NANOBIODEVICES FOR SINGLE DNA AND CELL ANALYSIS
Noritada Kaji1 and Yoshinobu Baba1,2
Department of Applied Chemistry, FIRST Research Center for Innovative nanobiodevices,
Nagoya University, JAPAN
2
Health Research Institute, National Institute of Advanced Industrial Science and Technology
(AIST), JAPAN
1
ABSTRACT
Nanopore-based DNA sequencing is an emerging technology that may soon overcome even the current
next-generation sequencing from the viewpoint of DNA sequencing cost and throughput. In this paper, we
will show that recent advances of the “pretreatment” process using various nanostructures such as
nanopillars, nanowires, and nanoslits structures inside microchannel. In addition, another type of micro and
nanostructures such as micro and nanometer-scale chamber array on a chip becomes a powerful new tool for
bioanalysis since it could stochastically capture and measure biomolecules at a single molecule level. The
applications of these chamber array structures for single cell analysis will be also described.
KEYWORDS: Nanopillars, Nanowires, Nanoslits, DNA, Electrophoresis
INTRODUCTION
In nanopore-based DNA sequencing technology, which is expected as a core technology of “nextnext” generation sequencer, control of DNA conformation and translocational velocity passing through
the nanopore is a key issue to obtain reliable and reproducible electrical signals derived from nucleic acid
bases. In addition, pretreatment processes of DNA sequencing including DNA extraction from the target
cells or organisms and purification might be another key issue to accelerate substantial “sequencing
speed” and enhance the total sequencing throughput.
So far, in collaboration with Profs. Kawai and Taniguchi in Osaka university, we have developed
nanopillars, nanowires and nanoslits array structures for DNA extraction, separation, conformational
manipulation and detection[1,2]. Various parameters affecting the analytical performances have been
intensively studied and some important findings were explored. For example, geometrical pattern of
nanopillar array structures could control DNA electrophoretic migration behavior and separation modes.
This unique phenomenon was observed only in precisely fabricated nanostructures, not in random gel or
polymer network systems. As a novel biomolecule detection system, we demonstrated nanoslits array
structures, which have comparable slit size to visible light[3]. Since this detection scheme requires no
pre-staining of DNA by fluorescent dye, it become possible to combine nanopore sequencing system.
More recently we introduced nanowires in microchannel as a DNA sieving matrix and showed its great
separation ability over a wide size range of DNA[4,5]. This kind of ‘nano spike’ structure could punch
the nano hole on cell surfaces and extract nucleic acids without any chemical detergent. This nanowire
structures might be compatible with nanopore DNA sequencer in which chemical additives generate
background noise. In nanopore DNA sequencer, control of translocational velocity of DNA is another
key factor to obtain high signal to noise ratio and discriminate each bases. To avoid DNA acceleration at
nanopore region by electric field concentration, we are trying to combine the shallow channel or
nanopillar array channel in front of a nanopore structure. These structures are expected to reduce entropy
gap and electric field concentration around the nanopore structure.
978-0-9798064-7-6/µTAS 2014/$20©14CBMS-0001
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18th International Conference on Miniaturized
Systems for Chemistry and Life Sciences
October 26-30, 2014, San Antonio, Texas, USA
Figure 1. A conceptual image of an innovative nanobiodevice. All necessary processes
including cell collection, DNA extraction, separation , manipulation and detection, should
be integrated on a single chip for future nanopore-based DNA sequencer.
EXPERIMENTAL
Two kinds of nanowires, composed of different materials, SnO2 and ZnO, were fabricated inside
microchannel for DNA extraction. SnO2 nanowires were fabricated using photolithography, electron
beam lithography and VLS(Vapor Liquid Solid) technique. The microchannel was etched by reactive ion
etching under the ambient of CF4 gas. The nanowire area was drawn in the microchannel by electron
beam lithography, and then, Au catalyst was sputtered by DC sputtering. SnO2 Nanowires which were
synthesized using Au arrays as catalysts for VLS growth mechanism in microchannel by Pulse Laser
Deposition system (PLD). In case of ZnO nanowires, ZnO particles (20 nm in diameter) were coated with
phosphonic acid cross-linker on PMMA substrate and annealed at 85 °C for 1 hour. The PMMA
substrates were dipped into growth solution (Hexamethylenetetramine and Zn(NO3)2) at low temperature
(85 °C) for 20 hours. Single-crystalline ZnO nanowires (100 nm in diameter and 2-3 m in length) were
grown on the PMMA substrates.
