NANO LETTERS
DNA Electrophoresis on Nanopatterned Surfaces
2004 Vol. 4, No. 4 659-664
Young-Soo Seo,†,| H. Luo,†,| V. A. Samuilov,† M. H. Rafailovich,*,† J. Sokolov,† D. Gersappe,† and B. Chu‡ Department of Materials Science and Engineering and Department of Chemistry, State UniVersity of New York, Stony Brook, New York 11794-2275 Received January 26, 2004; Revised Manuscript Received March 1, 2004
ABSTRACT We present a new technology that uses nanopatterned surfaces to separate DNA. This technology eliminates the need for disposable separation media (i.e., gels or polymer solutions that are susceptible to degradation and are difficult to load into small devices due to their inherent high viscosity). We demonstrate using molecular dynamics simulations and experiments that this method can simultaneously separate a broad band of DNA ranging from a few hundred base pairs (bp) through genomic size without a loss in resolution.
Current techniques for DNA separation are inadequate for the rapidly growing demand for accurate, versatile, and portable devices for rapid genetic identification. This limitation arises due to two main factors: First, current methods (based on gel or capillary electrophoresis) are tailored to separate DNA in a narrow size range; and second, these methods are limited to shorter fragments of DNA by the fundamental mesh size of the separation medium. To overcome these restrictions, in recent years, there has been a large proliferation of studies that have used corrugated inorganic two and three-dimensional matrices with larger mesh sizes to separate longer DNA molecules.1-7 These efforts have met with limited success. Even though they have shown the promise of being able to separate larger DNA fragments, their resolution and speed of operation has lagged far behind conventional electrophoretic methods. In this report we propose a new paradigm that uses chemical, rather than topological, restrictions to electrophoretically separate DNA. In particlar, we use a surface with a nanoscale chemical pattern to separate DNA in a size range from a few hundred basepairs to megabase pairs, without a loss in resolution. Since engineered surfaces are robust and reusable, this method offers the possibility of tailoring the surface for specific applications which can be easily integrated into a “lab on a chip” device. In earlier work,8-10 we were able to show that it was possible to electrophoretically separate DNA chains on a flat surface without any topological restrictions or any solution sieving media. In our case, we postulated that the source of the separation was a result of the differences in the * Corresponding author. E-mail:
[email protected]. † Department of Materials Science and Engineering. ‡ Department of Chemistry. | These authors contributed equally to the manuscript. 10.1021/nl0498435 CCC: $27.50 Published on Web 03/20/2004
© 2004 American Chemical Society
conformation of chains of different length adsorbed on an attractive surface. Thus, when we applied an electric field in the plane of the surface, DNA adsorbed on the surface had a length-dependent mobility and were able to be separated. This opened up a completely new way of approaching the design of devices using surface-directed separation since the ability to use chemical rather than physical means to separate molecules offers significant advantages. However, this reliance on conformational differences also resulted in limitations on the method. We found that if the surface had a strong attraction to DNA, we were restricted by the fact that long DNA chains are fully adsorbed and there is no difference between their conformations. On the other hand, if the surface had a weak attraction to DNA where conformal differences between longer chains persist, short chains desorb from the surface and cannot be separated. Hence, to optimize separation of DNA on a surface one must find a method to increase the sensitivity to conformational changes while allowing for the separation of a broad range of DNA chain lengths. An obvious solution to improve the method is to increase the complexity of the media such that small changes in conformational structures can be amplified without resulting in the desorption of shorter chains. This can be accomplished by introducing a nanopattern on the substrate surface where the additional length scale of the pattern will allow us one more control variable to separate chains of different lengths. Furthermore, by tuning the relative interaction strength of the domains, one can introduce dispersion while still keeping the smaller chains adsorbed onto the surface. Molecular dynamics (MD) simulations were carried out to probe the potential of a patterned surface on the separation of DNA molecules. In the simulation the DNA is modeled as a linear polymer chain with N segments.11 The substrate
Figure 1. Normalized time averaged segment density per site from the MD simulation. The density per site is calculated by counting the number of times that a site on the surface is visited by a DNA segment and is normalized by dividing by the maximum site density for each simulation. The color bar represents the normalized densities from the maximum value of 1 (red) to the minimum value of 0 (black). Snapshots (a-c) are for a chain of N ) 60 segments, and snapshots (d-f) are for a chain of N ) 120 segments. In a and d, the bare wall interaction is s ) 2.25, in b and e the bare wall DNA interaction is s ) 2.5, and in c and f the bare wall DNA interaction is s ) 3.0. In all cases the patch-DNA interaction is sp ) 2.0, thus the bare wall is more attractive than the patch. (g) SPM image in air of a λ-DNA chain adsorbed from buffer solution onto the nano patterened surface described in Figure 3.
