Single nucleotide detection using bilayer MoS2 nanopores with high efficiency
Authors: Payel Sen
Manisha Gupta
Journal: RSC Advances (2021)
Discipline: CHEMISTRY
Keywords: Single nucleotide detection; MoS2 nanopores; DNA sequencing; bilayer; monolayer
Affiliations: Department of Chemical and Materials Engineering, University of Alberta, Edmonton, Canada, Department of Electrical and Computer Engineering

Abstract:  Single nucleotide detection is important for early detection of diseases and for DNA sequencing. Monolayer (ML) MoS2 nanopores have been used to identify and distinguish single nucleotides with good signal-to-noise ratio in the recent past. Here, we use a bilayer (BL) MoS2 nanopore (∼1.3 nm thick) to detect distinct single nucleotides with high spatial resolution and longer dwell time. In this study, the performance of similar sized (&lt;3 nm) ML and BL MoS2 nanopores for detection of a single nucleotide has been compared. Both single nucleotide and single stranded DNA translocations through them are studied. For single nucleotide detection, we observe that BL MoS2 nanopores demonstrate twice the dwell time as compared to ML MoS2 nanopores with 95% confidence. Single nucleotide detection rate for BL MoS2 nanopores (50-60 nucleotides per s) is five-fold higher as compared to ML MoS2 nanopores (10-15 nucleotides per s) in 10 pM analyte concentration. For single stranded DNA, we observe 89% (for 60 DNA molecules detected) single nucleotide detection efficiency with BL MoS2 nanopores as compared to 85% for ML MoS2. The DNA sequencing efficiency through BL MoS2 nanopores is also found to be 8-10% better than through ML MoS2 nanopores, irrespective of DNA sequencing orientation. Thus, owing to improved analyte/nanopore charge interaction BL MoS2 nanopores can be used for single nucleotide detection with high resolution due to longer dwell time, detection rate and efficiency. This study demonstrates the improved ability of BL MoS2 nanopores in sequencing DNA with 8-10% higher efficiency, two-times temporally resolved single-nucleotide current signatures and five-times higher detection rate, compared to ML MoS2 nanopores. 

Retracted: No

PubPeer comments:
 In their paper entitled u201cSingle nucleotide detection using bilayer MoS2 nanopores with high efficiencyu201c (RSC Adv., 2021, 11, 6114), Sen and Gupta claim to have made a MoS2 nanopore that is capable of distinguishing among DNA nucleotides. The manuscript has numerous serious issues: 1)tIn figure 2c and 2d the y-axes for current are labeled nA and pA respectively, this may have been a careless typo and panel d should be labeled as nA, but it is surprising that reviewers did not notice this. More importantly, assuming this correction, the slopes of both curves show conductances of 10 nS (in the text the authors note a pore conductance of 10.5 and 17.2 nS) which is enormous for a 300 mM KCl electrolyte and a 2.7 nm diameter pore. One would expect a conductance value of a 1-2 nS for such a small pore and low electrolyte strength. 2)tIn Figure 3 several example traces are shown of transient current blocks purportedly corresponding to single nucleotide translocations. In these examples, the transitions in the currents (from high to low current levels and back from low to high levels) are almost perfect vertical lines (see blue arrow in figure below), indicating that the measured change in current is instantaneous. This cannot be the case in reality, because the current recording is subject to a low-pass filter u2013 the authors mention a 20 kHz 8-pole Bessel filter (actually, the Bessel filter of the multiclamp 700B-amplifier used is actually a 4-pole filter) u2013 so the minimum transition time from high to low current should be roughly u00bd of that inverse frequency (~25 u00b5s). E.g. in the case shown below (Fig. 3 a) the fall and rise times of the current should each take up more than a third of the duration of the total pulse. Based on the signals shown, the transition times from high-level to low-level current (and vice versa) are zero, which is outright impossible. Also, in the examples of Fig. 3 a and c the falling and rising steps of the current pulse appear to have exactly the same amplitude which is not possible in the presence of random noise. This clearly points to graphic manipulation (see below a superposition of the event as shown, and a vertical displacement of the trace shows the data manipulation). 3)tAlso in Fig 3, the authors claim that the events shown are 0.06-0.22 ms long. This is inconsistent with the traces shown, which imply that the bandwidth and sampling rates (maximum 500 kHz for the Digidata ADC digitizer used in this work) are much higher (~1-2 orders of magnitude), so that much shorter events would have been measurable. The traces shown are either fabricated or highly misrepresented as fast events when they really are a lot slower. 4)tIn Figure 3i, the authors plot the current blockade amplitudes for different nucleotides, and they show that these range from 500-2500 pA. These levels are absurdly high for a 200 mV applied voltage, especially given that the pore diameters are significantly larger than the analytes. For reference, expected current levels are in the range of 50-100 pA for nucleotides and these approximate pore geometries. Also, dwell times are surprisingly long, given that the pores are way larger than the small molecule analytes. Finally, the fact that there is zero overlap between nucleotides is remarkably peculiar, as different nucleotides are chemically quite similar, and further, all publications to date show significant overlap between nucleotides, except for this report. This result is surely not credible. 5)tFigure 4a and 4b contain the most ludicrous data. In this figure the authors claim that single nucleotides in a chain are measured. These two traces are only 800 u00b5s long, yet show extremely clearly separated current levels for each nucleotide. This violates basic physics, as it has been shown both experimentally and theoretically that single nucleotide resolution is not possible because of access resistance limitations, even in single layer MoS2 pores. Further, the 800 u00b5s event with 20 kHz bandwidth cannot possibly show ~30 distinct current levels for the 30-mer studied, each one being ~100 u00b5s long, on average. , 
 This paper is ridiculous on several levels :) and data looks made up. It is so good to be true :) especially the sequencing part :), people would have observed it already in previous papers, or inclings of it. Access resistance is not accounted for at all :). It is impossible currently to see difference in ionic current between number of layers (1, 2, 3) :) and this on it s own could be a paper if it can be engineered, and at best claim should be proven... :) - see previous papers on MoS2 pores and start of the art, and also check Feng paper (that you cite and rely heavily on, ref 13) for validity - see comments on Pubpeer. This paper is building on another Reference 13 that has issues which was brought up on PubPeer as well. This is  too good to be true ;), plus numbers are not consistent with basic physics laws, and therefore made up. , 
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 #3 Are you saying one event (rectangular current signal shown above) is from one nucleotide? , 
 Can you calculate the conductance expected from your pores given the pore diameter, pore thickness, access resistance? What do you get? This is not discussed in the paper and compared to your values? How come you can distinguish current from monolayer and bilayer pores, given the access resistance and other terms, while the thickness of bilayer vs. monolayer is really small, plus when you drill the pore may not even be bilayer - what s the edge structure for example - so many details that are not justified? Can you prove all this including expected current and elaborate - I don t see this discussion or explanation in the paper? , 
 The data treatment is completely unclear. What does up-sampling mean? Did they somehow extrapolate new data points? How can that result in steeper falls and rises of the signal? Increasing the bandwidth of a measurement in that way, without increasing noise would be an enormous invention in itself. ,