安田「一細胞分子計測」研究期間：平成 20 年 4 月～平成 24 年 3 月

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KAST 平成21年度研究概要　2010.7.16

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KAST 平成21年度研究概要　2010.7.16

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KAST 平成21年度研究概要　2010.7.16

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KAST 平成21年度研究概要　2010.7.16

FE-SEM

1

3 b, d

1

3f

1

FE-SEM

1. H. Takei, J. Vac. Sci. Technol., 17, 1906(1999).

2. M. Himmelhaus, H. Takei, Sens. Actuators B, 63, 24 (2000).

3. H. Kim, H. Takei, K. Yasuda, Jpn. J. Appl. Phys., 49, 048004

(2010).

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KAST 平成21年度研究概要　2010.7.16

target DNA

complementary

probe spot

non-complementary

probe spotmicrobeads

microcolumn

gold NP probe(a)

(c)

fluorescent probe(b)

complementary

probe spot

non-complementary

probe spot

－ 18 －

KAST 平成21年度研究概要　2010.7.16

DNA

1. M. Schena, D. Shalon, R.W. Davis, P.O. Brown, Science, 270,

467 (1995).

2. T.A. Taton, C.A. Mirkin, R.L. Letsinger, Science, 289, 1757

(2000).

3. H. Kim, , H. Takei, K. Yasuda, Sens. Actuators B, 144, 6

(2010).

(a)

(b)

(c)

1 0 -2

1 0 -1

1 0 0

1 0 1

1 0 2

1 0 3

1 0 -4 1 0 -3 1 0 -2 1 0 -1 1 0 0 1 0 1 1 0 2 1 0 3 1 0 4 1 0 5 1 0 6

C o m p le m e n ta ryN o n -c o m p le m e n ta ry

Target Concentration /pM

Num

berD

ensit

yof

Parti

cles

/um

-2

10 -2

10-1

100

101

102

103

10- 4 10-3 10-2 10-1 100 101 102 103 104 105 106

Complem entaryNon-com plementary

Target Concentration /pM

Num

berD

ensit

yof

Parti

cles

/um

-2

102

103

104

105

10-4 10-3 10-2 10-1 100 101 102 103 104 105 106

ComplementaryNon-compleme ntary

Target Concentration /pM

Rela

tive

Fluo

resc

ence

Inte

nsity

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KAST 平成21年度研究概要　2010.7.16

DNase

5’-FITC

a bc

d f g h

i j

e

0

2 10^6

4 10^6

6 10^6

8 10^6

Fluo

resc

ence

inte

nsity

(ar

b. u

.)

DNase Dnase

DNase DNase

(A)

(B)

(C)

(D)

0

2 10^6

4 10^6

6 10^6

8 10^6

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KAST 平成21年度研究概要　2010.7.16

: :

: : Dnase

: : Dnase

: : :

Fluorescence intensity (a.u.)

SS

C

-103 -102 103 104 105-300

64775

129850

194925

260000

Fluorescence intensity (a.u.)

SS

C

-103 -102 103 104 105-300

64775

129850

194925

260000

Fluorescence intensity (a.u.)

SS

C

-103 -102 103 104 105-300

64775

129850

194925

260000

Fluorescence intensity (a. u.)

SS

C

-103 -102 103 104 105-300

64775

129850

194925

260000

a b

c d

DNase-

(a) (c) (b)

-Qdot

4 C 30 37 C 104 C 60

(d)

DNase

1. P. B. Dirks, Nature, 444, 687 (2006)

2. J. E. Dick, Nat. Biotech., 27, 44 (2009)

3. H. Terazono, J. Nanobiotech., 8:8 (2010)

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KAST 平成21年度研究概要　2010.7.16

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KAST 平成21年度研究概要　2010.7.16

1. K. Matsumura, K. Orita, Y. Wakamoto, K. Yasuda, J

Nanobiotechnology. 4:7 (2006).

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KAST 平成21年度研究概要　2010.7.16

FE-SEM

Z I

Z - I

min (1)

Au Ag Ge

CuFe (Si)

(a) (b)

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KAST 平成21年度研究概要　2010.7.16

Z (I

Z - I

min) / (I

max - I

min) (2)

1. T. Sakurada, S. Hashimoto, Y. Tsuchiya, S. Tachibana, M.

Suzuki, K. Shimizu, J. Surf. Anal., 12, 118(2005).

2. H. Kim, T. Negishi, M. Kudo, H. Takei and K. Yasuda, J.

Electr. Microsc., doi:10.1093/jmicro/dfq012 (2010).

(a)

(b)

V / kV5 10 15 20 25 30

I (V

) / %

0

20

40

60

80

100AgGeCuFe

~ Z

AuAgGe

CuFe

V / kV5 10 15 20 25 30

I (V

)

0

50

100

150

200

250

~ Z

5 10 15 20 25 30R

atio

ofI

(V)

0

0.5

1.0

1.5

2.0

2.5

Au/AgAg/GeGe/CuCu/Fe

~ Z

3.0

V / kV

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KAST 平成21年度研究概要　2010.7.16

2

Low temperature High temperature

PCR PCR

100 bp75 bp

50 bp

25 bp

25 bpMarker Copper Polymer Aluminum

1. PCR

2.

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KAST 平成21年度研究概要　2010.7.16

0.3 sec 0.4 sec

50

60

70

80

90

100

0.0 0.4 0.8 1.2 1.6 2.0 2.4 2.8 3.2 3.6

Tem

pera

ture

(C)

Time (s)

5

10

15

20

25

30

35

40

0.0 0.7 1.3 2.0 2.6 3.3

Time (min)

Fluo

resc

ence

Inte

nsit

y (a

rb. U

nit)

0

5

10

15

20

25

30

35

40

45

50

1 6 11 16 21 26 31 36 41 46 51

Fluo

resc

ence

inte

nsit

y (a

.u.)

