Real-Time Fluorescence Imaging of His-Tag-Driven Conjugation of mCherry Proteins to Silver Nanowires

Round 1

Reviewer 1 Report

Summary:

    Metal-enhanced fluorescence (MEF) biosensors exploit the proximity of a fluorophore to a precisely designed metallic surface to boost fluorescent signal, increasing sensitivity and decreasing required detection times. This enhanced fluorescent signal is due to plasmonic interactions between the fluorophore and the metal surface, which are highly sensitive to the distance between the fluorophore and the surface. Thus, attachment strategies between fluorophores and metallic surfaces are very important to bring the desired molecule to the metal surface at the appropriate distance.

    In this study, Jankowska et al. demonstrate that Ni-NTA modified silver nanowires (AgNW) can be used to capture and enhance the fluorescent signal of a His tagged fluorescent protein, mCherry. The authors confirm via signal intensity and kinetics that the measured signals from the AgNW-mCherry conjugates are plasmon enhanced. Finally, the authors show that their system can be used in real time to detect the presence of mCherry.

General comments:

This is a nice study that I believe should be published, but it would be great if the authors could contextualize their results more. They state that their work provides a building block for future biosensors, which I don’t disagree with, but given the authors’ previous success with streptavidin-biotin linkers, I find it hard to understand what exactly this work enables. In what situations would it be beneficial to use this linker chemistry? I understand that it could just be seen as “another option” and it is valuable to know alternative methods, but this manuscript would be much more valuable if the authors could spend some time to discuss why this method would be preferable to others. With respect to the authors, the finding that His-tagged proteins can be localized to a Ni-NTA modified substrate is unsurprising since the use of His-tag Ni-NTA chemistry is well known. However, the study can answer the question of “How well does the His-tag Ni-NTA chemistry work for MEF applications?”. It would be great if the authors could do follow up experiments to characterize this system.

In addition, there are some issues with formatting and clarity; in particular, I find that the authors err a bit on the side of too much information. I have put some explicit examples in the comments, but in general I suggest the authors should go back through the paper and remove details that are not necessary for the study since inclusion of those details makes it difficult to parse out the important information.

Comments:

1. Pg 2: Although appreciated, it is not necessary to discuss the history of mCherry and the method of production. It would be clearer to just say “In this work, we purchased commercially available N-terminal His tagged mCherry (OriGene Technologies)”.
2. Pg 4. Similar to the above comment, figure 1A is not necessary; it is nice to see the structure of mCherry, but it is unclear what the structure has to do with the rest of the study. Unless there is some reason for it that I’m missing, it should be removed.
3. 4. The results and discussions section should be formatted to have subheadings that describe each finding (e.g. 3.1 mCherry in the vicinity of AgNW shows higher signal strength than mCherry alone).
4. On Pg. 4, Figure 2 is described as showing typical images for mCherry mixtures of unmodified AgNWs and mCherry-AgNW conjugates, but I think Figure 2 seems to only be showing unmodified AgNWs with high concentration mCherry (2A,B) and low concentration (2C,D). Is the conjugated mCherry-AgNW shown at all in Figure 2? It looks like the mCherry-AgNW conjugate is shown in Figure 4. Adding subheadings to describe the main result of each experiment will greatly help clarity.
5. Pg 6. When constructing the histogram of fluorescence intensities, the authors state that “in the selection process, we rejected agglomerates characterized with unusually high emission intensity”. Can the authors either provide some supplementary data or further specify the selection criteria? Given that the authors also make some statements that for the AgNW case, very large signals can be attributed to plasmon resonances, I’m wondering how the authors differentiate signals from aggregated fluorophores from MEF signals.
6. I would be interested to see a comparison of the signal from an mCherry-AgNW conjugate vs the signal from an mCherry conjugated to a non-metallic nanowire to get a sense of how much enhancement we get from the plasmonic interaction. Alternatively, if the AgNW can be Ni-NTA modified with the PVP coating, to reduce the plasmonic interaction, we could then get a better sense of how much plasmonic enhancement is boosting fluorescence (even better would be if the thickness of the PVP coating could be modulated to show how the precise distance of mCherry from the AgNW affects enhancement). I am mostly wondering because it seems that from Fig. 2, the His tag Ni-NTA interaction is not necessary to enhance signal strength. It is unclear to me how much benefit is gained from the linker chemistry – while it is nice to have a homogenous signal, if one is just trying to detect the presence of an analyte in solution, is it really necessary to do the modification? The linker chemistry may aid enrichment, but if the system is already sensitive enough for single molecule detection, could the concentration of AgNWs be increased to improve the likelihood of a fluorophore being in the vicinity of the nanowire?
7. It is unclear to me what the evaluation of this method is against biotin-streptavidin interactions; the paper suggests that biotin-streptavidin attachment is faster than His to Ni-NTA. This seems to suggest that biotin-streptavidin conjugation would be preferable than this method. Are there reasons why His tag Ni-NTA chemistry is desirable? The introduction describes wanting linkers for His as His tags are common in protein purification, but it is unclear what biosensing applications are being pursued here. Typically His tags are introduced at the DNA level – if making a sensor to detect analytes in patient blood, the Ni-NTA:His chemistry seems like it wouldn’t work well, whereas biotinylation can be done at the protein level. Can the authors explain the pros of this method over biotin-streptavidin interactions?

