Separation of DNA with Different Configurations on Flat and

We demonstrate that electrophoresis on a flat Si substrate is an effective method in separation of DNA with different configurations, e.g., linear, su...
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Separation of DNA with Different Configurations on Flat and Nanopatterned Surfaces Bingquan Li,*,† Xiaohua Fang,† Haobin Luo,† Young-Soo Seo,†,‡ Eric Petersen,§ Yuan Ji,† Miriam Rafailovich,*,† Jonathan Sokolov,† Dilip Gersappe,† and Benjamin Chu|

Department of Materials Science and Engineering, and Department of Chemistry, SUNY at Stony Brook, New York 11794, and Harvard University, Cambridge, Massachusetts 02138

We demonstrate that electrophoresis on a flat Si substrate is an effective method in separation of DNA with different configurations, e.g., linear, supercoiled, and relaxed or DNA of different length, e.g., supercoiled DNA ladder. The surface separation arises from the different number of contacts due to the conformational differences between adsorbed DNA chains. Imposing a Au nanopattern on the Si surface further improves the separation effect. The simulation of electric field on this patterned surface by the finite element method shows that Au nanodots act as local pinning points for DNA segments due to dielectrophoretic force. The results of molecular dynamics simulation showed that the conformational differences between adsorbed polymer chains were amplified on the patterned surface and enhanced separations were achieved, which are consistent with the experimental results.

Supercoiling of DNA is a general phenomenon in vivo and associated with many critical biological processes, such as transcription, replication, and recombination.1 The topologically closed circular DNAs also play very important roles in modern biotechnology. For example, plasmids are commonly used as genetic vectors in DNA recombinants, while bacterial artificial chromosomes in the circular form are the preferred vector for cloning of large DNA inserts (100-300 kbp).2 Moreover, interest * To whom correspondence should be addressed. E-mail: [email protected]; [email protected]. Fax: (631)-632-5764. † Department of Materials Science and Engineering, SUNY at Stony Brook. ‡ Current address: LG Chem Research Park, LG Chem, Ltd., Daejeon, Korea. § Harvard University. | Department of Chemistry, SUNY at Stony Brook. (1) Cozzarelli, N. R. Science 1980, 207, 953-960. (2) Monaco, A. P.; Larin, Z. Trends Biotechnol. 1994, 12, 280-286. 10.1021/ac060686z CCC: $33.50 Published on Web 06/17/2006

© 2006 American Chemical Society

in using plasmid DNA for in vivo delivery in gene therapy has increased rapidly due to the safety and regulatory concerns about viral vectors.3,4 Therefore, purification and characterization of such circular DNA can be quite important. Even though conventional gel or capillary electrophoresis is among the most common methods in analyzing DNA, surprisingly, very few studies using electrophoresis focused on circular DNA. Due to their closed ring structure, relaxed circular DNA becomes trapped more easily around topological constraints in gels than linear DNA.5 Even supercoiled circular DNA eventually becomes trapped at these locations, when the electric field strength is sufficiently high.6 While the trapping effect may be advantageous for removing contaminants of linear DNA, it is not helpful in the separation of topoisomers (supercoiled DNA with different degrees of twist). Furthermore, there is no general agreement on the time sequence of the migration of linear, supercoiled, and relaxed circular DNA, since it largely depends on the type and concentration of the intercalating dye, the coating of the capillary walls, and the operating conditions.7 Hence, identification of DNA of different forms could be difficult using conventional electrophoresis. We had previously shown that it was possible to separate linear double-stranded DNA fragments on a flat surface without any topological constraints.8-11 The mobility differences are due to the (3) Kelly, W. J. Biotechnol. Appl. Biochem. 2003, 37, 219-223. (4) Ferreira, G. N. M.; Monteiro, G. A.; Prazeres, D. M. F.; Cabral, J. M. S. Trends Biotechnol. 2000, 18, 380-388. (5) Akerman, B.; Cole, K. D. Electrophoresis 2002, 23, 2549-2561. (6) Cole, K. D.; Akerman, B. Biomacromolecules 2000, 1, 771-781. (7) Oana, H.; Hammond, R. W.; Schwinefus, J. J.; Wang, S. C.; Doi, M.; Morris, M. D. Anal. Chem. 1998, 70, 574-579. (8) Pernodet, N.; Samuilov, V.; Shin, K.; Sokolov, J.; Rafailovich, M. H.; Gersappe, D.; Chu, B. Phys. Rev. Lett. 2000, 85, 5651-5654. (9) Seo, Y. S.; Samuilov, V. A.; Sokolov, J.; Rafailovich, M.; Tinland, B.; Kim, J.; Chu, B. Electrophoresis 2002, 23, 2618-2625. (10) Luo, H. B.; Gersappe, D. Electrophoresis 2002, 23, 2690-2696.

