NANOWIRE LYSIS AND DNA EXTRACRTION FROM A SINGLE
BACTERIUM FOR BACTERIA ANALYSIS
Takao Yasui1,2*, Kohei Otsuka1, Masaki Takeuchi1, Takeshi Yanagida3,4, Noritada Kaji1,2,5,
Masaki Kanai3, Sakon Rahong1,2, Kazuki Nagashima3, Toyohiro Naito6, Tomoji Kawai4,
and Yoshinobu Baba1,2,7
1
Department of Applied Chemistry, Graduate School of Engineering, Nagoya University,
JAPAN
2
ImPACT Research Center for Advanced Nanobiodevices, Nagoya University, JAPAN
3
Laboratory of Integrated Nanostructure Materials Institute of Materials Chemistry and
Engineering, Kyushu University, JAPAN
4
Institute of Scientific and Industrial Research, Osaka University, JAPAN
5
ERATO Higashiyama Live-Holonics Project, Graduate School of Science, Nagoya University,
JAPAN
6
Department of Material Chemistry, Graduate School of Engineering, Kyoto University,
JAPAN
7
Health Research Institute, National Institute of Advanced Industrial Science and Technology
(AIST), JAPAN
ABSTRACT
Exploiting the method to extract DNA molecules from bacteria makes it possible to analyze bacteria
in a short time. Here, we realized DNA extraction from a single bacterium via physical interactions
between nanowires and bacteria; nanowire lysis. The combination of nanowire lysis and heating at 94 ºC
could help us to extract genome DNA from bacteria, efficiently.
KEYWORDS: Nanowire lysis, DNA extraction, Bacteria analysis
INTRODUCTION
Bacterial DNA analysis is still an essential task for identification of pathogenic bacteria, confirmation
of pathogenic bacteria acquiring drug resistance, and development of effective drug against pathogenic
bacteria. Analyzing single bacterial DNA provides an effective tool for rapid analysis of unculturable
bacteria. The realization of the single bacterial DNA analysi requires a technological bridge between
DNA extraction from a single bacterium and single DNA analysis. To the best of our knowledge, the
method to extract DNA molecules from a single bacterium has never achieved, while the method to
analyze single DNA molecule is on the way to accomplishment [1]. In this paper, we developed a method
to extract DNA molecules from a single bacterium via physical interactions between nanowires and
bacteria; nanowire lysis (Fig. 1).
EXPERIMENTAL
Vapor Liquid Solid (VLS) method allowed nanowires fabrication inside microcannels. After
fabrication of microchannel, we deposited Au layer as a catalyst for nanowire growth. And then, we set
the substrates into reaction chamber at 700 ºC under Ar/O2 gas ambient. Laser ablation of Sn target could
supply vapored Sn in the reaction chamber, and then, SnO2 nanowires was grown from Au catalyst. We
stained nucleic acid in bacteria using YOYO-1 (491/509 nm). We introduced the stained bacteria into
the microfluidic channels with nanowires by capillary force, and then, applied electric fields to move and
contact bacteria onto nanowires.
978-0-9798064-8-3/µTAS 2015/$20©15CBMS-0001
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19th International Conference on Miniaturized
Systems for Chemistry and Life Sciences
October 25-29, 2015, Gyeongju, KOREA
Figure 1. (a)&(b) Schematic illustrations for extraction of DNA molecules from a single bacterium via
physical interactions between nanowires and bacteria; (a) before interaction, and (b) after interaction.
RESULTS AND DISCUSSION
Key steps for the DNA extraction using nanowires are physical damage caused by interactions between
nanowires and bacteria. In 300 V applied condition, nanowires with 30 nm in diameter could damage
bacteria (Fig. 2). To investigate an effect of nanowire diameter on the bacteria damage, we demonstrated
nanowire penetration into bacteria by dropping bacteria suspension on the nanowires, and conclude that
nanowires with 30 nm in diameter could penetrate into bacteria efficiently. Since the exact mechanism for
physical damage of bacteria caused by nanowire interactions in the present work must be rather complex, it
seems that driving force of nanowires moving to bacteria greatly affect on the physical damage of bacteria.
Next, we fabricated a nanowire device for genome DNA extraction only by introducing bacteria, and
also we demonstrated easy and rapid DNA extraction from bacteria. The nanowire device consists of
nanowire structures on fused silica substrates and chaotic mixer on polydimethylsiloxane (PDMS) [2] (Fig.
3). The nanowire device, which has 30 nm nanowire diameter and 4 µm nanowire length, could extract
genome DNA from bacteria, when the device were set on Peltier device heating at 94 ºC.
Figure 2. Schematic illustrations for extraction of DNA molecules from a single bacterium via physical
interactions between nanowires and bacteria; nanowire lysis.
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Figure 3. (a) Schematic illustration of the nanowire device including nanowire structures on fused silica
substrates and chaotic mixer on PDMS. (b) Extracted DNA concentrations using the nanowire device.
CONCLUSION
We realized nanowire lysis to extract DNA from a single bacterium. The combination of nanowire
lysis and heating allowed researchers to extract genome DNA from bacteria.
ACKNOWLEDGEMENTS
This work was partly supported by the ImPACT Program of the Council for Science, Technology and
Innovation (Cabinet Office, Government of Japan), the Nanotechnology Platform Program (Molecule and
Material Synthesis) of the Ministry of Education, Culture, Sports, Science and Technology (MEXT), the
JSPS Grant-in-Aid for Scientific Research (A) 24241050, and a Grant-in-Aid for Scientific Research on
Innovative Areas “Innovative Materials Engineering Based on Biological Diversity” 15H01599 from
MEXT, Japan.
REFERENCES
[1] M. Tsutsui, M. Taniguchi, K. Yokota, and T. Kawai, “Identifying single nucleotides by tunnelling
current,” Nat. Nanotech., 5, 286-290, 2010.
[2] A. D. Stroock, S. K. Dertinger, A. Ajdari, I. Mezic, H. A. Stone, and G. M. Whitesides, “Chaotic
Mixer for Microchannels,” Science, 295, 647-651, 2002.
CONTACT
* Takao Yasui; phone: +81-52-789-4611; [email protected]
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