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生活與物理-奈米生技 - 中研院物理研究所

生活與物理-奈米生技 - 中研院物理研究所

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(cfchou@phys.sinica.edu.tw)

NCCU

12/19/08


or


10 -2 m

1 cm

10 mm

Head of a pin

1-2 mm

The Challenge

Ant

~ 5 mm

10 -3 m

1,000,000 nanometers =

1 millimeter (mm)

Dust mite

200 µm

Human hair

~ 60-120 µm wide

Red blood cells

(~7-8 µm)

~10 nm diameter

Fly ash

~ 10-20 µm

ATP synthase

Nanoworld

Microworld

10 -4 m

10 -5 m

10 -6 m

10 -7 m

10 -8 m

Ultraviolet Visible Infrared

Microwave

0.1 mm

100 µm

0.01 mm

10 µm

1,000 nanometers =

1 micrometer (µm)

0.1 µm

100 nm

0.01 µm

10 nm

Pollen grain

Red blood cells

Zone plate x-ray “lens”

Outer ring spacing ~35 nm

Self-assembled,

Nature-inspired structure

Many 10s of nm

MicroElectroMechanical

(MEMS) devices

10 -100 µm wide

Nanotube electrode

O

O

S

O

O

O

O O O O O O O

O

S

O

S

O

S

O

S

P O O

Fabricate and combine

nanoscale building

blocks to make useful

devices, e.g., a

photosynthetic reaction

center with integral

semiconductor storage.

O

O

S

O

O

S

O

S

DNA

~2-1/2 nm diameter

Atoms of silicon

spacing 0.078 nm

10 -9 m

10 -10 m

Soft x-ray

1 nanometer (nm)

0.1 nm

Quantum corral of 48 iron atoms on copper surface

positioned one at a time with an STM tip

Corral diameter 14 nm

Carbon

buckyball

~1 nm

diameter

Carbon nanotube

~1.3 nm diameter


Office of Basic Energy Sciences

Office of Science, U.S. DOE

Version 05-26-06, pmd


Nanotechnology’s Probable Business Impact

(CA, 2005)


DNA

Watson-Crick

DNA


Cell and Molecules

1 bp = 3.4 Å (0.2 e - )

Dia. = 2 nm

1 helical pitch = 10 bp

http://accessexcellence.org/RC/VL/GG/comparative.html


Reynolds number


Typical values of Reynolds number

* Spermatozoa ~ 1×10 −2

* Blood flow in brain ~ 1×10 2

* Blood flow in aorta ~ 1×10 3

For water, µ = 0.01 cm 2 /s

D h (µm) v (cm/s) Re (D h v/µ)

100 0.1 0.1

100 100

200 0.1 0.2

100 200

500 0.1 0.5

100 500

Onset of turbulent flow ~ 2.3×10 3 for pipe flow to 10 6 for boundary layers

* Typical pitch in Major League Baseball ~ 2×10 5

* Person swimming ~ 4×10 6

* Blue Whale ~ 3×10 8

* A large ship (RMS Queen Elizabeth 2) ~ 5×10 9


Laminar Flow -movie


Cavitation Microstreaming

• Flow streaming around bubbles in an acoustic field

• Optimized mixing conditions (waveform, amplitude, etc.)

PZT

PZT

bubble

microstreaming

f

=

3γP

o

/

2πa

ρ

R. Liu et al., Anal. Chem. 2003

Now (10 sec for 100 uL)

5 kHz, 40 Vpp, square wave


Manufacturing and prototyping

Fabrication methods Device components Assays Integrated

solutions

Valves

Channel hyb

Multi-layer devices

Molding, embossing, NanoImprinting

Pumps

Amplification - PCR

Multi-functional cartridges

Mixers

NIL Mask, 70 nm

Cell and DNA capture

Amplification-

Detection

On Chip


Integrated cartridge for low abundance bacteria detection

Crude Sample

Target cell capture

Cell Isolation

/enrichment

Objective: Integrate whole sample prep with microarray

for low abundance bacteria detection from blood (1 mL)

Cell Cell purification Lysis

DNA Cell Isolation Lysis

Amplification PCR

Hybridization Separation

Detection

Answer

Successfully demonstrated cell capture + purification + lysis + PCR

+ detection of 1000 E. coli K12 cells / 1mL sheep blood

Liu RH, Yang JN, et al. ANAL. CHEM. 76, 1824 (2004)


DNA Microarray (


DNA

Probe DNA

Substrate

A

T

T

G

G

C

C

T

G

G

A

A

T

T

G

G

C

C

T

G

G

A

A

T

T

G

G

C

C

T

G

G

A

A

T

T

G

G

C

C

T

G

G

A

Substrate

A

T

T

G

G

C

C

T

G

G

A

A

T

T

G

G

C

C

T

G

G

A

A

T

T

G

G

C

C

T

G

G

A

A

T

T

G

G

C

C

T

G

G

A

Optical/E-chem

Labeling

Signal DNA

A

G

G

A

G

T

A

C

G

A

A

T

A

A

C

C

G

G

A

C

C

T

Target DNA

T

C

C

T

C

A

T

G

C

T

T


Introduction to the F1-ATPase

• ATPase: Produce and hydrolyze ATP

• F1 Complex: α 3 β 3 hexamer and γ subunit

• γ subunit ( > 12nm) rotates due to the conversion of chemical to

mechanical energy (efficiency 80-100%, torgue 120-150

pN·nm).

