Voltage-driven translocation of DNA through a high throughput conical solid-state nanopore

Abstract

Nanopores have become an important tool for molecule detection at single molecular level. With the development of fabrication technology, synthesized solid-state membranes are promising candidate substrates in respect of their exceptional robustness and controllable size and shape. Here, a 30-60 (tip-base) nm conical nanopore fabricated in 100 nm thick silicon nitride (Si(3)N(4)) membrane by focused ion beam (FIB) has been employed for the analysis of λ-DNA translocations at different voltage biases from 200 to 450 mV. The distributions of translocation time and current blockage, as well as the events frequencies as a function of voltage are investigated. Similar to previously published work, the presence and configurations of λ-DNA molecules are characterized, also, we find that greater applied voltages markedly increase the events rate, and stretch the coiled λ-DNA molecules into linear form. However, compared to 6-30 nm ultrathin solid-state nanopores, a threshold voltage of 181 mV is found to be necessary to drive DNA molecules through the nanopore due to conical shape and length of the pore. The speed is slowed down ∼5 times, while the capture radius is ∼2 fold larger. The results show that the large nanopore in thick membrane with an improved stability and throughput also has the ability to detect the molecules at a single molecular level, as well as slows down the velocity of molecules passing through the pore. This work will provide more motivations for the development of nanopores as a Multi-functional sensor for a wide range of biopolymers and nano materials.

Figure 1. The Schematic illustrations of the microfluidic setup and nanopore detection. A:

Schematic illustration of the microfluidic setup. The ionic solution is separated into two isolated reservoirs by the insulating silicon nitride membrane containing a single nanopore. A couple of Ag/AgCl electrodes which connected to the patch clamp amplifier are placed in each of the two reservoirs. Inset is a SEM image of the nanopore fabricated by FIB, with a scale bar of 100 nm. The red circle of 30 nm is used to represent the pore at tip side, while the blue circle of 60 nm is for the pore at base side. B: Schematic diagram of single DNA molecule translocating through a nanopore, which results a downward spike in current trace (top inset). The sidewall angle (73°) is calculated and shown in red. Two main parameters: time duration of the blockage (t_d) and magnitude of the blockage (ΔI) are shown for a selected single molecule event. I–V curve of the conical nanopore is inserted at the bottom, which is smoothed using Savitzky-Golzy method (solid line), showing a typical non-linearity feature. C: Current trace recorded at 100 mV and 200 mV, after addition of the λ-DNA molecules, the current shows no spikes when 100 mV is applied (top), whereas a series of events is observed at 200 mV (down). It indicates there is a threshold of electric forces to impel the DNA chain through the pore.

Figure 2. Event scatter plot of current blockage vs translocation time of λ-DNA translocation events as a function of voltage.

A typical normalized histogram of 400 mV is inserted on the bottom-right. The experiments were all performed in 1 M KCl solution with 10 mM TrisHCl and 1 mM EDTA at pH of 8.0.

Figure 3. Current blockage histograms as a function of applied voltage.

A: For comparison all histograms are normalized as shown in the picture, by fitting all the histograms with Gaussian, a increase of the means of the histograms as a function of voltage can be clearly visualized. B: The plot of the means of the Gaussian fits of the current blockage histograms as a function of voltage, which is fitted by a line, with a slope of ∼0.85 pA/V and a intercept of 181 mV at voltage axis. It indicates that the current blockage increases with the applied voltage, and a threshold voltage of 181 mV.

Figure 4. Simulation of the electric potential and field distributions of the nanopore in two dimensions.

Left: Color coded potential distribution and electric field lines (white) of the 100 nm thick conical nanopore for a applied voltage of 300 mV. Right: A close-up view of the black rectangle area, integrated with the electric field strength () along the center (axis Z) of the pore, where the Z at the tip side of pore is set as 0. A asymmetric electric field is formed due to the conical shape of the pore, where the electric field strength () is higher at the tip side than the base side.

Figure 5. The simulations of the electric field as a function of the conical pore thickness. A:

Color coded potential distribution and electric field lines (white) of 50 nm, 100 nm, 150 nm, 200 nm, 250 nm thick conical nanopores with a same tip pore size (30 nm) and sidewall angle (73°), where the applied voltage is 300 mV. For a better comparison, all the Z at the tip pore center are set as 0. B: The electric field strengths () of the five pores as a function of Z (−300 nm–500 nm). C: The electric field strengths () at a set of distances from the base pore as a function of pore thickness. The inset is a schematic illustration of the nine distances from the pore.

Figure 6. Characteristic signals of translocation events.

Four types of typical multiple level translocation events generated by corresponding configurations of DNA molecule, left to right: linear, double local folded, single local folded and fully folded fragments of DNA molecules, respectively.

Figure 7. Translocation time histograms and the velocities as a function of applied voltage. A:

Normalized histograms of λ-DNA translocation events as a function of voltage are fitted by Gaussian, and aligned for better comparison. A typical current trace with interpretation of events judgment (red square line) of each voltage is insert on the right respectively, from which the events at each voltage can be well distinguished. The means of the Gaussian fits are plotted as a function of voltage (right axis), with a linear fit. B: The plot of the velocity (mm/s-left axis, bp/µs-right axis) as a function of voltage.

Figure 8. Translocation events frequencies (events per second) versus applied voltage.

Translocation events frequency (▪) of λ-DNA (∼300 µg/mL, 1 M KCl TE solution at pH of 8.0) through the pore as a function of voltage, which is fitted by a line (red). The inset is a pot of the percentage of linear form translocation events (•) as a function of voltage. the blue line is a exponential fit.