Continuous Confinement Fluidics: Getting Lots of Molecules into Small

Mar 25, 2016 - Confining DNA molecules with high throughput and structural integrity is an important challenge in nanochannel-based genomic mapping ...
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Continuous Confinement Fluidics: Getting Lots of Molecules into Small Spaces with High Fidelity Mohammed Jalal Ahamed,*,† Sara Mahshid,†,‡ Daniel J. Berard,† François Michaud,† Rob Sladek,‡ Walter W. Reisner,*,† and Sabrina R. Leslie*,† †

Department of Physics, McGill University, Montreal, QC H3A 2T8, Canada Department of Human Genetics, McGill University, Montreal, QC H3A 0G1, Canada

‡

ABSTRACT: Confining DNA molecules with high throughput and structural integrity is an important challenge in nanochannel-based genomic mapping technology. Here we demonstrate dynamic confinement and linearization of DNA polymers within an embedded nanogroove array with 95% channel occupation. In standard nanofluidic technology, the free energy of confinement experienced by the DNA molecules increases strongly with decreasing device dimensions, leading to a suppression of molecule concentration in nanoconfined spaces. We overcome this limitation by combining “convex lens-induced confinement” (CLiC) geometry with in situ electrophoresis, simultaneously establishing gentle and continuously adjustable nanoconfinement and precise electrokinetic control. Together, these capabilities enable trapping and visualization of extended DNA molecules with high yield over an extended range of conditions. We demonstrate 10-fold increased DNA concentration in a confined region from 10 to 500 nm. Moreover, we develop and validate a predictive model for describing electrokinetically controlled molecule enhancement in continuously varying nanoconfinement. We show that electrophoretic side loading, in continuously varying confinement, not only enhances DNA concentration but also contributes to extend to their full contour length.

1. INTRODUCTION The ability to confine single molecules in nanometer-size spaces provides an essential means for sensing, imaging, manipulating, and understanding the dynamics of molecular complexes.1−11 Developing technologies that can control and analyze single molecules is the key to discovering mechanistic insights into complex diseases like cancer, with myriad applications to genomics12 and drug discovery. Importantly, single-molecule approaches can capture rare events or states, including those that signal the onset of a disease state but are obscured in bulk analysis. Through parallel and continuous device operation, a large number of single-molecule measurements can be acquired to access not only ensemble-level information but also the variation of single-molecule properties across a biologically, chemically, or physically defined distribution of states. In genomic research, developing highfidelity methods to trap, linearize, and visualize hundreds of single DNA molecules with high throughput is key to enabling mapping of genomes extracted from as few as single. In particular, accessing heterogeneous and repetitive regions of the genome presents a challenge to existing techniques.12 In free solution DNA molecules adopt a random coil configuration. Confining a macromolecule into a small space alters its preferred conformation. In particular, when confined to a channel with dimensions below the molecule’s free solution gyration radius the DNA molecules will stretch out along the channel,6,13,14 creating a one-to-one correspondence between sequence position and position along the molecule’s extended length that is highly advantageous for efficient © XXXX American Chemical Society

mapping. In addition, the extended molecules are free to translate along the channel, permitting continuous cycling of molecules into and out of the channels. Lastly, nanochannels can be densely packed into a single optical field, enabling mapping of many molecules in one camera acquisition time. Nanochannel-confinement studies have achieved many scientific insights into polymer physics and taken greater steps toward DNA sequencing and mapping.6 While classic nanofluidic technology offers powerful tools for single-molecule confinement and manipulation, it has a significant Achilles’ heel: creating high macromolecular concentrations in small spaces is challenging. In a strongly nanoconfined region, macromolecules experience a high free energy penalty and the solution volume will be drastically depleted, leading to a lower effective 2D molecule concentration. In this paper, we combine the “convex lens-induced confinement” (CLiC) approach to achieve tunable nanoscale confinement with open-faced nanogrooves and controlled electrophoresis. This work newly introduces electrophoresis control within the CLiC platform and achieves up to 95% occupancy of nanochannel arrays in massively parallel DNA loading. Three design strategies can be envisioned to efficiently bring greater number of molecules from weak to strong confinement. In the first strategy, utilized by classical nanofluidic device Received: December 1, 2015 Revised: March 18, 2016

