Session P25: Focus Session: DNA and Protein Analysis with Micro and Nanofluidics

Learning from the Jersey Turnpike: Cell Lysis, Labeling and Washing with Microfluidic Metamaterials

Directing objects across functional streamlines at low Reynolds number is difficult but important since this motion can be used to label, lyse, and analyze complex biological objects on-chip without cross-contamination. Here we use an asymmeteric post array to move cells across coflowing reagents and show on-chip, immunofluorescent labeling of platelets with washing and \emph{E.Coli} cell lysis with simultaneous separation of bacterial chromosome from the cell contents. Furthermore, we develop the concept of a microfluidic metamaterial by using the basic asymmetric post array as a building block for complex particle handling modes. These modular array elements could be of great use for developing robust techniques for on-chip, continuous flow manipulation and analysis of cells, large bio-particles, and functional beads.   

DNA Docking with Functionalized Colloidal Probes

The docking of DNA with probe-functionalized microparticles remains inadequately understood, despite the significance of DNA hybridization-based technologies for high-throughput screening for genetic analysis and biomedical diagnostics. In this work, we employ fluorescence correlation spectroscopy (FCS) and confocal microscopy to examine DNA-colloid interaction and resulting conformational structures of DNA oligomers with oligonucleotide-functionalized colloidal probes, whose particle size varies from 100 nm to 3 um. We observe that the docking efficiency strongly depends on DNA length, colloid size and surface functionality. Optimal probe size and temperature are found for rapid hybridization. We conjecture that the resulting structure of DNA at the interface of colloidal probes is determined by both steric effects and DNA charge condensation. If time permits, we will discuss our recent work on the docking of elongated DNA with functional probes by imposed dielectrophoretic forces in the presence of AC fields.   

Confinement spectroscopy: A novel approach to force spectroscopy

In physics DNA is readily described by its mechanical properties, neglecting its chemical composition. By studying variations in these properties conclusions can be drawn about the interaction between DNA and both its environment and also ligand binding to DNA. These investigations are normally performed by force spectroscopy using optical or magnetic tweezers associated with an elaborate experimental setup. We introduce confinement spectroscopy, as a complementary technique, where a continuously variable degree of spatial restriction is applied to the molecule in a fluidic funnel-like geometry. By driving the molecule along the funnel, an extension versus confinement curve is obtained. This curve contains not only information regarding the molecule elasticity, but also new details concerning the self- and surface-interactions of the molecule. It is also easily integrated into lab-on-a-chip devices.   

Fluorescence microscopy studies of the DNA motion near voltage biased solid-state nanopores

A solid state nanopore in a Si-based thin insulating membrane works as a single DNA molecule sensing device that provides the information of the length and the folding configuration of the DNA by measuring ionic currents when the DNA translocates through the pore [1]. These nanopores may play a significant role in molecular electronics and rapid DNA sequencing. Now one of the issues related to this nanopore sensing technique is clogging the nanopores by DNA molecules because it significantly extends the DNA translocation time. To elucidate this issue, we use time-resolved fluorescence microscopy to observe the DNA motion near voltage biased nanopores. We will discuss the DNA motion near nanopores under the various applied voltages. [1]. J. Li M. Gershow, D. Stein, E. Brandin, and J.A. Golovchenko, Nature Materials 2: 611 (2003); T. Mitsui, D. Stein, Y.-R. Kim, D. Hoogerheide, and J.A. Golovchenko, Phys. Rev. Lett. 96: 036102-1 (2006)   

Influence of polymer-pore interactions on translocation

We investigate the influence of polymer-pore interactions on the translocation dynamics using Langevin dynamics simulations$^2$. An attractive interaction can greatly improve translocation probability. At the same time, it also increases translocation time slowly for weak attraction while exponential dependence is observed for strong attraction. For fixed driving force and chain length the histogram of translocation time has a transition from Gaussian distribution to long-tailed distribution with increasing attraction. Under a weak driving force and a strong attractive force, both the translocation time and the residence time in the pore show a non-monotonic behavior as a function of the chain length. Our simulations results are in good agreement with recent experimental data$^3$.\newline \newline $^1$ This work was supported by TransPoly Consortium grants.\newline $^2$ K. Luo, T. Ala-Nissila, S. C. Ying, and A. Bhattacharya, Phys. Rev. Lett. {\bf 99}, 148102 (2007).\newline $^3$ O. V. Krasilnikov {\em et al.}, Phys. Rev. Lett. {\bf 97}, 018301 (2006); A. Meller {\em et al.}, Proc. Natl. Acad. Sci. U.S.A. {\bf 97}, 1079 (2000).   

Nanofilters for high throughput DNA separation

Nanofilters are a novel class of microfabricated devices for rapidly separating short, rigid DNA. The succession of alternating narrow slits ($\sim $50nm) and deep wells ($\sim $300nm) is used to trap the DNA, which then escape at a size-dependent rate. Experiments and near-equilibrium theoretical arguments both indicate that smaller DNA travel faster in a weak field, but the separation fails at around 100V/cm. We theoretically show that the speed and performance of the device can be enhanced using high fields of several hundred V/cm. Based on scaling arguments, the separation of short, rod-like DNA molecules at high fields occurs via ``torque-assisted escape,'' which originates from the non-uniform electric field at the slit entrance. The quadratic dependence of the torque on the molecule size indicates that larger molecules will now emerge first; under a high field, the device operates in a band-inverted manner. Brownian dynamics simulation results confirm the mobility increase with size, with a quasi-plateau at very large fields.   