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differences in friction forces exerted on DNA chains adsorbed onto the surface. These differences arise from the nonlinear variation with chain length of the number of loops (segments away from the surface) and trains (segments in contact with the surface). Since the entropic penalty of confinement differs with chain structure, the number of surface contacts can also vary for chains with the same molecular weight, but different configurations.12 The resulting conformational differences of such chains adsorbed on an attractive surface have been investigated through theoretical calculations and computer simulations.13-17 Linear polymers will spread on the surface forming “trains”, “loops”, and “tails”.15,16 Star polymers and dendrimers will present the core away from the surface with segments at the end of branches, adsorbed. For comb polymers, depending on the branching density and relative length between backbone and branches, the backbone17 or branches15 will prefer adsorption on the surface. Therefore, we postulated that surface electrophoresis can differentiate not only between chains of the same form with different numbers of base pairs but also between chains of the same molecular weight but with different configurations, such as linear, supercoiled, and relaxed circular forms. The advantage of this technique is the absence of separation media used in standard gel or capillary electrophoresis and hence the preferential trapping of the circular forms is avoided. Theoretically, this technique provides a convenient method to separate DNA chains of different sizes, forms, and even topoisomers. Furthermore, since this technique relies on differences in surface adsorption, one can amplify its sensitivity by introducing chemical heterogeneity on the otherwise topographically flat surface. The statistical “match” between the pattern of heterogeneity and polymer internal structures can even give rise to additional conformational differences and larger mobility differences between chains of different architectures.18-22 In this paper, we first demonstrate that circular supercoiled and relaxed forms of plasmid DNA can be separated on a flat Si surface and that their electrophoretic mobilities are differently sensitive to the buffer ionic strength. Second, we show the fractionation of DNA fragments with the same form, e.g., supercoiled DNA ladder, on such a surface. The introduction of an Au nanopattern on the Si surface can further enhance the mobility dispersion and the resolution between the components in the ladder. Finally, finite element method (FEM) and molecular dynamics (MD) simulations were carried out to understand the electric field distribution and to probe the dynamics of polymer (11) Seo, Y. S.; Luo, H.; Samuilov, V. A.; Rafailovich, M. H.; Sokolov, J.; Gersappe, D.; Chu, B. Nano Lett. 2004, 4, 659-664. (12) Kosmas, M. K. Macromolecules 1990, 23, 2061-2065. (13) Semenov, A. N.; BonetAvalos, J.; Johner, A.; Joanny, J. F. Macromolecules 1996, 29, 2179-2196. (14) Moghaddam, M. S.; Vrbova, T.; Whittington, S. G. J. Phys. A 2000, 33, 4573-4584. (15) Striolo, A.; Prausnitz, J. M. J. Chem. Phys. 2001, 114, 8565-8572. (16) Striolo, A.; Jayaraman, A.; Genzer, J.; Hall, C. K. J. Chem. Phys. 2005, 123. (17) vanderLinden, C. C.; Leermakers, F. A. M.; Fleer, G. J. Macromolecules 1996, 29, 1000-1005. (18) Kriksin, Y. A.; Khalatur, P. G.; Khokhlov, A. R. J. Chem. Phys. 2005, 122. (19) Muthukumar, M. J. Chem. Phys. 1995, 103, 4723-4731. (20) Polotsky, A.; Schmid, F.; Degenhard, A. J. Chem. Phys. 2004, 121, 48534864. (21) Ellis, M.; Kong, C. Y.; Muthukumar, M. J. Chem. Phys. 2000, 112, 87238729. (22) Chakraborty, A. K. Phys. Rep: Rev. Sect. Phys. Lett. 2001, 342, 2-61.

