Immunomagnetic Separation of Microorganisms with Iron Oxide Nanoparticles
, Sonja Berensmeier and Sebastian P. Schwaminger *Round 1
Reviewer 1 Report
The authors present an immunomagnetic separation technique and its application in the detection of Legionella in water reservoirs.
a. A graphical representation for the preparation of nanoparticles and immobilization of the antibodies would help the readers better follow the manuscript.
b. Optimization methods for the amount of antibody immobilization have to be provided.
c. a tabular representation of the detection limits compared to other techniques should be provided.
d. a detailed explanation of control experiments for every characterization and difference in characteristic behavior of the particles needs to better represented
Author Response
Dear reviewer,
We appreciate your review and your time. You helped us to improve the manuscript and we answer your questions point by point:
The authors present an immunomagnetic separation technique and its application in the detection of Legionella in water reservoirs.
a. A graphical representation for the preparation of nanoparticles and immobilization of the antibodies would help the readers better follow the manuscript.
We added a graphical representation for the preparation of nanoparticles which complements our graphical abstract.
b. Optimization methods for the amount of antibody immobilization have to be provided.
We tested the binding capacity of our particles with BSA before using antibodies, where we performed an “adsorption” isotherm experiment to derive the amount of antibody which is optimal to be immobilized on our nanoparticles. Furthermore, we tried out multiple different nanoparticle coatings where we immobilized the antibodies for Legionella enrichment. We added the following sentence: “an adsorption test was performed with bovine serum albumin and antibodies, which yielded optimal binding conditions at 0.2 mg/ml for a nanoparticle concentration of 1 mg/ml.”
c. a tabular representation of the detection limits compared to other techniques should be provided.
We want to emphasize this technique as a proof of concept before developing the actual detection and thus setting up the detection limits. There is always the issue of a fine tuning of the detection limit versus an applicability of a method. Furthermore, the time requirements of different methods need to be considered as well. Such a table would go beyond the focus of this manuscript.
d. a detailed explanation of control experiments for every characterization and difference in characteristic behavior of the particles needs to better represented
We added the explanation of control experiments in the figure captions of the respective figure: The reference absorbance of water incubated with both nanoparticle species for all particle concentrations (pH 7.4) shows the same value as water after magnetic separation of 1 h (OD =0).
Sincerely,
Sebastian Schwaminger
Reviewer 2 Report
Thomas JA et al. report on the optimization and characterization of Legionella-specific antibody conjugated magnetic nanoparticles for the quantitation of Legionella in environmental samples. While the results are still preliminary, requiring a lower LOD and assessment of non-specific binding/matrix effects, the characterizations are well documented and the platform shows promise for future field deployment. Overall, the manuscript is well-written, with the results coming across as clear, concise, and following a logical progression. While I have only a couple of minor questions/concerns, I believe the article is suitable for publication in Chemosensors following minor revisions. Specific comments are below.
Line:
184: Can the zeta potentials of 1) APTES-functionalized, and 2) polyglutaraldehyde-activated nanoparticles at neutral pH be provided, and compared to the BIONs? It is expected that these zeta potentials would shift between negative and positive with each functionalization (given the functional group charges of each constituent), and will provide assurance that each step has been performed successfully.
188: Is it possible that glutaraldehyde is also covalently agglomerating the nanoparticles together, as it is a homobifunctional crosslinker?
266: (Figure 5) Is it possible to extract association constants from these binding curves? This could potentially provide more quantitative comparisons of binding between the BIONS and @Epoxy-Ab’s. Additonally, please provide replicate numbers (n) for this data.
291: (Figure 6) Please provide replicate numbers (n) and error for the data in panels (a) and (b).
Author Response
Dear reviewer,
We would like to thank you very much for your time and your valuable recommendations to improve the manuscript. In the following, we answer your recommendations point by point:
Thomas JA et al. report on the optimization and characterization of Legionella-specific antibody conjugated magnetic nanoparticles for the quantitation of Legionella in environmental samples. While the results are still preliminary, requiring a lower LOD and assessment of non-specific binding/matrix effects, the characterizations are well documented and the platform shows promise for future field deployment. Overall, the manuscript is well-written, with the results coming across as clear, concise, and following a logical progression. While I have only a couple of minor questions/concerns, I believe the article is suitable for publication in Chemosensors following minor revisions. Specific comments are below.
Line:
184: Can the zeta potentials of 1) APTES-functionalized, and 2) polyglutaraldehyde-activated nanoparticles at neutral pH be provided, and compared to the BIONs? It is expected that these zeta potentials would shift between negative and positive with each functionalization (given the functional group charges of each constituent), and will provide assurance that each step has been performed successfully.
The APTES coated nanoparticles show a zeta potential of 17.59 at pH 6.4 and the polyglutaraldehyde activated particles show a zeta potential of -23.88 at pH 7.3. We integrated these results to our discussion.
188: Is it possible that glutaraldehyde is also covalently agglomerating the nanoparticles together, as it is a homobifunctional crosslinker?
This is a good point. Yes, crosslinking might occur during the functionalization with polyglutaraldehyde and also later when the antibodies are coupled to the functionalized nanoparticles. However, the DLS data indicate no significant crosslinking during the functionalization with polyglutaraldehyde.
266: (Figure 5) Is it possible to extract association constants from these binding curves? This could potentially provide more quantitative comparisons of binding between the BIONS and @Epoxy-Ab’s. Additonally, please provide replicate numbers (n) for this data.
It was not possible to derive association constants from these binding curves. We want to show that a separation is possible for a distinct concentration but the separation is dependent on the particle concentration and the ratio between particles and bacteria. In order to determine the affinity further experiments would be necessary. However, we tested the affinity of the antibody to the Legionella cells with microscale thermophoresis.
291: (Figure 6) Please provide replicate numbers (n) and error for the data in panels (a) and (b).
The experiments in figure 6 were measured in triplicates which did not indicate differences. The measurement in triplicates is mentioned in the experimental part. Line 163 (in version with annotations)
Sincerely,
Sebastian Schwaminger
Round 2
Reviewer 1 Report
The authors have addressed the reviewers comments adequately and is acceptable for publication as is.
Reviewer 2 Report
The authors have addressed my minor concerns from the original submission, and I believe the manuscript is now suitable for publication in Chemosensors.
