, 30, 1 (2006) 2 7J. Inst. Electrostat. Jpn.

ಽሶᚻⴚߩߚ߼ߩિ㐳࿕ቯ DNA ߩ࠽ࡁࡊ࡝ࡦ࠹ࠖࡦࠣ⑔ኅ⌀ผ㧖,1㧘ዊⓣ⧷ᑝ㧖㧘㣐ᵤᱜᄦ㧖㧔2005 ᐕ 8 ᦬ 1 ᣣฃઃ㧘2005 ᐕ 9 ᦬ 18 ᣣฃℂ㧕

Nano-Printing of Stretch-and-Positioned DNA for Molecular Surgery
Masafumi FUKE*,1, Hidehiro OANA* and Masao WASHIZU*(Received August 1, 2005; Accepted September 18, 2005)

Molecular surgery refers to the space-resolved operation on a molecule, where the target molecule is immobilized
onto a solid surface and the operation is performed using position-controlled probes. In order to achieve a highresolution DNA surgery with enzyme-immobilized probes, the target DNA must be immobilized onto a solid surface
with a stretched conformation, yet in such a way that the surface itself does not cause steric hindrance to the activity ofthe enzyme. In this paper, we propose and experimentally demonstrate a new method to realize such immobilization
using nano-printing of DNA onto a microfabricated convex/concave structure. DNA is first stretched and anchoredonto a micro electrode system using electrostatic stretch-and-positioning method. A substrate having a surface
microstructure of periodical convex/concave texture is prepared separately, onto which the stretch-and-positioned
DNA is made into contact to transfer DNA onto the texture. It is found that the binding strength between DNA and thesurface must be carefully controlled for successful transfer, and is realized in this paper by a discharge plasma
treatment of PDMS. DNA strands, being securely held at multiple locations along its length onto the convexes, whileleaving other parts freely suspended above the concaves to allow interactions with enzymes, are obtained with themethod.
1.

ߪߓ߼ߦᓥ᧪ߩ↢ൻቇ⊛ᚻᴺߦ߅޿ߡ DNA ߪ᳓ṁᶧߣߒߡ㓸࿅ߢᛒࠊࠇࠆߚ߼㧘․ቯಽሶߩ․ቯ૏⟎ࠍᜰቯߒߡᠲ૞ࠍട߃ࠆߢ߈ࠆߎߣࠍ␜ߒ 1,2)㧘ߐࠄߦߘߩᔕ↪ߣߒߡ㧘࿕ቯߐࠇߚ
DNA ࠍᓸዊᎿౕߦࠃࠅಾᢿߒ㧘․ቯㇱ૏ߩ DNA ᢿ ࠍขᓧߔࠆᛛⴚߩ㐿⊒ࠍⴕߞߡ߈ߚ 3)㧚

ߎߣߪ࿎㔍ߢ޽ࠆ㧚଀߃߫㧘㐳޿ DNA ߩႮၮ㈩೉ࠍ⸃ᨆߔ

৻ᣇ㧘ߎߩᓸዊᎿౕߣߒߡ㧘㉂⚛ࠍ࿕ቯൻߒߚࡊࡠ࡯ࡉࠍࠆߚ߼ߦߪ㧘߹ߕ DNA ࠍ⍴޿ᢿ ߦಾᢿߒߡฦ‫⸃ࠍޘ‬ᨆߒ㧘↪޿ࠇ߫㧘ಾᢿὐߩಽሶ᭴ㅧ߇᣿ࠄ߆ߥಾᢿࠍⴕ߁ߎߣ߇นߘࠇࠄࠍߟߥ߉วࠊߖࠆߎߣߦࠃࠅో૕ߩ㈩೉ࠍౣ᭴▽ߔ⢻ߢ޽ࠅ㧘ߐࠄߦߪ㧘DNA ਄ߩ․ቯㇱ૏ࠍࡔ࠴࡞ൻߔࠆߥࠆ㧚ߎߩㆊ⒟ߦ߅޿ߡ㧘ߘࠇߙࠇߩᢿ ߇߽ߣߩ DNA ߩߤߤߩㇱ૏․⇣⊛ಽሶୃ㘼߳ߩᔕ↪߽ᦼᓙߐࠇࠆ㧚ߎߩࠃ߁ߥߩㇱಽ߆ࠄ߈ߚ߽ߩߢ޽ࠆߩ߆ߣ޿߁૏⟎ᖱႎ߇ᄬࠊࠇߡ㉂⚛෻ᔕࠍ↪޿ߚಽሶᚻⴚߦ߅޿ߡߪ㧘㉂⚛ߩᵴᕈㇱ૏ߣߒ߹߁ߚ߼㧘ౣ᭴▽ߩࡊࡠ࠮ࠬߪ⤘ᄢߥᚻ㑆ߩ߆߆ࠆ߽ߩߣ

DNA ߣ߇߈ߜࠎߣ፰วߐࠇࠆᔅⷐ߇޽ࠆߩߢ㧘଀߃߫㧘DNAߥࠆ㧚ߎࠇߦኻߒ㧘DNA ࠍ࿕૕⴫㕙ߦ࿕ቯߒߡߒ߹߃߫㧘߇࿕૕⴫㕙ߦో㕙⊛ߦๆ⌕ߒߡ޿ࠆߣ㧘࿕૕⴫㕙߇┙૕㓚ኂಽሶߩછᗧ૏⟎ߦࠕࠢ࠮ࠬߢ߈ࠆߩߢ㧘ಽሶߩ․ቯ૏⟎߳ߩߣߥࠅ෻ᔕ߇㒖ኂߐࠇࠆน⢻ᕈ߇޽ࠆ㧚ߘߩߚ߼㧘ࠊࠇࠊࠇᠲ૞㧔ಽሶᚻⴚ㧕߇น⢻ߣߥࠅ㧘ౣ᭴▽ߩࡊࡠ࠮ࠬ߇シᷫߐߪ㧘એ೨ߦ㧘࿑ 1 ߩࠃ߁ߥᓸ⚦㔚ᭂ♽㧔ࡈࡠ࡯࠹ࠖࡦࠣࡐ࠹

