Modulating DNA Translocation by a Controlled Deformation of a PDMS Nanochannel Device (original) (raw)
Related papers
Conformational Manipulation of DNA in Nanochannels Using Hydrodynamics
Macromolecules, 2013
The control over DNA elongation in nanofluidic devices holds great potential for large-scale genomic analysis. So far the manipulation of DNA in nanochannels has been mostly carried out with electrophoresis and seldom with hydrodynamics, although the physics of soft matter in nanoscale flows has raised considerable interest over the last decade. In this report the migration of DNA is studied in nanochannels of lateral dimension spanning 100 to 500 nm using both actuation principles. We show that the relaxation kinetics are 3-fold slowed down and the extension increases up to 3-folds using hydrodynamics. We propose a
Elongation and migration of single DNA molecules in microchannels using oscillatory shear flows
Lab on a Chip, 2009
Much of modern biology relies on the strategic manipulation of molecules for creating ordered arrays prior to high throughput molecular analysis. Normally, DNA arrays involve deposition on surfaces, or confinement in nanochannels; however, we show that microfluidic devices can present stretched molecules within a controlled flow in ways complementing surface modalities, or extreme confinement conditions. Here we utilize pressure-driven oscillatory shear flows generated in microchannels as a new way of stretching DNA molecules for imaging "arrays" of individual DNA molecules. Fluid shear effects both stretch DNA molecules and cause them to migrate away from the walls becoming focused in the centerline of a channel. We show experimental findings confirming simulations using Brownian dynamics accounting for hydrodynamic interactions between molecules and channel-flow boundary conditions. Our findings characterize DNA elongation and migration phenomena as a function of molecular size, shear rate, oscillatory frequency with comparisons to computer simulation studies.
The ability to precisely control the transport of single DNA molecules through a nanoscale channel is critical to DNA sequencing and mapping technologies that are currently under development. Here we show how the electrokinetically driven introduction of DNA molecules into a nanochannel is facilitated by incorporating a three-dimensional nanofunnel at the nanochannel entrance. Individual DNA molecules are imaged as they attempt to overcome the entropic barrier to nanochannel entry through nanofunnels with various shapes. Theoretical modeling of this behavior reveals the pushing and pulling forces that result in up to a 30-fold reduction in the threshold electric field needed to initiate nanochannel entry. In some cases, DNA molecules are stably trapped and axially positioned within a nanofunnel at sub-threshold electric field strengths, suggesting the utility of nanofunnels as force spectroscopy tools. These applications illustrate the benefit of finely tuning nanoscale conduit geometries, which can be designed using the theoretical model developed here.
Pressure-driven DNA transport across an artificial nanotopography
2009
The pressure-driven transport of DNA was studied in slit-like nanochannels with an embedded nanotopography consisting of linear arrays of nanopits. We imaged individual DNA molecules moving single-file down the nanopit array, undergoing sequential pit-to-pit hops using fluorescence video microscopy. Distinct transport dynamics were observed depending on whether a molecule could occupy a single pit, or was forced to subtend multiple pits. We interpret these results in terms of a scaling theory of the free energy of polymer chains in a linear array of pits. Molecules contained within a single pit are predicted to face an entropic free energy barrier, and to hop between pits by thermally activated transport. Molecules that subtend multiple pits, on the other hand, can transfer DNA contour from upstream to downstream pits in response to an applied fluid flow, which lowers the energy barrier. When the trailing pit completely empties, or when the leading pit reaches its capacity, the energy barrier is predicted to vanish, and the low-pressure, thermally activated transport regime gives way to a high-pressure, dissipative transport regime. These results contribute to our understanding of polymers in nanoconfined environments, and may guide the development of nanoscale lab-on-a-chip applications.
