Nanotechnology

Development and use of materials and devices for analysis and measurement on a nanometer scale

Diverse and interdisciplinary field : Biology/Biotechnology, Physics, Chemistry, Material sciences,

Electronics, Chemical Engineering, Information technology

Future implications - Creation of new materials and devices in medicine,

electronics, energy etc. - Social issues: toxicity, environmental impact of nano-

materials

Nanotechnology Plays by Different Rules

Normal scale Nanoscale

Nano-sized materials Unusual and different property - Semiconductor nanocrystals : Size-dependent optical property: Absorption and emission - Magnetic nanoparticles (iron oxide)

Ferromagnetic: Materials that can be magnetized by an

external magnetic filed and remain magnetized after the external field is removed The spin of the electrons in atoms is the main source of ferromagnetism Super-paramagnetic : In the absence of magnetic field Magnetization is zero An external magnetic field is able to magnetize the nanoparticles ex) MRI contrast agents

Nano-Bio Convergence

Nano-Bio Convergence

Molecular Switch

DNA barcode

Molecular Imaging

Biochip / Biosensor

Nanotherapy / Delivery

Bio-Technology Nano-Technol

Bionano-machine /Nano-Robot

Bio-inspired device and system

Nano-Biotechnology

Integration of nano-sized/structured materials, nano-scale analytical tools, and nano-devices into biological sciences including biotechnolgy for development of new biomaterials and analytical toolkits

Use of bio-inspired molecules or materials

- Typical characteristics of Biological events - Self assembly - Highly efficient : high energy yield - Very specific : extremely precise Bio-molecules DNA, Proteins, Antibody, RNA, Aptamers,,

Peptides,

Applications and perspectives of nanobiotechnology

Development of tools and methods - More sensitive - More specific - Multiplexed

- More efficient and economic

Implementation

- Diagnosis and treatment of diseases : Nanomedicine

Rapid and sensitive detection(Disease biomarkers, Imaging),

Targeted delivery of therapeutics

- Drug discovery

- Understanding of life science

Examples

Nano-Biodevices Nano-Biosensors Biomolecular motors Medical use : Targeting with Ab-magnetic beads Contrast agents for MRIAnalysis of a single molecule/ a single cell

Issues to be considered

Synthesis or selection of nano-sized/ structured materials : Biocompatibility, cytotoxicity

Functionalization with biomolecules or for biocompatibility

Integration with devices and/or analytical tools

Assessment : Reproducibility, Toxicity Implementation to human body

The size of Things

How are nano-sized materials fabricated ?

이전까지 방식은 이미 존재하는 큰 물질을 깎아서 원하는 작은 크기로 만드는 것 : top-

down

나노 물질은 원자나 분자를 벽돌처럼 쌓듯이 조합해 완전히 새로운 방식 : bottom-up

Top-down : Hard to fabricate materials less

than

50nm in size

Bottom-up : Self-assembly

현재 생체분자처럼 원자나 분자가 알아서 어떤 물질로 만들어지는 방법인 자기조립이 활발히 연구 중 . 사진은 원자들이 일정 형태를 갖추는 자기조립의 과정을 보여 줌

NanoBiotech was initiated by development of AFM and STM that enable imaging at atomic level in 1980

- Contacting mode- Non-contacting mode- Tapping mode

Foremost tool for imaging, measuring, and manipulating matter at the nanoscale

Atomic Force Microscope

- When the tip is brought into proximity of a sample surface, forces between the tip and the sample lead to a deflection of the cantilever according to Hooke's law

- If the tip was scanned at a constant height, a risk would exist that the tip collides with the surface, causing damage. -A feedback mechanism is employed to adjust the tip-to-sample distance to maintain a constant force between the tip and the sample. -Sample is mounted on a piezoelectric tube that can move the sample in the z direction for maintaining a constant force

- Deflection is measured using a laser spot reflected from the top surface of the cantilever into an array of photodiodes

VEECO

TESPA®

VEECO

TESPA-HAR®

NANOWORLDSuperSharpSilicon®

Tip length : 10 m

Radius : 15~20 nm

Tip length :10 m

(last 2 m 7:1)