ࠇࠆߚ߼ DNA ⸃ᨆߩ㜞ㅦൻ߳ߣߟߥ߇ࠆߎߣ߇ᦼᓙߐࠇࠆ㧚ᚒ‫⎇ߩޘ‬ⓥቶߢߪ㧘એ೨ࠃࠅ㧘ᓸ⚦ടᎿ㔚ᭂ㑆ߦ૞ࠅ಴ߐࠇࠆ㜞๟ᵄ㔚⇇ࠍ↪޿㧘DNA ࠍિ㐳࡮㈩ะߒၮ᧼਄ߦ࿕ቯ
ࠠ࡯ࡢ࡯࠼㧦 DNA㧘ಽሶᚻⴚ㧘⺃㔚ᵒേ㧘ࡑࠗࠢࡠࡑ࠾ࡇࡘ࡟࡯࡚ࠪࡦ㧘PCP* ᧲੩ᄢቇᎿቇ♽⎇ⓥ⑼ᯏ᪾Ꮏቇኾ᡹㧔113-0033 ᧲੩ㇺᢥ੩඙ᧄㇹ 7-3-1㧕
Department of Mechanical Engineering, The University ofTokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, JAPAN1
fuke@washizu.t.u-tokyo.ac.jp

DNAenergization electrodeglass substratefloating-potential electrodes࿑ 1 ࡈࡠ࡯࠹ࠖࡦࠣࡐ࠹ࡦࠪࡖ࡞㔚ᭂ
Fig.1 Floating-potential electrode system.

3DNAelectrodeDNA

cut80 Pm

electrodeglass substrate࿑ 2 ਔ┵࿕ቯ DNA ߩಾᢿ
Fig.2 Cutting DNA anchored at both ends.

PDMS
3 Pm

DNA1.5 Pm

࿑ 5 DNA ᄙὐ࿕ቯߩߚ߼ߩၮ᧼
Fig.5 Device for multi point DNA immobilization.

substrate࿑ 3 DNA ߩᄙὐ࿕ቯᴺ
Fig.3 Immobilization of DNA at multi point.

stretched DNA

10 Pm

microfabricated convex/concave structure࿑ 6 PDMS ᓸ⚦ಳಲ᭴ㅧ㧔෻኿㗼ᓸ㏜௝㧕Fig. 6 Reflecting microscopic image of PDMS
microfabricated convex/concave structure.

࿑ 4 ᓸ⚦ಳಲ᭴ㅧ਄߳ߩિ㐳 DNA ߩ࿕ቯ
Fig.4 Immobilization of stretched DNA on
microfabricated convex/concave structure.

ࡦࠪࡖ࡞㔚ᭂ㧕ࠍ↪޿ߡ㧘࿕૕⴫㕙ߦๆ⌕ߔࠆߎߣߥߊ DNAࠆ⁁ᘒߢ DNA ࠍ଻ᜬߒߟߟ㧘1 ▎ᚲߢಾᢿ߇↢ߓߡ߽િ㐳ࠍ଻ᜬߔࠆᚻᴺࠍ㐿⊒ߒߚ㧚ߎߩ♽ߢߪ㧘࿕ቯߐࠇࠆߴ߈

ߐࠇߚ࿕ቯ⁁ᘒࠍ⛽ᜬߔࠆߎߣ߇น⢻ߣߥࠆ㧚ߐࠄߦ㧘ಾᢿ

DNA ߩ㐳ߐࠃࠅࠊߕ߆ߦ⍴޿ࠡࡖ࠶ࡊࠍᜬߟ㔚ᭂߦ㜞๟ᵄᓟ㧘࿕ቯὐࠍ࡟࡯ࠩ࡯ࠕࡉ࡟࡯࡚ࠪࡦ╬ߢ೸㔌ߐߖࠆߎߣߦ㜞㔚⇇ࠍශടߔࠆߎߣߦࠃࠅ㧘DNA ࠍ㕒㔚િ㐳ߒߡ㧘ߘߩ

ࠃࠅ DNA ᢿ ࠍ࿁෼ߔࠆߎߣ߇ᦼᓙߐࠇࠆ㧚ਔ┵ࠍ㔚ᭂ㑆ࠍ߹ߚ߇ࠆᒻߢ࿕ቯߔࠆ߇㧘ߎߩᤨ㧘㔚ᭂߩࠡDNA ᄙὐ࿕ቯၮ᧼ߩ૞⵾ࡖ࠶ࡊㇱಽߦ⋧ᒰߔࠆၮ᧼⴫㕙߇ࠛ࠶࠴ࡦࠣߦࠃࠅជࠅਅ

2.

