Letter pubs.acs.org/NanoLett
Arrangement of a Nanostructure Array To Control Equilibrium and Nonequilibrium Transports of Macromolecules Takao Yasui,*,†,‡ Noritada Kaji,*,†,‡,△ Ryo Ogawa,§ Shingi Hashioka,§ Manabu Tokeshi,‡,∥ Yasuhiro Horiike,§ and Yoshinobu Baba*,†,‡,⊥,# †
Department of Applied Chemistry, Graduate School of Engineering and ‡ImPACT Research Center for Advanced Nanobiodevices, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan § National Institute for Materials Science, Tsukuba 305-0044, Japan ∥ Division of Biotechnology and Macromolecular Chemistry, Faculty of Engineering, Hokkaido University, Kita 13 Nishi 8, Kita-ku, Sapporo 060-8628, Japan ⊥ Institute of Innovation for Future Society, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan # Health Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Takamatsu 761-0395, Japan △ ERATO Higashiyama Live-Holonics Project, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8602, Japan S Supporting Information *
ABSTRACT: Exploiting the nonequilibrium transport of macromolecules makes it possible to increase the separation speed without any loss of separation resolution. Here we report the arrangement of a nanostructure array in microchannels to control equilibrium and nonequilibrium transports of macromolecules. The direct observation and separation of macromolecules in the nanopillar array reported here are the first to reveal the nonequilibrium transport, which has a potential to overcome the intrinsic trade-off between the separation speed and resolution. KEYWORDS: Nonequilibrium transport, macromolecules, nanostructure array, nanopillar Laachi et al.3 pointed out that the near-equilibrium separation process was not intrinsic to nanofluidic systems, and they theoretically predicted that the nonequilibrium separation process could occur and be applied in these systems. Developments of nanofabrication techniques since that prediction have made it possible to construct various nanofabricated structures for the nonequilibrium separation process. Precisely controlled structures provide ideal nanoenvironments for theoretical studies of polymer dynamics, and highly ordered nanostructures have been used to experimentally elucidate dynamics of macromolecules (e.g., DNA molecules) in confined spaces,4−8 which had been theoretically predicted by de Gennes.9 For the practical use of these nanofabricated structures as an alternative to conventional separation matrices such as gels or polymers, nanopillars,10−15 nanowalls, 16 nanofilters, 17−20 nanochannels, 21−23 nanowires,24,25 and nanoparticles26−30 have been developed to achieve high-speed separation under applied large driving
T
ransport processes of macromolecules in a separation column are near-equilibrium processes, for example, electrophoresis and gas/liquid chromatography, which accompanies a separation based on differences in physical or chemical characteristics.1,2 When the macromolecules move through the column, a separation occurs with accompanying the nearequilibrium transport process between the driving forces for transport and diffusion of the macromolecules. Because the driving force for transport of the macromolecules (electric field and pressure flow) is typically much weaker than that for diffusion of them, the separation of the macromolecules inherently has a limitation of separation speed. Therefore, in the near-equilibrium transport process, increasing the transport velocity results in lowering the occurrence of the physical or chemical interactions related to the resolution; an intrinsic trade-off between transport velocity and separation resolution. This trade-off is not unique to electrophoresis and chromatography, but rather spreads much of the separations, such as, distillation, adsorption, and extraction. In this study, we found a way to escape the intrinsic trade-off by using a nanopillar array in microchannels: a nonequilibrium transport process. © XXXX American Chemical Society
Received: February 26, 2015 Revised: April 13, 2015
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DOI: 10.1021/acs.nanolett.5b00783 Nano Lett. XXXX, XXX, XXX−XXX
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Figure 1. Nanopillars arranged in a square array pattern. (a) Schematic illustrations showing nanopillars embedded in a microchannel. The microchannel and nanopillars are formed on fused silica substrate. (b) Schematic illustrations showing the migration behavior of DNA molecules between nanopillars. The blue dotted arrow shows the migration direction of the molecules. (c) A photo of the fused silica substrate with fabricated nanopillars embedded in its microchannel. (d) SEM image of the nanopillars embedded in a microchannel; scale bar, 25 μm. Inside the 25 μm wide microchannel, nanopillar regions were patterned. Enlarged SEM images of the square array pattern with various spacings: (e) 300, (f) 500, (g) 700, and (h) 1000 nm; scale bars, 1 μm. Each nanopillar is 500 nm in diameter and 4 μm high.