Nanopillars and nanoslits structures were fabricated by combining EB lithography, photolithograpy,
and RIE.
RESULTS AND DISCUSSION
Since these nanowires have different diameters, 10-20 nm for SnO2 and 100-200 nm for ZnO
nanowires, more sharp-pointed SnO2 nanowires were applied to bacteria and ZnO nanowires were
applied to eukaryotic cells. Both of nanowires worked well and successfully extract genomic DNA from
the puncture holes. As a DNA separation matrix, nanopillar array structures, which have 500 nm in
diameter with various sizes of spacing, were fabricated inside microchannel and optimized parameters
affecting the separation performance. Finally, we achieved millisecond separation of miRNA and genomic
DNA molecules. As a novel biomolecule detection system, we demonstrated nanoslits array structures,
which have comparable slit size to visible light. Since this detection scheme requires no pre-staining of
DNA by fluorescent dye, it become possible to combine nanopore sequencing system. These
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nanobiodevices consists of nanofabricated structures could offer strong support to realize next next
generation sequencing using nanopore.
In this talk, the applications of micro and nanochamber array chip for a single cell, nucleus and enzyme analysis will be also described. In enzyme analysis, we have measured a single β-galactosidase activity in various sizes of micro and nanochambers, from 10 μm to 800 nm in diameter, and found that the
activity gradually decreased according to its chamber size. Another application of micro and nanochamber was for a single nucleus analysis. In the field of gene delivery systems, it is still unknown that virus
vector showed much higher transportation efficiency of plasmid DNA from cytoplasm to nucleus in contrast to non-virus vectors. To improve gene transportation efficiency, which directly lead to increase
transfection efficiency, gene transportation mechanism through nucleus membrane should be cleared.
But unfortunately there are no experimental protocol to quantitatively evaluate the transportation efficiency through the nucleus membrane. So, in this study, nuclei were extracted from cells by gentle detergent treatments and captured into a microchamber at a single nucleus level. PDMS and PMMA plastics were used for the microchamber array, and capturing efficiency of nucleus was counted and observed
by confocal microscope. After the confirmation of capturing a single nucleus into a microchamber, nucleotides and nuclear membrane stained nuclei, transportation of mRNA by molecular beacon were observed.
CONCLUSION
These nanobiodevices consists of nano-fabricated structures would offer strong support, not only to
realize ‘next-next’ generation nanopore DNA sequencing but also other novel bioanalytical techniques
such as single cell analysis.
ACKNOWLEDGEMENTS
This research is granted by the Japan Society for the Promotion of Science (JSPS) through the
“Funding Program for World-Leading Innovative R&D on Science and Technology (FIRST Program),”
initiated by the Council for Science and Technology Policy (CSTP). N. K. also acknowledge the financial support by JSPS KAKENHI Grant Number 24681027.
REFERENCES
[1] T. Yasui, N. Kaji, Y. Baba, Nanobiodevices for Biomolecule Analysis and Imaging, Annual review
of analytical chemistry, 6, 83-96 (2013)
[2] N. Kaji, Y. Okamoto, M. Tokeshi, Y. Baba, Nanopillar, nanoball, and nanofibers for highly efficient
analysis of biomolecules, Chemical Society Reviews, 39 (3), 948-956 (2010)
[3] T. Yasui, N. Kaji, R. Ogawa, S. Hashioka, M. Tokeshi, Y. Horiike, Y. Baba, DNA separation in
nanowall array chips, Analytical Chemistry, 83 (17), 6635-6640 (2011)
[4] T. Yasui, S. Rahong, K. Motoyama, T. Yanagida, Q. Wu, N. Kaji, M. Kanai, K. Doi, K. Nagashima,
M. Tokeshi, M. Taniguchi, S. Kawano, T. Kawai, Y. Baba, DNA Manipulation and Separation in
Sublithographic-Scale Nanowire Array, ACS Nano, 7 (4), 3029-3035 (2013)
[5] S. Rahong, T. Yasui, T. Yanagida, K. Nagashima, M. Kanai, A. Klamchuen, G. Meng, Y. He, F.
Zhuge, N. Kaji, T. Kawai, Y. Baba, Ultrafast and wide range analysis of DNA molecules using rigid
network structure of solid nanowires, Scientific Reports, 4, 5252 (2014)
CONTACT
* N. Kaji; phone: +81-52-789-4498; [email protected]
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