ߍࠄࠇߡ޿ࠆߩߢ㧘DNA ߪၮ᧼߳ๆ⌕ߔࠆߎߣߥߊ㧘㉂⚛

DNA ߩᄙὐ࿕ቯߪ㧘࿑ 4 ߦ␜ߔࠃ߁ߦ㧘ᓸ⚦ߥಳಲ᭴ㅧߣߩ⋧੕૞↪߇น⢻ߥ⁁ᘒߢ࿕ቯߐࠇࠆ㧚ታ㓙㧘ߎߩ㔚ᭂ♽਄ߦિ㐳 DNA ࠍ࿕ቯߔࠆᣇᴺߢⴕ߁㧚ߎࠇߦࠃࠅ㧘DNA ߪࠍ↪޿ߡ࿕ቯߐࠇߚ DNA ߩછᗧ૏⟎ࠍ㧘DNA ಾᢿ㉂⚛ࠍ࿕ⶄᢙߩಲㇱߢ࿕ቯߐࠇ㧘ಳㇱ਄ߢߪⓨ㑆ਛߦᶋ޿ߚ⁁ᘒߢ଻ቯൻߒߚࡊࡠ࡯ࡉࠍ᛼ߒ޽ߡߡಾᢿߔࠆᚻᴺ߇ታ⸽ߐࠇߡᜬߐࠇࠆ㧚ߎߩ㓙㧘㉂⚛෻ᔕࠍ↪޿ߡ DNA ࠍಾᢿߔࠆߎߣ4)޿ࠆ 㧚߇ߢ߈ࠆㇱ૏ߪಳㇱ਄ߩߺߢ޽ࠆߩߢ㧘࿁෼ߢ߈ࠆ DNA ᢿߒ߆ߒߥ߇ࠄ㧘ߎߩࠃ߁ߥ⁁ᘒߢ࿕ቯߐࠇߚ DNA ߦኻߒ㧘㐳ߪಲㇱ᏷ߦଐሽߔࠆߎߣߣߥࠆ㧚੹࿁ߩታ㛎ߢߪಲㇱ᏷

1 ▎ᚲߢಾᢿࠍ↢ߓߐߖࠆߣ㧘࿑ 2 ߩࠃ߁ߦ㧘DNA ߪࠛࡦ࠻

ࠍ 3 Pm ߣߒ㧘࿑ 5 ߦ␜ߔࠃ߁ߥᢿ㕙ᒻ⁁ߩᓸ⚦ಳಲ᭴ㅧࠍࡠࡇ࡯ᒢᕈߦࠃࠅ❗ࠎߢߒ߹߁ߚ߼㧘િ㐳ߒ࿕ቯߐࠇߚ⁁ᘒ૞⵾ߒߚ㧚ࠍ⛽ᜬߔࠆߎߣ߇ߢ߈ߥ޿㧚଀߃߫㧘㉂⚛࿕ቯൻࡊࡠ࡯ࡉߦ߹ߕ㧘ࡀࠟ࡟ࠫࠬ࠻ SU-8㧔MicroChem. Corp.㧕ࠍ૶↪ߒ㧘

ࠃࠅ DNA ࠍಾᢿߒ㧘ಾᢿ ࠍ࿁෼ߔࠆߚ߼ߦߪ㧘ಾᢿ߇↢ࡈࠜ࠻࡝࠰ࠣ࡜ࡈࠖ࡯ߦࠃࠅࠬ࡜ࠗ࠼ࠟ࡜ࠬ਄ߦಳಲߩ㍌ߓߡ߽િ㐳ߒ࿕ቯߐࠇߚ⁁ᘒࠍ⛽ᜬߢ߈㧘߆ߟ㉂⚛ߣߩ⋧੕ဳߣߥࠆ᭴ㅧࠍᓧࠆ㧚ߎߩ਄ߦ PDMS㧔KE-106㧘ା⿧ࠪ࡝ࠦ૞↪߇น⢻ߢ޽ࠆ DNA ࿕ቯᴺ߇ᔅⷐߣߐࠇࠆ㧚࡯ࡦ㧕ࠍṢਅߒ㧘ࠬ࡜ࠗ࠼ࠟ࡜ࠬࠍ߆߱ߖ㧘⎬ൻߐߖߚᓟߦߎߩ⋡⊛ߢ㧘ᧄ⺰ᢥߢߪ㧘࿑ 3 ߩࠃ߁ߥ DNA ᄙὐ࿕ቯᴺߩ㐿⊒ࠍⴕߞߚ㧚ߎߩ࿕ቯᴺߦࠃࠇ߫㧘㉂⚛ߣ⋧੕૞↪ߢ߈㍌ဳ߆ࠄ೸㔌ߔࠆߎߣߦࠃࠅ㧘ࠬ࡜ࠗ࠼ࠟ࡜ࠬ਄ߦ࿑ 6 ߩࠃ
߁ߥ PDMS ᓸ⚦ಳಲ᭴ㅧࠍ߽ߟၮ᧼ࠍ૞⵾ߒߚ㧚

4303.

ᓸ⚦ಳಲ᭴ㅧ਄߳ߩ DNA 㕒㔚િ㐳࿕ቯ

12006a) Apply voltage to stretch DNA and anchor one end.

ߎߩࠃ߁ߦߒߡᓧࠄࠇߚᓸ⚦ಳಲ᭴ㅧ਄ߦ㧘㕒㔚િ㐳ᴺ 2)

cover slipࠍ↪޿ߡ DNA ࠍિ㐳࿕ቯߐߖࠆߴߊ㧘ߎߩ᭴ㅧߩ਄߆ࠄࡔ࠲࡞ࡑࠬࠢࠍㅢߒߡࠕ࡞ࡒ࠾࠙ࡓࠍ⫳⌕ߔࠆߎߣߦࠃࠅ㧘࿑electrodeDNAsubstrate

5 ߦ␜ߔࠃ߁ߥࠡࡖ࠶ࡊ 80 Pm ߩ㔚ᭂࠍߟߌߚ㧚ߎߩ㓙㧘⫳⌕ᤨߦࠕ࡞ࡒ࠾࠙ࡓ߇ PDMS ⴫㕙ߦߟ߈߿ߔ޿ࠃ߁ߦ㧘޽ࠄb) Remove voltage. DNA is still anchored.

߆ߓ߼㉄⚛ࡊ࡜࠭ࡑߦࠃࠅ PDMS ⴫㕙ߩⷫ᳓ൻಣℂࠍⴕߞߚ㧚DNA ߩિ㐳࿕ቯߦߪ㧘㕒㔚િ㐳ᴺߦࠃࠅ㔚ᭂࠛ࠶ࠫߦ
DNA ߩ৻┵ࠍ࿕ቯ㧔࿑ 7-a㧕ߒߚᓟ㧘㔚⇇ࠍขࠅ෰ࠅ
㧔࿑ 7-b㧕㧘ߘߩᓟ molecular combing ᴺ 5)ߦࠃࠅ㧘ࠞࡃ࡯ࠟ࡜ࠬࠍၮ᧼⴫c) Move cover slip to stretch DNA by meniscus force.

