Peer review of: Rational design of DNA nanostructures for single molecule biosensing (2020)

Reviewers' Comments: 
Reviewer #1: 
Remarks to the Author: 
 

“Rational design of DNA nanostructures for diagnostic: towards single molecule biosensing” by 
Raveendran et al. is a well written and interesting manuscript that describes the use of 2-d DNA origami 
to act as a binding site for CRP marker.  They use glass nanopipette-based sensing to detect for the 
presence of CRP-bound origami and report an accurate means of detecting CRP down to ~3 nM.   

Given the importance of developing rapid and accurate protocols for detecting CRP (CRP is used in 
common bloodwork to check for inflammatory-based diseases (i.e. Crohn’s, colitis, lupus, arthritis, etc.)) 
and the interesting approach that the authors have described for doing so, I am enthusiastic in my 
recommendation of publication for this manuscript, provided they address my comments below.   

1.  The title is a little awkward and I also think it undersells this work.  “diagnostic” ends abruptly.  

Also, you are doing single molecule biosensing, I think you’ve moved beyond the “towards”.  
Also, I would strongly recommend you clearly state that you can detect CRP given its ubiquitous 
role in present day clinical bloodwork for inflammatory-based diseases.  Perhaps something like: 
“Rational design of DNA nanostructures for single molecule C-reactive protein detection”....Feel 
free to come up with something else...but I think you should think a bit more to maximize your 
google search hit rate. 
 

2.  A general question: Given the 2-D nature of the DNA structure, why do you not find different 
blockades for when the origami goes through the opening perpendicular to the interface (as 
shown in the cartoon in Figure 2A) versus parallel?   
 

3.  Please include a current and time scale on figures 4A and 4B.   

4. In the supplementary section, Fig. S6b (ii) seems to be the only long-time current trace I see that 
shows the blockades from the DNA nanostructures.  Please include some more traces that 
clearly show that the number of blockade events increases with increasing DNA structure 
concentration.  As it stands now, without the 1-8 number labels in Fig. S6b(ii), I could just as 
easily argue that you are observing spurious background.  In fact, I see two or three upward 
steps in that same trace that are on the some order of magnitude as the so-called DNA events. 
 
5.  Same figure as point 4 above.  When you zoom in on the 8 events, why does the #1 event have 
an equal magnitude blockade as the other events when clearly number 1 in the raw trace 
appears to be about 3-4 times deeper?  Also, you should include a current and time scale on 
these zoomed in traces.         


 
Reviewer #2: 
Remarks to the Author: 
The manuscript by Raveendran et al. targets the task of detecting a low concentration of 
biomarkers in a sample by conjugating highly-charged DNA carriers to the biomarkers, to assist 
with capture and detection of these complexes using a nanopipette. The electrical signatures from 
origami carrier are different from protein-loaded origami carrier. The authors demonstrate the 
detection of C-reactive protein in diluted human plasma, which is impressive since detection in a 
"dirty" sample using nanopores is typically difficult to accomplish. The authors are well established 
in single-molecule sensing using nanopores, and the paper was written very well. While the paper 
is well written, I feel that there are improvements that the authors should make, and note my 
suggestions below. I have combined minor and major comments: 
 
- In the abstract, I would change "small concentrations" to "low concentrations". 
- I have a comment about the way the paper begins: The first sentence of the introduction 
discusses "Rapid and low-cost detection of disease biomarkers...", but oddly, the references all 
point out to single-molecule analysis papers, which is highly inconsistent with the claim in the 
sentence. There are many diagnostics platforms that are currently employed and the state of the 
art should be introduced to include current practices, followed by an excursion into single-molecule 
detection. This will avoid confusing readers that biomarker detection is exclusively owned by 
single-molecule techniques. 
- In Figure 1d, it seems that for ConC particles there is no translocation, based on the evidence of 
a broad dwell-time population at a relatively shallow level. Have the authors considered that ConC 
does not go through the pore? A voltage dependent study can confirm or reject this hypothesis. 
Otherwise, it is strange that the same exterior dimensions of a particle as ConA and ConB 
produces such a broad distribution. 
- In figure 2c the dwell time axis has negative value, which should not be shown because it is 
physically impossible to have a negative dwell time. 
- The dynamic range of the measurement is a function of the DNA origami maximum 
concentration, as the authors show in figure 3c. 
- The apparently low number of events shown in figure 3b, as well as figure s6, is a bit concerning. 
Is it because the pore lifetime is compromised by protein sticking to the pore walls, or because the 
experiment is intentionally short to demonstrate quick accurate readout? It would be good if the 
number of overall events for each plot is specified, and perhaps also to mention the overall 
sampling time for each experiment (for example, n=xxx, T=yyy min in each panel where data is 
shown). 
- It is important to include a statement on how the error bars were calculated, particularly for the 
data shown in Figures 3c and 4c. Is it a measurement-to-measurement error, or just a standard 
error of a single measurement? How many times was each point measured? Based on the table in 
the SI it seems that the authors have repeated results with 3 pipettes, which is good, but this 
should be specified throughout the manuscript where data are shown. 
- Overall, it seems that detection of all structures is somewhat convoluted by the bandwidth of the 
measurement. This is evident by events being close to the ~100 µs timescale limit. The question 
is, how would sensitivity be affected by the exact pipet size? 
- Also, it is confusing to me why origami are pulled out from the pipette, rather than being sucked 
in from the sample chamber. Is this amenable to the study of serum, and possible serial studies 
where multiple samples are analyzed? 
- Finally, it is interesting that the two peaks are seen for the free origami, and would be great to 
study this further, perhaps in a separate publication.