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chains with different forms on both flat and patterned surfaces and compared with the experimental results. EXPERIMENTAL SECTION Materials. Plasmid DNA Φx174 RF I (5386 bp, circular supercoiled form) and Φx174 RF II (circular relaxed form) were purchased from New England Biolabs. Supercoiled DNA ladder (ranging from 2067 to 16 210 bp) was ordered from Sigma (St.Louis, MO). The concentration of running buffer Tris-borate EDTA (TBE) (Sigma) was varied at different values. Ethidium bromide dye (Sigma) was mixed with buffer at a constant concentration of 1 µg/mL for all runs. Separation Method. The surface electrophoresis method has been described elsewhere.9 Briefly, one clean substrate was placed in a separation cell. A droplet (∼0.5 µL) of DNA solution was loaded on the surface and let dry in air, followed by addition of buffer premixed with ethidium bromide dye. A homemade laser fluorescence microscope was used to detect signals from the migrating DNA chains on substrate. The separation length from the edge of the dried droplet to the detector is fixed typically from 3 to 7 mm. Surface Preparation. Since the surface quality will largely affect the interaction with DNA and thus the migration on the surface, all Si substrates were carefully cleaned according to a modified Shiraki method.23 The hexagonal Au nanopattern on the Si surface was made following a similar procedure in a previous report.11 A layer of Au (∼50 nm) was deposited onto a Si wafer through thermal evaporation with a layer of Cr (∼5 nm) to strengthen the adhesion between Au and Si. Poly(styrene-b-methyl methacrylate) (PSPMMA; Mw ) 193.6k-201k, Mw/Mn ∼1.14) diblock copolymer in chloroform at a concentration of 1 mg/mL was spread at the air/water interface in a Langmuir-Blogett (LB) trough. At the surface pressure of 5 dyn/cm, the copolymer formed a hexagonal array of surface micelles with PS as the core and PMMA as the corona. This LB film was then transferred vertically to the Aucoated Si wafer at a speed of 2 mm/min to serve as a mask for the following Ar ion etching process. With the process pressure 1.35 × 10-4 Torr, rf power 200 W, and zero degree etch angle, the etching rate for Au is almost 2 times faster than PS and PMMA. The etching process was stopped when the Si signal was detected by the optical end point detector. The Au nanopattern was characterized by SEM (LEO-1500), EDAX, and AFM (DI, Dimension 3000) as shown in Figure 1. The Au dot is about 35 nm in height, 175 nm in diameter, and 100 nm from nearest neighbors. This fabrication method allows us to make a nanoscale pattern of large area, and easily to adjust the geometry of pattern through many parameters, such as polymer molecular weight, concentration, and surface pressure. Molecular Dynamics Simulation. In this simulation, the DNA is modeled as a linear or ring polymer chain with N segments. Monomers with an effective charge q interact through a truncated Lennard-Jones (L-J) potential. The adjacent monomers along the chain are coupled by an additional FENE potential to prevent chain breaking and yield realistic dynamics for polymers.24 The flat substrate surface consists of atoms forming two (111) (23) Ishizaka, A.; Shiraki, Y. J. Electrochem. Soc. 1986, 133, 666-671. (24) Grest, G. S.; Kremer, K. Phys. Rev. A 1986, 3628-3631.