Cycle number

10ng/ul

1ng/ul

0.1ng/ul

non DNA

PCR

PCR

3

3

(59 ) (96 ) 0.3s

0.4s

PCR

2.1.1 PCR

PCR 97, 95, 93

70, 65, 60 100

PCR

PCR

PCR

1

1

1 PCR

PCR

PCR DNA

SYBR Green I Taqman Probe

PCR

SYBR Green I PCR

4

4 PCR

SYBR Green I

PCR

PCR 1.8

PCR DNA

PCR ( 5)

5 PCR

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KAST 平成21年度研究概要　2010.7.16

A

B

PCR

5μL

PCR

PCR

6

6

PCR

DNA

PCR

PCR

PCR 1/10

DNA

PCR

PCR

MRSA

O-157

1

1. P. B. Dirks, Nature, 444, 687 (2006)

2. J. E. Dick, Nat. Biotech., 27, 44 (2009)

3. H. Terazono, A. Hattori, H. Takei, K. Takeda and K.

Yasuda, Jpn. J. Appl. Phys. 47 no. 6 5212 (2008)

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KAST 平成21年度研究概要　2010.7.16

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KAST 平成21年度研究概要　2010.7.16

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KAST 平成21年度研究概要　2010.7.16

1. A. Kira, H. Kim, K. Yasuda

Homogeneous Immobilization of Probe DNAs on DNA

Chip using Polyurea Thin Film

e-Journal of Surface Science and Nanotechnology, 7,

728-730 (2009)

2. H. Kim, K. Yasuda, H. Takei

Production of nanoscopic metal labels for electron

microscopy: Specific detection of target DNA

Sensors and Actuators B: Chemical, 142, 1-6 (2009)

3. H. Kim, H. Takei, K. Yasuda

Quantitative evaluation of nano-particle-based labeling at

a measurement of DNA microarray

Sensors and Actuators B: Chemical, 144, 6-10 (2010)

4. H. Kim, H. Takei, K. Yasuda

Production of size-controlled nanoscopic cap-shaped

metal shells

Japanese Journal of Applied Physics, 49, 048004 (2010)

5. H. Terazono, H. Takei, A. Hattori, K. Yasuda

Development of a high-speed real-time PCR system using

a circulating water-based rapid heat-exchange

Japanese Journal of Applied Physics, in press.

6. H. Kim, A. Kira, K. Yasuda

Non-amplified quantitative detection of nucleic acid

sequences using a gold nanoparticle probe set and field

emission scanning electron microscopy

Japanese Journal of Applied Physics, in press.

7. H. Kim, T. Negishi, M. Kudo, H. Takei, K. Yasuda

Quantitative backscattered electron imaging of field

emission scanning electron microscopy for discrimination

of nano-scale elements with nm-order spatial resolution

Journal of Electron Microscopy, in press.

8. H. Terazono, H. Takei, A. Hattori, K. Yasuda

Development of a high-speed real-time PCR system for

rapid and precise nucleotide recognition

Proc. SPIE, Vol. 7673, 76730U (2010)

9. H. Terazono, Y. Anzai, K. Yasuda

Labelling of live cells using fluorescent aptamers: binding

reversal with DNA nucleases

Journal of Nanobiotechnology, 8:8 (2010)

10. H. Terazono, H. Takei, M. Hayashi, A. Hattori, K. Yasuda

Development of an integrated system for rapid detection

of biological agents

Proc. SPIE, Vol. 7665, 766503 (2010)

11. A. Hattori, K. Yasuda

Comprehensive study of micro gel electrode for on-chip

electrophoretic cell sorting

Japanese Journal of Applied Physics, in press.

1. H. Kim, H. Takei, K. Yasuda

2. H. Kim, H. Takei, K. Yasuda

"Multi-Color" Labeling in Scanning Electron Microscopy

3. H. Terazono, H. Takei, K. Yasuda

Minimized Non-specific Amplification with A Rapid

Denaturation to Annealing Temperature Transition Using

Novel Ultra Rapid Real-Time PCR system

4. M. Hayashi, K. Yasuda

Single Event Observation of Sequential Phagocytosis by

Macrophage

5. H. Kim, H. Takei, K. Yasuda

Production of homogeneous nano-particles created with

several materials

22nd International Microprocesses and Nanotechnology

Conference

6. H. Terazono, H. Takei, K. Yasuda

Micro droplet PCR: Development of high-speed real-time

PCR system for rapid and precise nucleotide recognition

22nd International Microprocesses and Nanotechnology

Conference

7. A. Hattori, M. Hayashi, K. Yasuda

A Novel Cell Purification Device based on Fast Image

Analysis in Cellomics Era.

22nd International Microprocesses and Nanotechnology

Conference

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KAST 平成21年度研究概要　2010.7.16

8. M. Hayashi, K. Yasuda

Single Event Observation of Sequential Phagocytosis by

a Mouse Alveolar Macrophage

The American Society for Cell Biology 49th Annual

Meeting

9. H. Kim, H. Takei, K. Yasuda

Quantitative and Sensitive Detection of Target DNA using

Nano-Particles as Labels

The American Society for Cell Biology 49th Annual

Meeting

10. H. Terazono, H. Takei, A. Hattori, K. Yasuda

Development of a high-speed and automatable real-time

PCR system for rapid nucleotide recognition

The American Society for Cell Biology 49th Annual

Meeting

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KAST 平成21年度研究概要　2010.7.16