Calin Plesa

Fast Translocation of Proteins through Solid State Nanopores

Measurements on protein translocation through solid-state nanopores reveal anomalous (non-Smoluchowski) transport behavior, as evidenced by extremely low detected event rates; that is, the capture rates are orders of magnitude smaller than what is theoretically expected. Systematic experimental measurements of the event rate dependence on the diffusion constant are performed by translocating proteins ranging in size from 6 to 660 kDa. The discrepancy is observed to be significantly larger for smaller proteins, which move faster and have a lower signal-to-noise ratio. This is further confirmed by measuring the event rate dependence on the pore size and concentration for a large 540 kDa protein and a small 37 kDa protein, where only the large protein follows the expected behavior. We dismiss various possible causes for this phenomenon and conclude that it is due to a combination of the limited temporal resolution and low signal-to-noise ratio. A one-dimensional first-passage time-distribution model supports this and suggests that the bulk of the proteins translocate on time scales faster than can be detected. We discuss the implications for protein characterization using solid-state nanopores and highlight several possible routes to address this problem.

Direct observation of DNA knots using a solid-state nanopore

Long DNA molecules can self-entangle into knots. Experimental techniques for observing such DNA knots (primarily gel electrophoresis) are limited to bulk methods and circular molecules below 10 kilobase pairs in length. Here, we show that solid-state nanopores can be used to directly observe individual knots in both linear and circular single DNA molecules of arbitrary length. The DNA knots are observed as short spikes in the nanopore current traces of the traversing DNA molecules and their detection is dependent on a sufficiently high measurement resolution, which can be achieved using high-concentration LiCl buffers. We study the percentage of molecules with knots for DNA molecules of up to 166 kilobase pairs in length and find that the knotting occurrence rises with the length of the DNA molecule, consistent with a constant knotting probability per unit length. Our experimental data compare favourably with previous simulation-based predictions for long polymers. From the translocation time of the knot through the nanopore, we estimate that the majority of the DNA knots are tight, with remarkably small sizes below 100 nm. In the case of linear molecules, we also observe that knots are able to slide out on application of high driving forces (voltage).

Data analysis methods for solid-state nanopores

We describe a number of techniques for the analysis of solid-state nanopore ionic current traces and introduce a new package of Matlab analysis scripts with GUI front ends. We discuss methods for the detection of the local baseline and propose a new detection algorithm that bypasses some of the classical weaknesses of moving-average detection. Our new approach removes detected events and re-creates an ideal event-free baseline subsequently used to recalculate the local baseline. Iterative operation of this algorithm causes both the moving average of the baseline current and its standard deviation to converge to their correct values. We explain different approaches to selecting events and building event populations, and we show the value of keeping track of the changes in parameters, such as the event rate and the pore resistance, throughout the course of the experiment. Finally, we introduce a new technique for separating unfolded events and detecting current spikes present within translocation events. This open source software package is available online at: http://ceesdekkerlab.tudelft.nl/downloads/

Detection of Individual Proteins Bound along DNA Using Solid-State Nanopores.

DNA in cells is heavily covered with all types of proteins that regulate its genetic activity. Detection of DNA-bound proteins is a challenge that is well suited to solid-state nanopores as they provide a linear readout of the DNA and DNA-protein volume in the pore constriction along the entire length of a molecule. Here, we demonstrate that we can realize the detection of even individual DNA-bound proteins at the single-DNA-molecule level using solid-state nanopores. We introduce and use a new model system of anti-DNA antibodies bound to lambda phage DNA. This system provides several advantages since the antibodies bind individually, tolerate high salt concentrations, and will, because of their positive charge, not translocate through the pore unless bound to the DNA. Translocation of DNA-antibody samples reveals the presence of short 12 μs current spikes within the DNA traces, with amplitudes that are about 4.5 times larger than that of dsDNA, which are associated with individual antibodies. We conclude that transient interactions between the pore and the antibodies are the primary mechanism by which bound antibodies are observed. This work provides a proof-of-concept for how nanopores could be used for future sensing applications.

Ionic Permeability and Mechanical Properties of DNA Origami Nanoplates on Solid-State Nanopores

While DNA origami is a popular and versatile platform, its structural properties are still poorly understood. In this study we use solid-state nanopores to investigate the ionic permeability and mechanical properties of DNA origami nanoplates. DNA origami nanoplates of various designs are docked onto solid-state nanopores where we subsequently measure their ionic conductance. The ionic permeability is found to be high for all origami nanoplates. We observe the conductance of docked nanoplates, relative to the bare nanopore conductance, to increase as a function of pore diameter, as well as to increase upon lowering the ionic strength. The honeycomb lattice nanoplate is found to have slightly better overall performance over other plate designs. After docking, we often observe spontaneous discrete jumps in the current, a process which can be attributed to mechanical buckling. All nanoplates show a nonlinear current-voltage dependence with a lower conductance at higher applied voltages, which we attribute to a physical bending deformation of the nanoplates under the applied force. At sufficiently high voltage (force), the nanoplates are strongly deformed and can be pulled through the nanopore. These data show that DNA origami nanoplates are typically very permeable to ions and exhibit a number of unexpected mechanical properties, which are interesting in their own right, but also need to be considered in the future design of DNA origami nanostructures.

