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Carbon Nanotube Biosensor for the Assessment of Personal

Quality of LifeKatsuyuki Murata1, Masuhiro Abe2, Yasuo Ifuku3, Atsuhiko Kojima4, Mitsuaki Shimuzu5, Masatoshi Maeda6, Tatsuaki Ataka1, Kazuhiko Matsumoto7

1 Olympus Corpration, CREST-JST and NEDO, 2 NEDO, 3 Mitsubishi Kagaku Iatron, 4 Mitsubishi

Chemical, 5 AIST, 6 Tsukuba University, 7 NEDO, Osaku University, CREST-JST, and AIST

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ProlegomenonQuotation from our HP

• Future Creation Laboratory of Olympus Corporation– Our goal: enhancement quality of life (QOL)

on mind and body– The Future Creation Laboratory is committed

to pursuing a variety of research projects aimed at identifying and creating future values for enriching people's lives.

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Our researchesThe Future Creation Laboratory conducts highly original

research in "HIKARI"-HI Technology, primarily focusing on four areas:Bioscience, Humanware, Optics and Nanotechnology and

Ubiquitous energy. Closely linked, these fields will yield new discoveries and values for the future.

Approaches taken from four research domains

`We explore what future generations want and what universal values will be like in the future.`

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Bioscience domain-Enhance QOL on mind and body-

Our future dreams and goal

In the area of carbon nanotube(CNT) biosensors research, we are attempting to establish a technology that allows the ultrasensitive measuring (so called “single molecule detection”) of biomolecules by applying the CNT single electron transistor to biosensors.This technology will open a new possibility for home physical check-ups and will contribute to preventive medicine and health management during recovery.

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IntroductionWhat is Personal QOL

This project is “Research and Development of Nanodevices for

Practical Utilization of Nanotechnology” in NEDO

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Challenge to nanotechnology of Olympus

1980

1990

2000

Reinforcement of core competence

A c

halle

nge

and

deve

lopm

ent t

o a

front

ier

2010Nano

measurementNano medical

device

Many functions Capsule

Precisely

microMicro machine

capsule endoscope

ＡＦＭcantilever

biolever

ＭＥＭＳ scanner

Nanobio

living body imitation sensor

end microscope

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ＷｅｂＰＤＦ file ；21C US security in the 21st centryCurrent Japan is

super aged society

Japan → World

Country of currentaged society: about 10

20 years past:about 20

We take the fastest problem of aging speed underhand

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Personal QOL system～Aims to reduce medical cost and promote healthy

intentions～

market size10trillion yenin Japan

200-300trillion yenin the world

Preventivemedicine Diagnosis Treatment

Postoperativecarecurrent

future

Personal QOL system

•Feature•24 hours monitor with a terminal in home•Biotip, microTAS

at home at hospital at home

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24 hours monitor in home

server at home

network

7209：10

wearable biosensor

Biotip

capsule type biosensor

PQOL system

Central server Medical care and welfare service institution

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抗体

カーボンナノチューブソース ドレイン

抗体

カーボンナノチューブソース ドレイン

CNTSource Drain

Target molecule

CNT-FET

Bioaffinity imaging MicroTAS

中央サーバ

Cupcel

diagnosis tip

Network

DNA direct reading NEWBiotip

Biosensor array

Enhancement of QOL (at-home distant place medical care)

PQOL systemClaim success for introducing

new measurement system

SPM for Bio

Social value

Economic value

Economic value72

09：1072

09：10

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• Fundamental studies of CNT-transistor

• PQOL system using CNT-Biosensor

Positioning of this study

CREST-JST project: Olympus, AIST, Mitsubishi Kagaku Iatron, & Osaka Univ.

NEDO project: Olympus, Osaka Univ. & JAIST

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• CREST-JST Project

• Mainly fundamental research– CNT-field effect

transistor (FET)– using CNT-FET in

biosensor

• NEDO Project

• Investigation for practical use– measure yield, ,

quantifiability, selectivity, sensitivity, stability, reliability Establishment of micro total analysis system (TAS) by MEMS technology

Relationship between JST & NEDO Project

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Position of CNT biosensors in CNT applications

Hydrogen storage

Catalyst

Electrode of Fuel cell

Capacitor

Filed emission

DDS

FET

STM tip

Mass-production method of CNTs must be established

CNTs not required in large quantities

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Contents of this presentation

• What is CNT biosensor?• Problems for practical use and solutions

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What is CNT-biosensor?