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Figure 1. (a) Schematic showing classical micro/nanoslit device with abrupt change in device dimension. (b) In this classic approach, molecules accumulate at the micro/nanointerface and (c) are loaded into the nanoslit only at low concentration. (d) Schematic showing device with gently varying nanoconfinement using CLiC technology. (e) Molecules are continuously distributed over the smoothly varying confinement region rather than built up at the micro/nano inlet. (f) Molecule loading at h = 100 nm is significantly higher than for the classical design using equivalent DNA initial concentrations. (g) Modified design with electrode placed at the center of the nanoconfinement area. (h) The applied electric field acts as an enhancing force for DNA concentration in confinement. (i) DNA concentration enhancement for h = 100 nm with applied electric field.

approach succeeds in introducing DNA into subpersistence length confinement, it does not lead to sample concentration. If the lid is lowered quickly, radially directed hydrodynamic flow is introduced that pushes molecules away from the confined region, so that higher DNA starting concentrations are required (∼50 μg/mL).1 If the lid is lowered very slowly, high free energy gradients between the confined and unconfined regions of the chamber will drive out molecules and lead to an exponentially depressed DNA concentration (Figure 1f). In the third strategy, devices can be created that incorporate a very gentle lateral increase in confinement. While there is a history of using grayscale lithography approaches to create gently ramping structures,24−27 the largest lateral variation of vertical confinement that can be achieved with these approaches is in microns scale.25,26 In comparison to this approach, CLiC technology accesses all confinement regimes (e.g, 0 nm to initial flow-chamber height of ∼10 μm). In this work, we combine CLiC microscopy with in situ electrophoresis to improve on these strategies. We utilize CLiC to create gently varying vertical confinement over a large lateral scale, e.g., varying from 10 to 900 nm over a 2 mm lateral distance. This corresponds to a confinement gradient which is an order of magnitude less steep than conventional approaches. We combine the gently varying confinement with electrokinetic driving, which we introduce by positioning an electrode in the center of the confined region (Figure 1g). Using this center electrode, we establish an electrophoretic field that “pulls” molecules back into the confined area (Figure 1h,i). Using this new approach, we first demonstrate that we can increase in DNA concentration by 10-fold in high confinement. Second, we show this new approach creates a substantial “prestretching effect” as the molecules thread in from regions of low to high

designs, an abrupt increase in confinement is imposed at the junction of microloading channels and the nanofluidic array (Figure 1a). This abrupt increase in channel dimension leads to a very rapid rise in free energy, leading to a large free energy barrier that prevents molecules from entering the nanoarray and consequently suppresses molecule concentration in the nanoconfined region (Figure 1b,c). In particular, the sharpness of the free energy increase leads to a throughput that is governed by entropic trapping at the barrier and thermally activated entrance statistics.15 In the second strategy, one can imagine varying nanoconfinement.3,16−20 In the varying confinement, molecules are pushed into the nanofeatures from the top (Figure 1d), which is achieved by the convex lens-induced confinement (CLiC) technique. CLiC technology enables macromolecules to be manipulated and analyzed in tunable confinement geometries, improving loading, and imaging. 21−23 In a flow cell implementation of CLiC technology, a convex “push lens” is used to deform a flow cell’s top coverslip, bringing it into controlled nanoscale contact with the bottom coverslip over an extended region, subsequently creating a continuous range of confinement heights varying from the initial flow cell thickness (tens of microns) to essentially zero (set by the surface roughness, typically ∼1 nm). Combining CLiC with nanolithography techniques enables the placement of many embedded open-face nanostructures within a single optical field, allowing many single-molecule states to be analyzed during a fixed camera acquisition time.1,2 The nanogroove structure affords an extended conformation ideal for genome mapping,1−3 which we have demonstrated by fluorescence denaturation mapping of the AT-rich regions of λ-phage DNA loaded into and extended along nanogrooves.1 While the CLiC B

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Macromolecules confinement. Note that this approach is distinct from our earlier work1 in that here we present side loading of molecules into nanofeatures by the driving field after the CLiC lens is lowered, rather than top loading by the CLiC lens. Moreover, while electrokinetic concentration has been employed previously in nanochannels,28−30 the smallest vertical dimension used was 150 nm. Our approach is effective down to vertical confinement height as low as 10 nm, leveraging the CLiC geometry and hence accessing new experimental regimes.

