Electrically facilitated translocation of protein through solid nanopore
© Wu et al.; licensee Springer. 2014
Received: 16 February 2014
Accepted: 14 March 2014
Published: 24 March 2014
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.
KeywordsProtein translocation Solid state nanopore Current blockage Translocation time
Over past decades, nanopores have been widely evolved in various devices for investigating unlabeled biopolymers at the single-molecule level [1, 2]. Although the focus is on nucleic acids, proteins are becoming a prime target for investigation [3, 4]. Protein transport through the cellular compartments is a very important physiological process for substance and energy metabolism of living cells [5–7]. Compared with DNA sequencing, protein translocation through nanopores is more challenging. First, proteins have a variety of charge profiles depending on the solvent environment. When pH is lower than the isoelectric point of proteins, the net charge of protein is positive, while the reverse case is negatively charged [8, 9]. Second, each protein has a unique structural architecture, including the primary peptide chain, secondary, tertiary, and quaternary structures, which are responsible for their biological functions. Yet the native protein conformation is only marginally stable. Once the protein’s physical and chemical environment is modestly changed, the rigid structure of a protein will unfold into random coils [8, 10]. These features of proteins are distinct from the linear DNA with a uniform negative charge. Thus, nanopore experiments on proteins are more complicated than the DNA sequencing.
Yet for all that, a set of experiments have demonstrated the unique and advantageous ability of nanopores to discriminate protein translocations [9–14], protein folding [10, 13, 15–18], and enzymatic kinetic reactions [19–26] in the context of single-molecule analysis. For example, nanopores have been used to discriminate the surface charge and size of proteins as a function of pH [27–29]. The unfolding transition and structural stability of proteins have also been studied by chemical and thermal denaturation, as well as electric field stretching [3, 10, 13, 15, 30]. An interesting phenomenon is non-specific adsorption interaction between proteins and nanopores, whereby the protein sticks to the pore for a prolonged dwell time [18, 31, 32]. These results closely depend on the quality and geometry of the nanopores used, most of which focus on the small nanopores with the dimension comparable to the analyzers to achieve an optimal solution. Even so, the capture rate of proteins is low in nanopore experiments, and the electroosmotic flow against electrophoretic mobilities of proteins through silicon nitride membranes is dominant in small nanopores [9, 10, 18, 27, 33, 34]. Meanwhile, the adsorption interaction of proteins easily makes the small pore plugged [31, 32]. Therefore, to reduce these negative effects, nanopores with a larger scale are an alternative choice to analyze the varied targets. First, the arriving probability of protein in pore mouth is governed by free diffusion in bulk, which is referred to the pore geometry [9, 35]. A higher capture rate is expected for large nanopores . And both electroosmotic effect and protein-pore interaction corresponding to the electric double layer along the charged inner wall will be weakened in large nanopores; thus, more proteins will freely pass through nanopores [36, 37]. Additionally, more space in large nanopores is in favor of the surface modification to change the physical and chemical properties of pores [38, 39], which will broadly expand the utility of nanopores for biological sensing. Certainly, the signal-to-noise ratio of the blockade current will inevitably deteriorate if the pore is too large. Hence, the choice of nanopore with a suitable dimension is critical for the design of nanopore devices to understand the physical mechanism of molecules translocating through nanopores.
Herein, bovine serum albumin (BSA), an important transport protein, is chosen to pass through a silicon nitride nanopore with a diameter of 60 nm. By applying a set of biased voltages, the protein swims through the large channel with a detectable signal-to-noise ratio of the blockage current. Comparing with small nanopores, a higher threshold voltage of 300 mV is observed to drive the protein into the nanopore. With the voltage increasing, the current blockage events are greatly enhanced and are classified as a function of voltages. At the medium-voltage region, the amplitude of blockage current increases linearly while the dwell time decreases exponentially with the increasing voltage. Despite more free space in our large nanopore, the adsorption and desorption phenomenon of proteins has also been detected with a prolonged dwell time, but it is greatly weakened compared with small nanopore cases. With further increasing voltage, the protein is more likely to be destabilized by the applied electric forces. And a couple of proteins can pass through the nanopore simultaneously. Together, the experiments yield a new aspect of protein transport through a solid-state nanopore with a large scale. The results may help in the future development of nanopore devices such as single-molecule sorting, dynamic molecular interaction, and controllable self-assembly of molecules.