㕙ߣᐔⴕߦേ߆ߒߡขࠅ෰ࠆ㧔࿑ 7-c㧕ߣ౒ߦ㧘ࡔ࠾ࠬࠞࠬmeniscusࡈࠜ࡯ࠬߦࠃࠅ DNA ࠍᓸ⚦ಳಲ᭴ㅧ਄߳ߣౣિ㐳࡮࿕ቯߔࠆ㧔࿑ 7-d㧕ߣ޿߁ᚻᴺ 3)ࠍ↪޿ߚ㧚ߎߩ molecular combing ᴺߦࠃࠆ DNA ౣિ㐳ᤨߦ㧘㔚ᭂ਄ߣ PDMS ਄ߩ⴫㕙ࠛࡀ࡞ࠡ࡯߇⇣ߥࠆߣ㧘ࡔ࠾ࠬࠞࠬ߇߁߹d) Fully stretch and immobilize DNA.

ߊ⒖േߢ߈ߥ޿ߚ߼㧘ၮ᧼⴫㕙ߪ㉄⚛ࡊ࡜࠭ࡑߦࠃࠅౣᐲⷫ᳓ൻಣℂࠍߒ㧘ߐࠄߦ㧘DNA ߩๆ⌕ࠍ㒐ᱛߔࠆߚ߼ߦ BSAimmobilized DNA

㧔10 mg/ml㧕ߢࠦ࡯࠻ߒߚ㧚DNA ߣߒߡߪ㧘O-DNA ṁᶧ㧔48kb㧘㐳ߐ 16.5 Pm, Ớᐲ 5 ng/Pl㧕ࠍ↪޿㧘Ⱟశ⦡⚛ YO-PRO-1
㧔Molecular Probes Inc.㧘5 PM㧕ߢᨴ⦡ߒนⷞൻߒߚ㧚એ਄ߩᚻ㗅ߢᓸ⚦ಳಲ᭴ㅧ਄߳࿕ቯߒߚ DNA ߩⰯశ㗼ᓸ

࿑ 7 DNA ߩિ㐳࿕ቯ
Fig.7 Immobilization of stretched DNA.

㏜௝ࠍ࿑ 8 ߦ␜ߔ㧚㔚ᭂࠛ࠶ࠫߦ ┵ㇱࠍ࿕ቯߐࠇߚ DNA߇㧘࿑ਛᮮᣇะ߳િ㐳࿕ቯߐࠇ㧘❑ᣇะߦߪߒߞߡ޿ࠆᓸ⚦stretched DNAಳಲ᭴ㅧ਄߳࿕ቯߐࠇߡ޿ࠆ߇㧘ಳㇱ਄㧔࿑ਛ⍫ශㇱ㧕ߢDNA ߇ಾᢿߐࠇߡߒ߹ߞߡ޿ࠆߩ߇ⷰኤߐࠇߚ㧚ߎࠇߪ㧘
molecular combing ᴺߦࠃࠆ DNA ౣિ㐳ᤨߦṁᶧ߇ಳㇱߦᱷࠅ㧘ߎࠇ߇ੇ῎ߔࠆ㓙ߩࡔ࠾ࠬࠞࠬࡈࠜ࡯ࠬߦࠃࠅ DNA ߇electrodeಳㇱౝߦᒁ߈ㄟ߹ࠇߡಾᢿߐࠇߡߒ߹߁ߚ߼ߢߪߥ޿߆ߣផ᷹ߐࠇࠆ㧚ߚߣ߃߫㧘Ⴎၮ㈩೉⸃ᨆߩ⋡⊛ߢ㧘නߦ DNA ࠍ޽ࠆ᳿߹

edge10 Pmߞߚ㐳ߐߦᢿ ൻߔࠆߩ߇⋡⊛ߢ޽ࠇ߫㧘ߎߩᚻᴺߦࠃࠅ⋡⊛ߪ㆐ᚑߐࠇࠆ㧚ߒ߆ߒߥ߇ࠄ㧘೙㒢㉂⚛╬ࠍ↪޿ߡಾᢿὐߩಽሶ᭴ㅧ߇᣿⏕ߦቯ⟵ߐࠇߚಾᢿࠍⴕ޿ߚ޿႐วߦߪ㧘ߎߩࠃ߁ߥಾᢿ߇↢ߓߥ޿ࠃ߁ߦߔࠆᔅⷐ߇޽ࠆ㧚

4.

PDMS ߣ㔚ᭂࠍ↪޿ߚિ㐳 DNA ߩォ౮

concaves࿑ 8 ᓸ⚦ಳಲ᭴ㅧ਄߳࿕ቯߐࠇߚ DNAFig.8 Immobilized DNA on microfabricatedconvex/concave structure.

೨▵ߩታ㛎ࠃࠅ㧘න⚐ߥ molecular combing ᴺߢߪ㧘ࡔ࠾ࠬߐߖ㧘ߎࠇࠍࠟ࡜ࠬၮ᧼߳᛼ߒઃߌࠆߎߣߦࠃࠅォ౮ߒߚႎࠞࠬࡈࠜ࡯ࠬߦࠃࠅ DNA ߇ಳಲᒻ⁁ߩḴㇱಽߦᒁ߈ㄟ߹ࠇ๔଀߇޽ࠆ 6)㧚ߎߩႎ๔ߢߪ㧘PDMS ⴫㕙ߪ⇹᳓⊛ߢ޽ࠅ㧘ಾᢿߐࠇߡߒ߹߁ߎߣ߇್᣿ߒߚ㧚ߘߎߢ㧘޽ࠄ߆ߓ߼೎ߩ