2. METHODS Our device fabrication is performed on 4 in. fused silica wafers. The nanogrooves were defined using electron-beam lithography (VB6 UHR EWF; Vistec Lithography) and etched into the fused silica using reactive ion etching (RIE). Via holes were then fabricated in the central region of the device using abrasive jet micromachining. After milling, the via holes were filled with conductive epoxy creating electrodes interfaced to the fluid cell. The device was then bonded to a glass coverslip using 30 or 10 μm thick double-sided adhesive tape, which sets the initial vertical dimension of the flow cell, prior to compression. The device was then mounted on a plastic chuck containing external electrodes and interfaced to a precision positioning stage on an inverted microscope (Nikon Ti-E). A push lens, controlled via a piezoelectric Z-actuator, was used to deform the coverslip inducing the vertical nanoconfinement, as described in ref 1. The DNA was then imaged using a 488 nm optically pumped semiconductor laser (Coherent Sapphire 488-150 CW CDRH), a 60× waterimmersion objective (Nikon CFI Apo 60XW NIR), and an EMCCD camera (Andor iXon Ultra). Syringe pumps were used to insert and retrieve the fluid from the imaging chamber through the complete chuck assembly. The chamber height profile was estimated using interferometry methodology described in ref 21. The chamber height h is a function of the radial distance (r) away from the lens center point and can be calculated from the radius of curvature of the confined chamber rc using methods established in ref 21. We have performed experiments with λ-phage and T4-phage DNA stained for visualization with YOYO-1 at a 10:1 intercalation ratio. The stain YOYO-1 is known to increase the full contour length of DNA. At this staining ratio, the contour increases from 16.5 to 19.0 ± 0.7 μm for λ-DNA and from 56.4 to 65 ± 2 μm for T4 DNA.31 The buffer used is a solution of 45 mM Tris-base, 45 mM boric acid, and 1 mM EDTA (0.5× TBE) with pH 8.1. In addition, β-mercaptoethanol (BME) is added as an anti-photobleaching agent (3% v/v). The bulk DNA concentration was diluted to 5 μg/mL for our experiments.

Figure 2. (a) Apparatus cross section showing different components of CLiC assembly: the device layer, chamber, spacer, and push lens. (b) Normalized molecule count (normalized with maximum molecule count c0 found at height 500 nm) as a function of chamber height and time, showing that count decreases as height decreases. Note that this data represents a short-time measurement (taken 10 min after lowering the lens), not an equilibrium profile (which takes at least 1 h to establish). (c) Sequence of fluorescence micrographs showing the decrease in molecule count as the push lens is lowered. Green speckles in image are YOYO-1 stained λ-DNA. (d) Molecule count with time at one field of view (at final confinement height 50−90 nm) showing no chance in concentration after about 10 min reaching a steady-state condition.

where kB is Boltzmann’s constant, T the temperature, and C(h)/C0 the ratio of molecular concentration at a height h to the bulk concentration. The quantity Fconf(h) is the free energy of confinement, the work required to transfer the polymer from a bulk environment (where h is very large) into a slit of finite h. As the confinement scale varies very gradually in the lateral dimensions, Fconf(h) should be well approximated by the confinement free energy of a polymer in a slit of constant h. In particular, it is well-known that for a semiflexible chain Fconf(h) has two distinct limits, depending on the ratio of the slit height to the chain persistence length (lP, for DNA lP = 50 nm). For large h/lP, the free energy is given by that of a confined ideal chain:32,33 Fconf(h) ∼ kBTlP + L/h2 (with L the chain contour length). For small h/lP, known as the “Odijk regime”, the free energy is determined by periodic deflections between the confining walls and is given by33,34 Fconf(h) ∼ kBTL/lP1/3h2/3. The crossover between these regimes occurs at h ∼ lP (although in practice the transition regime between these limits is quite broad, ranging from around 0.10lP33). While DNA is selfavoiding, simulations suggest that a purely semiflexible model gives a good estimate of the confinement free energy for slitconfined DNA with self-exclusion playing a negligible role.35 In our setup, when the push lens is completely lowered, continuously varying confinement is created as shown in Figure 2a. For a radial distance of 0 to about 100 μm (measured with respect to the lens center point) the confinement height is less than 50 nm (the DNA persistence length). Only a few molecules are present in this highly confined Odijk regime (Figure 2b). The majority of our measurements takes place in the transition and ideal chain regimes for which h > 50 nm. In this regime, the molecule count increases very strongly with h, in qualitative agreement with eq 1 and the theoretical prediction that Fconf(h) ∼ 1/h2 for large h. Our primary objective is to enable and characterize rapid, high-throughput loading of molecules into strongly confined spaces. To counteract the free energy penalty associated with