Figure 1. Characterization of Au nanopattern on Si surface. (A) Scanning electron microscope image. (B) Atomic force microscope image. (C) Energy dispersion analysis of X-ray when electron beam focused on area between and on Au nanodots, respectively.

the distance between them is about 2-3 persistence lengths, which corresponds to the geometry of Au nanopattern fabricated experimentally.

Figure 2. (A) Electropherogram of Φx174 RFI (supercoiled form) and Φx174 RFII (relaxed circular form) on a flat Si surface in 0.3 M TBE buffer at an electric field of 5 V/cm. (B) Electrophoretic mobilities of DNA with different forms as a function of buffer ionic strength. The solid lines for linear and relaxed DNA were fitted according to eq 3. For supercoiled DNA, the solid line is a linear fit.

planes of an fcc lattice. An L-J potential is used to model the interactions between the monomers and the surface atoms. The hexagonal patterned surface is modeled by introducing two types of atoms that are differentiated by the interaction with polymer atoms. The atoms with a higher interaction will be referred to as patch atoms with the other atoms as bare wall atoms. The size of each patch is about 3-4 persistence lengths of polymer chain and

RESULTS AND DISCUSSION Separation of DNA with Different Forms. In Figure 2A, we show the electropherogram of the 1:1 mixture of plasmid DNA Φx174 RF I (supercoiled form) and Φx174 RF II (relaxed form) with a concentration of 100 µg/mL at 0.3 M TBE buffer on a flat Si wafer. From the figure, we can see clearly that the supercoiled DNA moves much faster than the relaxed DNA, with a mobility that is nearly three times larger (Figure 2B). In contrast, the mobility of linear DNA of the same base pairs was calculated with previous data and shown to move with an intermediate value at the same buffer concentration (Figure 2B).9,25 We have also shown previously that the mobility of DNA on a surface was directly related to the average train ratio (number of segments in the trains over that of the full chain).25 For a given DNA chain, a higher train ratio, i.e., more surface contact points, produced a larger frictional force on the chain and resulted in a lower mobility. Therefore, the different mobilities for these three DNA forms could be ascribed to the different numbers of surface contacts associated with the different conformations on the surface. To further probe this model, we investigated the functional forms of DNA mobility varying with the buffer concentration. The counterions provide screening of the charges along the chain backbone and hence the persistence length P varies with buffer ionic strength I according to9

p ) 500 + 0.324I-1 Å

(1)

If we assume Gaussian statistics for the DNA chain, the number of surface contacts Nc is given by9

Nc ∼ Ns1/2 ∼ (aN)1/2/p1/2

(2)

(25) Li, B.; Fang, X.; Luo, H.; Petersen, E.; Seo, Y.; Samuilov, V.; Rafailovich, M.; Sokolov, J.; Gersappe, D.; Chu, B. Electrophoresis 2006, 27, 13121321.

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Figure 3. Electropherogram of supercoiled DNA ladder (ranging from 2067 to 16 210 bp) on (A) a flat Si surface and (B) a Au nanopatterned surface. (C) Calculated mobilities as a function of DNA length. (D) Resolution length calculated from electropherograms according to eq 4.

where Ns is the number of DNA segments, N the number of base pairs, a the length per base pairs, and P the persistence length. In our model, we assume the mobility of DNA chain on the surface is inversely proportional to the number of surface contacts. Therefore, combining eqs 1 and 2, we have

µ ∼ Nc-1 ∼ p1/2/(aN)1/2 ∼ (500 + 0.324I-1)1/2/(aN)1/2 (3)

