NanoBiotechnology

Lecture by Richard Feynman (1959)

“There is a plenty of room at the bottom”

• We could arrange the atoms one-by-one in a way we want them• High-resolution microscopes would allow a direct look at single molecules

in biological systems• It is very easy to answer many of these fundamental biological questions;

you just look at the thing!

• Unfortunately, the present microscope sees at a scale which is just a bit too crude.

• Make the microscope one hundred times more powerful, and many problems of biology would be made very much easier.

Nanotechnology

• Creation of useful materials, devices, and systems through the manipulation of matter on a nanometer scale.

- Generally nanotechnology deals with structures sized between 1 to 100 nanometer in at

least one dimension.

• Ability to design systems with defined structure and function on the nanometer scale.

• Involves developing materials, devices within that size, and analytical tools (methodology), which can be used for analysis and measurement on a molecular scale

• Interdisciplinary area :

Biology, Physics, Chemistry, Material science, Electronics, Chemical Engineering, Information technology

Nanotechnology Plays by Different Rules

Normal scale Nanoscale

- Nanoscale research is to reach a better understanding of how matter behaves on this small scale. - The factors that govern larger systems do not necessarily apply at the nanoscale. - Because nanomaterials have large surface areas relative to their volumes, phenomena like friction and sticking are more important than they are in larger systems.

Analytical methods and Nano-sized materials

• Analytical tools :

Atomic force microscopy(AFM), Electron microscopy (EM)

• Nano-sized materials

• Unusual and different property

- Semiconductor nanocrystals:

Size-dependent optical property

- Nanoparticles:

Magnetic nanoparticles (Ferromagnetic, superparamagnetic), Gold nanoparticles, Carbon nanotubes, Graphene

- Superparamegnetism: In the absence of an external magnetic field, magnetization is in average zero

Graphene

• Allotrope of carbon in the form of a two-dimensional, atomic-scale, hexagonal lattice

• Extraordinary properties.

- About 200 times stronger than steel by weight

- Conducts heat and electricity with great efficiency

- Nearly transparent

• Potential applications:

-Semiconductor, electronics, battery energy, and composites

• Andre Geim and Konstantin Novoselov at the Univ. Manchester won

the Nobel Prize in Physics in 2010

- Groundbreaking experiments regarding the two-dimensional material graphene

Scanning probe microscopy image

Graphene-Based Nanomaterials

Examples of nano-sized materials

Future implications of nanotechnology

• Nanotechnology may be able to create many new materials and devices with a vast range of applications, such as in medicine, biomaterials, electronics, and energy production.

• Nanotechnology raises many of the same issues as with any introduction of new technology, including concerns about the toxicity and environmental impact of nano-sized materials, and their potential effects on global economics.

Nano-Biotechnology

• Integration of nano-sized/structured materials, nano-scale analytical tools, and nano-devices with biological sciences for development of new biomaterials and analytical tool-box as well as for understanding life science

• Use of bio-inspired molecules or materials

• Typical characteristics of Biological events/materials

- Self assembly- Highly efficient : high energy yield- Very specific : extremely precise

• Bio-moleculesProteins, DNA, RNA, Aptamers, Peptides, Antibody, Virus

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

Applications and Perspectives of Nanobiotechnology

• Development of new tools and methods

- More sensitive

- More specific- Multiplexed

- More efficient and economic

• Implementation

- Diagnosis and treatment of diseases

- Rapid and sensitive detection (Biomarkers, Imaging)

- Targeted delivery of therapeutics (higher therapeutic efficacy, low

side-effects

- Drug development

- Drug target discovery

- Understanding of biology

Examples

• Nano-Biodevices

• Nano-Biosensors

• Drug and gene delivery using nanoparticles

• Imaging with nanoparticles

• Analysis of a single molecule/ a single cell

Issues to be considered

• Synthesis or selection of nano-sized/ structured materials: bottom-up or top-down

• Functionalization with biomolecules or for biocompatibility

• Integration with devices and/or analytical tools

• Assessment : Reproducibility, Toxicity

• Mass production and practical implementation

The size of things

NanoBiotech was initiated by the development of SPM(Scanning Probe Microscopy) that enables imaging at atomic level in 1980

• Scanning Tunneling Microscopy (STM)• Atomic Force Microscopy (AFM)

Scanning Tunneling Microscopy (STM)

• Instrument for imaging surfaces at the atomic level.

