Detecting single stranded DNA with a solid state nanopore

Abstract

Voltage biased solid-state nanopores are used to detect and characterize individual single stranded DNA molecules of fixed micrometer length by operating a nanopore detector at pH values greater than approximately 11.6. The distribution of observed molecular event durations and blockade currents shows that a significant fraction of the events obey a rule of constant event charge deficit (ecd) indicating that they correspond to molecules translocating through the nanopore in a distribution of folded and unfolded configurations. A surprisingly large component is unfolded. The result is an important milestone in developing solid-state nanopores for single molecule sequencing applications.

Figure 1

a) Schematic illustration of DNA molecule translocating through a solid-state nanopore. b) Experimental setup for single molecule measurements with a nanopore detector. c) TEM of a silicon nitride nanopore, in this case 4 nm in diameter in a ~ 5–10 nm thick local membrane.

Figure 2

a) Event density plot vs. translocation time and average blockage current for 3 kb ss-DNA in pH 7 electrolyte solution. b) pH 13 electrolyte solution. The dashed lines are hyperbolae of constant event charge deficit (see text). In figure 2(b) the green outline of the data from 2(a) is presented to aid comparison. The color scale represents the probability density for a single event to occur at a given translocation time and blockage. Insets show representative time traces for individual events (i – unfolded molecule, ii – partially folded molecule, iii – completely doubled over molecule). The bounding box on each inset event is 400 us wide and 500 pA tall.

Figure 3

Plot of mean current blockage (for simple, unfolded, single level) events and DNA optical absorbance as a function of pH showing transition from ds to ss-DNA at pH ~11.5. The square markers represent the data from figure 2, while the triangle and diamond markers represent experiments in two other pores including that in Figure 1(b). The dashed line is a guide to the eye and not a fit.