Consequently, decreasing the buffer ionic strength should decrease the number of surface contacts and therefore increase the mobility of the chain at a given surface. The mobilities of the three types of DNA are plotted as a function of buffer concentration in Figure 2B. From the figure, we can see that the differences between them become even more evident. Relaxed DNA is obtained from supercoiled DNA when one of the strands is nicked, releasing the stored energy of supercoiling. The resulting conformation is then similar to the linear DNA, except that the chain is now a circular ring. Kosmas et al.12,26 have calculated the density profiles of polymer chains of various architectures: linear, ring, and branched near a surface. They found that the average number of surface contacts of the ring polymer was higher near the surface than for the linear polymer. Vanlent et al.27 also found the same results using selfconsistent field theory, and this effect was possibly due to the more compact structure and less conformational entropy loss upon (26) Stratouras, G. K.; Kosmas, M. K. Macromolecules 1991, 24, 6754-6758. (27) Vanlent, B.; Scheutjens, J.; Cosgrove, T. Macromolecules 1987, 20, 366370.

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adsorption of ring polymers than linear ones.27 However, the entropy difference between rings and linear polymers will converge to zero in the limit of infinite chain length. Equation 3 was used to fit the data for both relaxed and linear DNA, and as can be seen from the solid lines, it provides a very good fit for both shapes with only two fitting parameters. The fit for the linear and relaxed DNA can be superimposed with a simple shift. This is in agreement with the calculations of Kosmas,12 which predicted that even though the magnitude of number of surface contacts is different, the function is unchanged with both linear and ring polymers scaling with the molecular weight M as ∼M1/2, and hence, the two types of chains can be related by a constant scale factor. The supercoiled DNA does not appear to obey this simple relationship. Compared with linear DNA, the supercoiled form has a higher total free energy, which, besides the elastic energy, also has contributions from entropy induced by topological constraints and the electrostatic energy due to interactions between the two spatially close helices.28 This relatively higher energy makes the supercoiled DNA chain a little stiffer and more difficult to be adsorbed on surface. Furthermore, the conformation of supercoiled DNA is largely dependent on the buffer concentration. At high ionic strength, supercoiled DNA is strongly interwound and behaves as a linear-like chain. When the ionic strength goes lower, the supercoiled chain will form branches.28-30 If the ionic strength becomes further lower, the repulsive force between (28) Marko, J. F.; Siggia, E. D. Phys. Rev. E 1995, 52, 2912-2938. (29) Cherny, D. I.; Jovin, T. M. J. Mol. Biol. 2001, 313, 295-307. (30) Vologodskii, A. V.; Cozzarelli, N. R. Annu. Rev. Biophys. Biomol. Struct. 1994, 23, 609-643.

Figure 4. Electric field distribution simulated by finite element method on a nanopatterned surface. (A) Top view and (B) side view. The electric field lines are shown in insets. In simulation, the electric field strength between two electrodes was set at 5 V/cm.

DNA helices will unwind the supercoil into a circular ring.29 Correspondingly, the motion of these chains along the surface obeys a somewhat different dynamics from that of linear or ring chains. Qualitatively, two opposing effects can act. Kosmas12 calculated that the DNA branching may increase the number of surface contacts, which in turn would decrease the mobility. On the other hand, as the ionic strength is decreasing, the higher stiffness due to larger persistence length would counteract this effect by decreasing the number of surface contacts. This combination of factors clearly produces a different functional dependence on the buffer concentration, which could be fitted with a linear function as shown in Figure 2B. Since the relative magnitudes of the two effects are not known, we can only provide a qualitative explanation at this point. Due to the different functions with ionic strength, a mobility crossover between supercoiled and linear DNA was observed at very low ionic strength. Nevertheless, the pronounced differences in the mobility with buffer concentration suggest that one way to improve the separation between DNA of different forms can be realized by tuning buffer ionic strength. Separation of Supercoiled DNA Ladder. It has been shown previously that a DNA ladder with linear form can be separated on a flat surface.8,9 Here we investigated whether this technique is applicable in the case of a DNA ladder with other forms, such