arranged in a tilted array pattern were for the equilibrium transport. The tilted array pattern could be a sieving matrix and could separate large DNA molecules, which are difficult to separate under direct current (dc) electric fields, by physical collisions between DNA molecules and nanopillars; the separation was a near-equilibrium process.12 In contrast, the square array pattern had fewer physical collisions between DNA molecules and the nanopillars. Actually, we observed that a long DNA molecule migrated straightforwardly through the spacing between the nanopillars with a linear conformation in the square array pattern (see Supporting Information Movie 1), although the DNA molecule had a collision at the interface between the nanopillar and nanopillar-free regions. Significantly, the square array pattern could separate DNA molecules with an increase of the transport velocity (separation speed) that enhanced the separation resolution; this separation was a nonequilibrium process.
forces. The separation processes using these nanostructures were, however, all fundamentally based on the near-equilibrium transport process between the driving forces for transport and diffusion. These separations involved the intrinsic trade-off between transport and diffusion; an increase of the transport velocity by an electric field or a pressure flow forced the inherent equilibrium consisting of physical or chemical interactions to shift farther away, resulting in degradation of the separation resolution. To the best of our knowledge, only one experimental report has elucidated the separation based on the nonequilibrium transport in nanofluidic systems.31 Here we report the arrangement of a nanopillar array in microchannels to control equilibrium and nonequilibrium transports of macromolecules. Previously, we fabricated two types of a nanopillar array pattern on a quartz substrate, tilted and square, in microchannels.14 In this study, we found the nonequilibrium transport of DNA molecules could be realized just by arranging a nanopillar square array, while nanopillars B
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Figure 2. Nonequilibrium transport of DNA molecules in the square array pattern with 300 nm spacing. Time course observation of a single 600 bp DNA molecule (a) every 1 frame at 70 V/cm (Per = 0.2) and (b) every 5 frames at 20 V/cm (Per = 0.06); scale bars, 10 μm. Tracking of a single 600 bp DNA molecule (c) at 70 V/cm and (d) at 20 V/cm. Each spot color corresponds to the behavior of the same one DNA molecule. Five DNA molecules were tracked and plotted. (e) Schematic of the simulated field potential in the nanopillars arranged in the square array pattern. In this nonuniform electric field, the rotational configuration of depicted DNA molecules is determined by two forces, the characteristic torque M subjected to the electric field and the Boltzmann factor kBT. The predicted trajectory and the rotational force of DNA molecules are depicted in white arrows and black arrows, respectively. (f) Magnified images for the white dotted box in Figure 2e. Fn and Fs are the exerted forces on the two ends of the DNA molecule. The predicted trajectory and the rotational force of DNA molecules are depicted in white arrows and red arrows, respectively. Larger DNA molecules occupying a wider electric field gradient receive a stronger torque M to align along the electric field. Smaller DNA molecules occupying a narrower electric field gradient do not receive enough torque M to align along the electric field.
