Detecting single stranded DNA with a solid state nanopore

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Original Entry: Peter Foster, AP 225, Fall 2011

General Information

Authors: Daniel Fologea, Marc Gershow, Bradley Ledden, David S. McNabb, Jene A. Golovchenko, & Jiali Li

Publication: Fologea et al. Detecting single stranded DNA with a solid state nanopore. Nano letters (2005) vol. 5 (10) pp. 1905-1909

Keywords: nanopore, DNA sequencing, biomolecule detection, electrophoresis

Summary

The point of this paper was to detail the behavior of DNA as it passes through a synthetic silicon nitride nanopore. Two reservoirs containing an aqueous ionic glycerol solution were connected by a pore with a 4 nm diameter. An electrode was placed in each reservoir and a constant bias voltage was applied across the nanopore causing an ionic current to flow and drawing the negatively charged DNA from the upper reservoir where it was initially loaded through the nanopore and into the lower reservoir. A schematic drawing of DNA transversing the nanopore and a drawing of the experimental setup is shown in Figure 1.


With the bias voltage applied, the flowing current was measured as a function of time. When DNA passed through the nanopore, it blocked curent flow. Several sample current vs. time diagrams are shown in the insets of figure 2. Figure 2 shows a two dimensional histogram with the vertical axis being the current blockage (the depth of the wells in the current vs. time in the insets) and the horizontal axis being the event duration (the width of the current vs. time wells in the insets.) The experiments were carried out at a pH of 7 and at a pH of 13. This was done because double stranded DNA is stable at pH 7, but at pH 13 double stranded DNA denatures into single stranded DNA. This was verified in the paper using optical absorbance measurements.


From the distribution shown in figure 2, it seems like most of the events that have event durations less than the most probable time have an event blockage that is much larger than the most probable. This is interpreted as having the DNA pass through the nanopore in a folded conformation. Thus, if we look at events that take half the most probable event duration, they for the most part have twice the most probable event blockage. This corresponds to the DNA being folded in half. The hyperbolae plotted in the figures are lines of constant event charge deficit (ecd) defined as the product between the event blockage and the event duration. Events with event durations less than the most probable duration seem to figure this curve fairly well.

Figure 1, taken from [1] (Fig 1 a & b).


Figure 2, taken from [1] (Fig 2).


Discussion

The point of this paper is to be a first step towards fast and cheap genetic sequencing. One of the paths towards this goal involves using a synthetic nanopore as in this paper. The idea is to pass DNA through the pore and use some change in electrical property (resistance, capacitance, etc.) to identify the base passing though the pore. Using a synthetic nanopore as opposed to a naturally occurring biological one comes with the advantage of stability. Synthetic materials are stable over a wider range of pH, temperatures, etc. then biological pores. This is important because for sequencing, single stranded DNA would have to be used. DNA is made of only four bases (A,C,T, and G) and they have unique pairings (A with T and C with G). Thus, if double stranded DNA passed through pore, and the sequence at some point had the signature of the AT base pair, there would be no way to discern which base was on which strand. In other words, you wouldn't be able to distinguish A,T,C, and G separately, only the combinations AT or CG. One can melt double stranded DNA into single stranded DNA with an increase of temperature or pH moving the problem to finding a stable pore. With the use of a synthetic (stable) nanopore, this approach is more feasible and progress can be made.


References

[1] Fologea et al. Detecting single stranded DNA with a solid state nanopore. Nano letters (2005) vol. 5 (10) pp. 1905-1909