• We studied– Amperometry

• High reliability– Potensiometry

• High Sensitivity

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抗体

カーボンナノチューブ

ソース ドレイン

CNT

抗体

Potensiometry

antibody

Target molecules

CNT

Source Drain

CNT

antibody

Amperometry（Large surface area electrode） （FET）

CNT biosensor of Amperometry or Potensiometry

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ProbePNA

Target DNA

DNA HybridizationDNA Hybridization

CNTV A

Au

Si substrateSiO2

We achieved detection of 6.8 fmol/L of DNA using a CNT-FET biosensor

Previous CNT-FET biosensor research

18microTAS

Biosensor array

マルチプレクサー

マルチプレクサー

多項目アレイ

抗体

カーボンナノチューブ

ソース ドレイン

抗体

Potentiometry

atnibody

Target molecule

CNT

Source Drain

CNT

antibody

Amperometry

pretreatment

specimen injection

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Why did we select a CNT biosensor for PQOL system?

• PQOL system requires– wearable biosensor

• small and light• microTAS

– real time sensing– non disposable

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CNT biosensor compared with conventional protein sensing methods

yesnoyessensor array

nonoyesNot disporsable?

nonoyesReal time sensing

noyesyesMobility

most high sensitivitynmol/l orderc.a. 1μmol/l

conventional study

Sensitivity

Chemical luminescence ChromatographyCNT Biosensor

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Problems for practical use of CNT-FET based biosensor

• CREST-JST– Fundamental design– Stability & reliability

• NEDO– Quantifiability– Sensitivity– Yield– Selectivity

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Contamination provides less stability of CNT-FET

Washing CNT using aproticsolvent

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What is DMSO & DMAC

• aprotic solvent– Dimethylsulfoxide（DMSO)– Dimethyl acetamide（DMAC)

O

S

Me Me

O

Me C N

Me Me

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Aprotic solvent features

• hydrophobic• hydrophilic

– strong dipole moment– strong nucleophilicity（basic）

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Experiments and sample preparation

Au/Ti Electrode• Photolithography

• CVD

• EtOH ７５０ｓｃｃｍ

• H2 ５００ｓｃｃｍ

• ＠９００℃ １０min.

• Wash by DMAC

CVD process contaminates the CNT with pitch, amorphous carbons and so on

Photolithography process makes lots of photoresistresidue.

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Vd-Id Characteristic of CNT-FET Before and After Washing

-1.5 10-6

-1 10-6

-5 10-7

0

5 10-7

1 10-6

-1 -0.5 0 0.5 1

-5V-4V-3V-2V-1V0V1V2V3V4V5V

Dra

in C

urre

nt I D

S/A

Drain Bias VDS

/V

VBGS

-2 10-6

-1.5 10-6

-1 10-6

-5 10-7

0

5 10-7

1 10-6

1.5 10-6

2 10-6

-1 -0.5 0 0.5 1

-5V=4V-3V-2V-1V0V1V2V3V4V5V

Dra

in C

urre

nt I D

S/A

Drain Bias VDS

/V

-2 10-6

-1.5 10-6

-1 10-6

-5 10-7

0

5 10-7

1 10-6

1.5 10-6

2 10-6

-1 -0.5 0 0.5 1

-5V=4V-3V-2V-1V0V1V2V3V4V5V

Dra

in C

urre

nt I D

S/A

Drain Bias VDS

/V

As Prep. Wash by DMSO Wash by DMAC

become sensitive to gate voltage

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Results: top gate is best structure

• CNT must be kept clean for optimal CNT-FET performance

• Top-gate CNT-FET biosensor– CNT kept clean because CNT is covered with

SiNx.

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Top-gate CNT-FET illustration

SiNx

SiO2

Si

Drain(AU/Ti) Source (Au/Ti)

metal cat. (Co)

Resin Top Gate (Au/Ti) 10000 mm2

Carbon nanotube

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Top-gate structure prevents adsorbing contamination on CNT

Water, oxygen and other contaminates cannot adsorb on CNTs

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Top-gate CNT-FET has high stability

open airPrevious work

Our top-gate device

time/sec.

Little change in drain current

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Top-gate CNT-FET produces no hysteresis

Little hysteresis occurred in our top-gate device

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Top-gate CNT biosensor has low sensitivity

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Why low sensitivity?

• No modulation by schottky barrier

Top-gate FET was modulated by only channel part, back gate FET was modulated by schottky barrier and channel part.

Schottky barrier

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Double gate structure enhances CNT-FET sensitivity

SiNx

SiO2

Back Gate

Si

Drain Source

Metal cat.

resinTop Gate

Carbon nanotube

top-gateCNT-FET

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Double gate enhances sensitivity

-6 10-9

-4 10-9

-2 10-9

0

2 10-9

4 10-9

6 10-9

-1.5 -1 -0.5 0 0.5 1 1.5

+0.10 V+0.08 V+0.06 V+0.04 V+0.02 V0 V-0.02 V-0.04 V-0.06 V-0.08 V-0.10 V

Dra

in C

urre

nt/ A

Tap gate voltage/ V

High sensitivity

Back gate voltage

-5 V 0 V 5 V

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Biosensing

CNT-FET can sense proteins based on antigen-antibody

reaction

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Immobilizing PSA on Top-gate

a-PSA + 10 mM Phosphate buffer (pH 7.4)Top Gate

Phosphate buffer, including a-PSA, was dropped on a top gate of CNT-FET.a-PSA was physically adsorbed on top-gate.