as supercoiled. Figure 3A shows the electropherogram of the supercoiled DNA ladder ranging from 2067 to 16 210 bp obtained on a flat Si surface at an ionic strength of 0.1 M. Distinct peaks were observed, indicating that a supercoiled DNA ladder can also be effectively separated on a bare surface without any topological constraints. The calculated mobility, µ versus DNA base pairs, N was plotted on a log-log scale shown in Figure 3C, and it suggests that µ scales with N as N-R. The exponential constant obtained, R ) 0.27, is similar to that for linear DNA on the same Si surface, or R ) 0.25. An Au nanopattern was introduced on the Si surface, and the electropherogram of the supercoiled DNA ladder is shown in Figure 3B. Comparison with Figure 3A indicates that the mobilities were slightly decreased but both the efficiency and selectivity of the peaks were improved. The resolution length (RSL) of the peaks can be calculated from the dispersion using eq 4, where w are the FWHM of peaks and ∆t and ∆N are the

RSL ) w/(∆t/∆N)

(4)

difference in migration time and number of base pairs between two peaks, respectively. Obviously, as shown in Figure 3D, the resolution becomes better on a flat surface than on a patterned surface than on the flat surface, which is consistent with the Analytical Chemistry, Vol. 78, No. 14, July 15, 2006

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Figure 5. Mobility of ring polymer chains as a function of the number of polymer segments on flat and patterned surfaces. For a flat surface, the interaction parameter between bare surface and polymer chains, s, was set at 2.25, and for a patterned surface, the interaction between patches and polymer chains, p, was 2.5 with the bare wall having the same value s at 2.25. The inset is the corresponding plots of center of mass of ring polymers along the electric field direction as a function of time on flat and patterned surfaces. (Circle, up-triangle, and downtriangle denote ring polymers with different lengths of 60, 120, and 200, respectively. Black symbols mean on flat surface, while red symbols mean on patterned surface.)

Figure 6. Mobility of linear and ring polymers as a function of interaction amplitude between polymers and patches. The interaction parameter between bare surface and polymers, s, was set at 2.25, while the interaction parameter between patches and polymers, p, varied from 2.25 to 2.75. The inset is the corresponding plots of center of mass of linear (circles) and ring (triangles) polymers along the electric field direction as a function of time. (Black, red, and green symbols correspond to the interactions between patches and polymers at 2.25, 2.5, and 2.75, respectively.)

exponential constant R being increased from 0.27 to 0.33 (Figure 3C). Note that the florescence intensity of the peaks on the patterned surface is significantly higher, implying that a larger fraction of the chains were adsorbed onto the surface, thereby increasing the efficiency. Equation 3 indicates that the scaling exponent is ∼ -0.5, which seems inconsistent with the values we obtained experimentally. 4748 Analytical Chemistry, Vol. 78, No. 14, July 15, 2006

In fact, the function for the number of contact points in eq 2 is very simple, which is only valid in the case of an ideal Gaussian chain. The contact number is largely dependent on the conformation of adsorbed chains, which is a complicated function of chain length, the interaction between DNA segments and surfaces, and the environment the DNA chains experience. The previous papers have shown that the scaling can range from -0.25 on a bare Si