• Developed in 1981 by Gerd Binnig and Heinrich Rohrer (at IBM Zürich)

• Nobel Prize in Physics in 1986.

• Resolution : 0.1 nm lateral resolution and 0.01 nm (10 pm) depth resolution.

• Used not only in ultra-high vacuum but also in air, water, and various other liquid or

gas ambients, and at temperatures ranging from near zero kelvin to over 1000°C.

• Operating principle: Concept of quantum tunneling. - When a conducting tip is brought very near to the surface to be examined,

a bias (voltage difference) applied between the two can allow electrons to tunnel

through the vacuum between them.

- The resulting tunneling current is a function of tip position, applied voltage, and local

density of states (LDOS) of the sample.

- Information is acquired by monitoring the current as the tip's position scans across the

surface, and is usually displayed in image form.

- Needs extremely clean and stable surfaces, sharp tips, excellent vibration control,

and sophisticated electronics

• A cantilever with a sharp tip (probe) at its end that is used to scan the specimen surface

• When the tip is brought into a close proximity of a sample surface, force between the tip and sample leads to a deflection of the cantilever according to the Hooke’s law (F= -kX)

• Deflection is measured using a laser spot reflected from the top surface of the cantileverinto an array of photodiode

One of the foremost tools for imaging, measuring, and manipulating matters at the nanoscale.

AFM (Atomic Force Microscope)

• Depending on the situation, forces that are measured in AFM include mechanical contact force, van der Waals forces, capillary forces, chemical bonding, electrostatic forces, magnetic forces (magnetic force microscope, MFM), Casimir forces, solvation forces.

Principle and mode of AFM

Modes of AFM

Contact mode:

- Tip is "dragged" across the surface of the sample and the contours of the surface are measured either using the deflection of the cantilever directly or, more commonly, using the feedback signal required to keep the cantilever at a constant position.

- Because the measurement of a static signal is prone to noise and drift, low stiffness cantilevers (i.e. cantilevers with a low spring constant, k) are used to achieve a large enough deflection signal while keeping the interaction force low.

- Close to the surface of the sample, attractive forces can be quite strong, causing the tip to "snap-in" to the surface.

- Thus, contact mode AFM is almost done at a depth where the overall force is repulsive, that is, in firm "contact" with the solid surface.

Tapping mode (Intermittent contact mode)

• In ambient conditions, most samples develop a liquid meniscus layer. • Keeping the probe tip close enough to the sample for short-range forces to become

detectable while preventing the tip from sticking to the surface presents a major problem for contact mode in ambient conditions.

• Dynamic contact mode (also called intermittent contact) was developed to bypass this problem.

• Currently, tapping mode is the most frequently used AFM mode when operating in ambient conditions or in liquids

• Cantilever is driven to oscillate up and down at or near its resonance frequency. • Amplitude of this oscillation usually varies from several nm to 200 nm. • Frequency and amplitude of the driving signal are kept constant, leading to a constant

amplitude of the cantilever oscillation as long as there is no drift or interaction with the surface.

• Interaction of forces acting on the cantilever when the tip comes close to the surface, Van der Waals forces, dipole-dipole interactions, electrostatic forces, etc. cause

the amplitude of the cantilever's oscillation to change (usually decrease) as the tip gets closer to the sample.

• This amplitude is used as the parameter that goes into the electronic servo that controls the height of the cantilever above the sample

- Tip of the cantilever does not contact the sample surface. - The cantilever is instead oscillated at either its resonant frequency (frequency modulation) or just above (amplitude modulation) where the amplitude of oscillation is typically a few nanometers(<10 nm) down to a few picometers.

- The van der Waals forces, which are strongest from 1 nm to 10 nm above the surface, or any other long-range force that extends above the surface acts to decrease the resonance frequency of the cantilever.

- This decrease in resonant frequency combined with the feedback loop system maintains a constantoscillation amplitude or frequency by adjusting the average tip-to-sample distance.