Pig Serum Albumin (PSA)anti-Pig serum albumin (a-PSA)

a-PSA was immobilized on CNT-FET, and PSA in solution was sensed.

Proteins

Experimentalfixation of protein

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・Silicone rubber wall surrounded top-gate.

・Test solutions added into pool on top-gate, PSA was trapped by a-PSA on top-gate.

・FET characterizations were measured, and PSA concentration represented a decrease in drain current.

Ag/AgCl

Back Gate

SourceTop Gate

: a-PSA: PSA

Wall

SiO2

n+-Si

SiNx

Tris buffer

CNTCNT-FET

Analyzer

Drain

ExperimentalSensing

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Top Gate Voltage [V]

-1 +10

25

20

15

10

5

0

24

20

16+0.5 +1+0.75

Top Gate Voltage [V]

Dra

in C

urr

ent

[nA

]

Dra

in C

urr

ent

[nA

]

BiosensingBack Gate Voltage: +5 VDrain Voltage: +0.1 V0.1 M Tris Buffer (pH = 8.0)

ID – VTG curve

拡大図

0.19 V

0 nmol/l PSA70 nmol/l PSA700 nmol/l PSA7000 nmol/l PSA15000 nmol/l PSA

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Drain Voltage: +0.1 VTop Gate Voltage: +1 V

Relationship between drain current change and PSA concentration

10 102

Added PSA conc. [nmol/l]

-⊿

I D[n

A]

2

1.5

1

0.5

0

-⊿ID vs. PSA concentration

sensitivity limit: 20 nmol/l

Back Gate Voltage: +5 V0.1 M Tris Buffer (pH = 8.0)

103 104 105

It is the highest sensitivity of the kinds type CNT biosensor.

Sensitivity is possibility to improve with amount of added PSA or top-gate electrode area.

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Our CNT biosensor obtained high sensitivity

• at least 20 nmol/L of sensitivity limit • In the case of top-gate area 10000mm2

and 10 micro L of test solution added.• Sensitivity limit is changed by above

situations.

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Our device can be used consecutively after washing process

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Selective protein sensing for CNT biosensor

First reported study of selective protein sensing

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CNT-FET Biosensor Problems Do we really observe antigen-antibody reaction?

Top GateTop Gate

A B

C

If protein selectivity is not confirmed, we cannot be sure of sensing by non-specific adsorption

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CNT-FET immobilized a-PSA(PSA biosensor)

CNT-FET immobilized a-MIgG(MIgG biosensor)

a-PSA a-MIgG

Biosensors

Selective protein detection

（MIgG：Mouse immunoglobulin G）

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・Test solution (shown below) was poured on top-gate of PSA biosensor or MIgG biosensor.

・FET drain current measured, and PSA or MIgGconcentration represented a decrease in drain current. .

Test solutions: Tris buffer without antigensTris buffer containing PSATris buffer containing MIgGTris buffer containing both PSA and MIgG

Selective detection of protein

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Drain current decrease at PSA biosensorDrain Voltage: +0.1 V Top Gate Voltage: +1 VBack Gate Voltage: +5 V Solution：0.1 M Tris Buffer (pH = 8.0)

Antigen concentration [nmol/l]

2

1

0

100 1000 10000

PSA

MIgG

PSA+MIgG

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PSA [nmol/l]

Table. Drain current of PSA biosensor which antigen solution was poured.

Drain current [nA]

MIgG [nmol/l]

23.840023.85700023.4707022.72070022.9770070021.9207000

a-PSA

PSA MIgG

PSA

MIgG

a-PSA

-⊿I D

[nA

]

-⊿ID - PSA concentration curve

At PSA biosensorOnly PSA detectedMIgG was not detected

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MIgG concentration [nmol/l]

-⊿I D

[nA

]

6

2

0

100 1000 10000

PSA

MIgGPSA+MIgG

20

4

Drain current decrease at MIgG biosensorDrain Voltage: +0.1 V Top Gate Voltage: +1 VBack Gate Voltage: +5 V Solution：0.1 M Tris Buffer(pH = 8.0)

PSA [nmol/l]

Table. Drain current of PSA biosensor which antigen solution was poured.

Drain current [nA]

MIgG [nmol/l]

106.5900103.087000101.4370000106.540700103.10700700

-⊿ID - MIgG concentration curve

a-MIgG

PSA MIgG PSA

MIgG

a-MIgG

At MIgG biosensorOnly MIgG was detectedPSA was not detected.

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Conclusion

・We successfully achieved real-time sensing and selective detection of proteins using top-gate CNT-FET.

• Our CNT biosensor demonstrated high sensitivity, high stability, and selectivity.

• CNT biosensors have potential uses as industrial product.