Chemicals and materials
Pure (>98%) crystallized BSA from Fraction V was purchased from Sigma-Aldrich (St. Louis, MO, USA) and used without further purification. All other chemical reagents used in our experiment were of analytical grade without further purification. All samples were prepared by Milli-Q super purified water with resistance >18 MΩ/cm (Millipore, Billerica, MA, USA). All solutions were filtered with 0.02-μm Anotop filter (Whatman, Maidstone, UK) before using. Nanopores were hydrated with the addition of degassed and filtered KCl electrolyte solution buffer. Electrolyte strength was typically 1 M/1 M KCl cis/trans in protein translocation studies.
The schematic of the experimental setup is shown in Figure 1a. The nanopore-containing chip encapsulated with two PDMS films was immersed in ionic solutions, which was then divided into two isolated reservoirs; 1 M KCl salt solution was added into the two isolated reservoirs. Two Ag/AgCl electrodes were inserted into the reservoirs, respectively, and connected to a patch clamp amplifier (Axon Instruments, Axopatch 700B, Molecular Devices, Sunnyvale, CA, USA). The ionic current was filtered at 10 kHz and sampled using a 16-bit DAQ card (National Instruments, Austin, TX, USA) for a better signal-to-noise ratio, operated with homemade LabVIEW software. The whole fluidic device was put in a Faraday cage for shielding electromagnetic noise. In order to clean the chip and increase the hydrophilicity of nanopores, silicon nitride chips were soaked in piranha solution (30% H2O2/H2SO4 1:3 (v/v)) for 30 min at 80°C and then rinsed with double-purified water. The dried chip is ready for nanopore experiments.
Results and discussion
Detection of protein translocations
Here dp and lp are the diameter and length of the pore, respectively, μ is the electrophoretic mobility, D is the protein diffusion coefficient, and φ is the biased voltage. This shows that the capture radius grows with the pore diameter and the biased voltages, and a bigger capture area can make more proteins trapped into the nanopore. Thus, a high throughput is expected in our nanopore device, which is also confirmed in our studies behind.
In addition, it is worth to mention the current noise in solid-state nanopores, which involves the 1/f-type excess noise and other contributions [45, 46]. The 1/f-type noise is related to the fluctuation of charge carriers. As the voltage increases, the accelerated motion of charge carries will cause local ion aggregation in the nanopore, resulting in the increase of 1/f-type excess noise. It can be confirmed from the noise power spectra observed in our experiment (not shown here) and other experiments [45, 46].
Protein transport at the medium-voltage region
where σ is the solution conductivity, φ is the applied voltage between the electrodes, Λ is the excluded volume of a translocation molecule inside the pore, Heff is the effective length of the nanopore, dm is the diameter and lm is the length of a particle molecule, Dp is the average diameter of a cylindrical nanopore, and is a correction factor that depends primarily on the relative geometry of the molecule and the pore [47, 48]. Since the spherical-shaped protein is much smaller than the large nanopore, contributes little to the current drop. Thus, ΔIb can be simplified as ΔIb(t) ~ Λφ, implying a linear dependence of the current blockade on the biased voltage. And the excluded volume of proteins in the pore can be calculated from the current drop. Based on the equation, the estimated volume of BSA in our experiments is about 260 nm3, which is very close to that of the native BSA structure (224 nm3) . The volume change is less than 15%; thus, the unfolding of the protein destabilized by electric field forces can be ignored in the medium voltage from 300 to 600 mV, which appears in small nanopores due to the intensive electric field inside the pore [10, 18].
