pH tuning of DNA translocation time through organically functionalized nanopores

Abstract

Controlling DNA translocation speed is critically important for nanopore sequencing as free electrophoretic threading is far too rapid to resolve individual bases. A number of promising strategies have been explored in recent years, largely driven by the demands of next-generation sequencing. Engineering DNA-nanopore interactions (known to dominate translocation dynamics) with organic coatings is an attractive method as it does not require sample modification, processive enzymes, or complicated and expensive fabrication steps. In this work, we show for the first time 4-fold tuning of unfolded, single-file translocation time through small, amine-functionalized solid-state nanopores by varying the solution pH in situ. Additionally, we develop a simple analytical model based on electrostatic interactions to explain this effect which will be a useful tool in designing future devices and experiments.

Figure 1

DNA translocation before and after APTMS coating. a. Schematic of an uncoated and an APTMS-coated solid-state nanopore with DNA threading from cis to trans. Inset is a TEM image of a 5.2 nm nanopore before coating. b. I-V curves before (black) and after (red) coating a 5.7 nm pore in 1M KCl pH 8.0. c. normalized blockade current (I_B) histograms measured using 4 kbp DNA under the same conditions as in panel b for uncoated and coated nanopores (black, n = 1525 and red, n = 1628).

Figure 2

Translocation time through a coated pore can be sped up or slowed down by changing pH. a. 1 kbp DNA translocating through a coated 5.2 nm pore at pH 6.0 (red), 7.0 (green), and 8.0 (blue) and characterized by open-pore current i_o, blocked-level current i_b, and dwell time t_D. b. I_B histograms at pH 6.0, 7.0, and 8.0. c. t_D histograms at pH 6.0, 7.0, and 8.0 with corresponding exponential timescales t_pH6 = 118 μs, t_pH7 = 46 μs, and t_pH8 = 29 μs (n = 1000-3000 events analyzed for each pH in the same pore).

Figure 3

Tuning translocation time for three lengths of DNA (1 kbp, 4 kbp, and 10 kbp) in 5.6 ± 0.1 nm coated pores at pH 6.0 (red) and 8.0 (blue). Main figure displays I_B vs. t_D event diagrams (>1000 events per experiment) and lower insets show normalized t_D histograms.

Figure 4

Relative dwell times vs. pH for 1 kbp DNA and different pore diameters. The dwell times are normalized to their corresponding value at pH 8.0 to allow comparison under different conditions. Squares represent a coated 5.2 nm pore, solid circles a coated 5.6 nm pore, and open circles an uncoated 5.4 nm pore. Lines serve as guides to the eye.

Figure 5

a. Stages of the DNA translocation process. b. Simple cylindrical model of DNA-nanopore electrostatic interactions.

Figure 6

a. Free-energy landscape during DNA translocation. b. Electrostatic barrier as a function of the number of bases inside the pore.

Figure 7

Translocation time τ (arbitrary units) as a function of ε.