3. RESULTS AND DISCUSSION The CLiC chamber is formed when the push lens deforms the coverslip (Figure 2a), decreasing the gap between the bottom substrate and the top coverslip. Because of the rigidity of the coverslip, the confinement changes very gradually, with h ranging from 10 nm immediately under the push lens to 900 nm over a lateral range of 2 mm (Figure 2b). Quantification of the molecule concentration directly beneath the push lens shows a strongly decreasing count with chamber height with radial symmetry (Figure 2b,c) around the push lens center point. As can be seen in Figures 2c and 2b, the concentration of λ-phage DNA in the CLiC chamber decreases ∼100-fold from the greatest chamber depth to the most confined regions. In equilibrium the depletion of molecules in the most confined regions of the chamber reflects the large free energy penalty from confinement. The molecule concentration satisfies the equation C(h) = e−Fconf (h)/ kBT C0

(1) C

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The quantity ξ is the friction factor of the chain in its steadystate conformation. When fext = 0, then the polyelectrolyte travels at a velocity v = μ0E. If the external force is strong enough to stall the polymer, i.e., v = 0, then fext = −ξμ0E. In our experiment, the external force is supplied by the entropic force, i.e., fext = −∇Fconf. The polymer drift velocity is then given by

strong confinement, we desire to introduce a force that can drive molecules back against the confinement gradient, increasing throughput into the nanoconfined region. One option is to apply a pressure drop across the confined region. A transverse applied pressure drop, however, will not have the effect of increasing molecule loading in confinement. The pressure drop will instead create flow that circulates around the confined region and fails to penetrate. In particular, the hydraulic resistance of the nanoslit is orders of magnitude higher than the surrounding unconfined regions, with the hydraulic resistance growing as h3. To bring the molecules toward the center of the confined area an inward, radially directed transport mechanism is required. We place an electrode in the center of the confined region as shown in Figure 3a. A voltage applied between this

v = μ0 E −

∇Fconf ξ

(3)

We estimate the friction factor as that of a slit-confined chain in the de Gennes regime, where h > lp, i.e., ξdeGennes = ηL(Pw)1/3/h2/3. The quantity w is the effective diameter of the molecule, and η is solution dynamic viscosity. We estimated an average for the mobility μ0 [(2.69 ± 0.2) × 10−4 cm2/(V s)] by fitting the velocity data due to electric field. Lastly, in order to apply the model, we need a form for Fconf that is valid for all slit heights. Chen and Sullivan33 proposed a now-classic interpolation formula for the confinement free energy of a worm-like chain in a slit:

Fconf =

⎡ ⎢⎣1.2865

2

( ha )

R0 2 h

⎤2/3

( ha ) + 1⎥⎦

+ 0.9920

(4)

