Design for DNA Separation Medium Using Bacterial
Cellulose Fibrils
Mari Tabuchi*,† and Yoshinobu Baba†,‡
Department of Molecular and Pharmaceutical Biotechnology, Graduate School of Pharmaceutical Science, The University of
Tokushima COE, 1-78 Shomachi, Tokushima 770-8505, Japan, and Department of Applied Chemistry, Graduate School of
Engineering, Nagoya University, Nagoya 464-8603, Japan and National Institute of Advanced Industrial Science and
Technology, Takamatsu 761-0395, Japan
In this paper, we present a novel DNA separation medium
using bacterial cellulose fibrils. Bacterial cellulose has an
intrinsic three-dimensional micrometer- to nanometer-
scale network structure. Addition of this material to a low-
concentration polymer solution (<5 cP) enables high-
resolution electrophoretic separation of DNA, even for
fragments of 10-100-bp or single-nucleotide polymor-
phism. The newly designed medium consists of a double
mesh: a 10-nm flexible mesh derived from a conventional
polymer medium containing 10-nm to 1-µm rigid pores
made up of 10-µm bacterial cellulose fragments.
Rapid and accurate analysis of biomolecules is especially
important for clinical diagnosis, and such clinical applications allow
no room for error. Electrophoresis, including capillary sequencer
or microchip techniques, is a powerful technique in this regard.
These methods take advantage of the sieving effect of gels or
polymer solutions. A variety of separation media have been
developed that utilize the sieving effect of polymer solutions.
1-4
Design of the optimal mesh size for DNA separation has been a
major focus (Figure 1Aa,b). Recently, a new concept for separation
has been developed that takes advantage of the nanospaces in
nanofluid structures, for example, pillars,
5-7
magnetic structures,
8
or nanoballs
9
(Figure 1Ac). We and Huang et al. recently
developed nanosphere mixed media (PEGylated latex
10
or gold
nanoparticles
11
), which combine these two effects (Figure 1Ad).
Herein, we describe our most recent refinement of this, which
we call the double-mesh concept combined with stereo (obstacle)
effect (Figure 1Ae).
Plant cellulose derivatives are often used as a polymer solution
in microchip electrophoresis buffers. Bacterial cellulose (BC),
12
which is produced by some strains of bacteria, offers a unique
alternative to plant cellulose. For example, Acetobacter produces
ultrafine cellulose fibrils (50-80 nm in width and 3-8nmin
thickness), which form a micrometer- to nanometer-scale three-
dimensional network structure
12,13
(Figure 1Ba). These properties
and the characteristics are substantially different from conven-
tional plant celluloses. The fibrils are difficult to dissolve in media
because of high molecular weight (>10 000 DPw), stiffness
(Young module of a dry sheet is 33.3 GPa),
14
and hydrogen-bond
network.
13,15,16
Many unique characteristics and applications of BC
have been exploited. For example, it is a useful food additive (it
is well known as a component of the “Nata de Coco”), and it is
also utilized in construction of a commercially available vibration
membrane in a speaker phone.
17
In the current studies, we investigated the use of underivatized
BC fragments as a component of an electrophoretic separation
medium. Our design for this new medium is presented in Figure
1Bb. Two different types of mesh structure coexist in the
medium: the mesh derived from the conventional polymer
solution and that due to the BC fibrils. Thus, the structure is
composed of ∼10-µm fragments containing 10-nm to 1-µm mesh
with BC rigid fibrils and the several 10-nm mesh of the flexible
polymer network. We speculated that this new structure would
allow high-resolution separation of a wide range of DNA fragments
and that it would be more effective than conventional high-
viscosity polymer solutions for the resolution of small DNAs
including single-nucleotide polymorphisms (SNPs). Herein, we
demonstrate the use of the novel nanostructure system using BC
nanofibrils for the separation of biomolecules.
†The University of Tokushima.
‡Nagoya University.
(1) Madabhushi, R. S. Electrophoresis 1998,19, 224-230.
(2) Buchholz, B. A.; Doherty, E. A. S., Albarghouthi, M. N.; Bogdan, F. M.;
Zahn, Z. M.; Barron, A. E. Anal. Chem. 2001,73, 157-164.
(3) Barron, A. E.; Blanch, H. W.; Soane, D. S. Electrophoresis 1994,15, 597-
615.
(4) Rill,R. L.; Locke, B. R.; Liu, Y.; Van Winkle, D. Proc. Natl. Acad. Sci. U.S.A.
1998,95, 1534-1539.
(5) Volkmuth, W. D.; Austin, R. H. Nature 1992,358, 600-602.
(6) Han, J.; Craighead, H. G. Science 2000,288, 1026-1029.
(7) Hung,L. R.; Tegenfeldt, J. O.; Kraeft, J. J.; Strum, J. C.; Austin, R. H., Cox,
E. C. Nat. Biotechnol. 2002,20 1048-1051.
(8) Doyle, P. S., Bibette, J., Bancaud, A.; J.-L. Science,2002,295, 2237.
(9) Tabuchi, M.; Ueda, M.; Kaji, N.; Yamasaki, Y.; Nagasaki, Y.; Yoshikawa,
K.; Kataoka, K.; Baba, Y. Nat. Biotechnol. 2004,22,3,337-340.
(10) Tabuchi,M., Katsuyama, Y., Nogami, K., Nagata, H., Wakuda, K., Fujimoto,
M., Nagasaki, Y., Yoshikawa, K., Kataoka, K., Baba, Y. Lab Chip 2005,5,
199-204.
(11) Huang, M.-F.; Kuo, Y.-C.; Huaang, C.-C.; Chang, H.-T. Anal. Chem. 2004,
76, 192-196.
(12) Brown, A. J. J. Chem. Soc. 1886,49, 432-439.
(13) Cousins, S. K.; Brown, R. M., Jr. Polymer 1997,38, 903-912.
(14) Tabuchi,M.; Watanabe, K.; Morinaga, Y,; Yoshinaga, F. Biosci, Biotechnol.
Biochem. 1998,62, 1451-1454.
(15) Kuga, S.; Takagi, S.; Brown, R. M., Jr. Polymer 1993,34, 3293-3297.
(16) Javis, M. Nature 2003,426, 611-612.
(17) Yamanaka,S.; Watanabe, K.; Kitamura, N.; Iguchi, M.; Mitsuhashi, S.; Nishi,
Y.; Uryu, M. J. Mater. Sci., 1989,25, 3141-3145.
Anal. Chem.
2005,
77,
7090-7093
7090
Analytical Chemistry, Vol. 77, No. 21, November 1, 2005
10.1021/ac0511389 CCC: $30.25 © 2005 American Chemical Society
Published on Web 09/30/2005
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Published on September 30, 2005 on http://pubs.acs.org | doi: 10.1021/ac0511389