electric field and nanopillar spacing, affect the nonequilibrium transport process and attempted to interpret the separation mechanism in the square array pattern by comparing the present results with those of other conventional separation mechanisms. To quantify the events of the nonequilibrium transport for DNA molecules, rotational Péclet number, Per, is derived as the following equation3 (details shown in the Supporting Information)
The nonequilibrium transport for DNA molecules, which has a potential to overcome the intrinsic trade-off between the separation speed and resolution of DNA separations, was first elucidated experimentally for the nanopillars arranged in the square array pattern in this paper. We found that the nonequilibrium transport could be achieved under a high electric field condition in the square array pattern. For the square array pattern, 500 nm diameter and 4000 nm high nanopillars were positioned parallel to the microchannel with various spacings from 300 to 1000 nm as shown in Figure 1. Using the condition to satisfy the nonequilibrium transport of DNA molecules in the square array pattern, we separated some specific sizes of DNA molecules, and we also saw the interesting phenomenon that the DNA migration order was reversed; larger DNA molecules migrated faster than smaller DNA molecules. We investigated which parameters, such as applied
Per =
⎛ 1 − δ ⎞⎛ qÊ av L2 ⎞ M ⎟⎜ ⎟ =⎜ ⎝ 1 + δ ⎠⎝ kBT ⎠ kBT
(1)
where kBT is the Boltzmann factor, M is the electric-fieldinduced torque estimated from the electric field gradient acting on a DNA molecule near an entry or an exit of the nanopillar C
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Figure 3. Nonequilibrium transport-based separation of DNA molecules in the square array pattern with 300 nm spacing. (a) Relationship between the rotational Péclet number and the electric field for 600 and 100 bp DNA molecules. The dotted line indicates Per = 0.1. Electropherograms of the mixture of 600 and 100 bp DNA molecules at (b) 70 V/cm and (c) 20 V/cm. Concentrations are 30 ng/μL (600 bp) and 30 ng/μL (100 bp). (d) Separation resolution of 1000 and 100 bp (blue) and 600 and 100 bp (red), and 300 and 100 bp (green) DNA molecules as a function of the electric field. Each plotted value is the average over five repeated runs. (e) Separation of the mixture of 48.5, 2.2, 1 kbp, and 100 bp DNA molecules at 70 V/ cm. Concentrations are 10 ng/μL (48.5 kbp), 15 ng/μL (2.2 kbp), 20 ng/μL (1 kbp), and 30 ng/μL (100 bp).
spacing, δ is the spacing ratio, q̂ is DNA charge per unit length, Eav is the averaged electric field, and L is the contour length of the DNA molecule. Because the rotational diffusion forces DNA molecules to take a forbidden conformation upon reaching the next nanopillar spacing, the DNA molecules do not simply migrate unhindered from one nanopillar spacing to another. To escape from the forbidden conformation, these DNA molecules must overcome the rotational diffusion through one of two transport processes: one is a continued rotational diffusion and the other is an electric-field-induced torque, that is, an electrorotation. Concerning semirigid DNA molecules of less than about 1500 base pair (bp),3,18,19 they are expected to migrate in the nonequilibrium manner via electrorotation between consecutive nanopillars under strong electric fields. Equation 1 suggests that a high electric field and large DNA molecules lead to a large rotational Péclet number, that is, the electrorotation overcomes the diffusion-based transport. To confirm the nonequilibrium transport of DNA molecules in the square array pattern (300 nm spacing), we carried out electrophoretic migrations of 600 bp DNA molecules under relatively low and high electric fields at a single DNA molecule level (Figure 2a−d). At 70 V/cm, which corresponds to Per = 0.2, DNA molecules migrated straightforward along the electric field line (Figure 2a,c, and Supporting Information Movie 2):
the nonequilibrium transport. In contrast, at 20 V/cm (Per = 0.06), DNA molecules migrated while diffusing perpendicular to the electrophoretic migration direction (Figure 2b,d and Supporting Information Movie 3): the diffusion-based transport. Therefore, Per might be a major index to determine transport conditions, such as the nonequilibrium transport regime or the diffusion-based transport regime, and the nonequilibrium transport of the DNA molecule would be controlled by the electric fields. Actually, Lacchi et al.3 performed simulations based on this model, and they found that there is a crossover regime from the diffusion-based transport to the nonequilibrium transport around Per ∼ 0.1 where the loss of