Here R0 is the polymer ideal chain radius and a the Kuhn length (a = 2lP with lP = 50 nm the persistence length). This formula interpolates from the anticipated ideal chain free energy in weak confinement (h ≫ a) to the Odijk weakly bending regime in strong confinement (h ≪ a) with numerical constants fitted from comparison to simulation. While this model likely breaks down for strongly subpersistence length channels due to electrostatic wall−DNA interactions, we anticipate it holds for scales above the persistence length (50 nm). The Chen and Sullivan model was recently corroborated using lambda-size DNA molecules in the ideal to Odijk transition region of h = 50−200 nm, via partitioning of single DNA molecules in nanopit arrays.37 The recoil term is estimated from eq 28 of ref 33 and the de Gennes38 form of the chain friction factor. In addition, the fitting procedure requires knowledge of how the electric field varies with radial position away from the chamber center. To determine this dependence, we perform COMSOL field simulations using a simulated deformation profile of the confinement that was validated against experiment (Figure 3a). Electrophoretic velocity with applied voltage showed a linear relationship (Figure 3b) as expected. Figure 3c shows the fit with (solid line) and without (dotted line) free energy correction. Evidently, including the confinement free energy term improves the agreement at large r (high h), but the velocity is dominated by the electrophoretic term (μ0E) at low h, yielding robust measurements of the μ0 parameter. Critically, the applied field also enhances the molecular concentration in confinement. Experimental measurements of the molecule concentration as a function of h are shown in Figure 3d. The molecule counts were obtained 20 min after sample insertion. The 20 min delay ensured that the chamber could reach an approximately steadystate condition prior to the measurement. Figure 3d shows the comparison between the experimental measurement and theoretical estimation. While the agreement between experiment and theory is not perfect, it systematically captures the trend. In order to establish a theoretical guide for the

Figure 3. (a) Integrated CLiC design shows electrode positioned near the center of the confined region. Inset: numerical simulation of electric field in the confined region. (b) Electrophoretic molecule velocity versus applied voltage for two spacer thicknesses (10 and 30 μm) show linear increase of velocity with voltage. (c) Theoretically and experimentally determined electrophoretic velocity versus height for two spacer thicknesses (10 and 30 μm) shows the fit with (solid line) and without free-energy correction (dotted line) at voltage V = 10 V. (d) Theoretically and experimentally determined normalized molecule count as a function of chamber height for three different applied voltages. The experimental data are normalized to the molecule count measured at the CLiC near the chamber edge (at height equals 800 nm) for the 30 V data set.

central electrode and the loading reservoirs on the chuck creates an electrophoretic force directed radially to the center of the CLiC lens. We expect this electrophoretic force to balance the confinement free energy gradient under optimal conditions, leading to enhanced concentration in the confined region. When an electric field is applied in the CLiC chamber, the molecules experience two forces: (1) an electrophoretic force that drives the molecules inward toward the chamber center and (2) an entropic force, arising from the free energy gradient, which pushes the molecules outward. Viovy et al. have suggested36 that the velocity v of a polyelectrolyte subjected to an electric field E and an external force fext is given by the following relation: fext − ξ(v − μ0 E) = 0

π2 6

( )( )

kBT

(2) D

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Figure 4. Sequence of fluorescence micrographs showing molecule count increasing with applied voltage: (a) 0, (b) 10, and (c) 30 V. (d) Plot of molecule count versus applied voltage.

field strength, with higher electric field lowering the time required to achieve maximum stretching (Figure 5b). Finally, to evaluate whether our approach increases the loading efficiency of low concentration DNA sample (5 μg/ mL), experiments were performed with devices that have nanochannels (100 nm cross section) together with electrodes for performing high throughput DNA nanotemplating into nanochannels (Figure 6a). In our previous nanotemplating demonstration1 we used an initial DNA concentration of 50 μg/mL, which has been reduced to 5 μg/mL in the current experiment. A comparison of loading efficiency with and without voltage is shown in Figure 6b,c. When DNA templating was performed with an application of 30 V, the channel occupancy increased drastically. The effects of templating DNA in parallel nanochannels for three different applied voltages are compared in Figure 6c. To estimate the nanochannel loading efficiency, we calculate the ratio of nanochannels occupied by DNA to the total number of nanochannels. Note that some DNA fragmentation and breakage occurred due to the mechanical shear from the push lens loading effect, leading to nonuniform stretching across the array. We find that at 30 V input voltage we can achieve 95% channel occupation in the nanochannels. A histogram of DNA extension (Figure 6b) shows that a higher proportion of molecules reached their full extension (full extension length is about 64 μm for T4 DNA) at higher electric field.

dependence of concentration on h and applied voltage, we have used equilibrium expressions and the following field-adjusted free energy, where r is the radial distance from center zero to outer radius r0: Fadj = Fconf −

∫0

r0

ξμ0 E dr

(5)