ᭂᕈಽሶߢ޽ࠆ DNA ߣߩⷫ๺ᕈ߇ૐ޿ߚ߼㧘DNA ߣߩⷫ๺ၮ᧼ߦ DNA ࠍિ㐳ߒߡ઒࿕ቯߒߡ߅߈㧘ߘߩ DNA ࠍಳಲၮᕈߩ㜞޿ࠟ࡜ࠬၮ᧼஥߳ォ౮ߐࠇࠆ㧘ߣߐࠇߡ޿ࠆ㧚ߚߛߒ᧼਄߳ォ౮ߔࠆᚻᴺࠍ㐿⊒ߒߚ㧚ߎߩ႐ว㧘නߦ⫳⊒ᤨߩࡔ࠾ࠬࠞࠬࡈࠜ࡯ࠬࠍ೑↪ߒߡ޿ࠆߎߩࠃ߁ߥォ౮ᴺߦ㑐ߒߡߪ㧘Ṣਅߒߚ DNA ṁᶧ߇⫳⊒ߚ߼㧘DNA ߩિ㐳ߪቢోߢߪߥߊ㧘࿕ቯߐࠇࠆ૏⟎੍߽᷹ߔࠆᤨߩࡔ࠾ࠬࠞࠬࡈࠜ࡯ࠬߦࠃࠅ DNA ࠍ PDMS ਄߳㈩ะਇ⢻ߢ㧘ᓧࠄࠇࠆ㈩ะ߽㧘߅߅ࠃߘ᡼኿⁁ߢ㧘৻ᣇะߦឥ߁DNA5floating-potential electrodesa) The device for transfer-printing of stretched DNA.

floating-potential electrodes
Teflon AFPDMSglass substrateb) Apply voltage to stretch DNA and anchor both ends.
DNA10 Pm

DNA anchored at both ends
Front

DNA anchored at one end࿑ 10 ਔ┵࿕ቯߐࠇߚ DNA
Fig.10 Anchoring DNA at both ends.

electrodesc) Press against aminosilanized glass and then remove.
Backtransfer-printed DNA anchored at both ends

Front
aminosilanized glassd) Transfer DNA onto glass substrate.
transfer-printed stretched DNA

10 Pm

aminosilanized glasstransfer-printed DNA anchored at one end࿑ 9 િ㐳 DNA ߩォ౮ᴺ
Fig.9 The process of transfer-printing for stretched DNA.

࿑ 11 ォ౮ߐࠇߚિ㐳 DNAFig.11 Transfer-printed DNA on aminosilanized glass.

ࠊߌߢߪߥ޿㧚৻ᣇ㧘ࠊࠇࠊࠇ߇᰼ߔࠆߩߪ㧘ቯ߼ࠄࠇߚ૏ߒߡ޿ࠆߩߪ㧘ߎࠇࠍ㒐ᱛߔࠆߚ߼ߢ޽ࠆ 7)㧚࠹ࡈࡠࡦߪ⇹⟎ߦ৻ᣇะߦ㈩ะߐࠇߚቢోߦિ㐳ߐࠇߚ⁁ᘒߩ DNA ߢ޽᳓ᕈߢ޽ࠆߚ߼㧘PDMS ߣห᭽ߩലᨐ߇ᓧࠄࠇ㧘߹ߚ㧘PDMSࠆ㧚ጀߪォ౮ߩ㓙ߩⵍォ౮஥ၮ᧼ߣߩኒ⌕ᕈߩߚ߼ߦᔅⷐߣߥߘߎߢ㧘ᚒ‫ߪޘ‬㧘PDMS ਄ߦࡈࡠ࡯࠹ࠖࡦࠣࡐ࠹ࡦࠪࡖ࡞ࠆ㧚㔚ᭂࠍ૞⵾ߔࠆߎߣߦࠃࠅ㧘PDMS ⴫㕙ߦિ㐳 DNA ࠍ઒࿕ߎߩ㔚ᭂ਄ߦ DNA ṁᶧ㧔5 ng/Pl O-DNA㧘5 PM YO-PRO-1㧕

ቯߒ㧘ߎࠇࠍォ౮ߔࠆᛛⴚࠍ㐿⊒ߒߚ㧚ᚻ㗅ࠍ࿑ 9 ߦ␜ߔ㧚ࠍṢਅߒ㧘㕒㔚િ㐳࿕ቯᴺࠍ↪޿ߡ㧘DNA ߩਔ┵ㇱࠍ㓞ࠅ߹ߕ㧘ࠬ࡜ࠗ࠼ࠟ࡜ࠬ਄ߦ㧘PDMS ࠍṢਅߒࠬࡇࡦࠦ࡯࠻޽߁㔚ᭂࠛ࠶ࠫ߳࿕ቯߔࠆ㧔࿑ 9-b㧕㧚ߎࠇࠍ㧘ォ౮ߐࠇࠆ஥

ߦࠃࠅෘߐ 10 Pm ߣߒߚᓟ㧘⎬ൻߐߖߚ㧚ᰴߦ㧘㉄⚛ࡊ࡜࠭ߩၮ᧼߳᛼ߒઃߌ㧔࿑ 9-c㧕㧘ขࠅ෰ࠆߎߣߦࠃࠅિ㐳 DNAࡑߦࠃࠅ PDMS ⴫㕙ߩⷫ᳓ൻಣℂࠍⴕ޿㧘ࠬࡇࡦࠦ࡯࠻ߦࠃ߇ォ౮ߐࠇࠆ㧔࿑ 9-d㧕㧚ߎߩ㓙㧘ੇ῎ߐߖࠆߎߣߦࠃࠅ DNAߞߡ⤑ෘ 2 Pm ߩ࠹ࡈࡠࡦጀ㧔Teflon AF 2400㧘DuPont㧕ࠍ߇࠹ࡈࡠࡦ਄ߦ࿕⌕ߒߡߒ߹߁ߚ߼㧘৻ㅪߩᚻ㗅ߪၮ᧼⴫㕙૞⵾ߒߚᓟ㧘⌀ⓨࠕ࡞ࡒ⫳⌕෸߮ࠛ࠶࠴ࡦࠣߦࠃࠆࡈࡠ࡯࠹ࠍੇ῎ߐߖࠆߎߣߥߊⴕߞߚ㧚߹ߚ㧘ォ౮ߐࠇࠆ஥ߩၮ᧼ߪ㧘ࠖࡦࠣࡐ࠹ࡦࠪࡖ࡞㔚ᭂߩ૞⵾ࠍⴕߞߚ㧔࿑ 9-a㧕㧚㔚ᭂࠡࡖDNA ߩๆ⌕ࠍ⏕ታߦߔࠆߚ߼ߦ㧘⴫㕙ࠍࠕࡒࡁࠪ࡜ࡦ