The effect of dextran nanoparticles on the conformation and compaction of single DNA molecules confined in a nanochannel was investigated with fluorescence microscopy. It was observed that the DNA molecules elongate and eventually condense into a compact form with increasing volume fraction of the crowding agent. Under crowded conditions, the channel diameter is effectively reduced, which is interpreted in terms of depletion in DNA segment density in the interfacial region next to the channel wall. Confinement in a nanochannel also facilitates compaction with a neutral crowding agent at low ionic strength. The threshold volume fraction for condensation is proportional to the size of the nanoparticle, due to depletion induced attraction between DNA segments. We found that the effect of crowding is not only related to the colligative properties of the agent and that confinement is also important. It is the interplay between anisotropic confinement and osmotic pressure which gives the elongated conformation and the possibility for condensation at low ionic strength. depletion ͉ dextran ͉ fluorescence ͉ nanofluidics ͉ nanoparticles A substantial fraction of the total volume of biological media is occupied by macromolecules, which do not directly participate in biochemical reactions. Nevertheless, it is now well established that these background species have an important effect on molecular transport, reaction rates, and chemical equilibrium (1). The steric repulsion between impenetrable macromolecules in a crowded medium is a major factor in determining the thermodynamic activities of the reactants. Crowding by an inert osmotic agent can also affect macromo-lecular structure. A well known example is the transition of DNA to a compact form (condensation) in the presence of overthresh-old concentrations of simple neutral polymers and simple salts (2, 3, 4). It has been proposed that macromolecular crowding is the basis for phase separation in the cytoplasm (5) and condensation of DNA into the nucleoid of bacterial cells (6). The latter hypothesis is supported by the observation that DNA can be condensed by cytoplasmic extracts from Escherichia coli at extract concentrations corresponding to about 1 ⁄2 the cellular concentration (7). Besides background species, the cytoplasm of most eukaryotic cells contains stationary elements such as fiber lattices and membranes. These structures affect macromolecular conformation through confinement in 1D or 2D. Accordingly, macromolecular crowding and confinement are intimately related and deserve an integrated approach to understand their modes of operation and how they couple. DNA condensation can be assisted and directed by a surface. In surface directed condensation, DNA is first adsorbed onto an interface, after which it is condensed with an agent. Examples that have been reported in the literature are the condensation of single molecules into rods and toroidal structures with prota-mine or ethanol (8, 9). Single DNA molecules can be confined and visualized with f luorescence microscopy in quasi 1D nanochannels. The extension in the longitudinal direction of these channels has been measured as a function of channel diameter (10, 11) and ionic strength of the supporting medium (12, 13). Other studies have focused on the dynamics to elucidate the variation in the extension due to thermal fluctuation (14) or the response of the molecular motion to entropic, electro-phoretic, and frictional forces (15). To the best of our knowledge, the effect of macromolecular crowding on the configurational properties of a single DNA molecule confined in a nanochannel has not been reported before. Here, we report the effect of the generic crowding agent dextran on the conformation and condensation of DNA confined in long, straight, and rectangular nanochannels with a depth of 300 nm and a width in the range 150-300 nm. Due to the cross-sectional diameter of a few hundred nm, the elongated DNA molecules remain coiled (16). The advantage of such a configuration is that the data can be interpreted using well established polymer theory, including the effects of the local bending rigidity (persistence length) and interaction of spatially close segments which are separated over a long distance along the contour (excluded volume or self-avoidance). Dextran is a neutral branched polysaccharide made of glucose monomers. The dextran molecules used in the present study have a radius of gyration R g in the range 2.6-17 nm. They behave as spherical nanoparticles, which readily dissolve in water. Because of their inert behavior, dextran is often used as crowding agent to mimic the intracellular crowded environment in vitro (1). Our experiments are done using nanofluidic devices made of poly-(dimethyl siloxane) (PDMS). We have used a lithography process with proton beam writing to fabricate a nanopatterned stamp (17, 18). The stamp was subsequently replicated in PDMS, followed by curing and sealing with a glass slide (19). The advantage of this method is that around 100 chips can be replicated using a single stamp, which allows the use of a fresh chip for every experiment. To visualize the DNA molecules with fluorescence microscopy, it is necessary to stain them with a dye. The intercalation ratio was 23 base-pairs per YOYO-1 molecule. For such a low level of intercalation, the distortion of the secondary DNA structure is minimal; the contour length has increased from 57 to 60 m and the DNA charge is reduced by a factor 42/46 (20, 21). Furthermore, there is no appreciable effect on the bending rigidity, as inferred from previously reported measurements of the extension of DNA in nanochan-nels with different concentrations of dye (13). T4-DNA (166 kbp) molecules were brought into the nanochannels with an electric field and their extensions were measured in buffer of various ionic strength and in the presence of various volume fractions of dextran. We have also monitored the condensation
Transverse electric field dragging of DNA in a nanochannel
Scientific Reports, 2012
Nanopore analysis is an emerging single-molecule strategy for non-optical and high-throughput DNA sequencing, the principle of which is based on identification of each constituent nucleobase by measuring trans-membrane ionic current blockade or transverse tunnelling current as it moves through the pore. A crucial issue for nanopore sequencing is the fact that DNA translocates a nanopore too fast for addressing sequence with a single base resolution. Here we report that a transverse electric field can be used to slow down the translocation. We find 400-fold decrease in the DNA translocation speed by adding a transverse field of 10 mV/nm in a gold-electrode-embedded silicon dioxide channel. The retarded flow allowed us to map the local folding pattern in individual DNA from trans-pore ionic current profiles. This field dragging approach may provide a new way to control the polynucleotide translocation kinetics. N anopore sequencing is an emerging non-optical technology for high-throughput real-time single-molecule sequencing 1-3 . The basic idea is to identify each nucleobase by the size (or the electronic structure) through detecting a trans-pore ionic current blockade 4-8 (or transverse tunnelling current 3,9-14 ) during DNA translocation through a pore. A longstanding challenge for nanopore sequencing has been to slow down the flow speed of polynucleotides in the pore so as to achieve single-base spatial resolution 6-17 . While retarding of translocation and concomitant Å ngstrom precision has recently been achieved in bioengineered nanopores by using a polymerase-DNA complex 6 , the system instability and limited pore-size selectivity of biological systems remains to be a critical issue for practical applications 1 . On the other hand, solid-state nanopores can serve as a robust and configurable single-molecule sensing platform. We present herein an electric-field dragging approach for retarding DNA translocation in a solid-state nanopore. We find that a transverse electric field of 10 mV/nm in an electrode-embedded silicon dioxide nanochannel slows down the biopolymer translocation velocity by more than two orders of magnitude. In addition, we observe field-induced unfolding of DNA. The results presented in this study suggest the usefulness of transverse field for providing essential conditions for ''sequencing by tunnelling'': slow translocation of unfolded DNA through an electrode gap.