- Measuring the tip-to-sample distance at each data point : to construct a topographic image of the sample surface

Non-contact mode

Imaging profiles

VEECO

TESPA®

VEECO

TESPA-HAR®

NANOWORLD

SuperSharpSilicon®

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

Resolution of protein structure by AFM

Image of ATP synthase composed of 14 subunits

Molecular imaging

• Biomedical & Biological 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, localization of proteins,

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

Properties and Biological Applications

Semiconductor Nanocrystals

Quantum Dots

Synthesis of CdSe/ZnS (Core/Shell) QDs

Step 1

CdO + Se CdSe

Step 2

ZnEt2 + S(TMS)2CdSe

ZnS

Solvent : TOPO, HAD, TOPSurfactant : TDPA, dioctylamine

Growth temperature 140 ℃ (green)

200 ℃ (red)

20 nm

CdSe/ZnS 5.5 nm

(red)

Bawendi et al. J. Am. Chem. Soc. (1994)

Optical Properties of Quantum Dots

a) Multiple colors with size b) Photostability

c) Wide absorption and narrow emission d) High quantum yield

Quantum Yield ≥ 60 ~ 70 %

Single source excitation

Coating of QD Surface for Biocompatibility

CdSe

ZnS

CdSe

ZnS

O P

O P

OPO

P

OP

OP

OP

CdSe O P

O PO

POP

OP

OP

OP

CdSe

ZnS

CdSe

ZnS

O P

O P

OPO

P

OP

OP

OP

NH2

NH2

NH2

NH2

+N

+N

+N

+N

NH2

NH2

NH2

NH2

+N

CdSe/ZnS core-shell Quantum Dots encapsulated in

phospholipid micelles

CdSe QDs

Dubertret et al. Science (2002)

PEG-PE (n-poly(ethylenglycol) phosphatidylethanolamine): micelle-forming hydrophilicpolymer-grafted lipids comparable to natural lipoproteins

PEG : low immunogenic and antigenic, low non-specific protein binding PC : Phosphatidylcholine

Encapsulation with the hydrophobic core of a micelle

Coating with PC

Coating of the outer Shell with ZnS

QD

Y

Organelle

+

QD-Antibodyconjugates

Antigen

QD

Y

Organelle

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

In vitro imaging

Wu et al. Nature Biotech. 2003 21 41

- Multiple Color Imaging- Stronger Signals

In vivo 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

• Inherent capabilities of molecular recognition and self-assembly

• Attractive template for constructing and organizing the nano-structures

• Proteins, toxin, coat proteins of virus etc.

Bio-inspired systems

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 lysis of red blood cells

• α-Hemolysin monomers bind to the outer membrane of the cells.

• 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 50 A beta-barrel stem

- Narrowest part (1.4 nm in diameter) of channel at the base of stem

- Magnitude of the current reduction : type of analyte

- Frequency of current reduction intervals: 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

Biotechnological applications : Stochastic sensors

Stochastic system: systems that are unpredictable due to the influence of a random variable

Construction of stochastic sensors

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

Cyclodextrins

• Family of compounds made up of sugar molecules bound together in a ring : cyclic oligosaccharides).

• Comprising hydrophobic inside and hydrophilic outside, they can form complexes with hydrophobic compounds.

• Enhance the solubility and bioavailability of such compounds. • Useful for pharmaceutical as well as dietary supplement applications in which

hydrophobic compounds shall be delivered.

DNA sequencing

Transmembrane pore can conduct big (tens of kDa) linear macromolecules like DNA or RNA

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

Changes in the ionic current by the chemical structure of individual strands

Nucleotide sequence directly from a DNA or RNA strand

A single nucleotide resolution

DNA sequencing by nanopore

Understanding Cancer and Related Topics

Understanding Nanodevices

Developed by:Jennifer Michalowski, M.S.Donna Kerrigan, M.S.Jeanne KellyBrian Hollen

Explains nanotechnology and its potential to improve cancer detection, diagnosis, and treatment. Illustrates several nanotechnology tools in development, including nanopores, quantum dots, and dendrimers.

These PowerPoint slides are not locked files. You can mix and match slides from different tutorials as you prepare your own lectures. In the Notes section, you will find explanations of the graphics.

The art in this tutorial is copyrighted and may not be reused for commercial gain.Please do not remove the NCI logo or the copyright mark from any slide. These tutorials may be copied only if they are distributed free of charge for educational purposes.