surface,9 -0.36 on a Ni nanopatterned surface,11 to -0.87 on a silane-coated surface.8 A more accurate mathematic model including such important parameters as ionic strength, surface interaction, DNA length, and DNA conformation is needed and is under development. Here we did not take the effect of electroosmotic flow (EOF) into account for two reasons. One is that the EOF is very small near surface and then increases to its bulk value at a distance of at least several times Debye length.31,32 Therefore, the effect of EOF on the DNA chains adsorbed on the surface is not strong compared with those in solution. The second reason is the Au nanopatterned surface has much fewer surface charges due to fewer SiOH groups and greater roughness than on a flat Si surface, which should result in much smaller EOF. Simulations of Electric Field Distribution on a Patterned Surface by FEM. In equilibrium, the adsorption of DNA from solution onto Si or Au surfaces is not appreciably different when no external electric field is applied.33 However, if there is an external electric field, since Au and Si have very different conductivities, the electric field distribution can be significantly altered by the presence of the Au nanopattern. To characterize this effect, we used the finite element method to simulate the electric field in the presence of the Au nanopattern. The geometry of each nanodot was constructed in accordance with that from AFM results. Panels A and B in Figure 4 show respectively the top and side views of electric field distribution with field lines illustrated in the insets. Along the electric field applied direction, there are areas with maximum field strength at both side walls of each nanodot, while the nanodot itself has minimum field strength. In the direction normal to the top surface of each nanodot, the electric field strength quickly rises up to a constant value within a distance of ∼200 nm, where the electric field lines are not disturbed by the Au nanodots. The existence of maximum field strength in a very local space means there is a strong electric field gradient directed to the side walls of the nanodot. On the other hand, the DNA chain as a polyelectrolyte will have a dipole in the presence of an external electric field due to the redistribution of counterions along the backbone of the DNA chain.34 Therefore, this electric field-induced dipole will experience a dielectrophoretic force in the gradient electric field and point to the side walls of each nanodot. In such a way, the dielectrophoretic trapping force makes the Au nanodots have higher adsorption capability and function as pinning points for DNA segments during electrophoresis. MD Simulations of Polymers on Flat and Patterned Surfaces. To probe the dynamics of polymer chains on flat or patterned surfaces, MD simulations were carried out. The strength of adsorption between polymer segments and patches, polymer and bare surface are characterized by parameters, p and s, respectively. The inset in Figure 5 shows the center of mass along the electric field direction as a function of time for ring polymer chains on flat and patterned surfaces, respectively. On the flat (31) Rice, C. L.; Whitehea. R J. Phys. Chem. 1965, 69, 4017-&. (32) Osuga, T.; Sakamoto, H.; Takagi, T. J. Phys. Soc. Jpn. 1996, 65, 18541858. (33) Petersen, E.; Li, B.; Fang, X.; Luo, H.; Seo, Y.; Samuilov, V.; Rafailovich, M.; Sokolov, J.; Gersappe, D.; Chu, B. DNA Migration on Electrically Inhomogeneous Surfaces, will be reported elsewhere. (34) Chou, C. F.; Tegenfeldt, J. O.; Bakajin, O.; Chan, S. S.; Cox, E. C.; Darnton, N.; Duke, T.; Austin, R. H. Biophys. J. 2002, 83, 2170-2179.

Figure 7. Ratio between the maximum length along the field direction to the maximum length normal to the field direction at a given conformation for both linear and ring polymers with the same length 60 segments on (A) flat, s ) 2.25 and (B) patterned surface, p ) 2.5s ) 2.25, as a function of time, respectively.

surface, the parameter s was kept at 2.25, while on patterned surface, p was set to be 2.5 with the bare wall having the same s value at 2.25. We can see that on both kinds of surfaces the separation between ring polymers of different lengths ranging from 60 to 200 segments was achieved and this was more evident on the surface with patterned structure. As shown in the main panel of Figure 5, the patterned surface has a larger mobility dispersion, which is in accordance with the experimental result shown in Figure 3C. The simulation results for polymer chains of the same size but different forms, such as linear and ring, were shown in Figure 6. We kept the surface attraction s at 2.25 and varied the interaction between patch and polymer p from 2.25 to 2.5 and 2.75. On all surfaces, ring polymers moved slower than linear ones and thus they could be separated. However, the maximum mobility difference was obtained in the intermediate interaction range between patch and polymer segments. Too strong or too weak interactions will impair the separation. This effect is similar to the results shown in Figure 2B, where the surface interaction was varied through ionic strength. Therefore, one advantage of a Au nanopatterned surface is that the possibility of modifying the Analytical Chemistry, Vol. 78, No. 14, July 15, 2006

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Figure 8. 3D snapshots of linear polymer and ring polymer of 60 segments on (A) flat, s ) 2.25 and (B) patterned surfaces, p ) 2.5s ) 2.25, respectively. Linear polymer chain is in black and ring polymer chain is in red.