separation resolution was observed in the entropic trapping-based separation. The nonequilibrium transport of DNA molecules in the square array pattern (300 nm spacing) was anticipated from a simulated field potential (Figure 2e). Under the nonuniform electric field gradient in the square array, the larger DNA molecule occupies a wider electric field gradient than the smaller DNA one, and consequently, the larger DNA molecule experiences a stronger torque M and tends to align along the electric field. This motion of the larger DNA molecule by the electric-field-induced torque prohibits excursion of the DNA molecule between the consecutive nanopillars and enhances the entry and the escape from the confined space formed by the D
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chromatography),33−35 and from a couple of hundred kbp down to 1.5 kbp (the crossover point to Ogston sieving-based separation),18,19,36 respectively. In slalom chromatography and Ogston sieving-based separation, the smaller DNA molecules migrate faster than the larger ones, and thus gel filtration chromatography and slalom chromatography and entropic trapping-based separation and Ogston sieving-based separation inherently cannot be employed at the same time. We noted that the nonequilibrium transport-based separation could possibly be used in combination with entropic trapping-based separation. To demonstrate the combination use in the square array pattern, we separated four different sizes of DNA molecules (48.5, 2.2, 1 kbp, 100 bp) within 60 s (Figure 3e). A striking characteristic of this separation was that the larger DNA molecules migrated faster than the smaller DNA molecules; no reversals of migration order were seen in this range. From these results, the nonequilibrium transport-based separation can be used in company with entropic trappingbased separation. The relationship between separation resolution and nanopillar spacing in the nonequilibrium transport-based separation showed that the narrower spacing provided better resolution (Figure 4). A mixture of 1 kbp and 100 bp DNA molecules was provided for separation experiments in the square array pattern with the nanopillar spacing from 300 to 1000 nm. We separated
surrounding nanopillars (Figure 2f). Thus, we would expect that the larger DNA molecule under higher electric field, that is, over a threshold of the rotational Péclet number, had a chance to transit from the diffusion-based transport to the nonequilibrium transport. On the other hand, under the threshold of the rotational Péclet number, the diffusion-based transport would be the dominant force for DNA molecules (Supporting Information Movie 4). These experimental and simulation results elucidated the nonequilibrium transport of rigid macromolecules in the nanometer-confined spaces between the nanopillars at high electric fields and also suggested the separation of DNA molecules based on the nonequilibrium transport in the square array pattern. In such a separation, larger DNA molecules should migrate faster than smaller DNA ones. The applicable size range of DNA molecules using this theory will be around 1500 bp DNA molecules, which are regarded as a semirigid DNA molecule. In the square array pattern, which has 500 nm diameter nanopillars with 300 nm spacing, the rotational Péclet numbers for 600 and 100 bp at 70 V/cm were estimated to be over and below 0.1, respectively, while both numbers for 600 and 100 bp at 20 V/cm were below 0.1 (Figure 3a). As expected, we could separate the mixture of 600 and 100 bp at 70 V/cm, but could not separate the mixture at 20 V/cm (Figure 3b,c). Also, Figure 3d and Supporting Information Figure S1 show that the resolution increased in response to an increase of the electric field. These results highlighted that nonequilibrium DNA transport had a potential to overcome the intrinsic trade-off between the separation speed and resolution of DNA separations; that is, we could increase the separation speed without any loss of resolution. Besides the separation of 600 and 100 bp DNA molecules, we could separate 1000, 300, and 100 bp DNA molecules under the high electric fields and they showed the same tendency for separation resolution (Figure 3d). The nonequilibrium transport-based separation had the same feature, faster migration of the larger DNA molecules, as gel filtration chromatography or entropic trapping-based separation; however, neither of them could satisfy the separation mechanism due to the following two reasons. For gel filtration chromatography, which has degraded resolution with increasing elution speed,32,33 the separation is the near-equilibrium process. For entropic trapping-based