Velocity of DNA during translocation through a solid state nanopore

While understanding translocation of DNA through a solid-state nanopore is vital for exploiting its potential for sensing and sequencing at the single-molecule level, surprisingly little is known about the dynamics of the propagation of DNA through the nanopore. Here we use linear double-stranded DNA molecules, assembled by the DNA origami technique, with markers at known positions in order to determine for the first time the local velocity of different segments along the length of the molecule. We observe large intramolecular velocity fluctuations, likely related to changes in the drag force as the DNA blob unfolds. Furthermore, we observe an increase in the local translocation velocity toward the end of the translocation process, consistent with a speeding up due to unfolding of the last part of the DNA blob. We use the velocity profile to estimate the uncertainty in determining the position of a feature along the DNA given its temporal location and demonstrate the error introduced by assuming a constant translocation velocity.

Rapid manufacturing of low-noise membranes for nanopore sensors by trans-chip illumination lithography

In recent years, the concept of nanopore sensing has matured from a proof-of-principle method to a widespread, versatile technique for the study of biomolecular properties and interactions. While traditional nanopore devices based on a nanopore in a single layer membrane supported on a silicon chip can be rapidly fabricated using standard microfabrication methods, chips with additional insulating layers beyond the membrane region can provide significantly lower noise levels, but at the expense of requiring more costly and time-consuming fabrication steps. Here we present a novel fabrication protocol that overcomes this issue by enabling rapid and reproducible manufacturing of low-noise membranes for nanopore experiments. The fabrication protocol, termed trans-chip illumination lithography, is based on illuminating a membrane-containing wafer from its backside such that a photoresist (applied on the wafers top side) is exposed exclusively in the membrane regions. Trans-chip illumination lithography permits the local modification of membrane regions and hence the fabrication of nanopore chips containing locally patterned insulating layers. This is achieved while maintaining a well-defined area containing a single thin membrane for nanopore drilling. The trans-chip illumination lithography method achieves this without relying on separate masks, thereby eliminating time-consuming alignment steps as well as the need for a mask aligner. Using the presented approach, we demonstrate rapid and reproducible fabrication of nanopore chips that contain small (12 μm × 12 μm) free-standing silicon nitride membranes surrounded by insulating layers. The electrical noise characteristics of these nanopore chips are shown to be superior to those of simpler designs without insulating layers and comparable in quality to more complex designs that are more challenging to fabricate.

Single-molecule sensing with nanopores

A 70-year-old idea for measuring blood cells has evolved into a powerful, versatile tool for studying DNA, proteins, and other biomolecules.

Self-Aligned Plasmonic Nanopores by Optically Controlled Dielectric Breakdown

We present a novel cost-efficient method for the fabrication of high-quality self-aligned plasmonic nanopores by means of an optically controlled dielectric breakdown. Excitation of a plasmonic bowtie nanoantenna on a dielectric membrane localizes the high-voltage-driven breakdown of the membrane to the hotspot of the enhanced optical field, creating a nanopore that is automatically self-aligned to the plasmonic hotspot of the bowtie. We show that the approach provides precise control over the nanopore size and that these plasmonic nanopores can be used as single molecule DNA sensors with a performance matching that of TEM-drilled nanopores. The principle of optically controlled breakdown can also be used to fabricate nonplasmonic nanopores at a controlled position. Our novel fabrication process guarantees alignment of the nanopore with the optical hotspot of the nanoantenna, thus ensuring that pore-translocating biomolecules interact with the concentrated optical field that can be used for detection and manipulation of analytes.

Nanofabrication Yields. Hybridization and Click-Fixation of Polycyclic DNA Nanoassemblies

We demonstrate the stepwise assembly of a fully addressable polycyclic DNA hexagon nanonetwork for the preparation of a four-ring system, one of the biggest networks yet constructed from tripodal building blocks. We find that the yield exhibits a distinct upper level <100%, a fundamental problem of thermodynamic DNA assembly that appears to have been overlooked in the DNA nanotechnology literature. A simplistic model based on a single step-yield parameter y can quantitatively describe the total yield of DNA assemblies in one-pot reactions as Y = y(duplex)(n), with n the number of hybridization steps. Experimental errors introducing deviations from perfect stoichiometry and the thermodynamics of hybridization equilibria contribute to decreasing the value of y(duplex) (on average y = 0.96 for our 10 base pair hybridization). For the four-ring system (n = 31), the total yield is thus less than 30%, which is clearly unsatisfactory if bigger nanoconstructs of this class are to be designed. Therefore, we introduced site-specific click chemistry for making and purifying robust building blocks for future modular constructs of larger assemblies. Although the present yield of this robust module was only about 10%, it demonstrates a first step toward a general fabrication approach. Interestingly, we find that the click yields follow quantitatively a binomial distribution, the predictability of which indicates the usefulness of preparing pools of pure and robust building blocks in this way. The binomial behavior indicates that there is no interference between the six simultaneous click reactions but that step-yield limiting factors such as topological constraints and Cu(I) catalyst concentration are local and independent.