DNA detection with a polymeric nanochannel device
Lab on a Chip, 2011
We present the development and the electrical characterization of a polymeric nanochannel device. Standard microfabrication coupled to Focused Ion Beam (FIB) nanofabrication is used to fabricate a silicon master, which can be then replicated in a polymeric material by soft lithography. Such an elastomeric nanochannel device is used to study DNA translocation events during electrophoresis experiments. Our results demonstrate that an easy and low cost fabrication technique allows creation of a low noise device for single molecule analysis.
Pressure-driven transport of confined DNA polymers in fluidic channels
Proceedings of the National Academy of Sciences, 2006
The pressure-driven transport of individual DNA molecules in 175-nm to 3.8-m high silica channels was studied by fluorescence microscopy. Two distinct transport regimes were observed. The pressure-driven mobility of DNA increased with molecular length in channels higher than a few times the molecular radius of gyration, whereas DNA mobility was practically independent of molecular length in thin channels. In addition, both the Taylor dispersion and the self-diffusion of DNA molecules decreased significantly in confined channels in accordance with scaling relationships. These transport properties, which reflect the statistical nature of DNA polymer coils, may be of interest in the development of ''lab-on-a-chip'' technologies. nanofluidics T ransport of DNA and proteins within microf luidic and nanof luidic channels is of central importance to ''lab-ona-chip'' bioanalysis technology. As the size of f luidic devices shrinks, a new regime is encountered where critical device dimensions approach the molecular scale. The properties of polymers like DNA often depart significantly from bulk behavior in such systems because statistical properties or finite molecular size effects can dominate there. DNA confinement effects have been exploited in novel diagnostic applications such as artificial gels (1), entropic trap arrays (2), and solidstate nanopores (3, 4). These advances underline the importance of exploring the fundamental behavior of f lexible polymers in f luid f lows and channels (5-10) that underlie current and future f luidic technologies.
DNA analysis by single molecule stretching in nanofluidic biochips
Microelectronic Engineering, 2011
Stretching single DNA molecules by confinement in nanofluidic channels has attracted a great interest during the last few years as a DNA analysis tool. We have designed and fabricated a sealed micro/nanofluidic device for DNA stretching applications, based on the use of the high throughput NanoImprint Lithography (NIL) technology combined with a conventional anodic bonding of the silicon base and
Amplified stretch of bottlebrush-coated DNA in nanofluidic channels
Nucleic acids research, 2013
The effect of a cationic-neutral diblock polypeptide on the conformation of single DNA molecules confined in rectangular nanochannels is investigated with fluorescence microscopy. An enhanced stretch along the channel is observed with increased binding of the cationic block of the polypeptide to DNA. A maximum stretch of 85% of the contour length can be achieved inside a channel with a cross-sectional diameter of 200 nm and at a 2-fold excess of polypeptide with respect to DNA charge. With site-specific fluorescence labelling, it is demonstrated that this maximum stretch is sufficient to map large-scale genomic organization. Monte Carlo computer simulation shows that the amplification of the stretch inside the nanochannels is owing to an increase in bending rigidity and thickness of bottlebrush-coated DNA. The persistence lengths and widths deduced from the nanochannel data agree with what has been estimated from the analysis of atomic force microscopy images of dried complexes on s...