Protein transport at the high-voltage region
An intriguing question is the origin of the multiple peaks of current blockage that occurred at high voltages. First, a possible mechanism is related to the unfolding state of the protein disrupted by the enhanced electrical force, which is a common phenomenon observed in small nanopores [3, 10]. Serum exhibits a heterogeneous charge distribution along its backbone, which allows for individual amino acids to be pulled in opposite directions. The electric force acting on each charged residue of the protein molecule is given by Fi = Qi × E, where Qi is the charge carried by each residue and E is the electric field strength , while the summation of Fi is the global driving force applied on the whole protein. The electric force acting on the protein is more than 10 piconewtons (pN) at high voltages above 700 mV. As proteins can be destabilized by elongation forces of several piconewtons based on the force spectroscopy measurements [50–52], the protein is potentially stretched into unfolding state with increasing voltages in the nanopore. Based on excluded volume values estimated from the main peaks at high voltages, the maximal volume change of protein is up to 50% in our high voltage experiments, which indicates that the protein has been stretched into an extended conformation by increased electric forces. Additionally, the excluded volume derived from the minor peak is about twofold of that from the main peak. The substantial growth of current amplitude is not merely the structural change of a single protein. Then we propose that the main peak with low magnitude is described by one protein (partial or full denatured state) entering the pore, and the minor peak with high magnitude is described by two molecules passing through the nanopore at the same time. The dimension of the nanopore is about five times as large as the protein, which allows multiple proteins to simultaneously pass through the nanopore. Especially, the stronger electric forces drive more molecules rapidly towards the nanopore. Thus, there is a higher probability of multiple molecules together entering into the pore at high voltages.
Protein capture rates depending on voltages
Here R0 ∝ f* exp(−U*/kBT) is the zero voltage capture rate controlled by an activation barrier U * of entropic and electrostatic effect (f * is a frequency factor). The ratio |V|/V0 is a barrier reduction factor due to the applied voltage. The potential V0 corresponds to the necessary applied potential to allow a charged protein to overcome the Brownian motion. From the fitted exponential function, we obtain R0 = 3.01 ± 1.1 Hz and V0 = 268 ± 8.9 mV. The voltage value is close to the threshold of 300 mV obtained in our measurement, which is necessary to drive the protein into the nanopore. It is known that the protein translocation through the nanopore is involved in the completion of the electroosmotic flow and electrophoretic mobility. The electroosmotic flow will suppress the penetration of the negatively charged proteins into silicon nitride pores, and its velocity increases with the electrical field. As the electroosmotic effect is dominant in small nanopores, the capture rate would decrease with the applied voltage increasing. However, an exponential increase of capture rate is observed as a function of voltages in our experiment. Thus, the electroosmotic effect is minor in our experiment with a large nanopore. With the increasing voltages, more protein is crowded at the pore entrance. Hence, the phenomenon of two molecules entering into the pore simultaneously occurs due to the high electric potential and large dimension of the nanopore.
In summary, electrically facilitated protein translocation through a large nanopore has been investigated in our work. A large number of current blockage events are detected above the voltage of 300 mV. The distribution of the current magnitude and dwell time of the transition events are characterized as a function of applied voltages. Major proteins rapidly pass through the pore in a short-lived form, while minor long-lived events are observed with a prolonged time. With the increase of voltages, the current amplitude linearly increases while the dwell time is exponentially decreased. Meanwhile, the capture rate of proteins is greatly enhanced with an exponential growth. The protein absorption phenomenon and electroosmotic flow, which are dominant in small pores, are also compared in our work. These phenomena are weakened in large nanopores, especially at high voltages. Based on the excluded volume theory, the conformational change of proteins is estimated during the translocation process, and the unfolding of the protein stretched by intensive electric forces is confirmed at high voltages. The results show a new aspect of protein transport through a solid-state nanopore with a large size, which can provide more motivation for the development of nanopore devices as multifunctional sensors to analyze a wide range of biopolymers and nanomaterials.
bovine serum albumin
focused ion beam
scanning electron microscopy.
The work was supported by the National Natural Science Foundation of China (Nos. 61071050, 61101056, and 61372031), National Basic Research Program of China (2011CB707600), China Postdoctoral Science Foundation (No. 20110491339), Tsinghua National Laboratory for Information Science and Technology (TNList) Cross-discipline Foundation, and Research Fund for the Doctoral Program of Higher Education of China (No. 20110092130003).
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