surface consists of atoms forming two (111) planes of an fcc lattice. The pattern on the surface is introduced by using two types of wall atoms which are differentiated by their strength of interaction with the DNA segments. The strength of attraction between the polymer segments and the surface is characterized by a parameter, s (larger values of s correspond to a more attractive surface, and the adsorption/ desorption transition occurs at a value of s ∼ 1.8). In the first set of simulations we used a surface with a hexagonal patch pattern (a pattern that can be easily produced experimentally by utilizing the self-assembly of diblock polymers at the air/water interface12,13). The periodicity of the patches is chosen to be on the order of 2-3 persistence lengths of the DNA moleclule to ensure that the DNA remains adsorbed on the surface. The strength of attraction between the patch and the polymer is defined by a parameter, sp. We make the patch to be less attractive to the DNA segments than the bare wall. We fixed the interaction between the patch and the polymer segments to be sp ) 2.0 and varied the interaction between the rest of the surface and the polymer segments s. When we applied an electric field in the plane of the surface, we monitored the frequency with which the adsorbed segments of the chain visit a particular site on the lattice. Then we could calculate a time averaged segment density as a function of the local position on the surface. We plot the time averaged density in Figure 1. From Figure 1 we see that at low values of s ) 2.25 (Figure 1a,d) the pattern on the surface can be seen in the density plots for the longer chain, while it disappears for 660
the shorter chain, and while at higher patch attractions, s g 2.5 the pattern appears to be the same for both chain lengths (Figure 1c, f). A possible explanation is that, for this range of interaction strength, short chains (e.g., N ) 60) tend to adopt a conformation in which “loops” predominate and the density profiles are averaged out over the whole surface, regardless whether the chain is on top of the patch or the bare surface. For longer chains, on the other hand, (e.g., N ) 120) “train” conformations are preferred and it is likely that the chains stay more on top of the bare wall surface because the higher attraction can lower the free energy and the patch pattern shows up in the density map. As we increase the attraction between the surface and the polymer segment, while keeping the patch-polymer interaction the same, the pattern now starts to appear in both the long and the short chain density maps. This implies that there is an optimal range of interactions, which will amplify the differences between the short and the long chains. Indeed, when we calculate the mobility on the surface we can clearly see that there is a length-dependent mobility which loses its length dependence when the interaction strength is increased (Figure 2a). From the simulation we can also see that separation between the chains is also observed if the patches on the surface are more attractive and thus serve as pinning points (Figure 2b). Thus, the pattern should, in theory, expand the range of DNA that can be separated by this method. To test this hypothesis, we designed a nanopattern of Ni patches superimposed upon a Si matrix, where the DNA Nano Lett., Vol. 4, No. 4, 2004
Figure 2. Mobility of DNA calculated by MD simulations. (a) Plot of the mobility of DNA on a hexagonal patch surface where the patches are less attractive than the bare wall. (b) Plot of the mobility of DNA on a square patch surface where the patches are more attractive than the bare wall. In (a) the dimensionless electric field was fixed at E ) 0.02 and in (b) E ) 0.25.