࠶ࡊ㐳ߪ㧘DNA ߇࿕ቯߐࠇ߿ߔ޿ࠃ߁㧘O-DNA 㐳ࠃࠅ⧯ᐓ

㧔KBE-903㧘ା⿧ࠪ࡝ࠦ࡯ࡦ㧕ߢࠦ࡯࠻ߒߚ㧚®

⍴޿ 13 Pm ߣߒߚ㧚PDMS ਄ߦ⋥ធࠕ࡞ࡒ㔚ᭂࠍ૞⵾ߒߚ႐

࿑ 10 ߪ૞⵾ߒߚၮ᧼ࠍ↪޿ߡ㧘ࡈࡠ࡯࠹ࠖࡦࠣࡐ࠹ࡦࠪ

ว㧘ࠛ࠶࠴ࡦࠣࡊࡠ࠮ࠬߦ઻߁ PDMS ߩᾲᄌᒻߦࠃߞߡ㔚ᭂࡖ࡞㔚ᭂߦ DNA ࠍਔ┵ߢ࿕ቯߒߚ߽ߩߢ޽ࠆ㧚㔚ᭂࠡࡖ࠶ߦࠢ࡜࠶ࠢ߇౉ߞߡߒ߹߁߇㧘਄⸥ᚻ㗅ߢ࠹ࡈࡠࡦጀࠍ૞⵾

ࡊ㐳߇ DNA 㐳ࠃࠅ⍴޿ߚ߼㧘ᢳ߼ߦ࿕ቯߐࠇߡ޿ࠆ DNA ߇

630

12006

DNA anchored at one end
concaves

glass substrate

PDMS
electrode

DNA anchored at both ends

࿑ 12 ࠡࡖ࠶ࡊ㑆ࠍជࠅਅߍߚ㔚ᭂࠍ↪޿ߚ DNA ߩォ౮
Fig.12 Transfer-printing DNA by floating-potential
electrode with trench structure.

10 2m

electrodes

Transfer-printed DNA

࿑ 15 ᓸ⚦ಳಲ᭴ㅧ਄߳ߩ DNA ߩォ౮
Fig.15 Transfer-printed DNA on microfabricatedconvex/concave structure.

concaves
10 2m

࿑ 13 ㉄⚛ࡊ࡜࠭ࡑಣℂߒߚ PDMS ߳ߩ DNA ߩォ౮
Fig.13 Transfer-printed DNA on oxygen plasma-treated
PDMS.

Cleavage ofDNA

PDMS microfabricated convex/concave structure
10 2m

glass substrateelectrodeDNA࿑ 16 ࿕ቯߐࠇߚ DNA ߩಳㇱ਄ߩಾᢿFig.16 Photocleavage of DNA immobilized above theconcaves.

objective lens

࿑ 14 ⷰኤᣇᴺ᭎⇛࿑
Fig.14 Schematic diagram of the device.

ᄙᢙ⷗ࠄࠇࠆ߇㧘㔚ᭂࠡࡖ࠶ࡊ㐳ߣ DNA 㐳ࠍ߶߷╬ߒߊߣߦߟ޿ߡ߽㧘PDMS ਄ߦ㔚ᭂࠍ૞⵾ߔࠆߚ߼㧘߆ߟ DNA ߣ

ࠇ߫㧘DNA ߇ห৻ᣇะߦ࿕ቯߐࠇࠆ߽ߩߣ⠨߃ࠄࠇࠆ㧚ߩⷫ๺ᕈ߇ૐ޿㧘ߣ޿߁ὐߢ↪޿ߡ޿ࠆ߇㧘ᶧਛߢ․ߦࠟ࡜ߎߩၮ᧼ࠍ㧘⴫㕙ߦࠕࡒࡁࠪ࡜ࡦୃ㘼ࠍᣉߒߚࠬ࡜ࠗ࠼ࠟࠬ⴫㕙ߦ DNA ߇ๆ⌕ߔࠆߎߣߪߥ޿ߚ߼㧘ߎࠇ߽ਇⷐߣߥ࡜ࠬ߳᛼ߒઃߌ㧘ォ౮ߩᠲ૞ࠍⴕߞߚ߽ߩࠍ࿑ 11 ߦ␜ߔ㧚ࠅ㧘ࠬ࡜ࠗ࠼ࠟ࡜ࠬ਄ߦ⋥ធ㔚ᭂࠍ૞⵾ߒߚߩߺߩၮ᧼ࠍ↪

࿑ 10 ߦ߅޿ߡ㔚ᭂࠛ࠶ࠫߦ ┵ߢ࿕ቯߐࠇߡ޿ߚ DNA ߇㧘޿ߡ߽㧘PDMS ⴫㕙߳ォ౮ࠍⴕ߁ߎߣ߇ߢ߈ࠆ㧚㔚ᭂᒻ⁁ߦᴪߞߡォ౮ߐࠇߡ߅ࠅ㧘㔚ᭂࠡࡖ࠶ࡊㇱ߇᛼ߒ޽