Now, while a steady-state condition holds, an equilibrium condition does not rigorously hold; therefore, the deviation is noticeable in Figure 3d. However, the equilibrium assumption provides a simplified theory that serves well as a baseline comparison to measurements, allowing us to build understanding and intuition for our results. In Figure 4a−c, we show images of molecules at h = 100 nm for a range of applied voltages. Figure 4d shows molecule count for a range of different applied voltages at the same chamber location. Clearly, increasing voltage leads to increasing molecule count, with overall concentration enhancement by over 10-fold. An additional advantage of our continuous confinement approach is an enhanced prestretching effect. Gradients in electrophoretic force can give rise to stretchingan effect observed in geometries with lateral “width” variation39−42 as well as electrophoretic stretching in classical lateral nanofluidic feature.42 In this paper, we demonstrate electrophoretic stretch in a vertically varying nanoconfinement. In our design, due to the rapidly increasing electric field strength with increasing confinement, molecules can prestretch as they thread into the confinement area. Figure 5a shows a time sequence of a molecule stretching as it moves from a region with h = 300 nm toward a central region with h = 10 nm. Figure 5b shows quantitative measurements of DNA prestretching for three applied voltages. Note that the degree of the stretching and stretching time scale can be controlled by tuning the electric

4. CONCLUSIONS We have shown that electric-field-assisted loading into a varying geometry can enhance DNA concentration 10-fold in strong confinement. Such molecular enhancement increases the number of nanofuidic features that can be used for singlemolecule manipulation and analysis in a device. This high loading efficiency cannot be achieved using classical nanofluidic approaches and is an essential requirement for effective use of nanofluidic single-molecule manipulation approaches. In addition, we have developed a guiding theoretical model, based on an electric-field-assisted free energy, which can describe how external electric fields can drive molecules up free energy gradients into continuously varying confined regions. Our model explains how both the electrophoretic velocity and the molecule concentration depend on chamber height. Finally, we show that the molecules tend to “prestretch” when they are threaded into the varying confinement area, a valuable technological contribution. While such electric-field-based stretching has been demonstrated before, here we show that it can occur in a nanofluidic context where the vertical confinement dimension (rather than the lateral dimension or channel “width”) is being varied. While we have demonstrated our approach for DNA, it can be translated to proteins, vesicles, and other biological analytes with use of the appropriate confinement free energy and applied fields.

Figure 5. (a) Image sequence showing one λ-DNA molecule being electrophoretically driven from low to high confinement. The molecule stretches when it enters the high confinement area. (b) Measured DNA prestretch length versus threading time for three different applied voltages (15, 20, and 30 V). E

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Figure 6. (a) A “CLiC” push lens deforms a thin coverslip inducing local nanoscale confinement for optical interrogation (inset: electron micrograph showing the nanochannels). (b) Histogram of measured DNA extension in nanochannels for three different applied voltages and (c) example micrographs of nanochannel extended T4 DNA. The images, taken at the device center, show that increasing voltage increases loading efficiency. The nanochannels have a spacing of 1 μm and width of 100 nm.

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The high throughput DNA nanochannel stretching presented in this paper is poised to enable further development of next-generation mapping and sequencing with myriad applications in oncology and agriculture as well as fundamental biophysical and biological research.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail [email protected] (S.R.L.). *E-mail [email protected] (W.W.R.). *E-mail [email protected] (M.J.A.). Present Address

M.J.A.: Department of Mechanical, Automotive and Materials Engineering, University of Windsor, ON N9B 3P4, Canada. Notes

The authors declare no competing financial interest.