chains were found to be preferentially adsorbed14 (see Figure 1g). In order for the chains to sense the pattern, we chose the dimensions to be roughly 2 or 3 DNA persistence lengths (similar to the simulations), or approximately 150 to 200 nm. The pattern was produced by a relatively simple technique developed in our laboratory for rapid, large area, nanoscale chemical patterning. This pattern can of course be optimized for specific DNA geometries (circular or supercoiled), and the details of this procedure will be described elsewhere.15 A Ni film, 25 nm thick, was sputtered onto a 100 mm diameter Si wafer under high vacuum conditions. Monodisperse symmetric poly(styrene-b-methyl mathacrylate) (PSb-PMMA, Mw ) 193.6-201 k, Mw/Mn = 1.14) diblock copolymers were spread at the air/water interface in a Langmuir-Blodgett trough (KSV 3000) where they are known to form a two-dimensional hexagonal pattern of surface micelles, whose dimensions can be adjusted by Nano Lett., Vol. 4, No. 4, 2004
varying the molecular weight and the composition ratio of the two blocks.12,13 In this case the pattern was chosen such that the PS cores were 13-15 nm in height with a centerto-center distance of 250-300 nm. The monolayer film was compressed with constant barrier speed at 5 mm/min until a target surface pressure of 5 dyn/cm was achieved. At this pressure the copolymer has been shown to self-assemble into a uniform hexagonal array of surface micelles with a PS core and stretched PMMA corona (approximately 1 nm thick). The film was then transferred onto a Ni coated Si wafer and inserted into an Ar ion mill (base pressure of 3.7 × 10-7 Torr) where a 200 W RF plasma was used to sputter the samples for 2.1 min at normal incidence. Since the Ni sputters approximately twice as fast as the PMMA with an average rate of ∼10 nm/min, which in turn sputters faster than PS, the thin PMMA regions and the underlying Ni were removed first, while the Ni under the thicker PS regions remained unsputtered (Figure 3 inset a). The total etching time, 2.1 min, was determined by surface electrical conductivity and optical end point detection of Si, which indicated exposure of the Si surface beneath the Ni layer. The sample was rinsed in toluene to remove organic contaminants before use. The conductivity of the Si wafer (F ) 2500 Ω-cm) was unchanged by the procedure, as expected, since the Ni regions are not continuous. The water contact angle on the sample, θ ) 42°, was not significantly different than unetched native oxide covered Si (θ ) 30-40°). The resulting pattern was imaged, as shown in Figure 3, using SFEG-SEM (Schottky field-emission gun on a scanning electron microscope, LEO-1550) where the dots appear much brighter than the surrounding native Si oxide background, consistent with the larger electron density of Ni. The topography of the pattern was measured using a Dimension 3000 scanning probe microscope (inset 3b) which shows that the height of the dots, 11-13 nm, is only slightly lower than the original PS cores. A chemical map of the surface was obtained using a focused electron beam (circle) and analyzing the fluorescence with elemental dispersion analysis (EDAX) (inset c). Comparing the two spectra we can see that the Ni peak is much higher when the dots are centered in the beam. The effectiveness of the pattern was tested using a series of commercial double stranded DNA capillary electrophoresis standards which span five decades: λ-Hind III digest, which is produced by digestion of λ-DNA into distinct fragments ranging from 125 bp to 23.1 kbp, λ-DNA (48.5kbp), T2 DNA (164 kbp) and S. Pombe chromosomal DNA consisting of three components at 3.5 Mbp, 4.7 Mbp, and 5.7 Mbp, gently extracted from a 1% agarose gel plug with a field of 1-2 V/cm. Droplets of 0.2-1.0 µL containing approximatly 0.04 µg of DNA for the first three standards and approximatley 1 ng of S. Pombe DNA were deposited onto separate Ni patterned substrates. 0.3 M tris-borateEDTA (TBE) buffer solution with 1 µg/mL ethidium bromide dye was added to the chamber. The mobility of the DNA chains was then measured in an external DC field of E ) 5 V/cm by detecting the electropherograms (the fluorescence intensity as a function of time, ∆t) at a fixed distance, l of 5 to10 mm from the surface injection point.10 At least four 661
Figure 3. Surface image of Ni nanopattern on Si wafer using SEM. Inset (a) shows schematic etching process fabricating Ni nanopattern from surface micelle array. Inset (b) The topography of Ni nanopattern was measured using SPM where the height of the dots is 11-13 nm and center-to-center distance is 250-300 nm. Inset (c) A chemical map of the surface was obtained using the fluorescence with elemental dispersion analysis (EDAX) where Ni peak intensity is much higher when a electron beams indicated by circle are focused on the Ni dots.