separation, no effect can be caused for DNA molecules smaller than 1.5 kbp.18,19 Our separation results showed the DNA molecules (less than 1.5 kbp) had better resolution in higher electric fields, and therefore, the DNA migration behaviors in the square array pattern were completely different from those in gel filtration chromatography or entropic trapping-based separation. Another different aspect between the nonequilibrium transport-based separation and gel filtration chromatography or entropic trapping-based separation is the applicable size range of DNA molecules. The nonequilibrium transport-based separation can be applied to only semirigid DNA molecules, while the gel filtration chromatography can be applied to only DNA molecules without conformation change in hydrodynamic flow and the entropic trapping-based separation can be applied to only coiled conformation DNA molecules. The applicable size ranges for these three kinds of separation methods, the nonequilibrium transport-based separation, the gel filtration chromatography, and the entropic trapping-based separation, should be from several bp to less than about 1.5 kbp, from several bp up to 7 kbp (the crossover point to slalom
Figure 4. Effect of different nanopillar spacings of the square array pattern on DNA separation. (a) Electropherograms of the mixture of 1 kbp and 100 bp DNA molecules at 40 and 20 ng/μL, respectively. All separations were run at 70 V/cm. (b) Separation resolution versus the nanopillar spacing. Each plotted value is the average for eight repeated runs. E
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(Takara Bio) digested by PstI (Takara Bio) was used for 2.2 kbp DNA, and 1 kbp, 600 bp, 300 bp, and 100 bp DNA molecules were purchased from Thermo Scientific. Observation and separation of DNA molecules were carried out on a custom-built laser-induced fluorescent microscope-based electrophoresis system. The system mainly consists of an optical imaging part and a high voltage power supply. The high voltage for electrophoresis was supplied from a high voltage control (HVS448, LabSmith) through a platinum electrode that was precisely positioned on an inverted microscope (Eclipse TE300, Nikon). A 20 mW blue laser (FLS-488-20, Sigma Koki Co., Ltd.) was used as an optical source to illuminate DNA bands at a detection point and emitted fluorescence was collected with an EB-CCD camera (C7190-43, Hamamatsu Photonics K.K.) through a 100×/1.40NA and a 10×/0.45NA objective lens. Electropherograms were taken at 5115 μm from the entrance of the nanopillar channel. All images were recorded on a DV tape (DSR-11, Sony) and then the fluorescence intensity was analyzed by image-processing software (Cosmos32, Library). DNA ladders were stained with bis-intercalating fluorescence dye, YOYO-1 (Invitrogen), at a dye-to-base pair ratio of 1:5 for single DNA observation or 1:15 for DNA separation. To reduce the influence of electroosmotic flow on the quartz surface, a concentrated buffer solution (3× TBE; 267 mM Tris-borate and 6 mM EDTA, pH 8.2) containing 10 mM dithiothreitol as an antiphotobleaching agent was used;14 the electro-osmotic flow mobility is around 10−5 cm2 V−1 s−1, which is ten times smaller than the electrophoretic mobility (approximately 10−4 cm2 V−1 s−1). Electric Field Simulation. Numerical simulations of the electric field profile in the nanopillars were performed using CoventorWare v2003.1 (Coventor Technologies). Nanopillars were 500 nm in diameter with a spacing of 500 nm and were arrayed in a square array pattern.
the mixture of 1 kbp and 100 bp even when the nanopillar spacing was expanded to 700 nm. Only the 1000 nm nanopillar spacing did not provide separation (Figure 4a). As could be expected from conventional gel electrophoresis, the separation resolutions were proportional to the nanopillar spacing and the 300 nm spacing gave the best resolution among these nanopillar spacings (Figure 4b). This was due to the gradient of the enhanced periodically repeated electric fields formed by the narrower nanopillar spacing and was unlike the general rule in gel electrophoresis, which indicates that relatively small DNA molecules are in smaller pore sizes formed by higher concentrations of gels and polymers.37 Actually, in nanowall array structures, which have less periodically repeated nanopillar spacing and electric field gradient, the mixture of 1 kbp and 100 bp DNA molecules was never separated by the nanowall array structures.16 For the separation of the mixture of 1 kbp and 100 bp DNA molecules in the nonequilibrium transport-based separation, the periodically repeated spacing and electric field gradient is essential for the 100 bp