Author Response

General comments:

This is a nice study that I believe should be published, but it would be great if the authors could contextualize their results more. With respect to the authors, the finding that His-tagged proteins can be localized to a Ni-NTA modified substrate is unsurprising since the use of His-tag Ni-NTA chemistry is well known. However, the study can answer the question of “How well does the His-tag Ni-NTA chemistry work for MEF applications?”. It would be great if the authors could do follow up experiments to characterize this system.

Response: We would like to thank the Reviewer for such a detailed review of the manuscript. We appreciate the comments and suggestions to improve the quality of our work. We agree that His-tag Ni-NTA chemistry is well known, however at the same time we found no report aimed at studying real-time fluorescence imaging using plasmonic nanostructures. The previous work of D. Kowalska et al. [Plasmon-induced absorption of blind chlorophylls in photosynthetic proteins assembled on silver nanowires] suggested that time of at least of 1 hour is required for complete conjugation, and the results of real-time imaging indicate this process in this geometry is more efficient. Moreover, we decided to determine the effect of plasmonic enhancement in a structure, where His-tag Ni-NTA chemistry is used. Clearly there is large variety of proteins, viruses or molecules, and each case needs to be considered separately. Depending on the distance between an emitter and metallic nanoparticle, fluorescence can be either enhanced or quenched. The experiments described in the manuscript are important and introductory experiments before carrying out studies of more advanced geometry, which are now ongoing in our laboratory, but are at a very early stage.

Comments:

1. Pg 2: Although appreciated, it is not necessary to discuss the history of mCherry and the method of production. It would be clearer to just say “In this work, we purchased commercially available N-terminal His tagged mCherry (OriGene Technologies)”.

1. Pg 4. Similar to the above comment, figure 1A is not necessary; it is nice to see the structure of mCherry, but it is unclear what the structure has to do with the rest of the study. Unless there is some reason for it that I’m missing, it should be removed.

Response: We appreciate the suggestion, but we are of the opinion that due to an interdisciplinary character of this work, such details should be provided.

1. 4. The results and discussions section should be formatted to have subheadings that describe each finding (e.g. 3.1 mCherry in the vicinity of AgNW shows higher signal strength than mCherry alone).

Response: We agree with the suggestion of adding subheadings. We have followed this in the revised manuscript.

1. On Pg. 4, Figure 2 is described as showing typical images for mCherry mixtures of unmodified AgNWs and mCherry-AgNW conjugates, but I think Figure 2 seems to only be showing unmodified AgNWs with high concentration mCherry (2A, B) and low concentration (2C, D). Is the conjugated mCherry-AgNW shown at all in Figure 2? It looks like the mCherry-AgNW conjugate is shown in Figure 4. Adding subheadings to describe the main result of each experiment will greatly help clarity.

Response: Thank you for paying attention to this description. Indeed, it might have been somewhat confusing. We have corrected this issue in the revised version of the manuscript.

1. Pg 6. When constructing the histogram of fluorescence intensities, the authors state that “in the selection process, we rejected agglomerates characterized with unusually high emission intensity”. Can the authors either provide some supplementary data or further specify the selection criteria? Given that the authors also make some statements that for the AgNW case, very large signals can be attributed to plasmon resonances, I’m wondering how the authors differentiate signals from aggregated fluorophores from MEF signals.

Response: In calculating histograms of fluorescence intensity we adopted the selection criteria that take into account the average intensity of emission spots with the size of 5x5 pixel. When the fluorescence intensity from a was twofold that value, we excluded such spots from the analysis ascribing them to agglomerates. The same approach was applied to analysis of mCherry on nanowires. An example of such an agglomerate can be noticed in Fig. 2A, where we have one spot on AgNWs with much higher fluorescence emission.

1. I would be interested to see a comparison of the signal from an mCherry-AgNW conjugate vs the signal from an mCherry conjugated to a non-metallic nanowire to get a sense of how much enhancement we get from the plasmonic interaction. Alternatively, if the AgNW can be Ni-NTA modified with the PVP coating, to reduce the plasmonic interaction, we could then get a better sense of how much plasmonic enhancement is boosting fluorescence (even better would be if the thickness of the PVP coating could be modulated to show how the precise distance of mCherry from the AgNW affects enhancement). I am mostly wondering because it seems that from Fig. 2, the His tag Ni-NTA interaction is not necessary to enhance signal strength. It is unclear to me how much benefit is gained from the linker chemistry – while it is nice to have a homogenous signal, if one is just trying to detect the presence of an analyte in solution, is it really necessary to do the modification? The linker chemistry may aid enrichment, but if the system is already sensitive enough for single molecule detection, could the concentration of AgNWs be increased to improve the likelihood of a fluorophore being in the vicinity of the nanowire?