runs per DNA solution were performed. Typical electrophoresis spectra obtained from λ-Hind III, λ, T2, and S. Pombe DNA runs are are shown in Figure 4a. λ-Hind III and λ-DNA gave similar results when the components were eluted separately or in mixtures. On the other hand, additional peaks appeared when T2 was added to the Hind III digest, possibly due to incomplete removal of the enzyme. The preparation of the S. Pombe DNA was very different from the others and hence it was difficult to form mixtures. Consequently, here we show only spectra obtained from separate elutions where identification of the peaks could be directly checked against the manufacturer’s specifications. For example, the assymtery in the peak for the T2 sample is consistent with the manufacturer’s gel electrophoresis results that indicate the presence of higher molecular weight impurities. It is interesting to compare the Hind III data to those reported by the group of Y. Kim using standard capillary electrophoresis with16 and without17 a sieving medium. The resolution of capillary electrophorsis is better, as expected, when a sieving medium is present. On the other hand, the medium is very selective for a specific moelcular weight band and both the two lowest and the highest peaks are not separated in ref 17. In the absence of sieving media, the spectrum reported in ref 17 is similar to ours where all peaks were observed. Hence, we postulate that this surfacedirected dispersion mechanism is a common phenomenon that may provide an alternative explanation to anomalies in the separation process reported by several groups on bare 662
capillary walls,17 microbead arrays,18 and microfluidic devices.7 In all these cases the authors indicate that the anomalies are affected by the surface charges which in turn are screened to different extents by the charges in the buffer media used. We also conducted alternative experiments where the buffer concentration or the surface corrugation was varied to reproduce surfaces in ref 10. Only the buffer concetration was found to affect mobility in the same manner as reported in ref 17. For low buffer concentration, the electroosmotic flow (EOF) became important and reverse molecuar weight elution similar to that reported in ref 7 was observed. Here we chose to use a relatively high buffer concentraion where more effective charge screening could be obtained and the EOF is minimized. In the lower inset we show the electropherogram of the S. Pombe DNA where we notice that the intensities of the three peaks are roughly equal, as expected. The reduction in the overall intensity compared to the other peaks reflects the far lower concentraion in the initial droplet. The sharpness of the λ-DNA peak, as well as the asymtery in the T2 peak in the upper inset, are consistent with the slab gel electrophoresis specification of the manufacturer which indicated the presence of higher molecular weight impurities in the latter. The mobilities, µ ) l/(∆tE), of the DNA chains were calculated from the averages of all the data and plotted on a log-log scale vs the number of base pairs (N) in Figure 4b. From the figure we can see that the data, spanning nearly five decades, can be fitted with the same power law dependence Nano Lett., Vol. 4, No. 4, 2004
Figure 4. (a) The fluoresence intensity was measured as a function of time in a confocal microscope positioned at a fixed distance from the injection point (5-10 mm). The mobility of the DNA was measured in an external field of 5 V/cm. The fluorescence intensity was detected as a function of time for λ-Hind III Digest, (insets) λ-, T2, and S. Pombe DNA. The labled peaks correspond to λ-Hind III Digest (1-124 bp, 2-564 bp, 3-2,027 bp, 4-2,322 bp, 5-4,361 bp, 6-6,557 bp, 7-9,416 bp, 8-23,130 bp), λ-DNA (48.5 kbp), T2 DNA (164 kbp), or chromosomal S. Pombe DNA (1-3.5 Mbp, 2-4.7 Mbp, and 3-5.7 Mbp). (b) The mobility of double stranded DNA, µ, as a function of the number of base pairs, N. The dashed line at the top of the figure corresponds to the mobility of free draining DNA. Inset: The fractional resolution, as defined in the text, versus the number of base pairs. The dashed line indicates (δt/t) ) 0.018 as a guide line.
µ ∼ NR where R ) -0.36 ( 0.05. This exponent is intermediate between -0.25 and -0.87 obtained on homogeneous bare and functionalized Si surfaces, respectively.8-10 As shown in the previous simulations, the homogeneous surfaces with either very strong or weak interactions, were suitable for either short or long DNA chains, respectively.8-9 These results show that imposing a chemical pattern can increase the exponent without compromising effects at the shorter end of the length scale. Since both the Ni and Si Nano Lett., Vol. 4, No. 4, 2004
could be further functionalized, we believe that the interactions as well as the length scale of the pattern could be further adjusted in order to increase the exponent and optimize the dispersion. In addition, since this method discerns the conformations of the adsorbed chains, the technique can in principle separate chains of identical moelcular weight but different structures, such as circular or supercoiled. Examination of the spectra in Figure 4a indicates that the resolution of the peaks does not degrade with increasing 663
molecular weight. The fractional resolution can be defined as (δt/t) ) wh(dN/dt)/N, where wh is the full width half maximum, obtained from fitting a Gaussian shape to the peaks in the electropherograms (Figure 4b). The fractional resolution is then plotted as a function of number of base pairs in the inset of Figure 4b, where we see that it is roughly constant in the range, (δt/t) ∼ 0.01-0.02, up to chromosomal length DNA. Hence, even though the resolution of this method is comparable to other separation techniques for low molecular weights, it is much better for the higher molecular weights. Furthermore, this technique requires very low loading amount and operating voltages, making it amenable for incorporation into chip-based portable detectors or microarrays where the separated bands are channeled in microfluidic capillaries with sieving media optimized for sequencing. Acknowledgment. Financial support was provided by the NSF MRSEC Program (DMR-9632525), NER Grant 0103470, SGER Grant 0244093, the Department of Energy, and the National Human Genome Research Institute (R01HG0138604).
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