Anal. Chem. 2002, 74, 3378-3385
Electrostretching DNA Molecules Using PolymerEnhanced Media within Microfabricated Devices Vijay Namasivayam,†,‡ Ronald G. Larson,† David T. Burke,§ and Mark A. Burns*,†
Department of Chemical Engineering, Department of Human Genetics, and Department of Electrical Engineering and Computer Science, The University of Michigan, Ann Arbor, Michigan 48109-2136
In this paper, we demonstrate immobilization and stretching of single λ-phage DNA molecules within microfluidic systems using ac fields. We present a novel “thiol-ongold”-based immobilization technique for fixing one specific end (3′ end) of a DNA molecule onto a gold electrode. A polymer-enhanced medium (∼3.75 wt % linear polyacrylamide in Tris-HCl) is used to obtain fully stretched configurations (21 µm) of fluorescently stained λ-DNA molecules. We also present an optimized microelectrode design with pointed electrodes and an electrode spacing of 20 µm for stretching DNA molecules with an ac field (1 MHz, 3 × 105 V/m). Finally, using these techniques, we immobilize a single DNA molecule at one electrode edge, stretch the molecule, and fix the other end at an adjacent electrode edge, forming a bridge between two electrodes within a microfabricated device. Single-molecule studies are becoming increasingly important in chemistry, biochemistry, and biotechnology. Recent developments in single-molecule analysis techniques allow the investigation of time-dependent reactions and understanding of molecular motors and also provide information on the structure and function of individual molecules.1 Among the molecules studied, biological macromolecules such as DNA and RNA are of tremendous interest to researchers from both fundamental and applied viewpoints.2 On a fundamental level, DNA manipulation studies can provide a wealth of information on the molecule’s elasticity and how it stretches under magnetic and flow fields.3 In addition, studies on how a DNA molecule interacts with enzymes that cut, copy, and splice can open doors for novel genetic analysis techniques.4,5 New techniques for mapping and sequencing have been reported that are based on stretching of single DNA molecules and splicing them with enzymes.6,7 * Corresponding author. Phone: (734) 764 4315. Fax: (734) 763 0459. E-mail:
[email protected]. † Department of Chemical Engineering. ‡ Department of Electrical Engineering and Computer Science. § Department of Human Genetics. (1) Bustamante, C.; Smith, S.; Liphardt, J.; Smith, D. Curr. Opin. Struct. Biol. 2000, 10, 279-285. (2) Mehta, A.; Simmons, M. A. Science 1999, 283, 1689-1695. (3) Smith, S. B.; Finzi, L.; Bustamante, C. Science 1992, 258, 1122-1126. (4) Strick, T. R.; Croquette, V.; Bensimon, D. Nature 2000, 404, 901-904. (5) Davenport, R. J.; Wuite, G. J.; Landick, R.; Bustamante, C. Science 2000, 287, 2497-2500.
