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Entrapment of Individual DNA Molecules and Nanoparticles in Porous Alumina Membranes Changbei Ma and Edward S. Yeung* Ames Laboratory-USDOE and Department of Chemistry, Iowa State University, Ames, Iowa 50011 Depth-resolved fluorescence imaging allows the motion of single DNA molecules and single nanoparticles at the liquid/solid interface to be recorded in real time. Porous alumina membranes were employed as model chromatographic packing material. Using a suitable pH and ionic strength, adsorptive interactions are suppressed. The effects of 3-dimensional topography, specifically the presence of nanopores, on DNA and nanoparticle migration across the surface are, thus, revealed. The residence times and the number of immobilized DNA molecules or particles increased as the pores size increased. Yet, we found that the pore diameter must be significantly larger than the particle diameter or the DNA short radius before entrapment can occur. Furthermore, the depth distribution of particles does not conform to one-dimensional diffusion in the pores, probably because of collisions with the walls. These observations provide new insights into conventional liquid chromatography as well as sizeexclusion chromatography and membrane separations. Single-molecule spectroscopy has proven itself to be a powerful tool in chemistry and biosciences.1 Observation of single molecules allow their dynamic behaviors to be studied and provide insight into molecular genetics,2-4 biochip assembly,5-7 biosensor design,8-10 DNA biophysics,11-18 enzyme dynamics,19-24 DNA/ * Corresponding author. E-mail:
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protein detection,25-27 and basic separation theory.28-40 In singlemolecule experiments with λ-DNA, Kang et al. analyzed their motion and adsorption/desorption behavior at the fused-silica/ water interface and the C18/water interface as a function of pH and buffer composition.36 Li et al. used λ-DNA as a single-molecule probe to study chromatographic retention on various self-assembled monolayer surfaces based on hydrogen bonding ability and hydrophobicity of the unpaired purine and pyrimidine bases at the ends of the molecule.39,40 So far, all of these studies have been on flat surfaces for mapping 2-dimensional motion. The primary goal of the present work is to employ nanoporous alumina membranes as the model surface for chromatographic retention. The pores on the surface provide 3-dimensional structures that more closely resemble real chromatographic packing materials and may provide additional insights beyond flat selfassembled monolayers. Such information would be valuable for understanding and controlling partition chromatography and sizeexclusion chromatography (SEC). SEC is a widely used technique for the separation and purification of large molecules or macromolecular complexes such (17) Xu, X. H.; Yeung, E. S. Science 1997, 276, 1106–1109. (18) Ma, Y.; Shortreed, M. R.; Yeung, E. S. Anal. Chem. 2000, 72, 4640–4645. (19) Kettling, U.; Koltermann, A.; Schwille, P.; Eigen, M. Proc. Natl. Acad. Sci. U. S. A. 1998, 95, 1416–1420. (20) Xue, Q.; Yeung, E. S. Nature 1995, 373, 681–683. (21) Li, H. W.; Yeung, E. S. Anal. Chem. 2005, 77, 4374–4377. (22) Lu, H. P.; Xun, L. Y.; Xie, X. S. Science 1998, 282, 1877–1882. (23) Gorris, H. H.; Rissin, D. M.; Walt, D. R. Proc. Natl. Acad. Sci. U. S. A. 2007, 104, 17680–17685. (24) Li, J. W.; Yeung, E. S. Anal. Chem. 2008, 80, 8509–8513. (25) Li, Z. H.; Hayman, R. B.; Walt, D. R. J. Am. Chem. Soc. 2008, 130, 12622– 12623. (26) Lee, J. Y.; Li, J. W.; Yeung, E. S. Anal. Chem. 2007, 79, 8083–8089. (27) Li, J. W.; Xie, W. J.; Fang, N.; Yeung, E. S. Anal. Bioanal. Chem. 2009, 394, 489–497. (28) Xu, X. H.; Yeung, E. S. Science 1998, 281, 