Soong et al., Science 290, 1555 (2000)


Biomotor-based single molecular sensors

DNA

streptavidin

γ

target DNA

Au nanobar

probe DNA signaling DNA

F1-ATPase

β

α

β

α

His-tag

C.F. Chou et al.,

US patent 6,989,235 (2006)


DEP for Manipulation of Biomolecules


• Dielectrophoresis (DEP) is a phenomenon in which a

force is exerted on a dielectric particle in a non-uniform

electric field –



Force acting on

polarizable particles:

Sphere of radius r,

ε m = 80 for water

C.F. Chou et al.,

Biophysical Journal (2002)

• DEP is a technique which can be used to separate cells

or molecules based on their difference in polarizability


Our Approach: Electrodeless DEP

()

Advantages ():

1) Simpler fabrication process ()

2) Structure chemically inert ()

3) High E fields without risk of electrolysis ()


Molecular Trap ()

Field focusing mostly occurs

at the tips of constriction



→ Increase of molecule

concentration in the trap


368 bp DNA

EDEP devicce



C.F. Chou et al., Biophys. J. 83: 2170 (2002);

US Patent # 6,824,664 (2004)


-movie


EDEP Array for Cell Trapping

E. coli

um constriction,

10 um deep

50-2M Hz, 100 Vpp/cm

Buffer salt concentration:

up to 100 mM

Enrichment ~ 10 3-4

C. F. Chou, F. Zenhausern (2003) IEEE Eng. Med. Biol. Mag. Nov/Dec. 62-67.


EDEP Array for Cell Trapping/Separation

E. coli (+DEP) RBC (-DEP)

C. F. Chou, F. Zenhausern (2003) IEEE Eng. Med. Biol. Mag. Nov/Dec. 62-67.


Stretching of DNA with buffer flow


Hydrodynamic flow

DNA

Elongation flow

Molecular combing

Perkins et al. Science (1995)

Optical tweezers

Perkins et al. Science (1997)

Bensimon, Science (1997)

Electrohydrodynamic flow

S. Chu; S. Block (1994-5)

Bakajin et al. PRL (1998)


DNA

PMMA

175 o C and 50 kg/cm 2

(4.9×10 6 Pa), 5 min

template

300(w)x500(h) nm 300(w)x130(h) nm 75(w)x120(h) nm

20um

20um

50um

T5 phage DNA

103 kb (35 µm)

Channel dimension Stretched DNA length Percentage of stretching

300nm x 700nm 6.2 ± 1.3 um 15%

300nm x 500nm 12.7 ± 4.5 um 30%

75nm x 120nm 39.8 ± 7.7 um 95%

L.J. Guo, X. Cheng, C.F. Chou, Nano Lett. 4, 69 (2004)


DNA

1D Nanochannels

Micro-to-nano Interface

Micro-to-nano

Interface

Nanochannel

Interface

PSQ

Microchannel

Fluid In

Fluid Out

Gu, Gupta, Chou, Wei,

Lab Chip, 7, 1198 (2007)

DNA strands going up the ramp in the

interfaced area (movie)


DNA



DNA


DNA


- DNA


1. DNA, ~

”entropy”

f 1

f 2

d

l p

2. DNA,

t=0 t


DNA

Walter Gilbert

Frederick Sanger

• Year 1977

• Maxem and Gilbert – “Chemical Cleavage Protocol”

• Sanger – Dideoxy sequencing or chain termination method.

• Gilbert and Sanger shared 1/2 Nobel Prize in 1980.

• Sanger method is used widely because of its practicality.


Organism Genome size Size

Status

relative to


human

HIV-1 9,750 0.0003% complete 1985, Wain-Hobson,et.al.

Mycoplasma genitalium 580,070 0.0171% complete 1995, TIGR

Mycoplasma pneumoniae 816,394 0.0240% complete 1995, Univ. of Heidelberg

Lyme disease spirochete 946,000 0.0278% complete 1997, TIGR

Methanococcus jannaschii 1,664,974 0.0490% complete 1996, TIGR

H. influenzae 1,830,137 0.0538% complete 1995, TIGR

Mycobacterium tuberculosis 4,397,000 0.1293% complete 1998, Sanger Centre

Escherichia coli 4,639,221 0.1364% complete 1997,Univ. of Wisc. + others

Yeast, Baker's 12,067,280 0.3549% complete, 1996, Stanford Genome Center

C. elegans 100,000,000 3% complete, 1999 Washington Univ. and Sanger Ctr.

Fruit fly 180,000,000 5% complete, 2000 European and US groups

Pufferfish, Japanese (Takifugu rubripes) 400,000,000 12% 90% complete, 2005

Rice 400,000,000 12% complete, 2002 International Rice Genome Seq. Proj.

Pig 2,700,000,000 79% Mapping complete – sequencing mid stages

Rat 2,900,000,000 85% Draft assembly

Cattle 3,000,000,000 88% Begun in April, 2003, draft assembly

Mouse 3,000,000,000 88% Complete, 2002

Rabbit 3,000,000,000 88%

Human 3,400,000,000 1.0 Complete, 2001,2002,2003

Onion 18,000,000,000 5.3

Marbled lungfish 139,000,000,000 40.9

Fern 160,000,000,000 47.1

Amoeba proteus 290,000,000,000 85.3


DNA


Capillary (Array) Electrophoresis


Cost:

~ $ 1M/genome

Time: months

goal

Cost: $1,000/

genome

Time: hours


A single α-hemolysin channel (Ø =2.5 nm) embedded in a lipid bilayer


Dekker group, Nat. Mater. 2003

D. Branton, J. Golovchenko, Harvard

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