Stefan W Kowalczyk

Delft University of Technology, Delft, South Holland, Netherlands

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Publications (15)146.85 Total impact

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    ABSTRACT: Measurements on protein translocation through solid-state nanopores reveal anomalous (non-Smoluchowski) transport behavior, as evidenced by extremely low detected event rates, i.e., 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 kDa 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 timescales 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.
    Nano Letters 01/2013; · 13.03 Impact Factor
  • Stefan W Kowalczyk, Cees Dekker
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    ABSTRACT: 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.
    Nano Letters 07/2012; 12(8):4159-63. · 13.03 Impact Factor
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    ABSTRACT: 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.
    Nano Letters 02/2012; 12(2):1038-44. · 13.03 Impact Factor
  • Biophysical Journal 01/2012; 102(3):728-. · 3.67 Impact Factor
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    Stefan W Kowalczyk, Timothy R Blosser, Cees Dekker
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    ABSTRACT: 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.
    Trends in Biotechnology 08/2011; 29(12):607-14. · 9.66 Impact Factor
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    ABSTRACT: 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.
    Nanotechnology 08/2011; 22(31):315101. · 3.84 Impact Factor
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    ABSTRACT: Nuclear pore complexes regulate the selective exchange of RNA and proteins across the nuclear envelope in eukaryotic cells. Biomimetic strategies offer new opportunities to investigate this remarkable transport phenomenon. Here, we show selective transport of proteins across individual biomimetic nuclear pore complexes at the single-molecule level. Each biomimetic complex is constructed by covalently tethering either Nup98 or Nup153 (phenylalanine-glycine (FG) nucleoporins) to a solid-state nanopore. Individual translocation events are monitored using ionic current measurements with sub-millisecond temporal resolution. Transport receptors (Impβ) proceed with a dwell time of ∼2.5 ms for both Nup98- and Nup153-coated pores, whereas the passage of non-specific proteins is strongly inhibited with different degrees of selectivity. For pores up to ∼25 nm in diameter, Nups form a dense and low-conducting barrier, whereas they adopt a more open structure in larger pores. Our biomimetic nuclear pore complex provides a quantitative platform for studying nucleocytoplasmic transport phenomena at the single-molecule level in vitro.
    Nature Nanotechnology 06/2011; 6(7):433-8. · 31.17 Impact Factor
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    Biophysical Journal 01/2011; 100(3). · 3.67 Impact Factor
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    ABSTRACT: 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.
    Nano Letters 08/2010; 10(8):3163-7. · 13.03 Impact Factor
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    ABSTRACT: 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.
    Nano Letters 03/2010; 10(4):1414-20. · 13.03 Impact Factor
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    Stefan W Kowalczyk, Adam R Hall, Cees Dekker
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    ABSTRACT: 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.
    Nano Letters 11/2009; 10(1):324-8. · 13.03 Impact Factor
  • A. R. Hall, S. W. Kowalczyk, C. Dekker
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    ABSTRACT: The translocation of molecules through nanometer-scale apertures has garnered much attention as a future sequencing method. Many challenges remain, including the high spatial and temporal resolution needed to do so. We examine the spatial limits of these measurements by translocating partially complexed RecA nucleoprotein filaments. These are dsDNA polymerized with discrete RecA protein patches of random length, ranging from a few monomers to full coverage (average length ˜10 kbp). With these molecules, we use nanopores for the first time to map the location of features along the length of a single molecule. We show that resolution of less than 500 bp is achieved and discuss the implications on translocation measurements.
    03/2009;
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    ABSTRACT: In recent years, solid state nanopores have emerged as a productive novel technique for molecular biophysics. The electrophoretic motion of single molecules through these small-scale structures can offer insights into both conformation and charge structure. Here, we apply the method to the RecA nucleoprotein filament - a conformation where proteins polymerize along the entire length of a double-stranded DNA. This offers a unique geometry and charge structure which we probe through a combination of translocation experiments and optical tweezer measurements. We discuss conductance blockade events that are notably larger (12 nS) than those measured for bare dsDNA (1 nS), and present force spectroscopy data showing a high level of charge screening in solution (>90%).
    03/2009;
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    ABSTRACT: 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.
    Nano Letters 01/2009; 9(9):3089-96. · 13.03 Impact Factor
  • Biophysical Journal 01/2009; 96(3). · 3.67 Impact Factor