Radius : 4~10 nm

Tip length :10 m

Radius : 2 nm

Image of ATP synthase

composed of 14 subunits

Example showing the resolution

of protein structure by AFM

Optical Properties Of Quantum Dots

a) Multiple colors b) Photostability

c) Wide absorption and narrow emissiond) High quantum yield

Quantum Yield ≥ 60 ~ 70 %

Single source excitation

QD

Y

Y

Y

Y

YYY

Y

Organelle

+

QD-Antibodyconjugates

Antigen

QD

Y

Y

Y

Y

YYY

Y

Organelle

▲ 3T3 cell nucleus stained with red QDs and microtubules with green QDs

In Vivo Cell Imaging

Wu et al. Nature Biotech. 2003 21 41

- Multiple Color Imaging- Stronger Signals

- Multiple Color Imaging- Stronger Signals

In Vivo Cell Imaging

Quantum Dot Injection

▶ Red Quantum Dot locating a tumor in a live mouse

Live Cell Imaging

Cell Motility Imaging

10um

◀ Green QD filled vesicles move toward to nucleus (yellow arrow) in breast tumor cell

Alivisatos et al., Adv. Mater., 2002 14 882

Förster (or Fluorescence) Resonance Energy Transfer (FRET)

Non-radiative energy transfer from an energy donor to an energy

acceptor

Dipole –dipole coupling

Energy transfer efficiency : - Degree of spectral overlap between donor fluorescence emission and

acceptor absorption - ~ 10 nm

- The transfer efficiency is dependent on the inter-fluorophore distance. -Calculation of the transfer efficiency allows the distance between fluorophores.

Use of FRET measurements

Detection of target analytes

Analysis of biomolecular interactions

Single molecule analysis

- Protein folding/unfolding

- Protein dynamics

Molecular ruler in the determination of inter- or intra-molecular distances

FRET probe based on Quantum dots and AuNPs

QDs as an energy donor - High quantum yield (~0.6) and less photo-bleaching Strong PL

intensity - Multiple acceptors per single QD Increased ET efficiency - Narrow and size-tunable emission Multiplexed assay

AuNPs as an energy acceptor (Quencher) - Higher quenching efficiency than conventional quenchers - Extended working distance by SET mechanism : ~ 22 nm - Applicable to various donors

QDAuAu

Oh et al., JACS (2006)Oh et al., Angewandte Chemi Intl Ed.(2007)

Förster Resonance Energy Transfer (FRET) using QDs

QD

Excitation FRET quenching

quencher

QD

Excitation Emission

QD with Quencher

Time (ns)

QD

QDquencher

PL intensity

A variety of proteases in cells Essential for the dynamic regulations of cell function and aberrations Involved in major human diseases (cancers, apoptosis, and inflammation) Protease inhibitors are known to be drug candidates Matrix metalloproteinases (MMPs), Caspase-3, Thrombin

Protease Assay Protease Assay

Gel- and LC-MS-based methods : accurate, but need a long analysis

time Limitation to high throughput assay Simple, fast, and high throughput method is required

“ Assay of proteases using FRET probe” - Simple and sensitive - Multiplexed and high-throughput assay

Protease

Conventional Methods for Protease Assay

Regulate cancer metastasis by degrading ECM components

or cytokines MMP-7 : Potential biomarker of human colorectal or breast cancer MMP-2 & 9 : Human carcinoma MMP-3 : Joint damage MMP inhibitors : Candidates for anticancer therapeutics

Steps in the process of metastasis Steps in the process of metastasis

Matrix metalloproteinases (MMPs)

Nat. Rev. Cancer 2002, 2, 161Nat. Rev. Cancer 2002, 2, 161

Construction of FRET probe for Protease Assay Construction of FRET probe for Protease Assay Construction of FRET probe for Protease Assay Construction of FRET probe for Protease Assay

Peptide-conjugated AuNP

+

biotincysteine

peptide

AuAuAuAu

monomaleimide AuNP

1.4 nm in diameter

Kim et al., Anal. Chem. (2009)

peptide(CRPLALWRSK-bio)

