Department of ChemistryNanoscale Materials Research Laboratory (NMRL)Pohang University of Science and Technology (POSTECH)

Hee Cheul Choi

NMRLElectrical and fluorescence based detection forbiospecific interactions using SWNTs

NMRL’s interests NMRL

Quantum structures

Synthetic methodology(Liquid-liquid phase, vapor-solid phase, liquid-solid phase)

Nature driven pathway; spontaneous chemical reaction

Electrical properties

NanotransistorsOptoelectronic devices

Photovoltaics

Applications

Nanobiosensor(Label free electrical biosensing)

Tunable catalyst

Contents NMRL

I. Electronic CNT-FET devices for biosensing application

II. Fluorescence-microarray based carbon nanotube protein chip

Graphite

• Nanotubes consist of graphenesheets of carbon

Single-walled nanotube (SWNT)

• Rolled into a cylinder

Multi-walled nanotube (MWNT)

• Some with multiple concentriccylinders

Structure of carbon nanotubes NMRL

• Particle size is corresponding to the nanotube diameter• Catalytic particles (active end) remain on support• The other end is dome-closed • Base growth (differs from the VLS growth mode)

Nanoparticle assisted growth of CNT NMRL

Y. Li, et al.,J. Phys. Chem. B, 105, 11424, 2001

Diameter controlled catalyst nanoparticles NMRL

Choi, H. C. et. al J. Phys. Chem. B 2002, 106, 12361.

Ferritin (iron containing protein)

PAMAM Dendrimer

250 nm

5 nm

1 μm

10 nm

1.3 nm250 nm

Diameters: 1-2 nm (1-5 nm with conventional supported catalyst)

CVD

Fe2O3 Nanoparticle on SiO2

NMRLNanotube growth using dendrimer driven nanoparticles

CH4

H2

C2H4

C2H2

900 oC

Vent or

MS

Pyrolysis CNT growth

Catalyst containing substrate

Controlled Gas Flows

Quartz tube

Chemical Vapor Deposition (CVD) NMRL

OH OH OHCalcination

Iron oxide nanoparticleFe(III) NH2OH

500 nm

SiO2 SiO2

250 nm

Choi, H. C. et al., Nano. Lett. 2003, 3, 157.

1 μm

NMRLThin film of pure SWNT by CVD – New catalyst NPs

P-type silicon wafer

500 nm - 2 mm SiO2

HP4156 Semiconductor Analyzer

VsVdVg

SWNTTi/Au

10 mm1 mm

Process for the fabrication of CNT-FET NMRL

VSD

30

25

20

15

10

5

0

I(nA

)

-10 -5 0 5 10Vg(V)

Applied bias = 10 mV

-Vg

- - - - -iSD

+ + + + +

+Vg

I-Vg characteristics of CNT-FET NMRL

- Change of IDS by the effect of VGS- VGS by electric field

Conventional CNT-FET

CNT-Chemical Effect Transistor (CET)- Change of IDS NOT by the effect of VGS- Why not by chemical effects?

* Charge transfer from molecules to CNT

CNT-FET as a smart sensor NMRL

Bias (V)

Counter electrode (Pt)Reference electrode (Ag/AgCl)

-Teflon based electrochemical cell-

200 μm

50 μm

5 μm

CNT-FET device for biosensor applications NMRL

100 nm

C

rinses

-200

-150

-100

-50

0

50

QC

M S

igna

l ΔF

(Hz)

1614121086420

t/103 (s)

1 μM Streptavidin

100 nM10 nM.1 nM

1 nM

(A

(B200 nm Turns out to be generic:

Streptavidin, Protein A, Glucosidase, Bovine Serum Albumin, IgG…

Chen et al, PNAS 2003, 100, 4984

Non-specific interaction of SWNT with proteins NMRL

A

OO

O

OO OHO

OOHO

OHw x

y

zOO

O

OO OHO

OOHO

OHw x

y

z

SA

E

IIIIII

B200 nm

repelled

-200

-150

-100

-50

0

50

QC

M Δ

F(H

z)

1612840t/10

3(s)

-200

-150

-100

-50

0

50

QC

M S

igna

l Δ

F (H

z)

543210

t /103(s)

C

.05% Tween 20

rinses

rinse100 nMSpA

100 nMBSA

100 nMSA

Non-covalent irreversible adsorption

Water solubility, highly stable

Protein resistant

Tween 20 & Pluronic block copolymer P103 are the best

Hydrophobic/vdW anchoring of Tween20/PEG NMRL Selective electronic biosensor NMRL

Where does the conductance change come from?