Response: In this comment the Reviewer points out the critical issue of having as appropriate reference as possible. In the manuscript we decided to stick with metallic nanowires applied in varied geometries instead of developing new and qualitatively different protocols of functionalizing non-metallic nanowires. One would need to assure the same chemistry to be applicable as well as comparable sizes. This is currently beyond our competences. Also applying PVP polymer is questionable as it would change the distance from metallic nanoparticle to the protein as well as dielectric surroundings. Comparison would be extremely difficult. In our opinion the best measure of plasmon induced fluorescence enhancement are the changes in decay characteristics, which in this case are rather dramatic.

1. It is unclear to me what the evaluation of this method is against biotin-streptavidin interactions; the paper suggests that biotin-streptavidin attachment is faster than His to Ni-NTA. This seems to suggest that biotin-streptavidin conjugation would be preferable than this method. Are there reasons why His tag Ni-NTA chemistry is desirable? The introduction describes wanting linkers for His as His tags are common in protein purification, but it is unclear what biosensing applications are being pursued here. Typically His tags are introduced at the DNA level – if making a sensor to detect analytes in patient blood, the Ni-NTA:His chemistry seems like it wouldn’t work well, whereas biotinylation can be done at the protein level. Can the authors explain the pros of this method over biotin-streptavidin interactions?

Response:

The work was aimed to show the kinetic reaction for different type of modification. We compare thesetwo  different linkers because it might seem that both the biotin-streptavidin and His to Ni-NTA attachment would behave in a similar way as far as dynamics is concerned. Despite similar dimensions and mass weights of the protein, the start of the process of His-tag driven conjugation is about 20 s compared to somewhat shorter time in the case of biotin-streptavidin conjugation (~ 10 s). This point requires more systematic experiments in order to quantify these differences in a more robust way.  

Reviewer 2 Report

Mackowski et. al reported work "Real-time fluorescence imaging of His-tag-driven conjugation of mCherry proteins to silver nanowires" is well written, however it needs minor revision (comments specified below) before it get published in Chemosensors. 

1) Some of the recent references regarding fluorescence quenching (decay response) of AgNw upon conjugation with protein units need to be cited to justify the hypothesis in current work. 

2) It is highly recommended to use IUPAC notations when author use analytical parameters in the research article most of the time random notations were used it need to be corrected. 

3) Surface functionalisation of AgNw with biomolecules need to be characterized atleast by one technique such as XPS, SEM or TEM. 

4) Author report bi-exponential decay profiles, therefore decay components need to tabulated with radiative rate constants. 

Author Response

Mackowski et. al reported work "Real-time fluorescence imaging of His-tag-driven conjugation of mCherry proteins to silver nanowires" is well written, however it needs minor revision (comments specified below) before it get published in Chemosensors. 

• Some of the recent references regarding fluorescence quenching (decay response) of AgNw upon conjugation with protein units need to be cited to justify the hypothesis in current work. 

Response:

First, we would like to thank the Reviewer for the detailed assessment of our work. We appreciate the comments and suggestions. In the revised version we added references concerned with fluorescence quenching. Information regarding such effects can be found in publications 15-18 of the revised manuscript.  

• It is highly recommended to use IUPAC notations when author use analytical parameters in the research article most of the time random notations were used it need to be corrected. 

Response: Thank you for this recommendation. We implemented the IUPAC notation in the revised version of the manuscript.

• Surface functionalisation of AgNWs with biomolecules need to be characterized at least by one technique such as XPS, SEM or TEM. 

Response: As our primary experimental technique is fluorescence microscopy, we design our research in such a way to be able to detect emission of photoactive proteins without any need to apply advanced and sophisticated methods suggested by the Reviewer,. The main experiment that confirms conjugation of AgNWs with proteins in real-time imaging experiment, and in our opinion, from the point of view of the scope of this work, no further confirmation is necessary. Nevertheless, neither TEM nor SEM would provide insight into functionalization of silver nanowires, as the functional groups are very small is size and perhaps would give next to none contrast.   

• Author report bi-exponential decay profiles, therefore decay components need to tabulated with radiative rate constants. 

Response: The decay times of fluorescence are discussed in the manuscript. We have made additional fitting with the assumption than the longer time constant is the same as for the mCherry protein on glass. The quality of the fit is sufficient and the obtained decay constant for the fast decay is also comparable to the previous procedure. This bi-exponential decay profile combined with observed increase of the fluorescence emission provide sufficient ground for concluding about the strong interaction between proteins and plasmon excitations in AgNWs.

Round 2

Reviewer 1 Report

I thank the authors for their effort in revision and addressing my concerns. I am satisfied with the manuscript.