What Is NanoBiotechnology?

NanodevicesNanoporesDendrimersNanotubesQuantum dotsNanoshells

Tennis ballA periodWhite blood cell

Water molecule

Designing Nano-devices for Use in the Body

Too BigToo Small

Manufacturing Nanodevices

Scattered X-rays

Atoms in crystal

Detector

NanodevicesCrystal White blood cell

CrystalX-ray beam

• Top-down approach: Molding or etching materials into smaller components

• Bottom-up approach :Assembling structures atom-by-atom or molecule-by-molecule, useful in manufacturing devices used in medicine.

Nanodevices Are Small Enough to Enter Cells

Cell (10,000~ 20,000 nm)

White blood cell

Water molecule

Nanodevices

Nanodevices

Nano-devices can improve cancer detection and diagnosis at early stages

ImagingNanoBiotechnology Physical Exam,Symptoms

Nanodevices can improve sensitivity

and determinewhich cells arecancerous orprecancerous.

Precancerous cells

Normal cells

Nanodevices could potentiallyenter cells

Precancerous cells

Normal cells

Nanodevices can preserve patients’ samples

Cells from patientCells preserved

Active state preserved

Cells altered Active state lost

Additional tests

Cells from patient

Nanotechnology Tests

Traditional Tests

Nanodevices can make cancer testsfaster and more efficient

Patient A Patient B

Many diagnostic tests

simultanelusly

Cantilevers can make cancer testsfaster and moreefficient

Cancer cell

Watermolecule

NanodevicesCantilevers

Antibodieswith proteins

Antibodies

Whiteblood cell

Bent cantilever

Nanopores

ASingle-strandedDNA molecule

Single-strandedDNA molecule

Whiteblood cell

Watermolecule

NanodevicesNanopores

Single-strandedDNA molecule

ATCGNanopore Nanopore

Nanopore

T

Quantum DotsUltraviolet

light off

Whiteblood cell

Watermolecule

NanodevicesQuantum dots

Quantum dots emit light

Ultravioletlight on

Quantumdots

Quantum dotbead

Quantum dots can find cancer signatures

Cancer cells

Healthy cells

Quantum dot beads

Quantum dot beads

Healthy cells

Cancer cells

Improving cancer treatment

Nanotechnology TreatmentTraditional Treatment

Intact noncancerous cells

Noncancerous cells

Toxins Nanodevices

Cancercells

Dead noncancerous cells

Noncancerous cells

Drugs

Deadcancercells

Deadcancercells

ToxinsCancercells

Nanoshells

Nanoshell

Whiteblood cell

Watermolecule

NanodevicesNanoshells

Gold

Near-infrared light onNear-infrared light off

Nanoshell absorbs heat

Nanoshells as cancer therapyNanoshells

Dead cancer cells

Nanoshells

Cancer cells

Healthy cells

Intact healthy cells

Near-infrared light

Healthy cells

Cancer cells

Nanodevices as a link betweendetection, diagnosis, and treatment

Nanodevice

Reporting

TargetingDetection

Imaging

NanoBiotechnologyCancer Treatment

Cancer cell

TraditionalCancer Treatment

Cancer cell

Drug

Dendrimers

Dendrimer

Whiteblood cell

Watermolecule

NanodevicesDendrimer

Cancer cell

Dendrimers : Highly Branched Dendritic

Macromolecules

● Characteristics

Monodisperse macromolecule

Globular (Spherical)

Facile surface bio-functionalization

Similar molecular size to biomolecules

(Glucose oxidase 65.27.7 nm)

Molecular carriers for chemical

catalysts

(Core, Peripheral)

Medical applications (MRI contrast

enhancer)

Biomimetic catalysts

(Peptides-, Glycodendrimers)

Vehicles for delivery of genes and

drugs

Poly (amido amine) Dendrimers

4.5 nm

G4 Poly(amidoamine)

Dendrimer

● Applications

Dendrimers as cancer therapy

Cell deathmonitor

Reporter

Therapeuticagent

Cancerdetector

Watermolecule

Whiteblood cell

NanodevicesDendrimer

Manipulate dendrimers to release their contents only in the

presence of certain trigger (molecules or light) caged molecules

NanoBiotechnology in Diagnosis and

Patient Care

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