rather than naked viral genomes, the use of these approaches together (IMS-qPCR) increases the possibility to detect viable viral particles, in other words, increase the detection of infectious virus and decrease the possibility of nonviable virus detection. These details are described on page 2 (lines 93 and 94) and page 12 (lines 436-439).
2. IMS provides better sensitivity due to higher specificity since the functionalized particles search and bind to the specific target due to antibody-antigen compatibility. Ultracentrifugation separates the particles due to their physical properties, which is not as specific as immune binding. It is true that ultracentrifugation allows for higher volume handling, but it might not be necessary to handle such large volumes, especially in contaminated waters by anthropic activities as Brazilian water resources. This info has been included in the revised manuscript (pages 13 and 14 – lines 484-491).
3. Both antibodies showed some differences in HAdV concentration when compared to each other. However, found results in tests using standard and water samples showed no significant statistical difference as described on page 12 (lines 420-422) and page 13 (lines 479-480). The slight difference found between polyclonal and monoclonal antibodies can be better understand because of the natural different characteristics that they have as described on page 12 (lines 408-420).
4. That has been done now (page 2 - line 90).
5. This mistake has been corrected (page 3 - line 138).
6. This has been reviewed and unified in the revised manuscript.
7. Thanks, the figure has been corrected and unified to GC/5µL.

Reviewer 3 Report

The paper reported on the comparison of functionalized and non-functionalized based methods in the detection of two types of Human mastadenovirus, species C (HAdV-C) and species F (HAdV-F), in water samples. The functionalization consists on applying magnetic bead surface in the form of immunomagnetic separation (IMS) combined with real-time polymerase chain reaction (qPCR) (IMS-qPCR). The paper is well written, readable and understandable also to non-experts in the field.

A couple of minor comments before the publication:

• At pag. 2, line 69, the authors write “PCR” which is defined later at line 81;
• The graphics of the figure could be improved in quality;
• (just an advice) The authors claim that “No conductive coatings were applied to the samples prior to imaging in order to preserve the sample surface information. Charge compensation was ensured through a low-energy electron beam…” The reviewer can be in agreement with that, but probably not if you consider low magnification SEM images as the ones reported in Fig. 2. In that case, I don’t think a low thickness metal coating would have changed the observed morphology (and probably, with metallization, you would obtain a better resolution in Fig.2 a)

• (a) and (b) are repeated in Fig. 2. It is clear that the magnification is different, but it would be better to refer to different images with different letters.

Author Response

Dear Reviewer,

Thanks for your valuable comments, we have addressed them in the manuscript:

1. It is now defined in the first mentioning of PCR in the manuscript (page 2 – line 68).
2. This has been done now.
3. Thanks for the advice, we agree with your comment.
4. We have now corrected it.