QDQD

1.4 nm

15-20 nm

(5-10 SA/ QD)

11.3-13.8 nm

AuAu

monomaleimide nanogold

QD-SA

3.8 nm

AuAu

FRET probe based on QDs and AuNPs

QD

+ protease

Wavelength

PL

Wavelength

PL

QD

Glass

Glass

+ protease+ inhibitor

QD

Glass Wavelength

PL

dequenching

quenchingquenching

Chip-based assay

Kim et al., Anal. Chem. (2009)

Chip-based Protease Assay

Chip-based Protease Assay

Fluorescence Silver-staining Relative PL (%)

(C)

(D)

(A)

(B)

0 20 40 60 80 100

0 20 40 60 80 100

QD

QD

QD

QD

SA-QD605+Pep-AuNPs

SA-QD605

SA-QD605+Pep-AuNPs+MMP-7 protease

SA-QD605+Pep-AuNPs+MMP-7 protease+Inhibitor

+ Thrombin

+ MMP-7 + Caspase-3

Specificity of FRET probe

Peptide sequences for proteases

Thrombin : Biotin-GKGGLVPR-GSGCMMP-7 : Biotin-KSRWLA-LPRCCasp-3 : Biotin-GRRGDEVD-GGGRRC

α -Hemolysin: Self-Assembling Transmembrane Pore

A self-assembling bacterial exo-toxin produced by some pathogens like Staphylococcus aureus as a way to obtain nutrients lyzes red blood cells

Alpha-hemolysin monomers bind to the outer membrane of susceptible cells.

The monomers oligomerize to form a water-filled

heptameric transmembrane channel that facilitates uncontrolled permeation of water, ions, and small organic molecules.

Rapid discharge of vital molecules, such as ATP,

dissipation of the membrane potential and ionic gradients, and irreversible osmotic swelling leading to the cell wall rupture (lysis), can cause death of the host cell.

- Mushroom-like shape with a cap and stem-50 A bets-barrel stem-Narrowest part : ~ 1.4 nm in diameter

Biotechnological applications

• Unique structural features make self-assembling membrane channel

suitable for biotechnological applications• Assembled alpha-hemolysin : stable over a wide range of pH and temperature• Transmembrane pore stays open at normal conditions• Hemolysin binds to various biological or synthetic lipid bilayer

Delivery system : facilitate controlled delivery of ions and small organic compounds such as sugars and oligonucleotides across the plasma membrane of cells or through the walls of synthetic lipid vesicles : Engineered pores Stochastic sensors DNA sequencing

Magnitude of the current reduction : type of analyte Frequency of current reduction : analyte concentration

A molecular adaptor is placed inside its engineered stem, influencing the transmembrane ionic current induced by an

applied voltage

Reversible binding of analytes to the molecular adaptor transiently

reduces the ionic current

Stochastic sensor

Stochastic Sensors

a : Histidine captured metal ions (Zn+2, Co+2, mixture ) b: CD captures anions ( promethazine, imipramine, mixture)c : biotin ligand

DNA sequencing

Eelectrophoretically-driven translocation of a 58-nucleotide DNA strand through the transmembrane pore of alpha-hemolysin

- Transmembrane potential drives translocation of DNA or RNA through the pore- Ionic current blockades reflect the chemical structure of individual strands- Discrimination of different sequences of DNA or RNA - Single resolution of purine and pyrimidine nucleotides

Single DNA Engineering  

유전자의 일부가 잘못되었을 때 잘못된 유전자를 제거하고 정상적인 유전자로 대체 .

단일 DNA 를 고정하기 위해서 광학적 포획(optical trapping) 기술을 이용 DNA 의 양단에 구슬 (bead) 을 붙이고 레이저를 이용하여 구슬을 잡으면 수용액상에서 끊임없이 움직이는 단일 DNA 를 고정시킬 수 있음 .

자르고 싶은 위치에 마이크로피펫(micropipet) 을 이용 제한효소를 넣어 DNA 조각을 잘라냄 .