1650–1653. (29) Shortreed, M. R.; Li, H.; Huang, W. H.; Yeung, E. S. Anal. Chem. 2000, 72, 2879–2885. (30) Smith, S. B.; Aldridge, P. K.; Callis, J. B. Science 1989, 243, 203–206. (31) Zullli, S. L.; Kovaleski, J. M.; Zhu, X. R.; Harris, J. M.; Wirth, M. J. Anal. Chem. 1994, 66, 1708–1712. (32) McCain, K. S.; Schluesche, P.; Harris, J. M. Anal. Chem. 2004, 76, 939– 946. (33) Swinton, D. J.; Wirth, M. J. Anal. Chem. 2000, 72, 3725–3730. (34) Wirth, M. J.; Swinton, D. J. Anal. Chem. 1998, 70, 5264–5271. (35) Honcius, A.; Schwartz, D. K. J. Am. Chem. Soc. 2009, 131, 5973–5979. (36) Kang, S. H.; Shortreed, M. R.; Yeung, E. S. Anal. Chem. 2001, 73, 1091– 1099. (37) Kang, S. H.; Yeung, E. S. Anal. Chem. 2002, 74, 6334–6339. (38) Isailovic, S.; Li, H. W.; Yeung, E. S. J. Chromatogr. 2007, 1150, 259–266. (39) Li, H. W.; Park, H. Y.; Porter, M. D.; Yeung, E. S. Anal. Chem. 2005, 77, 3256–3260. (40) Park, H. Y.; Li, H. W.; Yeung, E. S.; Porter, M. D. Langmuir 2006, 22, 4244–4249. 10.1021/ac902109g 2010 American Chemical Society Published on Web 12/16/2009
Figure 1. Scanning electron microscope images of a 200 nm porous alumina membrane used in our experiments. (a) Top-surface view. (b) Cross-sectional view.
as proteins, polysaccharides, and nucleic acids.41 The popular commercial chromatographic packing materials are polyacrylamide, dextran, agarose, cross-linked polystyrene, or porous silica. The underlying principle of SEC is that particles of different sizes will elute through a stationary phase at different rates based on differential degrees of excursion into the pores. In this study, we elucidate the interactions between single DNA molecules or single nanoparticles in aqueous solution and porous alumina membranes with various pore sizes. EXPERIMENTAL SECTION Buffer Solutions. CHES buffer solutions (pH 9.0 and 10.0) were prepared from 1.0 M solutions of CHES and sodium hydroxide (ACS grade or higher, Fisher Scientific, Fair Lawn, NJ) dissolved in ultra pure (18 MΩ) water. The molarity and the nominal ionic strength of the buffers were 25 mM. All solutions were photobleached overnight using a mercury UV lamp and filtered through a 0.2 µm filter prior to use. DNA Samples. λ-DNA (48 502 bp) was purchased from Promega (Madison, WI). All DNA samples were prepared in 10 mM Gly-Gly buffer, pH 8.2 (Sigma Chemical Co., St. Louis, MO). DNA samples were labeled with YOYO-1 (Molecular Probes, Eugene, OR) at a ratio of one dye molecule per five base pairs. The stained DNA has a persistence length of 66 nm and a contour length of 20 µm.42 DNA samples were prepared at a concentration of 200 pM. For single-molecule imaging, these samples were further diluted to 4 pM with appropriate buffer solutions immediately prior to the start of the experiment. Nanoparticles. Carboxylated polystyrene nanoparticles were purchased from Duke Scientific (Fremont, CA). The particle solution consists of suspended green fluorescent polystyrene nanoparticles 85 nm in diameter with refractive index n ) 1.59 in a 100 mM phosphate buffer solution. The aqueous suspensions are packaged as 1% solids. The particles were diluted 10 000-fold with ultrapure water prior to the start of the experiment. Nanoporous Alumina Membranes. Nanoporous alumina membranes with pore sizes of 20, 100, and 200 nm and thickness of 60 µm were obtained commercially from Whatman International Ltd. (Maidstone, UK). Typical scanning electron microscope images of the 200 nm porous alumina membrane are shown in Figure 1. Prior to the measurements, the membranes were immersed in a buffer at room temperature overnight. Epifluorescence Microscopy and CCD Camera. An upright Nikon Eclipse 80i microscope was used to perform single-molecule (41) Striegel, A. M. Anal. Chem. 2005, 77, 104A–113A. (42) Quake, S. R.; Babcock, H.; Chu, S. Nature 1997, 388, 151–154.
