Nanofluidic Channels for Biological Manipulation and Analysis

 

John T. Mannion and Stephen L. Levy

Department of Applied and Engineering Physics, Cornell University

 

Nanoscale fluid filled channels can be used as tools for manipulating and observing fluorescently labeled DNA molecules.  A DNA molecule with a radius of gyration larger than the nanochannel width can be forced into a channel using externally applied electric fields.  Once inside, it is subject to confinement induced forces which cause it to elongate in the direction of the nanochannel axis.  Here, we report on the dynamics of DNA molecules which initially enter the channel with a looped front end.  Such folded molecules are observed to  spontaneously unfold over a period of time ranging from seconds to minutes and depending on the length of the initially folded portion.
 
Nanofluidic channels have shown great promise as tools for the analysis of genomic length DNA molecules.  Assays for fragment length analysis, real time enzymatic degradation, and localization of hybridized probes have all been demonstrated.  Additionally, detailed studies of the conformation and dynamics of single molecules in nanochannels have corroborated models for self-excluding polymers in confined environments.  Recently, molecular dynamics and monte carlo simulations have been applied to the study of confined polymers and overlapping (but unconnected) polymer chains.  While it is experimentally difficult to position two DNA molecules in a nanochannel so that they partially overlap, it is relatively straightforward to insert a single molecule into a nanochannel such that the front end is folded over on itself.
 
We have achieved this using the strategy depicted in Figure 1.  Long DNA molecules (T4 bacteriophage) are electrophoretically driven through a microchannel towards an array of nanochannels.  Just before entering the channel the field is turned off and the molecules are allowed to sit near the entrance, where thermal agitation causes them to experience a number of different conformations.  When a molecule happens to be in an appropriate conformation and position relative to the channel entrance (as depicted in Figure 1A), the field is turned on and the molecule is driven into the channel, often with a folded front end.  After the entire DNA molecule has entered the channel, the field is switched off and the dynamics of the molecule are observed.  Here we pay particular attention to the spontaneous unfolding of the looped end, a process that we theorize to be entropically induced.
 
All unfolding events are observed with fluorescence microscopy.  Videos are taken using a cooled CCD camera, recorded to disk and analyzed using a cutstom program written in MATLAB.  A time trace graph, showing a single DNA molecule in a nanochannel as it unfolds over time is shown in Figure 2.  The part of the molecule that is looped over on itself is roughly twice as bright as the unlooped portion of the molecule.
 
A simple model, based on the balance of entropic and frictional forces, is depicted in Figure 3.  The entropic force causing separation is thought to be localized to the interface between the looped and unlooped segments, and is constant in magnitude throughout the unfolding process.  Hydrodynamic friction depends on both the length and velocity of a moving segment of DNA.  The equations resulting from a balance of forces were solved numerically, resulting in the plot seen in Figure 4.  The residual between the generated points and the data points was minimized by an algorithm which adjusted the model parameters, allowing for an estimate of the unfolding force magnitude.
  
The nanochannel devices were patterned on a mirror-polished fused silica wafer with a thickness of 500mm (MarkOptics, Santa Ana, CA) using a combination of electron beam and optical lithography.  Both micro- and nanochannels were etched simultaneously using a Reactive Ion Etch process.  Access holes were created by  alumina powder blasting from the backside of the wafer. Finally a 170mmfused silica cover wafer (MarkOptics) was touch bonded and  annealed at 1050_C to the device wafer, enclosing the channels. Nanoports (Upchurch Scientific, Oak Habor, WA) were sealed to the access holes forming buffer reservoirs.
 
 
 
Figure 1.  Overview of experimental procedure.  Left) A long DNA molecule sits in a microchannel, next to the entrance of a nanochannel.  Middle Left) The electric field (blue arrow) pulls the DNA into the nanochannel.  Because molecule’s entrance was initiated at some point along the backbone (not by one of the two ends), it enters the channel in a looped conformation. Middle Right) The electric field is turned off and the DNA strand is allowed to sit inside the nanochannel, in a high energy looped state.  It gradually unfolds, thereby reducing its conformational free energy.  Right) Molecule has completely unfolded and remains in the channel, extended in its equilibrium conformation.
 
 
Figure 2.  Time trace of a fluorescently labeled DNA molecule confined to a nanofluidic channel.  Each column of pixels in the time trace image represents the fluorescence intensity along the axis of the channel in one movie frame.  Many of these vertical lines are placed side by side to produce the image showing the position, length, and contour of the DNA molecule in the channel over the course of the entire movie clip.  The molecule in this time trace initially has a looped front end, with the folded segment of the molecule being more intense than the unfolded part.  Over the course of 35 seconds this molecule is observed to spontaneously unfold.  White dots show the end points of the folded segment of the molecule as identified by an automated image analysis routine.
 
 
 
Figure 3.  Unfolding model.  The points x1, x2, x3 are the endpoints of the folded and unfolded portions of the molecule.  The separation force is localized at the point x2, and results from the difference in free energy between a conformation in which the blue segment overlaps with the looped end and a conformation in which the blue segment slides left, no longer overlapping with the looped end.  The resulting forces, Fs and -Fs, are equal in magnitude and opposite in direction.  They act on the upper and lower segments of the molecule in a localized manner as depicted.  The upper and lower segments are accelerated until two frictional forces resist the motion.  The hydrodynamic frictional forces act on the entire upper and lower segments.  The forces are proportional to the length and velocity of each segment.
 
 
Figure 4.  Fit of numerical solution of the unfolding model to data. 
Plotted in red is the length of the folded part, for a given frame, and
plotted in blue is the end to end length the whole molecule.  By fitting the model, it is possible to extract the parameter which is the ratio of the unfolding force to the hydrodynamic friction factor per unit length of DNA.  Using a previously measured value for  (Mannion 2006), the unfolding force is determined to be on the order of .
 
Reference
 
Mannion, J. T., C. H. Reccius, et al. (2006). "Conformational analysis of single DNA molecules undergoing entropically induced motion in nanochannels." Biophysical Journal 90(12): 4538-4545.