nanobiotechnologybel.kaist.ac.kr/.../bs223/nanobiotechnology_2018.pdf · 2018-06-08 ·...
TRANSCRIPT
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
Today 2020