잘린 DNA 는 전극을 이용하여 펴고 정상적인DNA 를 넣어 효소를 이용하여 DNA 를 연결함 .

단일 DNA 는 형광물질로 염색 시키고 , CCD카메라가 장착된 형광현미경을 이용하여 관찰 .

Field Effect Transistor- A transistor is a linear semiconductor device that controls current with the application of a lower-power electrical signal. - The bipolar and field-effect transistors. - The bipolar transistors utilize a small current to control a large current. - The field-effect transistor utilizes a small voltage to control current : The junction field-effect transistor.

In a junction field-effect transistor, or JFET, the controlled current passes from source to drain, or from drain to source as the case may be. The controlling voltage is applied between the gate and source. With no voltage applied between gate and source, the channel is a wide-open path for electrons to flow. However, if a voltage is applied between gate and source of such polarity that it reverse-biases the PN junction, the flow between source and drain connections becomes limited, or regulated.

Carbon Nanotube FET

Chen et al., PNAS, 100, 4984-4989, 2003

Detection of biomolecular binding by using microcantilever

• Label-free detection of biomolecules

• Methods for measuring the microcantilever bending

- Optical Less amenable to monolithic integration and multiplexed detection because of difficulties in laser alignment and power management Interference with turbid or opaque fluidic and smoky environment

- Piezoresistive Compatible with aqueous media and parallel cantilever arrays The piezoresistors cover a large length of the cantilever, and high doping levels are required the stress measurement is not localized thermal and electronic noise, thermal drift, non-linearity in piezo- response More than 50 nm bending is required

Nanomechanical cantilever arrays

Mckendry et al., PNAS, 99, 9783-9788 (2002)

Linear position detector : beam-deflectionWith an accuracy of 0.1 nm

MOSFET-Embedded Microcantilever• Embedding a metal-oxide semiconductor FET(MOSFET) into the base of the

cantilever, and recording decreases in drain current with deflection as small as 5 nm.

• The specific biomolecular binding between ligands and receptors on the surface of a microcantilever beam

physical bending of the beam change in the surface stress altered channel resistance modulation of the channel current underneath the gate region.

• Embedding of MOSFET in the high-stress region of the cantilever to measure deflection.

Shekhawart et al., Science, 17, 1592-1595 (2006)

N-type FET

Molecular imaging

Biomedical Sciences : - Ultra-sensitive imaging of biological targets under non-invasive

in-vivo conditions

- Fluorescence, positron emission tomography, Magnetic resonance imaging

- Ultra-sensitive imaging

- Cancer detection, cell migration, gene expression, angiogenesis,

apotosis - MRI : powerful imaging tool as a result of non-invasive nature,

high spatial resolution and tomographic capability Resolution is highly dependent on the molecular imaging

agents signal enhancement by using contrast agents : iron oxide nanoparticles

Detection of tumors as small as ~ 50 mg cells

In-vivo MR detection of cancer using NP-Herceptin conjugates

Virus-enabled synthesis and assembly of nanowires for lithium ion battery electrodes

Nam et al., Science, 312, 885-888, 2006

• Smaller and more flexible Li ion batteries

• Dimensionally small batteries : Nanoparticles, nanotubes, nanowires as well as several assembly methods based on lithography, block copolymer, layer-by-layer deposition

• Nanostructured materials : Improvement of the electrochemical property of Li ion batteries

• Monodisperse, homogeneous nanomaterials and hierarchical organization control are needed to maximize the potential

M13 virus

• Helically wrapped by ~ 2700 major coat proteins(p8) and minor coat proteins ( p3, p6, p7, and p9) around its single-stranded DNA

• Coat proteins were used to form functional hetero-structured template for precisely positioned nano-materials

Predictive-based design Fusion of tetraglutamate(EEEE-) to the N-terminus of the major

coat p8 protein

- A general template for growing nanowires through the interaction of the

glutamate with various metal ions

- A blocking motif for gold nanoparticles due to the electrostatic repulsion,

reducing nonspecific gold NP binding to phage

Favorable interaction with the positively charged electrolyte polymer

(Ex) Design of cobalt oxide nanowires

- Incubation of the virus templates in aqueous cobalt chloride solution

- Cobalt oxide has large reversible storage capacity arising from displacement reactions : ~ three times larger capacity compared to carbon-based anodes currently used in commercial batteries

- Homogeneous and high-crystalline nanowires : 141.7 m2/g

Schematic diagram of the virus-enabled synthesis and assembly of nanowires as negative electrode materials for Li ion batteries. Rationally designed peptide and/or materials-specific peptides identified by biopanning were expressed on the major coat p8 proteins of M13 viruses to grow Co3O4 and Au-Co3O4 nanowires.