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Stretching of long DNA molecules has been demonstrated using a variety of techniques including hydrodynamic, electrostatic, and magnetic manipulations. Most of these techniques rely on immobilizing one end of the DNA and applying an external force onto the other end of the molecule. In these studies, immobilization of DNA is often accomplished by using optical tweezers and latex beads8 and stretching is done using external syringe pumps,9 magnets,3 and AFM tips.10 Although such techniques have elucidated the physics of stretching, they are relatively bulky and may not be the best choice for incorporation into practical, highly integrated DNA analysis devices. One technique for single-molecule analysis that may be beneficial to explore is dielectrophoresis. Dielectrophoresis, though widely used for trapping and manipulating large biological particles such as bacteria and blood cells,11 has only recently been applied to the area of DNA manipulations. Dielectrophoresis occurs due to interaction of induced dipoles with electric fields. In many ways, these forces may be viewed as electrostatic equivalents to optical tweezers in that they exert translational forces on a particle due to interaction between the particle and an imposed electric field gradient. The first device for DNA stretching based on dielectrophoresis was built by Washizu et al.12 In their study, DNA molecules were stretched by a highfrequency (1 MHz), high-intensity (1 MV/m) ac field applied between aluminum electrodes. No active immobilization technique was used, and the experiments were carried out on solutions sandwiched between glass slides with one side containing the electrodes. A simple technique to construct dielectrophoresis devices for DNA stretching is photolithographic micromachining. Bulk and surface micromachining techniques are becoming increasingly popular for construction of a variety of miniaturized biochemical analysis devices. Devices made out of silicon, glass, and plastic (6) Jing, J.; Reed, J.; Huang, J.; Hu, X.; Clarke, V.; Edington, J.; Housman, D.; Anantharaman, T. S.; Huff, E. J.; Mishra, B.; Porter, B.; Shenker, A.; Wolfson, E.; Hiort, C.; Kantor, R.; Aston, C.; Schwartz, D. C. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 8046-8051. (7) Dorre, K. Bioimaging 1997, 5, 139-152. (8) Perkins, T. T.; Smith, D. E.; Larson, R. G.; Chu, S. Science 1995, 268, 8387. (9) Perkins, T. T.; Smith, D. E.; Chu, S. Science 1997, 276. (10) Washizu, M.; Yamamoto, T.; Kurosawa, O.; Shimamoto, N. Proc. Int. Conf. Sold-State Sens. Actuators 1997, 2C1.01, 473-476. (11) Green, N. G.; Morgan, H.; Milner, J. J. J. Biochem. Biophys. Methods 1997, 35, 89-102. (12) Washizu, M.; Kurosawa, O.; Arai, I.; Suzuki, S.; Shimamoto, N. IEEE Trans.: Ind. Appl. 1995, 31, 447-456. 10.1021/ac025551h CCC: $22.00
© 2002 American Chemical Society Published on Web 06/11/2002
Figure 1. Schematic of process used for making a microfabricated electrostretching device. (a) Thin glass side with gold electrodes, (b) thick glass side with microfluidic channel, and (c) assembled device.
materials have been used to carry out a variety of DNA-based analyses.13,14 Devices for sorting single molecules and cells already exist.15 In this work, we demonstrate the working of a microfluidic device that utilizes a novel immobilization chemistry, an optimized polymer-enhanced medium, and a well-characterized electrode design for immobilizing and stretching single DNA molecules. MATERIALS AND METHODS Device Construction. Our device consists of two sidessa thick glass side (500 µm thick) with fluidic channels etched in it and a thin glass side (100 µm thick) with the fabricated microelectrodes. Figure 1 illustrates the complete process for device construction. On the thin glass wafers (Dow Corning Pyrex 7740, 100-mm diameter), fabrication of the microelectrodes is accomplished by the “liftoff” technique. A positive resist (PR 1827, Hoechst Celanese) is spun on, patterned, and developed. A 300Å-thick chromium metal layer followed by a 1000-Å-thick gold metal layer is then deposited on the substrate by electron beam evaporation. The resist and the overlying metal layers are then “lifted off” using Microposit 1112A remover in solution (Shipley Co., Newton, MA), leaving the metal (Cr/Au) electrodes in the patterned areas and bare glass elsewhere. The wafers are then rinsed and dried. (13) Burns, M. A.; Johnson, B. N.; Brahmasandra, S. N.; Handique, K.; Webster, J. R.; Krishnan, M.; Sammarco, T. S.; Man, P. M.; Jones, D.; Heldsinger, D.; Mastrangelo, C. H.; Burke, D. T. Science 1998, 282, 484-487. (14) Liu, Y.; Ganser, D.; Schneider, A.; Liu, R.; Grodzinski, P.; Kroutchinina, N. Anal. Chem. 2001, 73, 4196-4201. (15) Chou, H.-P.; Spence, C.; Scherer, A.; Quake, S. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 11-13.