DNA molecules to travel perpendicular to the field line by diffusion. In summary, we have demonstrated that equilibrium and nonequilibrium transports of DNA molecules could be controlled by arranging the nanopillar array pattern as tilted or square array patterns. Equilibrium transport of DNA molecules has been demonstrated in the titled array pattern previously,12 and nonequilibrium DNA transport was elucidated experimentally in the square array pattern in this paper. This nonequilibrium transport-based separation has a potential to overcome an intrinsic trade-off between the separation speed and resolution of DNA separations, resulting in a fast separation without any loss of resolution. Because the nanopillars are fabricated by a nanofabrication technique, their high design flexibility can contribute not only to fundamental studies such as on this nonequilibrium molecular transport, but also to applications such as separations for biomolecules. Ultimately, we anticipate that a highly integrated system on a single chip will be feasible if we can combine nanopillar array structures and an inkjet-based DNA sample injection system38 (Supporting Information Figure S2). Such a highly integrated system will offer new separation tools not only for biomolecules but also for macromolecules. Methods. Nanopillar Fabrication. Nanopillars were fabricated on a quartz substrate using basically the same procedures as described elsewhere.12,14,16 First a Pt layer and then a Cr layer (each 10 nm thick) were sputter-coated on a 0.5 mm thick quartz plate. A positive electron beam (EB) resist (ZEP-520A, Zeon) was spin-coated on the Cr layer, and then a nanopillar array pattern was delineated by EB lithography (ELS-7500, Elionix). Ni was electroplated into the holes of the nanopillar array pattern in the EB resist to provide strong resistance in the following etching process. After the EB resist removal, photoresist (OFPR8600, Tokyo Ohka Kogyo) was spin-coated and a microchannel pattern was transferred by photolithography. The substrate was etched by neutral loop discharge plasma using CF4. Inside a 25 μm wide microchannel, 500 nm diameter and 4 μm high nanopillars were fabricated with different nanopillar spacings of 300, 500, 700, and 1,000 nm. After removal of the remaining metal and resist layers on the quartz substrate, both the substrate and a 130 μm thick quartz cover plate were dipped into H2SiF6 and bonded under pressure of 5 MPa at 65 °C for 12 h. DNA Observation and Separation. Lambda DNA (Nippon Gene) was used for 48.5 kbp DNA molecules, and pKF3 DNA
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ASSOCIATED CONTENT
S Supporting Information *
DNA migration Movies 1−3, animation Movie 4, Figures S1 and S2, additional text, and references. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Authors
*(T.Y) E-mail:
[email protected]. Phone: +81-52789-4611. Fax: +81-52-789-4666. *(N.K) E-mail:
[email protected]. Phone: +81-52789-4498. Fax: +81-52-789-4666. *(Y.B) E-mail:
[email protected]. Phone: +8152-789-4664. Fax: +81-52-789-4666. Author Contributions
T.Y., N.K., R.O., S.H., M.T., Y.H., and Y.B. planned and designed the experiments. T.Y., N.K., R.O., and S.H. performed the research. T.Y. and N.K. analyzed data and wrote the paper. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This research was supported by the Cabinet Office, Government of Japan and the Japan Society for the Promotion of Science (JSPS) through the Funding Program for WorldLeading Innovative R&D on Science and Technology (FIRST Program), Nanotechnology Platform Program (Molecule and F
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(31) Strychalski, E. A.; Lau, H. W.; Archer, L. A. J. Appl. Phys. 2009, 106, 024915. (32) Kato, Y.; Sasaki, M.; Hashimoto, T.; Murotsu, T.; Fukushige, S.; Matsubara, K. J. Biochem. 1984, 95, 83−86. (33) Wu, C.-S. Handbook of Size Exclusion Chromatography and Related Techniques, 2nd ed.; Marcel Dekker: New York, 2004; pp xiv− 694. (34) Hirabayashi, J.; Kasai, K. Anal. Biochem. 1989, 178, 336−341. (35) Hirabayashi, J.; Ito, N.; Noguchi, K.; Kasai, K. Biochemistry 1990, 29, 9515−9521. (36) Viovy, J. L. Rev. Mod. Phys. 2000, 72, 813−872. (37) Buchholz, B. A.; Shi, W.; Barron, A. E. Electrophoresis 2002, 23, 1398−1409. (38) Yasui, T.; Inoue, Y.; Naito, T.; Okamoto, Y.; Kaji, N.; Tokeshi, M.; Baba, Y. Anal. Chem. 2012, 84, 9282−9286.
Material Synthesis) of the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan, the JSPS Grant-in-Aid for JSPS Fellows, the JSPS Grant-in-Aid for Scientific Research (A) 24241050, and the JSPS Grant-in-Aid for Young Scientists (B) 25790028.
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