Au surface chemically endows us with more choices to adjust such an interaction to get the optimum separation effects. The mobility difference between linear and ring polymers on flat or patterned surfaces should arise from their conformational differences. The polymer chains could stretch preferentially in one direction due to the driving and friction forces. For any conformation of a polymer chain at a given time, we can calculate from the simulations the maximum length along field direction and the maximum length normal to field direction. Figure 7 shows the ratio between these two values for both linear and ring polymer chains on a flat (Figure 7A) or patterned surface (Figure 7B) as a function of time. The fluctuations in data points indicate that the polymer chains follow a repeated process of stretching and shrinking to migrate on surfaces. We can see there is a slight biased orientation induced by an external field as the average ratios are not too much bigger than unit. Nonetheless, the linear chains have a higher average ratio and a bigger fluctuation, which means an easier deformation in the electric field than ring chains. The surface patterning does not have a noticeable effect on the chain deformation in this 2D picture. However, if we look at the polymer chains in a 3D picture, there are more obvious conformational differences as shown in Figure 8. The 3D snapshots of polymer chains on a flat surface (Figure 8A) illustrate that the ring chains are closer to the surface than the linear ones and the effect is more apparent on the patterned surface (Figure 8B). This is further confirmed by the average center of mass of polymer chains in the direction normal to surfaces, . The difference in between linear and ring chains is about 0.03 on the flat surface (Figure 9A) but goes up to 0.12 on the patterned surface (Figure 9B). From the above MD results, we can see that the flat surface can differentiate the conformational differences between linear and ring polymer chains in terms of deformation or center of mass in the normal direction. Due to the smaller fluctuation in deformation and the lower center of mass, ring polymer chains have a higher probability of forming surface contacts and move slower than 4750 Analytical Chemistry, Vol. 78, No. 14, July 15, 2006

Figure 9. Average center of mass of linear polymers and ring polymers of 60 segments in the direction normal to surface, on (A) flat, s ) 2.25 and (B) patterned surface, p ) 2.5s ) 2.25 as a function of time, respectively. Linear polymers are denoted in black and ring polymers in red.

linear polymer chains, which agrees with the migration order shown in Figure 2. Moreover, the conformational differences between linear and ring polymers were amplified on the patterned surface. It is the enhanced differences in conformations that give rise to a better separation on the patterned surface. CONCLUSIONS We show that Si surface electrophoresis is capable of separating DNA of different configurations, such as linear, supercoiled, and relaxed. The technique is based on detecting differences in mobility due to surface interactions between the chains and the substrate. Even though the numbers of base pairs of these samples were the same, the configuration differences give rise to different numbers of surface contacts. This can be a balance between the entropy penalty of confinement near the surface with the enthalpy gained from adsorption. It had been previously predicted12 that the number of surface contacts was proportional to M1/2, where M is molecular weight, with the proportionality constant being different for polymers of different forms. Our data are in good agreement with these predictions. The surface was also demonstrated to be effective in separation of DNA chains of the same configuration but different length, like the supercoiled DNA ladder. The superimposed Au nanopattern

onto the Si substrate improved both the efficiency and the separation. The patterned Au nanodots were found to act as sticky points due to dielectrophoretic force using finite element analysis. MD simulation of linear and ring polymers on flat and patterned surfaces revealed that the nanopattern can amplify the conformational differences between adsorbed polymer chains to achieve better separation effects.

ACKNOWLEDGMENT This work was supported by grants from NSF-MRSEC and Department of Energy. Received for review April 12, 2006. Accepted May 31, 2006. AC060686Z

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