ߚߛߒ㧘PDMS ⴫㕙ߪ⇹᳓ᕈߢ޽ࠅ DNA ߣߩⷫ๺ᕈ߇ૐߚߞߡ޿ߚㇱಽߦ㧘ਔ┵࿕ቯߐࠇߡ޿ߚિ㐳 DNA ߇ォ౮ߐ޿ߚ߼㧘DNA ࠍォ౮ߔࠆߎߣ߇ߢ߈ߥ޿㧚DNA ࠍォ౮ߔࠆࠇߡ޿ࠆ㧚ߚ߼ߦߪ㧘PDMS ⴫㕙ߦࠕࡒࡁࠪ࡜ࡦߩࠃ߁ߥᱜ㔚⩄ࠍᜬߟߎߩᚻᴺߦ߅޿ߡߪ㧘ࠟ࡜ࠬ਄߳ DNA ߩォ౮ࠍⴕ߁㓙ߦ㧘ಽሶࠍୃ㘼ߔࠆߎߣ߇᦭ലߣ⠨߃ࠄࠇࠆ߇㧘ᚒ‫ߪޘ‬㧘PDMSၮ᧼ห჻ߩኒ⌕ᕈߩߚ߼ߦォ౮↪ၮ᧼ߩ PDMS ጀ߇ᔅⷐߢ⴫㕙ࠍ㉄⚛ࡊ࡜࠭ࡑಣℂߔࠆߛߌߢ߽ DNA ࠍๆ⌕ߢ߈ࠆߎ޽ߞߚ߇㧘PDMS ᓸ⚦ಳಲ᭴ㅧߩࠃ߁ߦォ౮ߐࠇࠆ஥ߩၮ᧼

ߣࠍᣂߚߦ⷗಴ߒߚ㧚㉄⚛ࡊ࡜࠭ࡑಣℂࠍߒߚ PDMS ⴫㕙ߦ߇ᒢᕈᄌᒻߢ߈ࠆ⚛᧚ߢ޽ࠇ߫㧘ߘࠇߦࠃࠅኒ⌕ᕈ߇⏕଻ߢDNA ߇ๆ⌕ߔࠆේℂߦߟ޿ߡߪ㧘⃻ᤨὐߢߪ᣿ࠄ߆ߢߪߥ߈ࠆߚ߼㧘ォ౮↪ၮ᧼ߩ PDMS ጀߪਇⷐߣߥࠆ㧚࠹ࡈࡠࡦጀ޿㧚DNA7ߐࠄߦ㧘ࠟ࡜ࠬࠛ࠶࠴ࡦࠣߦࠃࠅ㔚ᭂࠡࡖ࠶ࡊ㑆ࠍជࠅਅࠬߦࠃࠅಳㇱߦᒁ߈ㄟ߹ࠇߡಾᢿߐࠇߡߒ߹߁ߎߣ߇ߍࠆߎߣߦࠃࠅ㧘ォ౮ࡊࡠ࠮ࠬਛ㧘ਔၮ᧼ࠍኒ⌕ߐߖߚ㓙ߦ㧘್᣿ߒߚ㧚࿑ 12 ߩࠃ߁ߦ ┵ߢ࿕ቯߐࠇߡ޿ߚ DNA ߇᛼ߒઃߌࠄࠇࠆ2) PDMS ጀ਄ߦ㔚ᭂࠍ㈩⟎ߒ㧘ߎߎߦ㕒㔚િ㐳࿕ቯߒߚߎߣߥߊၮ᧼㑆ߩⓨ㓗ߦㅏࠇࠆߎߣ߇ߢ߈㧘․ߦ㉄⚛ࡊ࡜࠭DNA ࠍ㧘೎ߩၮ᧼ߩ਄ߦォ౮ߔࠆᚻᴺࠍ㐿⊒ߒߚ㧚ࡑಣℂߒߚ PDMS ࠍ↪޿ߚ႐วߦߪ㧘DNA ߇Ⓧᭂ⊛ߦᒁ߈3) ਄⸥ᚻᴺࠍ↪޿ߡ㧘િ㐳 DNA ߩᓸ⚦ಳಲ᭴ㅧ਄߳ߩォߟߌࠄࠇࠆߎߣ߇ߥ޿ߩߢ㧘࿑ 13 ߦ␜ߔࠃ߁ߦਔ┵࿕ቯߐ౮ࠍታ⸽ߒߚ㧚ബ⿠శߦࠃࠆಾᢿߦࠃࠅಳㇱ਄ߩ DNAࠇߡ޿ߚિ㐳 DNA ߩߺࠍォ౮ߔࠆߎߣߦ߽ᚑഞߒߚ㧚ߩߺ߇ᄬࠊࠇࠆߎߣ߆ࠄ㧘ಲㇱ਄ߢ DNA ߇࿕ቯߐࠇ㧘ಳㇱ਄ߢߪⓨ㑆ਛߦ଻ᜬߐࠇࠆߣ޿߁ᄙὐ࿕ቯ⁁ᘒ߇

5.