Rapid DNA Idetification by Dielectrophoresis of Nanocolloids

Due to their size and number, dispersed oligo-functionalized nanocolloids can reduce the diffusion length/docking time and increase the sensitivity of ssDNA hybridization reactions by orders of magnitude compared to immobilized probes. We find that, for long target ssDNAs, their docked conformation is a sensitive function of the nanocolloid size, surface charge, functionalized probe density and number of docked DNAs per bead. Three distinct conformations (collapsed, stretched and condensed) are detected via independent light scattering, Zeta potential, dielectrophoresis (DEP) and electron micrograph techniques. By optimizing the hybridization conditions to produce a stretched conformation, we are able to significantly change the DEP cross-over frequency of hybridized beads, thus allowing rapid label-free detection of hybridization by simple impedance techniques down to pM concentrations.   

The non-driven polymer translocation through a nanopore: relaxation and translocation are not decoupled

Most theoretical models describing the translocation of a polymer chain through a nanopore use the hypothesis that the polymer remains in an equilibrium random coil conformation during the process. In other words, models generally assume that the characteristic relaxation time of the chain is small enough compared to the translocation time that non-equilibrium polymer conformations can be ignored. We present Molecular Dynamics simulations that directly test this hypothesis by looking at the escape time of unbiased polymer chains starting with different initial conditions. We find that the chains are deformed for the systems studied, even though the translocation time is about 10 times larger than the relaxation time. Our most striking result is the observation that the last half of the chain escapes in less than 12\% of the total escape time, which implies that there is a large entropy-driven acceleration of the chain at the end of its escape from the channel.   

Digital DNA: Physics of DNA in Nanopit Lattices

Controlling the on-chip organization and conformation of DNA is important for a number of interrelated nanotechnology disciplines. We introduce a new type of nanostructure consisting of a nanoslit with a built in spatial modulation of confinement created by arrays of embedded nanopits. Nanopits are square depressions in a 50-100 nm deep nanoslit with a width in the range of 100-500 nm and a depth of 100 nm. A DNA molecule placed in a nanopit lattice will spontaneously adopt a `digitized' conformation consisting of filled nanopits connected by fluctuating linkers. By adjusting the spacing, organization and placement of the nanopits it is possible to immobilize DNA at predetermined regions of device without additional chemical modification and achieve a high degree of control over local DNA conformation. We will present results from fluorescence microscopy experiments on the equilibrium behavior and dynamics of DNA in such structures and interpret these results in terms of a simple statistical mechanical model.   

Dynamics of DNA molecules confined to slit-like nanofluidic channels

We experimentally investigate the dynamics of DNA in confined spaces. This is not only important for a better understanding of the behavior of DNA as a biologically relevant molecule but also helps us to test general polymer dynamics models. Fluorescently stained DNA molecules are inserted into slit-like nanofluidic channels. The channel material is fused silica and the channels are fabricated using a bonding process. We take fluorescent images of the DNA in channels of different heights and measure the projected size of the DNA molecules fitted by an ellipse. Furthermore, we measure the relaxation times derived from the autocorrelation function of the size. If the channel height is smaller than twice the radius of gyration of the DNA molecules (R$_{g}$ = 700 nm) both parameters agree with the predictions of de Gennes. For even smaller channels with a height less than twice the persistence length of stained DNA (L$_{p}$ = 60 nm) the dynamics resemble the predictions made by Odijk for this regime.   

Electrokinetic transport at a nanocapillary/microchannel interface

Coupled electrokinetic transport phenomena play a central role in concentration polarization near the interface between a permselective nanocapillary membrane and a microchannel. Here the effects of ion concentration and potential distribution on transport through a finite-length nanocapillary are studied using both semi-empirical and fundamental models. The fundamental models are based on the coupled electrohydrodynamic transport equations for multiple charged species in aqueous solution. The semi-empirical models describe average species and fluid fluxes through the respective regions.   

Water-encapsulated protein source for x-ray serial crystallography

A reliable source of micron size water droplets has been constructed for the purpose of delivering water-encapsulated protein for x-ray serial crystallography. A linear stream of droplets of negligible divergence is produced by accelerating a liquid water jet through a high pressure gradient.......[1] inside a converging gas nozzle. Using a co-flowing gas rather than the nozzle walls to squeeze the liquid jet to smaller diameter eliminates the problem of clogging that has thus far limited the minimum size of Rayleigh nozzle jets [2]. We examine the nozzle shape effects on the dripping-jetting transition and drop size. Supported by NSF award IDBR 0555845 and ARO award DAAD190010500. [1] Ganan-Calvo, A.M. and A. Barrero, \textit{A novel pneumatic technique to generate steady capillary microjets.} Journal of Aerosol Science, 1999. \textbf{30}(1): p. 117-125. [2] http://arxiv.org/abs/physics/0701129   

Separation of DNA in nanoscale devices with alternating channel depth.

The size-dependent separation of DNA using nanofabricated devices consisting of alternating deep and shallow regions have been the subject of numerous experimental and theoretical works. Recent Brownian dynamics simulations suggest that the separation of rigid-rod DNA can be effected at high electric fields without a loss of resolution (PRL, 2007, 98, 098106). To study the dynamics of DNA separation at high fields, electrophoresis experiments were carried out using DNA fragments up to 753 bp in size. As the transport mechanism of DNA fragments in gels has been shown to be a strong function of topology (Electrophoresis, 2004, 25, 1772), electrophoresis of branched rigid-rod DNA molecules was performed to investigate the effects of analyte architecture on mobility in nanofabricated devices. By comparing the mobility of branched and linear DNA molecules of identical total molecular weight, we exclude the influence of size and charge and focus on the effects of branch size and location, and overall analyte topology. Our results help to elucidate the electrophoretic migration mechanism of DNA molecules with complex architecture in sieving media with precisely-controlled internal structures.