Stefan W. Kowalczyk

DNA translocation through graphene nanopores.

Nanopores--nanosized holes that can transport ions and molecules--are very promising devices for genomic screening, in particular DNA sequencing. Solid-state nanopores currently suffer from the drawback, however, that the channel constituting the pore is long, approximately 100 times the distance between two bases in a DNA molecule (0.5 nm for single-stranded DNA). This paper provides proof of concept that it is possible to realize and use ultrathin nanopores fabricated in graphene monolayers for single-molecule DNA translocation. The pores are obtained by placing a graphene flake over a microsize hole in a silicon nitride membrane and drilling a nanosize hole in the graphene using an electron beam. As individual DNA molecules translocate through the pore, characteristic temporary conductance changes are observed in the ionic current through the nanopore, setting the stage for future single-molecule genomic screening devices.

Modeling the conductance and DNA blockade of solid-state nanopores

We present measurements and theoretical modeling of the ionic conductance G of solid-state nanopores with 5-100 nm diameters, with and without DNA inserted into the pore. First, we show that it is essential to include access resistance to describe the conductance, in particular for larger pore diameters. We then present an exact solution for G of an hourglass-shaped pore, which agrees very well with our measurements without any adjustable parameters, and which is an improvement over the cylindrical approximation. Subsequently we discuss the conductance blockade ΔG due to the insertion of a DNA molecule into the pore, which we study experimentally as a function of pore diameter. We find that ΔG decreases with pore diameter, contrary to the predictions of earlier models that forecasted a constant ΔG. We compare three models for ΔG, all of which provide good agreement with our experimental data.

Slowing down DNA translocation through a nanopore in lithium chloride

The charge of a DNA molecule is a crucial parameter in many DNA detection and manipulation schemes such as gel electrophoresis and lab-on-a-chip applications. Here, we study the partial reduction of the DNA charge due to counterion binding by means of nanopore translocation experiments and all-atom molecular dynamics (MD) simulations. Surprisingly, we find that the translocation time of a DNA molecule through a solid-state nanopore strongly increases as the counterions decrease in size from K(+) to Na(+) to Li(+), both for double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA). MD simulations elucidate the microscopic origin of this effect: Li(+) and Na(+) bind DNA stronger than K(+). These fundamental insights into the counterion binding to DNA also provide a practical method for achieving at least 10-fold enhanced resolution in nanopore applications.

Detection of Local Protein Structures along DNA Using Solid-State Nanopores

Nanopores have been successfully employed as a new tool to rapidly detect single biopolymers, in particular DNA. When a molecule is driven through a nanopore by an externally applied electric field, it causes a characteristic temporary change in the trans-pore current. Here, we examine the translocation of DNA with discrete patches of the DNA-repair protein RecA attached along its length. Using the fact that RecA-coated DNA and bare DNA yield very different current-blockade signatures, we demonstrate that it is possible to map the locations of the proteins along the length of a single molecule using a solid-state nanopore. This is achieved at high speed and without any staining. We currently obtain a spatial resolution of about 8 nm, or 5 RecA proteins binding to 15 base pairs of DNA, and we discuss possible extensions to single protein resolution. The results are a crucial first step toward genomic screening, as they demonstrate the feasibility of reading off information along DNA at high resolution with a solid-state nanopore.

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.

Translocation of RecA-Coated Double-Stranded DNA through Solid-State Nanopores

We report translocation of double-stranded DNA (dsDNA) molecules that are coated with RecA protein through solid-state nanopores. Translocation measurements show current-blockade events with a wide variety in time duration (10-4-10-1 s) and conductance blockade values (3-14 nS). Large blockades (11.4+/-0.7 nS) are identified as being caused by translocations of RecA-dsDNA filaments. We confirm these results through a variety of methods, including changing molecular length and using an optical tweezer system to deliver bead-functionalized molecules to the nanopore. We further distinguish two different regimes of translocation: a low-voltage regime (<150 mV) in which the event rate increases exponentially with voltage, and a high-voltage regime in which it remains constant. Our results open possibilities for a variety of future experiments with (partly) protein-coated DNA molecules, which is interesting for both fundamental science and genomic screening applications.

Biomimetic nanopores: learning from and about nature

Through recent advances in nanotechnology and molecular engineering, biomimetics - the development of synthetic systems that imitate biological structures and processes - is now emerging at the nanoscale. In this review, we explore biomimetic nanopores and nanochannels. Biological systems are full of nano-scale channels and pores that inspire us to devise artificial pores that demonstrate molecular selectivity or other functional advantages. Moreover, with a biomimetic approach, we can also study biological pores, through bottom-up engineering approaches whereby constituent components can be investigated outside the complex cellular environment.

Unraveling Single-Stranded DNA in a Solid-State Nanopore

Solid-state nanopores are an emerging class of single-molecule sensors. Whereas most studies so far focused on double-stranded DNA (dsDNA) molecules, exploration of single-stranded DNA (ssDNA) is of great interest as well, for example to employ such a nanopore device to read out the sequence. Here, we study the translocation of long random-sequence ssDNA through nanopores. Using atomic force microscopy, we observe the ssDNA to hybridize into a random coil, forming blobs of around 100 nm in diameter for 7 kb ssDNA. These large entangled structures have to unravel, when they arrive at the pore entrance. Indeed, we observe strong blockade events with a translocation time that is exponentially dependent on voltage, tau approximately e(-V/V(0)). Interestingly, this is very different than for dsDNA, for which tau approximately 1/V. We report translocations of ssDNA but also of ssDNA-dsDNA constructs where we compare the conductance-blockade levels for ssDNA versus dsDNA as a function of voltage.

Measurement of the docking time of a DNA molecule onto a solid-state nanopore.

We present measurements of the change in ionic conductance due to double-stranded (ds) DNA translocation through small (6 nm diameter) nanopores at low salt (100 mM KCl). At both low (<200 mV) and high (>600 mV) voltages we observe a current enhancement during DNA translocation, similar to earlier reports. Intriguingly, however, in the intermediate voltage range, we observe a new type of composite events, where within each single event the current first decreases and then increases. From the voltage dependence of the magnitude and timing of these current changes, we conclude that the current decrease is caused by the docking of the DNA random coil onto the nanopore. Unexpectedly, we find that the docking time is exponentially dependent on voltage (t ∝ e(-V/V(0))). We discuss a physical picture where the docking time is set by the time that a DNA end needs to move from a random location within the DNA coil to the nanopore. Upon entrance of the pore, the current subsequently increases due to enhanced flow of counterions along the DNA. Interestingly, these composite events thus allow to independently measure the actual translocation time as well as the docking time before translocation.

Salt and Voltage Dependence of the Conductance Blockade Induced by Translocation of DNA and RecA Filaments Through Solid-state Nanopores

Amongst many other applications, solid-state nanopores – tiny holes in a thin membrane – can be used for rapid single-molecule high-throughput label-free detection of small samples of, for example, DNA or DNA-protein complexes. The interpretation of the current signal that one observes when DNA transverses a nanopore is a central point in all nanopore work. However, a complete and thorough understanding is still lacking.