experiments. A 100× objective lens (Nikon Plan Apo/1.40 oil) was used. The fine focusing adjustment of the microscope is 0.1 mm per rotation. A motorized rotary stage from Sigma Koki (model no. SGSP-60YAM) was coupled to the fine-adjustment knob on the microscope. The motor is controlled by Intelligent Driver, CSG-602R (Sigma Koki). The travel for each pulse is 0.0025° (0.69 nm). A CCD (Cool SNAP ES, Photometrics, Tucson, AZ, pixel size 6.4 µm) camera was mounted on the microscope. An X-Cite 120 PC (EXPO Photonic Solution, Inc.) 120 W metal halide short arc lamp was used as the light source. The CCD exposure frequency was 5 Hz (0.2 s/frame), and the exposure time for each frame was 10 ms for single-DNA molecule experiments and 100 ms for single-nanoparticle experiments. Winview 32 (Roper Scientific, Princeton, NJ) was used for image collection and data processing. RESULTS AND DISCUSSION Single-DNA Molecule Experiments. In order to demonstrate the steric effect of the pores on the interactions of single DNA molecules and porous alumina membranes, the outer surface must not favor the adsorption of DNA. First, we investigated singleDNA motion under bulk flow on the surface at pH 9.0 (point of zero charge, pzc, for AlOH) using a 20 nm membrane. We found that no DNA molecules stopped on this surface (data not shown). This indicates that alumina has low affinity for DNA at this pH. At the same time, it is difficult for DNA molecules to go into the pores because of the small size of the latter. Then, we monitored the motion of DNA molecules at pH 9.0 for different pore sizes (100 and 200 nm) under the same conditions. For the 100 nm pore membrane, most of the DNA molecules were freely moving on the surface, but some molecules stopped temporarily at seemingly random locations on the surface in the field of view. The 66 nm persistence length and the slight flexibility of the DNA chain allow partial penetration into the 100 nm pores. For the 200 nm pore membrane, more molecules stopped moving on the surface. Furthermore, once the molecules stopped, they stayed there for 13.0 s or more. Even more interestingly, about 20% of the DNA molecules could be seen with the free end rotating while the other end is inserted in the pore (Movie 1 in Supporting Information). Since the DNA strand has been shown not to adsorb onto alumina, the trapped end can be assumed to be free to allow rotation as opposed to being adsorbed to cause gyration. As can be seen from Figure 2, at pH 9.0, single λ-DNA molecules were immobilized in the 100 nm pores for 0.2-13.0 s (2 consecutive frames to 66 consecutive frames) and most molecules were immobilized for 4.8 or 5.2 s (25 or 27 consecutive Analytical Chemistry, Vol. 82, No. 2, January 15, 2010
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Figure 2. Histogram of single-DNA molecules recorded versus residence times for 4 pM DNA in a pH 9.0 buffer solution on various pore size membranes.
Figure 3. Histogram of single particles recorded versus residence time on various pore size membranes.