(A) TEM image of virus-templated Co3O4 nanowires. (B) High-resolution TEM image of a Co3O4 viral nanowire. Electron diffraction pattern (Inset, upper right) confirmed that the crystal structure was Co3O4. (C) Charging-discharging curves for a virus-mediated Co3O4/Li half cell cycled between 3 and 0.01 V at a rate of C/26.5. C was defined as eight Li ions per hour. (D) Specific capacity versus cycle number for the same cell. (E) TEM images of differently nanostructured Co3O4 viral nanowires (F) TEM images of the assembly of discrete Co3O4 nanocrystals on the p8 proteins.

Reversible capacity : 600 ~ 750 mA-hour/gTwice that of current carbon-based anodes

New hybrid material electrodes

• Systematic and controlled arrangement of two distinct nanomaterials

• Increase in the electrochemical properties through the cooperative contribution of each material

• Design of composite material

• Hybride Au-Co3O4,

- Gold NPs: high electronic conductivity

- Isolation and expression of gold-binding peptide motif

(LKAHLPPSRLPS) with major coat p8 protein

- Assembly of bifunctional virues expressing both Au- and Co3O4-

specific peptides with the virus coat

- Production and random package of two types p8 proteins :

Characterization of the hybrid nanostructure of Au nanoparticles incorporated into Co3O4 nanowires. (A) Visualization of the genetically engineered M13 bacteriophage viruses. P8 proteins containing a gold-binding motif (yellow) were doped by the phagemid method in E4 clones, which can grow Co3O4. (B) TEM images of the assembled gold nanoparticles on the virus. (C) TEM image of hybrid nanowires of Au nanoparticles/Co3O. (D) Specific capacity of hybrid Au-Co3O4 nanowires. (E) Cyclic voltammograms of hybrid Au-Co3O4 and Co3O4 nanowires at a scanning rate of 0.3 mV/s

Specific capacity of hybride : ~ 30 % greater than Co3O4 nanowires

Two-dimensional assembly of Co3O4 nanowires driven by liquid crystalline ordering of the engineered M13 bacteriophage viruses. (A and B) Phase-mode atomic force microscope image of macroscopically ordered monolayer of Co3O4-coated viruses. The Z range is 30° (C) Digital camera image of a flexible and transparent free-standing film of (LPEI/PAA)100.5 on which Co3O4 viral nanowires are assembled into nanostructured monolayer with dimensions of 10 cm by 4 cm. (D) Capacity for the assembled monolayer of Co3O4 nanowires/Li cell at two different charging rates.

Linear poly(ethylene imine)/Poly(acrylic acid)

◆ 나노기술 핵심은 구동력 = 마이크로 머신의 동력원은 배터리 크기를 극도로 줄이면 되지만 나노 머신의 경우는 배터리 개념 자체를 완전히 바꿔야 함 . 따라서 생체모터의 응용기술 개발이 필수 !!

나노 모터의 모습과 광학현미경으로 얻은 프로펠러의 회전모습 .

ATPase 를 이용한 나노 모터 제조 .

ATPase 는 모터처럼 돌면서 기능을 수행하는 모터분자 : ATP 의 화학 에너지를 이용해 기계적 일을 수행

ATPase 를 작은 기둥 위에 놓고 , 나노 공정 기술을 이용해 여기에 작은 프로펠러를 달았고 ATP 가 공급되었을 때 ATPase 의 모터분자에 부착된 프로펠러가 회전함 .

100% 효율의 단백질 모터