• Nanotube aspects: – Charge injection from biomolecules– Electric double layer field modulation caused by

biomolecules

• Metal-nanotube contact aspect: – Adsorbed chemical species may modulate work

function level of contact metals, which consequently change the Schottky barrier height resulting in the conductance change.

NMRLOrigin of the conductance change

S

OOMe

n

Pd/Au evaporation

mPEG-SH SAM

SiO2/Si

PMMA

SWNT

s

SiO2/Si

SiO2/Si

s s s s ss ss s ss s

SiO2/Sis s ss

Lift off

SiO2/Sis s ss

Tween 20

Lift offthen

SiO2/Si

Lift off

OO

O

OO OHO

OOHO

OH

w x

y

z11

S

OOMe

n

Device IIIDevice I

S

OOMe

nS

OOMe

n

Device II

Chen, Choi et al J. Am. Chem. Soc. 2004, 126, 1563

CNT-FET device fabrication

Both photolithography and electron-beam lithography can be used

.02x10-6

3.00

2.98

2.96

2.94

I ds (μ

Α)

12008004000Time (s)

.50x10-6

1.48

1.46

1.44

I ds (μ

Α)

12008004000Time (s)

.40x10-6

3.30

3.20

I ds (μ

Α)

12008004000Time (s)

.50x10-6

3.45

3.40

3.35

3.30

I ds (

μΑ)

10008006004002000Time (s)

100 nM

BSA HIgG

Device IMetal-nanotube

Device IINanotube only

100 nM

100 nM100 nM

Nanotube vs. metal-nanotube contact NMRL

.50x10-6

2.40

2.30

I ds (μ

Α)

12008004000Time (s)

3.5x10-6

3.4

3.3

3.2

3.1

I ds (

μΑ)

800600400200Time (s)

.50x10-6 3.453.403.353.303.253.20

I ds (μ

Α)

12008004000Time (s)

.65x10-6

2.60

2.55

2.50

2.45

I ds (

μΑ)

12008004000Time (s)

hCG HSA

100 nM

100 nM

100 nM100 nM

Nanotube vs. metal-nanotube contact NMRL

Device IMetal-nanotube

Device IINanotube only

Effective functionalization of metal surface with appropriate chemical species will lead high sensitive and selective nanotube-biosensor.

Device I

Device II

S

OOMe

nS

OOMe

n

Pd or Au

Pd or AuS

OOMe

n

1. hCG NSB

1. mPEG-SH on Pd or Au

2. hCG NSB

Conductance (G) Measurement

G

G

Time (s)

Time (s)SiO2/Si

SWNT-FET

Summary of biomolecule sensing mechanism NMRL

Target ssDNA

solid substrate

DNA Chip Protein Chip

“Specific Binding” via shape recognition assisted chemical interaction

Biochip: DNA vs. Protein Chip NMRL

ATTC

CTA

TCA

AC

TA

ATG

GA

GC

TAA

GG

AT

AG

TTGA

T

TAC

CTC

G

- Sequence specific hybridization via hydrogen bonding- Facile immobilization of probe ssDNA

- Conservation of the shape of probe protein is critical- Conservation of the active binding site is also critical

Importance of density control of probe biomolecules

Hurdles for protein chip applications NMRL

• Preservation of active sites– Introducing orientation directing linker molecules

• Preservation of protein shapes– Requires smart substrates providing only limited (minimum contact surface

area)– Highly porous inorganic, organic structures (polymers, porous silicon)

• Effective protection of the chip from nonspecific binding (NSB)– Bovine serum albumin (BSA) generally used by space filling and repelling

proteins– Protein resisting polymers (PEG, Triton X100, Tween20, etc.)

OO O

OO OH

O

OHO

HO

z

x

y

w

O

Spontaneous coating of Tween20

1-2 nmMacBeath, and Schreiber Science 2000, 289, 1760

Ressine, A. et al, Anal. Chem. 2003, 75, 6968

Kim, Y. D. et al, Biotechnol Bioeng. 2001, 73, 331

1 μm

SEM AFM

Choi, H. C. et al., Nano. Lett. 2003, 3, 157.

Nanotube strings formed as a pseudo-3D structures: providing minimum contact surface area

TEM

High yield SWNT for protein immobilization NMRL

How about carbon nanotubes?