ᓸ⚦ಳಲ᭴ㅧ਄߳ߩિ㐳 DNA ߩォ౮⏕⹺ߐࠇߚ㧚਄⸥ᚻᴺࠍ↪޿ߡ㧘PDMS ᓸ⚦ಳಲ᭴ㅧ਄߳ߩિ㐳 DNA੹ᓟ㧘ᄙὐ࿕ቯߒߚ DNA ߦኻߒߡ೙㒢㉂⚛ࠍ૞↪ߐߖ㧘ߩォ౮ࠍ⹜ߺߚ㧚࿑ 14 ߦ␜ߔࠃ߁ߦ㧘ォ౮ᓟߦਔၮ᧼ࠍ೸㉂⚛ࠍ↪޿ߚಽሶᚻⴚߩታ⸽ࠍⴕ߁ߣߣ߽ߦ㧘ಲㇱ਄ߦᱷߐ㔌ߖߕߦⷰኤࠍⴕߞߚߩ߇࿑ 15 ߢ޽ࠆ㧚࿑ਛ㧘❑ᣇะߦᓸࠇߚ DNA ᢿ ߩ࿁෼ᚻᴺߩ㐿⊒ࠍⴕ߁੍ቯߢ޽ࠆ㧚⚦ߥಳಲ߇ߪߒߞߡ߅ࠅ㧘㔚ᭂ㑆ߦᮮᣇะߦિ㐳ߐࠇߚ DNA⻢ㄉ߇࿕ቯߐࠇߡ޿ࠆ㧚ࢃࠕ࠼ࡃࡦࠬߩ㤥Ỉୃඳ჻ߩᓮ⼏⺰ߣᓮഥജߦ⻢ᗧࠍ⴫ߎߩᓟ㧘ബ⿠శࠍᒝߊᾖ኿ߔࠆߎߣߦࠃࠅ㧘࿑ 16 ߩࠃ߁ߦಳㇱ਄ߩ DNA㧔࿑ਛ⍫ශㇱ㧕ߩߺ߇ᶖᄬߔࠆߩ߇ⷰኤߐߒ߹ߔ㧚ࠇߚ㧚ߎࠇߪ㧘ബ⿠శߦࠃࠆ㕖․⇣⊛ಾᢿ߇ DNA ߦ⿠߈㧘ߎߩ⎇ⓥߩ৻ㇱߪ㧘↢‛♽․ቯ↥ᬺᛛⴚ⎇ⓥផㅴᯏ᭴㧔↢ಳㇱ਄ߢߪⓨ㑆ਛߦ଻ᜬߐࠇߡ޿ࠆߚ߼ߦಾᢿߦࠃࠅᵹߐ⎇ᯏ᭴㧕ᣂ੐ᬺഃ಴⎇ⓥ㐿⊒੐ᬺ‫ޟ‬ㆮવሶᎿቇߦ߅ߌࠆಽሶ

ࠇߡ DNA ߇ᄬࠊࠇߡߒ߹߁৻ᣇ㧘ಲㇱ਄ߢߪၮ᧼਄ߦๆ⌕ᠲ૞‫ޠ‬㧘ᢥㇱ⋭⑼⎇⾌㧔A14205037㧕
㧘NEDO ↥ᬺᛛⴚ⎇ⓥഥ

ߒߡ޿ࠆߚ߼㧘ಾᢿ߇↢ߓߡ߽ DNA ߇ၮ᧼਄ߦ⇐߹ߞߡ޿ᚑ੐ᬺ‫ޟ‬Intact genomic DNA නಽሶᠲ૞࡮⸃ᨆࠪࠬ࠹ࡓ㐿⊒‫ޠ‬ࠆߣ⺑᣿ߢ߈ࠆ㧚ߎߩ⚿ᨐࠃࠅ㧘DNA ߩᄙὐ࿕ቯ⁁ᘒ߇ታ

㧔04A01541㧕ߩഥᚑࠍฃߌߡⴕࠊࠇ߹ߒߚ㧚⃻ߢ߈ߡ޿ࠆߣ⏕⹺ߐࠇߚ㧚

6.

߅ࠊࠅߦ㉂⚛ࠍ↪޿ߚಽሶಾᢿᚻⴚࠍታ⃻ߔࠆߚ߼ߦߪ㧘DNA ߇㉂⚛ߣ⋧੕૞↪ߥ⁁ᘒߢ࿕ቯߢ߈㧘߆ߟಾᢿࠍⴕߞߡ߽િ㐳⁁ᘒ߇⛽ᜬߢ߈ࠆߎߣ߇ᔅⷐߢ޽ࠆ㧚ᧄ⎇ⓥߢߪ㧘ߎߩ⋡⊛ߩߚ߼㧘ᓸ⚦ಳಲ᭴ㅧࠍ↪޿ߚ DNA ᄙὐ࿕ቯᴺߩ㐿⊒ࠍⴕߞߚ㧚ᓧࠄࠇߚ⚿ᨐߪ㧘એਅߦⷐ⚂ߐࠇࠆ㧚1) ಲㇱ᏷ 3 Pm㧘ᷓߐ 3 Pm㧘ࡇ࠶࠴ 4.5 Pm ߩಳಲ᭴ㅧߩਔ஥ߦ㔚ᭂࠍᜬߟᓸ⚦᭴ㅧࠍ૞⵾ߒ㧘㕒㔚િ㐳࿕ቯᴺࠍ↪
޿ߡ㧘ᓸ⚦ಳಲ᭴ㅧ਄߳ߩિ㐳 DNA ߩ࿕ቯࠍታ⸽ߒߚ㧚ߒ߆ߒߥ߇ࠄ㧘િ㐳ߐࠇߚ DNA ߪ㧘ࡔ࠾ࠬࠞࠬࡈࠜ࡯

ෳ⠨ᢥ₂
M. Washizu and O. Kurosawa: IEEE Trans. Ind. Applicant., 26(1990) 11652) M. Washizu, O. kurosawa, I. Arai, S. Suzuki and N.
Shimamoto: IEEE Trans. Ind. Applicant., 31 (1995) 4473) 㤥Ỉୃ㧘㣐ᵤᱜᄦ㧦㔚᳇ቇળ⺰ᢥ⹹ E, 123 (2003) 1124) T. Yamamoto, O. Kurosawa, H. Kabata, N. Shimamoto and M.
Washizu: IEEE Trans. Ind. Applicant., 36 (2000) 1010
5) A. Bensimon, A. Shimon, A. Chiffaudel, V. Croquette, F.
Heslot, and D. Bensimon: Science, 265 (1994) 2096
6) H. Nakao, M. Gad, S. Sugiyama, K. Otobe and T. Ohtani: J.
Am. Chem. Soc, 125 (2003) 71627) M. Ishizuka, H. Hojo, M. Abe, K. Akahori, N. Honda, M.
Mori, T. Sekiguti, and S. Shoji: Micro Tot. Anal. Sys., 1 (2002)
413
1)