frames). However, almost all molecules were immobilized for 13.0 s or more (66 consecutive frames) on a 200 nm alumina membrane. Single Nanoparticles Experiments. As above, we first confirmed that nanoparticles are not immobilized on this surface at pH 9.0. Then, we monitored the motion of nanoparticles for different membrane pore sizes (100 and 200 nm) under the same conditions. For the 100 nm membrane, most of the nanoparticles were moving on or near the surface, but some nanoparticles were immobilized temporarily at seemingly random locations on the surface in the field of view. The particles were not held up for a long time, about 80% of them stayed there for only one second, probably because their diameters (85 nm) were close to the diameter of the pores (100 nm). For the 200 nm membrane, more nanoparticles were immobilized on the membrane. Furthermore, once the particles stopped moving, they stayed there for 13.0 s or more (Movie 2 in Supporting Information). As can be seen from Figure 3, single particles on a 100 nm alumina membrane were immobilized for 0.2-13.0 s (2 consecutive frames to 66 consecutive frames) and most were immobilized for 1.0 ± 0.2 s (5 to 7 consecutive frames) at the surface. In comparison to the behavior of DNA molecules on the same membrane, the residence times are generally shorter. This is consistent with the picture that DNA is not a rigid rod but a random coil that makes its effective short axis larger than its persistence length. However, almost all particles were immobilized for 13.0 s (66 consecutive frames) on the 200 nm alumina membrane, identical to the behavior of DNA molecules. In order to see how far the particles (85 nm) migrate into the 200 nm pores, the vertical positions of the particles were measured 656
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Figure 4. Histogram of single nanoparticles recorded versus pore depth (mean ( SD, n ) 3).
through optical sectioning accomplished by coupling the epifluorescence microscope with a motorized stage. Such depth-resolved particle distributions inside nanopores have profound implications on the mechanisms behind SEC and membrane separations. Even for conventional liquid chromatography, nanopores are inherent features in porous packing materials. The location of the surface of the membrane was determined by manually seeking the best focus for that plane. Then, the microscope stage was scanned vertically away from the surface in synchrony with the acquisition rate of the camera (Movie 3 in Supporting Information). By locating the image with the best focused spot for each particle, the vertical position of the particle was calculated. The precision stage has a resolution of 0.69 nm. The rotary rate was set as 500 steps/s, and the acquisition frequency was set as 5.2 Hz. So, each frame traveled 96 steps (500/5.2); that is 66 nm (0.69 × 96). The particle locations were measured to the nearest one or two frames, which was 66 or 132 nm. We examined each movie and counted all particles that were in focus somewhere within 30 consecutive frames. So, each bin is 2 µm wide. The median vertical positions are plotted against the particle number in Figure 4. It is clear that the particles were located at different depths from the membrane surface. As the depth increased, the number of particles in the pores decreased rapidly. However, since each pore is independent, linear diffusion theory predicts a Gaussian drop off of the molecule numbers as the depth increases.43 This is clearly not the case in Figure 4 (see analysis in Supporting Information). The fact that the nanoparticle size is not substantially smaller than the pore diameter may be contributing to this anomaly. This behavior is very different from those for proteins (with much smaller diameters) trapped in polycarbonate membranes,44 where a Gaussian distribution fits similar histograms very well. Further studies are needed to reveal the mechanism. CONCLUSIONS We demonstrated real-time 3-D imaging of single DNA molecules and single nanoparticles at the surface of and inside nanoporous alumina membranes. The residence times and the number of immobilized DNA molecules or particles increased as (43) Dill, K. A.; Bromberg, S. Molecular driving forces: statistical thermodynamics in chemistry and biology; Garland Science: New York, 2003; p 57. (44) Ma, C.; Yeung, E. S. Anal. Chem., in press.
the pores size increased, as expected. However, DNA molecules behaved as if their short axes were larger than the calculated persistence length. Furthermore, we demonstrated that the nanoparticles were actually distributed anomalously inside the pores at different depths. These results offer us valuable insights into the fundamental interactions governing single-molecule DNA sequencing, separation mechanisms in size-exclusion chromatography, and the application of nanoporous membranes in general. ACKNOWLEDGMENT We thank Dr. Hung-Ting (Tommy) Chen for the SEM images in Figure 1. E.S.Y. thanks the Robert Allen Wright Endowment for Excellence for support. The Ames Laboratory is operated for
the U.S. Department of Energy by Iowa State University under Contract No. DE-AC02-07CH11358. This work was supported by the Director of Science, Office of Basic Energy Science, Division of Chemical Sciences. SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.
Received for review September 21, 2009. Accepted December 4, 2009. AC902109G
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