On the thick glass wafers (Dow Corning Pyrex 7740, 100-mm diameter), channels are fabricated by depositing a 600-Å-thick chromium metal layer followed by a 4000-Å-thick layer of gold. Photoresist (PR 1827, Hoechst Celanese) is spun, patterned using a channel mask, and developed. The metal layers are etched in a commercial gold etchant (Gold Etchant TFA, Transene Co., Danvers, MA) and chromium etchant (CR-14, Cyantek Inc., Fremont, CA). The accessible glass is then etched in a freshly prepared solution of hydrofluoric and nitric acid (7:3 v/v). After etching to the desired depth (20 µm), the metal layers are removed using the respective etchants and the resist is stripped in PRS 2000. The wafer is then rinsed in DI water, air-dried, and oven-dried at 100 °C for 20 min. Holes (∼200-µm radius) are drilled on the processed wafer by electrochemical discharge drilling16 to access the microchannels. The final channel dimensions are 100-400 µm wide and 20 µm deep. The individual devices on the thick glass and thin glass wafers are then diced and bonded using optical adhesive (SK-9 Lens Bond, Summers Laboratories, Fort Washington, PA). Finally, the assembled devices are wired to printed circuit boards (PCBs). The circuit boards have holes drilled on them to permit backside illumination needed for fluorescence imaging using an inverted microscope. DNA Sample Preparation. λ-phage DNA (48 000 bp long) with a 12-base-long single-strand “overhang” on each end was obtained from Gibco BRL. Oligonucleotides complementary to this overhang sequence were custom synthesized by Sigma Genosys, (16) Shoji, S.; Esahi, M. Technical Digest of the 9th Sensor Symposium 1990, 27-30.
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with special requests for a thiol label at the 3′ end and dephosphorylation at the 5′ end (oligonucleotide sequence: 5′-AGG TCG CCG CCC-thiol-3′]. Prior to ligating the custom oligos onto λ-DNA molecules, the λ-DNA molecules were first dephosphorylated. Dephosphorylation was done by a standard reaction protocol that uses calf intestine alkaline phosphatase.17 The custom oligos were then ligated onto the λ-phage DNA following a standard molecular biology protocol that uses T4 ligase enzyme.18 The thiol-labeled λ-DNA molecules were then mixed with a fluorescent marker YOYO-1 (Molecular Probes), in the ratio of 5 bp/1 dye molecule and diluted in Tris-HCl (pH 8) to a concentration of 5000 pg/µL. This solution was further diluted to a DNA concentration of 50 pg/µL in an 80:20 mixture of Tris-HCl/βmercaptoethanol. β-Mercaptoethanol was used to prevent photobleaching of the DNA molecules. For studying DNA stretching in polymer-enhanced media, linear polyacrylamide (Beckman Coulter) was added in various proportions in the second (TrisHCl/β-mercaptoethanol) dilution solution. A typical polymerenhanced medium was prepared in two steps as follows: In the first step, λ-DNA (0.5 µg/µL from Gibco BRL) was stained with YOYO-1 (0.077 µL, 1 mM from Molecular Probes) and suspended in a Tris-HCl solution (49.4 µL, pH 8). In the second step, the premixed λ-DNA solution from the first step was diluted (100fold) in the polymer-enhanced medium that consists of 50 µL of linear polyacrylamide (for 3.75 wt % polymer solution), 30 µL of Tris-HCl (pH 8), and 20 µL of β-Mercaptoethanol. Fluorescence Microscopy. The microfabricated device was mounted on an inverted fluorescence microscope (Nikon TE 200) for visualizing DNA stretching and immobilization. Single-molecule observations were obtained using a high-magnification (100×) oil immersion objective lens. Images were acquired using a 12-bit high-resolution cooled Digital Interline CCD (Micromax: 1300 YHS, Princeton Instruments) and analyzed using Metaview software. A 100-W mercury lamp (Nikon) with the appropriate filter for YOYO-1/SYBR was used as an excitation source. To record the time-dependent behavior of the molecules, each sequence of images was taken with 100-ms exposure time per image. An electronic shutter (Uniblitz) was used to prevent photobleaching during imaging. Application of Electric Fields. Oscillating voltages were generated using a function generator (Elenco MX 9300A) with frequencies ranging from 1 Hz to 1 MHz. Amplification circuitry was built in-house to amplify the 2-V signals from the function generator to 30 V. At low frequencies (