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REFERENCES

(1) Berard, D. J.; Michaud, F.; Mahshid, S.; Ahamed, M. J.; McFaul, C. M. J.; Leith, J. S.; Berube, P.; Sladek, R.; Reisner, W.; Leslie, S. R. Convex Lens-induced Nanoscale Templating. Proc. Natl. Acad. Sci. U. S. A. 2014, 111, 13295−13300. (2) Mahshid, S.; Ahamed, M. J.; Berard, D. J.; Amin, S.; Sladek, R.; Leslie, S. R.; Reisner, W. Development of a Platform for Single Cell Genomics Using Convex Lens-Induced Confinement. Lab Chip 2015, 15, 3013−3020. (3) Leslie, S. R.; Fields, A.; Cohen, A. Convex Lens-Induced Confinement for Imaging Single Molecules. Anal. Chem. 2010, 82, 6224−6229. (4) Dai, L.; van der Maarel, J.; Doyle, P. S. Extended de Gennes Regime of DNA Confined in a Nanochannel. Macromolecules 2014, 47, 2445−2450. (5) Chen, Y.-L.; Lin, P. K.; Chou, C.-F. Generalized Force− Extension Relation for Wormlike Chains in Slit Confinement. Macromolecules 2010, 43, 10204−10207. (6) Reisner, W.; Pedersen, J. N.; Austin, R. H. DNA Confinement in Nanochannels: Physics and Biological Applications. Rep. Prog. Phys. 2012, 75, 106601−106610. (7) Reisner, W.; Larsen, N. B.; Silahtaroglu, A.; Kristensen, A.; Tommerup, N.; Tegenfeldt, J. O.; Flyvbjerg, H. Single-molecule Denaturation Mapping of DNA in Nanofluidic Channels. Proc. Natl. Acad. Sci. U. S. A. 2010, 107, 13294−13299. (8) Reisner, W.; Larsen, N. B.; Flyvbjerg, H.; Tegenfeldt, J. O.; Kristensen, A. Directed Self-organization of Single DNA Molecules in a Nanoslit via Embedded Nanopit Arrays. Proc. Natl. Acad. Sci. U. S. A. 2009, 106, 79−84. (9) Strychalski, E. A.; Geist, J.; Gaitan, M.; Locascio, L. E.; Stavis, S. M. Quantitative Measurements of the Size Scaling of Linear and Circular DNA in Nanofluidic Slitlike Confinement. Macromolecules 2012, 45, 1602−1611. (10) Moerner, W. E.; Fromm, P. D. Methods of Single-Molecule Fluorescence Spectroscopy and Microscopy. Rev. Sci. Instrum. 2003, 74, 3597−3619. (11) Moerner, W. E. A Dozen Years of Single-Molecule Spectroscopy in Physics, Chemistry, and Biophysics. J. Phys. Chem. B 2002, 106, 910−927. (12) Marie, R.; Pedersen, J. N.; Bauer, D. L. V.; Rasmussen, K. H.; Yusuf, M.; Volpi, E.; Flyvbjerg, H.; Kristensen, A.; Mir, K. U. Integrated view of genome structure and sequence of a single DNA molecule in a nanofluidic device. Proc. Natl. Acad. Sci. U. S. A. 2013, 110, 4893−4898. (13) Reisner, W.; Morton, K.; Riehn, R.; Wang, Y. M.; Yu, Z.; Rosen, M.; Sturm, J.; Chou, S.; Frey, E.; Austin, R. Statics and Dynamics of

ACKNOWLEDGMENTS

This work was supported by a Canadian Health Research Projects (CHRP) award with joint contributions from the National Science and Engineering Research Council of Canada (NSERC, 446656-13) and Canadian Institute for Health Research (CIHR, CPG-127762). We thank the Canadian Foundation for Innovation (CFI) and Genome Quebec Innovation Centre for financial support. In addition, the authors thank Laboratoire de Micro- et Nanofabrication (LMN) at INRS-Varennes, McGill Nanotools-Microfab and the Facility for Electron Microscopy Research at McGill (FEMR). Authors are thankful to Haig Djambazian for assistance in metal evaporation; to Christopher McFaul for data analysis software; to Andrew Caleb Guthrie and Alexander Verge for custom instrumentation control software; and to Dr. Jason Leith for CLiC microscopy instrumentation support and a critical reading of this manuscript. R.S. is grateful for a FRSQ Chercheur-Boursier Junior 2 Award and a CIHR New Investigator Award. D.B. is grateful to the NSERC Bionanomachines CREATE and NSERC PhD award programs for recognition and fellowship support. S.M. is grateful for a Dr. Lin Post Doctoral fellowship. F

DOI: 10.1021/acs.macromol.5b02617 Macromolecules XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.macromol.5b02617 Macromolecules XXXX, XXX, XXX−XXX