SWNT modified with CDI*-Tween20 as blocking reagent & covalent bonding with protein

* 1,1’-carbonyldiimidazole** CDI-Tween20 solution (1wt% in water) soaked SWNT film

O

NNO

CDI-Tween20 on SWNT film**(imidazolyl carbamate)

④ in vacuoovernight

Tween20 (2.25 ml)

+ CDI (1.09 g)

+ DMSO (6.08 ml)

② heating 40℃ for 2 h

③ yellow precipitation

ethyl ether

①

Non-covalent functionalization of SWNT NMRL

Chen, et al, PNAS 2003, 100, 4984

N

N

O

N

N

* with PBS (Phosphate Buffered Saline) buffer, pH=7.4 and DI water for 1m

Probe protein spot arrays formed using microarrayer NMRL

1. Growth High Yield SWNTs& anchor CDI-Tween20

2. Spotting Probe Protein using Microarray,then 3 h incubation with 80% humidity

3. Reaction with target proteinfor 1 h

target protein

Microarrayer (Cartesian™)

Protein A (SpA)-rabbit IgG

Representation of specific bindings

* diluted with PBS buffer, pH = 7.4

Specific binding and cross-reactivity NMRL

SA

OO O

OO OH

O

OO

HONHO

z

x

y

w

O

OO O

OO OH

O

OO

HONHO

z

x

y

w

O

SpA

IgG

SWNT SWNT

BSA

biotin-Streptavidin (SA)biotin-BSA

Protein A500 μm

Cy5-Streptavidin Cy3-rabbit IgGCy5-Streptavidin+ Cy3-rabbit IgG

target probe

no binding

biotin-Streptavidin

Cy5-Streptavidin

Protein A-rabbit IgGProtein A-rabbit IgGProtein A

biotin-Streptavidinno bindingbiotin-BSAProbe Protein*

Cy5-Streptavidin+ Cy3-rabbit IgG

Cy3-rabbit IgG

Target Protein*

Specific binding and cross-reactivity NMRL

Byun et al, submitted (2004)

Dual function of Tween20 NMRL

①①

③

②

① ② ③

- Non-specific binding blocking agents: BSA vs. Tween20

BSA problemBSA obscures the molecules of interaction due to SIZE ISSUE!1

aldehyde slide

protein

aldehyde slide

XH2N

XBSA

Space filling In case of small molecules

ShieldingBSA

MacBeath, G. et al, Science 2000, 289, 1760

BSA-free protein chip NMRL

Selective sensing of small biomolecules NMRL

biotin-Streptavidin (SA) 3X FLAG® peptide-anti FLAG

OO O

OO OH

O

OO

HONHO

z

x

y

w

O

OO O

OO OH

O

OO

HONHO

z

x

y

w

O

MetAspTyrLys

AspHis AspGlyAsp

Tyr

Lys

N

Asp HisAspIle

AspTyrLysAspAspAsp

AspAsp Asp

Lysc

anti FLAG

SWNT SWNT

SA

target probe

biotin-Streptavidin

no binding

Cy5-Streptavidin

Biotin-Streptavidinno bindingbiotin

3X FLAG-anti FLAG3X FLAG-anti FLAG3X FLAG® peptideProbe molecule*

Cy5-Streptavidin+ Cy3-anti FLAGCy3-anti FLAG

Target molecule*

Cy5-Streptavidin Cy3-anti FLAG Cy5-Streptavidin+ Cy3-anti FLAG

biotin

FLAGpeptide

500 μm

Selective sensing of small biomolecules NMRL

CDI-Tween20/LOW yield SWNTs(resistance 200 ~ 500 ㏀)

High yield SWNTs(no CDI-Tween20)

Standard

vs. vs.

500 μm

FLAGpeptide

biotin

target probe

Cy5-Streptavidin + Cy3-anti FLAG

Control experiments NMRL Control experiments with state-of-the-art substrates NMRL

- Increased background intensity in the absence of BSA- Decreased spot size in the presence of BSA (~75%)

Summary NMRL

Successful demonstration of CVD grown SWNT film substrate for fluorescence based microarray protein chip

- Reproducible formation micro-spots of proteins on SWNT film

- High specific/non-specific binding discrimination

- BSA-free protein chip system respecting to Tween20

Graduate StudentsMs. Hye Ryung ByunMs. Yoonmi LeeMr. Seok Min YoonMr. Hyunjae SongMs. Hynjin Yang

Undergraduate StudentsMr. Hyunsub LimMr. Yungjin Na

Dr. Bong Jin HongProf. Joon Won ParkProf. Yong Song Gho(Department of Life Science)

This work was supported by

Air Force Office of Scientific Research (AFOSR, US)Basic Research Program of the Korea Science & Engineering ProgramCenter for Integrated Molecular Systems (CIMS)

Acknowledgements NMRL