Detection of short single-strand DNA homopolymers with ultrathin $\mathrm{S}{\mathrm{i}}_3{\mathrm{N}}_4$ nanopores

A series of nanopores with diameters ranging from 2.5 to 63 nm are fabricated on a reduced $\mathrm{S}{\mathrm{i}}_3{\mathrm{N}}_4$ membrane by focused ion beam and high energy electron beam. Through measuring the blocked ionic currents for DNA strands threading linearly through those solid-state nanopores, it is found that the blockade ionic current is proportional to the square of the hydrodynamic diameter of the DNA strand. With the nanopore diameter reduced to be comparable with that of DNA strands, the hydrodynamic diameter of the DNA becomes smaller, which is attributed to the size confinement effects. The duration time for the linear DNA translocation events increases monotonically with the nanopore length. By comparing the spatial configurations of DNA strands through nanopores with different diameters, it is found that the nanopore with large diameter has enough space to allow the DNA strand to translocate through with complex conformation. With the decrease of the nanopore diameter, the folded part of the DNA is prone to be straightened by the nanopore, which leads to the increase in the occurrence frequency of the linear DNA translocation events. Reducing the diameter of the nanopore to 2.5 nm allows the detection and discrimination of three nucleotide “G” and three nucleotide “T” homopolymer DNA strands based on differences in their physical dimensions.

Figure 1

Ultrathin $\mathrm{S}{\mathrm{i}}_3{\mathrm{N}}_4$ nanopore fabrication and characterization. (a) Scheme of a nanopore sensor showing a DNA molecule translocating through the pore. The sensor consists of a $2.5\times 2.5\mathrm{m}{\mathrm{m}}^2$ silicon chip that contains a freestanding $\mathrm{S}{\mathrm{i}}_3{\mathrm{N}}_4$ membrane $(100\times 100\mu {\mathrm{m}}^2)$. After the process of FIB milling a circular area on the thick $\mathrm{S}{\mathrm{i}}_3{\mathrm{N}}_4$ membrane, a nanopore is sputtered using a TEM on the thinned area. (b) AFM topography image of a $4\times 2$ circle array following FIB milling process, as well as a line profile that shows the depth of the trenches. (c) The etched depth is a linear function of the etched time and the etched rate is totally up to the size of the ion beam. The red point is for the focused ion beam operated at 1.1 pA and the blue point is operated at 7.7 pA. (d) Transmission electron micrograph of two typical nanopores: the first one has a diameter at 2 nm fabricated by TEM; the second one is fabricated by FIB. It looks like an oval; the longest length is about 45 nm and the smallest length is about 35 nm.

Figure 2

DNA translocation through different size nanopores. Data are taken in 1 M NaCl solution at 1 V bias voltage and filtered at 10 kHz for display. (a). The spikelike current trace in a 63-nm-diameter, 100-nm-long nanopore. The upper inset shows the amplitude histograms of all events. The lower inset shows examples of amplified signals that are observed, accompanied with the interpretation of the DNA spatial configuration while they translocate the nanopore. (b) The data collected from a 28-nm-diameter, 37-nm-long nanopore. (c) The data correspond to a 12-nm-diameter, 17-nm-long nanopore. (d) The data for an 8-nm-diameter, 28-nm-long nanopore.

Figure 3

(a) Scatter diagram of the amplitude of the ionic current blockade versus translocation time for DNA translocation through four nanopores. (b) Scatter plot of the relative ionic current change versus the duration.

Figure 4

The ratio of the ionic current blockade to the baseline current for DNA translocating nanopores in a linear configuration versus the nanopore diameter. The best fit curve from our fitting procedure is the function of $5.316/x^2$ (black curve). The purple dash-dotted curve corresponds to the theoretical model with the diameter of dsDNA molecules estimated as 2 nm.

Figure 5

The mean duration of unfolded DNA translocation events for DNA translocation through five nanopores with different diameters and lengths measured in 1 M NaCl solution at 1000 mV.

Figure 6

(a) At the beginning, 48 kb $\lambda$-DNA can translocate through a 7-nm-diameter nanopore. (b) With the elapse of the time, 48 kb $\lambda$-DNA will crowd the mouth of the nanopore until they cannot pass anymore. (c) After the base ionic current is stabilized again, 30 nt “G” single-strand DNA homopolymers are added in the cis side chamber. The smaller homopolymers can still pass through the crowded nanopore. (d) Continuous current trace shows the baseline ionic currents decreased step by step due to the $\lambda$-DNA strands accumulated in the nanopore, which is reduced from 32 to 16 nA step by step. (e) Scatter plot of the relative blockade current versus the duration for 48 kb $\lambda$-DNA before the nanopore is crowded and for 30 nt “G” single-strand DNA homopolymers after the nanopore is crowded by the $\lambda$-DNA. The inset shows the $I\text{−}V$ curve before and after the 48 kb $\lambda$-DNA crowded the nanopore.

Figure 7

(a) Continuous current traces for 30 nt “G” (black), 3 nt “G” (red), and 3 nt “T” (blue) translocating through a 2.5-nm-diameter nanopore, respectively, measured at 1000 mV in 1 M NaCl solution. (b) Amplified blockade signals corresponding to the three kinds of DNA homopolymers across the nanopore in (a), respectively. (c) Schematic illustrations of the short DNA strands across the 2.5-nm-diameter nanopore.

Figure 8

Differentiation of short single-strand DNA homopolymers in a 2.5-nm-diameter and 8-nm-long nanopore. (a) Scatter plot of the current blockade versus the duration time of 30 nt “G” and 3 nt “G” homopolymers. (b) The frequency histogram of two kinds of homopolymer blockade amplitude with the Gaussian fit. (c) Histograms of 30 nt “G” and 3 nt “G” translocation time of events. (d) Scatter plot of the blockade current versus the translocation time of three nt “G” and three nt “T” homopolymers. (e) The amplitude histogram of the two kinds of homopolymer translocation events with the Gaussian fit. The amplitude of the blocked current of “G” is located in 0.51 nA, while “T” is located in 0.375 nA. (f) Histograms of three nt “G” and three nt “T” translocation time of events.