Electrically facilitated translocation of protein through solid nanopore

Abstract

Nanopores have been proven as versatile single-molecule sensors for individual unlabeled biopolymer detection and characterization. In the present work, a relative large nanopore with a diameter of about 60 nm has been used to detect protein translocation driven by a series of applied voltages. Compared with previous studied small nanopores, a distinct profile of protein translocation through a larger nanopore has been characterized. First, a higher threshold voltage is required to drive proteins into the large nanopore. With the increase of voltages, the capture frequency of protein into the nanopore has been markedly enhanced. And the distribution of current blockage events is characterized as a function of biased voltages. Due to the large dimension of the nanopore, the adsorption and desorption phenomenon of proteins observed with a prolonged dwell time has been weakened in our work. Nevertheless, the protein can still be stretched into an unfolded state by increased electric forces at high voltages. In consideration of the high throughput of the large nanopore, a couple of proteins passing through the nanopore simultaneously occur at high voltage. As a new feature, the feasibility and specificity of a nanopore with distinct geometry have been demonstrated for sensing protein translocation, which broadly expand the application of nanopore devices.

Figure 1

Schematic illustrations of the microfluidic setup and nanopore detection. (a) Schematic illustration of the microfluidic setup. A nanopore connects two compartments filled with an electrolyte solution (1 M/1 M KCl cis/trans), separated by a silicon nitride membrane. The application of an electric potential difference via two Ag/AgCl electrodes generates an ionic current through the pore. (b) A SEM image of approximately 60-nm nanopore fabricated by FIB, with a scale bar of 100 nm. (c) The schematic conformation of bovine serum albumin (BSA). Serum is a negatively charged globular protein with 583 residues and consists of three domains (I, II, III); the hydrodynamic diameter of the native state is about 10 nm measured with dynamic light scattering at neutral condition.

Figure 2

Time recording of current traces, contour of electric field distribution, and electric field strength. (a) Time recording of current traces recorded at 100, 300, and 600 mV of biased voltages. As a positive voltage was applied across the SiN membrane, a uniform, event-free open-pore current was recorded. The low noise in the baseline measurement allowed reliable identification of current blockages. After addition of protein in the cis reservoir, downward current pulses were observed at 300 and 600 mV. With the increase of voltages, the occurrence frequency of transition events was greatly improved. (b) Contour of electric field distribution of the cylindrical nanopore with a diameter of 60 nm as a function of biased voltages. (c) Electric field strength along the center axis of the pore.

Figure 3

Current blockage histograms as a function of applied voltage at medium voltages. The histograms of current amplitude are normalized by fitting with Gaussian distribution; a linear increase of the means of current amplitude as a function of voltage can be clearly visualized in the inset. The numbers of translocation events at 300, 400, 500, and 600 mV are 102, 123, 156, and 160, respectively.

Figure 4

Current blockage histograms as a function of applied voltage at medium voltages. The histograms of time duration are fitted by exponential distribution. An exponential function of dwell time versus voltage is defined in the inset.

Figure 5

Representative current blockades of translocation events at medium voltages. In type I, the negatively charged protein will flash past the nanopore under strong electric forces within the nanopore. In types II and III, the protein is absorbed in the pore and around the pore mouth, respectively, for several milliseconds and then driven through the nanopore.

Figure 6

Current blockage histograms as a function of applied voltage at high voltages. (a) The histograms of current amplitude are normalized at voltages of 700, 800, and 900 mV. Multiple peaks with greater amplitude appear. (b) The histograms of time duration are fitted by Gaussian distribution at voltages of 700, 800, and 900 mV.

Figure 7

Typical examples of translocation events at high voltages. In type I, the negatively charged protein fast passes through the nanopore driven by the strong electric forces. In type II, a couple of molecules simultaneously pass through the nanopore.

Figure 8

The capture rate as a function of voltages. The relationship of capture rate versus voltages is well fitted by an exponential function.