生活與物理－奈米生技 - 中研院物理研究所

(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
啓
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